Induced DNA DNA Double-strand Breaks Research Articles

Efficient oligo nucleotide mediated CRISPR-Cas9 gene editing in Aspergilli

CRISPR-Cas9 technologies are revolutionizing fungal gene editing. Here we show that survival of specific Cas9/sgRNA mediated DNA double strand breaks (DSBs) depends on the non-homologous end-joining, NHEJ, DNA repair pathway and we use this observation to develop a tool, TAPE, to assess protospacer efficiency in Aspergillus nidulans. Moreover, we show that in NHEJ deficient strains, highly efficient marker-free gene targeting can be performed. Indeed, we show that even single-stranded oligo nucleotides efficiently work as repair templates of specific Cas9/sgRNA induced DNA DSBs in A. nidulans, A. niger, and in A. oryzae indicating that this type of repair may be wide-spread in filamentous fungi. Importantly, we demonstrate that by using single-stranded oligo nucleotides for CRISPR-Cas9 mediated gene editing it is possible to introduce specific point mutations as well gene deletions at efficiencies approaching 100%. The efficiency of the system invites for multiplexing and we have designed a vector system with the capacity of delivering Cas9 and multiple sgRNAs based on polymerase III promoters and tRNA spacers. We show that it is possible to introduce two point mutations and one gene insertion in one transformation experiment with a very high efficiency. Our system is compatible with future high-throughput gene-editing experiments.

Induction and repair of DNA double-strand breaks in hippocampal neurons of miсe of different age after exposure to 60Со γ-rays in vivo and in vitro

One of the central problems of modern radiobiology is the study of DNA damage induction and repair mechanisms in central nervous system cells, in particular, in hippocampal cells. The study of the regularities of molecular damage formation and repair in the hippocampus cells is of special interest, because these cells, unlike most cells of the central nervous system (CNS), keep proliferative activity, i.e. ability to neurogenesis. Age-related changes in hippocampus play an important role, which could lead to radiosensitivity changes in neurons to the ionizing radiation exposure. Regularities in DNA double-strand breaks (DSB) induction and repair in different aged mice hippocampal cells in vivo and in vitro under the action of γ-rays 60Со were studied with DNA comet-assay. The obtained dose dependences of DNA DSB induction are linear both in vivo and in vitro. It is established that in young animals' cells, the degree of DNA damage is higher than in older animals. It is shown that repair kinetics is basically different for exposure in vivo and in vitro.

Cellular Uptake and Radio-sensitization Effect of Small Gold Nanoparticles in MCF-7 Breast Cancer Cells

Gold nanoparticles (AuNPs) are shown to increase cancer cells sensitization to ionizing radiation. Theoretical models predicted that due to the differences in absorption coefficient between gold and soft tissue, maximum sensitization can be achieved at kilovoltage (kV), with minimum to no effect at megavoltage (MV) range, which was indeed demonstrated by previous research on AuNPs radio-sensitization with kV radiation. However, AuNPs effect at MV energies is still not clear. Radio-sensitization depends on AuNPs’ internalization by cancer cells that is in turn affected by AuNPs size. The purpose of this study was to explore, in vitro, radio sensitization potential of AuNPs of 4 and 14 nm size with clinically relevant MV, as well as kV photons in MCF7 breast cancer cell line. We investigated the differences in cellular uptake and intracellular localization between 4 and 14 nm AuNPs by transmission electron microscopy (TEM). We also examined the effect of these AuNPs on MCF7 cells response to irradiation by MTT analysis of cell proliferation, and flow cytometry analysis of H2AX phosphorylation as an indicator of DNA double strand breaks (DSBs). Our results demonstrated significant AuNPs cellular uptake and a selective, size dependent intracellular localization with 4 nm AuNPs mostly residing in cytoplasm and 14 nm in nucleus. Nanoparticles of both sizes sensitized MCF-7 breast cancer cells to radiation at both kV and MV energies, independent of particle size. Radio-sensitization was detected as an increase in radiation induced inhibition of cell proliferation and radiation induced DNA DSBs. This work provides valuable information for further exploring mechanisms of AuNPs radio-sensitization in cancer cell lines that may be important in designing and optimizing AuNPs based radiotherapies.

