Sister Chromatid Exchange Levels Research Articles

Vitamin B12 Protects Against Genotoxicity Induced by Cisplatin.

Cisplatin is an effective synthetic chemotherapeutic drug used for cancer treatment. Vitamin B12 has been shown to possess anti-genotoxic activity. This study aimed to investigate the effect of vitamin B12 on chromosomal damage induced by cisplatin. The level of sister chromatid exchanges (SCEs) and chromosomal aberrations (CAs) were measured in cultured human blood lymphocytes treated with cisplatin and/or vitamin B12. The results showed a significantly elevated frequency of CAs and SCEs of cisplatin-treated cultures compared to the control (P < 0.05). The CAs and SCEs induced by cisplatin were significantly lowered by pretreatment of cell cultures with vitamin B12. In addition, cisplatin caused a slight reduction in the mitotic index (MI), while vitamin B12 did not modulate the effect of cisplatin on MI. Vitamin B12 can protect human lymphocytes against genotoxicity associated with cisplatin.

The meta-analysis of sister chromatid exchange as a biomarker in healthcare workers with occupational exposure to antineoplastic drugs.

Sister chromatid exchange (SCE) can be used to identify early occupational health status in health care workers. Our aim is to comprehensively assess the relationship between long-term exposure to antineoplastic drugs (ADs) and SCE in health care workers via meta-analysis. Five databases were systematically searched for relevant articles published from inception to November 30, 2022. Literature data are expressed as mean difference and 95% confidence intervals (CI) or relative risk and 95% CI. For I2 > 50% trials, random effect model is used for statistical analysis, otherwise fixed effect model is used. This review was registered in the International Prospective Register of Systematic Reviews (identifier CRD42023399914). Fourteen studies were included in this study. Results showed the level of SCE in healthcare workers exposed to ADs was significantly higher than in controls. The mean difference of the SCE trial was 0.53 (95% CI: 0.10-0.95, P = .01) under a random-effects model. The findings suggested a significant correlation between occupational exposure to ADs in health care workers and SCE, requiring the attention of health care workers in general.

Inactive PARP1 causes embryonic lethality and genome instability in a dominant-negative manner.

PARP1 (poly-ADP ribose polymerase 1) is recruited and activated by DNA strand breaks, catalyzing the generation of poly-ADP-ribose (PAR) chains from NAD+. PAR relaxes chromatin and recruits other DNA repair factors, including XRCC1 and DNA Ligase 3, to maintain genomic stability. Here we show that, in contrast to the normal development of Parp1-null mice, heterozygous expression of catalytically inactive Parp1 (E988A, Parp1+/A) acts in a dominant-negative manner to disrupt murine embryogenesis. As such, all the surviving F1 Parp1+/A mice are chimeras with mixed Parp1+/AN (neoR retention) cells that act similarly to Parp1+/-. Pure F2 Parp1+/A embryos were found at Mendelian ratios at the E3.5 blastocyst stage but died before E9.5. Compared to Parp1-/- cells, genotype and expression-validated pure Parp1+/A cells retain significant ADP-ribosylation and PARylation activities but accumulate markedly higher levels of sister chromatid exchange and mitotic bridges. Despite proficiency for homologous recombination and nonhomologous end-joining measured by reporter assays and supported by normal lymphocyte and germ cell development, Parp1+/A cells are hypersensitive to base damages, radiation, and Topoisomerase I and II inhibition. The sensitivity of Parp1+/A cells to base damages and Topo inhibitors exceed Parp1-/- controls. The findings show that the enzymatically inactive PARP1 dominant negatively blocks DNA repair in selective pathways beyond wild-type PARP1 and establishes a crucial physiological difference between PARP1 inactivation vs. deletion. As a result, the expression of enzymatically inactive PARP1 from one allele is sufficient to abrogate murine embryonic development, providing a mechanism for the on-target side effect of PARP inhibitors used for cancer therapy.

Genotoxicity of Glyphosate on Cultured Human Lymphocytes.

