DNA-damage-inducible p53 activity in SV40-transformed cells

Hess, Ralf D.; Brandner, Gerhard

doi:10.1038/sj.onc.1201404

Cited by 19 publications

(16 citation statements)

References 10 publications

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“…on May 10, 2018 by guest http://mcb.asm.org/ achieve near saturation of p53), with early reports that expression of exogenous mutant p53 could enhance transformation by T (42), and finally with a recent report of residual DNA damage-inducible p53 activity in SV40-transformed murine cell lines (30). Direct evidence for this notion comes from immunofluorescence analysis of the behavior in our model of a key downstream target of p53, p21…”

Section: Discussionmentioning

confidence: 72%

Reinitiation of DNA Synthesis and Cell Division in Senescent Human Fibroblasts by Microinjection of Anti-p53 Antibodies

Gire

Wynford-Thomas

1998

Molecular and Cellular Biology

154

115

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In human fibroblasts, growth arrest at the end of the normal proliferative life span (induction of senescence) is dependent on the activity of the tumor suppressor protein p53. In contrast, once senescence has been established, it is generally accepted that reinitiation of DNA synthesis requires loss of multiple suppressor pathways, for example, by expression of Simian virus 40 (SV40) large T antigen, and that even this will not induce complete cell cycle traverse. Here we have used microinjection of monoclonal antibodies to the N terminus of p53, PAb1801 and DO-1, to reinvestigate the effect of blocking p53 function in senescent human fibroblasts. Unexpectedly, we found that both antibodies induce senescent cells to reenter S phase almost as efficiently as SV40, accompanied by a reversion to the "young" morphology. Furthermore, this is followed by completion of the cell division cycle, as shown by the appearance of mitoses, and by a four-to fivefold increase in cell number 9 days after injection. Immunofluorescence analysis showed that expression of the p53-inducible cyclin/kinase inhibitor p21 sdi1/WAF1 was greatly diminished by targeting p53 with either PAb1801 or DO-1 but remained high and, moreover, still p53 dependent in cells expressing SV40 T antigen. As previously observed for induction, the maintenance of fibroblast senescence therefore appears to be critically dependent on functional p53. We suggest that the previous failure to observe this by using SV40 T-antigen mutants to target p53 was most probably due to incomplete abrogation of p53 function.Normal human fibroblasts are capable of only a finite number of cell divisions even under optimum culture conditions, after which they enter a state of viable but permanent growth arrest (29). This phenomenon of cellular senescence has been observed in many other normal cell types (15,23,54) and represents a natural obstacle to clonal expansion (14, 58) which is thought to be an important restriction on the progression of many (although probably not all) human cancers (19).One currently popular model for senescence proposes that an intrinsic cell division clock, possibly based on telomere erosion (4), triggers one or more signal pathways which inhibit key components of the cell cycle regulatory machinery. Two candidate inhibitors whose levels increase with proliferative life span are p16 (2, 25) and p21 sdi1/WAF1 (46); these proteins inhibit the cyclin-dependent kinases CDK4/6 and CDK2, which are required for passage through and exit from the G 1 phase of the cell cycle (51). A related protein, p24 (40), may represent a third inhibitor. One consequence (55) of this inhibition is the failure of senescent cells to phosphorylate a major downstream target of these enzymes-the product of the retinoblastoma (Rb) sensitivity gene, pRb-which in its unphosphorylated form sequesters transcription factors needed for G 1 -S progression (61). Not surprisingly, therefore, escape from senescence is often associated with deregulation of the Rb pathway, either directly throu...

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Section: Discussionmentioning

confidence: 72%

Reinitiation of DNA Synthesis and Cell Division in Senescent Human Fibroblasts by Microinjection of Anti-p53 Antibodies

Gire

Wynford-Thomas

1998

Molecular and Cellular Biology

154

115

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“…Both GM637 and GM5849 cells are transformed by the SV40 virus and express SV40Tag. SV40 Tag protein binds to and stabilizes p53, resulting in elevated levels of p53 (Hess and Brandner, 1997;Kohli and Jorgensen, 1999;. Changes in p53 protein levels after DNA damage are therefore not usually detected in SV40-transformed cells (Kohli and Jorgensen, 1999).…”

