DNA Damage Strand Research Articles

Abstract 2926: Nomination and characterization of TTK for radiosensitization in basal-like breast cancers

Abstract Background: Increased rates of locoregional recurrence have been observed in basal-like breast cancer despite the use of radiation therapy (RT), therefore approaches that result in radiosensitization of basal-like breast cancer are critically needed. Studies detailing the poor response of basal-like tumors to adjuvant RT underscore the biologic differences and as of yet undefined oncogenic drivers of these particular types of breast cancer. Methods: 4 independent datasets were used to correlate gene expression with local recurrence (LR) after 3 years. Kaplan-Meier analysis was used to validate the impact of TTK expression on LR. The TCGA and institutional breast cancer dataset were used to determine TTK expression in BC subtypes. TTK RNA and protein levels were measured using qPCR and western blot at baseline and correlated to intrinsic radiosensitivity. Clonogenic survival assays were used to determine the radiosensitization of cell lines after TTK inhibition (TTKi). Mice models were used to assess TTKi in combination with RT in vivo. DNA damage was quantified using γH2AX staining. HR and NHEJ efficiency assays were performed using HR/NHEJ specific reporter systems. HR competency was also assessed using RAD51 foci formation assays. Results: Ten genes were found to significantly correlate with early LR (≤3 years) across 4 distinct datasets (N=896 pts) (OR of recurrence > 2, p-value <0.000001), with TTK, a cell cycle kinase, ranked the highest. Kaplan-Meier survival analysis in 3 cohorts demonstrated that higher than median TTK expression correlates with decrease LR free survival after RT (HR 1.70-2.42, p<0.01 for all 3 cohorts). Subtype association analysis demonstrated that TTK expression was most elevated in basal-like breast cancer (p<0.0001). Clonogenic survival assays, using inducible shRNA models, show the combination of TTK knockdown and RT increases radiosensitivity in MDA-MB-231 and BT-549 basal-like breast cancer cell lines (rER 1.21-1.63). Additionally, TTKi using, Bayer 1161909 (B909), enhanced radiosensitivity in both cell lines (rER 1.10-1.39). In vivo, TTKi, using shRNA and B909 in combination with RT led to a significant increased time to tumor tripling. Using γH2AX foci formation assays, increased DNA damage was found after combination treatment of TTKi and RT compared to RT alone, indicating that DNA damage repair mechanisms may be compromised by TTKi. We found that the efficiency of the double strand DNA damage repair mechanism, homologous recombination (HR), but not non-homologous end joining (NHEJ), was reduced upon TTKi in HR/NHEJ specific reporter systems. Additionally, RAD51 foci formation was reduced by TTKi after RT compared to RT alone. Conclusion: These data support TTK inhibition as a rationale radiosensitizing strategy for clinical development in basal-like breast cancer patients and that the mechanism of radiosensitization is, at least in part, through impaired HR repair. Citation Format: Benjamin C. Chandler, Leah Moubadder, Cassie Ritter, Kari Wilder-Romans, Meleah Cameron, Meilan Liu, Shyam Nyati, Andrea Pesch, Anna Michmerhuizen, Eric Olsen, Yashar Niknafs, Arul Chinnaiyan, Corey Speers. Nomination and characterization of TTK for radiosensitization in basal-like breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2926.

Genetic deletion of the DNA damage repair protein, ATM kinase, is not sufficient to induce vascular dysfunction in young mice

