Sister Chromatid Exchange Rates Research Articles

The Determination of the Effects of Wild Thyme (Thymbra spicata L.) and Cumin (Cuminum cyminum L.) Extracts on SCE (Sister Chromatid Exchange) in Human Lymphocyte Chromosomes

The purpose of this work is to determine of T. spicata L. and C. cyminum L. plant extracts on sister chromatid exchange rate in human peripheral lymphocyte culture as in vitro . Human blood lymphocyte cells, T. spicata L. and C. cyminum L. plant extracts were allowed to interact with doses of 0.05 µL/mL, 0.10 µL/mL, 0.15 µL/mL and 0.20 µL/mL for 24 hours. When trial groups where plant extracts were applied were compared with negative control and mitomycin-C (MMC) which was used as positive control, it was determined that the extract doses applied led to an increase in sister chromatid exchange rate. Also, it was found that increasing concentrations of plant extracts caused cell replication index to decrease. Between C. cyminum L. doses and RI, a negative correlation (r = -0.95) was observed and between T. spicata L . doses and RI a negative correlation was observed (r = -0.94) respectively. As a result of this study; T. spicata L. and C. cyminum L. plant extracts were found to have genotoxic and clastogenic effects on human peripheral lymphocytes.

Cellular protection induced by genistein in mouse and its antioxidant capacity

Pharmacognosy Magazine ,2019,15,66,520-526.DOI:10.4103/pm.pm_78_19Published:November 2019Type:Original Article Authors:Rogelio Paniagua-Pérez, Susana Reyes-Cadena, Carlos Martínez-Canseco, Celia Reyes-Legorreta, Jesús Martínez-Castro, Eduardo O Madrigal-Santillán, José A Morales-González, José M Cristóbal-Luna, Isela Álvarez-González, and Eduardo Madrigal-Bujaidar Author(s) affiliations:Rogelio Paniagua-Pérez1, Susana Reyes-Cadena1, Carlos Martínez-Canseco1, Celia Reyes-Legorreta1, Jesús Martínez-Castro2, Eduardo O Madrigal-Santillán3, José A Morales-González3, José M Cristóbal-Luna4, Isela Álvarez-González4, Eduardo Madrigal-Bujaidar4 1Department of Biochemistry, National Institute of Rehabilitation, Mexico City, México 2Department of Computer, Computer Research Center, National Polytechnic Institute, Mexico City, México 3Department of Graduate, Conservation Medicine Laboratory, National Polytechnic Institute, Mexico City, México 4Department of Morphology, Genetics Laboratory, National Polytechnic Institute, Mexico City, México Abstract:Background: Genistein (GT) is an isoflavone phytoestrogen present in a number of plants. The chemical has been reported to have antioxidant, antigenotoxic, and cancer-preventive qualities; however, no studies against cisplatin (CP) have been reported. Objective: The main objective of the study is to determine the capacity of GT to inhibit the genotoxic and cytotoxic damage induced by CP in mouse, as well as its immunostimulant ability and its capacity to scavenge free radicals. Materials and Methods: We determined the effect of six doses of GT on the rate of sister chromatid exchanges (SCEs) and of micronuclei (MN) in mice administered with 5 mg/kg of CP. Besides, we determined its capacity to increase the amount of lymphocytes in mouse and to reduce oxidation with the 2,2-diphenyl-1-picrylhydrazyl assay. Results: Our results showed that GT (10–60 mg/kg) significantly decreased the frequency of SCE and of MN in mice. Furthermore, we also observed a moderate bone marrow cytotoxic correction of the damage induced by CP, as shown by an improvement in the rate of polychromatic erythrocytes. In addition, with 60 mg/kg, GT increased 69.6% the production of mouse lymphocytes over the control value throughout a 72-h trial. Moreover, the compound also showed a high capacity to trap free radicals (95.25%, with 250 μg/ml). Conclusion: Our results, therefore, established that GT is an effective cellular protective agent against the action of CP. Keywords:2, 2-diphenyl-1-picrylhydrazyl, cisplatin, genistein, lymphocytes, micronuclei, sister chromatid exchangeView:PDF (949.43 KB)

Cytoprotective and genotoxic effects of vitamins K1 and B1 on irinotecan in vitro

