DNA damage in cells is inevitable. It has been estimated that up to one million DNA changes occur per cell per day in response to environmental changes and byproducts of normal metabolism.(1) If not repaired, lesions in critical genes such as tumour suppressors can impede a cell’s normal functions and increase the likelihood of tumour formation, as in the case of skin cancer.
Genotoxicity describes the capacity of chemical agents to cause DNA damage within a cell, leading to mutations and potentially cancer. Genotoxicity tests are routinely used in the pharmaceutical industry to determine whether a pharmaceutical compound induces genetic damage, which can cause a wide range of problems including cancer and inherited birth defects.
A cell’s response to DNA damage involves many complex pathways and mechanisms, collectively called the DNA damage response. Once initiated, these pathways ultimately lead to repair of the DNA damage or initiation of apoptosis. The DNA damage response plays a crucial role in maintaining the function, genomic stability and viability of the cell and organism at large. Dysfunctions in the DNA damage response are implicated in many disease states, including cancer, premature aging, tissue toxicity and neurodegenerative disease.
FDA regulations require testing drug candidates for safety, efficacy, pharmacokinetics, toxicology, carcinogenicity and genotoxicity. The International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) guidelines on genotoxicity testing recommend two in vitro assays (such as the Ames test and the comet assay) and one in vivo assay (such as a micronucleus test (MNT)). These are the gold standards for genotoxicity testing and are offered as assay services from various companies. In addition, cell-based assays, such as the ATAD5 assay and high content assay/screening (HCA/HCS) services are also readily available.(2) Although these assays are broadly used to determine if a drug is genotoxic, they provide limited mechanistic understanding of the cellular response to genotoxic
compounds. To meet this need for mechanistic understanding of drug induced DNA damage response, a high-throughput assay to elucidate pathway changes within cells can be useful.
Signaling molecules in the DNA damage/genotoxicity pathway are regulated by phosphorylation, and understanding the role of this pathway requires the ability to simultaneously measure the phosphorylation status of multiple protein targets. Several assays to examine phosphorylation status are currently available, including Western blotting, ELISA, reverse phase arrays, quantitative cell imaging and mass spectrometry. Although some of these platforms yield absolute, quasi-quantitative data, the assays are either limited to measuring only one analyte at a time, or are excessively difficult or expensive. In recent years, bead-based multiplex assays, such as those using Luminex xMAP® technology, have enabled the high-throughput measurement of phosphorylation levels of multiple proteins simultaneously, which give the advantages of reduced sample volume, time and cost compared to traditional methods.
The MILLIPLEX® MAP DNA Damage/Genotoxicity Kit (EMD Millipore, Cat. No. 621MAG) is a magnetic bead-based immunoassay that simultaneously detects seven proteins in the DNA damage/genotoxicity pathway in a single sample, enabling the measurement of phosphorylation changes (Figure 1). This article demonstrates the utility of this assay in the analysis of DNA damage/genotoxicity in two cancer cell lines, HepG2 and HEK293. All analytes were detected with strong specificity, sensitivity and precision. In addition, genotoxic compound screening shows the utility of this kit in drug discovery and development research.
Methods
Tissue Culture
HepG2 and HEK293 cells were cultured according to ATCC® guidelines in recommended media. Cells were plated at 50,000 cells per well in a 96-well plate. Twenty-four hours after plating, cells were treated with fresh complete media. After another 24 hours, cells were treated with designated genotoxic and nongenotoxic compounds (listed in Table 1) for a predetermined time period.
Sample Preparation
Immediately prior to harvest, media were collected and centrifuged. Cells were lysed, and the samples were collected and incubated with gentle
rocking and centrifuged. Lysate supernatants were transferred into new tubes. Protein concentration in untreated samples was determined by a bicinchoninic acid (BCA) assay. Using unstimulated sample protein concentration as an estimator, samples were diluted in assay buffer to provide a concentration of approximately 20 μg/well of a 96-well plate. Signals from the compound screening studies were all normalized to
β-Tubulin signal using the β-Tubulin MAPmate™ assay (EMD Millipore, Cat. No. 46-713MAG).
Microspheres
We developed the MILLIPLEX® MAP 7-plex DNA Damage/Genotoxicity Kit by conjugating specific capture antibodies to magnetic microsphere
beads purchased from Luminex Corporation. Each set of beads is distinguished by different ratios of two internal dyes, yielding a unique fluorescent signature to each bead set. Capture antibodies were covalently coupled to the carboxylate-modified magnetic microsphere beads.
Immunoassay Protocol
The multiplex assay was performed in a 96-well plate according to product instructions. The plate was first rinsed with 100 μL assay buffer. 25μL of controls and samples and 25 of μL beads were added to each well. Plates were incubated overnight at 4°C. Beads were washed twice with assay
buffer, and then incubated for one hour at room temperature with the biotinylated detection antibody cocktail. The detection antibody cocktail was replaced with 25 μL of streptavidinphycoerythrin (SAPE) and incubated for 15 minutes. 25 μL of amplifi cation buffer was added and incubated for another 15 minutes. Then the SAPE/amplifi cation buffer was removed and beads were resuspended in 150 of μL assay buffer. The assay plate was read and analyzed in a Luminex 200™ system, a compact unit consisting of an analyzer, a computer and software.
