Carcinogenesis vol.13 no.3 pp.447-451, 1992
Altered regulation of arachidonic acid metabolism by SV40 immortalized human urothelial cells
Don J.Thomas, Ammini K.Jacob, Catherine A.Reznikoff1, Terry V.Zenser2 and Bernard B.Davis VA Medical Center, and Department of Biochemistry and Division of Geriatric Medicine, St Louis University School of Medicine, St Louis, MO 63125 and 'Department of Human Oncology, University of Wisconsin Clinical Cancer Center, Madison, Wl 53792, USA 2
To whom correspondence should be addressed
Introduction Arachidonic acid metabolism is involved in different steps in the carcinogenic process [reviewed in several volumes (1—4)], including bladder cancer. A two-stage model for carcinogenesis, as originally described for the mouse skin (5), has been demonstrated for chemically induced carcinoma of the rat urinary bladder (6,7). Arachidonic acid metabolites, i.e. prostaglandin E2 (PGE2*), stimulate the formation of cyclic AMP in cells and tissues, including urothelial (8,9). Cyclic AMP and its analogs •Abbreviations: PGE2, prostaglandin E2; TPA, 12-O-tetradecanoylphorbol13-acetate; SV-HUC, SV40 immortalized, non-tumongenjc human urothelial cell line.
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Regulation of arachidonic acid metabolism was investigated in an SV40 immortalized, non-tumorigenic human urotheliaJ cell line (SV-HTJC). This cell line is being used to evaluate the multistage carcinogenic process. Media from confluent cultures were analyzed for radioimmunoassayable prostaglandin E2 (PGE2). A variety of agonists, including 12-0-tetradecanoylphorbol-13-acetate (TPA) and A23187 were tested and did not increase PGE2 synthesis within 2 h of addition. This was not due to the lack of prostaglandin H synthase activity because addition of exogenous arachidonic acid increased PGEj synthesis. Cultures prelabeled overnight with [3H]arachidonk acid failed to increase the release of radioactivity following agonist addition. Thus, the lack of an early response in SV-HUC appears to be due to decreased release of endogenous arachidonic acid by phospholipase(s). After a 24 h incubation with 0.1 pM TPA or 1.0 /iM A23187, the addition of arachidonic acid elicited significantly more PGE2 synthesis in agonist-treated cells than it did in control cells. Microsomes from 24 h TPA-treated cells produced ~ 15-fold more PGE2 than did those from control cells. In addition, the PGE2 content of overnight media was significantly greater in TPA-treated cells than in control cells. The 24 h agonist response was blocked by cycloheximide and staurosporine—inhibitors of protein synthesis and protein kinase C respectively. Pretreatment of cells with aspirin, an irreversible inhibitor of prostaglandin H synthase, prior to addition of TPA did not prevent the late 24 h TPA-mediated increase in PGE2 synthesis. Results suggest that this late effect of TPA is due to de novo synthesis of prostaglandin H synthase. Thus, SV-HUC has lost the early but retains the late response to agonists.
effect the growth and differentiation of cells (10,11). The bladder tumor promoters sodium ascorbate and butylated hyroxyanisole elicit increases in bladder tissue PGE2 and cyclic AMP content (12). 12-OTetradecanoylphorbol-13-acetate (TPA) also induces urothelial cell arachidonic acid metabolism (13 — 16). Aspirin, an inhibitor of prostaglandin synthesis, inhibits both FANFT initiation and saccharin promotion of bladder cancer (17,18). These results are consistent with arachidonic acid metabolism being involved in bladder carcinogenesis. Regulation of arachidonic acid metabolism can be separated into two distinct areas (19,20). The first area relates to arachidonic acid, i.e. substrate, availability. Arachidonic acid is not found free but esterified to phospholipids within the cell. Thus, phospholipases are necessary for arachidonic acid release and availability. The second area relates to metabolism of free, unesterified arachidonic acid. Prostaglandin H synthase and lipoxygenases are the two enzymes most notable in this area. Cytochrome P450s can also metabolize arachidonic acid (21). De novo synthesis of these enzymes can be induced by certain agents and conditions. Arachidonic acid metabolism has been extensively studied in urothelial cells (13-16,22). PGEj is the major product (16,22). In normal and subcultured urothelial cells, TPA regulation of arachidonic acid metabolism can be separated into early (increased arachidonic acid availability inhibited by aspirin pretreatment) and late (increased de novo synthesis of prostaglandin H synthase not inhibited by aspirin pretreatment) responses (23). Neoplastic transformation models using human epithelial cells can assist in evaluating multistage carcinogenesis. The SV40 immortalized, non-tumorigenic human urothelial cell line (SVHUC) developed by Reznikoff et al. (24) is one model being developed. This line is clonal in origin and pseudodiploid. Cells exhibit a loss of differentiated functions, i.e. stratification. SVHUC have been neoplastically transformed by treatment with chemicals and with ras oncogene transfection in combination with additional stochastically occurring events (25,26). Transformed cell lines derived from SV-HUC may represent different stages that delineate the carcinogenic process: immortalization, transformation and metastases. The mechanism of SV40 immortalization is hypothesized to be similar to that of human papilloma viruses and adenovimses. Products from these three DNA viruses bind to the retinoblastoma gene protein pl05-Rb (27-29). Onethird of a sample of bladder carcinomas lacked intact RB alleles (30). Re-expression of RB in a bladder cell line that lacks RB expression demonstrated that this gene has growth and tumor suppressor activity (31). Viral transformation can have differing effects on the ability of cells to produce prostaglandins. Changes in PGE2 production are dependent upon the cell type and independent of the kind of virus used (32). For example, SV40 transformation increases PGE2 production by 3T3 cells and decreases the production by FRTL cells. To delineate potential changes in urothelial cell regulation of arachidonic acid metabolism, following SV40 immortalization and subsequent transformation, the current study tested a variety of agents for their potential to regulate arachidonic
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acid metabolism by SV-HUC cells. TPA and A23187 were studied in more detail than other agents because their effects on protein kinase C and calcium are involved in agonist-mediated arachidonic acid metabolism in many tissues. Phospholipase activity was assessed by prelabeling overnight cultures with [3H]arachidonic acid and measuring release of radioactivity. Prostaglandin H synthase activity was assessed directly with microsomes and indirectly after addition of exogenous arachidonic acid at a maximum effective concentration (100 /iM) to media. The mechanism by which SV40 alters urothelial cell PGE2 synthesis is described. Materials and methods
Prelabeling Cells were rinsed twice with serum-free media and incubated with 1 jiCi[3H]arachidonic acid (100 Ci/mmol; Du Pont NEN Research Products, Boston, MA) as previously described (15,16,22). After 1 h, fetal calf serum was added to a final concentration of 1 %, and cells were incubated overnight. Cells were then rinsed twice with a-MEM containing 5 mg/ml BSA and twice with a-MEM containing 1 mg/ml BSA. All subsequent conditions contained a-MEM with 1 mg/ml BSA. Agonists were added and media was analyzed for radioactivity over 2 h period.
Results To assess the regulation of arachidonic acid metabolism by SVHUC, different agonists were incubated with cells for 2 h (Figure 1). A23187 and TPA were two of the agonists tested. Both agonists, used over a wide concentration range, were ineffective in increasing PGE2 synthesis. Other agonists that were tested and found ineffective include bradykinin (800 nM), angiotension II (20 /tg/ml), platelet-activating factor (2 /tM), thrombin (1 U/ml), vasopressin (0.1 U/ml), mezerein (0.2 /tM), epinephrine (100 /tM), phorbol-12,13-didecanoate (0.1 /tM), phorbol-12,13-dibutyrate (0.1 /tM) and l-oleyl-2-acetylglycerol (200 /tM). Because the agonists tested have been shown to increase urothelial PGE2 synthesis by increasing endogenous arachidonic acid availability (15,16,22), cells were prelabeled with [3H]-arachidonic acid. When agonists such as TPA, mezerein or bradykinin were added to prelabeled cells, no increase in release of radioactivity was observed (not shown). This is consistent with the lack of effect of these agonists on PGE2 synthesis. Exogenous arachidonic acid was the only compound tested that elicited increases in PGE2 synthesis. Thus, SV-HUC do not elicit an early response to agonists but contain prostaglandin H synthase activity. Agonist-mediated de novo synthesis of prostaglandin H synthase has been reported (39-42). This possibility was evaluated (Figure 2). Following a 24 h incubation with agonists, media was removed and fresh media added. The basal level of PGE2 synthesis in cells treated with A23187 and TPA was similar to control. However, when fresh media contained 100 /tM arachidonic acid, agonists-treated cells elicited large increases in PGE2. TPA was also shown to increase PGE2 synthesis as early as 6 h after addition. The lack of difference in the basal level of PGE2 synthesis between control and these agonists is probably due to very low levels of free intracellular arachidonic acid. Experiments were conducted to determine whether TPA increases in PGE? were due to de novo synthesis of
Radioimmunoassay of PGE2 The PGE2 content of the media was measured by double-antibody radioimmunoassay (36). Rabbit amiserum to PGE2 was obtained from Regis Chemical Co., Morton Grove, IL. Tritium-labeled PGE2 was purchased from Du Pont NEN, and goat antiserum to rabbit -(-globulins was purchased from Antibodies Inc., Davis, CA. Each condition was evaluated in two or three separate experiments. In each experiment, conditions were evaluated in at least triplicate plates. The medium from each plate was analyzed in duplicate and the value of duplicate determinations averaged and considered as an n of one. Data were expressed as mean ± SEM of ng PGE2/IO3 cells. Differences between data were evaluated statistically by the Student's Mest for unpaired observations. Prostaglandin H synthase assay Following a 24 h incubation of cells in the absence and presence of TPA, media were saved for PGE2 radioimmunoassay and microsomes were prepared for direct assessment of synthase activity (37). All subsequent procedures were performed at 4°C. Cells were scraped into tubes, centrifuged at 2000 r.p.m. for 10 min to pellet cells, and were resuspended in buffer and sonicated. Buffer contained 25 mM HEPES (pH 7.4), 1 mM EGTA, 1 mM EDTA, 1 mM dithiothreitol, 250 mM sucrose, 4 jig/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride. Samples were centrifuged at 100 000 g for 60 min. The microsomal
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Fig. 1. Effect of test agents on arachidonic acid metabolism evaluated 2 h after their addition. Cells were incubated in the absence (control) and presence of arachidonic acid (AA, 100 )M), A23187 (1 /tM) and TPA (100 nM). Media were removed for PGEj determination (n = 3).
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Cells and culture methods A clonal line of SV-HUC was used in this study. SV-HUC is an immortalized, non-tumorigenic human urothelial cell line that has been previously characterized (24,25,33). Studies were initiated with cells at passage 26. These cells remain non-tumorigenic after 80 serial passages and > 2 years in continuous culture. SV-HUC were grown on plastic dishes in medium consisting of Ham's Nutrient Mixture F12 (GIBCO, Grand Island, NY) and incubated at 37°C in 5% CC>2/95% humidified air. Medium was supplemented with 1 % fetal calf serum and the following (final concentration in parentheses): insulin (10 /ig/mi), hydrocortisone (1 jig/ml), transferrin (5 /ig/ml) and amphotencin B (2.5 /tg/ml), which were obtained from Sigma Chemical Co., St Louis, MO; all non-essential amino acids (0.1 mM), L-glutamine (2.0 mM), streptomycin (100 /tg/ml) and penicillin (100 units/ml), which were purchased from GIBCO; and dextrose (2.7 mg/ml), obtained from Fisher Scientific. This medium was developed for human uroepithelial cells (34,35). Routine dispersion of cultures for serial passage was performed using 0.01% EDTA (Sigma) dissolved in Hank's balanced salt solution (HBSS, GIBCO) and adjusted to pH 7.4 with NaHCO3. Experiments were conducted with 80-90% confluent cultures (0.7-1.0 X 10* cells/60 mm plate). Growth media were aspirated and cells washed twice with 1 ml of HBSS. Serum-free a-MEM was then added with or without test agents as indicated in the Results. Cells were incubated at 37°C in 5% CO2/95% air. Test agents used were arachidonic acid purchased from Nu-Chek Prep, Inc., Elysian, MN; calcium ionophore A23187 and staurosporine were from CalBiochem, La Jolla.CA; platelet-derived growth factor (human) was from Collaborative Research, Inc., Bedford, MA; and cycloheximide, aspirin, bradykinin triacetate, epidermal growth factor, epinephrine bitartrate, thrombin (2000 NIH units/mg protein), EDTA (disodium salt), l-oleoyl-2-acetylglycerol, antidiuretic hormone (grade VI), mezerein, and TPA and the other phorbol esters were from Sigma. Each agonist was used at a maximally effective concentration previously determined (15,22).
pellet was rcsuspended in 100 mM phosphate buffer (pH 7.8) and immediately assayed. Reaction mixtures contained 1 mM epinephnne and 1 mM glutathione in 100 mM phosphate buffer (pH 7.8). Samples were preincubated for 1 min at 37°C before arachidonic acid (200 yM) was added to start the reaction. The total reaction volume was 0.25 ml. The reaction was stopped after 10 min by adding 0.5 ml of ice-cold methanol. Samples were centrifuged to remove protein, and the supernatant evaporated to dryness. Water (0.25 ml) was added to each sample, which was then sonicated to resuspended PGE2 prior to radioimmunoassay. Protein was determined using bovine serum albumin as a standard (38). Prostaglandin H synthase activity is expressed as mean ± SEM of ng protein/10 min.
