Signaling molecules in the genotoxicity and DNA damage pathway are regulated by phosphorylation. Understanding the role of this pathway requires the ability to simultaneously measure the phosphorylation status of multiple protein targets. Bead-based multiplex assays, such as MILLIPLEX® multiplex assays using Luminex® xMAP® technology, enable the high-throughput measurement of phosphorylation levels of multiple proteins simultaneously and reduce sample volume, time, and cost, compared to traditional methods.
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 insults and byproducts of normal metabolism.1 If not repaired, the lesions in critical genes (such as tumor suppressor genes) can impede the normal functions of a cell and increase the likelihood of tumor formation, as in the case of skin cancer. Similarly, 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 the repair of the DNA damage or the 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 Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) guidelines on genotoxicity testing methods 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 a limited mechanistic understanding of the cellular response to genotoxic compounds. To meet this need for a mechanistic understanding of drug-induced DNA damage response, a high-throughput assay to elucidate pathway changes within cells can be of high utility.
Signaling molecules in the DNA damage/genotoxicity pathway are coordinately 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 MILLIPLEX® multiplex assays 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.
Multiplexing the DNA damage and genotoxicity pathway allows researchers to easily measure multiple proteins at once. The MILLIPLEX® DNA Damage/Genotoxicity Multiplex Kit 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 in this pathway (Figure 1).
MILLIPLEX® DNA Damage/Genotoxicity Kit Analytes:
Figure 1.Simplified schematic showing the DNA damage/genotoxicity pathway. Analytes detected using the MILLIPLEX® DNA Damage/Genotoxicity multiplex kit are highlighted in the schematic.
The utility of this assay was demonstrated in the analysis of DNA damage and genotoxicity in two cancer cell lines: HepG2 and HEK293. All analytes were detected with good specificity, sensitivity, and precision. In addition, genotoxic compound screening shows the utility of this kit in drug discovery and development research.
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.
Immediately prior to harvest, media were collected and centrifuged (10,000 x g for 10 minutes at 4 °C). Cells were lysed and samples collected according to the MILLIPLEX® DNA Damage/Genotoxicity Kit protocol. Samples were then incubated with gentle rocking at 4°C for 15 minutes and centrifuged (10,000 x g for 10 minutes at 4 °C). Lysate supernatants were transferred into new tubes. Protein concentration in untreated samples was determined by 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.
The MILLIPLEX® DNA Damage/Genotoxicity multiplex kit was developed 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.
The multiplex assay was performed in a 96-well plate according to product instructions. The plate was first rinsed with 100 μL assay buffer. 25 μL of controls and samples and 25 μL beads were added to each well. Plates were incubated overnight at 4°C (alternatively, the plates may be incubated for 2 hours at room temperature [RT]). Beads were washed twice with assay buffer and then incubated for 1 hour at RT with the biotinylated detection antibody cocktail. The detection antibody cocktail was replaced with 25 μL streptavidin-phycoerythrin (SAPE) and incubated for 15 minutes at RT. 25 μL of amplification buffer was added and incubated for another 15 minutes at RT. Then, the SAPE/amplification buffer was removed, and beads were resuspended in 150 μL assay buffer. The assay plate was read and analyzed in a Luminex® 200™ system. This is a compact unit consisting of an analyzer, a computer, and software (Luminex® Corporation, Austin, TX).
The MILLIPLEX® DNA Damage/Genotoxicity multiplex kit enabled the detection of phosphorylated Chk1, Chk2, H2A.X, and p53, and total ATR, MDM2, and p21 with good specificity, sensitivity, and precision. 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 (Table 2) lent support to the robustness of this kit. All analytes in the MILLIPLEX® DNA Damage/Genotoxicity Kit could be detected in human cell lines and tissues (data not shown).
Figure 2.Specificity and sensitivity of the MILLIPLEX® DNA Damage/Genotoxicity Kit. Phosphorylated proteins were simultaneously detected in HeLa, Jurkat, and A549 cells. (2A) Immunoprecipitation (IP) of proteins was performed with capture beads and detected by Western blotting with the biotinylated detection antibodies. Lanes correspond to: (1) untreated HeLa cell lysate, (2) camptothecin-treated A549 cell lysate, and (3) anisomycin-treated Jurkat cell lysate. (2B) Lysate titrations were performed on Jurkat cells treated with 25 μM anisomycin (4 hours) and A549 cells treated with 5 μM camptothecin (overnight). The signal is represented as Median Fluorescent Intensity (MFI).
Using the DNA Damage/Genotoxicity Kit, 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, the varying effects of compounds that exerted their genotoxicity through various mechanisms were distinguished, 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
On the other hand, 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 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.
Figure 3.Dose response in HepG2 and HEK293 cells. DNA Damage/Genotoxicity Multiplex Panel analytes were detected in HepG2 (3A) and HEK293 (3B) cells treated with genotoxic and non-genotoxic compounds ranging in concentration from 1 μM to 1 mM (on diagram, decreasing from left to right; 0.01 μM to 10 μM for TAX) for 48 hours. Median Fluorescent Intensities (MFI) were normalized to β-Tubulin and reported as fold change over the untreated control.
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 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.
Figure 4.Time course in HepG2 and HEK293 cells. DNA Damage/Genotoxicity Panel analytes were detected in HepG2 (4A) and HEK293 (4B) cells treated with genotoxic and non-genotoxic compounds for 0, 0.5, 6, 16, and 24 hours. Median Fluorescent Intensities (MFI) were normalized to β-Tubulin and reported as fold change over the untreated control.
As expected, LIM and DIA (non-genotoxic carcinogens) did not result in any significant 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.
No significant changes in apoptosis were detected (data not shown) using the MILLIPLEX® Early Apoptosis Kit. Cell toxicity was also not observed in HEK293 cells (Figure 5) using the MILLIPLEX® Human Kidney Injury Panel 6, as shown by the absence of any increase in the measured analytes with respect to time.
Figure 5.Analytes were detected in HEK293 cells treated with 1% DMSO, non-genotoxic compound (DIA), or genotoxic compounds (CIS and ETO) for 0, 0.5, 6, 16, and 24 hours. Analyte protein concentrations are reported as fold change over the untreated control.
These studies demonstrate 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 the MILLIPLEX® DNA Damage/Genotoxicity Kit in elucidating the mechanism of action of DNA damaging and genotoxic compounds.
As shown by the DNA damage and genotoxicity data above, the complexity and number of protein targets involved in signaling events, as well as cellular responses, are best addressed using multiplexed analysis of samples to achieve a complete, accurate picture of a signaling network. Our MILLIPLEX® cell signaling multiplex assays enable the analysis of a greater number of intracellular analytes per well, saving valuable time and resources. Flexible assay formats include preconfigured multiplex kits, as well as singleplex MAPmate™ assays, which can be mixed and matched to meet individual needs.
MILLIPLEX® cell signaling multiplex assays offer: