Synergistic effect of retinoic acid and 1,25-dihydroxyvitamin D3 on the differentiation of the human monocytic cell line U937

Leukemia Research Vol. 15. No. 12, pp. 11-15 1152. 1991 Printed in Great Britain

ill.15 212e,'91 $3 00 s- O0 Pcrgam,m Press pie

SYNERGISTIC EFFECT OF RETINOIC ACID AND 1,25-DIHYDROXYVITAMIN D3 ON THE DIFFERENTIATION OF THE HUMAN MONOCYTIC CELL LINE U937 MOHAMM[-D TAIMI, MARIE-THERESE ClIATEAU,* SUZANNE CABANE and JACOUES MARTI Laboratoire de Biologie Cellulaire, INSERM U65, Ddpartemcnt de Biologie-Sant& C.P. 10(I. Universit~ de MontpeIlier II, F-34095 Montpellier Cedex 5, France and *Faculte de Pharmacie, Universit6 de Montpellier I, Montpellier, France (Received 27 March 1991. Accepted l June 1991) Abstract--The human-derived leukemia cell lines HL-60 and U937 are known to differentiate into more mature phagocytic cells in the presence of retinoic acid or 1,25-dihydroxyvitamin D> We studied the effects of combinations of these two agents on cell growth and differentiation. These treatments were found to increase inhibition of cell proliferation. A dramatic enhancement of functional properties was observed in U937, but not HL-60 cells exposed to combinations of the two inducers. We investigated the conditions required to obtain the highest synergistic effects on the differentiation of U937 cells. These effects were found to be highly dose-dependent. We found that synergism required the simultaneous presence of both inducers and did not occur upon sequential exposure to each agent used separately.

KQ' words'. U937, HL-60, retinoic acid, 1.25-dihydroxyvitamin I)~, differentiation.

with most agents, including R A and VD, they mature along the m o n o c y t e / m a c r o p h a g e pathway [1(>13]. However, the precise stage of normal development to which they correspond is ill-defined and a recent work suggests that these cells may have also the capacity to express granulocytic characteristics [14]. Recently, we observed that combinations of R A and VD could be at least as effective as phorbol myristate acetate, a potent inducer of U937 cells differentiation, in inducing specific functional properties [15]. This led us to study the conditions required to obtain these cooperative effects. We found that RA and VD could act synergistically to induce functional properties in U937, but not HL-N) cells and we observed that this synergism was lost when RA and VD are added sequentially. The fact that VD potentiates the effects of R A on some types of leukemic cells may have therapeutic implications.

INTRODUCTION LF,UKFM|A-derived human myeloid cell lines, such as U937 and I tL-60, are useful models for investigating the biology of myeloid differentiation [1,2]. These cells are characterized by a block in their ability to undergo terminal differentiation. As such, they remain in the proliferative pool and rapidly accumulate. However, this phenotype can be reverted by physiological or pharmacological inducers of differentiation. The fact that cells which differentiate lose their proliferative capacities provides an approach to treatment of acute promyelocytic leukemia [31. The 15937 cell line was established from the plcural fluid of a patient with diffuse histiocytic lymphoma [4] and HL-6(I from the blood of a patient with acute promyelocytic leukemia [5] I IL-60 cells are blocked at an early stage of the myelomonocytic lineage. The,,' can be induced to differentiate into more mature macrophage-likc cells by the hormonal form of vitamin D~, (1,25-(OH)zDx) I [6, 7] and into granulocytic cells by the active metabolite of vitamin A, retinoic acid [8, 9]. It is usually considered that U937 cells are blocked at a later stage of development since

MATERIALS AND METttOI)S ChemicaLs AII-trans retinoic acid (RA), propidium iodide, RNase A, phorbol 12-mvristate 13-acetate (PMA) and zymosan A were purchased from SIGMA Chimie Sarl {France). 1,25-Dihydroxyvitamin D 3 (VD) was a generous gift from l)r ( . l)amais (INSERM U313. Paris, France). Luminol (5-amino-2.3-dihydro-l,4-phthalazinedione} was from Boehringer Xlannheinl (France). Immunological reagents

Ahhreviations: RA, all-trans retinoic acid: VD, 1,25-dihydroxyvitamin D.~: RNase A, ribonuclease A: PMA, phorbol 12-mvristate 13-acetate; PBS, physiological butfered saline: I]~LI'. formyl-mel hionylleucylphcnylahmine. 1145

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for flow cytometry were purchascd from Immunotech (Marseille, France). RA and VD were dissolved in absolutc ethanol and stored at -70°C at an initial stock concentration of 10 ~ M. Dilutions were performed in RPMI 1640 medium. The final concentrations of ethanol had no effect on cell growth and diffcrcntiation. ('ells U937 and HL-60 cells (American Type Culture Collection. Rockville, MD) were cultured at 37°C, 5 ~ CO2, in complete RPMI 1640 medium (GIBCO BRL Sarl, France) with 10~/~ (v/v) fetal calf serum (SIGMA Chimie Sarl, France). The cells were regularly tested and found to be free of mycoplasma by 4.6-diamino-2-phenylindole (DAPI) tluorescence. (:ells were counted by hemocytometer and viability was determined by trypan blue exclusion. DNA synthesis was measured by incubation with 0.75 lxCi (5-methyl-3H)thymidine (25 Ci mmol; ( ' E A Saclay, France) for 4 h in 2001al quadruplicate cultures in 96-well platcs. The cells were harvested with a semiautomatic harvester and the dried filters were counted in a liquid scintillation counter.

Luminal-dependent luminescence Measurements were made in 75 x 11 mm Sarstedt tubes (Niimbrecht, F.R.G.) with a 6-channel Lumicon (tlamilton, Switzerland) automatic luminescence analyser. The temperature in the counting chamber was 37 '- 11.I°C. The reaction mixture contained 0.5 ml of cell suspension and (1.5 ml of luminol solution ( final concentration: 3 × 10 -s M) in physiological-buffered saline (PBS). The samples were equilibrated in the dark at 37°C for 4 min before stimulation. Stimulating agents were added in a volume of 0.05 ml with gentle agitation. Resting cell luminescence was measured in parallel runs. PMA, stored at -70°(7 at 1 mg/ml in dimethyl sulfoxide, was dilutcd in PBS to give a final concentration of 1 ug/ml in the assay. The solvent alone had no effect at this dilution. Zymosan particles. suspended in PBS at 10 mg/ml, were placed in a boiling water-bath for 30 min, then washed twice in PBS. Opsonization was achieved by incubation in fresh human serum for 30 rain at 37°C. After washing twice, opsonized zymosan was reconstituted at 10 mg/ml in PBS and stored at -70°C. The concentration in the assay was (1.5 mg/ml. Reaction times were adjusted differently for each agent, depending on the time elapsed between initiation of stimulation and peak chemiluminescence velocity. Counts were recorded at l-rain intervals for 15 min with PMA and for 50 min with zymosan. The programs provided with the Lumicon were used to calculate the integral of counts over the incubation period. This value was found to vary linearly with the number of cells in the assay, the magnitude of the standard error depending mainly on cell count incertitudes. The results are expressed as the integral of counts calculated for 105 cells.

Flow cytometry Quantitative fluorescence analyses were performed by C. Duperray (Service Commun INSERM de Cytom6trie, Montpellier) using a four-parameter Ortho Cytofluograph 50H (Westwood, MA) equipped with a 250 mW argon ion laser at 488 rim. Fluorescence signals were processed by using a logarithmic amplifier. Light-scattering signals were processed by linear amplification. Cell cycle analysis was performed by quantifying the

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Time(cloys) FIG. 1. Time-dependent inhibition of growth of U937 cells exposed to RA and VD. Cells (2 x 10~ per ml) were cultured with differentiation inducers ( I RA 100 nM, • VD 100 nM, • RA 100 nM + VD 100 nM) or control ((2)). Each point represents the mean ( ± S F ) of six individual cell counts. The data, corrected for cell dilutions, arc expressed as the number of cells per ml of initial culture.

DNA content of cells using the fluorescent intercalating agent propidium iodide. Aliquots of 2 × ll)~ cells were washed once with PBS and fixed in 4 ml of 70~4 ethanol in PBS at 40( TM. After 60 rain, the cells were washed and incubated with RNasc A (1 mg/ml) for 30 rain at 37°C. Propidium iodide (5 × 10 -5 g/ml) was added just before analysis and the cclls were washed and rcsuspended in PBS. A minimum of 2l)000 cclls per samplc were analysed Scattcr gates were set to discriminate single cells and cells clumps. Calculation of the cell cycle compartments was performed on a 256-channel DNA histogram from the gated cells population. Modulation of the expression of ccll surface antigens (CDI lb and CDI8 clusters) was studied by monoclonal antibody binding. Cells (5 × 10~) were washed twice with PBS containing 1~/~ bovine serum albumin and incubated for 30 min with the quantity of monoclonal antibody specitied by the producer. After two washes, the cells were incubated for 30min in the dark with tluoresccin-conjugated anti-mouse IgG. Cells were washed twice and tixed in 1% paraformaldehyde until fluorescence analysis. All operations were performed at 4°C.

