MOLECULAR AND CELLULAR BIOLOGY, Jan. 1992, p. 302-308

Vol. 12, No. 1

0270-7306/92/010302-07$02.00/0 Copyright © 1992, American Society for Microbiology

Phospholipase C-Mediated Hydrolysis of Phosphatidylcholine Is Target of Transforming Growth Factor 13 Inhibitory Signals

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MARIA T. DIAZ-MECO,1 ISABEL DOMINGUEZ,' LAURA SANZ,1 MARIA M. MUNICIO,1 EDURNE BERRA,' MARIA E. CORNET,' ANTONIO GARCIA DE HERREROS,1 TERJE JOHANSEN,2 AND JORGE MOSCATL.3*

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Cirugia Experimental, Hospital General "Gregorio Marano6n", Calle Dr. Esquerdo 46, 28007 Madrid'; Institute of Medical Biology, University of Tromso, 9001 Tromso, Norway2; and Centro de Biologia Molecular, CSIC-UAM, Canto Blanco, 28049 Madrid, Spain3 Received 1 July 1991/Accepted 10 October 1991

Cell growth and tumor transformation can be restrained in certain cell systems by the action of transforming growth factor (TGF- ). It has been established that the mechanism whereby TGF-IV1 inhibits cell growth does not interfere with the triggering of early mitogenic signal transduction mechanisms. Phospholipase C-catalyzed hydrolysis of phosphatidylcholine (PC) is a relatively late step in the cascade activated by growth factors. Therefore, conceivably activation of phospholipase C-catalyzed hydrolysis of PC could be the target of TGF-I1 action. In the study reported here, we demonstrate that TGF-131 inhibits the coupling of ras p21 to the activation of PC hydrolysis, which appears to be critical for the antiproliferative effects of TGF-41.

Intense research is unveiling the signal transduction pathways involved in the control of cell growth and tumor transformation (for recent reviews, see references 5, 8, and 13). The products of genes involved in these cascades appear to be subverted in the neoplastic process. These genes when altered give rise to oncogenes. Some of them code for growth factors or growth factor receptors (8, 10), while others, such as ras, appear to be linked either directly or indirectly in postreceptor mitogenic signalling pathways (1). Cell growth and tumor transformation can be restrained in certain cell systems by the action of peptide negative regulators. One example of these molecules is transforming growth factor a (TGF-P). This polypeptide is prototypic of a large family that includes closely related genes in mammals (TGF-1l, TGF-P2, and TGF-P3) (22). Although nearly all cell types display receptors for TGF-P, their cloning remains elusive, and the nature of the signals generated in response to the interaction with the ligand are still far from clear (24). The mechanism whereby TGF-1l inhibits cell growth has been the focus of a number of studies that established that TGF-p1 does not interfere with the binding of the growth factor to its receptor or with the triggering of very well

dence has accumulated showing that activation of phospholipase C (PLC)-catalyzed hydrolysis of PC is a relatively late step in the cascade activated by growth factors and is sufficient to mimic a significant portion of the plateletderived growth factor mitogenic response (18). PLC-mediated PC hydrolysis has also been shown to be rapidly stimulated in fibroblasts by the product of ras oncogene, ras p21 (21, 28), whose role in mitogenic cascades has been demonstrated (29). Since activation of PC-hydrolyzing PLC (PC-PLC) is a relatively late event in mitogenic signal transduction, and taking into account that TGF-,1l does not interfere with early signals generated following growth factor stimulation (6, 19), an attractive hypothesis is that TGF-,1l inhibits cell growth by negatively affecting PC hydrolysis. To test this possibility, in this study we used both a continuous cell line of mouse keratinocytes (BALB/MK) and oocytes from Xenopus laevis. These systems are suitable for investigating the effect of TGF-,1l on different enzymatic activities of relevant signal transduction mechanisms. First, the action of TGF-1l on the regulation of different growth factor-stimulated pathways has been investigated in BALB/MK keratinocytes (6, 27); second, the mitogenic signalling routes in Xenopus oocytes are beginning to be well characterized. Thus, Xenopus oocytes undergo a maturation program following stimulation with either insulin or progesterone, and several lines of evidence indicate the specific involvement of ras p21 in the maturation signalling cascades activated by insulinlike growth factor 1 (3, 15). The involvement and importance of PC-PLC in mitogenic activation in Xenopus oocytes has been documented (12). Thus, we have shown that PLCmediated hydrolysis of PC is both necessary and sufficient for activation of maturation in X. laevis oocytes by insulin or ras p21 but not by progesterone (12). In the study reported here, we demonstrate for the first time that Xenopus oocytes possess receptors for TGF-,1l and that this polypeptide inhibits the coupling of ras p21 to the activation of PC hydrolysis. These results are consistent with the notion that PC hydrolysis may be critical for the antiproliferative effects of TGF-11.

