Antisense RNA-mediated reduction of p53 induces malignant phenotype in nontumorigenic rat urothelial cells

p53 mutation is commonly associated with high-grade, high-stage humanurothelial carcinomas. Recent studies suggest that p53 mutation in low-grade, low-stage bladder carcinomas may be correlated with the progressionof the disease. In the present study, we used antisense RNA methodology invitro to evaluate the significance of the loss of p53 function at an earlystage of urinary bladder carcinogenesis. An immortalized nontumorigenic raturothelial cell line (MYP3) that strongly expresses wild-type (WT) p53 wastransfected with a plasmid (pcDL-SR alpha-296) containing a rat WT p53 cDNAin antisense orientation. The transfection resulted in a significantreduction in p53 mRNA expression and protein synthesis, in stimulation ofanchorage- dependent growth, and in acquisition of anchorage-independentgrowth potential. Three such clones, when tested in athymic nude mice, allformed muscle-invasive, high-grade transitional cell carcinomas at s.c.injection sites. When cells were inoculated into an orthotopic site(urinary bladder), one of two antisense transfectants tested formed bulkytumors in the bladder in all seven nude mice and metastases to lungs inthree of the seven mice. Analysis of these cells revealed a decrease in theexpression of p21 (WAF1, sdi1, or CIP1) and retinoblastoma (Rb) geneproduct. Phosphorylation of Rb protein was not inhibited when the cellswere starved. No significant difference was observed in the expression ofp16 protein. In cell cycle analysis, all antisense transfectants testedescaped from G1 arrest by starvation. Furthermore, secretion of interleukin(IL)-6 into culture medium was increased significantly. Treatment withanti-IL-6 antibody suppressed anchorage-dependent growth. This studydirectly demonstrates that the loss of p53 function at an early stage ofurothelial carcinogenesis may result in acquisition of a malignantphenotype by regulating IL-6 production as well as cell cycle relatedgenes.