Clustered DNA damages induced by x rays in human cells

Although DNA DSBs are known to be important in producing the damaging effects of ionizing radiation in cells, bistranded clustered DNA damages-two or more oxidized bases, abasic sites or strand breaks on opposing DNA strands within a few helical turns-are postulated to be difficult to repair and thus to be critical radiation-induced lesions. Gamma rays can induce clustered damages in DNA in solution, and high-energy iron ions produce DSBs and oxidized pyrimidine clusters in human cells, but it was not known whether sparsely ionizing radiation can produce clustered damages in mammalian cells. We show here that X rays induce abasic clusters, oxidized pyrimidine clusters, and oxidized purine clusters in DNA in human cells. Non-DSB clustered damages comprise about 70% of the complex lesions produced in cells. The relative levels of specific cluster classes depend on the environment of the DNA.     

Processing of clustered DNA damage generates additional double-strand breaks in mammalian cells post-irradiation

Clustered DNA damage sites, in which two or more lesions are formed within a few helical turns of the DNA after passage of a single radiation track, are signatures of DNA modifications induced by ionizing radiation in mammalian cells. Mutant hamster cells (xrs-5), deficient in non-homologous end joining (NHEJ), were irradiated at 37 degrees C to determine whether any additional double-strand breaks (DSBs) are formed during processing of gamma-radiation-induced DNA clustered damage sites. A class of non-DSB clustered DNA damage, corresponding to approximately 30% of the initial yield of DSBs, is converted into DSBs reflecting an artefact of preparation of genomic DNA for pulsed field gel electrophoresis. These clusters are removed within 4 min in both NHEJ-deficient and wild-type CHO cells. In xrs-5 cells, a proportion of non-DSB clustered DNA damage, representing approximately 10% of the total yield of non-DSB clustered DNA damage sites, are also converted into DSBs within approximately 30 min post-gamma but not post-alpha irradiation through cellular processing at 37 degrees C. That the majority of radiation-induced non-DSB clustered DNA damage sites are resistant to conversion into DSBs may be biologically significant at environmental levels of radiation exposure, as a non-DSB clustered damage site rather than a DSB, which only constitutes a minor proportion, is more likely to be induced in irradiated cells.     

Detection of complex DNA damage in gamma-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.     

Radiation induced DNA DSBs: Contribution from stalled replication forks?

When cells are exposed to radiation serious lesions are introduced into the DNA including double strand breaks (DSBs), single strand breaks (SSBs), base modifications and clustered damage sites (a specific feature of ionizing radiation induced DNA damage). Radiation induced DNA damage has the potential to initiate events that can lead ultimately to mutations and the onset of cancer and therefore understanding the cellular responses to DNA lesions is of particular importance. Using γH2AX as a marker for DSB formation and RAD51 as a marker of homologous recombination (HR) which is recruited in the processing of frank DSBs or DSBs arising from stalled replication forks, we have investigated the contribution of SSBs and non-DSB DNA damage to the induction of DSBs in mammalian cells by ionizing radiation during the cell cycle. V79-4 cells and human HF19 fibroblast cells have been either irradiated with 0–20 Gy of γ radiation or, for comparison, treated with a low concentration of hydrogen peroxide, which is known to induce SSBs but not DSBs. Inhibition of the repair of oxidative DNA lesions by poly(ADP ribose) polymerase (PARP) inhibitor leads to an increase in radiation induced γH2AX and RAD51 foci which we propose is due to these lesions colliding with replication forks forming replication induced DSBs. It was confirmed that DSBs are not induced in G₁ phase cells by treatment with hydrogen peroxide but treatment does lead to DSB induction, specifically in S phase cells. We therefore suggest that radiation induced SSBs and non-DSB DNA damage contribute to the formation of replication induced DSBs, detected as RAD51 foci.     

