Growth arrest of immortalized human keratinocytes and suppression of telomerase activity byp21WAF1 gene expression

Abstract

Because most non-melanocytic human skin cancers have p53 mutations, it is unclear whether the aberrant growth of these cancers is simply a result of the abrogation of a p53 downstream mediator, the universal cyclin-dependent kinase inhibitor p21WAF1. To investigate the role of p21WAF1 in human skin carcinogenesis, we studied its regulation in normal and p53-mutated immortalized human keratinocytes. In proliferating human normal keratinocytes (HNK), more wild-type p53 protein (wt p53) was expressed than in growth-arrested differentiating keratinocytes. However, the function of wt p53 as a transcriptional activator of the p21WAF1 gene was suppressed in proliferating keratinocytes. In response to ultraviolet B irradiation, expression of wt p53 increased in proliferating keratinocytes, but p21WAF1 transcriptional activation was not induced. Two isoforms of mdm2 (p57 and p90), which can bind to wt p53 and negatively regulate wt p53 function, were expressed in proliferating HNK, suggesting that mdm2 may play a role in the suppression of wt p53's function in proliferating HNK. Increased expression of p21WAF1 was detected in both Ca(2+)-induced growth-arrested and differentiating HNK, in which the wt p53 expression was down regulated. This reflects the complexity of the p53/p21WAF1 pathways of cell-cycle regulation and differentiation in keratinocytes. No p21WAF1 expression was detected in human immortalized keratinocytes (HaCaT) or in two ras-transformed variants, HaCaT ras I/7 and HaCaT ras II/3, which have two p53 mutations. Retrovirus-mediated expression of p21WAF1 stopped the growth of all these cell types, but expression of wt p53 did not affect the cells' growth properties. p21WAF1 also downregulated human telomerase RNA component mRNA expression in HaCaT cells. This novel function of p21WAF1 partly explains the suppression of telomerase activity by p21WAF1 expression in HaCaT. Taken together, these results are consistent with the idea that p21WAF1 successfully inhibits the growth of non-melanocytic skin cancers, even those with alterations in p53, p21ras, retinoblastoma gene product, and telomerase activity.

Downregulation of telomerase reverse transcriptase mRNA expression by wild type p53 in human tumor cells

“…Thus, hTERT expression does not strictly correlate with the proliferative status of cells. However, transfection of human immortal keratinocytes with p21 was shown to trigger cell cycle arrest accompanied by an inhibition of telomerase activity (Kallassy et al, 1998). A possible explanation for the discrepancy between this result and ours is that p21 is not involved in the early downregulation of hTERT expression observed in BL41-ts p53 cells but may be directly or indirectly involved in hTERT/telomerase regulation over a longer period.…”

Inactivation of Ret/Ptc1 oncoprotein and inhibition of papillary thyroid carcinoma cell proliferation by indolinone RPI-1

“…Nonetheless, the marked inhibition obtained in TPC-1 cells (85 ± 10 %) was not reached in other cell systems. Forced overexpression of p21WAF1 CDK inhibitor was reported to inhibit telomerase activity in immortalized human keratinocytes as a consequence of a reduced expression of the enzyme RNA subunit hTR [44]. However, results from our RT-PCR experiments did not support such an interpretation, since no significant reduction in the abundance of the different telomerase components was observed in TPC-1 cells exposed to the drug, despite up-regulation of p21WAF1, compared to control cells.…”

P16INK4A is implicated in both the immediate and adaptative response of human keratinocytes to UVB irradiation

“…Thus our data con®rm that the decrease in MDM2 after high UVB doses is independent of P53. We did not observe any P21 waf1/cip1 decrease in HaCat cells in these conditions as this protein is not expressed in this cell line (Kallassy et al, 1998). Unfortunately, hypermethylation of the p16 INK4A promoter in HaCat cells does not allow a study of UVB induction of P16 INK4A in this cell line (Chaturvedi et al, 1999).…”