Effects of dimaprit on growth and differentiation of human promyelocytic cell line, HL-60

Life Sciences, Vol. 36, pp. 1909-1916 Printed in the U.S.A.

Pergamon Press

EFFECTS OF DIMAPRIT ON GROWTH AND DIFFERENTIATION OF HUMAN PROMYELOCYTIC CELL LINE, HL-60 •

,

.

4-

.

Karen A. Kalinyak , Davld,G. Sawutz , Beatrlce ~4 Lampkin*, Carl L. Johnson-, Jeffrey A. Whitsett *Division of Hematology/Oncoko4gy, Children's Hospital Research Foundation, Cincinnati, Ohio; Departments of - P'-ediatrics, *Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Cincinnati, Ohio (Received in final form March 7, 1985)

Summary The human promyelocytic cell line HL-60, differentiates in response to a variety of agents including dibutyrl cAMP and agents which increase intracellular cAMP concentrations (phosphodiesterase inhibitors, PGE2, and cholera toxin). HL-60 is also known to be rich in H2 -histamine sensitive adenylate cyclase activity. The present study was therefore designed to test the effects of H2stimulation on growth and differentiation of HL-60 using the potent H 2 agonist dimaprit. Dimaprit markedly increased cAMP production in a dose-dependent manner reaching maximal levels after 30-60 minutes. Intraeellular cAMP levels decreased thereafter and by 24 hours were approximately 2-3 fold increased above control. Intracellular cAMP levels were not altered by dimaprit (10-7M to 10-4M) at 4 days in culture compared to either untreated HL-60 cells or dimethylsulfoxlde (DMSO) (1.3%) treated cells. While exponential growth was unaltered by dimaprit (10-7M to 10-~M) as compared to control, dimaprit induced i) morphologic maturation to the myelocyte and metamyeloeyte form with no differentiation seen beyond the metamyelocyte even after 6 days in culture, il) increased NBT reductase activity and iii) dose-dependent increase in lysozyme activity which could be completely blocked by cimetidine, a specific H2 antagonist. Dimapritinduced differentiation of HL-60 cells was associated with an initial but transient increase in intracellular cAMP production. Maturation beyond the metamyelocyte stage was not observed. Acquisition of NBT reductase and lysozyme activity correlated with morphologic maturation. The human promyelocytic cell line, HL-60, is an established human myeloid cell line derived from a patient with acute promyelocytic leukemia (1). HL-60 differentiates into more mature cells in response to a variety of agents. Dimethylsulfoxide (DMSO) and retinoic acid induce these cells to differentiate into myelocytes, metamyelocytes, banded and segmented neutrophils (2,3). Butyric acid induces differentiation of HL-60 cells limited to myelocytes and metamyelocytes. Phorbol diesters and 1,25 dihydroxyvitamin D3 on the other hand, induce differentiation of HL-60 cells into monocyte-andmacrophage-like cells (4,5). Increases in cAMP can influence cell growth and differentiation in many cell types (6,7). Agents which increase intracellular cAMP concentration in HL-60 cells enhance differentiation, however there are significant differences in the degree of maturational changes in response to these agents. For example, N6 02 dibutyryl adenosine 3'5' cyclic monophosphate (dbeAMP) will induce maturation of HL-60 cells to myelocyte and metamyelocyte forms, but not to forms more mature than the metamyelocyte. Exposure to dbcAMP induces these cells to synthesize chemotactic peptide receptor, reduce 0024-3205/85 $3.00 + .00 Copyright (c) 1985 Pergamon Press Ltd.