DNA repair of clustered lesions in mammalian cells: involvement of non-homologous end-joining

Clustered lesions are defined as ≥two lesions within 20 bps and are generated in DNA by ionizing radiation. In vitro studies and work in bacteria have shown that attempted repair of two closely opposed lesions can result in the formation of double strand breaks (DSBs). Since mammalian cells can repair DSBs by non-homologous end-joining (NHEJ), we hypothesized that NHEJ would repair DSBs formed during the removal of clustered tetrahydrofurans (furans). However, two opposing furans situated 2, 5 or 12 bps apart in a firefly luciferase reporter plasmid caused a decrease in luciferase activity in wild-type, Ku80 or DNA-PKcs-deficient cells, indicating the generation of DSBs. Loss of luciferase activity was maximal at 5 bps apart and studies using siRNA implicate the major AP endonuclease in the initial cleavage. Since NHEJ-deficient cells had equivalent luciferase activity to their isogenic wild-type cells, NHEJ was not involved in accurate repair of clustered lesions. However, quantitation and examination of re-isolated DNA showed that damage-containing plasmids were inaccurately repaired by Ku80-dependent, as well as Ku80-independent mechanisms. This work indicates that not even NHEJ can completely prevent the conversion of clustered lesions to potentially lethal DSBs, so demonstrating the biological relevance of ionizing radiation-induced clustered damage.     

Measurement of complex DNA damage induction and repair in human cellular systems after exposure to ionizing radiations of varying linear energy transfer (LET)

Abstract

Detrimental effects of ionizing radiation (IR) are correlated to the varying efficiency of IR to induce complex DNA damage. A double strand break (DSB) can be considered the simpler form of complex DNA damage. These types of damage can consist of DSBs, single strand breaks (SSBs) and/or non-DSB lesions such as base damages and apurinic/apyrimidinic (AP; abasic) sites in different combinations. Enthralling theoretical (Monte Carlo simulations) and experimental evidence suggests an increase in the complexity of DNA damage and therefore repair resistance with linear energy transfer (LET). In this study, we have measured the induction and processing of DSB and non-DSB oxidative clusters using adaptations of immunofluorescence. Specifically, we applied foci colocalization approaches as the most current methodologies for the in situ detection of clustered DNA lesions in a variety of human normal (FEP18-11-T1) and cancerous cell lines of varying repair efficiency (MCF7, HepG2, A549, MO59K/J) and radiation qualities of increasing LET, that is γ-, X-rays 0.3–1?keV/μm, α-particles 116?keV/μm and ³⁶Ar ions 270?keV/μm. Using γ-H2AX or 53BP1 foci staining as DSB probes, we calculated a DSB apparent rate of 5–16 DSBs/cell/Gy decreasing with LET. A similar trend was measured for non-DSB oxidized base lesions detected using antibodies against the human repair enzymes 8-oxoguanine-DNA glycosylase (OGG1) or AP endonuclease (APE1), that is damage foci as probes for oxidized purines or abasic sites, respectively. In addition, using colocalization parameters previously introduced by our groups, we detected an increasing clustering of damage for DSBs and non-DSBs. We also make correlations of damage complexity with the repair efficiency of each cell line and we discuss the biological importance of these new findings with regard to the severity of IR due to the complex nature of its DNA damage.     

Post-irradiation chemical processing of DNA damage generates double-strand breaks in cells already engaged in repair

In cells exposed to ionizing radiation (IR), double-strand breaks (DSBs) form within clustered-damage sites from lesions disrupting the DNA sugar-phosphate backbone. It is commonly assumed that these DSBs form promptly and are immediately detected and processed by the cellular DNA damage response (DDR) apparatus. This assumption is questioned by the observation that after irradiation of naked DNA, a fraction of DSBs forms minutes to hours after exposure as a result of temperature dependent, chemical processing of labile sugar lesions. Excess DSBs also form when IR-exposed cells are processed at 50°C, but have been hitherto considered method-related artifact. Thus, it remains unknown whether DSBs actually develop in cells after IR exposure from chemically labile damage. Here, we show that irradiation of 'naked' or chromatin-organized mammalian DNA produces lesions, which evolve to DSBs and add to those promptly induced, after 8-24 h in vitro incubation at 37°C or 50°C. The conversion is more efficient in chromatin-associated DNA, completed within 1 h in cells and delayed in a reducing environment. We conclude that IR generates sugar lesions within clustered-damage sites contributing to DSB formation only after chemical processing, which occurs efficiently at 37°C. This subset of delayed DSBs may challenge DDR, may affect the perceived repair kinetics and requires further characterization.     