Ataxia telangiectasia mutated (ATM) kinase orchestrates the cellular response to double stranded DNA breaks, phosphorylating and activating downstream targets that lead to cell cycle arrest or apoptosis, as well as by recruiting the machinery necessary for DNA repair. In the vasculature, ATM may play a critical role in maintaining genomic stability, thereby preserving vascular function, as aberrant DNA repair has been implicated in vascular dysfunction. Here, we evaluated several measures of vascular function in ATM knockout (ATM −/−) and wildtype (ATM +/+) mice (4–6 mo, n=7–9/group). Aortic pulse wave velocity, a measure of large artery stiffness, was similar between groups (P>0.05). Furthermore, using an automated capture and analysis system we assessed markers of microvascular perfusion and glycocalyx properties. Perfused microvascular density, a measure of the number of perfused microvessels in a given area, and red blood cell (RBC) fraction, a marker of the heterogeneity of RBCs between microvessel segments, were not significantly different between groups (P>0.05). Likewise, perfused boundary region, a measure of endothelial glycocalyx barrier function, was not significantly different between ATM −/− and ATM +/+ controls (P>0.05). Furthermore, we examined endothelium‐dependent dilation and nitric oxide (NO) bioavailability in mesenteric arteries by measuring vasodilation to acetylcholine (ACh) in the absence and presence of the nitric oxide synthase inhibitor, L‐NAME. Maximal vasodilation to ACh did not differ between groups (P>0.05). Likewise, the contribution of NO to maximal vasodilation was similar between ATM −/− and ATM +/+ mice (P>0.05). Next, we assessed smooth muscle responsiveness to NO by measuring vasodilation to the NO donor, sodium nitroprusside (SNP). Maximal vasodilation to SNP did not differ between groups (P>0.05). Lastly, we evaluated the passive mechanical properties of isolated mesenteric arteries and found no differences in vessel lumen diameter in response to increasing pressure between ATM −/− and ATM +/+ mice (P>0.05). In conclusion, these findings suggest that removal of a major kinase involved with double stranded DNA damage signaling does not affect vascular function in young mice. This could be due to several reasons. First, young mice may have not accumulated sufficient DNA damage and mutagenesis to induce dysfunction or second, signaling downstream from DNA damage may drive vascular dysfunction. Thus, future studies are warranted that examine the role of ATM kinase in vascular function in response to conditions that augment DNA damage.Support or Funding InformationFunding Information: Funded in part by awards from the NIH, R01 AG048366, R01 AG050238, K02 AG045339 and K99 AT010017, and by US Department of Veterans Affairs 1I01BX002151.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

High doses of synthetic antioxidants induce premature senescence in cultivated mesenchymal stem cells

Stress-induced premature senescence program is known to be activated in cells by various genotoxic stressors, and oxidative stress is considered to be the main of those. To this end, many studies discover antioxidants as protective anti-aging agents. In the current study, we examined the effects of different antioxidants (Tempol, resveratrol, NAC, DPI) on the mesenchymal stem cells maintained in normal physiological conditions. We used high, but non-cytotoxic antioxidant doses which are widely used in laboratory practice to protect cells from oxidative damage. We show that these substances induce reversible block of cell proliferation and do not cause any genotoxic effects when applied to the quiescent cells. However, the same doses of the same substances, when applied to the proliferating cells, can induce irreversible cell cycle arrest, DNA strand breaks accumulation and DNA damage response activation. As a consequence, antioxidant-induced DNA damage results in the stress-induced premature senescence program activation. We conclude that high doses of antioxidants, when applied to the proliferating cells that maintain physiological levels of reactive oxygen species, can cause DNA damage and induce premature senescence which suggests to re-estimate believed unconditional anti-aging antioxidant properties.

THE DIFFERENCES OF APOPTOSIS EFFECTS BETWEEN COMBINATION OF MELOXICAM WITH GEMCITABINE-CARBOPLATIN CHEMOTHERAPY COMPARED TO GEMCITABINE-CARBOPLATIN CHEMOTHERAPY ALONE IN UROTHELIAL CARCINOMA CULTURE CELLS

