Chapter XX
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In vitro methods for studying the mechanisms of resistance to DNA damaging therapeutic drugs
Abstract
Most commonly used anticancer drugsexert their effects mainly by causing DNA damage.The enhancement in DNA damage response is considered a key mechanism that enables cancer cells to survive througheliminating the damaged DNA lesions and thereby, developing resistance to DNA damaging agents. This chapter describes the four experimental approaches for studying DNA damage response and genotoxicdrug resistance, including the use of γ-H2AX and comet assays to monitor DNA damage and repair capacity as well as the use of clonogenic and β-galactosidase staining assays to assess long term cell fate after DNA damaging treatment. Finally, we also present examples of these methods currently used in our laboratory for studying the role of FOXM1 in DNA damage-induced senescence and epirubicin resistance.
Key words: γ-H2AX, comet assay, clonogenic assay, β-galactosidase staining, DNA damage, Resistance
1. Introduction
Genotoxic chemotherapy is one of the principal modes of cancer treatment. Most commonly used anticancer drugs, such as epirubicin, doxorubicin, 5-fluorouracil and cisplatin, target genomic DNA. They primarily function as DNA intercalators, blocking DNA synthesis and inducing DNA double-strand breaks (DSBs) leading to cancer cell death [1]. Clinically, these DNA damaging agents effectively inhibit the growth and spread of cancer cells. However, the majority of these treatments will eventually fail due to the development of drug resistance. Elucidation of the molecular mechanisms underlying chemoresistance is therefore needed to aid disease management and improve patient survival. As many chemotherapeutic agents mainly exert their effects by causing DNA damage, the enhancement in DNA damage response (DDR) signalling is considered as a key mechanism that can enable cancer cells to survive through eliminating the induced DNA lesions and thereby, developing resistance to these genotoxicagents.
At the molecular level, cells respond to DNA damage by activating the so-called DNA damage response (DDR), a complex molecular mechanism developed to repair DNA damage and maintaining genome integrity. Initially, the formation of DSBs triggers activation of ATM and phosphorylation of serine 139 on histone variant H2AX at the site of the DNA break to form ‘foci’. This process plays a key role in DDR and is required for the recruitment of DNA repair proteins, such as 53BP1, NBS1, and MDC1 to the sites of damage as well as for the activation of checkpoint proteins which arrest the cell cycle progression[2]. Once DNA lesions are completely repaired, DDR foci are disassembled and cells quickly resume normal proliferation. In contrast, severe or irreparable DNA damage induce more protracted DDR signalling, increase gamma(γ)-H2AX foci spreading and eventually cells may undergo apoptosis or cellular senescence to prevent transmission of the lesions to the daughter cells upon cell division[3]. The factors responsible for this differential outcome are still unclear, but the dose and duration of exposure to DNA damaging agents as well as the cell types are likely to be crucial determinants[3, 4].
In a recent study in our lab, we have found that breast cancer cell lines as well as immortalized MEFs can be induced to undergo senescence by DNA-damaging agents including epirubicin and γ-irradiation. Interestingly, the dose of DNA damaging agents required for triggering senescent phenotypes is a much lower dose than that required for induction of apoptosis[5, 6]. Therefore, the emerging knowledge about the pathways and the patterns of gene expression related to DNA damage-induced senescence in tumour cells may lead to more effective treatments for cancer patients with fewer side effects.
In this chapter, we will describe the four standard approaches for studying DNA damage response and their related drug resistance mechanisms, including the use of γ-H2AX and comet assays to monitor DNA damage and repair capacity as well as the use of clonogenic and senescence-associated β-Gal staining assays to assess long term cell survival following DNA damaging treatment. Finally, we also present examples of applying these methods to study the role of FOXM1 in DNA damage-induced senescence and epirubicinresponse.
2. Materials
2.1.Chemicals, Buffers and Equipment to be used in Comet Assay
1.Single-frosted glass microscope slides 70×26 mm and coverslips 24×40 mm
2.LMA: Agarose, Low Melting Point, Analytical Grade (Promega)
3.NMA: Agarose, LE, Analytical Grade (Promega)
4.Lysis buffer: 100 mM EDTA-Na2, 2.5 M NaCl, 10 mMTris-HCL, 250 mMNaOH, pH 10. Store at room temperature (RT). Before use, add immediately 1% v/v Triton X-100 and 10% v/v DMSO and store at 4°C.
5.Electrophoresis buffer: 250 mMNaOH, 1.5 mM EDTA-Na2, pH 13. Store at 4°C.
6.Neutralis[GAR1]ation buffer: 400 mMTris-HCL, pH 7.5. Sterilise by autoclaving and store at RT.
