Melphalan Transport, Glutathione Levels, and Glutathionc-5-transferase Activity in Human Medulloblastoma1

Melphalan transport, glutathione levels, and glutathione-S-transferase activity were measured in two continuous human medulloblastoma cell lines and transplantante xenografts in attiviminude mice, TE-671 and Dany. In vitro mean glutathione levels were 10.06 nmol/10' cells in TE671 and 2.96 nmol/106 cells in Daoy. In vitro mean glutathione-.?transferase values were 91.52 nmol/min/mg protein in TE-671 and 50.31 nmol/min/mg protein in Daoy. Transport studies revealed kinetic param eters of Km= 108.3 JIM,V„.x = 363.1 pmol/106 cells/min in TE-671 and A',,,= III.7 Ã-Ã-M, V,»,= 180.6 pmol/10' cells/min in Daoy. Melphalan transport was inhibited by both DL-a-2-aminobicyclo[2.2.1]heptane-2carboxylic acid and sodium ion depletion in TE-671 and Daoy cells in vitro, indicating that both systems of amino acid transport are functional in these medulloblastoma lines. In vivo s.c. xenograft glutathione values were lower (7.79 nmol/mg protein) in TE-671 than in Daoy (13.68 nmol/ mg protein). The mean plasma concentration in mice given a 10% lethal dose (71.3 mg/m2) of melphalan i.p. was 50.3 «IMat 10 min, with the half-life of 29.9 min. At this dose, s.c. xenograft levels were 2to 3-fold higher in TE-671 than in Daoy tumors for the 3-h period measured. These studies demonstrate transport parameters confirming facilitated transport of melphalan in human medulloblastoma, a mean murine plasma melphalan concentration (following treatment with melphalan) above the in vitro drug dose at which there is a 90% reduction in the number of colonies in comparison to controls for TE-671 and Daoy for 2 h, and glutathione and glutathione-5-transferase levels in the same range previously reported in other melphalan-sensitive and melphalan-resistant human tumors. Future work with spontaneous and acquired melphalanresistant human medulloblastoma cell lines and xenografts will define the role of these mechanisms in mediating drug resistance.