Mechanisms controlling sensitivity to platinum complexes: role of p53 and DNA mismatch repair

Although cisplatin is effective in the treatment of different types of tumors, resistance to treatment is a major limitation. In an attempt of overcoming resistance mechanisms, a large effort has been made to generate compounds with a different geometry. At present, the most clinically relevant compounds include mononuclear (i.e. oxaliplatin) as well as multinuclear platinum complexes (i.e. BBR 3464). The mechanisms of cellular response to platinum complexes have not been completely elucidated. Among the main pathways affecting cell sensitivity of these drugs a role for p53 has been proposed at least for cisplatin and BBR 3464. Our results indicate that, also in the case of oxaliplatin, cytotoxicity is modulated by this pathway. Indeed, the effect of oxaliplatin could be reduced in tumor cells expressing mutant p53. The DNA mismatch repair system also appears to be critical in regulating cellular sensitivity to cisplatin because the loss of DNA mismatch repair results in low level of resistance to cisplatin, but not to oxaliplatin. Thus, platinum compounds are endowed with differential capability to activate pathways of p53-dependent or independent apoptosis, and differential recognition by specific cellular systems is likely to be the critical determinant of the cell fate (death/survival) after drug exposure. Further molecular studies are required to better define the precise contribution of such pathways to the cellular responses of the clinically relevant platinum complexes. A complete understanding of the molecular basis of sensitivity to platinum drugs is expected to provide useful insights for the optimization of tumor treatment.     

血红素氧化酶在软骨藻酸对细胞DNA损伤中的作用

目的　　研究血红素氧化酶 (hemeoxygenase ,HO)系统在软骨藻酸 (domoicacid ,DA)对H4细胞DNA损伤中的作用。方法　应用单细胞凝胶电泳法 (SCGE)分别测定 0、0 0 64、0 64和 6 4μmol/LDA单独及与HO系统的活性阻断剂ZnPP 9(10 - 5mol/L)联合作用于H4细胞 6、12、2 4和 48h后彗星拖尾细胞率及拖尾尾长。结果　彗星拖尾细胞率 :中剂量和高剂量组各时间点均比对照组高 ,差异有显著性 (P <0 0 5 )。中剂量 +阻断剂组各时间点均相应高于中剂量组 ,差异有显著性 (P <0 0 5 )。彗星拖尾尾长 :中剂量组 12、2 4和 48h比对照组长 ,2 4、48h比低剂量组长 ,差异均有显著性 (P<0 0 5 )。高剂量组各时间点均比对照组、低剂量组和中剂量组长 ,差异有显著性 (P <0 0 5 )。高剂量 +阻断剂组和中剂量 +阻断剂组 2 4、48h分别高于高剂量和中剂量组 ,差异有显著性 (P <0 0 5 )。尾长 时间作Regression分析 ,各剂量组r差异值均有显著性 (P <0 0 1)。结论　软骨藻酸能诱导H4细胞DNA损伤 ,HO系统对这种损伤有一定的保护作用。     

Mammalian DNA base excision repair proteins: their interactions and role in repair of oxidative DNA damage

The DNA base excision repair (BER) is a ubiquitous mechanism for removing damage from the genome induced by spontaneous chemical reaction, reactive oxygen species (ROS) and also DNA damage induced by a variety of environmental genotoxicants. DNA repair is essential for maintaining genomic integrity. As we learn more about BER, a more complex mechanism emerges which supersedes the classical, simple pathway requiring only four enzymatic reactions. The key to understand the complete BER process is to elucidate how multiple proteins interact with one another in a coordinated process under specific physiological conditions.     

Recognition by the DNA repair system of DNA structural alterations induced by reversible drug-DNA interactions