Radiation-induced heat-labile sites that convert into DNA double-strand breaks

The yield of DNA double-strand breaks (DSBs) in SV40 DNA irradiated in aqueous solution was found to increase by more than a factor of two as a result of postirradiation incubation of the DNA at 50 degrees C and pH 8.0 for 24 h. This is in agreement with data from studies performed at 37 degrees C that were published previously. Importantly, similar results were also obtained from irradiation of mammalian DNA in agarose plugs. These results suggest that heat-labile sites within locally multiply damaged sites are produced by radiation and are subsequently transformed into DSBs. Since incubation at 50 degrees C is typically employed for lysis of cells in commonly used pulsed-field gel assays for detection of DSBs in mammalian cells, the possibility that heat-labile sites are present in irradiated cells was also studied. An increase in the apparent number of DSBs as a function of lysis time at 50 degrees C was found with kinetics that was similar to that for irradiated DNA, although the magnitude of the increase was smaller. This suggests that heat-labile sites are also formed in the cell. If this is the case, a proportion of DSBs measured by the pulsed-field gel assays may occur during the lysis step and may not be present in the cell as breaks but as heat-labile sites. It is suggested that such sites consist mainly of heat-labile sugar lesions within locally multiply damaged sites. Comparing rejoining of DSBs measured with short and long lysis procedure indicates that the heat-labile sites are repaired with fast kinetics in comparison with repair of the bulk of DSBs.     

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.     

Base excision repair by hNTH1 and hOGG1: a two edged sword in the processing of DNA damage in gamma-irradiated human cells

Using siRNA technology, we down-regulated in human B-lymphoblastoid TK6 cells the two major oxidative DNA glycosylases/AP lyases that repair free radical-induced base damages, hNTH1 and hOGG1. The down-regulation of hOGG1, the DNA glycosylase whose main substrate is the mutagenic but not cytotoxic 8-oxoguanine, resulted in reduced radiation cytotoxicity and decreased double strand break (DSB) formation post-irradiation. This supports the idea that the oxidative DNA glycosylases/AP lyases convert radiation-induced clustered DNA lesions into lethal DSBs and is in agreement with our previous finding that overexpression of hNTH1 and hOGG1 in TK6 cells increased radiation lethality, mutant frequency at the thymidine kinase locus and the enzymatic production of DSBs post-irradiation [N. Yang, H. Galick, S.S. Wallace, Attempted base excision repair of ionizing radiation damage in human lymphoblastoid cells produces lethal and mutagenic double strand breaks, DNA Repair (Amst) 3 (2004) 1323-1334]. Interestingly, cells deficient in hNTH1, the DNA glycosylase that repairs a major lethal single free radical damage, thymine glycol, were more radiosensitive but at the same time fewer DSBs were formed post-irradiation. These results indicate that hNTH1 plays two roles in the processing of radiation damages: repair of potentially lethal single lesions and generation of lethal DSBs at clustered damage sites. In contrast, in hydrogen peroxide-treated cells where the majority of free radical DNA damages are single lesions, the base excision repair pathway functioned to protect the cells. Here, overexpression of hNTH1 and hOGG1 resulted in reduced cell killing while suppression of glycosylase expression resulted in elevated cell death.     

