Role of prostaglandin H synthase-2-mediated conversion of arachidonic acid in controlling 3T6 fibroblast growth

Javier Martinez, Teresa Sanchez, and Juan J. Moreno

Department of Physiological Sciences, Faculty of Pharmacy, Unit of Physiology, Barcelona University, Barcelona E-08028, Spain

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The specific role(s) of arachidonic acid (AA) and its metabolites in the signaling pathways that regulated fibroblast growth was studied. A Western blot analysis demonstrated that prostaglandin H synthase-2 (PGHS-2) was expressed by 3T6 fibroblast cultures in RPMI 1640 supplemented with fetal calf serum (10%). Dexamethasone, which inhibits AA release and PGHS-2 expression, significantly reduced cell proliferation. Ketoprofen, a dual cyclooxygenase inhibitor, and CGP-28238, a specific PGHS-2 inhibitor, reduced fibroblast proliferation in a dose-dependent manner. These drugs also reduced [3H]thymidine incorporation into the DNA of fibroblasts. These effects were correlated with a decrease in prostaglandin (PG) E2 levels in the cell medium. However, piroxicam at doses that selectively inhibit PGHS-1 did not have a significant effect on fibroblast proliferation. Finally, we showed that the antiproliferative effect of dexamethasone and PGHS-2 inhibitors was significantly antagonized when PGE2 was added to the culture medium. Our results suggest that PGHS-2 and prostaglandins such as PGE2 might play an important role in the regulation of 3T6 fibroblast growth stimulated by growth factors of serum.

cyclooxygenase-2; prostaglandin E2; phospholipase A2; cell proliferation

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

CONSIDERABLE AMOUNTS OF arachidonic acid (AA) are found esterified in the plasma membranes of all types of mammalian cells; it appears predominantly at the sn-2 position of the glycerol backbone of phosphatidylinositol, phosphatidylcholine, and phosphatidylethanolamine. Under normal physiological conditions, the amount of free intracellular AA available is quite small. Liberation of AA from membrane phospholipids occurs through the action of phospholipases, primarily phospholipase A2 (PLA2). Then, free intracellular AA can be metabolized via cyclooxygenase, lipoxygenase, or cytochrome P-450 monooxygenase pathway. Thus this molecule is the precursor for the further metabolism of a large number of biologically active products, collectively termed eicosanoids (22).

Synthesis of AA metabolites via these pathways varies depending on the species, tissue, and hormonal background. Once synthesized, these AA metabolites exert many biological actions. Recent findings have demonstrated that two isoenzymes of prostaglandin endoperoxide H synthase (PGHS, also known as cyclooxygenase; EC 1.14.99.1.) catalyze the conversion of AA to prostaglandin (PG) G2 and then PGG2 to PGH2, the precursor for prostanoid biosynthesis. The isoforms PGHS-1 and PGHS-2 are encoded by separate genes and differ in their regulation and tissue distribution. Thus PGHS-1, a housekeeping gene expressed constitutively, is assumed to be responsible for producing prostanoids for physiological functions such as vascular homeostasis, regulation of renal blood flow, and maintenance of glomerular filtration rate (20), whereas PGHS-2, an immediate early gene, appears to be expressed only by specific stimulatory events. Thus inflammation mediators such as growth factors, cytokines, and endotoxins dramatically induce enzyme expression in different cellular systems (9, 19). This led to the hypothesis that a highly expressed PGHS-2 is responsible for the high levels of prostanoids present in inflamed tissues. Cross-species comparisons of PGHS isozymes show >85% amino acid identity and that PGHS-1 and PGHS-2 protein sequences from the same organism share, on average, ~60% identity (8). Thus the major difference between these isozymes appears to be their regulation.

