Inactivation of CACNA1G, a T-type calcium channel gene, by aberrant methylation of its 5' CpG island in human tumors.

Using a newly developed PCR-based technique called methylated CpG island amplification, we have identified several DNA fragments that are aberrantly methylated in a colon cancer cell line. One of the fragments, termed MINT31, mapped to human chromosome 17q21, where frequent loss of heterozygosity is detected in various human tumors. By characterizing the genomic sequence around this area, we identified a gene encoding a T-type calcium channel, CACNA1G, as a target for hypermethylation in human tumors. By reverse transcriptase-PCR we detected expression of CACNA1G in normal colon and bone marrow, but expression was absent in the five tumor cell lines in which methylation was found. After treatment with the methylation inhibitor 5-deoxyazacytidine, the expression of CACNA1G was restored in all five cell lines. Detailed methylation mapping of the 5' CpG island by bisulfite-PCR revealed that methylation of a region 300-800 bp upstream of the translation initiation site closely correlated with the inactivation of CACNA1G. This region contained the transcription start site, as determined by 5' rapid amplification of cDNA ends analysis. Aberrant methylation of CACNA1G was also examined in various human primary tumors and was detected in 17 of 49 (35%) colorectal cancers, 4 of 16 (25%) gastric cancers, and 3 of 23 (13%) acute myelogenous leukemia cases. Inactivation of CACNA1G may play a role in cancer development by modulating calcium signaling, which potentially affects cell proliferation and apoptosis.

[1]  M. Frommer,et al.  CpG islands in vertebrate genomes. , 1987, Journal of molecular biology.

[2]  A. Bird,et al.  High levels of De Novo methylation and altered chromatin structure at CpG islands in cell lines , 1990, Cell.

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

[4]  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.

[5]  Complement-induced Ca2+ influx in cultured fibroblasts is decreased by the calcium-channel antagonist nifedipine or by some bivalent inorganic cations. , 1993, The Biochemical journal.

[6]  A. Razin,et al.  DNA methylation and genomic imprinting , 1994, Cell.

[7]  S. Mori,et al.  Frequent loss of heterozygosity in the region including BRCA1 on chromosome 17q in squamous cell carcinomas of the esophagus. , 1994, Cancer research.

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

[9]  P. Zimmerman,et al.  Loss of heterozygosity frequently affects chromosome 17q in non-small cell lung cancer. , 1995, Cancer research.

[10]  B. Trump,et al.  Calcium‐mediated cell injury and cell death , 1995, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[11]  G. Chenevix-Trench,et al.  Colorectal carcinomas show frequent allelic loss on the long arm of chromosome 17 with evidence for a specific target region. , 1995, British Journal of Cancer.

[12]  D. Grignon,et al.  Loss of heterozygosity of the BRCA1 and other loci on chromosome 17q in human prostate cancer. , 1995, Cancer research.

[13]  S. Baylin,et al.  p53 activates expression of HIC-1, a new candidate tumour suppressor gene on 17p13.3 , 1995, Nature Genetics.

[14]  S. Baylin,et al.  Switch from monoallelic to biallelic human IGF2 promoter methylation during aging and carcinogenesis. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[15]  I. Petersen,et al.  Distinct patterns of chromosomal alterations in high- and low-grade head and neck squamous cell carcinomas. , 1996, Cancer research.

[16]  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.

[17]  E. Wang,et al.  Bcl-2 potentiates the maximal calcium uptake capacity of neural cell mitochondria. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[18]  G. Liang,et al.  Identification and characterization of differentially methylated regions of genomic DNA by methylation-sensitive arbitrarily primed PCR. , 1997, Cancer research.

[19]  T. Brent,et al.  Methylation hot spots in the 5' flanking region denote silencing of the O6-methylguanine-DNA methyltransferase gene. , 1997, Cancer research.

[20]  P. Laird,et al.  COBRA: a sensitive and quantitative DNA methylation assay. , 1997, Nucleic acids research.

[21]  Stephen B. Baylin,et al.  Mapping Patterns of CpG Island Methylation in Normal and Neoplastic Cells Implicates Both Upstream and Downstream Regions inde Novo Methylation* , 1997, The Journal of Biological Chemistry.

[22]  M. Klemsz,et al.  Methylation of an ETS site in the intron enhancer of the keratin 18 gene participates in tissue-specific repression , 1997, Molecular and cellular biology.

[23]  M. Estacion,et al.  PDGF-stimulated calcium influx changes during in vitro cell transformation. , 1997, Cellular signalling.

[24]  Y. Hosoya,et al.  Establishment of methylation-sensitive-representational difference analysis and isolation of hypo- and hypermethylated genomic fragments in mouse liver tumors. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[25]  A. Bird,et al.  MeCP2 Is a Transcriptional Repressor with Abundant Binding Sites in Genomic Chromatin , 1997, Cell.

[26]  Y. Hayashizaki,et al.  Restriction Landmark Genomic Scanning (RLGS) , 1997, Springer Lab Manuals.

[27]  D. Lubahn,et al.  Identification of DNA methylation markers for human breast carcinomas using the methylation-sensitive restriction fingerprinting technique. , 1997, Cancer research.

[28]  M. Loda,et al.  Methylation of the hMLH1 promoter correlates with lack of expression of hMLH1 in sporadic colon tumors and mismatch repair-defective human tumor cell lines. , 1997, Cancer research.

[29]  S. Weitzman,et al.  Methylation of the HIC-1 candidate tumor suppressor gene in human breast cancer , 1998, Oncogene.

[30]  P. Jones,et al.  Involvement of DNA methylation in human carcinogenesis. , 1998, Biological chemistry.

[31]  S. Semba,et al.  Frequent microsatellite instability and loss of heterozygosity in the region including BRCA1 (17q21) in young patients with gastric cancer. , 1998, International journal of oncology.

[32]  A. Bird,et al.  An Alternative Promoter in the Mouse Major Histocompatibility Complex Class II I-Aβ Gene: Implications for the Origin of CpG Islands , 1998, Molecular and Cellular Biology.

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

[34]  R. Fern.  Intracellular Calcium and Cell Death during Ischemia in Neonatal Rat White Matter Astrocytes In Situ , 1998, The Journal of Neuroscience.

[35]  Pier Paolo Di Fiore,et al.  Epsin is an EH-domain-binding protein implicated in clathrin-mediated endocytosis , 1998, Nature.

[36]  J. Rice,et al.  Aberrant methylation of the BRCA1 CpG island promoter is associated with decreased BRCA1 mRNA in sporadic breast cancer cells , 1998, Oncogene.

[37]  Methylation of the 5' CpG island of the FHIT gene is closely associated with transcriptional inactivation in esophageal squamous cell carcinomas. , 1998, Cancer research.

[38]  Jung-Ha Lee,et al.  Molecular characterization of a neuronal low-voltage-activated T-type calcium channel , 1998, Nature.

[39]  J. Herman,et al.  Incidence and functional consequences of hMLH1 promoter hypermethylation in colorectal carcinoma. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[40]  Peter Lipp,et al.  Calcium - a life and death signal , 1998, Nature.

[41]  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.

[42]  S. Baylin,et al.  Identification of differentially methylated sequences in colorectal cancer by methylated CpG island amplification. , 1999, Cancer research.