Upregulation of the cyclin-dependent kinase inhibitor p21 WAF1/CIP1 and subsequent cell growth arrest or senescence is one mechanism by which normal cells are believed to respond to stress induced by the constitutively activated GTPase Ras. We hypothesize that in the absence of p21, the onset of Ras-dependent oncogenesis is accelerated. To test this hypothesis, we crossed MMTV/v-Ha-ras transgenic mice into a p21-de®cient background. By 63 days of age, all 8 ras/p21 7/7 mice developed either malignant (mammary and/or salivary adenocarcinomas) or benign (Harderian hyperplasia) tumors. In contrast, by the same age, only one out of nine of the ras/p21 +/+ mice developed a tumor. Furthermore, by 94 days of age, half of the ras/p21 7/7 mice, but none of the ras/p21 +/+ mice, developed mammary tumors. p21-de®ciency also accelerated the development of salivary (T 50 =66 days for ras/p21 7/7 vs T 50 =136 days for ras/p21 +/+ ) and Harderian (T 50 =52 days for ras/p21 7/7 vs T 50 4221 days for ras/p21 +/+ ) tumors. Furthermore, two out of the eight ras/p21 7/7 mice had metastatic lesions, one in its lungs, the other in its abdomen. None of the nine ras/p21 +/+ mice had metastatic lesions. By 4 months of age, the mammary tumor multiplicity was 10-fold greater in ras/p21 7/7 (average 3.40 tumors/mouse) than in ras/p21 +/+ (average 0.33 tumor/mouse) mice. However, once the tumors appeared, their growth rate, apoptosis level, and mitotic index were not a ected by the loss of p21, suggesting that loss of p21 is critical in early but not late events of Ras oncogenesis. Altogether, the results show that tumor onset in MMTV/v-Ha-ras mice is p2l-dependent with loss of p2l associated with earlier tumor appearance and increased tumor multiplicity and aggressiveness.

Our studies examined the effects of p27 kip1 and p21 cip1 on the assembly and activity of cyclin D3-cdk4 complexes and determined the composition of the cyclin D3 pool in cells containing and lacking these cyclin-dependent kinase inhibitors. We found that catalytically active cyclin D3-cdk4 complexes were present in fibroblasts derived from p27 kip1 -p21 cip1-null mice and that immunodepletion of extracts of wild-type cells with antibody to p27 kip1 and/or p21 cip1 removed cyclin D3 protein but not cyclin D3-associated activity. Similar results were observed in experiments assaying cyclin D1-cdk4 activity. Data obtained using mixed cell extracts demonstrated that p27 kip1 interacted with cyclin D3-cdk4 complexes in vitro and that this interaction was paralleled by a loss of cyclin D3-cdk4 activity. In p27 kip1 -p21cip1 -deficient cells, the cyclin D3 pool consisted primarily of cyclin D3 monomers, whereas in wild-type cells, the majority of cyclin D3 molecules were complexed to cdk4 and either p27 kip1 or p21 cip1 or were monomeric. We conclude that neither p27 kip1 nor p21 cip1 is required for the formation of cyclin D3-cdk4 complexes and that cyclin D3-cdk4 complexes containing p27 kip1 or p21 cip1 are inactive. We suggest that only a minor portion of the total cyclin D3 pool accounts for all of the cyclin D3-cdk4 activity in the cell regardless of whether the cell contains p27 kip1 and p21 cip1.Cell cycle progression is regulated by an ordered sequence of events that includes the activation of the cyclin-dependent kinases (cdk's) (37, 43). Activation of the cdk's requires both their association with cyclins, whose levels fluctuate during the cell cycle, and their phosphorylation at specific threonine residues by cdk-activating kinase, a constitutively expressed enzyme (46). cdk's also interact with a group of proteins collectively termed cdk inhibitors (CKIs); CKI levels, like cyclin levels, vary during the cell cycle and thus contribute to the timing of cdk activation (44,45). Traverse of G 0 /G 1 and entry into S phase is controlled by the sequential activation of complexes containing the D cyclins and cdk4 or cdk6, cyclin E and cdk2, and cyclin A and cdk2. This series of events is initiated by mitogen-induced increases in the expression of the D cyclins (D1, D2, and D3) and the formation of active cyclin D-cdk4 (or cdk6) complexes in mid-G 1 (30,50). D cyclin-containing complexes phosphorylate the antioncogene Rb, as do cyclin E-cdk2 complexes, which become active in late G 1 due, at least in part, to decreases in CKI levels (16,20,22,29,49). When phosphorylated by these kinases, Rb no longer represses the activity of the E2F transcription factors, and a variety of E2F target genes, including those encoding cyclin E, cyclin A, and several DNA replication enzymes, are expressed (6,13,48). At this point, cells pass through the restriction point in late G 1 and, in a manner dependent on cyclin E-cdk2 and cyclin A-cdk2 activity, enter and traverse S phase (36).Two classes of CKIs have been defined: the INK pro...