Tao Gong-hua, Gong Chun-mei, Yang Lin-qing, Liu Qing-cheng, Liu Jian-dong, Wu De-sheng, Hu Xin-nan, Huang Hai-yan, Liu Jian-jun, Ke Yue-bin, Zhuang Zhi-xiong
Toxicology Laboratory, Shenzhen Center for Disease Control and Prevention, Shenzhen 518020, China.
Zhonghua Yu Fang Yi Xue Za Zhi. 2011 May;45(5):410-5.
To investigate DNA methylation variation in human cells induces by B(a)P, and to explore the role of PARP1 during this process.
The changes of DNA methylation of 16HBE and its PARP1-deficient cells exposed to B(a)P (1.0, 2.0, 5.0, 10.0, 15.0, 30.0 µmol/L) were investigated by immunofluorescence and high performance capillary electrophoresis, and simultaneously, the expression level of PARP 1 and DNMT 1 were monitored dynamically.
The percentage of methylated DNA of overall genome (mCpG%) in 16HBE and 16HBE-shPARP1 cells were separately (4.04 ± 0.08)% and (9.69 ± 0.50)%. After being treated by 5-DAC for 72 hours, mCpG% decreased to (3.15 ± 0.14)% and (6.07 ± 0.54)%. After both being exposed to B(a)P for 72 hours, the mCpG% in 16HBE group (ascending rank) were separately (5.10 ± 0.13), (4.25 ± 0.10), (3.91 ± 0.10), (4.23 ± 0.27), (3.70 ± 0.15), (3.08 ± 0.07); while the figures in 16HBE-shPARP1 group (ascending rank) were respectively (10.63 ± 0.60), (13.08 ± 0.68), (9.75 ± 0.55), (7.32 ± 0.67), (6.90 ± 0.49) and (6.27 ± 0.21). The difference of the results was statistically significant (F values were 61.67 and 60.91, P < 0.01). For 16HBE group, expression of PARP 1 and DNMT 1 were 141.0%, 158.0%, 167.0%, 239.0%, 149.0%, 82.9% and 108.0%, 117.0%, 125.0%, 162.0%, 275.0%, 233.0% comparing with the control group, whose difference also has statistical significance (t values were 11.45, 17.32, 32.24, 33.44, 20.21 and 9.87, P < 0.01). For 16HBE-shPARP1 group, expression of PARP 1 and DNMT 1 were 169.0%, 217.0%, 259.0%, 323.0%, 321.0%, 256.0% and 86.0%, 135.0%, 151.0%, 180.0%, 229.0%, 186.0% comparing with the control group, with statistical significance (t values were 9.06, 15.92, 22.68, 26.23, 37.19 and 21.15, P < 0.01). When the dose of B(a)P reached 5.0 µmol/L, the mRNA expression of DNMT 1 in 16HBE group (ascending rank) were 125.0%, 162.0%, 275.0%, 233.0% times of it in control group, with statistical significance (t values were 12.74, 24.92, 55.11, 59.07, P < 0.01); while the dose of B(a)P reached 2.0 µmol/L, the mRNA expression of DNMT 1 in 16HBE-shPARP1 group were 135.0%, 151.0%, 180.0%, 229.0%, 186.0% of the results in control group, and the differences were statistically significant (t values were 23.82, 40.17, 32.69, 74.85, 46.76, P < 0.01).
The hypomethylation of 16HBE cells induced by B(a)P might be one important molecular phenomenon in its malignant transformation process. It suggests that PARP1 could regulate DNA methylation by inhibiting the enzyme activity of DNMT1, and this effect could be alleviated by PARP1-deficiency.
研究苯并[a]芘（B(a)P）诱导的人细胞DNA甲基化变化，并探讨聚（ADP-核糖）聚合酶1（PARP1）在此过程中的作用。
采用免疫荧光和高效毛细管电泳技术，研究16HBE及其PARP1缺陷细胞在暴露于B(a)P（1.0、2.0、5.0、10.0、15.0、30.0 μmol/L）后DNA甲基化的变化，同时动态监测PARP1和DNA甲基转移酶1（DNMT1）的表达水平。
16HBE和16HBE-shPARP1细胞中全基因组甲基化DNA的百分比（mCpG%）分别为（4.04±0.08）%和（9.69±0.50）%。经5-氮杂-2'-脱氧胞苷（5-DAC）处理72小时后，mCpG%分别降至（3.15±0.14）%和（6.07±0.54）%。两组细胞暴露于B(a)P 72小时后，16HBE组（升序排列）的mCpG%分别为（5.10±0.13）、（4.25±0.10）、（3.91±0.10）、（4.23±0.27）、（3.70±0.15）、（3.08±0.07）；而16HBE-shPARP1组（升序排列）的相应数值分别为（10.63±0.60）、（13.08±0.68）、（9.75±0.55）、（7.32±0.67）、（6.90±0.49）和（6.27±0.21）。结果差异具有统计学意义（F值分别为61.67和60.91，P<0.01）。对于16HBE组，与对照组相比，PARP1和DNMT1的表达分别为141.0%、158.0%、167.0%、239.0%、149.0%、82.9%和108.0%、117.0%、125.0%、162.0%、275.0%、233.0%，差异也具有统计学意义（t值分别为11.45、17.32、32.24、33.44、20.21和9.87，P<0.01）。对于16HBE-shPARP1组，与对照组相比，PARP1和DNMT1的表达分别为169.0%、217.0%、259.0%、323.0%、321.0%、256.0%和86.0%、135.0%、151.0%、180.0%、229.0%、186.0%，具有统计学意义（t值分别为9.06、15.92、22.68、26.23、37.19和21.15，P<0.01）。当B(a)P剂量达到5.0 μmol/L时，16HBE组中DNMT1的mRNA表达（升序排列）是对照组的125.0%、162.0%、275.0%、233.0%，具有统计学意义（t值分别为12.74、24.92、55.11、59.07，P<0.01）；而当B(a)P剂量达到2.0 μmol/L时，16HBE-shPARP1组中DNMT1的mRNA表达是对照组结果的135.0%、151.0%、180.0%、229.0%、186.0%，差异具有统计学意义（t值分别为23.82、40.17、32.69、74.85、46.76，P<0.01）。
B(a)P诱导的16HBE细胞低甲基化可能是其恶性转化过程中的一个重要分子现象。提示PARP1可通过抑制DNMT1的酶活性来调节DNA甲基化，而PARP1缺陷可减轻这种作用。
