Using the comet assay to study DNA repair: progress in the past decade.

Transcription

1 Using the comet assay to study DNA repair: progress in the past decade. Dr. Sabine A.S. Langie Flemish Institute for Technological Research (VITO) UKEMS Young Scientist Award 2014

2 DNA Repair NER UV BER O 2 - DNA Repair Fixation Cell proliferation Promotion / Progression Normal Cell DNA damage Damaged / Ageing Cell Apoptosis Cell degradation

3 DNA repair processes There are various DNA repair pathways, that deal with different classes of damage: rejoining of single strand breaks rejoining of double strand breaks direct reversal of base damage (e.g. O6 alkylguanine) removal and replacement of bases with small changes (base excision repair, BER) removal and replacement of bulky adducts and helix distortions (nucleotide excision repair, NER) mismatch repair (Not all can be studied with the comet assay)

4 DNA repair processes Correction of small base changes, e.g. oxidation, alkylation Oxidized base Glycosylases are lesion-specific. They often incorporate an AP endonuclease/lyase OGG1

5 DNA repair processes Bulky adducts and helix distortions Many proteins involved Two nicks in DNA, on either side of the lesion Repair patch synthesised about 28 nucleotides

6 Measuring DNA repair There are various ways to assess repair with the comet assay: first, the cellular repair assay, in which cells are treated with damaging agent, incubated, and removal of lesions is monitored. Strand breaks, followed with the standard comet assay Damaged bases, followed using specific endonucleases: 8-oxoguanine and formamidopyrimidines with FPG oxidised pyrimidines with endonuclease III cyclobutane pyrimidine dimers with UV endonuclease V alkylated bases with AlkA

7 Following the decrease of DNA damage over time. Time

8 First report on the Comet Assay Biochem Biophys Res Commun Aug 30;123(1): Microelectrophoretic study of radiation-induced DNA damages in individual mammalian cells. Ostling O, Johanson KJ. Abstract Mammalian cells were after irradiation suspended in melted agarose, and casted on microscope slides. The slides were after gelling at 0 degree C immersed in a neutral detergent solution which lysed the cells. A weak electric field (5 V/cm) was then applied over the gel for 5 minutes. The DNA in the gel was stained with the fluorescent dye acridine orange and gives a green emission in a microscope photometer. DNA had migrated towards the anode and this migration was more pronounced in irradiated than in control cells. The differences in migration pattern were quantitatively measured. The lower detection limit was below 0.5 Gy and a plateau in the dose-effect curve was reached at about 3 Gy. In repair experiments residual DNA damage could be observed after post-irradiation incubation for 60 minutes. The advantages of the method is: no radioactive labelling and only a few number of cells is required.

9 Cellular repair assay Cellular repair in Chinese hamster ovary (CHO) cells DNA strand break repair: Cells were treated with H 2 O 2 to induce single strand breaks (SSB) ( ), and incubated for 2 h to allow repair. Most breaks are rejoined in 30 min or less. [Collins and Horváthová (2001) Bioch. Soc. Trans. 29(2), 337]

10 History of the in vitro comet-based repair assay Following the decrease of DNA damage over time. 1 st decade 2 nd decade 3 rd decade Ostling & Johanson Neutral comet assay Singh et al. Exp. Cell Res., 175: SSB repair (alkaline) Gedik et al. Int J Radiat Biol.; 62: Green et al. Mutat Res.; 273: NER (UVC-irradiation) Evans et al. Radiat Res., 134: DSB repair

11 History of the in vitro comet-based repair assay Following the decrease of DNA damage over time. 1 st decade 2 nd decade 3 rd decade Ostling & Johanson Neutral comet assay Singh et al. Exp. Cell Res., 175: SSB repair (alkaline) McKelvey-Martin et al. Mutagenesis. 13(1):1-8. FISH comet gene specific DNA repair Gedik et al. Int J Radiat Biol.; 62: Green et al. Mutat Res.; 273: NER (UVC-irradiation) Evans et al. Radiat Res., 134: DSB repair

