Uncoupling of p21WAF1/CIP1/SDI1 mRNA and protein expression upon genotoxic stress

Abstract

The p21WAF1/CIP1/SDI1 gene is an important regulator of crucial cellular processes, including cell cycle control, cellular dierentiation, and the response to genotoxic stress. Induction of p21 gene expression upon DNA damage is widely believed to be p53-dependent. In the present study we analysed the expression of p21 following genotoxic stress, using dierent DNA-damaging agents and cellular systems. We found that the p21 response markedly varied between dierent cell lines and also for dierent genotoxic agents within the same cell line. Genotoxic induction of p21 mRNA expression can occur in the presence of p53-antagonists, such as overexpressed mdm-2 or human papillomavirus (HPV) E6, and in cells harbouring mutated p53 genes. Moreover, upon genotoxic stress, p21 mRNA and protein expression were found to be uncoupled in several cell lines. Thus, transcriptional and postranscriptional changes in p21 expression following DNA damage are not necessarily linked to the intracellular p53 status but strongly depend on the individual cellular background and the type of DNA-damaging agent. Our ®ndings indicate that p21 expression following genotoxic stress underlies a complex control and can be substantially modulated on the posttranscriptional level in a cellspeci®c manner.

Expression of human p53 requires synergistic activation of transcription from the p53 promoter by AP-1, NF-κB and Myc/Max

“…This corresponds well with the induction of endogenous p53 protein as judged from Western blots with nuclear extracts from TPAtreated and -untreated HeLa tk 7 cells that can be detected in 50 mg of nuclear extracts after overnight exposure (Figure 2b, insertion), despite the p53-degrading activity of intrinsic HPV 18 E6 (Sche ner et al, 1990, Butz et al, 1995.…”

“…Due to the reported autoregulation in case of the mouse p53 promoter through sequences that overlap with the NF-kB motif (De e et al, 1993), we had ®rst chosen HeLa tk 7 cells, since HeLa they were reported to contain low levels of the p53-protein caused by HPV-E6-mediated degradation of p53 (Sche ner et al, 1990, Butz et al, 1995. To con®rm our results in cells that are free from viral sequences and express p53 wt, we examined transcription from the hp53-promoter in exponentially growing HepG2 hepatoma cells (Bressac et al, 1990;Puisieux et al, 1993;Ho et al, 1996;Jiang et al, 1996).…”

HPV-18 E6*I protein modulates the E6-directed degradation of p53 by binding to full-length HPV-18 E6

“…These results demonstrate that the E6*I inhibition of E6-directed degradation of p53 is largely dependent upon the binding between E6*I and the full-length E6 protein and that, at least in CaSKi cells, the antiproliferative e ects of HPV-18 E6*I overexpression are due in part to activation of apoptosis. At present we do not know whether a signi®cant percentage of the cells also enter into growth arrest, although it has been shown previously that p21 protein is not induced by p53 in CaSKi cells (Butz et al, 1998), suggesting that G1 arrest is unlikely to play a major role in this activity of E6*I in these cells.…”

“…Tracks 3 ± 6: 4 ml of in vitro translated, radiolabelled p53 incubated with 3 ml of in vitro translated HPV-18E6 which had been pre-incubated with 20 ml of in vitro translated wild-type (WT) E6*I or mutants D1, D2 or D3 as indicated. Residual p53 was visualized by immunoprecipitation and polyacrylamide gel electrophoresis readily activated in HeLa cells but that it can be activated in CaSKi cells (Butz et al, 1995;Pim et al, 1997;Mantovani and Banks, 1999). In addition, since the mutant E6*I proteins bound HPV-16 E6 in a similar manner to HPV-18 E6 (data not shown), we proceeded to investigate the antiproliferative capacities of the three mutants in a CaSKi cell colony-forming assay.…”

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

“…It still remains to be investigated whether the residual activity of p53 in SV40-transformed cells is a feature of murine cells or occurs also in cells from other vertebrates, especially of human origin. Remarkably, also in human papillomavirus-transformed cells basal DNA-damage-inducible p53 functions are intact (Butz et al, 1995) and the high-risk papillomavirus type 16 E6 gene product promotes the cell cycle per se in cervical cancer cells (Spitkovsky et al, 1996).…”