Ditercalinium (NSC 335153) was synthesized as a bifunctional DNA intercalator. It is made of two 7-H pyridocarbazole rings joined by a rigid bis-ethyl bispiperidine chain. It binds to DNA with high affinity and elicits anti-tumor activity on a variety of animal tumors. 1H n.m.r. studies of ditercalinium bis-intercalated into d(CpGpCpG)2 have shown that the intercalation process occurs from the large groove of the DNA helix while the two intercalated rings are separated by two base pairs. Because of the linking chain rigidity of ditercalinium, DNA conformation has to be altered to permit the intercalation of the two rings. DNA must be bent toward the minor groove. In E. coli, ditercalinium elicits a specific toxicity on polA strains which is suppressed by an additional uvrA mutation. In vitro, the purified UvrA and UvrB proteins bind to the DNA-ditercalinium complex in an ATP dependent manner. The UvrABC complex induces single-strand nicks, but only when ditercalinium is bound to negatively supercoiled DNA. The life-time of the UvrAB-DNA-ditercalinium complex is greater than 50 min when free ditercalinium concentration is maintained constant in the incubation medium. The cytotoxicity of ditercalinium in E. coli results from the induction of a futile and abortive DNA repair. The reversible ditercalinium-DNA complex mimics a bulky DNA lesion, yet the UvrABC endonuclease is unable to cope with a reversible lesion since it cannot eliminate the causative agent. The interaction of UvrA and UvrB proteins has also been studied with DNA and other DNA-binding drugs forming high-affinity complexes such as distamycin. The Uvr protein recognition process appears to be associated with specific DNA structural alterations. In eukaryotic cells, ditercalinium is concentrated in mitochondria. Mitochondrial DNA is rapidly and totally degraded. Mitochondrial DNA coded proteins being no longer synthesized, the respiratory chain is progressively inactivated. The stimulation of the glycolytic pathway allows the cells to continue growth for several generations. Dihydro-orotate dehydrogenase is located in the inner membrane of mitochondria and its activity is dependent on mitochondria energization. It becomes inactive after ditercalinium treatment. A drop of the pyrimidine pool is then observed. Complementation of treated cells with uridine decreases 10-fold the ditercalinium toxicity. The cellular delayed toxicity of ditercalinium results from the slow induction of a pyrimidineless state associated with the progressive inactivation of mitochondria. The results show that DNA structural alterations induced by reversible drug-DNA complexes can be recognized by DNA repair enzymes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Arsenic inhibits the repair of DNA damage induced by benzo(a)pyrene

In order to study the effect of arsenic on DNA damage, Sprague-Dawley rats were dosed with sodium arsenite (10 mg/kg) with or without 800 microg of benzo(a)pyrene (BP) by intramammilary injection. The animals were sacrificed on day 1, 3, 5, 10 and 27 and the mammary gland tissues were collected for DNA adduct measurement using a (32)P post-labeling assay. Animals dosed with arsenic alone did not show any DNA adducts. DNA adduct levels in rats dosed with BP alone reached a maximum level by day 5, reducing to 13% of this level by day 27. Adduct levels in rats dosed with arsenic and BP also reached a maximum by day 5 but only 80% of the level observed in the BP group. However, 84% of this amount still remained by day 27. The First Nucleotide Change (FNC) technique was used for the screening of 115 samples of various tissues from mice that had been chronically exposed to sodium arsenate for over 2 years revealed that inorganic arsenic did not attack the two putative hotspots (codons 131 and 154) of the hOGG1 gene. These results support the hypothesis that arsenic exerts its biological activity through DNA repair inhibition.     

Chemical screen identifies shikonin as a broad DNA damage response inhibitor that enhances chemotherapy through inhibiting ATM and ATR

DNA damage response (DDR) is a highly conserved genome surveillance mechanism that preserves cell viability in the presence of chemotherapeutic drugs. Hence, small molecules that inhibit DDR are expected to enhance the anti-cancer effect of chemotherapy. Through a recent chemical library screen, we identified shikonin as an inhibitor that strongly suppressed DDR activated by various chemotherapeutic drugs in cancer cell lines derived from different origins. Mechanistically, shikonin inhibited the activation of ataxia telangiectasia mutated (ATM), and to a lesser degree ATM and RAD3-related (ATR), two master upstream regulators of the DDR signal, through inducing degradation of ATM and ATR-interacting protein (ATRIP), an obligate associating protein of ATR, respectively. As a result of DDR inhibition, shikonin enhanced the anti-cancer effect of chemotherapeutic drugs in both cell cultures and in mouse models. While degradation of ATRIP is proteasome dependent, that of ATM depends on caspase- and lysosome-, but not proteasome. Overexpression of ATM significantly mitigated DDR inhibition and cell death induced by shikonin and chemotherapeutic drugs. These novel findings reveal shikonin as a pan DDR inhibitor and identify ATM as a primary factor in determining the chemo sensitizing effect of shikonin. Our data may facilitate the development of shikonin and its derivatives as potential chemotherapy sensitizers through inducing ATM degradation.     

p53 interacts with the DNA mismatch repair system to modulate the cytotoxicity and mutagenicity of hydrogen peroxide

This study focused on the question of how the DNA mismatch repair (MMR) system and p53 interact to maintain genomic integrity in the presence of the mutagenic stress induced by hydrogen peroxide (H(2)O(2)). The cytotoxic and mutagenic effects of H(2)O(2) were compared in four colon carcinoma sublines: HCT116, HCT116/E6, HCT116+ch3, and HCT116+ch3/E6, representing MMR(-)/p53(+), MMR(-)/p53(-), MMR(+)/p53(+), and MMR(+)/p53(-) phenotypes, respectively. Loss of p53 in MMR-proficient cells did not significantly alter cellular sensitivity to H(2)O(2), but disruption of p53 in MMR-deficient cells resulted in substantial resistance to H(2)O(2) (IC(50) values of 203.8 and 66.2 microM for MMR(-)/p53(-) and MMR(-)/p53(+) cells, respectively). The effect of loss of p53 and MMR function on sensitivity to the mutagenic effect of H(2)O(2) paralleled the effects on cytotoxic sensitivity. In MMR-deficient cells, loss of p53 resulted in a 3.5- and 2.2-fold increase in the generation of 6-thiogunaine and ouabain-resistant clones, respectively. Loss of MMR in combination with loss of p53 synergistically increased the frequency of frameshift mutations in the CA repeat tracts of the out-of-frame shuttle vector pZCA29 and further promoted instability of microsatellite sequences under H(2)O(2) stress. Flow cytometric analysis showed that H(2)O(2) treatment produced a G(l) and G(2)/M phase arrest in MMR(+)/p53(+) cells. Loss of MMR did not alter the ability of H(2)O(2) to activate either checkpoint; loss of p53 in either the MMR-proficient or deficient cells resulted in impairment of the G(l) arrest and a more pronounced G(2)/M arrest. H(2)O(2) caused a greater and more longed increase in p53 protein levels in MMR-proficient than in the MMR-deficient cells. The results demonstrate that the effect of disabling p53 function is modulated by the proficiency of the MMR system (and vice versa) and that there is an overlap between the functions of p53 and the MMR system with respect to the activation of apoptosis and mutagenesis after an oxidative stress.     

DNA damage induced by coexposure to PAHs and light

Polycyclic aromatic hydrocarbons (PAHs) are widely distributed in the environment as pollutants in air, water and soil, and some are carcinogenic, being associated with various types of cancer. A majority of the research concerning the biological effects of PAHs has focused on the metabolic activation and DNA adducts leading to mutation and transformation. Although the role of the PAHs as photosensitizers has received much less attention, investigators have shown that PAHs excited by sunlight induced significant cytotoxicity and several kinds of DNA damage. Some PAHs were recently proved to be photomutagenic. In this review, we discuss the influence of PAHs in combination with sunlight focusing on the phototoxicity and cellular DNA damage produced.     

Oxidative DNA damage induced by equine estrogen metabolites: role of estrogen receptor alpha

Excessive exposure to synthetic and endogenous estrogens has been associated with the development of cancer in several tissues. 4-Hydroxyequilenin (4-OHEN), a major metabolite of equine estrogens present in estrogen replacement formulations, has been shown to induce cytotoxic/carcinogenic effects. In the present study, we have found that 4-OHEN caused DNA damage in breast cancer cells, and cells that contain estrogen receptor alpha (S30) are more sensitive to 4-OHEN-mediated DNA damage as compared to estrogen receptor negative cells (MDA-MB-231). For example, concentration-dependent increases in 8-oxo-deoxyguanosine (8-oxo-dG), as measured by LC-MS-MS or by the Fpg comet assay, were only detected in the S30 cells, and the amount of this lesion could be enhanced by agents, which catalyze redox cycling (NADH) or deplete GSH (diethyl maleate). The role of the estrogen receptor in modulating DNA damage was further established in incubations with the ER antagonist tamoxifen, where decreases in 8-oxo-deoxyguanosine were observed. Another equine estrogen metabolite, 4,17 beta-hydroxyequilenin (4,17 beta-OHEN), was found to have the same cytotoxicity and a similar ability to induce reactive oxygen species (ROS), and caused the same oxidative DNA damage in S30 cells as compared to 4-OHEN. However, 4,17 beta-OHEN induced twice as much single strand DNA breaks in S30 cells compared to 4-OHEN. Also 4,17 beta-OHEN was more estrogenic than 4-OHEN as demonstrated by a higher binding affinity for ER alpha and an enhanced induction in activity of estrogen-dependent alkaline phosphatase in Ishikawa cells. These data suggest that the mechanism of DNA damage induced by equine estrogen metabolites could involve oxidative stress and that the estrogen receptor may play a role in this process.     