characterized early signal transduction mechanisms (6, 19). A more recent work indicates that TGF-p1 affects the generation of later events in the mitogenic pathways, such as c-myc expression (27) or the phosphorylation of the product of the retinoblastoma gene (16). These data suggest that the growth-inhibitory actions of TGF-,11 are mediated not by effects on early signals but by effects on late steps in the mitogenic cascade. Phospholipid degradation, which is potently activated following stimulation with growth factors (2, 11), has been proposed as a critical event in signal transduction pathways. Although most of the work has been focused on phosphoinositide (PI) turnover (2), a number of studies demonstrate the existence of phosphoinositide-independent mechanisms involving the phosphodiesterase-mediated hydrolysis of phosphatidylcholine (PC) (11). Recently, evi*

Corresponding author. 302

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MATERIALS AND METHODS Oocyte culture and labeling. Oocytes were prepared according to standard procedures (7). Briefly, ovaries from X. laevis frogs (Blades Biologicals, Oxford, United Kingdom) were incubated with collagenase (2 mg/ml; Boehringer, Mannheim, Germany) for 45 min in modified Barth solution without Ca2+ (110 mM NaCl, 2 mM KCI, 1 mM MgCl2, 1 mM CaCl2, 2 mM NaHCO3, 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid [HEPES], pH 7.8). After extensive washing, stage VI oocytes were selected and incubated overnight at 20°C. Selected oocytes were labeled with [methyl-14C]choline (12 jxCi/ml; specific radioactivity, 55 mCi/mmol; Amersham International) for 24 h in modified Barth solution, after which medium was removed and fresh, label-free medium was added; experiments were carried out after a 30-min equilibration period. Analysis of products of phospholipid metabolism. Labeled oocytes were treated or not with the corresponding agonists or were microinjected with Bacillus cereus PC-PLC or with ras p21. At different times, reactions were stopped by adding ice-cold methanol. Methanolic cell extracts were fractionated into chloroform and aqueous phases as previously described (4). The presence and levels of water-soluble choline metabolites were evaluated in the aqueous phases by thin-layer chromatography (9), followed by autoradiography of plates in which standards corresponding to the different water-soluble choline metabolites were included. Isolation of PC-PLC from B. cereus and preparation of affinity-purified antibody. PC-PLC was isolated from cultures of B. cereus SE-1 essentially as described by Myrnes and Little (26). In accordance with the protocol of Johansen et al. (14), the enzyme preparation was purified to complete homogeneity as confirmed by sodium dodecyl sulfate (SDS)polyacrylamide gel electrophoresis (PAGE) followed by silver staining. The specific activity of the purified enzyme was 1.5 U/,ug (20). Preparation of ras p21 proteins. Transforming and normal ras p21 proteins were expressed in bacteria as previously described (12). A final step of purification consisted of gel filtration chromatography through Sephadex G-100 column (2.5 by 90 cm); fractions containing the purified protein were pooled and dialyzed extensively against 20 mM Tris-HCl (pH 7.5) to remove urea and kept at -70°C until used. Histone 1 (H1) kinase assay. Twenty oocytes were homogenized in a buffer containing 20 mM HEPES (pH 7.0), 10 mM ,-glycerophosphate, 5 mM EGTA, 5 mM MgCl2, 50 mM NaF, 2 mM dithiothreitol, 100 ,ug of leupeptin per ml, and 100 ,uM phenylmethylsulfonyl fluoride. Following centrifugation at 13,000 x g for 15 min, extracts (1 to 2 mg per assay) were assayed for 10 min at 30°C in a final reaction volume of 50 ,ul containing 20 mM HEPES (pH 7.0), 5 mM P-mercaptoethanol, 10 mM MgCI2, 100 ,uM _-32p (2 to 5 dpm/fmol), 0.2 p.g of heat-stable inhibitor of cyclic AMP-dependent protein kinase, and 0.6 mg of Sigma type III-S calf thymus histone per ml. Reactions were terminated, spotted onto Whatman P81 phosphocellulose paper, washed, and quantitated as described previously (12). Cell culture. BALB/MK cells were grown on plastic dishes in low calcium (0.05 mM Ca2+) containing Dulbecco's modified Eagle's medium supplemented with 10% dialyzed fetal calf serum and 5 ng of epidermal growth factor (EGF) per ml. Cells were made quiescent by incubation in a chemically defined medium consisting of low-calcium minimum Eagle's medium supplemented with transferrin (5 ng/ml) and selenium (1 ,uM). Some experiments were performed with

