|
1
|
Wilkinson B, Hill MA, Parsons JL. The Cellular Response to Complex DNA Damage Induced by Ionising Radiation. Int J Mol Sci 2023; 24:4920. [PMID: 36902352 PMCID: PMC10003081 DOI: 10.3390/ijms24054920] [Citation(s) in RCA: 20] [Impact Index Per Article: 10.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/02/2023] [Revised: 02/24/2023] [Accepted: 03/01/2023] [Indexed: 03/08/2023] Open
Abstract
Radiotherapy (ionising radiation; IR) is utilised in the treatment of ~50% of all human cancers, and where the therapeutic effect is largely achieved through DNA damage induction. In particular, complex DNA damage (CDD) containing two or more lesions within one to two helical turns of the DNA is a signature of IR and contributes significantly to the cell killing effects due to the difficult nature of its repair by the cellular DNA repair machinery. The levels and complexity of CDD increase with increasing ionisation density (linear energy transfer, LET) of the IR, such that photon (X-ray) radiotherapy is deemed low-LET whereas some particle ions (such as carbon ions) are high-LET radiotherapy. Despite this knowledge, there are challenges in the detection and quantitative measurement of IR-induced CDD in cells and tissues. Furthermore, there are biological uncertainties with the specific DNA repair proteins and pathways, including components of DNA single and double strand break mechanisms, that are engaged in CDD repair, which very much depends on the radiation type and associated LET. However, there are promising signs that advancements are being made in these areas and which will enhance our understanding of the cellular response to CDD induced by IR. There is also evidence that targeting CDD repair, particularly through inhibitors against selected DNA repair enzymes, can exacerbate the impact of higher LET, which could be explored further in a translational context.
Collapse
Affiliation(s)
- Beth Wilkinson
- Department of Molecular and Clinical Cancer Medicine, University of Liverpool, 6 West Derby Street, Liverpool L7 8TX, UK
| | - Mark A. Hill
- MRC Oxford Institute for Radiation Oncology, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Oxford OX3 7DQ, UK
| | - Jason L. Parsons
- Institute of Cancer and Genomic Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK
| |
Collapse
|
|
2
|
Kang S, Guo Z, Zhao F, Song L, Lu L, Wang C, Liu Z, Zhao J. Lanzhou Lily polysaccharide fragment protects human umbilical vein endothelial cells from radiation-induced DNA double-strand breaks. Hum Exp Toxicol 2022; 41:9603271221140110. [DOI: 10.1177/09603271221140110] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
Background Radiotherapy is widely used in the treatment of tumors. However, while killing tumor cells, radiation may also cause damage to the surrounding normal tissues. Therefore, it is very important to find safe and effective radiation protection agents. Purpose To investgate the radiation protection effect of Lanzhou Lily polysaccharide fragments (LLP). Methods: The crude polysaccharides of Lanzhou Lily were extracted from the dried bulb powder of Lilium lilium by ultrasonic-assisted hot water method, and then five different fragments were separated from the polysaccharides by DEAE-52-cellulose column. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assay, neutral comet and immunofluorescent staining were used to investigate the effect of LPe fragment on Human Umbilical Vein Endothelial Cells (HUVEC) survival and the possible radioprotective mechanism. Results The LPe fragment (composing of mannose and glucose, with a ratio of 5.5:2.9, and the average molecular weight is 8629.8 Da), significantly promoted the proliferation of HUVECs and protected cells from X-ray-induced double-strand breaks (DSBs) in DNA, in which pretreatment with the LPe fragment at 100 μg/mL showed the most pronounced protection. In addition, the occurrence of X-ray-induced γH2AX foci was significantly reduced by treatment with the LPe fragment at 50, 100, and 200 μg/mL. Furthermore, caffeine or wortmannin in combination with the LPe fragment at 25 μg/mL significantly reduced the number of X-ray-induced γH2AX foci, indicating phosphoinositide-3 kinases (PI3K) is involved in H2AX phosphorylation in HUVECs. Conclusion These results indicate the LPe fragment has a protective effect against radiation-induced DSBs and may be used as a natural antioxidant agent.
Collapse
Affiliation(s)
- S Kang
- School of Chemical Engineering, Northwest Minzu University, Lanzhou, China
- Key Laboratory for Utility of Environment-Friendly Composite Materials and Biomass in Universities of Gansu Province, Lanzhou, China
| | - Z Guo
- Medical College of Northwest Minzu University, Lanzhou, China
- Key Laboratory of Environmental Ecology and Population Health in Northwest Minority Areas, Medical College of Northwest Minzu University, Lanzhou, China
| | - F Zhao
- Medical College of Northwest Minzu University, Lanzhou, China
| | - L Song
- Key Laboratory of Environmental Ecology and Population Health in Northwest Minority Areas, Medical College of Northwest Minzu University, Lanzhou, China
| | - L Lu
- School of Chemical Engineering, Northwest Minzu University, Lanzhou, China
- Key Laboratory for Utility of Environment-Friendly Composite Materials and Biomass in Universities of Gansu Province, Lanzhou, China
| | - C Wang
- Medical College of Northwest Minzu University, Lanzhou, China
| | - Z Liu
- School of Chemical Engineering, Northwest Minzu University, Lanzhou, China
- Key Laboratory for Utility of Environment-Friendly Composite Materials and Biomass in Universities of Gansu Province, Lanzhou, China
| | - J Zhao
- Medical College of Northwest Minzu University, Lanzhou, China
- Key Laboratory of Environmental Ecology and Population Health in Northwest Minority Areas, Medical College of Northwest Minzu University, Lanzhou, China
| |
Collapse
|
|
3
|
Use of Biological Dosimetry for Monitoring Medical Workers Occupationally Exposed to Ionizing Radiation. RADIATION 2021. [DOI: 10.3390/radiation1020009] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023] Open
Abstract
Medical workers are the largest group exposed to man-made sources of ionizing radiation. The annual doses received by medical workers have decreased over the last several decades, however for some applications, like fluoroscopically guided procedures, the occupational doses still remain relatively high. Studies show that for some procedures the operator and staff still use insufficient protective and dosimetric equipment, which might cause an underestimation of medical exposures. Physical dosimetry methods are a staple for estimating occupational exposures, although due to the inconsistent use of protection measures, an alternative method such as biological dosimetry might complement the physical methods to achieve a more complete picture. Such methods were used to detect exposures to doses as low as 0.1 mSv/year, and could be useful for a more accurate assessment of genotoxic effects of ionizing radiation in medical workers. Biological dosimetry is usually based on the measurement of the effects present in peripheral blood lymphocytes. Although some methods, such as chromosome aberration scoring or micronucleus assay, show promising results, currently there is no one method recognized as most suitable for dosimetric application in the case of chronic, low-dose exposures. In this review we decided to evaluate different methods used for biological dosimetry in assessment of occupational exposures of medical workers.
