Induction of HL-60 Leukemia Cell Differentiation by the Novel Antifolate 5,10- Dideazatetrahydrofolic Acid1

(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 reducÃase,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 reducÃase.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 reducÃaseby DDATHF. followed by an accumulation of cells in the G, and <. + M phases of the Reduced folates particÃpalein 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 prevenÃs 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 (50Â°Cfor accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 30 min) fetal calf serum (GIBCO). Cultures were maintained at 37Â°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 37Â°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 Â£: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

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 (â€¢)DDATHF. cell suspension was incubated for 30 min at 37Â°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 â€¢¿37.a termined microscopically. Nonspecific-esterase (a-naphthylbutyrate es S -442 -40.1 S â€¢¿40.0 G,Â»M- 19.1 â€ž¿.UC.Â¡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 4Â°Cfor up to 2 weeks prior to analysis. Histo grams of the relative DNA content of induced cells were obtained from Â» w* D. 30,000 cells stained with SO/Â¿I/mlofpropidium iodide after treatment O G, Â»14.8 ' 39.9 with 1 mg/ml of RNase for 30 min at 37Â°C.The percentage of cells in S -62.1 â€¢¿40.6 G,Â»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. At intervals, the cells were collected by centrifugation, washed twice in 2.0 ml of ice-cold PBS, and extracted for analysis by Fluorescence (DMA content) high-performance liquid chromatography as previously described (13). Fig. 3. DNA histograms of HL-60 leukemia cells after treatment with DDATHF. HL-60 cells were incubated with IO"6 M DDATHF for the indicated periods of time. Aliquots of 2 x IO6cells were removed, stained with propidium iodide, and relative DNA contents were measured with a Becton-Dickinson FACS RESULTS IV flow cytometer as described in "Materials and Methods." Data are from a representative experiment from three independent experiments. Control (A), 24 The incubation of HL-60 leukemia cells with DDATHF at h (B), 48 h (C), 72 h (D), 120 h (Â£),168 h (F). concentrations of from 10~8 M to 10~5 M resulted in a loss of cellular proliferative capacity that became apparent at 72 h, and parable degree of inhibition of HL-60 cellular replication were was followed by a complete cessation of cellular replication by associated with a progressive concentration-dependent increase 96 h (Fig. 2). This corresponded to results obtained with DNA in the percentage of cells capable of reducing NBT, to 80% of histograms of HL-60 cells exposed to IO"6 M DDATHF, which the cell population at 10 " M, the maximum concentration of remained comparable to those seen with untreated control DDATHF used. It is possible that a relationship exists between cultures for the initial 48 h (Fig. 3). By 72 h, however, a marked the effects of long-term exposure to DDATHF on the growth reduction in the G, and G2 + M cell compartments was ob and differentiation of HL-60 cells. Thus, it would appear that served, concomitant with an increase in the population of cells DDATHF, when present at optimal concentrations for differ in the early S phase of the cell cycle. Continued incubation of entiation, imposes a growth restraint on HL-60 cells. However, HL-60 cells with DDATHF for 5 days resulted in a progression the inhibition of cellular replication, which is essential for of the cohort of cells initially blocked in early S phase to late S differentiation, is not by itself sufficient to initiate the matura and G: + M, with a further reduction in the number of cells in tion program, since lower concentrations of DDATHF, which G,. By day 7, a marked decrease in the size of the early S phase limited cellular growth, were unable to induce significant dif population was evident, as cells accumulated in both the G, and ferentiation. Differentiation, however, was observed to increase the late S and G2 + M phases of the cell cycle. This contrasts in a progressive manner at higher concentrations of DDATHF, with the CCRF-CEM cell line which is blocked irreversibly in without further inhibition of cellular proliferation, suggesting the G i and early S phases of the cell cycle by 72 h after exposure that these levels of DDATHF were sufficient to trigger the to 1.0 MMDDATHF (18). signals necessary for the induction of maturation, in addition Exposure of HL-60 cells to DDATHF for 7 days resulted in to blocking growth. Under these conditions, DDATHF had a pronounced concentration-dependent increase in NBT posi- little or no effect on HL-60 cell viability, since greater than tivity, a phenotypic marker of the differentiated state. As shown 95% of treated cells were able to exclude trypan blue after 7 in Fig. 4A, concentrations of DDATHF which caused a com- days of exposure to DDATHF. In addition, DDATHF caused 4825