12 DNA repair at the molecular level Approach: Fluorescent in situ hybridisation (FISH) to comet DNA The two ends of the gene of interest are identified by hybridisation with specific oligonucleotides, tagged with different coloured labels. The appearance of the labels in the comet tail implies that there is damage within the DNA loop containing the gene. Repair of the labelled gene can be assessed relative to repair of total DNA

13 DNA repair at the molecular level [Horváthová et al. (2004) Mutagenesis 19, ]

14 History of the in vitro comet-based repair assay Following the decrease of DNA damage over time. Use of human cell extracts to study damage-specific incision 1 st decade 2 nd decade 3 rd decade Ostling & Johanson Neutral comet assay Singh et al. Exp. Cell Res., 175: SSB repair (alkaline) 1992 McKelvey-Martin et al. Mutagenesis. 13(1):1-8. FISH comet gene specific DNA repair Collins et al. Biochimica et Biophysica Acta 1219: BER assay 2001 Gedik et al. Int J Radiat Biol.; 62: Green et al. Mutat Res.; 273: NER (UVC-irradiation) 1993 Evans et al. Radiat Res., 134: DSB repair Collins et al. Mutagenesis, 16: BER assay

15 First report on the in vitro comet-based repair assay

16 History of the in vitro comet-based repair assay Following the decrease of DNA damage over time. Use of human cell extracts to study damage-specific incision 1 st decade 2 nd decade 3 rd decade Ostling & Johanson Neutral comet assay Singh et al. Exp. Cell Res., 175: SSB repair (alkaline) Gedik et al. Int J Radiat Biol.; 62: Green et al. Mutat Res.; 273: NER (UVC-irradiation) Evans et al. Radiat Res., 134: DSB repair McKelvey-Martin et al. Mutagenesis. 13(1):1-8. FISH comet gene specific DNA repair Collins et al. Biochimica et Biophysica Acta 1219: BER assay 2001 Collins et al. Mutagenesis, 16: BER assay Langie et al. Mutagenesis. 21: NER assay (BPDE adducts) Gaivão et al. Cell Biol Toxicol 25: NER assay (UVC - dimers)

17 First in vitro comet-based repair assay for NER

18 The importance of optimizing protein concentration 2x 5x Fibroblast cell line

19 The importance of adding ATP to study NER

20 History of the in vitro comet-based repair assay Following the decrease of DNA damage over time. Use of human cell extracts to study damage-specific incision In vitro repair assay with (animal) tissue extracts 1 st decade 2 nd decade 3 rd decade Ostling & Johanson Neutral comet assay Singh et al. Exp. Cell Res., 175: SSB repair (alkaline) Gedik et al. Int J Radiat Biol.; 62: Green et al. Mutat Res.; 273: NER (UVC-irradiation) Evans et al. Radiat Res., 134: DSB repair McKelvey-Martin et al. Mutagenesis. 13(1):1-8. FISH comet gene specific DNA repair Collins et al. Biochimica et Biophysica Acta 1219: BER assay 2001 Collins et al. Mutagenesis, 16: BER assay Langie et al. Mutagenesis. 21: NER assay (BPDE adducts) Gaivão et al. Cell Biol Toxicol 25: NER assay (UVC - dimers) Langie et al. Mutat Res. 695: NER assay (pigs) Mikkelsen et al. Free Radic. Biol. Med 47: BER assay (mice)

21 In vitro comet-based repair assay for BER in tissues

22 In vitro comet-based repair assay for NER in tissues Tissue extract: 0.3mg/ml

23 History of the in vitro comet-based repair assay Following the decrease of DNA damage over time. Use of human cell extracts to study damage-specific incision In vitro repair assay with (animal) tissue extracts 1 st decade 2 nd decade 3 rd decade Ostling & Johanson Neutral comet assay Singh et al. Exp. Cell Res., 175: SSB repair (alkaline) Gedik et al. Int J Radiat Biol.; 62: Green et al. Mutat Res.; 273: NER (UVC-irradiation) Evans et al. Radiat Res., 134: DSB repair McKelvey-Martin et al. Mutagenesis. 13(1):1-8. FISH comet gene specific DNA repair Collins et al. Biochimica et Biophysica Acta 1219: BER assay 2001 Collins et al. Mutagenesis, 16: BER assay Langie et al. Mutagenesis. 21: NER assay (BPDE adducts) Gaivão et al. Cell Biol Toxicol 25: NER assay (UVC - dimers) Langie et al. Mutat Res. 695: NER assay (pigs) Mikkelsen et al. Free Radic. Biol. Med 47: BER assay (mice) 2011 Langie et al. Mutagenesis. 26: BER assay (mice)

