[Effect of poly-ADP-ribosylation on the alteration of DNA methylation level of human bronchial epithelial cells induced by Cr (VI)].
OBJECTIVE
To reveal the role of poly-ADP-ribosylation and DNA methylation in carcinogenic process induced induced by Cr (VI), and to discuss the relations between them.


METHODS
The pre-established Poly (ADP-ribose) glycohydrolase (PARG) deficient cells and 16HBE cells were treated with different concentrations of Cr (VI), and the changes of total genomic DNA methylation level in different groups were detected by methylation immunofluorescent detection, as well as the changes of the activity of methyltransferases. Moreover, RT-PCR and western blotting method were applied to analyze the changes of expression of DNMT1, DNMT3a, DNMT3b and MBD2, upon the protein level.


RESULTS
After treated by Cr(VI) for 24 h, the healthy 16HBE cells showed a significant lower level of genomic DNA methylation; however, there was no significant changes (P > 0.05) found in PARG deficient cells by immunofluorescence assay. When the dose of Cr (VI) reached 5.0 µmol/L, the activity of methyltransferases in 16HBE cells and PARG deficient cells (49.33 ± 2.65, 80.05 ± 2.05) decreased by 20% and 50% comparing with contrast group (99.27 ± 1.10, 99.30 ± 0.60) . After treated by Cr (VI) for 24 h, the expression of mRNA and protein level among DNMT1, DNMT3a, DNMT3b and MBD2 decreased significantly in healthy 16HBE cells; and the expression of DNMT1 and DNMT3a decreased in PARG deficiency cells. The relevant expression levels of mRNA of DNMT1 were separately (0.99 ± 0.09), (0.79 ± 0.10), (0.59 ± 0.13) and (0.39 ± 0.02) (F = 247.17, P < 0.01), the expression levels of protein were separately (1.00 ± 0.03), (0.69 ± 0.15), (0.65 ± 0.10) and (0.55 ± 0.13) (F = 214.12, P < 0.01), the expression levels of DNMT3a mRNA were separately (1.00 ± 0.04) , (0.93 ± 0.11) , (0.79 ± 0.07) , (0.59 ± 0.05) (F = 498.16, P < 0.01) , and the expression levels of protein were separately (1.00 ± 0.14) , (0.97 ± 0.11) , (0.79 ± 0.17) , (0.57 ± 0.15) (F = 390.11, P < 0.01) when the dose of Cr (VI) at 0, 0.3, 1.2 and 5.0 µmol/L. However, there were no significant changes of expression found in DNMT3b and MBD2.


CONCLUSIONS
Poly-ADP-ribosylation could regulate the activity of DNMT3b and MBD2, protect cells against the DNA methylation alteration induced by Cr(VI) and maintain the global genomic DNA methylation level.
Key Laboratory of Modern Toxicology of Shenzhen, Shenzhen Center for Disease Control and Prevention, Shenzhen 518055, China.
Haiyan
H
Huang
Jianfeng
J
Cai
Gonghua
G
Hu
Bo
B
Xia
Linqing
L
Yang
Jianjun
J
Liu
Xinfeng
X
Huang
Desheng
D
Wu
Email: zxzhuang2007@126.com.
Zhixiong
Z
Zhuang
D002460:Cell Line; D002857:Chromium / Q000633:toxicity*; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000378:metabolism; D019175:DNA Methylation / Q000187:drug effects*; D000090205:DNA Methyltransferase 3A; D004268:DNA-Binding Proteins / Q000378:metabolism; D004847:Epithelial Cells / Q000378:metabolism*; D016678:Genome; D006801:Humans; D011064:Poly Adenosine Diphosphate Ribose / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D000097584:DNA Methyltransferase 3B
D002857:Chromium; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; D000090205:DNA Methyltransferase 3A; D004268:DNA-Binding Proteins; D011064:Poly Adenosine Diphosphate Ribose; D012333:RNA, Messenger; D000097584:DNA Methyltransferase 3B; C000717967:DNMT3A protein, human; C489325:MBD2 protein, human; C000619380:DNMT1 protein, human