JOURNAL OF CELLULAR PHYSIOLOGY 139:313-319 (1989)

Effects of Fluorescent Derivatives of TPA on HL60 Cells: Dissociation

Between the Differentiation-Induced and Protein Kinase C Activity

PHUONC LAN TRAN,* CHRISTIAN LE PEUCH, AND MICHELE BASSET lmmunogenetique Molkulaire, lnstitut des Cordeliers, 75006 Paris (P.L. J.), and Centre de

Recherche de Biochimie Macromoleculaire, 34033 Montpellier (C.L.P., M.B.), France

The four fluorescent derivatives of TPA-dansylaza-TPA, NBDaza-TPA, and (N)- and (P)-dansylamino-TPA-were synthesized and examined for their ability to induce differentiation in human promyelocytic leukemic HL60 cells. At a concentration of 20 nM, all the derivatives inhibited proliferation and induced 60-80% of the cells to differentiate into macrophage-like cells. Removal of dansylaza-TPA from the medium after 5 h did not arrest adherence or the expression of nonspecific esterase activity. However, upon removal of any of the other three compounds after 5 h, HL60 cells became nonadherent and expressed low nonspecific esterase activity after additional culture. To investigate the relationship between protein kinase C (PKC) activation and cell maturation, PKC activity and translocation were measured after 0.5, 5, 24, and 48 h of treatment with each compound. Cells induced to differentiate by dansylaza-TPA or (N)- or (P)-dansylamino-TPA exhibited enhanced PKC activity, 50-80% of which was located in the particulate fraction. In cells that differentiated with NBDaza-TPA, 65-70% of PKC activity remained in the cytosol. After removal of the TPA derivatives, all cells exhibited PKC activity in the cytosol. These results indicate that the fluorescent derivatives are as potent as TPA in inducing HL60 cell differentiation. However, in the case of NBDaza-TPA and (N)- or (P)-dansylamino- TPA, their continuous presence in the culture medium was required for the recruitment of cells to differentiate. Consequently, it i s suggested that activation and translocation of PKC are among the early biochemical events that trigger HL60 cell differentiation. Nevertheless, these two events alone are not sufficient to induce differentiation.

Human promyelocytic HL60 cells were induced to differentiate into either mature granulocytes or macro- phages in response to various chemical compounds (Breitman et al., 1978; Collins et al., 1978, 1980; Huberman and Callaham, 1979; Rovera et al., 1979a). This cell line therefore provides a useful model for the study of the biochemical events associated with cell differentiation.

Phorbol esters such as 12-0-tetradecanoyl phorbol 13-0-acetate (TPA) are potent tumor promoters in mouse skin (Berenblum, 1975). Both in mouse skin and in cultured cells, TPA elicits various biochemical and biological responses (Slaga et al., 1978; Weinstein et al., 1979; Blumberg, 1981). These responses have been suggested to be mediated by high-affinity specific re- ceptors (Driedger and Blumberg, 1981; Horowitz et al., 1981; Solanski et al., 1981; Tran et al., 1983a), which mainly appear to be associated with the calcium- and phospholipid-dependent protein kinase C (PKC) (Nishizuka, 1984). In HL60 cells, TPA induces a ma- ture macrophage-like phenotype (Lotem and Sachs, 1979; Rovera et al., 1979b; Huberman et al., 1982, Murao et al., 1983). Its effects were shown to be

accompanied by protein phosphorylation andlor de- phosphorylation (Feuerstein and Cooper, 1983, 1984; Braverman et al., 1986). Vandenbark et al. (1984) and Ebeling et al. (1985) suggested that PKC activation is involved in the differentiation of HL60 cells. Shoji et al. (1986) and Homma et al. (1986) further showed that treatment of intact HL60 cells with TPA results in the translocation of the PKC from the cytosolic to the membrane fraction.

To study the interactions of phorbol esters with their cellular targets, we synthesized various fluorescent derivatives of TPA. We obtained, first, a mixture of two diastereoisomers that was resolved into two isomers, (N)- and (PI-dansylamino-TPA, and second, optically pure dansylaza-TPA and NBDaza-TPA (Tran et al., 1983b, 1984, 1986a). These fluorescent derivatives were shown to displace [3H]PDBu binding to C3HI 10T1/2 cells and to increase [3H]choline release from

Received July 7, 1988; accepted November 18, 1988. *To whom reprint requestsicorrespondence should be addressed.