7.Phosphate Buffered Saline (PBS), pH 7.4
8.Vectashield mounting solution with DAPI (Vector Laboratories, Cat.#H-1200)
9.Large-bed gel electrophoresis boxes and Power pack
10. Fluorescence microscope.
11.Comet image analysis softwaresuch as CometScore software (Tritek Corp., USA) or Kinetic Imaging Komet assay software (Kinetic imaging, Liverpool, UK).
2.2. Chemicals, Buffers and Equipment to be used in γ-H2AX immunofluorescent staining
1. BD Falcon culture slides are used with four chambers each
2.Glass coverslips
2.PBS, pH 7.4
3.4% Paraformaldehyde (PFA) in PBS
4.0.2% Triton in PBS
5.Blocking solution: 5% goat serum. This should be kept on ice once made.
6.Primary Antibodies:Rabbit monoclonal anti-γ-H2AXSer139.Dilute in 0.2% goat
7.Secondary antibodies: Goat anti-rabbit Alexa Flour 488 (Molecular Probes, Cat.# A11034): dilute in PBS
8.Vectashield mounting solution with DAPI (Vector Laboratories, Cat.#H-1200)
10.Clear Nail Polish
11.Leica TCS SP5 confocal microscope, equipped with a 63X oil immersion objective
12. ImageJ, FociCounter or CellProfiler Image analysis software.
2.3Chemicals, Buffers and Equipment to Be Used for Clonogenic Assay
1.PBS
2.4% (v/v) paraformaldehyde (PFA) in PBS
3.0.5% (w/v)crystal violet in dH2O. Store in the dark at RT.
4.33% acetic acid
5.6-well plates
6.Pipettes
7.Tubes for dilution
8.Hemocytometer
9.Digital camera
10. Include also the plate reader Tecan here!
2.4 Chemicals, Buffers and Equipment to be used in β-galactosidase staining
1.PBS
2.Fixative solution: 2% (v/v) formaldehyde and 0.2% (v/v) glutaradehyde in PBS.
3.0.1 M citric acid (C6H8O7.H2O) in dH2O
4.0.2 M sodium phosphate (Na2HPO4) in dH2O
5.0.2 M citric acid/ sodium phosphate solution (100 mL): Mix 36.85 ml of 0.1 M citric acid solution with 63.15 ml of 0.2 M sodium phosphate solution. The pH of citric acid/ sodium phosphate solution must be adjusted to 6.0by adding either citric acid or sodium phosphate solution.
6.20 mg/ml X-gal solution in N-N-dimethylformamide (DMF);This solution should be prepared freshly or can be stored at -20°C for short term and should be protected from light. Always prepare and store in polypropylene or glass tubes.
7.50 mM potassium ferrocyanide and 50 mM potassium ferricyanide, these solutions should be stored at 4°C in the dark (cover the solution tubes with aluminium foil to protect from light).
8.5M NaCl
9.1M MgCl2
10.SA-β-gal staining solution: 40 mM citric acid /sodium phosphate solution, 150 mMNaCl, 2 mM MgCl2, 5mM potassium ferrocyanide, 5mM potassium ferricyanide and 1 mg/ml X-gal in dH2O. This solution must be prepared freshly just before staining.
11.6-well plates
2.5Chemicals, Buffers to be used in cell cultures
1.Cell lines: human breast carcinoma MCF-7 cells and epirubicin resistant MCF-7 breast carcinoma (MCF-7-EpiR) cells.
2.Complete medium (Dulbecco’s Modified Eagle’s Medium (DMEM), 10% foetal calf serum (FCS), 2 mM L-glutamine-PenStrep). Store at 4°C and warm to 37°C prior to use.
3.Trypsin-EDTA
4.PBS , pH 7.4. Store at RT after autoclaving.
5. DNA damaging agent: epirubicin (100 µM stock solution in the complete culture medium, store in the dark at 4°C)
3. Methods
3.1. Measurement of drug-induced DNA breaks using Comet Assay
The Comet assay, also known as the single cell gel electrophoresis assay, is a sensitive and simple method for detecting DNA strand breaks in individual eukaryotic cells. It is widely used in a broad variety of applications including bio-monitoring, genotoxicity assessment, and as a tool to investigate DNA damage and repair capacity in a wide range of tumour cells in response to DNA-damaging agents. In this assay, cells embedded in low melting point agarose on a microscope slide are lysed with a lysis buffer that removes cell membranes, cytoplasm and most of cellular proteins. When the leftover nucleoids are treated with high alkaline solution, the DNA supercoils start to unwind. During electrophoresis, the relaxed DNA breaks migrate towards the anode thereby forming a ‘comet’-like tail. The relative intensity of the tail reflects the frequency of damaged DNA breaks[7-9].
