Induction Of DNA Breaks Research Articles

Genotoxicity kinetics in murine normoblasts as an approach for the in vivo action of difluorodeoxycytidine.

This study analyzed the kinetics of in vivo micronucleus induction in normoblasts by determining the kinetics of difluorodeoxycytidine (dFdC)-induced micronucleated polychromatic erythrocytes (MN-PCEs) in the peripheral blood of mice. The kinetic indexes of MN-PCE induction of dFdC were correlated with the previously reported mechanisms DNA damage induction by this compound. In general, this study aimed to establish an in vivo approach for discerning the processes underlying micronucleus induction by antineoplastic agents or mutagens in general. The frequencies of PCEs and MN-PCEs in the peripheral blood of mice were determined prior to treatment and after treatment using dFdC at doses of 95, 190, or 380µmol/kg at 8h intervals throughout a 72h post-treatment. The area beneath the curve (ABC) for MN-PCE induction as a function of time, which is an index of the total effect, indicated that the dose response was directly proportional and that the effect of dFdC on micronucleus induction was reduced compared with that of aneuploidogens and monofunctional and bifunctional alkylating agents but increased compared with that of promutagens, which is consistent with our previous results. The ABC showed a single peak with a small broadness index, which indicates that dFdC has a single mechanism or concomitant mechanisms for inducing DNA breaks. The time of the relative maximal induction (T rmi) indicated that dFdC requires more time to achieve MN-PCE induction compared with aneugens and monofunctional and bifunctional alkylating agents, although it requires a similar time to achieve MN-PCE induction as azacytidine, which is consistent with evidence showing that both agents must be incorporated into DNA for their action to be realized. The timing of maximal cytotoxicity observed with the lowest dFdC dose was correlated with the timing of the main genotoxic effect. However, early and late cytotoxic effects were detected, and these effects were independent of the genotoxic response. A correlation analysis indicated that dFdC appears to induce MN-PCEs through only one mechanism or mechanisms that occur concomitantly, which could be explained by the previously reported concurrent inhibitory effects of dFdC on DNA polymerase alpha, polymerase epsilon, and/or topoisomerase. The timing of maximal cytotoxicity was correlated with the timing of maximal genotoxicity; however, an early cytotoxic effect that appeared to occur prior to the incorporation of dFdC into DNA was likely related to a previously reported inhibitory effect of dFdC on thymidylate synthase and/or ribonucleotide reductase.

Doxorubicin and vincristine affect undifferentiated rat spermatogonia.

Anticancer drugs, such as alkylating agents, can affect male fertility by targeting the DNA of proliferative spermatogonial stem cells (SSC). Therefore, to reduce such side effects, other chemotherapeutics are used. However, less is known about their potential genotoxicity on SSC. Moreover, DNA repair mechanisms in SSC are poorly understood. To model treatments deprived of alkylating agents that are commonly used in cancer treatment, we tested the impact of exposure to doxorubicin and vincristine, alone or in combination (MIX), on a rat spermatogonial cell line with SSC characteristics (GC-6spg). Vincristine alone induced a cell cycle arrest and cell death without genotoxic impact. On the other hand, doxorubicin and the MIX induced a dose-dependent cell death. More importantly, doxorubicin and the MIX induced DNA breaks, measured by the COMET assay, at a non-cytotoxic dose. To elucidate which DNA repair pathway is activated in spermatogonia after exposure to doxorubicin, we screened the expression of 75 genes implicated in DNA repair. Interestingly, all were expressed constitutively in GC-6spg, suggesting great potential to respond to genotoxic stress. Doxorubicin treatments affected the expression of 16 genes (>1.5 fold change; P < 0.05) involved in cell cycle, base/nucleotide excision repair, homologous recombination and non-homologous end joining (NHEJ). The significant increase in CDKN1A and XRCC1 suggest a cell cycle arrest and implies an alternative NHEJ pathway in response to doxorubicin-induced DNA breaks. Together, our results support the idea that undifferentiated spermatogonia have the ability to respond to DNA injury from chemotherapeutic compounds and escape DNA break accumulation.

