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  • 101
    Electronic Resource
    Electronic Resource
    Detection of chromosome instability of tissue fields at risk: In situ hybridization (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 63 (1996), S. 57-62 
    Details
    ISSN: 0730-2312
    Keywords: cancer risk ; genetic instability ; in situ hybridization ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Many human tumors are thought to develop along a multistep pathway in tissues that have encountered long periods of carcinogen exposure and thus have accumulated genetic hits in functional targets relevant to tumor evolution. The cumulative degree of genetic change is dependent on both exogenous (e.g., degree of carcinogen exposure) and endogenous factors (e.g., metabolism of procarcinogens, repair or misrepair capacity, proliferation properties of the tissue, capability of damaged cells to survive). Thus one approach to risk estimation is to measure the accumulated amount of genetic damage in a target tissue at risk for tumor development. Since one cannot predict the exact site of the future tumor, the risk assay must detect a generalized ongoing process of genetic instability from small, random biopsies. The technique of chromosome in situ hybridization involves the use of chromosome- or region-specific probes and provides an ability to directly visualize genetic change (e.g., random or clonal chromosome polysomy and monosomy) on thin tissue sections (where tissue architecture is maintained) or exfoliated cells. Analyses of normal and premalignant lesions adjacent to tumors (e.g., head and neck, lung, bladder, cervix, breast) have demonstrated that chromosome instability can be detected in the field of the tumor (i.e., in normal and premalignant cells in a tissue at 100% risk of tumor development) and the degree of chromosome instability increases with the degree of histologic progression toward cancer. Analyses of premalignant lesions (e.g., oral leukoplakia and erythroplakia from individuals at risk for aerodigestive tract cancer) by chromosome in situ hybridization have uncovered varying degrees of chromosome instability. However, approximately half of those individuals who showed a high degree of chromosome instability in biopsies subsequently developed aerodigestive tract cancer. Of interest, half of these tumors have developed away from the biopsied site, suggesting that the detection of a chromosome instability process in one aspect of the tissue might yield risk information for the total tissue field. These studies also suggest that chromosome in situ hybridization might be useful for identifying individuals with high tumor risk who might benefit from chemopreventive intervention. J. Cell. Biochem. 25S:57-62. © 1997 Wiley-Liss, Inc.
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  • 102
    Electronic Resource
    Electronic Resource
    Mutational specificity and cancer chemoprevention (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 63 (1996), S. 99-107 
    Details
    ISSN: 0730-2312
    Keywords: mutational assays ; mutational spectra ; monitoring mutation in people ; transgenic animals ; lacl ; hprt T-cell clonal assay ; chemoprevention ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Mutational specificity describes the composite of all of the genetic alterations in a collection of mutations arising from a specific treatment. The information includes not only the nature of the genetic change (e.g., a base substitution or a frameshift), but also information about nucleotide position and hence the DNA context. As both the type of DNA damage and its position can be expected to reflect the nature of the chemical and physical mutagen, mutational specificity can be expected to provide insights into mechanisms of mutation. Conversely, mutational spectra should also provide insights into the identity of the mutagen. Indeed, the pioneering work on mutational specificity in Escherichia coli indicates that each physical or chemical treatment produces a unique spectrum of mutations.With the application of biotechnology to the field of genotoxicology, the database of sequenced mutations has become quite substantial. Both in vitro and in vivo data has been obtained following exposure to a variety of agents. In this communication we will critically assess whether the reality of mutational specificity has fulfilled the expectations and to examine what potential remains to be explored, especially in the area of monitoring human populations. The usefulness of both mutational spectra analysis and population monitoring with regards to chemoprevention are discussed. J. Cell. Biochem. 25S:99-107. © 1997 Wiley-Liss, Inc.
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  • 103
    Electronic Resource
    Electronic Resource
    Identification of a chemoprevention cohort from a population of women at high risk for breast cancer (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 63 (1996), S. 112-122 
    Details
    ISSN: 0730-2312
    Keywords: biomarkers ; breast cancer ; chemoprevention ; high-risk ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: In a prospective pilot study, we performed breast fine needle aspirations (FNAs) on 213 high-risk and 30 low-risk women and analyzed these aspirates for cytologic changes and biomarker abnormalities of aneuploidy and overexpressed estrogen receptor (ER), epidermal growth factor receptor (EGFR), p53 and HER-2/neu. High-risk women were those with a first degree relative with breast cancer (73%), prior biopsy indicating premalignant breast disease (26%), a history of breast cancer (13%), or some multiple of these risk factors (11%). Median ages of the high-risk and low-risk groups were 44 and 42, respectively. Sixty-three percent of the high-risk and 73% of the low-risk group were premenopausal. Sixty-eight percent of the high-risk and 17% of low-risk women had cytologic evidence of hyperplasia with or without atypia (P 〈 .0001). Aneuploidy and overexpression of EGFR and p53 occurred in 25%, 36%, and 28% of high-risk subjects but in less than 4% of low-risk subjects (P 〈 .0002). Overexpression of ER and HER-2/neu occurred in 8% and 19%, respectively of high-risk women; no low-risk women had these abnormalities. Sixty-eight percent of high-risk women and 7% of low-risk women had abnormalities of one or more of these biomarkers exclusive of cytology. Thirty-one percent of high-risk women, but no low-risk women had abnormalities of two or more biomarkers (P = .0004). Biomarker abnormalities were more frequent with increasing cytologic abnormality. Eighteen percent of women with normal cytology, 29% of women with epithelial hyperplasia and 60% of women with hyperplasia with atypia had abnormalities of two or more biomarkers (P = .048 and 〈 .0001, respectively). Restricting the analysis to those three biomarkers most frequently overexpressed in the high-risk group (ploidy, EGFR, p53), 13% of high-risk women with normal cytology, 20% of high-risk women with epithelial hyperplasia and 51% of high-risk women with atypical hyperplasia had abnormalities of 2 or more of these 3 biomarkers. At a median follow up of two years, 8 of 213 women have been diagnosed with in situ (n = 5) or invasive (n = 3) cancer. Later detection of neoplasia was associated with prior FNA evidence of atypical hyperplasia (P 〈 .0001) and multiple biomarker abnormalities in the 5 test battery (P = .006) by univariate analysis. By multivariate analysis, development and/or detection of cancer was primarily predicted by atypical hyperplasia (P = .0047) and secondarily by multiple biomarker abnormalities (P = 0.021). Atypical hyperplasia, EGFR, and p53 in breast FNAs have promise as risk markers and as surrogate endpoint biomarkers for breast cancer chemoprevention trials. J. Cell. Biochem. 25S:112-122. © 1997 Wiley-Liss, Inc.
    Additional Material: 3 Ill.
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  • 104
    Electronic Resource
    Electronic Resource
    Large bowel adenomas: Markers of risk and endpoints (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 63 (1996), S. 142-148 
    Details
    ISSN: 0730-2312
    Keywords: adenoma ; clinical trial ; large bowel ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: In many large bowel chemoprevention trials adenomas have a double duty: they are used to identify subjects at risk for large bowel neoplasia, and also serve as endpoints. Many features of adenomas make them suitable for these tasks. Patients with adenomas are fairly numerous and easy to identify; further, the ‘adenoma-carcinoma’ sequence suggests that adenomas are logical endpoints. The high recurrence risk among adenoma patients means that a relatively modest number of subjects will suffice for adequate statistical power.The are some limitations to the use of adenomas, however. There is clearly heterogeneity of risk for subsequent cancer. Patients with only small adenomas may have rates of colorectal cancer that are not much greater than those of the general population. Certainly subjects with larger adenomas, and those with villous or highly dysplastic adenomas have a higher risk. Often, one would chose the high-risk patients for preventive interventions. Such a strategy makes sense from a risk-benefit point of view. However, from a population perspective, such a strategy may well have only a minor impact on the overall colorectal cancer burden. For more complete population-based prevention, efforts will have to be directed to the numerous individuals who are each at small risk, but who collectively account for most colorectal cancer. For this preventive approach, patients with any adenoma would certainly be part of the target population, and so are sensible subjects in chemoprevention trials.There are similar complexities in consideration of the use of adenomas as endpoints of chemoprevention trials. The adenomas that occur in prevention trials are generally small, and may not be associated with a greatly increased cancer risk. The issue for chemoprevention trials, however, is not whether the endpoints are truly intermediate in the causal chain - but whether the intervention under study alters the adenoma recurrence risk to the same extent as it does for colorectal cancer risk. This is a difficult matter to verify, but the limited data available are encouraging. The epidemiology of colorectal adenomas (largely small adenomas) is similar in many regards to that for colorectal cancer itself. Thus to the extent that data are available, one can tentatively conclude that external influences affect adenomas and colorectal cancer similarly.To date, more than ten adenoma prevention trials have reported results. The data have been fairly consistent. Vitamin C (with or without vitamin E) has provided at most a modest protective benefit, except in one small trial in which it was combined with vitamin E and preformed vitamin A. β-Carotene seems to be without any effect, and interventions to increase fiber and decrease fat intake have not indicated substantial effects. On the other hand, trials among familial polyposis patients have provided evidence for an impact of nonsteroidal anti-inflammatory drugs. Studies in progress have the potential to clarify greatly the preventive potential of the currently promising - but yet unproven - chemopreventive regimens. J. Cell. Biochem. 25S: 142-148. © 1997 Wiley-Liss, Inc.
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  • 105
    Electronic Resource
    Electronic Resource
    Genetic alterations as clonal markers for bladder cancer detection in urine (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 63 (1996), S. 191-196 
    Details
    ISSN: 0730-2312
    Keywords: bladder tumor ; cancer screening ; molecular markers ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Bladder cancer is the result of a clonal expansion of cancer cells in which multiple genetic alterations have accumulated. Point mutations of the p53 gene are frequently observed in bladder cancer. Loss of a retinoblastoma (Rb) allele is also common in bladder cancer. Recent data have shown frequent loss of heterozygosity (LOH) and homozygous deletion of 9p21, including the region of p16INK4A, a putative tumor suppressor gene, in bladder cancer. LOH is also observed frequently at several other chromosome regions in bladder cancer. These genetic changes have proved useful as clonal markers in the detection of cancer cells in urine. Because of their complexity, most molecular diagnostic approaches are not considered promising cancer screening tools in patients or high-risk populations. However, a new molecular approach, the examination of microsatellite alterations in bladder cancer and urine specimens, is a promising screening tool for the disease. The common genetic alterations in bladder cancer and their use as clonal markers in screening or diagnosis strategies will be discussed. J. Cell. Biochem. 25S: 191-196. © 1997 Wiley-Liss, Inc.
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  • 106
    Electronic Resource
    Electronic Resource
    Examination of p53 alterations and cytokeratin expression in sputa collected from patients prior to histological diagnosis of squamous cell carcinoma (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 63 (1996), S. 185-190 
    Details
    ISSN: 0730-2312
    Keywords: cytokeratins ; lung cancer ; p53 overexpression ; sputum ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Mutations in the p53 gene are detected in greater than 50% of squamous cell carcinomas of the lung and to a lesser extent in adenocarcinomas. The p53 protein is also overexpressed in a relatively high percentage of preinvasive lesions of the bronchial epithelium. However, unlike tumor tissue, immunoreactivity does not necessarily imply that cells in preinvasive lesions carry a mutant p53 allele. In some cases, overexpression may result from a cellular checkpoint reaction to a toxic or mutagenic substance such as exposure to tobacco smoke. In any case, p53 overexpression in preinvasive lesions may serve as a biomarker for high risk assessment of lung cancer and other tumors in the aerodigestive tract. A study was designed to retrospectively analyze p53 overexpression in cells from sputum samples collected prior to histological tumor diagnosis. The rationale was based on the observation that both preinvasive and tumor cells from the bronchial epithelium are exfoliated into the airways and can be detected based on morphology in sputa. Two sets of cases were chosen: 1) patients whose first primary tumor was a squamous cell carcinoma containing a mutant p53 allele with overexpression observed in most of the tumor cells; and 2) patients whose squamous cell tumor did not contain a mutant p53 allele. Cells which stained positive for p53 expression were observed in sputum samples collected from all six patients whose tumors were positive for a mutant p53 allele. Also p53 positive cells were detected on sputum slides for two of the five cases where the tumor DNA did not contain a mutation and/or tumor cells which overexpress p53 were not detected in tissue sections. Although cells which stained positive for p53 were present in sputum from patients whose tumors contained a missense mutation, the presence of p53 overexpression was not specific for tumors which contain an altered p53 allele since overexpression was detected in sputum cells from patients whose tumor DNA did not contain a p53 mutation and/or tumor cells which stained positive for p53 were not observed in tissue sections. However, the p53 positive cells in sputa collected from the latter group of patients could have been exfoliated from other lesions which contained a mutant p53 allele. The accumulation of p53 in some sputum cells was concomitant with expression of simple epithelial type cytokeratins (CK) 8 and 18 or at least one of the other cytokeratins detected by a broad spectrum (PAN) CK antibody mixture. These data imply that most of the sputum cells which overexpress p53 are epithelial cells. Moreover, our results are consistent, at least in part, with other observations that cells which overexpress p53 in dyplasias and hyperplasias express CK 8, 18. We will continue to explore the possibility that expression of cytokeratins 8, 18 and/or other cytokeratins in conjunction with p53 overexpression and/or morphological criteria could define a new class of atypical cells which are predisposed to cancer development. J. Cell. Biochem. 25S:185-190. © 1997 Wiley-Liss, Inc.
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  • 107
    Electronic Resource
    Electronic Resource
    G-actin as a risk factor and modulatable endpoint for cancer chemoprevention trials (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 63 (1996), S. 197-204 
    Details
    ISSN: 0730-2312
    Keywords: biomarkers ; chemoprevention ; cancer risk factor ; G-actin ; retinoids ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Because tumorigenesis is an ongoing process, biomarkers can be used to identify individuals at risk for bladder cancer, and treatment of those at risk to prevent or slow further progression could be an effective means of cancer control given accurate individual risk assessment. Tumorigenesis proceeds through a series of defined phenotypic changes, including those in genetically altered cells destined to become cancer as well as in surrounding normal cells responding to the altered cytokine environment. A panel of biomarkers for the changes can provide a useful system for individual risk assessment in cancer patients and in individuals exposed to carcinogens. The use of such markers can increase the specificity of chemoprevention trials by targeting therapy to patients likely to respond, and thereby markedly reduce the costs of the trials.Previous studies in our laboratories showed the cytoskeletal proteins G- and F-actin reflect differentiation-related changes in cells undergoing tumorigenesis and in adjacent “field” cells, and a pattern of low F-actin and high G-actin is indicative of increased risk. Actin changes may be a common feature in genetic and epigenetic carcinogenic mechanisms. In a group of over 1600 workers exposed to benzidine, G-actin correlated with exposure, establishing it as an early marker of effect. In another study, a profile of biomarkers was monitored in patients who underwent transurethral resection of bladder tumor (TURBT) and received Bacillus Calmette Guerin (BCG) and/or DMSO. The primary objective was to determine how the defined biomarkers expressed in the tumor and the field correlate with clinical response and recurrence. DMSO, known to modulate G-actin in vitro, was used as an agent. Results strongly support the hypothesis that cytosolic G-actin levels measured by quantitative fluorescence image analysis (QFIA) can be an important intermediate endpoint marker for chemoprevention and that the p300 (M344) and DNA ploidy markers identify a high-risk group that requires more aggressive therapy and recurrence monitoring. Further research with other markers has shown that DD23 and nuclear actin, both of which identify late, specific changes, may increase the battery of useful markers. Taken together these studies show how biomarkers are employed to study individuals at risk, aid in the selection of chemopreventive compounds and assist in the understanding of the pathogenesis of malignancy. J. Cell. Biochem. 25S:197-204. © 1997 Wiley-Liss, Inc.
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  • 108
    Electronic Resource
    Electronic Resource
    Conversion of dermal fibroblasts to a myogenic lineage is induced by a soluble factor derived from myoblasts (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 61 (1996), S. 363-374 
    Details
    ISSN: 0730-2312
    Keywords: MyoD ; myosin heavy chain ; muscle ; desmin ; mouse ; myogenesis ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The limb and axial skeletal muscles of mammals originate from somitic dermomyotome, which during early development separates to form two discrete structures, the dermatome and the myotome. The latter cell mass gives rise to the muscle-forming lineage while cells of the dermatome will form the skin dermal fibroblast population of the dorsal regions of the body. It has been generally accepted for some time that myotome-derived myoblasts were the sole source of muscle fibre nuclei, but evidence has recently been presented from several laboratories that fibroblasts can fuse with myoblasts to contribute active nuclei to the resulting myotubes.We report here an investigation into the myogenic capacity of fibroblasts. Confluent monocultures of mouse dermal fibroblasts, muscle fibroblasts, and C2C12 myoblasts each retain their individual phenotype when maintained for periods up to 7 days in culture. We also grew isolated colonies of fibroblasts and myoblasts in an arrangement which allowed free exchange of tissue culture medium between the 2 cell types. We found evidence of the conversion of dermal fibroblasts to a myogenic lineage as measured by the appearance of MyoD-positive cells expressing the muscle-specific intermediate filament desmin. In addition, dermal fibroblast cultures contained multinucleate syncytia positive for MyoD and containing sarcomeric myosin heavy chain. In contrast, muscle-derived fibroblasts showed no evidence of myogenic conversion when maintained in identical culture conditions. We prepared conditioned medium from confluent cultures of C2C12 myoblasts and added this material to confluent monocultures of either dermal or muscle fibroblasts. While muscle fibroblasts showed no phenotypic alterations, cultures of dermal fibroblasts responded to myoblast conditioned medium by converting to a myogenic lineage as judged by expression of MyoD and desmin. We conclude that a proportion of dermal fibroblasts retain a myogenic capacity into stages well beyond their early association with myoblasts in the dermomyotome. © 1996 Wiley-Liss, Inc.
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  • 109
    Electronic Resource
    Electronic Resource
    Regulation of urokinase-type plasminogen activator expression by an ERK1-dependent signaling pathway in a squamous cell carcinoma cell line (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 61 (1996), S. 430-443 
    Details
    ISSN: 0730-2312
    Keywords: urokinase-type plasminogen activator ; ERK ; MAPK ; c-raf ; AP-1 ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The urokinase-type plasminogen activator contributes to tissue remodeling by controlling the synthesis of the extracellular matrix-degrading plasmin. We undertook a study to determine the role of the extracellular signal-regulated kinases (ERKs) in the regulation of urokinase-type plasminogen activator expression in a squamous cell carcinoma cell line (UM-SCC-1) that contains a transcriptionally activated urokinase-type plasminogen activator gene. Transient transfection studies using a CAT reporter driven by the urokinase-type plasminogen activator promoter, which had progressive 5′ deletions or which had been point-mutated, indicated the requirement of binding sites for AP-1 (-1967) and PEA3 (-1973) for its maximal activation. Expression of a mutant jun protein, which lacks the transactivation domain, caused a dose-dependent repression of a CAT reporter driven by either the urokinase-type plasminogen activator promoter or three tandem AP-1 repeats upstream of a thymidine kinase minimal promoter indicating the importance of AP-1-binding transcription factor(s) in the regulation of urokinase-type plasminogen activator synthesis. Mobility shift assays with UM-SCC-1 nuclear extract revealed binding of fos and junD proteins to an oligonucleotide spanning the AP-1 site at -1967. In-gel kinase assays indicated the constitutive activation of ERK1, which regulates fos synthesis via phosphorylation of p621CT, but not ERK2, in UM-SCC-1 cells. Moreover, the expression of a dominant-negative ERK1, but not ERK2, repressed urokinase-type plasminogen activator promoter activity. Similarly, interfering with the function of the c-raf serine-threonine kinase, which lies upstream of ERK1, by the expression of a kinase-inactive c-raf repressed the activity of a CAT reporter driven by either the urokinase-type plasminogen activator promotor or tandem AP-1 repeats. These data suggest that urokinase-type plasminogen activator expression in UM-SCC-1 cells is regulated partly by an ERK1, but not ERK2, -dependent signaling pathway. © 1996 Wiley-Liss, Inc.
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  • 110
    Electronic Resource
    Electronic Resource
    Application of an in vitro system in the study of chemotherapeutic drug effects on DNA replication (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 61 (1996), S. 444-451 
    Details
    ISSN: 0730-2312
    Keywords: in vitro DNA replication ; mammalian ; doxorubicin ; araC ; progesterone ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: DNA replication machinery is an important target for chemotherapeutic drugs. We have used an in vitro system to study the effect of drugs on mammalian DNA replication, either by direct interaction with the DNA structure or with replication proteins and machinery. The anthracycline doxorubicin (Dox) showed a dose-dependent inhibitory effect on DNA replication, whether incubated with HeLa cell extracts or with DNA and nucleotides. Earliest-labeled fragment analysis revealed that inhibition of replication began within the origin-containing fragment in both control and Dox-containing reactions in vitro. AraC, a nucleoside analog, had no significant effect on DNA synthesis. In contrast, araCTP was able to inhibit DNA replication in vitro. Since metabolism is diminished in this in vitro system, the degree of phosphorylation of araC was apparently low. Progesterone showed an increase in nucleotide incorporation (sensitive to BuPdGTP inhibition of replication-specific polymerases α and δ) after preincubation with HeLa cell extracts, although progesterone receptors were not detectable in the HeLa cell extracts. In addition, we observed an inhibition in DNA replication when progesterone was preincubated with DNA and nucleotides. These results suggest that progesterone may have a mechanism of action that is different from any known to be mediated through progesterone receptors. In conclusion, these results indicate that this mammalian in vitro replication system will be useful for the study of mechanisms and design of therapeutic drugs that inhibit mammalian DNA replication. © 1996 Wiley-Liss, Inc.