Cultured human lymphocytes were treated with vitamins K1 and B1, potential anticancer agents, either alone or in combination with irinotecan, a semisynthetic analogue of camptothecin. The frequency of sister chromatid exchanges (SCEs) was measured as an indicator of genotoxicity and the proliferation rate index (PRI) and mitotic index (MI) was measured as indicators of cytostatic effect. Vitamin K1 alone did not induce SCEs at the concentrations tested and combined with irinotecan does not increase SCE rates induced by irinotecan alone.Vitamin B1 significantly increased SCEs and, in combination with irinotecan, increased rates further (p < 0.05). Vitamin K1 decreased PRI and MI in combination with irinotecan, there were further increases in MI. At a low concentration, vitamin B1 reduced the levels of SCE and increased PRI induced by irinotecan. The use of these vitamins in combination with antitumor agents might reduce clinical side effects of the antineoplastics.

Mutations in TOP3A Cause a Bloom Syndrome-like Disorder

Bloom syndrome, caused by biallelic mutations in BLM, is characterized by prenatal-onset growth deficiency, short stature, an erythematous photosensitive malar rash, and increased cancer predisposition. Diagnostically, a hallmark feature is the presence of increased sister chromatid exchanges (SCEs) on cytogenetic testing. Here, we describe biallelic mutations in TOP3A in ten individuals with prenatal-onset growth restriction and microcephaly. TOP3A encodes topoisomerase III alpha (TopIIIα), which binds to BLM as part of the BTRR complex, and promotes dissolution of double Holliday junctions arising during homologous recombination. We also identify a homozygous truncating variant in RMI1, which encodes another component of the BTRR complex, in two individuals with microcephalic dwarfism. The TOP3A mutations substantially reduce cellular levels of TopIIIα, and consequently subjects’ cells demonstrate elevated rates of SCE. Unresolved DNA recombination and/or replication intermediates persist into mitosis, leading to chromosome segregation defects and genome instability that most likely explain the growth restriction seen in these subjects and in Bloom syndrome. Clinical features of mitochondrial dysfunction are evident in several individuals with biallelic TOP3A mutations, consistent with the recently reported additional function of TopIIIα in mitochondrial DNA decatenation. In summary, our findings establish TOP3A mutations as an additional cause of prenatal-onset short stature with increased cytogenetic SCEs and implicate the decatenation activity of the BTRR complex in their pathogenesis.

Genome-wide mapping of sister chromatid exchange events in single yeast cells using Strand-seq.

Homologous recombination involving sister chromatids is the most accurate, and thus most frequently used, form of recombination-mediated DNA repair. Despite its importance, sister chromatid recombination is not easily studied because it does not result in a change in DNA sequence, making recombination between sister chromatids difficult to detect. We have previously developed a novel DNA template strand sequencing technique, called Strand-seq, that can be used to map sister chromatid exchange (SCE) events genome-wide in single cells. An increase in the rate of SCE is an indicator of elevated recombination activity and of genome instability, which is a hallmark of cancer. In this study, we have adapted Strand-seq to detect SCE in the yeast Saccharomyces cerevisiae. We provide the first quantifiable evidence that most spontaneous SCE events in wild-type cells are not due to the repair of DNA double-strand breaks.

Loss of RMI2 Increases Genome Instability and Causes a Bloom-Like Syndrome.

Bloom syndrome is a recessive human genetic disorder with features of genome instability, growth deficiency and predisposition to cancer. The only known causative gene is the BLM helicase that is a member of a protein complex along with topoisomerase III alpha, RMI1 and 2, which maintains replication fork stability and dissolves double Holliday junctions to prevent genome instability. Here we report the identification of a second gene, RMI2, that is deleted in affected siblings with Bloom-like features. Cells from homozygous individuals exhibit elevated rates of sister chromatid exchange, anaphase DNA bridges and micronuclei. Similar genome and chromosome instability phenotypes are observed in independently derived RMI2 knockout cells. In both patient and knockout cell lines reduced localisation of BLM to ultra fine DNA bridges and FANCD2 at foci linking bridges are observed. Overall, loss of RMI2 produces a partially active BLM complex with mild features of Bloom syndrome.

Mechanistic insight into cadmium-induced inactivation of the Bloom protein.