The role of ROS and subsequent DNA-damage response in PUMA-induced apoptosis of ovarian cancer cells.

PUMA is a member of the “BH3-only” branch of the BCL-2 family. Our previous study suggests a therapeutic potential of PUMA in treating ovarian cancer, however, the action mechanism of PUMA remains elusive. In this work, we found that in PUMA adenovirus-infected A2780s ovarian cancer cells, exogenous PUMA was partially accumulated in the cytosol and mainly located to the mitochondria. We further showed that PUMA induces mitochondrial dysfunction-mediated apoptosis and ROS generation through functional BAX in a ROS generating enzyme- and caspase-independent manner irrespective of their p53 status, and results in activation of Nrf2/HO-1 pathway. Furthermore, PUMA induces DNA breaks in γ-H2AX staining, and causes activation of DNA damage-related kinases including ATM, ATR, DNA-PKcs, Chk1 and Chk2, which are correlated with the apoptosis. PUMA also results in ROS-triggered JNK activation. Intriguingly, JNK plays a dual role in both DNA damage response and apoptosis, and has an additional contribution to apoptosis. Taken together, we have provided new insight into the action mechanism by which elevated PUMA first induces ROS generation then results in DNA damage response and JNK activation, ultimately contributing to apoptosis in ovarian cancer cells.

Abstract ES9-2: ES9-2 Metastatic TNBC: New targets/new strategies

Abstract Triple negative breast cancer (TNBC) is a diagnosis of exclusion as these tumors lack ER, PR and HER2. Through advances in molecular profiling, the heterogeneity of TNBC has been revealed as subsets with molecular features targetable with either investigational or existing FDA approved drugs. Basal-like TNBCs (BL1 and BL2) are associated with proliferative gene signatures. BL1 tumors demonstrate at higher rate of pCR to standard chemotherapy compared to other subtypes. The BL2 subset demonstrates gene ontologies involving growth factor signaling such as EGFR and IGF1R. Preclinical models demonstrate that basal-like TNBCs have high sensitivity to cisplatin and relative resistance to PI3K inhibitors in cell culture and xenografts. Germ line aberrations in BRCA1 and BRCA2 lead to a defect in DNA repair in cancers harboring such mutationsand these tumors appear to be more sensitive to therapy inducing DNA breaks, such as those induced by platinum based therapies or PARP inhibition. Approximately 30% of TNBCs display mesenchymal features and carry a high rate of molecular aberrations that activate the PI3K/Akt/mTOR axis with clinical data suggesting that this subgroup may be responsive to therapeutic regimens targeting this pathway. Approximately 10-15% of TNBCs are associated with expression of androgen receptor (AR+ or LAR) and are characterized by the expression of luminal genes and a high rate of PI3K activation/mutation. Response to AR and/or PI3K directed therapy has been demonstrated in preclinical models and AR inhibition has demonstrated response in early phase clinical trials. Lastly, though breast cancer is generally considered a non-immunogenic malignancy, some TNBCs have tumor infiltrating lymphocytes. Novel agents to enrich immune response have been associated with durable responses in a subset of patients with metastatic TNBC. In summary, molecular characterization has identified subsets of TNBC that show promising response to existing or emerging targeted agents. Citation Format: Moulder SL. ES9-2 Metastatic TNBC: New targets/new strategies [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr ES9-2.

Impact of RAD51C-mediated Homologous Recombination on Genomic Integrity in Barrett's Adenocarcinoma Cells.