Objective: To determine the differences of apoptosis effect between the combination of meloxicam and gemcitabine-carboplatin compared to gemcitabine-carboplatin alone as the standard of chemotherapy care in urothelial carcinoma culture cells. Material & Methods: This research is an in vitro experimental using human bladder cell carcinoma type 5637 which was cultured in the laboratory. In this study, the study group was divided into 3 groups: untreated control group, gemcitabine-carboplatin group, and the meloxicam-gemcitabine-carboplatin combination group, each group consist of 5 replications. To determine the dose of meloxicam, gemcitabine, carboplatin used and the time of apoptosis evaluation, cytotoxic tests were carried out using the MTT assay method. The time of apoptosis evaluation is carried out for 24 hours. Apoptosis was assessed using the Apoptotag reagent from Trevigen®. Observation of apoptosis characterized by a positive reaction (the color turning brown) against DNA strand damage using the TUNEL assay method. One Way ANOVA was used for comparative analysis of apoptosis between the group with a significant value of p<0.05. The analysis was continued with a post hoc test, to determine the differences in each group. Results: The mean of apoptosis in the control group, gemcitabine-carboplatin group, apoptosis and the meloxicam-gemcitabine-carboplatin combination group was 0.748%, 80.336%, and 83.312%, respectively. Post hoc Bonferroni analysis showed that the results had significant difference between the meloxicam-gemcitabine-carboplatin combination group compared to the gemcitabine-carboplatin group (p=0.026) and the control group (p=0.000). Conclusion: Meloxicam-gemcitabine-carboplatin combination therapy has a significantly higher apoptotic effect than gemcitabine-carboplatin alone.

Rhaponticum carthamoides transformed root extract inhibits human glioma cells viability, induces double strand DNA damage, H2A.X phosphorylation, and PARP1 cleavage

Rhaponticum carthamoides transformed root extract induces double strand DNA damage by increasing the number of phosphorylated H2A.X- and cleaved PARP1-positive U87MG cells and patient-derived IV grade glioma cells. Furthermore, treatment of these cells with root extract causes down-regulation of UHRF1 and DNMT1. Transformed root extract is rich in caffeoylquinic acid derivatives, especially tricaffeoylquinic acid derivatives. Our findings demonstrate that the R. carthamoides transformed root extract may trigger apoptosis in glioma cells by induction of DNA damage, PARP cleavage and epigenetic modification.

Biomarker responses of the freshwater clam Corbicula fluminea in acid mine drainage polluted systems

The environmental quality of an acid mine drainage polluted river (Odiel River) in the Iberian Pyrite Belt (SW Spain) was assessed by combining analyses of biomarkers (DNA strand breaks, LPO, EROD, GST, GR, GPx) in freshwater clams (Corbicula fluminea) exposed during 14 days and correlated with metal(loid) environmental concentrations.Results pointed that enzymatic systems are activated to combat oxidative stress in just 24 h. Along exposure, there were homeostatic regulations with the glutathione activity that influenced in lipid peroxidation oscillations, provoking significant DNA strand damage after 14 exposure days. EROD activity showed no changes throughout the exposure period.The Asian clam displayed balance biomarkers of exposure–antioxidant activity under non–stressfully environments; meanwhile, when was introduced into acid polymetallic environments, such as the acid mine drainage, its enzymatic activity was displaced towards biomarkers of effect and the corresponding antioxidant activity.

Abstract 4286: Tissue-specific preference for ATM or ATR pathway activation determines DNA damage outcome and provides insights in cancer predisposition