Akt1 and Akt3 but not Akt2 through interaction with DNA-PKcs stimulate proliferation and post-irradiation cell survival of K-RAS-mutated cancer cells

Akt1 through the C-terminal domain interacts with the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and stimulates the repair of DNA double-strand breaks (DSBs) in K-RAS-mutated (K-RASmut) cells. We investigated the interactions of distinct domain(s) of DNA-PKcs in binding to full-length Akt1. Similarly, we analyzed potential interactions of DNA-PKcs with Akt2 and Akt3. Finally the effect of Akt isoforms in cell proliferation and tumor growth was tested. We demonstrated that Akt1 preferentially binds to the N-terminal domain of DNA-PKcs using pull-down studies with distinct eGFP-tagged DNA-PKcs fragments that were expressed by plasmids in combination with mCherry-tagged full-length Akt isoforms. These binding studies also indicated an interaction with the intermediate and C-terminal domains of DNA-PKcs. In contrast, Akt3 interacted with all four DNA-PKcs fragments without a marked preference for any specific domain. Notably, we could not see binding of Akt2 to any of the tested DNA-PKcs fragments. In subsequent studies, we demonstrated that Akt inhibition interferes with binding of Akt1 to the N-terminal domain of DNA-PKcs. This indicated a correlation between Akt1 activity and the Akt1/DNA-PKcs complex formation. Finally, knockdown studies revealed that the depletion of endogenous Akt1 and Akt3, but not Akt2, inhibit clonogenic activity and repair of ionizing radiation (IR)-induced DNA DSBs, leading to radiosensitization. Furthermore, in a xenograft study the expression of shAkt1 or shAkt3, but not shAkt2 in K-RASmut breast cancer cell line MDA-MB-231 showed major tumor growth delay. Together, these data indicate that Akt1 and Akt3, but not Akt2, physically interact with DNA-PKcs, thus stimulating the repair of DSBs and therefore protecting K-RASmut cells against IR. Likewise, interaction of Akt isoforms with DNA-PKcs could be crucial for their role in regulating tumor growth.

Efficient repair of DNA double strand breaks in individuals from high level natural radiation areas of Kerala coast, south-west India

High level natural radiation areas (HLNRA) of Kerala coastal strip (55km long and 0.5km wide) in southwest India exhibit wide variations in the level of background dose (< 1.0–45.0mGy/year) due to thorium deposits in the beach sand. The areas with ≤1.5mGy/year are considered as normal level natural radiation area (NLNRA), whereas areas with >1.5mGy/year are HLNRA. Individuals belonging to HLNRA were stratified into two groups, Low dose group (LDG: 1.51–5.0mGy/year) and high dose group (HDG: >5.0mGy/year). The mean annual dose received by the individuals from NLNRA, LDG and HDG was 1.3±0.1, 2.7±0.9 and 9.4±2.3mGy/year, respectively. Induction and repair of DNA double strand breaks (DSBs) in terms of gamma-H2AX positive cells were analysed in peripheral blood mononuclear cells (PBMCs) using flow cytometry. Induction of DSBs was studied at low (0.25Gy) and high challenge doses (1.0 and 2.0Gy) of gamma radiation in 78 individuals {NLNRA, N=23; HLNRA (LDG, N=21 and HDG, N=34)}. Repair kinetics of DSBs were evaluated in PBMCs of 30 individuals belonging to NLNRA (N=8), LDG (N=7) and HDG (N=15) at low (0.25Gy) and high doses (2.0Gy) of gamma radiation. Transcription profile of DNA damage response (DDR) and DSB repair genes involved in non-homologous end joining (NHEJ) and homologous recombination repair (HRR) pathways was analysed after a challenge dose of 2.0Gy in PBMCs of NLNRA (N=10) and HDG, HLNRA (N=10) group. Our results revealed significantly lower induction and efficient repair of DSBs in HLNRA groups as compared to NLNRA. Transcription profile of DCLRE1C, XRCC4, NBS1 and CDK2 showed significant up-regulation (p≤0.05) in HDG at a challenge dose of 2.0Gy indicating active involvement of DDR and DSB repair pathways. In conclusion, lower induction and efficient repair of DNA DSBs in HLNRA groups is suggestive of an in vivo radio-adaptive response due to priming effect of chronic low dose radiation prevailing in this area.

The transcription factor GATA3 is required for homologous recombination repair by regulating CtIP expression.

