Formation Of DNA Breaks Research Articles

Interference Between DNA Replication and Transcription as a Cause of Genomic Instability

Replication and transcription are key aspects of DNA metabolism that take place on the same template and potentially interfere with each other. Conflicts between these two activities include head-on or co-directional collisions between DNA and RNA polymerases, which can lead to the formation of DNA breaks and chromosome rearrangements. To avoid these deleterious consequences and prevent genomic instability, cells have evolved multiple mechanisms preventing replication forks from colliding with the transcription machinery. Yet, recent reports indicate that interference between replication and transcription is not limited to physical interactions between polymerases and that other cotranscriptional processes can interfere with DNA replication. These include DNA-RNA hybrids that assemble behind elongating RNA polymerases, impede fork progression and promote homologous recombination. Here, we discuss recent evidence indicating that R-loops represent a major source of genomic instability in all organisms, from bacteria to human, and are potentially implicated in cancer development.

DNA damage defines sites of recurrent chromosomal translocations in B lymphocytes.

Recurrent chromosomal translocations underlie both haematopoietic and solid tumours. Their origin has been ascribed to selection of random rearrangements, targeted DNA damage, or frequent nuclear interactions between translocation partners; however, the relative contribution of each of these elements has not been measured directly or on a large scale. Here we examine the role of nuclear architecture and frequency of DNA damage in the genesis of chromosomal translocations by measuring these parameters simultaneously in cultured mouse B lymphocytes. In the absence of recurrent DNA damage, translocations between Igh or Myc and all other genes are directly related to their contact frequency. Conversely, translocations associated with recurrent site-directed DNA damage are proportional to the rate of DNA break formation, as measured by replication protein A accumulation at the site of damage. Thus, non-targeted rearrangements reflect nuclear organization whereas DNA break formation governs the location and frequency of recurrent translocations, including those driving B-cell malignancies.

Cytotoxic and Genotoxic Effects of the Quercetin/Lanthanum Complex on Human Cervical Carcinoma Cells In Vitro

Quercetin is the main flavonoid in diet with a potential in the treatment of cancer, cardiovascular, and neurodegenerative diseases. Due to its specific planar chemical structure, quercetin readily forms chelates with metal ions. Complexes of bioactive compounds and metal ions such as lanthanum often show strong cytotoxic and antitumour properties. The aim of this study was to compare the genotoxic effects of the quercetin/lanthanum complex on human cervical carcinoma cells with compare it to the effects of free ligands, quercetin, and lanthanum alone. The quercetin/lanthanum complex showed considerable cytotoxicity in the concentration range of (100 to 1000) mmol mL-1 and exposure time of three hours. The complex also induced a dose-dependent pro-oxidative effects and the formation of single-strand and double-strand DNA breaks. Although we obtained promising results on the cell level, future experiments should answer whether the quercetin/lanthanum complex is cancer-specific and stable enough in physiological conditions to make a potential new antitumour drug.

CK2 phosphorylation of XRCC1 facilitates dissociation from DNA and single-strand break formation during base excision repair

CK2 phosphorylates the scaffold protein XRCC1, which is required for efficient DNA single-strand break (SSB) repair. Here, we express an XRCC1 protein (XRCC1 ckm) that cannot be phosphorylated by CK2 in XRCC1 mutated EM9 cells and show that the role of this post-translational modification gives distinct phenotypes in SSB repair and base excision repair (BER). Interestingly, we find that fewer SSBs are formed during BER after treatment with the alkylating agent dimethyl sulfate (DMS) in EM9 cells expressing XRCC1 ckm (CKM cells) or following inhibition with the CK2 inhibitor 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT). We also show that XRCC1 ckm protein has a higher affinity for DNA than wild type XRCC1 protein and resides in an immobile fraction on DNA, in particular after damage. We propose a model whereby the increased affinity for DNA sequesters XRCC1 ckm and the repair enzymes associated with it, at the repair site, which retards kinetics of BER. In conclusion, our results indicate that phosphorylation of XRCC1 by CK2 facilitates the BER incision step, likely by promoting dissociation from DNA.