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  • 111
    Electronic Resource
    Electronic Resource
    Reproducibility of erythrocyte polyamine measurements and correlation with plasma micronutrients in an antioxidant vitamin intervention study (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 62 (1996), S. 19-26 
    Details
    ISSN: 0730-2312
    Keywords: polyamines ; erythrocyte ; biomarker ; reproducibility ; plasma micronutrients ; antioxidant ; intervention ; cancer prevention ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Erythrocyte polyamine measurements have been previously investigated as candidate biomarkers for hyperproliferation and recently as a potential intermediate endpoint in clinical chemoprevention trials with difluoromethylornithine, an inhibitor of polyamine biosynthesis. This study was performed to determine the reproducibility of erythrocyte polyamine measurements and their possible correlation with plasma micronutrients in seven healthy adults in an antioxidant vitamin intervention study. As part of this cross-over intervention study, three subjects took β-carotene (31.4 mg/day) plus D-α-tocopherol acetate (720 IU/day) supplements during the first 3 months and four subjects took the supplements during the second 3 months. Heparinized blood samples were collected at baseline and every month over total 6 months for simultaneous determination of erythrocyte polyamines and plasma micronutrients by the high-performance liquid chromatographic method. For all the measures of erythrocyte polyamines the intraindividual variation was smaller than that between subjects, and three or four measurements required to accurately characterize long-term erythrocyte polyamines for an individual. The intra-class correlations were moderately high for all erythrocyte polyamine measurements, indicating a good reproducibility for intra-individual erythrocyte polyamine measurements. Based on monthly values, significant inverse correlations were found between erythrocyte spermidine and the plasma levels of retinol (r = -0.50) and lutein (r = -0.52). There were also significant inverse associations between erythrocyte spermine and plasma levels of α-tocopherol (r = -0.29), lutein (r = -0.44), lycopene (r = -0.29), β-cryptoxanthin (r = -0.30), and total carotenoids (r = -0.29). The effects of supplementation upon the associations between erythrocyte polyamines and plasma nutrient levels were additionally addressed. The results indicate an acceptable longitudinal reproducibility of erythrocyte polyamine measurements, support the hypothesis that erythrocyte polyamine measurements may be correlated with plasma levels of certain nutrients, and suggest a further biomarker application in cancer prevention trials involving dietary modifications or specific relevant micronutrients. © 1996 Wiley-Liss, Inc.
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  • 112
    Electronic Resource
    Electronic Resource
    In vitro fusion of endocytic vesicles: Effects of reagents that alter endosomal pH (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 62 (1996), S. 27-39 
    Details
    ISSN: 0730-2312
    Keywords: ricin ; transferrin ; monensin ; bafilomycin A1 ; chloroquine ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Ricin, a plant toxin that binds to galactose-terminated glycoproteins and glycolipids on the cell surface, is internalized into endosomes before reaching the cytosol where it exerts its toxic activity. Fusion of early endosomes containing ricin or transferrin was demonstrated by using postnuclear supernatant fractions from K-562 cells. For both ligands, fusion depended on time, temperature, and ATP and was blocked by preincubation with N-ethylmaleimide. Some reagents that increase endosomal pH, the ionophores monensin and nigericin and the weak base chloroquine, stimulated the rate of fusion. However, bafilomycin A1, a specific inhibitor of vacuolar H+-ATPases, did not alter the rate of fusion. Moreover, it reduced or eliminated stimulation caused by monensin, nigericin, or chloroquine. Thus, the increased rate of fusion did not correlate with the higher lumenal pH of the endosome. The results suggest instead that fusion was stimulated by reagents that promoted accumulation of cations within the vesicles. © 1996 Wiley-Liss, Inc. This article is a U.S. Government work and, as such, is in the public domain in the United States of America.
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  • 113
    Electronic Resource
    Electronic Resource
    Protein kinase C activation inhibits stress-induced synthesis of heat shock protein 27 in osteoblast-like cells: Function of arachidonic acid (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 62 (1996), S. 69-75 
    Details
    ISSN: 0730-2312
    Keywords: heat shock protein 27 ; arachidonic acid ; protein kinase C ; osteoblast ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Exposure of osteoblast-like MC3T3-E1 cells to sodium arsenite (arsenite) increased the level of heat shock protein 27 (hsp27). The effect of arsenite was dose-dependent in the range of 50 to 200 μM. Arsenite also stimulated arachidonic acid release dose-dependently in the range between 50 and 200 μM in these cells. Both indomethacin, an inhibitor of cyclooxygenase, and nordihydroguaiaretic acid, a lipoxygenase inhibitor, significantly enhanced the arsenite-induced accumulation of hsp27. Melittin, an activator of phospholipase A2, significantly enhanced the arsenite-induced accumulation of hsp27. 12-O-Tetradecanoylphorbol-13-acetate (TPA), a protein kinase C (PKC)-activating phorbol ester, inhibited the arsenite-induced accumulation of hsp27. In contrast, 4α-phorbol 12, 13-didecanoate (4α-PDD), a PKC-nonactivating phorbol ester, had little effect. TPA suppressed the arsenite-induced arachidonic acid release, but 4α-PDD had little effect. Arsenite no longer affected cAMP accumulation, inositol phosphates formation nor the formation of choline and phosphocholine in these cells. These results suggest that the response to stress of hsp27 is coupled with the metabolic activity of the arachidonic acid cascade, and the activation of PKC inhibits the induction of hsp27 through the suppression of arachidonic acid release in osteoblast-like cells. © 1996 Wiley-Liss, Inc.
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    Active transforming growth factor-β in human melanoma cell lines: No evidence for plasmin-related activation of latent TGF-β (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 62 (1996), S. 113-122 
    Details
    ISSN: 0730-2312
    Keywords: transforming growth factor-β ; melanoma ; activation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Cultured human melanoma cells were found to secrete TGF-β mostly in latent biologically inactive form but in addition five of six melanoma cell lines studied produced in conditioned culture medium active TGF-β in the range from 370 to 610 pg per 106 cells per 24 h. A distinct characteristic of these melanoma cell lines is that they form active surface-bound plasmin by the activation of plasminogen with surface-bound tissue-type plasminogen activator. The present study was performed to assess the role of plasmin in the process of latent TGF-β activation in the melanoma cell lines. No direct correlation was found between cell-associated plasmin activity and the amount of active TGF-β present in the conditioned medium of individual cell lines. The melanoma cell lines exhibited diverse responses to exogenous active TGF-β1; three cell lines were growth-stimulated, two were growth-inhibited, and one had a very low sensitivity to the growth factor. The active TGF-β produced by the melanoma cells was found to inhibit the natural killer cell function of peripheral blood lymphocytes, suggesting that it may have an immunosuppressive effect and a role in the development of melanomas. © 1996 Wiley-Liss, Inc.
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    Regulation of P120 mRNA levels during lymphocyte stimulation: Evidence that the P120 gene shares properties with early and late genes (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 60 (1996), S. 458-468 
    Details
    ISSN: 0730-2312
    Keywords: nucleolar protein ; rRNA ; G1-phase ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: P120 is a growth-regulated nucleolar protein, the expression of which is required for G1- to S-phase transition in lymphocytes. P120 appears to be involved in ribosomal biogenesis presumptively through its putative role as a rRNA methyltransferase. To better understand the role of P120 in cell cycle progression, we examined the regulation of the P120 gene in resting lymphocytes and in mitogen-stimulated lymphocytes as they progress from G1-phase toward S-phase. P120 mRNA was detected after the immediate early gene c-fos and persisted as the cells approached S-phase. A decrease in P120 mRNA coincided with the expression of histone H3 mRNA. The level of P120 mRNA increased as cells proceeded through G1-phase, and this increase was attributed to a more than threefold increase in the P120 transcription rate and an increase in P120 mRNA stability. The P120 gene is transcribed in resting lymphocytes, although the steady-state level of P120 is small or nonexistent. P120 mRNA accumulates in resting cells in the presence of the protein synthesis inhibitor cycloheximide. Furthermore, the steady-state level of P120 mRNA increases in the presence of cycloheximide after PHA-stimulation; this level does not increase in cells not treated with this protein synthesis inhibitor. The presence of cycloheximide increases both the transcription rate of the P120 gene and the stability of P120 mRNA. These studies indicate that P120 expression is cell cycle regulated in a complex manner and that the P120 gene has properties of both early and late genes. This time ordered regulation for P120 expression may represent a necessary step for the cell cycle associated increase in ribosomal biogenesis that is required for G1- to S-phase transition. © 1996 Wiley-Liss, Inc.
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    Bar-Shavit R, Maoz M, Ginzburg Y, Vlodavsky I (1996): Specific involvement of glypican in thrombin adhesive properties. J Cell Biochem 61:278-291. (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 62 (1996), S. 144-144 
    Details
    ISSN: 0730-2312
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: No abstract.
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    Cohen NS (1996): Intracellular localization of the mRNAs of argininosuccinate synthetase and argininosuccinate lyase around liver mitochondria, visualized by high-resolution in situ reverse transcription-polymerase chain reaction. J Cell Biochem 61:81-96. (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 62 (1996), S. 143-143 
    Details
    ISSN: 0730-2312
    Keywords: mRNA sorting ; mRNA targeting ; urea cycle ; enzyme organization ; cell organization ; electron microscopy ; digoxigenin ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Argininosuccinate synthetase and argininosuccinate lyase, two cytoplasmic enzymes of the urea cycle, are released into the soluble phase in the absence of detergent when cells are disrupted. Yet previous biochemical studies, as well as immunocytochemistry at the electron microscope level, have shown that these enzymes are localized around mitochondria in situ. Such intracellular localization of soluble enzymes requires mechanisms to deliver the proteins to the appropriate sites, where they may then be anchored by specific protein-protein interactions. A method was developed to examine the intracellular distribution of the mRNA of argininosuccinate synthetase and argininosuccinate lyase in intact rat liver at the ultrastructural level by in situ reverse transcription and the polymerase chain reaction, using primers targeting regions of the coding sequences of the rat enzymes, digoxigenin-dUTP as the label, and anti-digoxigenin/1nm gold plus silver enhancement as the detection method. The tissue was fixed in 4% paraformaldehyde/0.1% glutaraldehyde and embedded in Lowicryl. Examination of the numbers and the location of the silver grains, coupled with morphometric analysis of the electron micrographs, permitted the calculation of the silver “enrichment ratio” for each type of cell structure. These ratios showed that the mRNAs for argininosuccinate synthetase and argininosuccinate lyase were located next to the cytoplasmic side of the mitochondrial membrane and in the nearby endoplasmic reticulum. Most of the silver grains that were observed in the endoplasmic reticulum were within 200 nm of the mitochondria; it was not possible, however, to determine if those grains were actually associated with the reticular membranes. These studies demonstrate that the mRNAs of these two soluble cytoplasmic proteins are localized to the same limited regions where the proteins are situated. Translation of the proteins, therefore, must occur at these specific sites. The targeting of argininosuccinate synthetase and argininosuccinate lyase mRNAs to the immediate vicinity of the mitochondria may be the first step of the mechanisms by which the spatial organization of these soluble proteins in situ is accomplished. The targeting of mRNAs for soluble cytoplasmic proteins of organized metabolic pathways has not been demonstrated previously. These studies also show that in situ reverse transcription and the polymerase chain reaction at the ultrastructural level, which has not been previously reported, can be used to detect specific mRNAs; it should be extremely valuable for the intracellular detection of low-abundance mRNAs. © 1996 Wiley-Liss, Inc.
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    Basic fibroblast growth factor selectively regulates ornithine decarboxylase gene expression in malignant H-ras transformed cells (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 60 (1996), S. 572-583 
    Details
    ISSN: 0730-2312
    Keywords: basic fibroblast growth factor ; ornithine decarboxylase ; H-ras transformed cells ; G-protein ; protein kinase C ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Cell growth regulation by fibroblast growth factors (FGFs) is highly complex. The present study demonstrates a novel link between alterations in bFGF regulation during malignant conversion and the expression of ornithine decarboxylase, a key rate-limiting and regulatory activity in the biosynthesis of polyamines. H-ras transformed mouse 10T½ cell lines exhibiting increasing malignant potential were investigated for possible bFGF-mediated changes in ornithine decarboxylase gene expression. Selective induction of ornithine decarboxylase gene expression was observed, since, in contrast to nontransformed 10T½ cells and cells capable of only benign tumor formation, H-ras transformed metastatic cells exhibited marked elevations in ornithine decarboxylase message levels. Evidence for regulation of ornithine decarboxylase gene expression by bFGF at both transcription and posttranscription was found. Actinomycin D pretreatment of malignant cells prior to bFGF exposure inhibited the increase in ornithine decarboxylase message. Furthermore, striking differences in the rates of ornithine decarboxylase message decay were observed when cells treated with bFGF were compared to untreated control cells, with the half-life of ornithine decarboxylase mRNA increasing from 2.4 h in untreated cells to 12.5 h in cells exposed to bFGF. Evidence was also obtained for a cycloheximide-sensitive regulator of ornithine decarboxylase gene expression whose effect, in combination with bFGF, resulted in a further augmentation of ornithine decarboxylase gene expression. Furthermore, evidence is presented to suggest a possible role for G-protein-coupled events in the bFGF-mediated regulation of ornithine decarboxylase gene expression. The bFGF regulation of ornithine decarboxylase expression in H-ras transformed malignant cells appeared to occur independent of protein kinase C-mediated events. These results show that bFGF can modulate ornithine decarboxylase gene expression in malignant H-ras transformed cells and further suggests a mechanism of growth factor stimulation of malignant cells wherein early alterations in the regulatory control of ornithine decarboxylase gene expression are critical. © 1996 Wiley-Liss, Inc.
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    Basic fibroblast growth factor: An autocrine growth factor for epiphyseal growth plate chondrocytes (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 62 (1996), S. 372-382 
    Details
    ISSN: 0730-2312
    Keywords: bFGF ; extracellular matrix ; in situ hybridization ; RT-PCR ; immunocytochemistry ; cell proliferation ; Western blotting ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Basic fibroblast growth factor (bFGF) is a permissive mitogen for cultured chondrocytes and has been localized in the specific zones of the epiphyseal growth plate. In this study, we demonstrate that bFGF present in cartilage originates from within the cellular constituents of this tissue. Utilizing reverse transcription coupled to the polymerase chain reaction (PCR), bFGF mRNA was found in extracts of cartilage tissue. Immunocytochemical studies revealed that bFGF was present intracellularly in freshly isolated proliferative chondrocytes and in the extracellular matrix (ECM) after 24 h of culture. Western blot analysis of protein extracts from isolated proliferative chondrocytes identified a bFGF immunoreactive species with a molecular weight of approximately 18 kDa. In situ hybridization confirmed the presence of bFGF mRNA in freshly isolated proliferative chondrocytes. The bFGF in the ECM seemed to be sequestered and not available for biological activity, since these cells still required exogenous bFGF for cell proliferation. This sequestered bFGF could be released to stimulate cell proliferation when cultures were treated with plasmin, a proteolytic enzyme. These data support the hypothesis that bFGF is synthesized by chondrocytes and functions as an autocrine/paracrine mitogen via its deposition into the ECM with subsequent release from the ECM of cartilage being a critical step in biological activity. In addition, the study provides further evidence that locally produced bFGF plays an important role in normal growth and development of cartilage tissue. © 1996 Wiley-Liss, Inc.
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    Interleukin 4 inhibits hepatocyte growth factor-induced invasion and migration of colon carcinomas (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 62 (1996), S. 443-453 
    Details
    ISSN: 0730-2312
    Keywords: cancer ; collagenase ; Met ; cytokine ; metastasis ; motility ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Hepatocyte growth factor (HGF) is known to have a number of biological properties including promoting tumor progression of human carcinomas. Metastasis involves a number of events that are attributed to induction by paracrine factors such as HGF. Identification of natural inhibitors of these events would allow better control of tumor progression. Recently we demonstrated that interleukin 4 (IL-4) can regulate proliferation of various human carcinoma cell lines. In the present study, we used established human colon carcinoma cell lines and primary colon carcinoma cell cultures to determine if IL-4 could regulate HGF-induced cell proliferation and other events of tumor progression such as MMP (matrix metalloproteinases)-1, -2, and -9 production, cell migration and cell-matrix invasive activity. All colon carcinoma cell lines expressed HGF and IL-4 receptors. IL-4 significantly inhibited HGF-induced proliferation of one cell line. Cell-matrix invasion was significantly enhanced by HGF (0.1-10 ng/ml); IL-4 (1-10 U/ml) significantly inhibited HGF-induced invasion in a dose-dependent manner. IL-4 also inhibited HGF-induced cell-matrix invasion of metastatic colon carcinoma cells and HGF-induced cell migration. HGF enhanced MMP-1, -2, and -9 production by cell lines. This effect could be inhibited by IL-4. These findings indicate that IL-4 is a potent inhibitor of HGF-induced invasion and metastasis-related functions of human colon carcinoma cells. © 1996 Wiley-Liss, Inc.
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    Dexamethasone regulation of marrow stromal-derived osteoblastic cells (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 62 (1996), S. 476-483 
    Details
    ISSN: 0730-2312
    Keywords: stromal osteoblasts ; dexamethasone ; attachment ; growth factors ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The clonal subtypes of cells in the osteogenic family represented by fibroblastoid MBA-15.33, preosteoblast MBA-15.4, and mature osteoblastic MBA-15.6 cells were used to study the effects of glucocorticoid (dexamethasone). The role of dexamethasone was monitored on cell attachment when plated on various protein substrata (BSA, collagen I, and Matrigel). A 24 h exposure of the cells to 10-6 M or 10-7 M dexamethasone differential affects their attachment preference. MBA-15.33 and MBA-15.4 cells increased their attachment capability on collagen I, while MBA-15.6 cells' attachment was inhibited. Pretreatment with (10-6 M) dexamethasone caused an increase in attachment on Matrigel by MBA-15.33 cells and to less extent by MBA-15.4 cells. Additionally, measurements of two enzymatic activities were monitored; one is alkaline phosphatase (ALK-P), and the second is neutral endopeptidase (CD10/NEP). MBA-15.33, MBA-15.4, and MBA-15.6 cells were exposed to dexamethasone or to various growth factors (bone morphogenic protein (BMP-2 and BMP-3), TGFβ, and IGF-I). In some experiments, pretreatment of cells by dexamethasone was followed by exposure to the growth factors. The cells' challenged cellular responses were not uniform and revealed a differential pattern when their ALK-P and CD10/NEP enzymatic activities were measured. © 1996 Wiley-Liss, Inc.
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    Inverted repeats, stem-loops, and cruciforms: Significance for initiation of DNA replication (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 63 (1996), S. 1-22 
    Details
    ISSN: 0730-2312
    Keywords: inverted repeats ; cruciform DNA ; secondary structure ; DNA replication ; cruciform binding proteins ; structure-specific recognition ; protein-DNA interactions ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Inverted repeats occur nonrandomly in the DNA of most organisms. Stem-loops and cruciforms can form from inverted repeats. Such structures have been detected in pro- and eukaryotes. They may affect the supercoiling degree of the DNA, the positioning of nucleosomes, the formation of other secondary structures of DNA, or directly interact with proteins. Inverted repeats, stem-loops, and cruciforms are present at the replication origins of phage, plasmids, mitochondria, eukaryotic viruses, and mammalian cells. Experiments with anti-cruciform antibodies suggest that formation and stabilization of cruciforms at particular mammalian origins may be associated with initiation of DNA replication. Many proteins have been shown to interact with cruciforms, recognizing features like DNA crossovers, four-way junctions, and curved/bent DNA of specific angles. A human cruciform binding protein (CBP) displays a novel type of interaction with cruciforms and may be linked to initiation of DNA replication. © 1996 Wiley-Liss, Inc.
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    Modulation of transcription of the rat fibronectin gene by cell density (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 63 (1996), S. 74-85 
    Details
    ISSN: 0730-2312
    Keywords: fibronectin ; gene regulation ; cell growth ; lacZ ; NIH/3T3 ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The fibronectin (FN) gene is under complex regulatory control in vitro and in vivo. Sequences from the rat FN gene directed efficient expression of a lacZ reporter gene product, β-galactosidase, in NIH/3T3 mouse fibroblasts. Stable transfectants were generated to facilitate studies of gene regulation by cell growth state. The expression of FN-lacZ constructs increased approximately twofold when cultures attained confluence, relative to total protein. The magnitude of this increase correlates well with that observed for FN mRNA levels and protein synthesis rate. Fragments containing 4.9, 0.9, or 0.3 kbp upstream of the transcription start site are equally responsive to cell density and/or cell contact. Deletion of a cAMP-responsive element enhanced the response, suggesting a negative role for this sequence motif and demonstrating that the FN gene is regulated by cell density at the transcriptional level. The effect of high cell density is apparently different from decreased growth rate, as incubation with low serum did not result in increased expression of the lacZ reporter. Finally, conditioned medium from dense cells did not enhance reporter gene expression in sparse cells, suggesting that the density signal is not transmitted via a soluble factor. © 1996 Wiley-Liss, Inc.
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    Erratum: Wang W, Kucuk O, Franke AA, Liu LQ, Custer LJ, Higuchi CM (1996): Reproducibility of erythrocyte polyamine measurements and correlation with plasma micronutrients in an antioxidant vitamin intervention study. J Cell Biochem 62:19-26. (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 63 (1996), S. 123-123 
    Details
    ISSN: 0730-2312
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: No abstract.
