Inhibition of phorbol ester-dependent differentiation of human

THEJOURNAL OF BIOLOGICAL CHEMISTRY 8 1986 by The American Societyof Bio1o@celChemists, Inc.

Vol. 261, No. 27, Iasue of September25, pp. 12610-12615,1986 Printed in U S A .

Inhibition of Phorbol Ester-dependent Differentiationof Human Promyelocytic Leukemic (HL-60) Cells by Sphinganine and Other Long-chain Bases* (Received for publication, February25, 1986)

Alfred H. Merrill, Jr.3, Anne M. SereniS, Victoria L. Stevens$, Yusuf-A. Hannung,Robert M. Bells, and Joseph M. Kinkade, Jr.S From the $Department of Biochemistry, Emory University School of Medicine, Attanta, Georgia 30322 and the §Department of Biochemistry, Duke University Medical Center, Durham, North Carolinu 27710

The effects of long-chain (sphingoid) bases on the pholipids, Ca2*,and diacylglycerols (phorbol esters) has been phorbol ester-dependent differentiation of HL-60 cells extensively investigated in uitro and in uiuo. In contrast, the were investigated since thesemolecules are potent in- ways in which protein kinase C is regulated negatively are hibitors of protein kinase C (Hannun, Y. A., Loomis, less well defined. Sphinganine and other long-chain (sphinC. R., Merrill, A. H., Jr., and Bell, R. M. (1886) J. goid) bases have been recently discovered to be potent inhibB i d . Chem. 261, 12604-12609). After 2 4 h, low con- itors of protein kinase C in uitro and in human platelets (6) centrations of sphinganine (1-5 p~ blocked both cell and to block the oxidative burst in human neutrophils (7). adherence and the inhibition of growth in response to These moleculeshave thepotential of serving as natural phorbol 12-myristate 13-acetate, as measured by cell inhibitors in viuo because they, like diacylglycerols, could be number and acid phosphatase activity. Sphinganine mobilized frommembrane lipids. and sphingosine decreased adherence by 60% at 1-3 The possible effects of sphinganine on protein kinase Cp ~ other ; long-chain bases were effective in parallel to dependent differentiation of HL-60 cells into macrophagetheir inhibition of protein kinase C. Sphinganine decreased the binding of [3HJphorboldibutyrate by the and monocyte-like cells was investigated. HL-60 cells are a phorbol receptor of HL-60 cells, protein kinaseC, and human promyeiocytic cell line that matures into a variety of inhibited the response of HL-60 cells to dioctanoyl- cell types depending on the stimulant (8-10). Since the phorglycerol, a cell permeable activator of this enzyme. bo1 ester receptor has been found to be protein kinase C (11) Long-chain base uptake by HL-60 cells was demon- and differentiation can be effected by lY2-dioctanoylglycerol and within 1-3 days (12), it is likely that protein kinase C is involved in differenstrated with [3-~H]sphing~ine much had been converted to ceramides. By day 3, most tiation, although other effects of phorbol esters cannot be of the cells had recovered the ability to adhere and excluded (13,14). exhibited macrophage characteristics, whereascells in When HL-60 cells were treated with s p h i n g ~ i n e ,there suspension did not differentiate.The level of free was pronounced inhibition of cell adherence and other marksphinganine inHL-60 cells was determined to be 12.3 ers of diffe~ntiationin response to phorbol 18-myristate 132 1.2 pmol/106cells. These results establish that sphinacetate (PMA’). The characteristics of the inhibition and the goid bases inhibit protein kinaseC in HL-60 cells and structural specificity suggested that these effects were due to may function physiolo~caIlyas negative e€fectors of inhibition of protein kinase C. Furthermore, sphinganine this enzyme. inhibited phorbol dibutyrate binding by HL-60 cells and differentiation of the cells in response to ~octanoylglycerol. These results suggest that long-chain bases can inhibit protein kinase C physiologically. Diverse biochemical and cellular changes occur in response to extracellular agents that stimulate phosphatidylinositol EXPERIMENTALPROCEDURES hydrolysis, mobilization of Ca2+, and activation of a lipidMaterials-RPMI 1640 medium was purchased fromGibco (Grand dependent protein kinase termed protein kinase C (1-3). Island, NY) and defined bovine calf serum was from HyClone LaboDiacylglycerols produced from phosphatidylinositols are nat- ratories (Logan, UT). Fatty acid-free bovine serum albumin and ail urally occurring activators of protein kinase C; but cellular other tissue culture reagents were obtained from Sigma. The sphinprocesses have been investigated using the cell permeable golipid standards: erythro-dihydrosphingosine(sphinganine), sphinN-palmitoyl~hy~sphingosine, and ceramides (from bovine , activators l-oleoy1-8-acetyIglycero1, l , Z - d i ~ t ~ o y l g l y ~ e r o lgosine, and phorbol diesters, which are structurally related (1-5). brain sphingomyelin) were purchased from Sigma; N-acetylsphinganResponses to these compounds range from acute metabolic ine and 3-ketosphinganine were synthesized according to Gaver and Sweeley (15).The other homologs were provided by Dr. DennisLiotta effects, such as activation of the oxidative burst in neutro- at Emory University. PMA was purchased from LC Services Corp. phils, to much more protracted responses, including stimula- (Woburn, MA) and [3H]phorbol dibutyrate (8.3 Ci/mmol) was from Amersham Corp.; 1,2-di~tanoylglycerolwas obtained from Avanti tion or inhibitionof growth and differentiation. The mechanism of activation of protein kinase C by phos- Polar Lipids (Birmingham, AL). The [3-3H]sphinganine was synthesized by the reduction of N(15)by NaB3& (AmershamCorp.) followed * This work was supported by National Institutesof Health Grants acety~-3-ketosph~ganine GM33369 (A. H. M.), CA22294 (J. M. K.), and AM20205 (R. M. B.) by acid hydrolysis, and purified by silicagel column chromatography The costs of publication of this article were defrayed in part by the (Unasil,ClarksonChemical Go., Williamsport, PA). Theproduct payment of pagecharges.This article mustthereforebehereby The abbreviations used are: PMA; phorbol 12-myristate 13-acemarked “&vertisernent” in accordance with 18 U.S.C. Section 1734 tate; GMa, NeuAco;2-+3Gal~l4Glcfll+Cer. solely to indicate this fact. 12610

