Cancer Letters, 66 (1992) 139 - 146 Efsevier Scientific Publishers Irefand Ltd.
139
Differential inhibition by staurosporine of phorbol bryostatin and okadaic acid effects on mouse skin Michael Gschwendt, Germon Cancer
Research
Walter Kittstein, Dieter Lindner Center,
Im Neuenheimer
ester,
and Friedrich Marks
Fe/d 280, D-6900 Heidelberg
(FRG)
(Received 11 May 1993 (Revision received 15 July 1992) (Accepted 15 July 199il)
Summary
The tumor promoters 12-O-tetradecanoylphorbol-13-acetate (TPA), a strong actiuator of protein kinase C (PKC) and okadaic acid, which is ineffectiue
in this respect,
induce
a
rapidiy developing (‘early’) edema of the mouse ear. Bryostatin, another potent activator of PKC, is unable to induce an ‘early’ edema but causes o more delayed deuelopment of edema at a time when most of the PKC is down-regulated. The PKC inhibitor staurosporine neither inhibits the early TPAnor the late bryodatin-induced edema, but suppresses the okadaic acid-induced edema very effectively. TPA as well as bryostatin, but not okadaic acid cause a down-regulation of PKC, which is not inhibited by staurosporine. The calmodulin antagonist cyclosporine A, which does not suppress PKC activity, very effectively inhibits the TPA-induced edema and down regulation of PKC. Hence we conclude that protein phosphory.ylation catalyzed by staurosporine-suppressable PKC is not inuolued in the induction of edema and PKC downCorrespondence to: Michael Gschwendt, German Research Center, Im Neuenheimer Feld 280, Heidelberg, FRG. Abbreviations: PKC, protein tetradecanoylphorbol-13-acetate; 0304-3835/92/$05.00 Printed and Published
kinase
C;
CsA, cyclosporin
TPA, A.
regulation by TPA but that a calmodulin dependent process may play a critical role in these and other TPA effects in mouse skin.
Cancer D-6900 12-O-
@ 1992 Elsevier Scientific Publishers in Ireland
Keywords: 12-0-tetradecanoylphorbol-13acetate; protein kinase C; okadic acid; early edema; bryostatin; staurosporine Introduction The protein kinase C (PKC) family, a group of several isoenzymes, plays a central role in intracellular signal transduction [ l] . Physiological activation of PKC is achieved by the second messenger diacylglycerol produced from phosphoinositides as a consequence of a receptor-mediated activation of phospholipase C. Tumor-promoting phorbol esters such as 12-0-tetradecanoylphorbol-13-acetate (TPA) have been shown to be much more potent activators of PKC than diacylglycerol [l]. Therefore, it is generally assumed that the numerous biological effects of TPA, including tumor promotion, are due to an activation of PKC resulting in the phosphorylation of certain substrate proteins. Recently, however, it was reported that the potent PKC inhibitor staurosporine was unable to suppress several effects of TPA on mouse skin [2] and in other Ireland Ltd
140
systems [3,4]. Here we compare the biological effects on mouse skin (edema formation and down-regulation of PKC) of two tumorpromoting compounds, one acting as a PKC activator (TPA) and the other ineffective in this respect (okadaic acid) [5,6] and of a nontumor-promoting PKC activator (bryostatin) [7 - lo] and their inhibition by PKC inhibitors as well as by calmodulin antagonists. The results indicate that TPA might induce some biological effects independently of protein phosphorylation catalyzed by staurosporinesensitive PKC but via a calmodulin-dependent process. Methods
and Materials
Materials TPA and 4-0-methyl-TPA were kindly provided by Dr. E. Hecker, German Cancer Research Center, Heidelberg, FRG. Bryostatin 1 (bryostatin) was a generous gift of Dr. G .R. Pettit, State University of Arizona, Tempe, USA. Okadaic acid and staurosporine were from Boehringer, Mannheim, FRG. Cyclosporine A (CsA) and the calpain inhibitor E64d were kindly provided by Sandoz, Basel, Switzerland and Taisho-Pharmaceutical Co., Ltd., Japan, respectively. The calmodulin antagonist R2457 1, the ionophore A23 187, dioctanoylglycerol, phosphatidyl serine and histone III-S were from Sigma, Munich, FRG. [y-32P]ATP (spec.act. 3000 Ci/mmol) was from NEN (Waltham, MA, USA). Animals Female NMRI mice (7 weeks old) were used and fed a standard diet ad libitum. The back skin was shaved 3 days before treatment. Methods Ear edema. A potential inducer of edema alone or together with an inhibitor was dissolved in acetone and applied at a volume of 20 ~1 to the outer surface of both mouse ears. Controls were treated with acetone alone. Edema were determined on the basis of ear plug weight as described previously [l 11.
Epidermis extracts. The shaved back skin of mice was treated with various compounds dissolved in 0.1 ml of acetone. The animals were sacrificed at the times indicated and epidermal specimens were prepared from frozen back skin as described previously [ 121. Epidermis was homogenized in a buffer containing Triton X-100 (20 mM Tris/HCl, pH 7.5,5 mM EGTA, 2mM EDTA, 1 mM PMSF, 50 mM 2-mercaptoethanol, 0.2% Triton X100) and applied to SDS polyacrylamide gel electrophoresis. lmmunoblotting Immunoblots using an cr,P,-yPKC-specific antiserum and 35S-labelled anti-rabbit-IgG as second antibody were performed as described previously [ 13,141. The immunoblots were visualized by autoradiography and quantitated by densitometry. Phosphorylation
of histone 111-S with
wP,rPKC
The assay mixture (total volume: 100 ~1) contained 40 ~1 Tris buffer (50 mM Tris - HCl, pH 7.5, 10 mM mercaptoethanol), 5 ~1 ATP in Tris-buffer (250 ~1 of 750 PM ATP + 10 ~1 of 1.7 TM [y-32P]ATP), 35 pg phosphatidyl serine, 4 mM MgC12, 60 pg histone III-S, lo-‘M TPA, 5 ~1 c~,fl,rPKC purified from mouse brain [13]. After the addition of ATP, the mixture was incubated at 30°C for 7 min. Fifty-microliter aliquots were then dropped onto 20-mm square pieces of phosphocellulose paper (Whatman p81), which were washed once with deionized water, once with 5% sodium pyrophosphate 9pH 8.0)) twice with deionized water, and once each with acetone and petrol ether. The radioactivity on each piece of paper was determined by scintillation counting. Results As reported earlier [ll], treatment of the mouse ear with 1 hmol of the tumorpromoting and PKC-activating phorbol ester TPA induced the formation of an edema. The
141
effect was maximal 6 h after treatment with TPA (Fig. 1). Okadaic acid, another tumor promoter with a structure entirely different from that of TPA and without PKC-activating capacity [5,6], also caused the development of an edema (Fig. 1). The time course of the okadaic acid-induced edema was identical with that of the TPA-induced edema. Upon application of bryostatin, another PKC-activator
300
250
2 -if zoo -2 %
150
% 2 .y
100
; 50
1
0 0
,
5
10 Hour!;
Table 1.
