Oxidative DNA Damage And Repair In Prostate Cancer

前列腺癌中的氧化 DNA 损伤和修复

• 批准号: 7132274
• 负责人: michele k evans
• 金额: --
• 依托单位: NATIONAL INSTITUTE ON AGING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7132274
• 关键词: DNA damage; DNA repair; carcinogenesis; cell line; enzyme activity; gas chromatography mass spectrometry; glutathione peroxidase; glutathione transferase; inflammation; liquid chromatography mass spectrometry; mitochondrial DNA; molecular oncology; neoplasm /cancer genetics; oxidative stress; prostate neoplasms; superoxide dismutase; thymine

Mutagenic oxidative DNA base damage increases with age in prostatic tissue. Various factors may influence this increase including: increased production of reactive oxygen species, increased susceptibility to oxidative stress, alterations in detoxifying enzyme levels or defects in DNA repair. Using LC/MS and GC/MS, we show increased levels of oxidative DNA base lesions, 8-hydroxyguanine (8-oxoG), 8-oxoadenine (8-oxoA) and 5-hydroxycytosine (5OHC) over the baseline in PC-3 and DU-145 prostate cancer cells following exposure to ionizing radiation and a repair period. Nuclear extracts from PC-3 and DU-145 prostate cancer cell lines are defective in the incision of 8-oxoG, 5OHC and thymine glycol (TG) relative to the non-malignant prostate cell line. Consistent with reduced expression of OGG1 2a, incision of 8-oxoG is reduced in PC-3 and DU-145 mitochondrial extracts. We also show a correlation between severely defective incision of TG and 5OHC and reduced levels of NTH1 in PC-3 mitochondria. The antioxidant enzymes, glutathione peroxidase (GPx), catalase, and superoxide dismutases (SOD1, SOD2), have altered expression patterns in these cancer cell lines. Genetic analysis of the OGG1 gene reveals that both PC-3 and DU-145 cell lines harbor polymorphisms associated with a higher susceptibility to certain cancers. These data suggest that the malignant phenotype in PC-3 and DU-145 cell lines may be associated with defects in base excision repair (BER) and alterations in expression of antioxidant enzymes. Prostate cancer, is the most prevalent form of cancer among American men and is the second leading cause of their cancer mortality. In the United States, prostate cancer is one of the fastest growing cancers in terms of incidence among American men. While certain factors including: dietary, genetic, lifestyle and environmental are associated with prostate cancer risk, the molecular mechanisms underlying the etiology of the disease are largely unknown. Several genes associated with heritable forms of prostate cancer have been identified and somatic alterations in these genes are presumed to set the stage for the development and/or progression of the disease. To this end, it has been shown that hypermethylation of the -class glutathione S-transferase gene (GSTP1) promoter region inhibits transcription of the gene and is associated with prostate cancer development [4-6]. The GSTP1 gene product probably protects genomic DNA in prostate cells from the deleterious effects of genotoxic agents. Environmental carcinogens such as polycyclic aromatic hydrocarbons may play a role in the etiology of prostate cancer since 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine has been shown to induce prostate cancer in rats. Reactive oxygen species (ROS), most notably the hydroxyl radicals, generated endogenously by cellular metabolism are known to cause oxidative DNA damage that has been implicated in prostate carcinogenesis. Research on the development of prostate cancer suggests that symptomatic and asymptomatic chronic and acute inflammation occurs in the prostate over the life span and acts in synergy with other factors to cause injury to prostatic epithelium. In response to this injury, cellular proliferation occurs followed by oxidative stress related to the ongoing inflammatory process that may in turn result in high rates of oxidative damage to DNA. Furthermore, Bostwick et al. has reported low levels of SOD1, SOD2, and catalase in prostate intraepithelial neoplasia and prostate cancer relative to benign prostate epithelium thereby implicating oxidative DNA damage in prostate carcinogenesis. There is also a significant increase in the proportion of mutagenic oxidatively induced DNA base lesions, 8-hydroxyadenine (8-oxoA), and 8-hydroxyguanine (8-oxoG) in malignant prostatic tissue as well as an increase in the levels of these lesions in benign prostatic tissue with aging. The existence of OGG1 genetic polymorphisms in prostate cancer patients further supports the notion that defective DNA repair may be associated with prostate cancer risk. Taken together these data suggest that reactive oxygen species and oxidative DNA damage may play a critical role in the development of prostate cancer. Oxidative DNA damage has been shown to be higher in the mitochondrial than in the nuclear genome due to the higher metabolic rate in the mitochondria relative to the nucleus. DNA lesions caused by ROS are numerous and include: DNA strand breaks, apurinic/apyrimidinic (AP) sites, modified DNA bases and DNA-protein cross-links. Thymine glycol (TG), 8-oxoG and other DNA base lesions may lead to deleterious biological consequences. TG is a cytotoxic lesion that blocks both DNA replication [21] and transcription [22], causing cell death. On the other hand, 8-oxoG is a pre-mutagenic lesion that results in GC to TA transversions, whereas, 8-oxoA causes both AT to GC transition and AT to CG transversion mutations [24]. Indeed, spontaneous transversion mutations have been observed in proto-oncogenes and the tumor suppressor gene, p53, a commonly mutated gene in cancer that has been shown to play a role in DNA repair. Oxidatively induced mutations in the mtDNA can lead to cellular dysfunction and have been implicated in degenerative diseases, cancer and aging. This damage must be repaired in order to maintain proper genetic integrity. Cells utilize BER pathway as the primary means of minimizing the deleterious effects associated with oxidative DNA damage. In addition to DNA repair, cells express antioxidants that detoxify ROS produced during aerobic respiration. The genome of cancer cells is more prone to oxidative damage due to the high rate of metabolism associated with increased cellular proliferation. This high metabolic rate in cancer cells may result in increased production of ROS that could significantly increase oxidative DNA damage and may be accompanied by alterations in antioxidant levels. It is plausible that increased oxidative DNA damage coupled with alteration in antioxidant levels in cancer cells may result in insufficient DNA repair. Indeed, this is the case in a few cancer cell lines studied showing defective BER in breast cancer cell lines. Recently, defects in DNA mismatch repair (MMR), the pathway that removes errors arising during DNA replication, have been reported in prostate cancer. Although, levels of oxidative DNA damage have been shown to be increased in cancerous cells or tissues relative to non-cancerous cells or tissues, the relationship between elevated oxidative DNA damage and DNA repair in carcinogenesis is still poorly understood. To our knowledge, there have been no reports addressing the repair of oxidatively induced DNA base lesions in prostate cancer cells. Therefore, we hypothesize that oxidative DNA repair may be defective in prostate tissue as a result of uncontrolled oxidative stress and increased oxidative DNA damage. We used two well-studied prostate cancer cell lines, PC-3, DU-145 and a non-malignant prostate cell line, RWPE-1 to address our hypothesis.

