Ectopic expression of pRB2/p130 suppresses the tumorigenicity of the c-erb-2-overexpressing SKOV3 tumor cell line

We investigated the in vitro and in vivo effects of the ectopic expression of the pRb2/p130 cell cycle regulator on c-erB-2-associated tumorigenicity. SKOV3 ovarian cancer cells, which display c-erB-2 gene amplification and oncoprotein (p185(HER2)) overexpression, were stably transfected with a plasmid containing the coding sequence for human wild-type pRb2/p130 (wtRb2), or with pcDNA3 empty vector. Three wtRb2-transfected clones (cl. 24, cl. 49, cl. 100) and one empty vector-transfected clone (cl. mock) were randomly picked and further analysed. Western blot analysis revealed high levels of pRb2/p130 in the three clones compared to mock cells. Levels of p185(HER2) and the extent of its tyrosine phosphorylation were similar in all transfectant clones, as were levels of pRb1 and p107. In anchorage-independent growth assays, the number of colonies from wtRb2 clone-transfectants was about 90% less than that arising from mock cells (P < 0.001). Tumor take rates of the three wtRb2-transfected clones xenografted in nu/nu mice were much lower than those of mock cells, and tumor was decreased by 80% (P < 0.001). A volume mutant version of pRb2/p130 deleted of the pocket region (mut-Rb2) was also transfected into SKOV3 cells and studied in parallel with the wtRb2-transfected and pcDNA empty vector-transfected bulk populations. mutRb2 transfected cells showed no inhibition of in vitro colony formation and were fully tumorigenic. Together, these findings indicate that Rb2 acts as a tumor suppressor gene in vivo and in vitro in SKOV3 cells and that the intact pocket region is required for the suppressor activity.