[CANCER RESEARCH 50, 222-226, January 15. 1990]

Modulation of Retinoic Acid-induced Differentiation of Human Leukemia (HL-60)Cells by Serum Factors and Sphinganine1

Victoria L. Stevens, Nancy E. Owens, Elliott F. Winton, Joseph M. Kinkade, Jr., and Alfred H. Merrill, Jr.2

Departments of Biochemistry ¡V.L. S., N. E. O., J. M. K., A. H. M.] and Medicine [E. F. W.], Emory University, Atlanta, Georgia 30322

ABSTRACT

The human cell line HL-60 was used to investigate the role of proteinkinase C in the regulation of retinole acid-induced maturation of promye-locytic leukemia cells by growth and differentiation factors found inserum. Cells grown in serum-containing medium differentiated less thancells in serum-free medium due to several factors, including albuminbinding of retinoic acid. Addition of an inhibitor (sphinganine) of proteinkinase C, an enzyme that participates in cellular responses to many serumfactors, facilitated the retinoic acid-induced differentiation. Cells treatedwith both retinoic acid and sphinganine produced more Superoxide whenstimulated by formylmethionylleucylphenylalanine; hence, this combination generated a more functional population of cells. The ability ofsphinganine to promote retinoic acid-induced differentiation suggests thatretinoic acid therapy might be improved by the concurrent use of amodulator of protein kinase C activity.

INTRODUCTION

Recent approaches to therapy for various types of cancer havefocused on drugs that induce the maturation of the aberrant,differentiation-resistant cells causing the disease (1, 2). Onecompound currently being tested clinically for its ability towork in this manner is retinoic acid (3). RA3 is a potent inducer

of differentiation in numerous established myeloid cell lines (4,5) as well as primary cultures of cells isolated from patientswith promyelocytic leukemia (6). Clinical trials and case reportsthus far show variable success of therapy with RA (7-10).Recent reports indicate that differentiation induced by RA maybe facilitated by concurrent treatment with other agents suchas DNA synthesis inhibitors, hormones, and other biologicalmodifiers (10-14).

The biochemical mechanism by which RA causes differentiation remains unclear. However, some evidence suggests thatthe calcium- and phospholipid-dependent protein kinase (protein kinase C) is involved in the response of cells to this drug(15-17). This kinase regulates some serum factor-induced processes (18, 19) as well as the differentiation of myeloid cellsinduced by phorbol esters (20). In this study, the human leukemia cell line HL-60 was used to investigate the effects ofdifferent sera as well as inhibition of protein kinase C ondifferentiation. HL-60 is a bipotent cell line (21) isolated froma patient with acute myelocytic leukemia (22, 23). These cellsundergo granulocytic differentiation when treated with RA (4)and have been used extensively to characterize hematopoieticcell differentiation (24).

The ability of RA to induce differentiation of HL-60 cellswas found to vary depending on the serum in which the cells

Received 10/31/88; revised 6/8/89; accepted 10/10/89.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1This work was supported by Grants CA46508 (A. H. M.) and CA22294 (J.M. K.) from the NIH. DCB-871083 from the NSF (A. H. M.), and Jill AndrewsMemorial Fund for Leukemia Research (E. F. W.).

1To whom requests for reprints should be addressed.'The abbreviations used are: RA. retinoic acid; PMA, phorbol 12-myristate

13-acetate; NBT. nitroblue tetrazolium; BSA, bovine serum albumin; PBS, phosphate-buffered saline; PCS, fetal calf serum; fMLP, jV-formyl-L-methionyl-L-leucyl-L-phenylalanine.

were grown. The possible role of protein kinase C in mediatingthese serum effects was investigated with the long-chain basesphinganine.4 Long-chain bases are among the most potent

inhibitors of this enzyme reported to date (25) and their effectiveness has been established in numerous cellular systems (26-37) including HL-60 cells (38). The presence of sphinganinewas found to increase the percentage of RA-treated HL-60 cellsthat exhibited mature morphology as well as the functionalcompetence of these cells.