RESULTS

Restriction of cell growth C o n s t a n t e x p o s u r e of U937 cells to R A o r V D i n h i b i t e d cell g r o w t h as assessed by the rates of increase in cell n u m b e r (Fig. 1). T h e c o m b i n a t i o n o f b o t h agents was m o r e efficient t h a n e a c h a g e n t a l o n e in r e d u c i n g U937 p r o l i f e r a t i o n a n d , as s h o w n in Fig. 1, the e x p o s u r e to 100 nM R A plus 1 0 0 n M V D for 3 days led to a c o m p l e t e inhibition o f cell g r o w t h . U n d e r these c o n d i t i o n s , the viability was h i g h e r t h a n 9 0 % . T h e d o s e - d e p e n d e n c e o f cell g r o w t h i n h i b i t i o n

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FIo. 2. Dose-dependent inhibition of (3H)thymidine incorporation. U937 cells were seeded at 2.5 x I(P cells per ml in 96-well plates, in the presence of various combinations of RA and VD, and incubated for 4 days prior to 4h (3tt)thymidine pulse. Resulls are expressed as the percentage of inhibition, relative to control cells. Each point represents the mean of three experiments with quadruplicate wells per point. was studied by measuring (3H)thymidine uptake in cells cultured with various combinations of RA and VD. Figure 2 shows that for any given dose of one agent, the presence of the other one increased the inhibition. The largest variations of the d o s e response curves were observed between 1 and 10 nM concentrations of both agents. Growth inhibition was correlated with an accumulation of cells in the G./G~ phase of the cell cycle and a decrease in the number of cells in the S and G2 phases (Table 1).

Increased expression of CDIIh/CD18 The heterodimeric glycoprotein M A C - I (CDI l b / C D I 8 , C3bi receptor), mediating leukocyte adherence reactions, is a m a r k e r of myeloid differentiation. Its expression at the cell surface of U937 cells is increased by differentiation inducers. The expression

of M A C - I , and more particularly of the CD1 lb subunit, was enhanced when both inducers were used together (Fig. 3). The mean fluorescence p a r a m e t e r , which correlates with the number of molecules present at the cell surface, increased from about 20 to 611 for CD1 lb, i.e. a 3-fold increase, when RA and VD were used in combination.

,~vnergistic effects on chemiluminescent re,~ponses triggered hv different stimuli A typical functional property of phagocytic cells is the production of reactive oxygen intermediates. The luminol-cnhanced chemiluminescence associated with the production of a superoxide anion was used to evaluate the extent of differentiation of U937 cells. Oxidative bursts were triggered by PMA (Fig. 4) or opsonized zymosan (Fig. 5). We found that cells treated by combinations of RA and VD gave far more than additive responses when c o m p a r e d to cells treated by a single agent. To compare the effectiveness of different combinations, we calculated the factor (S) obtained by dividing the luminescence response (integral of counts) of R A + VD treated cells by the sum of the responses of cells treated separately by the same agents used alone at the same concentration. Figure 6 shows that the S-value was dose-dependent. The highest synergistic effect was observed with 10nM V D in the presence of 10 nM RA (zymosan-induced burst) to 1(~)nM RA (PMAinduced burst).

TABLE 1. CEI.I. ('Y('LF- I)ISTRIBt'TION OF U937 AND I IL-60 (FI.LS IN('t;BATI!I) FOR FOUR DAYS WITtl (-)R WIFtI()t'T RA AND VD

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U937 55.9 34.3 9.8

ttl.-60 51.3 38.6 10.1

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VD 10 7M U937 711.9 24.4 4.7

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RA 10 : M + VD 111 7M U937 86.75 11.30 1.95

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Loss of synergistic effects by ,sequential treatments with the two differentiation inducer,s Experiments were performed to know if both inducers needed to be administrated at the same time in order to act synergistically. U937 cells were first exposed to one agent alone during 24 or 48 h. In a second step, the cells were washed twice by centrifugation, resuspended and cultured in fresh medium supplemented with the same or the other agent. The extent of functional differentiation was

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FIG. 6. Dose-dependence of the S value. S = (integral of luminescence counts for RA + VD treated cells)/(sum of the integrals of counts for cells treated separately by RA and VD). The experimental data are taken from Figs 4 (A: stimulation by PMA) and 5 (B: stimulation by opsonized zymosan). assessed by measuring the chemiluminescence triggered by zymosan. We did not find m a j o r differences between cells sequentially exposed to both inducers and cells which had been re-exposed to the same one. In all cases, the responses were substantially lower than those of cells treated at each step with similar concentrations of R A + VD. As illustrated in Fig. 7, cells incubated sequentially with both agents for a total of 96 h were even less differentiated than cells exposed only for 4 8 h to RA + VD.