303

BALB/MK cells carrying the v-K-ras oncogene (ras-BALB/ MK). This cell line was obtained by infection of normal BALB/MK keratinocytes with Kirsten murine sarcoma virus essentially as described previously (30). Estimation of the release of PCho. Cells grown to confluence in 30-mm culture dishes were labeled for 48 h with 2 ,uCi of [methyl-'4C]choline (specific radioactivity, 50 to 60 mCi/mmol; Amersham International) per dish. The last 24 h of labeling was performed in serum-free medium supplemented as described above. After labeling, cultures were incubated in the presence of EGF (10 ng/ml) for different times, and the release of phosphocholine (PCho) into the intracellular medium was determined (23). [3H]thymidine incorporation assays. Quiescent cells were incubated with the corresponding stimulants in the presence of [3H]thymidine (2 ,uCi/ml) for different times. Afterwards, de novo DNA synthesis was determined as previously described (18). Northern (RNA) hybridization. Total RNA (10 p.g) isolated from either control or stimulated cells was run on formaldehyde-agarose gels, blotted onto nitrocellulose filters, and hybridized overnight at 68°C with 32P-labeled mouse c-myc cDNA probe in 5 x SSC-5 x Denhardt's solution 0.5% SDS-0.1 mg of denaturated salmon sperm per ml. Filters were washed at 68°C in 0.1 x SSC-0.1% SDS. Northern blots were normalized by hybridization with a 28S rRNA oligonucleotide probe. TGF-0 receptor affinity labeling. Oocytes (10 per well) were incubated with 100 pM '25I-TGF-pl (specific radioactivity, 161 ,uCi/,ug; New England Nuclear) for 3 h either in the absence or in the presence of an excess of unlabeled TGF-pl and with 150 p.M disuccinidimyl suberate (Pierce Chemical Co.). Detergent extracts from affinity-labeled oocytes were separated by SDS-PAGE on 7% polyacrylamide gels under reducing conditions and subjected to autoradiography. Unlabeled TGF-pl and the neutralizing antibody anti-TGF-f1 were from R&D Systems. RESULTS TGF-t1 inhibits the activation of Hi kinase by insulin and ras p21 but not by progesterone in X. laevis oocytes. We initially determined whether the presence of TGF-pl affected the ability of insulin, ras p21, or progesterone to activate Hi kinase in Xenopus oocytes. This enzymatic activity has been thoroughly characterized as a component of the so-called maturation-promoting factor, which includes p34CDC28/cdc2+ (25), and is an excellent marker of the biochemical mechanisms controlling oocyte maturation (25). The presence of 10 ng of TGF-pl per ml dramatically reduced the activation of Hi kinase by insulin and ras p21 (Fig. 1A and 1B) but not by progesterone (Fig. 1C), indicating the specific interaction of TGF-,B1-triggered inhibitory signals with the mitogenic pathway activated by insulin. These results also suggest that the target of TGF-1l inhibitory action is a step situated downstream of ras. To locate the TGF-pl action in this mitogenic cascade, the following series of experiments was carried out. Xenopus oocytes were stimulated with insulin (1 ,ug/ml), TGF-pl (10 ng/ml) was added at different times thereafter, and Hi kinase activity was measured 6 h after the stimulation with insulin. Addition of TGF-41 either simultaneously or 2 h after stimulation of oocytes with insulin dramatically inhibited the activation of Hi kinase in response to this agonist (Fig. 2). However, little or no effect on insulin-stimulated Hi kinase was detected when TGF-3il was added 3 h after insulin