MATERIALS AND METHODS

Materials. Defined bovine calf serum (HyClone) was obtained fromHyClone Laboratories (Logan, UT). Wright-Giemsa stain (CameoQuik II) and RPMI 1640 medium were from American ScientificProducts (Atlanta, GA). Fetal calf serum was from GIBCO (GrandIsland, NY). ery/Aro-Dihydrosphingosine (sphinganine), PMA, NBT,and cytochrome c (type VI from horse heart), insulin, transferrin, all-trans-RA, and human serum (type AB from male donors) were purchased from Sigma Chemical Company (St. Louis, MO). Fatty acid-free BSA was from Boehringer Mannheim (Indianapolis, IN). [3H]RA

was from Dupont NEN (Wilmington, DE).Cell Culture. HL-60 cells (ATCC CCL240; obtained from the Amer

ican Type Culture Collection) were grown in RPMI 1640 mediumsupplemented with 2 rriMglutamine, 100 units/ml penicillin, 100 units/ml streptomycin, and either no (serum-free) or 10% serum (from calf,fetal calf, or human), at 37°Cin an atmosphere of 5% CO2. When

serum-free medium was used, 5 ¿ig/mlinsulin and 5 Mg/ml transferrinwere included. The cells were routinely passaged every 3 or 4 days andseeded at a density of 2.5 x IO5cells/ml. Cells used for differentiation

experiments were between passages 19 and 35. The cell numbers weremeasured with a Coulter counter. Viability was assessed by exclusionof 0.2% trypan blue.

Differentiation of HL-60 Cells. Cells (5 x 105/well) were plated in12-well culture dishes in 2 ml of medium containing the specified serumand were treated with l /ÃœMRA (added from a 10 ITIMstock solution indimethyl sulfoxide) or the appropriate concentration of sphinganine(prepared as the 1:1 molar complex with fatty acid-free BSA). Sphinganine was then added every 24 h for 4 days.

Morphology was judged on slides prepared with a Shandon SouthernCytospin and stained with Wright-Giemsa stain (Cameo Quik II). Thepercentage of cells capable of reducing NBT was determined by counting the number which contained precipitated formazan after a 30-min37°Cincubation with an equal volume of NBT (1 mg/ml in 140 mM

NaCl, 9.2 mM Na2HPO4, 1.3 mM NaH2PO4, pH 7.4) and 200 nMPMA.

Respiratory Burst Measurements. After the indicated treatments,cells were collected by centrifugation and washed with modified PBS(0.1 g/liter CaCl2, 0.2 g/liter KC1, 0.2 g/liter KH2PO4, 0.1 g/literMgCl2 6 H2O, 8.0 g/liter NaCl, and 2.16 g/liter Na2HPO4 7 H2O)containing glucose (1 g/liter). The cells were then resuspended inmodified PBS plus glucose at a density of 1 x IO7 cells/ml. The

respiratory burst of each group was quantitated by measuring thereduction of cytochrome c (25 mg/ml), as reflected by the change inabsorbance at 549 nm minus 540 nm measured with a SLM AmincoDW-2000 spectrophotometer in the dual wavelength mode. Each meas-

4The predominant long-chain bases in mammalian tissues arc sphingosine andsphinganine. Sphingosine refers to the 18-carbon long-chain base with a 4-trans-double bond (also called sphingenine). Sphinganine, which is used in this studybecause it is readily available with a defined alkyl chain length, is the saturatedform of sphingosine.

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RA-INDUCED DIFFERENTIATION OF HL-60 CELLS

urement was done with 2.4 x 10*cells in a total volume of 2.4 ml. The

respiratory burst was initiated with either fMLP (1 ^M) or PMA (100DM).

Uptake of |3H]RA by HL-60 Cells. [3H)RA (1 ^M; specific activity,20 mCi/iimol) was added to HL-60 cells (2.5 x 10' cells/ml) adapted

to growth in the indicated serum. At each time point, 0.5 ml of cellswas removed and collected on a Whatman glass fiber filter (Type GF/C) and the excess radiolabeled RA was removed by several washes withPBS. The filters were then dried and radioactivity incorporated by thecells was quantitated, after addition of 4 ml of Universo! cocktail, byscintillation counting with a Beckman LS7000 counter. Nonspecificbinding of the radiolabel to the filter was determined with 0.5 ml ofmedium (supplemented with the various sera) containing 1 UM[3H]RA

in the absence of cells. This quantity was then subtracted from thatdetermined in the presence of cells to give specific binding.

