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  • 1
    ISSN: 1573-4919
    Keywords: HSP70 ; HSP28 ; transfection ; compensatory interactions ; northern blots ; two-dimensional polyacrylamide gel electrophoresis ; gel mobility shift assay
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract We have previously reported the lack of HSP28 gene expression during acute and chronic thermotolerance development in L929 cells (J Cell Physiol 152: 118–125, 1992; Cancer Res 52: 5787, 1992). In contrast to HSP28, an extremely high level of inducible HSP70 synthesis was observed. These results led us to investigate the possibility of compensatory interactions between HSP70 and HSP28. To test the hypothesis, L929 cells were transfected with the human HSP28 gene contained in plasmid pCMV27. Data from Western blot and two-dimensional gel electrophoresis of [3H] leucine and [32P] orthophosphate-labeled proteins showed the synthesis and phosphorylation of HSP28 in transfected cells after heating at 45°C for 10 min. However, the expression of constitutive and inducible HSP70 genes, along with the synthesis of their proteins, was not decreased after heat shock. These results suggest an independent regulation of HSP28 and HSP70 gene expression.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1573-4919
    Keywords: Quercetin ; heat shock transcription factor ; heat shock element ; nuclear run-on assay ; thermotolerance
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Previous studies have shown that a combination of low pH and quercetin (QCT) treatment following heat shock markedly suppresses and delays the expression of heat shock protein genes, particularly the HSP70 gene (Lee et al., Biochem. Biophys. Res. Commun., 186:1121–1128, 1992). The possible mechanism for alteration of gene expression by treatment with QCT at low pH was investigated in human colon carcinoma cells. Cells were heated at 45°C for 15 min and then incubated at 37°C for various times (0–12 h) with QCT (0.05–0.2 mM) at pH 7.4 or 6.5. Gel mobility-shift analysis of whole cell extracts from heated cells showed the formation of the heat shock transcription factor (HSF)-heat shock element (HSE) complex. Dissociation of HSF from the HSE of the human HSP70 promotor occurred within 4 h under both pH conditions. The kinetics of recovery were not affected by treatment with 0.1% dimethyl sulfoxide (DMSO). However, the dissociation of HSF-HSE complex was markedly delayed during treatment with a combination of low pH and QCT. In addition,in vitro transcription assays showed a suppression of initiation and elongation of HSP70 mRNA. These results may explain why the combination of low pH and QCT treatment suppresses and delays the HSP70 gene expression as well as thermotolerance development.
    Type of Medium: Electronic Resource
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  • 3
    ISSN: 1573-4919
    Keywords: AP-1 binding activity ; basic fibroblast growth factor ; c-Jun mutant protein ; H-7
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract We investigated the effect of hypoglycemic treatment on the activation of the AP-1 transcription factors and the regulation of basic fibroblast growth factor (bFGF) gene expression in multidrug resistant human breast carcinoma MCF-7/ADR cells. Northern blot and gel mobility shift assays showed that hypoglycemic treatment induced c-jun and c-fos gene expression, AP-1 binding activity, as well as bFGF gene expression. Moreover, transfected cells expressing high levels of abnormal c-Jun protein exhibited a reduction in the bFGF protein levels compared to parental cells. A potent protein kinase C (PKC) inhibitor, H-7 (60 μg/ml) suppressed the stress-induced bFGF gene expression. Our study also demonstrated that H-7 did not facilitate the decay of bFGF mRNA. Thus, the suppression of bFGF gene expression by treatment with H-7 was due to the effect of the drug on the synthesis of bFGF mRNA rather than the stability of bFGF mRNA. Our data suggest that hypoglycemia-induced bFGF gene expression is mediated through the activation of PKC and the AP-1 transcription factors. (Mol Cell Biochem 155: 163–171, 1996)
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  • 4
    ISSN: 1573-4919
    Keywords: glucose deprivation ; MAPK ; MCF-7 ; MCF-7/ADR ; signal transduction
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract We have investigated the effect of glucose deprivation treatment on the activation of mitogen activated protein kinases (MAPKs) in the drug-sensitive human breast carcinoma cells (MCF-7) and its drug resistant variant (MCF-7/ADR) cells. Western blots and in-gel kinase assays showed that glucose free medium was a strong stimulus for the activation of MAPK in MCF-7/ADR cells. No activation was seen in MCF-7 cells. MAPK was activated within 3 min of being in glucose free medium and it remained activated for over 1 h in MCF-7/ADR cells. After being returned to complete medium, 1 h was required for the MAPK to become deactivated. To investigate whether alternative sources of ATP could inhibit glucose deprivation induced MAPK activation, we added glutamine and glutamate to glucose deprived medium. The addition of glutamine did not reverse glucose deprivation induced MAPK activation in MCF-7/ADR cells. The addition of glutamate, however, decreased the MAPK activation and the length of time of activation. We observed an increase greater than three fold in MEK, Raf, Ras, and PKC activity with glucose deprivation in MCF-7/ADR cells. This suggests that glucose deprivation-induced MAPK activation is mediated through this signal transduction pathway.
