Short Communication: The involvement of DNA topoisomerases in DNA repair and mutagenesis

DNA topoisomerase II (DNA gyrase) is the enzyme which insertsnegative supercoils into double-stranded DNA. In this paperit is shown that both subunits of this enzyme, and one subunitof the related DNA topoisomerase II' are involved in the repairof u.v.-induced damage and in u.v.-induced mutagenesis.     

Novobiocin inhibition of DNA excision repair may occur through effects on mitochondrial structure and ATP metabolism, not on repair topoisomerases

Novobiocin inhibits DNA topoisomerases. It also inhibits excisionrepair of DNA photodamage, blocking both repair synthesis andthe earlier step of incision at u.v. damage sites (as measuredby the accumulation of DNA strand breaks in u.v.-irradiatedinterphase cells treated with DNA synthesis inhibitors suchas hydroxyurea or cytosine arabinoside). It has been supposed,therefore, that novobiocin affects repair by blocking a putativetopoisomerase step prior to incision. But we find that novobiocinalso has a marked dose- and time-dependent effect on mitochondria:in cells exposed to novobiocin, mitochondria swell and theircristae become disrupted, and the intracellular ATP: ADP ratiois lowered, though the membrane potential is maintained as judgedby rhodamine 123 fluorescence. Mitotic cells are more resistantto mitochondrial disruption by novobiocin than are interphasecells. This correlates with a relative resistance of u.v.-irradiatedmitotic cells to the inhibition of incision by novobiocin. Thechromosomal decondensation that results from the accumulationof DNA breaks due to incision when u.v.-irradiated mitotic cellsare treated with hydroxyurea and cytosine arabinoside is largelysuppressed by novobiocin. Furthermore, the suppression of inducedstrand break accumulation is partly due to a suppression bynovobiocin of the uptake and phosphorylation of cytosine arabinoside;breaks accumulated in u.v.-irradiated cells in the presenceof aphidicolin, an inhibitor of DNA polymerase alpha that doesnot require phosphorylation, are less novobiocin-sensitive.We conclude that the effects of novobiocin on excision repairare more likely to be due to a non-specific effect on ATP metabolismthan to a specific effect on a repair-related topoisomerase.     

Heat shock proteins: role in thermotolerance, drug resistance, and relationship to DNA topoisomerases

The events following a mild heat exposure to cells that appear to be closely linked are: enhanced synthesis of hsp's and thermotolerance. In some cases, thermotolerance is also associated with drug resistance. We have recently examined the role that DNA topoisomerase II may play in the induction of these phenomena. VM-26 was found to both initiate hsp synthesis and to cause thermotolerance. Furthermore, the permanent heat resistant or transient thermotolerant cells were more resistant to VM-26 than controls. These results suggest that topoisomerase II, or more likely topoisomerase II-DNA complexes, are affected by heat or by VM-26 in a phenomenologically overlapping manner. Elevated levels of hsp's apparently protect cells against the cytotoxic action of both heat and VM-26.     

The function of DNA topoisomerases in UV-induced DNA excision repair: studies with specific inhibitors in permeabilized human fibroblasts.

Fifteen specific inhibitors of DNA topoisomerases I and II were used to elucidate whether these enzymes participate in the excision repair of UV-induced DNA damage, monitoring DNA repair synthesis in confluent saponin-permeabilized human fibroblasts. To achieve a sufficient degree of accuracy dose--response experiments were performed, analysed by linear regression, and the concentrations at which repair activity was reduced to 50% were calculated and designated K50. Camptothecin, a specific inhibitor of topoisomerase I did not markedly diminish DNA repair synthesis. Similarly, when combined with topoisomerase II inhibitors [nalidixic acid, oxolinic acid, 4'-demethylepipodophyllotoxin-9-(4,6-O-ethylidene-beta-D-glucop yra noside) (etoposide), 4'-demethylepipodophyllotoxin-thenylidene-beta-D-glucoside (teniposide), 1,4-dihydroxy-5,8-bis ((2-[(2-hydroxyethyl)amino]ethyl)amino)-9,10-anthracenedione (mitoxantrone), 5-(N-phenyl-carboxamido)-2-thiobarbituric acid (merbarone) or 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA)], it did not lower K50 values determined for topoisomerase II-specific drugs in separate experiments. The effects observed can be classified according to the mechanism of action the inhibitors exhibit. (i) Novobiocin and coumermycin, inhibitors of the ATPase subunit of topoisomerase II, completely reduced DNA repair synthesis. (ii) Inhibition of repair was also found for ethidium bromide, quinacrine and distamycin, drugs known to modify the DNA substrate by intercalation or binding to the DNA minor groove. (iii) Inhibitors acting through intercalation and, simultaneously, binding to the cleavable DNA-topoisomerase complex (m-AMSA, mitoxantrone, doxorubicin and daunorubicin) also suppressed reparative DNA synthesis. (iv) Only small effects were observed for etoposide, nalidixic acid and oxolinic acid, whereas teniposide caused marked inhibition of DNA repair synthesis. (v) Merbarone, a novel type of topoisomerase II inhibitor, blocked UV-induced DNA repair drastically. The results are best explained by assuming that in UV-irradiated human fibroblasts the 180 kDa form of topoisomerase II is the main target enzyme for inhibitors which suppressed DNA excision repair and that this isozyme is involved in steps preceding repair-specific DNA incision.     

