This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Huschtscha, L. I. Articles by Reddel, R. R. Search for Related Content PubMed PubMed Citation Articles by Huschtscha, L. I. Articles by Reddel, R. R.

Loss of p16^INK4 Expression by Methylation Is Associated with Lifespan Extension of Human Mammary Epithelial Cells¹

Children's Medical Research Institute, Westmead, Sydney, NSW 2145, Australia [L. I. H., J. R. N., A. A. N., E. L. M., R. R. R.]; Department of Surgery, Westmead Hospital, Westmead, NSW 2145, Australia [P. B.]; and Kanematsu Laboratories, CSIRO, Division of Molecular Science, North Ryde, NSW 2113, Australia [J. R. M., S. J. C.]

Inactivation of p16^INK4 tumor suppressor gene function is frequently observed in breast cancer. We examined p16^INK4 expression in human mammary epithelial cell (HMEC) cultures established from four normal donors. Normal HMECs divide a limited number of times before proliferation ceases in a state referred to as selection (or M₀). The cell subpopulation that emerges spontaneously from selection undergoes a further limited period of proliferation before senescence. By immunofluorescence and Western blot analysis of four independent cultures, we have shown loss of p16^INK4 expression in postselection HMECs. In contrast, p16^INK4 was present in both early and late passage fibroblasts from the same individuals. Bisulfite genomic sequencing revealed extensive methylation of the p16^INK4 CpG island in post- but not preselection cells. Thus, the extended period of growth observed in postselection HMECs is associated with hypermethylation of the p16^INK4 CpG island and loss of p16^INK4 expression. Although postselection HMECs are widely considered to be normal, these data indicate that they have sustained an epigenetic alteration.

¹ Supported by the Kathleen Cuningham Foundation for Breast Cancer Research and the Carcinogenesis Fellowship of the New South Wales Cancer Council.

² To whom requests for reprints should be addressed, at Children's Medical Research Institute, 214 Hawkesbury Road, Westmead, Sydney, NSW 2145, Australia. Phone: 61-2-9687-2800; Fax: 61-2-9687-2120; E-mail: rreddel@mail.usyd.edu.au.

Received 4/27/98. Accepted 7/ 1/98.

This article has been cited by other articles:

				K. Gosselin, S. Martien, A. Pourtier, C. Vercamer, P. Ostoich, L. Morat, L. Sabatier, L. Duprez, C. T'Kint de Roodenbeke, E. Gilson, et al. Senescence-Associated Oxidative DNA Damage Promotes the Generation of Neoplastic Cells Cancer Res., October 15, 2009; 69(20): 7917 - 7925. [Abstract] [Full Text] [PDF]

				R. A. Hinshelwood, J. R. Melki, L. I. Huschtscha, C. Paul, J. Z. Song, C. Stirzaker, R. R. Reddel, and S. J. Clark Aberrant de novo methylation of the p16INK4A CpG island is initiated post gene silencing in association with chromatin remodelling and mimics nucleosome positioning Hum. Mol. Genet., August 15, 2009; 18(16): 3098 - 3109. [Abstract] [Full Text] [PDF]

				L. I. Huschtscha, J. D. Moore, J. R. Noble, H. G. Campbell, J. A. Royds, A. W. Braithwaite, and R. R. Reddel Normal human mammary epithelial cells proliferate rapidly in the presence of elevated levels of the tumor suppressors p53 and p21WAF1/CIP1 J. Cell Sci., August 15, 2009; 122(16): 2989 - 2995. [Abstract] [Full Text] [PDF]

				L. L. Scurr, A. D. Guminski, Y.-E. Chiew, R. L. Balleine, R. Sharma, Y. Lei, K. Pryor, G. V. Wain, A. Brand, K. Byth, et al. Ankyrin Repeat Domain 1, ANKRD1, a Novel Determinant of Cisplatin Sensitivity Expressed in Ovarian Cancer Clin. Cancer Res., November 1, 2008; 14(21): 6924 - 6932. [Abstract] [Full Text] [PDF]

				X. Zhao, M. Goswami, N. Pokhriyal, H. Ma, H. Du, J. Yao, T. A. Victor, K. Polyak, C. D. Sturgis, H. Band, et al. Cyclooxygenase-2 Expression during Immortalization and Breast Cancer Progression Cancer Res., January 15, 2008; 68(2): 467 - 475. [Abstract] [Full Text] [PDF]