Induction
after
15
I
I
20
25
treatment
and inhibition of the mouse
Treatment
ear edema
Fig. 1. Induction of a mouse ear edema by TPA (A), okadaic acid (A) and bryostatin (W). Mice were treated with 1 nmol of the inducing compound dissolved in 20 ~1 acetone and the weight of the ear plug was determined as a measure of the edema at the indicated times after treatment as described previously [ll]. The values are given as percent of the control (treatment with the solvent acetone alone; 100% = 11.43 mg) and are the mean of at least two determinations.
by various compounds. Edema
(weight of ear plug)
(%)
Induction
Acetone (control) 1 nmol TPA (6h) 1 nmo] OkA (6h) 1 nmol Bry (13h) 10 nmol St (6h) 10 nmol St (19h) 1 nmo] TPA (23 h) followed by 1 nmo] TPA (6 h) 1 nmo] OkA (23 h) followed by 1 nmo] TPA (6h) 1 nmol TPA, 10 nmo] !St (6h) 1 nmol Bry, 10 nmol St (13h) 1 nmol TPA, 42 nmo] GA (6h) 1 nmol TPA, 72 nmo] Ii,,,,, (6h) 1 nmol OkA, 1 nmol St (6h) 1 nmol OkA, 5 nmo] St (6h) 1 nmol OkA, 10 nmol St (6h) 1 nmol OkA, 42 nmol ‘CsA (6h) 1 nmol OkA, 72 nmo] Rz4 571 (6h)
100 260 248 262 100 152
f 7 ziz 19 zt 16 ziz 20 ziz 6 ziz 10
lOO* 257
l
-
Inhibition of induction (%) -
6
-
20
0*2 O*l 93 + 92 f 67 f 82 f 100 f 100 f 85 f
6 4 4 5 3 5 4
The mouse ear was treated with an inducing compound alone or together with an inhibitor dissolved in acetone and the weight of the ear plug as a measure of the edema was determined as described previously [ll]. The duration of treatment is given in brackets. Induction of the edema is given as percent of control (acetone alone; 100%) and inhibition as percent of reduction of the induced edema (no reduction: 0% ; reduction to the control: 100%). The values are the mean of at least two determinations. OKA, okadaic acid; Bry, bryostatin; St, staurosporine.
142
Table 11. Activity of TPA-stimulated presence of CsA.
without tumor-promoting capacity [7 - 101, not even the slightest formation of an edema could be observed after 6 h. Instead, edema developed with a delayed kinetic maximal around 13 h after treatment (Fig. 1). As shown in Table I, 10 nmol of the PKC inhibitor staurosporine were unable to inhibit the TPA-and bryostatin-induced ear edema at 6 h and 13 h, respectively, i.e. at the time of maximal edema formation. At the same concentration, staurosporine suppressed the okadaic acid-induced edema completely. Even lower concentrations of staurosporine were effective in this respect. 1 nmol of staurosporine still caused a 67% inhibition. The calmodulin antagonists cyclosporine A (CsA) [15] and R24 571 (Van Belle, 1981) effectively inhibited both the TPA-and the okadaic acidinduced edema (Table I). Compared with staurosporine higher concentrations of CsA (42 nmol) and R24571 (72 nmol) were required though, as was expected from the I&, values obtained in cell-free systems [16,17]. Staurosporine alone did not induce an edema comparable to that seen after TPA, okadaic acid or bryostatin treatment. Only 19 h after staurosporine application a weak edema was observed (Table I). The calmodulin antagonist CsA was found to be absolutely inactive as an inhibitor of PKC activity. In a cell-free system,
t o--i 0
1
1 ‘0
LO
30
0 lo-’ 1O-6 1O-5 1O-4
Time
50
60
M M M M
27 25 27 26 28
130 740 810 930 100
(counts/min)
f 1970 zt 1430 ztz 690 zt 1840 f 1580
Phosphorylation of histone Ill-S with purified cr,@,yPKC and [Y-~‘P]ATP was performed in the presence of phosphatidyl serine and TPA as described in Methods. Various concentrations of CsA dissolved in acetone were added as indicated. Incorporation of 32P into histone is given as counts per minute (counts/min). The value for unstimulated kinase activity (without TPA) was 5430 counts/min. Values are the mean of two determinations.
up to 10 -4M CsA did not at all suppress the phosphorylation of histone III-S catalyzed by TPA-stimulated purified a,P,yPKC (Table II). Treatment of the back skin of mice with 10 nmol TPA caused down regulation of PKC in the epidermis as determined by immunoblotting using an a,P,yPKC-specific antiserum (Fig. 2). Lowest levels of (r,PPKC (mouse epidermis does not contain yPKC [18] were reached between 8 and 15 h after TPA (20% of control). Recovery of PKC was rather slow.
_i
.A-
40
PKC-activity
CsA
c~,fl,-rPKC in the
70
80
[h]
TPA-induced down-regulation of PKC in mouse epidermis. The shaved back skin of mice was treated with Fis. 2. 10 nmol of TPA dissolved in 0.1 ml acetone. At the times indicated the relative amount of PKC was determined in epidermal extracts by immunoblotting using an cy,@,yPKC-specific antiserum and an 35S-labelled anti-rabbit-IgG as second antibody as described in Methods. The values are the mean of two determinations.
143
C
TPA
Bry
Induction Fig. 3. regulation in mouse Animals were treated as described in Fig. Leibersperger et al. acid; Bry, bryostatin;
OkA
St
TPA +St
TPA +CsA
and inhibition of PKC downepidermis by various compounds. and immunoblots were performed 2 and by Gschwendt et al. [13] [la], respectively. OkA, okadaic St, staurosporine.
Even 72 h after TPA the epidermis contained only 40% of the amount of the normal PKC level. Previously we had shown that GPKC, a predominant PKC isoenzyme in mouse epidermis, is also down-regulated as a consequence of TPA treatment of the animals [14]. However, GPKC recovered much faster, almost reaching the control level 72 h after TPA. As shown in Fig. 3, down-regulation of (r,PPKC in mouse epidermis was also induced by 10 nmol of bryostatin, but not by 10 nmol of okadaic acid. Staurosporine (1 pmol) was unable to inhibit the TPA-induced downregulation of PKC, whereas 0.8 pmol of CsA completely prevented this TPA effect. Downregulation of PKC was suppressed in part also by 3.5 pmol of cycloheximide (52%) and by 2.9 pmol of the catpain inhibitor E64d (32%) 18 h after treatment (data not shown). Staurosporine by itself did not induce down regulation of PKC. Other compounds ineffective in inducing PKC down-regulation were the ionophore A23187 (0.1 pmol), the phorbol ester 4-0-methyl-Tl?A (0.4 pmol; inactive as tumor promoter in NMRI mouse skin) and dioctanoylglycerol (1 pmol) (data not shown). In order to test the requirement of PKC for the induction of an ear edema, the ears were pretreated with TPA for 23 h leading to a reduction of PKC by 80% (see Fig. 2). At this time point the edema induced by TPA had
decreased by 60% (Fig. 1). A second TPA treatment for 6 h did not result in reinducing the ear edema (Table I). When the ears were pretreated with okadaic acid for 23 h no downregulation of PKC was observed (see Fig. 3) and a subsequent TPA treatment for 6 h completely reinduced the edema (Table I).
