Repair of bleomycin-damaged DNA by human fibroblasts

The ability of human fibroblasts to repair bleomycin-damaged DNA was examined in vivo. Repair of the specific lesions caused by bleomycin (BLM) was investigated in normal cell strains as well as those isolated from patients with apparent DNA repair defects. The diseases ataxia telangiectasia (AT), Bloom syndrome (BS), Cockayne syndrome (CS), Fanconi anemia (FA), and xeroderma pigmentosum (XP) were those selected for study. The method used for studying the repair of DNA after BLM exposure was alkaline sucrose gradient centrifugation. After exposure to BLM, a fall in the molecular weight of DNA was observed, and after drug removal the DNA reformed rapidly to high molecular weight. The fall in molecular weight upon exposure to BLM was observed in all cells examined with the exception of some XP strains. Prelabeled cells from some XP complementation groups were found to have a higher percentage of low molecular weight DNA on alkaline gradients than did normal cells. This prelabeled low molecular weight DNA disappeared upon exposure to BLM.     

Molecular characterization of mutations at the hprt locus in V79 Chinese hamster cells induced by bleomycin in the presence of inhibitors of DNA repair.

The molecular basis of bleomycin (BLM)-induced mutations in the absence and presence of inhibitors of DNA repair was investigated in V79 cells with Southern hybridization techniques. 43% of the BLM-induced thioguanine-resistant mutants suffer from large alterations of hprt DNA sequences. To understand the role of DNA repair in the process of mutagenesis, the effect of inhibitors of DNA repair on the frequency and types of BLM-induced mutations was tested. The inhibitors used were arabinofuranosyl cytosine (araC), didesoxythymidine (ddThd) and 3-aminobenzamide (3AB), which inhibit different steps of excision repair. Only 3AB caused a comutagenic effect. The increased mutation frequency was mainly due to additionally induced gene deletions. In the presence of 3AB, 70% of the HPRT-deficient mutants revealed partial or total deletions of the hprt coding sequences. Thus, it could be shown that BLM induces a broad range of types of mutation and that inhibited repair of BLM-induced DNA damage leads to specific types of mutations.     

Werner protein protects nonproliferating cells from oxidative DNA damage

Werner syndrome, caused by mutations of the WRN gene, mimics many changes of normal aging. Although roles for WRN protein in DNA replication, recombination, and telomere maintenance have been suggested, the pathology of rapidly dividing cells is not a feature of Werner syndrome. To identify cellular events that are specifically vulnerable to WRN deficiency, we used RNA interference (RNAi) to knockdown WRN or BLM (the RecQ helicase mutated in Bloom syndrome) expression in primary human fibroblasts. Withdrawal of WRN or BLM produced accelerated cellular senescence phenotype and DNA damage response in normal fibroblasts, as evidenced by induction of gammaH2AX and 53BP1 nuclear foci. After WRN depletion, the induction of these foci was seen most prominently in nondividing cells. Growth in physiological (3%) oxygen or in the presence of an antioxidant prevented the development of the DNA damage foci in WRN-depleted cells, whereas acute oxidative stress led to inefficient repair of the lesions. Furthermore, WRN RNAi-induced DNA damage was suppressed by overexpression of the telomere-binding protein TRF2. These conditions, however, did not prevent the DNA damage response in BLM-ablated cells, suggesting a distinct role for WRN in DNA homeostasis in vivo. Thus, manifestations of Werner syndrome may reflect an impaired ability of slowly dividing cells to limit oxidative DNA damage.     

The kinetics of DNA damage by bleomycin in mammalian cells.

The kinetics of DNA damage by bleomycin (BLM) was assessed by measuring the amount of DNA breakage induced by BLM at different doses, treatment lengths, and treatment temperatures. DNA degradation was measured with the alkaline unwinding method. Comparison of the curves of DNA cleavage by BLM leads to the conclusion that low doses (1-5 micrograms/ml) and short treatments (5-15 min) produce marked damage in the DNA. High increases in BLM concentration produce relatively small increases in DNA damage above the levels obtained with low doses. Extension of treatment times does not increase the DNA degradation above the rate observed with 15-min treatments. The repair of DNA damage starts at about 15 min after the initiation of treatment. The mending of DNA breaks is very fast and extensive when BLM is no longer present. Repair not only implies the closing of DNA nicks, but very likely the degradation of the BLM molecules intercalated in the DNA interrupting the reactions responsible for the generation of free radicals. Persistence of BLM in the cell environment facilitates the replacement of degraded BLM molecules by new ones. This produces the persistent production of free radicals and the establishment of a balance between the processes of DNA damage and repair.     