GATA3, a critical transcription factor involved in the development of the mammary gland, also plays important roles in mammary tumorigenesis by regulating transcription in coordination with two essential DNA repair factors, PARP1 and BRCA1. However, whether and how GATA3 participates in the process of DNA repair, which is often associated with tumorigenesis, has not been investigated. Here we demonstrate that GATA3 is required for the repair of DNA double-strand breaks (DSBs) by homologous recominbation (HR). Mechanistic studies indicate that at both the protein and the mRNA level, depleting GATA3 leads to reduced expression of CtIP, an essential HR factor involved in end resection, thereby suppressing the repair of DSBs by HR and sensitizing cells to etoposide induced DNA DSBs. Further studies indicate that upon the occurrence of DNA DSBs GATA3 directly binds to the CtIP promoter at the region of -2119 to -2130 and -2274 to -2285, and promotes the transcription of CtIP. Overexpression of CtIP in GATA3 depleted cells rescues the decline of HR, and cell survival in the presence of etoposide. In addition, through data mining analysis, we observed an extremely strong correlation between the expression levels of GATA3 and CtIP in paratumors, but the correlation turned insignificant in mammary tumors. Using vectors encoding GATA3 with mutations frequently occurring in mammary tumors, we found that several mutations on GATA3 led to a dysregulation of CtIP, and therefore HR repair. In summary, our data delineates the regulatory mechanisms of GATA3 in DNA DSB repair and strongly suggests that it might act as a tumor suppressor by promoting CtIP expression and HR to stabilize genomes.

Generation of Gross Chromosomal Rearrangements by a Single Engineered DNA Double Strand Break

Gross chromosomal rearrangements (GCRs), including translocations, inversions amplifications, and deletions, can be causal events leading to malignant transformation. GCRs are thought to be triggered by DNA double strand breaks (DSBs), which in turn can be spontaneous or induced by external agents (eg. cytotoxic chemotherapy, ionizing radiation). It has been shown that induction of DNA DSBs at two defined loci can produce stable balanced chromosomal translocations, however, a single engineered DNA DSB could not. Herein, we report that although a single engineered DNA DSB in H2AX “knockdown” cells did not generate GCRs, repair of a single engineered DNA DSB in fibroblasts that had ablated H2ax did produce clonal, stable GCRs, including balanced translocations and megabase-pair inversions. Upon correction of the H2ax deficiency, cells no longer generated GCRs following a single engineered DNA DSB. These findings demonstrate that clonal, stable GCRs can be produced by a single engineered DNA DSB in H2ax knockout cells, and that the production of these GCRs is ameliorated by H2ax expression.

Photodynamic Therapy with Liposomal Zinc Phthalocyanine and Tirapazamine Increases Tumor Cell Death via DNA Damage.

The efficacy of photodynamic therapy (PDT) in some solid tumors is limited by the poor biodistributive properties of conventional photosensitizers and a natural predisposition of tumor cells to survive hypoxia and oxidative stress. This study investigated the therapeutic potential of a third-generation photosensitizer, liposomal zinc phthalocyanine (ZnPC), in combination with the hypoxic cytotoxin tirapazamine (TPZ). TPZ induces DNA double strand breaks (DSBs) under hypoxic conditions and subsequent apoptosis via p53 signaling. Experiments were performed in tumor cells with functional p53 (Sk-Cha1) and dysfunctional p53 (A431). The combination therapy of TPZ and PDT induced DNA DSBs and cell cycle stalling and enhanced the cytotoxicity of PDT by exacerbating apopotic and non-apoptotic tumor cell death. These phenomena occurred regardless of oxygen tension and the mechanism of cell death differed per cell line. Liposomes containing both ZnPC and TPZ exhibited no dark toxicity but were more lethal to both cell types after PDT compared to ZnPC-liposomes lacking TPZ—an effect that was more pronounced under hypoxic conditions. In conclusion, TPZ is a suitable pharmaceutical compound to increase PDT efficacy by exploiting the post-PDT tumor hypoxia. The inclusion of TPZ and ZnPC into a single liposomal delivery system was feasible. The PDT strategy described in this study may be valuable for the treatment of PDT-recalcitrant tumors.