    Type of Medium: Electronic Resource
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  • 5
    ISSN: 1573-4919
    Keywords: aB-Crystallin ; L929 cells ; methylation ; genomic footprinting
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract We have previously shown that murine L929 cells do not express the small heat shock protein αB-crystallin upon exposure to thermal stress (Mol Cell Biochem 155: 51–60, 1996). In these studies, we demonstrate that L929 cells also fail to express αB-crystallin upon exposure dexamethasone, whereas NIH 3T3 and Swiss 3T3 murine cells exhibit αB-crystallin expression under identical conditions. Mobility shift assays demonstrated heat-inducible binding, presumably by heat shock factor(s), to an αB-crystallin heat shock element (HSE) oligomeric sequence in total cellular extracts from L929 cells. Transient transfection of a plasmid containing the αB-crystallin promoter linked to a CAT reporter gene exhibited heat-inducible expression in L929 cells. In addition, L929 cells stably transfected with a plasmid containing the complete αB-crystallin gene showed expression of this gene following heat shock. The presence of the endogenous αB-crystallin gene was detected by Southern blot hybridization of genomic L929 DNA, and sequence analysis revealed identical nucleotide structure to published murine sequences throughout the entire promoter. Treatment of L929 cells with 5-azacytidine enabled heat-inducible expression of αB-crystallin from the endogenous gene, however, methylation of the putative heat shock element (HSE) and flanking promoter sequences of L929 cell genomic DNA was not detected. In vivo genomic footprinting demonstrated constitutive binding to the endogenous HSE of the αB-crystallin promoter in L929, L929/αB-crystallin transfectant cells, and Swiss 3T3 cells during unstressed and heat stressed conditions. Therefore, the genomic αB-crystallin HSE region in L929 cells appears to be available for binding of putative transcription factors, but methylation in other regions of the gene or genome repress the expression of αB-crystallin in L929 cells. In vitro culture of L929 cells appears to have rendered the αB-crystallin gene loci inactive through methylation, thus providing a unique system by which to study the function of transfected small heat shock proteins.
    Type of Medium: Electronic Resource
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  • 6
    ISSN: 1573-4919
    Keywords: chemical stress ; HSP70 synthesis ; gene regulation ; heat shock factors
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract It was reported that chemical stresses such as arsenite, cadmium or salicylate fail to induce synthesis of the inducible form of HSP70 (HSP70i). We report here that exposure of cells to higher doses of these chemical treatments induced significant synthesis of HSP70i in CHO cells as well as other cell lines. The synthesis of HSP70i is primarily regulated at the transcriptional level. Although all tested chemical treatments induced heat shock factor (HSF) binding to the heat shock element (HSE), HSP70i synthesis appears to be regulated by an alternative factor (CHBF) which constitutively binds to the HSE at 37°C. The treatments, which dissociate the HSE-CHBF complex, induced significant HSP70i synthesis. The treatments, which failed to induce HSP70i synthesis, still activated HSF binding to HSE but the HSE-CHBF complex remained as that of untreated control cells.
    Type of Medium: Electronic Resource
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  • 7
    ISSN: 1573-4919
    Keywords: αB-Crystallin ; transfection ; thermotolerance ; L929 cells ; thermoresistance
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract We investigated the role of αB-crystallin expression in the development of thermotolerance in murine L929 cells. An initial heat-shock of 10 min at 45°C induced thermotolerance in these cells to a heat challenge at 45°C administered 24 h later. The thermotolerance ratio at 10−1 isosurvival was 1.7. Expression of αB-crystallin gene was not detected during the 24 h incubation at 37°C following heat shock by either northern or western blots. In contrast, inducible HSP70 synthesis was observed during this time period. Thus, this cell line provided an unique system in which to examine the effects of transfected αB-crystallin on thermoresistance and thermotolerance. Cells stably transfected with αB-crystallin under the control of an inducible promoter did not show a significant increase in the ability to develop thermotolerance. However, a stably transfected L929 clone expressing high levels of constitutive αB-crystallin exhibited an approximately 50% increase in thermal resistance over parental and control cells. Though expression of αB-crystallin is not requisite for the development of thermotolerance in L929 cells, overexpression of transfected αB-crystallin can contribute to increased thermoresistance.