				R. A. Hinshelwood, L. I. Huschtscha, J. Melki, C. Stirzaker, A. Abdipranoto, B. Vissel, T. Ravasi, C. A. Wells, D. A. Hume, R. R. Reddel, et al. Concordant Epigenetic Silencing of Transforming Growth Factor- Signaling Pathway Genes Occurs Early in Breast Carcinogenesis Cancer Res., December 15, 2007; 67(24): 11517 - 11527. [Abstract] [Full Text] [PDF]

				N. Widodo, C. C. Deocaris, K. Kaur, K. Hasan, T. Yaguchi, K. Yamasaki, T. Sugihara, T. Ishii, R. Wadhwa, and S. C. Kaul Stress Chaperones, Mortalin, and Pex19p Mediate 5-Aza-2' Deoxycytidine-Induced Senescence of Cancer Cells by DNA Methylation-Independent Pathway J. Gerontol. A Biol. Sci. Med. Sci., March 1, 2007; 62(3): 246 - 255. [Abstract] [Full Text] [PDF]

				V. W. Wen, K. Wu, S. Baksh, R. A. Hinshelwood, R. B. Lock, S. J. Clark, M. A.S. Moore, and K. L. MacKenzie Telomere-Driven Karyotypic Complexity Concurs with p16INK4a Inactivation in TP53-Competent Immortal Endothelial Cells. Cancer Res., November 15, 2006; 66(22): 10691 - 10700. [Abstract] [Full Text] [PDF]

				J. Zhang, C. R. Pickering, C. R. Holst, M. L. Gauthier, and T. D. Tlsty p16INK4a Modulates p53 in Primary Human Mammary Epithelial Cells Cancer Res., November 1, 2006; 66(21): 10325 - 10331. [Abstract] [Full Text] [PDF]

				P. A. Reynolds, M. Sigaroudinia, G. Zardo, M. B. Wilson, G. M. Benton, C. J. Miller, C. Hong, J. Fridlyand, J. F. Costello, and T. D. Tlsty Tumor Suppressor p16INK4A Regulates Polycomb-mediated DNA Hypermethylation in Human Mammary Epithelial Cells J. Biol. Chem., August 25, 2006; 281(34): 24790 - 24802. [Abstract] [Full Text] [PDF]

				S.-H. Kim, J. Rowe, H. Fujii, R. Jones, B. Schmierer, B.-W. Kong, K. Kuchler, D. Foster, D. Ish-Horowicz, and G. Peters Upregulation of chicken p15INK4b at senescence and in the developing brain J. Cell Sci., June 15, 2006; 119(12): 2435 - 2443. [Abstract] [Full Text] [PDF]

				A. Ayyanan, G. Civenni, L. Ciarloni, C. Morel, N. Mueller, K. Lefort, A. Mandinova, W. Raffoul, M. Fiche, G. P. Dotto, et al. Increased Wnt signaling triggers oncogenic conversion of human breast epithelial cells by a Notch-dependent mechanism PNAS, March 7, 2006; 103(10): 3799 - 3804. [Abstract] [Full Text] [PDF]

				C J Fabian, B F Kimler, M S Mayo, and S A Khan Breast-tissue sampling for risk assessment and prevention Endocr. Relat. Cancer, June 1, 2005; 12(2): 185 - 213. [Abstract] [Full Text] [PDF]

				M. Strunnikova, U. Schagdarsurengin, A. Kehlen, J. C. Garbe, M. R. Stampfer, and R. Dammann Chromatin Inactivation Precedes De Novo DNA Methylation during the Progressive Epigenetic Silencing of the RASSF1A Promoter Mol. Cell. Biol., May 15, 2005; 25(10): 3923 - 3933. [Abstract] [Full Text] [PDF]

				H. BERMAN, J. ZHANG, Y.G. CRAWFORD, M.L. GAUTHIER, C.A. FORDYCE, K.M. McDERMOTT, M. SIGAROUDINIA, K. KOZAKIEWICZ, and T.D. TLSTY Genetic and Epigenetic Changes in Mammary Epithelial Cells Identify a Subpopulation of Cells Involved in Early Carcinogenesis Cold Spring Harb Symp Quant Biol, January 1, 2005; 70(0): 317 - 327. [Abstract] [PDF]

				H. Zhang and S. N. Cohen Smurf2 up-regulation activates telomere-dependent senescence Genes & Dev., December 15, 2004; 18(24): 3028 - 3040. [Abstract] [Full Text] [PDF]

