Reach for the sky

Genes & Development, 2004

Mammalian genes frequently present allelic variants that differ in their expression levels and that, in the case of tumor suppressor genes, can be of relevance for cancer susceptibility and aging. We report here the characterization of a novel mouse model with increased activity for the Ink4a and Arf tumor suppressors. We have generated a "super Ink4a/Arf" mouse strain carrying a transgenic copy of the entire Ink4a/Arf locus. Cells derived from super Ink4a/Arf mice have increased resistance to in vitro immortalization and oncogenic transformation. Importantly, super Ink4a/Arf mice manifest higher resistance to cancer compared to normal, nontransgenic, mice. Finally, super Ink4a/Arf mice have normal aging and lifespan. Together, these results indicate that modest increases in the activity of the Ink4a/Arf tumor suppressor result in a beneficial cancer-resistant phenotype without affecting normal viability or aging.

Cell, 1996

was first observed as a CDK4-associated protein in human cells (Xiong et al., 1993) and was subse-Carlos Cordon-Cardo, § David Beach, † quently cloned and characterized as a specific inhibitor and Ronald A. DePinho ‡ of the CDK4-6/D kinases (Serrano et al., 1993; Hannon † Howard Hughes Medical Institute and Beach, 1994). Three other members of the INK4 Cold Spring Harbor Laboratory family have since been isolated (p15 INK4b , p18 INK4c , and 1 Bungtown Road p19 INK4d). Each shares biochemical and biological prop-Cold Spring Harbor, New York 11724 erties with p16 INK4a but is subject to different transcrip-‡ Department of Microbiology and Immunology tional regulation (reviewed by Sherr and Roberts, 1995). Albert Einstein College of Medicine The critical substrate of the CDK4-6/D kinases in vivo Bronx, New York 10461 is the retinoblastoma-susceptibility tumor suppressor § Memorial Sloan-Kettering Cancer Center protein (Rb) (reviewed by Weinberg, 1995). Rb negatively 1275 York Avenue controls passage from G1 into S phase by sequestering New York, New York 10021 transcription factors that are required for the G1/S transition. The ability of Rb to bind transcription factors is abolished by phosphorylation that occurs in G1 and is Summary sustained to the end of mitosis. Overexpression of the CDK4-6 inhibitor p16 INK4a results in G1 arrest in cells with The cell cycle inhibitor p16 INK4a is inactivated in many functional Rb, but is without effect in cells with inactive human tumors and in families with hereditary mela-Rb (Guan et al., 1994; Serrano et al., 1995; Lukas et al., noma and pancreatic cancer. Tumor-associated alter-1995a; Koh et al., 1995; Medema et al., 1995). ations in the INK4a locus may also affect the overlap-The locus encoding p16 INK4a , named INK4a, has the ping gene encoding p19 ARF and the adjacent gene capacity to give rise to two distinct transcripts from encoding p15 INK4b , both negative regulators of cell prodifferent promoters (Duro et al., 1995; Stone et al., 1995a; liferation. We report the phenotype of mice carrying a Mao et al., 1995). Each transcript has a specific 5Ј exon, targeted deletion of the INK4a locus that eliminates E1␣ or E1␤, which is spliced into common exons E2 and both p16 INK4a and p19 ARF. The mice are viable but de-E3. The E1␣-containing transcript encodes p16 INK4a , and velop spontaneous tumors at an early age and are the E1␤-containing transcript encodes p19 ARF from a different reading frame initiated in the E1␤ exon (Quelle highly sensitive to carcinogenic treatments. INK4aet al., 1995a). p19 ARF has the ability to arrest cell proliferadeficient primary fibroblasts proliferate rapidly and tion at both G1 and G2, albeit through mechanisms not have a high colony-formation efficiency. In contrast involving direct inhibition of known CDK-cyclin comwith normal cells, the introduction of activated Ha-ras plexes (Quelle et al., 1995a). Another level of complexity into INK4a-deficient fibroblasts can result in neoplasat the INK4a locus results from its close physical proximtic transformation. These findings directly demonity to the gene encoding p15 INK4b , an inhibitor thought strate that the INK4a locus functions to suppress neoto play an integral role in transforming growth factor plastic growth. (TGF)␤-induced cell-cycle arrest (Hannon and Beach, 1994).

