Frequency Of Chromosome Breaks Research Articles

Karyological Study of Acanthocephalus lucii (Echinorhynchida): The Occurrence of B Chromosomes in Populations from PCB-Polluted Waters

In this study, we performed a cytogenetic analysis of Acanthocephalus lucii specimens from three sites with different levels of environmental pollution. Standard and fluorochrome staining (CMA3/DAPI), fluorescence in situ hybridization (FISH) with 18S rDNA and histone H3 probes, and silver impregnation were performed. Chromosome complements of 2n = 7/8 (male/female), n = 1m + 2sm + 1a (X), and CMA3-positive bands in all chromosomes were found in all three populations. FISH revealed one 18S rDNA locus on the X chromosome and one locus of H3 histone genes on the first chromosome pair. At the intraspecific level, the populations differed in the presence of supernumerary B chromosomes, which were found in all specimens from Zemplínska Šírava and in 89.4% of specimens from the Laborec River, but not at the reference site. The first two sites are considered to be water bodies with high toxin contamination. Based on this fact, we assume an increased frequency of chromosome breaks leading to the formation of DNA fragments that have the potential to form B chromosomes. The present results add to the very limited data on the organization of multigene families in the genome of Acanthocephala and suggest a possible causal link between water pollution and the occurrence of B chromosomes in fish parasites.

Low dose rate γ-irradiation protects fruit fly chromosomes from double strand breaks and telomere fusions by reducing the esi-RNA biogenesis factor Loquacious

It is still continuously debated whether the low-dose/dose-rate (LDR) of ionizing radiation represents a hazard for humans. Model organisms, such as fruit flies, are considered valuable systems to reveal insights into this issue. We found that, in wild-type Drosophila melanogaster larval neuroblasts, the frequency of Chromosome Breaks (CBs), induced by acute γ-irradiation, is considerably reduced when flies are previously exposed to a protracted dose of 0.4 Gy delivered at a dose rate of 2.5 mGy/h. This indicates that this exposure, which is associated with an increased expression of DNA damage response proteins, induces a radioadaptive response (RAR) that protects Drosophila from extensive DNA damage. Interestingly, the same exposure reduces the frequency of telomere fusions (TFs) from Drosophila telomere capping mutants suggesting that the LDR can generally promote a protective response on chromatin sites that are recognized as DNA breaks. Deep RNA sequencing revealed that RAR is associated with a reduced expression of Loquacious D (Loqs-RD) gene that encodes a well-conserved dsRNA binding protein required for esiRNAs biogenesis. Remarkably, loss of Loqs mimics the LDR-mediated chromosome protection as it decreases the IR-induced CBs and TFs frequency. Thus, our molecular characterization of RAR identifies Loqs as a key factor in the cellular response to LDR and in the epigenetic routes involved in radioresistance.

Chromosomal Instability at Fragile Sites in Blue Foxes, Silver Foxes, and Their Interspecific Hybrids.

Simple SummaryThe paper describes the karyotypes of blue and silver foxes and their hybrids, in terms of the numbers of A and B chromosomes and the frequency of fragile sites on chromosomes. Genome stability in these species is affected by Robertson translocations in the karyotype of the blue fox and by B chromosomes in the silver fox. The fragile sites assay was used as a biomarker to assess genome stability in foxes. This test enables the identification of breaks, chromatid gaps, and deletions. In healthy individuals, the number of these instabilities remains low. The test can be used to select individuals with the most stable genome for breeding of blue and silver foxes. The fewer an individual’s susceptible sites, the more likely it is to have good reproductive performance. This factor is extremely important in the case of blue foxes, which are an endangered species.A cytogenetic assay based on fragile sites (FS) enables the identification of breaks, chromatid gaps, and deletions. In healthy individuals, the number of these instabilities remains low. Genome stability in these species is affected by Robertsonian translocations in the karyotype of the blue fox and by B chromosomes in the silver fox. The aims of the study were to characterise the karyotype of blue foxes, silver foxes, and their hybrids and to identify chromosomal fragile sites used to evaluate genome stability. The diploid number of A chromosomes in blue foxes ranged from 48 to 50, while the number of B chromosomes in silver foxes varied from one to four, with a constant number of A chromosomes (2n = 34). In interspecific hybrids, both types of karyotypic variation were identified, with the diploid number of A chromosomes ranging from 40 to 44 and the number of B chromosomes varying from 0 to 3. The mean frequency of FS in foxes was 4.06 ± 0.19: 4.61 ± 0.37 in blue foxes, 3.46 ± 0.28 in silver foxes, and 4.12 ± 0.22 in hybrids. A relationship was identified between an increased number of A chromosomes in the karyotype of the hybrids and the frequency of chromosomal breaks. The FS assay was used as a biomarker for the evaluation of genomic stability in the animals in the study.