0 1989 ALAN R. LISS, INC

3 14 TRAN ET AL

these cells (Tran et al., 1984, submitted). They acti- vated purified rat brain PKC to the same degree as TPA, although they displayed differential binding behavior toward this PKC (Tran et al., submitted). Our previous studies with fluorescent unresolved dansylamino-TPA showed that in C3HilOT1/2 cells it displays tumor-promoting activity initiated by the chemical carcinogen N-methyl-N’-nitro-N-nitro- soguanidine (Tran et al., 1986b). In the present study, we evaluated the ability of four fluorescent TPA deriv- atives to induce differentiation of the human promy- elocytic HL60 cell line. We investigated PKC activa- tion and the role of PKC translocation in the presence and absence of these derivatives a t various stages of differentiation. Our results show that they had differ- ential effects on HL60 cells and suggest that the differentiation induced may be dissociated from PKC activation.

MATERIALS AND METHODS Cell culture

Human promyeloeytic HL60 leukemia cells, gener- ously supplied by Dr. A. Ladoux (INSERM U204, Centre Hayem, Paris), were grown at 37°C in RPMI 1640 medium supplemented with 15% fetal calf serum, 2 mM glutamine, 10 mM Hepes, pH 7.4, 100 IU/ml penicillin, and 100 pgiml streptomycin, in a humidified atmosphere containing 5% COz. Cells were seeded twice weekly a t a density of 3 x lo5 cells/ml. In the differentiation experiments, TPA or its fluorescent derivatives were diluted from a stock solution in etha- nol and added after 24 h of subculture; the cells were harvested 0.5, 5, 24, and 48 h later. The concentration of ethanol in control and treated cells did not exceed 0.04%. The concentration of viable cells was deter- mined by trypan blue exclusion. Morphological exam- ination was performed using Wright-Giemsa staining. Monocytelmacrophage differentiation was assessed by the expression of nonspecific a-naphtyl AS-D acetate esterase, determined by the method of Li et al. (1973).

PKC assays Cells were harvested and washed twice in Ca2 +,

Mg2 + -free phosphate-buffered saline. They were sus- pended a t a density of lo7 cells/ml and homogenized in 20 mM Hepes, pH 7.5,300 mM sucrose, 2 mM ethylene- diamine tetraacetate (EDTA), 10 mM ethylene glycol bis (2-aminoethyl ether) N,NS\r‘,N’ tetraacetate (EGTA), and 2 mM dithiothreitol and in the presence of 10 pg/ml each of the protease inhibitor leupeptin, soybean trypsin inhibitor, and pepstatin, 25 pg/ml aprotinin, and 2 mM phenylmethylsulfonylfluoride. The homogenate was centrifuged at 9OOg for 5 min to remove cell debris and nuclei, and the supernatant fluid was centrifuged at 100,OOOg for 1 h. This fluid was used as the source of cytosolic fraction enzyme. The pellet was resuspended in the same homogenization buffer containing 0.5% Triton X-100, incubated on ice for 30 min, and centrifuged a t 15,000 rpm for 20 min. The resulting supernatant constituted the particulate fraction enzyme.

Samples of both the cytosolic and particulate frac- tions were applied to a DEAE-cellulose DE-52 column preequilibrated with buffer A (20 mM Hepes, pH 7.5,2 mM EGTA, 2 mM EDTA, and 1 mM dithiothreitol).