BACKGROUNDIn normal cells, RAD51-mediated homologous recombination (HR) is a precise DNA repair mechanism which plays a key role in the maintenance of genomic integrity and stability. However, elevated (dysregulated) RAD51 is implicated in genomic instability and is a potential target for treatment of certain cancers, including Barrett’s adenocarcinoma (BAC). In this study, we investigated genomic impact and translational significance of moderate vs. strong suppression of RAD51 in BAC cells.METHODSBAC cells (FLO-1 and OE33) were transduced with non-targeting control (CS) or RAD51-specific shRNAs, mediating a moderate (40–50%) suppression or strong (80-near 100%) suppression of the gene. DNA breaks, spontaneous or following exposure to DNA damaging agent, were examined by comet assay and 53BP1 staining. Gene expression was monitored by microarrays (Affymetrix). Homologous recombination (HR) and single strand annealing (SSA) activities were measured using plasmid based assays.RESULTSWe show that although moderate suppression consistenly inhibits/reduces HR activity, the strong suppression is associated with increase in HR activity (by ~15 – ≥ 50% in various experiments), suggesting activation of RAD51-independent pathway. Contrary to moderate suppression, a strong suppression of RAD51 is associated with a significant induced DNA breaks as well as altered expression of genes involved in detection/processing of DNA breaks and apoptosis. Stronger RAD51 suppression was also associated with mutagenic single strand annealing mediated HR. Suppression of RAD51C inhibited RAD51-independent (SSA-mediated) HR in BAC cells.CONCLUSIONElevated (dysregulated) RAD51 in BAC is implicated in both the repair of DNA breaks as well as ongoing genomic rearrangements. Moderate suppression of this gene reduces HR activity, whereas strong or near complete suppression of this gene activates RAD51C-dependent HR involving a mechanism known as single strand annealing (SSA). SSA-mediated HR, which is a mutagenic HR pathway, further disrupts genomic integrity by increasing DNA breaks in BAC cells.

In vitro toxicological assessment of an organosulfur compound from Allium extract: Cytotoxicity, mutagenicity and genotoxicity studies

Garlic (Allium sativum) and onion (Allium cepa) are being used in the food industry as flavoring but also for their antimicrobial activities. These activities are mainly derived from the organosulfur compounds (OSCs). Propyl propane thiosulfinate (PTS) is an OSC with potential use in the active packaging, but its safety should be guaranteed before being commercialized. The aim of this work was to investigate for the first time the cytotoxicity of PTS as well as its in vitro mutagenic/genotoxic potential using the following battery of genotoxicity tests:(1)the bacterial reverse-mutation assay in S. typhimurium (Ames test, OECD 471, 1997); (2) the micronucleus test (MN, OECD 487, 2016); (3) the mouse lymphoma thymidine-kinase assay (MLA, OECD 476, 2015), and (4) the comet assay (standard and modified with restriction enzymes). The results revealed that PTS was not mutagenic neither in the Ames test nor in MLA. However, genotoxic effects were recorded in the MN test on mammalian cells (L5178YTk+/−cells) after PTS exposure at the highest concentration tested (17.25 μM) without S9, and also its metabolites (+S9, from 20 μM). Moreover, in the comet assay, PTS induced DNA breaks damage in Caco-2 cells at the highest concentration tested (280 μM) but it did not induce oxidative DNA damage.

Flap Structure-Specific Endonuclease 1 (FEN1) May be a Key Mediator of Genome Instability in Myeloma: A Cellular Vulnerability with Potential Therapeutic Significance

