Progressive Region-Specific De Novo Methylation of the p16 CpG Island in Primary Human Mammary Epithelial Cell Strains during Escape from M0 Growth Arrest

ABSTRACT CpG island methylation plays an important role in normal cellular processes, such as genomic imprinting and X-chromosome inactivation, as well as in abnormal processes, such as neoplasia. However, the dynamics of de novo CpG island methylation, during which a CpG island is converted from an unmethylated, active state to a densely methylated, inactive state, are largely unknown. It is unclear whether the development of de novo CpG island methylation is a progressive process, in which a subset of CpG sites are initially methylated with a subsequent increase in methylation density, or a single event, in which the initial methylation event encompasses the entire CpG island. The tumor suppressor gene p16/CDKN2a/INK4a (p16) is inactivated by CpG island methylation during neoplastic progression in a variety of human cancers. We investigated the development of methylation in the p16 CpG island in primary human mammary epithelial cell strains during escape from mortality stage 0 (M0) growth arrest. The methylation status of 47 CpG sites in the p16 CpG island on individual DNA molecules was determined by sequencing PCR clones of bisulfite-treated genomic DNA. The p16 CpG island was initially methylated at a subset of sites in three discrete regions in association with p16 transcriptional repression and escape from M0 growth arrest. With continued passage, methylation gradually increased in density and methylation expanded to sites in adjacent regions. Thus, de novo methylation in the p16 CpG island is a progressive process that is neither site specific nor completely random but instead is region specific. Our results suggest that early detection of methylation in the CpG island of the p16 gene will require methylation analysis of the three regions and that the identification of region-specific methylation patterns in other genes may be essential for an accurate assessment of methylation-mediated transcriptional silencing.

[1]  D. Wong,et al.  Inactivation of p16 in Human Mammary Epithelial Cells by CpG Island Methylation , 1998, Molecular and Cellular Biology.

[2]  V. Band,et al.  Distinctive traits of normal and tumor-derived human mammary epithelial cells expressed in a medium that supports long-term growth of both cell types. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[3]  A. Weissbach,et al.  Primary DNA sequence determines sites of maintenance and de novo methylation by mammalian DNA methyltransferases , 1986, Molecular and cellular biology.

[4]  J. Costello,et al.  Methylation of discrete regions of the O6-methylguanine DNA methyltransferase (MGMT) CpG island is associated with heterochromatinization of the MGMT transcription start site and silencing of the gene , 1997, Molecular and cellular biology.

[5]  P. Jones,et al.  The role of DNA methylation in expression of the p19/p16 locus in human bladder cancer cell lines. , 1998, Cancer research.

[6]  P. Jones,et al.  DNA methylation errors and cancer. , 1996, Cancer research.

[7]  S. Clark,et al.  Loss of p16INK4 expression by methylation is associated with lifespan extension of human mammary epithelial cells. , 1998, Cancer research.

[8]  F. Watt,et al.  Cytosine methylation prevents binding to DNA of a HeLa cell transcription factor required for optimal expression of the adenovirus major late promoter. , 1988, Genes & development.

[9]  M. Bartolomei,et al.  A 5' 2-kilobase-pair region of the imprinted mouse H19 gene exhibits exclusive paternal methylation throughout development , 1997, Molecular and cellular biology.

[10]  G. Hannon,et al.  A new regulatory motif in cell-cycle control causing specific inhibition of cyclin D/CDK4 , 1993, Nature.

[11]  Galloway Da,et al.  HUMAN PAPILLOMAVIRUS TYPE 16 E7 ALLEVIATES A PROLIFERATION BLOCK IN EARLY PASSAGE HUMAN MAMMARY EPITHELIAL CELLS , 1996 .

[12]  D. Wong,et al.  p16INK4a promoter is hypermethylated at a high frequency in esophageal adenocarcinomas. , 1997, Cancer research.

[13]  S. Kass,et al.  Inactive chromatin spreads from a focus of methylation , 1993, Molecular and cellular biology.