				V. A. Shamanin and E. J. Androphy Immortalization of Human Mammary Epithelial Cells Is Associated with Inactivation of the p14ARF-p53 Pathway Mol. Cell. Biol., March 1, 2004; 24(5): 2144 - 2152. [Abstract] [Full Text] [PDF]

				B.-S. Herbert, V. P. Pearce, L. S. Hynan, D. M. LaRue, W. E. Wright, L. Kopelovich, and J. W. Shay A Peroxisome Proliferator-activated Receptor-{gamma} Agonist and the p53 Rescue Drug CP-31398 Inhibit the Spontaneous Immortalization of Breast Epithelial Cells Cancer Res., April 15, 2003; 63(8): 1914 - 1919. [Abstract] [Full Text] [PDF]

				C. R. Holst, G. J. Nuovo, M. Esteller, K. Chew, S. B. Baylin, J. G. Herman, and T. D. Tlsty Methylation of p16INK4a Promoters Occurs in Vivo in Histologically Normal Human Mammary Epithelia Cancer Res., April 1, 2003; 63(7): 1596 - 1601. [Abstract] [Full Text] [PDF]

				H. Zhang, K.-H. Pan, and S. N. Cohen Senescence-specific gene expression fingerprints reveal cell-type-dependent physical clustering of up-regulated chromosomal loci PNAS, March 18, 2003; 100(6): 3251 - 3256. [Abstract] [Full Text] [PDF]

				S. J. Edwards, B. R. Dix, C. J. Myers, D. Dobson-Le, L. Huschtscha, M. Hibma, J. Royds, and A. W. Braithwaite Evidence that Replication of the Antitumor Adenovirus ONYX-015 Is Not Controlled by the p53 and p14ARF Tumor Suppressor Genes J. Virol., November 13, 2002; 76(24): 12483 - 12490. [Abstract] [Full Text] [PDF]

				R. F. Newbold The significance of telomerase activation and cellular immortalization in human cancer Mutagenesis, November 1, 2002; 17(6): 539 - 550. [Abstract] [Full Text] [PDF]

				R. Marcotte and E. Wang Replicative Senescence Revisited J. Gerontol. A Biol. Sci. Med. Sci., July 1, 2002; 57(7): B257 - 269. [Abstract] [Full Text]

				J. A. O'Shaughnessy, G. J. Kelloff, G. B. Gordon, A. J. Dannenberg, W. K. Hong, C. J. Fabian, C. C. Sigman, M. M. Bertagnolli, S. P. Stratton, S. Lam, et al. Treatment and Prevention of Intraepithelial Neoplasia: An Important Target for Accelerated New Agent Development : Recommendations of the American Association for Cancer Research Task Force on the Treatment and Prevention of Intraepithelial Neoplasia Clin. Cancer Res., February 1, 2002; 8(2): 314 - 346. [Abstract] [Full Text] [PDF]

				U. Lehmann, F. Langer, H. Feist, S. Glockner, B. Hasemeier, and H. Kreipe Quantitative Assessment of Promoter Hypermethylation during Breast Cancer Development Am. J. Pathol., February 1, 2002; 160(2): 605 - 612. [Abstract] [Full Text] [PDF]

				J. Duan, Z. Zhang, and T. Tong Senescence Delay of Human Diploid Fibroblast Induced by Anti-sense p16INK4a Expression J. Biol. Chem., December 14, 2001; 276(51): 48325 - 48331. [Abstract] [Full Text] [PDF]

				M. Gadd, C. Pisc, J. Branda, V. Ionescu-Tiba, Z. Nikolic, C. Yang, T. Wang, G. M. Shackleford, R. D. Cardiff, and E. V. Schmidt Regulation of Cyclin D1 and p16INK4A Is Critical for Growth Arrest during Mammary Involution Cancer Res., December 1, 2001; 61(24): 8811 - 8819. [Abstract] [Full Text] [PDF]

				I. E. Krop, D. Sgroi, D. A. Porter, K. L. Lunetta, R. LeVangie, P. Seth, C. M. Kaelin, E. Rhei, M. Bosenberg, S. Schnitt, et al. HIN-1, a putative cytokine highly expressed in normal but not cancerous mammary epithelial cells PNAS, July 24, 2001; (2001) 171138398. [Abstract] [Full Text] [PDF]

				J. F Costello and C. Plass Methylation matters J. Med. Genet., May 1, 2001; 38(5): 285 - 303. [Abstract] [Full Text]

				S. B. Baylin, M. Esteller, M. R. Rountree, K. E. Bachman, K. Schuebel, and J. G. Herman Aberrant patterns of DNA methylation, chromatin formation and gene expression in cancer Hum. Mol. Genet., April 1, 2001; 10(7): 687 - 692. [Abstract] [Full Text] [PDF]

