SELECTIVE EXPRESSION OF MUTATED P53 IN HUMAN-CELLS IMMORTALIZED WITH EITHER 4-NITROQUINOLINE 1-OXIDE OR CO-60 GAMMA-RAYS

Many studies on in vitro transformation of human cells indicate that t he cells must be immortalized before they can be neoplastically transf ormed, indicating that immortalization is a critical step in multistep neoplastic transformation of human cells. We immortalized three human cell lines by repeated treatment with either Co-60 gamma rays or a ch emical carcinogen, 4-nitroquinoline 1-oxide, and found that all three immortalized cell lines have mutations in the tumor suppressor gene, p 53. Direct sequencing of the reverse-transcribed mRNA and immunoprecip itation of p53 protein revealed that mutant p53 is selectively express ed in all the immortalized cell lines, whereas the genomic fragments o f the immortalized cells contain wild-type and mutated p53 alleles. Al though the mutated p53 is selectively expressed in the immortalized ce lls, expression of the wild-type p53 was induced by treatment of the c ells with a hypomethylating reagent, 5-azacytidine, indicating that th e wild-type p53 allele might be inactivated by hypermethylation of DNA . Actually, the entire genomic locus including the promoter region of p53 is hypermethylated in all the immortalized cell lines. Expression and phosphorylation of Rb was normal in these three cell lines. Thus, inactivation of both wild type p53 alleles and selective expression of mutated p53 seem to be key factors in the immortalization of human fi broblasts.