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Ihibitory effect of prostaglandin D2 on dna synthesis in nuclei
Prostaglandins, Leukotrienesand Medicine23: 253-265,1986
IHIBITORY EFFECT OF PROSTAGLANDIN ON DNA SYNTHESIS IN NUCLEI
D2
Yasuko KOSHIHARA and Mieko KAWAMURA Department of Pharmacology Tokyo Metropolitan Institute of Gerontology Itabashi-ku, Tokyo-173, Japan (reprint request to YK) ABSTRACT Prostaglandin (PG) D2 treatment inhibited DNA synthesis in isolated nuclei of mastocytoma P-815, 2-E-6 cells. On treatment with PGD2 (IO pg/ml), the inhibition was distinct by 8 hrs, and complete after 18 hrs. This effect of PGD on DNA synthesis in nuclei was not direct
or mediated % y cyclic AMP, but was a cell-mediated reaction. The cytoplasmic fractions of PGD2-treated and untreated cells both had stimulatory effects and their potencies were the same except for that of the cytoplasmic fraction of 8 hr-treated cells, which was less than that of the cytoplasmic fraction of untreated cells.. On treatment with PGD2, inhibition of DNA synthesis in the nuclei began after 8 hrs, and this inhibition could not be reversed, even by adding the cytoplasmic fraction from untreated cells to the assay system. Anuclear saltextractpreparedby adding 0.3 M NaCl to nuclei of cells that had been treated with PGD2 for 18 hrs had a much smaller stimulatory effect on DNA synthesis of salt-treated nuclei than an extract of nuclei from untreated cells. It is suggested that inhibition of cell growth by PGD2 is not mediated by intracellular cyclic AMP, but that
induces PGD synt?Iesis.
a factor(s)
that
inhibits
nuclear
DNA
INTRODUCTION We found that PGD2 strongly inhibited growth of mouse mastocytoma cells (1,2). Later, others reported similar inhibitory effects of PGD2 on several kinds of malignant 253
tumor cell lines, such as mouse leukemia L1210 cells (3), B-16 melanoma cells (4) and human malignant tumor cell lines Moreover, on its local daily injection, PGD2 (5). significantly inhibited tumor formation by neuroblastoma cells implanted subcutaneously into mice The (6). Other PGs, mechanism of this effect of PGD2 is unknown. such as PGA and PGE, that inhibit cell growth have been suggested to act by increasing the intracellular cyclic AMP level (7,8), since cyclic AMP is reported to inhibit cell growth (9,lO). However, the mechanism of the inhibitory effect of cyclic AMP on growth is also unknown. PGD was also found to increase the intracellular cyclic AMP 1 eve1 in platelets (11) and neuroblastoma NIE-115 cells (12 ). But it did not increase the cyclic AMP level in mouse leukemia L1210 cells (13), and increased the level in mastocytoma we investigated P-815 cells, only 1.5-fold. In this work the mechanism of growth inhibition by PGD2, by studies on nuclei from its effect on DNA synthesis in isolated mastocytoma cells. We also examined the effect of cytoplasm on nuclear DNA and untreated cells of PGD2 -treated synthesis. Results indicated that PGD2 probably induces some inhibitory factor(s) other than cyclic AMP and that the inhibition is caused by a cell-mediated reaction. MATERIALS and METHODS Materials. PGD2 (97% pure, as judged by high performance liquid chromatography) was obtained from Funakoshi Pharmaceutical Co. (Tokyo, Japan). PGD in aqueous media, determined by incubating it in phosp K ate buffer (pH 7.0) at 37'C, had a half life of 8.6 hrs as judged by high performance liquid chromatography ; 3H-dTTP was obtained from New England Nuclear (Boston, Mass), unlabelled deoxyribonucleoside triphosphate from P-L Biochemical, Inc. (Milwaukee, Wis), fetal bovine serum (mycoplasm- and virus-free) from Armour Pharmaceutical Co. ( Kankakee, Ill) and cyclic AMP from Sigma Chemical Co. (St. Louis, MO). Cell --A Culture Mastocytoma P-815, 2-E-6 cells were --established from cells from the ascites fluid of female DBA/2 mice by -in vitro culture and were cloned (14,15,16). Cells were cultured in suspension in Eagle's basal medium supplemented with 10% (v/v) fetal bovine serum under CO2 in air (1:19) at 37OC in a gyratory shaker (Model 2; New Brunswick Scientific Co., Edison, NJ). Treatment with PGD= Cells in the early logarithmic phase of growtGe=treated with PGD2 (10 pg/ml) in ethanol for 18 hrs. The final concentration of ethanol was less than 0.1% and the same amount of vehicle was added to control cultures. Cell numbers were counted a Coulter Counter (Model G, Coulter Electronics Inc., ,Hialeah, Fla).