Glyphosate-based herbicides are the most used herbicides in the world. Despite being widely used, a dispute exists whether glyphosate-based herbicides have a negative effect on human health, particularly genotoxic effects. Therefore, the aim of this study was to investigate glyphosate genotoxicity on cultured human lymphocytes. Cultured human lymphocytes were treated with different concentrations of glyphosate (20, 40, and 200µmol/L). Four toxicity measures were examined: frequency of chromosomal aberrations (CAs), frequency of sister-chromatid exchange (SCE), production of 8-OHdG, and cell kinetics analysis. The results show that glyphosate induced significant (P < 0.05) increases in the levels of SCE at the highest used concentration (200μmol/L). However, no significant elevation in SCE levels was observed at the lower examined concentrations (20 and 40μmol/L). No significant changes in CA were detected at all examined concentrations (P = 0.86). Also, glyphosate did not induce changes to the normal level of 8-OHdG at all examined concentrations (P = 0.98). Last, no significant changes in either mitotic index or proliferative index were observed at any examined concentrations (P > 0.05). The results collectively indicate a lack of genotoxicity and cytotoxicity of glyphosate in cultured human lymphocytes when dealing with environmentally relevant concentrations (20 and 40μmol/L). However, being exposed to higher concentrations (200μmol/L) led to slightly higher level of SCE. Therefore, we recommend cautionary measures when dealing with glyphosate-based herbicides for individuals, such as farmers, who may be extensively exposed to high concentrations of these herbicides.

Distinct pathways of homologous recombination controlled by the SWS1\u2013SWSAP1\u2013SPIDR complex

Homology-directed repair (HDR), a critical DNA repair pathway in mammalian cells, is complex, leading to multiple outcomes with different impacts on genomic integrity. However, the factors that control these different outcomes are often not well understood. Here we show that SWS1–SWSAP1-SPIDR controls distinct types of HDR. Despite their requirement for stable assembly of RAD51 recombinase at DNA damage sites, these proteins are not essential for intra-chromosomal HDR, providing insight into why patients and mice with mutations are viable. However, SWS1–SWSAP1-SPIDR is critical for inter-homolog HDR, the first mitotic factor identified specifically for this function. Furthermore, SWS1–SWSAP1-SPIDR drives the high level of sister-chromatid exchange, promotes long-range loss of heterozygosity often involved with cancer initiation, and impels the poor growth of BLM helicase-deficient cells. The relevance of these genetic interactions is evident as SWSAP1 loss prolongs Blm-mutant embryo survival, suggesting a possible druggable target for the treatment of Bloom syndrome.

Differential effects of cisplatin combined with the flavonoid apigenin on HepG2, Hep3B, and Huh7 liver cancer cell lines

The potential of apigenin (APG) to enhance cisplatin’s (CDDP) chemotherapeutic efficacy was investigated in HepG2, Hep3B, and Huh7 liver cancer cell lines. The presence of 20 μM APG sensitized all cell lines to CDDP treatment (degree of sensitization based on the MTT assay: HepG2>Huh7>Hep3B). As reflected by sister chromatid exchange levels, the degree of genetic instability as well as DNA repair by homologous recombination differed among cell lines. CDDP and 20 μM APG cotreatment exhibited a synergistic genotoxic effect on Hep3B cells and a less than additive effect on HepG2 and Huh7 cells. Cell cycle delays were noticed during the first mitotic division in Hep3B and Huh7 cells and the second mitotic division in HepG2 cells. CDDP and CDDP + APG treatments reduced the clonogenic capacity of all cell lines; however, there was a discordance in drug sensitivity compared with the MMT assay. Furthermore, a senescence-like phenotype was induced, especially in Hep3B and Huh7 cells. Unlike CDDP monotherapy, the combined treatment exhibited a significant anti-invasive and anti-migratory action in all cancer cell lines. The fact that the three liver cancer cell lines responded differently, yet positively, to CDDP + APG cotreatment could be attributed to variations they present in gene expression. Complex mechanisms seem to influence cellular responses and cell fate.