Dismssion It is well known that the phorbol ester TPA binds with high affinity to PKC and activates the enzyme for the phosphorylation of substrate proteins [ 11. Therefore, biological effects of TPA have always been interpreted in the literature as a consequence of stimulated PKC activity. In no case, however, was the involvement of PKC-catalyzed protein phosphorylation unequivocally proved. In accordance with a previous report [2], we obtained a number of data indicating that TPA induces certain biological effects in mouse skin independently of the activation of PKC, at least of staurosporine-sensitive PKC. Bryostatin 1, a macrocyclic lactone [7] structurally unrelated to TPA, activates Ca*+responsive as well as unresponsive PKC isoenzymes as effectively as TPA (see Ref. 8; Gschwendt et al., unpublished) and, like TPA, induces down-regulation of the enzyme in mouse epidermis. However, unlike TPA, bryostatin is unable to induce the formation of an early edema (with a maximum at 6 h after treatment) of the mouse ear. An edema begins to develop around 8 h after treatment with bryostatin, i.e. at a time when PKC is downregulated by about 70% (data not shown). Several other biological effects induced by TPA in mouse skin, including tumor promotion, are not seen at all after bryostatin treatment [9, lo] indicating that activation of PKC is at least not sufficient for the induction of these effects. Staurosporine, the most potent inhibitor of PKC activity available at present [ 191, is unable to suppress the TPA-induced edema and down-regulation of PKC. In this context it is important to note that staurosporine is able to
144
penetrate the mouse skin and to act as an inhibitor in vivo since it very effectively suppresses the okadaic acid-induced edema and, as shown by Chida et al. [20], inhibits the TPA-stimulated phosphorylation of two epidermal proteins in vivo. In contrast to its derivative K252a [21], staurosporine suppresses the Ca2+ -responsive c~,fi,rPKC, the Ca2+-unresponsive GPKC, PKC [22] and EPKC [23] equally well. Therefore, it is unlikely that its inability to suppress some TPAeffects is due to differences in the inhibition of PKC isoenzymes. However, no data on the inhibition of 7PKC by staurosporine are available as yet. Staurosporine has been reported to paradoxically induce PKC agonist effects [4,24,25]. Therefore, it may be argued that an inhibiting effect of staurosporine on TPA activity might be superimposed by an agonistic action resulting in an apparent lack of inhibition. However, staurosporine neither induces an early edema nor down-regulation of PKC. Moreover, we recently found that several synthetic staurosporine derivatives inhibiting PKC as strongly as staurosporine but with a much higher specificity and not inducing PKC agonist effects, did not suppress TPA-induced ear edema, epidermal DNA synthesis and tumor promotion. However, the okadaic acid induced edema was inhibited by these compounds (Gschwendt et al., unpublished). Pretreatment with TPA, but not with okadaic acid, renders mouse skin insensitive to TPA. This could be interpreted as a consequence of down-regulation of PKC and would indicate that at least the intact protein PKC, yet not necessarily its enzyme activity, is required for the action of TPA in mouse skin. In this context it is intriguing that, according to Kahn et al. [26], cells only overexpressing the regulatory domain of PKC@i show a dramatic morphologic response to TPA. Furthermore, it is possible that, beside PKC, other components essential for the action of TPA are downregulated as well. There is some evidence for the existence of non-PKC target proteins for TPA [27 - 291. It has to be emphasized in this context that the PKC inhibitors of the
staurosporine family seem to interact with the catalytic site rather than with the TPA/DAGbinding site of the enzyme [30]. Thus, any interaction of TPA with non-PKC target proteins may be expected to be staurosporineinsensitive. We conclude from these results that protein phosphorylation catalyzed by staurosporinesensitive PKC is not only insufficient but not even essential for TPA-induced edema, downregulation of PKC and possibly also other TPA-induced effects on mouse skin. Furthermore, okadaic acid and TPA might induce an edema via different routes, one being sensitive and the other insensitive to staurosporine. Since okadaic acid inhibits protein phosphatases [5,6], it can be expected to indirectly allow increased protein phosphorylation. However, the inhibition by staurosporine cannot be considered as an indication for the type of protein kinases possibly involved in the formation of the okadaic acid-induced edema, since beside PKC staurosporine also inhibits other kinases [30,31]. On the other hand, staurosporine derivatives more specific for PKC (see above) also inhibited okadaic acidinduced edema indicating PKC to play a role in this process (Gschwendt et al., unpublished). The TPA-induced edema as well as the down-regulation of PKC are not affected by staurosporine but are effectively inhibited by calmodulin antagonists such as CsA. We found previously that CsA [15,17,32,33] and other compounds acting as calmodulin antagonists [34,35] inhibit most if not all biological effects of TPA on mouse skin, including tumor promotion. In this context it is important to note that, contrary to many other calmodulin antagonists, CsA is absolutely ineffective in suppressing PKC activity (see Table II). Thus, a calmodulin-dependent process, possibly a Ca2+/calmodulin dependent, staurosporine-insensitive protein phosphorylation might play an essential role in the TPAinduced effects. This hypothesis is supported by a recent report of Chuprun et al. [36] on TPA induced smooth muscle contraction that can be inhibited by calmodulin antagonists
145
but not by staurosporine. In this context it is intriguing that in contrast to CsA [15,17] staurosporine barely inhibits the Ca2+/ calmodulin-dependent protein kinase III (I&: 10m5 M; Gschwendt et al., unpublished), which specifically catalyzes the phosphorylation of the elongation factor 2. Since this reaction seems to provide an early key event in the induction of de-novo protein synthesis, in particular of that related to mitogensis [37], it may well play a critical role also in the hyperplasticinflammatory response of skin to TPA [38]. The okadaic acid-induced edema is also inhibited by the calmodulin antagonists, indicating the initially different routes of the TPA- and okadaic acid-induced edema to meet at a common calmodulin-dependent stage. Acknowledgements The authors are grateful to Dr. G.R. Pettit, Arizona State University, Tempe, USA, to the Taisho-Pharmaceutical Company, Japan and to Sandoz, Basel, Switzerland for supplying bryostatin 1, E64d and cyclosporine A, respectively. This work was supported by the Mildred Scheel-Stiftung.
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References Nishizuka, Y. (1988) The molecular heterogeneity of protein kinase C and its implications for cellular regulation. Nature, 334, 661- 665 Yamamoto, S., Kiyoto, I., Aizu, E., Nakadate, T., Hosoda, T., Hosoda, Y. and Kato, R. (1989) Differential inhibition by staurosporine, a potent protein kinase C inhibitor, of 12.O-tetradecanoylphorbol-13-acetate-caused skin tumor promotion, epidermal ornithine decarboxylase induction, hyperplasia and inflammation. Carcinogenesis, 10, 1315- 1322. Kiyoto, I., Yamamoto, S., Aizu, E. and Kato, R. (1987) Staurosporine, a poent protein kinase C inhibitor fails to inhibit 12 - 0-tetradecanoylphorbol-1 3-acetate-caused ornithine decarboxylase induction in isolated mouse epidermal cells. Biochem. Biophys. Res. Commun., 148, 740 - 746. Sako, T.. Tauber, A.I., Jeng, A.Y.. Yuspa, S.H. and actions of P.M. (1988) Contrasting Blumberg, staurosporine, a prfotein kinase C inhibitor, on human neutrophils and primary mouse epidermal cells. Cancer Res., 48, 4646 - 4650.
