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CpG methylation reduces genomic instability
1Department of Radiation Oncology and Program in Cell and Molecular Biology, State University of New York Health Science Center, Syracuse, NY 13210, USA.
Abstract:
Hypomethylation of DNA in tumor cells is associated with genomic instability and has been suggested to be due to activation of mitotic recombination. We have studied the methylation patterns in two 650 kb double minute chromosomes present in two mouse tumor cell lines, resistant to methotrexate. Multiple copies of the double minute chromosomes amplifying the dihydrofolate reductase gene are present in both the cell lines. In one of the cell lines (Mut F), two unmethylated CpG islands in the double minute chromosomes are readily cleaved by methylation-sensitive rare-cutting restriction endonucleases. In the other cell line (Mut C), the cleavage sites in the double minute chromosomes are partially methylated and resistant to cleavage. The double minute chromosomes with the two unmethylated CpG islands undergo rapid dimerization, whereas the double minute chromosomes with the partially methylated CpG islands are unchanged in size for over a year in continuous culture. The partially methylated CpG islands can be demethylated by azacytidine treatment or naturally by extended time in culture, and become sensitive to cleavage with the rare-cutting restriction endonucleases. The Mut C double minute chromosomes, with the newly demethylated CpG islands, but not the double minute chromosomes with the partially methylated CpG islands, undergo deletions and dimerizations. These results suggest a role for CpG island methylation controlling mitotic recombination between and within large DNA molecules.
Insights
DNA hypomethylation in tumor cells drives genomic instability via mitotic recombination. CpG island methylation on double minute chromosomes controls this process, influencing tumor cell evolution and drug resistance.
Area of Science:
- Molecular Biology
- Cancer Genomics
- Epigenetics
Background:
- Tumor cells exhibit DNA hypomethylation, linked to genomic instability and mitotic recombination.
- Double minute chromosomes (DMs) are extrachromosomal DNA elements often found in cancer cells, amplifying oncogenes like dihydrofolate reductase (DHFR).
- Methotrexate resistance in tumor cells is associated with DHFR gene amplification on DMs.
Purpose of the Study:
- To investigate the role of DNA methylation patterns on double minute chromosomes in controlling genomic instability.
- To determine the relationship between CpG island methylation status and mitotic recombination frequency in DMs.
- To explore how epigenetic modifications on DMs influence tumor cell evolution and drug resistance.
Main Methods:
- Analysis of methylation patterns in double minute chromosomes from methotrexate-resistant mouse tumor cell lines (Mut F and Mut C) using methylation-sensitive rare-cutting restriction endonucleases.
- Assessment of DM size dynamics (dimerization, deletion) over time in continuous cell culture.
- Treatment with azacytidine to induce demethylation and subsequent analysis of DM structural changes.
Main Results:
- Unmethylated CpG islands on DMs in Mut F cells facilitated rapid dimerization, while partially methylated islands in Mut C cells were resistant to cleavage and structural changes.
- Demethylation of CpG islands in Mut C cells, induced by azacytidine or extended culture, sensitized DMs to cleavage and promoted deletions and dimerizations.
- The methylation status of CpG islands on DMs directly correlates with their susceptibility to mitotic recombination and subsequent structural alterations.
Conclusions:
- CpG island methylation status acts as a critical regulator of mitotic recombination frequency within and between large DNA molecules like DMs.
- Epigenetic control of recombination on DMs plays a significant role in driving genomic instability and adaptation in tumor cells.
- Targeting DNA methylation could offer novel therapeutic strategies to control tumor evolution and overcome drug resistance.