Aldehyde degradation deficiency (ADD) syndrome: discovery of a novel fanconi anemia-like inherited BMF syndrome due to combined ADH5/ALDH2 deficiency

We have recently described the identification of a novel inherited bone marrow failure syndrome. The first set of patients was diagnosed through the exome analysis of cells from Japanese patients with hypoplastic anemia, which have been deposited to the JCRB cell bank for quite some time previously. Originally, these cases were diagnosed with a novel disorder based on increased levels of sister chromatid exchanges in lymphocytes; however, causative genes were clarified only after applying the recently developed next-generation sequencing technology. Aldehyde degradation deficiency syndrome (ADDS) is caused by combined defects in two genes, ADH5 and ALDH2, which are both critical for degrading endogenously generated formaldehyde. Formaldehyde is highly reactive and toxic to biological molecules including DNA, and its endogenous generation in the absence of the degradation system results in DNA damage that overwhelms the DNA repair capacity, leading to the development of BMF with loss of hematopoietic stem cells and progression to MDS/leukemia. In this short review, we would like to summarize what is known today about ADDS for a wide readership of hematology clinicians in Japan.

Cytogenetic effects among workers exposed to formaldehyde. The possible role of some polymorphisms

Abstract Background Formaldehyde (FA) is a human carcinogen. It is commonly used in diluted form as formalin. The purpose of this work was to evaluate the frequencies of chromosomal aberrations (CAs) and sister chromatid exchanges (SCEs) as a consequence of the exposure to FA in a hospital setting, taking into account the role of some polymorphisms in developing genotoxic damage. Methods CAs and SCEs assays were performed on peripheral blood lymphocytes of 57 pathologists enrolled in Turin hospitals (Italy) and 48 unexposed enrolled as controls. All subjects were genotyped for CYP1A1 exon 7 (A&amp;gt;G), CYP1A1*2A (T&amp;gt;C), CYP2C19*2 (G&amp;gt;A), GSTT1 (Positive/Null), GSTM1 (Positive/null), GSTP1 (A&amp;gt;G), XRCC1 (G399A), XRCC1 (C194T), XRCC1 (A280G), XPD (A751C), XPC exon 15 (A939C), XPC exon 9 (C499T), TNFα -308 (G&amp;gt;A), IL10 -1082 (G&amp;gt;A), IL10 -819 (C&amp;gt;T) and IL6 -174 (G&amp;gt;C) gene polymorphisms. Due to the little number of recessive homozygotes, we compared the dominant genotype with the combined group of heterozygotes and recessive homozygotes. Results Among pathologists, the mean value of personal air-FA was 64.2 µg/m3, mean significantly higher (p = 0.000) than controls (19.1 µg/m3). Similarly, pathologists showed significantly higher values of SCEs (p = 0.009) and CAs (p = 0.000) with respect to controls. In the same group, CYP2C19*2 (p = 0.011), XRCC1 (G399A) (p = 0.007), and IL10 (-1082) (p = 0.042) gene polymorphisms influenced the CAS levels, as well as those of CYP1A1 exon 7 (p = 0.010), XPD A751C (p = 0.002), XPC C499T (p = 0.040), and IL10 (-819) (p = 0.005) the levels of SCEs. In control group, CAs resulted significantly lower in CYP1A1 exon 7 dominant homozygotes (p = 0.001). Conclusions Our study confirms the role of FA as an inductor of genotoxicity, even when, daily, workers are exposed to low FA levels. Some genetic polymorphisms seem to have an influence in modulating the effect of FA exposure, highlighting, in occupational health studies, the role of the individual susceptibility. Key messages FA exposure is confirmed to be a genotoxicity inductor even at low concentrations. Some genetic polymorphisms seem to have a role in the modulation of FA exposure-related damage.

EdU-Based Step-by-Step Method for the Detection of Sister Chromatid Exchanges for Application in Plant Genotoxicity Assessment.

This study is an example of using 5-ethynyl-2′-deoxyuridine (EdU) for detecting sister chromatid exchanges (SCEs) at chromosomal level. Here we report a detailed protocol for differential labeling sister chromatids in barley (Hordeum vulgare, 2n = 14) cells that is based on the incorporation and simple detection of EdU. The perfect distinguishing of sister chromatids enabled an analysis of the effects of two model agents—maleic acid hydrazide (MH) and gamma rays—on the formation of SCEs. Using this method, we demonstrated the high sensitivity of barley cells to maleic hydrazide, which is expressed as an increased level of SCEs. A gamma ray induced only slightly more SCEs than in the control cells. The possible mechanisms of MH and gamma ray action in respect to distinguishing chromatids using EdU are discussed. Recommendation for SCEs visualization using EdU as an easy and quick method that can be successfully adapted to other plant species and potentially for human genotoxicity studies is presented.

