The tumor-suppressor gene p16INK4/CDKN2 (p16) is a cyclin-dependent kinase (cdk) inhibitor and important cell cycle regulator. Here, we show that adenovirus-mediated gene transfer of p16 (AdCMV. p16) into colon cancer cells induces uncoupling of S phase and mitosis and subsequently apoptosis. Flow cytometric analysis revealed that cells infected with AdCMV. p16 showed an initial G2-like arrest followed by S phase without intervening mitosis (DNA> 4N). Using microscopic analysis, deformed polyploid cells were detectable only in cells infected with AdCMV. p16 but not in control-infected cells. Subsequently, AdCMV. p16-infected polyploid cells underwent apoptosis, as assessed by AnnexinV staining and DNA fragmentation, suggesting that cell cycle dysregulation is upstream of the onset of apoptosis. Treatment of mice with subcutaneously transplanted tumors of colorectal cancer cells with AdCMV. p16 but not AdCMV. p53 resulted in significantly reduced tumor volume and prolonged survival. Using an orthotopic model of liver metastasis, we observed both reduced local tumor growth and secondary intrahepatic metastasis after AdCMV. p16 treatment. Importantly, induction of apoptosis in vitro and reduction of tumor growth in vivo by p16 was p53-as well as bax-independent because identical results were obtained using cancer cells, either wild type or mutant for p53 or bax. The studies suggest that an AdCMV. p16-based treatment may be especially effective in patients with bax-negative colon cancer where overexpression of p53 appears not to be of therapeutic value.