Oxidative Clustered DNA Lesions Research Articles

Detection of DNA Double-Strand Breaks Using Pulsed-Field Gel Electrophoresis.

DNA is one of the most biologically important targets of exogenous and endogenous toxicants as well as carcinogens. Damage to DNA can be of different types (e.g., DNA adducts, DNA protein cross-links, single-strand breaks, oxidized bases, abasic sites, and double-strand breaks (DSBs)). DSBs are considered the most lethal form of DNA damage for eukaryotic cells, and if left unrepaired or misrepaired, can cause cell death, chromosome instability, and cancer. DSBs can arise in the cells through different sources and can be distinguished as endogenous or exogenous DSBs. Exogenous sources can be chemotherapeutic drugs, irradiation, and environmental chemicals. The endogenous causes of DNA DSBs in the cells are mainly reactive oxygen species and faulty repair of oxidative clustered DNA lesions. Qualitative and quantitative analysis of DNA DSBs is of utmost importance to understand physiologically relevant cellular processes as well as to investigate the genotoxic or clastogenic effects of toxicants. Pulsed-field gel electrophoresis (PFGE) is a widely used method for direct quantification of DNA DSBs. In this method, the cells exposed to DSB-inducing agents are embedded in the agarose blocks and lysed. These agarose blocks containing DNA are then run under multiple electric fields which are at 120° angle, to aid in the movement of large DNA strands. It gives a direct and specific measure of DSBs unlike the foci-based assays. This chapter provides a brief overview of the various commonly used approaches to analyze DNA DSBs and describes the theory, advantages and method of PFGE, for use in cells exposed to DNA DSB inducing agents.

Oxidative stress at low levels can induce clustered DNA lesions leading to NHEJ mediated mutations.

DNA damage and mutations induced by oxidative stress are associated with various different human pathologies including cancer. The facts that most human tumors are characterized by large genome rearrangements and glutathione depletion in mice results in deletions in DNA suggest that reactive oxygen species (ROS) may cause gene and chromosome mutations through DNA double strand breaks (DSBs). However, the generation of DSBs at low levels of ROS is still controversial. In the present study, we show that H2O2 at biologically-relevant levels causes a marked increase in oxidative clustered DNA lesions (OCDLs) with a significant elevation of replication-independent DSBs. Although it is frequently reported that OCDLs are fingerprint of high-energy IR, our results indicate for the first time that H2O2, even at low levels, can also cause OCDLs leading to DSBs specifically in G1 cells. Furthermore, a reverse genetic approach revealed a significant contribution of the non-homologous end joining (NHEJ) pathway in H2O2-induced DNA repair & mutagenesis. This genomic instability induced by low levels of ROS may be involved in spontaneous mutagenesis and the etiology of a wide variety of human diseases like chronic inflammation-related disorders, carcinogenesis, neuro-degeneration and aging.

PARP-1: Friend or Foe of DNA Damage and Repair in Tumorigenesis?

Oxidative stress induced by reactive oxygen species can result in DNA damage within cells and subsequently increase risk for carcinogenesis. This may be averted by repair of DNA damage through the base or nucleotide excision repair (BER/NER) pathways. PARP, a BER protein, is known for its role in DNA-repair. However, multiple lesions can occur within a small range of DNA, known as oxidative clustered DNA lesions (OCDLs), which are difficult to repair and may lead to the more severe DNA double-strand break (DSB). Inefficient DSB repair can then result in increased mutagenesis and neoplastic transformation. OCDLs occur more frequently within a variety of tumor tissues. Interestingly, PARP is highly expressed in several human cancers. Additionally, chronic inflammation may contribute to tumorigenesis through ROS-induced DNA damage. Furthermore, PARP can modulate inflammation through interaction with NFκB and regulating the expression of inflammatory signaling molecules. Thus, the upregulation of PARP may present a double-edged sword. PARP is needed to repair ROS-induced DNA lesions, but PARP expression may lead to increased inflammation via upregulation of NFκB signaling. Here, we discuss the role of PARP in the repair of oxidative damage versus the formation of OCDLs and speculate on the feasibility of PARP inhibition for the treatment and prevention of cancers by exploiting its role in inflammation.