Nitric oxide synthase-2 (NOS2) gene polymorphism c.1832C>T (Ser608Leu) associated with nitrosative stress in Fanconi anaemia.

Fanconi anemia (FA) occurs due to genomic instability with predisposition to bone marrow failure, phenotypic abnormalities and cancers. Though mutations in 22 genes leading to DNA repair defect have been identified, the cellular factor such as oxidative stress has also shown to be associated with FA. Nitrosative Stress (NS) is biochemically correlated to many oxidative stress related disorders and the NS as a pathological hallmark in FA has been so far overlooked. We carried out the study first time in Indian patients with FA with an objective to understand the role of NS in the pathogenesis of FA. The study was carried out in 70 FA subjects. The FA subjects were diagnosed by chromosomal breakage analysis. Molecular study was carried out by Next Generation Sequencing and Sanger sequencing. The 3-nitrotyrosine [3-NT] levels were estimated through enzyme-linked immuno-sorbent assay (ELISA) and the nitric oxide synthase genes- NOS1 (c.-420-34221G>A (rs1879417), c.-420-10205C>T (rs499776), c.4286+720G>C (rs81631)) and NOS2 (c.1823C>T (p. Ser608Leu) (rs2297518)) polymorphism were studied by direct sequencing. Chromosomal breakage analysis revealed a high frequency of chromosomal breaks (Mean chromosomal breakage-4.13 ± 1.5 breaks/metaphase) in 70 FA patients as compared to the control. Molecular studies revealed FANCA (58.34%), FANCG (18.34%) and FANCL (16.6%) complementation groups. The 3-nitrotyrosine [3-NT] levels showed to be significantly (p < 0.05) elevated in FA subjects when compared to the age match controls. Genotyping of the NOS2 gene c.1823C>T (p. Ser608Leu) (rs2297518), showed statistically significant (P < 0.05) association with FA. Elevated level of 3-NT is one of the cause of NS and NOS2 gene polymorphism associated with FA is an important target in the treatment regimen.

Severe telomere shortening in Fanconi anemia complementation group L.

Fanconi Anemia (FA) is a rare genetic disease with the incidence of 1 in 360,000 and is characterised by bone marrow failure, physical abnormalities, pancytopenia, and high frequency of chromosomal breakage and increased risk of evolving into malignancy. Telomere plays an important role in genomic stability, ageing process and cancers. Telomere shortening has been reported in FA. We studied telomere length in FA subjects and compared with complementation groups. Chromosomal breakage analysis from PHA stimulated, MMC induced peripheral blood culture was carried out in 37 clinically diagnosed FA. Molecular study of FANCA, G, and L was done through Sanger sequencing and next generation sequencing. Telomere length was estimated using real time quantitative polymerase chain reaction (qPCR) method. Student t-test was applied to test the significance. A high frequency of chromosomal breakage was observed in all the patients compared to healthy controls. We found significantly shorter telomere length in all the three complementation groups compare to age matched healthy controls. Among all complementation groups, FANCL showed severe telomere shortening (P value 0.0001). A negative correlation was observed between telomere length and chromosomal breakage frequency (R = -0.3116). Telomere shortening is not uncommon in FA subjects. However the telomere length shortening is different in complementation groups as FANCL showed severe telomere shortening in FA subjects. Though BM transplantation is essential for the management of the FA subjects, the telomere length can be considered as biological marker to understand the prognosis of the disease as FA subjects primarily treated with androgens.

