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Genetic changes in methotrexate-resistant mosquito cells
1Department of Entomology, University of Minnesota, St. Paul 55108.
Abstract:
A stepwise selection procedure was used to obtain from Mtx-5011 Aedes albopictus cells, variants with increased resistance to methotrexate (mtx). On the basis of growth, the Mtx-5011 derivatives were 270- to 3,000-fold more resistant to mtx than wild-type mosquito cells. Properties associated with mtx resistance in these cells were consistent with amplification of the dihydrofolate reductase (DHFR) gene. The cells overproduced DHFR protein, were enriched with DHFR mRNA, and DNA from resistant cells was enriched for a band that likely contained the DHFR coding sequence. Karyotype analysis indicated that high levels of resistance were accompanied by a conversion to tetraploidy, chromosome rearrangements, and an apparent duplication of one of the mosquito chromosomes.
Insights
Researchers developed methotrexate (mtx)-resistant Aedes albopictus mosquito cells. These cells showed significant mtx resistance due to dihydrofolate reductase (DHFR) gene amplification and chromosomal changes.
Area of Science:
- Molecular Biology
- Genetics
- Cell Biology
Background:
- Methotrexate (mtx) is a chemotherapy drug that inhibits dihydrofolate reductase (DHFR).
- Drug resistance in cancer cells often involves gene amplification and altered protein expression.
- Aedes albopictus cells provide a model for studying drug resistance mechanisms.
Purpose of the Study:
- To generate and characterize methotrexate-resistant variants of Aedes albopictus cells.
- To investigate the molecular mechanisms underlying methotrexate resistance in these cells.
Main Methods:
- Stepwise selection procedure to induce methotrexate resistance.
- Growth assays to quantify resistance levels.
- Gene amplification analysis (mRNA and DNA enrichment).
- Karyotype analysis to study chromosomal changes.
Main Results:
- Generated Aedes albopictus cell variants 270- to 3,000-fold more resistant to methotrexate.
- Observed amplification of the dihydrofolate reductase (DHFR) gene.
- Confirmed DHFR gene amplification through increased DHFR protein and mRNA levels.
- Identified chromosomal abnormalities including tetraploidy and rearrangements in resistant cells.
Conclusions:
- Dihydrofolate reductase (DHFR) gene amplification is a key mechanism for methotrexate resistance in Aedes albopictus cells.
- Chromosomal alterations, including tetraploidy and rearrangements, are associated with high-level methotrexate resistance.
- These resistant cell lines serve as valuable tools for studying drug resistance in insect cells.