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DNA Damage, PARP, and the Comet Assay

Society of Toxicology Exhibitor Hosted Session Trevigen®: Jay George, Ph.D. March, 2010
Trevigen, Inc. 8405 Helgerman Court Gaithersburg, MD P. 1 (800) 873-8443 F. 1 (301) 560-4973
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Background

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Background
• The Comet Assay is a single cell gel electrophoresis assay for evaluating DNA damage in cells
• The premise is that damaged DNA becomes fragmented. Increasing amounts of DNA damage results in
– Increased number of fragments – Smaller fragments based on gel electrophoresis.

• Applicable for the analysis of either single strand or double strand DNA breaks.

• Applications
• genotoxicity testing, human biomonitoring • cellular response to DNA damage, cancer risk assessment • cancer cell resistance to treatment
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The Comet Assay

Cells placed in LM Agaraose

Treat with lysis solution

Electrophorese and stain

Large Fragments

Small Fragments

Immobilize cells on CometSlide™

Alkali Treatment

Figure 1.

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com ● 1-800-873-8443 . • Neutral Comet is performed under non denaturing conditions and is used to analyze double strand breaks in DNA.There are Two CometAssay® Formats • Alkaline Comet is performed under denaturing conditions and is used to analyze single strand breaks in DNA.trevigen. ● www.com ● info@trevigen. • Analysis of DNA damaging agents V6 Trevigen. Inc.

Σ (Lx • % DNAx) • a damage measure combining the amount of DNA in the tail with the distance of migration (severity of damage) V6 Trevigen.Comet Definitions Percent DNA in the Tail • The integrated tail intensity x 100 divided by the total integrated cell intensity for a normalized measure of the percent of total cell DNA found in the tail Head Tail Tail Moment • The product of distance and normalized intensity integrated over the tail length.com ● info@trevigen.com ● 1-800-873-8443 . Inc.trevigen. ● www.

Comet Control Cells V6 Trevigen. Inc.com ● 1-800-873-8443 . ● www.trevigen.com ● info@trevigen.

• Used as Standards to Develop Comet Electrophoresis system • Four unsynchronized suspension cell preparations with incremental increases of DNA damage • Designed for long term storage • CometAssay® Control Cells – cat# 4256-010-CC for Alkaline Comet • Neutral CometAssay® Control Cells – cat# 4257-010-NC for Neutral Comet V6 .Control Cells • Control Cells to standardize and compare electrophoresis methods between users and laboratories.

0928 1.683 Median 1.240 75% CI of Median 1.0853 3.328 52. n 50 50 50 50 Mean 5.230 25.0080 21.905 IQR 8.8164 23.790 to 44.313 32.290 to 2.144 to 43.640 28.916 to 60.5893 SE 1.680 36.9810 3.7270 14.180 to 31.800 SD 7.029 26.736 56.Specifications of Alkaline Control Cells 90 70 % DNA in Tail 50 30 10 -10 CCO CC1 CC2 CC3 Control Cells % DNA by Etoposide CCO CC1 CC2 CC3 V6 • Four statistically distinct populations Figure 2.460 to 64.390 9 .3360 75% CI of Mean 4.990 37.925 20.050 51.068 to 30.757 28.374 39.840 27.183 40.485 to 7.630 45.

.Specifications of Neutral Control Cells NCO NC1 NC2 NC3 V6 Figure 3.

trevigen.com ● 1-800-873-8443 .Comet Electrophoresis System V6 Trevigen.com ● info@trevigen. ● www. Inc.

288 samples 2/20 Well Tray Tempered Glass 96 Well Tray Figure 4. V6 Water Chamber Ports .Comet Electrophoresis System • 2/20 well Tray Interlocking Safety Lid • Ten 2-well slides 2• Five 20-well slides 20- • 96 well Tray Overlay • 3 slides .

Non-homogenous electric field create alters Comet tail parameters. 2. V6 Trevigen. Buffer height above slides dramatically affects Comet electrophoresis. Inc. 3. ● www.4) assures consistent run to run temperatures. Variations in temperature are responsible for inconsistent Comet results. Critical positioning of electrodes assures consistent electric field. Overlay plate (Fig. Solution: 1.com ● info@trevigen. 4) assures identical run to run buffer height.trevigen.com ● 1-800-873-8443 .Factors that affect Comet electrophoresis Problem: 1. 3. Cooling chamber and glass plate (Fig. 2.

trevigen. Inc.com ● info@trevigen. ● www.com ● 1-800-873-8443 .Demonstration of well to well consistency • Tests performed on 96 well Comet slide • 2 rows of 12 for each control cell population was applied a 96 well Comet slide.5. • Data shown in Fig. V6 Trevigen. • 30 cells per well analyzed • Tail length and percent DNA in the tail were measured.

