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Epistatic role of base excision repair and mismatch repair pathways in mediating cisplatin cytotoxicity.

Kothandapani A, Sawant A, Dangeti VS, Sobol RW, Patrick SM - Nucleic Acids Res. (2013)

Bottom Line: MSH2 preferentially binds a cisplatin interstrand cross-link (ICL) DNA substrate containing a mismatch compared with a cisplatin ICL substrate without a mismatch, suggesting a novel mutagenic role of Polβ in activating MMR in response to cisplatin.Collectively, these results provide the first mechanistic model for BER and MMR functioning within the same pathway to mediate cisplatin sensitivity via non-productive ICL processing.In this model, MMR participation in non-productive cisplatin ICL processing is downstream of BER processing and dependent on Polβ misincorporation at cisplatin ICL sites, which results in persistent cisplatin ICLs and sensitivity to cisplatin.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Cancer Biology, University of Toledo - Health Science Campus, Toledo, OH 43614, USA, Department of Pharmacology & Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA, University of Pittsburgh Cancer Institute, Hillman Cancer Center, Pittsburgh, PA 15213, USA and Department of Human Genetics, University of Pittsburgh Graduate School of Public Health, Pittsburgh, PA 15213, USA.

ABSTRACT
Base excision repair (BER) and mismatch repair (MMR) pathways play an important role in modulating cis-Diamminedichloroplatinum (II) (cisplatin) cytotoxicity. In this article, we identified a novel mechanistic role of both BER and MMR pathways in mediating cellular responses to cisplatin treatment. Cells defective in BER or MMR display a cisplatin-resistant phenotype. Targeting both BER and MMR pathways resulted in no additional resistance to cisplatin, suggesting that BER and MMR play epistatic roles in mediating cisplatin cytotoxicity. Using a DNA Polymerase β (Polβ) variant deficient in polymerase activity (D256A), we demonstrate that MMR acts downstream of BER and is dependent on the polymerase activity of Polβ in mediating cisplatin cytotoxicity. MSH2 preferentially binds a cisplatin interstrand cross-link (ICL) DNA substrate containing a mismatch compared with a cisplatin ICL substrate without a mismatch, suggesting a novel mutagenic role of Polβ in activating MMR in response to cisplatin. Collectively, these results provide the first mechanistic model for BER and MMR functioning within the same pathway to mediate cisplatin sensitivity via non-productive ICL processing. In this model, MMR participation in non-productive cisplatin ICL processing is downstream of BER processing and dependent on Polβ misincorporation at cisplatin ICL sites, which results in persistent cisplatin ICLs and sensitivity to cisplatin.

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Cisplatin cytotoxicity. Colony survival assays in wild-type and Polβ /deficient MEFs (A) and MDA-MB-231 cells (B). Control and MSH2 siRNA transfected cells were treated with increasing doses of cisplatin, and cytotoxicity was determined by clonogenic assays. (C) Hec59 and Hec59+2 cells were treated with cisplatin and MX+cisplatin, and clonogenic assays were performed as described. Results are represented as mean ± SD from three independent experiments.
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gkt479-F1: Cisplatin cytotoxicity. Colony survival assays in wild-type and Polβ /deficient MEFs (A) and MDA-MB-231 cells (B). Control and MSH2 siRNA transfected cells were treated with increasing doses of cisplatin, and cytotoxicity was determined by clonogenic assays. (C) Hec59 and Hec59+2 cells were treated with cisplatin and MX+cisplatin, and clonogenic assays were performed as described. Results are represented as mean ± SD from three independent experiments.

Mentions: To address the effect of BER and MMR downregulation on cell viability in response to cisplatin treatment, clonogenic assays were performed in proficient and deficient cells (Figure 1). Wild-type and Polβ /deficient MEFs (Figure 1A) and MDA-MB-231 cells (Figure 1B) were transfected with siRNA directed against MSH2 to examine cisplatin sensitivity. Both Polβ and Polβ deficient cells displayed cisplatin resistance compared with wild-type cells, consistent with our previous report (30). Knockdown of MSH2 in wild-type cells enhanced cisplatin resistance by ∼2-fold, which is consistent with the literature (14–17,37). However, knockdown of MSH2 in Polβ /deficient cells did not give rise to any additional resistance to cisplatin (Figure 1A and B). To confirm these results, we used MX to inhibit BER in MSH2-proficient and -deficient cells (Figure 1C). MX binds to abasic sites and hinders Ape1 cleavage and therefore leads to the inhibition of BER (38). In parallel with previous reports, MSH2-deficient Hec59 cells showed cisplatin-resistant phenotypes compared with their proficient counterparts (14–17). In MSH2-proficient Hec59+2 (Figure 1C) cells, MX treatment led to cisplatin resistance. We recently showed that MX treatment results in a cisplatin-resistant phenotype in MEFs and human cancer cells (30). However, MMR-deficient cells, when treated with MX, exhibited no increased resistance to cisplatin. If BER and MMR operate in separate mechanistic pathways, an additive response in cisplatin resistance would be expected. The failure to observe an additive effect provides evidence that BER and MMR have epistatic roles in mediating cisplatin cytotoxicity.Figure 1.