Cadmium is a toxic metal that inactivates DNA-repair proteins via multiple mechanisms, including zinc substitution. In this study, we investigated the effect of Cd2+ on the Bloom protein (BLM), a DNA-repair helicase carrying a zinc-binding domain (ZBD) and playing a critical role to ensure genomic stability. One characteristics of BLM-deficient cells is the elevated rate of sister chromatid exchanges, a phenomenon that is also induced by Cd2+. Here, we show that Cd2+ strongly inhibits both ATPase and helicase activities of BLM. Cd2+ primarily prevents BLM-DNA interaction via its binding to sulfhydryl groups of solvent-exposed cysteine residues and, concomitantly, promotes the formation of large BLM multimers/aggregates. In contrast to previously described Cd2+ effects on other zinc-containing DNA-repair proteins, the ZBD appears to play a minor role in the Cd2+-mediated inhibition. While the Cd2+-dependent formation of inactive multimers and the defect of DNA-binding were fully reversible upon addition of EDTA, the inhibition of the DNA unwinding activity was not counteracted by EDTA, indicating another mechanism of inhibition by Cd2+ relative to the targeting of a catalytic residue. Altogether, our results provide new clues for understanding the mechanism behind the ZBD-independent inactivation of BLM by Cd2+ leading to accumulation of DNA double-strand breaks.

A high rate of telomeric sister chromatid exchange occurs in chronic lymphocytic leukaemia B-cells.

Cancer cells protect their telomere ends from erosion through reactivation of telomerase or by using the Alternative Lengthening of Telomere (ALT) mechanism that depends on homologous recombination. Chronic lymphocytic leukaemia (CLL) B cells are characterized by almost no telomerase activity, shelterin deregulation and telomere fusions. To characterize telomeric maintenance mechanisms in B-CLL patients, we measured their telomere length, telomerase expression and the main hallmarks of the ALT activity i.e. C-circle concentration, an extra-chromosomal telomere repeat (ECTR), and the level of telomeric sister chromatid exchange (T-SCE) rate. Patients showed relative homogenous telomere length although almost no TERT transcript and nearly no C-circle were evidenced. Nevertheless, compared with normal B cells, B-CLL cells showed an increase in T-SCE rate that was correlated with a strong down-regulation of the topoisomerase III alpha (TOP3A) expression, involved in the dissolution of Holliday Junctions (HJ), together with an increased expression of SLX1A, SLX4, MUS81 and GEN1, involved in the resolution of HJ. Altogether, our results suggest that the telomere maintenance mechanism of B-CLL cells do not preferentially use telomerase or ALT. Rather, the rupture of the dissolvasome/resolvasome balance may increase telomere shuffling that could homogenize telomere length, slowing telomere erosion in this disease.

Cytotoxicity and genotoxicity of iron oxide nanoparticles: An in vitro biosafety study

With the development of nanotechnology and the wide use of iron oxide nanoparticles, it has become necessary to assess the potential adverse biological effects of magnetite. This study investigated the cytotoxicity, genotoxicity and oxidative damage of different concentrations of magnetite (0 to 1000 mg/L) in human whole blood cultures. After supplementation of magnetite, the blood samples were incubated for 72 h. Cell viability was assessed by the 3-(4,5-dimethyl-thiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) release assays. The total antioxidant capacity (TAC) and total oxidant status (TOS) were determined to evaluate the dose-dependent effects of magnetite on the oxidant/antioxidant balance and to evaluate the potential oxidative injury due to increased oxidative stress. Genotoxicity was estimated by by the sister chromatid exchange (SCE), micronuclei (MN) and chromosome aberration (CA) assays and determination of 8-oxo-2-deoxyguanosine (8-OH-dG) levels. The results of MTT and LDH assays showed that the higher concentrations of magnetite (100, 150, 300, 500 and 1000 mg/L) decreased cell viability. Concentrations of magnetite higher than 10 mg/L increased TOS levels and decreased TAC levels in human blood cells. Increasing concentrations of magnetite caused significant increases in MN, SCE and CA rates and 8-OH-dG levels. The obtained results showed that magnetite exerted dose-dependent effects on oxidative damage, genotoxicity and cytotoxicity in human blood cells.