Cell, 1997

cycle arrest . While p16 INK4a is speci-2 Department of Tumor Cell Biology fied by three exons (designated 1␣, 2, and 3), an alterna-3 Department of Pathology and Laboratory Medicine tive first exon (1␤) maps ‫31ف‬ kb 5Ј to exon 1␣ in the 4 Department of Experimental Oncology mouse genome, and its coding sequences are spliced to 5 Department of Genetics the identical acceptor site of INK4a exon 2. The resulting St. Jude Children's Research Hospital mRNA specifies a p19 ARF protein of 169 amino acids, 65 332 North Lauderdale encoded by exon 1␤ and the remainder by a second Memphis, Tennessee 38105 reading frame in exon 2. p19 ARF shares no amino acid homology with p16 INK4a or other known proteins, and apart from being a highly basic protein that localizes to Summary nuclear speckles during interphase, its function remains unknown. Unlike p16 INK4a , p19 ARF overexpression induces The INK4a tumor suppressor locus encodes p16 INK4a , both G1 and G2 phase arrest in rodent fibroblasts, an inhibitor of cyclin D-dependent kinases, and p19 ARF , whether or not the cells retain INK4a. The unusual orgaan alternative reading frame protein that also blocks nization of the INK4a locus is conserved in humans (Duro cell proliferation. Surprisingly, mice lacking p19 ARF but et al., 1995; Mao et al., 1995; Stone et al., 1995) and rats expressing functional p16 INK4a develop tumors early in (Swafford et al., 1997), whereas three related but distinct life. Their embryo fibroblasts (MEFs) do not senesce INK4 genes do not encode analogous ARF proteins. and are transformed by oncogenic Ha-ras alone. Con-Disruption of INK4a exon 2 in mice predisposes young version of ARF ؉/؉ or ARF ؉/Ϫ MEF strains to continuanimals to tumor development (Serrano et al., 1996). Their cultured mouse embryo fibroblasts (MEFs) fail to p19 ARF or p53. p53-mediated checkpoint control is unundergo a senescence crisis and can be transformed perturbed in ARF-null fibroblast strains, whereas p53by oncogenic ras alleles, which, in the absence of collabnegative cell lines are resistant to p19 ARF -induced orating "immortalizing oncogenes," would otherwise ingrowth arrest. Therefore, INK4a encodes growth induce growth arrest (Lloyd et al., 1997; Serrano et al., hibitory proteins that act upstream of the retinoblas-1997). Although it was clear that elimination of exon 2 toma protein and p53. Mutations and deletions tarof INK4a would compromise expression of both p16 INK4a geting this locus in cancer cells are unlikely to be and p19 ARF , it has been widely assumed that the observed phenotype stemmed from p16 INK4a disruption functionally equivalent. alone. We have now selectively disrupted ARF function in mice by deleting exon 1␤ and leaving all p16 INK4a cod-

The EMBO Journal, 2002

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.

Oncogene, 2003

Inactivation of the INK4a/ARF (or CDKN2a) locus is a common and critical genetic event in the development of human and mouse melanoma. This locus engages the Rb and p53 tumor suppressor pathways through its capacity to encode two distinct gene products, p16 INK4a and p14 ARF. This review highlights the body of evidence supporting a role for both p16 INK4a and p14 ARF in the suppression of melanoma, and speculates as to why this locus is preferentially targeted in this tumor type. In addition, the potential importance of these two pathways in mediating UV-induced melanoma genesis will be addressed via genetic and molecular evidence in the mouse.

Oncogene, 2003

Deletion of the INK4a/ARF locus at 9p21 is detected with high frequency in human melanoma. Within a short genomic distance, this locus encodes several proteins with established tumor-suppressor roles in a broad spectrum of cancer types. Several lines of evidence support the view that p16INK4a and p19ARF exert the tumor-suppressor activities of this locus, although their relative importance in specific cancer types such as melanoma has been less rigorously documented on the genetic level. Here, we exploit a well-defined mouse model of RAS-induced melanomas to examine the impact of germline p16INK4a or p19ARF nullizygosity on melanoma formation. We demonstrate that loss of either Ink4a/Arf product can cooperate with RAS activation to produce clinically indistinguishable melanomas. In line with the common phenotypic end point, we further show that RAS+ p16INK4a-/- melanomas sustain somatic inactivation of p19ARF-p53 and, correspondingly, that RAS+ p19ARF-/- melanomas experience high-frequency loss of p16INK4a. These genetic studies provide definitive proof that p16INK4a and p19ARF cooperate to suppress the development of melanoma in vivo.