Multiple myeloma is associated with a marked genomic instability which leads to acquisition of mutational changes, some of which underlie disease progression including development of drug resistance and poor clinical outcome. Understanding mechanisms of genomic instability is, therefore, extremely important to develop novel improved therapeutic strategies. Since dysregulated nuclease activity can induce DNA breaks and genetic recombination eventually disrupting genomic integrity, we have evaluated nuclease activity and specific nucleases for their role in genomic instability in MM. We previously identified a nuclease gene signature correlating with genomic instability in a myeloma patient dataset and tested it for correlation with survival in two other datasets. We showed that expression of these genes associated with poor overall as well as event free survival in both datasets, IFM172 (P< 0.00005) and gse24080 (P< 0.0008). We have now further refined this signature to a nine gene signature and tested it for correlation with survival in three different MM patient datasets in which gene expression was evaluated either by microarray (GSE39754, n=170; gse24080; n=559) or RNASeq (n=300). Elevated expression of nine gene signature significantly correlated with poor overall survival in all three datasets (P ≤ 1e-06 for IFM70 and gse24080 and P = 0.002 for RNASeq). To biologically and molecularly validate this signature, we conducted an shRNA screen and evaluated impact of all nine genes in signature as well as four additional nucleases on homologous recombination (HR) activity, using a plasmid based assay in which HR produces a functional luciferase gene. Of thirteen nucleases tested, knockdown of seven was associated with ≥50% inhibition of HR activity; the strongest (~80%) inhibition of HR activity was observed by FEN1 knockdown. To further investigate FEN1, we confirmed the role of this nuclease in HR using a different (DRGFP) assay in which homology-based recombination between two mutated genes, generates a functional GFP gene. Using this assay, we showed that FEN1-knockdown in U2OS cells was also associated with a strong (71%) inhibition of HR activity, confirming the role of this nuclease in dysregulation of HR. Evaluation by Western blotting in three different normal PBMC samples and eleven MM cell lines showed that FEN1 was not detected in normal cells, whereas highly expressed in MM cells. Expression profile using microarray also showed that FEN1 is elevated in a subset of MM patient samples. Knockdown of FEN1 in two MM cell lines, RPMI and H929, led to reduction in overall nuclease activity (by ~40%) as assessed by a fluorescence based nuclease activity assay and a similar (~50%) reduction in the levels of gamma-H2AX, a marker of DNA breaks. These data indicate that FEN1 nuclease activity contributes to increased DNA breaks as well as elevated HR activity in MM cells. To further understand the role of FEN1 in dysregulated HR and genome stability in MM, using mass spectrometry, we have identified the interacting proteins. Role of FEN1 in acquisition of new genomic changes over time in MM cells is cuurently being investigated in our laboratory. In summary, we show that FEN1 is an important component of machinery maintaining genomic integrity and plays a significant role in genome dysregulation in myeloma. The FEN1 dysfunction may provide a cellular vulnerability that can be therapeutically exploited. DisclosuresNo relevant conflicts of interest to declare.

Toxicity of tannic acid-modified silver nanoparticles in keratinocytes: potential for immunomodulatory applications

Hydrolyzable tannins are known to exhibit anti-inflammatory activity, which can be used in combination with silver nanoparticles (AgNPs) for dermal uses. In this study, we investigated the effects of tannic acid-modified 13, 33, 46nm and unmodified 10–65nm AgNPs using the human-derived keratinocyte HaCaT and VK2-E6/E7 cell lines in the form of stationary and spheroids cultures. After exposition to tannic acid-modified AgNPs, VK2-E6/E7 cells showed higher toxicity, increased production of reactive oxygen species (ROS) and activity of JNK stress kinase, while HaCaT cell line demonstrated less ROS production and activation of ERK kinase. AgNPs internalization was detected both in the superficial and internal layers of spheroids prepared from both cell lines. Tannic acid modified AgNPs sized above 30nm did not induce DNA breaks in comet assay performed in both cell lines. Tannic acid-modified but not unmodified AgNPs down-regulated TNF-α and LPS-triggered production of IL-8 in VK2-E6/E7 but not in HaCaT cells. In summary, tannic acid-modified AgNPs sized above 30nm show good toxicological profile both in vitro and possess immunomodulatory properties useful for potential dermal applications in humans.

Histone Modification Is Involved in Okadaic Acid (OA) Induced DNA Damage Response and G2-M Transition Arrest in Maize.