Collapse
|
|
4
|
The Enzyme-Modified Neutral Comet (EMNC) Assay for Complex DNA Damage Detection. Methods Protoc 2021; 4:mps4010014. [PMID: 33669320 PMCID: PMC7931015 DOI: 10.3390/mps4010014] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/25/2021] [Revised: 02/12/2021] [Accepted: 02/13/2021] [Indexed: 12/14/2022] Open
Abstract
The comet assay is a versatile, simple, and sensitive gel electrophoresis-based method that can be used to measure and accurately quantify DNA damage, particularly single and double DNA strand breaks, in single cells. While generally this is used to measure variation in DNA strand break levels and repair capacity within a population of cells, the technique has more recently been adapted and evolved into more complex analysis and detection of specific DNA lesions, such as oxidized purines and pyrimidines, achieved through the utilization of damage-specific DNA repair enzymes following cell lysis. Here, we detail a version of the enzyme-modified neutral comet (EMNC) assay for the specific detection of complex DNA damage (CDD), defined as two or more DNA damage lesions within 1-2 helical turns of the DNA. CDD induction is specifically relevant to ionizing radiation (IR), particularly of increasing linear energy transfer (LET), and is known to contribute to the cell-killing effects of IR due to the difficult nature of its repair. Consequently, the EMNC assay reveals important details regarding the extent and complexity of DNA damage induced by IR, but also has potential for the study of other genotoxic agents that may induce CDD.
Collapse
|
|
5
|
Clinical Progress in Proton Radiotherapy: Biological Unknowns. Cancers (Basel) 2021; 13:cancers13040604. [PMID: 33546432 PMCID: PMC7913745 DOI: 10.3390/cancers13040604] [Citation(s) in RCA: 23] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/17/2020] [Revised: 01/27/2021] [Accepted: 02/01/2021] [Indexed: 02/07/2023] Open
Abstract
Simple Summary Proton radiation therapy is a more recent type of radiotherapy that uses proton beams instead of classical photon or X-rays beams. The clinical benefit of proton therapy is that it allows to treat tumors more precisely. As a result, proton radiotherapy induces less toxicity to healthy tissue near the tumor site. Despite the experience in the clinical use of protons, the response of cells to proton radiation, the radiobiology, is less understood. In this review, we describe the current knowledge about proton radiobiology. Abstract Clinical use of proton radiation has massively increased over the past years. The main reason for this is the beneficial depth-dose distribution of protons that allows to reduce toxicity to normal tissues surrounding the tumor. Despite the experience in the clinical use of protons, the radiobiology after proton irradiation compared to photon irradiation remains to be completely elucidated. Proton radiation may lead to differential damages and activation of biological processes. Here, we will review the current knowledge of proton radiobiology in terms of induction of reactive oxygen species, hypoxia, DNA damage response, as well as cell death after proton irradiation and radioresistance.
Collapse
|
|
6
|
Muruzabal D, Collins A, Azqueta A. The enzyme-modified comet assay: Past, present and future. Food Chem Toxicol 2020; 147:111865. [PMID: 33217526 DOI: 10.1016/j.fct.2020.111865] [Citation(s) in RCA: 41] [Impact Index Per Article: 8.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/25/2020] [Revised: 11/08/2020] [Accepted: 11/13/2020] [Indexed: 12/17/2022]
Abstract
The enzyme-modified comet assay was developed in order to detect DNA lesions other than those detected by the standard version (single and double strand breaks and alkali-labile sites). Various lesion-specific enzymes, from the DNA repair machinery of bacteria and humans, have been combined with the comet assay, allowing detection of different oxidized and alkylated bases as well as cyclobutane pyrimidine dimers, mis-incorporated uracil and apurinic/apyrimidinic sites. The enzyme-modified comet assay has been applied in different fields - human biomonitoring, environmental toxicology, and genotoxicity testing (both in vitro and in vivo) - as well as in basic research. Up to now, twelve enzymes have been employed; here we describe the enzymes and give examples of studies in which they have been applied. The bacterial formamidopyrimidine DNA glycosylase (Fpg) and endonuclease III (EndoIII) have been extensively used while others have been used only rarely. Adding further enzymes to the comet assay toolbox could potentially increase the variety of DNA lesions that can be detected. The enzyme-modified comet assay can play a crucial role in the elucidation of the mechanism of action of both direct and indirect genotoxins, thus increasing the value of the assay in the regulatory context.
Collapse
Affiliation(s)
- Damián Muruzabal
- Universidad de Navarra, Department of Pharmacology and Toxicology, Faculty of Pharmacy and Nutrition, Irunlarrea 1, 310008, Pamplona, Spain
| | - Andrew Collins
- Department of Nutrition, Institute for Basic Medical Sciences, University of Oslo, Sognsvannsveien 9, 0372, Oslo, Norway
| | - Amaya Azqueta
- Universidad de Navarra, Department of Pharmacology and Toxicology, Faculty of Pharmacy and Nutrition, Irunlarrea 1, 310008, Pamplona, Spain; IdiSNA, Navarra Institute for Health Research, Pamplona, Spain.
| |
Collapse
|
|
7
|
In vitro chromosomal radiosensitivity in patients with common variable immunodeficiency. Cent Eur J Immunol 2018; 43:155-161. [PMID: 30135627 PMCID: PMC6102621 DOI: 10.5114/ceji.2018.77385] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/01/2016] [Accepted: 12/05/2016] [Indexed: 01/03/2023] Open
Abstract
Common variable immunodeficiency (CVID) is one of the predominant antibody deficiency disorders, some evidence of which indicates that chromosome instability is present in these patients. An increased risk of cancer in patients with CVID has been documented. This study was undertaken to highlight radiation sensitivity in CVID patients and to clarify the genetic basis of this defect in these cases. Stimulated lymphocytes of the studied subjects were exposed to low-dose gamma-rays in the G2 phase or the G0 phase of the cell cycle and chromosomal aberrations were scored. Lymphocytes of healthy individuals, ataxia telangiectasia (AT) cases and a group of acute lymphoblastic leukemia (ALL) patients were investigated in the same way as controls. By two methods of analysis (one-way ANOVA and unpaired t-test), the CVID cases were significantly more radiosensitive than healthy controls based on the results of the G2 and the G0 assays. First-degree relatives of CVID patients were radiosensitive by the micronucleus assay which showed a significant difference as compared with normal controls (p = 0.001). In conclusion, this study may support that chromosomal radiosensitivity in CVID patients is a marker of genetic predisposition to the disease. The results might be a clue to describe the increased risk of cancer in CVID patients.
