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Effect of Uridine on Response of 5-Azacytidine-Resistant Human Leukemic Cells to Inhibitors of De Novo Pyrimidine Synthesis1

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Effect of Uridine on Response of 5-Azacytidine-Resistant Human Leukemic Cells to Inhibitors of De Novo Pyrimidine Synthesis1 [CANCER RESEARCH 44, 5505-5510, December 1984] Effect of Uridine on Response of 5-Azacytidine-resistant Human Leukemic Cells to Inhibitors of de Novo Pyrimidine Synthesis1 S. Grant,2 K. Bhalla,3 and M. Gleyzer Department of Medicine, Columbia University College of Physicians and Surgeons, New York, New York 10032 ABSTRACT activity is the most commonly encountered mode of resistance in animal systems (28). A uridine-cytidine kinase-deficient human promyelocytic leu- We have recently isolated a uridine-cytidine kinase-deficient, kemic subline (HL-60-5-aza-Cyd) has been isolated which is highly 5-aza-Cyd-resistant human promyelocytic leukemic sub- highly resistant to the antileukemic agent 5-azacytidine. Resist line (HL-60-5-aza-Cyd) (8) which is capable of surviving 5-aza- ant cells exposed to 10~5 M 5-azacytidine for 2 hr exhibit a Cyd concentrations (10~4 M) that exceed peak plasma levels in marked reduction in both the total ¡ntracellularaccumulation of humans (27). The purpose of the present studies was to assess 5-azacytidine (11.9 versus 156.0 pmol/106 cells) as well as its the metabolism of 5-aza-Cyd in these resistant cells and to incorporation into RNA (3.1 versus 43.4 pmol//ig o-ribose) com examine their response to a variety of clinically available inhibitors pared to the parent line. These biochemical changes are asso of de novo pyrimidine synthesis. Of the latter agents, PALA, an ciated with nearly a 100-fold decrease in sensitivity to the growth inhibitor of aspártele transcarbamylase (26), and pyrazofurin, an inhibitory effects of 5-azacytidine (concentration of drug associ ated with a 50% reduction in cell growth, 3.5 x 10~5 versus 5.0 inhibitor of orotidylate decarboxylase (5), are of particular inter x 10"7 M). 5-Azacytidine-resistant cells exhibit cross-resistance est, since their in vitro as well as in vivo effects have been shown to be reversible by Coadministration of exogenous uridine (4,16). to 3-deazauridine, 6-azauridine, and 5-fluorouridine, but not to This raises the theoretical possibility that drug-resistant cells 1-/3-D-arabinofuranosylcytosine, 2'-deoxyazacytidine, or 5-aza- impaired in their capacity to metabolize a naturally occurring 1-/3-D-arabinofuranosylcytosine. Coadministration of 50 ^M un nucleoside might be particularly vulnerable to a regimen using dine prevented depletion of pyrimidine nucleoside triphosphates such a nucleoside in conjunction with an appropriate pyrimidine and inhibition of colony formation of HL-60 cells exposed to 3 HIM A/-(phosphonacetyl)-L-aspartate) or 5 x 10~6 M pyrazofurin antagonist. Finally, examination of the effect of uridine on the response of normal and leukemic myeloid cells to inhibitors of but was not capable of protecting HL-60-5-azacytidine under the de novo pyrimidine biosynthesis might shed light on the role that same conditions. This uridine concentration was also able to salvage pathway enzymes play in overcoming the effects of de restore control colony formation to normal human bone marrow novo pyrimidine biosynthetic blockade. progenitor cells (granulocyte-macrophage colony-forming units) exposed to de novo pyrimidine biosynthetic blockade. These in vitro studies suggest that 5-azacytidine resistant cells lacking MATERIALS AND METHODS the pyrimidine salvage pathway enzyme uridine-cytidine kinase HL-60 cells are derived from the line originally isolated by Collins and may be selectively vulnerable to a regimen using pyrimidine Gallo (7) and are maintained in RPMI 1640 medium (Grand Island antagonists administered in conjunction with the naturally occur Biological Co., Grand Island, NY) supplemented with 10% heat-inacti ring nucleoside undine. vated fetal calf serum (Grand Island Biological Co.). Cells are subcultured twice weekly and examined routinely for Mycoplasme contamination. HL-60-5-aza-Cyd were obtained by subculturing HL-60 cells in progres INTRODUCTION sively higher concentrations of 5-aza-Cyd. Initially, 200 ml of HL-60 cells (approximate cell density, 106 cells/ml) were exposed to 5 x 10"8 M 5- The nucleoside analogue 5-aza-Cyd4 (NSC 102816) is an aza-Cyd for 4 days in a 37°, 5% CO2 incubator. The cells were then effective agent in the treatment of acute myelogenous leukemia washed free of medium and resuspended in 200 ml of fresh RPMI in humans (29). After conversion to its nucleoside monophos- medium containing 5 x 10"8 M 5-aza-Cyd. After an additional 4-day phate derivative by the pyrimidine salvage pathway enzyme incubation, the cell suspension was layered over a cushion of 3 ml uridine-cytidine kinase (21), 5-aza-Cyd is ultimately metabolized lymphocyte separation medium (specific gravity, 1.077 to 1.081 ; Bionet- to its lethal derivative 5-aza-Cyd-CTP, which