Base and nucleotide excision repair facilitate resolution of platinum drugs-induced transcription blockage

Abstract

Sensitivity and resistance of cells to platinum drug chemotherapy are to a large extent determined by activity of the DNA damage response (DDR). Combining chemotherapy with inhibition of specific DDR pathways could therefore improve treatment efficacy. Multiple DDR pathways have been implicated in removal of platinum-DNA lesions, but it is unclear which exact pathways are most important to cellular platinum drug resistance. Here, we used CRISPR/Cas9 screening to identify DDR proteins that protect colorectal cancer cells against the clinically applied platinum drug oxaliplatin. We find that besides the expected homologous recombination, Fanconi anemia and translesion synthesis pathways, in particular also transcription-coupled nucleotide excision repair (TC-NER) and base excision repair (BER) protect against platinum-induced cytotoxicity. Both repair pathways are required to overcome oxaliplatin- and cisplatin-induced transcription arrest. In addition to the generation of DNA crosslinks, exposure to platinum drugs leads to reactive oxygen species production that induces oxidative DNA lesions, explaining the requirement for BER. Our findings highlight the importance of transcriptional integrity in cells exposed to platinum drugs and suggest that both TC-NER and BER should be considered as targets for novel combinatorial treatment strategies.

Figure 1.

Oxaliplatin sensitivity of DLD-1 cells with CRISPR-induced loss-of-function mutations in DNA repair genes. Each dot represents a cell line stably expressing a unique sgRNA targeting a DNA repair gene as listed in Supplementary Table S1. Responses to an IC₂₀ concentration of oxaliplatin (15 μM) of all cell lines were evaluated by MTT assay in two independent screens and expressed as percentage cell death of treated versus untreated cells. Top candidate genes are represented by distinct colors corresponding to their main involvement in different DNA repair pathways (nucleotide and base excision repair, NER and BER; Fanconi anemia, FA; homologous recombination, HR; translesion synthesis, TLS). Most top hits were identified by one of two sgRNAs, except XPF, FANCA and RAD51, which are represented by both sgRNAs. Wild type cells are indicated by the star.

Figure 2.

Cells deficient in transcription-coupled NER are sensitive to platinum drugs. (A) Immunoblot showing loss of XPF, CSA and CSB protein expression in stable knockout (KO) DLD-1 cells as compared to wild type (WT) cells. Ku70 and Tubulin were used as loading controls. (B) MTT assay showing sensitivity of NER-deficient KO cells to 15 μM oxaliplatin. (C) MTT assay showing sensitivity of NER-deficient KO cells to 10 μM cisplatin. Cell viability in (B) and (C) was measured 3 days after drug treatment. Bars denote mean and SEM of 2 independent experiments each performed in triplicate. (D) Response of NER-proficient human C5RO fibroblasts (WT), TC-NER-deficient CS1AN fibroblasts (CSB-deficient) and GG-NER-deficient XP4PA fibroblasts (XPC-deficient) to UV-C irradiation and (E) oxaliplatin. Each experimental point represents mean and SEM of 3 (D) or 6 (E) independent experiments, each performed in triplicate. For all panels, *P < 0.05; **P < 0.01

Figure 3.

Transcription-coupled NER is required for transcription recovery in platinum-treated cells. (A) Recovery of RNA synthesis (RRS), as determined by quantification of EU incorporation at the indicated time points, in DLD-1 wild type (WT) and CSA knockout (CSA KO) cells after 2 h treatment with 100 μM of oxaliplatin. (B) Representative pictures of the oxaliplatin RRS. (C) RRS in DLD-1 WT and CSA KO cells after 2 h treatment with 100 μM of cisplatin. (D) Representative pictures of the cisplatin RRS. Bars represent mean EU signal and SEM of >100 cells from two independent experiments normalized to untreated control for each cell line, set at 100%. For all panels, *P < 0.05; **P < 0.01

Figure 4.

CSB is recruited to chromatin in platinum-treated cells in a transcription-dependent manner. (A) Fluorescence recovery after photobleaching (FRAP) analysis of YFP-CSB^del, showing reduced mobility after 15 J/m² UV-C or 200 μM cisplatin (cis; applied for 6 h), depending on transcription which was blocked by RNA polymerase II inhibitor (RNAP2i) flavopiridol (1 μM applied for 1 h). The reduced mobility represents the fraction of CSB bound to chromatin and involved in active DNA repair. (B) Percentage immobile fraction, i.e. chromatin-bound fraction, of CSB^del after DNA damage induction as determined by FRAP analyses such as shown in (A). Cells were irradiated with 15 J/m² UV-C or treated for 6 h with either 100 or 200 μM oxaliplatin (oxa) or cisplatin (cis) without or with 1 h pre-treatment with 1 μM RNA polymerase II inhibitor (RNAP2i) flavopiridol, as indicated. Bars represent mean and SEM of at least 24 cells analyzed in 2 independent experiments. (C) Immunofluorescence showing localization of FANCD2, XPA and YFP-CSB^del to sites of intra- and interstrand crosslinks (visualized by γH2AX staining), introduced by UVA-laser activation of 8-methoxypsoralen (50 μM). For all panels, *P < 0.05; **P < 0.01

Figure 5.

