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Epidermal prostaglandins after topical application of a tumor promotor
Cancer Letters, 7 (1979) 121--125 © Elsevier/North-Holland Scientlfm Publishers Ltd
121
E P I D E R M A L P R O S T A G L A N D I N S A F T E R TOPICAL APPLICATION OF A TUMOR PROMOTOR
EDWARD BRESNICK, PAUL MEUNIER and MERTON LAMDEN Vermont Regmnal Cancer Center and Department o f Bmchemzstry, ?'he Unwerstty of Vermont College o f Medicine, Burhngton, Vermont 05401 ( U S A )
(Received 24 January 1979) (Accepted 22 March 1979)
SUMMAR'L T h e topical apphcation of the p r o m o t o r , 12-O-tetradec~noyl-13-acetate, o n t h e shaved backs of m, ce was a t t e n d e d by a striking increase m t h e PGE c o n c e n t r a t i o n within epidermis as early as 1 h later. This increase peaked at 24 h b u t t h e prostaglandin value remained elevated for at least 48 h. PGF, o n t h e o t h e r hand, showed only a m o d e s t increase in epidermis with a marem u m at 12 h. By 48 h, t h e PGF c o n c e n t r a t i o n returned to normal. INTRODUCTION In t h e 2-stage mechanism of carcinogenesm, an initiator and p r o m o t o r are believed to act cooperative]ly in t h e f o r m a t i o n o f a neoplasm. A l t h o u g h an u n d e r s t a n d i n g o f t h e action o f initiators is gradually being reached, ~ e have little knowledge of t h e underlying events in t u m o r promo~,ion. Recently, evidence has been presented which suggests t h a t p r o m o t o r s m a y e.~ert their effects b y interaction with substances which comprise part o f t h e structure o f t h e plasma m e m b r a n e o f responsive cells [16,17]. Since prom o t o r s such as 12-O-tetr,~decanoyl-13-acetate (TPA) have striking i m t a n t properties, some investigators have suggested t h a t t h e prostaglandin~ m a y be revolved in t h e action o f t h e p r o m o t o r s [2,8,9,13]. This hypothesis is based u p o n t h e well known i n f l a m m a t o r y action o f a n u m b e r of 'the prostaglandins [ 5]. Additional information which bears u p o n this point was r ~.ported from BoutweU's laboratory [17]. I n d o m e t h a c i n , an inhibitor of pr,3tagLandm syntl~etase, blocked t h e TPA-mediated i n d u c t i o n of o m i t h i n e decarboxylase in epidermal cells; this blockage was alleviated b y p r o s t a g l ~ d m s o f the E series. We therefore set a b o u t to establish w h e t h e r T P A did in f~ct stimulate t h e synthesis o f t h e prostaglandins in epidermis. We wish to report m this m a n u s c r i p t t h a t topical application o f t h e p r o m o t o r to m o u s e sl~n results m a p r o f o u n d elevation of t h e PGE level in epiderrms and a s mailer increase in t h e PGF series. T h e former is sustained for a m u c h longer ~lme thau~ PGF.
