Pergamon

PII: SO145-2126(97)00010-6

L.eukemia Research Vol. 21, No. 7. PP. 589-594, 1997. 0 1997 Else&r Science Ltd. All rights reserved

printed in Great Britain 0145%2126/97 S.17.M) + 0.00

DIFFERENTIAL EFFECTS OF PHORBOL ESTER ON SIGNALING AND GENE EXPRESSION IN HUMAN LEUKEMIA CELLS

Ralf Hass*, Igor Prudovskyt and Mogens Kruh@ffer$

*Institute of Anatomy, University Clinic Charit& D-10098 Berlin, Germany; tEngelhardt Institute for Molecular Biology, 117984 Moscow, Russia and SAarhus University, Department of Molecular and

Structural Biology, DK-8000 Aarhus C, Denmark

(Received 3 September 1996. Accepted 28 December 1996)

Abstract-Human U937 myeloid leukemia cells were treated with different concentrations of 12-0-tetradecanoylphorbol-13-acetate (TPA) to determine signals that contribute to growth arrest and differentiation. While 0.5 nM TPA had little if any effect, exposure of U937 cells to higher TPA concentrations (5-500 nM) revealed a complete growth arrest after 48 h. Cytosolic PKC activity decreased by 50% after exposure to 0.5 nM TPA and by 80 and 95% after stimulation with 5 nM and 50 nM TPA, respectively. Simultaneously, the PKC activity in the particulate fraction of U937 cells increased accordingly. These events were associated with induction of a differentiated monocytic phenotype. Expression of the c-myc gene was down- regulated and c-jun and c-fms transcripts increased following exposure to 5-500 nM TPA. In contrast, exposure to 0.5 nM TPA decreased c-myc expression and increased c-jun transcripts only transiently between 4 and 8 h while little if any effect was detectable on c-fms mRNA expression and subsequent differentiation. Taken together, these data suggest that a certain initial threshold of PKC activation is required for induction of a differentiated monocytic phenotype while beyond this threshold, a growth-arrested and differentiated state in these human leukemic cells can be maintained regardless of TPA concentrations. 0 1997 Elsevier Science Ltd

Key words: Leukemia cells, phorbol ester, gene expression, cell signaling threshold.

Introduction

Suspension U937 cells with round and smooth surface attach to the matrix and to each other by extending pseudopodia and developing 3-dimensional aggregates upon exposure to 12-O-tetradecanoylphorbol- 13-acetate (TPA) [ 1,2]. This is accompanied by a growth arrest in GdG,. Moreover, U937 tumor cells acquire certain functional properties associated with maturating mono- cytes/macrophages [3]. These include the expression of a-naphthyl-acetate esterase and the capacity to reduce nitroblue tetrazolium. Furthermore, expression of c-fms (M-CSF receptor), macrophage colony stimulating factor (M-CSF), prostanoids [4], cytokines [S], and cytokine receptors [6] are increased during TPA treat- ment of U937 cells. However, the intracellular signals of this differentiation program remain unclear.

Exposure of a variety of different cell types to TPA is

Correspondence to: Ralf Hass, Institute of Anatomy, AG Cell Biology and Neurobiology, University Clinic Char&& D-10098 Berlin, Germany.

also known to activate protein kinase C (PKC) by translocating this enzyme from the cytosol to the particulate or membrane fraction [7-91. In this context, previous studies have demonstrated that TPA-induced translocation of PKC in U937 cells is one of the earliest events associated with induction of differentiation [lo]. These events are associated with a significantly increased expression of the c-fos [ll], c-&n [12, 131 and junB [14] early response genes prior to the differentiation process. The expression of these genes remains elevated until the cells enter the retrodiffer- entiation program whereby the cells return back to the undifferentiated phenotype [lo].

While the initial cascade of these events is still unknown we were interested in the strength and the duration of signals initiated by the phorbol ester. Thus, the present work investigated the effects of different concentrations of TPA on U937 cells to determine signals involved in growth arrest and differentiation. Our findings indicated that a certain initial threshold of PKC activation in human U937 myeloid leukemia cells is required to overcome a certain barrier for induction of

589

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a differentiated monocytic phenotype. Moreover, once the cells have reached a certain activation level, there is little if any additional contribution of higher TPA concentrations to relay signals for the development of a differentiated monocytic phenotype.

Material and Methods

Cell culture

U-937 cells were cultured in RPM1 1640 containing 10% of heat-inactivated fetal bovine serum, 100 U/ml penicillin, 100 mg/ml streptomycin, and 2 mM L-gluta- mine in a 37°C humidified atmosphere with 5% COZ. Differentiation of U-937 cells was induced with various concentrations of TPA (OS-500 &I). Cell viability was assessed by Trypan Blue exclusion for the non-adherent U-937 cells and by acridine orange staining of the adherent TPA-differentiated cells.

Proliferation assay

Proliferation of U-937 cells was measured as described [3]. Briefly, U-937 cells were plated in 96- well microtiter plates at a density of 2 x lo3 cells/well and treated with TPA (OS-500 nM) for up to 48 h. The cells were pulsed with 20 ul of a 1:40 dilution of 6.7 Ci/ mm01 [3H]thymidine (New England Nuclear, Boston, MA) for 1 h, harvested and the incorporated radio- activity measured in a p-counter.