254
PGDy Assay for DNA Synthesis & Isolated Nuclei. treated and untreated cells were ha$yestec$ and washed once , Mg +-free, pH 7.4). with phosphate-buffered saline (Ca Nuclei were isolated by the method of Friedman & Mueller (17). In brief, the ce 1 pellet was suspended at a final concentration of 7 x 10 i cells/ml in buffer A consisting of 10 mM tris-HCl (pH 7.8), 4 mM MgC12, 1 mM HDTA, 6 mM B and washed by mercaptoethanol and 0.25 M sucrose The resulting cell pellet was resuspended centrifu ation. at 7 x 1097 cells/ml in buffer Aand incubated at O'C. After by 10 strokes of a 10 min, the cells were homogenized The homogenate was centrifuged at Dounce glass homogenizer. 800 g for 5 min to separate the nuclei and cytoplasmic The nuclear fraction was washed 3 times b fractions. resuspensioninbuffer Aata final concentration of 3 x 10 Y nuclei/ml and centrifugation min. The final at 350 g for pellet was resuspended in buffer Aat 5 x10 5 nuclei/ml. The nuclear fraction did not contain any intact cells and was purified over 95 %. The cytoplasmic fraction was centrifuged at 105,000 g for 60 min to remove organelles. DNA synthesis in isolated nuclei was assayed as follows: Incubation mixture (0.6 ml) consisting of 0.1 ml of nuclear suspension (5 x IO6 nuclei), 0.3 ml of cytoplasmic fraction or buffer A and 0.2 ml of reaction mixture composed of 0.1 M Tris-HCl(pH 7.8), 0.02 M MgC12, 0.3 M NaCl, 0.015 M ATP, 0.3 mM dATP, dCTP, dGTP each and 0.03 mM f3H]dTTP (spec. act. 83.3 Ci/mole) was incubated at 37'C for 30 min. The reaction was terminated by chilling the mixture in an ice-water bath and adding 2 ml of chilled 10% TCA containing 2% Na-pyrophosphate. Acid-insoluble material was washed 3 times with 5% TCA containing 1% Na-pyrophosphate and counted in a liquid scintillation spectrophotometer.
Preparation of salt-treated nuclei and salt extract. Salt-treated nuclx and salt extract wereprepared by the method of Seki and Mueller (18). In brief, the washed nuclear pellet was resuspended in 5 vol. of buffer A containing 0.3 M NaCl. The suspension was incubated at 0°C for 1 hr and then centrifuged at 800 g for 10 min. The final precipitate was resuspended in buffer A at a concentration of 3.3 x lo7 nuclei/ml and used for further experiments as salt-treated nuclei. The supernatant was recentrifuged at 130,000 g for 20 min to yield salt extract. The extract was dialized against 35 vol. buffer A at O'C for 3 hrs. The mixture (0.6 ml) for assay of DNA synthesis consisted of 0.3 ml of salt-treated nuclear suspension, 0.1 ml of salt extract or buffer A and 0.2 ml of substrate mixture as described above. DNA synthesis activity in salttreated nuclei was assayed at 37'C for 30 min. Acidinsoluble material was then counted in a liquid scintillation spectrophotometer. Measurement of Intracellular cyclic AMP
intracellularcvclicAMP. was extracted and succinylated 255
by
the method of Honma et al. (19). In brief, cells with or for 5 min, 18 hrs and 40 hrs were without PGD2-treatment harvested by centrifugation and suspended in 6% TCA. The TCA-soluble material was obtained, TCA was removed by etherThe extraction and the aqueous solution was lyophilized. residue was succinylated and cyclic AMP was determined by radioimmunoassay (20). RESULTS DNA Synthesizing Activity of Isolated Nuclei. Cultured mastocytoma P-815, 2-E-6 ce?-1s were treated with PGD2 (10 us/ml) for 18 hrs. This treatment, completely The DNA synthesizing activity of inhibited cell growth. isolated nuclei was assayed in the presence of ATP as described in Materials and Methods. The nuclei from untreated cells had very high activity, but nuclei from PGD2-treated cells did not show any activity, as on arrest of cell growth. The activity in nuclei from treated cells was enhanced by addition of cytoplasm, but the enhanced level was less than, or equal to that of nuclei from untreated cells without cytoplasm. In contrast, the activity in nuclei from untreated cells was enhanced 3.2 fold by addition of the cytoplasm (Fig. 1). However, there was no difference in the stimulatory effects of cytoplasmic fractions from treated and untreated cells. This result implies that the inhibitory effect of PGD2 had already reached the nuclei when the cells were treated with PGD2 for 18 hrs. Inhibition of DNA Time-course of Cell-mediated Synthesizing A&iv= in Isolated Nuclei. The incubatz time necessary for complete loss of DNA synthesizing activity in isolated nuclei was determined. The nuclei and cytoplasm of cells with and without treatment with PGD2 for 1,2,4,8 and 18 hrs were prepared as (10 uglml) described in Materials and Methods. The DNA synthesizing activity in nuclei from untreated cells increased with the incubation time, and reached a maximum after 8 hrs (Fig. 2a). On the contrary, the activity in nuclei from treated cells decreased after 4 hr-treatment and had nearly disappeared after 18 hr-treatment (Fig. 2b). Addition of cytoplasm increased the nuclear synthesizing activities of treated and untreated nuclei. The cytoplasm from untreated cells had higher stimulatory activity than that of cytoplasm from cells treated with PGD2 for 8 hrs. But cytoplasm from cells after 18 hr treatment had similar activity to that of cytoplasm from untreated cells, and cytoplasm from cells after 4 hr treatment had similar activity to cytoplasm from untreated cells. Effects --of PGD2 and Cyclic AMP on DNA Synthesizinq -__Activity in Isolated-Nuclei. Next.we examined the effects of direcTaddition of PGD2 and cyclic AMP to the mixture for assay of DNA synthesis. PG is thought not to 256
penetrate the oell membrane except in a few cases (21), but can penetrate the cell it is uncertain whether PGD membrane, and then inhibit DN 3 synthesis. We found that addition of PGD2 at concentrations of 0.1-1.0 ug/ml to the DNA synthesizing system with isolated nuclei had no effect on their DNA synthesis (Fig. 3). It also had no direct effect on DNA synthesis in this system when supplemented with cytoplasm. In other words, PGD2 did not modify the stimulatory activity of the cytoplasmic fraction. PGD2 is said to increase the intracellular cyclic AMP level (12). We measured the intracellular cyclic AMP level
E
e -
2.
