ANTISENSE RNA-MEDIATED REDUCTION OF P53 INDUCES MALIGNANT PHENOTYPE IN NONTUMORIGENIC RAT UROTHELIAL CELLS

p53 mutation is commonly associated with high-grade, high-stage human urothelial carcinomas, Recent studies suggest that p53 mutation in low -grade, low-stage bladder carcinomas may be correlated with the progre ssion of the disease, In the present study, we used antisense RNA meth odology in vitro to evaluate the significance of the loss of p53 funct ion at an early stage of urinary bladder carcinogenesis. An immortaliz ed nontumorigenic rat urothelial cell line (MYP3) that strongly expres ses wild-type (WT) p53 was transfected with a plasmid (pcDL-SR alpha-2 96) containing a rat WT p53 cDNA in antisense orientation, The transfe ction resulted in a significant reduction in p53 mRNA expression and p rotein synthesis, in stimulation of anchorage-dependent growth, and in acquisition of anchorage-independent growth potential, Three such clo nes, when tested in athymic nude mice, all formed muscle-invasive, hig h-grade transitional cell carcinomas at s.c. injection sites, When cel ls were inoculated into an orthotopic site (urinary bladder), one of t wo antisense transfectants tested formed bulky tumors in the bladder i n all seven nude mice and metastases to lungs in three of the seven mi ce, Analysis of these cells revealed a decrease in the expression of p 21 (WAF1, sdi1, or CIP1) and retinoblastoma (Rb) gene product. Phospho rylation of Rb protein was not inhibited when the cells were starved, No significant difference was observed in the expression of p16 protei n, In cell cycle analysis, all antisense transfectants tested escaped from G(1) arrest by starvation, Furthermore, secretion of interleukin (LL)-6 into culture medium was increased significantly, Treatment with anti-IL-6 antibody suppressed anchorage-dependent growth, This study directly demonstrates that the loss of p53 function at an early stage of urothelial carcinogenesis may result in acquisition of a malignant phenotype by regulating IL-6 production as well as cell cycle related genes.