Comparison between U937 and HL-60 cells HL-60 differ from U937 cells by their capacity to differentiate along multiple pathways, depending on the inducing agent. RA induces neutrophilic and VD monocytic differentiation. As for U937, we found that combinations were more effective than each agent alone in reducing HL-60 cell growth, by increasing the proportion of cells in the G . / G I compartment of the cell cycle (Table 1). l'towever, the two cell lines were clearly different at the functional level: as illustrated in Fig. 8, the oxidative burst triggered by PMA or zymosan in HI,-60 cells was more intense when RA and VD were used in combination but the responses were merely additive and we did not observe the synergism typical of U937 cells. I) ISCUSSION Our results show that RA and VD cooperate

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FIG 8. Comparison between the responses of U937 and HL-60 cells after 3 days treatment with RA and VD at 10 nM. Luminescence produced by 105 cells triggered by PMA (A) and by opsonizcd zvmosan (B). efficiently in reducing cell growth and inducing differentiation of U937 cells. This study extends and corroborates previous observations. A synergism between RA and VD was first detected by measuring

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the number of cells able to reduce nitroblue tetrazolium after 2 days exposure to various combinations of RA + VD [12]. More recently, we found that functional properties induced by micromolar concentrations of RA were enhanced by the addition of 10 nM VD. The cells became more phagocytic and adherent to plastic and responded to the chemotactic peptide fMLP by an oxidative burst, an activity which is not shared by cells exposed to RA or VD alone 115, 161. llere we found a significant increase of the C D I l b / C D 1 8 antigen, accounting for increased adherence [17]. Under optimal conditions, we obtained a dramatic enhancement of the oxidative burst in response to PMA, opsonized zymosan and other stimuli such as Concanavalin A (data not shown). In addition, preliminary results from our laboratory show that synergism also affects the LPSinduced production of interlcukins 1 and 6 (Taimi M. & Dornand J., in preparation). Taken together, all these results show that the cooperation between RA and VD drives the cells towards a highly differentiated phenotype, characteristic of phagocytic cells. However, since U937 cells may be susceptible to express granulocytic characters [14]. it cannot be concluded that RA and VD cooperate to trigger a unique differentiation program, leading to the expression of strictly monocytic properties. It is possible that both agents activate complementary programs, leading to a mixed, atypical, phenotype. To be resolved, this question will require a better characterization of specific targets of each agent. Additional information can be expected from a study of the effects of RA + VD on normal cells of the monocyte lineage. It seems now well established that VD plays an essential role in promoting the maturation of monocytes to macrophages [18-21 ]. It will ~e of interest to examine how RA modulates this VD-induced maturation. The luminescence associated with the oxidative burst was used to monitor the extent of [;'937 cells differentiation. This method offers specific advantages. ( i ) T h e counting system used in this work, giving linear responses over live orders of magnitude, is well suited to the study of large amplitude responses. (ii) The membrane-associated NADPH oxidase producing 0 2 , the precursor of reactive oxygen metabolites, is a marker of "'professional" phagocytic cells [22]. (iii) In U937 cells, a functional oxidase system can be detected only after exposure to differentiation inducers and requires the synthesis of inducible components of the electron transport chain [23-26]. Their presence was explored by using the (_7kinase activator PMA as a stimulating agent and bv measuring the response to particles of zymosan, involving more complex cellular events. This work