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Suganuma, M., Fujiki. H., Suguri, H., Yoshizawa, S., Hirota, M., Nakayasu, M., Ojika, M., Watamatsu, K., Yamada, K. and Sugimura, T. (1988) Okadaic acid: an additional non phorbol-12.tetradecanoate-13-acetate type tumor promoter. Proc. Natl. Acad. Sci. U.S.A., 85, 1768- 1771. Cohen P., Holmes C.F.B. and Tsukitani, Y. (1990) Okadaic acid: a new probe for the study of cellular regulation. Trends Biochem. Sci, 15, 98- 102. Pet-tit, G.R., Herald, C.L., Doubeck, D.L. and Herald, D.L. (1982) Isolation and structure of bryostatin 1. J. Am. Chem. Sot., 104, 6846-6848. Kraft, A.S., Smith, J.B. and Berkow, R.L. (1986) Bryostatin, an activator of the calcium phospholipiddependent protein kinase, blocks phorbol ester-induced differentiaiton of human promyelocytic leukemia cells HL60. Proc. Natl. Acad. Sci. U.S.A., 83, 1334- 1338. Gschwendt, M., Fiirstenberger, G., Rose-John, S., Rogers, M., Kittstein, W., Pettit, G.R., Herald, C.L. and Marks F. (1988) Bryostatin 1, an activator of protein kinase C, mimics as well as inhibits biological effects of the phorbol ester TPA in viva and in vitro. Carcinogenesis. 9, 555 - 562. Hennings, H., Blumberg, P.M., Pettit, G.R., Herald, C.L., Shores, R. and Yuspa, S.H. (1987) Bryostatin 1, an activator of protein kinase C, inhibits tumor promotion by phorbol esters in SENCAR mouse skin. Carcinogenesis, 8, 1343 - 1346. Gschwendt, M., Kittstein, W., Ftirstenberger, G. and Marks, F. (1984) The mouse ear edema: a quantitatively evaluable assay for tumor promoting compounds and for inhibitors of tumor promotion. Cancer Lett., 25, 177- 185. Krieg, L., Ktihlmann, I. and Marks, F. (1974) Effect of tumor-promoting phorbol esters and of acetic acid on mechanisms controlling DNA synthesis and mitosis (chalones) and on biosynthesis of histidine rich protein in mouse epidermis. Cancer Res., 34, 3135 - 3146. Gschwendt, M., Kittstein, W., Horn, F., Leibersperger, H. and Marks, F. (1989) A phorbol ester and phospholipidactivated, calcium-unresponsive protein kinase in mouse epidermis: characterization and separation from protein kinase C. J. Cell. Biochem., 40, 295-307. Leibersperger, H., Gschwendt, M., Gernold, M. and Marks, F. (1991) Immunological demonstration of a calcium-unresponsive protein kinase C of the b-type in different species and murine tissues. Predominance in epidermis. J. Biol. Chem., 266, 1477814784. Gschwendt, M., Kittstein, W. and Marks, F. (1988) Ciclosporin inhibits phorbol ester-induced hyperplastic transformation and tumor promotion in mouse skin probproably by suppression of Ca 2+ /calmodulin-dependent cesses such as phosphorylation of elongation factor 2. Skin Pharmacol., 1, 84 - 92. Van Belle, H. (1981) R24571: a potent inhibitor of calmodulin activated enzymes. Cell Calcium 2, 483 -494. Gschwendt, M., Kittstein, W. and Marks, F. (1988) The weak immunosuppressant cyclosporine D as well as the
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immunologically inactive cyclosporine H are potent inhibitors in vivo of phorbol ester TPA-induced biological effects in mouse skin and of Ca2+/calmodulin-dependent EF-2 phosphorylation in vitro. Biochem. Biophys. Res. Commun., 150, 545-551. Kuroki, T., Hashimoto, Y., Osada, S. Tajima, O., Nose, K. and Ohno. S. (1991) Predominant expression of Ca2+-independent protein kinase C isoforms, nPKCq and nPKCG in mouse skin. J. Cell. Biochem.. Suppl. 15B, p. 159. Tamaoki, T., Komoto, H., Takahashi, I., Kato, Y ., Morimoto, M. and Tonita, F. (1986) Staurosporine, a potent inhibitor of phospholipid/Ca2+-dependent protein kinase. Biochem. Biophys. Res. Commun., 135, 397 - 402. Chida, K., Yamada, S., Katao, N. and Kuroki, T. (1988) Phosphorylation of M, 34 000 and 40 000 proteins by protein kinase C in mouse epidermis in vivo. Cancer Res., 48, 4018-4023. Gschwendt, M., Leibersperger, H. and Marks, F. (1989) Differentiative action of K252a on protein kinase C and a calcium-unresponsive, phorbol ester/phosphilipidactivated protein kinase. Biochem. Biophys. Res. Commun., 164, 974-982. Gschwendt, M., Leibersperger, H., Kittstein, W. and Marks, F. (1992) Protein kinase Ce and ri in murine epidermis. TPA induces down-regulation of PKCv but not of PKCt. Fed. Eur. Biochem. Sot. Lett.. in press. Koide, H., Ogita, K., Kikkawa, U. and Nishizuka, Y. (1992) Isolation and characterization of the t subspecies of protein kinase C from rat brain. Proc. Natl. Acad. Sci. U.S.A., 89. Dlugosz, A.A. and Yuspa, S.H. (1991) Staurosporine induces protein kinase C agonist effects and maturation of normal and neoplastic mouse keratinocytes in vitro. Cancer Res., 51, 4677-4684. Yoshiiawa, S., Fujiki, H., Suguri, H., Suganuma, M., Nakayasu, M., Matsushima. R. and Sugimura, T. (1990) Tumor-promoting activity of staurosporine, a protein kinase inhibitor on mouse skin. Cancer Res., 50, 4974 - 4978. Kahn, S.M., O’Driscoll, K.R., Blackwood, M.A., Jiang, W. and Weinstein, LB. (1991) Overexpression of rat PKCp, regulatory domain causes the disordered growth of rat 6 fibroblasts. Abstract A-30, book of abstracts of the First Joint Conference of the American Association for Cancer Research and the European Association for Cancer Research, Santa Margherita, Italy. Ahmed, S., Kozma, R., Monfries, C., Hall, C., Lim, H.H., Smith, P. and Lim, L. (1990) Human brain
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n-chimaerin cDNA encodes a novel phorbol ester receptor. Biochem. J. 272, 767-773. Maruyama, I.N. and Brenner, S. (1991) A phorbol ester/diacylglycerol-binding protein encoded by the unc13 gene of Caenorhobditis elegans. Proc. Natl. Acad. Sci. U.S.A., 88, 5729-5733. Hashimoto, Y. and Shudo, K. (1990) Cytosolic-nuclear tumor promoter-specific binding protein (CN-TDBP) in human promyeolocytic leukemia cells HL-60. Biochem. Biophys. Res. Commun.. 166, 1126- 1132. Riiegg, U.T. and Burgess, G.M. (1989) Staurosporine, K252 and UCN 01: potent but nonspecific inhibitors of protein kinases. Trends Biochem. Sci., 10, 218-220. O’Brian, C.A. and Ward, N.E. (1990) Staurosporine: a prototype of a novel class of inhibitors of tumor cell invasion? J. Natl. Cancer Inst., 82, 1734- 1735. Gschwendt, M., Kittstein, W.. Horn, F. and Marks, F. (1985) Cyclosporin A inhibits biological effects of tumor promoting phorbol esters. Biochem. Biophys. Res. Commun., 126, 327-332. Gschwendt, M., Kittstein W. and Marks, F. (1987) Cyclosporin A inhibits phorbol ester-induced cellular proliferation and tumor promotion as well as phosphorylation of a lOO- kDa protein in mouse epidermis. Carcinogenesis, 8, 203 - 207. Gschwendt, M., Kittstein W. and Marks, F. (1987) Didemnin B inhibits biological effects of tumor promoting phorbol esters on mouse skin, as well as phosphorylation of a 100 kDa protein in mouse epidermis cytosol. Cancer Len., 34, 187- 191. Gschwendt, M., Kittstein W. and Marks, F. (1989) The immunosuppressant FK-506, like cyclosporines and didemnin B, inhibits calmodulin-dependent phorphorylation of the elongation factor 2 in vitro and biological effects of the phorbol ester TPA on mouse skin in vivo. Immunobiol., 179, 1-7. Chuprun, J.K., Bazan, E., Chang, K.-C., Campbell, A.K. and Rapoport, R.M. (1991) Inhibition of phorbol esterinduced contraction by calmodulin antagonists in rat aorta. Am. J. Physiol., 261, C675-C684. Palfrey, H.C., Nairn, A.C., Muldoon, L.L. and Villereal, M.L. (1987) Rapid activation of calmodulin-dependent protein kinase III in mitogen-stimulated human fibroblasts. J. Biol. Chem., 262, 9785-9792. Gschwendt, M.. Kittstein, W. and Marks, F. (1988d) Effect of tumor promoting phorbol ester TPA on epidermal protein synthesis: stimulation of an elongation factor 2 phosphatase activity by TPA in vivo. Biochem. Biophys. Res. Commun., 153, 1129- 1135.
