Mechanisms of natural resistance to antifolates in human soft tissue sarcomas.
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.
Laboratory of Molecular Pharmacology, Memorial Sloan-Kettering Cancer Center, New York, New York 10021.
W W
WW
Li
J T
JT
Lin
W P
WP
Tong
T M
TM
Trippett
M F
MF
Brennan
J R
JR
Bertino
D000630:Aminopterin / Q000031:analogs & derivatives / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D004351:Drug Resistance; D005493:Folic Acid Antagonists / Q000378:metabolism*; D006801:Humans; D008727:Methotrexate / Q000378:metabolism; D010453:Peptide Synthases / Q000378:metabolism; D011099:Polyglutamic Acid / Q000378:metabolism; D012509:Sarcoma / Q000378:metabolism*; D012983:Soft Tissue Neoplasms / Q000378:metabolism*; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism; D013940:Thymidylate Synthase / Q000096:biosynthesis*; D016597:Trimetrexate / Q000378:metabolism; D014410:Tumor Stem Cell Assay
D005493:Folic Acid Antagonists; D011099:Polyglutamic Acid; D013762:Tetrahydrofolate Dehydrogenase; D013940:Thymidylate Synthase; D010453:Peptide Synthases; C023103:folylpolyglutamate synthetase; C040210:edatrexate; D000630:Aminopterin; D016597:Trimetrexate; D008727:Methotrexate