干扰survivin表达减少肿瘤细胞对紫外线诱导DNA损伤的修复作用

目的：研究生存素（survivin）对紫外线（UV）诱导的肿瘤细胞DNA损伤修复作用及其机制。方法：本研究构建和包装survivin蛋白shRNA慢病毒载体;依据逐孔稀释法确定转染效率及滴度;以实时荧光定量法确定干扰效果;采用宿主细胞复活法（HCR）检测survivin蛋白表达对细胞整体DNA损伤修复能力的影响。结果：干扰survivin的表达显著降低肺腺癌H1299细胞对UV诱导DNA损伤的修复能力。结论：Survivin在UV诱导DNA损伤的修复作用中扮演重要角色。本研究可以为以survivin为靶点的肿瘤治疗提供一定的理论依据。     

Evaluation of adriamycin induced DNA damage and repair in human and animal cells

Adriamycin induced DNA damage and repair were investigated by the comet assay in four human cell lines (CRL2088, ME18, Lu106, WISH) and five animal cell lines (L929, Balb/3T3, CHO. MDBK, Vero). The results indicated the concentration-dependent induction of DNA strand breaks in all cell lines after adriamycin treatment. Simultaneously, wide differences in sensitivity of cells to the damaging action of adriamycin were observed. The most sensitive were two human cell lines derived from epithelial tissues, Lu106 and WISH. In these lines the breaks induced by adriamycin were not repaired within 6 h postincubation. In two other human cell lines, CRL2088 and ME18, only a small number of DNA breaks appeared after adriamycin exposure and these were very quickly repaired. Among the animal cell lines only the mouse cell line L929 was very sensitive to adriamycin, but most of the induced breaks were repaired within 6 h postincubation. The remaining animal cell lines were less sensitive to DNA damage by adriamycin and all except 3T3, completely repaired the breaks within 6 h postincubation.     

DNA damage caused by metal nanoparticles: involvement of oxidative stress and activation of ATM

Nanotechnology is a fast growing emerging field, the benefits of which are widely publicized. Our current knowledge of the health effects of metal nanoparticles such as nanosized cobalt (Nano-Co) and titanium dioxide (Nano-TiO(2)) is limited but suggests that metal nanoparticles may exert more adverse pulmonary effects as compared with standard-sized particles. To investigate metal nanoparticle-induced genotoxic effects and the potential underlying mechanisms, human lung epithelial A549 cells were exposed to Nano-Co and Nano-TiO(2). Our results showed that exposure of A549 cells to Nano-Co caused reactive oxygen species (ROS) generation that was abolished by pretreatment of cells with ROS inhibitors or scavengers, such as catalase and N-acetyl-L(+)-cysteine (NAC). However, exposure of A549 cells to Nano-TiO(2) did not cause ROS generation. Nano-Co caused DNA damage in A549 cells, which was reflected by an increase in length, width, and DNA content of the comet tail by the Comet assay. Exposure of A549 cells to Nano-Co also caused a dose- and a time-response increased expression of phosphorylated histone H2AX (γ-H2AX), Rad51, and phosphorylated p53. These effects were significantly attenuated when A549 cells were pretreated with catalase or NAC. Nano-TiO(2) did not show these effects. These results suggest that oxidative stress may be involved in Nano-Co-induced DNA damage. To further investigate the pathways involved in the Nano-Co-induced DNA damage, we measured the phosphorylation of ataxia telangiectasia mutant (ATM). Our results showed that phosphorylation of ATM was increased when A549 cells were exposed to Nano-Co, and this effect was attenuated when cells were pretreated with catalase or NAC. Pretreatment of A549 cells with an ATM specific inhibitor, KU55933, significantly abolished Nano-Co-induced DNA damage. Furthermore, pretreatment of A549 cells with ROS scavengers, such as catalase and NAC, significantly abolished Nano-Co-induced increased expression of phosphorylated ATM. Taken together, oxidative stress and ATM activation are involved in Nano-Co-induced DNA damage. These findings have important implications for understanding the potential health effects of metal nanoparticle exposure.     

