DNA 修复酶 MTH1 抑制剂研究进展

MTH1 是一种 DNA 氧化损伤修复酶,主要负责“清理”核苷酸池中氧化损伤的脱氧核苷三磷酸(dNTPs),以防其掺入 DNA 复 制中而造成碱基错配。研究表明,MTH1 与肿瘤细胞的生存密切相关,而正常细胞的生长与存活则不依赖于 MTH1。所以,以 MTH1 为靶 点开展抗肿瘤新药研发,已逐渐受到人们的关注。抑制 MTH1,为肿瘤治疗开辟了一条新途经。简介 MTH1 的结构和功能及其与肿瘤的关联, 着重对近年来 MTH1 抑制剂的发现过程和研究进展作一综述,探究小分子 MTH1 抑制剂与 MTH1 蛋白的作用模式,为 MTH1 抑制剂的设 计提供思路。     

Synthesis of γ-N-modified 8-oxo-2’-deoxyguanosine triphosphate and its characterization

8-OxodGTP is generated by the reaction between dGTP and reactive oxygen species and a considered mutagenic nucleotide. It can be incorporated into the duplex DNA during replication processes by the DNA polymerase, and thus the repair enzyme removes oxodGTP from the nucleotide pools in living cells. On the other hand, the γ-modified triphosphates show interesting properties for use as biological tools. Therefore, the γ-N-pyrenylalkyl-oxodGTP derivatives were synthesized and their effect on the enzymatic reactions were evaluated. The γ-N-pyrenylmethyl-oxodGTP was found to be accepted by the DNA polymerase just like oxodGTP, but showed a competitive inhibition property for the human oxodGTPase.     

Interleukin 8 exhibits a pro-mitogenic and pro-survival role in radiation induced genomically unstable cells

Radiation induced genomic instability can be perpetuated over time by the transmission of soluble factors. This can occur via cell-to-cell gap junction communication or the secretion/shedding of soluble factors. We have investigated whether our radiation induced chromosomally unstable GM10115 human–hamster hybrid clones secrete factors that can perpetuate the instability phenotype over time. These clones do not have functional gap junctions, but do secrete significant amounts of Interleukin 8 (IL-8) into the culture medium. We then determined whether IL-8 could initiate and or perpetuate genomic instability over time in parental GM10115 cells. Contrary to our hypothesis, IL-8 could induce DNA damage, but was not responsible for the unstable phenotype. Instead it appears that IL-8 secretion provides a pro-survival function in cells that are chromosomally unstable and generally fail to thrive.     

Hypoxia-inducible factor-1 mediates the expression of DNA polymerase iota in human tumor cells

Hypoxia generated in tumors has been shown to contribute to mutations and genetic instability. However, the molecular mechanisms remain incompletely defined. Since reactive oxygen species (ROS) are overproduced immediately after reoxygenation of hypoxic cells and generate oxidized guanine, we assumed that the mechanisms might involve translesion DNA polymerases that can bypass oxidized guanine. We report here that hypoxia as well as hypoxia mimetics, desferrioxamine, and CoCl(2), enhanced the expression of DNA polymerase iota (pol iota) in human tumor cell lines. Searching the consensus sequence of hypoxia response element to which HIF-1 binds revealed that it locates in the intron 1 of the pol iota gene. These results suggest that HIF-1-mediated pol iota gene expression may be involved in the generation of translesion mutations during DNA replication after hypoxia followed by reoxygenation, thereby contributing to the accumulation of genetic changes in tumor cells.     

Mitochondrial DNA maintenance and bioenergetics

Oxidative phosphorylation requires assembly of the protein products of both mitochondrial and of nuclear genomes into functional respiratory complexes. Cellular respiration can be compromised when mitochondrial DNA (mtDNA) sequences are corrupted. Oxidative damage resulting from reactive oxygen species (ROS) produced during respiration is probably a major source of mitochondrial genomic instability leading to respiratory dysfunction. Here, we review mechanisms of mitochondrial ROS production, mtDNA damage and its relationship to mitochondrial dysfunction. We focus particular attention on the roles of mtDNA repair enzymes and processes by which the integrity of the mitochondrial genome is maintained and dysfunction prevented.     

Drug resistance to 5-FU linked to reactive oxygen species modulator 1

While acute oxidative stress triggers cell apoptosis or necrosis, persistent oxidative stress induces genomic instability and has been implicated in tumor progression and drug resistance. In a previous report, we demonstrated that reactive oxygen species modulator 1 (Romo1) expression was up-regulated in most cancer cell lines and suggested that increased Romo1 expression might confer chronic oxidative stress to tumor cells. In this study, we show that enforced Romo1 expression induces reactive oxygen species (ROS) production in the mitochondria leading to massive cell death. However, tumor cells that adapt to oxidative stress by increasing manganese superoxide dismutase (MnSOD), Prx I, and Bcl-2 showed drug resistance to 5-FU. To elucidate the relationship between 5-FU-induced ROS production and Romo1 expression, Romo1 siRNA was used to inhibit 5-FU-triggered Romo1 induction. Romo1 siRNA treatment efficiently blocked 5-FU-induced ROS generation, demonstrating that 5-FU treatment stimulated ROS production through Romo1 induction. Based on these results we suggest that cellular adaptive response to Romo1-induced ROS is another mechanism of drug resistance to 5-FU and Romo1 expression may provide a new clinical implication in drug resistance of cancer chemotherapy.     