Long-chain (Sphingoidl Bases and HL-60 Cell Differentiation

12611

yielded a single spot coincident with sphinganine when examined by typical for several different experiments. The results are expressed TLC with Silica Gel H plates developed in CHCl3/methanol/2 N as means zk S.D. and the significance of differences between groups NH,OH (40:lOI). The specific activity was adjusted to 17,000 cpm/ was evaluated with the Student's t test for unpaired data. nmol by quantitating the amount of sphinganine as the 2,4,6-trinitroRESULTS benzenesulfonic acid derivative (16). CeU Culture-The HL-60 cells (obtained from the American Type Effect of S ~ ~ ~ ~ on u Celt n Growth-The ~ n e effect of sphinCulture Collection, ATCC CCL240) were grownat 37 "C as a suspension culture in 175 cmz Nunc tissue cultureflasks (Vangard Interna- ganine on HL-60 cell viability and growth was investigated tional, Neptune, NJ). The cells were subcultured at a density of 0.25 because sphinganine has been reported to altergrowth and to X IOe cells/ml and used between passage numbers 30 and 40. be cytotoxic for Chinese hamster ovary cells (23). Sphinganine Incubation of HL-60 CeUs-Cells were centrifuged at 600 X g for 3 was chosen over sphingosine because the former is available min, and added at 1 X 106cells/wellin 12-wellculture dishes. Medium, commercially as a homogeneous compoundwhereas the latter PMA, and long-chain bases (prepared as the 1:l molar complex with fatty acid-free bovine serum albumin)at theindicated concentrations is a mixture of various homologs. Untreated cells doubled during the first 24 h (Fig. I), and were added for a total volume of 2.0 ml. After the desired times, the cells in suspension were counted and checked for viability (trypan this was not changed by 1 pM sphinganine, but both 2.5 and blue exclusion) with a hemacytometer. The attached cells were quan- 5 HM limited growth. None of the cells exhibited a loss of cell titated by measuring the DNA content (17) and by assaying for acid viability for the first 24 h. By the second day, all concentraphosphatase (18). tions of sphinganine were still somewhat inhibitory; 1and 2.5 When 1,2-dioctanoylglycerolwas used, the cells were treated essentially as described by Ebeling et al. (12). Sphinganine was added to pht inhibited growth without cytotoxicity whereas 5 JLM rethe cells, then ~ ~ ~ n o y l g l y c einr o1lpl of ethanol to yield 100 p ~ , sulted in significant cell death. The change intotal cell followed by additional maintenance additionsof 20 PM diacylglycerol numbers between days 2 and 3 indicated that growth inhibievery 2 h for 12 h. tion had ceased for the cells in 1 and 2.5 p~ sphinganine. tive ~ C p ~ e of~ Phorbot n t by ~ p h i ~ ~ ~ ~ - C o m p e t ibinding This may have beendue to removal of sphinganine by metabassays were conducted as described by Goodwin and Weinberg (19) olism (see below). and Ebeling et al. (12). Approximately 1 x IO6 cells/ml were incubated These effects d e ~ n d e don both the cell number, which with 12 nM [*H]phorbol dibutyrate (8.3 Ci/mmol) and varying concentrations of sphinganine (1:l with bovine serum albumin) for 1 h probably reflected surface dilution (6), and the sphinganine shown); therefore, these parameters at 37 "C. The cells were recovered on Millipore filters, washed, and to albumin ratio (data not counted. The datawere corrected for nonspecific binding by subtract- were kept constant except where noted. ing the counts/min obtained in thepresence of 300 nM PMA. Effectson PMA-induced Adherence and Growth InhibiKinetics of [3-3HlSphinganine Uptake by HL-60 Cells-Approxition-Upon adding 9 nM PMA, growthof the HL-60 cells was mately 1 X lo6 HL-60 