Collapse
|
|
8
|
Carter RJ, Nickson CM, Thompson JM, Kacperek A, Hill MA, Parsons JL. Complex DNA Damage Induced by High Linear Energy Transfer Alpha-Particles and Protons Triggers a Specific Cellular DNA Damage Response. Int J Radiat Oncol Biol Phys 2017; 100:776-784. [PMID: 29413288 PMCID: PMC5796827 DOI: 10.1016/j.ijrobp.2017.11.012] [Citation(s) in RCA: 80] [Impact Index Per Article: 10.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/14/2017] [Revised: 10/27/2017] [Accepted: 11/07/2017] [Indexed: 12/31/2022]
Abstract
Purpose To investigate the precise mechanism of recognition and processing of ionizing radiation (IR)-induced complex DNA damage (CDD), where two or more DNA lesions are in close proximity, in cellular DNA which is packaged with histones to form chromatin. Methods and Materials HeLa and oropharyngeal squamous cell carcinoma (UMSCC74A and UMSCC6) cells were irradiated with high linear energy transfer (LET) α-particles or protons, versus low-LET protons and X rays. At various time points after irradiation, site-specific histone post-translational modifications were analyzed by quantitative Western blotting; DNA damage and repair were measured by different versions of the comet assay; and cell survival was determined using clonogenic assays. Results Site-specific histone post-translational modifications after low- and high-LET radiation, particularly proton irradiation, were screened, aiming to identify those responsive to CDD. We demonstrate that histone H2B ubiquitylated on lysine 120 (H2Bub) is specifically induced several hours after irradiation in response to high-LET α-particles and protons but not by low-LET protons or X rays/γ-radiation. This is associated with increased levels of CDD, which contributes to decreased cell survival. We further discovered that modulation of H2Bub is under the control of two E3 ubiquitin ligases, MSL2 and RNF20/RNF40 complex, whose depletion leads to defective processing and further persistence of CDD, and to additional decreased cell survival after irradiation. Conclusion This study demonstrates that the signaling and repair of CDD, particularly induced by high-LET IR is co-ordinated through the specific induction of H2Bub catalyzed by MSL2 and RNF20/40, a mechanism that contributes significantly to cell survival after irradiation.
Collapse
Affiliation(s)
- Rachel J Carter
- Cancer Research Centre, Department of Molecular and Clinical Cancer Medicine, University of Liverpool, United Kingdom
| | - Catherine M Nickson
- Cancer Research Centre, Department of Molecular and Clinical Cancer Medicine, University of Liverpool, United Kingdom
| | - James M Thompson
- Gray Laboratories, Cancer Research UK/Medical Research Council Oxford Institute for Radiation Oncology, University of Oxford, Oxford, United Kingdom
| | - Andrzej Kacperek
- The National Eye Proton Therapy Centre, The Clatterbridge Cancer Centre NHS Foundation Trust, Bebington, United Kingdom
| | - Mark A Hill
- Gray Laboratories, Cancer Research UK/Medical Research Council Oxford Institute for Radiation Oncology, University of Oxford, Oxford, United Kingdom
| | - Jason L Parsons
- Cancer Research Centre, Department of Molecular and Clinical Cancer Medicine, University of Liverpool, United Kingdom.
| |
Collapse
|
|
9
|
Systemic DNA damage accumulation under in vivo tumor growth can be inhibited by the antioxidant Tempol. Cancer Lett 2014; 353:248-57. [PMID: 25069035 DOI: 10.1016/j.canlet.2014.07.030] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/29/2014] [Revised: 07/16/2014] [Accepted: 07/16/2014] [Indexed: 12/18/2022]
Abstract
Recently we found that mice bearing subcutaneous non-metastatic tumors exhibited elevated levels of two types of complex DNA damage, i.e., double-strand breaks and oxidatively-induced clustered DNA lesions in various tissues throughout the body, both adjacent to and distant from the tumor site. This DNA damage was dependent on CCL2, a cytokine involved in the recruitment and activation of macrophages, suggesting that this systemic DNA damage was mediated via tumor-induced chronic inflammatory responses involving cytokines, activation of macrophages, and consequent free radical production. If free radicals are involved, then a diet containing an antioxidant may decrease the distant DNA damage. Here we repeated our standard protocol in cohorts of two syngeneic tumor-bearing C57BL/6NCr mice that were on a Tempol-supplemented diet. We show that double-strand break and oxidatively-induced clustered DNA lesion levels were considerably decreased, about two- to three fold, in the majority of tissues studied from the tumor-bearing mice fed the antioxidant Tempol compared to the control tumor-bearing mice. Similar results were also observed in nude mice suggesting that the Tempol effects are independent of functioning adaptive immunity. This is the first in vivo study demonstrating the effect of a dietary antioxidant on abscopal DNA damage in tissues distant from a localized source of genotoxic stress. These findings may be important for understanding the mechanisms of genomic instability and carcinogenesis caused by chronic stress-induced systemic DNA damage and for developing preventative strategies.