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    Inhibition of endothelial cell proliferation and bFGF-induced phenotypic modulation by nitric oxide (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 63 (1996), S. 125-134 
    Details
    ISSN: 0730-2312
    Keywords: angiogenesis ; contact inhibition ; inhibitor ; SNAP ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: S-nitroso-N-acetyl-D,L-acetylpenicillamine (SNAP), a chemical donor of NO, inhibited serum- and basic fibroblast growth factor (bFGF)-stimulated cultured endothelial cell (EC) proliferation in a dose-dependent manner. The inhibitory effect of NO was reversible after washoff of SNAP-containing media. Measurement of nitrate and nitrite in the media of SNAP-treated EC indicated that decomposition of SNAP into NO reached a stable level at or before 24 h; proliferation of EC was significantly inhibited for another 48 h and recovered thereafter if no additional SNAP was added. The level of NO produced by inhibitory concentrations of SNAP was comparable to NO levels produced by the induction of inducible nitric oxide synthase (iNOS) in smooth muscle cells or retinal pigmented epithelial cells. The growth-inhibitory effect of NO was unlikely to be due to cytotoxicity since 1) cells never completely lost their proliferative capacity even after 10 days of exposure to repeated additions of SNAP, 2) the inhibitory effect was reversible upon removal of NO and with the passage of time, and 3) NO did not reduce the number of cells that were growth-arrested with TGF-β1. In addition to its mitogenic effect, bFGF induced pronounced phenotypic changes, including suppression of contact inhibition, altered cell morphology, and scattering of the cells, in BPAEC cultures, whereas cells treated simultaneously with bFGF and NO did not exhibit these changes. These observations suggest that NO contributes to the regulation of angiogenesis and reendothelialization, processes that require EC proliferation, migration, and differentiation. © 1996 Wiley-Liss, Inc.
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    Estrogen regulation of nuclear matrix-intermediate filament proteins in human breast cancer cells (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 63 (1996), S. 174-184 
    Details
    ISSN: 0730-2312
    Keywords: cytokeratins ; hormone independence ; T-47D5 ; nuclear matrix ; breast cancer ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The tissue matrix consists of linkages and interactions of the nuclear matrix, cytoskeleton, and extracellular matrix. This system is a dynamic structural component of the cell that organizes and processes structural and functional information to maintain and coordinate cell function and gene expression. We have studied estrogen regulation of nuclear matrix associated proteins, including the intimately connected cytoskeletal intermediate filaments, in T-47D5 human breast cancer cells. Three proteins (identified as cytokeratins 8, 18, and 19) present in the nuclear matrix-intermediate filament fraction (NM-IF) of cells grown in estrogen-replete conditions were dramatically reduced when the cells were grown in acute (1 week) estrogen-depleted conditions. Replacing estrogen in the medium of acute estrogen-depleted cells restored expression of these proteins. T-47D5 cells that are chronically depleted of estrogen (T5-PRF) are estrogen-nonresponsive in culture. These cells overexpressed these three proteins, compared to parent cells grown in the presence of estrogen. Treatment of the T5-PRF cells with estrogen did not lead to further up-regulation of these proteins. Treating T-47D5 cells in estrogen-replete conditions with the antiestrogens 4-hydroxytamoxifen and ICI 164 384 (100 nM, 3 days) resulted in a significant reduction in these proteins, while no effect was seen in long-term chronic estrogen-depleted T-47D5 cells. In conclusion, we have identified NM-IF proteins (cytokeratins 8, 18, and 19) in human breast cancer cells that are estrogen regulated and may play a role in estrogen action in human breast cancer cells. © 1996 Wiley-Liss, Inc.
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    Requirement of distal and proximal promoter sequences for chromatin organization of the osteocalcin gene in bone-derived cells (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 63 (1996), S. 221-228 
    Details
    ISSN: 0730-2312
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The osteocalcin (OC) gene encodes a 10 Kda bone-specific protein which is expressed with the onset of mineralization during the differentiation of normal diploid osteoblasts. We have previously reported that transcriptional activation of this gene is accompanied by the presence of two DNase I hypersensitive sites, both located in the promoter region spanning key basal (proximal site, -170 to -70) and steroid-dependent enhancer (distal site, -600 to -400) elements. Here, we have examined stably transfected ROS 17/2.8 cell lines carrying OC promoter-reporter transgenes which contain a series of 5′-deletions and determined the effects of these truncations on the chromatin organization. It has been found that: (1) DNase I hypersensitivity at -600 is not a requirement for vitamin D-dependent transcriptional upregulation; (2) basal transcriptional activity and proximal nuclease hypersensitivity depend exclusively on protein-DNA interactions occurring within the proximal promoter region, and (3) within the chromatin context, the proximal 100 bp promoter fragment, containing essential elements such as the OC box (-99 -to -76) and TATA box (-44 to -31), is insufficient to support formation of the proximal nuclease hypersensitive site and transcriptional activity. © 1996 Wiley-Liss, Inc.
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    Differential induction of cell-mediated mineralization in rat marrow stroma by sera from women of low and high risk for vertebral fracture (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 63 (1996), S. 115-122 
    Details
    ISSN: 0730-2312
    Keywords: serum ; osteoporosis ; alkaline phosphatase ; DEXA ; Z-scores ; mineralization ; marrow stroma ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The purpose of this study was to analyze the ability of sera to reflect the state of bone metabolism by testing the osteogenic response of mesenchymal cells in culture. Sera of 20 peri- and postmenopausal women were tested before the initiation of hormone replacement therapy. The responding cells were osteoprogenitors (OPC) of rat marrow stroma which normally respond to dexamethasone (DEX) and β-glycerophosphate (βGP) by proliferation, differentiation, and mineralization in culture. Instead of DEX, diluted sera (1:50) were applied to rat stromal cell cultures for analysis of their ability to affect cell proliferation, specific alkaline phosphatase (ALP) activity, and cell-mediated mineralization. The results were compared individually with the respective values of vertebral bone mineral density (BMD), expressed as the number of standard deviations above or below the mean BMD of reference populations (positive or negative Z-score). Serum donors were divided in two; the group with positive Z-scores was considered to have a low risk, and that with negative Z-scores was considered to have a higher risk for vertebral fractures. No significant difference was found between the two groups in the ability of their sera to induce cell proliferation or specific ALP activity. However, sera representing negative Z-scores induced sixteenfold less mineralization than those of positive Z-scores. The scatter of individual mineralization values was highly discriminatory between the two groups (α 〈 0.00). These results indicate that the serum-induced, cell-mediated mineralization in culture might be suitable for initial evaluation of fracture risk and thus deserve further investigation. © 1996 Wiley-Liss, Inc.
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    Acidic fibroblast growth factor inhibits osteoblast differentiation in vitro: Altered expression of collagenase, cell growth-related, and mineralization-associated genes (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 61 (1996), S. 152-166 
    Details
    ISSN: 0730-2312
    Keywords: acidic FGF ; osteoblast differentiation ; collagenase ; osteopontin ; osteocalcin ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Fibroblast growth factors (FGF) are osteoblast mitogens, but their effects on bone formation are not clearly understood. Most in vitro studies examining the effects of FGFs on osteoblasts have been performed only during the initial proliferative stage of osteoblast culture. In these studies, we examined the consequential effect of acidic FGF in cultures of rat fetal diploid osteoblasts that undergo a developmental differentiation program producing a mineralized bone-like matrix. During the initial growth period (days 1-10), addition of acidic FGF (100 μg/ml) to actively proliferating cells increased (P 〈 0.05) 3H-thymidine uptake (2,515 ± 137, mean ± SEM vs. 5,884 ± 818 cpm/104 cells). During the second stage of maturation (days 10-15), osteoblasts form multilayered nodules of cells and accumulate matrix, followed by mineralization (stage 3, days 16-29). Addition of acidic FGF to the osteoblast cultures from days 7 to 15 completely blocked nodule formation. Furthermore, addition of acidic FGF after nodule formation (days 14-29) inhibited matrix mineralization, which was associated with a marked increase in collagenase gene expression, and resulted in a progressive change in the morphology of the nodules, with only a few remnants of nonmineralized nodules present by day 29. Histochemical and biochemical analyses revealed a decrease in alkaline phosphatase and mineral content, confirming the acidic FGF-induced inhibition of nodule and matrix formation. To identify mechanisms contributing to these changes, we examined expression of cell growth and bone phenotypic markers. Addition of acidic FGF during the proliferative phase (days 7-8) enhanced histone H4, osteopontin, type 1 collagen, and TGF-β mRNA levels, which are coupled to proliferating osteoblasts, and blocked the normal developmental increase in alkaline phosphatase and osteocalcin gene expression and calcium accumulation. Addition of acidic FGF to the cultures during matrix maturation (days 14-15) reactivated H4, osteopontin, type I collagen, and TGF-β gene expression, and decreased alkaline phosphatase and osteocalcin gene expression. In an in vivo experiment, rats were treated with up to 60 μg/kg/day acidic FGF intravenously for 30 days. Proliferation of osteoblasts and deposition of bone occurred in the marrow space of the diaphysis of the femur in a dose-related fashion. The metaphyseal areas were unaffected by treatment. In conclusion, our data suggest that acidic FGF is a potent mitogen for early stage osteoblasts which leads to modifications in the formation of the extracellular matrix; increases in TGF-β and collagenase are functionally implicated in abrogating competency for nodule formation. Persistence of proliferation prevented expression of alkaline phosphatase and osteocalcin, also contributing to the block in the progression of the osteoblast developmental sequence. © 1996 Wiley-Liss, Inc.
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    Modulation of protein phosphorylation and stress protein expression by okadaic acid on heat shock cells (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 61 (1996), S. 255-265 
    Details
    ISSN: 0730-2312
    Keywords: glucose-regulated proteins ; heat shock proteins ; heat shock ; okadaic acid ; protein phosphorylation ; vimentin ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We have demonstrated that pretreatment but not post-treatment with okadaic acid (OA) can aggravate cytotoxicity as well as alter the kinetics of stress protein expression and protein phosphorylation in heat shocked cells. Compared to heat shock, cells recovering from 1 hr pretreatment of OA at 200 nM and cotreated with heat shock at 45°C for the last 15 min of incubation (OA→HS treatment) exhibited enhanced induction of heat shock proteins (HSPs) 70 and 110. In addition to enhanced expression, the attenuation of HSC70 and HSP90 after the induction peaks was also delayed in OA→HS-treated cells. The above treatment also resulted in the rapid induction of the 78 kDa glucose-regulated protein (GRP78), which expression remained constant in cells recovering from treatment with 200 nM OA for 1 hr, heat shocked at 45°C for 15 min, or in combined treatment in reversed order (HS→OA treatment). Enhanced phosphorylation of vimentin and proteins with molecular weights of 65, 40, and 33 kDa and decreased phosphorylation of a protein with a molecular weight of 29 kDa were also observed in cells recovering from OA→HS treatment. Again, protein phosphorylation in cells recovering from HS→OA treatment did not differ from those in cells treated only with heat shock. Since the alteration in the kinetics of stress protein expression and protein phosphorylation was tightly correlated, we concluded that there is a critical link between induction of the stress proteins and phosphorylation of specific proteins. Furthermore, the rapid induction of GRP78 under the experimental condition offered a novel avenue for studying the regulation of its expression. © 1996 Wiley-Liss, Inc.
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    Transcriptional regulation of the lysozyme gene in airway gland serous cells (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 61 (1996), S. 350-362 
    Details
    ISSN: 0730-2312
    Keywords: lysozyme ; gene regulation ; cell differentiation ; serous cells ; gland cells ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Lysozyme is expressed in serous, but not mucous, cells of the tracheobronchial glands and thereby constitutes a marker of the serous cell lineage in these glands. To identify DNA regulatory elements and transcription factors mediating the commitment of progenitor cells to the serous cell lineage, we have characterized the regulatory activity and DNA-protein interactions of the 5′-flanking region of the bovine lysozyme gene lys 5a. Results obtained from these studies indicate that although approximately 94 bp of 5′ flanking DNA are necessary for high level expression in transient transfection assays, an evolutionarily conserved promoter within 66 bp of the transcription start site is sufficient to confer serous cell-specific expression. Farther upstream, within 6.1 kb of the 5′ flanking region, are 4 silencers. Analysis of the serous cell-specific lysozyme promoter by electrophoretic mobility shift assay (EMSA) revealed the presence of binding sites for 3 serous cell nuclear proteins, designated LSF1, LSF2 and LSF3. Binding of LSF2 and LSF3 was localized to a 20-mer subdomain (-50/-30) of the cell-specific promoter using binding competition assays. More accurate identification of the protein binding site(s) was achieved through the use of mutagenesis, which implicated the motif 5′AAGGAAT 3′ (-46/-40) in both protein binding and serous cell-specific transcriptional activity. This motif has previously been identified as a binding site for ets protein transcription factors, suggesting that serous cell-specific regulation of lys 5a transcription is partly controlled by the binding of ets-like protein(s) to the motif 5′AGGAAGT3′. © 1996 Wiley-Liss, Inc.
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    Role of αvβ5 and αvβ6 integrin glycosylation in the adhesion of a colonic adenocarcinoma cell line (HT29-D4) (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 61 (1996), S. 266-277 
    Details
    ISSN: 0730-2312
    Keywords: integrins ; glycosylation ; adhesion ; colon ; adenocarcinoma ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We have previously characterized the expression of the αvβ5 and αvβ6 integrins as major receptors for the human colonic adenocarcinoma cell line (HT29-D4), on vitronectin and fibronectin, respectively [Lehmann et al. (1994): Cancer Res 54:2102-2107]. In the present work we investigated the glycosylation role of these integrins in their adhesive functions. To this end, we used glycohydrolases to show that cell surface integrins were N-glycosylated and sialylated, and that only the αv subunit carried some immature oligosaccharide side chains. To alter the glycosylation state of the cell surface αvβ5 and αvβ6 integrins, we used two oligosaccharide-processing inhibitors: 1-deoxymannojirimycin (dMNJ) and tunicamycin (TM). Following treatment of HT29-D4 cells with dMNJ, cell surface αvβ5 and αvβ6 carried only high-mannose-type sugar chains, while TM-treated cells expressed de-N-glycosylated integrins. Neither α/β heterodimers assembly nor cell surface expression were impaired in the presence of the drugs. Finally, we established that adhesion of dMNJ- or TM-treated cells was altered on both vitronectin and fibronectin substrata, whereas the adhesion of these cells on laminin or collagen type I was virtually unchanged. © 1996 Wiley-Liss, Inc.
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    Specific inhibition of c-fos proto-oncogene expression by triple-helix-forming oligonucleotides (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 61 (1996), S. 301-309 
    Details
    ISSN: 0730-2312
    Keywords: c-fos ; triplex ; transcriptional factors ; promoter ; gene regulation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The promoter region of the c-fos oncogene 5′ flanking sequence contains enhancer elements crucial for binding nuclear factors that regulate transcription following cell proliferation and differentiation. Single-stranded deoxyoligonucleotides were chosen for modulation of c-fos protooncogene expression because of their high-affinity binding to specific nucleotide sequences. We designed two oligonucleotides that form a triple-helix complex on the retinoblastoma gene product-responsible element of the c-fos oncogene.Modification of the DNA triplex with dimethyl sulfate and affinity cleaving assays demonstrate that the predicted oligonucleotides form a DNA triplex structure with the c-fos promoter in a sequence-specific manner. Tumorigenic and non-tumorigenic fibroblasts were transiently transfected with fos-CAT plasmid modified with alkylating triplex-forming oligonucleotide reagents. A dramatic depression of CAT activity was found when the cross-linked triple helix complex at the retinoblastoma gene product-related site of the c-fos promoter was used.These experiments suggest that transcription of individual genes can be selectively modulated in cell culture by sequence specific triplex formation in regulatory enhancer sequences. © 1996 Wiley-Liss, Inc.
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    Modulation of α5β1 and αVβ3 integrins on the cell surface during mitosis (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 61 (1996), S. 338-349 
    Details
    ISSN: 0730-2312
    Keywords: mitosis ; cell adhesion ; integrins ; extracellular matrix ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: One of the hallmarks of cells undergoing mitotic division is their rounded morphology and reduced adhesion to the substratum. We have studied and compared the attachment of interphase and mitotic cells to substrata coated with fibronectin and vitronectin. We have found that adhesion of mitotic cells, as compared to interphase cells, is significantly reduced to fibronectin, but is higher to vitronectin. These results correlate well with the expression of α5β1 and αVβ3 integrins, the respective receptors for fibronectin and vitronectin, on the cell surface. Mitotic cells show higher levels of αVβ3 and very low levels of α5β1 proteins on the cell surface as compared to interphase cells. This difference in the levels of these integrins also reflects in the total amounts of fibronectin and vitronectin present on the cell surface of these cells. We have further shown, by flow cytometry, that binding of vitronectin, or the synthetic peptide-GRGDSP-, causes an increase in the intracellular levels of Ca2- in mitotic cells, but no change is seen in the interphase cells. Binding of fibronectin to either of these cells fails to elicit any response. One interesting feature of our results is that the levels of total, i.e., cytoplasmic plus membrane bound, α5β1 and αVβ3 integrins of mitotic and interphase cells remain the same, thus implying an alteration in the distribution of integrin chains between the plasma membrane and the cytoplasm during the conversion of interphase cells into the mitotic phase. © 1996 Wiley-Liss, Inc.
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    Chondrocyte cultures express matrix metalloproteinase mRNA and immunoreactive protein; stromelysin-1 and 72 kDa gelatinase are localized in extracellular matrix vesicles (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 61 (1996), S. 375-391 
    Details
    ISSN: 0730-2312
    Keywords: metalloproteinases ; growth plate cartilage ; chondrocytes ; matrix vesicles ; RT-PCR ; zymography ; stromelysin-1 ; 72 kDa gelatinase ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Previous studies have shown that costochondral cartilage cell cultures produce extracellular matrix vesicles which contain metalloproteinase activity. In the present study, we examined whether two matrix metalloproteinases (MMPs) known to be present in cartilage, stromelysin-1 and 72 kDa gelatinase, are expressed by fourth passage resting zone and growth zone costochondral chondrocytes and whether they are specifically incorporated into matrix vesicles produced by the cells. We also examined whether the cells synthesize tissue inhibitor of metalloproteinase-1 and -2 (TIMP-1 and TIMP-2). Oligonucleotide primers for stromelysin-1, 72 kDa gelatinase, tissue inhibitor of metalloproteinases-1 and -2 (TIMP-1 and TIMP-2), and GAPDH were synthesized and optimized for use in the reverse transcription-polymerase chain reaction (RT-PCR). It was found that both resting zone and growth zone chondrocytes produced mRNA for both MMPs and the two TIMPs. Further, immunostaining of cell layers with antibodies to 72 kDa gelatinase and stromelysin-1 showed that both cell types produced these MMPs in culture. Substrate gel electrophoresis and Western analysis were used to characterize MMP activity in matrix vesicles, media vesicles, or plasma membranes as well as in conditioned media produced by the chondrocyte cultures. It was found that matrix vesicles but not plasma membranes or media vesicles were selectively enriched in stromelysin-1. Also, 72 kDA gelatinase was found in matrix vesicles, but to a lesser extent than seen in media vesicles. The relative activity of each enzyme detected was cell maturation-dependent. No MMP activity was detected in conditioned media produced by either cell type. The results of this study show that MMPs are expressed by resting zone and growth zone chondrocytes in culture and differentially distributed among three different membrane compartments. This suggests that, in addition to the well-known activators and inhibitors of MMP activity in the matrix, differential membrane distribution may enable more precise control over the site, rate, and extent of matrix degradation by the cell. © 1996 Wiley-Liss, Inc.
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    Effect of transient overexpression of Gqα on soluble and polymerized tubulin pools in GH3 and AtT-20 cells (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 61 (1996), S. 392-401 
    Details
    ISSN: 0730-2312
    Keywords: G proteins ; cytoskeleton ; pituitary cells ; signal transduction ; prolactin ; thyrotropin-releasing hormone ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: In order to study Gq-tubulin interaction in the cytosol, GH3 and AtT-20 cells (stably expressing TRH receptor) were transiently transfected with Gqα cDNA. Forty-eight hours after transfection, thyrotropin-releasing hormone (TRH)-stimulated prolactin (PRL) secretion by Gqα-transfected GH3 cells increased by 90% compared to mock-transfected cells. In addition, using immunocytochemistry it was observed that Gqα-specific staining was much more prominent in Gqα-transfected GH3 and AtT-20 cells (also transfected with Gqα) compared to mock-transfected cells. Thus, transfection resulted in successful overexpression of functional Gqα. Forty-eight hours after transfection, cells were processed to obtain soluble and polymerized tubulin fractions. Tubulin levels were determined in these fractions by immunoblotting using polyclonal anti-tubulin antibodies. Compared to mock-transfected cells soluble tubulin levels decreased in Gqα-transfected GH1 and AtT-20 cells, by 33 and 52%, respectively. Moreover, compared to mock-transfected cells a 50% reduction in the ratio (an index of the flux between tubulin pools) of soluble and polymerized tubulin levels was observed in Gqα-transfected GH3 and AtT-20 cells. To determine whether these effects on tubulin were mediated by Gq directly, we examined the influence of purified Gq on tubulin polymerization. Gq (0.5 μM) inhibited polymerization of crude tubulin (present in GH3 cell cytosol) by 53%. In contrast to its effects on GH3 cell cytosol tubulin, Gq stimulated purified tubulin polymerization by 160%. These results suggest that Gq modulates the polymerization and depolymerization cycles of tubulin and that this modulation is in turn influenced by other unknown cellular components. © 1996 Wiley-Liss, Inc.
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    Selenium-regulated translation control of heterologous gene expression: Normal function of selenocysteine-substituted gene products (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 61 (1996), S. 410-419 
    Details
    ISSN: 0730-2312
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: In eukaryotes, the synthesis of selenoproteins depends on an exogenous supply of selenium, required for synthesis of the novel amino acid, selenocysteine, and on the presence of a “selenium translation element” in the 3′ untranslated region of mRNA. The selenium translation element is required to re-interpret the stop codon, UGA, as coding for selenocysteine incorporation and chain elongation. Messenger RNA lacking the selenium translation element and/or an inadequate selenium supply lead to chain termination at the UGA codon. We exploited these properties to provide direct translational control of protein(s) encoded by transfected cDNAs. Selenium-dependent translation of mRNA transcribed from target cDNA was conferred by mutation of an in-frame UGU, coding for cysteine, to UGA, coding for either selenocysteine or termination, then fusing the mutated coding region to a 3′ untranslated region containing the selenium translation element of the human cellular glutathione peroxidase gene. In this study, the biological consequences of placing this novel amino acid in the polypeptide chain was examined with two proteins of known function: the rat growth hormone receptor and human thyroid hormone receptor β1. UGA (opal) mutant-STE fusion constructs of the cDNAs encoding these two polypeptides showed selenium-dependent expression and their selenoprotein products maintained normal ligand binding and signal transduction. Thus, integration of selenocysteine had little or no consequence on the functional activity of the opal mutants; however, opal mutants were expressed at lower levels than their wild-type counterparts in transient expression assays. The ability to integrate this novel amino acid at predetermined positions in a polypeptide chain provides selenium-dependent translational control to the expression of a wide variety of target genes, allows facile 75Se radioisotopic labeling of the heterologous proteins, and permits site-specific heavy atom substitution. © 1996 Wiley-Liss, Inc.