Results and Discussion
The multiplex assay enabled the detection of phosphorylated Chk1, Chk2, H2A.X and p53, and total ATR, MDM2 and p21 with strong specificity, sensitivity and precision (Figure 2). The assay provided high specificity, indicated by the detection of proteins at the expected molecular weights as shown by immunoprecipitation/Western blot (Figure 2A). In addition, demonstrations of high signal-to-noise ratios (data not shown), sample linearity (Figure 2B) and precision (Figure 2C) lent support to the assay’s robustness. In addition, all analytes could be detected in human cell lines and tissues.
Changes in levels of phosphorylated Chk1, Chk2, H2A.X and p53, and total ATR, MDM2 and p21 were tested in HepG2 and
HEK293 cells treated with genotoxic and nongenotoxic carcinogens (Table 1). Changes in the DNA damage response were detected in a
dose- (Figure 3) and time dependent (Figure 4) manner.
Because the panel enabled the simultaneous measurement of multiple proteins, we could distinguish the varying effects of compounds that exerted their genotoxicity through varying mechanisms, as has been reported using gene expression profiling.(3) For example, treatment with compounds that caused DNA double-strand breaks, such as ETO, HQU and CIS, resulted in greatly increased phosphorylation of the cell cycle regulating kinases Chk1 and Chk2, indicating activation of checkpoint-mediated pathways (Figure 3).(4,5)
Compounds that exerted genotoxic effects through other means, such as the microtubule-binding agent TAX or the DNA alkylators ENU and MMS, showed different patterns of pathway activation. Dose response data for ENU, MMS and TAX, for example, showed less dramatic phosphorylation of the Chk kinases accompanied by phosphorylation of p53 or histone H2A.X (Figure 3). The p53 and histone H2A.X proteins
are important players in the DNA repair pathway and can be phosphorylated in response to multiple types of DNA damage.
Measurement of time-dependent DNA damage response (Figure 4) also revealed differences in mechanism between different genotoxic
compounds. While the double-strand break-inducers, ETO and CIS, caused increasing phosphorylation of Chk1, Chk2, p53 and histone H2A.X with respect to time, the DNA alkylator, MMS, caused an initial spike in Chk kinase and histone H2A.X phosphorylation that then diminished over time, but was accompanied by increased activation of p53. Again, this pattern may indicate initial checkpoint activation, which cells might have overcome, but was followed by checkpoint-independent DNA damage response.
As expected, LIM and DIA (nongenotoxic carcinogens) did not result in any signifi cant changes in the analytes. Also as expected, little effect was seen with any of the compounds on total ATR, except at high doses of compounds that may have caused a general decline in cell health.
Finally, cell toxicity was not observed in HEK293 cells (Figure 5) using the MILLIPLEX® MAP Human Kidney Toxicity Panel 2 (EMD Millipore, Cat. No. RKTX2MAG-37K), as shown by the absence of any increase in the measured analytes with respect to time. These studies demonstrate that the DNA damage response is complex and involves more than the dysregulation of a single pathway.(6) This conclusion further underscores the importance of simultaneous measurement of multiple phosphoprotein targets and demonstrates the utility of a new magnetic bead-based immunoassay in elucidating the mechanism of action for DNA damaging compounds.
References
1. Lodish H et al. Molecular Biology of the Cell, 5th Edition. New York (NY): Freeman. (US); 2004.
2. Fox JT et al. High-throughput genotoxicity assay identifi es antioxidants as inducers of DNA damage response and cell death. Proc Natl Acad Sci U S A. 2012 Apr 3;109(14):5423-8.
3. Boehme K et al. Genomic profiling uncovers a molecular pattern for toxicological characterization of mutagens and promutagens in vitro. ToxicolSci. 2011 Jul;122(1):185-97.
4. Niida H, Nakanishi M. DNA damage checkpoints in mammals. Mutagenesis. 2006 Jan;21(1):3-9.
5. Zhou BB, Bartek J. Targeting the checkpoint kinases: chemosensitization versus chemoprotection. Nat Rev Cancer. 2004 Mar;4(3):216-25.
6. Kastan MB. DNA damage responses: mechanisms and roles in human disease: 2007 G.H.A. Clowes
About the author: Joseph Hwang, Ph.D., Senior Research Scientist, Cell Signaling Group, EMD Millipore Corporation, EMAIL: [email protected] Dr. Hwang develops new MILLIPLEX® MAP multiplex kits and single plex MAPmate™ assays for the Luminex xMAP® magnetic bead-based platform. He has more than 20 years of experience in cell signaling research, primarily in the area of insulin signaling.