Involvement of DNA polymerase beta in repair of ionizing radiation damage as measured by in vitro plasmid assays

Characteristic of damage introduced in DNA by ionizing radiation is the induction of a wide range of lesions. Single-strand breaks (SSBs) and base damages outnumber double-strand breaks (DSBs). If unrepaired, these lesions can lead to DSBs and increased mutagenesis. XRCC1 and DNA polymerase beta (polbeta) are thought to be critical elements in the repair of these SSBs and base damages. XRCC1-deficient cells display a radiosensitive phenotype, while proliferating polbeta-deficient cells are not more radiosensitive. We have recently shown that cells deficient in polbeta display increased radiosensitivity when confluent. In addition, cells expressing a dominant negative to polbeta have been found to be radiosensitized. Here we show that repair of radiation-induced lesions is inhibited in extracts with altered polbeta or XRCC1 status, as measured by an in vitro repair assay employing irradiated plasmid DNA. Extracts from XRCC1-deficient cells showed a dramatically reduced capacity to repair ionizing radiation-induced DNA damage. Extracts deficient in polbeta or containing a dominant negative to polbeta also showed reduced repair of radiation-induced SSBs. Irradiated repaired plasmid DNA showed increased incorporation of radioactive nucleotides, indicating use of an alternative long-patch repair pathway. These data show a deficiency in repair of ionizing radiation damage in extracts from cells deficient or altered in polbeta activity, implying that increased radiosensitivity resulted from radiation damage repair deficiencies.     

Complex interplay of lesion-specific DNA repair enzyme on bistranded clustered DNA damage harboring Tg:G mismatch in nucleosome core particles

5,6-Dihydroxy-5,6-dihydrothymine (thymine glycol) and 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-oxodG) are major DNA damage lesions produced by endogenous oxidative stress, as well as inflicted by carcinogens and ionizing radiation. The processing of Tg:G mismatch and 8-oxodG in close proximity of each other in a bistranded clustered environment in DNA oligomer duplexes as well as in a nucleosome core particle (NCP) model are reported here. The processing of the lesions was evaluated by purified enzyme cocktails of hNTH1 and hOGG1 as well as with a HeLa cell extract. Interestingly, the yield of double-strand breaks (DSBs) resulting from the processing of the bistranded lesions are appreciably lower when the DNA is treated with the HeLa cell extract compared with the relevant purified enzyme cocktail in both models. Clustered bistranded lesions become more repair refractive when reconstituted as an NCP. This indicates a complex interplay between the repair enzymes that influence the processing of the bistranded cluster damage positively to avoid the formation of DSBs under cellular conditions. In addition to position and orientation of the lesions, the type of the lesions in the cluster environment in DNA along with the relative abundance of the lesion-specific enzymes in the cells strongly prevents the processing of the oxidized nucleobases.     

Apex1 can cleave complex clustered DNA lesions in cells

Current data indicate that clustered DNA damage generated by ionizing radiation contains 2–5 damages within 20 bps. The complexity of clustered damage is also believed to increase as the linear energy transfer of the radiation increases. Complex lesions are therefore biologically relevant especially with the use of carbon ion beam therapy to treat cancer. Since two closely opposed AP site analogs (furans) are converted to a double strand break (DSB) in cells, we hypothesized that breakage could be compromised by increasing the complexity of the cluster. We have examined the repair of clusters containing three and four lesions in mouse fibroblasts using a luciferase reporter plasmid. The addition of a third furan did reduce but not eliminate cleavage, while a tandem 8-oxo-7,8-dihydroguanine (8oxoG) immediately 5′ to one furan in a two or three furan cluster decreased DSB formation by a small amount. In vitro studies using nuclear extracts demonstrated that the tandem 8oxoG was not removed under conditions where the furan was cleaved, but the presence of the 8oxoG reduced cleavage at the furan. Interestingly, a cluster of an 8oxoG opposite a furan did not form a DSB in cells. We have shown that Apex1 can cleave these complex clustered lesions in cells. This therefore indicates that Apex1 can generate complex DSBs from clustered lesions consisting of base damage and AP sites. Repair of these complex DSBs may be compromised by the nearby oxidative damage resulting in potentially lethal and biologically relevant damage.     