[14]  J. Laborda,et al.  36B4 cDNA used as an estradiol-independent mRNA control is the cDNA for human acidic ribosomal phosphoprotein PO. , 1991, Nucleic acids research.

[15]  R. Beart,et al.  Methylation of the 5' CpG island of the p16/CDKN2 tumor suppressor gene in normal and transformed human tissues correlates with gene silencing. , 1995, Cancer research.

[16]  V. Chapman,et al.  CpG island promoter region methylation patterns of the inactive-X-chromosome hypoxanthine phosphoribosyltransferase (Hprt) gene , 1994, Molecular and cellular biology.

[17]  M. Stanley,et al.  Association of CDKN2A/p16INK4A with human head and neck keratinocyte replicative senescence: relationship of dysfunction to immortality and neoplasia. , 1996, Oncogene.

[18]  T. Yang,et al.  Chromosomes: Correlation with Binding Sites for Transcription Factors , 2022 .

[19]  M. Beckmann,et al.  Frequent allele loss on 9p21–22 defines a smallest common region in the vicinity of the CDKN2 gene in sporadic breast cancer , 1996, Genes, chromosomes & cancer.

[20]  S. Baylin,et al.  De novo methylation of CpG island sequences in human fibroblasts overexpressing DNA (cytosine-5-)-methyltransferase , 1996, Molecular and cellular biology.

[21]  M. Surani.  Imprinting and the Initiation of Gene Silencing in the Germ Line , 1998, Cell.

[22]  D. Sidransky,et al.  Rates of p16 (MTS1) mutations in primary tumors with 9p loss. , 1994, Science.

[23]  A. Bird,et al.  Repression of genes by DNA methylation depends on CpG density and promoter strength: evidence for involvement of a methyl‐CpG binding protein. , 1992, The EMBO journal.

[24]  T. Kiyono,et al.  Both Rb/p16INK4a inactivation and telomerase activity are required to immortalize human epithelial cells , 1998, Nature.

[25]  C. Moskaluk,et al.  Abrogation of the Rb/p16 tumor-suppressive pathway in virtually all pancreatic carcinomas. , 1997, Cancer research.

[26]  C. Reznikoff,et al.  Elevated p16 at senescence and loss of p16 at immortalization in human papillomavirus 16 E6, but not E7, transformed human uroepithelial cells. , 1996, Cancer research.

[27]  J. Herman,et al.  Inactivation of the CDKN2/p16/MTS1 gene is frequently associated with aberrant DNA methylation in all common human cancers. , 1995, Cancer research.

[28]  Susan J. Clark,et al.  CpNpG methylation in mammalian cells , 1995, Nature Genetics.

[29]  T. Bestor,et al.  DNA (cytosine-5)-methyltransferases in mouse cells and tissues. Studies with a mechanism-based probe. , 1997, Journal of molecular biology.

[30]  R. Reddel,et al.  Association of extended in vitro proliferative potential with loss of p16INK4 expression. , 1996, Oncogene.

[31]  A. Bird,et al.  Sp1 sites in the mouse aprt gene promoter are required to prevent methylation of the CpG island. , 1994, Genes & development.

[32]  D. Galloway,et al.  Human papillomavirus type 16 E7 alleviates a proliferation block in early passage human mammary epithelial cells. , 1996, Oncogene.

[33]  C. Sherr.  Cancer Cell Cycles , 1996, Science.

[34]  A. Riggs,et al.  Polymerase chain reaction-aided genomic sequencing of an X chromosome-linked CpG island: methylation patterns suggest clonal inheritance, CpG site autonomy, and an explanation of activity state stability. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[35]  J. Herman,et al.  Alterations in DNA methylation: a fundamental aspect of neoplasia. , 1998, Advances in cancer research.

[36]  R. Hubert,et al.  Whole genome amplification from a single cell: implications for genetic analysis. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[37]  A. Riggs,et al.  X-chromosome inactivation and cell memory. , 1992, Trends in genetics : TIG.