Cellular and molecular effects of bleomycin are modulated by heat shock in Saccharomyces cerevisiae

To study some mechanisms underlying the stress responses in eukaryotic cells, we investigated the effect of heat shock (HS) on the induction of DNA double strand breaks as well as on potentially lethal and mutagenic events induced by the radiomimetic antibiotic bleomycin (BLM) in Saccharomyces cerevisiae. Haploid wild-type yeast cells in the logarithmic phase of growth were exposed to different concentrations of BLM (0-30 microg/ml, 1.5 h) without and with a previous HS (38 degrees C, 1 h). Immediately after treatments, survival as well as mutation frequency were determined, and quantitative analysis of chromosomal DNA by laser densitometry were performed both immediately after treatments and after incubation of cells during different time intervals in liquid nutrient medium free of BLM. Our results indicate that HS induces resistance to potentially lethal and mutagenic effects of BLM. Quantitative analysis of chromosomal DNA performed immediately after treatments showed the same DNA fragmentation, either upon BLM as single agent or preceded by HS. However, HS pretreated cells incubated during 4 h in liquid nutrient medium free of BLM repaired DNA double strand breaks more efficiently as compared to non-pretreated cells. On this basis, we propose that the observed HS-induced resistance to BLM depends on a regulatory network acting after DNA-induced damage, which includes genes involved in DNA repair, HS response and DNA metabolism.     

USP37 regulates DNA damage response through stabilizing and deubiquitinating BLM

The human RecQ helicase BLM is involved in the DNA damage response, DNA metabolism, and genetic stability. Loss of function mutations in BLM cause the genetic instability/cancer predisposition syndrome Bloom syndrome. However, the molecular mechanism underlying the regulation of BLM in cancers remains largely elusive. Here, we demonstrate that the deubiquitinating enzyme USP37 interacts with BLM and that USP37 deubiquitinates and stabilizes BLM, thereby sustaining the DNA damage response (DDR). Mechanistically, DNA double-strand breaks (DSB) promotes ATM phosphorylation of USP37 and enhances the binding between USP37 and BLM. Moreover, knockdown of USP37 increases BLM polyubiquitination, accelerates its proteolysis, and impairs its function in DNA damage response. This leads to enhanced DNA damage and sensitizes breast cancer cells to DNA-damaging agents in both cell culture and in vivo mouse models. Collectively, our results establish a novel molecular mechanism for the USP37–BLM axis in regulating DSB repair with an important role in chemotherapy and radiotherapy response in human cancers.     

The Organic Selenium Compound Selenomethionine Modulates Bleomycin-Induced DNA Damage and Repair in Human Leukocytes

The objective of this work was to evaluate the effects of selenomethionine (SeMet) on the induction, repair, and persistence of DNA damage in human leukocytes challenged with bleomycin (BLM). Comet assay was used to determine DNA strand breaks and hOGG1 for the specific recognition of oxidative damage. Leukocytes were (A) stimulated with phytohemagglutinin, (B) damaged with BLM, and (C) incubated to allow DNA repair. Comet assay was performed after each phase. SeMet (50 μM) was supplemented either during phase A, B, or C, or AB, or ABC. Treatment with SeMet decreased BLM-induced stand breaks when added during phase AB. Results obtained after the repair period indicate that SeMet favors repair of DNA damage especially when applied during phase AB. The comparison between DNA damage before and after repair showed that BLM-induced damage was repaired better in the presence of SeMet. Our results showed antigenotoxic effect of SeMet on BLM-induced DNA and also on repair and persistence of this damage when applied before and simultaneously with BLM.     

Chromatin condensation and differential sensitivity of mammalian and insect cells to DNA strand breaks induced by bleomycin

Bleomycin (BLM) induces DNA damage in living cells. In this report we analyzed the role of chromatin compactness in the differential response of mosquito (ATC-15) and mammalian (CHO) cells to DNA strand breaks induced by BLM. We used cells unexposed and exposed to sodium butyrate (NaB), which induces chromatin decondensation. By nucleoid sedimentation assay and digestions of nuclei with DNAse I, untreated mosquito cells (no BLM; no NaB) were shown to have more chromatin condensation than untreated CHO cells. By alkaline unwinding ATC-15 cells treated with NaB showed more BLM-induced DNA strand breaks than NaB-untreated CHO cells. The time-course of BLM-induced DNA damage to nuclear DNA was similar for NaB-untreated mammalian and insect cells, but with mosquito cells showing less DNA strand breaks, both at physiological temperatures and at 4 °C. However, when DNA repair was inhibited by low temperatures and chromatin was decondensed by NaB treatments, differences in BLM-induced DNA damage between these cells lines were no longer observed. In both cell lines, NaB did not affect BLM action on cell growth and viability. On the other hand, the low sensitivity of ATC-15 cells to BLM was reflected in their better growth efficiency. These cells exhibited a satisfactory growth at BLM doses that produced a permanent arrest of growth in CHO cells. The data suggest that mosquito cells might have linker DNAs shorter than those of mammalian cells, which would result in the observed both greater chromatin condensation and greater resistance to DNA damage induced by BLM as compared to CHO cells.     

