EFFECT OF ACTINOMYCIN-D ON THE CELL-CYCLE PROGRESSION AND THE EXPRESSION OF P53, WAF1/CIP1, GADD45, AND MDM-2 GENES IN HUMAN ORAL KERATINOCYTES - IMPLICATION OF HUMAN PAPILLOMAVIRUS INFECTION

Abstract

Exposure of human oral keratinocytes immortalized by transfection with 'high risk' HPV DNA to chemical carcinogens converts the cells to malignant phenotype, but it does not transform normal cells. To investigate the underlying mechanism for different chemical carcinogen susceptibility of normal and the HPV-immortalized oral keratinocytes to genotoxic agent, we studied the progression of cell cycle and the expression of p53, WAF1/CIP1, gadd45 and mdm-2 genes in normal and in the HPV-immortalized oral keratinocytes after exposing cells to actinomycin D. Normal oral keratinocytes demonstrated transient G(1) arrest after the exposure, but the HPV-immortalized cells did not. Actinomycin D significantly increased the levels of intranuclear wild-type p53 and mdm-2 proteins and the transcripts of WAF1/CIP1, gadd45 and mdm-2 in normal cells, but it did not increase them in the HPV-immortalized cells. These data indicate that actinomycin D-induced transient cell cycle arrest may be associated with enhanced level of wild-type p53 protein and the transcripts of WAF1/CIP1 and gadd45 in normal human oral keratinocytes. This study also suggests that overexpressed mdm-2 may not be enough to abrogate the cell cycle arrest mediated by wild-type p53. Our data support the idea that conversion of the HPV-immortalized oral keratinocytes to tumorigenic cells may, in part, be due to the incapability of the cells to arrest the cell cycle when exposed to genotoxic agents, which would result in improper repair of damaged DNA and subsequent inheritance of genetic error.