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, 39 (8), 1359-1372

Co-inhibition of Pol η and ATR Sensitizes Cisplatin-Resistant Non-Small Cell Lung Cancer Cells to Cisplatin by Impeding DNA Damage Repair

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Co-inhibition of Pol η and ATR Sensitizes Cisplatin-Resistant Non-Small Cell Lung Cancer Cells to Cisplatin by Impeding DNA Damage Repair

Xiao-Qin Li et al. Acta Pharmacol Sin.

Abstract

For the majority of patients with advanced non-small cell lung cancer (NSCLC), the standard of care remains platinum-based chemotherapy. However, cisplatin resistance is a big obstacle to the treatment, and elucidation of its mechanism is warranted. In this study, we showed that there was no difference in intracellular uptake of cisplatin or the removal of platinum-DNA adducts between a cisplatin-resistant NSCLC cell line (A549/DR) and a cisplatin-sensitive NSCLC cell line (A549). However, the capacity to repair DNA interstrand crosslinks (ICLs) and double-strand breaks (DSBs) was significantly enhanced in the A549/DR cell line compared to 3 cisplatin-sensitive cell lines. We found that the protein and mRNA expression levels of Pol η, a Y-family translesion synthesis (TLS) polymerase, were markedly increased upon cisplatin exposure in A549/DR cells compared with A549 cells. Furthermore, intracellular co-localization of Pol η and proliferation cell nuclear antigen (PCNA) induced by cisplatin or cisplatin plus gemcitabine treatment was inhibited by depleting ataxia telangiectasia mutated and Rad-3-related (ATR). Pol η depletion by siRNA sensitized A549/DR cells to cisplatin; co-depletion of Pol η and ATR further increased A549/DR cell death induced by cisplatin or cisplatin plus gemcitabine compared to depletion of Pol η or ATR alone, concomitant with inhibition of DNA ICL and DSB repair and accumulation of DNA damage. No additional sensitization effect of co-depleting Pol η and ATR was observed in A549 cells. These results demonstrate that co-inhibition of Pol η and ATR reverses the drug resistance of cisplatin-resistant NSCLC cells by blocking the repair of DNA ICLs and DSBs induced by cisplatin or cisplatin plus gemcitabine.

Keywords: ATR; DNA damage repair; cisplatin; gemcitabine; non-small cell lung cancer; polymerase η (Pol η); resistance.