cells in 1 ml of medium were mixed with an equal volume of medium containing 2.5 p~ [3-3H]sphinganine (equi- inhibited by 70% within 24 h and the majority of the cells molar with bovine serum albumin). After varying time intervals, an (61%) attached to the Petri dish (Table I), which is typical aliquot of the cells was removed and counted, and a portion was for this cell line (10).When 1p~ sphinganine was alsoadded, extracted as described below. The extracts were applied to Silica Gel the cells continued to grow and only 26% of the totaladhered. H plates and developed in CHCIJmethanol/S N NH,OH (40:10:1, These data establish that sphinganine prevented PMA-inv/v/v), air dried, sprayed with Amplify (Amersham Corp.), and sub- duced growth inhibition at a concentration where sphinganine jected to fluorography. Radiolabel was observed in only three regions of the chromatogram, coincident with ceramides near the solvent itself did not affect growth (cf. Table I and Fig. 1). Because the celis in sp~inganine cont~nued to grow, the front, sphinganine withan R p of approximately 0.45, and ina region near the origin that encompassed sphingomyelin and other more number available for adherence in response to PMA was polar complex sphingolipids (RFof 0.1-0.2). higher. This results in similar numbers of adherent cells (ie. AnalysC of ~ ~Bases-From - 1-4 X IO7 c cells were ~ recovered ~ 0.51 X~ IO6 for PMA plus sphinganine versus 0.76 X lo5 for by centrifugation, washed thrice with phosphate-buffered saline, and PMA alone, or a 33% difference) while the adherent cells as extracted immediately by a minor modification of the procedure of Bligh and Dyer (20): 1.5 ml of c~lorofo~/methanol(1:2) were added a percent of the total was much lower( i e . a 74% difference). and mixed thoroughly; 1ml each of chloroform and water were added Adherence has been expressed as the percent of the total and thetwo phases were separated by centrifugation; the upper phase viable cells to normalize for differences in growth. was discarded, and thechloroform phase was washed twice with water Adherence was further limited to 17.6 t 6.4% and 15.0 t and dried by passage through a small column containing Na,SO,. 2.1% by 2.5 and 5 PM sphinganine, respectively, without The extracts were saponified in methanolic KOH (0.1 M, and incubated at 37 "C for 1 h) to remove e s ~ r - c o n ~ i n i n g g ~ y c e r o l i p i ~ . T h e 2,4-dinitrophenyl derivatives were prepared according to Braun and Snell (21) as follows. The lipids were dissolved in 50 rl of methanol/ ether (l:l),then 0.5ml of0.2% f l u o r ~ i n i t r o ~ n z e n(Sigma) e in methanol/ether (1:l) and 0.5 ml of 2 M &BO3 (pH 9.6) were added. After incubating for 1 h at 37 "C, 2 ml of ether and 2 ml of water were added. The ether was collected and the aqueous layer was reextracted with an additional 1 mlof ether. The combined ether extracts were washed with 2 ml of water and dried through NaZSO,, and thesolvent was removed under a stream of NO.The recovery of 13H]sphinganine (60%) was used to correct for losses during extraction. For each experiment, the 2,4-dinitrophenyl derivatives of standard sphingosine, sphinganine (dihy~osphingosiRe), and ph~osphingosine (Sigma) were also prepared. The derivatives were dissolved in 50 pl of methanol, 5 mM potassium phosphate (pH 7.0) (%IO) and 10 ql were injected onto a 0.5 x 25-cm CIS column (ISCO) and eluted isocratically with this same solvent.2 The derivatives were detected at 360 nm with an ISCO v, Detector. The standardsphinganine eluted at 11.2 min and sphingosine at 9 min, and both were well resolved from other species. StatLtical Methods-Data given in tables and figures are results

