[show abstract][hide abstract] ABSTRACT: The cyclin-dependent kinase (CDK) inhibitors, p18(INK4c) and p16(INK4a), both have the credentials of tumor suppressors in human cancers and mouse models. For p16(INK4a), the underlying rationale is its role in senescence, but the selective force for inactivation of p18(INK4c) in incipient cancer cells is less clear. Here, we show that in human fibroblasts undergoing replicative or oncogene-induced senescence, there is a marked decline in the levels of p18(INK4c) protein and RNA, which mirrors the accumulation of p16(INK4a). Downregulation of INK4c is not dependent on p16(INK4a), and RAS can promote the loss of INK4c without cell-cycle arrest. Downregulation of p18(INK4c) correlates with reduced expression of menin and E2F1 but is unaffected by acute cell-cycle arrest or inactivation of the retinoblastoma protein (pRb). Collectively, our data question the idea that p18(INK4c) acts as a backup for loss of p16(INK4a) and suggest that the apparent activation of p18(INK4c) in some settings represents delayed senescence rather than increased expression. We propose that the contrasting behavior of the two very similar INK4 proteins could reflect their respective roles in senescence versus differentiation.
Cancer Research 11/2011; 72(1):165-75. · 8.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: Mutations have been identified in the aryl hydrocarbon receptor-interacting protein (AIP) gene in familial isolated pituitary adenomas (FIPA). It is not clear, however, how this molecular chaperone is involved in tumorigenesis.
AIP sequence changes and expression were studied in FIPA and sporadic adenomas. The function of normal and mutated AIP molecules was studied on cell proliferation and protein-protein interaction. Cellular and ultrastructural AIP localization was determined in pituitary cells.
Twenty-six FIPA kindreds and 85 sporadic pituitary adenoma patients were included in the study.
Nine families harbored AIP mutations. Overexpression of wild-type AIP in TIG3 and HEK293 human fibroblast and GH3 pituitary cell lines dramatically reduced cell proliferation, whereas mutant AIP lost this ability. All the mutations led to a disruption of the protein-protein interaction between AIP and phosphodiesterase-4A5. In normal pituitary, AIP colocalizes exclusively with GH and prolactin, and it is found in association with the secretory vesicle, as shown by double-immunofluorescence and electron microscopy staining. In sporadic pituitary adenomas, however, AIP is expressed in all tumor types. In addition, whereas AIP is expressed in the secretory vesicle in GH-secreting tumors, similar to normal GH-secreting cells, in lactotroph, corticotroph, and nonfunctioning adenomas, it is localized to the cytoplasm and not in the secretory vesicles.
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.
[show abstract][hide abstract] ABSTRACT: The CDKN2A locus encodes two distinct proteins, p16INK4a and p14ARF, 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 1alpha), 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 Research 11/2007; 67(19):9134-41. · 8.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: Replicative senescence of human diploid fibroblasts (HDFs) is largely implemented by the cyclin-dependent kinase (CDK) inhibitors p16(INK4a) and p21(CIP1). Their accumulation results in a loss of CDK2 activity, and cells arrest with the retinoblastoma protein (pRb) in its hypophosphorylated state. It has become standard practice to bypass the effects of p16(INK4a) by overexpressing CDK4 or a variant form that is unable to bind to INK4 proteins. Although CDK4 and CDK6 and their INK4-insensitive variants can extend the life span of HDFs, they also cause a substantial increase in the levels of endogenous p16(INK4a). Here we show that CDK4 and CDK6 can extend the life span of HDFs that have inactivating mutations in both alleles of INK4a or in which INK4a levels are repressed, indicating that overexpression of CDK4/6 is not equivalent to ablation of p16(INK4a). 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 p16(INK4a) deficiency, CDK4 expression, and p53 or p21(CIP1) ablation have additive effects on replicative life span, our results underscore the idea that senescence is an integrated response to diverse signals.
Molecular and Cellular Biology 07/2007; 27(12):4273-82. · 5.37 Impact Factor
[show abstract][hide abstract] ABSTRACT: In mammalian cells, products of the INK4a-ARF locus play major roles in senescence and tumour suppression in different contexts, whereas the adjacent INK4b gene is more generally associated with transforming growth factor beta (TGF-beta)-mediated growth arrest. As the chicken genome does not encode an equivalent of INK4a, we asked whether INK4b and/or ARF contribute to replicative senescence in chicken cells. In chicken embryo fibroblasts (CEFs), INK4b levels increase substantially at senescence and the gene is transcriptionally silenced in two spontaneously immortalised chicken cell lines. By contrast, ARF levels are unaffected by prolonged culture or immortalisation. These expression patterns resemble the behaviour of INK4a and ARF in human fibroblasts. However, short-hairpin RNA (shRNA)-mediated knockdown of chicken INK4b or ARF provides only modest lifespan extension, suggesting that other factors contribute to senescence in CEFs. As well as underscoring the importance of the INK4b-ARF-INK4a locus in senescence, these findings imply that the encoded products have assumed different roles in different evolutionary niches. Although ARF RNA is not detectable in early chicken embryos, the INK4b transcript is expressed in the roof-plate of the developing hind-brain, consistent with a role in limiting cell proliferation.