Abstract The DNA damage response (DDR) is an elaborate signaling network that protects against mutations and genomic instability. Despite its importance in preventing carcinogenesis, certain mutations in the DDR (e.g. BRCA1/2) predispose for specific cancers only. An explanation for this could be that the DDR is regulated differently across tissues; however, systematic analyses of the response to DNA damage in healthy human cells have not been carried out. We analyzed proteomic changes in response to single- and double strand DNA damage in non-immortalized human primary cells and related them to cell outcome. Primary breast and lung cells were treated with cisplatin (single-strand damage) or doxorubicin (double-strand breaks) at doses that caused a similar amount of DNA damage. Cell outcomes that were studied were apoptosis and cell cycle progression. Protein dynamics of 366 proteins were assessed using protein arrays and quantitative western blots. Inhibitors and an ODE-based mathematical model were used to determine the network topology and link it to outcome. Treatment of primary breast and lung epithelial cells with an equal amount of damage resulted in different viabilities. Primary mammary cells showed higher levels of apoptosis after single-strand damage, primary airway cells were more sensitive to double-strand breaks. The difference in outcome was reflected in distinct protein dynamics, including DNA repair pathways. Surprisingly, these differences originated at the earliest level of DNA damage detection by ATM and ATR. We demonstrated that differential activation of ATM and ATR pathways in breast and lung cells affects p53 and p21 dynamics and leads to distinct outcomes. We were able to identify key network nodes, providing new insights into the regulation of p53 and p21 and their relation to cell outcome. p53 and p21 dynamics are also linked to regulation of the cell cycle. In line with this, we found that cell cycle progression under normal conditions and after DNA damage is different. Importantly, we show that the double-strand break pathway plays a larger role in cell cycle control in breast cells than in lung cells. Since cell cycle checkpoints are a safeguard against genomic instability, differential regulation may affect cancer predisposition in a cell-type specific manner. Our work may provide an explanation for the fact that certain mutations in the DSB-repair pathway predispose for breast cancer, but not for lung cancer. In summary, we show that primary breast and lung cells activate distinct programs in response to equal doses of DNA damage, resulting in different p53 and p21 dynamics and, consequently, diverse outcomes. In addition, this work sheds light on the tissue-specific cancer predisposition of mutated DNA repair genes and may be the basis for further studies. Citation Format: Marijn T. Van Jaarsveld, Difan Deng, Erik A. Wiemer, Zhike Zi. Tissue-specific preference for ATM or ATR pathway activation determines DNA damage outcome and provides insights in cancer predisposition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4286.

SsDNA damage dependence from singlet oxygen concentration at photodynamic interaction

Single stranded DNA damage at photodynamic treatment with Radachlorin photosensitizer was investigated. Chemical trap method was used to evaluate generation of singlet oxygen in water solution. Interaction of singlet oxygen with ssDNA resulted into decrease of the replication activity of ssDNA. DNA stopped replicating during PCR at irradiation doses greater than 15 J/cm2 and concentration of photosensitizer [PS] = 3.8 μM. The dependence of replication activity of ssDNA on generated singlet oxygen concentration was identified.

TLR9 stimulation of B-cells induces transcription of p53 and prevents spontaneous and irradiation-induced cell death independent of DNA damage responses. Implications for Common variable immunodeficiency.

In the present study, we address the important issue of whether B-cells protected from irradiation-induced cell death, may survive with elevated levels of DNA damage. If so, such cells would be at higher risk of gaining mutations and undergoing malignant transformation. We show that stimulation of B-cells with the TLR9 ligands CpG-oligodeoxynucleotides (CpG-ODN) prevents spontaneous and irradiation-induced death of normal peripheral blood B-cells, and of B-cells from patients diagnosed with Common variable immunodeficiency (CVID). The TLR9-mediated survival is enhanced by the vitamin A metabolite retinoic acid (RA). Importantly, neither stimulation of B-cells via TLR9 alone or with RA increases irradiation-induced DNA strand breaks and DNA damage responses such as activation of ATM and DNA-PKcs. We prove that elevated levels of γH2AX imposed by irradiation of stimulated B-cells is not due to induction of DNA double strand breaks, but merely reflects increased levels of total H2AX upon stimulation. Interestingly however, we unexpectedly find that TLR9 stimulation of B-cells induces low amounts of inactive p53, explained by transcriptional induction of TP53. Taken together, we show that enhanced survival of irradiated B-cells is not accompanied by elevated levels of DNA damage. Our results imply that TLR9-mediated activation of B-cells not only promotes cell survival, but may via p53 provide cells with a barrier against harmful consequences of enhanced activation and proliferation. As CVID-derived B-cells are more radiosensitive and prone to undergo apoptosis than normal B-cells, our data support treatment of CVID patients with CpG-ODN and RA.