RNF8 promotes high linear energy transfer carbon-ion-induced DNA double-stranded break repair in serum-starved human cells

The cell-killing effect of radiotherapy largely depends on unrepaired DNA double-stranded breaks (DSBs) or lethal chromosome aberrations induced by DSBs. Thus, the capability of DSB repair is critically important for the cancer-cell-killing effect of ionizing radiation. Here, we investigated the involvement of the DNA damage signaling factors ataxia telangiectasia mutated (ATM), ring finger protein 8 (RNF8), and RNF168 in quiescent G0/G1 cells, which are expressed in the majority of cell populations in tumors, after high linear energy transfer (LET) carbon-ion irradiation. Interestingly, ATM inhibition caused a substantial DSB repair defect after high-LET carbon-ion irradiation. Similarly, RNF8 or RNF168 depletion caused a substantial DSB repair defect. ATM inhibition did not exert an additive effect in RNF8-depleted cells, suggesting that ATM and RNF8 function in the same pathway. Importantly, we found that the RNF8 RING mutant showed a similar DSB repair defect, suggesting the requirement of ubiquitin ligase activity in this repair pathway. The RNF8 FHA domain was also required for DSB repair in this axis. Furthermore, the p53-binding protein 1 (53BP1), which is an important downstream factor in RNF8-dependent DSB repair, was also required for this repair. Importantly, either ATM inhibition or RNF8 depletion increased the frequency of chromosomal breaks, but reduced dicentric chromosome formation, demonstrating that ATM/RNF8 is required for the rejoining of DSB ends for the formation of dicentric chromosomes. Finally, we showed that RNF8 depletion augmented radiosensitivity after high-LET carbon-ion irradiation. This study suggests that the inhibition of RNF8 activity or its downstream pathway may augment the efficacy of high-LET carbon-ion therapy.

Influence of Polymorphisms of DNA Repair and GST Genes on Genotoxic Damage and Mutagen Sensitivity in Workers Occupationally Exposed to Very Low Doses of Ionizing Radiation

The study investigated the influence of genetic polymorphisms of the enzymes for DNA repair and detoxification of reactive intermediates on spontaneous and bleomycin-induced (BLM) genotoxic damage in 43 workers exposed to very low doses of ionizing radiation (IR) (mean cumulative dose 5.31 mSv) and 43 subjects with no occupational exposure to IR (controls). In all the subjects examined, the frequency of chromosome aberrations (CAs) and micronuclei (MN), both spontaneous and BLM-induced, the Comet assay parameters (tail intensity), the genotypic variants of the DNA repair enzymes XRCC1 (Arg194Trp, Arg280His, Arg399Gln), XRCC3 (Thr241Met), XPD (Lys751Gln), and of the detoxification enzymes GSTM1 and GSTT1 (null genotype) and BLMH (A1450G) were determined. Among the biomarkers considered, only the frequency of total CAs (p &lt; 0.05), and in particular of chromosome breaks (p &lt; 0.01), was found to be significantly higher in the exposed workers than the controls. The frequency of spontaneous MN was higher in subjects with at least one allelic variant in XRCC1 than in carriers of the wild-type, but again only in exposed workers (p = 0.046). Linear regression analysis showed a positive dependency of the frequency of spontaneous chromosome breaks on occupational exposure, and a dependency of the frequency of BLM-induced MN negative on occupational exposure and positive on alcohol consumption and the null GSTM1 genotype. In conclusion, the frequency of chromosome breaks seems to be a useful cytogenetic biomarker for exposure to very low doses of IR, while only the combined effect of different gene variants or genetic, occupational, and lifestyle habits factors seems to be able to modulate the genotoxic effect of very low doses of IR.

Depletion of ATP-Citrate Lyase (ATPCL) Affects Chromosome Integrity Without Altering Histone Acetylation in Drosophila Mitotic Cells.

The Citrate Lyase (ACL) is the main cytosolic enzyme that converts the citrate exported from mitochondria by the SLC25A1 carrier in Acetyl Coenzyme A (acetyl-CoA) and oxaloacetate. Acetyl-CoA is a high-energy intermediate common to a large number of metabolic processes including protein acetylation reactions. This renders ACL a key regulator of histone acetylation levels and gene expression in diverse organisms including humans. We have found that depletion of ATPCL, the Drosophila ortholog of human ACL, reduced levels of Acetyl CoA but, unlike its human counterpart, does not affect global histone acetylation and gene expression. Nevertheless, reduced ATPCL levels caused evident, although moderate, mitotic chromosome breakage suggesting that this enzyme plays a partial role in chromosome stability. These defects did not increase upon X-ray irradiation, indicating that they are not dependent on an impairment of DNA repair. Interestingly, depletion of ATPCL drastically increased the frequency of chromosome breaks (CBs) associated to mutations in scheggia, which encodes the ortholog of the mitochondrial citrate carrier SLC25A1 that is also required for chromosome integrity and histone acetylation. Our results indicate that ATPCL has a dispensable role in histone acetylation and prevents massive chromosome fragmentation when citrate efflux is altered.

