(CANCER RESEARCH 49. 4824-4828. September I. 1989] Induction of HL-60 Leukemia Cell Differentiation by the Novel Antifolate 5,10- Dideazatetrahydrofolic Acid1 John A. Sokoloski, G. Peter Beardsley, and Alan C. Sartorelli2 Departments of Pharmacology and Pediatrics and Developmental Therapeutics Program, Comprehensive Cancer Center, Yale University School of Medicine, New Haven, Connecticut 06510 ABSTRACT as an inhibitor of folate metabolism which acted at an enzymatic level other than dihydrofolate reducíase,the site of action of The novel tetrahydrofolate, 5,10-dideazatetrahydrofolic acid most classical antifolates, including aminopterin and metho- (DDATHF), was designed as an inhibitor of folate metabolism at a site other than dihydrofolate reducíase.DDATHF has been shown to inhibit trexate (15, 16). DDATHF closely resembles tetrahydrofolate, glycinamide ribonucleotide transformylase, a folate-requiring enzyme that thereby retaining the structural properties necessary for mem catalyzes the first of two one-carbon transfer reactions in the de novo brane transport and for it to serve as a substrate for folylpoly- purine nucleotide biosynthetic pathway. Incubation of HL-60 promyelo- glutamate synthetase (16). The presence of a fully reduced ring cytic leukemia cells with 5 x 10~* to IO"5 M DDATHF resulted in a and the 2-amino-4-oxo-substituents rather than the 2,4-dia- marked inhibition of growth after 48 h, with a complete cessation of mino-substitution pattern found in classical antifolates presum cellular replication by day 4. Cell cycle analyses of DDATHF-treated ably are responsible for the lack of inhibition of dihydrofolate HL-60 cells demonstrated an initial block in early S phase by day 3 reducíaseby DDATHF. followed by an accumulation of cells in the G, and <. + M phases of the Reduced folates particípalein a variely of one-carbon transfer cell cycle. Inhibition of growth was accompanied by a concentration- reaclions important for cell growlh, such as Ihe production of dependent increase in the percentage of mature myeloid cells that ex pressed nitroblue tetrazolium positivity, and a small increase in nonspe melhionine from homocysteine, the interconversion of serine cific esterase activity. Induction of differentiation and inhibition of growth and glycine, and the calabolism of several amino acids, in by DDATHF were completely prevented by hypoxanthine and 5(4)- addition to Iheir role in Ihe de novo production of purine amino-4(5)-imidazole carboxamide, suggesting that depletion of intracel- nucleotides and Ihymidylale (17). The presence of carbon aloms lular purine nucleotide pools has an important role in the biological in place of nilrogens al posilions 5 and 10 necessarily prevenís effects of this inhibitor. This possibility was confirmed by the finding DDATHF from parlicipaling as a cofactor in Ihese reaclions, that DDATHF caused a pronounced reduction in intracellular GTP and and also provides for increased chemical slabilily. ATP levels within 2 h, with maximum decreases being observed by 24 h, Previous studies with DDATHF have indicated that the a time interval which preceded the inhibition of cellular proliferation by parenl compound or a polyglutamyl derivative is a potent this agent. Pyrimidine nucleoside triphosphate levels were markedly inhibitor of de novo purine nucleolide biosynlhesis (16, 18). increased under these conditions. The findings indicate the importance of purine nucleotides to both the inhibition of growth and the induction of Furthermore, evidence has been presenled lo suggesl lhal GAR differentiation of HL-60 leukemia cells by DDATHF. Iransformylase, Ihe first of Iwo folale-requiring enzymes lhal calalyze one-carbon Iransfer reaclions in Ihe de novo purine nucleolide biosynlhelic palhway is the primary cellular target INTRODUCTION of this agent (18). Since the cyloloxicily of DDATHF in cul- The HL-60 promyelocytic leukemia has been used extensively lured cells appears lo be dependeni upon Ihe depletion of as a model of hematopoietic development (1). HL-60 cells are inlracellular purine nucleolide levels, it was of interesl lo sludy multipotent and have been shown to be capable of differentiat Ihe effecls of DDATHF on Ihe growlh and differenlialion of ing in vitro to cells with characteristics of mature granulocytes HL-60 promyelocylic leukemia cells. (2, 3) or monocytes (4, 5) by a variety of different inducing The presenl sludy demonslrales lhal DDATHF is a polenl agents including several antitumor agents (6-9). The differen inducer of Ihe maluration of HL-60 leukemia cells. Further tiation of HL-60 cells to eosinophils and basophils has also more, Ihe findings indicale lhal Ihe induclion of differentiation been reported (10, 11). Among the initiators of maturation are by DDATHF is closely associated with Ihe inhibilion of de novo the novel