Data from Intrinsic Cooperation between p16<sup>INK4a</sup> and p21<sup>Waf1/Cip1</sup> in the Onset of Cellular Senescence and Tumor Suppression <i>In vivo</i>

Although the p16^INK4a and p21^Waf1/Cip1 cyclin-dependent kinase (CDK) inhibitors are known to play key roles in cellular senescence in vitro, their roles in senescence remain rather poorly understood in vivo. This situation is partly due to the possibility of compensatory effect(s) between p16^INK4a and p21^Waf1/Cip1 or to the upregulation of functionally related CDK inhibitors. To directly address the cooperative roles of p16^INK4a and p21^Waf1/Cip1 in senescence in vivo, we generated a mouse line simply lacking both p16^INK4a and p21^Waf1/Cip1 genes [double-knockout (DKO)]. Mouse embryonic fibroblasts (MEF) derived from DKO mice displayed no evidence of cellular senescence when cultured serially in vitro. Moreover, DKO MEFs readily escaped Ras-induced senescence and overrode contact inhibition in culture. This was not the case in MEFs lacking either p16^INK4a or p21^Waf1/Cip1, indicating that p16^INK4a and p21^Waf1/Cip1 play cooperative roles in cellular senescence and contact inhibition in vitro. Notably, we found the DKO mice to be extremely susceptible to 7,12-dimethylbenz(a)anthracene/12-O-tetradecanoylphorbol-13-acetate–induced skin carcinogenesis that involves oncogenic mutation of the H-ras gene. Mechanistic investigations suggested that the high incidence of cancer in DKO mice likely reflected a cooperative effect of increased benign skin tumor formation caused by p21^Waf1/Cip1 loss, with increased malignant conversion of benign skin tumors caused by p16^INK4a loss. Our findings establish an intrinsic cooperation between p16^INK4a and p21^Waf1/Cip1 in the onset of cellular senescence and tumor suppression in vivo. Cancer Res; 70(22); 9381–90. ©2010 AACR.