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  • 1
    Electronic Resource
    Electronic Resource
    Parameters for optimizing detection of transforming growth factors (1986)
    Springer
    Methods in cell science 10 (1986), S. 109-115 
    Details
    ISSN: 1573-0603
    Keywords: transforming growth factors ; anchorage-independent growth ; soft agar growth ; plasma ; epidermal growth factor ; NRK-49F cells
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Transforming growth factors (TGF) induce the soft agar growth of nontransformed cells, such as NRK-49F cells. This report describes culture conditions that optimize the detection of TGF by NRK-49F cells. Two aspects are dealt with in depth: the need to select an appropriate clone of NRK-49F cells and the need to select a culture medium that supports little or no background growth in the absence of added TGF. Evidence is presented to demonstrate that medium supplemented with heat-treated, dialyzed bovine plasma provides better conditions for assaying TGF than serum-supplemented medium provides.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01404601
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  • 2
    Electronic Resource
    Electronic Resource
    Specific down-regulation of annexin II expression in human cells interferes with cell proliferation (1999)
    Springer
    Molecular and cellular biochemistry 199 (1999), S. 139-147 
    Details
    ISSN: 1573-4919
    Keywords: annexin II ; DNA synthesis ; antisense ; Simian Virus 40 ; cell proliferation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract The protein-tyrosine kinase substrate annexin II is a growth regulated gene whose expression is increased in several human cancers. While the precise function of this protein is not understood, annexin II is proposed to be involved in multiple physiological activities, including DNA synthesis and cell proliferation. Targeted disruption of the annexin II gene affects calcium signaling, tyrosine phosphorylation and apoptosis, indicating the important physiological role of this protein. We used a transient co-transfection assay to regulate annexin II expression in human HeLa, 293 and 293T cells, and measured the effects of annexin II down regulation on DNA synthesis and proliferation. Transfection of cells with an antisense annexin II vector results in inhibition of cell division and proliferation, with concomitant reduction in annexin II message and protein levels. Cellular DNA synthesis is significantly reduced in antisense transfected cells. Replication extracts made from antisense transfected cells have significantly reduced efficiency to support SV40 in vitro DNA replication, while the extracts made from sense transfected cells are fully capable of replication. Our results indicate an important role of annexin II in cellular DNA synthesis and cell proliferation.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1006942128672
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  • 3
    Electronic Resource
    Electronic Resource
    Mouse genetics in the 21st century: using gene targeting to create a cornucopia of mouse mutants possessing precise genetic modifications (1993)
    Springer
    Cytotechnology 11 (1993), S. 79-99 
    Details
    ISSN: 1573-0778
    Keywords: gene targeting ; homologous recombination ; embryonic stem cells ; transgenic animals ; genetics
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Over 1500 mouse mutants have been identified, but few of the genes responsible for the defects have been identified. Recent developments in the area of gene targeting are revolutionizing the field of mouse genetics and our understanding of numerous genes, including those thought to be involved in cell proliferation and differentiation. Gene targeting was developed as a method for producing a predetermined mutation in a specific endogenous gene. Advances in the design of targeting vectors and in the use of embryonic stem cells have permitted the production of numerous mutant mice with null mutations in specific genes. These mutant mice will be critical for investigating thein vivo functions of many genes that have been cloned in recent years. This review discusses a wide range of new developments in the field of gene targeting with a focus on issues to be considered by those planning to use this new technology. It also examines some of the lessons learned from recent gene targeting studies and discusses different applications of the technology that are likely to generate scores of new animal models for a wide range of human diseases.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00748997
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  • 4
    Electronic Resource
    Electronic Resource
    Fibroblast growth factor complexed to the cytotoxin saporin is growth inhibitory but not cytotoxic for embryonal carcinoma cells (1993)
    Springer
    Cytotechnology 13 (1993), S. 69-78 
    Details
    ISSN: 1573-0778
    Keywords: cytotoxin ; embryonal carcinoma cells ; fibroblast growth factor ; FGF receptors ; saporin
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Fibroblast growth factors (FGFs) have been implicated in a number of proliferative lesions, including malignant tumor growth and vascularization. As a result, cytotoxic agents that target cell surface FGF receptors are currently under investigation. Previous reports have shown that conjugation of basic FGF with the ribosome inactivator, saporin, results in a potent cytotoxin specific for cells bearing high-affinity FGF receptors. In this report, we have used this FGF receptor-dependent cytotoxin to study receptor interactions at the surface of embryonal carcinoma cells, which express low numbers of high-affinity FGF receptors. The growth of three embryonal carcinoma cell lines and one embryonic stem cell line was shown to be inhibited by bFGF-saporin, suggesting that these cells are able to bind and internalize FGF through high-affinity FGF receptors. In addition, we determined that the responses of these cells to bFGF-saporin are qualitatively different than the responses of CHO-KI cells, which also exhibit low numbers of high-affinity FGF receptors. Specifically, pretreatment with bFGF-saporin reduces the cloning efficiency of CHO-KI cells 8- to 10-fold, whereas bFGF-saporin has little or no effect on the cloning efficiency of embryonal carcinoma cells. This finding suggests that bFGF-saporin is cytotoxic for CHO-KI cells, but not for embryonal carcinoma cells. Thus, our findings argue strongly that other factors, in addition to high-affinity FGF receptor number, are important in determining sensitivity of cells of bFGF-saporin.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00749933