1 -
N&i
Control
CF
t-1 C T
Treated (-1 C T
Fig. 1. DNA synthesizing activity in nuclei. The nuclei and cytoplasmic fraction were prepared from mastocytoma P815, 2-E-6 cells after their incubation with or without10 ug/ml of PGD2 for 18 hrs. Nuclear DNA synthesis was carried out in the presence or ab ence of the cytoplasmic fraction at 37°C for 30 min and 3H-TMP incorporated into the acid-insoluble materials was counted. Control: nuclei prepared from untreated cells. Treated: nuclei prepared from PGD2 -treated cells. CF: cytoplasmic fraction. (-1: without a cytoplasmic fraction. C: with the cytoplasmic fraction prepared from untreated cells. T: with the cytoplasmic fraction from PGD2-treated cells. 257
I-
2,,_*f::~:III:f:,,,\_ -_.
.--. -*-.“-* -_ ---r&w_ =.* ___+__:-='~ -.‘~__*.----*------______~ 0 2 4 6 8, ----'-‘_‘__d I I I
l-
incubation
Time (hr)
Time courseof cell-mediated inhibition of DNA Fig. 2. synthesizing activity in isolated nuclei. The nuclei and with or cytoplasmic fraction were prepared from cells without treatment with 10 pg/ml of PGDZ for the indicated periods. Nuclear DNA synthesis was carried out in the presence yr absence of cytoplasmic fraction at 37°C for 30 min and H-TMP incorporated into acid-insoluble materials was counted. a): nuclei prepared from PGD2-untreated cells. b): nuclei prepared from PGD2-treated cells. (*I, from nuclei only; (A), with the cytoplasmic fraction untreated cells ; ( A), with the cytoplasmic fraction from Values are means for duplicate PGD2 -treated cells. Similar results were obtained in three separate assays. experiments.
258
O-
0
I
1
I
I
I
2
4
6
6
10
Concentration
of PGD2 @g/m0
Fig. 3. Direct effect of PGD2 on DNA synthesis in nuclei. DNA synthesis in nuclei was carried out in the presence (-1 or absence (~1 of the cytoplasmic fraction with various concentration of PGD2 at 37’C for 30 min.
0
12
20
30
40
50
Time (hr) Fis. 4. Intracellular cyclic AMP level untreated cells. Cells were incubated or 10 pg/ml (l****J) of PGD2 or (o----O) min, 18, 24 and 48 hrs. The amount measured by radioimmunoassay. 259
of PGD2-treated and with 5 ug/ial(@-*.+ without PCD for 5 of cyclic A% P was
by radioimmunoassay as described in Materials and Methods. The cyclic AMP level increased only 40-50% after treatment of cells with PGD2 for 5 min and 18 hrs in the absence of cyclic nucleotide phosphodiesterase inhibitor (Fig. 4). We next tested the direct effect of cyclic AMP on nuclear DNA synthesis. Cyclic AMP at concentration of 10Vg M to10m2 M did not have anydirecteffecton DNA synthesis in nuclei in the presence of the cytoplasmic fraction, which contains protein kinase and some proteins (Fig. 5). This result suggests that cyclic AMP is not a mediator in cell-mediated inhibition of nuclear DNA synthesis by PGD2. Effects of Modulators of Adenylate Cyclase 2 Mastocytoma -Cell Growth. Forskolin (22) is a stimulator of adenylate cyclase. Forskolin at concentration of 0.1 and 1.0 JIM did not have any effect on cell growth, but at 10 pM it was slightly inhibitory owing to a non-specific drug effect. Moreover, 2',5'-dideoxyadenosine (23), which had no stimulatory effect on cell inhibits the cyclase, growth at 100 JIM. Cell growth was inhibited as strongly by PGD2 alone as by PGD2 p lus 2',5'-dideoxy adenosine (Fig. 6).
n-
01 0
I
-9
i
-a
-7 -6
-5 -4
-3 -2
Concentration of CAMP (1og.M)
Ficr. 5. Direct effect of cyclic AMP on DNA synthesis in nucler DNA synthesis in nuclei was carried out in the presence ( O---O) or absence (M) of the cytoplasmic fraction with various concentration of cyclic AMP at 37*C for 30 min. 260
DNA Synthesizing activity -_ of a Reconstituted System Consimnq of Salt-treated Nuclei and Salt Extract. Nuclei isolaGd from cells cultured withzwst PGD2 for 18 hrsweretreatedwith 0.3 M NaCland-separated into salttreated nuclei and salt extract as described in Materials and Methods. The salt-treated nuclei showed greatly reduced DNA synthesizing activity as reported previously (181, suggesting that some essential component(s) necessary for DNA synthesis had been extracted from them. However, addition of the nuclear salt extract from cells that had not beentreated with PGD enhanced the activity more than 4-fold. In contrast, ad J*ition of the salt extract from nuclei of PGD - treated cells enhanced the activity only 2fold (Fig. 7 i! . This result indicates that some DNA replication factor(s) is decreased or that an inhibitory factor for DNA replication is induced by PGD treatment. The salt extracts themselves exhibite c? negligible abilities to incorporate deoxyriboside triphosphate precursors into an acid-insoluble polymer unless supplied with activated DNA as a template. The DNA polymerase activities of the salt extract of nuclei of untreated cells were more than double those of the extract of nuclei of PGD2-treated cells (Table 1).