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addition. From these results, two conclusions can be drawn: (i) the presence of TGF-131 during the first 2 h following insulin addition is not required for inhibition of Hi kinase, and (ii) TGF-pl must be present from at least 2 h after insulin stimulation to effectively inhibit the activation of HI kinase. Therefore, the presence of TGF-,41 2 to 3 h after insulin addition is necessary, although perhaps not sufficient, to inhibit stimulation of Hi kinase. To establish which is the minimum time required, as well as the precise time frame, for TGF-pl to inhibit insulin-stimulated Hi kinase, we carried out the following experiment. After addition of TGF-pl to insulin-stimulated oocytes, a neutralizing antiTGF-pl antibody was added at different times, and Hi kinase activity was measured 6 h following insulin stimulation. The presence of TGF-pl for as long as 2 h after insulin addition was not sufficient to inhibit the stimulated Hi kinase

activity (Fig. 2). However, the presence of TGF-pl for 3 h after insulin addition blocked activation of Hi kinase by this agonist. Taken together, the results depicted in Fig. 2 permit one to establish that the presence of TGF-pl during the time interval between 2 and 3 h following insulin addition is both necessary and sufficient to block the mitogenic signalling cascade activated by this hormone in oocytes. Binding of TGF-31 to most mammalian cells is mediated by two cell surface proteins and by the N-glycosylated core of 3-glycan, a 200- to 400-kDa cell surface proteoglycan (17). To determine whether TGF-p1 mediates its effects in Xenopus oocytes through this classical receptor, we determined its presence by affinity labeling experiments. Results from Fig. 3 demonstrate the presence of type 11 (70 kDa) and type I (53 kDa) receptors as well as that of P-glycan (200 to 400 kDa) in oocytes. An excess of unlabeled TGF-,B1 added during the binding experiments completely and specifically competed for the cross-linked species. Therefore, these results indicate that TGF-1l mediates its biological effects in Xenopus oocytes most probably through its classical receptor molecules (17). Activation of PC-PLC is a target for TGF-I1 action in Xenopus oocytes. It has been demonstrated that PC-PLC is a critical late step in mitogenic signal transduction (11, 12, 18). Previous results from our laboratory demonstrate that activation of PC-PLC takes place by 2 to 3 h following the addition of insulin to Xenopus oocytes (12). Since the

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FIG. 3. TGF-01 receptor expression in X. Iaevis oocytes. Cell surface receptors were affinity labeled by cross-linking with 1251_ TGF-pl. Oocytes (10 per well) were incubated with 100 pM 1251_ TGF-f1 (specific radioactivity, 161 ,uCi/,ug; New England Nuclear) for 3 h either in the absence (lanes 1 and 2) or in the presence (lanes 3 and 4) of an excess of unlabeled TGF-pl (lane 3, 1.5 nM; lane 4, 20 nM) either with (lanes 2 to 4) or without (lane 1) disuccinidimyl suberate. Oocyte proteins were extracted and separated by SDSPAGE under reducing conditions. Labeled bands corresponding to TGF-P receptors I and II and ,B-glycan are indicated with arrows. Essentially identical results were obtained in three independent experiments.