Regulation of PGE^ synthesis
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Aspirin was used to evaluate further the mechanism by which TPA regulates de novo synthesis of prostaglandin H synthase (Figure 5). Aspirin is an irreversible inhibitor of prostaglandin H synthase (44,45). The dose of aspirin used and the time of exposure (1 h) were sufficient to inhibit completely the 2 h exogenous arachidonic acid-mediated increases in PGE2 (16), like those observed in Figure 1. However, cells pretreated with aspirin and then exposed to TPA for 24 h elicited significant increases in PGE2 in response to arachidonic acid (Figure 5). Thus, the results suggest that TPA can regulate arachidonic acid metabolism by increasing de novo synthesis of prostaglandin H synthase. Discussion These studies document unique changes in the regulation of PGE2 synthesis by SV-HUC compared to previous studies with primary and subcultured human or dog urothelial cells (13-16,22,23). In the latter cells, agonist regulation of PGE2 synthesis has been extensively evaluated. Some agonists, like bradykinin, elicit increases in PGE2 synthesis within 5 min which peak by 15 min. Others, such as TPA, elicit more gradual increases, which may not be evident for 15 min and may not peak for 2 h (13,15). While separate mechanisms appear to exist for bradykinin and TPA, there are also some similarities in this early response. Both responses are calcium dependent and require
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Fig. 2. Effect of test agents on arachidonic acid metabolism evaluated 24 h after their addition. Cells were incubated in the absence (control) and presence of A23187 (1 pM) or TPA (100 nM). After 24 h, fresh media in the absence (basal) and presence of 100 pM arachidonic acid were added and removed 2 h later for PGE2 determination (n = 3).
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Fig. 4. Effect of cycloheximide and staurosporine on TPA-mediated de novo synthesis of prostaglandin H synthase. Where indicated, cells were preincubated with cycloheximide (35 /iM) or staurosporine (1 /iM) for 60 min before sidditions of TPA (100 nM). After 6 h, fresh media containing 100 nM arachidonic acid were added and removed 2 h later for PGE2 determination (n = 3).
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; m• ; Fig. 3. De novo synthesis of prostaglandin H synthase by SV-HUC. Cells were incubated in the absence (control) and presence of TPA (100 nM). After 24 h, overnight media were saved for PGE2 determination, and microsomes were prepared from cells. Microsomes were used to measure prostaglandin H synthase (PHS) activity (n = 4 - 8 ) .
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Fig. 5. Effect of aspirin on TPA-mediated de novo synthesis of prostaglandin H synthase. Where indicated, cells were preincubated with 3 mM aspirin for 60 min, washed twice and TPA (100 nM) added. After 24 h, fresh media containing 100 /jM arachidonic acid were added and removed 2 h later for PGEj determination (n = 3).
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prostaglandin H synthase. Following a 24 h exposure to TPA, microsomes were prepared from both control and TPA-treated cells. Following addition of arachidonic acid, microsomes from TPA-treated cells produced - 15-fold more PGE2 than did those of control cells (Figure 3). PGE2 synthesis was completely prevented by prior incubation of microsomes with 0.1 mM indomethacin. In addition, the PGE2 content of overnight media from TPA-treated cells was significantly greater than control (Figure 3). Thus, increased de novo synthesis of prostaglandin H synthase results in increased urothelial cell media content of PGEj. The mechanism by which TPA regulates de novo synthesis of prostaglandin H synthase was evaluated (Figure 4). Cycloheximide, a protein synthesis inhibitor, and staurosporine, a protein kinase C inhibitor (43), were utilized. The concentration of each inhibitor was previously shown to be a maximum effective dose. Both inhibitors were effective in preventing 6 h TPA-mediated increases in PGE2. In separate experiments, prostaglandin H synthase activity in cells exposed to TPA for 24 h exhibited more activity than did corresponding cells pretreated with cycloheximide (187 ± 11 versus 51 ± 5 ng PGF^/mg protein/10 min). Potential toxic effects of cycloheximide and staurosporine were also assessed. At the concentrations used in Figure 4, neither cycloheximide nor staurosporine altered the number of viable cells or the arachidonic acid-mediated increase in PGE2 (Figure 1). Thus, both protein synthesis and protein kinase C appear to be involved in the mechanism by which TPA regulates de novo synthesis of prostaglandin H synthase.
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thought to be involved in different steps of the carcinogenic process, this has been difficult to assess. SV-HUC allows a direct assessment of parameters that regulate arachidonic acid metabolism. SV-HUC has lost the early, but retains the late PGE2 synthetic response. Both responses are dependent upon protein synthesis and protein kinase C. Loss of the early response does not appear to be due to acclimation of urothelial cells to culture conditions since subcultured urothelial cells retain the early response (23). The prostaglandin H synthase activity of SV-HUC is significantly less than that observed in primary cultures of urothelial cells. Thus, the ability of SV-HUC to metabolize arachidonic acid is significandy impaired. The relationship between altered regulation of arachidonic acid metabolism and the immortalization process is not clear. Many different human epithelial cell lines have been established by SV40 immortalization (53,54) and our results may relate to those cells. Acknowledgements The authors thank Dr Yun-Hua Huang Wong for helpful discussions and appreciate the expert technical assistance of Cindee Rettke, Alma Schisla and Nathan T Zenser. This work was supported by the Department of Veterans Affairs (B.B D., T.V.Z.) and by the USPHS Grant CA-29525 (C.A.R.) and CA-28015 (T.V.Z).