Abstract 3547: In vitro and ex vivo anti-leukemia activity of AR-67, a novel lipophilic camptothecin

Abstract BACKGROUND: The overall survival and disease-free rates for Acute Myelogenous Leukemia (AML) patients treated with the gold-standard therapy comprised of Cytarabine (Ara-C) and Daunorubicin (DNR) remain significantly low. Camptothecins are potent anticancer drugs that confer their cytotoxic effect by interfering with Topoisomerase-I (Top1). Semi-synthetic camptothecin analogues have been used in the past to treat AML patients with relatively low response rates. Here, we examined the anti-leukemia activity of AR-67, a novel camptothecin analogue, alone or in combination with standard agents for the treatment of AML. METHODS: The anti-leukemia activity of AR-67, TPT, Ara-C and DNR was studied in HL60, KG1 and K562 cells and in primary leukemia cell cultures after exposure to increasing drug concentrations for 48 hours. Cell proliferation was determined using a colorimetric assay (MTT) assay and IC50s were obtained using nonlinear-regression analysis. Drug combination treatments were assessed for synergy. The expression of Top1, phosphohistone (γ-H2AX), Bcl-2 and c-PARP were evaluated using biochemical assays. RESULTS: The potency of AR-67 was in the nM range in the leukemic cell lines. Consistent with the lower proliferation rates of primary cells grown in culture, their IC50 values were variable and in the µM range. IC50s estimated for TPT were higher than the respective ones for AR-67. The IC50 values for Ara-C and DNR were in the μM range for the HL60, K562 and primary cells, and higher than the ones for AR-67. However, the KG-1 cell line appeared to be more sensitive to the AML gold standard therapy, but not as sensitive as to AR-67. AR-67 treatment decreased Top1 expression, increased γ-H2AX expression in a concentration-dependent manner suggesting removal of DNA-Top1 complexes and formation of double strand DNA breaks. Induction of apoptosis was manifested by decreased Bcl-2 and increased c-PARP expression suggesting a caspace-3 mediated effect. AR-67 combination treatments that included DNR and Ara-C showed synergy in HL60 cells. Additionally, pretreatment with Ara-C revealed synergy between AR-67 and DNR in both the KG1 and K562 cells, accompanied by increased γ-H2AX and c-PARP expression. CONCLUSIONS: Our results indicate AR-67 as a potent anticancer agent that induces DNA damage and apoptosis. Using in vitro leukemia models we have shown that AR-67 is a potentially efficacious agent when combined with standard agents for the treatment of AML and may benefit AML patients resistant to Ara-C. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3547. doi:10.1158/1538-7445.AM2011-3547

Effects of Non-Thermal Plasma on Mammalian Cells

Thermal plasmas and lasers have been widely used in medicine to cut, ablate and cauterize tissues through heating; in contrast, non-thermal plasma produces no heat, so its effects can be selective. In order to exploit the potential for clinical applications, including wound healing, sterilization, blood coagulation, and cancer treatment, a mechanistic understanding of the interaction of non-thermal plasma with living tissues is required. Using mammalian cells in culture, it is shown here that non-thermal plasma created by dielectric barrier discharge (DBD) has dose-dependent effects that range from increasing cell proliferation to inducing apoptosis. It is also shown that these effects are primarily due to formation of intracellular reactive oxygen species (ROS). We have utilized γ-H2AX to detect DNA damage induced by non-thermal plasma and found that it is initiated by production of active neutral species that most likely induce formation of organic peroxides in cell medium. Phosphorylation of H2AX following non-thermal plasma treatment is ATR dependent and ATM independent, suggesting that plasma treatment may lead to replication arrest or formation of single-stranded DNA breaks; however, plasma does not lead to formation of bulky adducts/thymine dimers.

