Phorbol ester (12-O-tetradecanoylphorbol 13-acetate) prevents ornithine decarboxylase inhibition and apoptosis in L1210 leukemic cells exposed to TGF-β1

Inr. J. Biochem. Cell Bid. Vol. 28, No. 12,p p1327-1335, . 1996

Pergamon PII: S1357-2725(96)00083-O

C o p y r i g h t 0 1996 Elsevier Science Ltd Printed in Great Britain. All rights reserved 1357-2725/96 $15.00 + 0.00

Phorbol Ester (12~O-tetradecanoylphorbol139acetate) Prevents Ornithine Decarboxylase Inhibition and Apoptosis in Ll210 Leukemic Cells Exposed to TGF-j?,* T. MOTYL,’ MARIA KASTERKA,’ KATARZYNA GRZELKOWSKA,‘t J. OSTROWSKI,’ M. FILIPECKI; ELiBIETA MALICKA, T. PIOSZAJ’ ‘Department of Animal Physiology, Faculty of Veterinary Medicine, Warsaw Agricultural University, Nowoursynowska 166, 02-787 Warsaw, Poland, ‘Department of Gastroenterology, Medical Center for Postgraduate Education, Warsaw, Poland, 3Department of Plant Genetics and Breeding in Horticulture, Faculty of Horticulture, Warsaw Agricultural University, Warsaw, Poland and 4Department of Pathology, Faculty of Veterinary Medicine, Warsaw Agricultural University, Warsaw, Poland Previous studies have shown that growth suppression and apoptosis of leukemic cells exposed to TGF-j?,is associated with the inhibition of ornitbine decarboxylase (ODC) - the key enzyme of polyamine pathway. The aim of the present study was to evaluate the influence of 12-O-tetradecanoylphorbol1Iacetate (TPA) - a potent ODC inducer on antiproliferative and apoptotic effects of TGF-/I, in L1210 leukemic cells. Cells were incubated in Z%FCS/RPMI1640 medium, supplemented with TGF-j?,(2 rig/ml), TPA (100 rig/ml) or a-diliuoromethylornithine (DFMO) (5 mM). Cell proliferation, apoptosis and necrosis were evaluated using [methyl-3H]thymidhte,electron microscopy, electrophoresis of DNA and trypan blue exclusion. Expression and activity of ODC were determinated by RT-PCR and measurement of WO, release from L-1-14C ornithine, respectively. TGF-fl, inhibited proliferation and induced apoptotic and necrotic cell death in L1210 leukemic cells. The above effects were associated with the inhibition of ODC expression and activity, measured 2 and 4 hr after TGF-/I, administration, respectively. The presence of DFMO, an irreversible inhibitor of ODC, led to apoptotic fragmentation of DNA, similar to that observed in TGF-b,-treated cultures. Administration of TPA simultaneously with TGF-b, significantly reduced antiproliferative, apoptotic and necrotic effects of TGF-/?,, and prevented its inhibitory action on ODC expression and activity. It is concluded that: down-regulation of ODC expression may be one of the early events associated with TGF-&evoked suppression of growth and apoptosis; ODC is involved in the mechanism of protective action of TPA on TGF-&related growth inhibition of L1210 leukemic cells. Copyright 0 1996 Elsevier Science Ltd Keywords: TGF-fi, Apoptosis Ornitbine decarboxylase Polyamines Phorbol ester Int. J. Biochem. Cell Biol.

(1996) 28, 1327-1335

INTRODUCTION

Transforming growth factor-b, (TGF-jI,) is a ubiquitous homodimeric peptide (25 kDa) *Results included in this paper have been partly presented at the 1 lth International Symposium “Regulatory Peptides”, Copenhagen, September 1996. tFellowship winner of the Foundation for Polish Science in

1995. Received 11 March 1996; accepted 22 July 1996.

which is known as a multifactoral regulator of cellular metabolism (Attisano et al., 1994; Roberts et al., 1988; Newman, 1993; McCartney-Francis and Wahl, 1994). Its metabolic effects depend on concentration, cell type, cell density, culture conditions, as well as the presence of other growth factors. TGF-B, is a negative growth factor in many normal and neoplastic cells, including leukemic cells (Fynan and Reiss, 1993). Among the biological effects

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attributed to TGF-/I,, the induction of apoptosis-programmed cell death has received much attention. TGF$,-induced apoptosis was found in different leukemic cells (Lotem and Sachs, 1992; Taetle et al., 1993; Motyl et al., 1993; Grzelkowska et al., 1995a). Our previous reports have shown that apoptosis of K562 and L1210 leukemic cells evoked by TGF-fl, is associated with the inhibition of ornithine decarboxylase (ODC)---the key and rate limiting enzyme of polyamine pathway (Motyl et al., 1993; Grzelkowska et al., 1995a). It is generally accepted that ODC is involved in the signal transduction pathway of many hormones, growth factors and tumour promoters (Garnet et al., 1991; Blachowski et al., 1993; Butter et al., 1991; Pavlath et al., 1993). It is interesting that erotic acid, a known tumour promoter, abolishes the suppressive action of TGF-P, on transcription and activity of ODC as well as on growth of L1210 leukemic cells (Grzelkowska et al., 1995a). Orotic acid itself stimulates the proliferation of K562 and L1210 leukemic cells, which is dependent on the increase of ODC expression and activity (Grzelkowska et al., 1995a, 1995b). In the present study we examined the effect of another tumour promoter, 12-O-tetradecanoylphorbol 13-acetate (TPA), on TGF-b,induced growth inhibition and apoptosis, as well as ODC expression and activity in L1210 leukemic cells. We found that TPA, an activator of protein kinase C and ODC, diminishes the mitoinhibitory, apoptotic and necrotic effects of TGF-fl, in examined cells.

MATERIALS AND METHODS

Media and reagents RPMI-1640 medium containing L-glutamine, fetal calf serum (FCS), fungizone, penicillin/ streptomycin mixture, proteinase K, DNAse free RNAse A, SuperScriptTM (Preamplification System for First Strand cDNA Synthesis, and GeneAmp DNA Amplification Reagent Kit were obtained from Gibco BRL. L -[l14C]ornithine ( 5 7 mCi/mmol), [methyl-3H] thymidine (5 Ci/mmol) and [(y”P] ATP (30 Ci/ mmol) were purchased from Amersham International. a-Difluoromethylornithine (DFMO) was kindly supplied by the Merrel Down Research Institute (Cincinati, OH, U.S.A.). The TGF-/I,, 12-O-tetradecanoylphorbol 13-acetate (TPA) and other chemicals were supplied by

Sigma Chemical Co. (St Louis, MO, U.S.A.). Sterile “Primaria” Petri dishes were purchased from Falcon, Becton Dickinson (Lincoln Park, NJ, U.S.A.). Cell culture Mouse lymphocytic leukemia L1210 cell line was supplied by the European Collection of Animal Cell Cultures (Porton Down, U.K.). Cell cultures were maintained in RPMI-1640 medium supplemented with 10% (v/v) FCS, streptomycin (100 pg/ml), penicillin (100 U/ml) and fungizone (250 pg/ml) in an atmosphere of 5% CO,/95% humidified air at 37°C and routinely subcultured every 2 or 3 days. Prior to the experimental treatment, cells were incubated for 24 hr in 2% FCS/RPMI supplemented with 2% bovine serum albumin (BSA). Assessment of cell viability and proliferation The cells were seeded in triplicate at an initial density of approx. 3 x 10’ cells/ml. Control dishes contained 2% FCS/RPMI (basal medium). Appropriate cultures received basal medium supplemented with test compounds. As indicated at time 0 and at subsequent time points, cells were counted on a hemocytometer and their viability was determined by trypan blue dye exclusion. The mitogenic response of L1210 leukemia cells to examined compounds was measured using [methyl-3H] thymidine. The cells were seeded in 96-well multidishes (3 x lo5 ml - ‘) in 2% FCS/RPMI with or without examined agents and cultured for 24 hr. Two hours before the end of incubation [methyl-3H] thymidine (1 pCi/well) was added. Cells were harvested by a Skatron Combi Cell Harvester and activity of incorporated isotope was counted in a Packard TRI-CARB 1600 CA B-counter. DNA fragmentation assay To examine DNA fragmentation the cells were cultured for 48 hr in a medium with or without test compounds. Then the cells were harvested by pipetting, pelleted at 200 g for 10 min and washed twice with ice-cold TBS [137 mM NaCl, 2.7 mM KC1 and 25 mM tris (hydroxymethyl)aminomethane (TRIS), pH 7.41. Cell suspension was adjusted to an approx. density of 2 x lo6 cells/ml, centrifuged at 200 g for 5 min and resuspended in 0.5 ml lysis buffer (10 mM TRIS, pH 8.0, 100 mM EDTA, and 0.5% SDS, pH 7.8) with 0.2 mg/ml proteinase K. The samples were incubated overnight at

Phorbol ester TPA 37°C. The lysates were extracted twice with phenol/chloroform/isoamyl alcohol (50:49: 1) and the DNA was precipitated with 0.1 vol of 3 M sodium acetate and 0.6 vol of isopropanol. DNA was recovered by centrifugation (12 000 g, 5 min, 20°C) and pellets were resuspended in 0.5 ml TNE (10 mM TRIS, 10 mM NaCl, and 1 mM EDTA, pH 8.0) with 50 pg/ml RNAse A. After incubation at 50°C for 1 hr, DNA was again precipitated with 0.7 vol of isopropanol and then resuspended in 50 ~1 TNE. The concentration of DNA was determined spectrophotometrically. Equal amounts of DNA (approx. 5 pg) were mixed with a loading buffer consisting of 12% Ficoll 400, 0.25% bromophenol blue and 0.25% xylene cyanol. A lDNAEcoRI, Hind III digest was applied to each gel to provide a size marker. Electrophoresis of DNA was performed for 1 hr at 100 V in a 1.6% agarose gel containing 10 pgg/ml ethidium bromide, with TAE (10 mM TRIS-acetate, 1 mM EDTA, pH 8.5) as a running buffer. Then the gels were photographed with Polaroid 655 film by UV illumination. Electrophoregrams were scanned using a JX-330 Sharp-scanner and Diversity OneTM (version 1.3; pdi) software.

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L-ornithine (10 mCi/mmol), 0.4 mM pyridoxal5-phosphate, 4 mM dithiothreitol and 0.1 mM EDTA. The incubation period at 37°C was 30 min. The protein concentration was determined by the method of Lowry et al. (1951) with BSA as a standard. ODC mRNA measurement

Total RNA of L1210 cell cultures, incubated for 2 hr under various conditions, was purified with the guanidinum thiocyanate method using TRIzolTM (Total RNA Isolation) Reagent (Gibco BRL). The reverse transcriptase-polymerase chain reaction (RT-PCR) was used to measure the ODC RNA transcript with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) serving as house-keeping gene. 32P-labelled PCR products were separated using polyacrylamide gel electrophoresis and visualized by autoradiography. The details of the procedure have been described previously (Grzelkowska et al., 1995a). Autoradiograms were quantified by scanning densitometry using a JX-330 Sharp-scanner and Diversity OneTM (version 1.3; pdi) software. Statistical evaluation

Electron

microscopy

The pellets of cells were fixed in 1.25% glutaraldehyde in a cacodylate buffer, pH 7.4 for 1 hr. Cells were washed with a cacodylate buffer and post-fixed in 1% osmium tetroxide in the same buffer, dehydrated in graded ethanol solution and in propylene oxide and embedded in Epon 812. Ultrathin sections were stained with uranyl acetate and lead citrate and examined under a JEM 1OOC transmission microscope. ODC activity

Following 4 hr incubation, both control (2% FCS/RPMI) and treated cell cultures were washed twice with a buffer (50 mM Hepes, 4 mM dithiothreitol, 0.1 mM EDTA, pH 7.4). The cells were disrupted by freezing and thawing three times, centrifuged (100 000 g, 20 min, 4°C) and the resulting cytosol fraction used for the enzyme assay and for the protein determination. ODC activity was estimated in triplicate by measuring the amount of WO, liberated from L- 1 -14C ornithine. The incubation medium consisted of: 50 mM Hepes, 0.4 mM 14C-labeled

The results were statistically evaluated using ANOVA and Tukey’s multiple range test. The probability of differences at the level P < 0.05 was regarded as significant.

RESULTS

TGF-/I, concentrations (1 and 2 rig/ml) applied in the current study were selected on the basis of the dose-dependent effects of this growth factor from previous experiments (Grzelkowska et al., 1995a). Twenty-four hour incubation of L 1210 leukemic cells with TGF-/I, (2 rig/ml) significantly reduced (to 72% of control) cell proliferation measured by the incorporation of [methyl-3H] thymidine (Fig. 1). This mitoinhibitory effect of TGF-P, was abolished by the addition of TPA (100 rig/ml) to the incubation medium. Mitoinhibition (65% of control) was also observed when DFMO (5 mM), an irreversible inhibitor of ODC, was administered to the culture of L1210 cells (Fig. 1). Exogenous putrescine in a concentration of 25 PM did not significantly modify the inhibitory effects of either TGF-j?, or DFMO.

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The influence of TGF-/I, on cell viability and proliferation has also been assessed using the trypan blue exclusion method. TGF-p,, significantly and progressively with time of exposition (24 and 48 hr), decreased the number of viable cells and increased (P < 0.05) the number of those dyed with trypan blue (Fig. 2). Addition of TPA significantly prevented the cell death evoked by this growth factor. These results could also indicate that TGF-fl, inhibits cell proliferation, since the drop in the number of viable cells was not balanced by an increase in the number of cells which died. TGF-&induced apoptosis of L 1210 leukemic cells has been visualized as a characteristic “DNA ladder” resulting from internucleosomal DNA cleavage for fragments that are multiples of 180-200 bp (Fig. 3, compare lines 1 and 2). Scanning of the electrophoregram allowed for the measurement of the reflective density of particular bands. Apoptotic fragments appeared as peaks of the reflective density at lower parts of the band. TPA apparently reduced DNA

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Fig. 2. The number of viable (dotted line) and dead (solid line) L1210 leukemic cells exposed to: (0) basal medium (Z%FCS/RPMI); (A) TGF-/l, (2 rig/ml); (D) and TGF,!I, + TPA (100 rig/ml), for 48 hr. Each value represents the mean of two separate experiments performed in triplicate. Means described with different superscript letters (a-i) differ significantly (P < 0.05).

Fig. 1. The effect of TGF-fl, (2 rig/ml), DFMO (5 mM), TPA (100 rig/ml), putrescine (25 PM) and combination of these compounds on proliferation of Ll210 leukemic cells after 24 hr of incubation. Results are expressed as percent of [methyl-‘H] thymidine incorporation in Ll210 cells growing in basal medium (BM). Each bar represents a mean (*SEM) of three separate experiments performed in triplicate. Means described with different superscript letters (a,b,c) differ significantly (P < 0.05).