Histone modifications are involved in regulation of chromatin structure. To investigate the relationship between chromatin modification and cell cycle regulation during plant cell proliferation, Okadaic acid (OA), a specific inhibitor of serine/threonine protein phosphatase, was applied in this study. The results showed that OA caused the cell cycle arrest at preprophase, leading to seedling growth inhibition. Western blotting assay revealed that the spatial distribution of phosphorylation of Ser10 histone H3 tails (H3S10ph) signals was altered under OA treatment. Reactive oxygen species (ROS) was found to be at higher levels and TdT-mediated dUTP nick end labeling (TUNEL) assay displayed DNA breaks happened at the chromatin after treatment with OA, companied with an increase in the acetylation of histone H4 at lysine 5 (H4K5ac) level. From these observations, we speculated that the alteration of the spatial distribution of H3S10ph and the level of H4K5ac was involved in the procedure that OA induced DNA breaks and G2-M arrested by the accumulation of ROS, and that the histone H3S10ph and H4K5ac might facilitate DNA repair by their association with the chromatin decondensation.

Toxic and DNA damaging effects of a functionalized fullerene in human embryonic lung fibroblasts

Water-soluble fullerenes have been studied as potential nanovectors and therapeutic agents, but their possible toxicity is of concern. We have studied the effects of F-828, a soluble fullerene [C60] derivative, on diploid human embryonic lung fibroblasts (HELFs) in vitro. F-828 causes complex time-dependent changes in ROS levels. Inhibition of Nox4 activity by plumbagin blocks F-828-dependent ROS elevation. F-828 induces DNA breaks, as measured by the comet assay and γH2AX expression, and the activities of the transcription factors NF-kB and p53 increase. F-828 concentrations>25μM are cytotoxic; cell death occurs by necrosis. Expression levels of TGF-β, RHOA, RHOC, ROCK1, and SMAD2 increase following exposure to F-828. Our results raise the possibility that fullerene F-828 may induce pulmonary fibrosis in vivo.

Berberine in combination with cisplatin suppresses breast cancer cell growth through induction of DNA breaks and caspase-3-dependent apoptosis.

Berberine (BBR) is an isoquinoline alkaloid extracted from medicinal plants such as Hydrastis canadensis, Berberis aristata and Coptis chinensis. BBR displays a number of beneficial roles in the treatment of various types of cancers, yet the precise mechanisms of its action remain unclear. Cisplatin is an effective cancer chemotherapeutic agent and functions by generating DNA damage, promoting DNA damage-induced cell cycle arrest and apoptosis; however, its efficacy is challenged by the resistance of tumor cells in clinical application. The aim of the present study was to investigate the effects of BBR in combination with cisplatin on human breast cancer cells. MTT assay showed that BBR inhibited breast cancer MCF-7 cell growth with a 50%inhibitory concentration (IC50) value of 52.178±1.593µM and the IC50 value of cisplatin was 49.541±1.618µM, while in combination with 26µM BBR, the IC50 value of cisplatin was 5.759±0.76µM. BBR sensitized the MCF-7 cells to cisplatin in a time- and dose-dependent manner. After treatment of BBR and cisplatin, the cellular pro-apoptotic capase-3 and cleaved capspase-3 and caspase-9 were upregulated and the anti-apoptotic Bcl-2 was downregulated. Importantly, BBR restrained the expression of cellular PCNA, and immunofluoresence analysis of γH2AX showed that BBR increased the DNA damages induced by cisplatin. Taken together, the results demonstrated that BBR sensitized MCF-7 cells to cisplatin through induction of DNA breaks and caspase-3-dependent apoptosis.

