Showing papers on "Sister chromatid exchange published in 2011"

Aberrant chromosome morphology in human cells defective for Holliday junction resolution

Abstract: In somatic cells, Holliday junctions can be formed between sister chromatids during the recombinational repair of DNA breaks or after replication fork demise. A variety of processes act upon Holliday junctions to remove them from DNA, in events that are critical for proper chromosome segregation. In human cells, the BLM protein, inactivated in individuals with Bloom's syndrome, acts in combination with topoisomerase IIIα, RMI1 and RMI2 (BTR complex) to promote the dissolution of double Holliday junctions. Cells defective for BLM exhibit elevated levels of sister chromatid exchanges (SCEs) and patients with Bloom's syndrome develop a broad spectrum of early-onset cancers caused by chromosome instability. MUS81-EME1 (refs 4-7), SLX1-SLX4 (refs 8-11) and GEN1 (refs 12, 13) also process Holliday junctions but, in contrast to the BTR complex, do so by endonucleolytic cleavage. Here we deplete these nucleases from Bloom's syndrome cells to analyse human cells compromised for the known Holliday junction dissolution/resolution pathways. We show that depletion of MUS81 and GEN1, or SLX4 and GEN1, from Bloom's syndrome cells results in severe chromosome abnormalities, such that sister chromatids remain interlinked in a side-by-side arrangement and the chromosomes are elongated and segmented. Our results indicate that normally replicating human cells require Holliday junction processing activities to prevent sister chromatid entanglements and thereby ensure accurate chromosome condensation. This phenotype was not apparent when both MUS81 and SLX4 were depleted from Bloom's syndrome cells, suggesting that GEN1 can compensate for their absence. Additionally, we show that depletion of MUS81 or SLX4 reduces the high frequency of SCEs in Bloom's syndrome cells, indicating that MUS81 and SLX4 promote SCE formation, in events that may ultimately drive the chromosome instabilities that underpin early-onset cancers associated with Bloom's syndrome.

Assessment of genotoxicity of waterpipe and cigarette smoking in lymphocytes using the sister-chromatid exchange assay: a comparative study.

Abstract: Tobacco smoking is a major world health problem. Recently, waterpipe smoking has become more popular in many countries. Although the genotoxicity associated with cigarette smoking has been extensively investigated, studies evaluating such toxicity in waterpipe users are still lacking. In this study, we examined the genotoxicity of waterpipe smoking in lymphocytes compared with the genotoxicity of cigarette smoking. Genotoxicity was evaluated using the sister chromatid exchanges (SCEs) assay. Fifty waterpipe smokers and 18 healthy nonsmokers participated in this study. Additionally, 18 heavy cigarette smokers (CS) were recruited for comparison. The results show that waterpipe smoking and cigarette smoking significantly increase the frequencies of SCEs (P < 0.01) compared with those of nonsmokers, indicating the genotoxic effect of tobacco smoking. In addition, frequencies of SCEs were significantly higher among waterpipe smokers compared with CS (P < 0.01), indicating that waterpipe smoking is more genotoxic than cigarette smoking. Moreover, the frequency of SCEs increased with the extent of waterpipe use. In conclusion, waterpipe smoking is genotoxic to lymphocytes and the magnitude of its genotoxicity is higher than that induced by regular cigarette smoking.

Pyrimidine pool imbalance induced by BLM helicase deficiency contributes to genetic instability in Bloom syndrome

Abstract: Defects in DNA replication are associated with genetic instability and cancer development, as illustrated in Bloom syndrome. Features of this syndrome include a slowdown in replication speed, defective fork reactivation and high rates of sister chromatid exchange, with a general predisposition to cancer. Bloom syndrome is caused by mutations in the BLM gene encoding a RecQ helicase. Here we report that BLM deficiency is associated with a strong cytidine deaminase defect, leading to pyrimidine pool disequilibrium. In BLM-deficient cells, pyrimidine pool normalization leads to reduction of sister chromatid exchange frequency and is sufficient for full restoration of replication fork velocity but not the fork restart defect, thus identifying the part of the Bloom syndrome phenotype because of pyrimidine pool imbalance. This study provides new insights into the molecular basis of control of replication speed and the genetic instability associated with Bloom syndrome. Nucleotide pool disequilibrium could be a general phenomenon in a large spectrum of precancerous and cancer cells.