Novel technology identifies significant rise in sublethal single strand DNA damage in oocytes following chemotherapy exposure which correlates with increased fetal malformations and pregnancy failure

Novel technology identifies significant rise in sublethal single strand DNA damage in oocytes following chemotherapy exposure which correlates with increased fetal malformations and pregnancy failure

Antileukemic activity and cellular effects of the antimalarial agent artesunate in acute myeloid leukemia

The artimisinins are a class of antimalarial compounds whose antiparasitic activity is mediated by induction of reactive oxygen species (ROS). Herein, we report that among the artimisinins, artesunate (ARTS), an orally bioavailable compound has the most potent antileukemic activity in AML models and primary patients’ blasts. ARTS was most cytotoxic to the FLT3-ITD+ AML MV4-11 and MOLM-13 cells (IC50 values of 1.1 and 0.82μM respectively), inhibited colony formation in primary AML and MDS cells and augmented cytotoxicity of chemotherapeutics. ARTS lowered cellular BCL-2 level via ROS induction and increased the cytotoxicity of the BCL-2 inhibitor venetoclax (ABT-199). ARTS treatment led to cellular and mitochondrial ROS accumulation, double stranded DNA damage, loss of mitochondrial membrane potential and induction of the intrinsic mitochondrial apoptotic cascade in AML cell lines. The antileukemic activity of ARTS was further confirmed in MV4-11 and FLT3-ITD+ primary AML cell xenografts as well as MLL-AF9 syngeneic murine AML model where ARTS treatment resulted in significant survival prolongation of treated mice compared to control. Our results demonstrate the potent preclinical antileukemic activity of ARTS as well as its potential for a rapid transition to a clinical trial either alone or in combination with conventional chemotherapy or BCL-2 inhibitor, for treatment of AML.

Efflux Transporters Regulate Arsenite-Induced Genotoxicity in Double Negative and Double Positive T Cells.

Arsenite (As+3) exposure is known to cause immunotoxicity in human and animal models. Our previous studies demonstrated that As+3 at 50-500 nM concentrations induced both genotoxicity and nongenotoxicity in mouse thymus cells. Developing T cells at CD4-CD8- double negative (DN) stage, the first stage after early T cells are transported from bone marrow to thymus, were found to be more sensitive to As+3 toxicity than the T cells at CD4 + CD8 + double positive (DP) stage in vitro. Induction of Mdr1 (Abcb1) and Mrp1 (Abcc1), 2 multidrug resistance transporters and exporters of As+3, was associated with the reversal of As+3-induced double strand breaks and DNA damage. In order to confirm that the thymus cell populations have different sensitivity to As+3in vivo, male C57BL/6J mice were exposed to 0, 100, and 500 ppb As+3 in drinking water for 30 d. A significant decrease in DN cell percentage was observed with exposure to 500 ppb As+3. Low to moderate concentrations of As+3 were shown to induce higher genotoxicity in sorted DN than DP cells in vitro. Calcein AM uptake and Mdr1/Mrp1 mRNA quantification results revealed that DN cells not only had limited As+3 exporter activity, but also lacked the ability to activate these exporters with As+3 treatments, resulting in a higher accumulation of intracellular As+3. Knockdown study of As+3 exporters in the DN thymic cell line, D1 using siRNA, demonstrated that Mdr1 and Mrp1 regulate intracellular As+3 accumulation and genotoxicity. Taken together, the results indicate that transporter regulation is an important mechanism for differential genotoxicity induced by As+3 in thymocytes at different developmental stages.

Study of γ-H2AX as DNA double strand break biomarker in resident living in high natural radiation area of Mamuju, West Sulawesi

High expression of phospho histone γ-H2AX, a sensitive marker of double stranded DNA damage, is believed to be an indication of defective DNA repair pathway or genomic instability that may cause mutations and ultimately cancer. DNA damage can be caused by ionizing radiation exposure. Beside in medical treatment/diagnosis or industry, ionizing radiation exposure can also be found in naturally in regions of high natural back ground radiation. In this study we collect the blood from 45 volunteers living in Mamuju, a region with highest natural radiation in Indonesia (dose of ∼7 mSv/year). Subjects were grouped as high natural background area (HNBA) (n = 37) and control area (n = 8). The expression γ-H2AX foci were evaluated by one of researcher fluorescence microscope examination. Our results show that the average foci numbers per cell were in the normal range. While not statistical different, the average of γ-H2AX foci in exposed area higher in the exposed compared to the control area, 0.31 versus 0.13 (p > 0.05), respectively. Moreover, there was also no statistical difference of average γ-H2AX foci between man and woman, old and young people in exposed and control area (p > 0.05). In this preliminary study we find that γ-H2AX foci (and thus DNA double strand break) frequency in residents living in the HNBA of Mamuju, West Sulawesi, show a trend towards higher (albeit not significant) average values relative to the control area. More research is needed to further scrutinize these observations.