Mouse models to study the role of telomeres in cancer,aging and DNA repair

The chromosome ends have protective structures that distinguish them from broken chromosomes, known as telomeres. The function of telomeres, and that of the cellular activity that synthesises them, telomerase, are proposed to be biological determinants in the processes of cancer and aging. In this review, we will focus on mammalian telomeres and, in particular, on the analysis of different mouse models for proteins that are important for telomere function, such as telomerase and various telomere-binding proteins. These mouse models have allowed the relevance of telomeres and telomerase in tumour development and the aging of the organism to be directly tested.     

DNA polymerases and DNA topoisomerases as targets for the development of anticancer drugs

Studies of a variety of compounds designed as derivatives of prototype active molecules aphidicolin and doxorubicin are reported. So far none of the aphidicolin simpler analogues is as active as the parental molecule. Ten anthracycline analogues, characterized for their cytotoxicity, antitumor activity and inhibition of the relaxing activity of purified human DNA topoisomerase II can be divided into five groups. The majority of the tested compounds shows properties very similar to those of doxorubicin. Epirubicin shows extremely high inhibitory activity toward the relaxing property of topoisomerase II but its antitumor activity and cytotoxicity are similar to those of the former group. The third group includes a compound with extremely high cytotoxicity. The fourth group is represented by a compound which shows a cytotoxicity. The fourth group is represented by a compound which shows a cytotoxicity. The fourth group is represented by a compound which shows a cytotoxicity typical of anthracyclines and good antitumor activity but which has no specific inhibitory activity on topoisomerase II. A fifth group includes a totally inactive compound. Our results suggest that the inhibition of human DNA topoisomerase II is only partially correlated with antitumor activity.     

The role of mismatch repair in DNA damage-induced apoptosis

DNA mismatch repair plays a critical role in maintaining genomic integrity. Defects in human mismatch repair are the primary cause of certain types of cancer, including hereditary nonpolyposis colorectal cancer. In the past, the ability of mismatch repair proteins to correct DNA mismatches that occur during DNA replication, repair, and recombination was considered the primary mechanism by which it contributes to genomic stability. However, increasing evidence supports the idea that the mismatch repair system also contributes to genome stability by stimulating DNA damage-induced apoptosis as part of the cytotoxic response to physical and chemical agents. MutS/MutL homologues mediate the process of apoptosis by binding to DNA adducts and either provoking futile repair events or blocking steps in DNA metabolism (i.e., DNA replication and/or repair). This damage recognition step by mismatch repair (MMR) proteins stimulates a signaling cascade for apoptosis, resulting in activation of protein kinase(s) that phosphorylate p53 and/or the related protein p73. Activated p53 and p73 in turn transmit a signal to the apoptotic machinery to execute cell death. The goal of this commentary is to discuss the molecular mechanism(s) by which mismatch repair proteins stimulate apoptosis.     

DNA adduct levels and DNA repair polymorphisms in traffic-exposed workers and a general population sample.

Peripheral blood DNA adducts have been considered an acceptable surrogate for target tissues and possibly predictive of cancer risk. A group of 114 workers exposed to traffic pollution and a random sample of 100 residents were drawn from the EPIC cohort in Florence, a population recently shown to present increased DNA adduct levels (Palli et al., Int J Cancer 2000;87:444-51). DNA bulky adducts and 3 DNA repair gene polymorphisms were analyzed in peripheral leukocytes donated at enrollment, by using (32)P-postlabeling and PCR methods, respectively. Adduct levels were significantly higher for traffic workers among never smokers (p = 0.03) and light current smokers (p = 0.003). In both groups, urban residents tended to show higher levels than those living in suburban areas, and a seasonal trend emerged with adduct levels being highest in summer and lowest in winter. Traffic workers with at least 1 variant allele for XPD-Lys751Gln polymorphism had significantly higher levels in comparison to workers with 2 common alleles (p = 0.02). A multivariate analysis (after adjustment for age, season, area of residence, smoking, XPD-Lys751Gln genotype and antioxidant intake) showed a significant 2-fold association between occupational exposure and higher levels of adducts (odds ratio 2.1; 95% confidence interval 1.1-4.2), in agreement with recent pooled estimates of increased lung cancer risk for similar job titles. Our results suggest that traffic workers and the general population in Florence are exposed to high levels of genotoxic agents related to vehicle emissions. Photochemical pollution in warmer months might be responsible for the seasonal trend of genotoxic damage in this Mediterranean urbanized area.     