122
MATERIALS AND M E T H O D S Mater~ls [3H]PGE: (180 Ci/mmol) and [3H]PGF2~ (14.4 C1/mmol) were obtained from Amersham/Searle Co. 12-O-Tetradecanoyl-phorbo]-13-a~cetate was obtmned from Consolidated Midland Corp., Brewster, NY. The prostaglandin derivatives were kindly donated by Dr. John Pike of the Upjohn Company, Kalamazoo, Michigan. The antlprostaglandin E-bovine serum albumin antiserum was purchased from Miles-Yeda laboratories of Rehovot, Israel and the corresponding antiserum a ~ n s t the prostaglandin F series was kindly provided by Dr. Hal Behrman of Yale University School of Medicine, New Haven, Connecl~icut. Dextran, charcoal, bovine serum albumin, and silicm acid were purchased from Sigma Chemical Co., St. Louis, Missouri. All other chemicals were from commercial sources. Male CD-1 mice 8--10 weeks of age, were obtained from Charles Rwer Laboratories; the mice were fed Purina Lab Chow and water ad lib,turn. They were maintained on a constant light-clark cycle for the duration of the exper,Lment. Tissue preparation The backs of mice were shaved 24 h prior to the topical admimstration of the TPA (10 ug ha 100 ul of acetone) and only those mice which were not in the hair regrowth cycle were employed in the assay. ~¢~uitablecontrol mice were shaved, received acetone topically and were sacrificed at She same time as were the experimental mice. Periodically after topical treatraent, the mice were killed by cervical dislocation and the shaved area of skin, ".e., 2 )< 2 cm, was removed. The skin was immersed for 10 sec in a 5b°C water bath, then quickfly brought to 4°C and the epidermal layer scraped off with a scalpel blade. In preliminary studies, we showed that no ~teration in p'~ostaglandin values of the epidermic was noted by this manipulations. The ep ~dermi~ in water was honmgenized in a polytron apparatus for 30 sec at 4°C and this homo genate was passed through several layers of gauze to remove clumps. This homogenate served as the sourc~ for DNA, protein and prostaglandm ass'~ys. Chemical assays DNA and protein were assayed by the methods of Burton [3] and Lowry et al. [1], respectively. The prostaglandins in the epidermal homogenates were extracted into ethyl acetate; the ethyl acetate fraction was dried undelr a stream of N~, and the residue was dissolved in benzene/ethyl acetate/ methanol (70 : 40 : 10, v/v/v). An aliquot of the latter was applied to a column o f silicic acid and the prostaglandins were selectively eluted exactly as described by Jaffe and Behrman [7]. h this chromatographic scheme, the PGE and PGF series are elu~d with 60 : 40 : 2 and 60 : 40 : 20 of benzene/ ethyl acetate/methanol, respecthvely. Under these conditions, the PGE and PGF derivatives are clearly separated from PGA nd PGB. This elh-nirmtes any contribution of the latter subst~-mces to the radioimmunoassay. The
123 PGE and FGF derivatives were dried under a stream of N2, d~ssolved in ethanol and stored at --20°C. An a h q u o t of the latter was introduced into test tubes, the ethanol evaporated and bt>ffer G (0.01 M Na-phosphate buffer pH 7.4--0.15 M NaC1-0.01% NaN3-0A% bovine serum albumin) was added. The radioLmmunoassay for PGE a~d PGF was conducted as described by Jaffe and Behrman [7].~ The efficmncy of the column chromatog:'aphy and the recovery were monitored by employing ~3H~PGE2 and [~H]P, GF2~. The concentration of the PGE and PGF series was determined f r o m a standard curve and was expressed as ng/ug DNA. We had found concentrations to be more smtably expressed per pg DNA thar~ per u m t protein. The antiserum to PGE2 exhibited 60% cross reaction to PGEt {from l~.iles/Yeda) while 45% c ~ s s reaction of the antiserum to PGF2a was reported with P G F l a (H. Behrman, pers. comm.). RESULTS Mouse epidermis cont,&us PGE2 and PGF2a in a ratio of PGE2/,)GF2a = 3.8 under basal conditions. The topical application of the potent promotor, TPA, to the backs of mice is accompanied by a p r o f o u n d increase J~nPGE2 and to a ~esser extent, of PGF2a. These data are presented in Table 1. PGE2 is elevated by almost 10-fold at 24 h after the single application. The elevation is observed as early as 1 h, at which time the PGE2 concentration was 83% increased over comparable controls. By 48 h, the PGE2 concentration was still quite high, i.e., 600% over controls. PGF~a, on the other hand, was significantly elevated over control values by 12 h after topical application of TPA. By 48 h, the PGF2~ concentration had returned to control levels. DISCUSSION
The results presented in this manuscript clearly indicate a profound effect o f TPA upo~ the intracel]ular concentration of the prostaglandins. In tins regard, PGE was significantly elevated as early as 1 h after topical applica-
TABLE 1 EPIDERMAL PROSTAGLANDINS
Control TPA, 1 h TPA, 12 h TPA, 24 h TPA, 48 h
AFTER TPA APPLICATION
P G E 2 (ng/¢g D N A )
PGF:~ (ng/~g D N A )
FGEJPGF,+ c~
0.23 ± 0.0+i (9)a 0 42 -+0.09 (5) 1.50 ± 0.20 (4) 2.20 ± 0.50 (4) 1.50 ~ 0.44) (3)
0.05 0.09 0.19 0.16 0 07
3.8 4.7 7.9 13 8 21 4
± 0.01 ± 0.02 ± 0.06 ± 0.03 ± 0.0)
(9) (5) (4) (8) (3)
TPA, 10 ~g in acetone, was apphed to the shaved backs of CD-1 mice ~hd the mice ,~-ere killed at the times indicated in the table. Control mice whmh were treated with acel~,one alone were killed simultaneom~ly;the control values have been grouped aAverage ~ standard error (no. of mice).
