J Natl Cancer Inst 2003, 95: 540547. DNA repair as a human biomonitoring tool: comet assay approaches. Moreover, the compounds' effects on antioxidant defense systems of enzymes in cells: superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx . 195, 3134. The effect of oxidative stress on nucleotide-excision repair in colon tissue of newborn piglets. & Johnson, R. T. Correlations of DNA damage and repair with nuclear and chromosomal damage in HeLa cells caused by methylnitrosamides. The Creative Commons Public Domain Dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. doi: 10.1016/j.mrrev.2008.11.001, van Dyk, E., Steenkamp, A., Koekemoer, G., and Pretorius, P. J. You are using a browser version with limited support for CSS. 103, 490501 (2010). Mutagenesis 32, 173180 (2017). comet assay. Mutagenesis 23, 249260 (2008). The variety between these receptors may explain the different response to chemotherapy. 3. 35, 390400. [14]. doi: 10.1371/journal.pone.0079044, Nag, R., and Smerdon, M. J. Biotechnol. & Moller, P. Repair activity of oxidatively damaged DNA and telomere length in human lung epithelial cells after exposure to multi-walled carbon nanotubes. 10.1159/000265166, Haagenson KK, Wu GS: The role of MAP kinases and MAP kinase phosphatase-1 in resistance to breast cancer treatment. in Genotoxicity and DNA Repair: A Practical Approach (eds Sierra, L. M. & Gaivo, I.) The comet assay (single cell gel electrophoresis) was performed as described in . Appl. doi: 10.1016/j.mrgentox.2009.01.007, Tice, R. R., Agurell, E., Anderson, D., Burlinson, B., Hartmann, A., Kobayashi, H.,et al. Choi, S. W., Yeung, V. T., Collins, A. R. & Benzie, I. F. Redox-linked effects of green tea on DNA damage and repair, and influence of microsatellite polymorphism in HMOX-1: results of a human intervention trial. The cells were subsequently cultured in the presence of 0.01 mM 5-azacytidine (5-Aza-dcR; SigmaAldrich) for 24 h. Following treatment, cells were harvested using 1 trypsin (Lonza). (2010). The difference in methylation sensitivity of the isoschizomeric restriction endonucleases HpaII and MspI may be exploited to demonstrate the feasibility of using the comet assay to measure global DNA methylation level in individual cells. The original low-throughput and modified medium-throughput comet assays were then compared. In the meantime, to ensure continued support, we are displaying the site without styles Res. Cytogenetic markers, DNA single-strand breaks, urinary metabolites, and DNA repair rates in styrene-exposed lamination workers. Cancer Res. The use of the comet assay over other methods such as CEA for the measurement of global DNA methylation offers the advantage that it is less expensive. Recombinant Host. Res. Acta 1219, 724727 (1994). The comet assay is a simple and cost effective technique, commonly used to analyze and quantify DNA damage in individual cells. Comet assay lysis solution and flare slides were purchased from R&D systems (Minneapolis, USA FASEB J. 123, 291298 (1984). It grew through various acquisitions until it had over 9,600 employees and was listed on the Fortune 1000. Slyskova, J. et al. Assay Reagent to the first tube and mixing. The use of specific restriction endonucleases with the comet assay expands the flexibility of the method. Twelve-gel slide format optimised for comet assay and fluorescent in situ hybridisation. The pink represent the DNA in nucleus, while the orange the fragmented DNA. Crebelli, R. et al. The carotenoid beta-cryptoxanthin stimulates the repair of DNA oxidation damage in addition to acting as an antioxidant in human cells. The samples were transferred to Whatman DE-81 ion exchange filters (Whatman) and washed three times with 1 phosphate-buffered saline (PBS). Dose-limiting side effects, such as myelosuppression and cardiotoxicity, have been reported after treatment with doxorubicin [18]. By submitting a comment you agree to abide by our Terms and Community Guidelines. The use of methylation sensitive restriction endonucleases can modify the traditional alkaline comet assay to be methylation sensitive. Differences in repair of DNA cross-links between lymphocytes and epithelial tumor cells from colon cancer patients measured in vitro with the comet assay. Mutagen. 