Use of genetic suppressor elements to dissect distinct biological effects of separate p53 domains.

Ossovskaya, Valeria; Mazo, Ilya; Chernov, Michail V.; Chernova, Olga B.; Strezoska, Žaklina; Kondratov, Roman V.; Stark, George R.; Chumakov, Peter M.; Gudkov, Andrei V.

doi:10.1073/pnas.93.19.10309

Cited by 161 publications

(176 citation statements)

References 36 publications

(38 reference statements)

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“…We expressed a dominant p53 inhibitor, GSE-22 (ref. 15), in early and later-passage 3% oxygen MEFs and confirmed that it increased p53 levels, as previously reported 15 (see Supplementary Information, Fig. S1a).…”

supporting

confidence: 89%

Oxygen sensitivity severely limits the replicative lifespan of murine fibroblasts

et al. 2003

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Most mammalian cells do not divide indefinitely, owing to a process termed replicative senescence. In human cells, replicative senescence is caused by telomere shortening, but murine cells senesce despite having long stable telomeres 1 . Here, we show that the phenotypes of senescent human fibroblasts and mouse embryonic fibroblasts (MEFs) differ under standard culture conditions, which include 20% oxygen. MEFs did not senesce in physiological (3%) oxygen levels, but underwent a spontaneous event that allowed indefinite proliferation in 20% oxygen. The proliferation and cytogenetic profiles of DNA repair-deficient MEFs suggested that DNA damage limits MEF proliferation in 20% oxygen. Indeed, MEFs accumulated more DNA damage in 20% oxygen than 3% oxygen, and more damage than human fibroblasts in 20% oxygen. Our results identify oxygen sensitivity as a critical difference between mouse and human cells, explaining their proliferative differences in culture, and possibly their different rates of cancer and ageing.Replicative senescence limits the proliferation of many cell types in culture and in vivo 2 .Human cells senesce replicatively largely because telomeres -DNA structures that cap chromosome ends -shorten and malfunction when replicated in the absence of telomerase, which most human cells do not express 1 . Senescent cells adopt a distinctive morphology and pattern of gene expression 2,3 , a phenotype also induced by telomere-independent events 2,4 such as DNA damage and activation of certain oncogenes. The senescence response is thought to suppress cancer 2 .MEFs are used widely in the study of replicative senescence, despite notable differences between human and rodent cells 1,2 . MEFs spontaneously overcome replicative senescence (immortalization) 5,6 , whereas human fibroblasts rarely do so. Moreover, MEF senescence relies predominantly on the p19 ARF /p53 tumour suppressor pathway, whereas human fibroblasts require loss of both p53 and retinoblastoma (Rb) tumour suppressor functions for Correspondence should be addressed to: J.C. (jcampisi@lbl.gov). Note: Supplementary Information is available on the Nature Cell Biology website. COMPETING FINANCIAL INTERESTSThe authors declare that they have no competing financial interests. Here, we compared the phenotypes of senescent mouse and human fibroblasts. We cultured MEFs from C57Bl/6 (or FVB; data not shown) mice under standard conditions, which include atmospheric (20%) oxygen (Fig. 1a). As expected 5 , the cells grew well for approximately 1 week (2-3 population doublings) before proliferation began to decline. After 4-5 weeks (8-10 population doublings), the cultures senesced (arrow), showing no change in cell number for more than 1 week and a senescent morphology. Proliferation eventually resumed, owing to outgrowth of immortal variants 5 . HHS Public AccessWe monitored the capacity for DNA synthesis at each passage by labelling cells with 3 Hthymidine for 3 days and counting radiolabelled nuclei. As reported 9 , the percentage of labelled nuclei d...

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confidence: 89%

Oxygen sensitivity severely limits the replicative lifespan of murine fibroblasts

et al. 2003

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“…At 24 h, the population of G 2 /M cells was still predominant, as compared to the proportion of S-phase cells. To determine if p53 activity was required for the radiationinduced cell cycle arrest, C4-2 cells were stably transfected with a C-terminal fragment dominantnegative (DN) p53 mutant (Ossovskaya et al, 1996), such that following irradiation, its transcriptional activity was abrogated, as shown by a dramatic decrease from abundant to undetectable levels of p21 WAF1 (S. Ray et al, unpublished data). Similar to the parental C4-2, the derivative cells containing the stably expressed DNp53 had a robust 4C arrest following irradiation (Figure 1b).…”

