An Integrated Approach for Analysis of the DNA Damage Response in Mammalian Cells: NUCLEOTIDE EXCISION REPAIR, DNA DAMAGE CHECKPOINT, AND APOPTOSIS

Abstract

DNA damage by UV and UV-mimetic agents elicits a set of inter-related responses in mammalian cells, including DNA repair, DNA damage checkpoints, and apoptosis. Conventionally, these responses are analyzed separately using different methodologies. Here we describe a unified approach that is capable of quantifying all three responses in parallel using lysates from the same population of cells. We show that a highly sensitive in vivo excision repair assay is capable of detecting nucleotide excision repair of a wide spectrum of DNA lesions (UV damage, chemical carcinogens, and chemotherapeutic drugs) within minutes of damage induction. This method therefore allows for a real-time measure of nucleotide excision repair activity that can be monitored in conjunction with other components of the DNA damage response, including DNA damage checkpoint and apoptotic signaling. This approach therefore provides a convenient and reliable platform for simultaneously examining multiple aspects of the DNA damage response in a single population of cells that can be applied for a diverse array of carcinogenic and chemotherapeutic agents.

Keywords: DNA damage; DNA damage checkpoint; DNA damage response; DNA repair; apoptosis; carcinogenesis; cell signaling; checkpoint control; chemotherapy; nucleotide excision repair.

FIGURE 1.

An integrated approach for monitoring the repair of genomic DNA damage, the generation of excised oligonucleotide repair products, and the activation of DNA damage signaling pathways in a single culture of cells. Following the exposure of cells to DNA-damaging agents, cells are lysed with a non-ionic detergent and soluble and insoluble fractions separated by centrifugation. The insoluble pellet is subjected to extraction of genomic DNA to measure the presence of lesions via immuno-slot blot assay. The soluble cell lysate is processed for extraction and visualization of the oligonucleotide products of excision repair or used in immunoblot assays for monitoring DNA damage response signaling (checkpoint and apoptosis).

FIGURE 2.

Analysis of genomic DNA, excised oligonucleotide repair products, and DNA damage checkpoint signaling in UV-irradiated cells. A, analysis of excised oligomers released from genomic DNA following UV irradiation. HeLa cells were exposed to 10 J/m² of UV (254 nm) light and harvested at the indicated time points. Following cell lysis and centrifugation the soluble fractions of the cells were processed for isolation and labeling of excised oligonucleotide repair products. To measure the total excised oligomers, the purified small DNA molecules were directly labeled with biotin, separated by gel electrophoresis, transferred to a membrane, and then detected with HRP-conjugated streptavidin (left). For detection of excision products containing (6–4)PPs or CPDs, the purified small DNA molecules were immunoprecipitated with anti-(6–4)PP (middle) or anti-CPD (right) antibodies prior to biotinylation. Data are presented as the mean values ± S.D. obtained from at least three independent experiments. B, immuno-slot blot assay for measuring the removal of photoproducts from genomic DNA. Genomic DNA was isolated from the insoluble pellets in A and then used in immuno-slot blot assays with antibodies against (6–4)PPs and CPDs. The Sybr-Gold staining shows equal loading of total genomic DNA. A quantitative analysis of immuno-slot blot assays in which the signal at time zero was set to 100 is also shown. The average (and S.D.) level of photoproduct removal from three independent experiments is shown. C, analysis of UV-induced DNA damage checkpoint signaling. A small portion of the soluble fractions prepared in A were analyzed by SDS-PAGE and immunoblotting with the indicated antibodies.

FIGURE 3.

Detection of excised oligomers containing bulky base adducts in vivo. A, HeLa cells were treated with different concentrations of BPDE (0.01–10 μm), harvested 1 h later, and then processed for analysis of excision products and DNA damage checkpoint signaling. For UV-induced excision products, cells were exposed to 10 J/m² of UV and harvested after 1 h of incubation. Immunoblotting was performed with antibodies against the indicated proteins (bottom). B, CHO wild-type (CHO-WT) and XPG-deficient (CHO-XPG) cells were treated with different concentrations of BPDE (0.05, 0.1, 0.5, 1, 5 μm) for 1 h and then analyzed for excision and DNA damage checkpoint signaling. C, A375 cells were treated for 1 h with BPDE (0, 1.25, 2.5 μm) and analyzed for excision repair. For detection of UV-induced excision products, cells were exposed to 10 J/m² of UV and then harvested 1 h later. D, normal human fibroblasts (GM00200 cells) were treated for 1 h with BPDE (0, 0.25, 0.5, 1 μm) and analyzed for excision. E, HeLa cells were treated with AAF (1–10 μm) for 1 h and analyzed for excised oligonucleotides. Lane 6 represents the excised oligonucleotides generated in HeLa cells exposed to 10 J/m² of UV and then harvested 1 h later.

