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. 2015 Nov:35:126-36.
doi: 10.1016/j.dnarep.2015.10.003. Epub 2015 Oct 23.

Role of mismatch repair proteins in the processing of cisplatin interstrand cross-links

Akshada Sawant  1 , Anbarasi Kothandapani  1 , Anatoly Zhitkovich  2 , Robert W Sobol  3 , Steve M Patrick  4
Affiliations

Role of mismatch repair proteins in the processing of cisplatin interstrand cross-links

Akshada Sawant et al. DNA Repair (Amst). 2015 Nov.

Abstract

Mismatch repair (MMR) deficiency gives rise to cisplatin resistance and can lead to poor prognosis in cancers. Various models have been proposed to explain this low level of resistance caused due to loss of MMR proteins. We have shown that MMR proteins are required to maintain cisplatin interstrand cross-links (ICLs) on the DNA leading to increased cellular sensitivity. In our previous studies, we have shown that BER processing of the cisplatin ICLs is mutagenic. Polymerase β (Polβ) can generate mismatches which leads to the activation and the recruitment of mismatch repair proteins. In this paper, we distinguished between the requirement of different downstream MMR proteins for maintaining cisplatin sensitivity. We show that the MutSα (MSH2-MSH6) heterocomplex is required to maintain cisplatin sensitivity, whereas the Mutsβ complex has no effect. These results can be correlated with the increased repair of cisplatin ICLs and ICL induced DNA double strand breaks (DSBs) in the resistant cells. Moreover, we show that MLH1 proficient cells displayed a cisplatin sensitive phenotype when compared with the MLH1 deficient cells and the ATPase activity of MLH1 is essential to mediate this effect. Based on these results, we propose that MutSα as well as the downstream MMR pathway proteins are essential to maintain a cisplatin sensitive phenotype as a consequence of processing Polβ induced mismatches at sites flanking cisplatin ICLs.

Keywords: ATPase; BER; Cisplatin; MMR; Resistance.

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Conflict of interest statement

Conflict of interest:

None declared. RWS is a scientific consultant for Trevigen, Inc.

The authors’ state that there are no conflicts of. The work is not submitted to any other journal.

Figures

Figure 1
Figure 1. Cisplatin cytotoxicity in MSH6 and MSH3 knockdown cells
(A) Colony survival assay in MDA-MB-231 wildtype (WT) cells or following MSH6 knockdown: Control (closed circles), MSH6 (open triangles), Polβ (closed squares) and Polβ MSH6 KD (open circles). (B) Colony survival assay in DLD-1 (open circles) and DLD-1 + chr2 cells (closed circles) (C) Colony survival assay in MDA-MB-231 cells following MSH3 knockdown: Control (closed circles), MSH3 (open triangles), Polβ (closed squares) and Polβ MSH3 KD (open circles). (D) Colony survival assay in MDA-MB-231 cells following a double knockdown of MSH3 and MSH6: WT control (closed circles), MSH3 +MSH6 KD in WT cells (open triangles), Polβ KD control (closed squares) and MSH3 +MSH6 KD in Polβ KD cells (open circles). shRNA transfected cells were treated with increasing doses of cisplatin and cytotoxicity was determined by colony survival assay as described in Materials and Methods. Results are represented as mean ± SD from 3 independent experiments.
Figure 2
Figure 2
Repair of cisplatin ICLs in MDA-MB-231 cells. (A) MSH6 KD (B) MSH3 KD in MDA-MB-231. Cells were treated with cisplatin for 2 hrs and comet assays were performed as described in Materials and Methods at different time intervals (0, 24, 48 and 72 hr) to assess ICL levels. The data was collected using komet 5.5 software. The percentage of ICLs present at each time point was calculated using olive tail moments. Results are represented as mean ± SD of three independent experiments. Statistical analysis was performed by student’s t test and comparisons are made between wildtype and proficient cells vs deficient cells. NS – non-significant; * - P< 0.01. Repair of cisplatin ICL induced DSBs in MDA-MB-231 (C) MSH6 KD (D) MSH3 KD. Cells were treated with cisplatin for 2 hrs and immunofluorescence was performed as described in Materials and Methods at different time intervals (0, 24, 48 and 72 hr). A minimum of 200 cells were analyzed for each time point. The percentage of γH2AX foci positive cells at each time point was calculated. Results are represented as mean ± SD of three independent experiments. Statistical analysis was performed by student’s t test and comparisons are made between wildtype and proficient cells vs deficientcells. NS–nonsignificant;*-P<0.05.
Figure 3
Figure 3. Effect of MLH1 knockdown on cisplatin cytotoxicity and repair
(A) Colony survival assays in WT and Polβ deficient MDA-MB-231 cells with MLH1 KD. (B) ELISA for cisplatin GG intrastrand adduct repair in MLH1 KD MDA-MB-231 cells. Cells were treated with cisplatin for 2 hrs and genomic DNA was extracted at the indicated time points. ELISA was performed using an antibody specific for cisplatin GG intrastrand adduct and the percentage of GG adducts remaining was calculated as described. Results are represented as mean ± SD of three independent experiments. Statistical analysis was performed by student’s t test and comparisons are made between wildtype and proficient cells vs deficient cells. NS – non-significant (C) Cisplatin ICL DNA repair using alkaline comet assay performed in WT and Pol β deficient MDA-MB231 cells as described previously. (D) Immunofluorescence assay for γH2AX assessing DSB repair in WT, MLH1 KD and Polβ deficient MDA-MB-231 cells.
Figure 4
Figure 4. MLH1 ATPase mutants in mediating cisplatin cytotoxicity
(A) Schematic of MLH1 ATPase mutant, (B) Western blot analysis and (C) real time PCR were performed to check the expression levels of MLH1 protein and transcript. (D) MTS assays were performed in HCT116 cells using increasing concentrations of cisplatin for 2 hrs. WT cells (closed circles), Empty vector (closed squares), S44L cells (open squares) and S44F cells (open circles). Results are represented as mean ± SE from 3 independent experiments.
Figure 5
Figure 5. Repair of cisplatin ICLs in HCT116 MLH1 ATPase mutant cells
(A) Alkaline comet assay was performed as previously described to assess cisplatin ICL DNA repair. (B) Repair of DSBs in HCT116 cells was measured using an immunofluorescence assay with an antibody specific for γH2AX as previously described Materials and Methods. (C) MLH1 was knocked down in DLD-1 and DLD-1 + chr 2 cells using siRNA transfection: DLD-1 control (open circles), MLH1 KD in DLD-1 cells (closed squares), DLD-1 + chr 2 control (closed circles) and MLH1 KD in DLD-1 + chr 2 (open triangles). Colony survival assays were performed as described previously.
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