3.1.1Preparation of cell cultures and DNA damaging treatment: seed 2×106 of MCF-7 cells in 10 cm plates. 24h after, the cells are treated with a DNA damaging agent by replacing with the medium containing the desired concentration of the agent. Leave the plate in an incubator for an appropriate exposure time (see Note 1).
3.1.2 Slide preparation:
1.Prepare 1% NMA agarose in dH2O and 0.7% LMP agarose in PBS. Microwave until the agarose is fully melted. Place the 1% NMA agarose in a 55°C water bath and the LMP agarose in 37°C water bath.
2.Merge the microscope slides in a vertical staining jar containing melted 1% NMA agarose in water and wipe one side clean. Then, allow the slidesto dry overnight at room temperature.The slides must be dry before use.
3.Prepare the cell suspension for the 2ndlayer. After suitable drug exposure time, pellet cells by centrifuging at 1200 rpm for 5 min at 4°C (see Note 2). Resuspend cells to final concentration of ~ 2×106 cells/mL in complete medium and maintain at 4°C. Pipet 10 ul of resuspended cells (containing ~2×104 cells) onto the center of the slide. Then add 85 uL of 0.7% LMA on top of the cells and place a coverslip on top to spread the cells (avoid bubbles). The cells need to be well spread in order to measure the length of tail. Allow 2nd LMA layer to set by placing the slides in a tray on ice for10 min.
4.Prepare the 3rd LMA layer. Carefully remove the coverslip and add another 85ul of 0.7% LMA directly onto the cells and place the new coverslip on top. Again place the slide in a tray on ice for 10 min (see Note 3).
3.1.3Lysis: Carefully remove the coverslip and merge the control and treated slides in a vertical staining jar containing complete lysis mixture at 4°C. Incubate for 1h in the dark by covering with foilensuring that all slides are sufficiently covered.
3.1.4Alkaline treatment: Prior to electrophoresis, take the slides from the lysis buffer and remove all excess of buffer from slides by carefully blotting on tissue. Then, gently place slides to the gel shelf in the electrophoresis tank containing the prepared cold alkaline electrophoresis buffer (pH 13) to unwind the DNA supercoils. Ensure that all slides are sufficiently covered. Slides should be placed close together and the remaining free space should be filled with empty slides to prevent any movement. Incubate for 40 min in the dark (see Note 4).
3.1.5Electrophoresis: Start applying the electric current at 25V, ~300 mA for 20 min. This must be carried out on ice.
3.1.6Neutralisation: To stop electrophoresis, carefully remove slides from the electrophoresis tank and place on a horizontal slide rack. Gently rinse with neutralisation buffer for 5 min, drain, and repeat twice.
3.1.7DAPI Staining: Add 30 ul of mounting media with DAPI (Vector Laboratories, Cat.#H-1200) [GAR2]onto the surface of agarose of each slide and cover with a coverslip, avoiding bubbles. Slides should be analysed as soon as possible. For storage, keep slides in a humid dark chamber at 4°C until they are visualised (see Note 5).
3.1.8Analysis: Identify random fields containing comets under the microscope and score using computer image analysis. Programmes are designed to differentiate comet head from tail and to measure a variety of parameters including tail length, % DNA in tail and tail moment. These are calculated in different ways but essentially represent the product of tail length and relative tail intensity (Figure X.1).
3.2 Measuring DNA damage using γ-H2AX immunofluorescent staining
Phosphorylation of Histone 2A (gemma[GAR3]-H2AX) is one of the early events following DNA double strand breaks (DSB). ATR, ATM and DNA-PK are related DNA activated kinase responsible for this phosphorylation. γ-H2AX immunofluorescent staining is a method for quantification of γ-H2AX foci using a γ-H2AX specific antibody. This method can be used for in vivo studies monitoring patient response to irradiation, DNA damage-including chemotherapeutics, and for in vitro experiments with mammalian cells to study DNA damage and repair.
3.2.1Preparation of cell cultures and DNA damaging treatment: seed cells in four-well chamber slides at 20,000 - 40,000 cells/ well. After 24h, the cells are treated with a DNA damaging agent by replacing with complete medium containing the desired concentration of the agent. Leave the chamber slide in an incubator at 37°C for an appropriate exposure time (see Note 1).
3.2.2Fixation, permeabilisation and blocking: At the end of the incubation time, aspire the media and wash cells once with PBS. Fix cells by adding 250 μl of 4% PFA in each well and leave for 15 min at RT (see Note 6 and 7). Remove fixative[GAR4] solution and wash 3 times with PBS. Permeabilise with 250 μl of 0.2% Triton X-100 in PBS for 10 min at RT. Wash samples 3 times with PBS. Block the slides with 250 μl of 5% goat serum in PBS for 60 min at RT.