FIGURE 4.

Time-course analysis of excision generation following BPDE treatment. A, HeLa cells were treated with 1 μm of BPDE, harvested at the indicated time points, and then processed for analysis of excised oligonucleotides. Immunoblotting was performed with antibodies against the indicated proteins (bottom). B, quantitative analysis (average and standard deviation) of three independent experiments performed as described in A. The highest BPDE-excision product signal was arbitrarily set to a value of 100 for normalization of the signals from other time points.

FIGURE 5.

Examination of excision repair products induced by cisplatin. A, HeLa cells were treated for 4 h with cisplatin (0.1, 0.3, 0.6, 0.9 mm) and then analyzed for excision repair. Lane 1 shows the excised oligonucleotides that were generated in HeLa cells following exposure to 5 J/m² of UV and then harvested after 30 min incubation. Immunoblotting was performed with antibodies against the indicated proteins (bottom). B, quantitative analysis (average and standard deviation) of three independent experiments as shown in A. C, human melanoma cells (A375) were treated with different concentrations of cisplatin, harvested 4 h later, and then analyzed for excision products. D, CHO wild-type (CHO-WT) and XPG-deficient (CHO-XPG) cells were treated with different concentrations of cisplatin (0.1, 0.3, 0.6, 0.9 mm), harvested 4 h later, and then processed for analysis of excision repair. Small portions of cell lysates were analyzed by immunoblotting with antibodies against the indicated proteins (bottom). E, HeLa cells were treated with cisplatin (0.6 mm), harvested at the indicated time points, and then analyzed for excision products. Small portions of cell lysates were analyzed by immunoblotting with antibodies against the indicated proteins (bottom). F, quantitative analysis (average and standard deviation) of three independent assays as shown in E.

FIGURE 6.

Detection of excised oligonucleotides following treatment with the anti-cancer compounds mitomycin C (MMC) and bleomycin. A, HeLa cells were treated with the indicated concentrations of mitomycin C (1, 10, 30, 100 μm), harvested after 1 h incubation, and then analyzed for the generation of excised oligonucleotides. Immunoblotting was performed with antibodies against the indicated proteins (bottom). B, HeLa cells were treated with bleomycin (0.7, 3, 17, 83 μm) for 1 h and then processed for detection of excised oligonucleotides. Lane 7 in A and B show excision repair products generated within 1 h after the exposure HeLa cells to 5 J/m² of UV. Immunoblotting was performed with antibodies against the indicated proteins (bottom).

FIGURE 7.

Detection of excision repair products following treatment with formaldehyde. A, HeLa cells were treated with various concentrations of formaldehyde (0.1, 0.2, 0.4, 0.6, 0.8, 1 mm), harvested after 3 h incubation, and then analyzed for the generation of excision products. Immunoblotting was performed with antibodies against the indicated proteins (bottom). B, quantitative analysis (average and standard deviation) of three independent experiments as shown in A. C, detection of excised oligonucleotides in A375 cells treated for 3 h with formaldehyde at different concentrations (0.2, 0.4, 0.6, 0.8 mm). Lane 1 shows the generation of repair products 1 h after exposure of HeLa cells to 5 J/m² of UV. D, GM00200 cells were treated at different concentrations (0.2, 0.4, 0.6, 0.8 mm), harvested 3 h later, and analyzed for excised oligomers. E, HeLa cells were treated with formaldehyde (0.4 mm), harvested at the indicated time points, and then analyzed for excision repair products. Lane 8 shows the excised oligonucleotides induced 1 h following exposure to 5 J/m² of UV. F, quantitative analysis (average and standard deviation) of three independent experiments as shown in E.