The Ink4a/Arf locus encodes two distinct proteins, both of which may contribute to senescence and tumor suppression. We find that human diploid fibroblasts (HDFs) that are specifically deficient for p16INK4a achieve anchorage independence when transduced with retroviruses encoding telomerase (hTERT) and either Ras or Myc. Significantly, Ras and Myc together enable the cells to form tumors in nude mice but at a frequency that suggests additional genetic changes. All five tumors analyzed expressed high levels of Ras and retained functional p53, although two showed downregulation of Arf. Cytogenetic analyses identified clonal chromosomal alterations that may have contributed to tumorigenesis, but the tumor cells were essentially diploid.

Gain-of-function mutants of Ras and Rho family smallGTPases have proven to be important tools in analyzing signaling downstream of these small GTPases. The Ras-related GTPase Rheb has emerged as a key player downstream of TSC1-2 in activating signaling to mammalian target of rapamycin (mTOR) effectors of cell growth such as S6K and 4E-BP1. The TSC1-2 tumor suppressor complex has been shown to act as a RhebGAP, converting Rheb from a GTP-bound to a GDPbound form. Here we report the identification of a mutant Rheb (S16HRheb) that exhibits gain-of-function properties. At endogenous levels of expression S16HRheb exhibits increased GTP loading in vivo and is resistant to TSC1-2 GAP in vitro. Compared with wild-type Rheb, S16HRheb is more active at promoting the phosphorylation of the mTOR effectors S6K1 and 4E-BP1. Thus S16HRheb will help to identify proximal signaling events downstream of Rheb and allow potential Rheb-independent functions downstream of TSC1-2 to be investigated.

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...