Regulation of sororin by Cdk1-mediated phosphorylation

Abstract: Tumor cells are commonly aneuploid, a condition contributing to cancer progression and drug resistance. Understanding how chromatids are linked and separated at the appropriate time will help uncover the basis of aneuploidy and will shed light on the behavior of tumor cells. Cohesion of sister chromatids is maintained by the multi-protein complex cohesin, consisting of Smc1, Smc3, Scc1 and Scc3. Sororin associates with the cohesin complex and regulates the segregation of sister chromatids. Sororin is phosphorylated in mitosis; however, the role of this modification is unclear. Here we show that mutation of potential cyclin-dependent kinase 1 (Cdk1) phosphorylation sites leaves sororin stranded on chromosomes and bound to cohesin throughout mitosis. Sororin can be precipitated from cell lysates with DNA–cellulose, and only the hypophosphorylated form of sororin shows this association. These results suggest that phosphorylation of sororin causes its release from chromatin in mitosis. Also, the hypophosphorylated form of sororin increases cohesion between sister chromatids, suggesting that phosphorylation of sororin by Cdk1 influences sister chromatid cohesion. Finally, phosphorylation-deficient sororin can alleviate the mitotic block that occurs upon knockdown of endogenous sororin. This mitotic block is abolished by ZM447439, an Aurora kinase inhibitor, suggesting that prematurely separated sister chromatids activate the spindle assembly checkpoint through an Aurora kinase-dependent pathway.

Roles of Vertebrate Smc5 in Sister Chromatid Cohesion and Homologous Recombinational Repair

Abstract: The structural maintenance of chromosomes (Smc) family members Smc5 and Smc6 are both essential in budding and fission yeasts. Yeast smc5/6 mutants are hypersensitive to DNA damage, and Smc5/6 is recruited to HO-induced double-strand breaks (DSBs), facilitating intersister chromatid recombinational repair. To determine the role of the vertebrate Smc5/6 complex during the normal cell cycle, we generated an Smc5-deficient chicken DT40 cell line using gene targeting. Surprisingly, Smc5− cells were viable, although they proliferated more slowly than controls and showed mitotic abnormalities. Smc5-deficient cells were sensitive to methyl methanesulfonate and ionizing radiation (IR) and showed increased chromosome aberration levels upon irradiation. Formation and resolution of Rad51 and gamma-H2AX foci after irradiation were altered in Smc5 mutants, suggesting defects in homologous recombinational (HR) repair of DNA damage. Ku70−/− Smc5− cells were more sensitive to IR than either single mutant, with Rad54−/− Smc5− cells being no more sensitive than Rad54−/− cells, consistent with an HR function for the vertebrate Smc5/6 complex. Although gene targeting occurred at wild-type levels, recombinational repair of induced double-strand breaks was reduced in Smc5− cells. Smc5 loss increased sister chromatid exchanges and sister chromatid separation distances in mitotic chromosomes. We conclude that Smc5/6 regulates recombinational repair by ensuring appropriate sister chromatid cohesion.

Protective role of methanol extract of Cetraria islandica (L.) against oxidative stress and genotoxic effects of AFB1 in human lymphocytes in vitro

Abstract: In this study, the antigenotoxic and antioxidant effects of Cetraria islandica methanol (CME) extract were determined by using sister chromatid exchange (SCE), micronuclei (MN) assays and superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities and malondialdehyde (MDA) levels against effects of aflatoxin B1 (AFB 1) induced oxidative stress and genotoxicity in human lymphocytes in vitro The results showed that the frequencies of SCE, MN and MDA level decreased, SOD and GPx activities increased when 5 μg/mL and 10 μg/mL doses of CME were added to AFBi-treated cultures Also, the present results indicate that CME has strong antioxidative and the antigenotoxicity mechanisms of CME are associated with its antioxidant nature