RESULTS

Effect of Different Sera on the RA-induced Differentiation ofHL-60 Cells. The maturation of HL-60 cells grown in mediumsupplemented with sera from different sources was assessed bymorphology and NBT reduction (Table 1). Cells grown inserum-free medium showed greater maturation by morphological (only 10% remained promyelocytic) as well as functional(82% were NBT+) criteria after 4 days in RA. Serum had an

inhibitory effect on differentiation, which varied with thesource. PCS allowed the most differentiation (i.e., only 25%remained promyelocytic and 58% were NBT+), while in calfserum (HyClone) 43% were promyelocytic and 43% were NBT+

and in human serum 44% were promyelocytic and only 37%were NBT+. As is typically seen (4), a small percentage of thecells in each serum group exhibit a more differentiated pheno-type without addition of RA.

Effect of Different Sera on the Uptake of |3H|RA by HL-60

Cells. Although this is the first documentation of such serumeffects for HL-60 cells treated with RA, a similar observationhas been made with F9 embryonal carcinoma cells grown inserum-containing versus serum-free media (39). These investigators suggested that serum albumin, which is known to bindRA (40), was responsible for the decreased differentiation. Totest this possibility, the uptake of [3H]RA by HL-60 cells wasquantitated in serum-free medium, 10% PCS, 10% calf serum,or 10% human serum. The results, shown in Fig. 1, demonstratethat serum decreased the cellular uptake of RA by 40 to 80%,depending on the source. To determine if albumin could beresponsible for this decrease, uptake was also measured inserum-free medium supplemented with 100 J/M bovine serum

Table 1 RA-induced differentiation of HL-60 cells grown in different serumHL-60 cells adapted to growth in the KI'M I 1640 medium supplemented with

no serum, PCS, human serum, or calf serum (all at 10% final concentration) weregrown in the presence or absence of RA for 4 days. Differentiation was assessedby morphology and NBT reduction, as described in "Materials and Methods."

The results from a representative determination are shown. These findings wereconfirmed by repeating the measurements twice for cells in serum-free mediumand at least 4 times for those in serum-containing media. In each case, 200 cellswere scored.

SerumNonePCSCalfHuman[RA](MM)01010101Blast"00000100Pro8610882587438444Myelo103363311311135Meta433225117318Band014191812Seg010380111NBT*(%)1782758643737

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Fig. 1. Uptake of [>H|RA by HL-60 cells. The uptake of [JH]RA by HL-60cells adapted to growth in serum-free medium (O) or medium supplemented with10% human serum (A) calf serum (A) or PCS (•)was measured as described in"Materials and Methods."

10080604020ASerum-

PCS CalfHumanfreeSerumSerum,|Ifift•ftTLÌUilII

fl Blast myeloblast; Pro. promyelocyte; Myelo, myelocyte. Meta, metamyelo-

cyte; Band, banded neutrophil; Seg. segmented neutrophil.

Fig. 2. Effect of sphinganine on RA-induced differentiation of HL-60 cells indifferent sera. The degree of maturity of cells grown in medium supplementedwith no serum, 10% PCS, 10% calf serum, or 10% human serum was assessed bymorphology (G) (expressed as the percentage of cells which had matured beyondthe promyelocyte stage) and NBT reduction (•)(determined as described above).The concentrations of sphinganine received daily was: for cells in serum-freemedium, 1 I¿M',in 10% PCS, 2.5 ^M; in 10% calf serum and 10% human serum,4 f¿M.The results for the serum-containing groups are averages of at least fourexperiments shown ±SE. C, Control; RA. RA alone, + Sa. RA + sphinganine.

albumin, which decreased [3H]RA uptake by approximately

70% (data not shown).Effect of Sphinganine on the RA-induced Differentiation of

HL-60 Cells Grown in Different Sera. To determine if otherserum factors, such as those that act via protein kinase C, areinvolved in the observed serum effects, differentiation was assessed in the presence of the long-chain base sphinganine, whichis a potent inhibitor of this enzyme. Because it is a normalintermediate of sphingolipid biosynthesis, exogenously suppliedsphinganine is metabolized by cells to complex sphingolipidsthat do not inhibit protein kinase C (27, 38). Therefore, tomaintain cellular levels during the 4-day differentiation period,sphinganine was supplied daily. The cytotoxicity of the long-chain base was much greater in serum-free medium and variedsomewhat with the different sera; hence, each group was giventhe highest possible concentration of sphinganine which didnot decrease the viability below 70% (after 4 days).