The columns were washed with 5 vol of buffer A, and PKC was eluted with the same buffer plus 0.12 M NaC1. PKC activity was determined by measuring the transfer of 32P from [y3’P]ATP into lysine-rich histones at 23°C after 5-min incubation (Le Peuch et al., 1983). The standard reaction mixture (final vol- ume: 60 ~ 1 ) contained 20 mM Hepes buffer, pH 7.5, 10 mM MgC12, 1 mM CaCI2, 1 mM dithiothreitol, 1 mg of lysine-rich histoneiml, 100 pM [Y-~~PIATP (600 cpmipmol), and 20 pl of sample, plus or minus 80 IJ-g of phosphatidylserineiml and 8 Fg of dioleiniml. The reaction was terminated by transferring 40-pl aliquots of the incubation mixture onto 2-cm-square pieces of Whatman cellulose phosphate paper (P81). Papers were washed three times with cold water and dried. The radioactivity on each piece was determined by scintillation counting. Protein was determined with Coomassie Blue (Bradford, 1976). PKC activity was taken as the difference between the 32P counts for samples containing phospholipids and for those con- taining none (controls). 32P incorporation was linear with time and with the protein concentration under present assay conditions. One unit of PKC activity was defined as the amount able to incorporate 1 nmol of 32P per min into histones.

Reagents RPMI 1640 and fetal calf serum were obtained from

Gibco (Flobio) and Flow, respectively. TPA was pur- chased from P. Borchert (Chemicals for Cancer Re- search, Eden Prairie, MN). The fluorescent phorbol ester derivatives were dansylaza-TPA, (N)- and (P)- dansylamino-TPA and NBDaza-TPA. They were syn- thesized according to Tran et al. (1984, 1986a). Phos- phatidylserine, 1,2-diolein, histone (type 111-S), pepstatin, and aprotinin were purchased from Sigma. Soybean trypsin inhibitor was from Boehringer, and leupeptin, from Bachem. DEAE-cellulose DE-52 was obtained from Whatman. [Y-~~PIATP (3000 Ciimmol) was from the Radiochemical Centre Amersham. Pro- tein determination reagents were from Bio-Rad.

RESULTS Induction of HL60 cell adherence by fluorescent

TPA derivatives The ability of the four fluorescent TPA derivatives

to induce HL60 cell differentiation was compared to that of TPA itself. As shown in Figure 1, three of these derivatives-dansylaza-TPA, (N)-dansylamino-TPA, and (P)-dansylamino-TPA-were labeled with 5-di- methylamino-naphtalene-1-sulfonyl (dansyl), whereas the fourth, NBDaza-TPA, bore a 4-nitrobenzo-2-oxa- 1,3-diazole (NBD) group. All four derivatives proved to be potent inducers of HL60 cell adherence (Fig. a), which occurred for 50% of the cells after 24 h of treatment a t AS0 concentrations of 12, 22, 14, and 20 nM, respectively, compared to 10 nM for TPA. These results show that 14 nM (P)-dansylamino-TPA was more potent than was 22 nM (N)-dansylamino-TPA in inducing maximal cell adherence. Concentrations higher than 50 nM caused damage to the cells when used for more than 24 h. Therefore, in our experiments, we chose a single dose of 20 nM. We observed that only a few cells had adhered to the plastic substrate after 5 h of treatment. After 24 h, 60-80% of the cells had

FLUORESCENT PHORBOL ESTERS AND KINASE C 315

a "- C H 3

CH,/ 8 L 6 CO-PhO p a

& N& CO-P ho

C

+ N O * * o

Fig. 1. Chemical structure of the fluorescent TPA derivatives. a, (N)- or (P)-dansylamino-TPA b, dansylaza-TPA; c, NBDaza-TPA. All compounds were stored in ethanol at -30°C. The final ethanol concentration was always < 0.04% and did not affect the assays.

attached, mostly in small clumps, whereas control cells grew as single suspension cultures. These effects of the TPA derivatives on cell adhesion resembled those of TPA itself (Table 1). In all cases, the percentages of adherent cells did not increase after 48 h of continuous treatment (Table 1). No difference was detected be- tween the activity of (N)- and (P)-dansylamino-TPA. The differences between the maximal effects of the four derivatives (Table 1, Fig. 2) may have been due to the use of different batches of cells. Adherent cells were also compared to control cells for morphological changes. These included an eccentric nucleus, a de- crease in the nucleus to cytoplasm ratio, and loss of azurophilic granulation (data not shown). Morphologi- cal differentiation in response to the fluorescent TPA derivatives was accompanied by inhibition of cell growth (Table 1). The differentiated cells exhibited intracellular accumulation of nonspecific esterase ac- tivity a t 24 and 48 h (Table 1). This accumulation was already observed after 5-h exposure to the different compounds. It is consistent with the observation re- ported by Rovera et al. (197913) and Fontana et al. (1981) that differentiated cells expressed macrophage characteristics as soon as they were recruited to differ- entiate.