10
20
Incubation
30
40
Time (hr)
Fig. 6. Effect of adenylate cyclase modulators on cell growth. Cells were incubated with adenylate cyclase modulators at 37'C for the indicated periods and cell numbers were counted. o-_-o; no addition, -;lyMof forskolin, b--d; IOyM of forskolin, )-1; 100 UM of 2',5'-dideoxyadenosine (ddA), D----(II ; 100 YM of ddA and PGD2. O--O; PGD2. 261
Table 1.
of nuclear salt DNA polymerase activities extracts from PGD2-treated and untreated cells
Cells used for salt extract
Enzyme activities (dpm/106 nuclei) DNA polymerase c1 DNA polymerase p
untreated PGD2-treated
1775 (2.7) 662 (1.0)
-
3395 (1.7) 1962 (1.0)
DNA polymerase activities were assayed using activated calf thymus DNA as a template-primer as described previously Values are means for duplicate assayes. (24).
extract ’
WC T
WC
salt
T
Treated
F”%t!+’
Control
Fiq. 7. DNA synthesizing activity of a reconstituted system-consisting of salt-treated nuclei and salt extract. Nuclei were isolated from mastocytoma P-815, 2-E-6 cells after their incubation with or without PGD2 for 18 hrs. Salt-treated nuclei and salt extract were prepared from nuclei as described in Materials and Methods. Salt-treated nuclei were suspended in buffer A and salt extract was dialyzed for 3 hrs against bu fer A at O*C. Then each fraction was reconstituted and 5 H-TMP incorporated into acid-insoluble materials was measured. (-1: without a nuclear extract, C: with a nuclear extract from untreated cells, T: with a nuclear extract prepared from PGD2-treated cells, Control: salt-treated nuclei prepared from untreated cells, Treated: salt-treated nuclei from PGD2treated cells. 262
DISCU$SION The mechanism of the inhibitory effects of PGs on cell growth has yet to be clarified. Here, we used new approaches to study this mechanism. We found that cyclic AMP did not directly affect DNA synthesis in nuclei in the presence of a cytoplasmic fraction (Fig. 5). Moreover, by use of modulators of adenylate cyclase, we confirmed that there was no correlation between the effects of PGD2 and cyclic AMP on cell growth (Fig. 7). Thus we conclude that the inhibitory effect of PGD on cell growth is not dependent on an increased intracel2lular cyclic AMP level. This finding is contrary to the result on the effects of PGA and PGE in various in vitro experiments, and so cannot be extrapolated to otherPGr However, in the mechanism of inhibition of cell growth by PGD2 a mediator(s) other than cyclic AMP must be reconsidered. Reconstitution experiments on nuclear DNA synthesis using salt-treated nuclei and salt extracts of PGD2-treated and untreated cells showed that some mediator(s) is transferred to the nuclei (Fig. 61, because the stimulatory activity of a salt extract from untreated cells was less than that of a salt extract from PGD2-treated cells. In this system, the difference in the DNA syntheses of nuclear fractions from PGD -treated and untreated cells probably depended on the di*3ference in the DNA polymerase activities of the nuclear salt extracts of these cells (Table 1). Previously we found that the nuclear DNA polymerase activities of mastocytoma cells were inhibited by PGD2 in a cell-mediated manner, and that the activity of DNA polymerase c1 was especially strongly inhibited (approximately 50%) (24). Our data imply that PGD2 inhibits cell growth by attaching to the cell membrane, and then inducing some component that is transferred to the nucleus where it inhibits DNA synthesis and especially DNA polymerase a. We are now investigating the possible mediator(s) induced by PGD It is probably a very complicated mediator(s), an 2'may fluctuate in level depending on the interaction of PGD2 with membrane receptors. ACKNOWLEDGEMENT We wish to thank Dr. Sei-itsu Murota for advice and encouragement. REFERENCES 1.
Kawamura, M. and Koshihara, Y. Inhibitory effect of prostaglandin D on mastocytoma cell growth. Proc. Jap. Cancer Assoc. Tl? e 40th Annual Meeting p.233, 1981
2.
Kawamura, M. and Koshihara, Y. Prostaglandin D2 strongly inhibits growth of murine mastocytoma cells. Prost. Leuk, & Med.12: 85-93, 1983 263
3.
Fukushima, M., Kato, T., Ueda, R., Ota, K., Narumiya, 0. Prostaglandin D S. and Hayaishi, gi,",,",",'.""",'h",:hE:enEL antineoplastic agent. Biochem. 105: 956-964, 1982 Commun.
4.
Simmet, T. and Jaffe, B.M. Inhibition of B-16 melanoma growth in vitro by prostaglandin D2. Prostaglandins 25: 47-54, 1983
5.
Sakai, T., Yamaguchi, N., Shiroko, Y., Sekiguchi, M., Fujii, G. and Nishino, H. Prostaglandin D inhibits the proliferation of human malignant 2 umor cells. Prostaglandins 27: 17-26, 1984
6.
Higashida, H., Kano-Tanaka, K., Natsume-Sakai,S., Sudo, Fukami, H., Nakagawa, Y. and Miki, N. Cytotoxic ai)tion of prostaglandin D on mouse neuroblastoma cells. Int. J. Cancer 31: 737-802, 1983 K
7.
Claesson, H-E., Lindgran, J.A. and Hammarstrbm, S. Endogenous prostaglandin E2 production inhibits proliferation of polyoma virus-transformed 3T3 cells : Correlation with cellular levels of cyclic AMP. Adv. Prost. Throm. Res. 6: 541-545, 1980
8.
Penit, J., Cantau, B., Huot, J. and Jard, S. Adenylate cyclase from synchronized neuroblastoma cells : responsiveness to prostaglandin El, adenosine, and dopamine during the cell cycle. Proc. Natl. Acad. Sci. USA 74: 1575-1579, 1977
9.
Kram, R., Mamont, P. and Tomkins, G.M. Pleiotypic control by adenosine 3':5'-cyclic monophosphate: a model for growth control in animal cells. Proc. Natl. Acad. Sci. USA 70: 1432-1436, 1983
IO.
Davis,J. and Ralph, R.K.Regulation of growth of mouse mastocytoma cells. Cancer Res. 35: 1495-1504, 1975
11.
Smith, J.B., Ingerman, C.M. and Silver, M.J. Formation of prostaglandin D during endoperoxideinduced platelet aggregation. T% romb. Res. 9, 413-418, 1976
12.
Shimizu, T., Mizuno, N., Amano, T. and Hayaishi, 0. a neuromodulator. Proc. Natl. Acad. Prostaglandin D Sci. U.S. 76: 62!!?-6234, 1979
13.
