MGMT DOWN-REGULATION IN THE CARCINOGENICITY OF HEXAVALENT CHROMIUM

项目摘要/摘要 六价铬[Cr(VI)]暴露会对人体产生多种毒性效应。六价铬的主要关注点 毒性是其致癌性，因为铬(VI)是最广为人知的环境和职业之一 会导致人类肺癌和其他癌症的致癌物质。在积极研究的同时，铬(VI)的作用机理 致癌性仍然难以捉摸。先前的研究表明，接触铬(VI)会引起遗传毒性效应，这 被认为在铬(VI)致癌中起重要作用。另一方面，研究也表明，铬(VI) 暴露对表观遗传学的失调，如DNA甲基化增加和组蛋白翻译后异常 修改。然而，调节失调的表观遗传学如何影响铬(VI)仍有待确定 致癌性和如果铬(VI)引起的表观遗传变化在其遗传毒性效应中起作用。我们的初步研究 结果显示：(1)慢性铬(VI)暴露使组蛋白H3抑制甲基化标志(H3K9me2)水平增加 和H3K27me3)及其相关的组蛋白甲基转移酶(HMTase)(G9a、SUV39H1和EZH2)。(Ii)向上- HMTase的调节在铬(VI)诱导的肿瘤干细胞(CSC)样特性和细胞恶性中起因果作用 转型。(3)慢性铬(VI)暴露下调O6-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，而MGMT的下调起着因果作用 在铬(VI)诱导的细胞转化中。(Iv)高诱变性DNA O6-甲基鸟嘌呤(O6-Meg)的水平 在铬(VI)转化的细胞中，损伤显著增加，但稳定表达MGMT的细胞显著减少 铬(VI)暴露诱导的O6-Meg。(V)暴露于铬(VI)也可显著降低MGMT水平，但 增加小鼠和人肺组织中O6-Meg的水平。(Vi)稳定地击倒HMTase G9a或 SUV39H1可显著提高铬(VI)暴露细胞的MGMT水平，降低O6-Meg水平。(Vii)铬(Vi)- 转化细胞显示DNA损伤修复能力受损，但稳定表达MGMT 减少慢性铬(VI)暴露引起的DNA损伤修复缺陷。和(Viii)用天然的 复方二氢甲基粘菌素(DHM)能提高铬(VI)-的MGMT水平，降低O6-Meg水平 转化的细胞。()我们建立了一种条件性和肺特异性MGMT表达的转基因小鼠模型。 根据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 慢性铬(VI)暴露上调HMTase下调MGMT表达 高突变DNA损伤O6-Meg水平升高并促进铬(VI)致癌。三个目标是 建议：目标1将确定慢性铬(VI)暴露导致MGMT下降的机制。 监管。目的2将使用条件性和条件性研究来确定MGMT在C(VI)诱导的肺肿瘤形成中的作用 肺组织特异性MGMT表达转基因小鼠模型。目标3将确定 天然化合物DHM对铬(VI)诱导的小鼠细胞转化和肿瘤形成的影响。

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.