24 In vitro comet-based repair assay for BER in tissues ABSTRACT During the past two decades the comet-based in vitro DNA repair assay has been used regularly to measure base excision repair (BER) related DNA incision activity. Most studies focus on the assessment of BER in human lymphocytes or cultured cells by estimating the activity of a cell extract on substrate DNA containing specific lesions such as 8- oxoguanine. However, for many "real life" studies, it would be preferable to measure BER in the tissues of interest instead of using in vitro models or surrogate "tissues" such as lymphocytes. Various attempts have been made to use the comet-based repair assay for BER with extracts from rodent tissues, but high non-specific nuclease activity in such tissues were a significant impediment to robust estimates of BER. Our aim in this study was to optimise the in vitro repair assay for BER for use with rodent tissues using extracts from liver and brain from C57/BL mice. Because the DNA incision activity of an extract is dependent on its protein concentration, the first optimization step in preventing interference by nonspecific nuclease activity was to determine the protein concentration at which there is a maximal difference between the total and non-specific damage recognition. This protein concentration was 5mg/ml for mouse liver extracts and 1mg/ml for brain extracts. Next, we tested addition of proteinase inhibitors during the preparation of the tissue extracts, but this did not improve the sensitivity of the assay. However, addition of 1.5µM aphidicolin to the tissue extracts improved the detection of DNA repair incision activity by reducing non-specific nuclease activity and possibly by blocking residual DNA polymerase activity. Finally, the assay was tested on tissue samples from an ageing mouse colony and in mice undergoing dietary restriction and proved capable of detecting significant inter-animal differences and nutritional effects on BER-related DNA incision activity.

25 In vitro BER assay Exposure to Ro plus light Tissue Substrate cells embedded in gel Nucleoids containing 8-oxodG lesions Protein/enzyme extract Cell lysis - + Incubation at 37 C Denaturation / electrophoresis Analyze comets

26 % DNA in tail % DNA in tail The importance of optimizing protein concentration A) Brain B) Liver Extract protein concentration [mg/ml] noro Ro Aim for highest difference between specific (Ro/Extract) and non-specific signal (noro/extract)

27 % DNA in tail %DNA in tail Aphidicolin reduces non-specific nuclease activity A) Brain APC significantly reduced the non-specific signal in brain extracts (P=0.002) APC 1 1 +APC 2 2 +APC APC ~20 ~20 +APC APC inhibits DNA polymerase activity, also inhibition of nucleases! APC 1 1 +APC 2 2 +APC APC ~20 ~20 +APC B) Liver No significantly higher non-specific signal in highly concentrated liver extracts APC 5 5 +APC APC APC ~50 ~50 +APC Extract protein concentration [mg/ml] noro Ro

28 History of the in vitro comet-based repair assay Following the decrease of DNA damage over time. Use of human cell extracts to study damage-specific incision In vitro repair assay with (animal) tissue extracts 1 st decade 2 nd decade 3 rd decade Ostling & Johanson Neutral comet assay Singh et al. Exp. Cell Res., 175: SSB repair (alkaline) Gedik et al. Int J Radiat Biol.; 62: Green et al. Mutat Res.; 273: NER (UVC-irradiation) Evans et al. Radiat Res., 134: DSB repair McKelvey-Martin et al. Mutagenesis. 13(1):1-8. FISH comet gene specific DNA repair Collins et al. Biochimica et Biophysica Acta 1219: BER assay 2001 Collins et al. Mutagenesis, 16: BER assay Langie et al. Mutagenesis. 21: NER assay (BPDE adducts) Gaivão et al. Cell Biol Toxicol 25: NER assay (UVC - dimers) Langie et al. Mutat Res. 695: NER assay (pigs) Mikkelsen et al. Free Radic. Biol. Med 47: BER assay (mice) 2011 Langie et al. Mutagenesis. 26: BER assay (mice) 2013 Azqueta et al. DNA Repair 12: standard protocol Godschalk et al. Mutat Res. 757: Validation

29 Need for standardized and validated protocols In conclusion, we have shown that the incubation step of cell extracts with the substrate cells embedded in the gel can be identified as a major source of inter-laboratory variation in the assessment of DNA repair activity. The inter-laboratory variation in the measured DNA repair activity was not reduced by standardization of the results with the calibration curve samples.