Sister chromatid exchanges occur in G2-irradiated cells

Abstract: DNA double-strand breaks (DSBs) are arguably the most important lesions induced by ionizing radiation (IR) since unrepaired or mis-repaired DSBs can lead to chromosomal aberrations and cell death. The two major pathways to repair IR-induced DSBs are non-homologous end-joining (NHEJ) and homologous recombination (HR). Perhaps surprisingly, NHEJ represents the predominant pathway in the G1 and G2 phases of the cell cycle, but HR also contributes and repairs a subset of IR-induced DSBs in G2. Following S-phase-dependent genotoxins, HR events give rise to sister chromatid exchanges (SCEs), which can be detected cytogenetically in mitosis. Here, we describe that HR occurring in G2-irradiated cells also generates SCEs in ~50% of HR events. Since HR of IR-induced DSBs in G2 is a slow process, SCE formation in G2-irradiated cells requires several hours. During this time, irradiated S-phase cells can also reach mitosis, which has contributed to the widely held belief that SCEs form only during S phase. We describe procedures to measure SCEs exclusively in G2-irradiated cells and provide evidence that following IR cells do not need to progress through S phase in order to form SCEs.

Genotoxic effect of occupational exposure to cadmium

Abstract: Many studies proved the genotoxic effect of cadmium (Cd) exposure and highlighted the importance of the cytogenetic studies as a sensitive and effective means for early detection of Cd-induced mutagenicity. The relationship between occupational exposure to Cd and increased risk of cancer, particularly lung cancer, has been explored in number of epidemiological studies. The aim of this study is to assess the role of chromosomal abnormalities and sister chromatid exchange as sensitive indicators for the genotoxicity of occupational exposure to Cd. Cytogenetic studies was done for 40 workers (27 smokers and 13 non-smokers) exposed to Cd dust and fumes with 40 control subjects (28 smokers and 12 non-smokers) not exposed to Cd before. Both exposed and control groups were similar in age and other sociodemographic factors. Clinical examination, laboratory investigation including urinary and blood Cd, cytogenic analysis for detection of chromosomal aberrations and sister chromatid exchange and environmental study of the work places were done. Statistical analysis of cytogenetic studies revealed the presence of significant elevation of chromosomal aberrations and sister chromatid exchanges of the exposed group. Urinary and blood Cd of the exposed group were significantly higher than that of the control group. These abnormalities were not significantly affected with age, duration of exposure, smoking habits, blood and urinary Cd. In conclusion, the study adds more proof that Cd exposure has a genotoxic effect and highlighted the importance of using cytogenetic studies as a sensitive and effective means for early detection of Cd-induced mutagenicity.

Essential Roles of BCCIP in Mouse Embryonic Development and Structural Stability of Chromosomes

Abstract: BCCIP is a BRCA2- and CDKN1A(p21)-interacting protein that has been implicated in the maintenance of genomic integrity. To understand the in vivo functions of BCCIP, we generated a conditional BCCIP knockdown transgenic mouse model using Cre-LoxP mediated RNA interference. The BCCIP knockdown embryos displayed impaired cellular proliferation and apoptosis at day E7.5. Consistent with these results, the in vitro proliferation of blastocysts and mouse embryonic fibroblasts (MEFs) of BCCIP knockdown mice were impaired considerably. The BCCIP deficient mouse embryos die before E11.5 day. Deletion of the p53 gene could not rescue the embryonic lethality due to BCCIP deficiency, but partially rescues the growth delay of mouse embryonic fibroblasts in vitro. To further understand the cause of development and proliferation defects in BCCIP-deficient mice, MEFs were subjected to chromosome stability analysis. The BCCIP-deficient MEFs displayed significant spontaneous chromosome structural alterations associated with replication stress, including a 3.5-fold induction of chromatid breaks. Remarkably, the BCCIP-deficient MEFs had a ∼20-fold increase in sister chromatid union (SCU), yet the induction of sister chromatid exchanges (SCE) was modestly at 1.5 fold. SCU is a unique type of chromatid aberration that may give rise to chromatin bridges between daughter nuclei in anaphase. In addition, the BCCIP-deficient MEFs have reduced repair of irradiation-induced DNA damage and reductions of Rad51 protein and nuclear foci. Our data suggest a unique function of BCCIP, not only in repair of DNA damage, but also in resolving stalled replication forks and prevention of replication stress. In addition, BCCIP deficiency causes excessive spontaneous chromatin bridges via the formation of SCU, which can subsequently impair chromosome segregations in mitosis and cell division.