-100 100 < periods longer than 72 h proliferated slowly after removal of the inducer. As shown in Fig. 5, inset a minimum period of 48 h of exposure to DDATHF was required to irreversibly commit HL-60 cells to enter a differentiation pathway and form a mature phenotype in the absence of inducer. Previous studies using CCRF-CEM cells (18) have demon

o o strated that DDATHF, at concentrations producing cellular 5 O) toxicity, caused a marked decrease in the level of cellular purine nucleotides, presumably due to the inhibition of GAR transfor- oo mylase, an early step in the purine nucleotide biosynthetic pathway. When HL-60 cells were incubated in the presence of DDATHF at a concentration of 10~6 M, both GTP and ATP levels were significantly reduced within 4 h, with a maximum 20 decrease in the GTP content being observed at 24 h, followed by a partial rebuilding of the GTP pools by day 6 (Fig. 6). In contrast, a gradual reduction in the levels of ATP was observed

.1 1 10 with a maximum decrease on day 6. Under these conditions, UTP levels were markedly increased by 10 h, with a gradual DDATHF Concentration (\iM) return to control levels by day 6. A similar pattern was seen for CTP levels (data not shown). Thus, the decrease in intracellular 100 < purine nucleotide concentrations preceded the inhibition of cellular growth and the attainment of the differentiated state, suggesting that alterations in purine nucleotide pools are im portant in the regulation of the induction of HL-60 cell differ entiation by this agent. This conclusion was confirmed by studies which demonstrated that the inhibition of growth (Fig. o O 0> 7/1) and the induction of differentiation (Fig. IB) by DDATHF O were prevented by the simultaneous exposure of HL-60 cells to 8 the metabolic precursors of purine nucleotide formation, hy- C o o poxanthine and AICA. ~0

40-

20 .01 .1 1 100 DDATHF Concentration Fig. 4. Inhibition of cellular proliferation and induction of differentiation produced by DDATHF. HL-60 human leukemia cells (/(). and WEHI-3B D* 24 48 72 96 168 murine leukemia cells (fi), were incubated in the presence of various concentra Time (h) tions of DDATHF. Cell numbers (â€¢)andNBT positively (O) were determined on day 7 for HL-60 and day 3 for WEHI-3B D* cells. Data represent the mean of three independent experiments; bars. SE. a concentration-dependent increase in the number of differen tiated murine myelomonocytic WEHI-3B D+ cells, with 36% of the cells being NBT positive at 100 ^M DDATHF (Fig. 4B). Commitment was assessed by monitoring the growth and differentiation of treated cells cloned in fresh RPMI 1640 complete medium containing 0.35% agar. Attainment of the differentiated state by cultures exposed to 10~6 M DDATHF for 72 h was accompanied by the production of a population of relatively small, loosely dispersed colonies expressing NBT positivity after 12 days in semisolid medium (data not shown). Under these conditions, continuous exposure to 3 x 10~8 to 10~5 M DDATHF had little or no effect on the formation of 24 48 72 96 120 168 colonies by HL-60 cells, implying that the loss of replicative capacity observed after a 48-h period of exposure to this agent Time (h) was due to the irreversible differentiation of HL-60 cells and Fig. 5. Effects of DDATHF on the commitment of HL-60 leukemia cells to a not to an early cytotoxic event. As shown in Fig. 5, HL-60 cells differentiation pathway. Cells were incubated in the presence of 1.0 Â¿IMDDATHF. At various periods of time thereafter, aliquots of cells were removed, washed exposed to DDATHF for 24 h retained normal growth capacity twice with PBS, and recuttured in fresh RPMI 1640 medium. The effects of after removal of the drug, whereas cells exposed to DDATHF DDATHF on cell growth: control (D), 24 h (â€¢),48h (A), 72 h (A), and 96 h (â€¢) of exposure to DDATHF. Inset, the commitment of HL-60 cells to differentiate, for 48 h required a short lag period before resuming normal as measured by NBT positivity, was ascertained 7 days after removal of drug, and growth. In contrast, HL-60 cells exposed to DDATHF for compared to cultures that were exposed continuously to DDATHF for 168 h. 4826