While many studies of eicosanoids have focused on their role as intercellular messengers in physiopathological processes such as inflammation, more recent information provides strong evidence that AA or its metabolites play an important role in cell proliferation. Thus a key enzyme responsible for the cleavage of AA from the membrane pool, PLA2, is stimulated by several mitogens (15). On the other hand, we observed recently that the multiple cell contact with neighboring cells in confluent 3T6 fibroblast monolayer inhibits PLA2 activity and, consequently, PGE2 release and 3T6 fibroblast growth (16), a situation that can be reversed with a mechanical wound. Thus wound injury of confluent monolayer initiates a repair process that restores the integrity of the cell monolayer. In these previous experiments, we demonstrated that prostanoids, and specifically PGE2, play an important role in induced cell proliferation and wound repair, stimulated by fetal calf serum (FCS) or platelet-derived growth factor (PDGF) in 3T6 fibroblast cultures (21). Furthermore, we must consider that PGHS-2 together with another eicosanoid synthetic enzyme, the 5-lipoxygenase that is responsible for the formation of leukotrienes, has been localized to the nuclear membrane (8, 30), implying that their products could have functions within the nucleus. Finally, it should be noted that there is substantial evidence in the literature that nonsteroidal anti-inflammatory drugs (NSAIDs), which inhibit PGHS-1 and PGHS-2, can effectively inhibit tumor progression in humans and experimental animals (17, 18, 24, 25). In addition, these findings provide strong evidence that prostanoids could also play an intracellular or autocrine/paracrine role. Thus one possibility is that PGHS-2 produces prostanoids that act at the level of the nucleus in cellular growth, replication, and differentiation, as well as in response to hormones and cytokines with nuclear recognition sites, whereas PGHS-1 may generate prostanoids for secretion as intercellular mediators important to homeostasis. However, the specific role(s) of AA and its metabolites in the complex signaling pathways of cell growth remains unclear.

The aim of the present study was to evaluate the role of AA and its metabolites in 3T6 fibroblast proliferation, including the regulation of the enzymes that produce them. Our results show that PGE2, the most abundant arachidonate metabolite produced by fibroblasts, is important in FCS-induced proliferation of 3T6 cells. Thus dexamethasone and manoalide, which inhibit AA mobilization and PGE2 release, blocked cell proliferation. In addition, PGHS-2 has also been shown to be present in 3T6 fibroblast culture and to participate in the control of 3T6 fibroblast growth.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Materials. RPMI 1640, FCS, penicillin G, streptomycin, and trypsin/EDTA were purchased from GIBCO (Gaithersburg, MD). [5,6,8,9,11,12,14,15-3H]AA (180-240 Ci/mmol) and [methyl-3H]thymidine (20 Ci/mmol) were obtained from Du Pont-New England Nuclear (Boston, MA). Aprotinin, leupeptin, diethyldithiocarbamic acid, phenylmethylsulfonyl fluoride (PMSF), alpha 2-macroglobulin, ketoprofen, piroxicam, and bovine serum albumin (BSA) were acquired from Sigma Chemical (St. Louis, MO). Polyclonal antiserum directed against PGHS-1 or PGHS-2 as well as PGHS-1 and PGHS-2 were from Cayman Chemicals (Ann Arbor, MI). The enhanced chemiluminescence (ECL) kit was purchased from Amersham (Buckinghamshire, UK). CGP-28238, a selective PGHS-2 inhibitor (11, 29), was kindly provided by Dr. J. Queralt. All other reagents were of analytical grade.

Cell culture. Murine 3T6 fibroblasts were supplied by Dr. N. Suesa (Lab. Menarini SA, Badalona, Spain) and were grown and maintained as previously described (16). Thus fibroblasts were grown in RPMI 1640 containing 10% FCS and penicillin (100 U/ml) and streptomycin (100 µg/ml). Cells were harvested with trypsin/EDTA and passed to tissue-culture plates with a surface area of 5 cm2/well (tissue-culture cluster 12; Costar, Cambridge, MA). Cell cultures were maintained in a temperature- and humidity-controlled incubator at 37°C with 95% air-5% CO2. Cell viability tests were performed under all experimental conditions using trypan blue exclusion test.

Protein determination. Total protein was measured by the Bradford method (1) by means of the Bio-Rad detergent-compatible protein assay, using BSA as standard.

Western blot analysis. 3T6 fibroblast cultures were washed twice with ice-cold phosphate-buffered saline solution (PBS) and scraped off in PBS containing 2 mM EDTA and pelleted. Cell pellets were sonicated in PBS containing 2 mM EDTA, 20 µg/ml PMSF, 20 µg/ml aprotinin, 20 µg/ml leupeptin, 200 µg/ml dimethyldithyocarbamic acid, and 0.2 mg/ml alpha 2-macroglobulin.