'Merrill, A. H., Jr., Wang, E., Mullins, R. E., and Wertz, P. W., manuscript submitted for publication.

Long-chain (Sphingoid) Bases and HL-60 Cell Differentiation

12612

TABLEI Effects of sphinganine (Sa) on the response of HL-60 cells to PMA Cells were incubated for 24 h with 1 NM sphinganine, 8 nM PMA, or both, and then the total number of viable cells in suspension and attached to the Petri dish was determined. Period of incubation

N~~~

Sa

Treatment PMA

PMA

+ Sa

h

ceUs/dish ( X l W )

0 24

1.0 f 0.1 1.78f 0.18 1.91 f 0.14 1.25 f0.05" 1.95 +. 0.15 % adherence

24

4.5 f 1.5

3.7 f 0.7

61.3 f 1.0

25.5 f 7.7

% viability

24 97 95 " p< 0.05 compared to all other groups.

80

93

TABLEI1

No PMA 0 p~ sphinganine 1p~ sphinganine

2.5 p~ sphinganine 5 p~ sphinganine

10

Sphinganine

Acid phosphatase activities of HL-60 cells after treatment with PMA and sphinganine Cells were incubated for24 h with8 nM PMA and varyingconcentrations of sphinganine and the acid phosphatase activities of the cells in suspension and attachedto the Petri dishwere determined. Treatment

5

0

~ d h ~ ~ -

Suspended Adherent nmollhldish

?6 total

4 4 5 4

8 nM PMA 0 .uM mhinganine "

0.11 f 0.05 0.52 f 0.01 83 (60%) 0.22 f 0.07 0.44 f 0.11 67 1 p~ sphinganine (87%) 0.30 0.04 38 2.5 p~ sphinganine 0.49 f 0.28 (94%) 5 PM sphinganine 0.52 f 9.23 0.14 f 0.04 21 (80%) The activity of the suspendedcells on day 0 was 0.84 f 0.05 nmol/ h/dish. Percent viabilityof suspended cells.

*

decreasing cell viabilities (data not shown). Bovine serum albumin added alone at equivalent concentrations had no effect ongrowth or adherence. Acid Phosphatase Activities of Treated Cells-For a more quantitative index of differentiation, the acid phosphatase activities of suspended andattached cells were compared (Table 11).Essentially allof the acid phosphatase activity was associated with the cells in suspension until PMA treatment, when varying percentages were transferred from the media to the dish. Sphinganine caused a concentration-dependent increase in theactivity remaining in suspension and a decrease in the adherent activities, which reflect inhibition of cell adherence. Inhibition of Phrbol Dibutyrate Binding by SphiqanineThe effect of sphinganine on phorbol dibutyrate binding was investigated because the blockage of attachment may be due to inhibition of protein kinase C, which is thought to be the phorbol ester receptor of HL-60 cells (11). Spinganine blocked [3Hlphorbol dibutyrate binding (Fig. 2), with 50% inhibition a t approximately 15 phi. Additionof 20 pM bovine serum albumin alone did not alterbinding significantly (8%).