Collapse
|
|
10
|
Lee J, Kim KS, Lee MH, Kim YS, Lee MH, Lee SE, Kim YK, Ryu MJ, Kim SJ, Choi MJ, Jo YS. NAD(P)H: quinone oxidoreductase 1 and NRH:quinone oxidoreductase 2 polymorphisms in papillary thyroid microcarcinoma: correlation with phenotype. Yonsei Med J 2013; 54:1158-67. [PMID: 23918565 PMCID: PMC3743184 DOI: 10.3349/ymj.2013.54.5.1158] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
PURPOSE NAD(P)H:Quinone Oxidoreductase 1 (NQO1) C609T missense variant (NQO1*2) and 29 basepair (bp)-insertion/deletion (I29/D) polymorphism of the NRH:Quinone Oxidoreductase 2 (NQO2) gene promoter have been proposed as predictive and prognostic factors for cancer development and progression. The purpose of this study is to investigate the relationship between NQO1/NQO2 genotype and clinico-pathological features of papillary thyroid microcarcinoma (PTMC). MATERIALS AND METHODS Genomic DNA was isolated from 243 patients; and clinical data were retrospectively analyzed. NQO1*2 and tri-allelic polymorphism of NQO2 were investigated by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis. RESULTS PTMC with NQO1*2 frequently exhibited extra-thyroidal extension as compared to PTMC with wild-type NQO1 (p=0.039). There was a significant relationship between I29/I29 homozygosity of NQO2 and lymph node metastasis (p=0.042). Multivariate analysis showed that the I29/I29 genotype was associated with an increased risk of lymph node metastasis (OR, 2.24; 95% CI, 1.10-4.56; p=0.026). CONCLUSION NQO1*2 and I29 allele of the NQO2 are associated with aggressive clinical phenotypes of PTMC, and the I29 allele represents a putative prognostic marker for PTMC.
Collapse
Affiliation(s)
- Junguee Lee
- Department of Pathology, Daejeon St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Daejeon, Korea
| | - Koon Soon Kim
- Research Center for Endocrine and Metabolic Diseases, Chungnam National University Hospital, Daejeon, Korea
| | - Min Ho Lee
- College of Biological Sciences and Biotechnology, Department of Bioscience, Chungnam National University, Daejeon, Korea
| | - Yeon Soo Kim
- Cheong Shim International Academy, Gapyeong, Korea
| | - Min Hee Lee
- Department of Internal Medicine, Chungnam National University School of Medicine, Daejeon, Korea
| | - Seong Eun Lee
- Department of Internal Medicine, Chungnam National University School of Medicine, Daejeon, Korea
| | - Yong Kyung Kim
- Department of Internal Medicine, Chungnam National University School of Medicine, Daejeon, Korea
| | - Min Jeong Ryu
- Department of Internal Medicine, Chungnam National University School of Medicine, Daejeon, Korea
| | - Soung Jung Kim
- Department of Internal Medicine, Chungnam National University School of Medicine, Daejeon, Korea
| | - Min Jeong Choi
- Department of Internal Medicine, Chungnam National University School of Medicine, Daejeon, Korea
| | - Young Suk Jo
- Research Center for Endocrine and Metabolic Diseases, Chungnam National University Hospital, Daejeon, Korea
| |
Collapse
|
|
11
|
The potential value of the neutral comet assay and γH2AX foci assay in assessing the radiosensitivity of carbon beam in human tumor cell lines. Radiol Oncol 2013; 47:247-57. [PMID: 24133390 PMCID: PMC3794881 DOI: 10.2478/raon-2013-0045] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/21/2012] [Accepted: 05/19/2013] [Indexed: 01/12/2023] Open
Abstract
Background Carbon ions (12C6+) are high linear energy transfer (LET) radiation characterized by higher relative biological effectiveness than low LET radiation. The assessment of tumour radiosensitivity would be particularly useful in optimizing the radiation dose during radiotherapy. The aim of the current study was to evaluate the potential value of the neutral comet assay and γH2AX foci assay in assessing 12C6+ radiosensitivity of tumour cells. Materials and methods The doses of 12C6+ and X-rays used in the present study were 2 and 4 Gy. The survival fraction, DNA double-strand breaks (DSB) and repair kinetics of DSB were assayed with clonogenic survival, neutral comet assay and γH2AX foci assay in human cervical carcinoma HeLa cells, hepatoma HepG2 cells, and mucoepidermoid carcinoma MEC-1 cells at the time points of 0.5, 4, 16 and 24 h after 12C6+ and X-rays irradiation. Results The survival fraction for 12C6+ irradiation was much more inhibited than for X-rays (p < 0.05) in all three tumour cell lines tested. Substantial amounts of residual damage, assessed by the neutral comet assay, were present after irradiation (p < 0.05). The highest residual damage was observed at 0.5 or 4 h, both for 12C6+ and X-ray irradiation. However, the residual damage in HeLa and MEC-1 cells was higher for 12C6+ than X-rays (p < 0.05). The strongest induction of γH2AX foci was observed after 30 min, for all three tumour cell lines (p < 0.01). The franction of γH2AX foci persisted for at least 24 h after 12C6+ irradiation; in HeLa cells and MEC-1 was higher than after X-ray irradiation (p < 0.05). The correlation coefficients between the clonogenic survival, neutral comet assay and γH2AX foci assay were not statistically significant, except for some tumour cells at individual irradiation doses and types. Conclusions Our study demonstrated that the neutral comet assay and γ-H2AX foci assay could be used to assess the radiosensitivity of 12C6+ in human tumour cells.
Collapse
|
|
12
|
Pleiotrophic effects of natural products in ROS-induced carcinogenesis: The role of plant-derived natural products in oral cancer chemoprevention. Cancer Lett 2012; 327:16-25. [DOI: 10.1016/j.canlet.2012.02.025] [Citation(s) in RCA: 35] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/05/2011] [Revised: 02/18/2012] [Accepted: 02/20/2012] [Indexed: 12/14/2022]
|
|
13
|
Cadet J, Douki T, Ravanat JL, Wagner JR. Measurement of oxidatively generated base damage to nucleic acids in cells: facts and artifacts. ACTA ACUST UNITED AC 2012. [DOI: 10.1007/s12566-012-0029-6] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023]
|
|
14
|
Cadet J, Loft S, Olinski R, Evans MD, Bialkowski K, Richard Wagner J, Dedon PC, Møller P, Greenberg MM, Cooke MS. Biologically relevant oxidants and terminology, classification and nomenclature of oxidatively generated damage to nucleobases and 2-deoxyribose in nucleic acids. Free Radic Res 2012; 46:367-81. [PMID: 22263561 DOI: 10.3109/10715762.2012.659248] [Citation(s) in RCA: 100] [Impact Index Per Article: 7.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
Abstract
A broad scientific community is involved in investigations aimed at delineating the mechanisms of formation and cellular processing of oxidatively generated damage to nucleic acids. Perhaps as a consequence of this breadth of research expertise, there are nomenclature problems for several of the oxidized bases including 8-oxo-7,8-dihydroguanine (8-oxoGua), a ubiquitous marker of almost every type of oxidative stress in cells. Efforts to standardize the nomenclature and abbreviations of the main DNA degradation products that arise from oxidative pathways are reported. Information is also provided on the main oxidative radicals, non-radical oxygen species, one-electron agents and enzymes involved in DNA degradation pathways as well in their targets and reactivity. A brief classification of oxidatively generated damage to DNA that may involve single modifications, tandem base modifications, intrastrand and interstrand cross-links together with DNA-protein cross-links and base adducts arising from the addition of lipid peroxides breakdown products is also included.