Honokiol Inhibits DNA Polymerases β and λ and Increases Bleomycin Sensitivity of Human Cancer Cells.

A major concept to sensitize cancer cells to DNA damaging agents is by inhibiting proteins in the DNA repair pathways. X-family DNA polymerases play critical roles in both base excision repair (BER) and nonhomologous end joining (NHEJ). In this study, we examined the effectiveness of honokiol to inhibit human DNA polymerase β (pol β), which is involved in BER, and DNA polymerase λ (pol λ), which is involved in NHEJ. Kinetic analysis with purified polymerases showed that honokiol inhibited DNA polymerase activity. The inhibition mode for the polymerases was a mixed-function noncompetitive inhibition with respect to the substrate, dCTP. The X-family polymerases, pol β and pol λ, were slightly more sensitive to inhibition by honokiol based on the Ki value of 4.0 μM for pol β, and 8.3 μM for pol λ, while the Ki values for pol η and Kf were 20 and 26 μM, respectively. Next we extended our studies to determine the effect of honokiol on the cytotoxicity of bleomycin and temozolomide in human cancer cell lines A549, MCF7, PANC-1, UACC903, and normal blood lymphocytes (GM12878). Bleomycin causes both single strand DNA damage that is repaired by BER and double strand breaks that are repaired by NHEJ, while temozolomide causes methylation damage repaired by BER and O6-alkylguanine-DNA alkyltransferase. The greatest effects were found with the honokiol and bleomycin combination in MCF7, PANC-1, and UACC903 cells, in which the EC50 values were decreased 10-fold. The temozolomide and honokiol combination was less effective; the EC50 values decreased three-fold due to the combination. It is hypothesized that the greater effect of honokiol on bleomycin is due to inhibition of the repair of the single strand and double strand damage. The synergistic activity shown by the combination of bleomycin and honokiol suggests that they can be used as combination therapy for treatment of cancer, which will decrease the therapeutic dosage and side effects of bleomycin.

Markers of Heart, Lung and Dorsal Aorta Damage of Mother Rats and Their Neonates Post Therapeutic Treatment with Doxorubicin, Cisplatin and 5-Flurouracil

Aim: Recently, there is an increased average of developing cancers. Though, the chemotherapeutic-treatment is unfavorable during pregnancy due to its harmful effects on developing fetuses, physicians have two ways to minimize these effects either by termination of the pregnancy or minimizing its side effects. The present work aimed to illustrate the susceptibility of cardiac, lung and dorsal aorta function to the widely applicable drugs doxorubicin and cisplatin as well as 5-flurouracil. Materials and Methods: Mother albino rats were arranged into four-groups (control, doxorubicin, cisplatin and 5-flurouracil-treated groups). Each pregnant rat received intraperitoneal administration of 0.2 mg/kg body weight at 10th and 14th day of gestation and sacrificed at parturition (two doses). At parturition, serum of mother rats used to assess troponin I, heat shock protein 70, 8-hydroxydeoxyguanosine, vascular endothelial growth factor and adhesion molecules (ICAM-1 & VCAM-1). Isoenzyme electrophoresis of alkaline and acid phosphatases, glucose-6-phosphate dehydrogenase and lactic dehydrogenase were estimated in serum, myocardium and dorsal aorta of mother rats. The myocardium and lung were processed for histopathological investigations for both mothers and their offspring. Single strand (comet assay) and double strand DNA damage were carried out in heart and dorsal aorta of mother rats. Results: The present finding revealed that there are detected alterations of myocardial markers and lung amino acid metabolism as well as disruption of myocardial isoenzymes. DNA damage of myocardium and dorsal aorta were observed. Conclusions: The authors concluded that the metabolic activity of heart and lung is highly susceptible to doxorubicin and cisplatin treatment compared to 5-flurouracil and the therapeutic doses must be degraded.