inhibitory signals generated by TGF-,1 are effective between 2 to 3 h after stimulation of oocytes with insulin, conceivably PC-PLC activation could be the target of TGF-pl action. To test this possibility, we determined the effect of this polypeptide on insulin-activated PC-PLC in [methyl-14C]choline-labeled Xenopus oocytes. The results (Fig. 4A) demonstrate that the presence of TGF-,1 (10 ng/ml) dramatically inhibits the release of PCho (a good parameter of PC-PLC activation in this system [12, 18, 21]) in response to insulin. Since ras p21 potently and rapidly activates PC-PLC, we next sought to determine whether the stimulation of PCho release in response to microinjection of ras p21 is abolished by TGF-pl. Results from Fig. 4B demonstrate that this is actually the case: the presence of TGF-pl dramatically inhibited ras p21-induced PC-PLC in Xenopus oocytes. TGF-Il1 inhibits the activation of PC-PLC in BALB/MK keratinocytes. To demonstrate that the effect of TGF-,B1 on PC-PLC described here is not restricted to oocytes but is also detected in other systems in which inhibitory actions of TGF-pl on mitogenic cascades have been described (6, 27), the following series of experiments was carried out. Mouse BALB/MK keratinocytes were made quiescent by serum starvation, after which they were stimulated with EGF either in the absence or in the presence of TGF-1l, and DNA synthesis was determined at different times thereafter. TGF-1l significantly abolished EGF-induced DNA synthesis (Fig. SA), in agreement with previously published results (27). The addition of EGF to quiescent [methyl-14C]cholinelabeled BALB/MK keratinocytes promoted a potent release of PCho, which indicates that, like platelet-derived growth factor in fibroblasts (18), EGF activates a delayed hydrolysis of PC in keratinocytes. Interestingly, the presence of TGF-pl completely inhibited EGF-induced PCho release (Fig. 5B). We next analyzed the time course of the effect of TGF-pl on both DNA synthesis and PCho release in BALB/MK keratinocytes. TGF-pl inhibition of PCho release in actively proliferating mouse BALB/MK cells (Fig.

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6A) or in keratinocytes transformed by the Ha-ras oncogene (Fig. 7A) took place before any detectable effect on DNA synthesis (Fig. 6B and 7B). As a control, treatment with emetidine, a potent inhibitor of DNA replication, completely abolished DNA synthesis in both cell systems but remarkably did not affect PCho release (Fig. 6 and 7). TGF-I1 does not inhibit Hi kinase in Xenopus oocytes microinjected with PC-PLC. If the repression of PC-PLC activation of TGF-,B1 is critical for the inhibition of mitogenic signalling, the activation of PC hydrolysis independently of agonist action should bypass the inhibition of insulinstimulated Hi kinase by TGF-p1 in oocytes. To address this question, we used a highly purified, permanently activated PC-PLC from B. cereus that has been characterized extensively. This enzyme specifically hydrolyzes PC following microinjection into Xenopus oocytes (12) and promotes a dramatic stimulation of Hi kinase activity in this system (12). Results from Fig. 8 indicate that whereas TGF-,1 potently inhibits insulin or ras p21-induced Hi kinase, it does not affect the stimulation of this parameter by microinjection of B. cereus PC-PLC. This finding strongly suggest that the negative regulation of PC hydrolysis by TGF-,1 is important for inhibition of Hi kinase. Exogenous addition of B. cereus PC-PLC bypasses the inhibition of c-myc expression by TGF-jl1 in BALB/MK keratinocytes. It has recently been demonstrated that TGF-p1 inhibits the expression of c-myc in response to EGF

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in BALB/MK keratinocytes (27). It could be argued that inhibition of PC-PLC by TGF-r31 in EGF-stimulated keratinocytes is secondary to its effects on c-myc expression. To rule out this possibility, it should be demonstrated that PC-PLC is upstream of c-myc gene transcription and that the hydrolysis of PC independently of growth factors bypasses the inhibitory actions of TGF-pl on the induction of c-myc. The results shown in Fig. 9 clearly demonstrate that both requirements are fulfilled. Thus, the exogenous addition of B. cereus PC-PLC to quiescent BALB/MK keratinocytes promotes a potent induction of c-myc to an extent similar to that produced by EGF. The exogenous addition of B. cereus PC-PLC has been shown to specifically hydrolyze PC and mimic mitogenic signals generated by growth factors (18). Interestingly, Fig. 9 also shows that TGF-pl does not affect PC-PLC-triggered induction of c-myc, although it dramatically reduces the activation of this parameter by EGF. DISCUSSION Recent studies suggest that phosphodiesterase hydrolysis of PC through either PLC or PLD plays a significant role in