Collapse
Affiliation(s)
- Jean Cadet
- Direction des Sciences de Matière, Institut Nanosciences et Cryogénie, CEA/Grenoble, Grenoble Cedex 9, France.
| | | | | | | | | | | | | | | | | | | |
Collapse
|
|
15
|
Okayasu R. Repair of DNA damage induced by accelerated heavy ions--a mini review. Int J Cancer 2011; 130:991-1000. [PMID: 21935920 DOI: 10.1002/ijc.26445] [Citation(s) in RCA: 97] [Impact Index Per Article: 6.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/12/2011] [Accepted: 09/15/2011] [Indexed: 12/14/2022]
Abstract
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.
Collapse
Affiliation(s)
- Ryuichi Okayasu
- International Open Laboratory and Heavy-ion Radiobiology Research Group, Research Center for Charged Particle Therapy, National Institute of Radiological Sciences, Inage-ku, Chiba, Japan.
| |
Collapse
|
|
16
|
Georgakilas A. Detection of clustered DNA lesions: Biological and clinical applications. World J Biol Chem 2011; 2:173-6. [PMID: 21915368 PMCID: PMC3158867 DOI: 10.4331/wjbc.v2.i7.173] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/30/2011] [Revised: 05/07/2011] [Accepted: 05/14/2011] [Indexed: 02/05/2023] Open
Abstract
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.
Collapse
Affiliation(s)
- Alexandros Georgakilas
- Alexandros Georgakilas, DNA Damage and Repair Laboratory, Department of Biology, Thomas Harriot College of Arts and Sciences, Howell Science Complex, East Carolina University, Greenville, NC 27858, United States
| |
Collapse
|
|
17
|
Cadet J, Douki T, Ravanat JL. Measurement of oxidatively generated base damage in cellular DNA. Mutat Res 2011; 711:3-12. [PMID: 21329709 DOI: 10.1016/j.mrfmmm.2011.02.004] [Citation(s) in RCA: 94] [Impact Index Per Article: 6.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/15/2010] [Revised: 01/26/2011] [Accepted: 02/06/2011] [Indexed: 05/30/2023]
Abstract
This survey focuses on the critical evaluation of the main methods that are currently available for monitoring single and complex oxidatively generated damage to cellular DNA. Among chromatographic methods, HPLC-ESI-MS/MS and to a lesser extent HPLC-ECD which is restricted to a few electroactive nucleobases and nucleosides are appropriate for measuring the formation of single and clustered DNA lesions. Such methods that require optimized protocols for DNA extraction and digestion are sensitive enough for measuring base lesions formed under conditions of severe oxidative stress including exposure to ionizing radiation, UVA light and high intensity UVC laser pulses. In contrast application of GC-MS and HPLC-MS methods that are subject to major drawbacks have been shown to lead to overestimated values of DNA damage. Enzymatic methods that are based on the use of DNA repair glycosylases in order to convert oxidized bases into strand breaks are suitable, even if they are far less specific than HPLC methods, to deal with low levels of single modifications. Several other methods including immunoassays and (32)P-postlabeling methods that are still used suffer from drawbacks and therefore are not recommended. Another difficult topic is the measurement of oxidatively generated clustered DNA lesions that is currently achieved using enzymatic approaches and that would necessitate further investigations.
Collapse
Affiliation(s)
- Jean Cadet
- Laboratoire "Lésions des Acides Nucléiques", SCIB-UMR-E n°3 (CEA/UJF), FRE CNRS 3200, Département de Recherche Fondamentale sur la Matière Condensée, CEA/Grenoble, F-38054 Grenoble Cedex 9, France.
| | | | | |
Collapse
|
|
18
|
Sage E, Harrison L. Clustered DNA lesion repair in eukaryotes: relevance to mutagenesis and cell survival. Mutat Res 2011; 711:123-33. [PMID: 21185841 PMCID: PMC3101299 DOI: 10.1016/j.mrfmmm.2010.12.010] [Citation(s) in RCA: 175] [Impact Index Per Article: 12.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/14/2010] [Revised: 12/10/2010] [Accepted: 12/17/2010] [Indexed: 12/28/2022]
Abstract
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.
Collapse
Affiliation(s)
- Evelyne Sage
- Institut Curie, Bât. 110, Centre Universitaire, 91405 Orsay, France
- CNRS UMR3348, Bât. 110, Centre Universitaire, 91405 Orsay, France
| | - Lynn Harrison
- Department of Molecular and Cellular Physiology, LSUHSC-S, Shreveport, LA
| |
Collapse
|
|
19
|
Ziech D, Franco R, Pappa A, Panayiotidis MI. Reactive oxygen species (ROS)--induced genetic and epigenetic alterations in human carcinogenesis. Mutat Res 2011; 711:167-73. [PMID: 21419141 DOI: 10.1016/j.mrfmmm.2011.02.015] [Citation(s) in RCA: 374] [Impact Index Per Article: 26.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/19/2010] [Revised: 02/26/2011] [Accepted: 02/28/2011] [Indexed: 12/12/2022]
Abstract
Cancer is a multistage and complex process characterized by molecular alterations that underlie all three phases of its development: (i) initiation, (ii) promotion and (iii) progression. Some of these molecular events include alterations in gene expression that are regulated by both genetic and epigenetic mechanisms. On the other hand, "oxidative stress" implies a cellular state where ROS production exceeds the cell's ability to metabolize them resulting in excessive accumulation of ROS that overwhelms cellular defenses. Such state has been shown to regulate both genetic and epigenetic cascades underlying altered gene expression in human disease including cancer. Throughout this manuscript, we review the current state of knowledge on the role of ROS-induced oxidative stress in altering the genetic and epigenetic involvement during human carcinogenesis.