139
Differential inhibition by staurosporine of phorbol bryostatin and okadaic acid effects on mouse skin Michael Gschwendt, Germon Cancer
Research
Walter Kittstein, Dieter Lindner Center,
Im Neuenheimer
ester,
and Friedrich Marks
Fe/d 280, D-6900 Heidelberg
(FRG)
(Received 11 May 1993 (Revision received 15 July 1992) (Accepted 15 July 199il)
Summary
The tumor promoters 12-O-tetradecanoylphorbol-13-acetate (TPA), a strong actiuator of protein kinase C (PKC) and okadaic acid, which is ineffectiue
in this respect,
induce
a
rapidiy developing (‘early’) edema of the mouse ear. Bryostatin, another potent activator of PKC, is unable to induce an ‘early’ edema but causes o more delayed deuelopment of edema at a time when most of the PKC is down-regulated. The PKC inhibitor staurosporine neither inhibits the early TPAnor the late bryodatin-induced edema, but suppresses the okadaic acid-induced edema very effectively. TPA as well as bryostatin, but not okadaic acid cause a down-regulation of PKC, which is not inhibited by staurosporine. The calmodulin antagonist cyclosporine A, which does not suppress PKC activity, very effectively inhibits the TPA-induced edema and down regulation of PKC. Hence we conclude that protein phosphory.ylation catalyzed by staurosporine-suppressable PKC is not inuolued in the induction of edema and PKC downCorrespondence to: Michael Gschwendt, German Research Center, Im Neuenheimer Feld 280, Heidelberg, FRG. Abbreviations: PKC, protein tetradecanoylphorbol-13-acetate; 0304-3835/92/$05.00 Printed and Published
kinase
C;
CsA, cyclosporin
TPA, A.
regulation by TPA but that a calmodulin dependent process may play a critical role in these and other TPA effects in mouse skin.
Cancer D-6900 12-O-
@ 1992 Elsevier Scientific Publishers in Ireland
Keywords: 12-0-tetradecanoylphorbol-13acetate; protein kinase C; okadic acid; early edema; bryostatin; staurosporine Introduction The protein kinase C (PKC) family, a group of several isoenzymes, plays a central role in intracellular signal transduction [ l] . Physiological activation of PKC is achieved by the second messenger diacylglycerol produced from phosphoinositides as a consequence of a receptor-mediated activation of phospholipase C. Tumor-promoting phorbol esters such as 12-0-tetradecanoylphorbol-13-acetate (TPA) have been shown to be much more potent activators of PKC than diacylglycerol [l]. Therefore, it is generally assumed that the numerous biological effects of TPA, including tumor promotion, are due to an activation of PKC resulting in the phosphorylation of certain substrate proteins. Recently, however, it was reported that the potent PKC inhibitor staurosporine was unable to suppress several effects of TPA on mouse skin [2] and in other Ireland Ltd
140
systems [3,4]. Here we compare the biological effects on mouse skin (edema formation and down-regulation of PKC) of two tumorpromoting compounds, one acting as a PKC activator (TPA) and the other ineffective in this respect (okadaic acid) [5,6] and of a nontumor-promoting PKC activator (bryostatin) [7 - lo] and their inhibition by PKC inhibitors as well as by calmodulin antagonists. The results indicate that TPA might induce some biological effects independently of protein phosphorylation catalyzed by staurosporinesensitive PKC but via a calmodulin-dependent process. Methods
and Materials
Materials TPA and 4-0-methyl-TPA were kindly provided by Dr. E. Hecker, German Cancer Research Center, Heidelberg, FRG. Bryostatin 1 (bryostatin) was a generous gift of Dr. G .R. Pettit, State University of Arizona, Tempe, USA. Okadaic acid and staurosporine were from Boehringer, Mannheim, FRG. Cyclosporine A (CsA) and the calpain inhibitor E64d were kindly provided by Sandoz, Basel, Switzerland and Taisho-Pharmaceutical Co., Ltd., Japan, respectively. The calmodulin antagonist R2457 1, the ionophore A23 187, dioctanoylglycerol, phosphatidyl serine and histone III-S were from Sigma, Munich, FRG. [y-32P]ATP (spec.act. 3000 Ci/mmol) was from NEN (Waltham, MA, USA). Animals Female NMRI mice (7 weeks old) were used and fed a standard diet ad libitum. The back skin was shaved 3 days before treatment. Methods Ear edema. A potential inducer of edema alone or together with an inhibitor was dissolved in acetone and applied at a volume of 20 ~1 to the outer surface of both mouse ears. Controls were treated with acetone alone. Edema were determined on the basis of ear plug weight as described previously [l 11.
Epidermis extracts. The shaved back skin of mice was treated with various compounds dissolved in 0.1 ml of acetone. The animals were sacrificed at the times indicated and epidermal specimens were prepared from frozen back skin as described previously [ 121. Epidermis was homogenized in a buffer containing Triton X-100 (20 mM Tris/HCl, pH 7.5,5 mM EGTA, 2mM EDTA, 1 mM PMSF, 50 mM 2-mercaptoethanol, 0.2% Triton X100) and applied to SDS polyacrylamide gel electrophoresis. lmmunoblotting Immunoblots using an cr,P,-yPKC-specific antiserum and 35S-labelled anti-rabbit-IgG as second antibody were performed as described previously [ 13,141. The immunoblots were visualized by autoradiography and quantitated by densitometry. Phosphorylation
of histone 111-S with
wP,rPKC
The assay mixture (total volume: 100 ~1) contained 40 ~1 Tris buffer (50 mM Tris - HCl, pH 7.5, 10 mM mercaptoethanol), 5 ~1 ATP in Tris-buffer (250 ~1 of 750 PM ATP + 10 ~1 of 1.7 TM [y-32P]ATP), 35 pg phosphatidyl serine, 4 mM MgC12, 60 pg histone III-S, lo-‘M TPA, 5 ~1 c~,fl,rPKC purified from mouse brain [13]. After the addition of ATP, the mixture was incubated at 30°C for 7 min. Fifty-microliter aliquots were then dropped onto 20-mm square pieces of phosphocellulose paper (Whatman p81), which were washed once with deionized water, once with 5% sodium pyrophosphate 9pH 8.0)) twice with deionized water, and once each with acetone and petrol ether. The radioactivity on each piece of paper was determined by scintillation counting. Results As reported earlier [ll], treatment of the mouse ear with 1 hmol of the tumorpromoting and PKC-activating phorbol ester TPA induced the formation of an edema. The
141
effect was maximal 6 h after treatment with TPA (Fig. 1). Okadaic acid, another tumor promoter with a structure entirely different from that of TPA and without PKC-activating capacity [5,6], also caused the development of an edema (Fig. 1). The time course of the okadaic acid-induced edema was identical with that of the TPA-induced edema. Upon application of bryostatin, another PKC-activator
300
250
2 -if zoo -2 %
150
% 2 .y
100
; 50
1
0 0
,
5
10 Hour!;
Table 1.