fragmentation evoked by TGF-/I, (compare lines 2 and 6). Inhibition of ODC by DFMO led to fragmentation of DNA similar to that induced by TGF-8, (compare lines 2 and 4). Exogenous putrescine (product of ODC reaction and substrate for spermidine synthesis) supplemented in concentration 25 ,uM did not affect TGF-P, (compare lines 2 and 3) and DFMO (compare lines 4 and 5)-induced DNA fragmentation (Fig. 3). Apoptosis of TGF-b,-treated L 12 10 leukemic cells was confirmed by electron microscopy. Among morphological features characteristic of apoptosis the following conditions predominated: condensation and margination of chromatin, nuclear pycnosis and fragmentation of nucleus, sometimes with secondary degenerative lesions of cytoplasm and organelles (Fig. 4). As early as 2 hr after administration to the culture of L1210 leukemic cells, TGF-P, significantly decreased the expression of ODC, measured by the ODC mRNA/GAPDH mRNA ratio (Fig. 5). Furthermore, a significant suppression of ODC activity in cells incubated

Phorbol ester TPA

with TGF-/?, for 4 hr was also found. TPA supplementation prevented an inhibitory action of TGF-/?, both on transcription and activity of ODC (Fig. 5).

DISCUSSION

The growth inhibitory effect of TGF-/?, in malignant lymphoid cells is not a common feature and depends on cell type, culture conditions and the presence of other growth factors (Fynan and Reiss, 1993). The current study confirms our recent observations indicating the growth-inhibitory action of TGF-P, in cultures of L1210 leukemic cells (Grzelkowska et al., 1995a). The growth inhibition induced by TGF-/3, is a result of mitoinhibition as well as increased cell mortality. Mitoinhibition manifests itself in the reduced incorporation of [methyl-3H] thymidine into DNA (Fig. 1) and decreased number of viable cells (Fig. 2). Flow cytometry analysis of L1210 leukemic cells exposed to TGF-b, (2 rig/ml) revealed that the mitoinhibitory effect of this cytokine is associated with impaired progression from Gl to the S phase of the cell cycle (Grzelkowska et al., 1996). The detection and evaluation of apoptotic and necrotic cell death evoked by TGF-B, was possible with the use of electron microscopy kb

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Fig. 3. DNA fragmentation in L1210 leukemic cells after 48 hr of incubation. (M) EDNA EcoRI, Hind III (size marker); (1) basal medium (2%FCS/RPMI); (2) TGF$, (2 rig/ml); (3) TGF-/?, + putrescine (25 PM); (4) DFMO (5 mM); (5) DFMO + putrescine; (6) TGF-8, + TPA (100 rig/ml).

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(Fig. 4) electrophoresis of DNA (Fig. 3) and the trypan blue exclusion method (Fig. 2) respectively. As was described previously (Grzelkowska et al., 1995a), TGF-&induced growth inhibition of L1210 leukemic cells was accompanied by enhancement of cell mortality. Since TGF-P, (in contrast to other cytokines, e.g. IL-l, TNF-a) does not exert direct cytotoxic effects on tumour cells (Lahm et al., 1992) the apoptosis is probably a primary form of death evoked by this growth factor. It is impossible, however, to establish how many of the L1210 cells that take up trypan blue (necrotic cells) have reached apoptosis earlier. Apart from the typical morphological features (shrinkage of the cell, chromatin condensation and margination, nuclear pycnosis and fragmentation, Fig. 4) TGF-&induced apoptosis was visualized as a characteristic “DNA ladder” (Fig. 3) resulting from the internucleosomal DNA cleavage. The random DNA cleavage in necrosis led to a DNA smear on the gel. Using flow cytometry it has been shown that L1210 leukemic cells undergo apoptosis (from 1 to 8% of total cell number) even in optimal growth conditions (10% FCS/RPMI-1640), and the number of apoptotic cells in TGF-P,-treated (2 rig/ml) cultures for 48 hr varies between 11.3 and 27.8% (unpublished). For comparison the number of cells dyed with trypan blue after 48 hr exposition to TGF-/3, establishes 55.8 + 6.7% (Fig. 2). In spite of a great interest in the TGF-P, metabolic effect, no consistent or widely correlated second messenger or mediator of mitoinhibition and apoptosis evoked by TGF-/?, has been established. There are multiple independent mechanisms involved in cell growth inhibition by TGF-/?, (Newman, 1993; Theodorescu et al., 1993; Wright et al., 1993). Many of the diverse cellular effects of TGF-p, are the result of alterations in expression levels of growth factors, nuclear proteins, immune factors, extracellular matrix proteins, integrins and protease inhibitors (Newman, 1993). Results of the present (Fig. 5) and previous (Grzelkowska et al., 1995a) experiments on L1210 leukemic cells indicate that mitoinhibitory and apoptotic effects of TGF-/?, are associated with the inhibition of ODC-the rate-limiting enzyme in polyamine synthesis. It has been shown that the inhibition of ODC activity by TGF-b, occurs at the transcriptional level. The ODC expression measured by ODC mRNA/GAPDH mRNA ratio decreased just