Influence of reduced glutathione on end-joining of DNA double-strand breaks: Cytogenetical and molecular approach

Radiation induced DNA double-strand breaks (DSB) are the major initial lesions whose misrejoining may lead to exchange aberrations. However, the role of glutathione (GSH), a major cellular thiol, in regulating cell’s sensitivity to DNA damaging agents is not well understood. Influence of endogenous GSH on the efficiency of X-rays and bleomycin (Blem) induced DNA DSBs end-joining has been tested here cytogenetically, in human lymphocytes and Hct116 cells. In another approach, oligomeric DNA (75bp) containing 5′-compatible and non-compatible overhangs mimicking the endogenous DSB were for rejoining in presence of cell-free extracts from cells having different endogenous GSH levels. Frequency of aberrations, particularly exchange aberrations, was significantly increased when Blem was combined with radiation. The exchange aberration frequency was further enhanced when combined treatment was given at 4°C since DNA lesions are poorly repaired at 4°C so that a higher number of DNA breaks persist and interact when shifted from 4°C to 37°C. The exchange aberrations increased further when the combined treatment was given to Glutathione-ester (GE) pre-treated cells, indicating more frequent rejoining of DNA lesions in presence of higher cellular GSH. This is further supported by the drastic reduction in frequency of exchange aberrations but significant increase in incidences of deletions when combined treatment was given to GSH-depleted cells. End-joining efficiency of DNA DSBs with compatible ends was better than for non-compatible ends. End-joining efficiency of testicular and MCF7 cell extracts was better than that of lungs and Hct116 cells. Cell extract made from GE-treated MCF-7 cells provided more efficient end-joining than from untreated and GSH-depleted cells. However, direct addition of GSH to the cell-free extracts showed considerable reduction in end-joining efficiency. Present data indicate that higher endogenous GSH favours rejoining of DNA DSBs (both restitution and illegitimate reunion) which in turn produce more exchange aberrations.

260 - Mechanism of DNA Double-Strand Cleavage by a New Chlorination Disinfection Byproduct Trichloro-1,4-Benzoquinone and H2O2 and Protection by Thiol-Containing Drugs

Trichloro-1,4-benzoquinone (TriCBQ) has been recently identified as a new chlorination disinfection byproduct in drinking water and swimming pool water. We found previously that the highly reactive and biologically important • OH can be produced by halogenated quinones and H 2 O 2 independent of transition metal ions. However, it is not clear whether DNA double-strand breaks (DSBs) can be induced by TriCBQ in the presence of H 2 O 2 ; and if so, whether TriCBQ/H 2 O 2 induced DNA DSBs can be protected by thiol-containing drugs? Here we show that DNA DSBs can be generated only when pBR322 DNA was treated with the combination of both TriCBQ and H 2 O 2 , while either TriCBQ or H 2 O 2 alone cannot. Typical ● OH scavengers such as dimethyl sulfoxide and ethanol can markedly inhibit TriCBQ/H 2 O 2 -induced DSBs, and a good correlation was observed between DNA DSBs and ● OH generation by TriCBQ/H 2 O 2 as measured by ESR spin-trapping method. DNA damage induced by TriCBQ/H 2 O 2 could be inhibited by several thiol-containing drugs such as captopril, penicillamime, N -acetyl-cysteine and 2,3-dimercaptosuccinic acid. The protection was found to be due to their direct interactions with TriCBQ to form the less reactive quinone-thiol conjugates.

Bisnaphthalimidopropyl diaminodicyclohexylmethane induces DNA damage and repair instability in triple negative breast cancer cells via p21 expression

Bisnaphthalimidopropyl diaminodicyclohexylmethane (BNIPDaCHM) bisintercalates to DNA and is a potential anti-cancer therapeutic. In an attempt to elucidate the mechanism(s) underlying the potential of BNIPDaCHM; earlier work was extended to investigate its effect on DNA damage and repair as well as cell cycle modulation, in a triple negative breast cancer (TNBC) cell line in vitro.BNIPDaCHM significantly decreased cell viability in a concentration (≥5 μM) and time (≥24 h) dependent manner. The mechanism of this growth inhibition involved alterations to cell cycle progression, an increase in the sub-G1 population and changes to plasma membrane integrity/permeability observed by flow cytometry and fluorescence microscopy with acridine orange/ethidium bromide staining. Using single cell gel electrophoresis (Comet assay) and fluorescence microscopy to detect γ-H2AX-foci expression; it was found that after 4 h, ≥ 0.1 μM BNIPDaCHM treatment-induced significant DNA double strand breaks (DSBs). Moreover, exposure to a non-genotoxic concentration of BNIPDaCHM induced a significant decrease in the repair of oxidative DNA strand breaks induced by hydrogen peroxide. Also, BNIPDaCHM-treatment induced a significant time dependent increase in p21Waf/Cip1 mRNA expression but, did not alter p53 mRNA expression.In conclusion, BNIPDaCHM treatment in MDA-MB-231 cells was associated with a significant induction of DNA DSBs and inhibition of DNA repair at non-genotoxic concentrations via p53-independent expression of p21Waf1/Cip1. The latter may be a consequence of novel interactions between BNIPDaCHM and MDA-MB-231 cells which adds to the spectrum of therapeutically relevant activities that may be exploited in the future design and development of naphthalimide-based therapeutics.