Well Position V6 . 70 60 50 40 30 20 10 0 1 2 3 4 5 6 7 8 9 10 11 12 % DNA in Tail CO C1 C2 C3 Well Position (cathode -> anode) 60 50 Length 40 30 20 10 0 1 2 3 4 5 6 7 8 9 10 11 12 CO C1 C2 C3 Figure 5.Trevigen’s Comet Electrophoresis System provides minimal well to well variation.

Trevigen’s Comet Electrophoresis System provides minimal well to well variation. V6 Trevigen. Inc. • System permits comparison of both intra and inter laboratory experiments.com ● 1-800-873-8443 .trevigen. • Minimal well to well variation observed in both intra-and inter-unit experiments. ● www.com ● info@trevigen.

● www.Alkaline vs Neutral CometAssay® V6 Trevigen.trevigen.com ● info@trevigen. Inc.com ● 1-800-873-8443 .

V6 .Alkaline and Neutral Comet Assay Overview Electrophoresis Electrophoresis 5 3 3 5 5 3 3 5 5 3 + 3 5 Figure 6.

com ● info@trevigen. ● www.com ● 1-800-873-8443 . V6 Trevigen. Inc.The “Heads or Tails” of Neutral Comet The biggest problem with neutral Comet is that it is frequently difficult to make “heads or tails” of the data.trevigen.

Is it “Heads or Tails ?” TBE Method Tris Acetate Method • Tris Borate EDTA electrophoresis is the traditional system but gives poor resolution between the head and tail. • Tris-Acetate system provides better resolution. . V6 Figure 7.

In this buffer it is frequently difficult to differentiate between the comet head and tail. Inc. ● www.com ● info@trevigen.trevigen.Neutral CometAssay® Buffer systems • Neutral Comet electrophoresis is traditionally performed in a Tris-Borate EDTA buffer.com ● 1-800-873-8443 . • Tris-Acetate EDTA buffer systems provide better resolution of comet head and tail resulting in more accurate more reproducible Neutral Comet analysis V6 Trevigen.

trevigen.com ● 1-800-873-8443 V6 . Trevigen. ● www. Inc.com ● info@trevigen.Damage Measurements Alkaline Comet • Single Strand Breaks • Droplet shape • Tail intensity increases but finite Tail length due to gel resolution Neutral Comet • Double Strand Breaks • Elongated shape • Both Tail intensity and Tail length increase of migration Figure 8.

com ● 1-800-873-8443 .com ● info@trevigen. ● www.DNA Damaging Agents • Hydrogen Peroxide • Bleomycin • Single-strand cuts • Double-strand cuts ss cut 5' 3' 3' 5' 5' 3' 3' 5' 5' 3' 3' 5' 5' 3' ds cut 3' 5' V6 Trevigen. Inc.trevigen.

Alkaline Comet Bleomycin Healthy T1 T2 Hydrogen Peroxide Healthy T1 T2 Increase in damage seen with increasing concentrations of Bleomycin and Hydrogen Peroxide Figure 9. V6 .

V6 .Neutral Comet Bleomycin Healthy T1 T2 Hydrogen Peroxide Healthy T1 T2 Increase in damage only seen with increasing concentration of Bleomycin Figure 10.

V6 Figure 11.Neutral Comet Differentiates Between Double and Single Strand DNA Damaging Agents Neutral Comet 35 30 25 20 15 10 5 0 T0 0 H2O2 Treatment Bleomycin Treatment T a il M o m e n t 40 30 20 10 0 T1 101 T2 5x101 Treatment T3 102 50 Alkaline Comet H2O2 Treatment Bleomycin Treatment T a il M o m e n t 0 T0 101 T1 1 5x10T2 Treatment 102T3 A. B. .

Panel A). Panel A) • do not show dose response behavior when treated single strand DNA damaging agents (Fig .11. 11. Inc.com ● 1-800-873-8443 .com ● info@trevigen. (Fig. ● www. (Fig. 11.trevigen.Neutral Comet Differentiates Between Double and Single Strand DNA Damaging Agents • In the Neutral CometAssay® Comet tail parameters: • show dose response behavior when cells are treated with double strand damaging agents. • In the Alkaline CometAssay® Comet tail parameters: • Show dose response behavior when treated with either single or double strand damaging agents. Panel B) V6 Trevigen.