[show abstract][hide abstract] ABSTRACT: In standard conditions of tissue culture, human fibroblasts undergo a limited number of population doublings before entering a state of irreversible growth arrest termed replicative senescence or M1. The arrest is triggered by a combination of telomere dysfunction and the stresses inflicted by culture conditions and is implemented, at least in part, by the cyclin-dependent kinase inhibitors p21(CIP1) and p16(INK4a). To investigate the role of p16(INK4a), we have studied fibroblasts from members of melanoma prone kindreds with mutations in one or both copies of the CDKN2A locus. The mutations affect the function of p16(INK4a) but not of the alternative product, p14(ARF). The p16(INK4a)-defective fibroblasts have an above average life span, compared to the heterozygous and normal age-matched controls, but they arrest with characteristics typical of senescence. Using agents that are known to bypass M1, such as DNA tumor virus oncoproteins or the Bmi1 transcriptional repressor, we provide evidence that p16(INK4a) defective cells arrest at a stage that is operationally between M1 and M2 (crisis). As well as indicating that p16(INK4a) contributes to but is not essential for replicative senescence of human fibroblasts, our data reveal considerable heterogeneity in the levels and accumulation of p16(INK4a) in different strains.
Experimental Cell Research 09/2004; 298(2):549-59. · 3.56 Impact Factor
[show abstract][hide abstract] ABSTRACT: Fusion proteins containing the amino-terminal domain of human p14(ARF) 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.
[show abstract][hide abstract] ABSTRACT: Human cells, including fibroblast strains that have been immortalized by telomerase, are much more resistant to transformation than rodent cells. Most of the experimental evidence suggests that transformation of human fibroblasts requires inactivation of both the retinoblastoma (pRb) and p53 tumor suppressors as well as the addition of one or more dominant oncogenes. By starting with strains of primary fibroblast (Leiden and Q34 cells) that are genetically deficient for p16INK4a, we have been able to generate anchorage independent colonies simply by addition of telomerase (hTERT) and either Ras or Myc. Importantly, the transformed cells appear to retain pRb and p53 functions and are essentially diploid. Whereas Leiden cells expressing the individual oncogenes did not form tumors in mice, the combination of hTERT, Myc and Ras enabled them to become tumorigenic, albeit at a frequency suggestive of an additional genetic event. Significantly, we have obtained karyotypically stable tumors without the need to use DNA tumor virus oncoproteins and without deliberate ablation of p53.
[show abstract][hide abstract] ABSTRACT: 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.
Cancer Cell 11/2003; 4(4):301-10. · 24.76 Impact Factor
[show abstract][hide abstract] ABSTRACT: The INK4a/ARF tumor suppressor locus is implicated in the senescence-like growth arrest provoked by oncogenic Ras in primary cells. INK4a and ARF are distinct proteins encoded by transcripts in which a shared exon is decoded in alternative reading frames. Here we analyze dermal fibroblasts (designated Q34) from an individual carrying independent missense mutations in each copy of the common exon. Both mutations alter the amino acid sequence of INK4a and functionally impair the protein, although they do so to different degrees. Only one of the mutations affects the sequence of ARF, causing an apparently innocuous change near its carboxy terminus. Unlike normal human fibroblasts, Q34 cells are not permanently arrested by Ras or its downstream effectors Ets1 and Ets2. Moreover, ectopic Ras enables the cells to grow as anchorage-independent colonies, and in relatively young Q34 cells anchorage independence can be achieved without addition of telomerase or perturbation of the p53 pathway. Whereas ARF plays the principal role in Ras-induced arrest of mouse fibroblasts, our data imply that INK4a assumes this role in human fibroblasts.
Molecular and Cellular Biology 01/2003; 22(23):8135-43. · 5.37 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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 fibroblasts from a member of a melanoma-prone family who is homozygous for an intragenic deletion in CDKN2A. Analyses of the resultant gene products imply that the cells are p16(INK4a) deficient but express physiologically relevant levels of a frameshift protein that retains the known functions of p14(ARF). Although they have a finite 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 find that in human fibroblasts, ARF is not induced demonstrably by RAS, pointing to significant differences between the proliferative barriers implemented by the CDKN2A locus in different cell types or species.
The EMBO Journal 07/2002; 21(12):2936-45. · 9.82 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.