UV-induced photolesions elicit ATR-kinase-dependent signaling in non-cycling cells through nucleotide excision repair-dependent and -independent pathways

Activation of signaling pathways by UV radiation is a key event in the DNA damage response and initiated by different cellular processes. Here we show that non-cycling cells proficient in nucleotide excision repair (NER) initiate a rapid but transient activation of the damage response proteins p53 and H2AX; by contrast, NER-deficient cells display delayed but persistent signaling and inhibition of cell cycle progression upon release from G0 phase. In the absence of repair, UV-induced checkpoint activation coincides with the formation of single-strand DNA breaks by the action of the endonuclease Ape1. Although temporally distinct, activation of checkpoint proteins in NER-proficient and NER-deficient cells depends on a common pathway involving the ATR kinase. These data reveal that damage signaling in non-dividing cells proceeds via NER-dependent and NER-independent processing of UV photolesions through generation of DNA strand breaks, ultimately preventing the transition from G1 to S phase.

Redox status of equine seminal plasma reflects the pattern and magnitude of DNA damage in sperm cells

Antioxidant status of seminal plasma from 23 stallions was evaluated. We found a negative correlation between total antioxidant capacity (ABTS •+ decolorization assay) and thiol content of seminal plasma, and sperm DNA damage (8-oxoG immunostaining, TUNEL reaction, comet assay). Low seminal redox status was the strongest correlated with 8-oxoG level which may indicate that seminal total antioxidant capacity influences mainly the formation of single strand DNA breaks in sperm cells. Since inter-individual differences in seminal antioxidant status were reported, we postulated that the redox status of seminal plasma may be an additional important parameter, both with sperm quantitative and morphological analysis, for evaluation of equine semen quality.

DNA damage response induced by exposure of human lung adenocarcinoma cells to smoke from tobacco- and nicotine-free cigarettes

Cigarette smoke (CS) is the major cause of lung cancer and contributes to the development of other malignancies. Attempts have been made to construct reduced toxicity cigarettes, presumed to have diminished genotoxic potential. One such product on the market is the tobacco and nicotine free (T&N-free) cigarette type made from lettuce and herbal extracts. We have recently developed a sensitive assay of the genotoxicity of CS based on cytometric analysis of induction of the DNA damage response (DDR) in normal human pulmonary endothelial or A549 pulmonary adenocarcinoma cells. In the present study, we observed that exposure of A549 cells to CS from T/N-free cigarettes induced a smoke-dose dependent DDR as evidenced by phosphorylation (activation) of the Ataxia telangiectasia mutated (ATM) protein kinase and of the histone H2AX (γH2AX). The extent of DDR induced by T&N-free smoke was distinctly greater than that induced by comparable doses of CS from reference cigarettes (2R4F) containing tobacco and nicotine. The pattern of DDR induced by T&N-free smoke was similar to that of 2R4F cigarettes in terms of the cell cycle phase specificity and involvement of reactive oxygen species (ROS). The data also imply that similar to 2R4F exposure of cells to T/N-free smoke leads to formation of double-strand DNA breaks (DSBs) resulting from collapse of replication forks upon collision with the primary ssDNA lesions induced by smoke. Since DSBs are potentially carcinogenic our data indicate that smoking tobacco and nicotine-free cigarettes is at least as hazardous as smoking cigarettes containing tobacco and nicotine.

DNA breaks at fragile sites generate oncogenic RET/PTC rearrangements in human thyroid cells

Common fragile sites are regions of the genome that are prone to DNA breakage and frequently coincide with the location of genes involved in carcinogenic chromosomal translocations. In this study, we examined the direct involvement of fragile sites in the formation of RET/PTC rearrangements, which are commonly found in papillary thyroid carcinomas, and are all a result of a translocation event involving the RET gene. We demonstrated that fragile site‐inducing chemicals, including aphidicolin, can induce the formation of RET/PTC rearrangements in thyroid cells. We further studied fragile site‐induced DNA breakage within the genes (RET, CCDC6, and NCOA4) participating in the two major types of RET/PTC rearrangements, RET/PTC1 and RET/PTC3, through cytogenetic, molecular biology, and sequence analysis methods. Using FISH, we found the rate of DNA breakage at these genes to be similar to known fragile site‐induction levels of the common fragile sites containing these genes, FRA10C and FRA10G. We mapped the location of induced breakpoints using LM‐PCR and found them to be located near breakpoints observed in patients. Factors contributing to the formation of DNA breaks have been investigated using sequence analysis. These results provide the first direct evidence for the role of chromosomal fragile sites in the generation of cancer‐specific rearrangements in human cells. (Supported by the NCI, CA113863).