20

(rM)

FIG. 2. Competition of sphinganine for phorbol dibutyrate binding by HI,-60 cells. Sphinganine at the indicated concentrations or fatty acid-free bovine serum albumin at20 PM was added to the cells with 12 nM [3H]phorbol dibutyrate and binding (including correction fornonspecific binding)was determined as described under "Experimental Procedures."

Acid phosphatase activitf

1.57 f 0.08 0.06 f 0.02 (96%)b 1.53 f 0.01 0.06 f 0.03 (90%) 1.25 f 0.16 0.06 f 0.02 (98%) 0.91 -+ 0.16 0.04 f 0.01 (87%)

15

TABLEI11

Inhibition of dwctanoylglycerol-induceddifferentiation of HL-60 ceUs by sphinganine Cells were incubated with dioctanoylglycerol and varying concentrations ofsphinganineasdescribedunder"ExperimentalProcedures." [Sphinganine]

% viable cells attached"

BM

0 100 f 10 1.0 70 f 32 2.5 63 f 21b 5.0 50 f 2ijb a Compared to cells treated with diacylglycerol only. Significantly differentfrom control (p < 0.05).

-

This concentration of sphinganine was higher than that resulting in 50% inhibition of attachment, but a higher cell number and shorter incubation time was used for binding. Therefore, less sphinganine would have been taken upby the cells in the binding experiment, and the effective concentration in themembrane would probably also be lower. To test this possibility, the effects of sphinganine on growth, viability, and adherence were evaluated with the higher cell numbers used in the binding experiments. Beginning with lo6 cells/ml, 25 phi sphinganine had no effect on cell growth (after 24 h, cell numbers in the absence and presence of sphinganine were 1.71 f 0.18 and 1.75 k 0.15 x lo6cells/ml, respectively), and 50 phi was only slightly inhibitory (1.49 f 0.27 x lo6 cells/ml). All groups had viabilities >97%. Adherence was reduced by 30% at 25 p~ sphinganine and 65% at 50 phi. Therefore, as predicted by the surface dilution model of Hannun et al. (6), the requirement of higher concentrations of sphinganine to inhibit phorbol dibutyrate binding at IO6 cells/ml can be attributed to the higher cell number. Inhibition of Diucylglycerol-induced Cell Attachment by Sphinganine-Dioctanoylglycerol, a cell-permeant activator of protein kinase C, also induces HL-60 cell differentiation (12). Dioctanoylglycerolwas added at 100 p~ with or without sphinganine in an initial loading dose, and additional dioctanoylglycerol was given to the cells in maintenance doses (20 PM) every 2 h for 16 h total. Sphinganine reduced adherence by 50% at approximately 5 p~ (Table 111). This trend was observed in three separate experiments; however, more precise comparison were thwarted by variability in the response of

12613

Long-chain (Sphingoid) Bases and HL-60 Cell Differentiation

TABLE V the cells to dioctanoylglycerol, which must be added as deMarkers of HL-60 cell differentiation after treatment with PMA and scribed above to elicit differentiation (12). sphinganine Structural Specificityof the Inhibition-The concentration Cells were treated with 8 nM PMA and sphinganine for 3 days and dependence of sphinganine inhibition of PMA-induced atexamined for morphology, a-naphtholacetate esterase (ANA), and tachment is shown in Fig. 3; results of similar experiments acid phosphatase activity. using other long-chain bases are also shown. Sphinganine at [Sphinganinel ( p M ) 3 p~ caused 50% inhibition and sphingosine,the predominant Parameter 0 1 2.5 5 long-chain base found in mammalian sphingolipids (24), caused 50% inhibition at 1pM. Stearylamine, which is S t N C No PMA, suspended cells +++ Promyelocytic morphology +++ +++ +++ turally related but lacks the 1,3-dihydroxy groups, effected - + Macrophage morphology similar inhibition at 10 p ~ Other . evidence for the minimal + + ++ ANA activity NA" involvement of the 3-hydroxyl was similar inhibition by 30.20 0.22 0.25 O.l6* Acid phosphatase ketosphinganine (not shown). 8 nM PMA, suspended cells Both the free amino and the long alkyl chain were impor+ + NA + ANA activity tant. Ceramides from bovine brain and N-palmitoyldihydro0.30 0.19 0.12 0.06 Acid phosphatase sphingosinewere not inhibitory, nor was N-acetylsphinganine 8 nM PMA, adherent cells + + + Promyelocytic morphology + at up to 500 p ~ Octylamine . did not inhibit, nor did another +++ +++ +++ +++ Macrophagemorphology short-chain analog of sphinganine, 1,3-dihydroxy-2-amino-3++ ++ ++ +++ ANA activity phenylpropane. 0.60 0.64 0.60 0.64 Acid phosphatase Effects of Sphinganine on Cell Morphology and Histochema Too few viable cells to score. ical Parameters-Expression of most other signs of HL-60 Activity in nmol/h/106 cells. cell differentiation requires longer time periods after treatment with PMA. For these experiments, the cells were examined on day3 for adherence and acid phosphatase activity 1.5 1.5 (Table IV) and morphology and the marker enzymes a-nap3 tholacetate esterase and acid phosphatase (Table V). 0