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Fig. 4. Morphology of L1210 leukemic cells exposed to TGF-/S (2 rig/ml) for 48 hr. Transmission electron microscopy revealed morphological changes typical of cells undergoing apoptosis: (a) chromatin condensation and its margination along the nuclear envelope; (b) nuclear pycnosis; (c.d) fragmentation of the nucleus and secondary degenerative lesions in the cytoplasm and organelles. Bars. 2 kern.

2 hr after TGF-P, administration to the culture of L1210 leukemic cells (Fig. 5). The inhibitory effect of TGF-fl, on the polyamine pathway has also been observed in the cell culture of K.562 human chronic myelogenous leukemia (Motyl et ul., 1993). This effect of TGF-fl, manifested itself not only in the decrease of ODC activity but also in the suppression of the activity of S-adenosylmethionine decarboxylase-a second key enzyme in polyamine biosynthesis. Since polyamines interact with the double helix and stabilize the DNA molecule (Cozzi et al., 1991) the inhibition of polyamine synthesis may be one of the first symptoms of apoptosis evoked by TGF-j3,. In contrast to TGF-/I,-induced apoptosis in L 12 10 leukemic cells, the cell death triggered by enforced c-myc expression in 32D.3 murine myeloid cells, following IL-3 withdrawal, was associated with increased expression of ODC (Askew et al., 1991, 1993). It has been suggested that c-Myc-induced ODC

expression contributes to the induction of apoptosis by c-Myc in 32D.3 myeloid cells (Packham and Cleveland, 1994). An adverse, inhibiting effect of TGF-j?, on ODC expression and activity in L1210 cells (Fig. 5) may be due to down-regulation of c-myc by this cytokine. Reduction of c-myc expression, blocking the activity of cyclins and cyclin-dependent kinases, and inhibition of Rb phosphorylation, are the most important mechanisms thus far involved in inhibition of DNA synthesis and cell growth by TGF-fl, (Attisano et al., 1994; Newman, 1993; Wright et al., 1993). Since the ODC gene is a transcriptional target of c-Myc (Bello-Fernandez et al., 1993), the down-regulation of c-myc expression by TGF-/I, could be an important mechanism affecting ODC expression in cells exposed to TGF-P,. The differences in ODC response to apoptotic agents may suggest an occurrence of multiple and independent signaling pathways in the program of apoptotic cell

Phorbol ester TPA

death. The mechanism of apoptosis induction by TGF-/3, is still unknown, however there are data indicating down-regulation of bcl-2 expression in TGF-&treated human umbilical vein endothelial cells (Tsukada et al., 1995). It should be emphasized that the presence of DFMO, a specific and irreversible inhibitor of ODC, leads to apoptotic fragmentation of DNA in cultures of L1210 leukemic cells (Fig. 3). The depletion of intracellular polyamines can cause the alteration of DNA supercoiling and increase sensitivity of molecular DNA to digestion by endonucleases activated in the course of the apoptotic process (Orrenius, 1995). It has been shown that spermine can prevent DNA fragmentation and apoptosis in thymocytes exposed to glucocorticoids and Ca*+ ionophores (Briine et al., 1991), as well as in irradiated LY-TH lymphoma cells (Meyn et al., 1993). Polyamines as frequent by-products of tumour metabolism may synergize with TPA in an antiapoptotic effect (Sinkovics and Horvath.

