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

Departments of Pediatrics [H. S. F., J. K., S. X. S.], Pathology [H. S. F., D. D. B.], and Medicine [G. B. E., S. C. S.], Duke University Medical Center, Durham, North Carolina 27710; The Johns Hopkins Oncology Center, Baltimore, Maryland 21205 [O. M. C., J. H.]; and Burroughs Wellcome Co., Research Triangle Park, North Carolina 27709 [M. R. B., P. M. S.]

Melphalan transport, glutathione levels, and glutathione-S-transferase activity were measured in two continuous human medulloblastoma cell lines and transplantable xenografts in athymic nude mice, TE-671 and Daoy. In vitro mean glutathione levels were 10.06 nmol/10⁶ cells in TE-671 and 2.96 nmol/10⁶ cells in Daoy. In vitro mean glutathione-S-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 parameters of K_m = 108.3 µM, V_max = 363.1 pmol/10⁶ cells/min in TE-671 and K_m = 111.7 µM, V_max = 180.6 pmol/10⁶ cells/min in Daoy. Melphalan transport was inhibited by both DL--2-aminobicyclo[2.2.1]heptane-2-carboxylic 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 µM at 10 min, with the half-life of 29.9 min. At this dose, s.c. xenograft levels were 2- to 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-S-transferase levels in the same range previously reported in other melphalan-sensitive and melphalan-resistant human tumors. Future work with spontaneous and acquired melphalan-resistant human medulloblastoma cell lines and xenografts will define the role of these mechanisms in mediating drug resistance.

¹ This work was supported by NIH Grants CA11898, 1 NINCDS NS 20023, 1 K07 NS 00958, and CA44640, and by American Chemical Society Grant 403.

² To whom requests for reprints should be addressed at Department of Pediatrics, Duke University Medical Center, Durham, NC 27710.

Received 3/17/88. Revised 6/17/88. Accepted 6/28/88.

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