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Human p16γ, a novel transcriptional variant of p16INK4A, coexpresses with p16INK4A in cancer cells and inhibits cell-cycle progression

Abstract

The INK4A locus encodes two tumor suppressor genes, p16INK4A and p14ARF, transcribed using alternative exons 1α or 1β spliced onto the same exons 2 and 3. Both p16INK4A and p14ARF are capable of inhibiting the cell-cycle progression, albeit in different manner; p16INK4A is phosphorylation of retinoblastoma (pRB) dependent while p14ARF is p53-dependent. In this study, we report the discovery of a novel variant of p16INK4A, termed p16γ, in a primary T-cell acute lymphoblastic leukemia (T-ALL) patient sample and a neuroblastoma cell line, which was expressed at both the transcriptional and translational levels. Cloning and sequencing of the p16γ cDNA revealed that p16γ was identical to p16INK4A, except that it contained an in-frame insertion of 197 bp between exons 2 and 3. p16γ expression was detected in the majority of p16INK4A-expressing primary T-ALL and B-ALL patient samples and other p16INK4A-expressing tumor samples, but was only barely detectable in some normal mononuclear cells and other non-tumor samples. Structural analysis by nuclear magnetic resonance and circular dichroism confirmed that p16γ, like p16INK4A, is also an ankyrin-repeat protein. Functional analysis of p16γ revealed that p16γ protein interacted with cyclin D-dependent kinase4 and inhibited its kinase activity. Using a luciferase reporter assay, the transfection of p16γ repressed the E2F response, the downstream target of pRB, with an efficacy equivalent to that of p16INK4A. Moreover p16γ, like p16INK4A, induced cell-cycle arrest at G0/G1, and inhibited cell growth in colony formation assay.

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References

  • Andrade MA, Chacon P, Merelo JJ, Moran F . (1993). Evaluation of secondary structure of proteins from UV circular dichroism spectra using an unsupervised learning neural network. Protein Eng 6: 383–390.

    Article  CAS  PubMed  Google Scholar 

  • Aretz S, Uhlhaas S, Sun Y, Pagenstecher C, Mangold E, Caspari R et al. (2004). Familial adenomatous polyposis: aberrant splicing due to missense or silent mutations in the APC gene. Hum Mutat 24: 370–380.

    Article  CAS  PubMed  Google Scholar 

  • Batova A, Diccianni MB, Yu JC, Nobori T, Link MP, Pullen J et al. (1997). Frequent and selective methylation of p15 and deletion of both p15 and p16 in T-cell acute lymphoblastic leukemia. Cancer Res 57: 832–836.

    CAS  PubMed  Google Scholar 

  • Byeon IJ, Li J, Ericson K, Selby TL, Tevelev A, Kim HJ et al. (1998). Tumor suppressor p16INK4A: determination of solution structure and analyses of its interaction with cyclin-dependent kinase 4. Mol Cell 1: 421–431.

    Article  CAS  PubMed  Google Scholar 

  • Caldas C, Hahn SA, da Costa LT, Redston MS, Schutte M, Seymour AB et al. (1994). Frequent somatic mutations and homozygous deletions of the p16 (MTS1) gene in pancreatic adenocarcinoma. Nat Genet 8: 27–32.

    Article  CAS  PubMed  Google Scholar 

  • Chen YJ, Chang JG, Shih LS, Chen PH, Endo M, Whang-Peng J et al. (1997). Frequent detection of aberrant RNA transcripts of the CDKN2 gene in human gastric adenocarcinoma. Int J Cancer 71: 350–354.

    Article  CAS  PubMed  Google Scholar 

  • Cheng JQ, Jhanwar SC, Klein WM, Bell DW, Lee WC, Altomare DA et al. (1994). p16 alterations and deletion mapping of 9p21–p22 in malignant mesothelioma. Cancer Res 54: 5547–5551.