ATM-dependent phosphorylation of MRE11 controls extent of resection during homology directed repair by signalling through Exonuclease 1.

The MRE11/RAD50/NBS1 (MRN) complex plays a central role as a sensor of DNA double strand breaks (DSB) and is responsible for the efficient activation of ataxia-telangiectasia mutated (ATM) kinase. Once activated ATM in turn phosphorylates RAD50 and NBS1, important for cell cycle control, DNA repair and cell survival. We report here that MRE11 is also phosphorylated by ATM at S676 and S678 in response to agents that induce DNA DSB, is dependent on the presence of NBS1, and does not affect the association of members of the complex or ATM activation. A phosphosite mutant (MRE11S676AS678A) cell line showed decreased cell survival and increased chromosomal aberrations after radiation exposure indicating a defect in DNA repair. Use of GFP-based DNA repair reporter substrates in MRE11S676AS678A cells revealed a defect in homology directed repair (HDR) but single strand annealing was not affected. More detailed investigation revealed that MRE11S676AS678A cells resected DNA ends to a greater extent at sites undergoing HDR. Furthermore, while ATM-dependent phosphorylation of Kap1 and SMC1 was normal in MRE11S676AS678A cells, there was no phosphorylation of Exonuclease 1 consistent with the defect in HDR. These results describe a novel role for ATM-dependent phosphorylation of MRE11 in limiting the extent of resection mediated through Exonuclease 1.

Efficient analyses of DNA double-strand breaks and the cell cycle in the secretory epithelial cells of fallopian tube fimbriae

ObjectiveReactive oxygen species (ROS)-induced DNA double-strand breaks (DSBs) are a feature of cancer initiation. Recently, the cells of origin of ovarian high-grade serous carcinoma (HGSC) have been identified as secretory cells in fallopian tube fimbriae, which might be heavily exposed to ROS after ovulation. Establishing a sensitive detection method for measuring DSBs and the cell cycle in fallopian tube secretory cells after ovulation is essential for facilitating research on ovarian cancer formation. Materials and methodsFimbrial epithelial cells from a prophylactically removed human fallopian tube were primarily cultured and immortalized by TP53/Rb-null and hTERT overexpression (FE25 cell line). Hydrogen peroxide (H2O2) was used to treat the FE25 cells to induce DNA DSBs. The DSBs and cell cycle were analyzed using flow cytometry. In addition, human specimens of fimbrial scrapings were subjected to the flow cytometry-based analysis. ResultsWe report an efficient flow cytometry-based method for analyzing the DSBs and cell cycle in immortalized fallopian tube secretory cells as well as in clinical specimens. ConclusionThis analysis method would facilitate the investigation of cancer initiation in fallopian tube fimbriae and other tissues with DSBs.

Persistent γH2AX: A promising molecular marker of DNA damage and aging.

One of the earliest cellular responses to DNA double strand breaks (DSBs) is the phosphorylation of the core histone protein H2AX (termed γH2AX). Persistent γH2AX is the level of γH2AX above baseline, measured at a given time-point beyond which DNA DSBs are normally expected to be repaired (usually persist for days to months). This review summarizes the concept of persistent γH2AX in the context of exogenous source induced DNA DSBs (e.g. ionizing radiation (IR), chemotherapeutic drugs, genotoxic agents), and endogenous γH2AX levels in normal aging and accelerated aging disorders. Summary of the current literature demonstrates the following (i) γH2AX persistence is a common phenomenon that occurs in humans and animals; (ii) nuclei retain persistent γH2AX foci for up to several months after IR exposure, allowing for retrospective biodosimetry; (iii) the combination of various radiosensitizing drugs with ionizing radiation exposure leads to persistent γH2AX response, thus enabling the potential for monitoring cancer patients' response to chemotherapy and radiotherapy as well as tailoring cancer treatments; (iv) persistent γH2AX accumulates in telomeric DNA and in cells undergoing cellular senescence; and (v) increased endogenous γH2AX levels may be associated with diseases of accelerated aging. In summary, measurement of persistent γH2AX could potentially be used as a marker of radiation biodosimetry, evaluating sensitivity to therapeutic genotoxins and radiotherapy, and exploring the association of unrepaired DNA DSBs on telomeres with diseases of accelerated aging.