loo'

O L I I J I 10 100

( n M ) Fig. 2. Effect of various concentrations of fluorescent TPA deriva- tives on adherence of HL60 cells. Cells were used a t a density of 3 x lo5 cellsiml in 10 ml of medium in 100-mm tissue culture dishes. The various compounds were diluted 1:1,000 in medium from stock solu- tions in ethanol and added to the cells after 24 h of subculture a t the final concentrations indicated; control cultures were treated with 0.04% ethanol. After 24 h of culture, the adherent cells were detached by scraping with a rubber policeman and counted. (0 ) TPA; ( X ) dansylaza-TPA; (A) (N)-dansylamino-TPA; (A) (P)-dansylamino- TPA; and ( +) NBDaza-TPA.

Intracellular distribution of PKC in HL60 cells treated with fluorescent TPA derivatives

As specified in Materials and Methods, the cells underwent subcellular fractionation for analysis of the subcellular distribution of PKC activity. The total amount of this activity in control cells (supernatant + particulate fractions) was 1.3 U/mg of protein, distrib- uted as follows: 70-80% of PKC activity was located in the cytosolic fraction, and 20-30%, in the particulate fraction (Table 2). We also noticed that the control cells displayed lower PKC activity in the confluent cultures than in the exponential phase cultures (Table 2).

As previously reported (Kreutter et al., 1985; Homma et al., 1986; Shoji et al., 19861, brief exposure of HL60 cells to TPA enhanced PKC activity, which was translocated to the particulate fraction. We there- fore examined the ability of the fluorescent derivatives to activate PKC and to change its subcellular distribu- tion in HL60 cells. For this purpose, cells were exposed to the fluorescent compounds for 0.5, 5, 24, and 48 h. Results are given in Table 2 and Figure 3. Those treated with 20 nM of either TPA or dansylaza-TPA for 0.5 or 5 h displayed a 40% decrease in supernatant PKC activity. Concomitantly, the activity in the par- ticulate fraction increased, from five- to 12-fold. Cells treated with 20 nM of either (N)- or (P)-dansylamino- TPA for 0.5 h displayed a three-fold increase in super- natant PKC activity, which slightly decreased when exposure was prolonged to 5 h. Again, a concomitant 10- to 16-fold increase in PKC activity was observed in the particulate fraction. No significant difference was observed between the increases in PKC activity in-

3 16 TRAN ET AL.

TABLE 1. Induction of macrophage characteristics by fluorescent TPA derivatives ~ ~ ~ ~

Time of treatment Cell adherence Nonspecific Growth inhibition Compound (h) (%) Morphology esterase (% of control) Dansylaza-TPA

TPA

24 48 24 48

Dansvlamino-TPA (N) 24

63 ? 6 Macrophage-like + 75 2 9 + .~

77 2 4 Macrophage-like + 81 i- 8 51 i 11

~~

MacroDhage-like + +

~

58 -t 3 62 ? 4 60 + 3 42 2 3 86 i- 5 ~-

48 75 -t 8 + 48 i- 4 Dansylamino-TPA (PI 24 67 k 8 Macrophage-1 i ke + 83 i- 4

48 65 i- 10 + 60 ? 4 NBDaza-TPA 24 38 2 5 Macrophage-like + 76 i- 2

48 56 2 8 + 86 + 3

HL60 cells, a t an initial concentration of 3 x lo5 cellsiml, were subcultured for 24 h and treated with each compound a t a concentration of 20 nM. At the times indicated, adherent cells were scraped off the culture dishes and assessed for viability, morphological change, and nonspecific esterase activity. Results are means 2 SD of four determinations.

TABLE 2. Effect of fluorescent TPA derivatives on the distribution of Ca2 +, phospholipid dependent protein kinase activity in the supernatant and particulate fraction.

