MGMT down-regulation in the carcinogenicity of hexavalent chromium

项目总结/摘要 六价铬[Cr（VI）]暴露会对人体产生多种毒性作用。Cr（VI）的主要问题 毒性是其致癌性，因为Cr（VI）是最公认的环境和职业危害之一。 致癌物质导致肺癌和其他人类癌症。目前，Cr（VI）的作用机理尚处于研究阶段， 致癌性仍然难以捉摸。先前的研究表明，Cr（VI）暴露会引起遗传毒性效应， 被认为在Cr（VI）致癌性中起重要作用。另一方面，研究还表明，Cr（VI） 暴露使表观遗传学失调，如DNA甲基化增加和组蛋白翻译后异常 修改.然而，它仍然有待确定如何失调的表观遗传学有助于铬（六） 致癌性以及Cr（VI）引起的表观遗传变化是否在其遗传毒性效应中发挥作用。我们的初步研究 显示：（i）慢性Cr（VI）暴露增加组蛋白H3抑制性甲基化标记（H3 K9 me 2）的水平 和H3 K27 me 3）及其相关的组蛋白甲基转移酶（HMT酶）（G9 a、SUV 39 H1和EZH 2）。(ii)向上- HMT酶的调节在Cr（VI）诱导的癌干细胞（CSC）样性质和细胞恶性化中起因果作用。 转型(iii)慢性Cr（VI）暴露下调O 6-甲基鸟嘌呤DNA的表达 甲基转移酶（MGMT）是DNA修复网络中的关键基因，MGMT的下调起着因果作用 在Cr（VI）诱导的细胞转化中。(iv)O 6-甲基鸟嘌呤（O 6-MeG），一种高度致突变的DNA 在Cr（VI）转化的细胞中，MGMT的表达显著增加;但稳定表达MGMT的细胞中， Cr（VI）离子交换诱导的O 6-MeG。(v)Cr（VI）暴露也显著降低MGMT水平， 增加小鼠和人肺组织中的O 6-MeG水平。(vi)稳定敲除HMT酶G9 a或 在Cr（VI）暴露的细胞中，SUV 39 H1显著增加MGMT水平，但降低O 6-MeG水平。(vii)Cr（VI）- 转化细胞的DNA损伤修复能力受损，但稳定表达MGMT， 减少慢性Cr（VI）中毒引起的DNA损伤修复缺陷。和（八）用天然的 化合物二氢麻醉椒苦素（DHM）能够增加Cr（VI）中的MGMT水平并降低O 6-MeG水平。 转化细胞（viii）我们产生了条件性和肺特异性MGMT表达转基因小鼠模型。 基于我们的初步研究和文献报道的这些发现，我们的中心假设是， 慢性Cr（VI）暴露上调HMT酶下调MGMT的表达，导致 高致突变性DNA损伤O 6-MeG水平增加，促进Cr（VI）致癌作用。三个目标是 建议：目标1将确定慢性Cr（VI）暴露导致MGMT下降的机制- 调控目的2将使用一种条件性的， 肺特异性MGMT表达转基因小鼠模型。目标3将决定 天然化合物DHM处理对Cr（VI）诱导的小鼠细胞转化和肿瘤发生的影响。

PROJECT SUMMARY/ABSTRACT Hexavalent chromium [Cr(VI)] exposure causes multiple toxic effects in humans. The main concern of Cr(VI) toxicity is its carcinogenicity as Cr(VI) is one of the most well-recognized environmental and occupational carcinogens causing lung and other cancer in humans. While it is under active study, the mechanism of Cr(VI) carcinogenicity remains elusive. Previous studies showed that Cr(VI) exposure causes genotoxic effects, which are thought to play important roles in Cr(VI) carcinogenicity. On the other hand, studies also showed that Cr(VI) exposure dysregulates epigenetics such as increased DNA methylation and abnormal histone posttranslational modifications. However, it remains to be determined how dysregulated epigenetics contributes to Cr(VI) carcinogenicity and if Cr(VI)-caused epigenetic changes play a role in its genotoxic effect. Our preliminary studies showed: (i) Chronic Cr(VI) exposure increases the levels of histone H3 repressive methylation marks (H3K9me2 and H3K27me3) and their related histone methyltransferases (HMTases) (G9a, SUV39H1, and EZH2). (ii) Up- regulation of HMTases play a causal role in Cr(VI)-induced cancer stem cell (CSC)-like property and cell malignant transformation. (iii) Chronic Cr(VI) exposure down-regulates the expression of O6-methylguanine DNA methyltransferase (MGMT), a key gene in DNA repair network; and MGMT down-regulation plays a causal role in Cr(VI)-induced cell transformation. (iv) The level of O6-methylguanine (O6-MeG), a highly mutagenic DNA lesion, is significantly increased in Cr(VI)-transformed cells; but stably expressing MGMT greatly reduces Cr(VI) exposure-induced O6-MeG. (v) Cr(VI) exposure also significantly decreases the level of MGMT but increases O6-MeG level in mouse and human lung tissues. (vi) Stably knocking down HMTases G9a or SUV39H1 significantly increases MGMT level but reduces O6-MeG level in Cr(VI)-exposed cells. (vii) Cr(VI)- transformed cells display impaired DNA damage repair capacity but stably expressing MGMT significantly reduces chronic Cr(VI) exposure-caused DNA damage repair deficiency. And (viii) Treatment with a natural compound dihydromethysticin (DHM) is capable of increasing MGMT level and reducing O6-MeG level in Cr(VI)- transformed cells. (viii) We generated a conditional and lung specific MGMT expression transgenic mouse model. Based on these findings from our preliminary studies and that from literature reports, our central hypothesis is that up-regulation of HMTases by chronic Cr(VI) exposure down-regulates the expression of MGMT leading to increased level of highly mutagenic DNA lesion O6-MeG and promoting Cr(VI) carcinogenesis. Three aims are proposed: Aim 1 will determine the mechanism by which chronic Cr(VI) exposure causes MGMT down- regulation. Aim 2 will determine the role of MGMT in C(VI)-induced lung tumorigenesis using a conditional and lung specific MGMT expression transgenic mouse model. And Aim 3 will determine the protective effect of the natural compound DHM treatment on Cr(VI)-induced cell transformation and tumorigenesis in mice.