Repair of DNA damage induced by the novel nucleoside analogue CNDAG through homologous recombination.

We postulate that the deoxyguanosine analogue CNDAG [9-(2-C-cyano-2-deoxy-1-β-D-arabino-pentofuranosyl)guanine] likely causes a single-strand break after incorporation into DNA, similar to the action of its cytosine congener CNDAC, and that subsequent DNA replication across the unrepaired nick would generate a double-strand break. This study aimed at identifying cellular responses and repair mechanisms for CNDAG prodrugs, 2-amino-9-(2-C-cyano-2-deoxy-1-β-D-arabino-pentofuranosyl)-6-methoxy purine (6-OMe) and 9-(2-C-cyano-2-deoxy-1-β-D-arabino-pentofuranosyl)-2,6-diaminopurine (6-NH2). Each compound is a substrate for adenosine deaminase, the action of which generates CNDAG. Growth inhibition assay, clonogenic survival assay, immunoblotting, and cytogenetic analyses (chromosomal aberrations and sister chromatid exchanges) were used to investigate the impact of CNDAG on cell lines. The 6-NH2 derivative was selectively potent in T cell malignant cell lines. Both prodrugs caused increased phosphorylation of ATM and its downstream substrates Chk1, Chk2, SMC1, NBS1, and H2AX, indicating activation of ATM-dependent DNA damage response pathways. In contrast, there was no increase in phosphorylation of DNA-PKcs, which participates in repair of double-strand breaks by non-homologous end-joining. Deficiency in ATM, RAD51D, XRCC3, BRCA2, and XPF, but not DNA-PK or p53, conferred significant clonogenic sensitivity to CNDAG or the prodrugs. Moreover, hamster cells lacking XPF acquired remarkably more chromosomal aberrations after incubation for two cell cycle times with CNDAG 6-NH2, compared to the wild type. Furthermore, CNDAG 6-NH2 induced greater levels of sister chromatid exchanges in wild-type cells exposed for two cycles than those for one cycle, consistent with increased double-strand breaks after a second S phase. CNDAG-induced double-strand breaks are repaired mainly through homologous recombination.

Anticancer and antiangiogenesis activities of novel synthesized 2-substitutedbenzimidazoles molecules

In this study, novel 2-substituted benzimidazoles molecules having triazole, thiadiazole, and oxadiazole rings were synthesized and were evaluated by anticancer, antioxidant/oxidant status, genotoxicity, and antiangiogenesis assays. Anticancer activity of the compounds was determined by MTT (0.5, 5, and 50 $\mu $g/mL) and lactate dehydrogenase (LDH) release assays against human prostate and breast cancer cells. Oxidative status of cells was elicited by total oxidative stress and total antioxidant capacity methods. Chick chorioallantoic membrane assay was used to evaluate the antiangiogenic activity. Genotoxicity was evaluated by the sister chromatid exchange (SCE) and micronucleus (MN) tests in lymphocyte cultured human blood. Our results showed that some of the compounds synthesized had significant antiproliferative activity against both cancer cell lines (between 4.54 $\pm $ 0.35 and 20.17 $\pm $ 3.15 $\mu $g/mL), with higher inhibition of the breast cancer, and caused inhibition of LDH release with a linear correlation to MTT results. Moreover, the 5 $\mu $g/mL dose of these molecules led to an increase in antioxidant levels. Compounds had antiangiogenic effectiveness in a dose-dependent manner. Additionally, all of the compounds did not affect SCE and MN levels compared to controls. In conclusion, these newly synthesized molecules can be a resource of new anticancer agents with their nongenotoxic, antiproliferative, and antiangiogenic properties.

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.