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revealed that synergistic effects were highly dosedependent. Optimal synergism was achievcd in a narrow range of concentrations between 1 and 100 nM for both inducers. The highest synergistic effect on the zymosan-induced response was obtained with 10nM concentrations of RA and VD. Under these conditions, we found that synergism was lost when both inducers were added sequentially. To get further insight on this phenomenon, we compared the responses of U937 and HL-60 cells, for which the effects of sequential additions of RA and VD had already been investigated [27]. This study had led to the proposal that a first exposure to any one of both agents induces a common precommitmcnt state, with no apparent lineage specificity. Following these early events, the specific terminal differentiation program, along the granulocytic lineage with RA or the monocytic lineage with VD, would bc driven by late events occurring in precommittcd cells [271. Assuming such a two-step model for U937 cells, our results should be consistent with syncrgy occurring at the first step, during the establishment of the precommitment state. Wc therefore examined if simultaneous application of RA and VD to 111,-60 cells also induced synergistic effects. Wc found that, although RA and VD cooperate in reducing I-IL-60 cell proliferation, their effects on the induction of functional properties were additive rather than synergistic. Difficulties in rationalizing the present findings arc linked to the complexity of the underlying molecular mechanisms. The presence of specific nuclear receptors for RA and VD in leukemic cells is well established [7, 28-31]. Therc is no doubt that thcv act as nuclear transcription factors, playing a central role in cell differentiation, and recent results demonstratcd that RA-induccd differentiation of llL-60 cells is mcdiatcd directly through the nuclear RA reccptor [32]. High affinity binding of the RA receptor to its cognate response element sequences is modulated by interaction with additional nuclear proreins [33]. RA and VD receptors share very high homology in the region of their l)NA-binding domain and gcnc sequences conferring responsiveness to both RA and VD have becn identified [34]. Thcrefore, nuclear mechanisms alone may be rcsponsible for the synergism of both agents. However, more complex pathways are probably involved. In addition to a nuclear protein, several other polypcptides can be rctinoylatcd in tlL-6() [35]. In apparent contradiction with the hypothesis of a unique nuclear target for RA, other experiments, using covalentlv immobilized RA, have shown that growth arrest and differentiation of HL-60 can be initiated by a signal originating at the cell membrane [36]. G-proteins

appear to bc implicated in the transduction pathway, on the basis of experiments showing that l IL-6() cells previously treated by pertussis toxin fail to respond to RA [37, 38 I. Therefore, it is still not clear whether the multiple events associated with RA treatment are a consequence of membranous or nuclear mechanisms or combinations of both. ()ur results, showing that RA-induccd differentiation can bc experimentally modulated by addition of VI), provide additional means for studying these mechanisms in U937 cells. The efficacy of RA in treatment of acute promyclocytic lcukcmia is clearly demonstrated [39-42]. It is possible that this efficacy lies in part on synergism with endogenous factors. Our results suggest that VD could be such a potentiating agent. However, available data indicate that normal serum levels of VD arc in the rangc of 10 "~M [21], a value about 100-fold lower than required here to achieve optimal syncrgy. Attempts to increase VD levels are limited by the adverse action of VD, inducing intcstinal calcium absorption and bone calcium mobilization. l lowever, synthetic analogs, less potent than VD in inducing hypercalcemia, proved to be efficient differentiation inducers [43, 44]. It would be interesting to examine if these derivatives cooperate with RA in inducing cell growth arrest and differentiation.

REFERENCES 1. Harris P. & Ralph P. (1985) lluman leukemic models of myelomonocytie development: a review of the IlL60 anti U937 cell lines. ,I. Leukocyte Biol. 37, 407. 2. Collins S. J. (1987) The HL-60 promyelocytic leukemia cell line: proliferation, differentiation and cellular oncogene expression. Blood 70, 1233. 3. Waxman S. (1990) Differentiation therapy. ('ancer Res. 50, 3453. 4. Sundstrom ('. & Nilsson K. (1976) Establishment and characterization of a human histiocytic lymphoma cell line (U-937). Int. J. Cancer 17, 565. 5. Gallagher R., Collins S.. Trujillo J.. Mc-Credie K., Ahearn M., Tsai S.. Metzgar R., Aulakh G.. Ting R., Rucetti F. & Gallo R. (1979) Characterization of the continuous, differentiating myeloid cell (HI,-60) from a patient with acute promyelocytic leukemia. Blood 54, 713. 6. Tanaka |i., Abe E.. Miyaura C., Kuribayashi T., Konno K.. Nishi Y. & Suda T. (1982) 1.25-Dihydroxycholecalciferol and a human myeloid leukemia cell line (ilL-60). Biochem. J. 204, 713. 7. Mangelsdorf D. J.. Koefflcr ! I. P., Donaldson C. A.. Pikc J. W. & Haussler M. R. (1984) 1,25-Dihydroxyvitamin D vinduced differentiation in a human promyelocytic leukemia cell line (HL-60): receptormediated maturation to macrophagc-like cells. J. cell. Biol. 98, 391. 8. Brcitman T. R., Sclonick S. E. & Collins S..I. (1980)

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