As illustrated in Fig. 2, sphinganine had little effect on theRA-induced differentiation of HL-60 cells in serum-free medium; however, it increased the percentage of mature cells inall of the serum-containing media. The cells in PCS treatedwith RA and sphinganine differentiated to levels close to thoseseen with RA alone in serum-free medium (as judged by morphology). Likewise, the percentage of mature cells after RA andsphinganine treatment in calf serum was similar to those obtained with RA in PCS. The percentage of NBT-positive cells

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RA-INDUCED DIFFERENTIATION OF HL-60 CELLS

was also increased with human serum, although to a lesserextent. Treatment with sphinganine alone (i.e., without RA;data not shown) slightly increased the percentage of differentiated cells (usually between 10 and 20%).

Effect of Sphinganine on the Respiratory Burst of HL-60 CellsDifferentiated by RA. During granulocytic maturation, cellsnormally acquire the ability to produce Superoxide when activated by physiological stimuli such as the chemotactic peptidefMLP. Whereas phorbol ester-stimulated Superoxide production requires only a functional NADPH oxidase, the responseto fMLP entails the presence of the fMLP receptor and thecoupling mechanisms to the oxidase. HL-60 cells induced todifferentiate by dimethyl sulfoxide and dimethyl formamideappear to develop all these components and produce Superoxidewhen stimulated by fMLP (41,42). However, cells treated withRA show little activation in response to this peptide (42),suggesting that maturation induced by this agent is incomplete.To determine whether sphinganine treatment affects the abilityof RA-differentiated HL-60 cells to be activated by fMLP, therate of Superoxide production in response to both this peptideand PMA was quantitated.

For these experiments, the cells were given sphinganine onlyfor the first 2 days, because activation of the NADPH oxidaseby either phorbol esters or fMLP is a protein kinase C-depend-ent event of which sphinganine is a potent inhibitor (27).Treatment for only 2 days allows subsequent metabolism of thelong-chain base (38) so that protein kinase C is not inhibitedby day 4, when this process was measured. When conducted inthis way, the initial rate of Superoxide production in responseto fMLP was increased in cells treated with RA alone (2.2-fold)relative to the undifferentiated cells (Fig. 3). The rate of thefMLP-initiated respiratory burst was further increased (4.1-fold, relative to control) in HL-60 cells treated with both RAand sphinganine. Some variability in the fMLP-stimulated rate

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Fig. 3. The respiratory burst of differentiated HL-60 cells. Cells were grownin RPMI 1640 medium supplemented with 10% calf serum with no additions(Control), 1 JIM RA, 4.0 JIMsphinganine (Sa) (added on days 1 and 2 only), orboth 1 ¡,,M RA and 4.0 I/Msphinganine (added on days 1 and 2). After 4 days, therespiratory burst was measured as described in "Materials and Methods." The

respiratory burst shown in the top panel was initiated by fMLP (1 /*M)while thatin the bottom panel was initiated with PMA (100 nM).

of Superoxide production was seen between different groups ofcells; averages of results from five separate experiments showthat this rate increased 75 ±30% for RA alone and 299 ±100% for RA plus sphinganine.

The rate of Superoxide production in response to PMA (Fig.3) was increased in cells differentiated with RA (2.1-fold) orRA and sphinganine (2.3-fold), relative to the control rate, inamounts consistent with the increases in NBT reduction (Fig.2). The average increase (calculated from five experiments) was82 ±24% for RA-treated cells and 123 ±60% for RA plussphinganine; hence, the effects of sphinganine are seen withboth agonists but are most evident with fMLP.

DISCUSSION

In this study, the differentiation of HL-60 cells induced byRA was shown to be sensitive to the medium in which the cellswere maintained. The presence of serum decreased the percentage of cells that were morphologically mature and capable ofreducing NBT. Uozumi et al. (43) have also reported that thepresence of serum or BSA decreases the differentiation of HL-60 cells induced by RA as well as the cellular uptake of [3H]-RA. The reduced uptake of RA by cells in serum-containingmedium may be partially responsible for the lesser differentiation. However, this does not fully account for the variation indifferentiation between the serum groups, because the level ofRA uptake (serum-free > human serum > calf serum = PCS)does not correspond to the degree to which either morphological or functional maturation was acquired (serum-free > PCS> calf serum > human serum). Other studies, including one inwhich uptake of RA is shown not to be necessary for inductionof differentiation (44) and one in which the increase in trans-glutaminase expression caused by RA in HL-60 cells was unaffected by albumin (45), suggest that many factors other thanthe level of RA uptake regulate the cellular effects of thiscompound. Another factor to be considered is the growth rateof the cells in the various sera, since it has been suggested thatthis parameter influences differentiation (11). The cells thatdifferentiated more (those grown in serum-free medium) grewsomewhat more slowly than those in the serum-supplementedmedium, consistent with the hypothesis that serum componentsinfluenced both of these processes.