DNA Polymerases as Potential Therapeutic Targets for Cancers Deficient in the DNA Mismatch Repair Proteins MSH2 or MLH1

SummarySynthetic sickness/lethality (SSL) can be exploited to develop therapeutic strategies for cancer. Deficiencies in the tumor suppressor proteins MLH1 and MSH2 have been implicated in cancer. Here we demonstrate that deficiency in MSH2 is SSL with inhibition of the DNA polymerase POLB, whereas deficiency in MLH1 is SSL with DNA polymerase POLG inhibition. Both SSLs led to the accumulation of 8-oxoG oxidative DNA lesions. MSH2/POLB SSL caused nuclear 8-oxoG accumulation, whereas MLH1/POLG SSL led to a rise in mitochondrial 8-oxoG levels. Both SSLs were rescued by silencing the adenine glycosylase MUTYH, suggesting that lethality could be caused by the formation of lethal DNA breaks upon 8-oxoG accumulation. These data suggest targeted, mechanism-based therapeutic approaches.

Medicaments: Gifts from the jungle

Medicaments: Gifts from the jungle

Suberoylanilide Hydroxyamic Acid Modification of Chromatin Architecture Affects DNA Break Formation and Repair

Chromatin-modifying compounds that inhibit the activity of histone deacetylases have shown potency as radiosensitizers, but the action of these drugs at a molecular level is not clear. Here we investigated the effect of suberoylanilide hydroxyamic acid (SAHA) on DNA breaks and their repair and induction of rearrangements. The effect of SAHA on both clonogenic survival and repair was assessed using cell lines SCC-25, MCF7, and TK6. In order to study unique DNA double-strand breaks, anti-CD95 antibody was employed to introduce a DNA double-strand break at a known location within the 11q23 region. The effects of SAHA on DNA cleavage and rearrangements were analyzed by ligation-mediated PCR and inverse PCR, respectively. SAHA acts as radiosensitizer at 1 microM, with dose enhancement factors (DEFs) at 10% survival of: SCC-25 - 1.24 +/- 0.05; MCF7 - 1.16 +/- 0.09 and TK6 - 1.17 +/- 0.05, and it reduced the capacity of SCC-25 cells to repair radiation induced lesions. Additionally, SAHA treatment diffused site-specific fragmentation over at least 1 kbp in TK6 cells. Chromosomal rearrangements produced in TK6 cells exposed to SAHA showed a reduction in microhomology at the breakpoint between 11q23 and partner chromosomes. SAHA shows efficacy as a radiosensitizer at clinically obtainable levels. In its presence, targeted DNA strand breaks occur over an expanded region, indicating increased chromatin access. The rejoining of such breaks is degraded by SAHA when measured as rearrangements at the molecular level and rejoining that contributes to cell survival.

Effect of silver nanoparticle and glycyrrhizic acid (SN-GLY) complex on repair of whole body radiation-induced cellular DNA damage and genomic instability in mice

Silver nanoparticles (SN) were redispersed in aqueous solution of Pluronic F127 and complexed with the phytoceutical, glycyrrhizic acid (GLY) to obtain SN-GLY complex. The ability of the SN-GLY complex to offer protection against ionising radiation in post-irradiation scenarios was evaluated in ex vivo and in vivo models using Swiss albino mice. Treatment of mouse blood leucocytes with SN-GLY immediately after 4 Gy gamma radiation exposure, ex vivo, enhanced the rate of repair of cellular DNA damages as revealed by comet assay. Exposure of mice to 4 Gy whole body gamma radiation induced formation of strand breaks in cellular DNA and the unrepaired double strand breaks eventually caused the formation of micronuclei. The post-irradiation administration of SN-GLY resulted in a faster decrease in the comet parameters indicating enhanced cellular DNA repair process and reduction in micronucleus formation. Thus the studies showed effective radiation protection by SN-GLY in post-irradiation conditions.