人DNA错配修复系统hMSH2蛋白在口腔扁平苔藓中的表达

目的观察错配修复基因系统中hMSH2蛋白在口腔扁平苔藓（OLP）中的表达。方法应用免疫组化的方法检测正常黏膜及OLP中hMSH2蛋白的表达。结果hMSH2蛋白在正常口腔黏膜组织和OLP二者中的阳性表达率之间差异有统计学意义（x2=7．1993,P〈0．05）。结论hMSH2蛋白在OLP的发生发展中发挥一定的作用。OLP可能有潜在的恶变能力。     

DNA mismatch repair deficiency, resistance to cancer chemotherapy and the development of hypersensitive agents

DNA Mismatch Repair (MMR) deficiency results in resistance to platinating and alkylating agents, DNA minor groove binders, inhibitors of topoisomerases and antimetabolites. The cellular MMR pathway, involving hMLH1 and MSH2, detects and repairs DNA frame shifts replication errors and regulates recombination events. Tumour cells are able to cope with DNA damage caused by chemotherapy as long as the MMR-process is disabled and hence there is a need to develop agents that (i) restore MMR proficiency or (ii) are hypersensitive in cells that are irreversibly MMR deficient. Decitabine is suggested to restore MMR function by reversal of gene promoter hypermethylation of hMLH1. However, when MMR is deficient due to gene mutation it is not feasible to design agents, since the absence of functional proteins that constitute the MMR machinery are not available as targets. The evidence that resistance to chemotherapy is associated with hMSH2 and/or hMLH1 deficiency has revealed a new paradigm for drug discovery of agents that positively exploit this phenotype to therapeutic advantage. Even more attractive is the development of agents that are hypersensitive in the absence of functional MMR to enable even more effective treatment. In this regard, established agents such as mitomycin C, camptothecin or novel hydroxyethylaminoanthraquinones may represent opportunities for exploitation of MMR-deficiency in tumour cells.     

Gastrointestinal damage induced by platelet-activating factor: role of leukotrienes

The role of leukotriene synthesis in the gastrointestinal damage induced by platelet-activating factor (PAF) was examined in the rat. The effects of a 20-min infusion of PAF (100 ng/kg per min) on leukotriene B4 (LTB4) and leukotriene C4 (LTC4) synthesis were examined in samples of the stomach, duodenum, jejunum, ileum and colon. Administration of PAF resulted in marked hemoconcentration and extensive hemorrhagic damage which was only observed in the corpus region of the stomach and in the small intestine. However, LTB4 synthesis was increased significantly in all regions studied, while LTC4 synthesis was increased significantly only in the duodenum. Pretreatment of the rats with dexamethasone significantly reduced the PAF-induced increase in LTB4 synthesis in all tissues studied. However, a reduction of PAF-induced damage following dexamethasone treatment was observed in the small intestine, but not the stomach. To further investigate the role of leukotrienes as mediators of PAF-induced gastrointestinal damage, the effects of a 10-min infusion of PAF (100 ng/kg per min i.v.) were compared to those of similar infusions of LTB4, LTC4 or leukotriene D4 (LTD4) (0.3-3 micrograms/kg per min). None of the doses of leukotrienes tested produced hemoconcentration or gastrointestinal damage comparable to that observed with the much lower dose of PAF, with the single exception of significant hemoconcentration observed with the highest dose of LTC4. The results of this study therefore suggest that leukotrienes are unlikely to play a major role as mediators of PAF-induced gastrointestinal damage in the rat.     