Inc.trevigen.com ● info@trevigen.com ● 1-800-873-8443 . ● www.Application of the CometAssay® to Study DNA Repair V6 Trevigen.

trevigen. • PARG: Poly (ADP-Ribose) Glycohydrolase is for the degradation of the PAR polymer. Inc.com ● 1-800-873-8443 .Two Regulators of Base Excision Repair • PARP: Poly (ADP-Ribose) Polymerase binds to DNA strand breaks and polymerizes NAD+ into polymers of [ADP]ribose (PAR) on itself and other DNA associated proteins. ● www. The PAR polymer recruits DNA repair proteins to the site of damage. V6 Trevigen.com ● info@trevigen.

captured PAR is quantified using a PAR directed rabbit polyclonal antibody. • Subsequently.com ● info@trevigen. ● www. tissues or cultured cells.com ● 1-800-873-8443 . • Free PAR and PAR bound to proteins is captured by anti-PAR monoclonal antibody attached to microtiter plates. Inc. • Lysates are prepared from lymphocytes.The PARP Pharmacodynamic Assay II is a Capture ELISA In order to measure PAR levels in cells we developed a PARP Pharmacodynamic Assay. V6 Trevigen.trevigen.

Inc. ● www.trevigen. Trevigen.com ● 1-800-873-8443 V6 .com ● info@trevigen.PARP Pharmacodynamic Assay II Figure 12.

Action of PARP and PARG Figure 13. V6 .

Repair enzymes recruited to DNA break STEP 4B. Repair enzymes bind to PAR.The Role of PARP and PARG in Base Excision Repair PARP STEP 1. Reduced PARG activity results in excessive PAR synthesis STEP 6.PARG digests PAR Figure 14. Polymer continues to grow STEP 4A. STEP 4. PARP associated with DNA Poly (ADP) ribose (PAR) DNA is damaged PARP PARP binds to broken strands Repair Enzyme PARG ADP Ribose PARP Inhibitor STEP 3A. V6 . DNA strand repaired STEP 9. STEP 2. PARP dissociates from DNA as a result of charge repulsion STEP 4C. PARP synthesizes PAR. Reduced PARG activity STEP 5. At the same time PARG hydrolyzes PAR to control polymer size. DNA damage is not repaired by BER pathway STEP 7. Due excessive PAR and charge repulsion PAR/PARP complex dissociate from DNA prior to recruitment of repair enzymes + + STEP 8 . STEP 3. PAR is not made and BER is stopped.

Cells Deficient in Homologous Recombination are Sensitive to PARP Inhibitors Single strand break Step 1 Replication fork Replication fork (n) DNA Repair (n) Step 2 PARP inhibitor and DNA synthesis Step 3 Growing Replication fork (n) PARP inhibitor Replication fork collides with single strand break resulting in double break in DNA Homologous recombination deficiency Homologous recombination + (n) Step 5 Step 4 (n) (n) Figure 15. V6 Cell Death .

Cell Treatment for PAR and Comet Analysis Samples +/.PARP inhibitor 1 hr 37°C Treated.PARP inhibitor 100 µM H202 15 min 37°C +/. 5 x 105 cells/ml Jurkat and CCRF-CEM +/. Determine Comet tail parameters +/. V6 +/.PARP Inhibitor Healthy. Determine Comet tail parameters.PARP inhibitor Pre-Warm Media 30 and 60 min 37°C .PARP inhibitor PBS rinse Measure recovery by CometAssay® Figure 17.

Do PAR Levels Reflect DNA Repair Capacity? Panel A Panel B Jurkatt-150 pg/m/ PAR CCR-CEM PAR below detection limits DNA repair measured by the Comet Assay V6 Figure 18. .

PARP inhibitor potentiates effect of Hydrogen Peroxide Basal PAR Level: 150 pg/ml Figure 19. Basal PAR Level: ND Comet DNA Repair measured by Comet Assay V6 .

V6 Trevigen. ● www. Cells with high PAR levels more readily repair their DNA compared to cells with low levels of PAR (Figure 18).com ● info@trevigen.com ● 1-800-873-8443 . • PARP inhibitors potentiate the effect of Hydrogen Peroxide on DNA damage (Figure 19).Relationship between PAR levels and DNA repair measured by the CometAssay® • DNA repair levels measured by the CometAssay® appear to be related to cellular PAR levels.trevigen. Inc.

Standardized CometAssay® System Instrumentation Slides Control Cells Kits V6 .

trevigen.com ● info@trevigen. ● www.Ordering Information CometAssay® Kit CometAssay® Control Cells Neutral CometAssay® Control Cells CometAssay® Electrophoresis System 4250-050-K 4256-010-CC 4257-010-NC 4250-050-ESK CometAssay® Electrophoresis System Starter Kit 4250-050-ESK PARP in vivo Pharmacodynamic Assay II 4520-096-K V6 Trevigen.com ● 1-800-873-8443 . Inc.