30 The importance of gel concentration Substrates with H 2 O 2 -induced DNA damage FPG incubation (slope=122.83) Liver extract incubation (slope=130.7) Standard comet assay (slope=79.18) Buffer incubation (slope=73.42) The effect of the LMP agarose concentration on the DNA migration (P ANOVA = 1.131) nd detection of DNA repair incision activity (P ANOVA = 0.032). ***P=0.014 and **P=0.026 versus 0.65%; *P=0.044 versus 0.65% Langie et al. Mutagenesis. 2011;26:

31 Need for standardized protocols

32 History of the in vitro comet-based repair assay Following the decrease of DNA damage over time. Use of human cell extracts to study damage-specific incision In vitro repair assay with (animal) tissue extracts 1 st decade 2 nd decade 3 rd decade Ostling & Johanson Neutral comet assay Singh et al. Exp. Cell Res., 175: SSB repair (alkaline) Gedik et al. Int J Radiat Biol.; 62: Green et al. Mutat Res.; 273: NER (UVC-irradiation) Evans et al. Radiat Res., 134: DSB repair McKelvey-Martin et al. Mutagenesis. 13(1):1-8. FISH comet gene specific DNA repair Collins et al. Biochimica et Biophysica Acta 1219: BER assay 2001 Collins et al. Mutagenesis, 16: BER assay Langie et al. Mutagenesis. 21: NER assay (BPDE adducts) Gaivão et al. Cell Biol Toxicol 25: NER assay (UVC - dimers) Langie et al. Mutat Res. 695: NER assay (pigs) Mikkelsen et al. Free Radic. Biol. Med 47: BER assay (mice) 2011 Langie et al. Mutagenesis. 26: BER assay (mice) Slyskova et al. Front Genet. 5:116 human biopsies 2013 Azqueta et al. DNA Repair 12: standard protocol Godschalk et al. Mutat Res. 757: Validation

33 Applications Clinical applications Human Biomonitoring Ageing studies Nutritional interventions

34 DNA repair as a biomarker Can the in vitro comet repair assay be used in population monitoring? 1. Is it reliable? Reproducible? 2. Does it reveal differences between individuals? 3. How constant is the repair activity of each individual? What intrinsic factors affect repair activity? Age? Sex? Genetic polymorphisms? Is repair activity affected by environment? Nutrition?

35 Reliability Langie et al. Mutagenesis (2006) Langie et al. Mutagenesis (2011)

36 Inter- and Intra-individual variability Inter-individual variations variations of ~8-fold Inter-individual variations variations of ~4-fold Inter-individual variations variations of ~10-fold 4-weeks between samplings Langie et al. Br J Nutr (2010) Gaivao et al. Cell Biol Toxicol (2009)

37 Effect of genetic polymorphisms Langie et al. Br J Nutr (2010) Sum of 10 SNPs in 7 genes

38 Questions? There is one thing even more vital to science than Intelligent Methods; and that is, the sincere desire to find out the Truth, whatever it may be." Charles Sanders Pierce

39 Acknowledgments (former) Department of Health Risk Analysis and Toxicology in Maastricht Currently Department of Toxicology Frederik-Jan Van Schooten Roger Godschalk Department of Nutrition in Oslo Andrew Collins Centre for Brain Ageing and Vitality Human Nutrition Research Centre John Mathers My colleagues Centre for Integrated Systems Biology of Ageing and Nutrition Kerry Cameron Thomas von Zglinicki Flemish Institute for Technological Research (VITO) Environmental Risk and Health unit