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release of endogenous arachidonic acid for PGE2 synthesis. The TPA response requires protein synthesis and protein kinase C (13,15,16). In contrast, SV-HUC did not demonstrate an early agonist-mediated response. All of the agonists tested increase PGE2 synthesis in either human and dog urothelial cells or other cell types. The lack of SV-HUC responsiveness appears to be due to its failure to release endogenous arachidonic acid following agonist addition. The reason for decreased release of arachidonic acid (phospholipase activity) is not clear and was not further investigated. Possible explanations include the lack of or the inability to activate a phospholipase(s). Aspirin played an important role in demonstrating de novo synthesis of prostaglandin H synthase. Aspirin is an irreversible inhibitor of prostaglandin H synthase (44,45). The concentration of aspirin used and the time of exposure were sufficient to inhibit maximally arachidonic acid-mediated increases in PGE2 seen within 2 h (Figure 1). Thus, aspirin completely destroyed all prostaglandin H synthase activity prior to addition of TPA. New enzyme synthesis was required to restore activity. Taken together, the effects of cycloheximide and aspirin are consistent with TPAmediated de novo synthesis of prostaglandin H synthase. Because A23187, like TPA, increases PGF^ after 24 h (Figure 2), A23187 might also elicit an increase in the de novo synthesis of prostaglandin H synthase. Protein kinase C appears to be involved in both the early and late urothelial cell responses elicited by TPA because of the inhibition observed with staurosporine (16,23). This is not surprising since most effects of TPA are mediated by protein kinase C (46—48). However, it should be noted that while staurosporine is one of the most widely used inhibitors of protein kinase C, staurosporine is also an inhibitor of calcium/calmodulindependent protein kinase and protein tyrosine kinases (49,50). Because SV-HUC retain the late TPA response, altered protein kinase C activity may not be responsible for the lack of an early response. Inhibition by cycloheximide indicates that additional proteins are necessary for arachidonic acid release. Agonistmediated arachidonic acid release by endothelial cells requires synthesis of a phospholipase A2 stimulatory peptide like melitin (51). A similar mechanism may regulate the early response in primary and subcultured urothelial cells, and may be altered in SV-HUC. TPA regulates—by a protein kinase C-dependent mechanism—forskolin-responsive cyclic AMP synthesis in urothelial cells. This TPA response is also not detected in SVHUC (52). The relationship between SV-HUC alterations in the regulation of arachidonic acid metabolism and cyclic AMP synthesis is not known. The capacity of SV-HUC to produce PGE2 is severely limited in comparison to primary urothelial cells in culture. Primary, as well as subcultured urothelial cells exhibit an early response to agonists that is either absent or not detectable in SV-HUC (23). In addition, the prostaglandin H synthase activity of control and TPA-treated primary cultured cells is 140 ± 10 and 395 ± 35 ng PGFVmg protein/10 min respectively (23). Values of 8.7 ± 0.7 and 140 ± 6 ng PGF^/mg protein/10 min are reported for control and TPA-treated SV-HUC respectively (Figure 3). The prostaglandin H synthase activity of TPA-stimulated SV-HUC is similar to the basal activity in primary cultures. Basal prostaglandin H synthase activity of SV-HUC is ~ 15-fold less than that in primary cultured cells. Prior studies with primary and subcultured cells used mixed cell populations and not clones. In addition, the particular clone from which SV-HUC was derived is unknown. In summary, although metabolism of arachidonic acid is
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by aspirin of Af-[4-{njtro-2-furyl)-2-thiazolyl]formamklc-induccd bladder carcinogenesis and enhancement of forestomach carcinogenesis. Cardnogenesis, 5, 53-55. 18. Sakata.T., Hasegawa.R., Johansson,S.L., Zenser.T.V. and Cohen,S.M. (1986) Inhibition by aspirin of A'-[4-(5-nitro-2-furyr)-2-thiazolyl]formamide initiation and sodium saccharin promotion of urinary bladder carcinogenesis in male F344 rats. Cancer Res., 46, 3903-3906. 19. Samuelsson.B., GoWyne.M., Granstrom.E., Hamberg.M., Hammarstrom.S. and Malmsten,C. (1978) Prostaglandins and thromboxanes. Annu. Rev. Biochem., 47, 997-1029. 20. Smith,W.L. (1989) The eicosanoids and their biochemical mechanisms of action. Biochem. J., 259, 315-324. 21. Fitzpatrick,F. and Murphy.R. (1989) Cytochrome P-450 metabolism of arachidonic acid: formation and biological actions of 'epoxygenase'-derived eicosanoids. Pharmacol. Rev., 40, 229-241. 22. Danon,A., Zenser.T.V., Thomasson.D.L. and Davis,B.B. (1986) Eicosanoid synthesis by cultured human urothelial cells: potential role in bladder cancer. Cancer Res., 46, 5676-5681. 23. Zenser.T.V., Thomas.D.J., Jacob.A.K. and Davis.B.B. (1992) Regulation of prostaglandin H synthase activity in dog urothelial cells. J. Cell. Physiol., 150, 214-219. 24. Christian.B.J., Loretz.L.J., Oberley.T.D. and Reznikoff.C.A. (1987) Characterization of human uroepithelial cells unmortalized in vitro by Simian virus 40. Cancer Res., 47, 6066-6073. 25. Reznikoff.C.A., Loretz.L.J., Christian.B.J., Wu,S.-Q. and Meisner.L.F. (1988) Neoplastic transformation of SV40-immortalized human urinary tract epithelial cells by in vitro exposure to 3-methylcholanthrene. Carcinogenesis, 9, 1427-1436. 26. Christian.B.J., Kao,C, Wu,S.-Q., Meisner.L.F. and Reznikoff.C.A. (1990) EJ/ras neoplastic transformation of SV40-immortalized human uroepithelial cells: a rare event. Cancer Res., 50, 4779-4786. 