[CANCER RESEARCH 46, 1754-1758, April 1986] Noninvasive Spectroscopic Analysis of Fluoropyrimidine Metabolism in Cultured Tumor Cells1 Max Keniry, Chris Benz,2 Richard H. Shafer, and Thomas L. James Departments of Pharmaceutical Chemistry [M. K., R. H. S., T. L. JJ and Radiology ¡T.L. J.J and Cancer Research Institute [C. B.], University of California, San Francisco, California 94143 ABSTRACT Nuclear magnetic resonance spectroscopy is a technique that may be used noninvasively to follow the intracellular metabolism of fluorinated antimetabolites such as 5-fluorouracil (FUra) and S-fluorouridine. Intra cellular "F spectral peaks are assigned by comparison with the pH- dependent chemical shifts measured for eight commercially available fluoropyrimidine metabolites as well as by comparison with the literature recorded values of five known catabolites of FUra. Five murine and human tumor cell lines (N,S„ Sarcoma 180, LI210, HI,-60, and Mia- PaCa) were exposed in vitro for 24 h to cytostatic doses of FUra or 5- fluorouridine. Treated cells were harvested and analyzed immediately or following a subsequent incubation under either nutrient-rich or nutrient- poor conditions. A major narrow component peak at 4.6-4.9 ppm was observed in all cell samples analyzed immediately after treatment. This peak was identified as intracellular FUra nucleotides, and its T, value was approximately 800 ms. No fluoropyrimidine catabolites were detect able in any of the treated cell lines. Free FUra could be measured in cells only after subsequent incubation under nutrient-poor conditions, and this was associated with a decline in the prominent FUra nucleotide peak. In treated cells chased with drug-free media containing 1 pM thymidine, spectra revealed a broad component signal underlying and downfield from the narrow nucleotide-containing peak. By biochemically fractionating treated cells into an acid-soluble fraction and phenol-purified cytoplasmic and nuclear RNA extracts, we were able to completely separate the nucleotide peak from the broad component signal resulting from FUra incorporation into RNA. Thymidine produced a marked enhancement of this "F signal into both cytoplasmic and nuclear RNA without affecting the nucleotide signal from the acid-soluble fraction. The present ability of nuclear magnetic resonance to monitor the metabolic channeling of fluoropyrimidines in intact tumor cells suggests that future spectroscopic imaging of patients treated with fluorinated antimetabolites may provide clinically important information about tumor biochemistry and drug sensitivity. INTRODUCTION Fluorinated pyrimidines are antimetabolites frequently used in the treatment of breast and gastrointestinal cancers (1); they are also occasionally used in the treatment of refractory lym- phomas and leukemias (2). The metabolism of these drugs has been extensively studied in murine and human tumor cell lines, and it now is well established that the rate limiting intracellular uptake of FUra,3 FUrd, or FdUrd is determined by three individual phosphorylating enzymes involved in pyrimidine synthesis (3-5). Once phosphorylated in the cell, the three fluoropyrimidines may interconvert or become anabolized into the primary cytotoxic nucleotides, FUTP and FdUMP. The former ribonucleotide is incorporated into RNA, disrupting Received 8/14/85; revised 12/30/85; accepted 1/2/86. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1Supported in part by grants CA-36769, CA-36773, and CA-27343 from the National Cancer Institute and CH-235B and NP-437 from the American Cancer Society. 2To whom requests for reprints should be addressed. 3 The abbreviations used are: FUra, 5-fluorouracil; FUrd, 5-fluorouridine; FdUrd, 5-fluorodeoxyuridine; FdUMP, 5-fluorodeoxyuridine monophosphate; FUDP, 5-fluorouridine diphosphate; FUTP, 5-fluorouridine triphosphate; NMR, nuclear magnetic resonance; FUMP, fluorouridine monophosphate. RNA processing and protein synthesis; the latter deoxynucleo- tide is a suicide inhibitor of thymidylate synthetase, an enzyme essential for DNA synthesis. The relative importance of these two anabolhes in the clinical antitumor effects of FUra remains a matter of dispute (6-8). Virtually all available data on the intracellular metabolism of fluoropyrimidines derive from the biochemical analysis of bro ken (lysed) cell preparations, usually following addition of some radiolabeled precursor. NMR is an alternative technique that may be used noninvasively to follow the metabolism of natural "F-containing compounds. We and others have shown that it is possible to monitor fluoropyrimidine metabolism within bac terial cells (9) or from the biological fluids of drug-treated animals (10, 11). The present study extends these results by utilizing "F-NMR to monitor in vitro the metabolism of FUra and FUrd in a variety of murine and human tumor cell lines. MATERIALS AND METHODS Drugs, Cells, Cytotoxicity, and Biochemical Assays. The commer cially obtained fluoropyrimidines used in this study were FUra, FUrd, FdUMP, 5-fluorouridylate, 5-fluorocytosine (Sigma, St. Louis, MO), 5-fluorocytidine (Hoffman-LaRoche, courtesy of A. F. Cook), 5-fluo- roorotate (Pharmacia, NJ), and FUTP (Sierra Bioresearch, Tucson, AZ). Tumor and leukemia cell lines were maintained as stock cultures in 5% CO2 incubators at 37°Cusing RPMI 1640 medium (Gibco Labo ratories, NY) supplemented with 5-10% fetal calf serum (HyClone Laboratories, UT). The NtSi (Novikoff). Sarcoma 180, and L1210 are well-characterized, rodent-derived hepatoma, sarcoma, and leukemia cell lines, respectively, which grow as cell suspensions in vitro. The MiaPaCa cells are a long-established monolayer cell line derived from an undifferentiated human pancreatic carcinoma, and the HI. 60 cells are a suspension culture of a widely available human promyelocytic leukemia cell line. For drug treatment prior to NMR analysis, or for assessment of drug Cytotoxicity, exponentially growing cells (in 75-enr sterile plastic flasks: Costar, Cambridge, MA) were exposed for 24 h to either FUra or FUrd at the indicated concentrations. All the cell lines studied were growth inhibited by 24-h exposure to either 100 MMFUra or 2.5 tiM FUrd. Complete growth curves were obtained for treated NiSi and Sarcoma 180 cells, as shown in Fig. 1. In most experiments, thymidine (1 pM) was added to the culture media 6 h following the addition of fluoropyrimidine to circumvent FdUMP inhibition of DNA synthesis and maximize incorporation of FUTP into RNA. After 24 h, the cells were harvested, rinsed free of drug, and then resuspended in fresh, thymidine-supplemented media. To measure the growth-inhibit ing effect of drug exposure, treated and untreated cells were then counted on a Coulter Counter (Coulter Electronics, Inc., Hialeah, FL) at frequent intervals for an additional 96 h, and these cell counts were plotted on log-linear graphs. For NMR analysis, the freshly harvested cells were chilled to 4*C and placed in a glass 10-mm diameter NMR tube for immediate spectroscopy. Biochemical fractionation was per formed on harvested Sarcoma 180 cells. Pellets of approximately 5x 10* cells were either treated with 0.5 M cold perchloric acid resulting in an acid-soluble extract containing catabolites, bases, nucleosides, and nucleotides (12) or alternatively, the chilled cells were gently lysed and separated into nuclear and cytoplasmic fractions for RNA purifi cation. Isolation and Purification of Cytoplasmic and Nuclear RNA. Sarcoma 1754 Research. on November 13, 2021. © 1986 American Association for Cancer cancerres.aacrjournals.org Downloaded from