Clustered DNA lesion repair in eukaryotes: relevance to mutagenesis and cell survival

A clustered DNA lesion, also known as a multiply damaged site, is defined as ≥ 2 damages in the DNA within 1-2 helical turns. Only ionizing radiation and certain chemicals introduce DNA damage in the genome in this non-random way. What is now clear is that the lethality of a damaging agent is not just related to the types of DNA lesions introduced, but also to how the damage is distributed in the DNA. Clustered DNA lesions were first hypothesized to exist in the 1990s, and work has progressed where these complex lesions have been characterized and measured in irradiated as well as in non-irradiated cells. A clustered lesion can consist of single as well as double strand breaks, base damage and abasic sites, and the damages can be situated on the same strand or opposing strands. They include tandem lesions, double strand break (DSB) clusters and non-DSB clusters, and base excision repair as well as the DSB repair pathways can be required to remove these complex lesions. Due to the plethora of oxidative damage induced by ionizing radiation, and the repair proteins involved in their removal from the DNA, it has been necessary to study how repair systems handle these lesions using synthetic DNA damage. This review focuses on the repair process and mutagenic consequences of clustered lesions in yeast and mammalian cells. By examining the studies on synthetic clustered lesions, and the effects of low vs high LET radiation on mammalian cells or tissues, it is possible to extrapolate the potential biological relevance of these clustered lesions to the killing of tumor cells by radiotherapy and chemotherapy, and to the risk of cancer in non-tumor cells, and this will be discussed.     

The repair of environmentally relevant DNA double strand breaks caused by high linear energy transfer irradiation – No simple task

High linear energy transfer (LET) ionising radiation (IR) such as radon-derived alpha particles and high mass, high energy (HZE) particles of cosmic radiation are the predominant forms of IR to which humanity is exposed throughout life. High-LET forms of IR are established carcinogens relevant to human cancer, and their potent mutagenicity is believed, in part, to be due to a greater incidence of clustered DNA double strand breaks (DSBs) and associated lesions, as ionization events occur within a more confined genomic space. The repair of such DNA damage is now well-documented to occur with slower kinetics relative to that induced by low-LET IR, and to be more reliant upon homology-directed repair pathways. Underlying these phenomena is the relative inability of non-homologous end-joining (NHEJ) to adequately resolve high-LET IR-induced DSBs. Current findings suggest that the functionality of the DNA-dependent protein kinase (DNA-PK), comprised of the Ku70-Ku80 heterodimer and the DNA-PK catalytic subunit (DNA-PKcs), is particularly perturbed by high-LET IR-induced clustered DSBs, rendering DNA-PK dependent NHEJ less relevant to resolving these lesions. By contrast, the NHEJ-associated DNA processing endonuclease Artemis shows a greater relevance to high-LET IR-induced DSB repair. Here, we will review the cellular response to high-LET irradiation, the implications of the chronic, low-dose modality of this exposure and molecular pathways that respond to high-LET irradiation induced DSBs, with particular emphasis on NHEJ factors.     

Activation of ataxia telangiectasia mutated by DNA strand break-inducing agents correlates closely with the number of DNA double strand breaks

The protein kinase ataxia telangiectasia mutated (ATM) is activated when cells are exposed to ionizing radiation (IR). It has been assumed that ATM is specifically activated by the few induced DNA double strand breaks (DSBs), although little direct evidence for this assumption has been presented. DSBs constitute only a few percent of the IR-induced DNA damage, whereas the more frequent single strand DNA breaks (SSBs) and base damage account for over 98% of the overall DNA damage. It is therefore unclear whether DSBs are the only IR-induced DNA lesions that activate ATM. To test directly whether or not DSBs are responsible for ATM activation, we exposed cells to drugs and radiation that produce different numbers of DSBs and SSBs. We determined the resulting ATM activation by measuring the amount of phosphorylated Chk2 and the numbers of SSBs and DSBs in the same cells after short incubation periods. We found a strong correlation between the number of DSBs and ATM activation but no correlation with the number of SSBs. In fact, hydrogen peroxide, which, similar to IR, induces DNA damage through hydroxyl radicals but fails to induce DSBs, did not activate ATM. In contrast, we found that calicheamicin-induced strand breaks activated ATM more efficiently than IR and that ATM activation correlated with the relative DSB induction by these agents. Our data indicate that ATM is specifically activated by IR-induced DSBs, with little or no contribution from SSBs and other types of DNA damage. These findings have implications for how ATM might recognize DSBs in cells.     