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  • 5
    Electronic Resource
    Electronic Resource
    Regulation and expression of transforming growth factor type-ß during early mammalian development (1990)
    Springer
    Cytotechnology 4 (1990), S. 227-242 
    Details
    ISSN: 1573-0778
    Keywords: transforming growth factor type-beta ; embryonal carcinoma cells ; mammalian embryogenesis ; polymerase chain reaction
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract We have examined the effect of differentiation on the expression of different members of the transforming growth factor type-beta (TGF-β) family using embryonal carcinoma (EC) cells and early mammalian embryos. We determined that TGF-β activity increases approximately 25–100% when the mouse EC cell line, F9, is induced to differentiate with retinoic acid (RA). Interestingly, the increased TGF-β activity reflects the induction of TGF-β2 secretion following differentiation of both F9 EC cells and the human EC cell line, NT2/D1. Using the technique of reverse transcription-polymerase chain reaction (RT-PCR), we have verified that differentiation induces the expression of TGF-β2 as well as a distant member of the TGF-β family, Vgr-1. Transcripts for TGF-β2 and Vgr-1 were readily detected in the differentiated cells of F9 and PC-13 but not in their undifferentiated counterparts. Moreover, TGF-β2 mRNA was readily detected in NT2/D1 cells following differentiation. In addition, transcripts for TGF-β2 were detected by RT-PCR in mouse morulae, preimplantation blastocysts and cultured blastocysts. Based on the data presented, it appears that the expression of both TGF-β2 and Vgr-1 is closely associated with the induction of differentiation during early development.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00563783
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  • 6
    Electronic Resource
    Electronic Resource
    Effects of cell density and phorbol esters on the expression of epidermal growth factor receptors (1991)
    Springer
    Cytotechnology 7 (1991), S. 85-92 
    Details
    ISSN: 1573-0778
    Keywords: phorbol esters ; growth factor receptors ; cell density
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Previously, it has been shown that the binding of epidermal growth factor (EGF) by a wide range of cells decreases as cell density increases. In this report, we demonstrate that KB cells treated chronically with phorbol esters continue to exhibit decreases in EGF receptor binding as cell density increases. This finding suggests that protein kinase-C may not be essential for density-induced down regulation of EGF receptors, since phorbol esters are known to down regulate protein kinase-C. We also report that short-term and long-term effects of phorbol esters on the binding of EGF are affected by density. As shown previously for several cell lines, the phorbol ester 12-0-tetradecanoylphorbol-13-acetate transiently reduces EGF binding. We now show that the magnitude of this reduction diminishes as cell density increases. In addition, we determined that long-term treatment of KB cells with phorbol ester increases EGF binding. Again, this effect is diminished at high cell densities. Finally, we report that the increases in EGF binding induced by long-term treatment with phorbol esters are due to increases in the number of EGF receptors.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00350914
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  • 7
    Electronic Resource
    Electronic Resource
    Introduction (1989)
    Springer
    Cytotechnology 2 (1989), S. 255-257 
    Details
    ISSN: 1573-0778
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00364991
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  • 8
    Electronic Resource
    Electronic Resource
    Recent developments in the structure, function and regulation of platelet-derived growth factor and its receptors (1989)
    Springer
    Cytotechnology 2 (1989), S. 333-350 
    Details
    ISSN: 1573-0778
    Keywords: platelet-derived growth factor ; post-transcriptional control ; receptor down regulation ; receptors ; transcriptional control
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Recent evidence strongly suggests that production of platelet-derived growth factor (PDGF) is regulated by several mechanisms, including noncoordinate expression of the PDGF A- and B-chain genes, alternative transcript splicing, differential mRNA stability, and translational control. Considerable progress has also been made in our understanding of PDGF receptors. Recent studies demonstrate that there are two distinct PDGF receptor genes. PDGF receptors can undergo dimerization and it has been postulated that dimerization can lead to the formation of three different receptor dimers. In addition, it appears that dimerization is required for activation of PDGF receptors and the biological activity of PDGF. Although relatively little is known about the regulation of PDGF receptors, PDGF receptors are known to be down regulated by PDGF, and expression of PDGF receptors begins relatively early during embryogenesis. Furthermore, the binding of PDGF is regulated by cell density. Unexpectedly, recent evidence suggests that PDGF can localize to the nucleus, which suggests an intranuclear function for PDGF may exist. Together, these findings imply a complex and multi-leveled regulatory scheme for controlling the production of PDGF and its receptors.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00364997