Protective role of eugenol on arsenic induced oxidative DNA damage and modulatory effect of GSTO2 polymorphism

Arsenic exposure is one of the major health problem worldwide. It is related with various cancers and numerous other pathological effects. The present work was aimed to study the ameliorative effects of eugenol against arsenic mediated oxidative DNA damage using Sister chromatid exchanges (SCE), comet, and lipid peroxidation assays as biomarkers. For the genotyping of GSTO2, PCR RFLP method was used. The treatment with sodium arsenite showed dose dependent increase in frequency of SCE, comet tail moment (Tm), and lipid peroxidation (TBARS levels). On administration of eugenol (10 and 20 µM) significant dose dependent decrease in the number of SCE, Tm value, and TBARS levels observed in arsenic treated cultured peripheral blood lymphocytes. Nonsignificant effects of GSTO2 polymorphism perceived on arsenic genotoxicity and eugenol antigenotoxicity. The results of the present work suggest a protective role of eugenol against arsenic induced genotoxic and oxidative damage in cultured human peripheral blood lymphocytes. Practical application Arsenic contamination of groundwater is one of the leading environmental issues worldwide. Chronic exposure of arsenic cause several health problems including tumor manifestation. The results of the present study suggest the use of indigenous dietary elements, herbs, shrubs as a safer and effective preventative medicine against heavy metal induced genotoxicity and oxidative stress.

Genoprotective Effects of Thai Royal Jelly against Doxorubicin in Human Lymphocytes in Vitro

Genoprotective effects of royal jelly (RJ) treatments against doxorubicin (DXR), a potent genotoxic chemotherapeutic compound in human lymphocytes were investigated using the sister chromatid exchange (SCE) assay, and their molecular mechanisms were examined by Western blot. Results showed that RJ pretreatments at 0.005 and 0.05 mg/mL significantly decreased DXR-induced SCE levels by 1.2-fold (p&lt;0.05), compared to DXR treatment alone. Co-treatment of RJ (5 mg/mL) with DXR (0.2 μg/mL) increased the ratios of BCL2/BAX (1.5-fold), NRF2/BAX (1.3-fold), and hTERT/BAX (1.1-fold) compared to the DXR alone, suggesting its power in enhancing cell survival, antioxidative potentials, and longevity over cell death. The study suggested that RJ protected human cells from DXR-induced genotoxicity, possibly mediated through anti-apoptotic, anti-oxidative, and anti-aging properties of RJ. However, lower doses of RJ co-treatments enhanced DXR toxicity. Further, in vivo molecular study is required to validate this in vitro study.

Effects of β-Carotene and Its Cleavage Products in Primary Pneumocyte Type II Cells.

β-Carotene has been shown to increase the risk of developing lung cancer in smokers and asbestos workers in two large scale trails, the Beta-Carotene and Retinol Efficacy Trial (CARET) and the Alpha-Tocopherol Beta-carotene Cancer Prevention Trial (ATBC). Based on this observation, it was proposed that genotoxic oxidative breakdown products may cause this effect. In support of this assumption, increased levels of sister chromatid exchanges, micronuclei, and chromosomal aberrations were found in primary hepatocyte cultures treated with a mixture of cleavage products (CPs) and the major product apo-8′carotenal. However, because these findings cannot directly be transferred to the lung due to the exceptional biotransformation capacity of the liver, potential genotoxic and cytotoxic effects of β-carotene under oxidative stress and its CPs were investigated in primary pneumocyte type II cells. The results indicate that increased concentrations of β-carotene in the presence of the redox cycling quinone dimethoxynaphthoquinone (DMNQ) exhibit a cytotoxic potential, as evidenced by an increase of apoptotic cells and loss of cell density at concentrations > 10 µM. On the other hand, the analysis of micronucleated cells gave no clear picture due to the cytotoxicity related reduction of mitotic cells. Last, although CPs induced significant levels of DNA strand breaks even at concentrations ≥ 1 µM and 5 µM, respectively, β-carotene in the presence of DMNQ did not cause DNA damage. Instead, β-carotene appeared to act as an antioxidant. These findings are in contrast with what was demonstrated for primary hepatocytes and may reflect different sensitivities to and different metabolism of β-carotene in the two cell types.