The effects of the mycotoxin austdiol on cell cycle progression, cytotoxicity and genotoxicity in Chinese hamster ovary (CHO-K1) cells

Austdiol is a mycotoxin mainly produced byAspergillus ustus andMycoleptodiscus indicus. These fungi are found in rye, oats, barley, corn and feed grains; thus, as a potential contaminant of human food and animal feed, this mycotoxin is of great concern. As such, the elucidation of the cytotoxicity and mutagenicity of austdiol is important. In this study, austdiol was purified from a rice-oat solid medium culture ofM. indicus using chromatographic separation techniques. Chinese hamster ovary (CHO-K1) cells were then used to study the effect of austdiol on mammalian cell cycle, clonogenicity and DNA damage. Austdiol induced cell cycle arrest in G2/M phase, with a decreased S phase population and increased sub-G1 population. Austdiol also increased the polyploid population. These events resulted in cell death detected 7 days after treatment by clonogenic assay. DNA damage represents the main mechanism of action of austdiol, which induces DNA breaks and increases the frequency of micronuclei and nucleoplasmic bridges in binucleated cells in a CHO-K1 cell line. Moreover, cells exposed to austdiol and doxorubicin (DXR) combined treatments presented a reduced number of colonies and increased frequencies of micronuclei and nucleoplasmic bridges compared with negative control and cells treated with austdiol or DXR alone.

Chemical and Biophysical Modulation of Cas9 for Tunable Genome Engineering.

The application of the CRISPR-Cas9 system for genome engineering has revolutionized the ability to interrogate genomes of mammalian cells. Programming the Cas9 endonuclease to induce DNA breaks at specified sites is achieved by simply modifying the sequence of its cognate guide RNA. Although Cas9-mediated genome editing has been shown to be highly specific, cleavage events at off-target sites have also been reported. Minimizing, and eventually abolishing, unwanted off-target cleavage remains a major goal of the CRISPR-Cas9 technology before its implementation for therapeutic use. Recent efforts have turned to chemical biology and biophysical approaches to engineer inducible genome editing systems for controlling Cas9 activity at the transcriptional and protein levels. Here, we review recent advancements to modulate Cas9-mediated genome editing by engineering split-Cas9 constructs, inteins, small molecules, protein-based dimerizing domains, and light-inducible systems.

Dihydropyrimidine Dehydrogenase Is a Prognostic Marker for Mesenchymal Stem Cell-Mediated Cytosine Deaminase Gene and 5-Fluorocytosine Prodrug Therapy for the Treatment of Recurrent Gliomas.

We investigated a therapeutic strategy for recurrent malignant gliomas using mesenchymal stem cells (MSC), expressing cytosine deaminase (CD), and prodrug 5-Fluorocytosine (5-FC) as a more specific and less toxic option. MSCs are emerging as a novel cell therapeutic agent with a cancer-targeting property, and CD is considered a promising enzyme in cancer gene therapy which can convert non-toxic 5-FC to toxic 5-Fluorouracil (5-FU). Therefore, use of prodrug 5-FC can minimize normal cell toxicity. Analyses of microarrays revealed that targeting DNA damage and its repair is a selectable option for gliomas after the standard chemo/radio-therapy. 5-FU is the most frequently used anti-cancer drug, which induces DNA breaks. Because dihydropyrimidine dehydrogenase (DPD) was reported to be involved in 5-FU metabolism to block DNA damage, we compared the survival rate with 5-FU treatment and the level of DPD expression in 15 different glioma cell lines. DPD-deficient cells showed higher sensitivity to 5-FU, and the regulation of DPD level by either siRNA or overexpression was directly related to the 5-FU sensitivity. For MSC/CD with 5-FC therapy, DPD-deficient cells such as U87MG, GBM28, and GBM37 showed higher sensitivity compared to DPD-high U373 cells. Effective inhibition of tumor growth was also observed in an orthotopic mouse model using DPD- deficient U87MG, indicating that DPD gene expression is indeed closely related to the efficacy of MSC/CD-mediated 5-FC therapy. Our results suggested that DPD can be used as a biomarker for selecting glioma patients who may possibly benefit from this therapy.