Section: Resultsmentioning

confidence: 99%

E2F4 regulates a stable G2 arrest response to genotoxic stress in prostate carcinoma

et al. 2006

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The retinoblastoma (pRB) family proteins regulate the E2F transcription factors; their complexes regulate critical transitions through the cell cycle. The function of these pRB family/E2F complexes, which includes p130/ E2F4, in response to genotoxic agents, is not well understood. We investigated the role of E2F4 in the genotoxic stress response. Following radiation treatment, E2F4 colocalized with p130 in the nucleus during a radiation-induced stable G 2 -phase arrest. Arrested cells had significantly decreased expression of Cyclins A2 and B1 and decreased phosphorylation of mitotic protein monoclonal-2 (MPM-2) mitotic proteins. Small interference RNA (siRNA)-mediated knockdown of E2F4 sensitized cells to subsequent irradiation, resulting in enhanced cellular DNA damage and cell death, as determined by caspase activation and decreased clonogenic cell survival. Downstream E2F4 targets potentially involved in the progression from G 2 into M phase were identified by oligonucleotide microarray expression profiling. Chromatin immunoprecipitation localized E2F4 at promoter regions of the Bub3 and Pttg1 mitotic genes following irradiation, which were among the downregulated genes identified by the microarray. These data suggest that in response to radiation, E2F4 becomes active in the nucleus, enforces a stable G 2 arrest by target gene repression, and thus provides increased cell survival ability by minimizing propagation of cells that have irreparable DNA damage.

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“…REF52 sublines expressing N-ras asp12 transgene along with either dominant-negative p53 cDNA fragment (GSE22, Genetic Suppressive Element #22 ± Ossovskaya et al, 1996) or mutant R175H p53 (REF52/GSE-ras and REF52/His175-ras cell lines, respectively) were obtained by infection with retroviral vectors carrying the N-ras asp12 cDNA (pPS/hygroras), mutant human p53 cDNA (pPS/neo-p53 175 ) and p53-GSE (pLXSN/GSE22) which were described by us earlier Kopnin et al, 1995;Ossovskaya et al, 1996). Also we used (i) wt-p53-positive Rat-2 sublines bearing human N-ras proto-oncogene or H-ras gene mutated at codon 12 (Rat-2/5 and Rat2-HT1 cell lines, respectively) driven by inducible mouse mammary tumor virus promoter (McKay et al, 1986;Alexandrova et al, 1993), and (ii) human pseudodiploid hereditary non-polyposis colon carcinoma LIM1215 (Whitehead et al, 1985) containing two wtp53 gene alleles (Zaichuk et al, 1993) and its sublines expressing transduced N-ras asp12 oncogene and/or dominantnegative p53 mutants (Agapova et al, 1996;.…”

Section: Cellsmentioning

confidence: 99%

p53-dependent effects of RAS oncogene on chromosome stability and cell cycle checkpoints

Ls¹,

Sablina³

et al. 1999

Oncogene

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Mutations activating the function of ras proto-oncogenes are often observed in human tumors. Their oncogenic potential is mainly due to permanent stimulation of cellular proliferation and dramatic changes in morphogenic reactions of the cell. To learn more on the role of ras activation in cancerogenesis we studied its e ects on chromosome stability and cell cycle checkpoints. Since the ability of ras oncogenes to cause cell transformation may be dependent on activity of the p53 tumorsuppressor the cells with di erent p53 state were analysed. Ectopic expression of N-ras asp12 caused in p53-de®cient MDAH041 cell line an augmentation in the number of chromosome breaks in mitogenic cells, signi®cant increase in the frequency of metaphases showing chromosome endoreduplication and accumulation of polyploid cells. Similar e ects were induced by di erent exogenous ras genes (N-ras asp12 , H-ras leu12 , N-ras proto-oncogene) in Rat1 and Rat2 cells which have a defect in p53-upstream pathways. In contrast, in REF52 and human LIM1215 cells showing ras-induced p53 upregulation, ras expression caused only slight increase in the number of chromosome breaks and did not enhance the frequency of endoreduplication and polyploidy. Inactivation in these cells of p53 function by transduction of dominant-negative C-terminal p53 fragment (genetic suppressor element #22, GSE22) or mutant p53s signi®cantly increased the frequency of both spontaneous and ras-induced karyotypic changes. In concordance with these observations we have found that expression of ras oncogene caused in p53-defective cells further mitigation of ethyl-metansulphonate-induced G1 and G2 cell cycle arrest, but did not abrogate G1 and G2 cell cycle checkpoints in cells with normal p53 function. These data indicate that along with stimulation of cell proliferation and morphological transformation ras activation can contribute to cancerogenesis by increasing genetic instability.

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Use of genetic suppressor elements to dissect distinct biological effects of separate p53 domains.

Cited by 161 publications

References 36 publications

Oxygen sensitivity severely limits the replicative lifespan of murine fibroblasts

Oxygen sensitivity severely limits the replicative lifespan of murine fibroblasts

E2F4 regulates a stable G2 arrest response to genotoxic stress in prostate carcinoma

p53-dependent effects of RAS oncogene on chromosome stability and cell cycle checkpoints

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