Comuound Fraction

Ca2+, PL protein kinase aactivity 32p incorporation ( d m g of protein) Time after addine the agents (h)

0.5 5 24 48 None Supernatant 0.96 0.96 0.94 0.40

Particulate 0.32 0.32 0.06 0.24 Dans ylaza-TPA Supernatant 0.56 1.03 0.17 0.35

Particulate 2.26 4.16 1.44 1.20 TPA Supernatant 0.62 0.54 0.07 0.59

Particulate 3.92 1.29 3.54 3.44 Dansylamino-TPA (N) Supernatant 2.89 1.59 1.50 1.66

Particulate 3.14 4.64 3.36 1.56 Dansylamino-TPA (PI Supernatant 2.95 0.68 1.82 2.98

Particulate 4.97 2.97 1.52 3.19 None Supernatant 5.04 5.04 6.44 4.62

Particulate 3.53 3.53 2.52 4.27

Particulate 5.40 7.2 4.43 1.69

HL60 cells were seeded at a density of 3 x lo5 cellsiml, subcultured for 24 h, and exposed to 20 nM of each compound. Control cells were treated with 0.046 ethanol. At the times indicated, cells were washed twice with 10 ml of Ca2+, MgZ +-free phosphate-buffered saline, scraped off the culture dishes, and homogenized as indicated in Materials and Methods. Cytosols and 0.5% Triton-extracted of particulate fractions were analyzed for their Ca2+, PL-dependent protein kinase activity after DEAE-cellulose chromatography. Studies were performed on two batches of HL60 cells.

NBDaza-TPA Supernatant 3.50 6.31 8.08 4.88

duced by (N)- and (P)-dansylamino-TPA. Cells treated with 20 nM of NBDaza-TPA for 0.5 h exhibited a slight decrease in cytosolic PKC activity, which then in- creased after 5 h of exposure, and particulate PKC activity also rose 1.2- to two-fold.

For the treatments lasting 24 and 48 h, that is, for differentiated cells, PKC activity persisted for as long as one of the four derivatives or TPA was present in the culture medium. In the case of dansylaza-TPA, (N)- and (P)-dansylamino-TPA, and TPA itself, most of this activity was found in the particulate fraction, and with NBDaza-TPA, in the cytosolic fraction (Table 2, Fig. 3). These results therefore indicate that although the stimulation of PKC activity was rapid, its intracellular translocation does not seem essential to differentiation. Our results also show that contrary to the reports of several authors (Ballester and Rosen, 1985; Darbon et al., 1986; Fournier and Murray, 1987; Stabel et al., 1987) no decrease in PKC activity was observed for up to 24 h of treatment with TPA or its fluorescent derivatives. However, at 48 h, a decrease of 2540% was observed. The differences between the distribution of PKC activity in the cells after treatment with the three dansylated derivatives of TPA (Fig. 3) might be

due to the difference between the chemical structure of dansylaza-TPA and dansylamino-TPA, which might affect the interaction between PKC and these three derivatives. The fourth derivative, NBDaza-TPA, acted differently on PKC activity, perhaps because NBD is labile and deteriorates with time in aqueous solution (Andrews et al., 1982).

Effects of removal from the medium of TPA or its fluorescent derivatives on PKC activity and

HL60 cell differentiation HL60 cell adherence in response to TPA has been

reported to be irreversible and independent of the continuous presence of TPA (Rovera et al., 1979b). To test the reversibility of its changes induced by the four TPA derivatives and TPA itself, we exposed HL60 cells to 20 nM of each one for 5 h, removed the agents, and washed the cells. Adherent and nonadherent cells were then counted after an additional 24 or 48 h of culture. Table 3 shows that 24 and 48 h after removal of the agents, only cells treated with TPA or dansylaza-TPA adhered to the substrate. However, adhesion of these cells diminished after drug removal, since only 40-50% of the whole population differentiated. Under these

FLUORESCENT PHORBOL ESTERS AND KINASE C 317

," 15 .- >

0 0

0

0) In 0

r c 0 0

.- c

.c 3

.- c

L a

a 2 + 0 3 0

- .- c

u a \ c C I 0

0 C

Q) Q 3 m

c

L

0

/'

0 1 5 2 4 48 Time ( h r s )

