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Development of an assay system for the detection and classification of methotrexate resistance in fresh human
Abstract:
An assay system was developed for the detection and classification of methotrexate resistance in fresh human leukemic cells. Mechanisms of resistance to be identified were: overexpression of dihydrofolate reductase, decreased cellular uptake of methotrexate, decreased affinity of dihydrofolate reductase for methotrexate, decreased polyglutamylation of methotrexate, and low thymidylate synthase activity. The initial screening procedure utilizes 3H release after addition of [5-3H]-2'-deoxyuridine as a measure of intracellular activity of thymidylate synthase and of DNA synthesis; 3H release is assayed after 3-h incubations with methotrexate, trimetrexate, or gamma-fluoromethotrexate and after 4-h incubations with these agents followed by a 6-h incubation in drug free medium. The pattern of DNA synthesis inhibition and recovery under these two sets of conditions establishes the presence or absence of methotrexate resistance and allows a tentative classification of the resistance mechanism involved. In combination with determinations of dihydrofolate reductase activity, methotrexate titration studies, and the determination of intracellular drug accumulations in vitro, the system is readily able to classify CCRF-CEM human leukemia cell lines possessing well defined mechanisms of resistance. The findings in seven leukemia patients are also reported. Applying tentative reference values, four patients showed biochemical evidence of methotrexate resistance: two patients had only a transport defect, one patient had evidence of both a transport defect and low thymidylate synthase activity, and one patient appeared to have decreased methotrexate polyglutamylation. Application of the assay system in larger numbers of patients is feasible and is required to establish adequate reference values for the evaluation of biochemical-clinical correlates.
Insights
A new assay detects and classifies methotrexate resistance in leukemia cells by measuring DNA synthesis inhibition. This method identifies specific resistance mechanisms, aiding in patient treatment strategies.
Area of Science:
- Biochemistry
- Molecular Biology
- Oncology
Background:
- Methotrexate resistance is a significant challenge in leukemia treatment.
- Understanding resistance mechanisms is crucial for optimizing therapeutic outcomes.
- Existing methods for classifying resistance can be complex and time-consuming.
Purpose of the Study:
- To develop and validate a novel assay system for detecting and classifying methotrexate resistance in human leukemic cells.
- To identify specific molecular mechanisms underlying methotrexate resistance, including dihydrofolate reductase (DHFR) activity, drug uptake, DHFR-methotrexate affinity, methotrexate polyglutamylation, and thymidylate synthase (TS) activity.
- To evaluate the assay's applicability in clinical settings by analyzing samples from leukemia patients.
Main Methods:
- Development of an assay system utilizing [5-3H]-2'-deoxyuridine to measure intracellular thymidylate synthase activity and DNA synthesis.
- Assessment of DNA synthesis inhibition and recovery patterns following incubation with methotrexate and its analogs under varying conditions.
- Integration of DHFR activity assays, methotrexate titration studies, and in vitro drug accumulation measurements.
- Analysis of leukemic cell lines (CCRF-CEM) with known resistance mechanisms and clinical samples from seven leukemia patients.
Main Results:
- The assay successfully detected and classified methotrexate resistance in CCRF-CEM cell lines with defined resistance mechanisms.
- In seven leukemia patients, four exhibited biochemical evidence of methotrexate resistance.
- Identified resistance mechanisms included transport defects, low thymidylate synthase activity, and decreased methotrexate polyglutamylation.
Conclusions:
- The developed assay system is effective for detecting and classifying methotrexate resistance in human leukemic cells.
- The assay can identify specific resistance mechanisms, providing valuable information for clinical decision-making.
- Further studies with larger patient cohorts are needed to establish reference values for clinical correlation.