Collapse
Affiliation(s)
- Dominique Ziech
- Department of Student Success Services, University of Nevada, Reno, NV 89557, USA
| | | | | | | |
Collapse
|
|
20
|
Georgakilas AG, Holt SM, Hair JM, Loftin CW. Measurement of oxidatively-induced clustered DNA lesions using a novel adaptation of single cell gel electrophoresis (comet assay). ACTA ACUST UNITED AC 2011; Chapter 6:Unit 6.11.. [PMID: 21154553 DOI: 10.1002/0471143030.cb0611s49] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/17/2023]
Abstract
The two basic groups of complex DNA damage are double-strand breaks (DSBs) and non-DSB oxidatively-induced clustered DNA lesions (OCDLs). The single-cell gel electrophoresis (SCGE) or comet assay has been widely used for the detection of low levels of various types of DNA lesions including single-strand breaks (SSBs), DSBs, and oxidized bases per individual cell. There are limited data on the use of the comet assay for the detection of non-DSB clustered DNA lesions using different repair enzymes as enzymatic probes. This unit discusses a novel adaptation of the comet assay used to measure these unique types of lesions. Until now OCDL yields have been measured using primarily pulsed-field agarose gel electrophoresis. The advantages offered by the current approach are: (1) measurement of OCDL levels per individual cell; (2) use of a small number of cells (∼10,000) and relatively low doses of ionizing radiation (1 to 2 Gy) or low levels of oxidative stress, which are not compatible with standard agarose gel electrophoresis; and finally, (3) the assay is fast and allows direct comparison with pulsed-field gel electrophoresis results.
Collapse
Affiliation(s)
- Alexandros G Georgakilas
- Thomas Harriot College of Arts and Sciences, East Carolina University, Greenville, North Carolina, USA
| | | | | | | |
Collapse
|
|
21
|
Baird BJ, Dickey JS, Nakamura AJ, Redon CE, Parekh P, Griko YV, Aziz K, Georgakilas AG, Bonner WM, Martin OA. Hypothermia postpones DNA damage repair in irradiated cells and protects against cell killing. Mutat Res 2010; 711:142-9. [PMID: 21185842 DOI: 10.1016/j.mrfmmm.2010.12.006] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/15/2010] [Revised: 12/16/2010] [Accepted: 12/17/2010] [Indexed: 01/03/2023]
Abstract
Hibernation is an established strategy used by some homeothermic organisms to survive cold environments. In true hibernation, the core body temperature of an animal may drop to below 0°C and metabolic activity almost cease. The phenomenon of hibernation in humans is receiving renewed interest since several cases of victims exhibiting core body temperatures as low as 13.7°C have been revived with minimal lasting deficits. In addition, local cooling during radiotherapy has resulted in normal tissue protection. The experiments described in this paper were prompted by the results of a very limited pilot study, which showed a suppressed DNA repair response of mouse lymphocytes collected from animals subjected to 7-Gy total body irradiation under hypothermic (13°C) conditions, compared to normothermic controls. Here we report that human BJ-hTERT cells exhibited a pronounced radioprotective effect on clonogenic survival when cooled to 13°C during and 12h after irradiation. Mild hypothermia at 20 and 30°C also resulted in some radioprotection. The neutral comet assay revealed an apparent lack on double strand break (DSB) rejoining at 13°C. Extension of the mouse lymphocyte study to ex vivo-irradiated human lymphocytes confirmed lower levels of induced phosphorylated H2AX (γ-H2AX) and persistence of the lesions at hypothermia compared to the normal temperature. Parallel studies of radiation-induced oxidatively clustered DNA lesions (OCDLs) revealed partial repair at 13°C compared to the rapid repair at 37°C. For both γ-H2AX foci and OCDLs, the return of lymphocytes to 37°C resulted in the resumption of normal repair kinetics. These results, as well as observations made by others and reviewed in this study, have implications for understanding the radiobiology and protective mechanisms underlying hypothermia and potential opportunities for exploitation in terms of protecting normal tissues against radiation.
Collapse
Affiliation(s)
- Brandon J Baird
- Laboratory of Molecular Pharmacology, CCR, NCI, Bethesda, MD 20892, USA
| | | | | | | | | | | | | | | | | | | |
Collapse
|
|
22
|
Baird BJ, Dickey JS, Nakamura AJ, Redon CE, Parekh P, Griko YV, Aziz K, Georgakilas AG, Bonner WM, Martin OA. Hypothermia postpones DNA damage repair in irradiated cells and protects against cell killing. Mutat Res 2010. [PMID: 21185842 DOI: 10.1016/j.mrfmmm.2010.1012.1006] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023]
Abstract
Hibernation is an established strategy used by some homeothermic organisms to survive cold environments. In true hibernation, the core body temperature of an animal may drop to below 0°C and metabolic activity almost cease. The phenomenon of hibernation in humans is receiving renewed interest since several cases of victims exhibiting core body temperatures as low as 13.7°C have been revived with minimal lasting deficits. In addition, local cooling during radiotherapy has resulted in normal tissue protection. The experiments described in this paper were prompted by the results of a very limited pilot study, which showed a suppressed DNA repair response of mouse lymphocytes collected from animals subjected to 7-Gy total body irradiation under hypothermic (13°C) conditions, compared to normothermic controls. Here we report that human BJ-hTERT cells exhibited a pronounced radioprotective effect on clonogenic survival when cooled to 13°C during and 12h after irradiation. Mild hypothermia at 20 and 30°C also resulted in some radioprotection. The neutral comet assay revealed an apparent lack on double strand break (DSB) rejoining at 13°C. Extension of the mouse lymphocyte study to ex vivo-irradiated human lymphocytes confirmed lower levels of induced phosphorylated H2AX (γ-H2AX) and persistence of the lesions at hypothermia compared to the normal temperature. Parallel studies of radiation-induced oxidatively clustered DNA lesions (OCDLs) revealed partial repair at 13°C compared to the rapid repair at 37°C. For both γ-H2AX foci and OCDLs, the return of lymphocytes to 37°C resulted in the resumption of normal repair kinetics. These results, as well as observations made by others and reviewed in this study, have implications for understanding the radiobiology and protective mechanisms underlying hypothermia and potential opportunities for exploitation in terms of protecting normal tissues against radiation.