前列腺组织中的诱变性氧化 DNA 碱基损伤随着年龄的增长而增加。多种因素可能会影响这种增加，包括：活性氧产生增加、对氧化应激的敏感性增加、解毒酶水平的改变或 DNA 修复缺陷。使用 LC/MS 和 GC/MS，我们发现在暴露于电离辐射和修复期后，PC-3 和 DU-145 前列腺癌细胞中氧化 DNA 碱基损伤、8-羟基鸟嘌呤 (8-oxoG)、8-氧代腺嘌呤 (8-oxoA) 和 5-羟基胞嘧啶 (5OHC) 水平较基线增加。相对于非恶性前列腺细胞系，PC-3 和 DU-145 前列腺癌细胞系的核提取物在 8-oxoG、5OHC 和胸腺嘧啶乙二醇 (TG) 切割方面存在缺陷。与 OGG1 2a 表达减少一致，PC-3 和 DU-145 线粒体提取物中 8-oxoG 的切口减少。我们还显示了严重有缺陷的 TG 和 5OHC 切口与 PC-3 线粒体中 NTH1 水平降低之间的相关性。抗氧化酶、谷胱甘肽过氧化物酶 (GPx)、过氧化氢酶和超氧化物歧化酶（SOD1、SOD2）改变了这些癌细胞系中的表达模式。 OGG1 基因的遗传分析表明，PC-3 和 DU-145 细胞系都具有与某些癌症较高易感性相关的多态性。这些数据表明，PC-3 和 DU-145 细胞系中的恶性表型可能与碱基切除修复 (BER) 缺陷和抗氧化酶表达的改变有关。 前列腺癌是美国男性中最常见的癌症形式，也是导致癌症死亡的第二大原因。在美国，就美国男性发病率而言，前列腺癌是增长最快的癌症之一。虽然饮食、遗传、生活方式和环境等某些因素与前列腺癌风险相关，但该疾病病因的分子机制在很大程度上尚不清楚。 已经鉴定出与前列腺癌的遗传形式相关的几种基因，并且推测这些基因中的体细胞改变为疾病的发生和/或进展奠定了基础。为此，研究表明β-类谷胱甘肽S-转移酶基因(GSTP1)启动子区域的高甲基化会抑制该基因的转录，并与前列腺癌的发展相关[4-6]。 GSTP1 基因产物可能保护前列腺细胞中的基因组 DNA 免受基因毒剂的有害影响。环境致癌物如多环芳烃可能在前列腺癌的病因学中发挥作用，因为2-氨基-1-甲基-6-苯基咪唑并[4,5-b]吡啶已被证明可诱发大鼠前列腺癌。 众所周知，细胞代谢内源性产生的活性氧 (ROS)，尤其是羟基自由基，会导致氧化性 DNA 损伤，从而导致前列腺癌发生。对前列腺癌发展的研究表明，在整个生命周期中，前列腺中都会发生有症状和无症状的慢性和急性炎症，并与其他因素协同作用，导致前列腺上皮损伤。为了应对这种损伤，细胞增殖发生，随后发生与持续炎症过程相关的氧化应激，这反过来可能导致 DNA 氧化损伤率很高。此外，Bostwick 等人。据报道，相对于良性前列腺上皮，前列腺上皮内瘤变和前列腺癌中 SOD1、SOD2 和过氧化氢酶水平较低，因此表明前列腺癌发生中存在氧化 DNA 损伤。随着年龄的增长，恶性前列腺组织中诱变氧化诱导的 DNA 碱基病变、8-羟基腺嘌呤 (8-oxoA) 和 8-羟基鸟嘌呤 (8-oxoG) 的比例显着增加，良性前列腺组织中这些病变的水平也随之增加。