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    Arachidonic acid-induced down-regulation of protein kinase C δ in beta-cells (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 62 (1996), S. 543-552 
    Details
    ISSN: 0730-2312
    Keywords: islets ; free fatty acids ; indomethacin ; PKC ; arachidonic acid ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We have previously identified expression of multiple protein kinase C (PKC) isoforms in insulinoma-derived beta-cells and whole islets. Both PKC γ and PKC α appear to be the more abundantly expressed isoforms. In this report we studied the effects of arachidonic acid (AA) on the subcellular distribution of PKC α and PKC γ. AA has been reported to activate both PKC α and PKC γ and it is thought to be an important second messenger in beta-cells. Here we report that AA interacted with and altered beta-cell pools of PKC γ preferentially over PKC α. AA (100 μM) over the course of 45 min reduced cytosolic levels of PKC γ (to 40 ± 15%, compared to time zero control) leaving membrane-and cytoskeleton-associated levels near control levels. Analysis of whole cell homogenates showed a slight down-regulation of PKC γ indicating proteolysis. The down-regulation of cytosolic PKC γ appeared to be isoform specific since cytosolic PKC α remained at control levels over the time course. The response was dose-dependent and negligible at concentrations below 30 μM and occurred, at least partially, in the cytosolic compartment of the cell. Indomethacin also down-regulated cytosolic PKC γ preferentially over PKC α possibly through accumulation of AA. These findings suggest that cytosolic PKC γ may be a downstream target of this beta-cell second messenger. © 1996 Wiley-Liss, Inc.
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    Strategies for manipulating apoptosis for cancer therapy with tumor necrosis factor and lymphotoxin (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 60 (1996), S. 56-60 
    Details
    ISSN: 0730-2312
    Keywords: TNF ; LT ; MnSOD ; cancer ; radioprotection ; radiosensitization ; Fas ; apoptosis ; ICE proteases ; oxygen free radicals ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Tumor necrosis factor (TNF) and lymphotoxin (LT), initially described as tumoricidal proteins, may be useful as adjuncts in cancer therapy. Treatment with TNF or LT was found to protect cells and animals against damage mediated by radiation or cytotoxic anticancer drugs. By contrast, tumor cells treated with TNF or LT were sensitized to these insults. We present a model in which TNF or LT induces both the synthesis of “protective” proteins such as manganous superoxide dismutase (MnSOD) and the activation of “killing” proteins, such as proteases, depending on the level of the inducing signal. Although the p55-TNF/LT receptor is structurally related to the Fas receptor, they can each signal apoptosis by distinct pathways. Furthermore, activation of both receptors acts synergistically in stimulating apoptosis. © 1996 Wiley-Liss, Inc.
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    Diverse expression of G-proteins in human sarcoma cell lines with different osteogenic potential: Evidence for the involvement of Gi2 in cell proliferation (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 60 (1996), S. 95-106 
    Details
    ISSN: 0730-2312
    Keywords: osteosarcomas ; adenylate cyclase ; phospholipase C ; G-proteins ; growth rate ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Previously, it has been shown that the GTP-binding protein Gi2 is implicated in cellular growth [1,2] and differentiation [2,3]. In the present paper we demonstrate that this is also the case for human sarcoma cells.Six human osteosarcoma and three soft tissue sarcoma clonal cell lines were analyzed for levels of G-protein mRNA and polypeptide expression and effector enzyme (i.e., adenylate cyclase and phospholipase C) activation, which were all compared with individual growth rates. Unexpectedly, it appeared that the various strains exhibited large inter-individual variations in G-protein expression and signaling system activation. However, cell doubling time in the exponential phase of growth was inversely correlated (r = 0.71, P 〈 0.05) to immunodetected levels of intrinsic Gi2α. Furthermore, cells stably transfected with a retroviral (pZipNeo(SV)X) construct containing the activating or inactivating Gi2α-R179E or Gi2α-G204A point mutations consistently reduced or enhanced individual cell strain doubling time, respectively.It appeared that other parameters investigated, including cellular alkaline phosphatase and monoclonal antibody epitope binding, both being markers of the proliferating osteoblast, did not correlate with cell doubling times. © 1996 Wiley-Liss, Inc.
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    Characterization of senescence- and apoptosis-dependent forms of terminin as derived from a precursor found in replicating and nonreplicating cells (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 60 (1996), S. 107-120 
    Details
    ISSN: 0730-2312
    Keywords: immunoprecipitation ; in vitro translation ; V-8 digestion ; peptide mapping ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Previously we have reported the production of a monoclonal antibody (Mab 1.2) which recognizes a cytoplasmic protein, terminin, in three different molecular weights: 90 (Tp90), 60 (Tp60), and 30 kDa (Tp30) forms. Further characterization shows that Tp90 is found in young growing and nongrowing quiescent fibroblasts, while Tp60 is found in permanently growth-arrested senescent fibroblasts and Tp30 in cells committed to undergo programmed cell death (apoptosis). In tissue, Tp90 is found in embryonic brain; later, in neonatal brain after terminal differentiation is completed, only Tp60 is found. Tp30 is found in crude liver fractions extracted without the protective action of protease inhibitors. In all these circumstances, Tp90 is mostly seen in the detergent-soluble fraction, while Tp60 and Tp30 are detergent-insoluble. We now report that in cultured fibroblasts, as well as in tissues such as brain and liver, Tp60 and Tp30 are derived from the Tp90 polypeptide, indicated by the fact that only the Tp90 species is identified by both immunoblotting and immunoprecipitation assays, when the cell or tissue extracts are prepared in the presence of protease inhibitors. Further evidence shows that immunoprecipitation of in vitro translation products from brain, liver, and cultured fibroblasts also present a single band of Tp90 polypeptide. Pulse-chase experiments show that during apoptosis, Tp90 is processed to Tp60, and eventually to Tp30. However, when the total protein extracts are fractionated, only Tp90 is found in the detergent-soluble fraction, with diminishing quantities during the time course of apoptosis, and Tp30, in contrast, is found as the only protein species in the insoluble fraction, with increasing quantity during the same time course. Newly processed Tp60 is not found in either of the fractions, reflecting its loss during the fractionation procedure. Limited one-dimensional peptide mapping of Tp90 yields three different bands at 30, 28, and 25 kDa, but only the one at 30 kDa is recognized by Mab 1.2. These results lead us to suggest that terminin protein is synthesized in the Tp90 form, and cleaved to lower molecular weight forms depends upon different physiologic conditions, with Tp60 processed in the terminally differentiated or senescent state and rapidly to Tp30 in apoptosis. Our findings further suggest that Tp90's processing to either Tp60 or Tp30 produces insoluble protein forms. Furthermore, the presence of Tp90 in nonapoptotic (either replicating or nonreplicating) cells may reflect the absence of necessary proteolytic action required for the execution of apoptosis. Future experiments will allow us to determine the nature of this proteolytic action, as well as whether this action is due to the autocatalytic action of Tp90 or by other endogenous proteases, and then to determine the significance of this biochemical action in cells. © 1996 Wiley-Liss, Inc.
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    Genetic susceptibility to cancer from exogenous and endogenous exposures (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 63 (1996), S. 15-22 
    Details
    ISSN: 0730-2312
    Keywords: bladder cancer ; breast cancer ; ethnicity ; polymorphism prostate cancer ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The past four decades of epidemiological research have yielded valuable information on the risks of populations to environmental exposures such as tobacco, asbestos, and dietary components. Prevention efforts have been focused on large-scale population-based interventions to minimize exposure to such external carcinogens. While some cancers are beginning to show a decline from changing environmental exposures, hormone-related cancers, such as breast and prostate, are becoming more prevalent. The development of these cancers appears to be closely related to endogenous exposures to circulating steroid hormones. Although prevention trials using antihormone agents are proving successful in some instances, the long-term control of these cancers necessitates a clearer understanding of the metabolism and transport of the relevant hormone in vivo.The revolution in molecular biology has provided powerful genetic tools for evaluating mechanisms of cancer causation as well as the potential to better define individual susceptibility. Using tobacco exposure as an example, we and others have demonstrated that polymorphisms in genes controlling aromatic amine metabolism provide at least a partial explanation for ethnic and individual susceptibility to bladder cancer. Similar studies have examined genetic polymorphisms in the metabolism of tobacco smoke and lung cancer risk, red meat and colorectal cancer, and aflatoxin and liver cancer.Our current studies have pursued a similar paradigm of genetic polymorphism and individual cancer susceptibility in prostate and breast carcinogenesis. We are evaluating polymorphisms in the steroid 5α-reductase type II and androgen receptor genes in relation to prostate cancer based on the evidence that intracellular dihydrotestosterone is the critical “carcinogen.” We are pursuing genetic polymorphisms affecting estradiol metabolism, including those in the 17β-hydroxysteroid dehydrogenase 2 and estrogen receptor genes as they relate to susceptibility to breast cancer. The potential role of a polymorphism in the cytochrome P450c17α gene in both breast and prostate cancers is also being examined. J. Cell. Biochem. 25S:15-22. © 1997 Wiley-Liss, Inc.
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    Cancer risk factors for selecting cohorts for large-scale chemoprevention trials (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 63 (1996), S. 29-36 
    Details
    ISSN: 0730-2312
    Keywords: biomarkers ; cancer risk assessment ; gene-environment interactions ; large-scale trials ; prevention trials decision network ; twin studies ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Many anticipate that application of findings in molecular genetics will help to achieve greater precision in defining high-risk populations that may benefit from chemopreventive interventions. We must recognize, however, that genetic susceptibility, environmental factors, and complex gene-environment interactions are all likely to be risk determinants for most cancers. Cohort studies of twins and cancer indicate that having “identical” genes is generally not a very accurate predictor of cancer incidence. Data from twin studies support the suggestion that environmental factors such as tobacco use significantly influence cancer risk. The complexities of the genetic contribution to disease risk are exemplified by the development of Duchenne muscular dystrophy in only one of monozygotic twin girls, hypothesized to be the result of X chromosome inactivation, with the distribution patterns of the X chromosome being skewed to the female X in the manifesting twin and to the male X in the normal twin. Evidence from transgenic and genetic-environmental studies in animals support the possibility of genetic-environmental interactions. Calorie restriction modifies tumor expression in p53 knockout mice; a high-fat, low-calcium, low-vitamin D diet increases prepolyp hyperplasia formation in Apc-mutated mice; and calorie restriction early in life influences development of obesity in the genetically obese Zucker rat (fafa). Such environmental modulation of gene expression suggests that chemoprevention has the potential to reduce risk for both environmentally and genetically determined cancers.In view of the growing research efforts in chemoprevention, the NCI has developed a Prevention Trials Decision Network (PTDN) to formalize the evaluation and approval process for large-scale chemoprevention trials. The PTDN addresses large trial prioritization and the associated issues of minority recruitment and retention; identification and validation of biomarkers as intermediate endpoints for cancer; and chemopreventive agent selection and development. A comprehensive database is being established to support the PTDN's decision-making process and will help to determine which agents investigated in preclinical and early phase clinical trials should move to large-scale testing. Cohorts for large-scale chemoprevention trials include individuals who are determined to be at high risk as a result of genetic predisposition, carcinogenic exposure, or the presence of biomarkers indicative of increased risk. Current large-scale trials in well-defined, high-risk populations include the Breast Cancer Prevention Trial (tamoxifen), the Prostate Cancer Prevention Trial (finasteride), and the N-(4-hydroxyphenyl) retinamide (4-HPR) breast cancer prevention study being conducted in Milan. Biomarker studies will provide valuable information for refining the design and facilitating the implementation of future large-scale trials. For example, potential biomarkers are being assessed at biopsy in women with ductal carcinoma in situ (DCIS). The women are then randomized to either placebo, tamoxifen, 4-HPR, or tamoxifen plus 4-HPR for 2-4 weeks, at which time surgery is performed and the biomarkers reassessed to determine biomarker modulation by the interventions. For prostate cancer, modulation of prostatic intraepithelial neoplasia (PIN) by 4-HPR and difluoromethylornithine is being investigated; similar studies are being planned for oltipraz, dehydroepiandrosterone, and vitamin E plus selenomethionine. The validation of biomarkers as surrogate endpoints for cancer incidence in high-risk cohorts will allow more agents to be evaluated in shorter studies that use fewer subjects to achieve the desired statistical power. J. Cell. Biochem. 25S:29-36. © 1997 Wiley-Liss, Inc. This article is a U.S. Government work and, as such, is in the public domain in the United States of America.
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    Inherited susceptibility and acquired allelic imbalance in rat mammary carcinogenesis (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 63 (1996), S. 37-40 
    Details
    ISSN: 0730-2312
    Keywords: mammary cancer ; cancer genetics ; epigenetic ; chemoprevention ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Individual genetically determined susceptibility to cancer as well as acquired epigenetic and genetic organ specific alterations are important considerations in choosing target populations for chemopreventive trials. These individual epigenetic and genetic alterations can also serve as potential biomarkers for chemoprevention clinical trials. In order to model these potential markers for chemoprevention investigations, we are examining a series of interrelated rat models.Inbred rats vary in their susceptibility to mammary cancer induction by environmental agents. For example, the WF strain is highly susceptible to chemically induced mammary cancer while the Cop rat is almost completely resistant. The F344 is intermediate in susceptibility to chemically induced mammary cancer. These differential susceptibilities are inherited in a dominant pattern. For example, resistance is due to the inheritance of Mcs gene(s) which likely act by altering the differentiation lineage of mammary epithelial cells.As tumors form in the mammary glands of these rats, they acquire additional epigenetic and genetic alterations. Epigenetic initiation is a very frequent cellular event following carcinogen exposure which may predispose cells to genetic change including allelic imbalance. For example, following a standard dose of NMU or DMBA over 1% of cells are epigenetically initiated. During the carcinogenesis process, initiated cells may acquire genetic change such as oncogene activation and allelic imbalance. Interestingly, the pattern of allelic imbalance appears to be an inherited trait. For example, a non-random loss of heterozygosity (LOH) in rat chromosome 1 following DMBA only occurs in certain strains, such as Cop rats. Interestingly this change does not occur following initiation by ionizing radiation.It will thus be important to identify these epigenetic and genetic events which underlie mammary carcinogenesis as well as determine their patterns of inherited predisposition and temporal occurrence. Such knowledge is critical if we are to develop new molecular markers for chemoprevention trials. J. Cell. Biochem. 25S:37-40. © 1997 Wiley-Liss, Inc.
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    DNA quantification in cervical intraepithelial neoplasia thick tissue sections by confocal laser scanning microscopy (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 63 (1996), S. 49-56 
    Details
    ISSN: 0730-2312
    Keywords: cervical intraepithelial neoplasia ; confocal microscopy ; DNA quantitation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Image analysis of tissue biopsies for determination of DNA content as an early marker of neoplasia is hampered by the complexity of corrections necessary to deal with nuclear truncation and overlap in thin sections. The use of confocal laser scanning microscopy (CLSM) for measurement of cellular DNA content on whole cells within thick tissue sections offers the advantage of preservation of cellular architecture, capacity for 3-dimensional analysis, and absence of sectioning artifacts. We have applied this technique to pararosaniline-Feulgen stained human cervical tissues graded from normal to cervical intraepithelial neoplasia (CIN) III. For the purpose of comparison, 15 μm sections were stained and mapped so that the same cell population could be analyzed by both integrated optical density and fluorescence intensity. Distribution of DNA content from normal cervical epithelial cells 2-3 layers out from the basal cell layer measured by both methodologies showed a stable G0/G1 population with no observable S-phase or G2 cells. Cells measured from areas of increasing CIN grade showed progressively higher DNA content values that were not observable in normal tissue. Although these data are preliminary they suggest that CLSM can be used to identify aneuploid states within defined structural areas of pre-invasive neoplasia. J. Cell. Biochem. 25S:49-56. © 1997 Wiley-Liss, Inc.
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    Normal and transforming Ras are differently regulated for posttranslational modifications (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 61 (1996), S. 172-181 
    Details
    ISSN: 0730-2312
    Keywords: c-H-ras ; Ras ; posttranslational modification ; NIH3T3 ; c-myc ; p53 ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Point mutation of the c-H-ras gene significantly increases cellular transforming activities of Ras. Since posttranslational modification and subsequent membrane localization are essential for the biological activities of Ras, we examined whether or not the mutation also affects these two factors. The normal (Gly12) or the transforming (Val12) c-H-ras gene was expressed in NIH3T3 cells using a metallothionein promoter. Expression of either type of Ras was efficiently induced by the cadmium treatment of these cells, and immunoprecipitation of metabolically labeled cell extracts revealed that both normal and transforming Ras were expressed as four differently migrating forms on SDS-polyacrylamide gels, two of which were slower migrating cytosolic precursors and the other two were faster migrating membrane-bound forms. There was no significant difference in half lives between normal and transforming Ras; however, posttranslational modification was quite different between the two types of Ras. Transforming Ras was processed and became membrane-bound forms much more efficiently than normal Ras. Interestingly, posttranslational modification and membrane localization of Ras was significantly inhibited when the c-myc oncogene was co-expressed with Ras. In contrast to the c-myc oncogene, expression of either wild type or mutant p53 did not affect the posttranslational modification of Ras, suggesting that the c-myc oncogene specifically impairs the posttranslational modification of Ras. © 1996 Wiley-Liss, Inc.
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    Specific involvement of glypican in thrombin adhesive properties (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 61 (1996), S. 278-291 
    Details
    ISSN: 0730-2312
    Keywords: endothelial cells ; heparan sulfate ; cryptic RGD ; cell attachment ; thrombin ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We have previously demonstrated that thrombin possesses an active yet cryptic Arg-Gly-Asp (RGD) site which upon exposure induces endothelial cell (EC) adhesion via αvβ3 integrin [Bar-Shavit et al. (1991): J Cell Biol 112:335]. This was achieved in the presence of cell surface-associated heparan sulfate proteoglycans (HSPG) and exceedingly low concentrations of plasmin [Bar-Shavit et al. (1993): J Cell Biol 123:1279]. A portion of the cell surface-associated HSPG (glypican) is anchored via a covalently linked glycosyl-phosphatidylinositol (PI) residue, which can be released by treatment with glycosyl-Pl-specific phospholipase C (PI-PLC). We report here that exposure of either bovine aortic EC, smooth muscle cells (SMC), or wild-type CHO cells to PI-PLC released HSPG involved in the conversion of thrombin to an adhesive molecule. The adhesion-promoting activity of the released HSPG was abolished following treatment with heparinase but not chondroitinase ABC. Incubation of thrombin with heparan sulfate-deficient CHO cells or cells that were pretreated with PI-PLC failed to induce its conversion to an adhesive molecule, indicating that glypican was playing a major role in this conversion. Moreover, affinity-purified glypican, but not syndecan or fibroglycan, elicited efficient conversion of plasmin-treated thrombin into an adhesive molecule. Antibodies raised against the RGD site in thrombin failed to interact with native thrombin, prothrombin, or the RGD site in other adhesive proteins such as vitronectin, fibrinogen, or fibronectin. Anti-thrombin-RGD antibodies which blocked the adhesion-promoting activity of thrombin were also capable of recognizing thrombin that was first incubated with a suboptimal concentration of plasmin in the presence of PI-PLC-released HSPG. Heparin, heparan sulfate, and PI-PLC-released HSPG had no effect on other cellular properties of thrombin such as receptor binding and growth-promoting activity. Altogether we have demonstrated that the heparin binding domain in thrombin plays a specific role in promoting thrombin adhesive properties and that membrane-associated glypican is likely to be the major physiological inducer of this property. © 1996 Wiley-Liss, Inc.
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    Bone tissue-specific transcription of the osteocalcin gene: Role of an activator osteoblast-specific complex and suppressor hox proteins that bind the OC box (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 61 (1996), S. 310-324 
    Details
    ISSN: 0730-2312
    Keywords: osteocalcin ; transcriptional regulation ; homeodomain protein ; Msx ; bone-specific ; OC box ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Bone-specific expression of the osteocalcin gene is transcriptionally controlled. Deletion analysis of osteocalcin promoter sequences by transient transfection of osseous (ROS 17/2.8) and nonosseous (R2 fibroblast) cells revealed that the most proximal 108 nucleotides are sufficient to confer tissue-specific expression. By gel mobility shift assays with wild-type and mutated oligonucleotides and nuclear extracts from several different cell lines we identified a novel transcription factor complex which exhibits sequence-specific interactions with the primary transcriptional element, the OC box (nt -99 to -76). This OC box binding protein (OCBP) is present only in osteoblast-like cells. Methylation interference demonstrated association of the factor with OC box sequences overlapping the Msx homeodomain consensus binding site. By assaying several mutations of the OC box, both in gel shift and transient transfection studies using ROS 17/2.8, we show the following. First, binding of OCBP correlates with osteocalcin promoter activity in ROS 17/2.8 cells. Increased binding leads to a 2-3-fold increase in transcription, while decreased binding results in transcription 30-40% of control. Second, homeodomain protein binding suppresses transcription. However, Msx expression is critical for full development of the bone phenotype as determined by antisense studies. Last, we show that one of the mutations of the OC box permits expression of osteocalcin in non-osseous cell lines. In summary, we demonstrate association of at least two classes of tissue-restricted transcription factors with the OC box element, the OCBP and Msx proteins, supporting the concept that these sequences contribute to defining tissue specificity. © 1996 Wiley-Liss, Inc.