Bleomycin-induced DNA-strand damage in isolated male germ cells

DNA strand damage in isolated male germ cells (MGC) was evaluated after in vitro exposure to bleomycin (BLM), a known genotoxin. The alkaline elution technique was used to determine DNA-strand breaks. Concentration-dependent strand damage was established following exposure to bleomycin for 1 h at 37 degrees C. Exposure at 0 degrees C resulted in an increase in the frequency of strand breaks as compared to those observed at 37 degrees C. Pretreatment of cells with deferoxamine (DM), an iron-selective chelating agent, abolished the DNA damage induced by bleomycin. Isolated male germ cells responded in a predictable and reproducible manner thus supporting their use in mechanistic studies of genotoxicity.     

Formation of single-stranded breaks in newly-synthesized Escherichia coli DNA following treatment with bleomycin

Changes in molecular weight of newly synthesized DNA was studied after bleomycin treatment of Escherichia coli cells. The treatment by this drug causes only the increase of dispersion in sedimentation profiles of daughter DNA strands in wild type cells. There are two alternative explanation of this fact. First, single-strand breakage does not occur in newly synthesized DNA, i.e. bleomycin-induced athyminic sites do not block cellular DNA polymerases. Second, it is possible to explain it by quick rejoining of given breaks by cell repair systems. The sedimentation profile of daughter DNA strands of recA mutant rules out the first possibility. Observed shift to low molecular weight fractions region strongly indicates the formation of single-strand breaks in newly synthesized DNA. Extensive daughter DNA degradation in xthA mutant supports the idea of the existence of very effective excision repair in the case of apyrimidinic sites. Thus, non-eliminated bleomycin-induced damage causes the formation of single-strand breaks in newly synthesized DNA strands. These breaks may be repaired in the course of recA-dependent post-replication repair.     

Opposite responses in two DNA repair capacity tests in lymphocytes of head and neck cancer patients

Genomic instability has long been recognized as the main feature of neoplasia and a factor modulating individual cancer susceptibility. There are attempts to find effective assays of both individual DNA repair capacity and genetic instability, and their relation to the cancer risk. Genetic predisposition plays an important role in the etiology and development of head and neck squamous cell carcinoma (HNSCC). The aim of our study was to search for a correlation between chromosomal instability and DNA repair capacity in HNSCC patients and healthy controls. The chromosomal instability was measured by the number of bleomycin (BLM)-induced chromosomal aberrations and diepoxybutane (DEB)-induced sister chromatid exchanges. The DNA repair capacity was assessed using the DEB-induced adaptive response (AR). The HNSCC patients in our study showed a significant increase in chromosomal instability after a preterminal exposure of their lymphocytes to either BLM for the last 5 h or DEB for the last 24 h of incubation. However, the AR was higher in HNSCC patients than in the control group, suggesting an increase in the DNA repair capacity in the cancer patients as compared to the control. There is no correlation between the DNA repair capacity estimated on the basis of preterminal exposures to BLM and DEB and the DNA repair capacity estimated on the basis of the adaptive response to DEB. The preterminal exposure and the adaptive response test may activate different DNA repair mechanisms.     

Increased sensitivity to chromatid aberration induction by bleomycin and neocarzinostatin results from alterations in a DNA damage response pathway