前列腺癌患者中 OGG1 基因多态性的存在进一步支持了 DNA 修复缺陷可能与前列腺癌风险相关的观点。综上所述，这些数据表明活性氧和氧化性 DNA 损伤可能在前列腺癌的发展中发挥关键作用。 由于线粒体相对于细胞核的代谢率较高，线粒体中的氧化性 DNA 损伤已被证明比核基因组中更高。 ROS 引起的 DNA 损伤有很多，包括：DNA 链断裂、脱嘌呤/脱嘧啶 (AP) 位点、修饰的 DNA 碱基和 DNA-蛋白质交联。胸腺嘧啶乙二醇 (TG)、8-oxoG 和其他 DNA 碱基损伤可能会导致有害的生物学后果。 TG 是一种细胞毒性损伤，可阻断 DNA 复制 [21] 和转录 [22]，导致细胞死亡。另一方面，8-oxoG 是一种诱变前病变，导致 GC 向 TA 颠换，而 8-oxoA 会导致 AT 向 GC 转换和 AT 向 CG 颠换突变[24]。事实上，在原癌基因和肿瘤抑制基因 p53 中已经观察到自发颠换突变，p53 是癌症中常见的突变基因，已被证明在 DNA 修复中发挥作用。线粒体 DNA 中氧化诱导的突变可导致细胞功能障碍，并与退行性疾病、癌症和衰老有关。这种损伤必须得到修复，以保持适当的遗传完整性。 细胞利用 BER 途径作为最大程度减少与氧化 DNA 损伤相关的有害影响的主要手段。除了 DNA 修复之外，细胞还表达抗氧化剂，可以解毒有氧呼吸过程中产生的 ROS。由于与细胞增殖增加相关的高代谢率，癌细胞的基因组更容易受到氧化损伤。癌细胞中的高代谢率可能导致 ROS 产生增加，从而显着增加 DNA 氧化损伤，并可能伴随抗氧化剂水平的变化。癌细胞中氧化性 DNA 损伤的增加加上抗氧化剂水平的改变可能会导致 DNA 修复不足，这似乎是合理的。事实上，在研究的一些癌细胞系中确实存在这种情况，显示乳腺癌细胞系中存在 BER 缺陷。最近，据报道，前列腺癌中存在 DNA 错配修复 (MMR) 缺陷，MMR 是消除 DNA 复制过程中出现的错误的途径。尽管已证明癌细胞或组织中的氧化性 DNA 损伤水平相对于非癌细胞或组织有所增加，但致癌过程中氧化性 DNA 损伤升高与 DNA 修复之间的关系仍知之甚少。 据我们所知，目前还没有关于修复前列腺癌细胞中氧化诱导的 DNA 碱基损伤的报道。因此，我们推测，由于氧化应激失控和氧化 DNA 损伤增加，前列腺组织中的氧化 DNA 修复可能存在缺陷。我们使用两种经过充分研究的前列腺癌细胞系 PC-3、DU-145 和非恶性前列腺细胞系 RWPE-1 来验证我们的假设。

项目成果

Loss of functional prion protein: A role in prion disorders?

• DOI: 10.1016/s1074-5521(96)90128-3
• 发表时间: 1996-08-01
• 期刊: CHEMISTRY & BIOLOGY
• 作者: Borchelt, DR;Sisodia, SS
• 通讯作者: Sisodia, SS