γH2AX is a biomarker of modulated cytostatic drug resistance.

ONE reason for reduced therapeutic efficiency of cancer chemotherapies is the appearance of multidrug resistance. Plasma membrane glycoprotein (P-gp) is recognized as one of the best characterized drug efflux pumps playing a central role in the absorption and disposition of many drugs, including chemotherapeutic agents. Increased P-gp activity has been suggested and reported to be an indicator of chemotherapeutic failure. Therefore, P-gp activity has been used as a potential target in being able to reverse multidrug resistance. Cytostatic drugs such as etoposide in combination with the immunosuppressive cyclosporine A may inhibit P-gp activity, ultimately resulting in intracellular enrichment of the targeted drug (1). One issue in cancer therapy involves the highly varying drug response among patients. To assess the multidrug resistance status for personalized cancer therapy, an assay that can provide results to monitor the cytostatic effect of antineoplastic agents in combination with different drugs is highly desirable. Many of these drugs, like etoposide, cause DNA double strand breaks (DSBs) that may eventuate in clonogenic death. DSBs are one of the most biologically significant DNA damage lesions that leads to chromosome breakage and/or rearrangement, mutagenesis and loss or gain of genetic information (2). The phosphorylation of H2AX histone proteins which are located in the vicinity of the DSBs is known as one of the earliest responses to DNA DSBs in cells. Induction of DNA DSBs in live mammalian cells triggers the phosphorylation of Ser139 in the SQ motif near the C-terminal of H2AX protein, which results in the phosphorylated form of H2AX, termed cH2AX. cH2AX signals appear as discrete nuclear “foci” which can be visualized and quantified by a number of methods including fluorescence microscopy and flow cytometry, following immunostaining procedures with fluorescence-coupled antibodies. The cH2AX foci counting approach has been used in numerous studies to assess the relationship between cH2AX foci removal and the rate of DSBs repair (3). Each DSB is represented by an individual cH2AX focus with a ratio of 1:1 and after successful repair of DSBs, the level of cH2AX foci decline over time in the presence of normal physiological repair mechanisms. The presence of persistent cH2AX indicates impaired DNA repair. Therefore, cH2AX quantification may prove to be a sensitive biomarker of DNA DSBs in human cells and has been suggested for biomonitoring tumor progression and treatment response (4). DSBs are directly generated by exogenous agents such as ionizing radiation and antitumor drugs (bleomycin, mitoxantrone, etoposide). Because mammalian cells respond to DSBs by activating a multitude of proteins involved in signalling and DNA repair pathways, the very nature of DSBs poses such a threat to cell survival that DNA damage checkpoint proteins may be activated to initiate cellular division arrest. This provides time for DNA repair to proceed before mitosis is completed or in the case of overwhelming damage, apoptosis ensues (5). Therefore, detection of accumulated cH2AX foci may be an efficient method for assessing multidrug resistance in cancer therapy as presented by Reddig et al. in this issue of Cytometry A (page XXX).

Comparison of the biological effects of 18F at different intracellular levels

We herein examined the biological effects of cells treated with 18F labeled drugs for positron emission tomography (PET). The relationship between the intracellular distribution of 18F and levels of damaged DNA has yet to be clarified in detail. We used culture cells (Chinese Hamster Ovary cells) treated with two types of 18F labeled drugs, fluorodeoxyglucose (FDG) and fluorine ion (HF). FDG efficiently accumulated in cells, whereas HF did not. To examine the induction of DNA double strand breaks (DSB), we measured the number of foci for 53BP1 that formed at the site of DNA DSB. The results revealed that although radioactivity levels were the same, the induction of 53BP1 foci was stronger in cells treated with 18F-FDG than in those treated with 18F-HF. The clonogenic survival of cells was significantly lower with 18F-FDG than with 18F-HF. We concluded that the efficient accumulation of 18F in cells led to stronger biological effects due to more severe cellular lethality via the induction of DNA DSB.