A

1.0

-a

c)

-

e .5

*'

c.

0 CI

0 E

1

10

Compound

(pM)

1

TABLE IV

Adherence and acid phsphutme activitiesof HL-60 cells nfter treatment with PMA and sphinganine for 72 h Cells were incubated for 72 h with 8 nM PMA and varying concentrations of sphinganine and the acid phosphatase activities of the cells in suspension and attached to the Petri dish were determined.

Azrt

Acid phosphatase

SusDended

%

No PMA 0 p M sphinganine 1 p~ sphinganine 2.5 phi sphinganine 5 #M sphinganine 8 XIM PMA 0 p M sphinganine 1 pM sphinganine 2.5 pM sphinganine 5 pM sphinganine

8 9

11 6

96 85

74 70

3

6

9 2 Hourr

4

FIG. 4. Kinetic@ of sphinganine uptake by HL-60 cells. Approximately 1 X 10' cells were suspended in medium containing 1 pM [3:H]sphqanine and after different intervals of incubation at 37 "C, ahquota wereremoved for measurement of the radiolabel in the medium and associated with the cells.

FIG.3. Effect of different long-chain bases on HL-60 cell attachment. The different compounds were added to the cells with 8 nM PMA and the percent attachment determined as described under "Experimental Procedures." The compounds used were sphingosine (So), sphinganine (Sa), stearylamine (St), octylamine (Oct), and ceramide (Cer) (similar results were obtained with bovine brain ceramidea and N-palmitoylsphinganine).

Treatment activity

O

Adherent

percent

nmol/h/dish

2.31 f 0.40 0.08f 0.03 2.77 0.25 0.05 2 0.03 2.35 0.11 0.05 f 0.02 0.24 f 0.01 0.04 f 0.01

2 14

0.05 f 0.01 2.20 f 0.09 0.05 f 0.01 2.95 f 0.62 0.06 f 0.01 3.10 f 0.30 0.12 f 0.08 0.82 f 0.38

98 98 98 87

* *

3 2

By day 3, most of the viable cells and acid phosphatase activities were adherent (Table IV). The majority of the cells had lost promyelocytemorphology, and resembledmacrophages with visiblyhigher a-naphtholacetate esterase activity (Table V). Acid phosphatase activities were higher foradherent cells when expressed as activity per l@cells, increasing 3-fold upon addition of PMA with and without sphinganine. This isa typical response of HL-60 cells to PMA (25). None of the few viable cells in suspension had clear signs of differentiation. Kinetics of Sphinganine Uptake and Metabolism-The rate of disappearance of [3H]sphinganinefrom the culture medium and its appearance in the cells is shown in Fig. 4. Approximately half of the sphinganine was taken up fromthe medium by the cells within 6 h. Free sphinganine accounted for most of the cellular radiolabel (Fig. 5).A large fraction was rapidly incorporated into ceramidesbut littlewas found in more polar sphingolipids (sphingomyelin and glycolipids).By days 2 and 3, only 0.07 and 0.06 nmol of free [3H]sphinganinewas associated with the cells. The radioactivity that could not be accounted for in lipids was in the aqueous phase and may reflect degradation, which produces 3H20. The removal of sphinganine by metabolism probably accounts for the absence of inhibition by day 3. Free Long-chain Base Contentof HL-60 Cells-The endog-