200 â€¢¿20

o 100 Ãœ o

O 24 48 72 96 120 144

Time (h)

Fig. 6. Effects of DDATHF on the nuclcoside triphosphate levels of HL-60 cells. HL-60 leukemia cells were incubated in the presence of 10"* M DDATHF for the indicated periods of time. Duplicate aliquots were removed, washed with PBS. extracted, and analyzed for nucleoside triphosphates by high-performance liquid chromatography as described in "Materials and Methods." ATP (O), GTP (â€¢), DTP (D). Data are expressed as the average percentage of control values from two experiments run in duplicate.

DISCUSSION DDATHF Concentration (

DDATHF was originally synthesized as an inhibitor of en Fig. 7. Effects of precursors of purine nucleotide biosynthesis on the inhibition of HL-60 Icukemic cell growth (A) and the induction of differentiation (B) by zymes of folate metabolism other than dihydrofolate reducÃase DDATHF. Cells were incubated with 10"" M DDATHF in the absence (A) or (15, 16). DDATHF possesses potent antitumor activity against presence of various concentrations of hypoxanthinc (A) or AICA (â€¢).Cell a variety of experimental tumor models both in vivo and in numbers and NBT positively were determined on day 7. Each value represents vitro, including the B-16 melanoma and other solid tumors the mean of three independent experiments for each precursor; bars, SE. which are relatively resistant to other antifolates, such as meth- otrexate (16). depletion of guanine nucleotides was associated with the induc In this report, we demonstrate that DDATHF is an effective tion of the maturation of the HL-60 leukemia by several inhib inducer of the terminal maturation of HL-60 promyelocytic itors of IMP dehydrogenase, including tiazofurin, its selenium leukemia cells, causing a progressive concentration-dependent analogue selenazofurin, ribavirin and mycophenolic acid, an increase in the development of a differentiated phenotype over effect also observed using the inhibitor of de novo purine nu a concentration range of from 10 * M to 10"5 M. Interestingly, cleotide biosynthesis, 6-methylmercaptopurine ribonucleoside each concentration of DDATHF was shown to have a corre (13, 14). In the present study, DDATHF caused a pronounced sponding effect on HL-60 cell growth which resulted in an reduction in the intracellular levels of GTP and ATP within 4 inhibition of proliferative capacity on day 3 without a loss in h, similar to previous findings with other cell lines (16, 18). cellular viability. Shorter periods of exposure had no effect on This suggests that the induction of the differentiation of HL- the growth and cell cycle characteristics of HL-60 cells. Fur 60 cells by DDATHF was also the consequence of the inhibition thermore, the complete inhibition of cellular replicative capa of purine nucleotide biosynthesis. This was confirmed by studies bility at day 3 coincided with the time interval required for cells demonstrating that both the inhibition of growth and the in to become irreversibly committed to the differentiation path duction of HL-60 differentiation by DDATHF was completely way, whereas cells exposed to DDATHF for shorter periods of prevented by the simultaneous exposure of cells to hypoxan- time continued to proliferate after removal of the inducer from thine or AICA. the medium without attainment of the differentiated state. DDATHF is believed to block production of the purine Taken together, these results imply that the loss of cellular precursor formylglycinamide ribonucleotide by GAR transfor- replicative capacity after exposure to DDATHF, which by itself mylase (16). GAR transformylase is a folate-requiring enzyme was not sufficient for cells to initiate the commitment to enter that catalyses the first of two one-carbon transfer reactions in the differentiation pathway, was due to an essential event(s) the de novo purine nucleotide biosynthetic pathway. Since which accompanied the differentiation process and not to cy- AICA can be converted intracellularly to AICA ribonucleotide todestruction resulting from the depletion of intracellular pur- and is able to enter the purine nucleotide biosynthetic pathway ine nucleotide levels. Thus, although the proliferative potential proximal to the second folate-requiring enzyme, 5(4)-amino- of HL-60 cells exposed to DDATHF is compromised at a point 4(5)-imidazole carboxamide ribonucleotide transformylase in time immediately preceding commitment, differentiation was (22), the finding that AICA protected HL-60 cells from the only possible if an additional stimulus, resulting from the inhibition of growth and the differentiation-inducing capacity further effects of DDATHF on purine nucleotides, occurred. of DDATHF provides evidence that inhibition of GAR trans In earlier studies, we have demonstrated that the selective formylase is involved in these actions in these cells. 4827