Different fates of oocytes with DNA double-strand breaks in vitro and in vivo

In female mice, despite the presence of slight DNA double-strand breaks (DSBs), fully grown oocytes are able to undergo meiosis resumption as indicated by germinal vesicle breakdown (GVBD); however, severe DNA DSBs do reduce and delay entry into M phase through activation of the DNA damage checkpoint. But little is known about the effect of severe DNA DSBs on the spindle assembly checkpoint (SAC) during oocyte maturation. We showed that nearly no first polar body (PB1) was extruded at 12 h of in vitro maturation (IVM) in severe DNA DSBs oocytes, and the limited number of oocytes with PB1 were actually at telophase. However, about 60% of the severe DNA DSBs oocytes which underwent GVBD at 2 h of IVM released a PB1 at 18 h of IVM and these oocytes did reach the second metaphase (MII) stage. Chromosome spread at MI and MII stages showed that chromosomes fragmented after GVBD in severe DNA DSBs oocytes. The delayed PB1 extrusion was due to the disrupted attachment of microtubules to kinetochores and activation of the SAC. At the same time, misaligned chromosome fragments became obvious at the first metaphase (MI) in severe DNA DSBs oocytes. These data implied that the inactivation of SAC during the metaphase-anaphase transition of first meiosis was independent of chromosome integrity. Next, we induced DNA DSBs in vivo, and found that the number of superovulated oocytes per mouse was significantly reduced; moreover, this treatment increased the percentage of apoptotic oocytes. These results suggest that DNA DSBs oocytes undergo apoptosis in vivo.

DNA-PK inhibition by NU7441 sensitizes breast cancer cells to ionizing radiation and doxorubicin

DNA-dependent protein kinase (DNA-PK) plays a key role in the repair of DNA double-strand breaks (DSBs) that are probably the most deleterious form of DNA damage. Inhibition of DNA-PK has been considered as an attractive approach to decrease resistance to therapeutically induced DNA DSBs. Ionizing radiation (IR) and doxorubicin, which induce DSBs, are used in the treatment of breast cancer. We determined the cellular concentration of DNA-PK and other DSB-activated kinases: ATM and ATR and the effect of DNA-PK inhibition by NU7441 on DNA repair, cell cycle, and survival after IR or doxorubicin treatment in three human breast cancer cell lines (MCF-7, MDA-MB-231, and T47D) representing different breast cancer subtypes. T47D cells had the highest expression of DNA-PKcs, ATM, and ATR and the most rapid rate of DNA DSB repair. IR caused a 10- to 16-fold increase in DNA-PK activity and two to threefold induction of ATM in all 3 cell lines. NU7441 inhibited IR-induced DNA-PK activity in all cell lines with IC50s in the range 0.17-0.25 μM. NU7441 retarded the repair of DSB and significantly increased the sensitivity of all cell lines to IR (4- to 12-fold) and doxorubicin (3- to 13-fold). The greatest sensitization by NU7441 was observed in MDA-MB-231 cells. NU7441 affected the cell cycle distribution in all studied cell lines; increasing accumulation of cells in G2/M phase after DNA damage. Our data indicate that DNA-PK might be an effective target for chemo- and radio-potentiation in breast cancer and suggest that further development of DNA-PK inhibitors for clinical use is warranted.

Repair Of DNA Double Strand Breaks By Insertion Of Nucleotide Sequences Derived From Distant Regions Of The Genome