l

p

havebeen seen in the work of Pagano and Sleight (31). Ebeling et al. (12) found that dioctanoylglycerol displacedall of the phorbol dibutyrate from its receptor in HL-60 cells, whereas 1-oleoyl-2-acetylglycerolonly displaced half. Our results do not establish whether or not all of the effects of PMA involved protein kinase C. Since adherent cells differentiated in dishes containing sphinganine, protein kinase C may be responsible for adherence and growth inhibition and the otherphenotypic markers arise from other effects of PMA. Alternatively, the lack of differentiation of cells in suspensionmay indicate that growth inhibition and adherence are prerequisites for these changes. If so, oncecells have adhered, sphinganine would have no further effect on differentiation. 3 6 9 24 Since several amine inhibitors are now known, the releHours vance of these molecules to the normal in vivo regulation of FIG. 5. Levels of different [ ‘ H J s p h i ~ g o ~in p iHL-60 ~ cells at various time points. The lipids from the experiment described protein kinase C is worth considering. The levels of free iongin Fig. 4 were separated by thin layer c h ~ ~ a t o ~ a p(see h y“Experi- chain bases in HL-60 cells suggest that these molecules are mental Procedures”) and visualizedby fluorography. The radiolabel endogenous inhibitors of protein kinase C in uiuo.4 Unfortuwas coincidentwithceramide(Cer),sphinganine (Sa), andpolar nately, we do not know the subcellular localization of the sphingolipidstandardsand was quantitated by liquidscintilIation sphinganine which makes further comparison of intracellular counting. and extracellular concentrations ambiguous. We have recently demonstrated that free long-chain bases enous level oflong-chain bases in HL-60 celIs wasquantitated are not detected early in the biosynthesis of sphingolipids by high performanceliquid chromatography. Sphingosine was from [14C]serine,2but appear at later times. This indicates the majorfree long-chain base detected (Le. >80% of the that they arise from sphingolipid breakdown, rather than as total) and was present at 12.3 f 1.2 pmol/106cells. Since intermediates of long-chain base formation. Furthermore, the leukocytes contain approximately 5 nmol of sphingolipids/1O6 majority of the newly synthesized long-chain bases were decells, this corresponds to about 0.2% of the total long-chain graded with a half-life of approximately 8 h, which is much bases present in the cells (26). Previous studies have found more rapid than the presumed rate of sphingolipid turnover that free long-chain bases are not artifactsof the isolation or (22). derivatization procedures (22). The source of the free long-chain bases of HL-60 cells is unknown, but might be GM3 ganglioside,which increases DISCUSSION during ~fferentiationof HL-60 cells and affects differentiaNaturally occurring long-chain (sphingoid) bases inhibit tion when added exogenously (32,331. Since the level of free the phorbol ester- and diacylglycerol-induced adherence of long-chain bases fornormal, immature leukocytes cellsis not HL-60cells. Since sphinganine has been found to inhibit known, it i s possible that theamount observed in HL-60 cells protein kinase C in uitro, and the structural specificity (6) is high and accounts for its arrested d~ferentiat~on. Sphingolipids provide a logical counterbalance to the actiwas similar to the in~bition of differentiation, it appears that vation of protein kinase C by diacylglycerols becausethey are the effects of long-chain bases on HL-60 cells are due to inhibition of this enzyme? Further evidence forthis was the primarily found in theplasma membrane, often interact with displacement by sphinganine of phorbol dibutyrate from its receptors, and arewell known to undergo changes with differentiationand transformation (34). Long-chain bases may receptor, protein kinase C (11). Inhibition of protein kinase C by other “lipoidal amines,” additionally provide an endogenous inhibitor to prevent the which include palmitoylcarnitine, polyamines, and CP- “accidental”activation of the C kinase by diacylglycerolsthat 46,665-1, an antineoplastic compound,have been reported arise from biosynthetic or degradatory pathways and thereby (27, 28). Treatment of intact HL-60 cells with palmitoylcar- influence the level of diacylglycerol necessary to overcome nitine blocked PMA-induced celladhesion but notacid phos- inhibition. phatase activity (291, which suggests that only some of the A c k ~ ~ ~ ~ ~ e ~ thank t s - Dm. W e J. David ~ r n ~ Carson ~ h , effects of PMA on HL-60 cells are mediated via protein kinase discussions C. A similar conclusion has been drawn from the different Loomis, and Barry Ganong, and Emily Wilson for helpful concerning this work, and Dr. Lee Winton for the histological scoring effects of PMA and 1-oleoyl-2-acetylglycerolon HL-60 cells, of the cells. We also thank Marion Little for help in preparing this since both activate the C kinase but only the former induced manuscript. differentiation (13, 14). RE~E~NCES In contrast to these findings, di~tanoylglycerol acts as a good analog of naturally occurring diacylglycerol activators of 1. Nishizuka, Y.(1984) Science 225, 1365-1370 protein kinase C (12) and caused differentiation of HL-60 2. Ashendel, C. L. (1985) Bwchim. Biophys. Acta 822,219-242 cells much like PMA (8).This suggests that PMA is inducing 3, Noki, L. E. (1985) Annu. Rev. Biochem. 54, 205-235 4, Kishimoto, A., Takai, Y., Mori, T., Kikkawa, W., and Nishizuka, differentiation via protein kinase C activation. A possible Y. (1980) J. Bid. C k m . 255,2273-2276 ~ x p ~ a n a t for ~ o nthe differences is that PMA and dioctanoyl- 5. Hannun, Y. A., Loomis, C. R., and Bell, R. M. (1985) J. Biol. glycerol may be accessible to protein kinase C in all cellular Chen. 260, 10039-10043 compartments (phosphopro~ins are found in cytoplasm and 6. Hannun, Y. A., Loomis,, C., Merrill, A. H., Jr., and Bell, R. M. in or around the nucleus) (30) whereas 1-oleoyl-2-acetylglycero1 is not. Precedents for suchintracellular sorting of lipids * If one assumesthat the cellular volumeis approxima~ly1 pl/106 0.3

