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Evolution of methotrexate resistance of human acute lymphoblastic leukemia cells in vitro
Abstract:
A human acute lymphoblastic T-cell line, MOLT-3, was fed with Roswell Park Memorial Institute Medium 1640 supplemented with 10% fetal bovine serum and antibiotics which contained increasing concentrations of methotrexate (MTX). The development of drug resistance was associated initially with progressive decrease in MTX transport. When the cells became 200-fold resistant, a rise in the dihydrofolate reductase was noted which was short-lived in the absence of the drug. A 10,000-fold increase in MTX resistance was accompanied, in addition to further decrease in MTX transport, by a 10-fold increase in the dihydrofolate reductase activity. While the purely transport-related resistant cell lines had a collateral sensitivity to lipid-soluble antifols, the sublines which had both transport- and enzyme-related MTX resistance contained a subpopulation highly resistant to these antifols. Chromosome analysis of the subline with increased dihydrofolate reductase activity showed an expanded abnormally banded region in chromosome 5.
Insights
Methotrexate (MTX) resistance in T-cells involves decreased MTX transport and increased dihydrofolate reductase. Chromosome 5 abnormalities were observed in highly resistant cells.
Area of Science:
- Cell Biology
- Pharmacology
- Genetics
Background:
- Methotrexate (MTX) is a key chemotherapy agent.
- Drug resistance mechanisms are crucial for treatment efficacy.
- Understanding resistance in T-cell acute lymphoblastic leukemia (T-ALL) is vital.
Purpose of the Study:
- To investigate the mechanisms of MTX resistance in MOLT-3 T-cells.
- To characterize the genetic and biochemical changes associated with MTX resistance.
- To explore collateral sensitivity and resistance patterns to other antifolates.
Main Methods:
- Culturing MOLT-3 cells with increasing MTX concentrations.
- Assessing MTX transport kinetics.
- Measuring dihydrofolate reductase (DHFR) activity.
- Analyzing collateral sensitivity to other antifolates.
- Performing chromosome analysis.
Main Results:
- Initial MTX resistance correlated with decreased MTX transport.
- Higher resistance (200-fold) showed a transient increase in DHFR activity.
- Extreme resistance (10,000-fold) involved reduced transport and a 10-fold DHFR increase.
- Cell lines with transport-only resistance showed collateral sensitivity to lipid-soluble antifols.
- Sublines with combined resistance exhibited subpopulations resistant to other antifolates.
- Chromosome analysis revealed an abnormal region on chromosome 5 in DHFR-overexpressing cells.
Conclusions:
- MTX resistance in MOLT-3 cells is multifactorial, involving both transport and enzyme alterations.
- DHFR activity and MTX transport are key determinants of resistance.
- Chromosome 5 aberrations may be linked to DHFR gene amplification.
- Understanding these resistance mechanisms can inform future therapeutic strategies for T-ALL.