Narumiya, S. and Fukushima, M. A2-Prostaglandin J2, An ultimate metabolite of prostaglandin D2 exerting cell growth inhibition. Biochem. Biophys. Res. Commun. 127: 739-745, 1985
14.
Koshihara, Y., Senshu, T., Kawamura, M. and Murota, S. Sodium n-butyrate induces prostaglandin synthetase 264
activity in mastocytoma P-815 cells. Acta 617: 536-539, 1980
Biochim. Biophys.
15.
Kawamura, M., Koshihara, Y., Senshu, T. and Murota, S. Enhancement of prostaglandin synthesizing activity by the treatment with sodium n-butyrate on cloned mastocytoma cells and various other tissue cell lines Prostaglandins. 19: 659-669, 1980
16.
Koshihara, Y., Kawamura, M., Senshu, T. and Murota, S. Effect of sodium n-butyrate on induction of prostaglandin synthase activity in cloned mastocytoma P-815, 2-E-6 cells. Biochem. J. 194: 111-117, 1981
17.
Friedman, D.L. and Mueller, G.C. A nuclear system for DNA replication from synchronized Hela cells. Biochim.
Biophys. Acta 161: 455-468, 1968 18.
Seki, S. and Mueller, G.C. Dissociation and reconstitution of the DNA replicase system of Hela cell nuclei. Biochim. Biophys. Acta 435: 236-250, 1976
19.
Honma. M., Satoh, T. Takezawa, J. and Ui, M. An ultrasensitive method for the simultaneous determination of cyclic AMP and cyclic GMP in smallvolume samples from blood and tissue. Biochem. Med. 18: 257-273, 1977
20.
Steiner, A.L., Parker, C.W. and Kipnis, D.M. Radioimmunoassay for cyclic nucleotides. I. Preparation of antibodies and iodinated cyclic nucleotides. J. 1972 Biol. Chem. 247: 1106-1113,
21.
Rao, C.V. and Mitra, S. Gonadotropin and prostaglandins binding sites in nuclei of bovine corpora lutea; Biochim.Biophys.Acta584:454-466, 1979
22.
Seamon, K.B., Padgett, W. and Daly, J.W. Forskolin : unique diterpene activator of adenylate cyclase in membranes and in intact cells. Proc. Natl. Acad. Sci, U.S.A. 78: 3363-3367, 1981
23.
Haslam, R.J., Davidson, M.L. and Desjardins, J.V. Inhibition of adenylate cyclase by adenosine analogues in preparations of broken and intact human platelets. Evidence for the unidirectional control of platelet function by cyclic AMP. Biochem. J. 176: 83-93, 1978
24.
Kawamura, M. and Koshihara, Y. Prostaglanchin D lowers nuclear DNA polymerase activity in cultured masz ocytoma cells. Prostaglandins 27: 517-525, 1984
265
IHIBITORY EFFECT OF PROSTAGLANDIN ON DNA SYNTHESIS IN NUCLEI
D2
Yasuko KOSHIHARA and Mieko KAWAMURA Department of Pharmacology Tokyo Metropolitan Institute of Gerontology Itabashi-ku, Tokyo-173, Japan (reprint request to YK) ABSTRACT Prostaglandin (PG) D2 treatment inhibited DNA synthesis in isolated nuclei of mastocytoma P-815, 2-E-6 cells. On treatment with PGD2 (IO pg/ml), the inhibition was distinct by 8 hrs, and complete after 18 hrs. This effect of PGD on DNA synthesis in nuclei was not direct
or mediated % y cyclic AMP, but was a cell-mediated reaction. The cytoplasmic fractions of PGD2-treated and untreated cells both had stimulatory effects and their potencies were the same except for that of the cytoplasmic fraction of 8 hr-treated cells, which was less than that of the cytoplasmic fraction of untreated cells.. On treatment with PGD2, inhibition of DNA synthesis in the nuclei began after 8 hrs, and this inhibition could not be reversed, even by adding the cytoplasmic fraction from untreated cells to the assay system. Anuclear saltextractpreparedby adding 0.3 M NaCl to nuclei of cells that had been treated with PGD2 for 18 hrs had a much smaller stimulatory effect on DNA synthesis of salt-treated nuclei than an extract of nuclei from untreated cells. It is suggested that inhibition of cell growth by PGD2 is not mediated by intracellular cyclic AMP, but that
induces PGD synt?Iesis.