Trex2 Enables Spontaneous Sister Chromatid Exchanges Without Facilitating DNA Double-Strand Break Repair

Abstract: Trex2 is a 3′ → 5′ exonuclease that removes 3′-mismatched sequences in a biochemical assay; however, its biological function remains unclear. To address biology we previously generated trex2null mouse embryonic stem (ES) cells and expressed in these cells wild-type human TREX2 cDNA (Trex2hTX2) or cDNA with a single-amino-acid change in the catalytic domain (Trex2H188A) or in the DNA-binding domain (Trex2R167A). We found the trex2null and Trex2H188A cells exhibited spontaneous broken chromosomes and trex2null cells exhibited spontaneous chromosomal rearrangements. We also found ectopically expressed human TREX2 was active at the 3′ ends of I-SceI–induced chromosomal double-strand breaks (DSBs). Therefore, we hypothesized Trex2 participates in DNA DSB repair by modifying 3′ ends. This may be especially important for ends with damaged nucleotides. Here we present data that are unexpected and prompt a new model. We found Trex2-altered cells (null, H188A, and R167A) were not hypersensitive to camptothecin, a type-1 topoisomerase inhibitor that induces DSBs at replication forks. In addition, Trex2-altered cells were not hypersensitive to γ-radiation, an agent that causes DSBs throughout the cell cycle. This observation held true even in cells compromised for one of the two major DSB repair pathways: homology-directed repair (HDR) or nonhomologous end joining (NHEJ). Trex2 deletion also enhanced repair of an I-SceI–induced DSB by both HDR and NHEJ without affecting pathway choice. Interestingly, however, trex2null cells exhibited reduced spontaneous sister chromatid exchanges (SCEs) but this was not due to a defect in HDR-mediated crossing over. Therefore, reduced spontaneous SCE could be a manifestation of the same defect that caused spontaneous broken chromosomes and spontaneous chromosomal rearrangements. These unexpected data suggest Trex2 does not enable DSB repair and prompt a new model that posits Trex2 suppresses the formation of broken chromosomes.

The template choice decision in meiosis: is the sister important?

Abstract: Recombination between homologous chromosomes is crucial to ensure their proper segregation during meiosis. This is achieved by regulating the choice of recombination template. In mitotic cells, double-strand break repair with the sister chromatid appears to be preferred, whereas interhomolog recombination is favoured during meiosis. However, in the last year, several studies in yeast have shown the importance of the meiotic recombination between sister chromatids. Although this thinking seems to be new, evidences for sister chromatid exchange during meiosis were obtained more than 50 years ago in non-model organisms. In this mini-review, we comment briefly on the most recent advances in this hot topic and also describe observations which suggest the existence of inter-sister repair during meiotic recombination. For instance, the behaviour of mammalian XY bivalents and that of trivalents in heterozygotes for chromosomal rearrangements are cited as examples. The “rediscovering” of the requirement for the sister template, although it seems to occur at a low frequency, will probably prompt further investigations in organisms other than yeast to understand the complexity of the partner choice during meiosis.

The Assessment of Cytotoxicity and Genotoxicity of Tetracycline Antibiotic in Human Blood Lymphocytes Using CBMN and SCE Analysis, in Vitro

Abstract: Tetracycline antibiotic, a widely used antimicrobial drug was tested for nuclear DNA damage in cultured peripheral blood lymphocytes in terms of chromosome alterations. The extent of cytogenetic da...