    CAS  PubMed  Google Scholar 

  • Cho JW, Jeong YW, Han SW, Park JB, Jang BC, Baek WK et al. (2003). Aberrant p16INK4A RNA transcripts expressed in hepatocellular carcinoma cell lines regulate pRb phosphorylation by binding with CDK4, resulting in delayed cell cycle progression. Liver Int 23: 194–200.

    Article  CAS  PubMed  Google Scholar 

  • Diccianni MB, Chau LS, Batova A, Vu TQ, Yu AL . (1996). The p16 and p18 tumor suppressor genes in neuroblastoma: implications for drug resistance. Cancer Lett 104: 183–192.

    Article  CAS  PubMed  Google Scholar 

  • Diccianni MB, Omura-Minamisawa M, Batova A, Le T, Bridgeman L, Yu AL . (1999). Frequent deregulation of p16 and the p16/G1 cell cycle-regulatory pathway in neuroblastoma. Int J Cancer 80: 145–154.

    Article  CAS  PubMed  Google Scholar 

  • Diccianni MB, Yu J, Meppelink G, de Vries M, Shao L, Gebauer S et al. (2004). 3-amino thioacridone inhibits DNA synthesis and induce DNA damage in T-cell acute lymphoblastic leukemia (T-ALL) in a p16-dependent manner. J Exp Ther Oncol 4: 223–237.

    CAS  PubMed  Google Scholar 

  • Dyson N . (1998). The regulation of E2F by pRB-family proteins. Genes Dev 12: 2245–2262.

    Article  CAS  PubMed  Google Scholar 

  • ElSharawy A, Manaster C, Teuber M, Rosenstiel P, Kwiatkowski R, Huse K et al. (2006). SNPSplicer: systematic analysis of SNP-dependent splicing in genotyped cDNAs. Hum Mutat 27: 1129–1134.

    Article  CAS  PubMed  Google Scholar 

  • Fu GH, Wang Y, Xi YH, Shen WW, Pan XY, Shen WZ et al. (2005). Direct interaction and cooperative role of tumor suppressor p16 with band 3 (AE1). FEBS Lett 579: 2105–2110.

    Article  CAS  PubMed  Google Scholar 

  • Harland M, Mistry S, Bishop DT, Bishop JA . (2001). A deep intronic mutation in CDKN2A is associated with disease in a subset of melanoma pedigrees. Hum Mol Genet 10: 2679–2686.

    Article  CAS  PubMed  Google Scholar 

  • Hillman RT, Green RE, Brenner SE . (2004). An unappreciated role for RNA surveillance. Genome Biol 5: R8.

    Article  PubMed  PubMed Central  Google Scholar 

  • Hussussian CJ, Struewing JP, Goldstein AM, Higgins PA, Ally DS, Sheahan MD et al. (1994). Germline p16 mutations in familial melanoma. Nat Genet 8: 15–21.

    Article  CAS  PubMed  Google Scholar 

  • Jen J, Harper JW, Bigner SH, Bigner DD, Papadopoulos N, Markowitz S et al. (1994). Deletion of p16 and p15 genes in brain tumors. Cancer Res 54: 6353–6358.

    CAS  PubMed  Google Scholar 

  • Kim WY, Sharpless NE . (2006). The regulation of INK4/ARF in cancer and aging. Cell 127: 265–275.

    Article  CAS  PubMed  Google Scholar 

  • Komata T, Kanzawa T, Takeuchi H, Germano IM, Schreiber M, Kondo Y et al. (2003). Antitumour effect of cyclin-dependent kinase inhibitors (p16(INK4A), p18(INK4C), p19(INK4D), p21(WAF1/CIP1) and p27(KIP1)) on malignant glioma cells. Br J Cancer 88: 1277–1280.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Krek W, Livingston DM, Shirodkar S . (1993). Binding to DNA and the retinoblastoma gene product promoted by complex formation of different E2F family members. Science 262: 1557–1560.

    Article  CAS  PubMed  Google Scholar 

  • Li J, Byeon IJ, Ericson K, Poi MJ, O’Maille P, Selby T et al. (1999). Tumor suppressor INK4: determination of the solution structure of p18INK4C and demonstration of the functional significance of loops in p18INK4C and p16INK4A. Biochemistry 38: 2930–2940.