Immunoblot analysis for both cyclooxygenase isoforms was performed as follows: cell lysates with equal amounts of protein (20 µg) were separated by a 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) gel (13) and blotted for 1 h with a constant current of 250 mA onto a nitrocellulose membrane (Trans-blot, Bio-Rad, 0.4 µm pore size) using a MiniProtean II system (Bio-Rad, Hercules, CA). PGHS-1 from ovine seminal vesicles and sheep PGHS-2 purified from placenta were also loaded on the gels as positive control. A prestained SDS-PAGE protein standard (Bio-Rad) was used to check transfer efficiency. The membranes were blocked with 5% nonfat milk powder in PBS-0.1% Tween 20 for 1 h. A rabbit polyclonal antiserum directed against PGHS-1 (sheep seminal vesicular) or PGHS-2 (synthetic peptide from murine PGHS-2) was applied in a dilution of 1:2,000 for 1 h. The specificity of antibodies used for immunodetection of cyclooxygenases was determined in presence of two purified isoforms of prostaglandin synthase. Rabbit polyclonal antiserum against PGHS-2 did not cross-react with PGHS-1 (see Fig. 1). The blot was washed several times with PBS-Tween 20 and incubated with a goat anti-rabbit antibody in a 1:2,000 dilution for 1 h. Antibody binding was visualized by the ECL technique (ECL system), according to the instruction of the supplier, using Kodak X-OMAT LS film.


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Fig. 1.   Western blot analysis of prostaglandin H synthase (PGHS) isoforms in cultured murine 3T6 fibroblasts. Lane 1, ovine PGHS-2 (100 ng) and PGHS-1 (200 ng); lane 2, other isoform; lane 3, fibroblast cultured with fetal calf serum (FCS) starvation overnight; lane 4, fibroblast lysate from cells cultured 4 days in RPMI 1640 with FCS (10%); lane 5, fibroblast lysate from cells cultured in RPMI 1640 with FCS (10%) and treated with dexamethasone (2.5 µM) overnight; lane 6, fibroblast lysate from cells cultured in RPMI 1640 (10% FCS) and treated with phorbol 12-myristate 13-acetate (PMA; 1 µM, 6 h); lane 7, lysate from cells cultured in RPMI 1640 (10% FCS) plus PMA (1 µM, 6 h) in presence of dexamethasone (2.5 µM). Data are representative of 3 experiments.

Cell growth. The influence of drugs was assessed on 3T6 fibroblasts plated at 103 cells/well in 12-well plates (Costar, Cambridge, MA) and cultured for 3 days in RPMI 1640 supplemented with 10% FCS in presence of different treatments. Finally, the cells were washed, trypsinized, and counted.

Analysis of DNA synthesis. DNA synthesis was measured by a [3H]thymidine incorporation assay. This involved culturing 3T6 fibroblasts in 96-well plates (Costar) in RPMI 1640 with 10% FCS at a density of 400 cells/well. Six hours later, cells were incubated with the drugs and [3H]thymidine (1 µCi/well) for 24 h. [3H]thymidine-containing media were aspirated, cells were overlaid with 1% Triton X-100, and then cells were scraped off the dishes. Finally, radioactivity present in the cell fraction was measured by scintillation counting, using a Packard Tri-Carb 1500 counter.

Incorporation and release of [3H]AA. After a period of fibroblast replication (3-4 days) and a period of FCS starvation (6 h), the medium was removed and replaced with 0.5 ml RPMI 1640 containing 0.1% fatty acid-free BSA and 0.1 µCi [3H]AA for 24 h. Cells were then washed three times with medium containing 0.5% BSA to remove unincorporated [3H]AA. After a study period, the medium was removed for analysis of radioactivity release. The amount of [3H]AA released into the medium was expressed as a percentage of cell-incorporated [3H]AA, which was determined in solubilized cells. Background release from untreated cells (~9 ± 2% of 3H incorporated) was subtracted from all data.