Cancer chromosome breakpoints cluster in gene-rich genomic regions.

Cancer cells are characterized by chromosome abnormalities, of which some, in particular balanced rearrangements, are associated with distinct tumor entities and/or with specific gene rearrangements that represent important steps in the carcinogenic process. However, the vast majority of cytogenetically detectable structural aberrations in cancer cells have not been characterized at the nucleotide level; hence, their importance and functional consequences are unknown. By ascertaining the chromosomal breakpoints in 22 344 different clonal structural chromosome abnormalities identified in the karyotypes of 49 626 cases of neoplastic disorders we here show that the distribution of breakpoints is strongly associated (P < 0.0001) with gene content within the affected chromosomal bands. This association also remains highly significant in separate analyses of recurrent and nonrecurrent chromosome abnormalities as well as of specific subtypes of cancer (P < 0.0001 for all comparisons). In contrast, the impact of band length was negligible. The breakpoint distribution is thus not stochastic—gene‐rich regions are preferentially affected. Several genomic features relating to transcription, replication, and chromatin organization have been found to enhance chromosome breakage frequencies; this indicates that gene‐rich regions may be more break‐prone. The salient finding in the present study is that a substantial fraction of all structural chromosome abnormalities, not only those specifically associated with certain tumor types, may affect genes that are pathogenetically important. If this interpretation is correct, then the prevailing view that the great majority of cancer chromosome aberrations is cytogenetic noise can be seriously questioned.

Hypothermia modulates the DNA damage response to ionizing radiation in human peripheral blood lymphocytes

Purpose: Low temperature at exposure has been shown to act in a radioprotective manner at the level of cytogenetic damage. It was suggested to be due to an effective transformation of DNA damage to chromosomal damage at low temperature. The purpose of the study was to analyze the kinetics of aberration formation during the first hours after exposing human peripheral blood lymphocytes to ionizing radiation at 0.8 °C and 37 °C.Materials and methods: To this end, we applied the technique of premature chromosome condensation. In addition, DNA damage response was analyzed by measuring the levels of phosphorylated DNA damage responsive proteins ATM, DNA-PK and p53 and mRNA levels of the radiation-responsive genes BBC3, FDXR, GADD45A, XPC, MDM2 and CDKN1A.Results: A consistently lower frequency of chromosomal breaks was observed in cells exposed at 0.8 °C as compared to 37 °C already after 30 minutes postexposure. This effect was accompanied by elevated levels of phosphorylated ATM and DNA-PK proteins and a reduced immediate level of phosphorylated p53 and of the responsive genes.Conclusions: Low temperature at exposure appears to promote DNA repair leading to reduced transformation of DNA damage to chromosomal aberrations.

Clinical profile of children with Fanconi anemia from a tertiary center in Southern India

Objectives: The aim of this study is to observe the clinical spectrum of presentation of Fanconi anemia (FA) in children presenting to the Hematology Department of Victoria Hospital, Bangalore Medical College and Research Institute. Materials and Methods: This observational study was carried out from February 2010 to December 2016 on all the 16 consecutive children &lt;18 years of age, with FA. Those with high frequency of chromosomal breakage in G-banded metaphases from mitomycin-treated cultures were considered as positive cases. Results: The mean age at presentation was 6.79±1.34 years, but for those with dysmorphic featureson examination, it was 4.77±0.98 years. Symptoms of anemia were seen in 14 (87.5%) and of bleeding in 8 (50%) cases. There was a history of prolonged fever at presentation in 5 (31.2%) cases. Dysmorphic features on examination were seen in 10 (62.5%) cases. Short stature and microcephaly and microphthalmia were the most common features. Pancytopenia was seen in 68.7% of the subjects at presentation and anemia in 87.5% of cases while thrombocytopenia was universal (100%). The mean hemoglobin was 5.33±0.36 g/dL, mean white blood cell count was 3429.37±477.65/mm3, and mean platelet count was 28156.25±4199.44/mm3. The bone marrow was hypoplastic in 14 (87.5%). Pelvic kidney seen in 5 (31%) was the most common ultrasound finding. Parentalconsanguinity was seen in 50% of cases. Conclusion: Parental consanguinity was common, and there was a male preponderance in our study. The most common presenting manifestations were anemia followed by bleeding. Two-third of the patients had pancytopenia at diagnosis while thrombocytopenia was universal (100%). In the presence of a family history and/or dysmorphic features, even mono- or bi-cytopenias should be evaluated for inherited bone marrow failure syndromes.