Section: Resultsmentioning

confidence: 99%

ATM's leucine-rich domain and adjacent sequences are essential for ATM to regulate the DNA damage response

2003

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The ATM protein kinase regulates the DNA damage response by phosphorylating proteins involved in cell cycle checkpoints and DNA repair. We report here on the function of the predicted leucine zipper (LZ) motif, and sequences adjacent to this, in regulating ATM activity. The predicted LZ sequence was deleted from ATM, generating ATMDLZ, and expressed in an ATM-negative AT cell line. ATM increased cell survival following exposure to ionizing radiation, whereas expression of ATMDLZ failed to increase cell survival. ATMDLZ retained in vitro kinase activity, but was unable to phosphorylate p53 in vivo. Leucine zippers mediate homoand heterodimerization of proteins. However, the predicted LZ of ATM did not mediate the formation of ATM dimers. We examined if the predicted LZ of ATM was a dominant-negative inhibitor of ATM function in SW480 cells. Expression of amino acids 769-1436 of ATM, including the predicted LZ, sensitized SW480 cells to ionizing radiation, but did not inhibit ATM's kinase activity or its ability to phosphorylate Brca1. Further, this dominant-negative activity was not dependent on the predicted LZ domain. The central region of the ATM protein therefore contains multiple sequences which regulate cell survival following DNA damage.

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“…The human lens epithelial cell line used in this study is immortalized from primary cultures through transfection with a plasmid containing the gene encoding simian virus 40 large-T antigen (SV40-TAg) (Ibaraki et al, 1998). Although SV40-TAg is well known to interact with tumor suppressor proteins p53 and RB, and therefore may disable their normal cellular signaling pathways in immortalized cells (Chen et al, 1992;Bryan and Reddel, 1994), a number of recent studies have demonstrated that, in SV40-Tag-transformed murine and human cells, p53 is still functional in responses to DNA damage (Hess and Brandner, 1997;Kohli and Jorgensen, 1999). Our present study also reveals that p53 and Rb are not completely inactivated by SV40-TAg in HLE cells.…”

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confidence: 99%

Human telomerase accelerates growth of lens epithelial cells through regulation of the genes mediating RB/E2F pathway

et al. 2002

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Telomerase is a specialized reverse transcriptase that extends telomeres of eukaryotic chromosomes. The catalytic core of human telomerase is composed of an RNA template known as hTER (human telomerase RNA) and a protein subunit named hTERT (human telomerase reverse transcriptase). We have been studying other functions of the telomerase besides its major role in telomere maintenance. In our previous work, we have demonstrated that the hTERT can functionally interact with a rabbit TER to regulate expression of other genes and also attenuate the induced apoptosis. Here we report that overexpression of hTERT in a human lens epithelial cell line accelerates growth of the transfected lens epithelial cells. Associated with the acceleration of cell growth, expression of p53, p21 and GCIP (Grap2 cyclin-D interacting protein) is downregulated in the hTERTtransfected cells. With the downregulation of p21 and GCIP, the retinoblastoma protein (RB) is completely hyperphosphorylated in the hTERT-transfected cells. As expected, in the presence of RB hyperphosphorylation, the E2F transactivity is upregulated. Inhibition of telomerase activity abolishes the observed growth acceleration and also the related molecular changes. Furthermore, expression of hTERT in telomerasenegative human lens epithelial cells derived from primary cultures also accelerates growth of the transfected cells. Taken together, our results suggest that hTERT, when overexpressed in human lens epithelial cells, accelerates cell growth rate through regulation of RB/E2F pathway and possibly other genes.

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DNA-damage-inducible p53 activity in SV40-transformed cells

Cited by 19 publications

References 10 publications

Reinitiation of DNA Synthesis and Cell Division in Senescent Human Fibroblasts by Microinjection of Anti-p53 Antibodies

Reinitiation of DNA Synthesis and Cell Division in Senescent Human Fibroblasts by Microinjection of Anti-p53 Antibodies

ATM's leucine-rich domain and adjacent sequences are essential for ATM to regulate the DNA damage response

Human telomerase accelerates growth of lens epithelial cells through regulation of the genes mediating RB/E2F pathway

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