10.1002/1097-0142(197509)36:3<881::AID-CNCR2820360309>3.0.CO;2-B, Rosenberg B, VanCamp L, Trosko JE, Mansour VH: Platinum compounds: a new class of potent antitumour agents. No. The values were expressed as disintegrations per minute (dpm). (2010). The influence of scoring method on variability in results obtained with the comet assay. PubMed Central (2010). 58, 74657471 (2010). 12, 10071010 (2013). Mutagenesis 26, 461471 (2011). Experiments were performed in triplicate. Nutr. Contrary to lymphocytes or white blood cells (Moller et al., 2021) no study has been done about the impact of the time between collection of colonic samples and the comet assay. & Sierra, L. M. Drosophila comet assay: insights, uses, and future perspectives. Biophys. Ann Oncol 2013, 24: 19992004. 10.1586/erm.12.46, Article Dusinska, M. et al. The comet assay is a simple and cost effective technique, commonly used to analyze and quantify DNA damage in individual cells. Biochem. statement and Below are the links to the authors original submitted files for images. 25, 4552 (2009). Methods of DNA methylation analysis. Res. 34, 717723. SCGE is a relatively quick versatile, simple-to-perform technique with a few requirements. Age (Dordr.) 10.1093/annonc/mds339, Kim MM, Allen P, Gonzalez-Angulo AM, Woodward WA, Meric-Bernstam F, Buzdar AU, Hunt KK, Kuerer HM, Litton JK, Hortobagyi GN, Buchholz TA, Mittendorf EA: Pathologic complete response to neoadjuvant chemotherapy with trastuzumab predicts for improved survival in women with HER2-overexpressing breast cancer. doi: 10.1016/0014-4827(88)90265-0, Speit, G., Vasquez, M., and Hartmann, A. Borst P, Wessels L: Do predictive signatures really predict response to cancer chemotherapy? DNA Repair (Amst.) In May 2023, Frontiers adopted a new reporting platform to be Counter 5 compliant, in line with industry standards. However, identifying optimal treatment for an individual patient can be difficult, time-consuming and expensive. A simple technique for quantitation of low levels of DNA damage in individual cells. The majority of CpGs in the genome are methylated, with the exception of CpG-islands which tend to remain hypomethylated in adult cells except on the inactivated X chromosome (French et al., 2009; Espada and Esteller, 2010). Are glutathione S transferases involved in DNA damage signalling? Interactions with DNA damage and repair revealed from molecular epidemiology studies. Curr. Anglique Lewies, Etresia Van Dyk, Johannes F. Wentzel, and Pieter J. Pretorius (principal investigator) designed the study. These challenges and limitations are; however, not unique to the methylation sensitive comet assay but are encountered in other adaptions of the comet assay as well. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). Hanova, M. et al. As the mode of action depends on the genetic structure, a means of detecting changes in DNA structure is needed [11]. The nucleoids were exposed by adding 150 l of lysis solution directly to each well and incubated at 4C for 1 h. Following cell lysis, each well was washed with 1x PBS (SigmaAldrich) at least twice. Slyskova, J. et al. 10.2174/187152012803529646. Statistical analysis was done with Prism 5 (GraphPad). 