DNA damage response pathways coordinate the cellular response to DNA damage. To investigate the roles of tumor suppressor genes in these pathways, human lymphoblastoid cells (wild-type, p53-/-, ATM-/-) were treated for 1 h with 0-3 microg/ml of the radiomimetic compound bleomycin (BLM), and cells treated in G(2) were analyzed for chromatid aberrations. BLM-induced aberration frequencies were significantly increased, to the greatest extent in the ATM-/- cells and, to a lesser extent, in the p53-/- cells compared to wild-type cells. These observations are consistent with p53 and ATM acting in a damage response pathway activated by DNA strand breaks. The consequences of disrupting this pathway were further investigated by studies using wortmannin, a PI-3 kinase and DNA repair inhibitor. Wortmannin significantly increased the BLM-induced aberration frequencies in all but the ATM-/- cells, elevating the sensitivity of p53-/- cells to ATM-/- levels and that of wild-type cells to intermediate levels. These differential sensitivities suggest that the ATM phenotype is the result of dual cellular defects, one involving p53 and the other a wortmannin-sensitive component. Similar studies in Brca1+/- and Brca2+/- human lymphoblasts showed no increased sensitization to BLM in the absence of inhibitor, and differential sensitization by wortmannin. To determine if there was any substrate specificity for p53- and ATM-mediated DNA damage responses, chromatid aberrations were assessed in wild-type, p53-/-, and ATM-/- cells exposed to 0-0.4 microg/ml neocarzinostatin (NCS) for 1 h. In contrast to results with BLM, the p53-/- cells exhibited a low sensitivity to NCS-induced aberrations, similar to wild-type, while ATM-/- cells remained highly sensitive. This suggests that the response to BLM- and NCS-induced lesions involves different mechanisms.     

Filamin-A as a marker and target for DNA damage based cancer therapy

Filamin-A, also called actin binding protein 280 (ABP-280), cross-links the actin filaments into dynamic orthogonal network to serve as scaffolds in multiple signaling pathways. It has been reported that filamin-A interacts with DNA damage response proteins BRCA1 and BRCA2. Defects of filamin-A impair the repair of DNA double strand breaks (DSBs), resulting in sensitization of cells to ionizing radiation. In this study, we sought to test the hypothesis that filamin-A can be used as a target for cancer chemotherapy and as a biomarker to predict cancer response to therapeutic DNA damage. We found that reduction of filamin-A sensitizes cancer cells to chemotherapy reagents bleomycin and cisplatin, delays the repair of not only DSBs but also single strand breaks (SSBs) and interstrand crosslinks (ICLs), and increases chromosome breaks after the drug treatment. By treating a panel of human melanoma cell lines with variable filamin-A expression, we observed a correlation between expression level of filamin-A protein and drug IC(50). We further inhibited the expression of filamin-A in melanoma cells, and found that this confers an increased sensitivity to bleomycin and cisplatin treatment in a mouse xenograft tumor model. These results suggest that filamin-A plays a role in repair of a variety of DNA damage, that lack of filamin-A is a prognostic marker for a better outcome after DNA damage based treatment, and filamin-A can be inhibited to sensitize filamin-A positive cancer cells to therapeutic DNA damage. Thus filamin-A can be used as a biomarker and a target for DNA damage based cancer therapy.     

DNA damage evaluated by alkaline single cell gel electrophoresis (SCGE) in children of Chernobyl, 10 years after the disaster

Using the alkaline single cell gel electrophoresis (Comet) assay, the extent of DNA damage was evaluated in leukocytes of 43 Belarussian children (16 healthy and 27 affected by thyroid cancer). Thirty-nine healthy children from Pisa (Italy) were enrolled in the study as controls. In addition to basal levels of DNA damage, leukocytes were treated in vitro with bleomycin (BLM), a radiomimetic drug, to evaluate a possible adaptive response in different groups of children. Results with the Comet assay indicated an increased level of DNA damage (P=0.037) in leukocytes of Belarussian children compared to the Italian control group. In addition, within the Belarus group, lower basal levels of DNA damage (P<0.001) were found in children with cancer compared to healthy children. Tumor affected children were living in less radiocontaminated areas (P<0.04) than the healthy children and there was a significant relationship (P=0.03) between the amount of environmental radiocontamination and DNA damage in leukocytes. There were no differences in the sensitivity of leukocytes from different groups of children to BLM, indicating the absence of an adaptive response. The lack of an adaptive response may have been due to the use of noncycling cells and/or the bleomycin dose chosen. Tests for the presence of clastogenic factors (CF) in the blood serum of children showed that 39% of the tumor affected children and 19% of the healthy children in the exposed group were positive as compared to the Italian control group (0%) (Chi-square test, P<0.04). The higher levels of genomic damage in children evaluated 10 years after the Chernobyl disaster could be related to the increased incidence of individuals with CF.     