27. DeCapnoJ-. LudtowJ., FiggeJ., ShewJ., Huang.C, Lee.W., Marsiolio.E., Paucha.E. and Livingston.D. (1988) SV40 large tumor antigen forms a specific complex with the product of the retinoblastoma susceptibility gene. Cell, 54, 275-283. 28. Whyte.P., Buchkovich.K., Horowitz J., Fnend.S., Raybuck.M., Weinberg.R. and Hariow.E. (1988) Association between an pncogene and an anti-oncogene: the adenovirus El A proteins bind to the retinoblastoma gene product. Nature, 334, 124-129. 29. Dyson.N., Howley.P., Mungcr.K. and Hariow.E. (1989) The human papilloma virus-26 E7 oncoprotein is able to bind to the retinoblastoma gene product. Science, 243, 934-937. 30. Horowitz.J., Park.S., Bogenmann.E., ChengJ., Yandell.D., Kaye.F., Minna.J., Dryja.T. and Weinberg.R. (1990) Frequent inactivation of the retinoblastoma anti-oncogene is restricted to a subset of human tumor cells. Proc. Nail. Acad. Sci. USA, 87, 2775-2779. 31. Takahashi,R., Hashimoto.T., Xu,H.-J., Hu,S.-X., Matsui.T., Miki.T., BigoMarshall.H., Aaronson.S.A. and Benedict.W.F. (1991) The retinoblastoma gene functions as a growth and tumor suppressor in human bladder carcinoma cells. Proc. Nail. Acad. Sci. USA, 88, 5257-5261. 32. Lin.M.C, Segawa.K., Ito.Y. and Beckner.S. (1986) The effect of viral transformation on prostaglandin production depends on cell type. Virology, 155, 19-26. 33. Meisner.L., Wu,S.-Q., Christian,B. and Reznikoff.C. (1988) Cytogenetic instability with balanced chromosome changes in an SV40 transformed human uroepithelial cell line. Cancer Res., 48, 3215-3220. 34. Reznikoff.C.A., Johnson.M.D., Norback.D.H. and Bryan.G.T. (1983) Growth and characterization of normal human orothelium in vitro. In Vitro, 19, 326-343. 35. Reznikoff.C.A., Loretz.L.J., Pesciotta.D.M., Oberley.T.D. and Ignjatovic.M.M. (1987) Growth kinetics and differentiation in vitro of normal human uroepithelial cells on collagen gel substrates in defined medium. J. Cell. Physiol., 131, 285-301. 36. Zenser.T.V. and Davis.B.B. (1978) Effects of calcium on prostaglandin E2 synthesis by rat inner medullary slices. Am. J. Physiol., 235, F213-F218. 37. Brown.W.W., Zenser.T.V. and Davis.B.B. (1980) Prostaglandin E2 production by rabbit urinary bladder. Am. J. Physiol., 239, F452-F458. 38. Bradford,M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72, 248-254. 39. Goerig.M., Habenicht.AJ.R., Heitz.R., Zeh.W., Katus.H., Kommerell.B., Ziegler.R. and GlomseU.A. (1987) sn-1,2-Diacylglycerols and phorbol diesters stimulate thromboxane synthesis by de novo synthesis of prostaglandin H synthase in human promyelocytic leukemia cells. J. Clin. Invest., 79, 903-911. 40. Beaudry.G.A., Waite.M. and Daniel,L.W. (1985) Regulation of arachidonic acid metabolism in Madin-Darby canine kidney cells: stimulation of synthesis
Altered regulation of arachidonic acid metabolism by SV40 immortalized human urothelial cells
Don J.Thomas, Ammini K.Jacob, Catherine A.Reznikoff1, Terry V.Zenser2 and Bernard B.Davis VA Medical Center, and Department of Biochemistry and Division of Geriatric Medicine, St Louis University School of Medicine, St Louis, MO 63125 and 'Department of Human Oncology, University of Wisconsin Clinical Cancer Center, Madison, Wl 53792, USA 2
To whom correspondence should be addressed
Introduction Arachidonic acid metabolism is involved in different steps in the carcinogenic process [reviewed in several volumes (1—4)], including bladder cancer. A two-stage model for carcinogenesis, as originally described for the mouse skin (5), has been demonstrated for chemically induced carcinoma of the rat urinary bladder (6,7). Arachidonic acid metabolites, i.e. prostaglandin E2 (PGE2*), stimulate the formation of cyclic AMP in cells and tissues, including urothelial (8,9). Cyclic AMP and its analogs •Abbreviations: PGE2, prostaglandin E2; TPA, 12-O-tetradecanoylphorbol13-acetate; SV-HUC, SV40 immortalized, non-tumongenjc human urothelial cell line.
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Regulation of arachidonic acid metabolism was investigated in an SV40 immortalized, non-tumorigenic human urotheliaJ cell line (SV-HTJC). This cell line is being used to evaluate the multistage carcinogenic process. Media from confluent cultures were analyzed for radioimmunoassayable prostaglandin E2 (PGE2). A variety of agonists, including 12-0-tetradecanoylphorbol-13-acetate (TPA) and A23187 were tested and did not increase PGE2 synthesis within 2 h of addition. This was not due to the lack of prostaglandin H synthase activity because addition of exogenous arachidonic acid increased PGEj synthesis. Cultures prelabeled overnight with [3H]arachidonk acid failed to increase the release of radioactivity following agonist addition. Thus, the lack of an early response in SV-HUC appears to be due to decreased release of endogenous arachidonic acid by phospholipase(s). After a 24 h incubation with 0.1 pM TPA or 1.0 /iM A23187, the addition of arachidonic acid elicited significantly more PGE2 synthesis in agonist-treated cells than it did in control cells. Microsomes from 24 h TPA-treated cells produced ~ 15-fold more PGE2 than did those from control cells. In addition, the PGE2 content of overnight media was significantly greater in TPA-treated cells than in control cells. The 24 h agonist response was blocked by cycloheximide and staurosporine—inhibitors of protein synthesis and protein kinase C respectively. Pretreatment of cells with aspirin, an irreversible inhibitor of prostaglandin H synthase, prior to addition of TPA did not prevent the late 24 h TPA-mediated increase in PGE2 synthesis. Results suggest that this late effect of TPA is due to de novo synthesis of prostaglandin H synthase. Thus, SV-HUC has lost the early but retains the late response to agonists.