Preparation of particulate and cytosol fractions About 2 x 10’ untreated or 30 min TPA-treated U937

cells were washed three times in ice-cold PBS and hypotonically swelled in homogenization buffer contain- ing 20 mM Hepes, pH 7.4; 1 mM ethyleneglykoltetra- acetic acid (EGTA); 1 mM ethylenediaminetetra-acetic acid (EDTA); 5 mM 2,3-dihydroxybutane-1 ,Cdithiol; 1 mM phenylmethylsulfonyl fluoride (PMSF). The cells were homogenized with 15-20 strokes of a Dounce cell homogenizer using an A-type pestle. The crude particulate and cytosolic fractions were separated by centrifugation of the cell homogenate at 100 OOOg for 30 min and the resulting supernatant was used as cytosolic fraction. The pellet was resuspended and solubilized in homogenization buffer containing 0.5% (v/v) Triton-X-100 for 30 min on ice. The solubilized particulate fraction was prepared by centrifugation again at 100 000s for 30 min.

Protein kinase C assay Aliquots containing 10 ug of protein from cytosol or

the particulate fraction were used to measure PKC activity in an appropriate assay system (Gibco BRL, Grand Island, NY). The protein samples were incubated for 5 min at 30°C in phospholipid (0.28 mg/ml phos- phatidylserine and 1 mM TPA in 0.3% Triton X-100

mixed micelles), [Y-~*P]ATP (20 @/ml; New England Nuclear, Boston, MA) and 50 mM PKC synthetic peptide substrate derived from myelin basic protein. All assays were performed in the presence or absence of a PKC pseudosubstrate inhibitor peptide (Gibco, BRL). The samples were dried on phosphocellulose, washed in 1% HsP04 and the amount of phosphorylated peptide quantified by scintillation counting.

Northern blot analysis

Total cellular RNA from untreated and TPA-differ- entiated U-937 cells was prepared according to Chirg- win et al. [ 151. Aliquots of 20 ug of RNA were separated in agarose-formaldehyde gels and transferred to Hybond N nylon membranes (Amersham). The filters were hybridized to the following random primer [32P]dCTP-labeled cDNA probes:

(1) A 1.8 kb BamIWEcoRI insert of a human c-jun DNA probe containing a 1.0 kb cDNA and 0.8 kb 3’-untranslated sequences purified from a pBluescript SK(+) plasmid [ 161;

(2) The 1.6 kb Cla.I/EcoRI c-myc insert in the pMC41-3RC plasmid [17]; and

(3) The 4.0 kB EcoRI c-fms insert in the pc-fms 102 plasmid [18].

The filters were washed and signal intensity deter- mined by autoradiographic exposure.

Results

Human U937 myeloid leukemia cells started to adhere to the substrate and by forming three-dimensional cell clusters after treatment with 0.5-500 nM TPA for 30 min. These observations are in agreement with changes in morphology described previously for 5 nM TPA [3]. After 4-6 h, however, the adherent cells treated with 0.5 nM TPA detached again and continued to proliferate as single cells (data not shown). In contrast, U937 cells treated with 5-500 nM TPA remained adherent until the previously described retrodifferentia- tion process (data not shown and Refs [lo, 20,211).

In order to investigate the proliferative capacity, exposure of U937 cells to 0.5 nM TPA partially slowed growth for only 24 h while the proliferation rate increased again after 48 h (Fig. 1). In contrast, treatment with higher TPA concentrations (2 5 nM) was asso- ciated with complete inhibition of cell growth after 48 h (Fig. 1).

Previous studies have suggested that TPA-induced activation of PKC contributes to induction and main- tenance of U937 cell differentiation [lo]. This issue was also addressed at different TPA concentrations. Ex- posure to 0.5 nM TPA was associated with a partial

Threshold signaling and gene expression in leukemia cells 591

respectively (Fig. 2). These effects were also associated

--C 0.5 nM TPA with certain changes in gene expression. While treat- & SnMTPA ment with 0.5 nM TPA resulted in a transient down- -t 50nMTPA

+ 500nMTPA regulation of c-myc transcripts between 4 and 24 h, expression of this gene was down-regulated after 4 h and remained undetectable for at least 72 h when U-937 cells were exposed to higher TPA concentrations (Fig. 3A).

---- -4 Moreover, treatment with 0.5 nM TPA was associated

incubation time [h]

Fig. 1. Concentration-dependent effects of TPA on the proliferation of U937 cells. The cells were exposed to the indicated concentrations of TPA for 4,24 and 48 h. During the last hour of incubation, the cells were pulsed with [3H]thymi- dine for determination of incorporated radioactivity. Data represent the mean f S.D. of four independent experiments. Statistical analysis using Student’s t-test revealed high

significance for the data.