Base excision repair helps to resolve platinum-induced transcription blockage. (A) Immunoblot showing loss of POLB, PARP1 and XRCC1 protein expression in stable knockout (KO) DLD-1 cells as compared to wild type (WT) cells. Ku70 and Tubulin were used as loading controls. (B) MTT assay showing sensitivity of BER-deficient KO cells to 15 μM oxaliplatin. (C) MTT assay showing sensitivity of BER-deficient KO cells to 10 μM cisplatin. (D and E) MTT assays showing sensitivity of DLD-1 cells treated with 10 μM of PARP inhibitor olaparib (PARPi) or 500 μM POLB inhibitor pamoic acid (POLBi) to 15 μM oxaliplatin or 10 μM cisplatin, respectively. Cell viability in (B-E) was measured 3 days after drug treatment. Bars denote mean and SEM of 2 independent experiments each performed in triplicate. (F) Recovery of RNA synthesis (RRS) in DLD-1 wild type (WT) and BER-deficient cells, either by POLB knockout (KO) or by application of the POLB inhibitor pamoic acid (POLBi). EU incorporation was quantified at the indicated time points following 2 h treatment with 100 μM of oxaliplatin. (G) Representative pictures of the oxaliplatin RRS. (H) RRS in DLD-1 WT and BER-deficient cells exposed to 100 μM of cisplatin for 2 h. (I) Representative pictures of the cisplatin RRS. Bars represent mean EU signal and SEM of >100 cells from 2 independent experiments normalized to untreated control for each cell line, set at 100%. Figures show representative RRS pictures. For all panels, * P < 0.05; ** P < 0.01

Figure 6.

XRCC1 chromatin retention upon platinum treatment is partially dependent on transcription. (A) Percentage XRCC1-YFP immobile fraction, i.e. chromatin-bound fraction, as determined by FRAP in MRC-5 cells, after treatment for 5 min with 10 mM hydrogen peroxide (H₂O₂), 6 h with 400 μM oxaliplatin (oxa), 6 h with 200 μM cisplatin (cis) without or with 1 h pre-treatment with 1 μM RNA Polymerase II inhibitor (RNAP2i) flavopiridol, as indicated. Bars represent mean and SEM of at least 24 cells analyzed in two independent experiments. (B) Percentage XRCC1-YFP immobile fraction determined by FRAP after 6 h exposure to 400 μM oxaliplatin of MRC-5 cells treated with control siRNA (siCTRL) and siRNA against XPA (siXPA) and OGG1 (siOGG1). Bars represent mean and SEM of at least 24 cells analyzed in 2 independent experiments. (C) Induction of reactive oxygen species (ROS) by 10 mM H₂O₂ applied for 5 min, and 100 μM oxaliplatin or cisplatin applied for 6 h in VH10 cells, as measured by H₂DCFDA fluorescence. Figure shows representative confocal pictures and quantification of fluorescence signal of >100 cells from 2 independent experiments represented by the mean and SEM. (D) Percentage XRCC1-YFP immobile fraction, as determined by FRAP in MRC-5 cells, after treatment for 5 min with 10 mM H₂O₂, 6 h with 400 μM oxaliplatin (oxa) or 6 h with 200 μM cisplatin (cis) without or with 24 h pre-treatment with 600 μM trolox, as indicated. Bars represent mean and SEM of at least 24 cells analyzed in two independent experiments. For all panels, *P < 0.05; **P < 0.01; ***P < 0.001

Figure 7.

Model depicting the DNA damage response to platinum drug exposure. Platinum drugs like cisplatin and oxaliplatin induce DNA interstrand and intrastrand crosslinks and lead to the production of reactive oxygen species (ROS) that induce oxidative lesions. Bulky DNA crosslinks can directly block RNA polymerase II, inhibiting transcription, and need to be dealt with by Fanconi anemia (FA), homologous recombination (HR), transcription-coupled nucleotide excision repair (TC-NER) and translesion synthesis (TLS) DNA repair and tolerance pathways. Oxidative lesions are processed by base excision repair (BER). Transcription is inhibited either directly by oxidative lesions or by BER intermediate abasic sites or single-strand breaks.