effect the growth and differentiation of cells (10,11). The bladder tumor promoters sodium ascorbate and butylated hyroxyanisole elicit increases in bladder tissue PGE2 and cyclic AMP content (12). 12-OTetradecanoylphorbol-13-acetate (TPA) also induces urothelial cell arachidonic acid metabolism (13 — 16). Aspirin, an inhibitor of prostaglandin synthesis, inhibits both FANFT initiation and saccharin promotion of bladder cancer (17,18). These results are consistent with arachidonic acid metabolism being involved in bladder carcinogenesis. Regulation of arachidonic acid metabolism can be separated into two distinct areas (19,20). The first area relates to arachidonic acid, i.e. substrate, availability. Arachidonic acid is not found free but esterified to phospholipids within the cell. Thus, phospholipases are necessary for arachidonic acid release and availability. The second area relates to metabolism of free, unesterified arachidonic acid. Prostaglandin H synthase and lipoxygenases are the two enzymes most notable in this area. Cytochrome P450s can also metabolize arachidonic acid (21). De novo synthesis of these enzymes can be induced by certain agents and conditions. Arachidonic acid metabolism has been extensively studied in urothelial cells (13-16,22). PGEj is the major product (16,22). In normal and subcultured urothelial cells, TPA regulation of arachidonic acid metabolism can be separated into early (increased arachidonic acid availability inhibited by aspirin pretreatment) and late (increased de novo synthesis of prostaglandin H synthase not inhibited by aspirin pretreatment) responses (23). Neoplastic transformation models using human epithelial cells can assist in evaluating multistage carcinogenesis. The SV40 immortalized, non-tumorigenic human urothelial cell line (SVHUC) developed by Reznikoff et al. (24) is one model being developed. This line is clonal in origin and pseudodiploid. Cells exhibit a loss of differentiated functions, i.e. stratification. SVHUC have been neoplastically transformed by treatment with chemicals and with ras oncogene transfection in combination with additional stochastically occurring events (25,26). Transformed cell lines derived from SV-HUC may represent different stages that delineate the carcinogenic process: immortalization, transformation and metastases. The mechanism of SV40 immortalization is hypothesized to be similar to that of human papilloma viruses and adenovimses. Products from these three DNA viruses bind to the retinoblastoma gene protein pl05-Rb (27-29). Onethird of a sample of bladder carcinomas lacked intact RB alleles (30). Re-expression of RB in a bladder cell line that lacks RB expression demonstrated that this gene has growth and tumor suppressor activity (31). Viral transformation can have differing effects on the ability of cells to produce prostaglandins. Changes in PGE2 production are dependent upon the cell type and independent of the kind of virus used (32). For example, SV40 transformation increases PGE2 production by 3T3 cells and decreases the production by FRTL cells. To delineate potential changes in urothelial cell regulation of arachidonic acid metabolism, following SV40 immortalization and subsequent transformation, the current study tested a variety of agents for their potential to regulate arachidonic
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acid metabolism by SV-HUC cells. TPA and A23187 were studied in more detail than other agents because their effects on protein kinase C and calcium are involved in agonist-mediated arachidonic acid metabolism in many tissues. Phospholipase activity was assessed by prelabeling overnight cultures with [3H]arachidonic acid and measuring release of radioactivity. Prostaglandin H synthase activity was assessed directly with microsomes and indirectly after addition of exogenous arachidonic acid at a maximum effective concentration (100 /iM) to media. The mechanism by which SV40 alters urothelial cell PGE2 synthesis is described. Materials and methods
Prelabeling Cells were rinsed twice with serum-free media and incubated with 1 jiCi[3H]arachidonic acid (100 Ci/mmol; Du Pont NEN Research Products, Boston, MA) as previously described (15,16,22). After 1 h, fetal calf serum was added to a final concentration of 1 %, and cells were incubated overnight. Cells were then rinsed twice with a-MEM containing 5 mg/ml BSA and twice with a-MEM containing 1 mg/ml BSA. All subsequent conditions contained a-MEM with 1 mg/ml BSA. Agonists were added and media was analyzed for radioactivity over 2 h period.
Results To assess the regulation of arachidonic acid metabolism by SVHUC, different agonists were incubated with cells for 2 h (Figure 1). A23187 and TPA were two of the agonists tested. Both agonists, used over a wide concentration range, were ineffective in increasing PGE2 synthesis. Other agonists that were tested and found ineffective include bradykinin (800 nM), angiotension II (20 /tg/ml), platelet-activating factor (2 /tM), thrombin (1 U/ml), vasopressin (0.1 U/ml), mezerein (0.2 /tM), epinephrine (100 /tM), phorbol-12,13-didecanoate (0.1 /tM), phorbol-12,13-dibutyrate (0.1 /tM) and l-oleyl-2-acetylglycerol (200 /tM). Because the agonists tested have been shown to increase urothelial PGE2 synthesis by increasing endogenous arachidonic acid availability (15,16,22), cells were prelabeled with [3H]-arachidonic acid. When agonists such as TPA, mezerein or bradykinin were added to prelabeled cells, no increase in release of radioactivity was observed (not shown). This is consistent with the lack of effect of these agonists on PGE2 synthesis. Exogenous arachidonic acid was the only compound tested that elicited increases in PGE2 synthesis. Thus, SV-HUC do not elicit an early response to agonists but contain prostaglandin H synthase activity. Agonist-mediated de novo synthesis of prostaglandin H synthase has been reported (39-42). This possibility was evaluated (Figure 2). Following a 24 h incubation with agonists, media was removed and fresh media added. The basal level of PGE2 synthesis in cells treated with A23187 and TPA was similar to control. However, when fresh media contained 100 /tM arachidonic acid, agonists-treated cells elicited large increases in PGE2. TPA was also shown to increase PGE2 synthesis as early as 6 h after addition. The lack of difference in the basal level of PGE2 synthesis between control and these agonists is probably due to very low levels of free intracellular arachidonic acid. Experiments were conducted to determine whether TPA increases in PGE? were due to de novo synthesis of
Radioimmunoassay of PGE2 The PGE2 content of the media was measured by double-antibody radioimmunoassay (36). Rabbit amiserum to PGE2 was obtained from Regis Chemical Co., Morton Grove, IL. Tritium-labeled PGE2 was purchased from Du Pont NEN, and goat antiserum to rabbit -(-globulins was purchased from Antibodies Inc., Davis, CA. Each condition was evaluated in two or three separate experiments. In each experiment, conditions were evaluated in at least triplicate plates. The medium from each plate was analyzed in duplicate and the value of duplicate determinations averaged and considered as an n of one. Data were expressed as mean ± SEM of ng PGE2/IO3 cells. Differences between data were evaluated statistically by the Student's Mest for unpaired observations. Prostaglandin H synthase assay Following a 24 h incubation of cells in the absence and presence of TPA, media were saved for PGE2 radioimmunoassay and microsomes were prepared for direct assessment of synthase activity (37). All subsequent procedures were performed at 4°C. Cells were scraped into tubes, centrifuged at 2000 r.p.m. for 10 min to pellet cells, and were resuspended in buffer and sonicated. Buffer contained 25 mM HEPES (pH 7.4), 1 mM EGTA, 1 mM EDTA, 1 mM dithiothreitol, 250 mM sucrose, 4 jig/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride. Samples were centrifuged at 100 000 g for 60 min. The microsomal
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Fig. 1. Effect of test agents on arachidonic acid metabolism evaluated 2 h after their addition. Cells were incubated in the absence (control) and presence of arachidonic acid (AA, 100 )M), A23187 (1 /tM) and TPA (100 nM). Media were removed for PGEj determination (n = 3).