Site-specific DNA damage induced by cobalt(II) ion and hydrogen peroxide: role of singlet oxygen

The effect of Co(II) ion on the reaction of hydrogen peroxide with DNA was investigated by a DNA sequencing technique using 32P-5'-end-labeled DNA fragments obtained from human c-Ha-ras-1 protooncogene. Co(II) induced strong DNA cleavage in the presence of hydrogen peroxide even without alkali treatment. Guanine residues were the most alkali-labile site, and the extent of cleavages at the positions of thymine and cytosine was dependent on the sequence. Adenine residues were relatively resistive. Diethylenetriaminepentaacetic acid, present in excess over Co(II), inhibited DNA cleavage. Singlet oxygen scavengers (dimethylfuran, sodium azide, 1,4-diazabicyclo[2.2.2]octane, dGMP), sulfur compounds (methional, methionine), and superoxide dismutase inhibited DNA cleavage completely. Hydroxyl radical scavengers were not so effective as singlet oxygen scavengers. ESR studies using 2,2,6,6-tetramethyl-4-piperidone as a singlet oxygen trap suggest that Co(II) reacts with hydrogen peroxide to produce singlet oxygen or its equivalent. ESR studies using 5,5-dimethylpyrroline N-oxide (DMPO) showed that the hydroxyl radical adduct of DMPO was also formed. The results suggest that Co(II) ion binds to DNA and subsequently reacts with hydrogen peroxide to produce singlet oxygen and hydroxyl radicals and that singlet oxygen plays a more important role in the DNA damage than hydroxyl free radicals.     

Oxidative stress and DNA damage induced by cadmium in the human keratinocyte HaCaT cell line: role of glutathione in the resistance to cadmium

Cadmium affects the cellular homeostasis and generates damage via complex mechanisms involving interactions with other metals and oxidative stress induction. In this work we used a human keratinocyte cell line (HaCaT) as a model to study the oxidative damage induced by cadmium to cellular macromolecules, its effect on the antioxidant systems and the role of glutathione in cell protection toward cadmium toxicity. The cells were incubated for 24 and 48 h with cadmium (3, 15, 50 and 100 microM). High doses of cadmium were required to induce a cytotoxicity: 100 microM lead to 30% mortality after 24h and 50% after 48 h. The oxidation of lipids and proteins and the DNA damage, respectively, assessed by thiobarbituric acid reactants determination, thiol group measurement and comet assay, were observed for 50-100 microM cadmium. The cytotoxic effects were strongly correlated to the cellular cadmium content. The glutathione peroxidase and the catalase activities were decreased, while the glutathione reductase activity and the glutathione concentration were increased after cadmium treatment. The superoxide dismutases activities were unchanged. A depletion in glutathione prior to cadmium exposure increased the cytotoxic effects and provoked DNA damage. Our results suggested that the hydroxyl radical could be the major compound involved in the oxidative stress generated by cadmium and that glutathione could play a major role in the protection of HaCaT cells from cytotoxicity but mostly from DNA damage induced by cadmium.     

Oxidative damage to bacterial DNA and evidence for its repair

Oxidative damage to DNA can be caused by excited oxygen species, which are produced by radiation or are by-products of aerobic metabolism. Endogenous levels of 8-hydroxy-2′-deoxyguanosine (8-OH-dG), an adduct that results from the damage of DNA caused by hydroxyl radical, have been detected inE. coli andS. typhimurium. Treatment of bacterial cells with various concentrations of hydrogen peroxide caused a moderate increase in the 8-OH-dG content. The enzymatic release of 8-OH-dG from asocorbate/Cu(II)-treated DNA was effected by an extract ofE. coli cells. These results indicate that 8-OH-dG is formedin vivo in bacterial DNA through endogenous oxidative mechanisms and on treatment with an oxygen radical-producing agent and that it is repairable.     

应用彗星试验检测乌头类生物碱致细胞DNA损伤作用

目的:研究乌头类生物碱对细胞DNA的损伤作用,以期在分子水平进一步阐明乌头类生物碱的毒效作用特征及其分子机制.方法:以500、100、50和10μg·mL(-1)乌头碱、次乌头碱或新乌头碱分别染毒HepG2细胞1.5 h,应用24孔板彗星试验检测细胞DNA损伤.结果:乌头类生物碱作用组细胞平均拖尾长度和拖尾细胞率与生物碱浓度存在剂量反应关系,且100和50μg·mL(-1)作用组细胞平均拖尾长度与阴性对照组之间差异具有统计学意义(P<0.05).结论:乌头类生物碱具有细胞DNA损伤作用.