The RNF8/RNF168 ubiquitin ligase cascade facilitates class switch recombination

An effective immune response requires B cells to produce several classes of antibodies through the process of class switch recombination (CSR). Activation-induced cytidine deaminase initiates CSR by deaminating deoxycytidines at switch regions within the Ig locus. This activity leads to double-stranded DNA break formation at the donor and recipient switch regions that are subsequently synapsed and ligated in a 53BP1-dependent process that remains poorly understood. The DNA damage response E3 ubiquitin ligases RNF8 and RNF168 were recently shown to facilitate recruitment of 53BP1 to sites of DNA damage. Here we show that the ubiquitination pathway mediated by RNF8 and RNF168 plays an integral part in CSR. Using the CH12F3-2 mouse B cell line that undergoes CSR to IgA at high rates, we demonstrate that knockdown of RNF8, RNF168, and 53BP1 leads to a significant decrease in CSR. We also show that 53BP1-deficient CH12F3-2 cells are protected from apoptosis mediated by the MDM2 inhibitor Nutlin-3. In contrast, deficiency in either E3 ubiquitin ligase does not protect cells from Nutlin-3-mediated apoptosis, indicating that RNF8 and RNF168 do not regulate all functions of 53BP1.

Kinetics of the UV‐induced DNA damage response in relation to cell cycle phase. Correlation with DNA replication

It has been reported that exposure to UV light triggers DNA damage response (DDR) seen as induction of gammaH2AX not only in S- but also in G(1)-phase cells. In the present study, in addition to gammaH2AX, we assessed other markers of DDR, namely phosphorylation of ATM on Ser1981, of ATM/ATR substrate on Ser/Thr at SQ/TQ cluster domains and of the tumor suppressor p53 on Ser15, in human pulmonary carcinoma A549 cells irradiated with 50 J/m(2) of UV-B. Phosphorylation of these proteins detected with phospho-specific Abs and measured by laser scanning cytometry in relation the cell cycle phase was found to be selective to S-phase cells. The kinetics of phosphorylation of ATM was strikingly similar to that of ATM/ATR substrate, peaking at 30 min after UV irradiation and followed by rapid dephosphorylation. The peak of H2AX phosphorylation was seen at 2 h and the peak of p53 phosphorylation at 4 h after exposure to UV light. Local high spatial density of these phospho-proteins reported by intensity of maximal pixel of immunofluorescence in the DDR nuclear foci was distinctly more pronounced in the early compared to late portion of S-phase. Exposure of cells to UV following 1 h pulse-labeling of their DNA with 5-ethynyl-2'deoxyuridine (EdU) made it possible to correlate the extent of DNA replication during the pulse with the extent of the UV-induced H2AX phosphorylation within the same cells. This correlation was very strong (R(2) = 0.98) and the cells that did not incorporate EdU showed no evidence of H2AX phosphorylation. The data are consistent with the mechanism in which stalling of DNA replication forks upon collision with the primary UV-induced DNA lesions and likely formation of double-strand DNA breaks triggers DDR. The prior reports (including our own) on induction of gammaH2AX in G(1) cells by UV may have erroneously identified cells initiating DNA replication following UV exposure as G(1) cells due to the fact that their DNA content did not significantly differ from that of G(1) cells that had not initiated DNA replication.

Structure-Specific Endonucleases in DNA Repair

Nucleotide excision repair (NER) is an essential DNA repair pathway that addresses a variety of DNA lesions formed by UV light, environmental mutagens and agents used in cancer chemotherapy. NER involves the concerted action of over 30 proteins and operates by the sequential assembly of the involved factors at sites of damage. NER culminates in the removal of an oligonucleotide containing the damage by two incision reactions 5' to the lesion by ERCC1-XPF and 3' to the lesion by XPG. ERCC1-XPF and XPG are structure-specific endonucleases, a large class of enzymes involved in a variety of DNA repair, recombination and replication pathways. This review focuses on some of our recent work on how the activities of the ERCC1-XPF and XPG proteins are regulated to incise DNA only at defined times during the NER pathway, thereby preventing the formation of unwanted and cytotoxic DNA breaks by random incision of DNA. The implications of this work for our understanding of endonuclease enzymology, carcinogenesis and anti-tumor therapy are discussed.