642, 3742 (2008). Cimbala, J. M. (2011). Neurotoxicity in the form of local-regional sensory has been observed especially in combination with cisplatin (CDDP). Comets created by the treating nucleoids with the isoschizomeric enzymes MspI and HpaII. Novel Chinese hamster ultraviolet-sensitive mutants for excision repair form complementation groups 9 and 10. Lorenzo, Y. et al. Our results here reinforced the heterogeneity of cancer by showing how the effectiveness of each drug varied in each cell line, even when derived from the same cancer type. Moller, P. et al. Mol. Alkaline lysis of the cells was performed according to manufacturers instructions. 195, 3134 (2010). Cell Res. Muruzabal, D., Langie, S. A. S., Pourrut, B. Mutagenesis 21, 153158 (2006). Single Cell Gel lectrophoresis - also known as a comet assay - is used to detect DNA damage at the level of individual eukaryotic cells. Biol. Slyskova, J., Langie, S. A., Collins, A. R. & Vodicka, P. Functional evaluation of DNA repair in human biopsies and their relation to other cellular biomarkers. We assessed the use of comet assays in determining response to chemotherapeutic drugs that are widely used in breast and colon cancer. Br. We used human breast cancer cell lines MCF-7, MDA-MB 231 and T47D, and colon cancer cell lines HCT-116, HT55, HCT-15 and LoVo, which came from the European Collection of Cell Cultures (ECACC - HPA cultures, UK). 516, 6370 (2002). 10.1093/toxsci/kfu032. Together with the still widely used standard comet assay, as described by Singh et al. In some cases, Comet assay was performed on cells pulse-labelled with 50 IdU 30 min before treatment to analyse DSBs in S . Comet assays are a sensitive means of detecting both single strand and double strand DNA damage, using only a few cells. Clin. To improve the low-throughput methylation sensitive comet assay, a 12-well gasket was used for the preparation of the comet slides and enzyme digestion. & Azqueta, A. 10.1038/222385a0, Prados J, Melguizo C, Ortiz R, Vlez C, Alvarez PJ, Arias JL, Ruiz MA, Gallardo V, Aranega A: Doxorubicin-loaded nanoparticles: new advances in breast cancer therapy. Outliers were removed using the modified Thompson Tau method (Cimbala, 2011). J. Biol. volume15,pages 38443878 (2020)Cite this article. 66, 1168311689 (2006). 22, 303324 (1976). Res. By doing this it is possible to simultaneously measure global as well as CpG island DNA methylation and DNA damage and repair in a variety of cells (Wentzel et al., 2010). Comet assay evaluation of DNA single- and double-strand breaks induction and repair in C3H10T1/2 cells. Genotoxic effects of asbestos in humans. The in situ proximity-ligation assay (PLA, Duolink kit from Sigma-Aldrich) was performed according to Iannascoli et al. 634, 101111 (2007). Mutat. The CEA reaction mixture consisted of 5 Taq buffer, 25 mM MgCl2, 5 U of GoTaq enzyme (Promega), and 0.1 l of [3H] deoxycytidine triphosphate (dCTP; GE Healthcare) in a final volume of 15 l. Google Scholar, Cunningham D, Atkin W, Lenz HJ, Lynch HT, Minsky B, Nordlinger B, Starling N: Colorectal cancer. Genome 20, 604611. Paz-Elizur, T. et al. Biochem. Caple, F. et al. Drug activities varied even in the same cancer types, further demonstrating the heterogeneity of different cancer types. Med. A comparative performance test of standard, medium- and high-throughput comet assays. In short, HepG2 cells were exposed to the demethylating agent 5-Aza-dcR for 24 h as exposure to this demethylating agent causes a decrease in the percentage global DNA methylations. Introduction Exposure to hydroquinone (1,4-benzenediol, HQ) is ubiquitous as it is a common, naturally occurring antioxidant in plants used for food and beverages. The global 5-CCGG-3 methylation can be calculated by the HpaII/MspI ratio. 