A Study of The Gene Control of Glycine N-methyltransferase

• Main Authors: Cheng-Ming Lee, 李建銘
• Other Authors: Yi-Ming A. Chen
• Format: Others
• Language: en_US
• Published: 2006
• Online Access: http://ndltd.ncl.edu.tw/handle/67997731795755042112

博士 === 國立陽明大學 === 公共衛生研究所 === 94 === Glycine N-methyltransferase (GNMT) is a key protein in the liver that its function is not only an enzyme regulating the concentration of the methyl-group donor, S-adenosylmethionine, but also the 4S polycyclic aromatic hydrocarbon-binding protein participating in the detoxification pathway of cells. We identify the transcription start site of gnmt that is located at 14 bases upstream from the ATG translation start codon by 5’ RACE. Base on 1.8-kb GNMT promoter luciferase construct, a series of 5’ deletion analysis is used to define the promoter region. The data show that the core promoter region is located at -133 to +14. It is a TATA-less promoter with a Sp1 site and a CCAAT box in the reverse orientation. EMSA demonstrates that the CCAAT box is the binding site for NF-Y and the -56/-47 is for a putative factor. Mutation of CCAAT or the -56/-47 results in the total loss of GNMT promoter activity. In addition, our studies indicate that GNMT promoter activity could be induced by benzo[a]pyrene (BaP) through the xenobiotic responsive element located at -104 to -82. The expression level of GNMT is diminished in human hepatocellular carcinoma. Aberrant cytosine methylation at CpG dinucleotide of the 5’ CpG island is associated with transcriptional repression of tumor suppressor genes. In order to elucidate the methylation status of GNMT in HCC and hepatoblastoma cell lines and patient’s tissues, methylation-sensitive restriction endonucleases digestion combined with PCR and bisulphate-conversion sequencing methods are performed. Both results show that the methylation status of GNMT promoter is partially associated with the expression levels. There is a trend that the more GNMT expression, the less methylation in promoter region. The methylation of promoter region might participate partially in repressing the expression of the GNMT. In addition, the methylation status of the 14th CpG site located at -52 maybe affect the GNMT expression through interfering the binding between the putative transcription factor and the -56/-47 region. BaP is one of many polycyclic aromatic hydrocarbons that have been identified as major risk factors for developing various cancers. We previously demonstrated that the liver cancer susceptibility gene GNMT is capable of binding with BaP and protecting cells from BaP-7,8-diol 9,10-epoxide-DNA adduct formation. In this study, we use a cytotoxicity assay to demonstrate that the higher expression level of GNMT, the lower cytotoxicity occurred in the cells treated with BaP. In addition, a cDNA microarray containing 7,597 human genes is used to examine gene expression patterns in BaP-treated HepG2 (a liver cancer cell line that expresses very low levels of GNMT) and SCG2-1-1 (a stable HepG2 clone that expresses high levels of GNMT) cells. The results show that among 6,018 readable HepG2 genes, 359 (6.0%) are up-regulated more than 1.5 fold and 768 (12.8%) are down-regulated. Overexpression of GNMT in SCG2-1-1 cells results in the down-regulation of genes related to the detoxification, kinase/phosphatase pathways, and oncogenes. Furthermore, real-time PCR is used to validate microarray data from 21 genes belonging to the detoxification pathway. Combining both microarray and real-time PCR data, the results show that among 89 detoxification pathway genes analyzed, 22 (24.7%) are up-regulated and 6 (6.7%) are down-regulated in BaP-treated HepG2 cells, while in the BaP-treated SCG2-1-1 cells, 12 (13.5%) are up-regulated and 26 (29.2%) are down-regulated (p < 0.001). Therefore, GNMT sequesters BaP, diminishes BaP’s effects to the liver detoxification pathway and prevents subsequent cytotoxicity.