Rtt107 Phosphorylation Promotes Localisation to DNA Double-Stranded Breaks (DSBs) and Recombinational Repair between Sister Chromatids

Abstract: Efficient repair of DNA double-stranded breaks (DSB) requires a coordinated response at the site of lesion. Nucleolytic resection commits repair towards homologous recombination, which preferentially occurs between sister chromatids. DSB resection promotes recruitment of the Mec1 checkpoint kinase to the break. Rtt107 is a target of Mec1 and serves as a scaffold during repair. Rtt107 plays an important role during rescue of damaged replication forks, however whether Rtt107 contributes to the repair of DSBs is unknown. Here we show that Rtt107 is recruited to DSBs induced by the HO endonuclease. Rtt107 phosphorylation by Mec1 and its interaction with the Smc5–Smc6 complex are both required for Rtt107 loading to breaks, while Rtt107 regulators Slx4 and Rtt101 are not. We demonstrate that Rtt107 has an effect on the efficiency of sister chromatid recombination (SCR) and propose that its recruitment to DSBs, together with the Smc5–Smc6 complex is important for repair through the SCR pathway.

Sister chromatid exchange in selected horse breeds (Equus caballus)

Abstract: . In studies of chromosome instability, the sister chromatid exchange (SCE) test is a particularly sensitive cytogenetic assay for detecting DNA damage. SCE tests of chromosome instability were performed in the group of 6 horse breeds (Pure-bred Arabian, Malapolski horse, Polish noble half-bred, Polish cold-blooded, Hucul and Polish Konik). The chromosome preparations were obtained from our in vitro culture of peripheral blood lymphocytes stained using the FPG technique. The mean number of SCEs/cell in the analysed population of horses was 5.14±1.44. The mean frequency of SCEs in the 6 analysed horse breeds varied depending on the breed. Statistically significant differences were observed between the horse breeds (P No statistically significant differences in the number of SCEs per cell were found between the males and females (5.10±1.34 and 5.20±1.52, respectively). The horses were also assessed for the number of SCEs/cell in relation to the age of the animals. The differences between the age groups were statistically significant (P

A role for GPN-loop GTPase yGPN1 in sister chromatid cohesion.

Abstract: Sister chromatid cohesion and separation, involving the cohesin complex, are crucial for accurate inheritance of genetic information. This complex is also fundamental for efficient post-replicative repair of DNA double-strand breaks and has a key role in the mechanisms of gene transcription control. Cohesin is subjected to many post-translational modifications but the regulators implicated in the control of its activity have been poorly described. Here, we show that the conserved and essential GPN loop GTPase yGPN1 in Saccharomyces cerevisiae is involved in sister chromatid cohesion mechanisms. Based on a sister chromatid cohesion assay, we found that over-expression of the yGPN1 gene promotes sister chromatid separation during anaphase. The sharp slowdown in progression of the S phase observed in cells where yGPN1 expression is down-regulated strongly suggests that yGPN1 is necessary for DNA replication. Moreover, analysis of yGPN1 protein-protein interaction network highlights the yGPN1 links with DNA replication, sister chromatid cohesion/separation and the gene expression process.

Cytogenetic study of the effect of Schistosoma mansoni infection on human peripheral blood lymphocytes and the role of β-carotene and vitamin E in modulating this effect.

Abstract: This study has been made to determine the potential genotoxicity of Schistosoma mansoni on lymphocytes of infected patients using different mutagenic end points. The protective role of antioxidants pro vitamin β-carotene and vitamin E in minimizing these genotoxic effect was also studied. The study focused on the effect of schistosomiasis on the induction of sister chromatid exchange (SCEs) and other chromosomal aberrations. This work was conducted on 24 Schistosoma mansoni infected patients and 10 healthy adults as a control group. Lymphocytes from peripheral blood of patients and control group were used for culture and subsequent cytogenetic studies. The results indicated that schistosomiasis was genotoxic in all examined tests. It induced a significant increase in the percentage of structural chromosomal aberrations and the frequency of SCEs. It also inhibited cell division and caused cell cycle delay. Lymphocyte cultures of S. mansoni patients treated with 10 μg/ml β-carotene or 20 mg/ml vitamin E showed a significant decrease in the percentage of structural chromosomal aberrations and the frequency of SCEs. Schistosomiasis has a genotoxic effect on peripheral blood lymphocytes. The use of the antioxidants β-carotene and vitamin E can be considered a promising approach not only toward inhibiting the genetic damage of schistosomiasis but also as prophylactic agents against infection with S mansoni. Furthermore, higher doses of antioxidant drugs, β-carotene and vitamin E, should be tried as an adjuvants to conventional therapy in a trial to improve treatment of schistosomiasis.