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  • 9
    Electronic Resource
    Electronic Resource
    Regulation of the transforming growth factor-β2 gene promoter in embryonal carcinoma cells and their differentiated cells: Differential utilization of transcription factors (1995)
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 40 (1995), S. 135-145 
    Details
    ISSN: 1040-452X
    Keywords: Embryonal carcinoma cells ; Differentiation ; Transcription ; Transforming growth factor-beta ; Activating transcription factor ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Previous studies demonstrated that differentiation of embryonal carcinoma (EC) cells increases the expression of the TGF-β2 gene and identified a CRE/ATF-like motif in the TGF-β2 promoter that is necessary for its activity. This suggested that differentiation may increase the transcription of this gene by differential binding of transcription factors to the CRE/ATF-like motif. To test this possibility, we performed gel mobility shift analysis using double-stranded oligodeoxynucleotides containing the TGF-β2 CRE/ATF-like motif and nuclear extracts prepared from F9 EC cells and F9-differentiated cells. We determined that the DNA/protein complexes formed by the EC nuclear extracts, but not the complexes formed by differentiated cell nuclear extracts, are recognized and supershifted by an ATF-1 specific antibody. This observation is consistent with our Western immunoblot analysis that detects ATF-1 in the EC cells, but not in their differentiated counterparts. In addition, we provide evidence that protein phosphorylation influences the formation of complexes between F9 nuclear proteins and the CRE/ATF-like motif. Together, our studies identify a likely role for the CRE/ATF-like motif in the regulation of TGF-β2 and suggest that this site binds one set of nuclear proteins in EC cells, where the gene is not expressed, and a different set of nuclear proteins in the differentiated cells, where the gene is expressed. © 1995 Wiley-Liss, Inc.
    Additional Material: 7 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/mrd.1080400202
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  • 10
    Electronic Resource
    Electronic Resource
    Transcriptional regulation of the murine k-fgf gene (1994)
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 39 (1994), S. 106-111 
    Details
    ISSN: 1040-452X
    Keywords: Fibroblast growth factor ; Transcription ; Embryonal carcinoma cells ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Embryonal carcinoma (EC) cells provide a useful model system for studying the roles of growth factors during early mammalian development. In 1988, we determined that EC cells express a member of the fibroblast growth factor (FGF) family that cannot be detected after EC cells undergo differentiation. Attempts to understand how differentiation regulates the production of FGFs led to the finding that EC cells express the fibroblast growth factor k-FGF (FGF-4), whereas there is a large decrease in the steady state levels of k-FGF mRNA when EC cells differentiate. This suggested that transcription of the k-fgf gene is repressed when EC cells differentiate. To investigate this possibility, we prepared a series of reporter gene constructs containing various regions of the murine k-fgf gene. These constructs were transfected into two mouse EC cell lines and one mouse embryonic stem (ES) cell line. We determined that the mouse 5′ flanking region cannot support expression of the reporter gene. In both EC and ES cell lines, expression of the reporter gene is elevated greatly by the addition of a 316 bp region from the third exon of the murine k-fgf gene. Sequence analysis of the 316 bp region identified one and possibly two conserved octamer binding motifs. These sequences are likely to be involved in regulation of the k-fgf gene, because differentiation of EC cells is known to reduce the expression of octamer binding proteins, including Oct-3. To test the possible role of octamer binding proteins, we examined the expression of our reporter gene constructs in F9-differentiated cells and in PYS-2 cells. In these cells, the expression of our k-fgf/reporter gene constructs is very low. However, expression of the reporter gene is elevated significantly when these cells are cotransfected with a construct that contains an Oct-3 cDNA under the control of a strong viral promoter. To test further the importance of the octamer motifs found in the 316 bp enhancer-like region, we replaced the 316 bp region in our constructs with a smaller region (48 bp) that contains the downstream octamer motif and flanking sequences. Like the 316 bp region, this 48 bp region elevated the expression of the reporter gene, but it does so at a much lower level. Thus, it appears that the downstream octamer motif may be involved in the regulation of the k-fgf gene, but that at least one other cis-regulatory element present in the 316 bp enhancer is likely to be involved in the expression of the k-fgf gene in EC cells. Lastly, we have identified two potentially important regulatory regions upstream of the transcription start site. One appears to regulate positively the expression of the murine k-fgf gene in EC cells and in ES cells, and the other appears to regulate negatively the expression of the k-fgf gene in these cells. © 1994 Wiley-Liss, Inc.
    Additional Material: 2 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/mrd.1080390116
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