Cytotoxicity Malignant Human Cells for Protection against Methotrexate Thymidine and Hypoxanthine Requirements of Normal and Updated Version

The thymidine (dThd) and hypoxanthine (Hyp) requirements for protection of normal human granulocyte-macrophage col ony-forming units, B-, and T-cells against methotrexate-induced inhibition of proliferation were compared to require ments of three malignant solid-tumor cell lines recently estab lished from human tumor xenografts and of the CCRF-CEM Tcell leukemia. In the presence of excess Hyp, the concentration of dThd that provided 50% protection (PC50) was 0.2 JUMfor granulocyte-macrophage colony-forming units and normal Tcells; the dThd PC50for malignant cells was fiveto seven-fold higher, while that for B-cells was 10to 40-fold higher. An accurate Hyp PC50 for granulocyte-macrophage colony-form ing units could not be established; however, the Hyp PC50 for T-cells was 0.8 ¡IM, while that for malignant cells was four to 30 times greater and that for B-cells, two to six times greater. All cells tested were dependent on both salvage pathway metabolites for protection. Physiological ranges for dThd and Hyp in humans were established using newly developed highpressure liquid Chromatographie techniques to measure con centrations in normal subjects and solid-tumor cancer patients. In normal subjects, the geometric mean serum dThd was 0.13 fiM (range, 0.04 to 0.6 ¿IM), and the mean plasma Hyp, 0.51 UM (range, 0.2 to 1.9 ¡UM); cancer patients did not differ signif icantly. Neither excess dThd nor excess Hyp provided signifi cant protection for any cell in the presence of average physi ological concentrations of the second metabolite. The Hyp concentration in 14 freshly aspirated marrow specimens aver aged 11 ¿IMwhich was high enough to make protection of marrow dependent only on dThd. Relative to the wide range of physiological dThd and Hyp concentrations, the differences in PCso were not large enough to suggest that administration of protective doses of dThd or Hyp would increase the therapeutic ratio. These results provide an explanation for why, in vivo, dThd alone is sufficient to protect patients against myelo-