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the control of cell functionality (11). Particularly, PC-PLC appears to be critically involved in signal transduction mechanisms controlling cell growth and tumor transformation (12, 18, 21, 28). Our previous results demonstrate that this novel phospholipid signal transduction pathway is activated relatively late in the mitogenic cascade triggered by growth factors (18). The fact that accumulating evidence suggests that the mechanism whereby TGF-,1 restrains cell growth does not involve inhibition of early signalling permits one to speculate on the possibility that PC-PLC activation is either one of the direct or one of the indirect important targets of TGF-pl inhibitory actions. We demonstrate here for the first time the presence of receptors for TGF-pl in Xenopus oocytes. The results presented in this study demonstrate that PC-PLC activation by insulin and ras p21 in oocytes and by EGF and ras p21 in BALB/MK keratinocytes is potently inhibited by TGF-pl. Previous studies have demonstrated that TGF-pl interferes with the stimulation of other late steps in the mitogenic cascade, such as the expression of c-myc (27) or the phosphorylation of the product of the retinoblastoma gene (16). Although the relationship between the two phenomena requires further clarification (24), these findings constitute a

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significant advance in the definition of the mechanisms of action of TGF-131. The results presented here, demonstrating that PC hydrolysis is inhibited by TGF-pl, define a potentially novel target of TGF-pl action. The effect of this polypeptide on PC-PLC is not secondary to its actions on other relatively late events in the mitogenic cascade, such as the inhibition of c-myc expression. Proof for this notion comes from the results showing that (i) addition of B. cereus PC-PLC to BALB/MK cultures potently activates c-myc expression and (ii) TGF-p1 is unable to block c-myc expression by PC-PLC although it completely abolishes the induction of this gene by EGF. Taken together, these data strongly support a model whereby PC-PLC is located proximal to and downstream of ras and upstream of c-myc expression. All of these data are also consistent with the idea that the inhibition of this phospholipid degradative pathway is critical for the repression of cell proliferation by TGF-,B1. Further support to this notion are the results demonstrating that microinjection of a permanently activated PC-PLC from B. cereus overcomes the inhibitory actions of TGF-p1 on Hi kinase, a good marker of mitogenic activity in Xenopus oocytes (25). These results together with our previous data, demonstrating that the specific blockade of PC-PLC with a

neutralizing antibody inhibits mitogenic signalling through insulin and ras p21, pinpoints PC degradation as a pivotal step for the regulation of both positive and negative mitogenic signals. From all these results, however, it is not possible to determine whether TGF-pl inhibits PC-PLC by directly affecting this enzyme or whether TGF-,l-derived signals influence a still unidentified step in the chain of events leading to PC-PLC activation. Although a pertussis toxininsensitive G protein appears to be involved in the modulation of PC hydrolysis by at least some stimuli (references 9 and 11 and references therein), the mechanisms used by growth factors to trigger this novel phospholipid signalling cascade are far from clear. In this regard, the results showing that ras p21 rapidly activates PC-PLC in several systems (12,

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FIG. 9. Lack of effect of TGF-41 on PC-PLC-stimulated c-myc expression in BALB/MK keratinocytes. Quiescent BALB/MK keratinocytes were either untreated or incubated with EGF (10 ng/ml) or B. cereus PC-PLC (0.5 U/ml) either in the absence or in the presence of TGF-pl (10 ng/ml) for 2 h, after which total RNA was prepared, run in a formaldehyde-agarose gel, blotted onto a nitrocellulose filter, and hybridized to a mouse c-myc-specific probe. Ethidium bromide staining of each RNA sample electrophoresed is shown. Results are representative of three other experiments with essentially identical results.

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21, 28), together with the fact that TGF-,1 inhibits the activation of PC-PLC by this oncogene, permit one to speculate that TGF-pl affects a step linking ras p21 to PC-PLC activation.

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ACKNOWLEDGMENTS This work was supported in part by grant SAL90-0070 from CICYT and by Glaxo Espafia. I.D. and M.E.C. are fellows from Gobierno Vasco and Comunidad de Madrid, respectively. M.T.D.-M. and L.S. are fellows from Ministerio de Educacion. E.B. is the recipient of an award from Fundaci6n Cientifica Asociaci6n Espafiola contra el Cancer. The technical assistance of Jesds Sanchez is greatly appreciated.

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Phospholipase C-mediated hydrolysis of phosphatidylcholine is a target of transforming growth factor beta 1 inhibitory signals.

Cell growth and tumor transformation can be restrained in certain cell systems by the action of transforming growth factor beta (TGF-beta). It has bee...
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