Induction
after
15
I
I
20
25
treatment
and inhibition of the mouse
Treatment
ear edema
Fig. 1. Induction of a mouse ear edema by TPA (A), okadaic acid (A) and bryostatin (W). Mice were treated with 1 nmol of the inducing compound dissolved in 20 ~1 acetone and the weight of the ear plug was determined as a measure of the edema at the indicated times after treatment as described previously [ll]. The values are given as percent of the control (treatment with the solvent acetone alone; 100% = 11.43 mg) and are the mean of at least two determinations.
by various compounds. Edema
(weight of ear plug)
(%)
Induction
Acetone (control) 1 nmol TPA (6h) 1 nmo] OkA (6h) 1 nmol Bry (13h) 10 nmol St (6h) 10 nmol St (19h) 1 nmo] TPA (23 h) followed by 1 nmo] TPA (6 h) 1 nmo] OkA (23 h) followed by 1 nmo] TPA (6h) 1 nmol TPA, 10 nmo] !St (6h) 1 nmol Bry, 10 nmol St (13h) 1 nmol TPA, 42 nmo] GA (6h) 1 nmol TPA, 72 nmo] Ii,,,,, (6h) 1 nmol OkA, 1 nmol St (6h) 1 nmol OkA, 5 nmo] St (6h) 1 nmol OkA, 10 nmol St (6h) 1 nmol OkA, 42 nmol ‘CsA (6h) 1 nmol OkA, 72 nmo] Rz4 571 (6h)
100 260 248 262 100 152
f 7 ziz 19 zt 16 ziz 20 ziz 6 ziz 10
lOO* 257
l
-
Inhibition of induction (%) -
6
-
20
0*2 O*l 93 + 92 f 67 f 82 f 100 f 100 f 85 f
6 4 4 5 3 5 4
The mouse ear was treated with an inducing compound alone or together with an inhibitor dissolved in acetone and the weight of the ear plug as a measure of the edema was determined as described previously [ll]. The duration of treatment is given in brackets. Induction of the edema is given as percent of control (acetone alone; 100%) and inhibition as percent of reduction of the induced edema (no reduction: 0% ; reduction to the control: 100%). The values are the mean of at least two determinations. OKA, okadaic acid; Bry, bryostatin; St, staurosporine.
142
Table 11. Activity of TPA-stimulated presence of CsA.
without tumor-promoting capacity [7 - 101, not even the slightest formation of an edema could be observed after 6 h. Instead, edema developed with a delayed kinetic maximal around 13 h after treatment (Fig. 1). As shown in Table I, 10 nmol of the PKC inhibitor staurosporine were unable to inhibit the TPA-and bryostatin-induced ear edema at 6 h and 13 h, respectively, i.e. at the time of maximal edema formation. At the same concentration, staurosporine suppressed the okadaic acid-induced edema completely. Even lower concentrations of staurosporine were effective in this respect. 1 nmol of staurosporine still caused a 67% inhibition. The calmodulin antagonists cyclosporine A (CsA) [15] and R24 571 (Van Belle, 1981) effectively inhibited both the TPA-and the okadaic acidinduced edema (Table I). Compared with staurosporine higher concentrations of CsA (42 nmol) and R24571 (72 nmol) were required though, as was expected from the I&, values obtained in cell-free systems [16,17]. Staurosporine alone did not induce an edema comparable to that seen after TPA, okadaic acid or bryostatin treatment. Only 19 h after staurosporine application a weak edema was observed (Table I). The calmodulin antagonist CsA was found to be absolutely inactive as an inhibitor of PKC activity. In a cell-free system,
t o--i 0
1
1 ‘0
LO
30
0 lo-’ 1O-6 1O-5 1O-4
Time
50
60
M M M M
27 25 27 26 28
130 740 810 930 100
(counts/min)
f 1970 zt 1430 ztz 690 zt 1840 f 1580
Phosphorylation of histone Ill-S with purified cr,@,yPKC and [Y-~‘P]ATP was performed in the presence of phosphatidyl serine and TPA as described in Methods. Various concentrations of CsA dissolved in acetone were added as indicated. Incorporation of 32P into histone is given as counts per minute (counts/min). The value for unstimulated kinase activity (without TPA) was 5430 counts/min. Values are the mean of two determinations.
up to 10 -4M CsA did not at all suppress the phosphorylation of histone III-S catalyzed by TPA-stimulated purified a,P,yPKC (Table II). Treatment of the back skin of mice with 10 nmol TPA caused down regulation of PKC in the epidermis as determined by immunoblotting using an a,P,yPKC-specific antiserum (Fig. 2). Lowest levels of (r,PPKC (mouse epidermis does not contain yPKC [18] were reached between 8 and 15 h after TPA (20% of control). Recovery of PKC was rather slow.
_i
.A-
40
PKC-activity
CsA
c~,fl,-rPKC in the
70
80
[h]
TPA-induced down-regulation of PKC in mouse epidermis. The shaved back skin of mice was treated with Fis. 2. 10 nmol of TPA dissolved in 0.1 ml acetone. At the times indicated the relative amount of PKC was determined in epidermal extracts by immunoblotting using an cy,@,yPKC-specific antiserum and an 35S-labelled anti-rabbit-IgG as second antibody as described in Methods. The values are the mean of two determinations.
143
C
TPA
Bry
Induction Fig. 3. regulation in mouse Animals were treated as described in Fig. Leibersperger et al. acid; Bry, bryostatin;
OkA
St
TPA +St
TPA +CsA
and inhibition of PKC downepidermis by various compounds. and immunoblots were performed 2 and by Gschwendt et al. [13] [la], respectively. OkA, okadaic St, staurosporine.
Even 72 h after TPA the epidermis contained only 40% of the amount of the normal PKC level. Previously we had shown that GPKC, a predominant PKC isoenzyme in mouse epidermis, is also down-regulated as a consequence of TPA treatment of the animals [14]. However, GPKC recovered much faster, almost reaching the control level 72 h after TPA. As shown in Fig. 3, down-regulation of (r,PPKC in mouse epidermis was also induced by 10 nmol of bryostatin, but not by 10 nmol of okadaic acid. Staurosporine (1 pmol) was unable to inhibit the TPA-induced downregulation of PKC, whereas 0.8 pmol of CsA completely prevented this TPA effect. Downregulation of PKC was suppressed in part also by 3.5 pmol of cycloheximide (52%) and by 2.9 pmol of the catpain inhibitor E64d (32%) 18 h after treatment (data not shown). Staurosporine by itself did not induce down regulation of PKC. Other compounds ineffective in inducing PKC down-regulation were the ionophore A23187 (0.1 pmol), the phorbol ester 4-0-methyl-Tl?A (0.4 pmol; inactive as tumor promoter in NMRI mouse skin) and dioctanoylglycerol (1 pmol) (data not shown). In order to test the requirement of PKC for the induction of an ear edema, the ears were pretreated with TPA for 23 h leading to a reduction of PKC by 80% (see Fig. 2). At this time point the edema induced by TPA had
decreased by 60% (Fig. 1). A second TPA treatment for 6 h did not result in reinducing the ear edema (Table I). When the ears were pretreated with okadaic acid for 23 h no downregulation of PKC was observed (see Fig. 3) and a subsequent TPA treatment for 6 h completely reinduced the edema (Table I).