Association between occupational exposure to benzene and chromosomal alterations in lymphocytes of Brazilian petrochemical workers removed from exposure.

We aimed to investigate the association between chronic exposure to benzene and genotoxicity in the lymphocytes of workers removed from exposure. The study included 20 workers with hematological disorders who had previously worked in the petrochemical industry of Salvador, Bahia, Brazil; 16 workers without occupational exposure to benzene served as the control group. Chromosomal analysis was performed on lymphocytes from peripheral blood, to assess chromosomal breaks and gaps and to identify aneuploidy. The Kruskal-Wallis test was used to compare the mean values between two groups, and Student's t test for comparison of two independent means. The frequency of gaps was statistically higher in and the exposed group than in the controls (2.13 ± 2.86 vs. 0.97 ± 1.27, p = 0.001). The frequency of chromosomal breaks was significantly higher among cases (0.21 ± 0.58) than among controls (0.12 ± 0.4) (p = 0.0002). An association was observed between chromosomal gaps and breaks and occupational exposure to benzene. Our study showed that even when removed from exposure for several years, workers still demonstrated genotoxic damage. Studies are still needed to clarify the long-term genotoxic potential of benzene after removal from exposure.

The Polymerase Activity of Mammalian DNA Pol ζ Is Specifically Required for Cell and Embryonic Viability.

DNA polymerase ζ (pol ζ) is exceptionally important for maintaining genome stability. Inactivation of the Rev3l gene encoding the polymerase catalytic subunit causes a high frequency of chromosomal breaks, followed by lethality in mouse embryos and in primary cells. Yet it is not known whether the DNA polymerase activity of pol ζ is specifically essential, as the large REV3L protein also serves as a multiprotein scaffold for translesion DNA synthesis via multiple conserved structural domains. We report that Rev3l cDNA rescues the genomic instability and DNA damage sensitivity of Rev3l-null immortalized mouse fibroblast cell lines. A cDNA harboring mutations of conserved catalytic aspartate residues in the polymerase domain of REV3L could not rescue these phenotypes. To investigate the role of REV3L DNA polymerase activity in vivo, a Rev3l knock-in mouse was constructed with this polymerase-inactivating alteration. No homozygous mutant mice were produced, with lethality occurring during embryogenesis. Primary fibroblasts from mutant embryos showed growth defects, elevated DNA double-strand breaks and cisplatin sensitivity similar to Rev3l-null fibroblasts. We tested whether the severe Rev3l-/- phenotypes could be rescued by deletion of DNA polymerase η, as has been reported with chicken DT40 cells. However, Rev3l-/- Polh-/- mice were inviable, and derived primary fibroblasts were as sensitive to DNA damage as Rev3l-/- Polh+/+ fibroblasts. Therefore, the functions of REV3L in maintaining cell viability, embryonic viability and genomic stability are directly dependent on its polymerase activity, and cannot be ameliorated by an additional deletion of pol η. These results validate and encourage the approach of targeting the DNA polymerase activity of pol ζ to sensitize tumors to DNA damaging agents.

Comparative genome integrity of riverine buffalos (Bubalus bubalis) under tropical climatic conditions

The purpose of the present study was to evaluate the genome integrity of indigenous buffalo breeds- Murrah and Nilli-Ravi (same age, sex and environmental conditions) in the northern states Punjab (Patiala) and Haryana (Hisar & Karnal) of India. The climographs based on the monthly mean temperature (30 years) and monthly mean relative humidity of Hisar, Karnal and Patiala regions were prepared for assessment of climatic conditions of particular regions in which animals were reared throughout the year. Seventy five buffaloes of the two breeds were used in this study. All animals were kept in organized herd at three different farm locations. Comparative cytogenetic analysis was made between the two breeds of buffalo. The cytogenetic studies of peripheral lymphocytes included chromosomal aberrations (CAs) and sister chromatid exchange (SCE). The mean number of gaps in Murrah buffalo of Karnal and Hisar regions were 12.48±4.12 and 10.86±3.65 respectively. Similarly, the mean number of gaps in Nilli-Ravi breed of buffalo from Patiala was 7.0±2.27. The mean frequency of chromosomal breaks in Murrah buffalo from Hisar and Karnal were 5.96±1.92 and 5.44±2.72 and 3.1±1.97 in Nilli-Ravi respectively, the difference was statistically significant. The frequency (mean±SD) of sister chromatid exchanges/per cell in Murrah buffaloes from Hisar and Karnal were 6.64±2.21 and 6.26±1.92 and for Nilli-Ravi was 3.52±1.87. The results of cytogenetic analysis between the two buffalo breeds reared under same climatic conditions at different farm locations (Haryana and Punjab) reveal significant ( P ≤ 0.01 ) differences in their genome integrity as detected by CAs and SCEs.

