[CANCER RESEARCH 44, 3355-3359, August 1984] Enhancement of the Sensitivity of Human Colon Cancer Cells to Growth Inhibition by Acivicin Achieved through Inhibition of Nucleic Acid Precursor Salvage by Dipyridamole1 Paul H. Fischer,2 Rifat Pamukcu, Gerard Bittner, and James K. V. Willson2-3 Department of Human Oncology, University of Wisconsin School of Medicine, Madison, Wisconsin 53792 [P. H. F., R. P., J. K. V. W.J, and William S. Middleton Memorial Veterans Medical Center, Madison, Wisconsin 53705 [G. B., J. K. V. W.] ABSTRACT mammary and lung xenografts (8). Although acivicin inhibits numerous enzyme reactions involving the transfer of nitrogen This study was undertaken to determine if salvage of nucleic from the 7-carboxamide of L-glutamine (10), including L-aspara- acid precursors might constitute a mechanism of resistance to gine synthetase (3), carbamoyl phosphate synthetase II (1, 10), acivicin in human colon cancer cells and, if so, to establish phosphoribosylformylglycinamidine synthetase (10), and amido- whether dipyridamole, an inhibitor of nucleoside and nucleobase phosphoribosyl transferase (4, 10), inhibition of 2 other L-gluta- transport, can block the salvage process and restore sensitivity mine-dependent enzymes, CTP synthetase (4,10,18) and XMP to acivicin. Acivicin inhibited the replication of human colon aminase (10), appears to be most closely associated with the cancer cells (VACO 5) in vitro in a dose- and time-dependent cytotoxicity of acivicin. Depletion of the CTP and GTP pools and fashion. In addition, marked cell lysis was evident after a 24-hr expansion of the DTP pool have typically been seen (10,13, 14, exposure to acivicin at concentrations greater than 1 ng/m\. The 30). primary metabolic effect of acivicin was depletion of the cytidine In rodent cancer cells, perturbation of both purine and pyrimi- triphosphate and guanosine triphosphate pools. Adenosine tri- dine metabolism is evidently critical, since cytosine and guanine phosphate levels were also reduced, but apparently as a con nucleosides antagonize acivicin cytotoxicity much more effec sequence of the guanosine triphosphate depletion. VACO 5 cells tively when they are used in combination than when they are exposed to acivicin (3 /iQ/ml) efficiently salvaged low levels (1 used alone (18,30). These data suggest the potentially important í¿m)ofcytidine, guanosine, and guanine and could, therefore, role which salvage of nucleic acid precursors might play in restore the depleted nucleotide pools. The combination of cyti modifying the actions of acivicin. In particular, studies on rat dine and guanosine, but not either nucleoside alone, provided hepatoma 3924A cells have documented the ability of nucleo significant protection against the growth-inhibitory properties of sides to circumvent the growth-inhibitory effects of acivicin (14, acivicin. Dipyridamole, at a noncytotoxic concentration (5 /¿M), 24,25,30). Furthermore, dipyridamole, an inhibitor of nucleoside blocked repletion of the cytidine triphosphate and guanosine transport (11, 19-21), antagonized the protection afforded by triphosphate pools in cells exposed to acivicin and the nucleic salvage mechanisms and substantially enhanced the cytotoxicity acid precursors. As a result, the growth-inhibitory effects of of acivicin (24, 25, 30). Since the antitumor activity of acivicin in acivicin were maintained. The salvage of cytidine was particularly humans is now being evaluated in Phase II trials, including colon sensitive to inhibition by dipyridamole, and no restoration of cancer, we felt that several important questions regarding the cytidine triphosphate pools was evident. The cellular uptake of actions of this drug in human cells should be addressed. Does a variety of nucleic acid precursors was differentially sensitive to acivicin inhibit the growth of a human colon cancer cell line, such inhibition by dipyridamole. The 50% inhibitory dose values ranged as VACO 5? Are the cytotoxic effects seen at clinically achievable from 0.01 to 2.5 fiM for cytidine and uridine, respectively. The concentrations and times of exposure to acivicin? Does salvage results of this study indicate that, although the replication of of nucleic acid precursors appear to be a likely mechanism of VACO 5 cells was inhibited by acivicin, low levels of nucleosides resistance to acivicin? Does the use of dipyridamole block reple and nucleobases can circumvent the cytotoxicity. Dipyridamole tion of the nucleotide pools and provide a reasonable therapeutic effectively blocked the salvage pathways and restored the sen approach for maintaining the growth-inhibiting properties of aciv sitivity of the cancer cells to the antiproliferative actions of icin? acivicin. MATERIALS AND METHODS INTRODUCTION Acivicin,4 a structural analogue of L-glutamine, is an antitumor Materials. Acivicin (NSC 163501) was obtained from the National Cancer Institute, Investigational Drugs division. Dipyridamole, nucleo- agent isolated from Streptomyces sviceus (6). The