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Decreased cisplatin damage-dependent DNA synthesis in cellular extracts of mismatch repair deficient cells
K V Ferry1, D Fink, S W Johnson
1Fox Chase Cancer Center, Philadelphia, PA 19111, USA.
Abstract:
The proficiency of both nucleotide excision repair (NER) and DNA mismatch repair (MMR) influences cellular sensitivity to cisplatin (cis-diamminedichloroplatinum). To gain further insight into how MMR may influence platinum drug sensitivity, the effect of loss of MMR on repair synthesis was measured in vitro by a commonly used method that relies on whole-cell extracts to drive [alpha-32P]dATP incorporation into cisplatin-damaged plasmid DNA. Extracts evaluated include those from cells with or without functional hMLH1 (HCT116+ch2 versus HCT116+ch3, respectively) and hMSH2 (HEC59 versus HEC59+ch2, respectively). Loss of MMR in the HCT116 system was associated with a 2.8-fold reduction in cisplatin damage-specific DNA synthesis, whereas it was associated with a 3.0-fold reduction in the HEC59 system, suggesting that a decrease in the ability to repair cisplatin-damaged DNA accompanies loss of MMR. An in vitro DNA excision assay that utilized a substrate containing a site-specific cisplatin adduct was performed. Using this highly NER-specific assay, no significant difference was apparent between the extracts derived from NER-proficient versus -deficient cells. These and other data lead us to suggest that the increase in apparent repair synthesis in platinum-damaged plasmids by extracts from MMR-proficient versus -deficient cellular extracts may reflect a distinct and possibly adverse DNA synthetic process rather than productive NER.
Insights
Loss of DNA mismatch repair (MMR) reduces the cell
Area of Science:
- Molecular Biology
- DNA Repair Mechanisms
- Cancer Therapeutics
Background:
- Cellular sensitivity to platinum-based chemotherapy drugs like cisplatin is influenced by DNA repair pathways.
- Nucleotide excision repair (NER) and DNA mismatch repair (MMR) are critical in processing DNA damage.
- Understanding the role of MMR in platinum drug response is essential for optimizing cancer treatment.
Purpose of the Study:
- To investigate the impact of DNA mismatch repair (MMR) deficiency on DNA repair synthesis in response to cisplatin damage.
- To differentiate between productive DNA repair and other DNA synthetic processes in MMR-deficient cells.
Main Methods:
- In vitro repair synthesis assays using whole-cell extracts from MMR-proficient and MMR-deficient cell lines (HCT116 and HEC59).
- Measurement of [alpha-32P]dATP incorporation into cisplatin-damaged plasmid DNA to assess repair synthesis.
- In vitro DNA excision assays using a site-specific cisplatin adduct to evaluate NER activity.
Main Results:
- Loss of MMR function in HCT116 and HEC59 cell systems resulted in a significant reduction (2.8-fold and 3.0-fold, respectively) in cisplatin damage-specific DNA synthesis.
- NER-specific assays showed no significant difference in repair activity between NER-proficient and NER-deficient cell extracts.
- Reduced DNA synthesis in MMR-proficient extracts acting on platinum-damaged DNA may indicate a non-productive or adverse DNA synthetic process.
Conclusions:
- A decrease in the ability to repair cisplatin-damaged DNA is associated with the loss of DNA mismatch repair (MMR).
- The observed increase in DNA synthesis in MMR-proficient extracts may not represent efficient nucleotide excision repair (NER) but rather a different DNA synthetic event.
- These findings suggest MMR plays a role in cellular response to cisplatin, potentially through mechanisms beyond direct NER pathway involvement.