Two-Tailed Comet Assay (2T-Comet): Simultaneous Detection of DNA Single and Double Strand Breaks.

A modification of the original comet assay was developed for the simultaneous evaluation of DNA single strand breaks (SSBs) and double strand breaks (DSBs) in human spermatozoa. The two-dimensional perpendicular tail comet assay (2T-comet) combines non-denaturing and denaturant conditions to the same sperm nucleoid. In this case, the species-specific deproteinized sperm is first subjected to an electrophoretic field under non-denaturing conditions to mobilize isolated free discrete DNA fragments produced from DSBs; this is then followed by a second electrophoresis running perpendicular to the first one but under alkaline conditions to produce DNA denaturation, exposing SSBs on the same linear DNA chain or DNA fragments flanked by DSBs. This procedure results in a two dimensional comet tail emerging from the core where two types of original DNA affected molecule can be simultaneously discriminated. The 2T-comet is a fast, sensitive, and reliable procedure to distinguish between single and double strand DNA damage within the same cell. It is an innovative method for assessing sperm DNA integrity, which has important implications for human fertility and andrological pathology. This technique may be adapted to assess different DNA break types in other species and other cell types.

Base excision repair pathway: PARP1 genotypes as modulators of therapy response in cervical cancer patients

Context: Genetic polymorphisms in genes of the base excision repair (BER) pathway appear to modulate the therapy response of cancer patients. PARP1 protein recognizes the DNA strand damage and facilitates the subsequent recruitment of BER proteins. Few studies have reported an association between PARP1 Val762Ala polymorphism (rs1136410) and cancer therapy response.Objective: The purpose of our study was to determine whether PARP1 Val762Ala polymorphism have prognostic value in patients with cervical cancer.Materials and methods: Two hundred and sixty adult patients, with histologically confirmed cervical cancer, at FIGO-stages IB2-IVA, primarily treated with concurrent chemotherapy (cisplatin) and radiotherapy. Overall survival (OS) and disease-free survival (DFS) were the primary end points of the analysis. The PARP1 Val762Ala genetic variants were analyzed by allelic discrimination by real-time PCR.Results: We observed that peri- and postmenopausal women carrying the C-allele present a statistically significant lower OS and DFS (log-rank test, p = 0.008 and p = 0.006, respectively) among those with early stage cervical cancer. Cox regression analysis confirmed these results, after adjustment for other prognostic factors (for OS: HR, 3.70; 95%CI, 1.32–10.38; p = 0.013 and for DFS: HR, 3.97; 95%CI, 1.59–9.93; p = 0.003).Conclusions: This is the first study evaluating the effect of PARP1 Val762Ala polymorphism in treatment response in cervical cancer patients. PARP1 genotypes may contribute as an independent prognostic factor in cervical cancer, being useful in predicting the clinical outcome.

A Versatile Bioreactor for Dynamic Suspension Cell Culture. Application to the Culture of Cancer Cell Spheroids.

A versatile bioreactor suitable for dynamic suspension cell culture under tunable shear stress conditions has been developed and preliminarily tested culturing cancer cell spheroids. By adopting simple technological solutions and avoiding rotating components, the bioreactor exploits the laminar hydrodynamics establishing within the culture chamber enabling dynamic cell suspension in an environment favourable to mass transport, under a wide range of tunable shear stress conditions. The design phase of the device has been supported by multiphysics modelling and has provided a comprehensive analysis of the operating principles of the bioreactor. Moreover, an explanatory example is herein presented with multiphysics simulations used to set the proper bioreactor operating conditions for preliminary in vitro biological tests on a human lung carcinoma cell line. The biological results demonstrate that the ultralow shear dynamic suspension provided by the device is beneficial for culturing cancer cell spheroids. In comparison to the static suspension control, dynamic cell suspension preserves morphological features, promotes intercellular connection, increases spheroid size (2.4-fold increase) and number of cycling cells (1.58-fold increase), and reduces double strand DNA damage (1.5-fold reduction). It is envisioned that the versatility of this bioreactor could allow investigation and expansion of different cell types in the future.