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    Molecular genetic biomarkers in hematological malignancies (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 63 (1996), S. 165-171 
    Details
    ISSN: 0730-2312
    Keywords: biomarkers ; leukemia ; lymphoma ; molecular genetics ; risk factors ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Most hematopoietic malignancies are widely disseminated even in their “early” stages and often do not have a well-defined localized phase. This makes them less amenable to conventional early screening methods such as imaging and observation. Furthermore, the staging systems for lymphomas are not particularly useful prognostically, with the possible exception of Hodgkin's disease. However, as currently compared with solid tumors, the extensively detailed understanding of the acquired (somatic) genetic lesions in leukemias and lymphomas provide useful molecular biomarkers for early detection. Moreover, well described high risk groups have been identified. These include individuals who are immunosuppressed, for example, iatrogenically following organ transplantation or those with AIDS. Also at high risk are patients treated with certain chemotherapeutic agents who are at risk for the development of acute non-lymphoblastic leukemia. Accordingly, these clinical settings might prove to be good models for evaluating molecular cancer risk markers and the possible introduction of chemoprevention. Here, we outline the biological basis for the application of biomarkers for the early detection of hematological neoplasia. These concepts may provide the stage for the creation of chemoprevention studies in leukemia and lymphoma. J. Cell. Biochem. 25S:165-171. © 1997 Wiley-Liss, Inc.
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    Clinical detection of lung cancer progression markers (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 63 (1996), S. 177-184 
    Details
    ISSN: 0730-2312
    Keywords: lung cancer ; progression markers ; sputum cells ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Lung cancer is the leading cause of cancer-related deaths in western countries. The prognosis for patients with lung cancer depends primarily on the stage of the tumor at the time of clinical diagnosis. New understanding of tumor biology has turned attention away from detection of clinical lung cancer, usually metastatic at presentation, toward recognition of genetic and protein markers which precede malignancy. Mutations of four types of genes contribute to the process of epithelial carcinogenesis by modifying control of cell growth. Examples of three of these changes have been detected in pre-malignant sputum, and validated in subsequent tumor. We have identified gene products (tumor associated and differentiation protein antigens), mutations of k-ras and p53, and microsatellite alterations as potential markers of subsequent malignancy.We consider the morphologic progression seen in archived sputum cells as the paradigm of neoplastic development in the lung. Although the NCI collaborative trials had shown that this progression is not recognized sufficiently often (sensitive) to be useful for lung cancer screening, this progression may be used to assess the timing of gene and peptide markers of carcinogenesis. Previous work has shown that at the time Johns Hopkins Lung Project sputum cells express moderately atypical metaplasia, 53% (8/15) of sputum specimens expressed common (codon 12) k-ras or (codons 273 or 281) p53 mutations. Other investigators have reported that earlier morphologic changes (metaplasia) accompany 3p and 9p losses of heterozygosity. These observations suggest that 3p and 9p loss likely precede k-ras or p53 mutations. Our preliminary data demonstrate that over-expression of a 31 kD tumor associated antigen recently purified, sequenced, and identified as heterogeneous nuclear ribonucleoprotein (hnRNP) A2 (with cross reactivity to splice variant B1), is expressed in most lung cancer cases before any morphologic abnormality. Comparison of the accuracy of this marker with sputum cytology will determine its value for early lung cancer detection. Preliminary evidence confirms this marker greatly improves the accuracy of standard sputum cytology for detection of lung carcinogenesis. Clinical intervention trials must be undertaken to determine whether modulation of hnRNP overexpression is useful as an intermediate endpoint for chemoprevention. J. Cell. Biochem. 25S:177-184. © 1997 Wiley-Liss, Inc.
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    High activity, soluble, bacterially expressed human vitamin D receptor and its ligand binding domain (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 61 (1996), S. 325-337 
    Details
    ISSN: 0730-2312
    Keywords: vitamin D receptor ; 1α,25(OH)2vitamin D3 ; pMal ; ligand binding ; gel shift analysis ; VDRE ; osteocalcin gene promoter ; fibronectin gene promoter ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The effects of 1α,25(OH)2vitamin D3 on cell growth and differentiation are primarily mediated by the nuclear vitamin D receptor (VDR). In order to study aspects of receptor function and ultimately the structural basis of the VDR-ligand interaction, it is necessary to produce large quantities of purified VDR. To achieve this, we have expressed the human VDR and its ligand binding domain in E. coli as fusion proteins with the maltose binding protein using the expression vector pMal-c2. In this system high level expression of both fusion proteins in a soluble form was achieved, whereas previous attempts to express the VDR in E. coli have resulted in an insoluble product. After affinity purification on amylose resin, the fusion proteins were isolated with yields of 10-20 mg/l of culture. Both forms of the recombinant receptor bound 1α,25(OH)2vitamin D3 with high affinity; estimated Kd values from Scatchard analysis for the purified full-length receptor and the ligand binding domain were 0.16 ± 0.07 nM and 0.04 ± 0.02 nM, respectively. The nonhypercalcemic analogs of vitamin D, MC903 and Δ22-1,25S,26(OH)3vitamin D3, bound the recombinant fusion proteins with a similar affinity to the native ligand, 1α,25(OH)2vitamin D3. In addition, the full-length VDR fusion protein was shown by gel shift analysis to bind weakly to the human osteocalcin gene vitamin D response element, an interaction greatly facilitated by addition of RXRα. These results show that the bacterial expression system detailed here is readily able to produce soluble and functional VDR and its ligand binding domain in high yield. These proteins are easily purified and should be suitable for further structural and functional analysis. © 1996 Wiley-Liss, Inc.
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    Low-molecular-weight variants of osteopontin generated by serine proteinases in urine of patients with kidney stones (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 61 (1996), S. 402-409 
    Details
    ISSN: 0730-2312
    Keywords: sialoglycoproteins ; ELISA for urine osteopontin ; kidney stones ; phenylmethylsulfonyl fluoride ; proteolytic cleavage ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Osteopontin (OPN) is a multifunctional glycosylated phosphoprotein found in body fluids, including urine, and has been implicated in urinary stone formation. We tested the hypothesis that OPN levels in urine of patients with kidney stones differed from normal individuals. To quantify OPN levels in the urine, we developed an ELISA using a combination of a mouse monoclonal and rabbit polyclonal antibodies raised against a recombinant glutathione-S-transferase-human OPN fusion protein. In a group of 34 patients diagnosed with kidney stones compared with a control group of 23 normal individuals, we found that OPN levels in urine of the patient and control groups ranged from 0.01 to 2.7 μg/ml, with no significant difference in their medians (P 〉 0.8, Mann-Whitney test). OPN in urine was qualitatively assessed by Western blotting using a biotinylated monoclonal antibody to detect various molecular forms. The urine of most individuals contained OPN species within in the 55- to 66-kDa electrophoretic mobility range. However, a significantly higher proportion of individuals in the patient group (13 of 34) was found to have aberrant urine OPN species (≤ 40 kDa) compared to 2 of 23 for the control group (P 〈 0.03, x2 test). Mixing experiments indicated that urine samples with aberrant OPN contain proteases inhibitable with phenylmethylsulfonyl fluoride. Such proteases could break down normal urine OPN in vitro. Therefore, urine from a high frequency of kidney stone patients contains serine proteases that contribute to proteolytic cleavage of OPN. © 1996 Wiley-Liss, Inc.
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    Phorbol myristate acetate transactivates the avian β3 integrin gene and induces αvβ3 integrin expression (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 61 (1996), S. 420-429 
    Details
    ISSN: 0730-2312
    Keywords: cell attachment ; gene transcription ; AP1 site; fos/jun ; 1,25(OH)2D3 ; macrophage-like cells ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: 1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) transactivates the avian β3 integrin gene whose promoter contains at least two vitamin D response elements, one of which is in close proximity to a candidate AP1 site (TGACTCA). Since fos/jun and steroid hormones interact to regulate gene expression, we asked whether phorbol-12-myristate-13-acetate (PMA), which stimulates binding of fos/jun to AP1 sites, transactivates the avian β3 integrin gene and, if so, does the phorbol ester modulate 1,25(OH)2D3 induction of the gene. We find the candidate AP1 sequence comigrates with the consensus AP1 sequence on electromobility shift assay when incubated with recombinant c-jun protein. Furthermore, PMA prompts expression of β3 integrin mRNA in the avian monocytic line, HD11. The increase in message reflects transactivation of the β3 gene and is mirrored by plasma membrane appearance of the integrin heterodimer αvβ3. Moreover, attesting to the functional significance of PMA-enhanced αvβ3 expression, cells treated with concentrations of the phorbol ester that induce the β3 gene, spread extensively on plastic, an event blocked by an anti-αv antibody and a peptide mimetic known to inhibit αvβ3-mediated cell attachment. Interestingly, co-addition of 1.25(OH)2D3 and PMA prompts greater expression of αvβ3 than when the cells are exposed to either agent alone and PMA enhances 1,25(OH)2D3-induced β3 integrin mRNA expression. Thus, PMA and 1,25(OH)2D3 impact on the avian β3 integrin gene independently and in combination. © 1996 Wiley-Liss, Inc.
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    Elucidation of the molecular actions of NCAM and structurally related cell adhesion molecules (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 61 (1996), S. 502-513 
    Details
    ISSN: 0730-2312
    Keywords: NCAM ; glycoproteins ; nervous system ; adhesion molecules ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The Neural Cell Adhesion Molecule (NCAM) is a founder member of a large family of cell surface glycoproteins that share structural motifs related to immunoglobulin and fibronectin type III (FN III) domains [Walsh and Doherty (1991) (Fig. 1). These glycoproteins have been grouped based on the respective number of each type of domain. In vertebrates members of this family of glycoproteins include L1/NILE, NgCAM, axonin-1/TAG-1, and Thy-1 as well as NCAM. In addition structural homologs of NCAM and L1 have been identified in Drosophila and Grasshoppers [Walsh and Doherty (1991)]. These insect homologs are called fasciclins and a series of mutants corresponding to these genes have been isolated. A homologue of NCAM has been identified in Aplysia where it may play a role in regulating aspects of synaptic plasticity [Mayford et al. (1992) Science 256:638-644]. In vertebrates all of these glycoproteins are expressed in the developing nervous system where they have been identified as candidate molecules for mediating axon outgrowth, fasciculation, regeneration, and target recognition. In addition, NCAM is expressed in a number of different tissues and cell types. For example, NCAM is expressed in a dynamic pattern in developing and regenerating adult muscle. In this review we aim to describe important aspects of the role of these CAMs in development of the nervous system, including the neuromuscular junction. Furthermore, we will explore the prospective use of molecular biology, cell biology, and molecular genetic techniques, such as transgenic mice, to understand the role and molecular action of this family of cell adhesion molecules in vivo. © 1996 Wiley-Liss, Inc.
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    Cadherin/catenin complex: A target for antiinvasive therapy? (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 61 (1996), S. 524-530 
    Details
    ISSN: 0730-2312
    Keywords: invasion ; cadherin ; catenins ; IGF-1 ; tamoxifen ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Invasion is a major challenge for cancer therapy. Invasion or noninvasion results from the cross talk between cancer cells and host cells, building molecular invasion-promoter and invasion-suppressor complexes. The E-cadherin/catenin invasion-suppressor complex is attractive as a target for a putative antiinvasive therapy because of its multifactorial regulation at multiple levels and sometimes in a reversible way. Mutations in the E-cadherin gene combined with loss of the wild type allele causes irreversible downregulation in some human cancers. Posttranslational and reversible downregulation may occur by tyrosine phosphorylation of β-catenin. Phosphorylation is implicated also in transmembrane receptor signal transduction through the E-cadherin/catenin complex. Homophilic interaction with E-cadherin on another cell through a dimeric adhesion zipper, involving the HAV sequence of the first extracellular domains, is the major extracellular link of the E-cadherin/catenin complex. Intracellularly, the list of proteins that bind to or signal through the complex or one or more of its elements is growing. In vitro, insulin-like growth factor-I, and tamoxifen may upregulate the functions of the E-cadherin/catenin complex and inhibit invasion, demonstrating that this complex may serve as a target for antiinvasive therapy. © 1996 Wiley-Liss, Inc.
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    Cadherin-catenin complex: Protein interactions and their implications for cadherin function (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 61 (1996), S. 514-523 
    Details
    ISSN: 0730-2312
    Keywords: cadherin ; catenin ; plakoglobin ; armadillo ; APC ; p120cas ; protein interactions ; gene targeting ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Cadherins comprise a family of calcium-dependent glycoproteins that function in mediating cell-cell adhesion in virtually all solid tissues of multicellular organisms. In epithelial cells, E-cadherin represents a key molecule in the establishment and stabilization of cellular junctions. On the cellular level, E-cadherin is concentrated at the adherens junction and interacts homophilically with E-cadherin molecules of adjacent cells. Significant progress has been made in understanding the extra- and intracellular interactions of E-cadherin. Recent success in solving the three-dimensional structure of an extracellular cadherin domain provides a structural basis for understanding the homophilic interaction mechanism and the calcium requirement of cadherins. According to the crystal structure, individual cadherin molecules cooperate to form a linear cell adhesion zipper. The intracellular anchorage of cadherins is regulated by the dynamic association with cytoplasmic proteins, termed catenins. The cytoplasmic domain of E-cadherin is complexed with either β-catenin or plakoglobin (γ-catenin). β-catenin and plakoglobin bind directly to α-catenin, giving rise to two distinct cadherin-catenin complexes (CCC). α-catenin is thought to link both CCC's to actin filaments. The anchorage of cadherins to the cytoskeleton appears to be regulated by tyrosine phosphorylation. Phosphorylation-induced junctional disassembly targets the catenins, indicating that catenins are components of signal transduction pathways. The unexpected association of catenins with the product of the tumor suppressor gene APC has led to the discovery of a second, cadherin-independent catenin complex. Two separate catenin complexes are therefore involved in the cross-talk between cell adhesion and signal transduction. In this review we focus on protein interactions regulating the molecular architecture and function of the CCC. In the light of a fundamental role of the CCC during mammalian development and tissue morphogenesis, we also discuss the phenotypes of embryos lacking E-cadherin or β-catenin. © 1996 Wiley-Liss, Inc.
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    Mucosal polyamine measurements and colorectal cancer risk (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 63 (1996), S. 252-257 
    Details
    ISSN: 0730-2312
    Keywords: polyamines ; biomarkers ; proliferation ; colorectal cancer risk ; mucosa ; biopsy ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Polyamines are short-chain aliphatic amines required for normal cellular growth that are ubiquitously found in all living tissues. Polyamine content has been shown to correlate with cellular proliferation. Quantitation of polyamines may thus provide a biochemical measure of proliferation in the colorectal mucosa where dysregulated epithelial proliferation is associated with colorectal cancer risk. A case-control study was conducted to validate the hypothesized association between mucosal polyamine measurements and colorectal cancer risk. Polyamines were measured in 4-6 multiple rectal mucosal biopsies from 11 normal control subjects and seven case patients with colon cancer. Compared with the controls, mean polyamine measurements, after adjustment for age and sex, were significantly increased for spermidine (P 〈 0.003) and spermine (P 〈 0.017). Subsequent analyses indicated that in controls 1-4 biopsies appeared adequate to characterize an individual. However, mucosal polyamines in the cases exhibited more sampling variability, requiring 4-8 biopsies to achieve an acceptable level of reliability. After adjustment for age and sex, the odds ratios for spermidine and spermine levels, compared to the controls, were 4.8 (95% confidence interval: 1.6-33.7) and 2.3 (1.2-6.3), respectively. The results of this study indicate that increases of mucosal polyamine measurements, after taking the sampling and methodological variability into account, are significantly associated with colorectal cancer risk, and suggest that polyamine measurements in rectal mucosa may play an important role as biomarkers for identifying high-risk individuals and/or for using as intermediate endpoints in prevention trials. © 1996 Wiley-Liss, Inc.
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    PML-containing nuclear bodies: Their spatial distribution in relation to other nuclear components (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 63 (1996), S. 280-291 
    Details
    ISSN: 0730-2312
    Keywords: nuclear bodies ; PML ; confocal microscopy ; image restoration ; RNA ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The PML protein is a human growth suppressor concentrated in 10 to 20 nuclear bodies per nucleus (PML bodies). Disruption of the PML gene has been shown to be related to acute promyelocytic leukaemia (APL). To obtain information about the function of PML bodies we have investigated the 3D-distribution of PML bodies in the nucleus of T24 cells and compared it with the spatial distribution of a variety of other nuclear components, using fluorescence dual-labeling immunocytochemistry and confocal microscopy. Results show that PML bodies are not enriched in nascent RNA, the splicing component U2-snRNP, or transcription factors (glucocorticoid receptor, TFIIH, and E2F). These results show that PML bodies are not prominent sites of RNA synthesis or RNA splicing. We found that a large fraction of PML bodies (50 to 80%) is closely associated with DNA replication domains during exclusively middle-late S-phase. Furthermore, in most cells that we analysed we found at least one PML body was tightly associated with a coiled body. In the APL cell line NB4, the PML gene is fused with the RARα gene due to a chromosomal rearrangement. PML bodies have disappeared and the PML antigen, i.e., PML and the PML-RAR fusion protein, is dispersed in a punctated pattern throughout the nucleoplasm. We showed that in NB4 cells the sites that are rich in PML antigen significantly colocalize with sites at which nascent RNA accumulates. This suggests that, in contrast to non-APL cells, in NB4 cells the PML antigen is associated with sites of transcription. The implications of these findings for the function of PML bodies are consistent with the idea that PML bodies are associated with specific genomic loci. © 1996 Wiley-Liss, Inc.
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    Prostaglandin E2 up-regulates insulin-like growth factor binding protein-3 expression and synthesis in human articular chondrocytes by a c-AMP-independent pathway: Role of calcium and protein kinase A and C (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 63 (1996), S. 320-333 
    Details
    ISSN: 0730-2312
    Keywords: chondrocytes ; calcium ; protein kinase C ; calphostin C ; gene expression ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Insulin-like growth factor-1, IGF-1, is believed to be an important anabolic modulator of cartilage metabolism and its bioactivity and bioavailability is regulated, in part, by IGF-1 binding protein 3 (IGFBP-3). Prostaglandin E2 (PGE2) stimulates IGF-1 production by articular chondrocytes and we determined whether the eicosanoid could regulate IGFBP-3 and, as such, act as a modifier of IGF-1 action at a different level. Using human articular chondrocytes in high density primary culture, Western and Western ligand blotting to measure secreted IGFBP-3 protein, and Northern analysis to monitor IGFBP-3 mRNA levels, we demonstrated that PGE2 provoked a 3.9 ± 1.1 (n = 3) fold increase in IGFBP-3 mRNA and protein. This effect was reversed by the Ca++ channel blockers, verapamil and nifedipine, and the Ca++/calmodulin inhibitor, W-7. The Ca++ ionophore, ionomycin, mimicked the effects of PGE2 as did the phorbol ester PMA, which activates Ca++-phospholipid-dependent protein kinase C (PKC). Cyclic AMP mimetics, such as forskolin, IBMX, Ro-20-1724, and Sp-cAMP, inhibited the expression and synthesis of the binding protein. PGE2 did not increase the levels of cAMP or protein kinase A (PKA) activity in chondrocytes. The PGE2 secretagogue, IL-1β, down-regulated control levels of IGFBP-3 which could be completely abrogated by pre-incubation with the tyrosine kinase inhibitor, erbstatin, and partially reversed (50 ± 8%) by KT-5720, a PKA inhibitor. These observations suggested that PGE2 does not mediate the effect of its secretagogue and that IL-1β signalling in chondrocytes may involve multiple kinases of diverse substrate specificities. Dexamethasone down-regulated control, constitutive levels of IGFBP-3 mRNA and protein eliminating the previously demonstrated possibility of cross-talk between glucocorticoid receptor (GR) and PGE2 receptor signalling pathways. Taken together, our results suggest that PGE2 modulates IGFBP-3 expression, protein synthesis, and secretion, and that such regulation may modify human chondrocyte responsiveness to IGF-1 and influence cartilage metabolism. © 1996 Wiley-Liss, Inc.
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    Final checkpoint in the drug-promoted and poliovirus-promoted apoptosis is under post-translational control by growth factors (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 63 (1996), S. 422-431 
    Details
    ISSN: 0730-2312
    Keywords: actinomycin D ; cycloheximide ; DNA degradation ; chromatin fragmentation ; serum factors ; epidermal growth factor ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The treatment of HeLa subline (HeLa-B) cells with cycloheximide or Actinomycin D resulted in a rapid (∼ 1.5 h and ∼ 2.5 h, respectively) development of morphological and biochemical signs of apoptosis. The addition of fetal bovine serum to the cycloheximide-treated or Actinomycin D-treated cells suppressed the apoptotic reaction, as evidenced by the postponement of the DNA fragmentation for at least 9 and 5 h, respectively. A similar suppressive effect was observed upon the serum addition to cells undergoing abortive infection with poliovirus, which died of apoptosis in the absence of the serum. The serum appeared to exert its anti-apoptotic effect without any appreciable lag and even immediately blocked further progress of ongoing DNA fragmentation. The epidermal growth factor also suppressed, although less efficiently and more transiently, the apoptotic reaction promoted by the metabolic inhibitors. It is concluded that growth factors may affect, without modulating either transcription or translation, the balance of pro-apoptotic and anti-apoptotic activities at a final checkpoint, just preceding the irreversible effector step of apoptosis. © 1996 Wiley-Liss, Inc.