Epistatic role of base excision repair and mismatch repair pathways in mediating cisplatin cytotoxicity.

Kothandapani A, Sawant A, Dangeti VS, Sobol RW, Patrick SM - Nucleic Acids Res. (2013)

Cisplatin cytotoxicity. Colony survival assays in wild-type and Polβ /deficient MEFs (A) and MDA-MB-231 cells (B). Control and MSH2 siRNA transfected cells were treated with increasing doses of cisplatin, and cytotoxicity was determined by clonogenic assays. (C) Hec59 and Hec59+2 cells were treated with cisplatin and MX+cisplatin, and clonogenic assays were performed as described. Results are represented as mean ± SD from three independent experiments.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3753620&req=5

gkt479-F1: Cisplatin cytotoxicity. Colony survival assays in wild-type and Polβ /deficient MEFs (A) and MDA-MB-231 cells (B). Control and MSH2 siRNA transfected cells were treated with increasing doses of cisplatin, and cytotoxicity was determined by clonogenic assays. (C) Hec59 and Hec59+2 cells were treated with cisplatin and MX+cisplatin, and clonogenic assays were performed as described. Results are represented as mean ± SD from three independent experiments.
Mentions: To address the effect of BER and MMR downregulation on cell viability in response to cisplatin treatment, clonogenic assays were performed in proficient and deficient cells (Figure 1). Wild-type and Polβ /deficient MEFs (Figure 1A) and MDA-MB-231 cells (Figure 1B) were transfected with siRNA directed against MSH2 to examine cisplatin sensitivity. Both Polβ and Polβ deficient cells displayed cisplatin resistance compared with wild-type cells, consistent with our previous report (30). Knockdown of MSH2 in wild-type cells enhanced cisplatin resistance by ∼2-fold, which is consistent with the literature (14–17,37). However, knockdown of MSH2 in Polβ /deficient cells did not give rise to any additional resistance to cisplatin (Figure 1A and B). To confirm these results, we used MX to inhibit BER in MSH2-proficient and -deficient cells (Figure 1C). MX binds to abasic sites and hinders Ape1 cleavage and therefore leads to the inhibition of BER (38). In parallel with previous reports, MSH2-deficient Hec59 cells showed cisplatin-resistant phenotypes compared with their proficient counterparts (14–17). In MSH2-proficient Hec59+2 (Figure 1C) cells, MX treatment led to cisplatin resistance. We recently showed that MX treatment results in a cisplatin-resistant phenotype in MEFs and human cancer cells (30). However, MMR-deficient cells, when treated with MX, exhibited no increased resistance to cisplatin. If BER and MMR operate in separate mechanistic pathways, an additive response in cisplatin resistance would be expected. The failure to observe an additive effect provides evidence that BER and MMR have epistatic roles in mediating cisplatin cytotoxicity.Figure 1.

Bottom Line: MSH2 preferentially binds a cisplatin interstrand cross-link (ICL) DNA substrate containing a mismatch compared with a cisplatin ICL substrate without a mismatch, suggesting a novel mutagenic role of Polβ in activating MMR in response to cisplatin.Collectively, these results provide the first mechanistic model for BER and MMR functioning within the same pathway to mediate cisplatin sensitivity via non-productive ICL processing.In this model, MMR participation in non-productive cisplatin ICL processing is downstream of BER processing and dependent on Polβ misincorporation at cisplatin ICL sites, which results in persistent cisplatin ICLs and sensitivity to cisplatin.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Cancer Biology, University of Toledo - Health Science Campus, Toledo, OH 43614, USA, Department of Pharmacology & Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA, University of Pittsburgh Cancer Institute, Hillman Cancer Center, Pittsburgh, PA 15213, USA and Department of Human Genetics, University of Pittsburgh Graduate School of Public Health, Pittsburgh, PA 15213, USA.

ABSTRACT
Base excision repair (BER) and mismatch repair (MMR) pathways play an important role in modulating cis-Diamminedichloroplatinum (II) (cisplatin) cytotoxicity. In this article, we identified a novel mechanistic role of both BER and MMR pathways in mediating cellular responses to cisplatin treatment. Cells defective in BER or MMR display a cisplatin-resistant phenotype. Targeting both BER and MMR pathways resulted in no additional resistance to cisplatin, suggesting that BER and MMR play epistatic roles in mediating cisplatin cytotoxicity. Using a DNA Polymerase β (Polβ) variant deficient in polymerase activity (D256A), we demonstrate that MMR acts downstream of BER and is dependent on the polymerase activity of Polβ in mediating cisplatin cytotoxicity. MSH2 preferentially binds a cisplatin interstrand cross-link (ICL) DNA substrate containing a mismatch compared with a cisplatin ICL substrate without a mismatch, suggesting a novel mutagenic role of Polβ in activating MMR in response to cisplatin. Collectively, these results provide the first mechanistic model for BER and MMR functioning within the same pathway to mediate cisplatin sensitivity via non-productive ICL processing. In this model, MMR participation in non-productive cisplatin ICL processing is downstream of BER processing and dependent on Polβ misincorporation at cisplatin ICL sites, which results in persistent cisplatin ICLs and sensitivity to cisplatin.

Show MeSH
Related in: MedlinePlus