				M.K. Kang and M.-H. Park Conversion of Normal To Malignant Phenotype: Telomere Shortening, Telomerase Activation, and Genomic Instability During Immortalization of Human Oral Keratinocytes Critical Reviews in Oral Biology & Medicine, January 1, 2001; 12(1): 38 - 54. [Abstract] [Full Text] [PDF]

				T. Davis, C. Kennedy, Y.-E. Chiew, C. L. Clarke, and A. deFazio Histone Deacetylase Inhibitors Decrease Proliferation and Modulate Cell Cycle Gene Expression in Normal Mammary Epithelial Cells Clin. Cancer Res., November 1, 2000; 6(11): 4334 - 4342. [Abstract] [Full Text] [PDF]

				I. H. N. Wong, Y. M. Dennis Lo, W. Yeo, W. Y. Lau, and P. J. Johnson Frequent p15 Promoter Methylation in Tumor and Peripheral Blood from Hepatocellular Carcinoma Patients Clin. Cancer Res., September 1, 2000; 6(9): 3516 - 3521. [Abstract] [Full Text]

				C. J. Jones, D. Kipling, M. Morris, P. Hepburn, J. Skinner, A. Bounacer, F. S. Wyllie, M. Ivan, J. Bartek, D. Wynford-Thomas, et al. Evidence for a Telomere-Independent "Clock" Limiting RAS Oncogene-Driven Proliferation of Human Thyroid Epithelial Cells Mol. Cell. Biol., August 1, 2000; 20(15): 5690 - 5699. [Abstract] [Full Text]

				M. Widschwendter, J. Berger, M. Hermann, H. M. Muller, A. Amberger, M. Zeschnigk, A. Widschwendter, B. Abendstein, A. G. Zeimet, G. Daxenbichler, et al. Methylation and Silencing of the Retinoic Acid Receptor-{beta}2 Gene in Breast Cancer J Natl Cancer Inst, May 17, 2000; 92(10): 826 - 832. [Abstract] [Full Text] [PDF]

				I. H. N. Wong, M. H. L. Ng, D. P. Huang, and J. C. K. Lee Aberrant p15 promoter methylation in adult and childhood acute leukemias of nearly all morphologic subtypes: potential prognostic implications Blood, March 15, 2000; 95(6): 1942 - 1949. [Abstract] [Full Text] [PDF]

				T. R. Yeager, A. A. Neumann, A. Englezou, L. I. Huschtscha, J. R. Noble, and R. R. Reddel Telomerase-negative Immortalized Human Cells Contain a Novel Type of Promyelocytic Leukemia (PML) Body Cancer Res., September 1, 1999; 59(17): 4175 - 4179. [Abstract] [Full Text] [PDF]

				S. Pavey, S. Conroy, T. Russell, and B. Gabrielli Ultraviolet Radiation Induces p16CDKN2A Expression in Human Skin Cancer Res., September 1, 1999; 59(17): 4185 - 4189. [Abstract] [Full Text] [PDF]

				D. J. Wong, S. A. Foster, D. A. Galloway, and B. J. Reid Progressive Region-Specific De Novo Methylation of the p16 CpG Island in Primary Human Mammary Epithelial Cell Strains during Escape from M0 Growth Arrest Mol. Cell. Biol., August 1, 1999; 19(8): 5642 - 5651. [Abstract] [Full Text] [PDF]

				L. I. Huschtscha and R. R. Reddel p16INK4a and the control of cellular proliferative life span Carcinogenesis, June 1, 1999; 20(6): 921 - 926. [Abstract] [Full Text] [PDF]

				L. Xu, B. J. Flynn, S. Ungar, H. I. Pass, K. Linnainmaa, K. Mattson, and B. I. Gerwin Asbestos induction of extended lifespan in normal human mesothelial cells: interindividual susceptibility and SV40 T antigen Carcinogenesis, May 1, 1999; 20(5): 773 - 783. [Abstract] [Full Text] [PDF]

				I. E. Krop, D. Sgroi, D. A. Porter, K. L. Lunetta, R. LeVangie, P. Seth, C. M. Kaelin, E. Rhei, M. Bosenberg, S. Schnitt, et al. HIN-1, a putative cytokine highly expressed in normal but not cancerous mammary epithelial cells PNAS, August 14, 2001; 98(17): 9796 - 9801. [Abstract] [Full Text] [PDF]