Intercellular Communication in Response to Radiation Induced Stress: Bystander Effects in Vitro and in Vivo and Their Possible Clinical Implications

Abstract: Communication between cells is important for maintaining homeostasis, the physiological regulatory processes that keep the internal environment of a system in a constant state. A disease can disturb the internal equilibrium of cells, and this can be further disrupted by various therapies. Malignances are the diseases that need to be treated by highly aggressive methods, such as radiotherapy, which affects not only tumor cells but also normal cells adjacent to the tumor and usually included in the radiation field. This treatment may interfere with normal intercellular communication. It has been a central radiobiological dogma for decades that damaging effects of ionizing radiation are the result of direct ionization of cell structures, particularly DNA, or are due to indirect damage via water radiolysis products. Indeed, DNA damage such as chromosomal aberrations, micronuclei, sister chromatid exchange and mutagenesis result from ionizing radiation. All of these types of damage, if unrepaired, can lead to cell death or, if misrepaired, can lead to genomic instability and carcinogenesis. Recently however, the attention was focused on the third mechanism, a phenomenon termed “radiation induced bystander effect” (RIBE). This phenomenon is a non-targeted effect where molecular signal(s) produced by directly irradiated cells elicit subsequent responses in unirradiated neighbors. These responses are manifested as decreased survival, increased sister chromatid exchanges (SCE), chromosomal aberrations (CA), micronucleus (MN) formation, gene mutations, apoptosis, genomic instability, neoplastic transformation and a variety of damage-inducible stress responses (reviewed in Morthersill and Seymour, 2001, Lorimore et al., 2003, Morgan, 2003a, 2003b, Little, 2006a,b, Chapman et al. 2008, Rzeszowska-Wolny et al., 2009a). Bystander effect accompanies very low doses of alpha particles (mGy and cGy), (Nagasawa and Little, 1992, Lorimore et al., 1998), as well as irradiation of cells with a low LET radiation (Xand gamma rays), even at conventionally used higher clinical doses (Morthersill and Seymour, 1997, 1998, 2002b, Przybyszewski et al., 2004). The mechanisms responsible for RIBE are complex and not quite well-known. Mechanisms by which bystander signals may be transmitted from irradiated to non-irradiated cells involve direct cell-to-cell contact mediated by gap

Cytogenetic Analysis Of Micronuclei, Sister Chromatid Exchange And Chromosomal Aberrations In Pan Masala Chewers

Abstract: Pan Masala (PM) chewing is very dangerous for health but it is becoming very popular day by day. PM is a dried powdered mixture containing ingredients like areca nut, catechu, lime, cardamom and flavouring agents. It is consumed abundantly by Indians and is also exported to Western countries. A cytogenetic study to assess the micronucleus (MN), sister chromatid exchange (SCE) levels and chromosomal aberrations among (CA) 60 pan chewers and 60 non-chewers was conducted in Chennai, Tamil Nadu. In the present cytogenetic monitoring study, analysis of MN was significantly higher (15.82 ± 1.31) in chewers than controls (4.82 ± 1.47) (P < 0.001) and SCE also was significantly higher in chewers (9.23± 2.12) than controls (4.80±1.11) (P < 0.001). In exfoliated buccal mucosa and chromosome analysis (CA), frequency of chromatid type aberrations is lower in controls than chewers such as gaps (0.90% v. 1.83%) breaks (0.47% v. 1.77%), exchanges (0.02% v. 0.18) and acentric fragments (0.20% v. 0.90%). The increased percentage of aberrations found among pan chewers is significantly higher when compared to that of the controls. Isochromatid aberrations also increased significantly such as gaps (0.12% v. 0.97%) breaks (0.07% v. 0.80%), acentric fragments (0.05% v. 0.23%), dicentrics (0.02% v. 0.63%), and these were estimated in the peripheral blood lymphocytes to assess possible DNA damage. All three cytogenetic endpoints demonstrated a statistically significant increase (P < 0.001) among the pan masala chewers as compared with the non-consuming controls. In conclusion, we propose the use of the cytokinesisblock micronucleus assay, sister chromatid exchange and chromosome aberrations for getting a sensitive information on cell cycle progression and cytotoxicity of chewing pan masala. V. RAMAKRISHNAN Department of Genetics, Chettinad University, Kelambakkam, Chennai603103, India. Mail: rkgenes@gmail.com

