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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. (A) Polβ KD, KD/wt Polβ and KD/D256A cells were treated with cisplatin for 2 h, and then cell sensitivity was determined by MTS assay. (B) Cells were treated with cisplatin and MX+cisplatin, and clonogenic assays were performed as described. (C) Cells were transfected with control or MSH2 siRNA, and cisplatin cytotoxicity was determined by clonogenic assays. Results are represented as mean ± SD from three independent experiments.
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gkt479-F3: Cisplatin cytotoxicity. (A) Polβ KD, KD/wt Polβ and KD/D256A cells were treated with cisplatin for 2 h, and then cell sensitivity was determined by MTS assay. (B) Cells were treated with cisplatin and MX+cisplatin, and clonogenic assays were performed as described. (C) Cells were transfected with control or MSH2 siRNA, and cisplatin cytotoxicity was determined by clonogenic assays. Results are represented as mean ± SD from three independent experiments.

Mentions: DNA Polβ is a 39 kDa single polypeptide consisting of a 8 kDa dRP lyase domain and a 31 kDa polymerase domain (46,47). Asp256 is one of the three active site aspartates in the polymerase domain, which is critical for the nucleotidyltransferase mechanism. Menge et al. (48) showed that mutation of Asp256 to alanine (D256A) completely abolished the polymerase activity of Polβ. Although the D256A Polβ mutant completely lacks the gap-filling DNA synthesis, it retains the dRP lyase activity (49). Several mutations in polymerase or the lyase domain of Polβ showed the importance of these domains in the cytotoxicity of certain DNA-damaging agents (49–55). Therefore, we re-expressed wild-type Polβ and a variant deficient in polymerase activity (D256A) in Polβ knockdown cells to determine the importance of the polymerase domain/activity in mediating cisplatin sensitivity. Consistent with a previous report, Polβ KD cells displayed cisplatin resistance (30). Re-expressing wild-type Polβ in the KD cells restored cisplatin sensitivity in both MTS (Figure 3A) and colony survival assays (Figure 3B). Interestingly, expression of the D256A Polβ mutant in the KD cells resulted in sensitivity to cisplatin as well. These data indicate that loss of Polβ protein can drive cisplatin resistance, but re-expression of wildtype or the presence of a polymerase dead mutant can mediate cisplatin sensitivity.Figure 3.


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. (A) Polβ KD, KD/wt Polβ and KD/D256A cells were treated with cisplatin for 2 h, and then cell sensitivity was determined by MTS assay. (B) Cells were treated with cisplatin and MX+cisplatin, and clonogenic assays were performed as described. (C) Cells were transfected with control or MSH2 siRNA, and cisplatin cytotoxicity was determined by clonogenic assays. Results are represented as mean ± SD from three independent experiments.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt479-F3: Cisplatin cytotoxicity. (A) Polβ KD, KD/wt Polβ and KD/D256A cells were treated with cisplatin for 2 h, and then cell sensitivity was determined by MTS assay. (B) Cells were treated with cisplatin and MX+cisplatin, and clonogenic assays were performed as described. (C) Cells were transfected with control or MSH2 siRNA, and cisplatin cytotoxicity was determined by clonogenic assays. Results are represented as mean ± SD from three independent experiments.
Mentions: DNA Polβ is a 39 kDa single polypeptide consisting of a 8 kDa dRP lyase domain and a 31 kDa polymerase domain (46,47). Asp256 is one of the three active site aspartates in the polymerase domain, which is critical for the nucleotidyltransferase mechanism. Menge et al. (48) showed that mutation of Asp256 to alanine (D256A) completely abolished the polymerase activity of Polβ. Although the D256A Polβ mutant completely lacks the gap-filling DNA synthesis, it retains the dRP lyase activity (49). Several mutations in polymerase or the lyase domain of Polβ showed the importance of these domains in the cytotoxicity of certain DNA-damaging agents (49–55). Therefore, we re-expressed wild-type Polβ and a variant deficient in polymerase activity (D256A) in Polβ knockdown cells to determine the importance of the polymerase domain/activity in mediating cisplatin sensitivity. Consistent with a previous report, Polβ KD cells displayed cisplatin resistance (30). Re-expressing wild-type Polβ in the KD cells restored cisplatin sensitivity in both MTS (Figure 3A) and colony survival assays (Figure 3B). Interestingly, expression of the D256A Polβ mutant in the KD cells resulted in sensitivity to cisplatin as well. These data indicate that loss of Polβ protein can drive cisplatin resistance, but re-expression of wildtype or the presence of a polymerase dead mutant can mediate cisplatin sensitivity.Figure 3.

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