301, 283315. (2013). Soares, J. P. et al. We thank the hCOMET project (COST Action, CA 15132) for support. Mutagenesis 33, 919 (2018). Top. Toxicol. Lett. After incubation, the frosted glass plate was removed from the gasket and placed in electrophoresis buffer at 4C. Res. doi: 10.1016/j.mrgentox.2011.05.010, Azqueta, A., Gutzkow, K. B., Priestley, C. C., Priestley, C. C., Meier, S., Meier, S.,et al. 682, 1320. DNA methylation patterns can be established on a global or gene-specific level in accordance with regulatory needs (Bird, 2002). Mutagenesis 21, 205211 (2006). Stock solutions of AFB1 (3 mM), MMS (1 mM) and Cyto B (1.5 mg/ml) were prepared in DMSO and stored at 20C. Mutat. Taiwan J Obstet Gynecol 2009, 48: 239244. DNA Repair (Amst.) Finally, the nucleoids were stained with ethidium bromide (10 g/ml) for 1 h at 4C and rinsed with distilled water. & Skoog, L. Age-related decrease of ultraviolet light-induced DNA repair synthesis in human peripheral leukocytes. All the colorectal cancer cell lines except LoVo, were also successfully affected (Tables1, 2, 3 and 4; Figures1, 2 and 3). Turker, M. S., and Bestor, T. H. (1997). 386, 119130. Cytosines within these regions tend to be methylated whereas cytosines in the CpG islands tend to be unmethylated (Shen and Waterland, 2007). Applied Filters: Keyword:'comet assay' Showing 1-1 of 1 result for "comet assay" within Products. 10.1016/0014-4827(88)90265-0, DeSantis C, Ma J, Bryan L, Jemal A: Breast cancer statistics, 2013. Cell Sci. Food Res. A.O., I.G. Res. Excluding side effects can greatly improve healing of carcinomas. During the harvesting process, cells are exposed to trypsin which may negatively influence the integrity of cells. Jensen, A. et al. Twelve-gel slide format optimised for comet assay and fluorescent in situ hybridisation. Epigenetics 1, 4548. Langie, S. A. et al. High-throughput comet assay using 96 minigels. (1988), the variety of modifications made to the comet assay perfectly showcase the adaptability and applicability of this technique. J of Biol Res-Thessaloniki 21, 14 (2014). D: Treatment with mechlorethamine. Alkylation of DNA in male pre-meiotic germ cells leading to heritable mutations. Slyskova, J. et al. doi: 10.1002/(SICI)1098-2280(2000)35:3<206::AID-EM8>3.0.CO;2-J, Tost, J. Biomarkers of oxidative damage and antioxidant defence and associations with DNA damage and repair. The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. Conversely, MspI will digest methylated DNA, but only 5-CmCGG-3 and not 5-mCCGG-3 (Tost and Gut, 2010). FIGURE 3. Resistance to chemotherapy agents may exist before, or develop during, therapy [3]. provided anticipated results and managed the manuscript preparation; S.V., A.A. and S.A.S.L. Health Perspect. Sabine A. S. Langie. Between HCT-15 and LoVo, which both represent colon adenocarcinoma, completely different data were observed. Med. Recent studies have indicated that MAP kinases play an important role in resistance to treatment with doxorubicin, mechlorethamine, paclitaxel and proteasome inhibitors [19]. On the search for an intelligible comet assay descriptor. The percentage of DNA in tail has been reduced, indicating the effect from the drugs. (2013). Cancer Res. The filters were air-dried at room temperature overnight. MT participated in cell lines culturing. Toxicol Sci 2014, 139: 108120. Management of cancer focuses on cure (achievable through surgery, chemotherapy or radiation) or palliation (an important consideration in incurable disease) [9, 10]. Lett. Natl Acad. Higher assay sensitivity and some information on the nature of the DNA lesions are achieved by the recent inclusion of DNA repair enzymes to treat nucleoids after cell lysis. 10.1055/s-0029-1242458, Article : 148-53-8; Synonyms: 2-Hydroxy-3-methoxybenzaldehyde; 2-Hydroxy-m-anisaldehyde; 3-Methoxysalicylaldehyde; Linear Formula . Int J Oncol 2004, 24: 647655. DNA methylation is not only important for maintaining genome stability but also plays an important role in gene regulation (Bird, 2002; Nag and Smerdon, 2009). Toxicol. 