~-24
tion o f t h e p r o m o t e r , reached its m a ~ i m u n i c o n c e n t r a t i o n by 24 h, and was still substantially increased at 24 h later. PGF, on t h e o t h e r hand, was elevated b y 12 h b u t r e t u r n e d to control levels by 48 h. This increase in t h e PGE series reported herein represents o n e o f t h e earliest effects o f TPA noted in t h e m o u s e system. It is interesting to n o t e that Aso et al. [1] have reported t h e intracelluT~Lr concentratlo ~s o f PGE2 and PGF2a in guinea pig and h u m a n epidermis, with a ratio of PGE2/PGF2a o f approximately 5. This value closely approxhrJates t h a t which we have o b s e ~ e d m m o u s e epidermis, Le., approx. 4. TPA h a s been reported to affect prostaglandin levels in several systems. Thus, Levin, e and Hassid [9] have sl'own t h a t this p r o m o t e r stimulates PGE2 release f r o m a canine kidney cell line. Levine [10] has also repovLed the s t i m u l a t i o n o f PGE2 release from tl~ese cells by addition o f certain polycyclic h y d r o c a r b o n s to t h e medium. T h e effect o f TPA u p o n the.;e kidney cells is preceded by an increased deacy]lation o f hpids ~9,13]. In a recent report f r o m Hecker's laboratory [ 2], T P A b u t n o t polycyclic h y d r o c a r b o n s evused a release o f PGE f r o m macrophages m vitro. Furthermore, Tashjian et al. [15] have studied t h e production o f t h e p m s t a g i a n d i n s m bone organ culture s y s t e m s as well as bone resorption. TPA stimulated both prostaglandin E2 release and bone resorptmn. T h e s e effects of T P A were completely inhibited by indomethacin. Recently, p r o m o t e r s such as TPA have been reported to affect a n u m b e r o f differentiating f:ellular systems. For example, t h e differel,tiation o f t h e Friend ery'throleukemia cells is markedly iuhibited by TPA [14,19], t h e conversion o f pre~',dlpose cells to m a t u r e adipocytes is reduced by TPA [4[ while it is accelerated by m d o m e t h a c i n [18] T h e above data all sugges~ t h a t p r o m o t e r s m a y exert part or all o f their action t h r o u g h t h e intervention o f PGE2. Our results are in accord with this h y p o t h e s i s b u t convincing proof m u s t await further experimentatLon. As a final n o t e t h e results o f Lupulescu 1112] should be presented since t h e y bear u p o n tins hypothesis. SquamovLs cell carcinoma in 3-methylcholanthrene-painted mine was markedly elevated by injected prostaglandlns E 2 or F2~. ACKNOWLEDGEMENTS
This research was supported by a grant f r o m the NIH (CA 20711) and by a s u m m e r fellowship to Paul Meunier s u p p o r t e d by NIH graa~t (R25 19381). The a u t h o r s w~sh to express their sincere t h a n k s to Dr. Hal Behrman for helping u s set up t h e assays, providing u s with reagents and for helping with some o f t h e p~oble ms. REFERENCES Aso, K, D,,neau, D.G., Kruhg, L., Wdkerson, O.I. and Farber, E.M. (19~5) Epidermal synthesis of prostaglandms and their effect on levels of cychc adenosine 3',5-monophosphate. J Invest. Dermatol, 64, 3'26 331