Measurement of PGE2 production by fibroblasts in culture. An aliquot of culture supernatant medium (0.25 ml) was acidified with 1 ml of 1% formic acid. PGE2 was extracted in ethyl acetate (5 ml), and, after the aqueous phase was discarded, the organic phase was evaporated in a stream of nitrogen. The overall recovery for the extraction procedure was established by including [3H]PGE2 and was found to be 80%. PGE2 levels in the medium were determined using a PGE2-monoclonal enzyme immunoassay kit (Cayman Chemicals), following the manufacturer's protocol.

Statistics and data analysis. Results are expressed as means ± SE. Differences between control cultures and treated cultures were tested by using either Student's t-test or one-way analysis of variance followed by the least significant difference test as appropriate.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Expression of PGHS-2 in 3T6 fibroblast cultures. Immunoblot analysis with a polyclonal antibody reacting exclusively with PGHS-2 revealed a significant band of ~70 kDa in 3T6 fibroblast cultured 4 days in RPMI 1640 supplemented with 10% FCS (Fig. 1). However, PGHS-1 was only barely detectable using an anti-PGHS-1 antibody. This 70-kDa protein band corresponding to PGHS-2 was significantly reduced by coincubation with 2.5 µM dexamethasone or overnight serum starvation and was significantly increased by phorbol 12-myristate 13-acetate (PMA; 1 µM, 6 h) (Fig. 1).

Effect of PLA2 and cyclooxygenase inhibitors on 3T6 fibroblast proliferation. To determine the role of AA release and its metabolites on 3T6 fibroblast proliferation, the cells were cultured in 10% FCS in the presence of PLA2 and cyclooxygenase inhibitors. We used a glucocorticoid such as dexamethasone that inhibited AA mobilization and was able to release PGHS-2 expression in these cells, manoalide as an enzymatic PLA2 inhibitor, ketoprofen as a dual inhibitor of both cyclooxygenase isoforms, and, finally, piroxicam at low concentrations and CGP-28238 as specific inhibitors of PGHS-1 and PGHS-2, respectively. The proliferative response of 3T6 fibroblasts was significantly reduced by the addition of dexamethasone, manoalide, ketoprofen, and CGP-28238 to the cell media. However, piroxicam in doses that inhibit PGHS-1 did not significantly decrease 3T6 growth (Fig. 2). To confirm the role of these treatments on AA cascade in our experimental conditions, we determined the effect of these drugs on [3H]AA release and PGE2 formation. In line with the literature, dexamethasone and manoalide decreased [3H]AA mobilization, and all drugs were able to inhibit significantly the levels of PGE2 in fibroblast culture media (Table 1).


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Fig. 2.   Effect of phospholipase A2 and cyclooxygenase inhibitors on 3T6 fibroblast growth stimulated by 10% FCS. Approximately 103 cells/well were plated and cultured for 4 days in RPMI 1640 with 10% FCS in presence of dexamethasone (Dex, 2.5 µM), manoalide (Mld, 1 µM), ketoprofen (Kp, 0.5 µM), piroxicam (Pir, 0.05 µM), and CGP-28238 (CGP, 0.5 µM). Finally, cells were trypsinized and counted. Results are means ± SE of 3 experiments performed in triplicate. C, control. Effect of treatments was statistically compared using paired Student's t-test. * P < 0.05.

                              
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Table 1.   Effect of treatments on [3H]AA release and PGE2 levels induced by cell culture in RPMI 1640 with 10% FCS

It was also observed that the inhibitory effect on cell proliferation of ketoprofen and CGP-28238 was dose dependent and reached a plateau. Thus the higher concentrations of both cyclooxygenase inhibitors exhibited a 50-60% inhibition of cell growth as a maximum effect (Fig. 3).


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Fig. 3.   Dose-dependent effect of ketoprofen (down-triangle) and CGP-28238 (bullet ) on 3T6 fibroblast proliferation. Approximately 103 cells/well were plated and cultured for 4 days in RPMI 1640 with 10% FCS in presence of ketoprofen or CGP-28238. Finally, cells were trypsinized and counted. Results are means ± SE of 3 experiments performed in triplicate.

Finally, some experiments were conducted to confirm that the effects of drugs were because of growth inhibition and not cytotoxicity due to long-term exposure to drugs. Thus it was observed that once drugs were removed from the 3T6 fibroblast cultures, the cells resumed growth at approximately the original rate (data not shown).