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    Nuclear membrane cholesterol can modulate nuclear nucleoside triphosphatase activity (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 63 (1996), S. 442-452 
    Details
    ISSN: 0730-2312
    Keywords: cholesterol ; NTPase ; nuclear membrane ; pore complex ; hepatic tissue ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Previous work has suggested that changes in nuclear membrane cholesterol may induce a stimulation in nuclear nucleoside triphosphatase (NTPase) activity. The purpose of the present study was to directly investigate if nuclear membrane cholesterol can stimulate nuclear NTPase activity. The cholesterol content of nuclei was altered with a liposomal methodology. The cholesterol content of nuclei isolated from hepatic tissue was relatively low in comparison to that typically exhibited by other membrane fractions. Because of this, it was difficult to further deplete the nuclear membrane of cholesterol, but we could successfully increase the cholesterol content after exposure to cholesterol-enriched liposomes. Nuclear NTPase activity was potently stimulated (∼ 150-200% of control) by an increase in the nuclear membrane cholesterol content. The Vmax of the NTPase activity in the presence of ATP or GTP was significantly increased after cholesterol enrichment without altering the affinity of the enzyme for these moieties. Mg2+ dependency of NTPase activity was also altered by cholesterol incorporation into the nuclear membrane. Cholesterol enrichment of the nuclear membrane also left the nuclei more susceptible to damage by salt-induced lysis than control nuclei. Our results clearly demonstrate that the cholesterol content of the nuclear membrane will have significant, direct effects on nuclear integrity and NTPase activity. © 1996 Wiley-Liss, Inc.
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    Basic fibroblast growth factor stimulates phosphatidylcholine-hydrolyzing phospholipase D in osteoblast-like cells (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 63 (1996), S. 491-499 
    Details
    ISSN: 0730-2312
    Keywords: tyrosine kinase ; protein kinase C ; proliferation ; osteoblasts ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We examined the effect of basic fibroblast growth factor (bFGF) on the activation of phosphatidylcholine-hydrolyzing phospholipase D in osteoblast-like MC3T3-E1 cells. bFGF stimulated both the formations of choline (EC50 was 30 ng/ml) and inositol phosphates (EC50 was 10 ng/ml). Calphostin C, an inhibitor of protein kinase C (PKC), had little effect on the bFGF-induced formation of choline. bFGF stimulated the formation of choline also in PKC down regulated cells. Genistein and methyl 2,5-dihydroxycinnamate, inhibitors of protein tyrosine kinases, significantly suppressed the bFGF-induced formation of choline. Sodium orthovanadate, an inhibitor of protein tyrosine phosphatases, enhanced the bFGF-induced formation of choline. In vitro kinase assay for FGF receptors revealed that FGF receptor 1 and 2 were autophosphorylated after FGF stimulation. bFGF dose-dependently stimulated DNA synthesis of these cells. These results strongly suggest that bFGF activates phosphatidylcholine-hydrolyzing phospholipase D through the activation of tyrosine kinase, but independently of PKC activated by phosphoinositide hydrolysis in osteoblast-like cells. © 1996 Wiley-Liss, Inc.
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    PTH/PTHrP receptor is temporally regulated during osteoblast differentiation and is associated with collagen synthesis (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 61 (1996), S. 638-647 
    Details
    ISSN: 0730-2312
    Keywords: osteoblast ; bone ; parathyroid hormone ; receptor ; differentiation ; collagen ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The temporal sequence of PTH/PTHrP receptor mRNA, binding, biologic activity, and its dependence on matrix synthesis was determined using MC3T3-E1 preosteoblast-like cells and primary rat calvarial cells in vitro. Osteoblastic cells were induced to differentiate and form mineralized nodules with the addition of ascorbic acid and β-glycerophosphate, and samples were collected from 0-26 days of culture. DNA levels as determined by fluorometric analysis increased 12- and 17-fold during the collection period for both MC3T3-E1 and primary calvarial cells respectively. Steady state mRNA levels for the PTH/PTHrP receptor as determined by northern blot analysis, were initially low for both cell types, peaked at day 4 and 5 for MC3T3-E1 and primary calvarial cells respectively, and declined thereafter. Competition binding curves were performed during differentiation using 125I-PTHrP. The numbers of receptors per μg DNA were greatest at days 3 and 5 for MC3T3-E1 and primary calvarial cells respectively. The biologic activity of the receptor was evaluated by stimulating the cells with 10 nM PTHrP and determining cAMP levels via a binding protein assay. The PTHrP-stimulated cAMP levels increased 5-fold to peak values at day 5 for MC3T3-E1 cells and 6-fold to peak values at day 4 for the primary calvarial cells. Ascorbic acid was required for maximal development of a PTH-dependent cAMP response since ascorbic acid-treated MC3T3-E1 cells had twice the PTH-stimulated cAMP levels as non-treated cells. When the collagen synthesis inhibitor 3,4-dehydroproline was administered to MC3T3-E1 cultures prior to differentiation, there was a subsequent diminution of the PTH/PTHrP receptor mRNA gene expression and numbers of receptors per cell; however, if administered after the initiation of matrix synthesis there was no reduction in PTH/PTHrP receptor mRNA. These findings indicate that the PTH/PTHrP receptor is associated temporally at the level of mRNA, protein, and biologic activity, with a differentiating, matrix-producing osteoblastic cell in vitro. © 1996 Wiley-Liss, Inc.
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    Ultrastructural localization of active genes in nuclei of A431 cells (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 62 (1996), S. 10-18 
    Details
    ISSN: 0730-2312
    Keywords: 5-bromouridine 5′-triphosphate ; electron microscopy ; domain ; nuclear matrix ; RNA polymerase II ; transcription ; ultrasmall gold ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We have studied the ultrastructural localization of active genes in nuclei of the human epidermoid carcinoma cell line A431. Nascent RNA was labeled by incorporation of 5-bromouridine 5′-triphosphate, followed by pre-embedment or postembedment immunogold labeling and electron microscopy using ultrasmall gold-conjugated antibodies and silver enhancement. This combination of techniques allowed a sensitive and high resolution visualization of RNA synthesis in the nucleus. Transcription sites were identified as clusters of 3-20 gold particles and were found throughout the nucleoplasm. The clusters had a diameter of less than 200 nm. The distribution of clusters of gold particles in nuclei is preserved in nuclear matrix preparations. Nascent RNA is associated with fibrillar as well as with granular structures in the matrix. A431 nuclei contained on average about 10,000 clusters of gold particles. This means that each cluster represents transcription of probably one active gene or, at most, a few genes. Our study does not provide evidence for aggregation of active genes. We found transcription sites distributed predominantly on the surface of electron-dense nuclear material, probably lumps of chromatin. This supports a model of transcription activation preferentially on the boundary between a chromosome domain and the interchromatin space. © 1996 Wiley-Liss, Inc.
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    Nitric oxide releasing reagent, S-nitroso-N-acetylpenicillamine, enhances the expression of manganese superoxide dismutase mRNA in rat vascular smooth muscle cells (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 62 (1996), S. 50-55 
    Details
    ISSN: 0730-2312
    Keywords: nitric oxide ; SNAP ; Mn-SOD ; inducible NOS ; vascular smooth muscle cells ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Cultured rat vascular smooth muscle cells (VSMCs) produce nitric oxide (NO) under stimulation by lipopolysaccharide (LPS) and interferon-γ (IFN-γ). NO synthase (NOS) and manganese superoxide dismutase (Mn-SOD) mRNA expressions are simultaneously induced by these stimulants in rat VSMCs. In VSMCs, S-nitroso-N-acetyl penicillamine (SNAP), one of the NO releasing reagents, induces Mn-SOD mRNA which may protect the VSMCs themselves. This suggests that NO itself may enhance the expression of Mn-SOD to protect the VSMC themselves against NO radicals in cultured rat VSMCs. © 1996 Wiley-Liss, Inc.
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    Remodelling of the nuclear periphery during muscle cell differentiation in vitro (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 62 (1996), S. 76-89 
    Details
    ISSN: 0730-2312
    Keywords: lamina ; lamins ; antigen P1 ; L6E9 ; nuclear matrix ; immunofluorescence ; immunoblotting ; electron microscopy ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We have examined the composition and ultrastructure of the nuclear periphery during in vitro myogenesis of the rat myoblast cell line, L6E9. Immunofluorescence labelling and immunoblotting showed that lamins A/C and B were all present in undifferentiated cells, but that they increased significantly before extensive cell fusion had occurred, with lamins A/C increasing proportionately more. Electron microscopic observations were consistent with these results, showing an increase in the prominence of the lamina during differentiation. On the other hand, immunofluorescence labelling suggested that the P1 antigen began to disappear from the nuclear periphery as the cells were fusing, after the increase in lamin quantity, and was no longer detectable in multinucleated cells. Unexpectedly, however, P1 was readily detected in isolated nuclei, whether prepared from myoblast or differentiated cultures, as well as in both myoblast and myotube nuclear matrices. It appears probable, therefore, that the fading of P1 labelling is due to masking of the epitope by a soluble factor recruited to the nuclear periphery as cells differentiate. These data, together with evidence that the genome is substantially rearranged during L6E9 myogenesis [Chaly and Munro, 1996], suggest that L6E9 cells are a useful model system in which to study the interrelationship of nuclear envelope organization, chromatin spatial order, and nuclear function. © 1996 Wiley-Liss, Inc.
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    Effect of organ site on nuclear matrix protein composition (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 62 (1996), S. 132-141 
    Details
    ISSN: 0730-2312
    Keywords: MAT-LyLu (MLL) ; gene expression ; two-dimensional electrophoresis ; orthotopic ; cancer ; extracellular matrix ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The nuclear matrix has been linked to several important cellular functions within cells, such as DNA organization and replication, as well as regulation of gene expression. It has been reported that the nuclear matrix protein composition is altered in cells grown on different extracellular matrices in vitro. This study examined the nuclear matrix protein composition of tumors produced by MAT-LyLu (MLL) rat prostate tumor cells implanted at different organ sites within the rat. When high resolution two-dimensional gels were utilized to compare nuclear matrix protein composition to the prostate orthotopic tumor, it was found that there were distinct protein differences depending upon where the tumor grew. In particular, there were 14 proteins found in the lung, six proteins found in intramuscular, 17 proteins is the heart, and five proteins in the tail vein tumor tissue that were not present in the prostate orthotopic tumor tissue. Therefore, this study adds evidence to support that the nuclear matrix composition of a cell is dependent, at least in part, by the extracellular matrix and/or different cellular environments and may have a role in site-specific differences in tumor properties. © 1996 Wiley-Liss, Inc.
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    Non-RGD domains of osteopontin promote cell adhesion without involving αv integrins (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 62 (1996), S. 123-131 
    Details
    ISSN: 0730-2312
    Keywords: OPN ; binding site ; integrin ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Osteopontin (OPN) is an integrin-binding secreted protein that contains an Arg-Gly-Asp (RGD) amino acid sequence and binds to various cell types via RGD-mediated interaction with the αvβ3 integrin. We have identified a cell line whose binding to OPN does not require RGD or αv interactions. We compared the ability of two murine cell lines, L929 fibroblastic cells and B16-BL6 melanoma cells, to interact with OPN (from human milk, and recombinant human and mouse OPN) as well as recombinant OPN prepared to include either the N-terminal or C-terminal halves but lacking the RGD sequence. Both cell lines adhered to GRGDS peptides coupled to BSA, and these interactions were inhibited by addition of GRGDS (but not GRGES) peptides or a monoclonal antibody specific to the αv integrin subunit. Adhesion of L929 cells to OPN was also dependent on the RGD sequence and the αv integrin subunit. However, the binding of B16-BL6 cells was not inhibited by either GRGDS peptides or the anti-αv antibody. B16-BL6 (but not L929) cells were also able to adhere to and spread on both N-terminal and C-terminal OPN proteins that lack the RGD sequence, and these interactions were not inhibited by either GRGDS peptides or anti-αv antibody. Together these results indicate that B16-BL6 cells can adhere to OPN by interactions that are independent of either the RGD sequence or the αv integrin subunit, and suggest that some cells can interact with additional, non-RGD binding sites in OPN. © 1996 Wiley-Liss, Inc.
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    Risk biomarkers and current strategies for cancer chemoprevention (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 63 (1996), S. 1-14 
    Details
    ISSN: 0730-2312
    Keywords: chemoprevention ; genetic/regulatory biomarkers ; high-risk cohorts ; intraepithelial neoplasia ; phase II clinical trials, risk biomarkers ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Quantifiable, well-characterized cancer risk factors demonstrate the need for chemoprevention and define cohorts for chemopreventive intervention. For chemoprevention, the important cancer risk factors are those that can be measured quantitatively in the subject at risk. These factors, called risk biomarkers, can be used to identify cohorts for chemoprevention. Those modulated by chemopreventive agents may also be used as endpoints in chemoprevention studies. Generally, the risk biomarkers fit into categories based on those previously defined by Hulka: 1) carcinogen exposure, 2) carcinogen exposure/effect, 3) genetic predisposition, 4) intermediate biomarkers of cancer, and 5) previous cancers.Besides their use in characterizing cohorts for chemoprevention trials, some risk biomarkers can be modulated by chemopreventive agents. These biomarkers may be suitable surrogate endpoints for cancer incidence in chemoprevention intervention trials. The criteria for risk biomarkers defining cohorts and serving as endpoints are the same, except that those defining cohorts are not necessarily modulated by chemopreventive agents. A primary criterion is that the biomarkers fit expected biological mechanisms of early carcinogenesis - i.e., differential expression in normal and high-risk tissue, on or closely linked to the causal pathway for the cancer, and short latency compared with cancer. They must occur in sufficient number to allow their biological and statistical evaluation. Further, the biomarkers should be assayed reliably and quantitatively, measured easily, and correlated to cancer incidence. Particularly important for cancer risk screening in normal subjects is the ability to use noninvasive techniques that are highly specific, sensitive, and quantitative.Since carcinogenesis is a multipath process, single biomarkers are difficult to correlate to cancer, as they may appear on only one or a few of the many possible causal pathways. As shown in colorectal carcinogenesis, the risks associated with the presence of biomarkers may be additive or synergistic. That is, the accumulation of genetic lesions is the more important determinant of colorectal cancer compared with the presence of any single lesion. Thus, batteries of biomarker abnormalities, particularly those representing the range of carcinogenesis pathways, may prove more useful than single biomarkers both in characterizing cohorts at risk and defining modulatable risks.Risk biomarkers are already being integrated into many chemoprevention intervention trials. One example is the phase II trial of oltipraz inhibition of carcinogen-DNA adducts in a Chinese population exposed to aflatoxin B1. Also, urine samples from subjects in this trial will be screened for the effect of oltipraz on urinary mutagens. A second example is a chemoprevention protocol developed for patients at high risk for breast cancer; the cohort is defined both by hereditary risk and the presence of biomarker abnormalities. Modulation of the biomarker abnormalities is a proposed endpoint. Also, dysplastic lesions, such as prostatic intraepithelial neoplasia, oral leukoplakia and colorectal adenomas, have been used to define high-risk cohorts and as potential modulatable surrogate endpoints in chemoprevention trials. J. Cell. Biochem. 25S:1-14. © 1997 Wiley-Liss, Inc. This article is a U.S. Government work and, as such, is in the public domain in the United States of America.
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    Defining and analyzing cohorts using molecular markers of cancer risk (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 63 (1996), S. 69-79 
    Details
    ISSN: 0730-2312
    Keywords: biological markers ; cancer ; gastric cancer ; genetics ; log rank test ; lung cancer ; molecular biology ; molecular epidemiology ; polymorphisms ; p53 ; prevention ; power ; sample size ; survival analyses ; trials ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Cancer is currently regarded to be the phenotypic expression of an accumulation of heritable alterations in the regulators of cell growth and differentiation. Though detailed knowledge of the sequence and in vivo mechanistic effects of these alterations is rudimentary for most, if not all, cancers, their identification does offer the potential for classifying groups of individuals who are heterogeneous with respect to their cancer risks, into more nearly homogeneous subgroups. In this paper, we illustrate the value of using markers, which we define as any manifestation of cellular molecular diversity, to increase subgroup homogeneity. In the context of time-to-event data, we demonstrate for both somatic mutations (acquired p.53 abnormalities in gastric mucosal cells) and inherited polymorphisms (polymorphisms in the phase 1 and 2 detoxifying enzymes) how knowledge regarding the population frequency of the marker, the effect of the marker on the risk of cancer development, and/or the effect of the marker on response to therapy, can be used to plan and analyze such trials. Using as paradigms demographic features of the recently begun Shandong precancerous gastric lesion intervention trial, and the recently completed α-tocopherol β-carotene (ATBC) lung cancer prevention study, we review the information, assumptions, and mathematical structure required for planning cancer prevention trials. We graphically demonstrate how informative markers make available strategies for selection, stratification, and optimal weighing, which, when properly implemented, increase the power of tests of effective cancer prevention agents. J. Cell. Biochem. 25S:69-79. © 1997 Wiley-Liss, Inc. This article is a U.S. Government work and, as such, is in the public domain in the United States of America.
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    Carcinogen-DNA and protein adducts: Biomarkers for cohort selection and modifiable endpoints in chemoprevention trials (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 63 (1996), S. 85-91 
    Details
    ISSN: 0730-2312
    Keywords: biologically effective dose ; aflatoxin-N7-guanine ; aflatoxin-albumin adducts ; oltipraz ; hepatocarcinogenesis ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Chemical-specific markers have been developed for a number of environmental carcinogens for use as molecular dosimeters of individual exposure. In addition to contributing substantially to the specificity and sensitivity of epidemiological studies aimed at determining the role of environmental agents in the etiology of human cancers, some of these biomarkers may prove to be useful endpoints for assessing the efficacy of preventive interventions, including exposure avoidance or remediation and chemoprevention. Biomarkers of the biologically effective dose may be particularly useful in this context in that they provide a mechanistic linkage between exposure and disease outcome. The biologically effective dose reflects the amount of toxicant that has interacted with its critical molecular target and can be measured through a variety of analytical techniques as either carcinogen-DNA or -protein adducts. Approaches for the development and validation of aflatoxin adduct biomarkers are presented as a paradigm for the application of carcinogen-specific markers for cohort selection and as modifiable endpoints for assessing efficacy in chemoprevention trials. J. Cell. Biochem. 25S:85-91. © 1997 Wiley-Liss, Inc.
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    Smokers and urinary genotoxins: Implications for selection of cohorts and modulation of endpoints in chemoprevention trials (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 63 (1996), S. 92-98 
    Details
    ISSN: 0730-2312
    Keywords: biomarkers ; chemoprevention ; DNA repair ; mutagenicity ; N-acetylcysteine ; oltipraz ; urinary mutagens ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Urinary genotoxicity assays measure the internal dose of genotoxic carcinogens, thereby providing a particularly sensitive endpoint for selecting cohorts of individuals exposed to cigarette smoke or other mutagens excreted with urines, as well as for evaluating the modulation of this parameter after administration of chemopreventive agents. Mutagenicity of urines was investigated in smoking Italian volunteers, who received oral N-acetylcysteine (NAC) at the same doses which are usually prescribed for the long-term treatment of chronic bronchitis. The daily excretion of mutagens, concentrated on XAD-2 columns and tested in Salmonella typhimurium YG1024 with S9 mix, was significantly and remarkably decreased by NAC in the majority of the subjects examined so far. Time-course experiments showed that this effect starts since the first day of drug administration and reverses when treatment is withdrawn. In addition, NAC administration almost totally prevented urinary genotoxicity in one subject whose concentrated urines induced a differential lethality in Escherichia coli strains having distinctive DNA repair capacities. The decrease of urinary genotoxicity produced by NAC in the majority of smokers correlates with the ability of this thiol to prevent tumors and to affect a variety of intermediate biomarkers in animal models. Modulation of the urinary excretion of mutagens is one of the biomarkers evaluated in two ongoing Phase II chemoprevention trials. One study involves the oral administration of NAC in Dutch smokers. The pretreatment urine samples of all the subjects so far recruited are clearly mutagenic. The other study involves the oral administration of the dithiolethione oltipraz to individuals living in the Qidong County of the People's Republic of China, an area of high endemy for HBV infection and of high exposure to aflatoxins. Additionally, a large proportion of the recruited male subjects are smokers. A total of 500 urine specimens will be assayed from 240 subjects according to a complex protocol arranged in three consecutive phases. J. Cell. Biochem. 25S:92-98. © 1997 Wiley-Liss, Inc.
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    Cohorts with familial disposition for colon cancers in chemoprevention trials (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 63 (1996), S. 131-135 
    Details
    ISSN: 0730-2312
    Keywords: adenomatous polyp ; colon cancer ; colonic polyp ; familial polyposis ; familial risk ; inherited risk ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Colon cancer provides an attractive setting for chemoprevention trails because of the frequency and variation of familial predisposition that is observed in this malignancy. Additionally, the adenomatous polyp, the precursor of colon cancer, is a valuable intermediate marker for judging the effectiveness of candidate chemopreventive agents.Inherited colon cancer susceptibility varies from mild to severe. Conditions with extreme susceptibility include the autosomal dominantly inherited syndromes of familial adenomatous polyposis (FAP) and hereditary nonpolyposis colorectal cancer (HNPCC). These are highly penetrant syndromes with extreme cancer risk. FAP arises from mutations of the APC gene and HNPCC from mutations of the mismatch repair genes. Specific and individual genetic diagnosis is now possible in both syndromes, thus allowing identification of genetically affected individuals for chemoprevention trials.FAP accounts for less than 1% of colon cancers, while HNPCC may be present in up to 5% of cases. Familial clustering is common in the remainder of cases, which are often referred to as sporadic, but probably arise in part from inherited susceptibility. Epidemiologic studies have shown that first-degree relatives have a two- to four-fold increased risk of acquiring colon cancer compared to the general population. Ten percent of individuals in the U.S. have a first-degree with colon cancer. This clinically identifiable higher risk group thus constitutes a large potential cohort for chemoprevention trials.The common familial cases of colon cancer can be further stratified by severity. A relative diagnosed under the age of 50 or two first-degree relatives affected with colon cancer confers an even greater risk for this malignancy, estimated to be four to six times that of the general population. Adenomatous polyps also precede the development of colon cancer in these categories, thereby providing a readily identifiable clinical endpoint to judge the effectiveness of chemoprevention.It is expected that genetic markers will soon be available for more precise identification of common colon cancer susceptibility. Candidate markers include mild mutations of the APC and mismatch repair genes, glutathione transferase isoenzymes, acetylator status, and phospholipase A2 expression. Bile acid concentrations of the bowel may be genetically and/or environmentally determined and likely have a role in colon cancer susceptibility.We recently identified a large kindred with polyp and cancer susceptibility arising from a mild mutation of the APC gene. There are over 4,000 kindred members and mutational testing has demonstrated 140 gene carriers to date. We expect to institute chemoprevention trials in this kindred using adenomatous polyp number as an endpoint of effectiveness. J. Cell. Biochem. 25S:131-135. © 1997 Wiley-Liss, Inc.