” ”

Inhibition of protein kinase C by sphinganine and steawiamine may also explainthe toxicity of these compounds (23,35). 3

cells (based on an average diameterof 13 pm), 12.3 pmol/106cells is equivalent to about 10&M sphinganine, oreven higherconcentration^ if the long-chain basesare located in a particular region of the cell.

Long-chain (Sphingoid) Bases and HL-60 Cell Differentiation (1986) J. Biol. Chem. 261, 12604-12609 20. 7. Wilson, E. Olcott, M.C., Bell, R. M., Merrill, A. H., Jr., and Lambeth, J. D.(1986) J. Biol.Chem. 261, 12616-1262321. 8. Rovera, G., Santoli, D., and Damsky, C. (1979) Proc. Natl. Acad. Sci. U. 5'.A . 76,2779-2783 9. Harris, P.9 and Ralph, P. (19'35) J. h w o ~ ~ t e 3 7 9 407-422 10. Miyaura, C., Abe, E., Suds, T., and Kuroki, T. (19%) Cancer Res. 45,4244-4248 11. Vanderbark, G. R., Kuhn, L. J., and Niedel, J. E. (1984) J. Clin. Invest. 73,448-457 12. Ebeling, J. G., Vanderbark, G . R., Kuhn, L. J., Ganong, B. R., Bell, R. M., and Niedel, J. E. (1985) Proc.Natl.Acad.Sci. U. 5'.A. 82,815-819 13. Kreutter, D., Caldwell, A. B., and Morin, M. J. (1985) J. Biol. Chem. 260,5979-5983 Commun. 14. Yamamoto, S., Gotoh, H., Aim, E., and Kato, R. (1985) J. Biol. Chem. 260, 14230-14234 15. Gaver, R., and Sweeley, C.C. (1966)J. Am. Chem. SOC.88,36433647 16. Yamamoto, A., and Rouser, G. (1970) Lipids 5,442-444 17. West, D. C., Sattar, A., and Kumar, S. (1985) A d . &&hem. 147,289-295 18. Schnyder, J., and Baggliolini, M. (1978) J. Exp. Med. 148, 435-34. 445 19. Goodwin, B. J., and Weinberg, J. B. (1982) J. Clin. Invest. 70, 35. 699-706

22. 23. 24. 25.

26. 27. 28. 29. 30. 31. 32. 33.

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