Methylation levels of P16 and TP53 that are involved in DNA strand breakage of 16HBE cells treated by hexavalent chromium.

The correlations between methylation levels of p16 and TP53 with DNA strand breakage treated by hexavalent chromium [Cr(VI)] remain unknown. In this research, Human bronchial epithelial cells (16HBE cells) in vitro and bioinformatics analysis were used to analyze the epigenetic role in DNA damage and potential biomarkers. CCK-8 and single cell gel electrophoresis assay were chosen to detect the cellular biological damage. MALDI-TOF-MS was used to detect the methylation levels of p16 and TP53. qRT-PCR was used to measure their expression levels in different Cr(VI) treatment groups. The transcription factors with target sequences of p16 and TP53 were predicted using various bioinformatics software. The findings showed that the cellular toxicity and DNA strand damage were Cr(VI) concentration dependent. The hypermethylation of CpG1, CpG31 and CpG32 of p16 was observed in Cr(VI) treated groups. There was significant positive correlation between the CpG1 methylation level of p16 and cell damage. In Cr(VI) treated groups, the expression level of p16 was lower than that in control group. The expression level of TP53 increased when the Cr(VI)concentration above 5μM. About p16, there was significant negative correlation between the CpG1 methylation levels with its expression level. A lot of binding sites for transcription factors existed in our focused CpG islands of p16. All the results suggested that the CpG1 methylation level of p16 could be used as a biomarker of epigenetic effect caused by Cr(VI) treatment, which can enhance cell damage by regulating its expression or affecting some transcription factors to combine with their DNA strand sites.

Differential genotoxic and epigenotoxic effects of graphene family nanomaterials (GFNs) in human bronchial epithelial cells

The widespread applications of graphene family nanomaterials (GFNs) raised the considerable concern over human health and environment. The cyto-genotoxic potentiality of GFNs has attracted much more attention, albeit the potential effects on the cellular epigenome remain largely unknown. The effects of GFNs on cellular genome were evaluated with single and double stranded DNA damage and DNA repair gene expressions while the effects on epigenome was accomplished by addressing the global DNA methylation and expression of DNA methylation machineries at non-cytotoxic to moderately cytotoxic doses in in vitro system. We used five different representatives of GFNs-pristine (GNP-Prist), carboxylated (GNP-COOH) and aminated (GNP-NH2) graphene nanoplatelets as well as single layer (SLGO) and few layer (FLGO) graphene oxide. The order of single stranded DNA damage was observed as GNP-Prist≥GNP-COOH>GNP-NH2≥FLGO>SLGO at 10mg/L and marked dose dependency was found in SLGO. The GFNs possibly caused genotoxicity by affecting nucleotide excision repair and non-homologus end joining repair systems. Besides, dose dependent increase in global DNA methylation (hypermethylation) were observed in SLGO/FLGO exposure and conversely, GNPs treatment caused hypomethylation following the order as GNP-COOH>GNP-NH2≥GNP-Prist. The decrements of DNA methyltransferase (DNMT3B gene) and methyl-CpG binding domain protein (MBD1) genes were probably the cause of global hypomethylation induced by GNPs. Conversely, the de novo methylation through the up-regulation of DNMT3B and MBD1 genes gave rise to the global DNA hypermethylation in SLGO/FLGO treated cells. In general, the GFNs induced genotoxicity and alterations of global DNA methylation exhibited compounds type specificity with differential physico-chemical properties. Taken together, our study suggests that the GFNs could cause more subtle changes in gene expression programming by modulating DNA methylation status and this information would be helpful for their prospective use in biomedical field.