1'.15 2 Brune, K., Kalm, H., Scbmidt, R. and Hecker, E. (1978) Inflammatory, tumor initiating and promotmg activities of polycyclle aromahe hydrocarboa~ and dlterpene esters in mouse skin as compared with t]~elr prostaglandm releasing potency m vlgro Cancer Letters, 4, 333- 342. 3 Burton, K. (1956) A study of the conditions and mechamsm of ~he dlphenylamme reaction for the colorimetrie estimation of deoxyribonucleic acid Blochem J , 62, 315--322. 4 Diamond, L , O'Brien, T G. and Rovera, G. (1977) Inhibition of adipose conversion of 3T3 flbroblasts by tumor promotors. Nature, 269,247--249 5 Ferrlera, S.H. and Vane, J.R (1974) New aspects of the mode of action of nonsteroid anti-inflammatory drugs Ann Rev Pharmacol , 14, 57--73 6 Hecker, E. (1978) Structure-activity relationships ~n dlterpene esters irritant and carcinogenic to mouse skin In. Carcinogenesis -- A Comprehensive Survey. Vol 2, Mechanisms of Tumor Promotion and Cocarcmogenesis, pp 11--48. Ed,'~ors T G Slaga, A. Sl~rak and R K Boutwell Raven Press, N Y. 7 Jaffe, B.M and Behrman, H.R (1974) Prostaglandins E, A, and F, In. Methods of Hormone Radiolmmunoassay, pp 19--34, Academic Press, N Y 8 Karm, S.M M. and Rao, B (1976) Prostaglandms and Turnouts In Prostaglandms Physiologlc-'d and Pathological Aspects, pp 303--325 Editor S M.M Karin. MTP Press, Lancaster. 9 Levine, L. and Hassid, A (1977) Effects of phorbol-12,13-dlesters on prostaglandms production and phosphohpase activity ~n canine kidney (MDCK) coils Blochem, Blophys. Res. Commun. '78, 477--484. 10 Lewne, L (1977) Chemical carcinogen stimulate canine kidney (MDCK) cells to produce prestaglandins Nature, 268, 447--448. 11 Lowry, D.H., Rosebrough, N.J., Farr, A L. and Rand~l, R.J. (1951) Protein measurement with tire folln phenol reagent. J Biol. Chem., 193, 265--275. 12 Lupulescu, A (1978) En~.ancement of carcinogenesis by prostaglandms in male albine Swiss mice. J. Natl. Cancer Inst., 61, 97--101 13 Ohueh~, K. and Levine, L (1~78) Shmulation of prostaglandm syntbesxs by tumorpromoting phorbol 12,13-dlesters in canine kidney (MDCK) cells. Cycloheximlde inhibit5 the stimulated prostagiandin synthesis, deacylation of hpids and morphological changes J. Biol Chem., 253, 4783--4790. 14 Rovera, G., O'Brien, T G and Diamond, L. (1977) Tumor promotors inhibit spontaneous differentiation of Friend erythroleukemia cells in culture Pros. Natl Acad SeL, 74, 2894--2898. 15 Tashjlan, A.H , Ivey, J.L., Declos, B and Levme, L. (1978) Shmulatlon of pro~taglandm production in hone by phorbol dlesters and mebttm Prostaglandms, 16, 221--232 16 Van Duuren, B L , Banerjee, S and Witz, G (1976) Fluorescence studies on the interaction of the tumor promoter phosbol myristate acetate and related compounds with rat liver plasma membranes Chem.-Biol Interact, 15, 233--246 17 Verma, A K , Rice, H.M. and Boutwell, R.K. (1977) Prostaglandlns and skin tumor promotion of tumor promoter-reduced ormthme decarboxylase activity m epidermis by inhlb~tors of prostaglandm synthesis. Biochem. Biophys Res. C o m m u n , 79, 1160--1166. 18 Williams, I H and Polakis, S E. (1977) Differentmtion of 3T3-L~ fibroblasts to adipocytes the effect of mdomethacm, prostaglandm, E, and cychc AMP on the process of differentiation. Bioehem. Blophys. Res. Commun., 77, 175--~ 86 19 Yamasaki, H , Fibacb, E , Nudel, U , Wemstem, I B , Rlfkmd, R A ancl Marks, P A (1977) Tumor promotors inhibit spontaneous and induced differentiation ~f ranrme erythroleukemia cells in culture Proc Natl. Acad. Sol USA, 74, 3451--34.55.