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Cells and culture methods A clonal line of SV-HUC was used in this study. SV-HUC is an immortalized, non-tumorigenic human urothelial cell line that has been previously characterized (24,25,33). Studies were initiated with cells at passage 26. These cells remain non-tumorigenic after 80 serial passages and > 2 years in continuous culture. SV-HUC were grown on plastic dishes in medium consisting of Ham's Nutrient Mixture F12 (GIBCO, Grand Island, NY) and incubated at 37°C in 5% CC>2/95% humidified air. Medium was supplemented with 1 % fetal calf serum and the following (final concentration in parentheses): insulin (10 /ig/mi), hydrocortisone (1 jig/ml), transferrin (5 /ig/ml) and amphotencin B (2.5 /tg/ml), which were obtained from Sigma Chemical Co., St Louis, MO; all non-essential amino acids (0.1 mM), L-glutamine (2.0 mM), streptomycin (100 /tg/ml) and penicillin (100 units/ml), which were purchased from GIBCO; and dextrose (2.7 mg/ml), obtained from Fisher Scientific. This medium was developed for human uroepithelial cells (34,35). Routine dispersion of cultures for serial passage was performed using 0.01% EDTA (Sigma) dissolved in Hank's balanced salt solution (HBSS, GIBCO) and adjusted to pH 7.4 with NaHCO3. Experiments were conducted with 80-90% confluent cultures (0.7-1.0 X 10* cells/60 mm plate). Growth media were aspirated and cells washed twice with 1 ml of HBSS. Serum-free a-MEM was then added with or without test agents as indicated in the Results. Cells were incubated at 37°C in 5% CO2/95% air. Test agents used were arachidonic acid purchased from Nu-Chek Prep, Inc., Elysian, MN; calcium ionophore A23187 and staurosporine were from CalBiochem, La Jolla.CA; platelet-derived growth factor (human) was from Collaborative Research, Inc., Bedford, MA; and cycloheximide, aspirin, bradykinin triacetate, epidermal growth factor, epinephrine bitartrate, thrombin (2000 NIH units/mg protein), EDTA (disodium salt), l-oleoyl-2-acetylglycerol, antidiuretic hormone (grade VI), mezerein, and TPA and the other phorbol esters were from Sigma. Each agonist was used at a maximally effective concentration previously determined (15,22).
pellet was rcsuspended in 100 mM phosphate buffer (pH 7.8) and immediately assayed. Reaction mixtures contained 1 mM epinephnne and 1 mM glutathione in 100 mM phosphate buffer (pH 7.8). Samples were preincubated for 1 min at 37°C before arachidonic acid (200 yM) was added to start the reaction. The total reaction volume was 0.25 ml. The reaction was stopped after 10 min by adding 0.5 ml of ice-cold methanol. Samples were centrifuged to remove protein, and the supernatant evaporated to dryness. Water (0.25 ml) was added to each sample, which was then sonicated to resuspended PGE2 prior to radioimmunoassay. Protein was determined using bovine serum albumin as a standard (38). Prostaglandin H synthase activity is expressed as mean ± SEM of ng protein/10 min.
Regulation of PGE^ synthesis
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Aspirin was used to evaluate further the mechanism by which TPA regulates de novo synthesis of prostaglandin H synthase (Figure 5). Aspirin is an irreversible inhibitor of prostaglandin H synthase (44,45). The dose of aspirin used and the time of exposure (1 h) were sufficient to inhibit completely the 2 h exogenous arachidonic acid-mediated increases in PGE2 (16), like those observed in Figure 1. However, cells pretreated with aspirin and then exposed to TPA for 24 h elicited significant increases in PGE2 in response to arachidonic acid (Figure 5). Thus, the results suggest that TPA can regulate arachidonic acid metabolism by increasing de novo synthesis of prostaglandin H synthase. Discussion These studies document unique changes in the regulation of PGE2 synthesis by SV-HUC compared to previous studies with primary and subcultured human or dog urothelial cells (13-16,22,23). In the latter cells, agonist regulation of PGE2 synthesis has been extensively evaluated. Some agonists, like bradykinin, elicit increases in PGE2 synthesis within 5 min which peak by 15 min. Others, such as TPA, elicit more gradual increases, which may not be evident for 15 min and may not peak for 2 h (13,15). While separate mechanisms appear to exist for bradykinin and TPA, there are also some similarities in this early response. Both responses are calcium dependent and require
1 BASAL ARACHIDONIC
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If. TPA
Fig. 2. Effect of test agents on arachidonic acid metabolism evaluated 24 h after their addition. Cells were incubated in the absence (control) and presence of A23187 (1 pM) or TPA (100 nM). After 24 h, fresh media in the absence (basal) and presence of 100 pM arachidonic acid were added and removed 2 h later for PGE2 determination (n = 3).
1 ••" HO TPA
Fig. 4. Effect of cycloheximide and staurosporine on TPA-mediated de novo synthesis of prostaglandin H synthase. Where indicated, cells were preincubated with cycloheximide (35 /iM) or staurosporine (1 /iM) for 60 min before sidditions of TPA (100 nM). After 6 h, fresh media containing 100 nM arachidonic acid were added and removed 2 h later for PGE2 determination (n = 3).
I MEDIA P G K 2 | P H = ACTIVITY
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; m• ; Fig. 3. De novo synthesis of prostaglandin H synthase by SV-HUC. Cells were incubated in the absence (control) and presence of TPA (100 nM). After 24 h, overnight media were saved for PGE2 determination, and microsomes were prepared from cells. Microsomes were used to measure prostaglandin H synthase (PHS) activity (n = 4 - 8 ) .
i i'
I
I
Fig. 5. Effect of aspirin on TPA-mediated de novo synthesis of prostaglandin H synthase. Where indicated, cells were preincubated with 3 mM aspirin for 60 min, washed twice and TPA (100 nM) added. After 24 h, fresh media containing 100 /jM arachidonic acid were added and removed 2 h later for PGEj determination (n = 3).