Dismssion It is well known that the phorbol ester TPA binds with high affinity to PKC and activates the enzyme for the phosphorylation of substrate proteins [ 11. Therefore, biological effects of TPA have always been interpreted in the literature as a consequence of stimulated PKC activity. In no case, however, was the involvement of PKC-catalyzed protein phosphorylation unequivocally proved. In accordance with a previous report [2], we obtained a number of data indicating that TPA induces certain biological effects in mouse skin independently of the activation of PKC, at least of staurosporine-sensitive PKC. Bryostatin 1, a macrocyclic lactone [7] structurally unrelated to TPA, activates Ca*+responsive as well as unresponsive PKC isoenzymes as effectively as TPA (see Ref. 8; Gschwendt et al., unpublished) and, like TPA, induces down-regulation of the enzyme in mouse epidermis. However, unlike TPA, bryostatin is unable to induce the formation of an early edema (with a maximum at 6 h after treatment) of the mouse ear. An edema begins to develop around 8 h after treatment with bryostatin, i.e. at a time when PKC is downregulated by about 70% (data not shown). Several other biological effects induced by TPA in mouse skin, including tumor promotion, are not seen at all after bryostatin treatment [9, lo] indicating that activation of PKC is at least not sufficient for the induction of these effects. Staurosporine, the most potent inhibitor of PKC activity available at present [ 191, is unable to suppress the TPA-induced edema and down-regulation of PKC. In this context it is important to note that staurosporine is able to
144
penetrate the mouse skin and to act as an inhibitor in vivo since it very effectively suppresses the okadaic acid-induced edema and, as shown by Chida et al. [20], inhibits the TPA-stimulated phosphorylation of two epidermal proteins in vivo. In contrast to its derivative K252a [21], staurosporine suppresses the Ca2+ -responsive c~,fi,rPKC, the Ca2+-unresponsive GPKC, PKC [22] and EPKC [23] equally well. Therefore, it is unlikely that its inability to suppress some TPAeffects is due to differences in the inhibition of PKC isoenzymes. However, no data on the inhibition of 7PKC by staurosporine are available as yet. Staurosporine has been reported to paradoxically induce PKC agonist effects [4,24,25]. Therefore, it may be argued that an inhibiting effect of staurosporine on TPA activity might be superimposed by an agonistic action resulting in an apparent lack of inhibition. However, staurosporine neither induces an early edema nor down-regulation of PKC. Moreover, we recently found that several synthetic staurosporine derivatives inhibiting PKC as strongly as staurosporine but with a much higher specificity and not inducing PKC agonist effects, did not suppress TPA-induced ear edema, epidermal DNA synthesis and tumor promotion. However, the okadaic acid induced edema was inhibited by these compounds (Gschwendt et al., unpublished). Pretreatment with TPA, but not with okadaic acid, renders mouse skin insensitive to TPA. This could be interpreted as a consequence of down-regulation of PKC and would indicate that at least the intact protein PKC, yet not necessarily its enzyme activity, is required for the action of TPA in mouse skin. In this context it is intriguing that, according to Kahn et al. [26], cells only overexpressing the regulatory domain of PKC@i show a dramatic morphologic response to TPA. Furthermore, it is possible that, beside PKC, other components essential for the action of TPA are downregulated as well. There is some evidence for the existence of non-PKC target proteins for TPA [27 - 291. It has to be emphasized in this context that the PKC inhibitors of the
staurosporine family seem to interact with the catalytic site rather than with the TPA/DAGbinding site of the enzyme [30]. Thus, any interaction of TPA with non-PKC target proteins may be expected to be staurosporineinsensitive. We conclude from these results that protein phosphorylation catalyzed by staurosporinesensitive PKC is not only insufficient but not even essential for TPA-induced edema, downregulation of PKC and possibly also other TPA-induced effects on mouse skin. Furthermore, okadaic acid and TPA might induce an edema via different routes, one being sensitive and the other insensitive to staurosporine. Since okadaic acid inhibits protein phosphatases [5,6], it can be expected to indirectly allow increased protein phosphorylation. However, the inhibition by staurosporine cannot be considered as an indication for the type of protein kinases possibly involved in the formation of the okadaic acid-induced edema, since beside PKC staurosporine also inhibits other kinases [30,31]. On the other hand, staurosporine derivatives more specific for PKC (see above) also inhibited okadaic acidinduced edema indicating PKC to play a role in this process (Gschwendt et al., unpublished). The TPA-induced edema as well as the down-regulation of PKC are not affected by staurosporine but are effectively inhibited by calmodulin antagonists such as CsA. We found previously that CsA [15,17,32,33] and other compounds acting as calmodulin antagonists [34,35] inhibit most if not all biological effects of TPA on mouse skin, including tumor promotion. In this context it is important to note that, contrary to many other calmodulin antagonists, CsA is absolutely ineffective in suppressing PKC activity (see Table II). Thus, a calmodulin-dependent process, possibly a Ca2+/calmodulin dependent, staurosporine-insensitive protein phosphorylation might play an essential role in the TPAinduced effects. This hypothesis is supported by a recent report of Chuprun et al. [36] on TPA induced smooth muscle contraction that can be inhibited by calmodulin antagonists
145
but not by staurosporine. In this context it is intriguing that in contrast to CsA [15,17] staurosporine barely inhibits the Ca2+/ calmodulin-dependent protein kinase III (I&: 10m5 M; Gschwendt et al., unpublished), which specifically catalyzes the phosphorylation of the elongation factor 2. Since this reaction seems to provide an early key event in the induction of de-novo protein synthesis, in particular of that related to mitogensis [37], it may well play a critical role also in the hyperplasticinflammatory response of skin to TPA [38]. The okadaic acid-induced edema is also inhibited by the calmodulin antagonists, indicating the initially different routes of the TPA- and okadaic acid-induced edema to meet at a common calmodulin-dependent stage. Acknowledgements The authors are grateful to Dr. G.R. Pettit, Arizona State University, Tempe, USA, to the Taisho-Pharmaceutical Company, Japan and to Sandoz, Basel, Switzerland for supplying bryostatin 1, E64d and cyclosporine A, respectively. This work was supported by the Mildred Scheel-Stiftung.