Abstract PR19: Targeting DNA double-strand break repair to potentiate radio- and chemo-therapy of glioblastoma

Abstract Glioblastomas (GBMs) are lethal brain tumors which are treated with ionizing radiation (IR) in combination with the DNA alkylating agent temozolomide (TMZ). Unfortunately, not all GBMs respond to therapy, and most of them quickly acquire resistance to TMZ and recur. In order to better understand the molecular basis for therapy-driven TMZ resistance, mice bearing orthotopic GBM xenografts were subjected to protracted TMZ treatment, and cell lines were generated from the primary (untreated) and recurrent (TMZ-treated) tumors. We found that cell lines derived from recurrent tumors were more resistant to TMZ in vitro compared to cell lines derived from primary tumors. We also found that the increased resistance to TMZ was due to augmented repair of TMZ-induced DNA double strand breaks (DSBs). TMZ induces DNA replication-associated DSBs that are primarily repaired by the homologous recombination (HR) pathway. Augmented HR appears to underpin TMZ resistance in the recurrent lines as these cells were cross-resistant to other agents that induce replication-associated DSBs, exhibited faster resolution of damage-induced Rad51 foci, and displayed higher levels of sister chromatid exchanges (SCEs). We have recently shown that CDKs 1 and 2 promote HR in S and G2 phases of the cell cycle, in part, by phosphorylating the exonuclease EXO1. We hypothesized, therefore, that blocking CDKs 1 and 2 might be a viable strategy for re-sensitizing recurrent tumors to TMZ. Indeed, we found that CDK inhibitors, AZD5438 and Roscovitine, could attenuate HR in the recurrent TMZ-resistant cell lines, resulting in significant chemo-sensitization. While TMZ-induced DSBs are primarily repaired by HR, IR- induced DSBs are mainly repaired by the non-homologous end joining (NHEJ) pathway. We, therefore, developed another approach to sensitize GBMs to both radiation and TMZ by using a dual PI3K/mTOR inhibitor, NVP-BEZ235, to target both DNA-PKcs and ATM, key enzymes in the NHEJ and HR pathways, respectively. We found that NVP-BEZ235 inhibited both DNA-PKcs and ATM in orthotopic GBM xenografts, thus blocking the repair of TMZ- and IR-induced DSBs in these tumors. Hence NVP-BEZ235, administered with IR, could attenuate tumor growth, and extend survival of tumor-bearing mice. Importantly, inhibition of DSB repair was more pronounced in tumor cells compared to normal brain cells, thereby providing a larger therapeutic window. In sum, these studies indicate that augmented DSB repair may underlie radio- and chemo-resistance in GBM, and provide support for DNA repair inhibition as an effective strategy for improving the efficacy of GBM therapy. This abstract is also being presented as Poster B22. Citation Format: Bipasha Mukherjee, Carlos Gil del Alcazar, Pavlina Todorova, Sandeep Burma. Targeting DNA double-strand break repair to potentiate radio- and chemo-therapy of glioblastoma [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr PR19.

Coordination of the Ser2056 and Thr2609 Clusters of DNA-PKcs in Regulating Gamma Rays and Extremely Low Fluencies of Alpha-Particle Irradiation to G0/G1 Phase Cells.

The catalytic subunit of DNA dependent protein kinase (DNA-PKcs) and its kinase activity are critical for mediation of non-homologous end-joining (NHEJ) of DNA double-strand breaks (DSB) in mammalian cells after gamma-ray irradiation. Additionally, DNA-PKcs phosphorylations at the T2609 cluster and the S2056 cluster also affect DSB repair and cellular sensitivity to gamma radiation. Previously we reported that phosphorylations within these two regions affect not only NHEJ but also homologous recombination repair (HRR) dependent DSB repair. In this study, we further examine phenotypic effects on cells bearing various combinations of mutations within either or both regions. Effects studied included cell killing as well as chromosomal aberration induction after 0.5-8 Gy gamma-ray irradiation delivered to synchronized cells during the G0/G1 phase of the cell cycle. Blocking phosphorylation within the T2609 cluster was most critical regarding sensitization and depended on the number of available phosphorylation sites. It was also especially interesting that only one substitution of alanine in each of the two clusters separately abolished the restoration of wild-type sensitivity by DNA-PKcs. Similar patterns were seen for induction of chromosomal aberrations, reflecting their connection to cell killing. To study possible change in coordination between HRR and NHEJ directed repair in these DNA-PKcs mutant cell lines, we compared the induction of sister chromatid exchanges (SCEs) by very low fluencies of alpha particles with mutant cells defective in the HRR pathway that is required for induction of SCEs. Levels of true SCEs induced by very low fluence of alpha-particle irradiation normally seen in wild-type cells were only slightly decreased in the S2056 cluster mutants, but were completely abolished in the T2609 cluster mutants and were indistinguishable from levels seen in HRR deficient cells. Again, a single substitution in the S2056 together with a single substitution in the T2609 cluster abolished SCE formation and thus also effectively interferes with HRR.