C-nucleoside, tiazofurin, and related inhibitors of purine nucleotide biosynthesis, presumably al Ihe reaclion cal- IMP dehydrogenase (12, 13). The induction of the differentia alyzed by GAR Iransformylase. tion of HL-60 cells by these agents, which diminishes intracel lular GTP pools, suggests that GTP depletion may be involved MATERIALS AND METHODS in modifying intracellular signals which increase the probability of a cell entering a differentiation pathway. The de novo inhib Cell Culture. HL-60 promyelocytic leukemia cells were originally itor of purine nucleotide biosynthesis, 6-methylmercaptopurine supplied by Dr. Robert C. Gallo of the National Cancer Institute, ribonucleoside, which produces depletion of both GTP and Bethesda, MD. The HL-60 cells used in these experiments were ex ATP, is also an effective inducer of HL-60 maturation (14). panded from stocks frozen in liquid nitrogen, and consisted of cells The novel tetrahydrofolate, DDATHF' (Fig. 1), was designed between passages 25 and 65. Cell stocks were routinely checked for Mycoplasma contamination by the gene probe method (Gen-Probe, Received 11/8/88; revised 5/11/89: accepted 6/7/89. Inc., San Diego, CA). The costs of publication of this article were defrayed in part by the payment Cells were routinely passaged in RPMI 1640 medium (GIBCO, of page charges. This article must therefore be hereby marked advertisement in Grand Island, NY) supplemented with 15% heat-inactivated (50°Cfor accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 30 min) fetal calf serum (GIBCO). Cultures were maintained at 37°C 1This research was supported in part by USPHS Grants CA-02817 and CA- 42300 from the National Cancer Institute. in a humidified atmosphere containing 5% COs in air. Experiments - To whom requests for reprints should be addressed, at Department of were performed by resuspending cultures of HL-60 cells at a density of Pharmacology. N'aie University School of Medicine, P. O. Box 3333. New Haven. 5 x IO4 cells/ml in the presence of DDATHF for 7 days at 37°C. CT 06510-8066. Solutions of DDATHF were prepared in 0.1 N NaOH and adjusted to 'The abbreviations used are: DDATHF. 5.10-didea/atetrahydrofolic acid; IO"3 M in PBS using an £:7,nm (0.1 N NaOH) value of 9.15 x 10"* PBS, phosphate-buffered saline: NBT. nitroblue tetra/olium: AICA. 5(4)-amino- 4(5)-imida/ole carboxamide: GAR, glycinamide ribonucleotide. M~' cm~' (18). Cell numbers were determined daily with a Coulter 4824 Downloaded from cancerres.aacrjournals.org on October 4, 2021. © 1989 American Association for Cancer Research. DDATHF INDUCTION OF DIFFERENTIATION COOH | Fig. 1. Chemical structure of DDATHF. particle counter equipped with a channelizer (Coulter Electronics, Hialeah, FL). Viable cells were ascertained by using a 0.1 % try pun blue solution and a hemocytometer. Smears were prepared with a Shandon- Southern Cytospin (Johns Scientific, Toronto, Ontario, Canada) and were stained with May-Grunwald-Giemsa. 0 48 96 144 Assessment of Differentiation. The capacity of HL-60 cells exposed Time (h) to DDATHF to undergo functional maturation was determined by NBT dye reduction (19). Approximately 1 x IO6cells were collected by Fig. 2. Effects of DDATHF on the proliferation of HL-60 leukemia cells. Five x 10* cells/ml were incubated with various concentrations of DDATHF for 7 centrifugation and resuspended in 1.0 ml of RPMI 1640 medium days. Cell numbers were measured daily with a Model ZBI Coulter particle containing 0.1% NBT (Sigma Chemical Co., St. Louis, MO) and 1.0 counter. Treatment: none (x), 0.01 >IM(D), 0.1 >iM(T), 1.0 >IM(A), and 10 >IM /ig/ml of 12-0-tetradecanoylphorbol-13-acetate (Sigma) in ethanol. The (•)DDATHF. cell suspension was incubated for 30 min at 37°C,and the percentage of cells containing blue-black formazan granules, indicative of a 12-0- B.S teiradecanoylphorbol-13-acctale-stimulated respiratory burst, was de G, -36.7 •¿37.a termined microscopically. Nonspecific-esterase (a-naphthylbutyrate es S -442 -40.1 S •¿40.0 G,»M- 19.1 „¿.UC.¡G,az*M'23.l GjtM'22.7y terase) activity was monitored as described by Yam et al. (20). Flow Cytometry. Flow cytometry was performed by using a FACS IV fluorescence-activated cell sorter (Becton Dickinson, Mountain View, CA). Cells were washed twice in ice-cold PBS, fixed in 70% ethanol, and stored at 4°Cfor up to 2 weeks prior to analysis. Histo grams of the relative DNA content of induced cells were obtained from » w* D. 30,000 cells stained with SO/¿I/mlofpropidium iodide after treatment O G, »14.8 ' 39.9 with 1 mg/ml of RNase for 30 min at 37°C.The percentage of cells in S -62.1 •¿40.6 G,»M'23.I the G,, S, and G2 + M compartments of the cell cycle was determined by the method of Jett (21). Analysis of Ribonucleotide Pools. HL-60 leukemia cells (1 to 2 x 10' cells/ml) were suspended in fresh RPMI 1640 medium containing 10 '' M DDATHF.