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    The role of prostate-specific antigen in the chemoprevention of prostate cancer (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 63 (1996), S. 149-155 
    Details
    ISSN: 0730-2312
    Keywords: age-specific PSA reference ranges ; complexed PSA ; free PSA ; prostate-specific antigen ; PSA velocity ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: An understanding of the natural history of changes in prostate-specific antigen (PSA) may be valuable as a surrogate view of prostate dynamics, as a method to differentiate between benign and malignant growth, and as a means to assess the use of PSA as a tool for monitoring activity of chemoprevention agents. Although PSA appears to be useful as a noninvasive marker of prostatic growth, PSA changes should not be confused with a direct measure of tumor growth. Serum PSA levels are a function of tumor volume but are also influenced by the volume of benign epithelium, grade of carcinoma (if any), inflammation, androgen levels, growth factors, and the extracellular matrix.The biological functions of PSA in the prostate and in its secretions need to be more completely elucidated in order that PSA measurements may more accurately describe prostate dynamics. The expression of PSA is androgen-regulated. It is one of the most abundant prostate-derived proteins in the seminal fluid. Seminogelin, a major protein in seminal fluid, is cleaved by PSA, and this cleavage is important in the liquefaction of semen. Less is known about other PSA substrates.Current PSA studies indicate that cancer cases exhibit an early slow linear PSA phase followed by a rapid exponential phase, and that PSA levels begin to increase exponentially approximately 7-9 years before diagnosis. The establishment of age-specific PSA reference ranges (ASRR) and of PSA velocity (PSAV) rates provide elements of a baseline from which prediction models could measure malignant potential of a prostatic carcinoma. Moreover, recent discoveries of different molecular forms of PSA in serum may allow a much more accurate differentiation of benign and malignant growth as well as a more potent measure of the impact of chemoprevention agents.If PSA doubling time is approximately 2.4-3.0 years and accurately reflects tumor doubling time, and if the average man has less than 0.5 ml of latent prostatic tumor tissue and the average stage T2 cancer is approximately 4 ml when detected, then the available PSA data suggest that the 3 doublings necessary to change from 0.5-4.0 ml. would take 7-12 years for a typical small volume tumor to reach the size of most stage T2 tumors. The findings that histologic cancers appear at much younger ages than previously known is disturbing. It indicates that disease initiation may begin sooner than ever thought likely. “Normal” PSA levels for younger men (〈 40 years of age) may need to be studied, and an emphasis upon premalignant lesions in this age group may be necessary. Younger men may represent the most appropriate population and premalignant lesions the most relevant clinical factor for prostate cancer chemoprevention studies and trials.The molecular composition and molecular changes of PSA derived from premalignant lesions have yet to be elucidated, but such investigations may lead to a more complete understanding of the possible progression or transformation of normal prostate cells to premalignancy and subsequently to carcinoma. High grade prostatic intraepithelial neoplasia (PIN) in and of itself does not account for elevated serum PSA levels, but subtle changes in the molecular dynamics of PSA may reveal the influence of androgens and the impact of chemopreventive agents. J. Cell. Biochem. 25S:149-155. © 1997 Wiley-Liss, Inc.
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    K-ras mutation in sputum of patients with or without lung cancer (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 63 (1996), S. 172-176 
    Details
    ISSN: 0730-2312
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: K-ras mutation appears in about 60% of patients with non-small-cell lung cancer (NSCLC). This frequency and its presence in normal appearing tissues point to the potential of ras oncogene mutation to serve as a good biomarker. Using enriched PCR (EPCR), which enables the detection of one mutant allele in the presence of 10,000 normal alleles, we have determined the frequency of mutant ras alleles in the sputum samples of patients with or without lung cancer. Samples were collected from 37 patients with NSCLC and from 4Q controls who suffered from non-oncological lung diseases, including bronchitis, asthma, and pneumonia. Of the 37 samples obtained from patients with lung cancer, 18 were found to harbor ras oncogene mutations (48%). Of the 40 cases that were free of lung cancer, five were found to harbor this mutation (12.5%). The difference between the two frequencies was found to be significant (P 〈 0.01). These findings indicate that (a) K-ras oncogene mutation can be identified in routinely obtained sputum samples of patients who may be at risk of developing lung cancer and (b) the higher frequency of these mutations in samples of patients with lung cancer points to the potential use of the ras mutation as a biomarker for either exogenous or endogenous exposure to carcinogens. Thus, the ability to examine sputum provides a powerful and convenient source of sampling and may be adapted for future large-scale screening. J. Cell. Biochem. 25S:172-176. © 1997 Wiley-Liss, Inc.
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    Nuclear and cytoplasmic expressions of the carbohydrate-binding protein CBP70 in tumoral or healthy cells of the macrophagic lineage (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 62 (1996), S. 529-542 
    Details
    ISSN: 0730-2312
    Keywords: carbohydrate-binding protein 70 ; HL60 ; nucleus ; macrophages ; lectins ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The expression of the carbohydrate-binding protein CBP70 was analyzed in undifferentiated HL60 cells, HL60 cells differentiated into monocytes/macrophages or granulocytes, healthy monocytes and in vitro monocyte-derived macrophages (MDM) using an anti-CBP70 serum. This study was performed by immunoblotting analysis of nuclear and cytoplasmic extracts before and after N-acetylglucosamine affinity chromatography and by indirect immunofluorescence microscopy. The results of this study show, for the first time, that CBP70 is expressed in the nucleus and the cytoplasm of healthy or leukemic cells of the macrophagic lineage. However, striking differences were observed depending upon the leukemic or normal state of cells and cell differentiation. Indeed, the level of expression and the intracellular distribution of CBP70 were found to be different in undifferentiated HL60 cells and monocytes/macrophages differentiated from these cells. Major differences were also observed according to whether macrophages differentiated from leukemic HL60 cells or healthy monocytes. Thus, the total cellular expression of CBP70 was markedly lower in MDM than in HL60-derived macrophages and the intracellular distribution of the protein was different. Nevertheless, in both cases, the total cellular expression of CBP70 was enhanced during cell differentiation. Another important result is the finding that CBP70 behaviour was totally different when HL60 cells were induced to differentiate into macrophages or granulocytes. These data could therefore suggest that CBP70 is involved in phagocytic cell differentiation. Moreover, we show that an additional 60 kDa polypeptide (p60), recognized by the anti-CBP70 serum, is expressed in HL60 cells differentiated into macrophages or granulocytes as well as in healthy monocytes or MDM but not expressed in undifferentiated HL60 cells. Although CBP70 und p60 appeared to be closely related polypeptides, their relationship remains to be precised. These findings are discussed with regard to data available on galectin-3. © 1996 Wiley-Liss, Inc.
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    Characterization of a new high-temperature-induced 66-kDa heat-shock protein, antigenically related to heat-shock protein 72 (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 63 (1996), S. 51-60 
    Details
    ISSN: 0730-2312
    Keywords: hyperthermia ; heat-shock proteins ; melanoma cells ; human cell lines ; Xenopus ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: M-14 human melanoma cells, following severe hyperthermic exposures, synthesized a heat-shock protein of 66 kDa (hsp 66), in addition to the major “classic” heat-shock proteins. This hsp 66 was not expressed following mild hyperthermic exposures sufficient to trigger the synthesis of the other heat-shock proteins. The induction of hsp 66 was observed also in Li human glioma cells treated at 45°C for 20 min. By contrast, hsp 66 was not induced in seven other human cell lines (both melanoma and nonmelanoma) when they were subjected to the same hyperthermic treatment. Immunological recognition experiments showed that hsp 66 cross-reacted with the inducible hsp 72, but not with the constitutive hsp 73. The possibility that hsp 66 is a breakdown product of hsp 72 was ruled out by the fact that Poly(A)+ RNA extracted from cells treated at 45°C for 20 min was able to direct the synthesis of hsp 66 (together with hsp 72) in a message-dependent rabbit reticulocyte lysate, as well as in microinjected Xenopus oocytes. By contrast, only the hsp 72 was expressed using Poly(A)+ RNA extracted from cells heated at 42°C for 1 h. Affinity chromatography experiments on ATP-agarose showed that hsp 66 did not bind ATP in vitro, hsp 66 was localized both in the cytoplasm (cytosol, mitochondria, and microsome fraction) and in the nuclei of cells recovered from a severe heat shock: this intracellular distribution closely corresponded to that of hsp 72. The nuclear-associated hsp 66 was found to be tightly bound to nuclear structures and could not be extracted by incubation in ATP-containing buffer. © 1996 Wiley-Liss, Inc.
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    Differential and multiple binding of signal transducing molecules to the ITAMs of the TCR-ζ chain (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 63 (1996), S. 94-103 
    Details
    ISSN: 0730-2312
    Keywords: ITAMs ; SH2 domains ; peptides ; TCR-ζ ; tyrosine phosphorylation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: A biotin-streptavidin-based technique was developed for high affinity, unidirectional, and specific immobilization of synthetic peptides to a solid phase. Biotinylated 23-mer carboxamide peptides corresponding to the three immunoreceptor tyrosine-based activation motifs (ITAMs) of the T cell antigen receptor associated ζ-chain (TCR-ζ) in their bis-, mono-, or unphosphorylated forms were used to study the binding of cellular proteins from human Jurkat T cells to these signal transduction motifs. The protein tyrosine kinase ZAP-70 bound specifically to all bisphosphorylated peptides but not to the mono- or unphosphorylated peptides. In contrast, Shc, phosphatidylinositol 3-kinase (P13K), Grb2, and Ras-GTPase activating protein (GAP) bound with different affinities to the bis- or monophosphorylated peptides, while the Src family protein tyrosine kinase (PTK) Fyn did not bind specifically to any of the tested peptides. The different preferences of the studied signaling molecules for distinct ITAMs, and in particular the binding of some of them preferentially to monophosphorylated peptides, suggests that the TCR-ζ may bind multiple signaling molecules with each ITAM binding a unique set of such molecules. In addition, partial phosphorylation of the ITAMs may result in recruitment of different proteins compared to double phosphorylation. This may be crucial for coupling of the TCR to various effector functions under different conditions of receptor triggering. © 1996 Wiley-Liss, Inc.
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    Genes that regulate apoptosis in the mouse thymus (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 60 (1996), S. 18-22 
    Details
    ISSN: 0730-2312
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Elimination of self-reactive T lymphocytes occurs during T-cell development in the thymus by a process known as negative selection. The mechanism that drives negative selection is apoptosis. To identify genes that regulate apoptosis in the mouse thymus, a library of negatively selected T cells was constructed and, by subtractive screening, several differentially regulated genes were isolated. Two transcripts that are repressed during cell death were identified, in addition to two induced transcripts. Further experiments demonstrated that cell death in thymocytes can occur via several induction pathways and each pathway appears to be regulated by a unique cascade of genes. © 1996 Wiley-Liss, Inc.
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    Immunoregulatory effects of Fas-mediated signalling (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 60 (1996), S. 39-46 
    Details
    ISSN: 0730-2312
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: No abstract.
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    Apoptosis mediated by the TNF-related cytokine and receptor families (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 60 (1996), S. 47-55 
    Details
    ISSN: 0730-2312
    Keywords: death domain ; ring finger ; signal transduction ; serine kinase ; T lymphocytes ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: T lymphocytes use several specialized mechanisms to induce apoptotic cell death. The tumor necrosis factor (TNF)-related family of membrane-anchored and secreted ligands represent a major mechanism regulating cell death and cell survival. These ligands also coordinate differentiation of tissue to defend against intracellular pathogens and regulate development of lymphoid tissue. Cellular responses are initiated by a corresponding family of specific receptors that includes two distinct TNFR (TNFR60 and TNFR80), Fas (CD95), CD40, p75NTF, and the recently identified lymphotoxin β-receptor (LTβR), among others. The MHC-encoded cytokines, TNF and LTα, form homomeric trimers, whereas LTβ assembles into heterotrimers with LTα, creating multimeric ligands with distinct receptor specificities. The signal transduction cascade is initiated by transmembrane aggregation (clustering) of receptor cytoplasmic domains induced by binding to their multivalent ligands. The TRAF family of Zn RING/finger proteins bind to TNFR80; CD40 and LTβR are involved in induction NFκB and cell survival. TNFR60 and Fas interact with several distinct cytosolic proteins sharing the “death domain” homology region. TNF binding to TNFR60 activates a serine protein kinase activity and phosphoproteins are recruited to the receptor forming a multicomponent signaling complex. Thus, TNFRs use diverse sets of signaling molecules to initiate and regulate cell death and survival pathways. © 1996 Wiley-Liss, Inc.
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    Host genotype controls the ability of the ISGF3 complex to activate transcription of IFN-inducible genes (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 60 (1996), S. 83-94 
    Details
    ISSN: 0730-2312
    Keywords: IFNs ; ISGF3 complex ; host genotype ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: C57BL/6 mice are unable to express the lfi 202 type genes upon injection in vivo of multiple dsRNA, poly rl:rC, or IFN-treatment in vitro. For this purpose the 5′ terminal flanking region (called the b segment of 804 bp) was linked to a heterologous reporter gene chloramphenicol acetyl transferase (CAT) and transfected into NIH3T3 cells or BLK cells derived from the C57BL/6 strain. IFN-α induced strong CAT activity in NIH3T3 but not in BLK cells. This lack of transcription activation was not due to a defect in STAT factor activity, since IFN-α treatment in the presence of IFN-γ priming induced translocation of the ISGF3 into the nucleus, and binding to the ISRE (IFN-Stimulated Response Element) of the 202 gene even in C57BL/6 derived cells. Surprisingly when three tandem copies of the 202 ISRE (42 bp) were linked to a heterologous promoter (c-fos promoter) driving the reporter CAT gene, activation was also observed in C57BL/6 cells upon IFN-treatment. Finally, another IFN-inducible gene, namely the Mx, was activated in C57BL/6 mice. Thus, the primary defect of the C57BL/6 strain leading to an impaired lfi 202 type gene response to IFN appears to be an inability of the ISGF3 complex to activate the endogenous promoter. Altogether these results suggest that unidentified nuclear factors related to the host genotype control the ability of the STAT factors to activate transcription upon IFN-treatment. © 1996 Wiley-Liss, Inc.
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    Regulation of distinct pools of protein kinase C δ in beta cells (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 60 (1996), S. 130-138 
    Details
    ISSN: 0730-2312
    Keywords: islets ; oleic acid ; cytoskeleton ; insulin ; free fatty acids ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Previous studies from our laboratory have demonstrated the presence of several isoforms of protein kinase C (PKC), Ca2+-independent and Ca2+-dependent, in both whole islets and tumor-derived beta cells. In the basal state, a major proportion of the isoform was found in the crude membrane fraction with smaller amounts found in both the cytosolic and cytoskeletal fractions. Whole islets showed a similar distribution of the isoform. These studies were done to analyze the effects of insulin secretagogues on the distribution of PKC δ to different cellular pools in isolated insulinoma beta cells. The phorbol ester, phorbol 12-myristate 13-acetate (PMA), produced a transient association of PKC δ with the beta cell cytoskeleton along with sustained decreases in cytosolic enzyme and transient increases in membrane enzyme. Neither glucose nor carbachol could acutely affect the subcellular distribution of PKC δ. Oleic acid decreased the amount of the enzyme associated with the cytoskeleton and led to a sustained decrease of cytosolic enzyme and a transient increase in membrane enzyme. Oleic acid was also able to prevent the increase in cytoskeletal enzyme induced by PMA. Both oleic acid and PMA potentiated glucose-induced insulin release but oleic acid, in contrast to PMA, was unable to initiate insulin release in the presence of substimulatory concentrations of glucose. These data demonstrate that different activators of PKC may have different effects on localization of the enzyme within the cells and suggest that there are at least three apparently distinct pools of PKC δ within the beta cell which may be important in insulin secretion or other aspects of beta cell function. © 1996 Wiley-Liss, Inc.
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    Alterations in endothelial cell proteinase and inhibitor polarized secretion following treatment with interleukin-1, phorbol ester, and human melanoma cell conditioned medium (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 60 (1996), S. 148-160 
    Details
    ISSN: 0730-2312
    Keywords: endothelium ; polarization ; proteinases ; IL-1α ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Polarized secretion of matrix metalloproteinases and plasminogen activators by monkey aortic endothelial cells was studied in vitro, using transwell inserts. The endothelial cells constitutively expressed matrix metalloproteinase-2, tissue inhibitors of metalloproteinases 1 and 2, urokinase, and tissue plasminogen activator, all with basal preference. Matrix metalloproteinase-9 activity was induced by phorbol 12-myristate 13-acetate (apical), interleukin-1α (basal), and by conditioned medium from DX3 human melanoma cells (basal). The DX3 melanoma conditioned medium also stimulated basal secretion of matrix metalloproteinase-2, urokinase, tissue plasminogen activator, and tissue inhibitors of metalloproteinases. The rise in proteolytic activity in the basal direction was reflected by increased capacity to degrade subendothelial basement membrane type IV collagen, shown immunohistologically, using monkey kidney tissue sections and basement membrane deposited by endothelial cells into the transwell membrane. Thus, IL-1α and DX3 melanoma conditioned medium can stimulate endothelial cells in vitro to concentrate secretion of proteinases spatially onto the underlying basement membrane. We suggest that the stimulation of endothelial cell proteinase activity by tumor cells may facilitate tumor cell extravasation. © 1996 Wiley-Liss, Inc. This article is a US Government work and, as such, is in the public domain in the United States of America.
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    Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin, 12-O-tetradecanoylphorbol-13-acetate and 17β-estradiol on estrogen receptor regulation in MCF-7 human breast cancer cells (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 60 (1996), S. 173-184 
    Details
    ISSN: 0730-2312
    Keywords: occupied nuclear estrogen receptors ; estrogen metabolism ; P450 ; environmental ; endocrine-modulating ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) exhibits remarkably potent antiestrogenic activity. To further elucidate the role of estrogen receptor (ER) regulation in this response, we examined the effects of exposure to TCDD in MCF-7 human breast cancer cells on ER mRNA levels by using an RNase protection assay, on ER accumulation by using an ER immunocytochemical essay (ER-ICA), and on ER function by competitive binding assays under conditions of saturating 17β-estradiol (E2). Comparative studies were conducted with E2 and 12-O-tetradecanoylphorbol-13-acetate (TPA), as both compounds are known to suppress ER expression. Our results indicate that 1 nM E2 and 100 nM TPA both suppress ER mRNA levels as early as 4 h after exposure and to 33.6% and 16.5% of control levels, respectively, after 72 h. In contrast, no significant effect on ER mRNA levels was attributed to exposure to 10 nM TCDD. A greater than 50% reduction in positive staining was observed by ER-ICA after 72 h exposure to 1 nM E2 and to 100 nM TPA, while only an 11% reduction in positive staining was observed with 10 nM TCDD. Specific binding of [3H]E2 under saturating conditions (10 nM E2) in whole cells was reduced by 50% in cultures exposed to 100 nM TPA, although no effect on binding was observed with exposure to 10 nM TCDD. In contrast, specific binding using subsaturating 1 nM [3H]E2 was depressed by 49% in MCF-7 cells exposed to 10 nM TCDD for 72 h. This depression was inhibited by a 1-h treatment with 5 μM α-naphthoflavone, which inhibits TCDD-induced, P450-mediated, E2 metabolism, and subsequent E2 depletion. In conclusion, while TPA and E2 effectively down-regulate ER expression, TCDD, under antiestrogenic conditions, has little if any effect on total ER levels in MCF-7 cells, and thus ER modulation is probably not necessary for the suppression of estrogenic activity in MCF-7 cells by TCDD. © 1996 Wiley-Liss, Inc.
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    Contraction of fibrillar type I collagen by endothelial cells: A study in vitro (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 60 (1996), S. 185-197 
    Details
    ISSN: 0730-2312
    Keywords: angiogenesis ; basic fibroblast growth factor ; bovine ; capillary ; collagen gel ; phorbol ester ; traction ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The formation of microvascular sprouts during angiogenesis requires that endothelial cells move through an extracellular matrix. Endothelial cells that migrate in vitro generate forces of traction that compress (i.e., contract) and reorganize vicinial extracellular matrix, a process that might be important for angiogenic invasion and morphogenesis in vivo. To study potential relationships between traction and angiogenesis, we have measured the contraction of fibrillar type I collagen gels by endothelial cells in vitro. We found that the capacity of bovine aortic endothelial (BAE) cells to remodel type I collagen was similar to that of human dermal fibroblasts - a cell type that generates high levels of traction. Contraction of collagen by BAE cells was stimulated by fetal bovine serum, human plasma-derived serum, bovine serum albumin, and the angiogenic factors phorbol myristate acetate and basic fibroblast growth factor (bFGF). In contrast, fibronectin and immunoglobulin from bovine serum, several nonserum proteins, and polyvinyl pyrrolidone (a nonproteinaceous substitute for albumin in artificial plasma) were not stimulatory. Contraction of collagen by BAE cells was diminished by an inhibitor of metalloproteinases (1, 10-phenanthroline) at concentrations that were not obviously cytotoxic. Zymography of proteins secreted by BAE cells that had contracted collagen gels revealed matrix metalloproteinase 2. Subconfluent BAE cells that were migratory and proliferating were more effective contractors of collagen than were quiescent, confluent cells of the same strain. Moreover, bovine capillary endothelial cells contracted collagen gels to a greater degree than was seen with BAE cells. Collectively, our observations indicate that traction-driven reorganization of fibrillar type I collagen by endothelial cells is sensitive to different mediators, some of which, e.g., bFGF, are known regulators of angiogenesis in vivo. © 1996 Wiley-Liss, Inc.
