To investigate the mode of action of the p16INK4a tumor suppressor protein, we have established U2-OS cells in which the expression of p16INK4a can be regulated by addition or removal of isopropyl-␤-D-thiogalactopyranoside. As expected, induction of p16INK4a results in a G 1 cell cycle arrest by inhibiting phosphorylation of the retinoblastoma protein (pRb) by the cyclin-dependent kinases CDK4 and CDK6. However, induction of p16INK4a also causes marked inhibition of CDK2 activity. In the case of cyclin E-CDK2, this is brought about by reassortment of cyclin, CDK, and CDK-inhibitor complexes, particularly those involving p27 KIP1 . Size fractionation of the cellular lysates reveals that a substantial proportion of CDK4 participates in active kinase complexes of around 200 kDa. Upon induction of p16INK4a , this complex is partly dissociated, and the majority of CDK4 is found in lower-molecular-weight fractions consistent with the formation of a binary complex with p16INK4a . Sequestration of CDK4 by p16 INK4a allows cyclin D1 to associate increasingly with CDK2, without affecting its interactions with the CIP/KIP inhibitors. Thus, upon the induction of p16 INK4a, p27KIP1 appears to switch its allegiance from CDK4 to CDK2, and the accompanying reassortment of components leads to the inhibition of cyclin E-CDK2 by p27 KIP1 and p21 CIP1 . Significantly, p16 INK4a itself does not appear to form higher-order complexes, and the overwhelming majority remains either free or forms binary associations with CDK4 and CDK6.

Cellular senescence is now recognized as a general response to a variety of oncogenic and genotoxic stresses but was originally observed in cultures of primary human diploid fibroblasts (HDFs) as they reached the end of their proliferative life span (21). After what appears to be a predetermined number of population doublings (PDs), HDFs enter a permanent state of growth arrest, termed M1, and develop a characteristic phenotype (49, 59). In HDFs, a critical determinant of M1 is the erosion of the telomeres that occurs with each division (20), but it is clear that there are additional telomere-independent mechanisms that limit proliferative life span, collectively referred to as culture stress (11,51,62). In the classical HDF system, it was found that senescence could be delayed by interfering with the retinoblastoma (pRb) and p53 tumor suppressor pathways, for example, by using DNA tumor virus oncoproteins that bind to either or both pRb and p53 (49). This results in a significant increase in the maximum number of PDs, but the continued erosion of telomeres during this period eventually leads to chromosome fusion and breakage and the cultures reach a state referred to as M2 or crisis, where cell division is still occurring but is offset by extensive cell death (49,59).A distinctive feature of senescent HDFs is that they express elevated levels of the p16INK4a and p21 CIP1 cyclin-dependent kinase (CDK) inhibitors (1,19,35,54,61). The expression of p21 CIP1 peaks as cells approach M1, presumably reflecting a p53-mediated signal from the damaged telomeres (9, 22), whereas the accumulation of p16INK4a is more pronounced after cell proliferation has ceased (1, 6, 54). It is tacitly assumed that these CDK inhibitors are responsible for implementing the senescence arrest by preventing the CDK-mediated phosphorylation of pRb and its relatives. Whereas p16INK4a interacts specifically with CDK4 and CDK6 and blocks their association with D-type cyclins (40, 47), p21 CIP1 interacts with multiple cyclin-CDK complexes (reviewed in reference 50). When bound to cyclin E-CDK2 and cyclin A-CDK2, the CIP/KIP proteins act as potent inhibitors of catalytic activity (42), but their impact on the cyclin D-dependent kinases is more enigmatic.Various pieces of evidence suggest that the CIP/KIP proteins promote the assembly of cyclin D-CDK complexes, and indeed, most of the D-type cyclins in the cell are present in these stable ternary complexes (8,27,31,36,41). However, it remains a matter of debate whether such complexes have catalytic activity or simply provide a buffering system that controls the availability of CIP/KIP proteins to inhibit CDK2 (37, 52). In either case, p16INK4a has the capacity to inhibit CDK2 as well as CDK4 and CDK6 by causing the redistribution of CIP/KIP proteins onto CDK2-containing complexes, where they function as inhibitors, and by promoting the formation of inactive cyclin D-CDK2 complexes (26,(31)(32)(33)54).The prospect that the D-type cyclins have both kinase-dependent and kinase-independent functions ha...

The CDKN2A locus encodes two distinct proteins, p16 INK4a and p14 ARF , both of which are implicated in replicative senescence and tumor suppression in different contexts. Here, we describe the characterization of a novel strain of human diploid fibroblasts (designated Milan HDFs) from an individual who is homozygous for the R24P mutation in p16INK4a . As this mutation occurs in the first exon of INK4a (exon 1A), it has no effect on the primary sequence of p14 ARF . Based on both in vitro and in vivo analyses, the R24P variant is specifically defective for binding to CDK4 but remains able to associate with CDK6. Nevertheless, Milan HDFs behave as if they are p16INK4a deficient, in terms of sensitivity to spontaneous and oncogene-induced senescence, and the R24P variant has little effect on proliferation when ectopically expressed in normal fibroblasts. It can, however, impair the proliferation of U20S cells, presumably because they express more CDK6 than primary fibroblasts. These observations suggest that CDK4 and CDK6 are not functionally redundant and underscore the importance of CDK4 in the development of melanoma. [Cancer Res 2007;67(19):9134-41]

Cellular senescence, the stable cell cycle arrest elicited by various forms of stress, is an important facet of tumor suppression. Although much is known about the key players in the implementation of senescence, including the pRb and p53 axes and the cyclin dependent kinase inhibitors p16INK4a and p21CIP1, many details remain unresolved. In studying conditional senescence in human fibroblasts that express a temperature sensitive SV40 large T-antigen (T-Ag), we uncovered an unexpected role for CDK4. At the permissive temperature, where pRb and p53 are functionally compromised by T-Ag, cyclin D-CDK4 complexes are disrupted by the high p16INK4a levels and reduced expression of p21CIP1. In cells arrested at the non-permissive temperature, p21CIP1 promotes reassembly of cyclin D-CDK4 yet pRb is in a hypo-phosphorylated state, consistent with cell cycle arrest. In exploring whether the reassembled cyclin D-CDK4-p21 complexes are functional, we found that shRNA-mediated knockdown or chemical inhibition of CDK4 prevented the increase in cell size associated with the senescent phenotype by allowing the cells to arrest in G1 rather than G2/M. The data point to a role for CDK4 kinase activity in a G2 checkpoint that contributes to senescence.