Suppression of growth in vitro and tumorigenicity in vivo of human carcinoma cell lines by transfectedp16INK4

The function of p16INK4 as a putative tumor suppressor gene was examined by investigating its ability to inhibit the growth of cancer cell lines in vitro and tumor formation in vivo. A p16INK4 cDNA expression vector was transfected into five human cancer cell lines that varied in their p16INK4 and retinoblastoma (Rb) status. Suppression of colony-forming efficiency was seen in four cell lines. Of two cell lines wild type for p16INK4 but null for Rb protein expression, one (Hep 3B) showed inhibition of colony formation, whereas the other (Saos-2) did not. This observation may demonstrate involvement of p16INK4 independent of Rb. The transfected p16INK4 gene was frequently selected against and lost during continued growth in vitro. When compared to the colon carcinoma cell line (DLD-1),p16INK4-transfected DLD-1 clone 1 cells were less tumorigenic in athymic nude mice. Tumors arising from p16INK4-transfected DLD-1 clones, which were growth suppressed in vitro, either lost the integrated exogenous p16INK4 or expressed reduced amounts of p16INK4 protein. Therefore, p16INK4 was also selected against during tumor formation in vivo. These data are consistent with the hypothesis that p16INK4 is a tumor suppressor gene.