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  • 187
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    Instability of endogenous MRP/proliferin transcripts in the nucleus of mouse embryo fibroblasts contrasts with their stability when produced during transient transfections (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 60 (1996), S. 198-210 
    Details
    ISSN: 0730-2312
    Keywords: post-transcriptional regulation ; RNA stability ; mRNA export ; gene regulation ; transient transfections ; immortalization ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The mitogen regulated protein/proliferin (MRP/PLF) gene is transcribed in primary mouse embryo fibroblasts (MEFs), but the pre-mRNA is not properly converted into a stable cytoplasmic mRNA and instead is rapidly degraded, apparently in the nucleus [Malyankar et al. (1994): Proc Natl Acad Sci USA 91:335-359]. In 3T3 cells derived from the MEFs by the standard 3T3 immortalization protocol, stable MRP/PLF mRNA is produced. We show here that the processing of intron sequences is similar in the two cell types and that some of the MRP/PLF transcripts are polyadenylated in the MEFs. We also document the production of stable MRP/PLF mRNA generated by transcription of various plasmid constructs containing different portions of the MRP/PLF3 gene after calcium phosphate-mediated transfection into the MEFs. We conclude that the inability of the MRP/PLF mRNA to accumulate in the MEFs is unlikely to result solely from a single localized sequence in the primary transcript (or the mRNA) that causes it to be subject to rapid breakdown; possibly export of the mRNA from the MEF nucleus is defective or some aspect of the transcriptional process marks the transcript for degradation. © 1996 Wiley-Liss, Inc.
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    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 60 (1996), S. 1-3 
    Details
    ISSN: 0730-2312
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: No abstract.
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  • 189
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    Heterogeneous distribution and organization of cytoskeletal proteins drive differential modulation of metabolic fluxes (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 60 (1996), S. 271-278 
    Details
    ISSN: 0730-2312
    Keywords: cytoskeleton ; actin ; microtubular protein ; metabolic fluxes ; cytoplasmic segregation ; topological regulation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: On the basis of experimental data obtained in vitro, we propose that differential segregation of actin and tubulin in the cytoplasm may be a regulatory mechanism of metabolic fluxes. The results presented point out that the same enzymes may be differentially modulated at different locations in the cytoplasm, depending on the cytoskeletal protein present at that location, its concentration, polymeric status, or geometric arrangement. Essentially, actin or microtubular protein would exert their effect on enzymatic catalysis through displacement of the equilibrium of enzyme oligomers either to active or less active species. The latter was corroborated by mathematical modeling of the dynamic coupling between microtubular protein assembly-disassembly and pyruvate kinase activity. From these results, a precise biochemical meaning can be given to the putative linkage existing between the mechanisms by which cells rearrange their cytoplasmic architecture and the dynamics of biochemical reactions taking place concomitantly. © 1996 Wiley-Liss, Inc.
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  • 190
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    Loss and shedding of surface markers from the leukemic myeloid monocytic line THP-1 induced to undergo apoptosis (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 60 (1996), S. 246-259 
    Details
    ISSN: 0730-2312
    Keywords: apoptosis ; CD13 ; CD14 ; CD33 ; aminopeptidase N shedding ; ethanol/THP-1 cultures ; ectopeptidase activation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Previous studies have established that a relationship exists between apoptosis and cell surface (ecto-) peptidase activity. Thus dose-dependent increases were found both in ectopeptidase activities and in the proportion of cells undergoing apoptosis in HeLa cell monolayers after exposure to UV and other perturbants causing arrest of DNA synthesis (indirectly or directly as a result of DNA damage). The nature of the correlation made no distinction as to whether an increase in peptidase activity was causal of, or consequential to apoptosis, nor whether the increase was a general response by all cells. As a wider approach to understanding the possible role played by ectopeptidases in apoptosis, we report the effect on expression of a known ectopeptidase, aminopeptidase N (CD13), by a myelomonocytic cell line induced to undergo apoptosis. Using THP-1 cultures exposed to low concentrations of ethanol, we used FACS technology to sort for early apoptotic cells that have an increased ability to sequester the vital dye Hoechst 33342 while excluding nonvital dyes. Apoptosis was verified by light, fluorescence, and transmission electron microscopy, and the presence of DNA fragmentation. These early apoptotic cells showed a significant loss in CD13 labeling. Another surface marker, CD33, behaved similarly, whereas CD14 was lost globally, and not just by the apoptotic cells. Peptidase assays confirmed that an aminopeptidase was shed into the bathing media and that this activity was inhibitable both by bestatin and by a CD13 neutralizing monoclonal antibody. In treated cells, there was no evidence for an increase in cell surface protease activity directed toward a highly aliphatic nonapeptide substrate used as a model for TGF-α scission from its precursor form. However, other cell surface proteases of different specificity are presumably responsible for the observed shedding of CD13. © 1996 Wiley-Liss, Inc.
    Additional Material: 8 Ill.
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  • 191
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    Common structural features of replication origins in all life forms (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 60 (1996), S. 297-316 
    Details
    ISSN: 0730-2312
    Keywords: replication origins ; DNA repeats ; initiator protein ; DNA distortion ; inverted repeat ; enhancer ; silencer ; transcription factor ; SV40 ORI ; HSV-1 ORI nuclear matrix ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Origins of replication (ORIs) among prokaryotes, viruses, and multicellular organisms appear to possess simple tri-, tetra-, or higher dispersed repetitions of nucleotides, AT tracts, inverted repeats, one to four binding sites of an initiator protein, intrinsically curved DNA, DNase I-hypersensitive sites, a distinct pattern of DNA methylation, and binding sites for transcription factors. Eukaryotic ORIs are sequestered on the nuclear matrix; this attachment is supposed to facilitate execution of their activation/deactivation programs during development. Furthermore, ORIs fall into various classes with respect to their sequence complexity: those enriched in AT tracts, those with GA- and CT-rich tracts, a smaller class of GC-rich ORIs, and a major class composed of mixed motifs yet containing distinct AT and polypurine or GC stretches. Multimers of an initiator protein in prokaryotes and viruses that might have evolved into a multiprotein replication initiation complex in multicellular organisms bind to the core ORI, causing a structural distortion to the DNA which is transferred to the AT tract flanking the initiator protein site; single-stranded DNA-binding proteins then interact with the melted AT tract as well as with the DNA polymerase α-primase complex in animal viruses and mammalian cells, causing initiation in DNA replication. ORIs in mammalian cells seem to colocalize with matrix-attached regions and are proposed to become DNase I-hypersensitive during their activation. © 1996 Wiley-Liss, Inc.
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  • 192
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    Argininosuccinate synthetase and argininosuccinate lyase are localized around mitochondria: An immunocytochemical study (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 60 (1996), S. 334-340 
    Details
    ISSN: 0730-2312
    Keywords: urea cycle ; liver ; enzyme organization ; cell organization ; electron microscopy ; immunogold ; immunocytochemistry ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Argininosuccinate synthetase and argininosuccinate lyase are soluble cytoplasmic enzymes of the urea cycle. Previous biochemical studies using permeabilized hepatocytes showed that these enzymes are organized in situ, and function as if they are located next to the outer membrane of mitochondria. We have now confirmed and extended those observations in intact liver by means of immunocytochemistry at the electron microscope level. Morphometric analysis of the electron micrographs shows that argininosuccinate synthetase and argininosuccinate lyase are located in the immediate vicinity of the mitochondria, predominantly next to the cytoplasmic surface of the outer membrane. Some immuno-specific protein is also observed in the endoplasmic reticulum in the immediate vicinity of the mitochondria. These results support our previous biochemical findings, and additionally suggest that virtually all of the argininosuccinate synthetase and argininosuccinate lyase of the liver parenchymal cell are located just outside the mitochondria. © 1996 Wiley-Liss, Inc.
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  • 193
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    Binding of nuclear proteins to a conserved histone H1t promoter element suggests an important role in testis-specific transcription (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 60 (1996), S. 348-362 
    Details
    ISSN: 0730-2312
    Keywords: histone H1t ; gene regulation ; tissue-specific gene expression ; tissue-specific transcription ; TE ; DNA binding protein ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The testis-specific histone H1t gene is transcribed only in primary spermatocytes during spermatogenesis. Recently, expression of the rat gene was shown to be limited to primary spermatocytes in transgenic mice, revealing that promoter elements sufficient for regulating tissue-specific transcription were present in the cloned rat gene. In this study the mouse histone H1t gene has been cloned, and sequenced and its promoter region has been compared to the rat H1t promoter with regard to conserved elements and protein binding activity. The amino acid sequence of each of the three H1t coding region domains is conserved when compared to the homologous domain in H1t derived from other species. H1t mRNA is found only in testis, where it accumulates to a high steady-state level, and examination of enriched testis cell populations shows that expression is limited to primary spermatocytes. Protein binding assays using nuclear extracts from various mouse tissues reveal testis-specific binding to TE1 and TE2, imperfect inverted repeat elements within the larger TE element. Although the H1t promoter contains an Sp1 consensus motif within the H1t/TE element, binding of testis Sp1 to the motif could not be detected using specific anti-Sp1 antibodies. © 1996 Wiley-Liss, Inc. This article is a US Government work and, as such, is in the public domain in the United States of America.
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  • 194
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    Temporal study of the activity of matrix metalloproteinases and their endogenous inhibitors during wound healing (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 60 (1996), S. 379-386 
    Details
    ISSN: 0730-2312
    Keywords: wound healing ; metalloproteinase ; metalloproteinase inhibitor ; connective tissue remodeling ; chronic wounds ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The restoration of functional connective tissue is a major goal of the wound healing process. This regenerative event requires the deposition and accumulation of collagenous and noncollagenous matrix molecules as well as the remodelling of extracellular matrix (ECM) by matrix metalloproteinases (MMPs). In this study, we have utilized substrate gel electrophoresis, radiometric enzyme assays, and Western blot analyses to determine the temporal pattern of appearance and activity of active and latent MMPs and their inhibitors during the entire healing process in a partial thickness wound model. Through the use of substrate gel electrophoresis, we studied the appearance of proteolytic bands whose molecular weight was consistent with their being members of the MMP family of enzymes. Proteolytic bands whose molecular weight is consistent with both the active and latent forms of MMP-2 (72 kDa, Type IV gelatinase) were detected in wound fluid of days 1-7 after wounding. The number of active MMP-2 species detectable in wound fluid was greatest during days 4-6 after wounding. The most prominent proteolytic band detected each day migrated with a molecular weight consistent with it being the latent form of MMP-9 (92 kDa, Type V pro-collagenase). In contrast to MMP-2, the active form of this enzyme was never detected. The presence of MMP-1 (interstitial collagenase) was detected by immunoblot in the wound fluid from days 1-6 post-injury. Using a radiometric enzyme assay for collagenase inhibitory activity we have also determined the time course of activity of endogenous matrix metalloproteinase inhibitors. We have correlated these data to the known cellular events occurring in the wound during this time period as well. This study establishes a prototypical pattern of MMP appearance in normal wound healing. It may also provide potential intervention sites for the therapeutic use of inhibitors of aberrant MMP activities which characterize chronic wounds. © 1996 Wiley-Liss, Inc.
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  • 195
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    Regulation of cytoplasmic tubulin carboxypeptidase activity in vitro by cations and sulfhydryl-modifying compounds (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 60 (1996), S. 424-436 
    Details
    ISSN: 0730-2312
    Keywords: enzyme regulation ; microtubule ; post-translational modification ; detyrosination ; inhibitor ; neuronal tissue ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: α-tubulin subunits within microtubules (MTs) can be post-translationally detyrosinated by a tubulin-specific carboxypeptidase (TCP) activity to form biochemically distinct MTs. Attempts to characterize and purify TCP have suffered from the inability to detect low levels of activity and to distinguish TCP from other, competing enzyme activities. We recently developed an assay for TCP [Webster et al. (1992) Biochemistry 31:5849] that uses taxol-stabilized MTs as the substrate. In this study, we exploited the increased sensitivity and specificity of this new assay to explore the effects of various agents that might act to either stimulate or inhibit this enzyme in vitro. We tested a variety of both monovalent and divalent cations for their ability to affect TCP, and tested whether the cations were affecting the enzyme, the substrate, or both. We found that TCP displayed salt-sensitive binding to MTs, characteristic of other, more well characterized MT-associated proteins. While both calcium and magnesium stimulated TCP activity over a narrow concentration range (2-10 mM), they inhibited activity at higher concentrations. Other divalent cations tested, including zinc, copper, and cobalt, inhibited TCP at virtually all concentrations tested, but to different levels (zinc 〉 copper 〉 cobalt). Most of the zinc-induced TCP inhibition was attributed to the interference with the normal binding of TCP to MTs. In addition, we examined the involvement of free sulfhydryl groups (which are important for the activities of many types of enzymes) in TCP activity by the addition of sulfhydryl-modifying compounds during the assay, and found that their addition reduced TCP activity mainly (but not solely) by their action on the extract that contained the TCP. Finally, we tested the ability of DL-benzylsuccinic acid, a potent inhibitor of carboxypeptidase A, to inhibit TCP. While carboxypeptidase A has been found, in other studies, to be inhibited by micromolar concentrations, TCP was affected only at concentrations above 20 mM, adding another proof that carboxypeptidase A and TCP are distinct enzyme activities. © 1996 Wiley-Liss, Inc.
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  • 196
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    Proinflammatory agents, IL-8 and IL-10, upregulate inducible nitric oxide synthase expression and nitric oxide production in avian osteoclast-like cells (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 60 (1996), S. 469-483 
    Details
    ISSN: 0730-2312
    Keywords: lipopolysaccharide ; interleukin-1 ; tumor necrosis factor ; interferon ; transforming growth factor β ; dexamethasone ; RT-PCR ; NADPH diaphorase ; bone ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Nitric oxide synthase (NOS) isoenzymes generate nitric oxide (NO), a sensitive multifunctional intercellular signal molecule. High NO levels are produced by an inducible NOS (iNOS) in activated macrophages in response to proinflammatory agents, many of which also regulate local bone metabolism. NO is a potent inhibitor of osteoclast bone resorption, whereas inhibitors of NOS promote bone resorption both in vitro and in vivo. The possibility that osteoclasts, like macrophages, express a regulated iNOS and produce NO as a potential autocrine signal following inflammatory stimulation was investigated in well-characterized avian marrow-derived osteoclast-like cells. NO production (reflected by medium nitrite levels) was markedly elevated in these cells by the proinflammatory agents lipopolysaccharide (LPS) and the synergistic action of IL-1α, TNFα, and IFNγ. Inhibitors of NOS activity (aminoguanidine, L-NAME) or iNOS induction (dexamethasone, TGFβ) reduced LPS-stimulated nitrite production. LPS also increased the NOS-associated diaphorase activity of these cells and their reactivity with anti-iNOS antibodies. RT-PCR cloning, using avian osteoclast-like cell RNA and human iNOS primers, yielded a novel 900 bp cDNA with high sequence homology (76%) to human, rat, and mouse iNOS genes. In probing osteoclast-like cell RNA with the PCR-derived iNOS cDNA, a 4.8 kb mRNA species was detected whose levels were greatly increased by LPS. Induction of iNOS mRNA by LPS, or by proinflammatory cytokines, occurred prior to the rise of medium nitrite in time course studies and was diminished by dexamethasone. Moreover, osteoclast-like cells demonstrated an upregulation of NO production and iNOS mRNA by IL-8 and IL-10, regulatory mechanism's not previously described. It is concluded that osteoclast-like cells express a novel iNOS that is upregulated by inflammatory mediators, leading to NO production. Therefore, NO may serve as both a paracrine and autocrine signal for modulating osteoclast bone resorption. © 1996 Wiley-Liss, Inc.
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  • 197
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    LFA-1/ICAM-3 mediates neutrophil homotypic aggregation under fluid shear stress (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 60 (1996), S. 550-559 
    Details
    ISSN: 0730-2312
    Keywords: shear stress ; homotypic aggregation ; LFA-1 ; ICAM-3 ; NiCl2 sensitive Ca2+ channel ; Ca2+ influx ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We found that human neutrophils undergo homotypic aggregation by loading the physiological range of fluid shear stress (12-30 dynes/cm2). Under the fluid shear stress, an increase of intracellular Ca2+ concentration of neutrophils was observed. This increase of intracellular Ca2+ concentration was caused by Ca2+ influx, and the blockage of the flux by NiCl2 suppressed the neutrophil homotypic aggregation. Furthermore, this neutrophil aggregation under fluid shear stress was completely inhibited by pretreatment with antibody against LFA-1 or ICAM-3. These results suggested that NiCl2-sensitive Ca2+ channel played an important role in LFA-1/ICAM-3-mediated neutrophil homotypic aggregation under fluid shear stress. © 1996 Wiley-Liss, Inc.
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  • 198
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    Effect of acidic phospholipids on sphingosine kinase (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 60 (1996), S. 529-537 
    Details
    ISSN: 0730-2312
    Keywords: phospholipids ; phosphatidylserine ; sphingosine ; sphingosine-1-phosphate ; sphingosine kinase ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Sphingosine-1-phosphate (SPP) is a unique sphingolipid metabolite involved in cell growth regulation and signal transduction. SPP is formed from sphingosine in cells by the action of sphingosine kinase, an enzyme whose activity can be stimulated by growth factors. Little is known of the mechanisms by which sphingosine kinase is regulated. We found that acidic phospholipids, particularly phosphatidylserine, induced a dose-dependent increase in sphingosine kinase activity due to an increase in the apparent Vmax of the enzyme. Other acidic phospholipids, such as phosphatidylinositol, phosphatidic acid, phosphatidylinositol bisphosphate, and cardiolipin stimulated sphingosine kinase activity to a lesser extent than phosphatidylserine, whereas neutral phospholipids had no effect. Diacylglycerol, a structurally similar molecule which differs from phosphatidic acid in the absence of the phosphate group, failed to induce any changes in sphingosine kinase activity. Our results suggest that the presence of negative charges on the lipid molecules is important for the potentiation of sphingosine kinase activity, but the effect does not directly correlate with the number of negative charges. These results also support the notion that the polar group confers specificity in the stimulation of sphingosine kinase by acidic glycerophospholipids. The presence of a fatty acid chain in position 2 of the glycerol backbone was not critical since lysophosphatidylserine also stimulated sphingosine kinase, although it was somewhat less potent. Dioleoylphosphatidylserine was the most potent species, including a fourfold stimulation, whereas distearoyl phosphatidylserine was completely inactive. Thus, the degree of saturation of the fatty acid chain of the phospholipids may also play a role in the activation of sphingosine kinase. © 1996 Wiley-Liss, Inc.
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  • 199
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    Analysis of human breast cancer nuclear proteins binding to the promoter elements of the c-myc gene (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 60 (1996), S. 560-571 
    Details
    ISSN: 0730-2312
    Keywords: human c-myc ; transcription factors ; promoters ; human breast cancer ; nuclear proteins ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The expression of the c-myc gene is essential for the proliferation of both hormone-dependent and -independent human breast cancer cells. The regulation of c-myc gene expression in MCF-7 (hormone-dependent, estrogen-receptor (ER)-positive) and MDA MB 231 (hormone-independent, ER-negative) human breast cancer cells differs, with the c-myc gene of MCF-7 but not MDA MB 231 cells being regulated at the transcriptional level by estrogen. We have shown previously that the DNAase I hypersensitive (DH) sites in the c-myc chromatin of hormone-dependent and -independent human breast cancer cells were similar, with the exception of DH site II2, DH site II2, which maps near the P0 promoter, was less sensitive in hormone-dependent than in hormone-independent cells. As DH sites generally indicate the presence of sequence-specific DNA-binding proteins, we undertook a study to identify the nuclear proteins isolated from MCF-7 and MDA MB 231 cells that bound to the P0 and P2 promoter regions of the c-myc gene in vitro. The studies presented here provide evidence that Sp1 and/or Sp1-like proteins bind to the P0 and P2 promoter regions of the c-myc gene of MCF-7 and MDA MB 231 cells. Furthermore, evidence is presented for the presence of several previously unidentified sequence-specific DNA-binding proteins binding to these promoters. The DNA-binding activities of these latter proteins differed in the nuclear extracts of the MCF-7 and MDA MB 231 human breast cancer cells. © 1996 Wiley-Liss, Inc.
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  • 200
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    Influence of subunit transcript and protein levels on formation of a mitochondrial multienzyme complex (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 61 (1996), S. 118-126 
    Details
    ISSN: 0730-2312
    Keywords: mitochondria ; multienzyme complex formation ; subunit transcript ; protein levels ; post-translational events ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Constitutive expression of nuclear genes encoding mitochondrial proteins raises the question of whether these proteins are present in similar amounts in mitochondria of different tissues. We report that amounts of a single multienzyme complex can vary on a per mitochondrion basis depending on the number of mitochondria per cell. Human branched-chain α-keto acid dehydrogenase (BCKD) expression is used as a paradigm in these studies. Expression is compared and contrasted in HepG2 and DG75 cells in which mitochondrial content is twofold higher in the hepatocarcinoma line than in the lymphoblastoid line. Per cell, BCKD activity is equal in the two cell types, but BCKD protein concentration per mitochondrion is twofold higher in DG75 cells. Steady-state mRNA levels do not appear to be directly related to amounts of protein in the two cell lines. To test whether one subunit is limiting in formation of complex, overexpression of each BCKD subunit was elicited by plasmid transfection of the DG75 cells. Only overexpression of the β-subunit of the decarboxylase component induced more BCKD activity without apparent increase in mRNA for the other endogenously expressed subunits. This implies that free BCKD subunits exist in a cell and can be recruited into an active complex when the limiting subunit becomes available. © 1996 Wiley-Liss, Inc.
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