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mtroblue tetrazolium (NBT), release lysosomal enzymes, demonstrate chemotaxm and adhere to substrate. Prostaglandin E2 (100 nM) and theophylline (500 uM) increase cAMP concentrations in HL-60 in association with similar functional changes, but the cells do not mature beyond the myelocyte stage. Choleratoxin induces formyl-peptlde receptor expression and adherence, but does not induce NBT reduction or morphologic differentlation (8). HL-60 cells have recently been shown to be rich in histamine H2-stimulated adenylate cyclase activity. Increases in cAMP concentration (20-30 fold) are seen after I0 minute incubation with histamine. Mature neutrophils are less responsive wlth a 10 fold increase observed only in the presence of a phosphodiesterase inhibltor (9). Because of the marked H2-stimulated adenylate cyclase activity and the effect of cAMP on differentiation of HL-60, the present study was done to determine the effects of histaminergic stimulation on growth and differentiation of HL-60 using the potent H2 agonist, dimaprit. Methods HL-60 cells were obtained from Dr. Irwin Bernstein (Seattle, Washington) and had been stored in hquid nitrogen at passage 37 until 1 month prior to use. These cells were originally established and described by Dr. R. Gallo et al., NIH (Bethesda, MD) (1). The cells were maintained in suspensionculture in RPMI-1640 medium (Grand Island Biological Co.) supplemented with 15% heat inactivated fetal calf serum (Sterile Systems Inc., Logan, UT), 2mM glutamine, 1raM sodium pyruvate, MEM amino acids, MEM vitamins and antl-PPLO agent (60 ug/ml) in an humidified atmosphere of 5% CO2 m air at 37 0. Cell cultures were divided every 3-4 days to maintain a density between 0.5 x 10 6 cells/ml and 1.0 x 106 cells/ml. For differentiation experiments, HL-60 cells were washed wlth phosphate buffered saline (PBS) and resuspended in 5 ml of fresh media at a density of 0.3-0.5 x 10 ~ cells/ml in triplicate 25 cm 2 flasks (Falcon). After 24 hour incubation, the inducing agent was added to give the appropriate final concentration. A similar volume of PBS was added to control flasks. Viability was assessed by trypan-blue exclusion. Doubling time was calculated as the number of population doubhngs during the initial 48 hours of exponential growth. Morphology Coverslip slides were made on days 2, 4 and 6 of cell growth and were stained wlth Wright-Giemsa. Differential counts were performed under light microscopy on a minimum of i00 cells. Morphologic maturity was determined by multiple factors including decreased azurophilic granules, decreased nuclear-cytoplasmic ratio, loss of nucleoli, increased nuclear density and indentations of the nucleus. Nltroblue tetrazolium Approximately 1 x 10 5 cells were pelleted by centrlfugatlon (250 g x 5 min), washed twice with phosphate buffered saline (PBS), resuspended in 0.5 ml of RPMI 1640 medium with 15% fetal calf serum to which was added the phorbol diester 12-0-tetradecanayl phorbol 13-acetate (TPA) (Sigma) at a final concentration of I00 ug/ml. Cells were incubated for 15 minutes at 37o C. At the end of the incubation, 0.5 ml of a 0.2% NBT solution in PBS was added and the cells incubated for 30 minutes at 37°C. Cells were then washed twice and resuspended in albumin for microscopic examination. Preparations were stained with Wright-Giemsa and the percent of NBT-treated cells containing blueblack formazan deposits was determined on a minimum of 100 cells and repeated in duplicate for each score. Lysozyme Cells were harvested by centrifugation, washed with PBS, and resuspended in 0.1 M sodium phosphate buffer (pH 7.0). Cells were disrupted by sonication (2-5 sec bursts at setting 60 with a Branson sonifier). Lysozyme was assayed by the decrease in turbidity of a suspensionof micrococcus lysodeikticus at 450 nm. Results are expressed as the change in optical density/minute/10 o cells.

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cAMP assay Cyclic AMP was assayed in a manner similar to that of Gespach et al (9). HL-60 cells were harvested at various times from the tissue culture. A 0.5 ml aliquot, representing total cAMP was incubated with 0.5 ml boiling 50 mM sodium acetate buffer (pH 5.6) for I0 minutes in a hot water bath. A second 0.5 ml aliquot was centrifuged for 30 seconds in a Beckman microfuge at 4 °C. The supernatant was removed and treated as described above. The pellet was resuspended in 1.0 ml boiling 50 mM sodium acetate and incubated for 10 minutes in a hot water bath. Samples were then centrifuged at 2800 rpm for 7 minutes and the supernatants frozen and stored for determination of cAMP. Levels of cAMP were determined by using an 1251-TME-SeAMP radioimmunoassay procedure. The RIA standard curve and experimental data were fit using an Apple Pascal computer program based on the ALLFIT algorithm of DeLean et al (10). All data were analyzed by one way analysis of variance. Significance is defined as p<0.05. Antibody to cAMP was the kind gift of S. Ganguh, Ph.D., Children's Hospital, Cincinnati. Results Morphology The morphologic changes induced by dimaprit in the HL-60 culture are illustrated in Fig. 1. In the absence of any additives, the HL-60 cells are predominantly promyelocytes (Fig.l, top) with large round nuclei, each containing 2-4 nucleoli and fine nuclear chromatin. The cytoplasm is basophilic with prominent azurophilic granules. Approximately 10-20% of control cells are more mature myeloid cells, consisting mostly of myelocytes, a few metamyelocytes and an occasional banded neutrophil. Dimaprit (10-7 M to 10-~ M) induced morphologic changes in HL-60 cells characteristic of more mature myeloid cells, with the predominant cell type being myelocytes and metamyelocytes (Fig. 1, bottom). Significant changes in morphology were observed by day 4 of treatment at all concentrations of dimaprit tested (i0-7M-10-~ M) (Table I). Differentiation to banded or segmented neutrophils was not generally observed in the presence of T a b l e I.

