Retinoblastoma-related protein pRb2/p130 and suppression of tumor growth in vivo

Abstract

The RB/p105 and p107 genes of the retinoblastoma family are tumor suppressor genes whose proteins are inactivated by interaction with T- antigen proteins encoded by polyomaviruses (e.g., simian virus 40 and human JC virus), which have been found to be highly tumorigenic in animals. A variety of indirect evidence suggests that another member of the retinoblastoma gene family, RB2/p130, is also a tumor suppressor gene. To investigate the putative tumor suppressor activity of RB2/p130 more directly, we utilized a tetracycline-regulated gene expression system to control expression of the encoded protein pRb2/p130 in JC virus-induced hamster brain tumor cells and to study the effects of pRb2/p130 on the growth of such tumor cells in nude mice. The ability of pRb2/p130 to interact with JC virus T antigen was also studied. Methods: Northern blot hybridization analyses were performed on samples of total cellular RNA to measure RB2/p130 and β-actin messenger RNA levels. Immunoprecipitation and western blot analyses were used to determine T-antigen and pRb2/p130 protein levels and to assess the phosphorylation status of these proteins. Tumor cells were injected subcutaneously into nude mice, and tumor growth, with or without induced expression of pRb2/p130, was monitored. Results: Induction of pRb2/p130 expression brought about a 3.2-fold, or 69% (95% confidence interval = 64%- 73%), reduction in final tumor mass in nude mice. We also demonstrated that JC virus T antigen binds hypophosphorylated pRb2/p130 and that stimulation of pRb2/p130 expression overcomes cellular transformation mediated by this antigen. Conclusion: Our findings support the hypothesis that RB2/p130 is a tumor suppressor gene.