compound tides, nucleosides, and nucleobases were purchased from either Sigma has activity against several murine tumors, the L1210 and P388 Chemical Co. (St. Louis, MO) or P-L Biochemicals (Milwaukee, Wl). leukemias (6) and M5076 ovarian carcinoma (8), and human Aldrich Chemical Co. (Milwaukee, Wl) supplied the Alamine (tri-n-octyla- 1 Supported in part by CA 14520 and the Veterans Administration Medical mine) and Freon (1,1,2-trichlorotrifluoromethane). [8-3H]Guanosine (5 Ci/ mmol), [5-3H]cytidine (30 Ci/mmol), and [8-"C]guanine sulfate (51 nCi/ Research Service. Portions of this study have been presented at the Central Society for Clinical Research, November 2,1983, Chicago, IL (28). mmol) were purchased from Amersham/Searie Corp. (Arlington Heights, 2 To whom requests for reprints should be addressed. IL). [5-3H]Uridine (20 Ci/mmol) and [5-3H]deoxycytidine (25 Ci/mmol) 3 Recipient of an American Cancer Society Junior Faculty Clinical Fellowship. 'The abbreviation used is: acivicin, L-(aS,5S)-a-amino-3-chloro-4,5-dihydro-5- were obtained from Moravek Biochemcials, Inc. (Brea, CA). isoxazolacetic acid. Cell Culture. VACO 5 cells, a human colorectal cell line, were grown Received November 14,1983; accepted May 9,1984. in Eagle's minimal essential medium supplemented with 2 mM u-gluta- AUGUST1984 3355 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1984 American Association for Cancer Research. P. H. Fischer et al. mine, 0.1 mu nonessential amino acids (Grand Island Biological Co., Grand Island, NY), insulin (2 //g/ml), transferrin (2 ¿jg/ml),and 7.5 nM sodium selenite (Sigma), gentamicin (50 ^g/ml) (Schering Co., Kenilworth, NJ), and 8% heat-inactivated fetal bovine serum, Lot 100374 (Hyclone, O.Ijig/ml Logan, UT). The contributions from the serum yielded final medium concentrations of less than 0.1 /JM guanosine and cytidine and 2.1 ^M guanine. Dissociation medium was identical to growth medium, except that a Ca2*- and Mg2+-free minimal essential medium was used and fetal bovine serum was excluded. VACO 5 cells were established from a poorly differentiated adenocarcmoma of the cecum and characterized as described previously (16). Stock cultures are passaged weekly and have maintained morphological, karyotypic, and drug sensitivity characteristics during the 12-month course of this study. VACO 5 cells were determined to be negative for Mycoplasme contamination, as demonstrated by culture under aerobic and anaerobic conditions (Wisconsin State Labo ratory of Hygiene, Madison, Wl). Cytotoxicity. Exponentially growing cells, initially plated at a density of 2 x 10* cells/ml in 25-sq cm tissue culture flasks, were maintained at 37°in a humidified 5% CO2 atmosphere. Medium containing the various additives was added 12 hr later. In the experiment described in Chart 1, 24 48 72 96 the exposure to acivicin was continued for an additional 2 to 96 hr. The EXPOSURE TO ACIVICIN (hr) cells were then washed twice, fresh medium was added, and the incubation was continued such that the entire growth period was 96 hr. Chart 1. Effects of acivicin on the replication of VACO 5 cells. Exponentially growing cells were plated at a density of 2 x 104 cells/ml and incubated for 12 hr For the experiment shown in Chart 3, the cells were exposed to acivicin, the nucleosides, or dipyridamole for 24 hr. The cells were then washed prior to the addition of acivicin. The cells were then exposed to acivicin at the indicated concentrations for increasing periods of time (2 to 96 hr). Following drug twice and incubated for 72 hr in fresh medium containing the nucleosides treatment, the cells were washed twice, and fresh medium was added. Incubation and dipyridamole as shown. Cells were harvested after a 1- to 2-hr was continued for a total of 96 hr for all conditions. The data are expressed as the exposure to a Ca2+ and Mg2* free dispersion medium. Cells excluding percentage of control cell number present at the end of 96 hr. trypan blue were counted using a hemocytometer. Measurement of Nucleoside Triphosphate Pools. Nucleoside tri- 200r UTP phosphate pool extractions were carried out on duplicate flasks in parallel with the cell growth studies. VACO 5 cells (2.5 to 12 x 106) were collected by centrifugation (10 min, 200 x g), the medium was removed, and the cells were resuspended in 1 ml of cold phosphate-buffered saline. The suspension was transferred to a 1.5-ml microfuge tube and centrifugea (30 sec, 11,500 rpm). The supernatant was removed, and the cell pellet was extracted in 0.1 ml of 0.5 M HCIO4 for 10 min at 4°. The extract was neutralized with an equal volume of 0.5 M Alamine in Freon (12). The neutralized samples were stored at -20° until analyzed 1 I 10 1 I 10 I 1 10 I I 10 by high-pressure liquid chromatography. Chromatography of the samples ACIVICIN(pg/ml) was performed on a system consisting of a Spectra-Physics 8700 solvent Chart 2. Effects of acivicin on ribonucleotide triphosphate pools in VACO 5 delivery system, a Whatman Partisil 10/25 SAX anion-exchange column cells.