Platinum resistance: the role of DNA repair pathways.

Although platinum chemotherapeutic agents such as carboplatin, cisplatin, and oxaliplatin are used to treat a broad range of malignant diseases, their efficacy in most cancers is limited by the development of resistance. There are multiple factors that contribute to platinum resistance but alterations of DNA repair processes have been known for some time to be important in mediating resistance. Recently acquired knowledge has provided insight into the molecular mechanisms of DNA repair pathways and their effect on response to chemotherapy. This review will discuss the most important DNA repair pathways known to be involved in the platinum response, i.e., nucleotide excision repair (NER) and mismatch repair (MMR), and will briefly touch on the role of BRCA in DNA repair. The therapeutic implications of alterations in DNA repair which affect response to platinum in the treatment of patients with malignant disease, such as excision repair cross-complementation group 1 (ERCC1) deficiency and mismatch repair deficiency, will be reviewed.     

The role of PARP in DNA repair and its therapeutic exploitation

Historically, PARP inhibitors (PARPi) were developed to potentiate the cytotoxic effect of certain chemotherapeutic agents and are currently being investigated in combination with chemotherapy in diverse cancer types. These agents are also radiosensitisers and clinical trials of PARPi with concurrent radiation are required. It has long been recognised that defective DNA repair pathways lead to tumour susceptibility. Recent studies indicate that tumour cells with defective homologous recombination (HR) repair pathways, the classic example being BRCA mutations, are exquisitely sensitive to PARPi. Defects in HR are not restricted to BRCA-associated tumours and other cancer types may be enriched for HR defects and hence susceptible to PARP inhibition. The identification of predictive markers for sensitivity to PARP inhibition is a priority area for research.     

A role for mismatch repair in control of DNA ploidy following DNA damage

Many reports have shown a link between mismatch repair (MMR) deficiency and loss of normal cell cycle control, particularly loss of G2 arrest. However almost all of these studies utilized transformed cell lines, and thus the involvement of other genes in this phenotype cannot be excluded. We have examined the effects of cisplatin treatment on primary embryo fibroblasts (MEFs) derived from mice in which the MMR gene Msh2 had been inactivated (Msh2(-/-)). This analysis determined that both primary Msh2(-/-) and wild type (WT) fibroblasts exhibited an essentially identical G2 arrest following cisplatin treatment. Similarly, we observed a cisplatin-induced G2 arrest in immortalized MMR deficient (Mlh1(-/-) and Pms2(-/-)) and WT MEFs. p53 deficient primary MEFs (p53(-/-)) exhibited both a clear G2 arrest and an increase in cells with a DNA content of 8N in response to cisplatin. When the Msh2 and p53 defects were combined (p53(-/-)/Msh2(-/-)) the G2 arrest was essentially identical to the p53(-/-) fibroblasts. However, the p53(-/-)/Msh2(-/-) fibroblasts demonstrated a further increase in cells with an 8N DNA content, above that seen in the p53(-/-) fibroblasts. These results suggest that loss of MMR on its own is not enough to overcome G2 arrest following exposure to cisplatin but does play a role in preventing polyploidization, or aberrant DNA reduplication, in the absence of functional p53.     

Effect of phenazine compounds XR11576 and XR5944 on DNA topoisomerases

Purpose  Previous in vitro cleavage data showed that XR11576 and XR5944 stabilised topoisomerase I and topoisomerase II complexes on DNA in a dose-dependent fashion. However, some studies indicated a possible topoisomerase-independent mechanism of action for these drugs. Methods  Three methods, the TARDIS assay, immunoband depletion and the K⁺/SDS assay have been used to assess topoisomerase complex formation induced by XR11576 or XR5944 in human leukaemic K562 cells. Results  TARDIS and immunoband depletion assays demonstrated that XR11576 and XR5944 induced complex formation for both topoisomerase I and topoisomerase II (alpha and beta) in a dose- and time-dependent manner, following exposure times of 24 and 48 h at concentrations of 1 or 10 μM. The K⁺/SDS assay showed the formation of protein/DNA complexes after a 1 h exposure to 1 or 10 μM XR11576. Conclusion  Our data confirm that XR11576 or XR5944 can form topoisomerase complexes, after long periods of exposure.     