a factor(s)
that
inhibits
nuclear
DNA
INTRODUCTION We found that PGD2 strongly inhibited growth of mouse mastocytoma cells (1,2). Later, others reported similar inhibitory effects of PGD2 on several kinds of malignant 253
tumor cell lines, such as mouse leukemia L1210 cells (3), B-16 melanoma cells (4) and human malignant tumor cell lines Moreover, on its local daily injection, PGD2 (5). significantly inhibited tumor formation by neuroblastoma cells implanted subcutaneously into mice The (6). Other PGs, mechanism of this effect of PGD2 is unknown. such as PGA and PGE, that inhibit cell growth have been suggested to act by increasing the intracellular cyclic AMP level (7,8), since cyclic AMP is reported to inhibit cell growth (9,lO). However, the mechanism of the inhibitory effect of cyclic AMP on growth is also unknown. PGD was also found to increase the intracellular cyclic AMP 1 eve1 in platelets (11) and neuroblastoma NIE-115 cells (12 ). But it did not increase the cyclic AMP level in mouse leukemia L1210 cells (13), and increased the level in mastocytoma we investigated P-815 cells, only 1.5-fold. In this work the mechanism of growth inhibition by PGD2, by studies on nuclei from its effect on DNA synthesis in isolated mastocytoma cells. We also examined the effect of cytoplasm on nuclear DNA and untreated cells of PGD2 -treated synthesis. Results indicated that PGD2 probably induces some inhibitory factor(s) other than cyclic AMP and that the inhibition is caused by a cell-mediated reaction. MATERIALS and METHODS Materials. PGD2 (97% pure, as judged by high performance liquid chromatography) was obtained from Funakoshi Pharmaceutical Co. (Tokyo, Japan). PGD in aqueous media, determined by incubating it in phosp K ate buffer (pH 7.0) at 37'C, had a half life of 8.6 hrs as judged by high performance liquid chromatography ; 3H-dTTP was obtained from New England Nuclear (Boston, Mass), unlabelled deoxyribonucleoside triphosphate from P-L Biochemical, Inc. (Milwaukee, Wis), fetal bovine serum (mycoplasm- and virus-free) from Armour Pharmaceutical Co. ( Kankakee, Ill) and cyclic AMP from Sigma Chemical Co. (St. Louis, MO). Cell --A Culture Mastocytoma P-815, 2-E-6 cells were --established from cells from the ascites fluid of female DBA/2 mice by -in vitro culture and were cloned (14,15,16). Cells were cultured in suspension in Eagle's basal medium supplemented with 10% (v/v) fetal bovine serum under CO2 in air (1:19) at 37OC in a gyratory shaker (Model 2; New Brunswick Scientific Co., Edison, NJ). Treatment with PGD= Cells in the early logarithmic phase of growtGe=treated with PGD2 (10 pg/ml) in ethanol for 18 hrs. The final concentration of ethanol was less than 0.1% and the same amount of vehicle was added to control cultures. Cell numbers were counted a Coulter Counter (Model G, Coulter Electronics Inc., ,Hialeah, Fla).
254
PGDy Assay for DNA Synthesis & Isolated Nuclei. treated and untreated cells were ha$yestec$ and washed once , Mg +-free, pH 7.4). with phosphate-buffered saline (Ca Nuclei were isolated by the method of Friedman & Mueller (17). In brief, the ce 1 pellet was suspended at a final concentration of 7 x 10 i cells/ml in buffer A consisting of 10 mM tris-HCl (pH 7.8), 4 mM MgC12, 1 mM HDTA, 6 mM B and washed by mercaptoethanol and 0.25 M sucrose The resulting cell pellet was resuspended centrifu ation. at 7 x 1097 cells/ml in buffer Aand incubated at O'C. After by 10 strokes of a 10 min, the cells were homogenized The homogenate was centrifuged at Dounce glass homogenizer. 800 g for 5 min to separate the nuclei and cytoplasmic The nuclear fraction was washed 3 times b fractions. resuspensioninbuffer Aata final concentration of 3 x 10 Y nuclei/ml and centrifugation min. The final at 350 g for pellet was resuspended in buffer Aat 5 x10 5 nuclei/ml. The nuclear fraction did not contain any intact cells and was purified over 95 %. The cytoplasmic fraction was centrifuged at 105,000 g for 60 min to remove organelles. DNA synthesis in isolated nuclei was assayed as follows: Incubation mixture (0.6 ml) consisting of 0.1 ml of nuclear suspension (5 x IO6 nuclei), 0.3 ml of cytoplasmic fraction or buffer A and 0.2 ml of reaction mixture composed of 0.1 M Tris-HCl(pH 7.8), 0.02 M MgC12, 0.3 M NaCl, 0.015 M ATP, 0.3 mM dATP, dCTP, dGTP each and 0.03 mM f3H]dTTP (spec. act. 83.3 Ci/mole) was incubated at 37'C for 30 min. The reaction was terminated by chilling the mixture in an ice-water bath and adding 2 ml of chilled 10% TCA containing 2% Na-pyrophosphate. Acid-insoluble material was washed 3 times with 5% TCA containing 1% Na-pyrophosphate and counted in a liquid scintillation spectrophotometer.
Preparation of salt-treated nuclei and salt extract. Salt-treated nuclx and salt extract wereprepared by the method of Seki and Mueller (18). In brief, the washed nuclear pellet was resuspended in 5 vol. of buffer A containing 0.3 M NaCl. The suspension was incubated at 0°C for 1 hr and then centrifuged at 800 g for 10 min. The final precipitate was resuspended in buffer A at a concentration of 3.3 x lo7 nuclei/ml and used for further experiments as salt-treated nuclei. The supernatant was recentrifuged at 130,000 g for 20 min to yield salt extract. The extract was dialized against 35 vol. buffer A at O'C for 3 hrs. The mixture (0.6 ml) for assay of DNA synthesis consisted of 0.3 ml of salt-treated nuclear suspension, 0.1 ml of salt extract or buffer A and 0.2 ml of substrate mixture as described above. DNA synthesis activity in salttreated nuclei was assayed at 37'C for 30 min. Acidinsoluble material was then counted in a liquid scintillation spectrophotometer. Measurement of Intracellular cyclic AMP
intracellularcvclicAMP. was extracted and succinylated 255
by
