Mercurio, S

Mercurio, S. p16INK4a. Here we show that CDK4 and CDK6 can extend the life span of HDFs that have Mouse monoclonal to IFN-gamma inactivating mutations in both alleles of or in which levels are repressed, indicating that overexpression of CDK4/6 is not equivalent to ablation of p16INK4a. However, catalytically inactive versions of these kinases are unable to extend the replicative life span, suggesting that the impact of ectopic CDK4/6 depends on their ability to phosphorylate as yet unidentified substrates rather than to sequester CDK inhibitors. Since p16INK4a deficiency, CDK4 expression, and p53 or p21CIP1 ablation have additive effects on replicative life span, our results underscore the idea that senescence is an integrated response to diverse signals. 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 Ondansetron HCl (GR 38032F) 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 Ondansetron HCl (GR 38032F) 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 p21CIP1 cyclin-dependent kinase (CDK) inhibitors (1, 19, 35, 54, 61). The expression of p21CIP1 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), p21CIP1 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 Ondansetron HCl (GR 38032F) 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, 31-33, 54). The prospect that the D-type cyclins have both kinase-dependent and kinase-independent functions has important implications (for example, see references 28 and 63). In the context of senescence, it has not been rigorously established whether the catalytic activity of cyclin D-CDK complexes is extinguished at M1, when the levels of p21CIP1 and p16INK4a are changing dramatically. In early senescence, the peak of p21CIP1 might be expected to promote the formation of ternary cyclin D-CDK-p21CIP1 complexes while simultaneously inhibiting cyclin E-CDK2. As the total levels of p21CIP1 start to decline in late senescence, the accumulating p16INK4a would potentially ensure that enough CIP/KIP proteins are redistributed onto cyclin E-CDK2 by displacing them from the CDK4 and CDK6.

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