Enterococcus faecalis, Colorectal Cancer, and Bystander Effects

描述（由申请人提供）： 结肠微生物群被怀疑在散发性结直肠癌（CRC）的病因学中起重要作用。然而，没有研究令人信服地确定细菌如何促进这种常见的肿瘤。在这项研究中，我们将这种常见的肠道寄生虫粪肠球菌的氧化生理学与染色体不稳定性（CIN）的起源联系起来。在肠道寄生虫E.粪肠球菌在产生细胞外超氧化物、促进氧化应激和损伤DNA方面是独特的。我们提出了一个新的假设，散发性CRC涉及先天性免疫反应引发的氧化还原活性大肠杆菌产生旁观者效应（BSE），导致CIN的隐窝干细胞。旁观者效应是指产生染色体断裂因子（或染色体断裂因子）的激活细胞对邻近细胞的附带DNA损伤。将评价脂质醛及其代谢对上皮靶细胞对CIN易感性的作用。E.将测定粪肠球菌在白细胞介素（IL）-10敲除小鼠的结肠粘膜中诱导环氧合酶-2、前列腺素类、TNF 1、丙二醛和4-羟基壬烯醛的能力。巨噬细胞在E.将评价粪菌定殖的小鼠。将测试丁硫克百威亚砜亚胺（BSO）和N-乙酰基-L-半胱氨酸（NAC）分别作为谷胱甘肽和谷胱甘肽S-转移酶的抑制剂或促进剂的作用。将使用LC/MS/MS、32 P-后标记技术和3 H2 AX免疫组织化学测定巨噬细胞诱导的BSE在靶细胞中产生的致突变性环外DNA加合物和DNA双链断裂（DSB）。大肠杆菌引起的结肠突变。将使用杂交IL-10-/-/Big Blue定量粪肠球菌定殖。变种人模型。最后，将使用裸鼠异种移植模型进行巨噬细胞诱导的BSE转化原代上皮细胞系的能力。将在转化细胞中评估通常与散发性CRC相关的遗传和表观遗传变化。这项资助的目的是阐明涉及结肠癌的潜在机制。IL-10-/-小鼠在E.粪便将被用来测试NAC，谷胱甘肽前体，作为一种潜在的化学预防。通过这种方式，我们希望能够确定散发性CRC致癌的机制和分子过程，并确定化学预防的新靶点。散发性结直肠癌是全球癌症和癌症死亡的主要原因。这些肿瘤被认为部分是由于结肠中的细菌引起的。在这个项目中，一种常见的肠道细菌，粪肠球菌，不寻常的氧化性质将被测试其破坏结肠内衬细胞中DNA的能力，从而促进导致癌症的突变的发展。这些结果将确定预防散发性结直肠癌的新方法和靶点。散发性结直肠癌是全球癌症和癌症死亡的主要原因。这些肿瘤被认为部分是由于结肠中的细菌引起的。在这个项目中，一种常见的肠道细菌，肠球菌， 粪便，将测试其破坏结肠内衬细胞DNA的能力，从而促进导致癌症的突变的发展。这些结果将确定预防散发性结直肠癌的新方法和靶点。

DESCRIPTION (provided by applicant): The colonic microbiota is suspected of playing an important role in the etiology of sporadic colorectal cancer (CRC). However, no studies have convincingly defined how bacteria might promote this common tumor. In this grant we link the oxidative physiology of this common intestinal commensal, Enterococcus faecalis, to the origin of chromosomal instability (CIN). Among intestinal commensals E. faecalis is unique in generating extracellular superoxide, promoting oxidative stress, and damaging DNA. We propose a novel hypothesis for sporadic CRC that involves innate immune responses triggered by redox-active commensal bacteria to produce bystander effects (BSE) that lead to CIN in crypt stem cells. The bystander effect refers to collateral DNA damage in neighboring cells from activated cells that produce clastogens (or chromosome breaking factors). The role of lipid aldehydes and their metabolism on the susceptibility of epithelial target cells to CIN will be evaluated. The ability of E. faecalis to induce cyclooxygenase-2, prostanoids, TNF1, malondialdehyde and 4- hydroxynonenal in the colonic mucosa of interleukin (IL)-10 knockout mice will be determined. The role of macrophages in the production of inflammation, dysplasia, and tumor formation in E. faecalis colonized mice will be evaluated. The effect of buthionine sulfoximine (BSO) and N-acetyl-L-cysteine (NAC) as inhibitors or promoters, respectively, of glutathione and glutathione S-transferases will be tested. The generation of mutagenic exocyclic DNA adducts and DNA double strand breaks (DSBs) in target cells by the macrophage-induced BSE will be measured using LC/MS/MS, 32P-postlabeling techniques, and immunohistochemistry for 3H2AX. Mutations in the colon due to E. faecalis colonization will be quantified using a hybrid IL-10-/-/Big Blue. Mutant model. Finally, the ability of macrophage-induced BSE to transform a primary epithelial cell line will be performed using a nude mouse xenograft model. Genetic and epigenetic changes typically associated with sporadic CRC will be assessed in transformed cells. The goal of this grant is to elucidate potential mechanisms of colonic carcinogenesis that involve commensals. Colonization of IL-10-/- mice by E. faecalis will be used to test NAC, a glutathione precursor, as a potential chemopreventive. In this fashion we hope to define mechanistic and molecular processes involved in sporadic CRC carcinogenesis and identify agents to novel targets for chemoprevention. Sporadic colorectal cancer is a leading cause of cancer and cancer death worldwide. These tumors are believed to arise, in part, because of bacteria in the colon. In this project the unusual oxidative nature of a common intestinal bacterium, Enterococcus faecalis, will be tested for its ability to damage DNA in cells lining the colon and thereby promote the development of mutations that lead to cancer. These results will identify new approaches and targets for preventing sporadic colorectal cancer. PUBLIC HEALTH RELEVANCE Sporadic colorectal cancer is a leading cause of cancer and cancer death worldwide. These tumors are believed to arise, in part, because of bacteria in the colon. In this project the unusual oxidative nature of a common intestinal bacterium, Enterococcus faecalis, will be tested for its ability to damage DNA in cells lining the colon and thereby promote the development of mutations that lead to cancer. These results will identify new approaches and targets for preventing sporadic colorectal cancer.