the method of Honma et al. (19). In brief, cells with or for 5 min, 18 hrs and 40 hrs were without PGD2-treatment harvested by centrifugation and suspended in 6% TCA. The TCA-soluble material was obtained, TCA was removed by etherThe extraction and the aqueous solution was lyophilized. residue was succinylated and cyclic AMP was determined by radioimmunoassay (20). RESULTS DNA Synthesizing Activity of Isolated Nuclei. Cultured mastocytoma P-815, 2-E-6 ce?-1s were treated with PGD2 (10 us/ml) for 18 hrs. This treatment, completely The DNA synthesizing activity of inhibited cell growth. isolated nuclei was assayed in the presence of ATP as described in Materials and Methods. The nuclei from untreated cells had very high activity, but nuclei from PGD2-treated cells did not show any activity, as on arrest of cell growth. The activity in nuclei from treated cells was enhanced by addition of cytoplasm, but the enhanced level was less than, or equal to that of nuclei from untreated cells without cytoplasm. In contrast, the activity in nuclei from untreated cells was enhanced 3.2 fold by addition of the cytoplasm (Fig. 1). However, there was no difference in the stimulatory effects of cytoplasmic fractions from treated and untreated cells. This result implies that the inhibitory effect of PGD2 had already reached the nuclei when the cells were treated with PGD2 for 18 hrs. Inhibition of DNA Time-course of Cell-mediated Synthesizing A&iv= in Isolated Nuclei. The incubatz time necessary for complete loss of DNA synthesizing activity in isolated nuclei was determined. The nuclei and cytoplasm of cells with and without treatment with PGD2 for 1,2,4,8 and 18 hrs were prepared as (10 uglml) described in Materials and Methods. The DNA synthesizing activity in nuclei from untreated cells increased with the incubation time, and reached a maximum after 8 hrs (Fig. 2a). On the contrary, the activity in nuclei from treated cells decreased after 4 hr-treatment and had nearly disappeared after 18 hr-treatment (Fig. 2b). Addition of cytoplasm increased the nuclear synthesizing activities of treated and untreated nuclei. The cytoplasm from untreated cells had higher stimulatory activity than that of cytoplasm from cells treated with PGD2 for 8 hrs. But cytoplasm from cells after 18 hr treatment had similar activity to that of cytoplasm from untreated cells, and cytoplasm from cells after 4 hr treatment had similar activity to cytoplasm from untreated cells. Effects --of PGD2 and Cyclic AMP on DNA Synthesizinq -__Activity in Isolated-Nuclei. Next.we examined the effects of direcTaddition of PGD2 and cyclic AMP to the mixture for assay of DNA synthesis. PG is thought not to 256
penetrate the oell membrane except in a few cases (21), but can penetrate the cell it is uncertain whether PGD membrane, and then inhibit DN 3 synthesis. We found that addition of PGD2 at concentrations of 0.1-1.0 ug/ml to the DNA synthesizing system with isolated nuclei had no effect on their DNA synthesis (Fig. 3). It also had no direct effect on DNA synthesis in this system when supplemented with cytoplasm. In other words, PGD2 did not modify the stimulatory activity of the cytoplasmic fraction. PGD2 is said to increase the intracellular cyclic AMP level (12). We measured the intracellular cyclic AMP level
E
e -
2.
1 -
N&i
Control
CF
t-1 C T
Treated (-1 C T
Fig. 1. DNA synthesizing activity in nuclei. The nuclei and cytoplasmic fraction were prepared from mastocytoma P815, 2-E-6 cells after their incubation with or without10 ug/ml of PGD2 for 18 hrs. Nuclear DNA synthesis was carried out in the presence or ab ence of the cytoplasmic fraction at 37°C for 30 min and 3H-TMP incorporated into the acid-insoluble materials was counted. Control: nuclei prepared from untreated cells. Treated: nuclei prepared from PGD2 -treated cells. CF: cytoplasmic fraction. (-1: without a cytoplasmic fraction. C: with the cytoplasmic fraction prepared from untreated cells. T: with the cytoplasmic fraction from PGD2-treated cells. 257
I-
2,,_*f::~:III:f:,,,\_ -_.
.--. -*-.“-* -_ ---r&w_ =.* ___+__:-='~ -.‘~__*.----*------______~ 0 2 4 6 8, ----'-‘_‘__d I I I
l-
incubation
Time (hr)
Time courseof cell-mediated inhibition of DNA Fig. 2. synthesizing activity in isolated nuclei. The nuclei and with or cytoplasmic fraction were prepared from cells without treatment with 10 pg/ml of PGDZ for the indicated periods. Nuclear DNA synthesis was carried out in the presence yr absence of cytoplasmic fraction at 37°C for 30 min and H-TMP incorporated into acid-insoluble materials was counted. a): nuclei prepared from PGD2-untreated cells. b): nuclei prepared from PGD2-treated cells. (*I, from nuclei only; (A), with the cytoplasmic fraction untreated cells ; ( A), with the cytoplasmic fraction from Values are means for duplicate PGD2 -treated cells. Similar results were obtained in three separate assays. experiments.
258
O-
0
I
1
I
I
I
2
4
6
6
10
Concentration
of PGD2 @g/m0
Fig. 3. Direct effect of PGD2 on DNA synthesis in nuclei. DNA synthesis in nuclei was carried out in the presence (-1 or absence (~1 of the cytoplasmic fraction with various concentration of PGD2 at 37’C for 30 min.
0
12
20
30
40
50
Time (hr) Fis. 4. Intracellular cyclic AMP level untreated cells. Cells were incubated or 10 pg/ml (l****J) of PGD2 or (o----O) min, 18, 24 and 48 hrs. The amount measured by radioimmunoassay. 259
of PGD2-treated and with 5 ug/ial(@-*.+ without PCD for 5 of cyclic A% P was
by radioimmunoassay as described in Materials and Methods. The cyclic AMP level increased only 40-50% after treatment of cells with PGD2 for 5 min and 18 hrs in the absence of cyclic nucleotide phosphodiesterase inhibitor (Fig. 4). We next tested the direct effect of cyclic AMP on nuclear DNA synthesis. Cyclic AMP at concentration of 10Vg M to10m2 M did not have anydirecteffecton DNA synthesis in nuclei in the presence of the cytoplasmic fraction, which contains protein kinase and some proteins (Fig. 5). This result suggests that cyclic AMP is not a mediator in cell-mediated inhibition of nuclear DNA synthesis by PGD2. Effects of Modulators of Adenylate Cyclase 2 Mastocytoma -Cell Growth. Forskolin (22) is a stimulator of adenylate cyclase. Forskolin at concentration of 0.1 and 1.0 JIM did not have any effect on cell growth, but at 10 pM it was slightly inhibitory owing to a non-specific drug effect. Moreover, 2',5'-dideoxyadenosine (23), which had no stimulatory effect on cell inhibits the cyclase, growth at 100 JIM. Cell growth was inhibited as strongly by PGD2 alone as by PGD2 p lus 2',5'-dideoxy adenosine (Fig. 6).