Genome Instability at Common Fragile Sites: Searching for the Cause of Their Instability

Common fragile sites (CFS) are heritable nonrandomly distributed loci on human chromosomes that exhibit an increased frequency of chromosomal breakage under conditions of replication stress. They are considered the preferential targets for high genomic instability from the earliest stages of human cancer development, and increased chromosome instability at these loci has been observed following replication stress in a subset of human genetic diseases. Despite their biological and medical relevance, the molecular basis of CFS fragility in vivo has not been fully elucidated. At present, different models have been proposed to explain how instability at CFS arises and multiple factors seem to contribute to their instability. However, all these models involve DNA replication and suggest that replication fork stalling along CFS during DNA synthesis is a very frequent event. Consistent with this, the maintenance of CFS stability relies on the ATR-dependent checkpoint, together with a number of proteins promoting the recovery of stalled replication forks. In this review, we discuss mainly the possible causes that threaten the integrity of CFS in the light of new findings, paying particular attention to the role of the S-phase checkpoint.

Chromosome Instability and Oxidative Stress Markers in Patients with Ataxia Telangiectasia and Their Parents

Ataxia telangiectasia (AT) is a rare neurodegenerative disorder, inherited in an autosomal recessive manner. Total blood samples were collected from 20 patients with AT, 13 parents of patients, and 17 healthy volunteers. This study aimed at evaluating the frequency of chromosomal breaks in spontaneous cultures, induced by bleomycin and ionizing radiation, and further evaluated the rates of oxidative stress in AT patients and in their parents, compared to a control group. Three cell cultures were performed to each individual: the first culture did not receive induction to chromosomal instability, the second was exposed to bleomycin, and the last culture was exposed to ionizing radiation. To evaluate the rates of oxidative stress, the markers superoxide dismutase (SOD), catalase (CAT), and thiobarbituric acid (TBARS) were utilized. Significant differences were observed between the three kinds of culture treatments (spontaneous, bleomycin, and radiation induced) and the breaks and chromosomal aberrations in the different groups. The oxidative stress showed no significant differences between the markers. This study showed that techniques of chromosomal instability after the induction of ionizing radiation and bleomycin are efficient in the identification of syndrome patients, with the ionizing radiation being the most effective.

Persistent genomic instability in peripheral blood lymphocytes from hodgkin lymphoma survivors

Advances in cancer treatment have led to an increase in patient survival. However, exposure to genotoxic chemotherapeutic agents and ionizing radiation may induce persistent genetic damage in cancer survivors. In this study, we detected genomic instability in chromosomes of peripheral blood lymphocytes from Hodgkin lymphoma patients, 2-17 years after MOPP (nitrogen mustard, Oncovin, procarbazine, and prednisone) chemotherapy with or without radiotherapy. Samples were obtained from 11 healthy individuals, 5 pretreatment patients, and 20 posttreatment patients. Cytogenetic analysis with GTG banding was performed on 1,000 lymphocyte metaphases per donor to identify genomic instability, including numerical and structural chromosomal aberrations, at a resolution of 10 Mb across the entire genome. Our results showed that anticancer treatment did not induce significant differences in the frequency of aneuploidy among the three study groups. However, 1 of the 11 healthy individuals, and 13 of the 20 posttreatment patients had a high frequency of chromosomal breaks and gross chromosomal rearrangements. The types of aberrations observed were random and complex, consistent with persistent genomic instability that was induced by cancer treatment. Clonal expansion of cells with chromosomal lesions was observed in one posttreatment patient only. These findings show that anticancer treatments induce persistent genomic instability, but not aneuploidy. Chemotherapy may affect genes with a role in DNA damage surveillance or repair, which in turn allows the accumulation of nontargeted structural chromosomal damage in future generations of cells. This genomic instability may facilitate the development of second malignancies in Hodgkin lymphoma survivors.