Proceedings of the National Academy of Sciences, 2007

American Journal of Pathology, 2000

INK4a/ARF locus codes for two different proteins, p16 INK4a and p14 ARF , involved in cell cycle regulation. p14 ARF is considered an upstream regulator of p53 function. To determine the role of these genes in the pathogenesis of human non-Hodgkin's lymphomas we have analyzed exon 1␤, 1␣, and 2 of the INK4a/ ARF locus and p53 gene aberrations in 97 tumors previously characterized for p16 INK4a alterations. p53 alterations were detected in four of 51 (8%) indolent lymphomas but in 15 of 46 (33%) aggressive tumors. Inactivation of p14 ARF was always associated with p16 INK4a alterations. Exon 1␤ was concomitantly deleted with exon 1␣ and 2 in eight tumors. One additional lymphoblastic lymphoma showed deletion of exon 1␣ and 2 but retained exon 1␤. No mutations were detected in exon 1␣ and 1␤ in any case. Two of the three mutations detected in exon 2 caused a nonsense mutation in the p16 INK4a reading frame and a missense mutation in the ARF reading frame involving the nucleolar transport domain of the protein.

British Journal of Cancer, 2004

Germline anomalies of the INK4a-ARF and Cdk4 genes were sought in a series of 89 patients suspected of having a genetic predisposition to melanoma. Patients were selected based on the following criteria: (a) familial melanoma (23 cases), (b) multiple primary melanoma (MPM; 18 cases), (c) melanoma and additional unrelated cancers (13 cases), (d) age at diagnosis less than 25 years (21 cases), and (e) nonphoto-induced melanoma (NPIM; 14 cases). Mutations of INK4a-ARF and Cdk4 were characterised by automated sequencing, and germline deletions of INK4a-ARF were also examined by real-time quantitative PCR. Seven germline changes of INK4a-ARF, five of which were novel, were found in seven patients (8%). Four were very likely to be pathogenic mutations and were found in three high-risk melanoma families and in a patient who had a pancreatic carcinoma in addition to melanoma. Three variants of uncertain significance were detected in one MPM patient, one patient o25 years, and one NPIM patient. No germline deletion of INK4a-ARF was found in 71 patients, and no Cdk4 mutation was observed in the 89 patients. This study confirms that INK4a-ARF mutations are infrequent outside stringent familial criteria, and that germline INK4a-ARF deletions are rarely involved in genetic predisposition to melanoma.

Journal of Pharmaceutical Sciences, 2012

The utility of pigs as preclinical animals for pharmaceutical development was assessed by evaluating the pharmacokinetics and pharmacodynamics of glipizide (Glucotrol R) following oral administration of immediate-release (IR) and modified-release (MR) formulations. Doses of 10 and 30 mg were administered to six male pigs in a crossover design. Blood samples were collected at selected time-points up to 48 h after dose. Relative to the IR formulation, the time to reach the maximum concentration (t max) was delayed with the MR formulation from 1.3 to 8.7 h with the 10 mg dose and to 6.2 h with the 30 mg dose. The relative bioavailability (BA) was approximately 92% at 10 mg and 79% at 30 mg dose. The area under the curve of the plasma concentration versus time curve (AUC) increased nearly proportionally with the dose. Interanimal coefficient of variation (CV) in AUC ranged from approximately 40% to 60%. Blood glucose results suggest that pigs demonstrate formulationdependent response to glipizide. Compared with the pigs, the 10 mg MR formulation in dogs showed a higher AUC CV of approximately 80%, a t max of 5.5 h, and a lower relative BA of 18%. These data indicate that the MR formulation performed less consistently in dogs as compared with humans, whereas the porcine absorption kinetics and BA were consistent with published clinical data.