Cell division is required for de novo methylation of CpG islands in bladder cancer cells.

Cell division is essential for tumor development and progression. Methylation-mediated silencing caused by aberrant de novo methylation of CpG islands located in the promoter regions of growth regulatory genes occurs frequently in human cancers. We investigated the relationship between cell division and de novo methylation to determine whether de novo methylation can occur in the absence of cell division in cancer cells. We treated T24 bladder carcinoma cells with 5-Aza-2'-deoxycytidine to induce a transient demethylation and then compared the timing and kinetics of remethylation of the p16 gene locus under conditions of either G(0)-G(1) growth arrest induced by serum starvation and confluence or continuous cell proliferation in complete medium. Variable levels of remethylation were detected in CpG poor regions of DNA, as well as repetitive DNA elements in the absence of cell division, yet no remethylation occurred at CpG islands under these conditions. This correlated with continuous expression of p16 protein in these cells. DNA methyltransferase (DNMT)1 and DNMT3b3 proteins were undetectable in 5-Aza-2'-deoxycytidine-treated and untreated nondividing cells, and their mRNA transcripts were down-regulated in these cells. Although DNMT3a mRNA levels were also reduced, they recovered to original levels in nondividing cells after drug treatment. Our results suggest that cell division is required for de novo methylation of CpG islands and that DNMT3a may play a role in methylating CpG poor regions or repetitive DNA elements outside of the S phase of the cell cycle.

[1]  T. Meitinger,et al.  DNA, FISH and complementation studies in ICF syndrome: DNA hypomethylation of repetitive and single copy loci and evidence for a trans acting factor , 1995, Human Genetics.

[2]  Michael Krawczak,et al.  Cytosine methylation and the fate of CpG dinucleotides in vertebrate genomes , 1989, Human Genetics.

[3]  Gangning Liang,et al.  Cooperativity between DNA Methyltransferases in the Maintenance Methylation of Repetitive Elements , 2002, Molecular and Cellular Biology.

[4]  S. Groshen,et al.  Susceptibility of nonpromoter CpG islands to de novo methylation in normal and neoplastic cells. , 2001, Journal of the National Cancer Institute.

[5]  A. Jeltsch,et al.  Enzymatic properties of recombinant Dnmt3a DNA methyltransferase from mouse: the enzyme modifies DNA in a non-processive manner and also methylates non-CpA sites 1 1 Edited by J. Karn , 2001 .

[6]  N. Ahuja,et al.  Accelerated age-related CpG island methylation in ulcerative colitis. , 2001, Cancer research.

[7]  K. Robertson,et al.  Differential mRNA expression of the human DNA methyltransferases (DNMTs) 1, 3a and 3b during the G(0)/G(1) to S phase transition in normal and tumor cells. , 2000, Nucleic acids research.

[8]  S. Groshen,et al.  Progressive increases in de novo methylation of CpG islands in bladder cancer. , 2000, Cancer research.

[9]  C. Wijmenga,et al.  The DNMT3B DNA methyltransferase gene is mutated in the ICF immunodeficiency syndrome. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[10]  N. Tommerup,et al.  Chromosome instability and immunodeficiency syndrome caused by mutations in a DNA methyltransferase gene , 1999, Nature.

[11]  R. Jaenisch,et al.  Mammalian (cytosine-5) methyltransferases cause genomic DNA methylation and lethality in Drosophila , 1999, Nature Genetics.

[12]  D. Haber,et al.  DNA Methyltransferases Dnmt3a and Dnmt3b Are Essential for De Novo Methylation and Mammalian Development , 1999, Cell.

[13]  Peter A. Jones,et al.  Roles of Cell Division and Gene Transcription in the Methylation of CpG Islands , 1999, Molecular and Cellular Biology.

[14]  E. Li,et al.  Cloning, expression and chromosome locations of the human DNMT3 gene family. , 1999, Gene.

[15]  C. Fanton,et al.  Confluence-induced alterations in CpG island methylation in cultured normal human fibroblasts. , 1999, Nucleic acids research.

[16]  J. Herman,et al.  CpG island methylator phenotype in colorectal cancer. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[17]  Peter A. Jones,et al.  Cancer-epigenetics comes of age , 1999, Nature Genetics.

[18]  K. Robertson,et al.  The human DNA methyltransferases (DNMTs) 1, 3a and 3b: coordinate mRNA expression in normal tissues and overexpression in tumors. , 1999, Nucleic acids research.

[19]  S. Baylin,et al.  Aging and DNA methylation in colorectal mucosa and cancer. , 1998, Cancer research.

[20]  A. Razin,et al.  CpG methylation, chromatin structure and gene silencing—a three‐way connection , 1998, The EMBO journal.

[21]  E. Li,et al.  Cloning and characterization of a family of novel mammalian DNA (cytosine-5) methyltransferases , 1998, Nature Genetics.

[22]  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.

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

[24]  P. Jones,et al.  Inhibition of DNA methylation by 5-aza-2'-deoxycytidine suppresses the growth of human tumor cell lines. , 1998, Cancer research.

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

[26]  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.

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

[28]  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.

[29]  A. Niveleau,et al.  Abnormal methylation pattern in constitutive and facultative (X inactive chromosome) heterochromatin of ICF patients. , 1994, Human molecular genetics.

[30]  S. Baylin,et al.  Stabilization of DNA methyltransferase levels and CpG island hypermethylation precede SV40-induced immortalization of human fibroblasts. , 1994, Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research.

[31]  S. Baylin,et al.  Methylation of the oestrogen receptor CpG island links ageing and neoplasia in human colon , 1994, Nature Genetics.

[32]  A. Bird,et al.  Number of CpG islands and genes in human and mouse. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[33]  J. Herman,et al.  Expression of an exogenous eukaryotic DNA methyltransferase gene induces transformation of NIH 3T3 cells. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[34]  S. Baylin,et al.  Increased cytosine DNA-methyltransferase activity during colon cancer progression. , 1993, Journal of the National Cancer Institute.

[35]  F. Ledeist,et al.  An embryonic-like methylation pattern of classical satellite DNA is observed in ICF syndrome. , 1993, Human molecular genetics.

[36]  Adrian Bird,et al.  The essentials of DNA methylation , 1992, Cell.

[37]  J. Steitz,et al.  Telomere shortening associated with chromosome instability is arrested in immortal cells which express telomerase activity. , 1992, The EMBO journal.

[38]  M. Pike,et al.  Increased cell division as a cause of human cancer. , 1990, Cancer research.

[39]  A. Bird.  CpG-rich islands and the function of DNA methylation , 1986, Nature.

[40]  P. Jones,et al.  DNA methyltransferase levels in tumorigenic and nontumorigenic cells in culture. , 1986, The Journal of biological chemistry.

[41]  M. Kastan,et al.  Methylation of deoxycytidine incorporated by excision-repair synthesis of DNA , 1982, Cell.