DNA损伤应答机制异常与MDS发生、发展的关系

在生命过程中,生物体的基因组不断受到攻击.识别并修复基因组的损伤对保证生物遗传性状稳定的重要性不言而喻.DNA损伤应答机制异常能够使错误的、甚至是有害的遗传信息在子代细胞中得以表达、并不断扩大,从而促进恶性肿瘤的发生、发展.在包括骨髓增生异常综合征(MDS)在内的血液系统恶性肿瘤中,染色体结构和数量异常比较常见.这些现象提示,在它们的起病和进展过程中,可能像实体肿瘤那样,存在着DNA损伤应答的缺陷,进而导致异常细胞克隆的形成和扩增.在诸多DNA修复机制中,错配修复缺陷的作用比较突出,并且与实体肿瘤的关系比较肯定.但在血液系统肿瘤中进行的类似研究尚未得出公认的结果.     

LGI1基因对胶质瘤细胞作用的研究

目的:研究富亮氨酸胶质瘤失活基因1(LGI1)与胶质瘤细胞生长及胶质瘤细胞凋亡的关系。方法:构建pcDNA3.1/LGI1质粒,采用脂质体介导的方法转染LGI1缺失的胶质瘤细胞系。RT-PCR法和免疫组化法检测转染细胞的LGI1基因表达,MTT法检测转染细胞的增殖活性,AnnevinV-FITC法检测转染细胞的凋亡。结果:pcDNA3.1/LGI1转染胶质瘤细胞系A172后,A172细胞LGI1mRNA和蛋白表达阳性;细胞增殖活性受到抑制,MTT吸收值在转染后24和48小时下降,与正常对照组相比有统计学意义(P<0.05);转染后24小时早期凋亡细胞较多,48小时后早期凋亡减少。结论:LGI1基因可抑制肿瘤细胞的增殖,诱导细胞凋亡。     

Various inhibitors of DNA topoisomerases diminish repair-specific DNA incision in UV-irradiated human fibroblasts

A function for topoisomerases I and II in DNA excision repaircan be postulated from the organization of the mammalian chromosome,involving nucleosomal structures and matrix-attached DNA loops.To analyse this function we determined UV-induced DNA incisionin confluent human fibroblasts in the presence of 16 inhibitorsof topoisomerases I and II which belonged to at least five differentdrug categories, based on their mechanism of action. Dose"responseexperiments were performed, analysed by linear regression andthe concentrations at which DNA-incising activity was reducedto 50% were calculated (K₅₀ values). The majority of these valuesrepresent concentrations for which interfering cell toxicitycould be excluded. K₅₀ concentrations, which were determinedby extrapolating dose-response data, may hit the toxicity range,nevertheless, we deem our K₅₀ scale useful for making biochemicalcomparisons. With respect to topoisomerase I, camptothecin andtopotecan diminished repair-specific DNA incision to a smallextent, whereas distamycin, which binds to the minor grooveof DNA, caused a stronger effect. With respect to topoisomerasen the results were as follows, (i) The DNA intercalator ethidiumbromide decreased DNA-incising activity at rather low concentrations,which indicates marked inhibitory potency. Quinacrine was lesseffective, (ii) Inhibitors intercalating and binding to the‘cleavable’ DNA-topoisomerase complex (m-AMSA, mitoxantrone,doxorubicin and daunorubicin) strongly suppressed reparativeDNA incision, (iii) Only small effects were observed using severaldrugs which act by trapping the ‘cleavable’ DNA-enzymecomplex, namely nalidixic add and oxolinic acid. In contrast,etoposide and teniposide inhibited post-UV DNA cleavage sizeably.(iv) Merbarone had to be applied at very high concentrationsto reduce UV-induced DNA incision, (v) Novobiocin, an inhibitorof the ATPase subunit of topoisomerase II, markedly diminishedrepair-specific DNA cleavage. A comparison of the K₅₀ valuesfor DNA incision with those for DNA repair synthesis (1) showsthat the majority of the investigated drugs inhibited both repairparameters. There were, however, differences in the concentrationsrequired to achieve the 50% inhibition level. The results arebest explained by assuming that in UV-irradiated human fibroblaststhe 180 kd form of topoisomerase II is a target enzyme for inhibitorswhich suppressed repair and that this isozyme is involved insteps preceding repair-specific DNA incision.