Our functional evaluation of AIP mutations is consistent with a tumor-suppressor role for AIP and its involvement in familial acromegaly. The abnormal expression and subcellular localization of AIP in sporadic pituitary adenomas indicate deranged regulation of this protein during tumorigenesis.

The alternative product of the human INK4a/ARF locus, p14ARF, has the potential to act as a tumour suppressor by binding to and inhibiting the p53 antagonist MDM2. Current models propose that ARF function depends on its ability to sequester MDM2 in the nucleolus. Here we describe situations in which stabilization of MDM2 and p53 occur without relocalization of endogenous MDM2 from the nucleoplasm. Conversely, forms of ARF that do not accumulate in the nucleolus retain the capacity to stabilize MDM2 and p53. We therefore propose that nucleolar localization is not essential for ARF function but may enhance the availability of ARF to inhibit MDM2.

The CDKN2A tumour suppressor locus encodes two distinct proteins, p16 INK4a and p14 ARF , both of which have been implicated in replicative senescence, the state of permanent growth arrest provoked in somatic cells by aberrant proliferative signals or by cumulative population doublings in culture. Here we describe primary ®broblasts from a member of a melanomaprone family who is homozygous for an intragenic deletion in CDKN2A. Analyses of the resultant gene products imply that the cells are p16 INK4a de®cient but express physiologically relevant levels of a frameshift protein that retains the known functions of p14 ARF . Although they have a ®nite lifespan, the cells are resistant to arrest by oncogenic RAS. Indeed, ectopic expression of RAS and telomerase (hTERT) results in outgrowth of anchorage-independent colonies that have essentially diploid karyotypes and functional p53. We ®nd that in human ®broblasts, ARF is not induced demonstrably by RAS, pointing to signi®cant differences between the proliferative barriers implemented by the CDKN2A locus in different cell types or species.

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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.

Fusion proteins containing the amino-terminal domain of human p14ARF linked to green fluorescent protein are able to bind MDM2 and stabilize p53 without localization in the nucleolus. However, these fusion proteins are inherently unstable, with half-lives considerably shorter than either authentic ARF or chimaeras containing the entire coding domain, both of which are predominantly nucleolar. We present evidence that the unstable fusion proteins are significantly stabilized if redirected to the nucleolus by addition of a basic motif based on the nuclear localization signal of SV40 T-antigen. Moreover, the stability of these proteins can be enhanced by modulating the functions of MDM2 and p53. These data are consistent with a model in which ARF interacts with MDM2 in the nucleoplasm but is consequently subject to proteasomal degradation. Nucleolar localization may serve to store or stabilize ARF.

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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...