Augmented HR Repair Mediates Acquired Temozolomide Resistance in Glioblastoma.

Augmented HR repair is a novel mechanism underlying acquired temozolomide resistance in GBM, and this raises the possibility of improving the therapeutic response to temozolomide by targeting HR with small-molecule inhibitors of CDK1/2. Mol Cancer Res; 14(10); 928-40. ©2016 AACR.

Evaluation of genotoxic activity of tenofovir disoproxil fumarate in human peripheral lymphocytes

Purpose: Antiretroviral drugs used in the treatment of HIV (Human Immunodeficiency Virus) iinfection, treat by preventing the proliferation of HIV in human body. People with HIV have to use this drugs for lifelong because of inability of the drugs to eradicate the viruses. In this study, we investigated the in vitro genotoxic activity of tenofovir disoproxil fumarate one of the antiretroviral drugs, in human peripheral lymphocytes. Material and Methods: The cells were treated with four different concentrations of tenofovir disoproxil fumarate for 24 and 48 hours. The levels of sister chromatid exchanges, chromosomal aberrations, and micronucleus in the cells were examined for the genotoxic activity of tenofovir disoproxil fumarate. Mitotic index, proliferation index, and nucleer division index of treated cells were also determined for the cytotoxic effect of tenofovir disoproxil fumarate. Results: There was no significant differences in the level of sister chromatid exchanges, chromosomal aberrations, and micronucleus in human lyphocytes treated with all concetrations of tenofovir disoproxil fumarate for all treatment period as compared to control group. Similarly, it was observed that treatment of tenofovir disoproxil fumarate did not affect the mitotic index, proliferation index, and nucleer division index values. Conclusion: As a result, in this study, it is demonstrated that tenofovir disoproxil fumarate did not have genotoxic or cytotoxic effect in the human peripheral lymphocytes.

Sister chromatid exchange assay as a predictor of tumor chemoresponse

Sister Chromatid Exchanges (SCEs) are known to enhance as a consequence of exposure to various mutagenic agents and appear to indicate DNA damaging effects and/or subsequent repair by homologous recombination (HR). DNA damage plays an interesting role in the majority of mechanisms underlying the effects of antitumor drugs, since the genetic activity of the plethora of these agents is due to their ability to damage the DNA. The DNA-effects of antitumor agents towards normal cells (genotoxicity) are great drawbacks of antitumor therapy and are connected to important adverse health effects in cancer patients undergoing chemotherapy. On the other hand, failure of chemotherapy in many cases is due to the DNA repair ability which cancer, like normal cells, also possess. As both DNA repair and genotoxic exposure are expected to vary among patients, correlating SCEs frequencies with only individual repair capacity may be feasible to predict. Cancer risk has not been observed to be associated with high SCEs levels. Since the administration of effective antitumor drugs with limited adverse effects is of great importance in the success of anticancer therapy, a lot of interest has been directed toward the development of methods and approaches that would enable the correct selection of appropriate drugs prior to the initiation of therapy on an individual basis. To this effect, more than 30 years ago, an investigation of the ability of the in vitro and the in vivo SCEs-assay to predict the in vitro and in vivo sensitivity of tumor cells to newly synthesized drugs or to those already in use began. In this short review a critical appraisal of the SCEs-assay as an important biomarker used for predicting cancer chemo-response as well as a summary of the key findings from several studies published within the last 20 years in this field is performed.