Atypically low spontaneous sister chromatid exchange formation in uveal melanoma.

Abstract: Uveal melanoma (UM) is the most common primary intraocular cancer of adults and is characterized by several well-established chromosomal changes. More recently, a specific mutation of guanine nucleotide binding protein Gq alpha subunit (GNAQ) has also been identified in a proportion of UM. Although some of these alterations have been suggested to be early changes, the genetic alterations responsible for the development of UM have yet to be clearly determined. Cancers are characterized by increased genetic instability, and analysis of established cancer cell lines and blood from cancer patients has universally been associated with an increased level of sister chromatid exchange (SCE). We have observed that the spontaneous frequency of SCE in primary cultures of UM and UM-derived cell lines is decreased below normal baseline levels, a phenomenon unique to UM when compared with multiple other cancers. This finding was specific to the tumor and not found in lymphocytes from the patients. Although we cannot exclude the possibility that low SCE (LSCE) is peculiar to the uveal melanocytes lineage, as it was consistently observed in all UM studied, regardless of other genetic defects, we propose that this phenomenon contributes to the molecular pathogenesis of UM. © 2010 Wiley-Liss, Inc.

Recombination phenotypes of the NCI-60 collection of human cancer cells.

Abstract: The NCI-60 is a collection of tumor cell lines derived from a variety of human adult cancer tissue types and is commonly used for genetic analysis and screening of potential chemotherapeutic agents. We wanted to understand the contributions of specific mechanisms of genomic instability to the etiology of cancers represented by the NCI-60. We screened the NCI-60 for dysregulated homologous recombination by using the gene cluster instability (GCI) assay we pioneered, and for defects in base excision repair by sensitivity to 5-hydroxymethyl-2'-deoxyuridine (hmdUrd). We identified subsets of the NCI-60 lines that either displayed the characteristic molecular signature of GCI or were sensitive to hmdUrd. With the exception of the NCI-H23 lung cancer line, these phenotypes were not found to overlap. None of the lines examined in either subset exhibited significant changes in the frequency of sister chromatid exchanges (SCE), neither did any of the lines in either subset exhibit microsatellite instability (MSI) indicative of defects in DNA mismatch repair. Gene cluster instability, sensitivity to hmdUrd and sister chromatid exchange are mechanistically distinct phenomena. Genomic instability in the NCI-60 appears to involve only one mechanism of instability for each individual cell line.

p53 null Fluorescent Yellow Direct Repeat (FYDR) mice have normal levels of homologous recombination

Abstract: The tumor suppressor p53 is a transcription factor whose function is critical for maintaining genomic stability in mammalian cells. In response to DNA damage, p53 initiates a signaling cascade that results in cell cycle arrest, DNA repair or, if the damage is severe, programmed cell death. In addition, p53 interacts with repair proteins involved in homologous recombination. Mitotic homologous recombination (HR) plays an essential role in the repair of double-strand breaks (DSBs) and broken replication forks. Loss of function of either p53 or HR leads to an increased risk of cancer. Given the importance of both p53 and HR in maintaining genomic integrity, we analyzed the effect of p53 on HR in vivo using Fluorescent Yellow Direct Repeat (FYDR) mice as well as with the sister chromatid exchange (SCE) assay. FYDR mice carry a direct repeat substrate in which an HR event can yield a fluorescent phenotype. Here, we show that p53 status does not significantly affect spontaneous HR in adult pancreatic cells in vivo or in primary fibroblasts in vitro when assessed using the FYDR substrate and SCEs. In addition, primary fibroblasts from p53 null mice do not show increased susceptibility to DNA damage-induced HR when challenged with mitomycin C. Taken together, the FYDR assay and SCE analysis indicate that, for some tissues and cell types, p53 status does not greatly impact HR.

