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Mechanisms of natural resistance to antifolates in human soft tissue sarcomas
1Laboratory of Molecular Pharmacology, Memorial Sloan-Kettering Cancer Center, New York, New York 10021.
Abstract:
Efforts to use fresh human sarcoma cells for evaluating antifolate resistance with an in situ thymidylate synthesis assay using 5-[3H] deoxyuridine were unsuccessful because of low thymidylate synthesis activity in enzymatically disaggregated tumors. By incubating tumor cell suspensions in supplemented RPMI-1640 medium with 10% fetal bovine serum for 3 days, activity of the in situ thymidylate synthesis assay markedly increased (1.42 versus 0.03 pmol/h/10(7) cells), thus allowing 75% of samples to be evaluated for antifolate sensitivity. By criteria developed with a methotrexate-resistant and -sensitive cell line, this assay indicated that most sarcomas are naturally resistant to methotrexate (12 of 15). Natural resistance to 10-ethyl-10-deazaaminopterin and trimetrexate was also observed in 60% of the samples (nine of 15, respectively). The results from the 3-day in situ assay were confirmed by specific tests for resistance mechanisms in most sarcoma samples. The resistance mechanisms detected were impaired polyglutamylation, an increased level of dihydrofolate reductase, and amplification of this gene. These results encourage further exploration of this assay to predict response to antifolates in individual patients and to evaluate efficacy of new antifolates as candidates for clinical trial.
Insights
An improved in situ assay enhances antifolate resistance testing in human sarcoma cells. This method allows for the evaluation of antifolate sensitivity, aiding in predicting patient response and developing new antifolate drugs.
Area of Science:
- Oncology
- Pharmacology
- Biochemistry
Background:
- Evaluating antifolate resistance in human sarcoma cells using traditional in situ thymidylate synthesis assays with 5-[3H] deoxyuridine proved challenging due to low enzymatic activity in disaggregated tumors.
- Low thymidylate synthesis activity in tumor cell suspensions limited the effectiveness of antifolate resistance evaluations.
Purpose of the Study:
- To optimize an in situ thymidylate synthesis assay for evaluating antifolate resistance in human sarcoma cells.
- To assess the sensitivity of sarcoma samples to various antifolates, including methotrexate, 10-ethyl-10-deazaaminopterin, and trimetrexate.
- To identify resistance mechanisms contributing to antifolate unresponsiveness in sarcomas.
Main Methods:
- Tumor cell suspensions were incubated in supplemented RPMI-1640 medium with 10% fetal bovine serum for 3 days to enhance thymidylate synthesis activity.
- An optimized in situ thymidylate synthesis assay using 5-[3H] deoxyuridine was employed to measure antifolate sensitivity.
- Resistance mechanisms, including polyglutamylation, dihydrofolate reductase (DHFR) levels, and DHFR gene amplification, were investigated.
Main Results:
- Incubation for 3 days significantly increased thymidylate synthesis activity (1.42 vs. 0.03 pmol/h/10(7) cells), enabling assay evaluation in 75% of samples.
- The assay revealed natural resistance to methotrexate in most sarcomas (12 of 15) and significant resistance to 10-ethyl-10-deazaaminopterin and trimetrexate (60% of samples).
- Confirmed resistance mechanisms included impaired polyglutamylation, elevated DHFR levels, and DHFR gene amplification.
Conclusions:
- The optimized 3-day in situ assay effectively evaluates antifolate sensitivity in human sarcoma samples.
- Most sarcomas exhibit natural resistance to common antifolates, mediated by specific molecular mechanisms.
- This assay holds promise for predicting individual patient responses to antifolates and for evaluating new antifolate drug candidates.