121
E P I D E R M A L P R O S T A G L A N D I N S A F T E R TOPICAL APPLICATION OF A TUMOR PROMOTOR
EDWARD BRESNICK, PAUL MEUNIER and MERTON LAMDEN Vermont Regmnal Cancer Center and Department o f Bmchemzstry, ?'he Unwerstty of Vermont College o f Medicine, Burhngton, Vermont 05401 ( U S A )
(Received 24 January 1979) (Accepted 22 March 1979)
SUMMAR'L T h e topical apphcation of the p r o m o t o r , 12-O-tetradec~noyl-13-acetate, o n t h e shaved backs of m, ce was a t t e n d e d by a striking increase m t h e PGE c o n c e n t r a t i o n within epidermis as early as 1 h later. This increase peaked at 24 h b u t t h e prostaglandin value remained elevated for at least 48 h. PGF, o n t h e o t h e r hand, showed only a m o d e s t increase in epidermis with a marem u m at 12 h. By 48 h, t h e PGF c o n c e n t r a t i o n returned to normal. INTRODUCTION In t h e 2-stage mechanism of carcinogenesm, an initiator and p r o m o t o r are believed to act cooperative]ly in t h e f o r m a t i o n o f a neoplasm. A l t h o u g h an u n d e r s t a n d i n g o f t h e action o f initiators is gradually being reached, ~ e have little knowledge of t h e underlying events in t u m o r promo~,ion. Recently, evidence has been presented which suggests t h a t p r o m o t o r s m a y e.~ert their effects b y interaction with substances which comprise part o f t h e structure o f t h e plasma m e m b r a n e o f responsive cells [16,17]. Since prom o t o r s such as 12-O-tetr,~decanoyl-13-acetate (TPA) have striking i m t a n t properties, some investigators have suggested t h a t t h e prostaglandin~ m a y be revolved in t h e action o f t h e p r o m o t o r s [2,8,9,13]. This hypothesis is based u p o n t h e well known i n f l a m m a t o r y action o f a n u m b e r of 'the prostaglandins [ 5]. Additional information which bears u p o n this point was r ~.ported from BoutweU's laboratory [17]. I n d o m e t h a c i n , an inhibitor of pr,3tagLandm syntl~etase, blocked t h e TPA-mediated i n d u c t i o n of o m i t h i n e decarboxylase in epidermal cells; this blockage was alleviated b y p r o s t a g l ~ d m s o f the E series. We therefore set a b o u t to establish w h e t h e r T P A did in f~ct stimulate t h e synthesis o f t h e prostaglandins in epidermis. We wish to report m this m a n u s c r i p t t h a t topical application o f t h e p r o m o t o r to m o u s e sl~n results m a p r o f o u n d elevation of t h e PGE level in epiderrms and a s mailer increase in t h e PGF series. T h e former is sustained for a m u c h longer ~lme thau~ PGF.