Evaluation of DNA damage induced by norcantharidin in human cultured lymphocytes

Norcantharidin (NCTD) is currently used in the treatment of several cancers such as leukemia, melanoma and hepatoma. The mechanism of action of NCTD is suggested to involve induction of apoptosis of cancer cells via production of reactive oxygen species. In this study, the genotoxic effect of different concentrations of NCTD (1, 10 and 20 μm) in human lymphocytes was investigated using sister chromatid exchanges (SCEs) and chromosomal aberrations (CAs) assays. The results revealed that NCTD significantly increased the rate of SCEs (p < 0.05) in a dose-dependent manner. In addition, NCTD significantly increased the number of high-frequency cells (SCEs ≥ 8, p < 0.05). However, NCTD did not have any significant effect on the rate of CAs (p > 0.05). In addition, no significant differences were detected in the mitotic index or proliferative index at examined doses (up to 20 μm). In conclusion, NCTD is genotoxic to human cultured lymphocytes as measured by SCE assay.

Effects of infliximab on sister chromatid exchanges and chromosomal aberration in patients with rheumatoid arthritis.

The aim of this study was to evaluate in a 24-weeks the effect of anti-TNF-alpha, infliximab, on cytogenetic biomarkers in peripheral lymphocytes of patients with rheumatoid arthritis (RA). A total of 40 patients with RA met the criteria to be treated with methotrexate (15mg/week) were evaluated. Twenty patients, randomly selected, were treated with infliximab in addition to methotrexate (group I), whereas the other 20 patients continued with only methotrexate treatment (group M). Twenty healthy volunteers matched for age, gender and smoking habits served as control group (group C). At baseline, sister chromatid exchange rate was 7.20±2.21 in group I, 7.40±1.60 in group M and 4.97±1.32 in group C (P<0.01 vs group I and M). After 24-weeks, sister chromatid exchange rate was 7.87±2.54 in group I and 7.81±1.95 in group M (P=ns). High frequency cells count was 4.9% and 4.7% in the groups I and M, respectively, at the end of the study (P=ns). The basal chromosomal aberration frequency was 4.90% in group I and 5.20% in groups M; after 24-weeks, this was 5.10% in group I and 5.10% in groups M (P=ns). Infliximab treatment, for 24weeks, did not increase the cytogenetic biomarkers in patients with RA. Our data show that the use of infliximab has not a genotoxic effect in patients with RA.

The Association between Anti - Oxidant , Redox Status and Sister Chromatid Exchanges in down Syndrome Individuals

The main objective of this project is to study the possible association between anti-oxidant/redox status and DNA instability in Down syndrome. The activities of 5 antioxidant enzymes were studied in 19 Down syndrome (DS) cases and in age- and sex-matched normal controls. Sister Chromatid Exchanges (SCE) were measured in lymphocyte cultures derived from all DS and control subjects. All DS and control individuals had normal hematological parameters, but the proliferation and mitotic indices were significantly lower in the DS- than in the controls-derived lymphocyte cultures, while the average generation time was higher than that in the controls. The specific activity of superoxide dismutase in the DS individuals was 40% higher than that in the controls, while the specific activity of glutathione S-transferase in the DS group was significantly lower than that in the controls (P ≤ 0.05). Catalase and glutathione peroxides’ activities were not different between the two groups (P > 0.05). SCE rate in the DS derived cultures was significantly higher (P < 0.001) than that of the controls. DS individuals have a higher oxidative stress, higher superoxide dismutase activities and higher rates of SCE in their derived lymphocyte cultures compared to those of the controls. We claim that such differences may have resulted from the over expression of superoxide dismutase gene, leading to imbalanced cellular antioxidant mechanisms and, consequently, resulted in a high concentration of free radicals that destabilized the DNA as expressed by the high rate of SCE.