    Article  CAS  PubMed  Google Scholar 

  • Li J, Joo SH, Tsai MD . (2003). An NF-kappaB-specific inhibitor, IkappaBalpha, binds to and inhibits cyclin-dependent kinase 4. Biochemistry 42: 13476–13483.

    Article  CAS  PubMed  Google Scholar 

  • Li J, Mahajan A, Tsai MD . (2006). Ankyrin repeat: a unique motif mediating protein-protein interactions. Biochemistry 45: 15168–15178.

    Article  CAS  PubMed  Google Scholar 

  • Li J, Poi MJ, Qin D, Selby TL, Byeon IJ, Tsai MD . (2000). Tumor suppressor INK4: quantitative structure-function analyses of p18INK4C as an inhibitor of cyclin-dependent kinase 4. Biochemistry 39: 649–657.

    Article  CAS  PubMed  Google Scholar 

  • Li J, Tsai MD . (2002). Novel insights into the INK4-CDK4/6-Rb pathway: counter action of gankyrin against INK4 proteins regulates the CDK4-mediated phosphorylation of Rb. Biochemistry 41: 3977–3983.

    Article  CAS  PubMed  Google Scholar 

  • Lin YC, Peng JM, Wang WB . (2000). The N-terminal common domain of simian virus 40 large T and small t antigens acts as a transformation suppressor of the HER-2/neu oncogene. Oncogene 19: 2704–2713.

    Article  CAS  PubMed  Google Scholar 

  • Mao L, Merlo A, Bedi G, Shapiro GI, Edwards CD, Rollins BJ et al. (1995). A novel p16INK4A transcript. Cancer Res 55: 2995–2997.

    CAS  PubMed  Google Scholar 

  • Merlo A, Herman JG, Mao L, Lee DJ, Gabrielson E, Burger PC et al. (1995). 5′ CpG island methylation is associated with transcriptional silencing of the tumour suppressor p16/CDKN2/MTS1 in human cancers. Nat Med 1: 686–692.

    Article  CAS  PubMed  Google Scholar 

  • Okamoto A, Demetrick DJ, Spillare EA, Hagiwara K, Hussain SP, Bennett WP et al. (1994). Mutations and altered expression of p16INK4 in human cancer. Proc Natl Acad Sci USA 91: 11045–11049.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Omura-Minamisawa M, Diccianni MB, Batova A, Chang RC, Bridgeman LJ, Yu J et al. (2000). Universal inactivation of both p16 and p15 but not downstream components is an essential event in the pathogenesis of T-cell acute lymphoblastic leukemia. Clin Cancer Res 6: 1219–1228.

    CAS  PubMed  Google Scholar 

  • Omura-Minamisawa M, Diccianni MB, Chang RC, Batova A, Bridgeman LJ, Schiff J et al. (2001). p16/p14(ARF) cell cycle regulatory pathways in primary neuroblastoma: p16 expression is associated with advanced stage disease. Clin Cancer Res 7: 3481–3490.

    CAS  PubMed  Google Scholar 

  • Ortega S, Malumbres M, Barbacid M . (2002). Cyclin D-dependent kinases, INK4 inhibitors and cancer. Biochim Biophys Acta 1602: 73–87.

    CAS  PubMed  Google Scholar 

  • Pace CN . (1986). Determination and analysis of urea and guanidine hydrochloride denaturation curves. Methods Enzymol 131: 266–280.

    Article  CAS  PubMed  Google Scholar 

  • Quelle DE, Zindy F, Ashmun RA, Sherr CJ . (1995). Alternative reading frames of the INK4a tumor suppressor gene encode two unrelated proteins capable of inducing cell cycle arrest. Cell 83: 993–1000.

    Article  CAS  PubMed  Google Scholar 

  • Robertson KD, Jones PA . (1999). Tissue-specific alternative splicing in the human INK4a/ARF cell cycle regulatory locus. Oncogene 18: 3810–3820.