A novel cell-cycle-regulated interaction of the Bloom syndrome helicase BLM with Mcm6 controls replication-linked processes

The Bloom syndrome DNA helicase BLM contributes to chromosome stability through its roles in double-strand break repair by homologous recombination and DNA replication fork restart during the replication stress response. Loss of BLM activity leads to Bloom syndrome, which is characterized by extraordinary cancer risk and small stature. Here, we have analyzed the composition of the BLM complex during unperturbed S-phase and identified a direct physical interaction with the Mcm6 subunit of the minichromosome maintenance (MCM) complex. Using distinct binding sites, BLM interacts with the N-terminal domain of Mcm6 in G1 phase and switches to the C-terminal Cdt1-binding domain of Mcm6 in S-phase, with a third site playing a role for Mcm6 binding after DNA damage. Disruption of Mcm6-binding to BLM in S-phase leads to supra-normal DNA replication speed in unperturbed cells, and the helicase activity of BLM is required for this increased replication speed. Upon disruption of BLM/Mcm6 interaction, repair of replication-dependent DNA double-strand breaks is delayed and cells become hypersensitive to DNA damage and replication stress. Our findings reveal that BLM not only plays a role in the response to DNA damage and replication stress, but that its physical interaction with Mcm6 is required in unperturbed cells, most notably in S-phase as a negative regulator of replication speed.     

Reactions of oxyl radicals with DNA

The importance of radical-induced damage to DNA is apparent from the ever-increasing number of publications in this area. This review focuses on the damage caused to DNA by reactive oxygen-centred radicals, however formed. These may be hydroxyl radicals, which arise either from the radiolysis of water by ionizing radiation (γ-rays or X-rays), or from a purely chemical source. Alternatively, metal-bound oxyl radicals (M–O·) are also active intermediates in DNA-cleaving reactions and may be formed from synthetic compounds or from natural products such as bleomycin (BLM). Chemical mechanisms leading to the observed degradation products are covered in detail. The biological effects of some of the DNA base lesions formed are touched upon, concentrating on the molecular mechanisms behind the initial events that lead to mutagenesis.     

Sensitivity of RECQL4-deficient fibroblasts from Rothmund–Thomson syndrome patients to genotoxic agents

RECQ helicase protein-like 4 (RECQL4) is a member of the human RECQ family of DNA helicases. Two-thirds of patients with Rothmund–Thomson syndrome (RTS) carry biallelic inactivating mutations in the RECQL4 gene. RTS is an autosomal recessive disorder characterized by poikiloderma, sparse hair, small stature, skeletal abnormalities, cataracts, and an increased risk of cancer. Mutations in two other RECQ helicases, BLM and WRN, are responsible for the cancer predisposition conditions Bloom and Werner syndromes, respectively. Previous studies have shown that BLM and WRN-deficient cells demonstrate increased sensitivity to hydroxyurea (HU), camptothecin (CPT), and 4-nitroquinoline 1-oxide (4NQO). Little is known about the sensitivity of RECQL4-deficient cells to these and other genotoxic agents. The purpose of this study was to determine if RTS cells display any distinct cellular phenotypes in response to DNA damaging agents or replication blocks that could provide insight into the molecular function of the RECQL4 protein. Our results show that primary fibroblasts from RTS patients carrying two deleterious RECQL4 mutations, compared to wild type (WT) fibroblasts, have increased sensitivity to HU, CPT, and doxorubicin (DOX), modest sensitivity to other DNA damaging agents including ultraviolet (UV) irradiation, ionizing radiation (IR), and cisplatin (CDDP), and relative resistance to 4NQO. The RECQ family of DNA helicases has been implicated in the regulation of DNA replication, recombination, and repair. Because HU, CPT, and DOX exert their effects primarily during S phase, these results support a greater role for the RECQL4 protein in DNA replication as opposed to repair of exogenous damage. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The repair of bleomycin-induced DNA damage and its relationship to chromosome aberration repair.

Previous studies using the technique of premature chromosome condensation indicated that nearly one-half of the bleomycin-induced chromatid breaks and gaps in CHO cells could be repaired within 1 h (repair starting at 30 min) after treatment. Cycloheximide and streptovitacin A (but not hydroxyurea or hycanthone) inhibited chromosome repair. The purpose of this study was to measure the kinetics of DNA repair after bleomycin treatment using the alkaline elution technique and to determine whether various inhibitors could block this repair. After bleomycin treatment, the major proportion of the repair of DNA damage occurred within 15 min, with significant repair evident by 2 min. This fast repair component was inhibited by 0.2% EDTA. A slower repair component was observed to occur up to 60 min after bleomycin treatment. None of the inhibitors tested were found to have a significant effect on the repair of bleomycin damage at the DNA level. Since chromosome breaks were observed not to begin repair until after 30 min while over 50% of the DNA was repaired by 15 min, these results suggest that the DNA lesions that are repaired quickly are not important in the formation of chromosome aberrations. Further, since cycloheximide and streptovitacin A blocked chromosome repair but had little measurable effect on DNA repair, these results suggest that the DNA lesions responsible for chromosome damage represent only a small proportion of the total DNA lesions produced by bleomycin.