Assessment of genotoxicity associated with Behcet’s disease using sister-chromatid exchange assay: vitamin E versus mitomycin C

Behcet's disease (BD) is a multisystemic chronic inflammatory disorder that presents throughout the world with high frequency in Turkey and Middle East. BD has been shown to be associated with genotoxicity as patients with the disease have demonstrated high rates of sister chromatid exchange (SCE) and oxidative DNA damage. In this study, we examined the effect of vitamin E, which is known for its strong antioxidant activity, on the rate of SCE in cultured lymphocytes obtained from BD patients. In addition, the susceptibility of patient lymphocytes to the mutagenic agent mitomycin C (MMC) was also investigated. The results showed significant elevation in the rate of SCE in lymphocytes obtained from patients compared to those from healthy subjects (P<0.01). Treatment with vitamin E normalized the elevated rate of SCE to a comparable level observed in the control group (P<0.01). Finally, treatment of cultures with MMC significantly increased the rate of SCE in the lymphocytes of both patients and controls (P<0.001). The magnitude of change in the rate of SCE induced by MMC was equivalent in both groups. This result suggests similar sensitivity of BD lymphocytes and control ones to MMC. In conclusion, genotoxicity associated with BD can be overcome by treatment with vitamin E. Lymphocytes of BD have normal sensitivity to the mutagenic agent MMC.

Mycotoxins’ Activity at Toxic and Sub-Toxic Concentrations: Differential Cytotoxic and Genotoxic Effects of Single and Combined Administration of Sterigmatocystin, Ochratoxin A and Citrinin on the Hepatocellular Cancer Cell Line Hep3B

Food safety organizations indicate the likelihood of constant human and animal exposure to mycotoxin mixtures as a possible negative public health impact. Risk assessment demonstrates that certain mycotoxins of Aspergillus and Penicillium spp. are toxic and hold a significant genotoxic efficacy at nanomolar concentrations. The aim of the current study was to investigate the potential cytogenetic effects of sterigmatocystin (STER), ochratoxin A (OTA) and citrinin (CTN) alone or in combination, at pM to μΜ concentrations, on the human hepatocellular cancer cell line Hep3B. MTT reduction, mitotic divisions, cell cycle delays and sister chromatid exchange rates (SCE) were determined as endpoints of metabolic activity, cytotoxicity, cytostaticity, and genotoxicity, respectively. All mycotoxin treatments induce SCE rates from 10−12 M, while their cytotoxic and cytostatic potential varies. In PRI and MI assays, but not at MTT, STER alone or in combination with OTA + CTN appeared cytostatic and cytotoxic, even at 10−12 M, while CTN alone and all other combinations displayed substantial cellular survival inhibition in doses ≥ 10−8 M. Co-administration of STER + OTA or STER + CTN in concentrations ≤ 10−1 M, increased the MI and MTT activity, while it did not affect the PRI. Mycotoxin co-treatments revealed in general similar-to-additive or antagonistic genotoxic and cytotoxic effects. Our results for the first time describe that STER alone or in combination with OTA and/or CTN share a cytotoxic and cytogenetic potential even at picoMolar concentrations on human hepatoma cells in vitro.

The evaluation of long-term effects of ionizing radiation through measurement of current sister chromatid exchange (SCE) rates in radiology technologists, compared with previous SCE values

Ionizing radiation is a strong physical mutagen, causing breakage of phosphodiester bonds in DNA at any stage of the mitotic cycle. Analysis of sister chromatid exchange (SCE) has come into use as a sensitive DNA-damage indicator. We investigated the SCE rates in radiology technologists who are occupationally and chronically exposed to ionizing radiation. The study included 39 radiology technologists and 35 sex- and age-matched healthy controls.There was a statistically significant difference in the SCE frequency between radiology technologists and controls (p<0.0001). Additionally, previous SCE data of 10 radiology technologists were compared with current results regarding radiation exposure time. There was statistically significant difference between previous and current SCE values (p=0.005).The significant increase in the frequency of SCE in radiology technologists emphasizes the importance of radiation–protection procedures in order to minimize radiation exposure and avoid possible genotoxic effects. Comparison of two studies that measured SCE values of radiology technologists after 8 years also suggests that the genotoxic effect is reversible.In conclusion, radiation is still an important mutagenic agent despite improvements in daily working hours and conditions.

