Mechanism Of DNA Meythylaiton In Malignant Transformation Human Bronchial Epithelial Cells Induced By Glycidyl Methacrylate

DNA methylation, which is catalysed by to DNA ethyltransferase (DNMT) and then transfer the S-adenosine was a sulfur acid (SAM) to the five carbon atoms on the cytosine, form the process of5-methyl cytosine chemical modification. It is the important way of adjusting eukaryotes gene expression. Different from of the classical genetic theory, DNA methylation which could change for outside factors can’t change the sequence of DNA and play an important role of malignant cells transformation process, gene regulation, cell proliferation, gene function and so on. It is considered to be the new tumor molecular biological markers (biomarker) of carcinogenic substance always exposure and cancer risk before. In normal cell, the whole genome expresses low methylation but such as tumor-suppressor genes, cancer gene, apoptosis related genes, mismatch genes, repair gene and so on express high methylation. The change of DNA methylaiton can affect the normal cell function abnormal, genetic imprinting and human tumors development. It provides a new treatment mechanism path of studying of tumors mechanism.Glycidyl methacrylate (GMA), as a comonomer, which contains double carbon bonds and expoxy clusters, has good characteristics of anti-UV, water resistance and heat resistance, it is widely used in resin, coating, adhesive and plastic industries. Previous researches have been reported that GMA is a low toxicity, with obvious irritation, causing rapod onset and delayed type hypersensitivity, the result of mutagenicity is positive, and it could induce malignant transformation in several types of mammalian or human cells. All these results have demonstrated its potential carcinogenicity for it can induce SHE, KMB-13and other mammalian cells to malignant transformation. Compared to in vivo-based assays, in vitro cell transformation tests which mimic some stages of in vivo multistep carcinogenesis are fast, cost efficient and provide a mean for initial screening of carcinogenesis potential. Furthermore, epithelial tissue-derived tumors account for about80%of all cancer cases, which gives epithelial cells all advantage over those derived from animal or human fibrocyte. Researches on tumorigenesis and tumor development using human epithelial cells will ideally imitate the natural progress in human.16HBE, an SV40large T antigen-immortalized human airway epithelial cell line, retains the phenotype of the differentiated lineage of the parent. This cell line retains contact inhibition and rarely produces tumors when injected into nude mice, which provides us an ideal model for detecting carcinogenic potential of chemicals and for studying on the mechanisms of malignant transformation.Cells were expoused to8μg/ml GMA72h, interval24h, continuous poisonous three times. In the study, biological characteristics of malignant transformation were dynamicly identified by the tests of conA and colony forming frequency on soft agar. Then cell was harvested on different times, protophase (the10th generation), metaphase(the20th generation) and anaphase (the30th generation) transformed cells. Using methylation chip of "NimbleGen HG18CpG Promoter Microarray Methlation" to detect the changes of genes DNA methylation at different stages and investigate the mechanism of its DNA metylation.1. malignant transformation human bronchial epithelial cells induced by glycidyl methacrylate and relative biological indexThe cells of at the14th generation in test group started to agglutinating in the conA solution, and at the20th generation, it became more faster than the control group. The30th generation cell transformed cells being anchorage independence could grow in semi-solid agar.2. Change of gene DNA methylaiton in malignant transformation of16HBE cells induced by GMA2.1DNA methylaiton at different stages The result is that cell expoused GMA had1374genes in protophase;825genes in metaphase;1149genes in anaphase;30genes are all methylation in the3stages.2.2Change of gene DNA methylation among different transformation process The result is that cell expoused GMA had318genes in protophase but not in metaphase and anaphase;272genes in metaphase but not in protophase and anaphase;683genes in anaphase but not in metaphase and protophase;73genes in protophase and metaphase but not in anaphase;67genes in protophase and anaphase but not in metaphase;59genes in metaphase and anaphase but not in protophase.3. The alteration DNA methylation of genes during malignant transforming stages of16HBE cells induced by GMA. According to the result before, we selected P15, OPCML and THBS1and then detected by MSP during malignant transforming stages of16HBE cells induced by GMA. The results showed that abnormally methylation in CpG island of OPCML can be considered it as an specific genes in the process of16HBE occured malignant transformation by GMA; THBS1can be considered as an early sensitive index; P15can be considered as an cell malignant relative biological index of16HBE occured malignant transformation by GMA.In summary, according to the result of methylation chip, we found that the pattern of DNA methylaion could change in the process of16HBE cells induced by GMA. Then we choose some genes detected their DNA methylation patterns found that was the same to the result before. The results showed that abnormally methylation in CpG island of OPCML can be considered it as an specific genes in the process of16HBE occured malignant transformation by GMA; THBS1can be considered as an early sensitive index; P15can be considered as an cell malignant relative biological index of16HBE occured malignant transformation by GMA. It provides new cues on carcinogenicity risk evaluation of GMA, epidemiologic monitoring and mechanism study on chemical carcinogenesis.