[38]  Robert L. Tanguay,et al.  In vivo footprint and methylation analysis by PCR-aided genomic sequencing: comparison of active and inactive X chromosomal DNA at the CpG island and promoter of human PGK-1. , 1990, Genes & development.

[39]  R. White,et al.  Mosaic methylation in clonal tissue. , 1993, Developmental biology.

[40]  L. E. McDonald,et al.  A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[41]  J. Herman,et al.  Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[42]  J. Mandel,et al.  Instability of a 550-base pair DNA segment and abnormal methylation in fragile X syndrome , 1991, Science.

[43]  Z. Siegfried,et al.  Spl elements protect a CpG island from de novo methylation , 1994, Nature.

[44]  A. Brenner,et al.  Increased p16 expression with first senescence arrest in human mammary epithelial cells and extended growth capacity with p16 inactivation , 1998, Oncogene.

[45]  P. Jones,et al.  Rapid quantitation of methylation differences at specific sites using methylation-sensitive single nucleotide primer extension (Ms-SNuPE). , 1997, Nucleic acids research.

[46]  R. Strom,et al.  Influence of pre-existing methylation on the de novo activity of eukaryotic DNA methyltransferase. , 1998, Biochemistry.

[47]  J. Bartek,et al.  High frequency of p16 (CDKN2/MTS-1/INK4A) inactivation in head and neck squamous cell carcinoma. , 1996, Cancer research.

[48]  W. Doerfler,et al.  Genomic sequencing reveals a 5-methylcytosine-free domain in active promoters and the spreading of preimposed methylation patterns. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[49]  G. Demers,et al.  The E7 gene of human papillomavirus type 16 is sufficient for immortalization of human epithelial cells , 1991, Journal of virology.

[50]  P. Lavia,et al.  In vitro methylation of CpG-rich islands. , 1989, Nucleic acids research.

[51]  J. Herman,et al.  5′ CpG island methylation is associated with transcriptional silencing of the tumour suppressor p16/CDKN2/MTS1 in human cancers , 1995, Nature Medicine.

[52]  Henry A. Erlich,et al.  PCR Technology: Principles and Applications for DNA Amplification , 1989 .

[53]  R. Hruban,et al.  Frequent somatic mutations and homozygous deletions of the p16 (MTS1) gene in pancreatic adenocarcinoma , 1994, Nature Genetics.

[54]  C. Hsieh,et al.  Dependence of transcriptional repression on CpG methylation density , 1994, Molecular and cellular biology.

[55]  S. Iguchi-Ariga,et al.  CpG methylation of the cAMP-responsive enhancer/promoter sequence TGACGTCA abolishes specific factor binding as well as transcriptional activation. , 1989, Genes & development.

[56]  W. Brown,et al.  Epigenetic variation illustrated by DNA methylation patterns of the fragile-X gene FMR1. , 1997, Human molecular genetics.

[57]  Kathleen R. Cho,et al.  Frequency of homozygous deletion at p16/CDKN2 in primary human tumours , 1995, Nature Genetics.

[58]  S. Clark,et al.  Extensive DNA methylation spanning the Rb promoter in retinoblastoma tumors. , 1997, Cancer research.

[59]  T. Bestor.  Supercoiling-dependent sequence specificity of mammalian DNA methyltransferase. , 1987, Nucleic acids research.

[60]  G. Hannon,et al.  Involvement of the cyclin-dependent kinase inhibitor p16 (INK4a) in replicative senescence of normal human fibroblasts. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[61]  Liang Zhu,et al.  Requirement of Cyclin E-Cdk2 Inhibition in p16INK4a-Mediated Growth Suppression , 1998, Molecular and Cellular Biology.

[62]  Martha R. Stampfer,et al.  Isolation and growth of human mammary epithelial cells , 1985 .

[63]  A. Riggs,et al.  Methylation dynamics, epigenetic fidelity and X chromosome structure. , 1998, Novartis Foundation symposium.