7. Bodner, A. J., Ting, R. C., and Gallo, R. C. Induction of differentiation of In conclusion, the novel tetrahydrofolate, DDATHF, has human promyelocytic leukemia cells (HL-60) by nucleosides and methotrex- been demonstrated to be an effective inducer of the maturation ate. J. Nati. Cancer Inst., 67: 1025-1030, 1981. of HL-60 leukemia cells. The induction of cellular differentia 8. Sartorelli, A. C. Malignant cell differentiation as a potential therapeutic approach. Br. J. Cancer, 52: 293-303, 1985. tion by this agent appears to be associated with the depletion 9. Reiss, M., Gamba-Vitalo, C., and Sartorelli, A. C. Induction of tumor cell of purine nucleotides. These findings stress the importance of differentiation as a therapeutic approach: preclinical models for In-mum purine nucleotides to both the growth and differentiation of poietic and solid neoplasms. Cancer Treat. Rep., 70: 201-217, 1986. 10. Fishkoff, S. A., Pollak, A., Gleich, G. J., Testa, J. R., Misawa, S., and Reber, HL-60 leukemia cells. Since guanine nucleotides have an im T. J. Eosinophilic differentiation of the human promyelocytic cell line HL- portant role in the transduction of intracellular signals across 60. J. Exp. Med., 160:179-196, 1984. 11. Hunt-Taylor, S. R., Harnish, D., Richardson, M., Ishizaka, T., and Denbury, the cell membrane, it is possible that the induction of differen J. A. Sodium butyrate and a T lymphocyte cell line-derived differentiation tiation of HL-60 cells by agents which deplete purine nucleotide factor induce basophilic differentiation of the human promyelocytic leukemia pools may represent a response to "metabolic stress" (23, 24). cell line HL-60. Blood, 71: 209-215, 1988. 12. Lucas, D. L., Webster, H. K., and Wright, D. G. Purine metabolism in The result of this action may lead to a diminished activation of myeloid precursor cells during maturation. Studies with the HL-60 cell line. signals from ras and other G proteins. Therefore, it would be J. Clin. Invest., 72: 1889-1900, 1983. 13. Sokoloski, J. A., Blair, O. C., and Sartorelli, A. C. Alterations in glycoprotein of interest to further study the relationship between the induc synthesis and guanosine triphosphate levels associated with the differentia tion of differentiation by these agents and the "stress response" tion of HL-60 leukemia cells produced by inhibitors of inosine 5'-phosphate in HL-60 cells. dehydrogenase. Cancer Res., 46: 2314-2319, 1986. 14. Sokoloski, J. A., and Sartorelli, A. C. Inhibition of the synthesis of glycopro- teins and induction of the differentiation of HL-60 promyelocytic leukemia cells by 6-methylmercaptopurine ribonucleoside. Cancer Res., 47: 6283- ACKNOWLEDGMENTS 6287, 1987. 15. Taylor, E. C., Harrington, P. J., Fletcher, S. R., Beardsley, G. P., and Moran, The authors gratefully acknowledge the assistance of Rocco Carbone R. G. Synthesis of the antileukemic agents, 5,10-deazaaminopterin and of the Yale Comprehensive Cancer Center in the flow cytometry meas 5,6,7,8-dideazatetrahydroaminopterin. J. Med. Chem., 28:914-921, 1985. 16. Beardsley, G. P., Taylor, E. C., Grindey, G. B., and Moran, R. G. Deaza urements. We also thank Dr. Owen C. Blair for performing the com derivatives of tetrahydrofolic acid. A new class of folate antimetabolites. In: puter analyses of DNA histograms. B. A. Cooper and V. W. Whitehead (eds.), Chemistry and Biology of the Pteridines, pp. 953-957. Berlin: DeGruyter & Co., 1986. 