is incorporated into ics, Kensington, MD) in sterile 50-ml centrifuge tubes. The tubes were leukemic cell RNA and to a lesser extent DNA (17). The bulk of centrifugea at 400 x g for 30 min. Viable cells at the interface layer were evidence suggests that 5-aza-Cyd-mediated cytotoxicity results extracted with a sterile Pasteur pipet and resuspended in fresh RPMI medium containing 10% fetal calf serum at an approximate concentration from interference with RNA production and processing and inhi of 2 x 10s cells/ml. The cells were placed in sterile tissue culture flasks bition of protein synthesis (20). Although no information is avail (Corning Glass Works, Corning, NY), to which was added sufficient 5- able concerning the mechanism by which leukemic patients aza-Cyd to yield a final concentration of 10~7 M. The flasks were placed become refractory to this agent, loss of uridine-cytidine kinase in the incubator, and the medium (containing fresh 5-aza-Cyd) was changed twice weekly. At approximately 2- to 3-week intervals, the 5- 1This work was supported by Grants CA-13696-12 and CA-35601-01 awarded aza-Cyd concentration was doubled and maintained at that level until by the National Cancer Institute, NIH, and by the William J. Matheson Foundation. 2 Special Fellow of the Leukemia Society of America. To whom requests for cells reached a growth rate nearly equivalent to that of the parent line. reprints should be addressed. After 6 months of subculturing, HL-60-5-aza are currently being main 3 Fellow of the Leukemia Society of America. tained in 10~* M 5-aza-Cyd, which is added to the medium twice weekly. 4 The abbreviations used are: 5-aza-Cyd, 5-azacytidine; PALA, N-(phosphona- cetylK-aspartate; CFU-GM, granulocyte-macrophage colony-forming unit. Cells maintained separately in drug-free medium for 3 months retained Received February 15,1984; accepted September 5,1984. 75% of their resistance to 5-aza-Cyd. More than 90% of the cells in CANCER RESEARCH VOL. 44 DECEMBER 1984 5505 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1984 American Association for Cancer Research. -~~:"ì 5-aza-Cyd-RESISTANT CELLS suspension culture are viable as determined by trypan blue exclusion. Soft-Agar Colony Growth: Normal and Leukemic Cells. The tech Drugs and Chemicals. PALA and 2,3-dihydro-1H-imidazo-[1,2b]pyr- nique for culturing human leukemic cells in soft agar has been described azole were furnished by Dr. Robert Engle, Drug Development Branch, in detail elsewhere (11). Logarithmically growing HL-60 and HL-60-5- National Cancer Institute (Bethesda, MD). 5-Aza-1 -/3-o-arabinofuranosyl- aza-Cyd were plated in the presence of 5 x 10"6 M pyrazofurin:3 mw cytosine was provided by Dr. John Douros, Drug Development Branch, PALA or both combined, along with uridine at concentrations ranging National Cancer Institute. Acivicin (AT-125) was provided by the Upjohn from 0 to 10"* M. At the end of a 10-day incubation in a 37°,5% CO2 Co., Kalamazoo, Ml. Pyrazofurin was purchased from Calbiochem, La fully humidified incubator, colonies consisting of groups of 50 or more Jolla, CA. Deoxyazacytidine, 5-aza-Cyd, 1-j3-o-arabinofuranosylcytosine, cells were enumerated with the aid of an inverted microscope. Values hydroxyurea, thymidine, 3-deazauridine, 6-azacytidine, 5-fluorouridine, for each condition were expressed as a percentage of untreated control- undine, and cytidine were purchased from Sigma Chemical Co., St. cell colony formation. Louis, MO. All agents were stored as dry powders at -20°. Prior to use, Parallel studies were performed utilizing a previously described drugs were reconstituted in RPMI medium and filter sterilized utilizing method for culturing normal human bone marrow myeloid progenitors 0.1 microfilters (Millipore Corp., Bedford, MA). (CFU-GM) (11). Samples were obtained with informed consent from [3H]Uridine (25 to 30 Ci/mol), [3H]cytidine (25 to 30 Ci/mmol), and patients undergoing routine diagnostic bone marrow aspirations for [3H]leucine (20 Ci/mmol) were purchased from Amersham/Searie Corp., nonneoplastic disorders. These studies have been sanctioned by the Arlington Heights, IL. [3H]5-aza-Cyd (1.4 Ci/mmol) was purchased from Human Investigation Institutional Review Board Committee, Columbia Morvek Biochemicals, Brea, CA. It was determined to be greater than University College of Physicians and Surgeons. Cells were plated in the 98% pure by high-pressure liquid Chromatographie analysis. presence of pyrazofurin, PALA, and uridine in the same manner as Intracellular Drug Accumulation Studies. The intracellular accumu cultured leukemic cells, and colonies were scored at Day 10. The ability lation of [3H]uridine, [3H]cytidine, and [3H]5-aza-Cyd was determined in of uridine to reverse the inhibitory effect of pyrazofurin or PALA on HL-60-5-aza-Cyd cells by a rapid centrifugation technique (10). Loga normal bone marrow progenitors was determined as for HL-60 and HL- rithmically growing cells were exposed to nucleoside or nucleoside 60-5-aza-Cyd cells.
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