GROWTH ARREST OF IMMORTALIZED HUMAN KERATINOCYTES AND SUPPRESSION OF TELOMERASE ACTIVITY BY P21(WAF1) GENE-EXPRESSION

Because most non-melanocytic human skin cancers have p53 mutations, it is unclear whether the aberrant growth of these cancers is simply a r esult of the abrogation of a p53 downstream mediator, the universal cy clin-dependent kinase inhibitor p21(WAF1). TO investigate the role of p21(WAF1) in human skin carcinogenesis, we studied its regulation in n ormal and p53-mutated immortalized human keratinocytes. In proliferati ng human normal keratinocytes (HNK), more wild-type p53 protein (wt p5 3) was expressed than in growth-arrested differentiating keratinocytes . However, the function of wt p53 as a transcriptional activator of th e p21(WAF1) gene was suppressed in proliferating keratinocytes. In res ponse to ultraviolet B irradiation, expression of wt p53 increased in proliferating keratinocytes, but p21(WAF1) 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 pro liferating HNK, suggesting that mdm2 may play a role in the suppressio n of wt p53's function in proliferating HNK. Increased expression of p 21(WAF1) was detected in both Ca2+-induced growth-arrested and differe ntiating HNK, in which the wt p53 expression was downregulated. This r eflects the complexity of the p53/p21(WAF1) pathways of cell-cycle reg ulation and differentiation in keratinocytes. No p21(WAF1) expression was detected in human immortalized keratinocytes (HaCaT) or in two I-a s-transformed variants, HaCaT ras 1/7 and HaCaT ras II/3, which have t wo p53 mutations. Retrovirus-mediated expression of p21(WAF1) stopped the growth of all these cell types, but expression of wt p53 did not a ffect the cells' growth properties. p21(WAF1) also downregulated human telomerase RNA component mRNA expression in HaCaT cells. This novel f unction of p21(WAF1) partly explains the suppression of telomerase act ivity by p21(WAF1) expression in HaCaT. Taken together, these results are consistent with the idea that p21(WAF1) successfully inhibits the growth of non-melanocytic skin cancers, even those with alterations in p53, p21(ras), retinoblastoma gene product, and telomerase activity. (C) 1998 Wiley-Liss, Inc.