DIFFERENTIAL COUNTS OF HL-60 CELLS AFTER INCUBATIONWITH DMSO OR DIMAPRIT2-6 DAYS % of Total Cells CONTROL

DAY 2

DAY 4

DAY 6

Blasts Promyelocytes Myelocytes Metamyelocytes Banded Neutrophils

. 90 10 -.

Blasts Promyelocytes Myelocytes Metamyelocytes Banded Neutrophils Blasts Promyelocytes Myelocytes Metamyelocytes Banded Neutrophils

.

1.3%DMSO .

.

.

10 -7 .

32 66 2 .

.

.

. 84 16 ---

.

.

.

. 73 24 3 --

.

. -46 48 6

. . . 76 72 24 28 . . . . . . . .

.

.

.

.

. . . 20 22 80 78 . . . . . . . .

.

.

.

-66 28 6 .

DIMAPRIT 10-6 10 -5

9 88 3 . .

.

. 11 88 1 . . .

. 80 20 . .

10-4 .

. 46 50 4

.

.

. . . 12 10 82 78 6 12 . . . . . 1 78 21 .

.

. 11 58 28 3

Morphological Analysis of HL-60 Cells After Incubation with DMSO or Dimaprit. HL-60 cells were incubated with dimaprit at concentrations of 10-7M to 10-4M. Differential cell counts were done on days 2, 4 and 6. Above values represent the mean of 3 experiments.

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4 FIG. I: Morphologicalappearance of uninduced and induced HL-60 cells. Covershp slide p r ~ a t i o n s of cells from suspension cultures were stained with Wright-Giemsa before and after treatment with dimaprit, DMSO and no reducing agents. (Top) HL-60 cells cultured without inducing agent. (Bottom) Cells cultured in 10-5 M dimaprit for 6 days.

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dimaprit even after 6-7 days in culture although an occasional banded form was observed by day 6 in the 10-4 M dimaprit treated cultures. This is in contrast to DMSO treated cells in which metamyelocyte and banded neutrophils were the predominant cell types by day 6 in culture (Table I). Cell growth Dimethylsulfoxide (DMSO) slightly stimulated cell growth during the first two days in culture as compared to the control, with subsequent slowing of growth. Final saturation density in the presence of DMSO was less than that seen in the control group (2.5 x i0 6 cel!s/ml vs. 3.5 x 106 cells/ml). Dimaprit, in concentrations of 10-'M to 10-SM, had no effect on cell growth, population doubling time, or final cell density compared to untreated cells (data not shown). Cells treated with 10-4M dimaprit had a slightly longer population doubling time, 39 hours compared to 32 hours in the control group (not siagnificantlydifferent, df = 2 p >0.05). Final cell density was also less in dimaprit (10-~ M) than the control (2.5 x 106 cell/ml). Cell viability, as assessed by trypan blue dye exclusion, was less in the DMSO treated and 10-~M dimaprit treated cultures by day 5 in culture (85% vs. 95%). Cyclic AMP concentrations Dimaprit (10-5M) increased intracellular cAMP production 30-40 fold after a 10 minute incubation, confirming previous studies (9,11,12). Cyclic AMP production reached maximal levels 20-60 minutes after treatment (Fig. 2) and thereafter decreased to 2-3 times that of basal by 24 hours. Longterm treatment of HL-60 (4 and 6 days) in various

DIMAPRIT-STIMULATED cAMP LEVELS IN HL-60 CELLS AND MEDIA 150

~

120

30

FIG 2

~,

0

2

4

6

8

10 12 14

Dimaprit-stimulated cAMP levels in HL-60 cell and media. HL-60 cells were cultured with 10 -s M dimaprit for 20 and 40 minutes; I, 2, 3, 4, 8 and 12 hours. At each time point, total (o-o), media (D-Q) and cellular (o-o) cAMP concentrations w e r e determined as described in methods. Levels of cAMP were measured in triplicate for each sample and expressed as pmoles cAMP/106 cells. The data represent one of three similar experiments.