Effect of maternal smoking during pregnancy on fetus: a cytogenetic perspective

Objective: The examination of the genotoxic, cytostatic and cytotoxic effects of smoking during pregnancy.Method: Lymphocyte cultures of peripheral blood were received from 20 women who smoked during pregnancy as well as umbilical cord blood of their newborns. Fluorescence Plus Giemsa staining technique was used in order to perform cytogenetic analyses for three indices, Sister Chromatid Exchanges (SCEs), Proliferation Rate Index (PRI) and Mitotic Index (MI). To reveal any underlying chromosome instability, CPT-11 was used as a positive control.Results: Newborns whose mothers smoke during pregnancy had increased SCEs levels on their lymphocytes when they were exposed to the mutagenic agent CPT-11 (p < 0.01) compared with newborns lymphocytes exposed to the same agent with non-smoking mothers. Also, mothers smoking during pregnancy had increased SCE levels when their lymphocytes were exposed to CPT-11 (p < 0.01) compared with non smoking mothers whose lymphocytes were exposed to the same agent. In both groups newborns appeared as having decreased (p < 0.01) spontaneous SCEs levels compared with the corresponding SCE rates of their mothers. Decreases of PRIs and MIs are observed in mothers compared to their newborns.Conclusion: Smoking during pregnancy can promote cytogenetic damage in newborn’s DNA, causing chromosome instability. The clinical importance of this indirect damage lies in the fact that this type of damage can act synergistically with other environmental and/or chemical mutagenic substances possibly leading to carcinogenicity.

The genoprotective activity of resveratrol on permethrin-induced genotoxic damage in cultured human lymphocytes

The aim of this work was to investigate the genetic effects of resveratrol (RSV) at concentrations of 10, 15, 25, 40, 75 and 100 µM and its activities on the genotoxicity induced by the permethrin (PM) (200 µM). After the application of PM and RSV, separately and together, cultured human lymphocytes were assessed by chromosome aberrations (CA) and sister chromatid exchange (SCE) tests. According to results, the frequencies of CA and SCE rates in the peripheral lymphocytes were significantly increased by PM compared with the controls. However, RSV had no genotoxic effect. Furthermore, the findings revealed that PM-induced increases in the mean frequencies of both genotoxic indices were diminished by RSV in a clear dose dependent manner, indicating its protective role towards the cells from PM exerted injury. In conclusion, these effects of RSV should be considered while evaluating the possible use of RSV as a therapeutic agent.

Time course changes of anti- and pro-apoptotic proteins in apigenin-induced genotoxicity

BackgroundApigenin (4′,5,7-trihydroxyflavone, AP), an active component of many medicinal Chinese herbs, exhibits anticancer properties in vitro and in vivo. This study aims to investigate the genotoxic, cytostatic, and cytotoxic effects of AP and time course changes in the levels of anti- and pro-apoptotic proteins involved in the DNA damage response in HepG2 cells.MethodsThe genotoxic potential of AP was determined by sister chromatid exchanges (SCEs) and chromosomal aberrations (CAs) analysis. The levels of cytostaticity and cytotoxicity were evaluated by the proliferation rate and mitotic indices, respectively. MTT was used to study cytotoxicity, while the induction of apoptosis and the expression of apoptosis-related proteins were determined by ELISA.ResultsAt concentrations greater than 10 μM, AP decreased cell survival in a dose- (48 h: 10 vs. 20 μΜ, P < 0.001 and 20 vs. 50 μΜ, P = 0.005; 72 h: 10 vs. 20 μΜ, P < 0.001 and 20 vs. 50 μΜ, P = 0.001) and time-dependent manner (20 μΜ: 24 vs. 48 h, P < 0.001 and 48 vs. 72 h, P = 0.003; 50 μΜ: 24 vs. 48 h, P < 0.001 and 48 vs. 72 h, P < 0.001; 100 μΜ: 24 vs. 48 h, P < 0.001 and 48 vs. 72 h, P < 0.001). SCEs rates, cell proliferation, and mitotic divisions were also affected in a dose-dependent manner (P < 0.001). There was no change in the frequency of aberrant cells (1 μΜ ΑP: P = 0.554; 10 μM AP: P = 0.337; 20 μΜ AP: P = 0.239). Bcl-2 levels were reduced 3 h after AP administration (P = 0.003) and remained reduced throughout the 48 h observation period (6 h, P = 0.044; 12 h, P = 0.001; 24 h, P = 0.042; 48 h, P = 0.012). Bax and soluble Fas exhibited a transient upregulation 24 h after AP treatment. The Bax/Bcl-2 ratio was also increased at 12 h and remained increased throughout the 48 h observation period.ConclusionAP exhibited dose-dependent genotoxic potential in HepG2 cells. The protein levels of sFas, Bcl-2, and Bax were affected by AP to promote cell survival and cell death, respectively.

