| Cell cycle progression from G1 to S-phase depends on phosphorylation of pRb by complexes containing a cyclin (D-type or E-type) and cyclin-dependent kinase (e.g. cdk2, cdk4, or cdk6). Growth arrest is achieved by activation of the Arf-p53 pathway, or through activation of Ink4 family proteins, which specifically inhibit the activity of cyclin D-cdk4/6 complexes. We employed genetic and pharmacologic approaches to study the roles of the Ink4 family of proteins and the Arf-p53 pathway in regulating proliferation of cultured murine embryonic fibroblasts (MEFs) and murine tissues. While the loss of the Ink4 genes p16Ink4a and p18 Ink4c did not affect growth kinetics of MEFs despite an increase in cdk4 activity, reduction of Arf mRNA and protein through genetic inactivation or culture at 3% oxygen resulted in immortalization. The decrease in Arf mRNA at 3% O2 was due to reduced mRNA stability, which was regulated by the 3'UTR of the Arf transcript. However, Arf was still functional at 3% O2, as senescence could be induced through over-expression of oncogenic Ras, which increased Arf mRNA levels. In contrast to the lack of effect of p16Ink4a and p18Ink4c loss on MEFs, in vivo, germline deficiency of p16Ink4a and p18Ink4c resulted in increased proliferation in the intermediate pituitary and pancreatic islets of adult mice, and survival of p16Ink4a-/-; p18Ink4c-/- mice was significantly reduced due to aggressive pituitary tumors. Compensation among the Ink4 proteins was observed both in vivo in p18Ink4c -/- mice and in MEFs from p16Ink4a -/-, p18Ink4c -/- or p16Ink4a -/- -/-; p18 Ink4c -/- mice. Treatment with PD 0332991, a specific cdk4/6 kinase inhibitor, abrogated proliferation in those compartments where Ink4-deficiency was associated with enhanced proliferation: islets, pituitary and B-lymphocytes, but had no effect on proliferation in other tissues such as the small bowel. These data suggest that while Arf is the dominant regulator of proliferation in MEFs, p16Ink4a and p18Ink4c have a significant in vivo role in coordinately regulating the catalytic activity of cdk4/6 in specific compartments of adult mice. In addition, these data demonstrate a novel, oxygen-dependent mechanism of the regulation of senescence by the Ink4a/Arf locus. |