5
6
7
8
9
10
11
12
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Suganuma, M., Fujiki. H., Suguri, H., Yoshizawa, S., Hirota, M., Nakayasu, M., Ojika, M., Watamatsu, K., Yamada, K. and Sugimura, T. (1988) Okadaic acid: an additional non phorbol-12.tetradecanoate-13-acetate type tumor promoter. Proc. Natl. Acad. Sci. U.S.A., 85, 1768- 1771. Cohen P., Holmes C.F.B. and Tsukitani, Y. (1990) Okadaic acid: a new probe for the study of cellular regulation. Trends Biochem. Sci, 15, 98- 102. Pet-tit, G.R., Herald, C.L., Doubeck, D.L. and Herald, D.L. (1982) Isolation and structure of bryostatin 1. J. Am. Chem. Sot., 104, 6846-6848. Kraft, A.S., Smith, J.B. and Berkow, R.L. (1986) Bryostatin, an activator of the calcium phospholipiddependent protein kinase, blocks phorbol ester-induced differentiaiton of human promyelocytic leukemia cells HL60. Proc. Natl. Acad. Sci. U.S.A., 83, 1334- 1338. Gschwendt, M., Fiirstenberger, G., Rose-John, S., Rogers, M., Kittstein, W., Pettit, G.R., Herald, C.L. and Marks F. (1988) Bryostatin 1, an activator of protein kinase C, mimics as well as inhibits biological effects of the phorbol ester TPA in viva and in vitro. Carcinogenesis. 9, 555 - 562. Hennings, H., Blumberg, P.M., Pettit, G.R., Herald, C.L., Shores, R. and Yuspa, S.H. (1987) Bryostatin 1, an activator of protein kinase C, inhibits tumor promotion by phorbol esters in SENCAR mouse skin. Carcinogenesis, 8, 1343 - 1346. Gschwendt, M., Kittstein, W., Ftirstenberger, G. and Marks, F. (1984) The mouse ear edema: a quantitatively evaluable assay for tumor promoting compounds and for inhibitors of tumor promotion. Cancer Lett., 25, 177- 185. Krieg, L., Ktihlmann, I. and Marks, F. (1974) Effect of tumor-promoting phorbol esters and of acetic acid on mechanisms controlling DNA synthesis and mitosis (chalones) and on biosynthesis of histidine rich protein in mouse epidermis. Cancer Res., 34, 3135 - 3146. Gschwendt, M., Kittstein, W., Horn, F., Leibersperger, H. and Marks, F. (1989) A phorbol ester and phospholipidactivated, calcium-unresponsive protein kinase in mouse epidermis: characterization and separation from protein kinase C. J. Cell. Biochem., 40, 295-307. Leibersperger, H., Gschwendt, M., Gernold, M. and Marks, F. (1991) Immunological demonstration of a calcium-unresponsive protein kinase C of the b-type in different species and murine tissues. Predominance in epidermis. J. Biol. Chem., 266, 1477814784. Gschwendt, M., Kittstein, W. and Marks, F. (1988) Ciclosporin inhibits phorbol ester-induced hyperplastic transformation and tumor promotion in mouse skin probproably by suppression of Ca 2+ /calmodulin-dependent cesses such as phosphorylation of elongation factor 2. Skin Pharmacol., 1, 84 - 92. Van Belle, H. (1981) R24571: a potent inhibitor of calmodulin activated enzymes. Cell Calcium 2, 483 -494. Gschwendt, M., Kittstein, W. and Marks, F. (1988) The weak immunosuppressant cyclosporine D as well as the
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immunologically inactive cyclosporine H are potent inhibitors in vivo of phorbol ester TPA-induced biological effects in mouse skin and of Ca2+/calmodulin-dependent EF-2 phosphorylation in vitro. Biochem. Biophys. Res. Commun., 150, 545-551. Kuroki, T., Hashimoto, Y., Osada, S. Tajima, O., Nose, K. and Ohno. S. (1991) Predominant expression of Ca2+-independent protein kinase C isoforms, nPKCq and nPKCG in mouse skin. J. Cell. Biochem.. Suppl. 15B, p. 159. Tamaoki, T., Komoto, H., Takahashi, I., Kato, Y ., Morimoto, M. and Tonita, F. (1986) Staurosporine, a potent inhibitor of phospholipid/Ca2+-dependent protein kinase. Biochem. Biophys. Res. Commun., 135, 397 - 402. Chida, K., Yamada, S., Katao, N. and Kuroki, T. (1988) Phosphorylation of M, 34 000 and 40 000 proteins by protein kinase C in mouse epidermis in vivo. Cancer Res., 48, 4018-4023. Gschwendt, M., Leibersperger, H. and Marks, F. (1989) Differentiative action of K252a on protein kinase C and a calcium-unresponsive, phorbol ester/phosphilipidactivated protein kinase. Biochem. Biophys. Res. Commun., 164, 974-982. Gschwendt, M., Leibersperger, H., Kittstein, W. and Marks, F. (1992) Protein kinase Ce and ri in murine epidermis. TPA induces down-regulation of PKCv but not of PKCt. Fed. Eur. Biochem. Sot. Lett.. in press. Koide, H., Ogita, K., Kikkawa, U. and Nishizuka, Y. (1992) Isolation and characterization of the t subspecies of protein kinase C from rat brain. Proc. Natl. Acad. Sci. U.S.A., 89. Dlugosz, A.A. and Yuspa, S.H. (1991) Staurosporine induces protein kinase C agonist effects and maturation of normal and neoplastic mouse keratinocytes in vitro. Cancer Res., 51, 4677-4684. Yoshiiawa, S., Fujiki, H., Suguri, H., Suganuma, M., Nakayasu, M., Matsushima. R. and Sugimura, T. (1990) Tumor-promoting activity of staurosporine, a protein kinase inhibitor on mouse skin. Cancer Res., 50, 4974 - 4978. Kahn, S.M., O’Driscoll, K.R., Blackwood, M.A., Jiang, W. and Weinstein, LB. (1991) Overexpression of rat PKCp, regulatory domain causes the disordered growth of rat 6 fibroblasts. Abstract A-30, book of abstracts of the First Joint Conference of the American Association for Cancer Research and the European Association for Cancer Research, Santa Margherita, Italy. Ahmed, S., Kozma, R., Monfries, C., Hall, C., Lim, H.H., Smith, P. and Lim, L. (1990) Human brain
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n-chimaerin cDNA encodes a novel phorbol ester receptor. Biochem. J. 272, 767-773. Maruyama, I.N. and Brenner, S. (1991) A phorbol ester/diacylglycerol-binding protein encoded by the unc13 gene of Caenorhobditis elegans. Proc. Natl. Acad. Sci. U.S.A., 88, 5729-5733. Hashimoto, Y. and Shudo, K. (1990) Cytosolic-nuclear tumor promoter-specific binding protein (CN-TDBP) in human promyeolocytic leukemia cells HL-60. Biochem. Biophys. Res. Commun.. 166, 1126- 1132. Riiegg, U.T. and Burgess, G.M. (1989) Staurosporine, K252 and UCN 01: potent but nonspecific inhibitors of protein kinases. Trends Biochem. Sci., 10, 218-220. O’Brian, C.A. and Ward, N.E. (1990) Staurosporine: a prototype of a novel class of inhibitors of tumor cell invasion? J. Natl. Cancer Inst., 82, 1734- 1735. Gschwendt, M., Kittstein, W.. Horn, F. and Marks, F. (1985) Cyclosporin A inhibits biological effects of tumor promoting phorbol esters. Biochem. Biophys. Res. Commun., 126, 327-332. Gschwendt, M., Kittstein W. and Marks, F. (1987) Cyclosporin A inhibits phorbol ester-induced cellular proliferation and tumor promotion as well as phosphorylation of a lOO- kDa protein in mouse epidermis. Carcinogenesis, 8, 203 - 207. Gschwendt, M., Kittstein W. and Marks, F. (1987) Didemnin B inhibits biological effects of tumor promoting phorbol esters on mouse skin, as well as phosphorylation of a 100 kDa protein in mouse epidermis cytosol. Cancer Len., 34, 187- 191. Gschwendt, M., Kittstein W. and Marks, F. (1989) The immunosuppressant FK-506, like cyclosporines and didemnin B, inhibits calmodulin-dependent phorphorylation of the elongation factor 2 in vitro and biological effects of the phorbol ester TPA on mouse skin in vivo. Immunobiol., 179, 1-7. Chuprun, J.K., Bazan, E., Chang, K.-C., Campbell, A.K. and Rapoport, R.M. (1991) Inhibition of phorbol esterinduced contraction by calmodulin antagonists in rat aorta. Am. J. Physiol., 261, C675-C684. Palfrey, H.C., Nairn, A.C., Muldoon, L.L. and Villereal, M.L. (1987) Rapid activation of calmodulin-dependent protein kinase III in mitogen-stimulated human fibroblasts. J. Biol. Chem., 262, 9785-9792. Gschwendt, M.. Kittstein, W. and Marks, F. (1988d) Effect of tumor promoting phorbol ester TPA on epidermal protein synthesis: stimulation of an elongation factor 2 phosphatase activity by TPA in vivo. Biochem. Biophys. Res. Commun., 153, 1129- 1135.