3.2.3Immunostaining: Add 250 µl of primary antibody anti-γ-H2AX Ser139 (Rabbit, monoclonal, 1:250 dilution) in 0.2% goat serum in PBS and incubate in a humidified chamber overnight at 4°C (or 60 min at RT). After incubation, wash samples 3 times with PBS. Add secondary antibody anti-rabbit Alexa Flour 488 (Molecular Probes, Cat.# A11034) at 1:500 dilution in PBS and incubate in the dark for 60 min at RT. Avoid exposing the secondary antibody to the light. After incubation, wash sample 3 times with PBS.
3.2.4Counterstaining and mounting: detach the chamber wells from the glass slide and add 1 drop of Vectorshield mounting medium with DAPI (Vector Laboratories Cat.#H-1200) to the center of each chamber (see Note 8). Gently place a cover slip on the slide avoiding air bubbles. Seal the edges of each coverslip with regular transparent nail polish and allow for it to dry for 5 min. Cover slides with foil and store at 4°C (see Note 9).
3.2.5Imaging and Image Analysis: capture image using a Leica TCS SP5 confocal microscope. Capture images from at least 100 cells and count the number of γ-H2AX foci manually by using Image J counting analysis. Alternatively, specific image analysis software can be used such as FociCounter, or CellProfiler cell image analysis software (see Note 10) (Figure X.2).
3.3 Clonogeniccell survival assay
Clonogenic assay is a useful long-term survival assay in cancer research laboratories. It is used to determine the ability of a single cell to proliferate and form a colony. The colony is defined as a group of at least 50 cells, which can be counted under a microscope. This method is now widely used to examine the effectiveness of anticancer cytotoxic agents on colony forming ability in several cancer cell lines. Before and after exposing to the treatment, a small number of cells are seeded onto plates containing different concentrations of the drug and are allowed to form colonies for 14-21 days. A final cell survival rate is referred to a relationship between the dose of the agent used to produce an insult and the fraction of cells retaining their ability to proliferate [10, 11].
3.3.1 Plating:
1.Trypsinise and collect cells in medium containing 10% foetal calf serum. Centrifuge the cell suspension at 1200 rpm for 4 min to pellets cells and resuspend in fresh medium.
2.Count the cells by using a hemocytometer (see Note 12).
3.Dilute the cell suspension into the appropriate seeding concentration (low densities of cells) and seeded equally into 6-well plate in a total volume of 2ml medium, at least in duplicate (see Note 12). Number of cells seeded per well depends on cell type and its growth rate as well as the toxicity of the treatment. For example: MCF-7 cells are seeded at a density of 1000-2000 cells/well in a six-well plate. Cells are then incubated at 37⁰C and 5% CO2 for 24h to allow the cells to attach to the plate prior to drug treatment.
3.3.2Treatment with DNA damaging agents:
1.After attachment of the cells to the plate surface, they are treated with different concentrations of DNA damaging agent. The drug concentration range should be varied and include higher concentrationsthat kill most of the cells as well as the lowest concentration that kills none of the cells. Leave the plate in an incubator at 5% CO2 for the appropriate exposure time, which depends on the cell type and the toxicity of the treatment. For example, MCF-7 cells are treated with epirubicin (concentration range from 0-10nM) for 48 hours (see Note 1).
2.After the appropriate exposure time, DNA damaging agent is removed byrinsing the cells with PBS and replacing with 2ml fresh medium.
3.Plates are left to incubate at 37ºC in 5% CO2 for 7 – 20 days or until cells in control (untreated) plates have formed sufficiently large clones consisting of 50 or more cells. Monitoring is essential to prevent colonies from merging together (see Note 13). In the example, the control wells for MCF-7 cells requires 9 days to form sufficiently large clones consisting of 50 or more cells.
3.3.3Fixation and Staining:
1.Following incubation, remove the medium from the cells and wash with PBS.
2.Fix the cells in each well with 1ml of 4% PFA for 15 minutes at RT.
3.Wash 3 times with PBS
4.Add 1ml of 0.5% (w/v) crystal violet (diluted in dH2O) for staining and incubate for 1 hour at RT.
5.Remove crystal violet gently and immerse the plates in tap water to remove excess crystal violet.
6.Leave the plates with colonies to dry overnight at RT.
3.3.4Colony Counting and Assay Analysis:
1.Digital images of the colonies (at least 5 random fields) are obtained using a camera. Adjust the microscope and lighting to avoid shadowing on the edges of the images. Do not change the focus and light settings because the image should have the same relative background throughout (Figure X.3A).
2.Colonies are counted using imaging analysis software packages such as imageJ.Average the colony counts (5 random fields) for each condition and divide the mean by the number of cell seeded. This will give the plating efficiency(PE)[10-12].