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prostaglandin H synthase. Following a 24 h exposure to TPA, microsomes were prepared from both control and TPA-treated cells. Following addition of arachidonic acid, microsomes from TPA-treated cells produced - 15-fold more PGE2 than did those of control cells (Figure 3). PGE2 synthesis was completely prevented by prior incubation of microsomes with 0.1 mM indomethacin. In addition, the PGE2 content of overnight media from TPA-treated cells was significantly greater than control (Figure 3). Thus, increased de novo synthesis of prostaglandin H synthase results in increased urothelial cell media content of PGEj. The mechanism by which TPA regulates de novo synthesis of prostaglandin H synthase was evaluated (Figure 4). Cycloheximide, a protein synthesis inhibitor, and staurosporine, a protein kinase C inhibitor (43), were utilized. The concentration of each inhibitor was previously shown to be a maximum effective dose. Both inhibitors were effective in preventing 6 h TPA-mediated increases in PGE2. In separate experiments, prostaglandin H synthase activity in cells exposed to TPA for 24 h exhibited more activity than did corresponding cells pretreated with cycloheximide (187 ± 11 versus 51 ± 5 ng PGF^/mg protein/10 min). Potential toxic effects of cycloheximide and staurosporine were also assessed. At the concentrations used in Figure 4, neither cycloheximide nor staurosporine altered the number of viable cells or the arachidonic acid-mediated increase in PGE2 (Figure 1). Thus, both protein synthesis and protein kinase C appear to be involved in the mechanism by which TPA regulates de novo synthesis of prostaglandin H synthase.
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thought to be involved in different steps of the carcinogenic process, this has been difficult to assess. SV-HUC allows a direct assessment of parameters that regulate arachidonic acid metabolism. SV-HUC has lost the early, but retains the late PGE2 synthetic response. Both responses are dependent upon protein synthesis and protein kinase C. Loss of the early response does not appear to be due to acclimation of urothelial cells to culture conditions since subcultured urothelial cells retain the early response (23). The prostaglandin H synthase activity of SV-HUC is significantly less than that observed in primary cultures of urothelial cells. Thus, the ability of SV-HUC to metabolize arachidonic acid is significandy impaired. The relationship between altered regulation of arachidonic acid metabolism and the immortalization process is not clear. Many different human epithelial cell lines have been established by SV40 immortalization (53,54) and our results may relate to those cells. Acknowledgements The authors thank Dr Yun-Hua Huang Wong for helpful discussions and appreciate the expert technical assistance of Cindee Rettke, Alma Schisla and Nathan T Zenser. This work was supported by the Department of Veterans Affairs (B.B D., T.V.Z.) and by the USPHS Grant CA-29525 (C.A.R.) and CA-28015 (T.V.Z).
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release of endogenous arachidonic acid for PGE2 synthesis. The TPA response requires protein synthesis and protein kinase C (13,15,16). In contrast, SV-HUC did not demonstrate an early agonist-mediated response. All of the agonists tested increase PGE2 synthesis in either human and dog urothelial cells or other cell types. The lack of SV-HUC responsiveness appears to be due to its failure to release endogenous arachidonic acid following agonist addition. The reason for decreased release of arachidonic acid (phospholipase activity) is not clear and was not further investigated. Possible explanations include the lack of or the inability to activate a phospholipase(s). Aspirin played an important role in demonstrating de novo synthesis of prostaglandin H synthase. Aspirin is an irreversible inhibitor of prostaglandin H synthase (44,45). The concentration of aspirin used and the time of exposure were sufficient to inhibit maximally arachidonic acid-mediated increases in PGE2 seen within 2 h (Figure 1). Thus, aspirin completely destroyed all prostaglandin H synthase activity prior to addition of TPA. New enzyme synthesis was required to restore activity. Taken together, the effects of cycloheximide and aspirin are consistent with TPAmediated de novo synthesis of prostaglandin H synthase. Because A23187, like TPA, increases PGF^ after 24 h (Figure 2), A23187 might also elicit an increase in the de novo synthesis of prostaglandin H synthase. Protein kinase C appears to be involved in both the early and late urothelial cell responses elicited by TPA because of the inhibition observed with staurosporine (16,23). This is not surprising since most effects of TPA are mediated by protein kinase C (46—48). However, it should be noted that while staurosporine is one of the most widely used inhibitors of protein kinase C, staurosporine is also an inhibitor of calcium/calmodulindependent protein kinase and protein tyrosine kinases (49,50). Because SV-HUC retain the late TPA response, altered protein kinase C activity may not be responsible for the lack of an early response. Inhibition by cycloheximide indicates that additional proteins are necessary for arachidonic acid release. Agonistmediated arachidonic acid release by endothelial cells requires synthesis of a phospholipase A2 stimulatory peptide like melitin (51). A similar mechanism may regulate the early response in primary and subcultured urothelial cells, and may be altered in SV-HUC. TPA regulates—by a protein kinase C-dependent mechanism—forskolin-responsive cyclic AMP synthesis in urothelial cells. This TPA response is also not detected in SVHUC (52). The relationship between SV-HUC alterations in the regulation of arachidonic acid metabolism and cyclic AMP synthesis is not known. The capacity of SV-HUC to produce PGE2 is severely limited in comparison to primary urothelial cells in culture. Primary, as well as subcultured urothelial cells exhibit an early response to agonists that is either absent or not detectable in SV-HUC (23). In addition, the prostaglandin H synthase activity of control and TPA-treated primary cultured cells is 140 ± 10 and 395 ± 35 ng PGFVmg protein/10 min respectively (23). Values of 8.7 ± 0.7 and 140 ± 6 ng PGF^/mg protein/10 min are reported for control and TPA-treated SV-HUC respectively (Figure 3). The prostaglandin H synthase activity of TPA-stimulated SV-HUC is similar to the basal activity in primary cultures. Basal prostaglandin H synthase activity of SV-HUC is ~ 15-fold less than that in primary cultured cells. Prior studies with primary and subcultured cells used mixed cell populations and not clones. In addition, the particular clone from which SV-HUC was derived is unknown. In summary, although metabolism of arachidonic acid is
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