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Effect of Intracellular Folate Concentration on the Modulation of 5-Fluorouracil Cytotoxicity by the Elevation of Phosphoribosylpyrophosphate in Cultured Human KB Cells¹

Chemotherapy Foundation Laboratory and Departments of Medicine and Neoplastic Diseases, Mt. Sinai School of Medicine, New York, New York 10029

The effects of extracellular folate concentration on intracellular folate and phosphoribosylpyrophosphate (PRPP) levels and the cytotoxicity of methotrexate and 5-fluorouracil were studied in human KB cells grown in fetal bovine serum-supplemented Eagle's minimum essential medium, which contained standard high folic acid levels (2.3 µM) (standard or S medium), or folic acid-free serum-supplemented medium containing approximately 4 nM 5-methyltetrahydrofolate (physiological or P medium), a folate level and form more comparable to that in normal human serum. Macrocytosis and prolongation of the doubling time by 150% were observed after 5–10 serial passes in P medium, but after 10–15 serial passes, KB cells became "adapted" to P medium with return of size and doubling time to values indistinguishable from cells maintained in S medium. Cellular folate levels fell, and marked elevations in PRPP levels from 68 ± 43 to 642 ± 287 pmol/mg cell protein (mean ± SD) were observed as KB cells were serially passed through P medium. Human leukemia HL-60 and K562 cells and MJY- mouse mammary tumor cells serially passed in P medium also exhibited 10- to 20-fold elevations in PRPP levels. Glucose consumption, glucose decarboxylation, thymidine and adenosine specific uptake, thymidine incorporation into DNA, and 5-fluorouracil uptake were studied in KB cells with elevated and control PRPP levels. As determined by clonal assay, despite elevated PRPP levels, KB cells cultured in P medium were less sensitive to 5-fluorouracil than cells cultured in S medium unless exogenous folate was added. These data support the concept that endogenous folate levels may be inadequate for optimal 5-FU pharmacological action in KB cells with a modulated increase in PRPP levels.

¹ Supported in part by an NIADDK NRSA AM07300 02 (M. A. K.), a Charles Revson Foundation Fellowship (M. A. K.), the Chemotherapy Foundation, and the Samuel Waxman Cancer Research Foundation. Portions of this work were presented as an abstract at the annual meeting of the American Association for Cancer Research, May 3–7, 1986, Los Angeles, CA.

² Present address: Denver VA Medical Center (111F), 1055 Clermont Street, Denver, CO 80220. To whom requests for reprints should be addressed.

³ Present address: Montefiore Medical Center, Albert Einstein College of Medicine, 111 East 210th Street, Bronx, NY 10467.

Received 3/ 4/87. Revised 9/21/87. Accepted 9/22/87.