p16 super(INK4A) participates in a G sub(1) arrest checkpoint in response to DNA damage

Members of the INK4 protein family specifically inhibit cyclin-dependent kinase 4 (cdk4) and cdk6-mediated phosphorylation of the retinoblastoma susceptibility gene product (Rb). p16 super(INK4A), a prototypic INK4 protein, has been identified as a tumor suppressor in many human cancers. Inactivation of p16 super(INK4A) in tumors expressing wild-type Rb is thought to be required in order for many malignant cell types to enter S phase efficiently or to escape senescence. Here, we demonstrate another mechanism of tumor suppression by implicating p16 super(INK4A) in a G sub(1) arrest checkpoint in response to DNA damage. Calu-1 non-small cell lung cancer cells, which retain Rb and lack p53, do not arrest in G sub(1) following DNA damage. However, engineered expression of p16 super(INK4A) at levels compatible with cell proliferation restores a G sub(1) arrest checkpoint in response to treatment with gamma -irradiation, topoisomerase I and II inhibitors, and cisplatin. A similar checkpoint can be demonstrated in p53 super(-/-) fibroblasts that express p16 super(INK4A) . DNA damage-induced G sub(1) arrest, which requires the expression of pocket proteins such as Rb, can be abrogated by overexpression of cdk4, kinase-inactive cdk4 variants capable of sequestering p16 super(INK4A), or a cdk4 variant incapable of binding p16 super(INK4A). After exposure to DNA-damaging agents, there was no change either in overall levels of p16 super(INK4A) or in amounts of p16 super(INK4A) found in complex with cdks 4 and 6. Nonetheless, p16 super(INK4A) expression is required for the reduction in cdk4- and cdk6-mediated Rb kinase activity observed in response to DNA damage. During tumor progression, loss of p16 super(INK4A) expression may be necessary for cells with wild-type Rb to bypass this G sub(1) arrest checkpoint and attain a fully transformed phenotype.