5, 304 (2014). doi: 10.1016/S1383-5742(96)00048-8, Valverde, M., and Rojas, E. (2009). Front. The comet images were captured with an Olympus IX70 fluorescence microscope (200 magnification) and scored using the Comet IV computer software version 4.3.1 (Perceptive Instruments Ltd). Concentrations that were chosen for further experiments were 0.1M for mechlorethamine (122564, Sigma-Aldrich), 1M for melphalan (M2011, Sigma-Aldrich), 1M for doxorubicin (D1515, Sigma-Aldrich) and 1M for cisplatin (P4394, Sigma-Aldrich). The deployment the Fast Digest versions of the restriction enzymes HpaII and MspI, further also improved nucleoid digestion and reduced incubation time. Exp Cell Res 1988, 175: 184191. 16, 621. 18 Articles, This article is part of the Research Topic, https://www.mne.psu.edu/me345/Lectures/outliers.pdf, Creative Commons Attribution License (CC BY). Further studies with a wider variety of tumor cell types and drugs are required to establish the clinical utility of comet assays in selecting cancer treatments. Mutagenesis 25, 113123 (2010). Dusinska, M., Dzupinkova, Z., Wsolova, L., Harrington, V. & Collins, A. R. Possible involvement of XPA in repair of oxidative DNA damage deduced from analysis of damage, repair and genotype in a human population study. (2006). Both techniques are non-invasive, however 18-FDG-PET involves exposure to ionizing radiation and the MRI environment may cause harm in patients by using strong magnetic fields and radiowaves. Front. Herrera, M. et al. GM participated in comet assay. Finally the nucleoids were stained with ethidium bromide (10 g/ml) for one hour at 4C and thoroughly rinsed with distilled water. Teratog. Tofilon PJ, Basic I, Milas L: Prediction of in vivo tumor response to chemotherapeutic agents by the in vitro sister chromatid exchange assay. This study describes an approach to running slides in representative variants of the comet assay. Mutagenesis 25, 2532 (2010). Lancet 2010, 375: 10301047. Correspondence to Res. Immunol. All experiments were at least performed in triplicate with two independent repeats. 175, 184191. Res. J. Nutr. Med. Commun. Mutat. Cancer Res. Mutat. The comet assay is an affordable and flexible method which can be easily adapted for the measurement of global DNA methylation. 21, 238246. Measurement of cellular repair activities for oxidative DNA damage. On the other hand, MspI digests 5-CmCGG -3but not 5-mCCGG-3. Since, Ostling and Johanson (1984) showed that DNA from -irradiated cells migrate toward the anode due to the relaxation of the DNA supercoils the comet assay has been modified numerous times. Products Genes Papers Technical Documents Site Content Chromatograms. First, we need high amounts of proteins, which mean a lot of genetic material. After 24hrs of incubation, cells were detached by trypsinization (Trypsin-0.25% EDTA, 25200-072; Invitrogen) and 20000 cells were used for single cell gel electrophoresis with IKZUS comet assay kit (0905-050-K, IKZUS). Thank you for visiting nature.com. The CEA was performed according to the method described by (Wentzel et al., 2010). Mechlorethamine also known as mustine (HN2) is a derivative of mustard gas. Performance of the comet assay in a high-throughput version. Enzymatic MPG DNA repair assays for two different oxidative DNA lesions reveal associations with increased lung cancer risk. The comet assay is also widely used for genotoxicity studies and determining DNA repair capacity and a variety of DNA lesions can be detected, including DNA double strand breaks (DSB) and single strand breaks (SSB), as well as alkali-labile sites (Fairbairn et al., 1995; Collins and Gaivao, 2007). Biol. Genet. Data were analyzed with Comet Score software (TriTek Corp., USA). Comet assay lysis solution and flare slides were purchased from R&D systems (Minneapolis, USA). However, fast, reliable results are urgently needed; moreover, a drugs effect cannot always be predicted by measuring gene expression [5]. 