DNA损伤修复酶OGG1/MTH1抑制剂对非洲猪瘟病毒复制的影响

非洲猪瘟(African swine fever, ASF)是由非洲猪瘟病毒(African swine fever virus, ASFV)引起的一种动物疫病。2018年传入我国后迅速波及国内多个省份,给我国养猪业带来了巨大的冲击。由于对ASFV致病机制了解有限,导致ASF疫苗及抗病毒药物的研发遇到了极大挑战。本课题组前期研究发现,ASFV感染能引起宿主细胞内的氧化损伤应答,并且DNA氧化损伤修复酶在ASFV的感染中发挥重要作用,因此本研究采用RNA干扰、RT-qPCR、Western blotting、红细胞吸附(hemadsorption, HAD)、流式细胞技术等生物学方法,验证8-羟基鸟嘌呤DNA糖苷酶1 (8-oxoguanine DNA glycosylase 1, OGG1)/8-羟基鸟嘌呤核苷酸酶1 (8-oxoguanine nucleoside triphos-phatase, MTH1)小分子抑制剂对ASFV复制的影响,以评估靶向DNA氧化损伤修复酶的抗ASFV感染效果。本研究为抗非洲猪瘟药物的研发提供了借鉴及参考。     

The role of the Fanconi anemia network in the response to DNA replication stress

Fanconi anemia is a genetically heterogeneous disorder associated with chromosome instability and a highly elevated risk for developing cancer. The mutated genes encode proteins involved in the cellular response to DNA replication stress. Fanconi anemia proteins are extensively connected with DNA caretaker proteins, and appear to function as a hub for the coordination of DNA repair with DNA replication and cell cycle progression. At a molecular level, however, the raison d’être of Fanconi anemia proteins still remains largely elusive. The thirteen Fanconi anemia proteins identified to date have not been embraced into a single and defined biological process. To help put the Fanconi anemia puzzle into perspective, we begin this review with a summary of the strategies employed by prokaryotes and eukaryotes to tolerate obstacles to the progression of replication forks. We then summarize what we know about Fanconi anemia with an emphasis on biochemical aspects, and discuss how the Fanconi anemia network, a late acquisition in evolution, may function to permit the faithful and complete duplication of our very large vertebrate chromosomes.     

Preferential recognition of peroxynitrite modified human DNA by circulating autoantibodies in cancer patients

Peroxynitrite (ONOO⁻) has been vastly implicated in mutagenesis and cancer development. Present study probes the antigenicity of peroxynitrite damaged DNA (ONOO⁻-DNA) in cancer patients. Purified human placental DNA was damaged by the synergistic action of sodium nitroprusside (SNP) and Pyrogallol for 3 h at 37 °C. Binding characteristics of cancer autoantibodies as well as experimentally induced anti-peroxynitrite-DNA (anti-ONOO⁻-DNA) antibodies were assessed by ELISA and band shift assay. DNA modifications produced single strand breaks, decreased melting temperature (T_m), hyperchromicity in UV spectrum and decreased fluorescence intensity. The ONOO⁻-DNA induced high titre antibodies in experimental animals. Cancer autoantibodies exhibited enhanced binding with the modified DNA as compared to the native form. Lymphocyte DNA from cancer patients showed appreciable recognition of anti-ONOO⁻-DNA IgG as compared to the DNA from healthy subjects. The peroxynitrite modified DNA presents unique epitopes which may be one of the factors for the autoantibody induction in cancer patients.     

A novel protein, Romo1, induces ROS production in the mitochondria

The majority of endogenous reactive oxygen species (ROS) are produced in the mitochondrial respiratory chain. An imbalance in ROS production alters the intracellular redox homeostasis, triggers DNA damage, and contributes to cancer development and progression. This study identified a novel protein, reactive oxygen species modulator 1 (Romo1), which is localized in the mitochondria. Romo1 was found to increase the level of ROS in the cells. Increased Romo1 expression was observed in various cancer cell lines. This suggests that the increased Romo1 expression during cancer progression may cause persistent oxidative stress to tumor cells, which can increase their malignancy.     

Metal specificity in DNA damage prevention by sulfur antioxidants

Metals such as Cu^I and Fe^II generate hydroxyl radical (OH) by reducing endogenous hydrogen peroxide (H₂O₂). Because antioxidants can ameliorate metal-mediated oxidative damage, we have quantified the ability of glutathione, a primary intracellular antioxidant, and other biological sulfur-containing compounds to inhibit metal-mediated DNA damage caused hydroxyl radical. In the Cu^I/H₂O₂ system, six sulfur compounds, including both reduced and oxidized glutathione, inhibited DNA damage with IC₅₀ values ranging from 3.4 to 12.4 μM. Glutathione and 3-carboxypropyl disulfide also demonstrated significant antioxidant activity with Fe^II and H₂O₂. Additional gel electrophoresis and UV-vis spectroscopy studies confirm that antioxidant activity for sulfur compounds in the Cu^I system is attributed to metal coordination, a previously unexplored mechanism. The antioxidant mechanism for sulfur compounds in the Fe^II system, however, is unlike that of Cu^I. Our results demonstrate that glutathione and other sulfur compounds are potent antioxidants capable of preventing metal-mediated oxidative DNA damage at well below their biological concentrations. This novel metal-binding antioxidant mechanism may play a significant role in the antioxidant behavior of these sulfur compounds and help refine understanding of glutathione function in vivo.