The rcbA Gene Product Reduces Spontaneous and Induced Chromosome Breaks in Escherichia coli

Elevated levels of DnaA cause excessive initiation, which leads to an increased level of double-strand breaks that are proposed to arise when newly formed replication forks collide from behind with stalled or collapsed forks. These double-strand breaks are toxic in mutants that are unable to repair them. Using a multicopy suppressor assay to identify genes that suppress this toxicity, we isolated a plasmid carrying a gene whose function had been unknown. This gene, carried by the cryptic rac prophage, has been named rcbA for its ability to reduce the frequency of chromosome breaks. Our study shows that the colony formation of strains bearing mutations in rep, recG, and rcbA, like recA and recB mutants, is inhibited by an oversupply of DnaA and that a multicopy plasmid carrying rcbA neutralizes this inhibition. These and other results suggest that rcbA helps to maintain the integrity of the bacterial chromosome by lowering the steady-state level of double-strand breaks.

An evaluation, using the comet assay and the micronucleus test, of the antigenotoxic effects of chlorophyll b in mice

We investigated the effects of the dietary pigment chlorophyll b (CLb) on cisplatin (cDDP)-induced oxidative stress and DNA damage, using the comet assay in mouse peripheral blood cells and the micronucleus (MN) test in bone marrow and peripheral blood cells. We also tested for thiobarbituric acid reactive substances (TBARS) and reduced glutathione (GSH) in liver and kidney tissues, as well as catalase (CAT) activity and GSH in total blood. CLb (0.2 and 0.5mg/kg b.w.) was administrated by gavage every day for 13 days. On the 14th day of the experiment, 6mg/kg cDDP or saline was delivered intraperitoneally. Treatment with cDDP led to a significant decrease in DNA migration and an increase in MN frequency in both cell types, bone marrow and peripheral blood cells. In the kidneys of mice treated with cDDP, TBARS levels were increased, whereas GSH levels were depleted in kidney and liver. In mice that were pre-treated with CLb and then treated with cDDP, TBARS levels maintained normal concentrations and GSH did not differ from cDDP group. The improvement of oxidative stress biomarkers after CLb pre-treatment was associated with a decrease in DNA damage, mainly for the highest dose evaluated. Furthermore, CLb also slightly reduced the frequency of chromosomal breakage and micronucleus formation in mouse bone marrow and peripheral blood cells. These results show that pre-treatment with CLb attenuates cDDP-induced oxidative stress, chromosome instability, and lipid peroxidation.

Small Ubiquitin-related Modifier Ligase Activity of Mms21 Is Required for Maintenance of Chromosome Integrity during the Unperturbed Mitotic Cell Division Cycle in Saccharomyces cerevisiae

The SUMO ligase activity of Mms21/Nse2, a conserved member of the Smc5/6 complex, is required for resisting extrinsically induced genotoxic stress. We report that the Mms21 SUMO ligase activity is also required during the unchallenged mitotic cell cycle in Saccharomyces cerevisiae. SUMO ligase-defective cells were slow growing and spontaneously incurred DNA damage. These cells required caffeine-sensitive Mec1 kinase-dependent checkpoint signaling for survival even in the absence of extrinsically induced genotoxic stress. SUMO ligase-defective cells were sensitive to replication stress and displayed synthetic growth defects with DNA damage checkpoint-defective mutants such as mec1, rad9, and rad24. MMS21 SUMO ligase and mediator of replication checkpoint 1 gene (MRC1) were epistatic with respect to hydroxyurea-induced replication stress or methyl methanesulfonate-induced DNA damage sensitivity. Subjecting Mms21 SUMO ligase-deficient cells to transient replication stress resulted in enhancement of cell cycle progression defects such as mitotic delay and accumulation of hyperploid cells. Consistent with the spontaneous activation of the DNA damage checkpoint pathway observed in the Mms21-mediated sumoylation-deficient cells, enhanced frequency of chromosome breakage and loss was detected in these mutant cells. A mutation in the conserved cysteine 221 that is engaged in coordination of the zinc ion in Loop 2 of the Mms21 SPL-RING E3 ligase catalytic domain resulted in strong replication stress sensitivity and also conferred slow growth and Mec1 dependence to unchallenged mitotically dividing cells. Our findings establish Mms21-mediated sumoylation as a determinant of cell cycle progression and maintenance of chromosome integrity during the unperturbed mitotic cell division cycle in budding yeast.