Repair of DNA damage induced by accelerated heavy ions—A mini review

Increasing use of heavy ions for cancer therapy and concerns from exposure to heavy charged particles in space necessitate the study of the basic biological mechanisms associated with exposure to heavy ions. As the most critical damage induced by ionizing radiation is DNA double strand break (DSB), this review focuses on DSBs induced by heavy ions and their repair processes. Compared with X- or gamma-rays, high-linear energy transfer (LET) heavy ion radiation induces more complex DNA damage, categorized into DSBs and non-DSB oxidative clustered DNA lesions (OCDL). This complexity makes the DNA repair process more difficult, partially due to retarded enzymatic activities, leading to increased chromosome aberrations and cell death. In general, the repair process following heavy ion exposure is LET-dependent, but with nonhomologous end joining defective cells, this trend is less emphasized. The variation in cell survival levels throughout the cell cycle is less prominent in cells exposed to high-LET heavy ions when compared with low LET, but this mechanism has not been well understood until recently. Involvement of several DSB repair proteins is suggested to underlie this interesting phenomenon. Recent improvements in radiation-induced foci studies combined with high-LET heavy ion exposure could provide a useful opportunity for more in depth study of DSB repair processes. Accelerated heavy ions have become valuable tools to investigate the molecular mechanisms underlying repair of DNA DSBs, the most crucial form of DNA damage induced by radiation and various chemotherapeutic agents.

BRCA1 role in the mitigation of radiotoxicity and chromosomal instability through repair of clustered DNA lesions

Oxidatively-induced clustered DNA lesions are considered the signature of any ionizing radiation like the ones human beings are exposed daily from various environmental sources (medical X-rays, radon, etc.). To evaluate the role of BRCA1 deficiencies in the mitigation of radiation-induced toxicity and chromosomal instability we have used two human breast cancer cell lines, the BRCA1 deficient HCC1937 cells and as a control the BRCA1 wild-type MCF-7 cells. As an additional control for the DNA damage repair measurements, the HCC1937 cells with partially reconstituted BRCA1 expression were used. Since clustered DNA damage is considered the signature of ionizing radiation, we have measured the repair of double strand breaks (DSBs), non-DSB bistranded oxidative clustered DNA lesions (OCDLs) as well as single strand breaks (SSBs) in cells exposed to radiotherapy-relevant γ-ray doses. Parallel measurements were performed in the accumulation of chromatid and isochromatid breaks. For the measurement of OCDL repair, we have used a novel adaptation of the denaturing single cell gel electrophoresis (Comet assay) and pulsed field gel electrophoresis with Escherichia coli repair enzymes as DNA damage probes. Independent monitoring of the γ-H2AX foci was also performed while metaphase chromatid lesions were measured as an indicator of chromosomal instability. HCC1937 cells showed a significant accumulation of all types of DNA damage and chromatid breaks compared to MCF-7 while BRCA1 partial expression contributed significantly in the overall repair of OCDLs. These results further support the biological significance of repair resistant clustered DNA damage leading to chromosomal instability. The current results combined with previous findings on the minimized ability of base clusters to induce cell death (mainly induced by DSBs), enhance the potential association of OCDLs with breast cancer development especially in the case of a BRCA1 deficiency leading to the survival of breast cells carrying a high load of unrepaired DNA damage clusters.

Role of oxidatively induced DNA lesions in human pathogenesis

Genome stability is essential for maintaining cellular and organismal homeostasis, but it is subject to many threats. One ubiquitous threat is from a class of compounds known as reactive oxygen species (ROS), which can indiscriminately react with many cellular biomolecules including proteins, lipids, and DNA to produce a variety of oxidative lesions. These DNA oxidation products are a direct risk to genome stability, and of particular importance are oxidative clustered DNA lesions (OCDLs), defined as two or more oxidative lesions present within 10 bp of each other. ROS can be produced by exposure of cells to exogenous environmental agents including ionizing radiation, light, chemicals, and metals. In addition, they are produced by cellular metabolism including mitochondrial ATP generation. However, ROS also serve a variety of critical cellular functions and optimal ROS levels are maintained by multiple cellular antioxidant defenses. Oxidative DNA lesions can be efficiently repaired by base excision repair or nucleotide excision repair. If ROS levels increase beyond the capacity of its antioxidant defenses, the cell's DNA repair capacity can become overwhelmed, leading to the accumulation of oxidative DNA damage products including OCDLs, which are more difficult to repair than individual isolated DNA damage products. Here we focus on the induction and repair of OCDLs and other oxidatively induced DNA lesions. If unrepaired, these lesions can lead to the formation of mutations, DNA DSBs, and chromosome abnormalities. We discuss the roles of these lesions in human pathologies including aging and cancer, and in bystander effects.

