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Suppression of gene amplification and chromosomal DNA integration by the DNA mismatch repair system
1National Health Research Institute, Cancer Research Division, Cooperative Laboratory, Veterans General Hospital, 201 Shih-Pai Road, Sec. 2, Taipei 112, Taiwan, Republic of China. linct@nhri.org.tw
Abstract:
Mismatch repair (MMR)-deficient cells are shown to produce >15-fold more methotrexate-resistant colonies than MMR normal cells. The increased resistance to methotrexate is primarily due to gene amplification since all the resistant clones contain double-minute chromosomes and increased copy numbers of the DHFR gene. In addition, integration of linearized or retroviral DNAs into chromosomes is also significantly elevated in MMR-deficient cells. These results suggest that in addition to microsatellite instability and homeologous recombination, MMR is also involved in suppression of other genome instabilities such as gene amplification and chromosomal DNA integration.
Insights
Mismatch repair deficiency dramatically increases resistance to methotrexate by promoting gene amplification. This highlights the crucial role of mismatch repair in preventing genome instability and DNA integration.
Area of Science:
- Genetics
- Molecular Biology
- Cancer Research
Background:
- Mismatch repair (MMR) is critical for maintaining genome stability.
- MMR deficiency is linked to microsatellite instability and homeologous recombination.
- The role of MMR in suppressing other forms of genome instability is less understood.
Purpose of the Study:
- To investigate the impact of MMR deficiency on methotrexate resistance.
- To determine the mechanisms underlying increased methotrexate resistance in MMR-deficient cells.
- To explore the role of MMR in suppressing gene amplification and chromosomal DNA integration.
Main Methods:
- Culturing and treating MMR-deficient and MMR-normal cells with methotrexate.
- Quantifying the formation of methotrexate-resistant colonies.
- Analyzing resistant clones for gene amplification (DHFR copy number) and chromosomal abnormalities (double-minute chromosomes).
- Assessing the integration of linearized and retroviral DNA into chromosomes.
Main Results:
- MMR-deficient cells produced over 15-fold more methotrexate-resistant colonies than MMR-normal cells.
- Increased methotrexate resistance was primarily driven by gene amplification, evidenced by double-minute chromosomes and elevated DHFR gene copy numbers.
- Integration of chromosomal DNA, including linearized and retroviral DNA, was significantly increased in MMR-deficient cells.
Conclusions:
- MMR deficiency substantially enhances genome instability, leading to increased gene amplification and DNA integration.
- MMR plays a vital role in suppressing gene amplification and chromosomal DNA integration, beyond its known roles in microsatellite stability.
- These findings underscore the importance of MMR in preventing broader genomic alterations that can contribute to cancer development.