122
MATERIALS AND M E T H O D S Mater~ls [3H]PGE: (180 Ci/mmol) and [3H]PGF2~ (14.4 C1/mmol) were obtained from Amersham/Searle Co. 12-O-Tetradecanoyl-phorbo]-13-a~cetate was obtmned from Consolidated Midland Corp., Brewster, NY. The prostaglandin derivatives were kindly donated by Dr. John Pike of the Upjohn Company, Kalamazoo, Michigan. The antlprostaglandin E-bovine serum albumin antiserum was purchased from Miles-Yeda laboratories of Rehovot, Israel and the corresponding antiserum a ~ n s t the prostaglandin F series was kindly provided by Dr. Hal Behrman of Yale University School of Medicine, New Haven, Connecl~icut. Dextran, charcoal, bovine serum albumin, and silicm acid were purchased from Sigma Chemical Co., St. Louis, Missouri. All other chemicals were from commercial sources. Male CD-1 mice 8--10 weeks of age, were obtained from Charles Rwer Laboratories; the mice were fed Purina Lab Chow and water ad lib,turn. They were maintained on a constant light-clark cycle for the duration of the exper,Lment. Tissue preparation The backs of mice were shaved 24 h prior to the topical admimstration of the TPA (10 ug ha 100 ul of acetone) and only those mice which were not in the hair regrowth cycle were employed in the assay. ~¢~uitablecontrol mice were shaved, received acetone topically and were sacrificed at She same time as were the experimental mice. Periodically after topical treatraent, the mice were killed by cervical dislocation and the shaved area of skin, ".e., 2 )< 2 cm, was removed. The skin was immersed for 10 sec in a 5b°C water bath, then quickfly brought to 4°C and the epidermal layer scraped off with a scalpel blade. In preliminary studies, we showed that no ~teration in p'~ostaglandin values of the epidermic was noted by this manipulations. The ep ~dermi~ in water was honmgenized in a polytron apparatus for 30 sec at 4°C and this homo genate was passed through several layers of gauze to remove clumps. This homogenate served as the sourc~ for DNA, protein and prostaglandm ass'~ys. Chemical assays DNA and protein were assayed by the methods of Burton [3] and Lowry et al. [1], respectively. The prostaglandins in the epidermal homogenates were extracted into ethyl acetate; the ethyl acetate fraction was dried undelr a stream of N~, and the residue was dissolved in benzene/ethyl acetate/ methanol (70 : 40 : 10, v/v/v). An aliquot of the latter was applied to a column o f silicic acid and the prostaglandins were selectively eluted exactly as described by Jaffe and Behrman [7]. h this chromatographic scheme, the PGE and PGF series are elu~d with 60 : 40 : 2 and 60 : 40 : 20 of benzene/ ethyl acetate/methanol, respecthvely. Under these conditions, the PGE and PGF derivatives are clearly separated from PGA nd PGB. This elh-nirmtes any contribution of the latter subst~-mces to the radioimmunoassay. The
123 PGE and FGF derivatives were dried under a stream of N2, d~ssolved in ethanol and stored at --20°C. An a h q u o t of the latter was introduced into test tubes, the ethanol evaporated and bt>ffer G (0.01 M Na-phosphate buffer pH 7.4--0.15 M NaC1-0.01% NaN3-0A% bovine serum albumin) was added. The radioLmmunoassay for PGE a~d PGF was conducted as described by Jaffe and Behrman [7].~ The efficmncy of the column chromatog:'aphy and the recovery were monitored by employing ~3H~PGE2 and [~H]P, GF2~. The concentration of the PGE and PGF series was determined f r o m a standard curve and was expressed as ng/ug DNA. We had found concentrations to be more smtably expressed per pg DNA thar~ per u m t protein. The antiserum to PGE2 exhibited 60% cross reaction to PGEt {from l~.iles/Yeda) while 45% c ~ s s reaction of the antiserum to PGF2a was reported with P G F l a (H. Behrman, pers. comm.). RESULTS Mouse epidermis cont,&us PGE2 and PGF2a in a ratio of PGE2/,)GF2a = 3.8 under basal conditions. The topical application of the potent promotor, TPA, to the backs of mice is accompanied by a p r o f o u n d increase J~nPGE2 and to a ~esser extent, of PGF2a. These data are presented in Table 1. PGE2 is elevated by almost 10-fold at 24 h after the single application. The elevation is observed as early as 1 h, at which time the PGE2 concentration was 83% increased over comparable controls. By 48 h, the PGE2 concentration was still quite high, i.e., 600% over controls. PGF~a, on the other hand, was significantly elevated over control values by 12 h after topical application of TPA. By 48 h, the PGF2~ concentration had returned to control levels. DISCUSSION
The results presented in this manuscript clearly indicate a profound effect o f TPA upo~ the intracel]ular concentration of the prostaglandins. In tins regard, PGE was significantly elevated as early as 1 h after topical applica-
TABLE 1 EPIDERMAL PROSTAGLANDINS
Control TPA, 1 h TPA, 12 h TPA, 24 h TPA, 48 h
AFTER TPA APPLICATION
P G E 2 (ng/¢g D N A )
PGF:~ (ng/~g D N A )
FGEJPGF,+ c~
0.23 ± 0.0+i (9)a 0 42 -+0.09 (5) 1.50 ± 0.20 (4) 2.20 ± 0.50 (4) 1.50 ~ 0.44) (3)
0.05 0.09 0.19 0.16 0 07
3.8 4.7 7.9 13 8 21 4
± 0.01 ± 0.02 ± 0.06 ± 0.03 ± 0.0)
(9) (5) (4) (8) (3)
TPA, 10 ~g in acetone, was apphed to the shaved backs of CD-1 mice ~hd the mice ,~-ere killed at the times indicated in the table. Control mice whmh were treated with acel~,one alone were killed simultaneom~ly;the control values have been grouped aAverage ~ standard error (no. of mice).