Compromised repair of clustered DNA damage in the human acute lymphoblastic leukemia MSH2-deficient NALM-6 cells

Ionizing radiation (IR) induces two classes of complex DNA damage, double-strand breaks (DSBs) and non-DSB bi-stranded oxidative clustered DNA lesions (OCDLs). OCDLs may consist of single strand breaks (SSBs), oxidized purines/pyrimidines and abasic sites within 5–10 bp. These significant biological lesions are hypothesized to challenge the repair machinery and carry a high mutagenic potential. MSH2, a classical DNA mismatch repair protein, has been also implicated in other repair pathways associated with DSB and base lesion processing. MSH2 mutations have been identified in acute lymphoblastic leukemia (ALL) patients as well as in other types of cancers. Our research model involves two precursors B (pre-B) ALL human cell lines, NALM-6 cells, homozygous null for MSH2, and wild type 697 cells. Using a modified version of neutral and alkaline single cell gel electrophoresis (SCGE) with Escherichia coli repair enzymes as damage probes, the processing capacity of single strand breaks (SSBs), DSBs and OCDLs was assessed in NALM-6 and 697 cells exposed to a radiotherapy relevant γ-ray dose of 5 Gy. Using reverse transcriptase PCR and Western blotting we verified the complete lack of expression of MSH2 in the NALM-6 cells at the transcriptional and translational level. No differences were measured between NALM-6 and 697 cells in the induction levels of SSBs, DSBs and OCDLs after exposure to γ-rays. However, 697 cells repaired each lesion more efficiently with significant differences observed after 1–3 h post-irradiation. Lastly, our results indicate a significantly higher population of apoptotic 697 cells compared to NALM-6 cells 6–24 h post-irradiation. Our studies suggest that MSH2 is probably involved in the processing of the biologically significant clustered DNA damages as well as the execution of apoptosis induced by ionizing radiation.

Processing of clustered DNA damage in human breast cancer cells MCF-7 with partial DNA-PKcs deficiency

Complex DNA damage such as double strand breaks (DSBs) and non-DSB bistranded oxidative clustered DNA lesions (OCDL) (two or more DNA lesions within a short DNA fragment of 1–10 bp on opposing DNA strands) are considered the hallmark of ionizing radiation. Clustered DNA lesions are hypothesized to be repair-resistant lesions challenging the repair mechanisms of the cell. The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) plays an important role during the processing of DSBs. To evaluate the role of DNA-PKcs in the processing of complex DNA damage in human MCF-7 breast cancer cells we used small interfering RNAs (siRNAs) to target the silencing of the gene Prkdc coding for DNA-PKcs. MCF-7 cells with knockdown DNA-PKcs expression showed a marked decrease in their efficiency to process DSBs and OCDL after exposure to radiotherapy-relevant γ ray doses. For the detection and measurement of complex DSBs and OCDL, we used the γ-H2AX assay and an adaptation of pulsed field gel electrophoresis with Escherichia coli repair enzymes as DNA damage probes. An accumulation of all types of DNA damage was detected for the siRNA-treated MCF-7 cells compared to controls. These findings point to the important role of DNA-PKcs in the processing of complex DNA damage and its potential association with breast cancer development.

Formation of Clustered DNA Damage after High-LET Irradiation: A Review

Radiation can cause as well as cure cancer. The risk of developing radiation-induced cancer has traditionally been estimated from cancer incidence among survivors of the atomic bombs in Hiroshima and Nagasaki.(1)) These data provide the best estimate of human cancer risk over the dose range for low linear energy transfer (LET) radiations, such as X- or gamma-rays. The situation of estimating the real biological effects becomes even more difficult in the case of high LET particles encountered in space or as the result of domestic exposure to alpha-particles from radon gas emitters or other radioactive emitters like uranium-238. Complex DNA damage, i.e., the signature of high-LET radiations comprises of closely spaced DNA lesions forming a cluster of DNA damage. The two basic groups of complex DNA damage are double strand breaks (DSBs) and non-DSB oxidative clustered DNA lesions (OCDL). Theoretical analysis and experimental evidence suggest an increased complexity and severity of complex DNA damage with increasing LET (linear energy transfer) and a high mutagenic or carcinogenic potential. Data available on the formation of clustered DNA damage (DSBs and OCDL) by high-LET radiations are often controversial suggesting a variable response to dose and type of radiation. The chemical nature and cellular repair mechanisms of complex DNA damage have been much less characterized than those of isolated DNA lesions like an oxidized base or a single strand break especially in the case of high-LET radiation. This review will focus on the induction of clustered DNA damage by high-LET radiations presenting the earlier and recent relative data.