Fig. 3. Time course of PKC distribution after addition of fluorescent TPA to HL60 cells. Cells were exposed to 20 nM of each compound for different periods. PKC activity was measured in the cytosol and 0.5% Triton-extracted particulate fractions after DEAE-cellulose chroma- tography. The ratios of supernatant to particulate PKC activity were plotted. Studies were performed on two batches of HL60 cells. (0) control; (0) TPA; ( x dansylaza-TPA; (A) (Nbdansylamino-TPA; (A) (P)-dansylamino-TPA; and ( + ) NBDaza-TPA.

conditions, cell growth was inhibited by less than 25%. In contrast, no such inhibition was observed after removal of (N)- or (P)-dansylamino-TPA or NBDaza- TPA from the culture medium, and the cells prolifer- ated in the same way as control cells. They also displayed lower nonspecific esterase activity. When we increased cell exposure to the compounds from 5 to 8 h, we obtained the same growth as after 5 h (data not shown). Consequently, the effects of (N)- or (PI- dansylamino-TPA and NBDaza-TPA on HL60 cell ad- herence were reversible after removal of the agents from the culture medium. However, unlike cell adher- ence, the morphological changes appear irreversible.

The distribution of PKC activity was examined in adherent and nonadherent cells. In adherent cells treated with dansylaza-TPA, the activity observed 24 h after removal of this agent was distributed fairly evenly between the cytosolic and particulate fractions. However, at 48 h postremoval, most of this activity was cytosolic (Table 3). Similar effects were observed in cells treated with TPA.

In nonadherent cells treated with (N)- or (PI- dansylamino-TPA, PKC activity was still high in the cytosol24 and 48 h after removal of these agents (Table 3), whereas nonadherent cells treated with NBDaza- TPA displayed enhanced PKC activity in both the cytosolic and particulate fractions. These results indi-

cate that stimulation of PKC activity did persist until 48 h after removal of the TPA agents in both adherent and nonadherent cells. However, maintenance of this activity in the particulate fraction required the contin- uous presence of either TPA or its fluorescent deriva- tives.

DISCUSSION In the present study, we demonstrated that the

ability of the fluorescent derivatives of TPA to induce HL60 cell maturation was of the same order of magni- tude as that of TPA. As regards dansylaza-TPA and TPA itself, the continuous presence of one or the other was not required for induction of cell adhesion and expression of macrophage-like characteristics, in agreement with the results reported by Rovera et al. (1979b). However, this was not the case for the three derivatives (N)- or (P)-dansylamino-TPA and NBDaza- TPA because their removal from the culture medium reversed both cell adhesion and inhibition of cell growth. The reversible properties of these compounds will therefore be useful for studying the mechanism of action of phorbol esters.

Dansylaza-TPA was the only derivative whose be- havior resembled that of TPA, since it changes both the subcellular distribution of PKC activity and cell mat- uration. These observations are consistent with those reported by Shoji et al. (19861, Homma et al. (19861, and Rovera et al. (1979b). Thus, the insertion of a nitrogen atom into the acyl chain and the linkage of the dansyl group to this atom do not appear to modify the biological activity of dansylaza-TPA. On the other hand, when, in the absence of such insertion, the nitrogen atom to which the dansyl group is attached is linked to the p-carbon of the acyl chain, the derivatives (N)- and (P)-dansylamino-TPA remain potent inducers of cell maturation but behave a little differently from dansylaza-TPA as regards PKC activation. With these derivatives, the -N-dansyl linked to the @-carbon of the acyl chain may therefore induce more perturbations than dansylaza-TPA in the environment of the sites a t which they bind to PKC and may affect the latter's subcellular distribution. This might also be the case for NBDaza-TPA, which was expected to behave like dansylaza-TPA. However, our results show that al- though i t was potent in inducing cell maturation, it did not significantly change the subcellular distribution of PKC activity. We cannot exclude the possibility that the lability of the NBD group might affect this distri- bution. Note that in previous in vitro activation of purified rat brain PKC, we found no significant differ- ence in the PKC activation induced by dansylaza-TPA and the three other compounds (Tran et al., submitted). It is conceivable that fluorescent compounds exhibit rather different behavior in intact cells and in vitro assays. However, i t is interesting to note that in labeling experiments on C3H/lOT1/2 fibroblasts, no difference was observed between the distribution of the fluorescent derivatives inside the cell (Tran and Deugnier, 1985; Tran and Grouselle, unpublished results).