(50%) translocation of PKC from the cytosol to the particulate fraction at 30 min (Fig. 2). In contrast, treatment with 5 and 50 nM TPA revealed that 80 and 95% of PKC activity was translocated to that fraction,

i r u-937 0,5 nM 5 nM 50 nM

concentration of TPA

Fig. 2. Concentration-dependent effects of TPA on PKC activation. U937 cells were treated with the indicated concentrations of TPA for 30 min. PKC activity was measured in the cytosol and particulate fraction. The data represent the

mean of one experiment performed in triplicate.

with transient increases in c-jun transcripts between 4 and 8 h. Exposure to 5 and 50 r&I TPA also increased c- jun expression after 4-8 h. However, following a slight reduction after 24 h, c-jun progressively increased again until 72 h. In contrast, exposure to 500 nM TPA resulted in equally enhanced levels of c-jun after 4-72 h (Fig. 3B).

Expression of the c-fms gene, a marker of monocytic differentiation, was undetectable in U937 control cells and there was little if any induction of this gene following exposure to 0.5 nM TPA (Fig. 3C). In con- trast, treatment of U937 cells with 5-500 nM TPA was associated with continuously increasing c-fms mRNA levels between 24 and 72 h (Fig. 3C). Control staining of the 28s and 18s RNAs with ethidium bromide demonstrated equal loading of RNA within all of the appropriate blots (data not shown).

Taken together, these findings suggested that while 0.5 nM TPA is insufficient to induce differentiation, exposure to higher TPA concentrations is associated with growth arrest and the appearance of a monocytic phenotype.

Discussion

Previous studies have demonstrated that treatment of U937 cells with TPA induces monocytic differentiation and cell cycle exit in GdGi [l-3]. However, our present data demonstrate that 0.5 nM TPA is insufficient to induce growth arrest and differentiation of U937 cells. While recent work has demonstrated that even TPA concentrations as low as 0.1 nM can affect certain gene expressions in human myeloid leukemia cells [ 191, these findings indicate that a certain initial threshold of TPA is required to overcome a signaling barrier for growth arrest and differentiation. Indeed, higher TPA concen- trations (25 nM) are associated with a complete down- regulation of c-myc in U937 cells and cell cycle arrest in GdGr. These effects are paralleled by induction of the c- jun and c--s genes. The products of the c-&n gene are components of the AP-1 transcription factor which can activate genes involved in mono&c differentiation. It is thus interesting to note that exposure of U937 cells to 5-50 nM TPA is associated with a biphasic induction of the c-jun gene. However, there is no difference in the induction of monocytic differentiation and growth arrest at TPA concentrations between 5 and 500 nM.

592 R. Hass et al.

(a)

28 S -

18 S-

28 S -

18S-

28 S -

18 S-

28 S -

18S-

c-myc

0.5 nM

5 nM

50 nM

500 nM

28 S -

18 S-

28 S -

18 S-

28 S -

18 S-

28 S -

18S-

c-fms

28 S -

18S-

28 S -

18 S-

28 S -

18S-

28 S -

18S-

0.5 nM

5 nM

50 nM

500 nM

0.5 nM

5 nM

50 nM

500 nM

Fig. 3. Effects of varying TPA concentrations on c-myc (Fig. 3A), c-&n (Fig. 3B) and c-fms (Fig. 3C) expression. U937 cells were exposed to the indicated TPA concentrations for 4-72 h. Total cellular RNA (20 pg) was hybridized to the 2P-labeled cDNA probes.

Threshold signaling and gene expression in leukemia cells 593

The activation of PKC as a crucial signaling coor- dinator in these leukemic cells is also supported by previous studies using non-specific PKC-inhibitors, such as staurosporine and the isoquinoline sulfonamide H7, which abrogate at least partially TPA-induced increases in c-jun transcripts, growth arrest and appearance of the monocytic phenotype [ 101. Furthermore, the reversibil- ity of the differentiation process which is known as retrodifferentiation [20,21] is accompanied by a PKC inactivation and results in redistribution of PKC isozymes. In this context, the human HL-6OR, HL525 and ZPA-U937-Resistant (TUR) myeloid leukemia cell variants exhibit a significantly decreased expression of PKC genes and consequently, demonstrate resistance to TPA-induced monocytic differentiation [ 141. In this context, the difference observed at 5 and 50 nM corresponded to a more pronounced PKC translocation due to the ten-fold higher TPA concentration. This effect supports the threshold of PKC activation required for the induction of a differentiation program.

Taken together, our results suggest that PKC asso- ciated with the particulate fraction in human leukemic cells requires a certain threshold of activation in order to confer signals for a complete down-regulation of c-myc mRNAs and persistently enhanced expression of the c- jun and c-fms genes. Thus, a certain initial threshold of PKC activation is required for induction of a differ- entiated monocytic phenotype while beyond this threshold, a growth-arrested and differentiated state in these human leukemic cells can be maintained regard- less of the initial TPA concentration until the cells enter the retrodifferentiation program.

Acknowledgements-This work was supported by Grant No. OlKX9611 from the German Ministry for Science and Education (BMBF). MK acknowledges support from the Danish Centre for Respiratory Physiological Adaptation.

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