Effect of PLA2 and cyclooxygenase inhibitors on DNA synthesis in 3T6 fibroblast cultures. To determine whether DNA synthesis is regulated by AA or its metabolites in 3T6 fibroblast cultures, the influence of PLA2 and cyclooxygenase pathway inhibitors on [3H]thymidine incorporation was evaluated. Figure 4 shows the inhibitory effect of dexamethasone, manoalide, ketoprofen, and CGP-28238, all employed at concentrations that induced cell growth decrease, on 3T6 fibroblast [3H]thymidine incorporation.


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Fig. 4.   Effect of phospholipase A2 and cyclooxygenase inhibitors on DNA synthesis in 3T6 fibroblast cultures. DNA synthesis was measured using [3H]thymidine incorporation in presence of dexamethasone (Dex, 2.5 µM), manoalide (Mld, 1 µM), ketoprofen (Kp, 0.5 µM), or CGP-28238 (CGP, 0.5 µM). Results are means ± SE of 2 experiments performed in triplicate. Dpm, disintegrations per minute. Effect of drugs was statistically compared using paired Student's t-test. * P < 0.05.

Effect of cyclooxygenase pathway on 3T6 fibroblast proliferation. Products of the cyclooxygenase pathway have been implicated in the control of cell growth. This study initially investigated the effect of PLA2 and cyclooxygenase pathway inhibitors on FCS-stimulated 3T6 fibroblast proliferation as well as DNA synthesis. As shown previously, inhibition of 3T6 growth by ketoprofen was dose dependent, with a maximum growth inhibition at 5 µM; at higher concentrations, cell numbers remained constant with no further inhibition or cell death. Interestingly, the same response was observed when a specific PGHS-2 inhibitor such as CGP-28238 was used. PGE2 is one of the major cyclooxygenase pathway metabolites produced by fibroblasts, and a correlation between cyclooxygenase inhibition and cell growth decrease was observed. Considering these findings, we finally determined the role of PGE2 on DNA synthesis and 3T6 fibroblast growth. For this purpose, PLA2 inhibitors or cyclooxygenase inhibitors were incubated together with PGE2. These experiments revealed that the inhibition of DNA synthesis and cell proliferation was reverted when PGE2 was present in culture medium (Table 2).

                              
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Table 2.   Cell growth and DNA synthesis were affected by the inhibition of PGE2 formation

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The release of AA from cellular phospholipids by PLA2 occurs as a result of a variety of physiological stimuli and is the rate-limiting step in the metabolism of AA by the cyclooxygenase and lipoxygenase pathways. Our results reveal a pivotal role for AA and its metabolites in 3T6 fibroblast proliferation. However, neither dexamethasone, a glucocorticoid that induced proteins with anti-PLA2 activity (6), nor manoalide, a specific enzymatic inhibitor of PLA2 (10), totally blocked AA mobilization and the cell growth stimulated by serum. It should also be considered that serum factors may also activate other phospholipases such as phospholipase C, which generates the second messengers diacylglycerol and inositol 1,4,5-trisphosphate via the hydrolysis of phosphatidylinositol 4,5-bisphosphate, leading to the activation of protein kinase C and calcium mobilization, which could also have a role in the control of cell growth. Thus increase in phospholipase C activity was associated with PDGF-stimulated DNA synthesis in 3T3 fibroblasts (14). Moreover, diacylglycerol generated by this pathway can also be deacylated to produce free fatty acids such as AA, which in turn can be acted on by cyclooxygenase and lipoxygenase. This could explain both the ability of serum-stimulated fibroblasts to maintain a steady level of growth in the presence of PLA2 inhibitors and also that PLA2 inhibitors did not produce a complete inhibition of [3H]AA mobilization in our experimental conditions.

Our findings of growth inhibition of 3T6 cells after blocking the cyclooxygenase pathway with several cyclooxygenase inhibitors suggested a role for prostanoids as autocrine or paracrine factors with a regulatory effect on 3T6 cell growth. If we consider that indomethacin, a cyclooxygenase pathway inhibitor, has only a slight inhibitory effect on human umbilical artery smooth muscle cell proliferation (2) and no effect on the growth of rat aortic smooth muscle cell (3), and that it has been demonstrated that lipoxygenase metabolites regulate the proliferation of endothelial cells (29) and smooth muscle vascular cells (4), we can suppose that each AA cascade pathway could be involved in the control of proliferation of a determined cell type.

