EVALUATION OF METHOTREXATE SENSITIVITY IN HUMAN LEUKEMIA-CELL LINES BY AN ADENOSINE-TRIPHOSPHATE BIOLUMINESCENCE ASSAY

To verify a recently developed in vitro tumor chemosensitivity assay ( ICA) based on adenosine triphosphate (ATP) measurement for detection o f methotrexate (MTX) sensitivity or resistance in human leukemias and solid tumors in which the antifol is indicated, we investigated the ab ility of the assay to discriminate between sensitivity and resistance to this antifolate in human leukemia cell lines sensitive (parental CC RF-CEM/S) and resistant (CCRF-CEM/E, CCRFC-CEM/T and CCRF-CEM/P sublin es) to MTX by virtue of known biochemical mechanisms, Correlation expe riments with a standard cell growth inhibition assay and a radiometric method for measurement of thymidylate (dTMP) synthesis ([5-H-3]-2'-de oxyuridine tritium release assay) were performed, No significant diffe rences were observed in the ICS, values for the four cell lines tested as determined by cell growth evaluation (cell number counts) and the ATP-TCA after a 72 h MTX exposure. After short-term (4 h) high-dose MT X exposure, no significant correlation between ATP-TCA and the classic [5-H-3]-2'-deoxyuridine tritium release assay was observed in both CC RF-CEM/S and CCRF-CEM/P cells, CCRF-CEM/T and CCRF-CEM/E displayed, in stead, complete resistance with both methods, When using conditions pr oposed for clinical application (long-term exposure, i.e. 144 h) the A TP-TCA permitted the identification of cell lines highly resistant to MTX (CCRF-CEM/T and CCRF-CEM/E), while intermediate MTX resistance due to altered polyglutamylation was not detectable. Detection of this ki nd of resistance was obtained, as expected, using a short-term exposur e (4 h) to MTX followed by a long-term efflux (72 h) in drug-free medi um, On the basis of these results, ATP-TCA appears to be a suitable me thod for the evaluation of cytotoxicity induced by MTX.