Genotoxicity assessment of propyl thiosulfinate oxide, an organosulfur compound from Allium extract, intended to food active packaging

Essential oils from onion (Allium cepa L.), garlic (Allium sativum L.), and their main components, such as propyl thiosulfinate oxide (PTSO) are being intended for active packaging with the purpose of maintaining and extending food product quality and shelf life. The present work aims to assess for the first time the potential mutagenicity/genotoxicity of PTSO (0–50 µM) using the following battery of genotoxicity tests: (1) the bacterial reverse-mutation assay in Salmonella typhimurium (Ames test, OECD 471); (2) the micronucleus test (OECD 487) (MN) and (3) the mouse lymphoma thymidine-kinase assay (OECD 476) (MLA) on L5178YTk+/−, cells; and (4) the comet assay (with and without Endo III and FPG enzymes) on Caco-2 cells. The results revealed that PTSO was not mutagenic in the Ames test, however it was mutagenic in the MLA assay after 24 h of treatment (2.5–20 µM). The parent compound did not induce MN on mammalian cells; however, its metabolites (in the presence S9) produced positive results (from 15 µM). Data from the comet assay indicated that PTSO did not induce DNA breaks or oxidative DNA damage. Further in vivo genotoxicity tests are needed to confirm its safety before it is used as active additive in food packaging.

E2F1 controls germ cell apoptosis during the first wave of spermatogenesis.

SummaryCell cycle control during spermatogenesis is a highly complex process owing to the control of the mitotic expansion of the spermatogonial cell population and following meiosis, induction of DNA breaks during meiosis and the high levels of physiological germ‐cell apoptosis. We set out to study how E2F1, a key controller of cell cycle, apoptosis, and DNA damage responses, functions in the developing and adult testis. We first analyzed the expression pattern of E2f1 during post‐natal testis development using RNA in situ hybridization, which showed a differential expression pattern of E2f1 in the adult and juvenile mouse testes. To study the function of E2f1, we took advantage of the E2F1−/− mouse line, which was back‐crossed to C57Bl/6J genetic background. E2f1 loss led to a severe progressive testicular atrophy beginning at the age of 20 days. Spermatogonial apoptosis during the first wave of spermatogenesis was decreased. However, already in the first wave of spermatogenesis an extensive apoptosis of spermatocytes was observed. In the adult E2F1−/− testes, the atrophy due to loss of spermatocytes was further exacerbated by loss of spermatogonial stem cells. Surprisingly, only subtle changes in global gene expression array profiling were observed in E2F1−/− testis at PND20. To dissect the changes in each testicular cell type, an additional comparative analysis of the array data was performed making use of previously published data on transcriptomes of the individual testicular cell types. Taken together, our data indicate that E2F1 has a differential role during first wave of spermatogenesis and in the adult testis, which emphasizes the complex nature of cell cycle control in the developing testis.

Bacterial Genotoxins: Merging the DNA Damage Response into Infection Biology.

Bacterial genotoxins are unique among bacterial toxins as their molecular target is DNA. The consequence of intoxication or infection is induction of DNA breaks that, if not properly repaired, results in irreversible cell cycle arrest (senescence) or death of the target cells. At present, only three bacterial genotoxins have been identified. Two are protein toxins: the cytolethal distending toxin (CDT) family produced by a number of Gram-negative bacteria and the typhoid toxin produced by Salmonella enterica serovar Typhi. The third member, colibactin, is a peptide-polyketide genotoxin, produced by strains belonging to the phylogenetic group B2 of Escherichia coli. This review will present the cellular effects of acute and chronic intoxication or infection with the genotoxins-producing bacteria. The carcinogenic properties and the role of these effectors in the context of the host-microbe interaction will be discussed. We will further highlight the open questions that remain to be solved regarding the biology of this unusual family of bacterial toxins.