Multiple Mechanisms for Hydrogen Peroxide–Induced Apoptosis

The mechanisms of cell killing by oxidative stress, in particular by hydrogen peroxide, are not yet well clarified. Here, we show that during recovery after H(2)O(2) treatment, apoptosis occurs in two different waves, peaking at 8 h (early) and 18 h (late) of recovery from oxidative stress. The two peaks are differentially modulated by a set of inhibitors of metabolic processes, which suggests that the first peak depends on DNA break formation, whereas the second may be correlated with H(2)O(2)-induced mitochondrial alterations.

In vitro and in vivo anti-leukemia activity of CHR-2845, a cell-targeted HDAC inhibitor for use in monocytic leukemia

e14579 Background: Histone deacetylase inhibitors alter gene expression and induce apoptosis in a wide range of cancer cells including those derived from human leukemias. CHR-2845 is a novel hydroxamic acid derivative histone deacetylase inhibitor (HDACi) which is a selective substrate for the intracellular carboxylesterase hCE-1, whose expression is restricted to cells of the monocyte- macrophage lineage. Methods: We studied the in vitro and in vivo anti-leukemia activity of CHR-2845 using cell proliferation assay, annexin V binding assay, cell cycle analysis, western blot and in vitro primary leukemia cell culture. Results: Both U937 and THP1 cells express high levels of hCE-1 whereas the myeloid cell line, HL60, does not. In comparison to vorinostat, CHR-2845 showed increased anti-proliferative potency (IC50) against monocytic cell lines (THP1, 30 nM vs 700 nM and U937, 30 nM vs 475 nM), compared to a myeloid cell line (HL60, 700nM vs 470 nM). In a broad panel of leukemic cell lines, the potency of CHR-2845 over vorinostat correlated completely with hCE-1 expression. In monocytic cell lines, CHR-2845 induced more apoptosis than vorinostat (THP1: 45±5% vs 11±1% and U937: 23±14% vs 6±1%), as measured by flow cytometry using Annexin V. Biochemical assessment of histone H3 and H4 protein acetylation by Western blot also indicateed that CHR-2845 is at least 10 times more potent than vorinostat in monocytic cell lines but not in HL-60 cells. This increase in histone acetylation was associated with increased phosphohistone H2AX, indicating formation of double-strand DNA breaks induced by this compound. These data for CHR-2845 and vorinostat on apoptosis and histone acetylation in THP1 and U937 versus HL60 cells, confirmed the selectivity of this novel compound for cells of the monocytic lineage. We also studied the anti-leukemia activity of CHR-2845 in primary leukemia cells from 8 patients with acute or chronic myelomonocytic leukemia. CHR-2845 decreased proliferation and induced apoptosis more than an equivalent dose of vorinostat in some of the patients we studied. Conclusions: These results indicated that CHR-2845 has potential to be efficacious in the treatment of patients with monocytic leukemia. [Table: see text]

Cytotoxicity and inhibitory properties against topoisomerase II of doxorubicin and its formamidine derivatives.

This work was undertaken to compare cytotoxicity, DNA damaging properties and effect on DNA cleavage by topoisomerase II of the anthracycline drug doxorubicin (DOX) and its two derivatives with a formamidino group containing a cyclic amine moiety such as morpholine (DOXM) or hexamethyleneimine (DOXH). The tetrazolium dye colorimetric assay was used to determine the cytotoxic activity of anthracyclines toward L1210 leukemia cells. DNA damage was measured by alkaline elution technique. The effect of anthracyclines on DNA cleavage was studied in a cell-free system containing supercoiled pBR322 DNA and purified human topoisomerase II. The cytotoxicity data and the results of studies on the mechanism of DNA break formation by anthracyclines at the cellular level and in the cell-free system showed that the presence of the formamidino group in the doxorubicin molecule reduced its ability to stimulate DNA cleavage by DNA topoisomerase II. DNA topoisomerase II is not a primary cellular target for DOXM or DOXH. An advantageous feature of formamidinoanthracyclines is their mechanism of cytotoxic action which is not related to the inhibition of DNA topoisomerase II. Therefore this class of anthracyclines seems to be a good source for selection of an anticancer drug directed toward cancer cells with the developed multidrug resistance attributed to the presence of altered DNA topoisomerase II.