Figures

Figure 1
Figure 1
Cisplatin resistance in A549/DR cells is not associated with the decrease of drug intracellular uptake and the increase of Pt-DNA adduct removal. (A) Cell viability measurement, A549, A549/DR, LOU-NH91 and HCC4006 cell lines growing in 96-well plates were treated with cisplatin, (B) carboplatin, (C) oxaliplatin, and (D) gemcitabine at indicated dose. The CCK-8 assay was used to determine cell viability. After treatment with drug as indicated for 2–4 h, cell proliferation reagent CCK-8 (DOJNDO, Japan) was added into media in each well and the cells were incubated for 2 h at 37 °C. The absorbance of each well was measured with a spectrophotometer reading at a wavelenth of 450 nm. Absorbance is assumed to be directly proportional to the number of viable cells (* P<0.05, ** P<0.01 vs A549, LOU-NH91 and HCC4006 cell lines). (E) Formation of platinum-DNA adducts in A549, A549/DR, LOU-NH91 and HCC4006 cell lines after a 2-h exposure to cisplatin as measured by the FAAS. (F) The rate of disappearance of platinum from total cellular DNA was measured in the four NSCLC cell lines after a 2-h exposure to cisplatin (10 μmol/L). Each datum represents the mean of three experiments.
Figure 2
Figure 2
A549/DR cells show decreased γ-H2AX foci formation, diminished tail moment, and rapider resumption of replication arrest after cisplatin treatment. (A) A549, A549/DR, LOU-NH91 and HCC4006 cell lines were treated with 10 μmol/L cisplatin for 2 h, fixed and immunostained using anti-γ-H2AX antibody. (B) The percentage of γ-H2AX positive cells, defined as cells with a fluorescence intensity>500 units (control value) was quantified using Metafer software (* P<0.05 vs A549, LOU-NH91 and HCC4006 cell lines). (C) The four cell lines were treated with 10 μmol/L cisplatin, fixed and immunostained using anti-γ-H2A antibody. The percentages of γ-H2AX positive cells were quantified at indicated time points (* P<0.05 vs A549, LOU-NH91 and HCC4006 cell lines). (D) The four cell lines were treated with 10 μmol/L cisplatin for 2 h. The alkaline comet assay was performed to measure ICLs, and the images show detectable comet tails visualized under a fluorescent microscope. (E) Tail moments in the cells were quantified using Comet Score software version 1.5 (* P<0.05 vsA549, LOU-NH91, and HCC4006 cell lines). (F) DNA replication was measured by BrdU incorporation assay 24, 48, and 72 h after cisplatin (10 μmol/L) treatment (* P<0.05 vs A549, LOU-NH91, and HCC4006 cell lines).
Figure 3
Figure 3
Expressions of pol η were upregulated in A549/DR cells upon exposure to cisplatin. (A) Whole cell lysate was prepared from A549, A549/DR, LOU-NH91 and HCC4006 cell lines and subject to Western blotting with specific antibodies as indicated to measure the protein levels of various factors. (B and C) Total RNA was isolated from the four NSCLC cell lines, and subject to real-time quantitative PCR to measure the mRNA levels of various factors as indicated (* P<0.05, ** P<0.01 vs A549, LOU-NH91 and HCC4006 cell lines). (D) Protein expressions of TLS polymerases as indicated were analyzed by Western blotting using specific antibodies in whole cell lysate of A549 and A549/DR cells at different time points after cisplatin (10 μmol/L) treatment. β-actin was used as loading control. (E and F) Real-time quantitative PCR was performed to measure mRNA expression of TLS polymerases as indicated in A549 and A549/DR cells at different time points after cisplatin (10 μmol/L) treatment. The mRNA expressions of TLS polymerases were normalized to GAPDH; the untreated control was set to one (** P<0.01 vs POLK, POLB, POLM and POLN).
Figure 4
Figure 4
PCNA monoubiquitination and intracellular location of Pol η. (A) PCNA monoubiquitination induced by cisplatin. A549, A549/DR, LOU-NH91 and HCC4006 cell lines were treated with cisplatin (10 μmol/L), and ubiquitinated PCNA (Ub-PCNA) from the cell extracts was detected using a monoclonal anti-PCNA antibody by western blotting. (B) A549/DR cells were treated with cisplatin (Cis, 10 μmol/L), or carboplatin (Car, 10 μmol/L), or oxaliplatin (Oxa, 15 μmol/L), or gemcitabine (Gem, 10 nmol/L), and Ub-PCNA was detected as described above. (Con: negative control). (C) Western blotting was performed to verify the efficiency of transfection with siRAD18 or siATR in A549 or A549/DR cells. siCon: control siRNA. (D) After transfection with siRNAs as indicated, intracellular location of Pol η in the four NSCLC cell lines was analyzed by immunostaining using anti-Pol η body after treatment with cisplatin at indicated doses. The lower panel shows the quantification of the mean fluorescent intensity of Pol η antibody staining as calculated by Image software (** P<0.01 vs A549, LOU-NH91, and HCC4006 cell lines). (E) Intracellular colocalizations of Pol η and PCNA in A549/DR cells post-transfection with siRNAs as indicated were analyzed by immunostaining using anti-Pol η and anti-PCNA bodies after treatment with cisplatin and cisplatin plus gemcitabine. (F and G) PCNA monoubiquitinations in A549 and A549/DR cell lines post-transfection with siRNAs as indicated were analyzed by Western blotting after cisplatin (10 μmol/L) treatment.
Figure 5
Figure 5
Co-depletion of POLH and ATR hypersensitize A549/DR cells to cisplatin, and cisplatin plus gemcitabine. (A) Western blotting was performed to verify the efficiency of the transfections with siPOLH, siPOLB, siPOLN and siATR in A549 and A549/DR cell lines siCon: control siRNA. (B and C) The viability analysis of A549 cells depleted of POLH or ATR alone, or double depleted of POLH and RAD18, or double depleted of POLH and ATR after treatment with cisplatin or cisplain plus gemcitabine as indicated doses. (D and E) The viability analysis of A549/DR cells depleted of the genes as indicated after treatment with cisplatin or cisplatin plus gemcitabine. (G and H) A549/DR cells depleted of POLH, or POLB, or POLN alone, or co-depleted of POLH and ATR were treated with cisplatin and cisplatin plus gemcitabine for 4 h and apoptotic cells was measured as sub-G1 fraction by flow cytometry (* P<0.05 vs siPOLB and siPOLN; ## P<0.01 vs siPOLH). (F) Representative imagines of cell cycle analysis show that sub-G1 peak was higher in A549/DR cells depleted of POLH alone and co-depleted of POLH and ATR than in the cells depleted of POLB or DOLN alone.
Figure 6A–C
Figure 6A–C
Co-depletion of POLH and ATR in A549/DR cell resulted in marked impairment of ICL and DSB repair and increase of DNA damage accumulation. (A) After transfection with siRNAs as indicated, A549/DR cells were treated with cisplatin or cisplatin plus gemcitabine in indicated doses for 4 h, cultured in fresh medium for another 24 h, fixed and immunostained with an anti-γ-H2AX antibody. (B) The percentage of γ-H2AX foci positive cells was quantified using Metafer software (** P<0.01 vs siPOLB or siPOLN; # P<0.05 vs siPOLH). (C) The percentages of γ-H2AX positive cells were quantified at indicated time points. Each time point represents the mean of γ-H2AX positive/total cells (%) derived from five independent fields in each culture (* P<0.05 vs siPOLH).
Figure 6D–F
Figure 6D–F
Co-depletion of POLH and ATR in A549/DR cell resulted in marked impairment of ICL and DSB repair and increase of DNA damage accumulation. (D) A549/DR cells were transfected with siRNAs as indicated, and then treated with cisplatin or cisplatin plus gemcitabine for 4 h. An alkaline comet assay was performed to measure ICLs, and the images show detectable comet tails visualized under a fluorescent microscope. (E) Tail moments in A549/DR cells were quantified using Comet Score software version 1.5 (** P<0.01 vs siPOLB or siPOLN; # P<0.05 vs siPOLH). (F) Western blotting for phosphorylated-Chk1, -KAP1, -RPA2, and -H2AX in A549/DR cells depleted of POLH and ATR alone or co-depleted of POLH and ATR following treatment with cisplatin as indicated doses, GAPDH was used as a loading control. The intensity of protein bands was quantified by densitometry and presented in Figures S4.

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