DNA break-induced sumoylation is enabled by collaboration between a SUMO ligase and the ssDNA-binding complex RPA

Upon genome damage, large-scale protein sumoylation occurs from yeast to humans to promote DNA repair. Currently, the underlying mechanism is largely unknown. Here we show that, upon DNA break induction, the budding yeast SUMO ligase Siz2 collaborates with the ssDNA-binding complex RPA (replication protein A) to induce the sumoylation of recombination factors and confer damage resistance. Both RPA and nuclease-generated ssDNA promote Siz2-mediated sumoylation. Mechanistically, the conserved Siz2 interaction with RPA enables Siz2 localization to damage sites. These findings provide a molecular basis for recruiting SUMO ligases to the vicinity of their substrates to induce sumoylation upon DNA damage.

The Genotoxin Colibactin Is a Determinant of Virulence in Escherichia coli K1 Experimental Neonatal Systemic Infection.

Escherichia coli strains expressing the K1 capsule are a major cause of sepsis and meningitis in human neonates. The development of these diseases is dependent on the expression of a range of virulence factors, many of which remain uncharacterized. Here, we show that all but 1 of 34 E. coli K1 neonatal isolates carried clbA and clbP, genes contained within the pks pathogenicity island and required for the synthesis of colibactin, a polyketide-peptide genotoxin that causes genomic instability in eukaryotic cells by induction of double-strand breaks in DNA. Inactivation of clbA and clbP in E. coli A192PP, a virulent strain of serotype O18:K1 that colonizes the gastrointestinal tract and translocates to the blood compartment with very high frequency in experimental infection of the neonatal rat, significantly reduced the capacity of A192PP to colonize the gut, engender double-strand breaks in DNA, and cause invasive, lethal disease. Mutation of clbA, which encodes a pleiotropic enzyme also involved in siderophore synthesis, impacted virulence to a greater extent than mutation of clbP, encoding an enzyme specific to colibactin synthesis. Restoration of colibactin gene function by complementation reestablished the fully virulent phenotype. We conclude that colibactin contributes to the capacity of E. coli K1 to colonize the neonatal gastrointestinal tract and to cause invasive disease in the susceptible neonate.

Telomere length in blood cells is related to the chronicity, severity, and recurrence rate of schizophrenia.

IntroductionTelomere shortening is strongly associated with higher mortality rates and has been shown in a number of age-related diseases, such as cardiovascular disorders, diabetes mellitus, Alzheimer’s disease, and psychiatric disorders. Oxidative stress is known to induce DNA breaks and genome instability. Telomeric DNA rich in guanosine is particularly sensitive to such oxidative damages. Psychosis is associated with a disequilibrium between free radical production and antioxidative defense. Although telomere attrition has been demonstrated in schizophrenia, no relationship has been reported between telomere length and severity of schizophrenia.AimThe aim of the present study was to identify differences in telomere length in peripheral blood cells between patients with chronic schizophrenia (C-SCZ) and early schizophrenia (E-SCZ) and to identify any relationship between telomere length and disease chronicity and severity.MethodsRelative average telomere lengths were determined using qPCR assay in patients with E-SCZ (n=42) and C-SCZ (n=44) hospitalized due to schizophrenia exacerbation. E-SCZ was diagnosed when less than 2 years had passed since the beginning of psychotic symptoms. The severity of symptoms was assessed using appropriate scales.ResultsThe severity of schizophrenia symptoms, as well as the number of psychotic episodes and hospital admissions, correlated significantly with telomere length in univariate analyses. Regression analysis revealed that a model incorporating study group (E-SCZ or C-ECZ), sex, and age, as well as the combined number of documented psychotic episodes and hospital admissions, can significantly predict the length of telomeres in patients with schizophrenia, with over 50% of variance in telomere length explained by the model (adjusted R2=0.512).ConclusionThe results of the current study indicate that the recurrence of psychotic symptoms as well as their intensity and chronicity may be correlated with telomere attrition, which is well known to contribute to the development of premature senescence and age-related diseases.