Abstract 4055: Non-canonical telomere maintenance mechanism in B-cell chronic lymphocytic leukemia.

Abstract Unlimited cancer cells proliferation requires mechanisms that counteract telomere attrition. In the majority of cancer cells this is possible through up-regulation of telomerase activity. Alternatively, cancer cells use homologous recombination between telomeres. This second mechanism called ALT (Alternative Lengthening of Telomere) implicates proteins that either elongates telomeres or that either prevents telomere loss (maintenance). Among these proteins a complex composed of the topoisomerase III alpha (hTopoIIIα), BLM, RMI1 and 2 is require to repair stalled replication forks. Little is known about the involvement of the ALT mechanism in B-cell chronic lymphocytic leukemia (B-CLL), which shows low telomerase activity, shelterin defect and telomeric dysfunctions. We found that hTopoIIIα gene expression is downregulated in almost all B-CLL patients tested (94%; n=31), with 60% of B-CLL patients presenting a two time decreased compared to control samples. That prompted us to build the first CpG islands map of the hTopoIIIα promoter and to characterize those that are methylated in B-CLL patients. To investigate the methylation impact, we first treated CLL cell lines with 5-Azacytidine, a chemical analogue of cytidine that induces hypomethylation, and demonstrated that this treatment increases hTopoIIIα expression. In line, luciferase experiments revealed that SSSI-induced CpG islands methylation inside the TopoIIIα promoter leads to a transcriptional inhibition. Nevertheless, deletion mutants of the TopoIIIα promoter suggested that CpG islands found to be methylated in B-CLL patients are not implicated in TopoIIIα expression regulation through methylation. Interestingly, we didn't observe a good correlation between mRNA and protein expressions in all B-CLL patients (n=6). Indeed some patients with a moderate amount of mRNA showed low level of the corresponding protein hTopoIIIα (&amp;lt;50% compared to controls; n=3). Downregulation of hTopoIIIα may increase recombination rate between sister chromatid. Hence, experiments aiming to evaluate sister chromatid exchange and telomeric sister chromatid exchange rate on B-CLL patients samples according to their hTopoIIIα expression level have been performed and the data will be discuss. Our results suggest that there is no canonical mechanism maintaining telomeres in B-CLL but rather an overall recombination arising during replication, due to hTopoIIIα downregulation, leading to a silenced genetic instability in the early step of the disease. Citation Format: Sandrine Medves, Morgan Auchter, Laetitia Chambeau, Sophie Gazzo, Aurélie Verney, Etienne Moussay, Wim Ammerlaan, Hamid Morjani, Vincent Géli, Valérie Palissot, Gilles Salles, Guy Berchem, Thomas Wenner. Non-canonical telomere maintenance mechanism in B-cell chronic lymphocytic leukemia. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4055. doi:10.1158/1538-7445.AM2013-4055

Scaffolding protein SPIDR/KIAA0146 connects the Bloom syndrome helicase with homologous recombination repair

The Bloom syndrome gene product, BLM, is a member of the highly conserved RecQ family. An emerging concept is the BLM helicase collaborates with the homologous recombination (HR) machinery to help avoid undesirable HR events and to achieve a high degree of fidelity during the HR reaction. However, exactly how such coordination occurs in vivo is poorly understood. Here, we identified a protein termed SPIDR (scaffolding protein involved in DNA repair) as the link between BLM and the HR machinery. SPIDR independently interacts with BLM and RAD51 and promotes the formation of a BLM/RAD51-containing complex of biological importance. Consistent with its role as a scaffolding protein for the assembly of BLM and RAD51 foci, cells depleted of SPIDR show increased rate of sister chromatid exchange and defects in HR. Moreover, SPIDR depletion leads to genome instability and causes hypersensitivity to DNA damaging agents. We propose that, through providing a scaffold for the cooperation of BLM and RAD51 in a multifunctional DNA-processing complex, SPIDR not only regulates the efficiency of HR, but also dictates the specific HR pathway.