Enterococcus faecalis, Colorectal Cancer, and Bystander Effects

描述(由申请人提供)： 结肠微生物区系在散发性结直肠癌(CRC)的病因学中起重要作用。然而，还没有研究令人信服地定义细菌如何促进这种常见的肿瘤。在这项授权中，我们将这种常见的肠道共生菌粪肠球菌的氧化生理学与染色体不稳定(CIN)的起源联系起来。在肠道共生菌中，粪肠球菌在产生细胞外超氧化物、促进氧化应激和破坏DNA方面是独一无二的。我们提出了一种新的假设，即散发性CRC涉及氧化还原活性共生细菌触发的先天性免疫反应，从而产生旁观者效应(BSE)，从而导致隐窝干细胞中的CIN。旁观者效应是指产生碎裂原(或染色体断裂因子)的激活细胞对邻近细胞的附带DNA损伤。脂醛及其代谢在上皮细胞对CIN易感性中的作用将被评估。将测定粪肠球菌诱导IL-10基因敲除小鼠结肠粘膜环氧合酶-2、前列腺素、肿瘤坏死因子1、丙二醛和4-羟基壬烯醛的能力。巨噬细胞在粪肠球菌定植小鼠的炎症、不典型增生和肿瘤形成中的作用将被评估。考察丁硫氨酸亚磺胺和N-乙酰-L半胱氨酸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。用LC/MS/MS、~(32)P-后标记技术和3H_2AX免疫组织化学技术检测巨噬细胞诱导的BSE靶细胞产生的致突变外环DNA加合物和DNA双链断裂(DSB)。粪肠球菌定植引起的结肠突变将使用混合IL-10-/-/Big Blue进行量化。变种人模型。最后，巨噬细胞诱导的BSE转化原代上皮细胞系的能力将使用裸鼠异种移植模型进行。通常与散发性结直肠癌相关的遗传和表观遗传学变化将在转化的细胞中进行评估。这笔赠款的目的是阐明涉及共生性的结肠癌发生的潜在机制。粪肠球菌对IL-10-/-小鼠的定植将被用于测试谷胱甘肽前体NAC作为潜在的化学预防措施。通过这种方式，我们希望定义散发性结直肠癌发生的机制和分子过程，并确定新的化学预防靶点的药物。散发性结直肠癌是世界范围内癌症和癌症死亡的主要原因。据信，这些肿瘤的产生部分是因为结肠中的细菌。在这个项目中，一种常见的肠道细菌--粪肠球菌--的不同寻常的氧化性质将被测试，以确定它是否能够破坏结肠细胞中的DNA，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目标。与公共卫生相关的散发性结直肠癌是全世界癌症和癌症死亡的主要原因。据信，这些肿瘤的产生部分是因为结肠中的细菌。在这个项目中，一种常见的肠道细菌肠球菌的不同寻常的氧化性质 粪便杆菌将接受测试，以确定其破坏结肠衬里细胞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目标。

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.