Detection of Complex DNA Damage in γ-Irradiated Acute Lymphoblastic Leukemia Pre-B NALM-6 Cells

Bistranded complex DNA damage, i.e., double-strand breaks (DSBs) and non-DSB oxidative clustered DNA lesions, is hypothesized to challenge the repair mechanisms of the cell and consequently the genomic integrity. The oxidative clustered DNA lesions may be persistent and may accumulate in human cancer cells for long times after irradiation. To evaluate the detection and possible accumulation of oxidative clustered DNA lesions in leukemia cells exposed to doses equivalent to those used in radiotherapy, we measured the induction of DSBs and three different types of oxidative clustered DNA lesions in NALM-6 cells, a human acute lymphoblastic leukemia (ALL) pre-B cell line, after exposure to (137)Cs gamma rays. For the detection and measurement of DSBs and oxidative clustered DNA lesions, we used an adaptation of the neutral comet assay (single-cell gel electrophoresis) using E. coli repair enzymes (Endo IV, Fpg and Endo III) as enzymatic probes. We found a linear dose response for the induction of DSBs and oxidative clustered DNA lesions. Clustered DNA lesions were more prevalent than prompt DSBs. For each DSB induced by radiation, approximately 2.5 oxidative clustered DNA lesions were detected. To our knowledge, this is the first study to demonstrate the detection and linear induction of oxidative clustered DNA lesions with radiation dose in an ALL cell line. These results point to the biological significance of clustered DNA lesions.

Induction and Processing of Oxidative Clustered DNA Lesions in56Fe-Ion-Irradiated Human Monocytes

Space and cosmic radiation is characterized by energetic heavy ions of high linear energy transfer (LET). Although both low- and high-LET radiations can create oxidative clustered DNA lesions and double-strand breaks (DSBs), the local complexity of oxidative clustered DNA lesions tends to increase with increasing LET. We irradiated 28SC human monocytes with doses from 0-10 Gy of (56)Fe ions (1.046 GeV/ nucleon, LET = 148 keV/microm) and determined the induction and processing of prompt DSBs and oxidative clustered DNA lesions using pulsed-field gel electrophoresis (PFGE) and Number Average Length Analysis (NALA). The (56)Fe ions produced decreased yields of DSBs (10.9 DSB Gy(-1) Gbp(-1)) and clusters (1 DSB: approximately 0.8 Fpg clusters: approximately 0.7 Endo III clusters: approximately 0.5 Endo IV clusters) compared to previous results with (137)Cs gamma rays. The difference in the relative biological effectiveness (RBE) of the measured and predicted DSB yields may be due to the formation of spatially correlated DSBs (regionally multiply damaged sites) which result in small DNA fragments that are difficult to detect with the PFGE assay. The processing data suggest enhanced difficulty compared with gamma rays in the processing of DSBs but not clusters. At the same time, apoptosis is increased compared to that seen with gamma rays. The enhanced levels of apoptosis observed after exposure to (56)Fe ions may be due to the elimination of cells carrying high levels of persistent DNA clusters that are removed only by cell death and/or "splitting" during DNA replication.

Detection of Oxidative Clustered DNA Lesions in X-Irradiated Mouse Skin Tissues and Human MCF-7 Breast Cancer Cells

Bistranded oxidative clustered DNA lesions are closely spaced lesions (1-10 bp) that challenge the DNA repair mechanisms and are associated with genomic instability. The endogenous levels of oxidative clustered DNA lesions in cells of human cancer cell lines or in animal tissues remain unknown, and these lesions may persist for a long time after irradiation. We measured the different types of DNA clusters in cells of two human cell lines, MCF-7 and MCF-10A, and in skin obtained from mice exposed to either 12.5 Gy or sham X radiation. For the detection and measurement of oxidative clustered DNA lesions, we used adaptations of number average length analysis, constant-field agarose gel electrophoresis, putrescine, and the repair enzymes APE1, OGG1 (human) and Nth1 (E. coli). Increased levels of all cluster types were detected in skin tissue from animals exposed to radiation at 20 weeks postirradiation. The level of endogenous (no radiation treatment) oxidative clustered DNA lesions was higher in MCF-7 cells compared to nonmalignant MCF-10A cells. To the best of our knowledge, this is the first study to demonstrate persistence of oxidative clustered DNA lesions for up to 20 weeks in animal tissues exposed to radiation and to detect these clusters in human breast cancer cells. This may underscore the biological significance of clustered DNA lesions.