CHK1 Affecting Cell Radiosensitivity is Independent of Non-Homologous End Joining

CHK1 is one of the most important checkpoint proteins in mammalian cells for responding toDNA damage. Cells defective in CHK1 are sensitive to ionizing radiation (IR). The mechanismby which CHK1 protects cells from IR-induced killing remains unclear. DNA double strandbreaks (DSBs) induced by IR are critical lesions for cell survival. Two major complementaryDNA DSBs repair pathways exist in mammalian cells, homologous recombination repair (HRR)and non-homologous end joining (NHEJ). By using CHK1 kinase dead human cell linesestablished in our laboratory, we show here that although these human cell lines have differentCHK1 activities with different sensitivities to IR-induced killing and G2 accumulation, all thesecell lines show similar inductions and rejoining rates of DNA DSBs. These results indicate thatthe different radiosensitivities and G2 checkpoint responses in these cell lines are independent ofNHEJ, suggesting that CHK1-regulated checkpoint facilitates HRR and therefore protects cellsfrom IR-induced killing.     

A model of interactions between radiation-induced oxidative stress, protein and DNA damage in Deinococcus radiodurans

Ionizing radiation triggers oxidative stress, which can have a variety of subtle and profound biological effects. Here we focus on mathematical modeling of potential synergistic interactions between radiation damage to DNA and oxidative stress-induced damage to proteins involved in DNA repair/replication. When sensitive sites on these proteins are attacked by radiation-induced radicals, correct repair of dangerous DNA lesions such as double strand breaks (DSBs) can be compromised. In contrast, if oxidation of important proteins is prevented by strong antioxidant defenses, DNA repair may function more efficiently. These processes probably occur to some extent even at low doses of radiation/oxidative stress, but they are easiest to investigate at high doses, where both DNA and protein damage are extensive. As an example, we use data on survival of Deinococcus radiodurans after high doses (thousands of Gy) of acute and chronic irradiation. Our model of radiogenic oxidative stress is consistent with these data and can potentially be generalized to other organisms and lower radiation doses.     

Detection of clustered DNA lesions: Biological and clinical applications

Humans are daily exposed to background radiation and various sources of oxidative stress. My research has focused in the last 12 years on the effects of ionizing radiation on DNA, which is considered as the key target of radiation in the cell. Ionizing radiation and endogenous cellular oxidative stress can also induce closely spaced oxidatively induced DNA lesions called "clusters" of DNA damage or locally multiply damage sites, as first introduced by John Ward. I am now interested in the repair mechanisms of clustered DNA damage, which is considered as the most difficult for the cell to repair. A main part of my research is devoted to evaluating the role of clustered DNA damage in the promotion of carcinogenesis in vitro and in vivo . Currently in my laboratory, there are two main ongoing projects. (1) Study of the role of BRCA1 and DNA-dependent protein kinase catalytic subunit repair proteins in the processing of clustered DNA damage in human cancer cells. For this project, we use several tumor cell lines, such as breast cancer cell lines MCF-7 and HCC1937 (BRCA1 deficient) and human glioblastoma cells MO59J/K; and (2) Possible use of DNA damage clusters as novel cancer biomarkers for prognostic and therapeutic applications related to modulation of oxidative stress. In this project human tumor and mice tissues are being used.