    Article  CAS  PubMed  Google Scholar 

  • Russo AA, Tong L, Lee JO, Jeffrey PD, Pavletich NP . (1998). Structural basis for inhibition of the cyclin-dependent kinase Cdk6 by the tumour suppressor p16INK4a. Nature 395: 237–243.

    Article  CAS  PubMed  Google Scholar 

  • Schutte M, Hruban RH, Geradts J, Maynard R, Hilgers W, Rabindran SK et al. (1997). Abrogation of the Rb/p16 tumor-suppressive pathway in virtually all pancreatic carcinomas. Cancer Res 57: 3126–3130.

    CAS  PubMed  Google Scholar 

  • Serrano M . (1997). The tumor suppressor protein p16INK4a. Exp Cell Res 237: 7–13.

    Article  CAS  PubMed  Google Scholar 

  • Sherr CJ . (2006). Divorcing ARF and p53: an unsettled case. Nat Rev Cancer 6: 663–673.

    Article  CAS  PubMed  Google Scholar 

  • Stone S, Jiang P, Dayananth P, Tavtigian SV, Katcher H, Parry D et al. (1995). Complex structure and regulation of the P16 (MTS1) locus. Cancer Res 55: 2988–2994.

    CAS  PubMed  Google Scholar 

  • Suh SI, Cho JW, Baek WK, Suh MH, Carson DA . (2000a). Lack of mutation at p16INK4A gene but expression of aberrant p16INK4A RNA transcripts in human ovarian carcinoma. Cancer Lett 153: 175–182.

    Article  CAS  PubMed  Google Scholar 

  • Suh SI, Pyun HY, Cho JW, Baek WK, Park JB, Kwon T et al. (2000b). 5-Aza-2′-deoxycytidine leads to down-regulation of aberrant p16INK4A RNA transcripts and restores the functional retinoblastoma protein pathway in hepatocellular carcinoma cell lines. Cancer Lett 160: 81–88.

    Article  CAS  PubMed  Google Scholar 

  • Tevelev A, Byeon IJ, Selby T, Ericson K, Kim HJ, Kraynov V et al. (1996). Tumor suppressor p16INK4A: structural characterization of wild-type and mutant proteins by NMR and circular dichroism. Biochemistry 35: 9475–9487.

    Article  CAS  PubMed  Google Scholar 

  • Xiong Y, Zhang H, Beach D . (1993). Subunit rearrangement of the cyclin-dependent kinases is associated with cellular transformation. Genes Dev 7: 1572–1583.

    Article  CAS  PubMed  Google Scholar 

  • Yu Y, Panhuysen C, Kranzler HR, Hesselbrock V, Rounsaville B, Weiss R et al. (2006). Intronic variants in the dopa decarboxylase (DDC) gene are associated with smoking behavior in European-Americans and African-Americans. Hum Mol Genet 15: 2192–2199.

    Article  CAS  PubMed  Google Scholar 

  • Zhang Y, Xiong Y, Yarbrough WG . (1998). ARF promotes MDM2 degradation and stabilizes p53: ARF-INK4a locus deletion impairs both the Rb and p53 tumor suppression pathways. Cell 92: 725–734.

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

This work was supported by funds from the Genomics Research Center, Academia Sinica, a grant from Leukemia & Lymphoma Society of America, #R6184-02 (ALY) and in part by a grant from the National Institutes of Health (CA69472) to MDT. Also supported in part by an American Cancer Society/Institutional Research Grant #IRG-70-002-29, the San Diego Padres Cindy Matters Foundation (ALY/MBD) and the Children's Oncology Group (COG). DNA sequencing was performed by the DNA Sequencing Shared Resource, UCSD Cancer Center, which is funded in part by NCI Cancer Center Support Grant #2 P30CA23100-18.

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Lin, YC., Diccianni, M., Kim, Y. et al. Human p16γ, a novel transcriptional variant of p16INK4A, coexpresses with p16INK4A in cancer cells and inhibits cell-cycle progression. Oncogene 26, 7017–7027 (2007). https://doi.org/10.1038/sj.onc.1210507

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