Since sera from different sources vary significantly in manycomponents including growth and differentiation factors (46),these differences in maturation may arise from other signalsthat affect this process. In fact, the presence of serum has beenshown to inhibit the differentiation of HL-60 cells caused byphorbol esters (compared to that in serum-free medium), withcalf serum being more inhibitory than fetal calf serum (47).Since protein kinase C is believed to be the primary regulatorof phorbol ester-induced differentiation (20) and participates inthe regulation of other serum factor-induced processes (18, 19),a serum component could be modulating differentiationthrough the activity of this enzyme. To probe the role of proteinkinase C in these serum effects on RA-induced differentiationof HL-60 cells, the long-chain base sphinganine, a potentinhibitor of this enzyme in vitro (26) and in intact cells (26-38), was used. Earlier studies using sphinganine showed thatthe phorbol ester-induced differentiation of HL-60 cells intomonocyte/macrophage-like cells is inhibited by this long-chainbase (38, 48). Conversely, the maturation of these cells alongthe same pathway caused by la,25-dihydroxyvitamin D3, forwhich a regulatory role for this enzyme is unclear, is not blockedby this long-chain base (48). While these results indicate that

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the involvement of protein kinase C differs in these two pathways, they also demonstrate that treatment of HL-60 cells with

sphinganine does not nonspecifically block maturation or causecell death.

The percentage of cells displaying the mature morphologyafter 4 days of RA treatment was increased by sphinganine inthe groups grown in serum-supplemented medium, demonstrating that the regulation of this differentiation program is different than that involved in either phorbol ester- or l«,25-dihy-droxyvitamin D.,-induced differentiation. Furthermore, it suggests that protein kinase C participates in the serum-dependentinhibition of RA-induced development. Whether this enzymeregulates the differentiation stimulated by RA is unknown. Aswith many other compounds that cause maturation of thesecells, the activity of protein kinase C increases as cells maturein response to RA ( 15). This agent also increases the expressionof a phosphoprotein believed to be a substrate of this enzymein these cells ( 16). While these findings suggest that this vitaminA analogue stimulates protein kinase C, RA inhibition of thisenzyme has also been demonstrated (17).

As expected from the increased percentage of NBT+ cells,

the production of Superoxide in response to PMA stimulationby cells treated with both RA and sphinganine was greater thanthat of cells given RA alone. Because the increase in Superoxideproduction when RA and sphinganine were used together wasgreater when stimulated by fMLP than by PMA, it appearsthat the long-chain base aids the HL-60 cells to differentiatemore completely, so that they become responsive to physiological agonists. A similar increased responsiveness to fMLP hasbetween reported when HL-60 cells were induced to differentiate with the combination of RA and a T-cell-derived lympho-kine differentiation-inducing activity (13).

The success of RA therapy is usually predicted by differentiating cells isolated from a patient's bone marrow and cultured

in medium supplemented with PCS with RA (49, 50). Whilethis method has proven effective in some studies (9), the decrease in differentiation in human serum relative to that in PCSobserved here suggests these conditions may not be comparable.The finding that treatment of HL-60 cells with sphinganine, aninhibitor of protein kinase C, increases the percentage of cellsdifferentiated by RA as well as the ability of the cells to beactivated by a physiological stimulus suggests that RA therapymay be improved by concurrent treatment with a modulator ofprotein kinase C activity.

ACKNOWLEDGMENTS

We thank Anni Sereni for assisting in the initial experiments of thisstudy and Georgianna Guzman for maintaining the HL-60 cells.

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1990;50:222-226. Cancer Res   Victoria L. Stevens, Nancy E. Owens, Elliott F. Winton, et al.   Leukemia (HL-60) Cells by Serum Factors and SphinganineModulation of Retinoic Acid-induced Differentiation of Human

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