17. Chabner, B. A., Allegra, C. J., and Karum. J. Antifolates: expanding horizons REFERENCES in 1986. In: B. A. Cooper and V. W. Whitehead (eds.), Chemistry and Biology of the Pteridines, pp. 945-951. Berlin: DeGruyter & Co., 1986. 18. Beardsley, G. P., Moroson, B. A., Taylor, E. C., and Moran, R. G. A new 1. Harris. P., and Ralph, P. Human leukemic models of myelomonocytic folate antimetabolite, 5,10-dideaza-5,6,7,8-tetrahydrofolate, is a potent in development: a review of the HL-60 and U-937 cell lines. J. Leukocyte Biol., hibitor of de novo purine synthesis. J. Biol. Chem., 264: 328-333, 1989. 37:401-422, 1985. 19. Collins, S. J., Bodner, A., Ting, R., and Gallo, R. C. Induction of morpho 2. Collins, S. J., Ruscetti, F. W., Gallagher, R. E., and Gallo, R. C. Terminal logical and functional differentiation of human promyelocytic leukemia cells differentiation of human promyelocytic leukemia cells induced by dimethyl by compounds which induce differentiation of murine leukemia cells. Int. J. sulfoxide and other polar compounds. Proc. Nati. Acad. Sci. USA, 75:2458- Cancer, 25:213-218, 1980. 2462, 1978. 20. Yam, L. T., Li, C. Y., and Crosby, W. W. Cytochemical identification of 3. Breitman, T. R., Selonick, S. W., and Collins, S. J. Induction of differentia monocytes and granulocytes. Am. J. Pathol., 5: 675-686, 1971. tion of the human promyelocytic leukemia cell line (HL-60) by retinoic acid. 21. Jett, J. H. Mathematical analysis of DNA histograms from asynchronous Proc. Nati. Acad. Sci. USA, 77: 2936-2940, 1980. and synchronous populations. In: D. Lutz (ed.), Pulse-cytophotometry, pp. 4. Miyaura, C., Abe, E., Kuribayashi, T., Tanaka, H., Kanno, K., Nishii, Y., 93-102. Ghent, Belgium: European Press, 1978. and Suda, T. la,25-Dihydroxyvitamin Dj induces differentiation of human 22. Hakala, M. T. Homofolate and tetrahydrofolate inhibitors of purine synthe myeloid leukemia cells. Biochem. Biophys. Res. Commun., 702; 937-943, sis. Cancer Res., 31:813-816, 1971. 1981. 23. Langdon, S. P., and Hickman, J. A. Correlation between the molecular 5. Rovera, <. . Santoli, D., and Damsky. C. Human promyelocytic cells in weight and potency of polar compounds which induce the differentiation of culture differentiate into macrophage-like cells when treated with a phorbol HL-60 human promyelocytic leukemia cells. Cancer Res., 47:140-144,1987. ester. Proc. Nati. Acad. Sci. USA, 76: 2779-2783, 1979. 24. Langdon, S. P., Richards, F. M., and Hickman, J. A. Investigation of the 6. Schwartz, E. L., and Sartorelli, A. C. Structure-activity relationships for the relationship between cell proliferation and differentiation of HL-60 cells induction of differentiation of HL-60 promyelocytic leukemia cells by an- induced to differentiate by iV-methylformamide. Leukemia Res., 12: 211- thracyclines. Cancer Res., 42: 2651-2655, 1982. 216, 1988.

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Downloaded from cancerres.aacrjournals.org on October 4, 2021. © 1989 American Association for Cancer Research. Induction of HL-60 Leukemia Cell Differentiation by the Novel Antifolate 5,10-Dideazatetrahydrofolic Acid

John A. Sokoloski, G. Peter Beardsley and Alan C. Sartorelli

Cancer Res 1989;49:4824-4828.

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