PKC activation constitutes a major pathway of trans- membrane signaling. In several types of cells stimu- lated by phorbol esters (Kraft et al., 1982; Kraft and

318 TRAN ET AL.

TABLE 3. Effect of removal of fluorescent TPA derivatives on HL60 cell differentiation and the distribution of Ca2+, PL-dependent protein kinase activity in the supernatant and particulate fraction

Ca2 + , PL protein kinase 32P Growth Nonspecific incorporation (Uimg of protein)

Particulate

Time after

agents (h) (%) (%I esterase Supernatant removing the Cell adherence inhibition

~~

None 24 0 48 0

Dansylaza-TPA 24 48

TPA 24 48

Dansylamino-TPA (N) 24 48

Dansylamino-TPA (P) 24 48

26 34 46 36 0.1 0.3 4.0 0.8

0 - 0.94l 0.03 0 - 0.40 0.24

15 18 23 26

0 0 0 n

+ 0.30 + 1.08 + 3.12' + 0.66 - 2.71

1.63 -

2.97 4.55

0.50 0 0.27 0 0.16 0 0 0

~~ ~~ ~

0 - 6.44 2.52 - 4.62 4.27

None 24 0 0

- 1.15 3.96 48 0

co.10 0 - 5.96 2.21

NBDaza-TPA 24 48 10 .1 0

HL60 cells were seeded a t a density of 3 x lo5 cellsiml, subcultured for 24 h, and exposed for 5 h to 20 nM of each compound. Control cells were treated wtih 0.04%' ethanol. Cells were then washed and cultured in fresh medium for another 24 or 48 h. Adherent and nonadherent cells were harvested and studied for morphological changes, nonspecific esterase activity, and PKC activity. Studies were performed on two batches of HL60 cells. 'PKC activity in adherent cells. 'PKC activity in nonadherent cells.

Anderson, 1983; Wooten and Wrenn, 1984) and hor- monal ligands (Vilgrain et al., 1984; Naor et al., 1985), the mechanism of activation appears to involve the translocation of PKC to the membrane. However, in HL60 cells exposed to retinoic acid (Durham et al., 1985) and in human monocytes exposed to concanava- lin (Costa-Casnellie et al., 1985, 1986), this transloca- tion was not observed. Our data are relevant to situa- tions in which the PKC activity in cells differentiated by all four fluorescent compounds is translocated to the membrane or remains in the cytosol. As suggested by Shoji et al. (19861, PKC translocation may be related to the antiproliferative action of phorbol esters. Our re- sults indicate that it does not seem essential to late- stage differentiation. This is substantiated by our demonstration that after removal of TPA or its deriv- atives, the HL60 cells exposed to dansylaza-TPA or to TPA continued to differentiate and to display PKC activity in the cytosol. Activation of PKC (this report; Kreutter et al., 1985; Homma et al., 1986; Shoji et al., 1986), stimulation of the Na + -dependent H' efflux (Tran and Ladoux, unpublished results; Besterman and Cuatrecasas, 1984), and induction of c-fos expres- sion (Muller et al., 1985; Mitchell et al., 1985) appear to be among the early events that are initiated by phorbol esters and coordinate the recruitment of HL60 cells for differentiation. This raises the question of the signifi- cance of the continuous activation of PKC observed here in differentiated cells both in the presence and absence of phorbol esters. This activation constrasts with stimulation of the Na'-dependent H' efflux and c-fos expression by phorbol esters, which is transient. Moreover, the present demonstration that removal of (N)-, (P)-dansylamino-TPA, or NBDaza-TPA, which are as potent as TPA in inducing cell differentiation, can reverse cell maturation proves that PKC activation alone is not sufficient to induce cell differentiation. Further experiments are in progress with (N)- and (P)-dansylamino-TPA and dansylaza-TPA to clarify the mechanism of action of these compounds in HL60 and other hematopoietic cells.

ACKNOWLEDGMENTS The authors wish to thank Chehrazade Brick-

Gannam for assaying the nonspecific esterase activity.

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