TIME (HOURS)

concentrations of dimaprit (10-7M to 10-4 M) resulted in intracellular cAMP concentrations which were no dlfferent than untreated or DMSO (1.3%) treated cells. Extracellular cAMP levels increased coincidental with the decreasing intracellular cAMP levels (Fig.2) and gradually decreased over the 6 days in culture such that by day 6 the extracellular cAMP levels of treated cells were no different than the untreated culture except in the 10-4 dimaprit treated cells, which were 2-3 times higher than the untreated cells (data not shown). NBT reduction NBT reductase activity was measured on days 2, 4 and 6 in culture. Dimaprit increased the percentage of NBT positive cells after 2 days in culture in the cells treated with 10-4M dimaprit and 10-~ M dbcAMP. By day 6 in culture, 34-52% of cells treated wlth dimaprit (10-4M to 10-v M) were positive for reduced NBT as compared to 18% in

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control cells (Table II). Approximately 40% of DMSO (1.3%) treated cells were able to reduce NBT by day 4 and 60% had NBT reductase activity by day 6 in culture. The timing of the acquisition of NBT reductase activity correlated well with morphologic evidence of maturation. Table U.

NBT REDUCTASEACTIVITYIN HL-60 CELLS (% Cells with Positive Reaction) DIMAPRIT (10-4M) (1.3%) CONTROL dbcAMP D M S O

10-7M

10-6M

10-5M

10-~ M

DAY 2

16

45

33

16

28

24

32

DAY 4

23

41

39

28

26

28

32

DAY 6

23

63

53

38

34

52

42

Effects of DMSO, Dimaprit and Dibutyryl cAMP on NBT Reductase Activity m HL-60 Cells. NBT reductase activity was compared after treatment with dimaprit, DMSO, dbcAMP or no treatment on days 2, 4 and 6. Values represent the percent of cells containing reduced NBT. Results represent the mean value from three experiments. Variability of values is --<5%for all points.

Lysozyme activity Lysozyme activity was measured on days 2, 4 and 6 in culture. Lysozyme activity was undetectable in untreated cultures but was detectable in dimaprlt treated (i0-7M to 10-4 M) as well as DMSO treated cells by day 4 m culture. Increased lysozyme activity in the dimaprit treated cells was dose-dependent and completely blocked by the H2 specific antagonist, cimetidine (Fig. 3). A similar dose dependent relationship was observed after the cells were exposed to dimaprit (10-7 M to 10-~M) for 6 days (data not shown). Discussion Dimaprit, a histamine H2 -.agonist, reduces dlfferentiation of HL-60 cells into more mature myeloid cells as evidenced by morphology, NBT reduction, and lysozyme production. Terminal differentiation beyond the metamyelocyte stage was not seen at any concentration of dimaprit tested. These findings are similar to the incomplete morphologic maturation reported w i t h dbcAMP and other agents which elevate intracellular cAMP productlon (8). Higher concentrations of dimaprit were not tested because of mild toxic effects and decreased vlability seen by day 6 in culture at 10-4M dimaprit. Dimaprit markedly increased cAMP concentration in HL-60 cells, reaching maximal levels after 20-60 minutes. Intracellular cAMP levels decreased thereafter and by 24 hours were approximately 2-3 fold above basal levels. There were no differences observed in the intracellular cAMP concentrations between the dimaprit treated, DMSO treated or untreated cells by day 4 in culture. The total cAMP concentration (intracellular plus extracellular) was also maximal at 20-60 minutes and then gradually decreased over the four days in culture. Morphologic evidence of maturation was seen as early as the second day in culture in cells treated with 10-#M dimaprit, and in DMSO treated cells, and by day 4 in all other concentrations of dimaprit tested. Expression of NBT reductase activlty did not precede