a

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a 1.5

a

z I 1.3 ::

a

2 h 1.1

4

z E g 0.9

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TGF-I31 TGF-I31 +TPA

& ODC expression 0 ODC activity

Fig. 5. The ODCmRNA/GAPDHmRNA ratio and activity of ODC in L1210 leukemic cells exposed to: basal medium (2%FCS/RPMI), TGF-B, (1 rig/ml) and TGF-p, + TPA (100 rig/ml). The ODCmRNA/GAPDHmRNA ratio in the basal medium was assumed to be 1. The results are expressed as a mean If: SEM of three separate experiments. Means described with different superscript letters (a,b,c) differ significantly (P < 0.05).

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1994). Polyamines as biologically important antioxidants (Lovaas, 1995; Pavlovic et al., 1992) may protect cells from apoptosis in these cases, where reactive oxygen species are mediators in apoptotic cell death (Sarafian and Bredesen, 1994). On the other hand, polyamines are utilized in transglutaminase-mediated protein cross-linking, leading to the formation of insoluble protein scaffold, which temporarily stabilizes the integrity of apoptotic cells and prevents the release of potentially harmful intracellular components and development of inflammatory responses (Melino et al., 1988, 1994; Piacentini et al., 1992). It has been suggested that a specific induction of the cytosolic tissue transglutaminase may be involved in the TGF-&-induced pathways of apoptotic cell death in hepatoma cells (Fukada et al., 1993). We have proved that TPA, a known tumour promoter, diminishes mitoinhibitory (Figs 1 and 2) apoptotic (Fig. 3) and necrotic (Fig. 2) effects of TGF-1, in L 12 10 leukemic cells. These results are in agreement with data of Chuang et al. (1994) indicating TPA protection from TGF/?,-evoked mitoinhibition and apoptosis in human hepatoma cells. Similar effects were observed when another tumour promoter, erotic acid, was administered to the culture of L1210 cells treated with TGF-8, (Grzelkowska et al., 1995a). Both TPA (Butler et al., 1991) and erotic acid (Grzelkowska et al., 1995a, 1995b) are potent ODC inductors in neoplastic cells. The increase of ODC activity belongs to the earliest changes in gene expression caused by TPA (Butler et al., 1991; Pavlath et al., 1993). Thus, the induction of ODC by TPA is regarded as a biochemical marker for tumour promotion (Mitra et al., 1992). It has also been shown that TPA prevents DFMO-related apoptotic fragmentation of DNA in L1210 leukemic cells (unpublished). In the present study, TPA completely abolished the inhibitory influence of TGF-/I, on ODC expression and activity in L1210 leukemic cells (Fig. 5). According to previous data (Butler et al., 1991; Pavlath et al., 1993; Warholm, 1992), the induction of ODC by TPA is mediated by protein kinase C (PKC), an intracellular enzyme involved in the signal transduction pathway of many cytokines and growth factors. PKC is also an important link in the apoptotic process, usually considered as an anti-apoptotic enzyme (Perandones et al., 1993; Chuang et al., 1994). However, the ability of PKC to modulate apoptosis depends on the

T. Mot) :I et al.

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expression of PKC isoforms (de Vente et al., 1995). Assuming that the influence of TGF-,!!I, on cells may be controlled via postreceptor mechanisms (Wilding, 1991) many factors (e.g. hormones, cytokines, growth factors. tumour promoters) affecting PKC-ODC activity could modulate the biological effects of TGF-/?,, including apoptosis. Acknowledgements-This work was supported by a grant from the State Committee of Scientific Research (No. 50102020030). The authors wish to express their thanks to the Foundation for Polish Science for supplying the equipment for automatic electrophoresis and scanning-Program “Nutris” 1995.

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