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Rapid emergence of methotrexate resistance in cultured mouse cells
Abstract:
We have examined the time required for mouse 3T6 cells to become resistant to 200 nM methotrexate by three selection protocols: (a) a single-step 0 to 200 nM dose; (b) a two-step 0 to 80 to 200 nM dose; and (c) a multistep 0 to 40 to 80 to 120 to 160 to 200 nM dose. An initial inoculum of 5 X 10(5) cells was grown to 10(6) cells at each increment of methotrexate, reduced to 5 X 10(5) cells, and again grown to 10(6) cells at the next increment. The total elapsed time required for an initial inoculum of 5 X 10(5) cells to grow to 1 X 10(6) cells resistant to 200 nM methotrexate was 45, 21, and 6.5 days, respectively, for the three drug dosage schedules. The single-step resistant variants did not contain amplified dihydrofolate reductase genes, whereas cells resistant to 200 nM methotrexate by the two stepwise selections were resistant as a result of a 6-fold amplification of the dihydrofolate reductase gene. We conclude that the resistance to 200 nM methotrexate resulting from gene amplification did not preexist in the initial population but was generated during the selection process. These results are discussed in terms of the emergence of drug resistance during the course of chemotherapy of tumors.
Insights
Developing drug resistance in mouse cells to methotrexate occurs faster with stepwise increases in dosage. Gene amplification drives resistance, and this amplification is generated during the selection process, not pre-existing.
Area of Science:
- Cell biology
- Molecular biology
- Genetics
Background:
- Methotrexate is a chemotherapy drug that inhibits dihydrofolate reductase.
- Acquired resistance to chemotherapy drugs is a major challenge in cancer treatment.
- Understanding the mechanisms and kinetics of drug resistance is crucial for optimizing therapeutic strategies.
Purpose of the Study:
- To investigate the time required for mouse 3T6 cells to develop resistance to methotrexate under different selection protocols.
- To determine the role of dihydrofolate reductase gene amplification in methotrexate resistance.
- To elucidate whether gene amplification pre-exists or is generated during drug selection.
Main Methods:
- Three selection protocols were used: single-step (0 to 200 nM), two-step (0 to 80 to 200 nM), and multistep (0 to 40 to 80 to 120 to 160 to 200 nM) methotrexate exposure.
- Cell populations were serially selected and grown to a specific density at each drug concentration increment.
- Dihydrofolate reductase gene copy number was assessed in resistant cell lines.
Main Results:
- The time to achieve 200 nM methotrexate resistance varied significantly: 45 days (single-step), 21 days (two-step), and 6.5 days (multistep).
- Single-step selection did not result in dihydrofolate reductase gene amplification.
- Two-step and multistep selections led to a 6-fold amplification of the dihydrofolate reductase gene in resistant cells.
Conclusions:
- The emergence of methotrexate resistance through dihydrofolate reductase gene amplification is dependent on the selection protocol.
- Gene amplification is generated during the drug selection process rather than pre-existing in the cell population.
- These findings have implications for understanding drug resistance development in cancer chemotherapy and designing more effective treatment regimens.