Collapse
Affiliation(s)
- Brandon J Baird
- Laboratory of Molecular Pharmacology, CCR, NCI, Bethesda, MD 20892, USA
| | | | | | | | | | | | | | | | | | | |
Collapse
|
|
23
|
The role of reactive oxygen species and oxidative stress in environmental carcinogenesis and biomarker development. Chem Biol Interact 2010; 188:334-9. [PMID: 20637748 DOI: 10.1016/j.cbi.2010.07.010] [Citation(s) in RCA: 169] [Impact Index Per Article: 11.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/17/2010] [Revised: 07/07/2010] [Accepted: 07/07/2010] [Indexed: 01/16/2023]
Abstract
Although we have greatly benefited from the use of traditional epidemiological approaches in linking environmental exposure to human disease, we are still lacking knowledge in to how such exposure participates in disease development. However, molecular epidemiological studies have provided us with evidence linking oxidative stress with the pathogenesis of human disease and in particular carcinogenesis. To this end, oxidative stress-based biomarkers have proved to be essential in revealing how oxidative stress may be mediating toxicity induced by many known carcinogenic environmental agents. Therefore, throughout this review article, we aim to address the current state of oxidative stress-based biomarker development with major emphasis pertaining to biomarkers of DNA, lipid and protein oxidation.
Collapse
|
|
24
|
Peddi P, Loftin CW, Dickey JS, Hair JM, Burns KJ, Aziz K, Francisco DC, Panayiotidis MI, Sedelnikova OA, Bonner WM, Winters TA, Georgakilas AG. DNA-PKcs deficiency leads to persistence of oxidatively induced clustered DNA lesions in human tumor cells. Free Radic Biol Med 2010; 48:1435-43. [PMID: 20193758 PMCID: PMC2901171 DOI: 10.1016/j.freeradbiomed.2010.02.033] [Citation(s) in RCA: 51] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/30/2009] [Revised: 01/22/2010] [Accepted: 02/23/2010] [Indexed: 02/07/2023]
Abstract
DNA-dependent protein kinase (DNA-PK) is a key non-homologous-end-joining (NHEJ) nuclear serine/threonine protein kinase involved in various DNA metabolic and damage signaling pathways contributing to the maintenance of genomic stability and prevention of cancer. To examine the role of DNA-PK in processing of non-DSB clustered DNA damage, we have used three models of DNA-PK deficiency, i.e., chemical inactivation of its kinase activity by the novel inhibitors IC86621 and NU7026, knockdown and complete absence of the protein in human breast cancer (MCF-7) and glioblastoma cell lines (MO59-J/K). A compromised DNA-PK repair pathway led to the accumulation of clustered DNA lesions induced by gamma-rays. Tumor cells lacking protein expression or with inhibited kinase activity showed a marked decrease in their ability to process oxidatively induced non-DSB clustered DNA lesions measured using a modified version of pulsed-field gel electrophoresis or single-cell gel electrophoresis (comet assay). In all cases, DNA-PK inactivation led to a higher level of lesion persistence even after 24-72h of repair. We suggest a model in which DNA-PK deficiency affects the processing of these clusters first by compromising base excision repair and second by the presence of catalytically inactive DNA-PK inhibiting the efficient processing of these lesions owing to the failure of DNA-PK to disassociate from the DNA ends. The information rendered will be important for understanding not only cancer etiology in the presence of an NHEJ deficiency but also cancer treatments based on the induction of oxidative stress and inhibition of cluster repair.
Collapse
Affiliation(s)
- Prakash Peddi
- Department of Biology, Thomas Harriot College of Arts and Sciences, East Carolina University, Greenville, NC 27858, USA
| | - Charles W. Loftin
- Department of Biology, Thomas Harriot College of Arts and Sciences, East Carolina University, Greenville, NC 27858, USA
| | - Jennifer S. Dickey
- Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20895, USA
| | - Jessica M. Hair
- Department of Biology, Thomas Harriot College of Arts and Sciences, East Carolina University, Greenville, NC 27858, USA
| | - Kara J. Burns
- Department of Biology, Thomas Harriot College of Arts and Sciences, East Carolina University, Greenville, NC 27858, USA
| | - Khaled Aziz
- Department of Biology, Thomas Harriot College of Arts and Sciences, East Carolina University, Greenville, NC 27858, USA
| | - Dave C. Francisco
- Department of Biology, Thomas Harriot College of Arts and Sciences, East Carolina University, Greenville, NC 27858, USA
| | - Mihalis I. Panayiotidis
- Department of Pathology, Medical School, University of Ioannina, University Campus, 45110, Ioannina, Greece
| | - Olga A. Sedelnikova
- Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20895, USA
| | - William M. Bonner
- Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20895, USA
| | - Thomas A. Winters
- Nuclear Medicine Department, Warren Grant Magnuson Clinical Center, National Institutes of Health, Bethesda, MD 20892, USA
| | - Alexandros G. Georgakilas
- Department of Biology, Thomas Harriot College of Arts and Sciences, East Carolina University, Greenville, NC 27858, USA
- Corresponding author: Alexandros G. Georgakilas, Address: Biology Department, Thomas Harriot College of Arts and Sciences, Howell Science Complex N418, East Carolina University, Greenville, NC 27858. Tel: 252-328-5446, Fax: 252-328-4178,
| |
Collapse
|
|
25
|
Au WW, Giri AK, Ruchirawat M. Challenge assay: A functional biomarker for exposure-induced DNA repair deficiency and for risk of cancer. Int J Hyg Environ Health 2009; 213:32-9. [PMID: 19818682 DOI: 10.1016/j.ijheh.2009.09.002] [Citation(s) in RCA: 35] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/27/2009] [Revised: 08/31/2009] [Accepted: 09/09/2009] [Indexed: 11/28/2022]
Abstract
A variety of biomarkers have been used to monitor exposed populations to determine potential health hazards from their exposure to environmental toxic agents. However, the majority of these biomarkers have been focused onto the identification of biological damage from the exposure. Therefore, there is a need to develop functional biomarkers that can identify exposure-induced functional deficiencies. More importantly, these deficiencies should be positioned along pathways that are responsible for the development of specific diseases. One of such pathways belongs to the extensive and complex DNA-repair machinery. The machinery thus becomes a large target for damage from environmental toxic agents. The hypothesis is that damage to any component of a repair pathway will interfere with the pathway-specific repair activities. Therefore, when cells from exposed populations are challenged with a DNA-damaging agent in vitro, the in vivo exposure-induced repair deficiency will be dramatically amplified and the deficiency will be detectable in a challenge assay as increased chromosome aberrations, micronuclei or un-repaired DNA strand breaks. The challenge assay has been used in different laboratories to show that a variety of exposed populations (with exposure to air pollutants, arsenic, benzene, butadiene, cigarette smoke, incense smoke, lead, mercury, pesticides, uranium or xylene but not to low concentrations of air pollutants or butadiene) expressed abnormal challenge response. The predicted health consequences of some of these studies have also been validated. Therefore, the challenge assay is a useful functional biomarker for population studies. Details of the challenge assay and its application will be presented in this review.