In our experiments, the use of various inhibitors showed that cyclooxygenase metabolites play an essential role in the mechanism by which serum stimulates 3T6 cell growth. Furthermore, we demonstrated that PGE2 is an important AA metabolite to 3T6 fibroblast proliferation. Thus the impairment of cell growth induced by PLA2 inhibitors or cyclooxygenase inhibitors was reverted by the adding of PGE2 to culture medium. In addition, this reversibility of the inhibitory effects of these drugs leads us to be quite confident that the inhibition induced by these drugs, observed in these experiments, is not due to cell death or cytotoxicity.

Prostaglandin endoperoxide H synthase catalyzes the formation of prostanoids from arachidonate. Two isoforms of PGHS have been described, PGHS-1 and PGHS-2 (27). PGHS-2 is often referred to as the inducible form of prostaglandin synthase because it is not expressed normally by unstimulated cells, but can be induced in fibroblasts, monocytes, or endothelial cells by mitogens, cytokines, and tumor promoters (12). Western blot analysis was used to examine whether 3T6 fibroblasts cultured in RPMI medium supplemented with FCS expressed PGHS-2. Our results showed that this isoform was expressed more clearly than PGHS-1 under our experimental conditions. These observations agree with the expression of PGHS-2 in all nonstimulated tissues, except the intestine and the lung (7, 28) as a result of stimulation by a lot of factors in serum, and with the results reported by DeWitt and Meade (5), who demonstrated that serum induced PGHS-2 expression in 3T3 fibroblast cultures. Moreover, we observed that PGHS-2 levels were reduced by serum starvation and increased by PMA and that dexamethasone inhibited serum- and PMA-induced PGHS-2 expression, as reported previously by DeWitt and Meade (5). Interestingly, these experimental conditions that reduced PGHS-2 levels also decreased 3T6 fibroblasts growth.

The differential inhibition of PGHS-1 and PGHS-2 by NSAIDs in clinical use was investigated. Thus ketoprofen had a similar action on both isoforms, whereas piroxicam at low concentrations showed a preferential inhibition of PGHS-1 (23). On the other hand, CGP-28238 was described as a powerful NSAID with an improved gastric tolerance, which did not inhibit PGHS-1 in a bovine seminal vesicle preparation (29). A relative lack of activity against PGHS-1 was also found when using human washed platelets [half-maximal inhibitory concentration (IC50) 70 µM], whereas PGHS-2 activity from interleukin-1-stimulated mesangial cells was inhibited with an IC50 of 25 nM. This yields a PGHS-2-to-PGHS-1 ratio of 0.0002 (11).

The ability of ketoprofen and CGP-28238 to inhibit the 3T6 fibroblast growth rate may reflect the role of PGHS-2 in the signaling pathways involved in the regulation of cell proliferation. Interestingly, ketoprofen and CGP-28238 were not able to block totally cell growth, which suggested, however, that other elements are involved in the control of 3T6 cell growth.

In conclusion, the early expression of PGHS-2 after serum and/or growth factors and the subsequent conversion of AA to prostaglandins could have an important role in the control of 3T6 fibroblast growth.

    ACKNOWLEDGEMENTS

We are very grateful to Robin Rycroft for valuable assistance in the preparation of the English manuscript.

    FOOTNOTES

T. Sánchez and J. Martínez had a grant from the Spanish Ministry of Education and Science. This study was supported by Spanish Ministry of Education and Science Grant DGICYT PB-94-0934.

Address for reprint requests: J. J. Moreno, Departamento de Ciencias Fisiológicas, Facultad de Farmacia, Unidad de Fisiología, Universidad de Barcelona, Avda. Joan XXIII s/n, Barcelona E-08028, Spain.

Received 24 September 1996; accepted in final form 3 July 1997.

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Abstract
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Results
Discussion
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AJP Cell Physiol 273(5):C1466-C1471
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