    Type of Medium: Electronic Resource
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  • 8
    Electronic Resource
    Electronic Resource
    Springer
    Molecular and cellular biochemistry 202 (1999), S. 1-8 
    ISSN: 1573-4919
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Although the synthesis of angiogenic factors in hypoxic regions of solid tumors is recognized as one of the critical steps in tumor growth and metastasis, the signal transduction pathway involved in hypoxic induction of basic fibroblast growth factor (bFGF) gene expression is still obscure. In the study described here, we investigated the intracellular responses to hypoxia and the mechanisms triggering the initiation of angiogenic activity in drug-resistant human breast carcinoma MCF-7/ADR cells. Northern blots showed an increase in the level of c-jun, c-fos, and bFGF mRNA during hypoxia. Gel mobility-shift analysis of nuclear extracts from hypoxia-exposed cells showed an increase in AP-1 binding activity. In addition, hypoxic treatment strongly activated c-Jun N-terminal kinase 1 (JNK1), leading to phosphorylation and activation of c-Jun. Expression of a dominant negative mutant of JNK1 suppressed hypoxia-induced JNK1 activation as well as bFGF gene expression. Taken together, hypoxia-induced bFGF gene expression is mediated through the stress-activated protein kinase (SAPK) signal transduction pathway.
    Type of Medium: Electronic Resource
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  • 9
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: In this study, we investigated the mechanism of synergistic effects of cytokine and hyperthermia on cytotoxicity in HT-29. When cells were heated at 42°C in the presence of recombinant human tumor necrosis factor (rhTNF-α), recombinant interferon-gamma (rhIFN-γ), or in a combination of both, a synergistic increase in the cytotoxic effects of the respective drugs was observed. We hypothesized that alteration of cytokine or heat-induced polypeptides synthesis was responsible for a synergistic interaction between heat and cytokine.Five heat shock proteins (HSPs, Mr 110,000, 100,000, 90,000, 70,000, and 28,000) were preferentially synthesized during chronic heating at 42°C. In contrast, the synthesis of two proteins (Mr 60,000 and 29,000) was induced by treatment with rhIFN-γ (1,000 U/ml). Although the combination of chronic hyperthermia (42°C) with TNF-α, IFN-γ, or TNF-α + IFN-γ increased cytotoxicity, alteration/induction of polypeptides was not correlated with the synergistical cytotoxic effects of cytokine and heat. Thus, the synergistic effects of cytokine and hyperthermia are not mediated through an induction of polypeptides. © 1993 Wiley-Liss, Inc.
    Additional Material: 7 Ill.
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  • 10
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Journal of Cellular Physiology 164 (1995), S. 404-413 
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Basic fibroblast growth factor (bFGF) has been shown to be a potent mitogen and a promoter of angiogenesis. It has been hypothesized that the expression of the bFGF gene may be induced by stress of various types. To test that hypothesis, we investigated the expression of the bFGF gene during heat treatment in adriamycin-resistant (MCF-7/ADR) and-sensitive (MCF-7) human breast carcinoma cells. Under normal growth conditions, the bFGF mRNA was detected in MCF-7/ADR cells, while it was not detectable in MCF-7 cells by Northern blot analysis. During heating at 41°C, the level of bFGF mRNA increased in MCF-7/ADR cells and the message became detectable in the MCF-7 cell line. However, after continuous heating at 41°C for 24 h, the bFGF mRNA level decreased to control level in MCF-7/ADR cells. Interestingly, simultaneous treatment with heat and 60 m̈g/ml H-7 (1-(isoquinolinylsulfonyl)-2-methylpiperzine, a potent PKC inhibitor) decreased the level of bFGF mRNA in MCF-7/ADR cells. These results suggest that a protein kinase, likely PKC, is involved in the transcriptional regulation of the heat-enhanced bFGF gene expression in human breast carcinoma cells. Although no heat shock element can be identified in the promoter of the bFGF gene, we observed that the AP-1 binding activity to a TPA responsive element (TRE)-like sequence in the promoter of bFGF gene was enhanced by heat, as tested by mobility shift assay. Antibody developed against the c-Jun and c-Fos proteins inhibited the AP-1 binding activity to TRE. Therefore, the AP-1 complex appears to be responsible for the heat-enhanced binding to the TRE-like motif of the bFGF gene. Furthermore, the increased AP-1 binding activity does not require new protein synthesis but activation of the preexisting c-Jun proteins. © 1995 Wiley-Liss, Inc.
    Additional Material: 11 Ill.
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