5:215. doi: 10.3389/fgene.2014.00215. Al-Serori, H. et al. Merck. CAS PubMed Food Chem. Chatterjee K, Zhang J, Honbo N, Karliner JS: Doxorubicin cardiomyopathy. The enzyme digestions are performed in individual wells and a silicon cover is placed over the gasket forming a lid over each individual well during incubation. Google Scholar. 2 The resulting image that is obtained resembles a "comet" with a distinct head and tail. Then 50 l of this enzyme mixture was applied to each well and sealed with the silicone cap. Archives of Toxicology For each cancer type cell lines with different properties were tested. 32, 177182 (2014). A: Treatment with cisplatin in colorectal cancer cell lines. 736, 130137 (2012). In the later method the enzyme/buffer mixture tends to evaporate, which changes the enzymatic reaction conditions. Guarnieri, S. et al. Pharmacol. IV participated in cell line culturing. Genes (Basel) 8, https://doi.org/10.3390/genes8020075 (2017). 695, 7580 (2010). Results 115, 11771182 (2007). Toxicol. (2009). DNA crosslinks, DNA damage and repair in peripheral blood lymphocytes of non-small cell lung cancer patients treated with platinum derivatives. (Lond.) The experimental data demonstrated that personalized treatment is essential in cancer treatment. Azqueta, A., Langie, S. & Collins, A. R. The effect of extract concentration and time of incubation in the comet based in vitro DNA repair assay. The results are expressed as percentage CpG methylation and are calculated using the ratio between the average percentage tail DNA of HpaII- and MspI-digested DNA. Tost, J., and Gut, I. G. (2010). Base excision repair capacity in chronic renal failure patients undergoing hemodialysis treatment. Front. These authors contributed equally: Sona Vodenkova, Amaya Azqueta. Langie, S. A. et al. The DNA repair efficiency was measured by neutral comet assay as well as extrachromosomal assay for DNA DSBs repair (TAK assay). 601, 5060 (2008). Microbiol. doi: 10.1016/j.semcdb.2009.10.006, Fairbairn, D. W., Olive, P. L., and Oneill, K. L. (1995). Mol. Prediction of response to chemotherapy drugs is a major concern in cancer treatment [2]. Protein extracts from samples are incubated with agarose-embedded substrate nucleoids (naked supercoiled DNA) containing specifically induced DNA lesions (e.g., resulting from oxidation, UVC radiation or benzo[a]pyrene-diol epoxide treatment). The ageing brain: effects on DNA repair and DNA methylation in mice. Results Personalized medicine requires that therapy should be customized to individual patients, using genetic or other information [1]. Springer Nature. Most techniques to predict response entail analysis of expression of different genes [4]. 401, 3236 (2010). https://doi.org/10.1038/s41596-020-0401-x. doi: 10.1016/0165-1110(94)00013-3, French, H. J., Attenborough, R., Hardy, K., Shannon, M. F., and Williams, R. B. Modulation of nucleotide excision repair in human lymphocytes by genetic and dietary factors. Lett. Percentage CpG methylation was calculated using the ratio between the average percentage tail DNA of HpaII- and MspI-digested DNA, that is, [(100HpaIIMspI 100) control], where HpaII and MspI are the average percentage tail DNA of HpaII- and MspI-digested nucleoids, respectively. Surg Oncol 2007, 16: 299310. Association between age and repair of oxidatively damaged DNA in human peripheral blood mononuclear cells. Genet. Dusinska, M. et al. No use, distribution or reproduction is permitted which does not comply with these terms. Res. C: The same cell line post-incubation with melphalan. Curr. Environ. All authors read and approved the final manuscript. Mikkelsen, L. et al. Mutat.
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