In vitro genotoxicity of rocuronium bromide in human peripheral lymphocytes

Abstract: Rocuronium bromide (RB), an aminosteroid type neuromuscular blocking agent, acts by reducing or inhibiting the depolarising effect of acetylcholine on the terminal disc of the muscle cell. To our knowledge, there is no adequate information on the genotoxic effects of RB, up to now. In the present study, possible genotoxic effects of RB have been determined by means of sister chromatid exchange (SCE), chromosome aberration (CA) and micronucleus (MN) analyses in human peripheral blood lymphocytes. The human peripheral blood lymphocytes were exposed to three different concentrations of RB (60, 80 and 100 μg/mL) for 24- and 48-h. In this study, RB increased the frequency of CAs, however, did not increase the frequency of SCEs. RB did not decrease the proliferation index (PI) and mitotic index (MI). Accordingly, RB increased the frequency of micronucleus (MN) but did not decrease the nuclear division index (NDI). Findings from this study suggest that rocuronium bromide is clastogenic but not cytotoxic to cultured human peripheral blood lymphocytes.

Chromosomal Damage Risk Assessment to Benzene Exposure among Gasoline Station Workers in Bangkok Metropolitan, Thailand

Abstract: This study was a cross-sectional survey study to assess relative risk (RR) of chromosomal damage through benzene exposure in 45 gasoline stations workers compared to 30 controls in central area of Bangkok Sister chromatid exchange (SCE) is as genotoxic biomarker, performed in white blood cells, and blood benzene level (BBL) is a biological marker of benzene exposure was performed by gas chromatography-flame ionization detector (GC-FID) using modified headspace solid-phase micro-extraction (HS-SPME) technique The results showed that the average blood benzene level of these workers was significantly higher than in the controls ( p < 0001) as well as the frequency of sister chromatid exchange The sister chromatid exchange was strongly and positively associated with blood benzene level of gasoline workers ( p < 0001) with the chromosomal damage relative risk at 250 ( p < 0001) Keywords : gasoline worker, benzene, sister chromatid exchange, chromosomal damage

Comparison of the frequency of sister chromatid exchange in pan chewers and oral submucous fibrosis patients.

Abstract: Sister chromatid exchange (SCE) test is a sensitive, biomarker of genotoxic substances. The frequency of SCE in lymphocytes of ten pan chewing patients, oral submucous fibrosis (OSF) patients and age matched healthy controls were investigated. The frequency of mean SCE/cell was found to be 10.428 ± 0.755 in OSF patients, 8.752 ± 0.383 in case of pan chewers as compared to 5.912 ± 0.310 in controls. These values show a significant increase in frequency of SCE/cell in OSF patients and pan chewers when compared with that of healthy controls. There is a positive correlation co-efficient of SCE/cell with frequency, quantity, duration, intensity and period of exposure of pan-parag to oral mucosa in pan chewers and OSF patients indicating genotoxic effect of pan. Thus SCE could be used as a biomarker in chewers also to assess the level of genomic damage and to advocate efficient control measures.

The evaluation of genotoxic potential of ornidazole, nitroimidazole, in lymphocyte culture of patients with amebiasis.

Abstract: The genotoxicity study of ornidazole (ONZ) was carried out on human lymphocyte chromosomes, using sister chromatid exchange (SCE) and micronucleus (MN). Thirty-two patients with Entemoeba histolitica infection who received 1000 mg/day for 10 days were included in this study. SCE and MN were measured before and after therapy. A statistically significant increase was observed in the SCE (P < 0.001) and MN frequencies (P < 0.001) after ornidazole therapy. It was concluded that ONZ has a potential geno- and cytotoxic effect in human peripheral lymphocyte cultures. For this reason, further, detailed studies are needed to elucidate the ONZ mechanism of genotoxicity and its carcinogenic potential.