n-
01 0
I
-9
i
-a
-7 -6
-5 -4
-3 -2
Concentration of CAMP (1og.M)
Ficr. 5. Direct effect of cyclic AMP on DNA synthesis in nucler DNA synthesis in nuclei was carried out in the presence ( O---O) or absence (M) of the cytoplasmic fraction with various concentration of cyclic AMP at 37*C for 30 min. 260
DNA Synthesizing activity -_ of a Reconstituted System Consimnq of Salt-treated Nuclei and Salt Extract. Nuclei isolaGd from cells cultured withzwst PGD2 for 18 hrsweretreatedwith 0.3 M NaCland-separated into salttreated nuclei and salt extract as described in Materials and Methods. The salt-treated nuclei showed greatly reduced DNA synthesizing activity as reported previously (181, suggesting that some essential component(s) necessary for DNA synthesis had been extracted from them. However, addition of the nuclear salt extract from cells that had not beentreated with PGD enhanced the activity more than 4-fold. In contrast, ad J*ition of the salt extract from nuclei of PGD - treated cells enhanced the activity only 2fold (Fig. 7 i! . This result indicates that some DNA replication factor(s) is decreased or that an inhibitory factor for DNA replication is induced by PGD treatment. The salt extracts themselves exhibite c? negligible abilities to incorporate deoxyriboside triphosphate precursors into an acid-insoluble polymer unless supplied with activated DNA as a template. The DNA polymerase activities of the salt extract of nuclei of untreated cells were more than double those of the extract of nuclei of PGD2-treated cells (Table 1).
10
20
Incubation
30
40
Time (hr)
Fig. 6. Effect of adenylate cyclase modulators on cell growth. Cells were incubated with adenylate cyclase modulators at 37'C for the indicated periods and cell numbers were counted. o-_-o; no addition, -;lyMof forskolin, b--d; IOyM of forskolin, )-1; 100 UM of 2',5'-dideoxyadenosine (ddA), D----(II ; 100 YM of ddA and PGD2. O--O; PGD2. 261
Table 1.
of nuclear salt DNA polymerase activities extracts from PGD2-treated and untreated cells
Cells used for salt extract
Enzyme activities (dpm/106 nuclei) DNA polymerase c1 DNA polymerase p
untreated PGD2-treated
1775 (2.7) 662 (1.0)
-
3395 (1.7) 1962 (1.0)
DNA polymerase activities were assayed using activated calf thymus DNA as a template-primer as described previously Values are means for duplicate assayes. (24).
extract ’
WC T
WC
salt
T
Treated
F”%t!+’
Control
Fiq. 7. DNA synthesizing activity of a reconstituted system-consisting of salt-treated nuclei and salt extract. Nuclei were isolated from mastocytoma P-815, 2-E-6 cells after their incubation with or without PGD2 for 18 hrs. Salt-treated nuclei and salt extract were prepared from nuclei as described in Materials and Methods. Salt-treated nuclei were suspended in buffer A and salt extract was dialyzed for 3 hrs against bu fer A at O*C. Then each fraction was reconstituted and 5 H-TMP incorporated into acid-insoluble materials was measured. (-1: without a nuclear extract, C: with a nuclear extract from untreated cells, T: with a nuclear extract prepared from PGD2-treated cells, Control: salt-treated nuclei prepared from untreated cells, Treated: salt-treated nuclei from PGD2treated cells. 262
DISCU$SION The mechanism of the inhibitory effects of PGs on cell growth has yet to be clarified. Here, we used new approaches to study this mechanism. We found that cyclic AMP did not directly affect DNA synthesis in nuclei in the presence of a cytoplasmic fraction (Fig. 5). Moreover, by use of modulators of adenylate cyclase, we confirmed that there was no correlation between the effects of PGD2 and cyclic AMP on cell growth (Fig. 7). Thus we conclude that the inhibitory effect of PGD on cell growth is not dependent on an increased intracel2lular cyclic AMP level. This finding is contrary to the result on the effects of PGA and PGE in various in vitro experiments, and so cannot be extrapolated to otherPGr However, in the mechanism of inhibition of cell growth by PGD2 a mediator(s) other than cyclic AMP must be reconsidered. Reconstitution experiments on nuclear DNA synthesis using salt-treated nuclei and salt extracts of PGD2-treated and untreated cells showed that some mediator(s) is transferred to the nuclei (Fig. 61, because the stimulatory activity of a salt extract from untreated cells was less than that of a salt extract from PGD2-treated cells. In this system, the difference in the DNA syntheses of nuclear fractions from PGD -treated and untreated cells probably depended on the di*3ference in the DNA polymerase activities of the nuclear salt extracts of these cells (Table 1). Previously we found that the nuclear DNA polymerase activities of mastocytoma cells were inhibited by PGD2 in a cell-mediated manner, and that the activity of DNA polymerase c1 was especially strongly inhibited (approximately 50%) (24). Our data imply that PGD2 inhibits cell growth by attaching to the cell membrane, and then inducing some component that is transferred to the nucleus where it inhibits DNA synthesis and especially DNA polymerase a. We are now investigating the possible mediator(s) induced by PGD It is probably a very complicated mediator(s), an 2'may fluctuate in level depending on the interaction of PGD2 with membrane receptors. ACKNOWLEDGEMENT We wish to thank Dr. Sei-itsu Murota for advice and encouragement. REFERENCES 1.
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265