Collapse
Affiliation(s)
- William W Au
- Department of Preventive Medicine and Community Health, 700 Harborside Drive, University of Texas Medical Branch, Galveston, TX 77555-1110, USA.
| | | | | |
Collapse
|
|
26
|
Preston TJ, Henderson JT, McCallum GP, Wells PG. Base excision repair of reactive oxygen species-initiated 7,8-dihydro-8-oxo-2'-deoxyguanosine inhibits the cytotoxicity of platinum anticancer drugs. Mol Cancer Ther 2009; 8:2015-26. [PMID: 19567822 DOI: 10.1158/1535-7163.mct-08-0929] [Citation(s) in RCA: 52] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
Anticancer therapy with cisplatin and oxaliplatin is limited by toxicity and onset of tumor resistance. Both drugs form platinum-DNA cross-linked adducts, and cisplatin causes oxidative DNA damage including the 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxodG) lesion. To assess oxidative DNA damage as a mechanism of cisplatin and oxaliplatin cytotoxicity, 8-oxodG-directed base excision repair was stably enhanced in human embryonic kidney cells by FLAG-tagged expression of human oxoguanine glycosylase 1 (alpha-OGG1) or its functional homologue, Escherichia coli formamidopyrimidine glycosylase (fpg). Both drugs increased reactive oxygen species and 8-oxodG levels, and cytotoxicity was decreased by antioxidant pretreatment. Ectopic expression of alpha-OGG1 or fpg in cell clones increased nuclear and mitochondrial 8-oxodG repair, and reduced death by reactive oxygen species initiators (H(2)O(2), menadione) and both platinum drugs. Exposure to oxaliplatin caused a more marked and sustained block of cell proliferation than exposure to cisplatin. We conclude that the 8-oxodG lesion is cytotoxic, and base excision repair a likely determinant of risk. The greater antitumor efficacy of oxaliplatin seems unrelated to oxidative DNA damage, suggesting a novel strategy for improving the therapeutic index in cancer therapy.
Collapse
|
|
27
|
Holt SM, Scemama JL, Panayiotidis MI, Georgakilas AG. Compromised repair of clustered DNA damage in the human acute lymphoblastic leukemia MSH2-deficient NALM-6 cells. MUTATION RESEARCH-GENETIC TOXICOLOGY AND ENVIRONMENTAL MUTAGENESIS 2009; 674:123-30. [DOI: 10.1016/j.mrgentox.2008.09.014] [Citation(s) in RCA: 30] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/19/2008] [Accepted: 09/23/2008] [Indexed: 01/03/2023]
|
|
28
|
Peddi P, Francisco DC, Cecil AM, Hair JM, Panayiotidis MI, Georgakilas AG. Processing of clustered DNA damage in human breast cancer cells MCF-7 with partial DNA-PKcs deficiency. Cancer Lett 2008; 269:174-83. [PMID: 18550272 DOI: 10.1016/j.canlet.2008.04.049] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/12/2008] [Revised: 04/22/2008] [Accepted: 04/22/2008] [Indexed: 12/25/2022]
Abstract
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 1-10bp 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 gamma ray doses. For the detection and measurement of complex DSBs and OCDL, we used the gamma-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.
Collapse
Affiliation(s)
- Prakash Peddi
- Department of Biology, Thomas Harriot College of Arts and Sciences, East Carolina University, Howell Science Complex, Greenville, NC 27858, USA
| | | | | | | | | | | |
Collapse
|
|
29
|
Hada M, Georgakilas AG. Formation of clustered DNA damage after high-LET irradiation: a review. JOURNAL OF RADIATION RESEARCH 2008; 49:203-10. [PMID: 18413977 DOI: 10.1269/jrr.07123] [Citation(s) in RCA: 320] [Impact Index Per Article: 18.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/23/2023]
Abstract
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.
Collapse
Affiliation(s)
- Megumi Hada
- NASA Johnson Space Center, Houston, Texas 77058, USA
| | | |
Collapse
|
|
30
|
Francisco DC, Peddi P, Hair JM, Flood BA, Cecil AM, Kalogerinis PT, Sigounas G, Georgakilas AG. Induction and processing of complex DNA damage in human breast cancer cells MCF-7 and nonmalignant MCF-10A cells. Free Radic Biol Med 2008; 44:558-69. [PMID: 18005669 DOI: 10.1016/j.freeradbiomed.2007.10.045] [Citation(s) in RCA: 50] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/29/2007] [Revised: 10/12/2007] [Accepted: 10/13/2007] [Indexed: 02/04/2023]
Abstract
Oxidatively induced stress and DNA damage have been associated with various human pathophysiological conditions, including cancer and aging. Complex DNA damage such as double-strand breaks (DSBs) and non-DSB bistranded oxidatively induced clustered DNA lesions (OCDL) (two or more DNA lesions within a short DNA fragment of 1-10 bp on opposing DNA strands) are hypothesized to be repair-resistant lesions challenging the repair mechanisms of the cell. To evaluate the induction and processing of complex DNA damage in breast cancer cells exposed to radiotherapy-relevant gamma-ray doses, we measured single-strand breaks (SSBs), DSBs, and OCDL in MCF-7 and HCC1937 malignant cells as well as MCF-10A nonmalignant human breast cells. For the detection and measurement of SSBs, DSBs, and OCDL, we used the alkaline single-cell gel electrophoresis, gamma-H2AX assay, and an adaptation of pulsed-field gel electrophoresis with E. coli repair enzymes as DNA damage probes. Increased levels for most types of DNA damage were detected in MCF-7 cells while the processing of DSBs and OCDL was deficient in these cells compared to MCF-10A cells. Furthermore, the total antioxidant capacity of MCF-7 cells was lower compared to their nonmalignant counterparts. These findings point to the important role of complex DNA damage in breast cancer and its potential association with breast cancer development especially in the case of deficient BRCA1 expression.
Collapse
Affiliation(s)
- Dave C Francisco
- Department of Biology, Thomas Harriot College of Arts and Sciences, East Carolina University, Greenville, NC 27858, USA
| | | | | | | | | | | | | | | |
Collapse
|