It has been suggested that errors in the repair of DNA double strand breaks (DSB) can result in gross chromosomal rearrangements (GCR), including chromosomal amplifications, deletions, inversions, and translocations. To study repair of DNA DSB in vivo, we established a subclone of the U937 cell line designated F5, in which a single copy of the rare-cutting meganuclease I-SceI recognition site has been integrated into chromosome 7. We transfected an I-SceI expression vector into these cells and analyzed misrepair events. We recovered no GCR, but instead found that most DSBs were repaired by small (<50bp) indels (91.8%). We also noticed some DSBs were repaired by large (<50bp) deletions (6.4%) and rare DSBs were repaired by large (100-500 bp) insertions (1.8%). Surprisingly, the nucleotide sequence of these insertions matched distant regions of the genome. To determine if this templated sequence insertion (TSI) was peculiar to I-SceI induced DNA DSB, we repeated the experiment using TALENs to produce DNA DSB. Again, we found insertions that were derived from distant genomic regions.To determine whether this form of DNA repair was restricted to experimentally induced DNA DSB, we analyzed structural variations identified by whole genome sequence (WGS) of two myeloma cell lines (KP6, MC1286PE1), and identified 15 independent TSIs. These TSIs were verified by Sanger sequencing, and lengths of inserted fragment were 38 to 666 bp (median 189 bp). Six insertions were noted to be identical between the 2 cell lines, suggesting that these TSIs were germline polymorphic events. These TSIs were compared to a catalog of known structural variations identified by WGS of 52 healthy individuals; 13 of 15 TSI junctions were identified, suggesting that these represented polymorphic insertions in the human genome. Two TSIs were potential somatic mutations in the KP6 cell line, including one that had 85bp insertion at the junction of an acquired t(6;12) chromosomal translocation. Six TSIs showed the hallmarks of L1 retrotransposon-mediated insertions, with a 5'-TTTT/A-3' integration site, a target site duplication (TSD), a polyA tract at the insertion site, and a polyadenylation signal. However, the inserted sequence was neither a LINE nor a SINE, but instead was a transcribed, genic region mapping to a distant genomic region. The presence of these L1 ORF1 hallmarks strongly suggests that these insertions were caused by the L1 integrase and reverse-transcriptase acting on nuclear pre-mRNA. A second class of TSI showed no preference for a 5'-TTTT/A-3' integration site, no TSD, no poly A tract, but instead showed several bp of microhomology at the insertion junctions. This second class is similar to TSI detected at I-SceI induced DNA DSB, and we predict that these TSI were generated by reverse transcription of pre-mRNA creating a “patch” for a spontaneous DNA DSB that occurred in a germ cell.To determine if TSI at I-SceI induced DNA DSB could be repaired by RNA-templated sequences, we cotransfected murine RNA and an I-SceI expression vector into F5 cells. Approximately 10% of the TSI recovered from these experiments contained mouse sequences, indicating that they were derived from mouse RNA. To further support the contention that TSIs were derived from RNA, we again used I-SceI to induce a DNA DSB, and treated the cells with reverse transcriptase inhibitors. The TSI frequency was reduced by 70%. Finally, a subset of insertions were due to telomere repeat sequences, lending further support for the hypothesis that RNA could serve as a template for the repair of DNA DSB. TSI is novel form of potentially mutagenic DNA DSB repair, which should be considered as an alternative to a balanced chromosomal translocation in the interpretation of reciprocal interchromosomal structural variations identified by short-read deep sequencing. Disclosures:No relevant conflicts of interest to declare.

In vivo γ-irradiation low dose threshold for suppression of DNA double strand breaks below the spontaneous level in mouse blood and spleen cells

There is a considerable controversy as to whether DNA damage induced by low doses and low dose rates of ionizing radiation is treated by cellular defence mechanisms in ways similar to that induced at high doses and high dose rates, and what downstream delayed effects may be caused by low doses compared to moderate and high doses. This constitutes the major challenge for the linear no-threshold model currently used for radiological risk estimates. Among the various DNA lesions induced by ionizing radiation, DNA double strand breaks (DSBs) are considered the most important due to their potential to cause cell death, mutagenesis and carcinogenesis. This study examined the accumulation of DNA DSBs in mouse blood leucocytes and splenocytes after long-term, chronic low dose γ-irradiation in vivo, and how this exposure may alter cell sensitivity to acute high dose irradiation. Animals were irradiated for 40, 80 or 120 days at a dose rate of 0.15mGy/h, with total accumulated doses of 144, 288 and 432mGy. DNA DSBs were measured in blood leucocytes and splenocytes using the neutral comet assay. We found that after an initial slight increase in the level of DNA DSBs at 40 days of exposure compared to controls, there was a subsequent drop after either 80 (P<0.01) or 120 days of exposure (P=0.066 for blood leucocytes; P=0.024 for splenocytes). Interestingly, the DNA breaks level after both 80 and 120 days of exposure was lower than in control. Similarly, the cells exposed to the chronic radiation for 80 and 120 days were less sensitive to the induction of DNA DSBs by acute 4Gy irradiation, whereas 40 days of exposure did not significantly modify the radiosensitivity. Our results indirectly indicate that low level ionizing radiation in vivo may trigger inducible repair of both endogenous and exogenous DNA DSBs, and that there is a dose threshold for this inducible defence mechanism, below which it does not occur. These data provide new evidence, now at the molecular level in vivo, that the dose–response for DNA DSBs at very low doses and dose rates is not linear.