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Isolation and partial characterization of three methotrexate-resistant phenotypes from Chinese hamster ovary cells
Abstract:
Three mechanisms for resistance to methotrexate (Mtx) have been identified in Chinese hamster ovary (CHO) cells selected from resistance to this drug. First-step selections produce cells with either an apparent structural alteration in the enzyme dihydrofolate reductase (class I), or a decreased permeability to the drug (class II). Mutagenesis with ethyl methanesulfonate increases the proportion of Mtx-resistant cells 5-10-fold. Second-step selections to higher resistance using class I resistant cells as parents results in cells with an increased activity of the reductase enzyme (class III) with no apparent further qualitative alterations in the enzyme. All three classes of resistant cells retain their Mtx-resistant phenotype when cultured under nonselectivve conditions.
Insights
Chinese hamster ovary cells exhibit three distinct mechanisms for developing resistance to methotrexate (Mtx). These involve changes in dihydrofolate reductase structure, drug permeability, or enzyme activity, all stably maintained under non-selective conditions.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Methotrexate (Mtx) is a chemotherapy drug that inhibits dihydrofolate reductase.
- Understanding drug resistance mechanisms is crucial for improving cancer treatment efficacy.
- Chinese hamster ovary (CHO) cells are a common model system for studying drug resistance.
Purpose of the Study:
- To identify and characterize the mechanisms of methotrexate resistance in CHO cells.
- To investigate the genetic and biochemical basis of Mtx resistance.
- To determine if Mtx resistance phenotypes are stable.
Main Methods:
- Selection of Mtx-resistant CHO cell lines.
- Biochemical assays to analyze dihydrofolate reductase activity and structure.
- Permeability studies to assess drug uptake.
- Mutagenesis using ethyl methanesulfonate to enhance resistance acquisition.
Main Results:
- Three distinct classes of Mtx resistance were identified: altered dihydrofolate reductase (Class I), decreased drug permeability (Class II), and increased reductase activity (Class III).
- First-step selection yielded Class I or Class II resistant cells.
- Second-step selection of Class I cells resulted in Class III resistance.
- All identified resistance mechanisms were stable when cells were cultured without Mtx.
Conclusions:
- CHO cells can acquire methotrexate resistance through at least three independent mechanisms.
- These mechanisms involve alterations in the target enzyme, drug transport, or enzyme expression levels.
- The stable nature of these resistance phenotypes has implications for long-term treatment strategies.