~-24
tion o f t h e p r o m o t e r , reached its m a ~ i m u n i c o n c e n t r a t i o n by 24 h, and was still substantially increased at 24 h later. PGF, on t h e o t h e r hand, was elevated b y 12 h b u t r e t u r n e d to control levels by 48 h. This increase in t h e PGE series reported herein represents o n e o f t h e earliest effects o f TPA noted in t h e m o u s e system. It is interesting to n o t e that Aso et al. [1] have reported t h e intracelluT~Lr concentratlo ~s o f PGE2 and PGF2a in guinea pig and h u m a n epidermis, with a ratio of PGE2/PGF2a o f approximately 5. This value closely approxhrJates t h a t which we have o b s e ~ e d m m o u s e epidermis, Le., approx. 4. TPA h a s been reported to affect prostaglandin levels in several systems. Thus, Levin, e and Hassid [9] have sl'own t h a t this p r o m o t e r stimulates PGE2 release f r o m a canine kidney cell line. Levine [10] has also repovLed the s t i m u l a t i o n o f PGE2 release from tl~ese cells by addition o f certain polycyclic h y d r o c a r b o n s to t h e medium. T h e effect o f TPA u p o n the.;e kidney cells is preceded by an increased deacy]lation o f hpids ~9,13]. In a recent report f r o m Hecker's laboratory [ 2], T P A b u t n o t polycyclic h y d r o c a r b o n s evused a release o f PGE f r o m macrophages m vitro. Furthermore, Tashjian et al. [15] have studied t h e production o f t h e p m s t a g i a n d i n s m bone organ culture s y s t e m s as well as bone resorption. TPA stimulated both prostaglandin E2 release and bone resorptmn. T h e s e effects of T P A were completely inhibited by indomethacin. Recently, p r o m o t e r s such as TPA have been reported to affect a n u m b e r o f differentiating f:ellular systems. For example, t h e differel,tiation o f t h e Friend ery'throleukemia cells is markedly iuhibited by TPA [14,19], t h e conversion o f pre~',dlpose cells to m a t u r e adipocytes is reduced by TPA [4[ while it is accelerated by m d o m e t h a c i n [18] T h e above data all sugges~ t h a t p r o m o t e r s m a y exert part or all o f their action t h r o u g h t h e intervention o f PGE2. Our results are in accord with this h y p o t h e s i s b u t convincing proof m u s t await further experimentatLon. As a final n o t e t h e results o f Lupulescu 1112] should be presented since t h e y bear u p o n tins hypothesis. SquamovLs cell carcinoma in 3-methylcholanthrene-painted mine was markedly elevated by injected prostaglandlns E 2 or F2~. ACKNOWLEDGEMENTS
This research was supported by a grant f r o m the NIH (CA 20711) and by a s u m m e r fellowship to Paul Meunier s u p p o r t e d by NIH graa~t (R25 19381). The a u t h o r s w~sh to express their sincere t h a n k s to Dr. Hal Behrman for helping u s set up t h e assays, providing u s with reagents and for helping with some o f t h e p~oble ms. REFERENCES Aso, K, D,,neau, D.G., Kruhg, L., Wdkerson, O.I. and Farber, E.M. (19~5) Epidermal synthesis of prostaglandms and their effect on levels of cychc adenosine 3',5-monophosphate. J Invest. Dermatol, 64, 3'26 331
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