Enhancement of trimetrexate cytotoxicity in vitro and in vivo by carboxypeptidase G2.
Carboxypeptidase G2 (CPG2), an enzyme produced by Pseudomonas strain RS-16, hydrolyzes the glutamate residue from methotrexate and other folates. The possibility of enhancing trimetrexate cytotoxicity by CPG2 induced folate depletion was investigated in vitro in a human leukemia cell line, CCRF-CEM, and in three sublines of these cells each with a different methotrexate resistance phenotype. The cytotoxic effect in vitro was detected using a colorimetric assay with a tetrazolium salt, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. Dose-effect relationships of drugs alone and in combination were analyzed by the median effect principle and by the combination indices for quantitation of synergy or antagonism with the aid of a computer program. Trimetrexate alone was cytotoxic against the parent and all the resistant cell lines with the drug concentrations required to decrease the cell count to 50% of control in the nanomolar range (1.4, 1.6, 1.5, and 0.7 nM in CCRF-CEM, CCRF-CEM/E, CCRF-CEM/P, and CCRF-CEM/T, respectively) following 5 days of exposure. The concentration of CPG2 required to decrease the cell count to 50% control for these cell lines was 3.5, 2.6, 26.6, and 7.9 x 10(-5) units/ml for CCRF-CEM, CCRF-CEM/E, CCRF-CEM/P, and CCRF-CEM/T, respectively. A synergistic cytotoxic effect of trimetrexate after simultaneous continuous exposure with CPG2 was observed with CCRF-CEM cells and with the three resistant cell lines. This drug combination given to BALB/c x DBA/2 F1 mice bearing L1210 cells also produced synergy over a narrow range of drug doses. The activity of this combination in both methotrexate sensitive and methotrexate resistant cell lines indicates that clinical trials of this combination should be undertaken.
Romanini
A
A
Laboratory of Molecular Pharmacology, Memorial Sloan-Kettering Cancer Center, New York, New York 10021.
Sobrero
A F
AF
Chou
T C
TC
Sherwood
R F
RF
Bertino
J R
JR
D000818:Animals; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects*; D002999:Clone Cells; D003546:Cysteine Endopeptidases / Q000494:pharmacology*; D004351:Drug Resistance; D004357:Drug Synergism; D005492:Folic Acid / Q000032:analysis; D006801:Humans; D007939:Leukemia L1210 / Q000188:drug therapy*; D051379:Mice; D008815:Mice, Inbred Strains; D011799:Quinazolines / Q000494:pharmacology* / Q000627:therapeutic use; D016597:Trimetrexate; D014410:Tumor Stem Cell Assay; D011623:gamma-Glutamyl Hydrolase / Q000494:pharmacology* / Q000627:therapeutic use
D011799:Quinazolines; D005492:Folic Acid; D011623:gamma-Glutamyl Hydrolase; D003546:Cysteine Endopeptidases; D016597:Trimetrexate