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morphologic maturation as was described in dbcAMP treated cells (8). Lysozyme activity was seen only after evidence of morphologic maturation was observed by day 4 in culture. The induction of lysozyme production was dose-dependent and could be completely blocked by cimetidine, an histamine H2 antagonist. Since dimaprit acts via the H2 receptor-cyclase system and has been shown to cause an increase in intracellular cAMP (9,11,12), it is possible that the trigger to begin the differentiation process is an increase in cAMP production within the cell. The fact that the induction of cAMP synthesis is associated with dose-dependent increased lysozyme activity which is inhibited by cimetidine would support this hypothesis. However, the inhibitory effect of cimetidine on NBT reduction and morphologic changes in the cells treated with dimaprit was not tested. Many agents which have no effect on cAMP production can cause morphologic as well as functional changes in HL60 cells similar to those seen with dimaprit. In addition, agents known to cause an increase in cAMP production such as cholera toxin and PGE2 have little or no effect on morphology of HL-60 (8). Therefore it is unlikely that the morphologic changes seen with dimaprit can be ascribed solely to the elevated cAMP levels. Rather it is more likely that the morphologic and functional changes seen with differentiation represent complex biochemical processes and that many factors, including cAMP, act to modulate and regulate these processes. 10

q) O >~QD

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Em >,

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4

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Control I

I

I

I

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5

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-log [Dimaprit] FIG. 3

Intracellular lysozyme activity. H L - 6 0 cells w e r e incubated in various concentrations of dimaprit. On day 4 the cells were washed and lysozyme activity measured as described in Methods. Each point represents the mean +-S.E. from three separate experiments. Viability, as measured by trypan blue exclusion, was >90% except for 10-~M dimaprit group which was> 80%. All data were analyzed by one-way analysis of variance, *p < 0.05. We have previously demonstrated that HL-60 cells cultured with dimaprit for increasing lengths of time had a decreasing ability to produce cAMP when rechallenged with dimaprit (11,12). Preliminary experiments suggest that this desensitization occurs as a result of uncoupling of the recepfor from the adenylate cyclase system rather than as a result of non-specific effect of dimaprit, increased cAMP phosphodiesterase activity, or decreased receptor number (12). The extracellular cAMP levels increased rapidly as intracellular levels decreased. The active extrusion of cAMP from the cell into the media

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has been described for other cell types and has been shown to persist for long tlmes from cells under continuous stimulation, remaining perceptible even when intracellular levels are only two or three-fold higher than basal concentration (13). In this study, however, passive diffusion of cAMP out of HL-60 cells cannot be excluded since the calculated intracellular concentration of cAMP was more than fivefold higher than the extracellular cAMP levels. Increased intracellular cAMP is associated with inhibition of cell proliferation in a variety of normal and malignant cell lines thus raising the possibility that decreased proliferation and induction of dlfferentiation are coupled events (14). When HL-60 cells are cultured wlth dimaprit, morphologic as well as functional evidence of maturation (at least partial maturation) was noted at concentrations of dimaprit which had no inhibitory effect on proliferation (10-SM to 10-7M). O n l y at the more toxic concentration of 10-4 M dimaprit was there an inhibitory effect on cell proliferation and a decrease in final saturation density. Studies by Fibach et al. suggest that loss of proliferative capacity was required for and precedes expression of differentiated characteristics in HL-60 treated with DMSO (15). In contrast, our study demonstrates that the proliferative ability of HL-60 was unaffected at concentrations of dimaprit (10-SM to 10-7 M) which induced partial expression of differentiation. In summary, dlmaprit, an hmtamine H2 agonist, enhances cAMP synthesis and reduces differentiation of HL-60 cells. The differentiation Is incomplete with the cells maturing only to the myelocyte and metamyelocyte form. As described previously, desensitization of the H2-response to dimaprit occurs rapidly with respect to cAMP synthems, however morphologic maturation progresses in spite of return of cAMP concentration to near basal levels (12). This would suggest that the early rise in cAMP production may be an important trigger in the differentiation process.

Acknowledgements Supported by grants from the National Instituties of Health (HL-22136 and HL-07382), The Lindner Fund and the Children's hospital Research Foundation, Cincinnati, Ohio References I) 2) 3) 4) 5)

6) 7) 8) 9) i0) ii) 12) 13) 14) 15)

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