Znt. J. Cancer: 51, 144-148 (1992) 0 1992 Wiley-Liss, Inc.
Publication of the International Union Against Cancer Publication de I Union Internationale Contre le Cancer
MODULATION BY STAUROSPORINE OF PHORBOL-ESTER-INDUCED EFFECTS ON GROWTH AND PROTEIN KINASE C LOCALIZATION IN A549 HUMAN LUNG-CARCINOMA CELLS Tracey D. BRADSHAW', Andreas G ~ S C H F Rand ' George R. PFTTIT* Cancer Research Campaign Expenmental Chemotherapy Group, Pharmaceutical Sciences Institute, Aston University, Birmingham B47ET, U E and 2Cancer Research Institute, Anzona State University, Tempe, AZ, USA. I 2-0-tetradecanoylphorbol- I 3-acetate (TPA) and bryostatin I are activators of protein kinase C (PKC). TPA is a potent inhibitor of the growth of A549 cells, while bryostatin I exerts a weak antiproliferative effect upon this cell line. We tested the hypothesis that the PKC inhibitor staurosporine (STAU) can interfere with the effects of TPA or bryostatin I on A549 cells. STAU alone arrested A549 cell growth effectively with an ICs0of 0.65 nM as determined by cell counting after incubation for 96 hr. It also caused the release of lactate dehydrogenase from cells with an lCsoof 18.4 nM. On incubation with cells for up to 8 hr, STAU (I 00 nM) alone did not reduce thymidine incorporation into cells. However, it partially abrogated the inhibition of DNA synthesis caused by TPA or bryostatin I (I 0 nM). The IC,, for inhibition by STAU of the activity of PKC purified from A549 cells was 6. I nM. Localizationand levels of PKC were studied by Western blot and phorbol ester receptor binding analyses. STAU (I 00 nM) did not prevent the TPA-induced rapid redistribution of PKC to the cell membrane, but instead increased it by 25%. The PKC downregulation caused by TPA was not reduced in the presence of STAU. The results suggest that (i) PKC activation is involved in growth inhibition caused by TPA or bryostatin I in A549 cells, and (ii) subcellular localization or levels of PKC can be pharmacologically manipulated even under conditions of inhibited kinase function.
Activators of PKC, such as TPA (Gescher and Reed, 1985) or bryostatin 1 (Dale and Gescher, 1989), interfere with the growth of human-derived A549 adenocarcinoma cells at exquisitely low, non-toxic concentrations. The mechanism of this growth arrest, and the precise role which PKC activation plays in this mechanism, are unclear. The study described here was designed to help elucidate the mechanisms by which PKC activators disturb cell proliferation. Activation of PKC in cells is thought to involve a tight association with membranes of soluble PKC or of enzyme which is loosely connected with the membrane (Kraft et al., 1982). Furthermore, chronic exposure to PKC activators generally results in the downregulation of cellular enzyme activity (Rodriguez-Pena and Rozengurt. 1984). Both TPA and bryostatin 1 are very similar in their ability to cause the redistribution and downregulation of PKC in A549 cells (Dale et al., 1989). However, considerable differences exist between TPA and bryostatin 1 in their effects on cell growth (Dale and Gescher, 1989). Bryostatin 1 exerts growth arrest only at concentrations within the narrow range of to lo-' M, and interference with proliferation is temporary, lasting for only 18 hr (Dale and Gescher, 1989), whereas TPA inhibits cell growth at concentrations of > lo-'" M for up to 6 days (Gescher and Reed, 1985). The aim of the work described here is to gain a better understanding of the link between growth inhibition induced by PKC activators and PKC redistribution and downregulation. To that end we tested the hypothesis that effects caused by TPA or bryostatin 1 can be abrogated by STAU. STAU is a potent, but not particularly selective, inhibitor of PKC, mediating its effect by interacting with the catalytic site of the enzyme (Tamaoki et al., 3986).
(Kyowa Hakko, Tokyo). Bryostatin 1was isolated and purified as described previously (Pettit et al., 1982). Stock solutions of TPA, STAU or bryostatin 1 were prepared in DMSO and stored at -20°C. The final concentration of DMSO in the medium did not exceed 0.5%, which alone did not affect cell growth. A549 human lung adenocarcinoma cells, which originated from the ATCC (Rockville, MD) were cultured in Ham's F-12 medium supplemented with 10% fetal calf serum; these conditions ensure optimal growth (Gescher and Reed, 1985). Cells (5 x lo4) were seeded in Nunclon 6-well multidishes (3.5 cm diameter, 3 ml medium), and became attached to the plastic within 4 hr. Medium was aspirated and replenished every other day. Cells were counted with a Coulter Counter (model ZM) (Luton, UK) after incubation for 4 days. Incorporation of 3H-TdR into cells was measured as described previously (Dale and Gescher, 1989). Cytotoxicity was assessed by determination of LDH released into the medium, according to Leathwood and Plummer (1969).
Measurement of PKC activity and cytosolic phorbol ester receptor binding The cytosolic fraction was prepared by disruption of cells via sonication and centrifugation for 30 min at 100,000 g. The particulate (membrane) fraction was obtained by suspending the pellet in H8 buffer containing Nonidet P40 (1%) and recentrifugation (l00,000g, 30 min) (Dale et al., 1989). Protein concentration of the fractions was determined by the assay of Bradford (1976). PKC activity was measured using a commercially available kit (Amersham, Aylesbury, UK), where the TPA-induced Ca2+ and phospholipid-dependent transfer of the y-phosphate group of 32P-ATPto a PKC-specific peptide was measured. Formation of mixed micelles from a semi-purified extract of the cytosolic fraction and measurement of phorbol ester receptor binding were performed as described by Hannun and Bell (1986) with some specifications as outlined by Bradshaw et al. (1991) using 3H-PDBu (New England Nuclear, Stevenage, UK) as ligand. Non-specific binding was < l o % of total binding. Westem-blot analysis For Western-blot analyses, proteins in the cytosolic or particulate fractions from approximately 4 x los cells were separated by SDS-PAGE on gels containing 8% (wiv) polyacrylamide, by the method of Laemmli (1970) in a Mini-Protean apparatus (Bio-Rad, Watford, UK). Rainbow markers (molecular weight range 14.3-20.0 kDa) were used and mouse
'To whom correspondence and reprint requests should be sent. Fax: 021-333 3172.
MATERIAL AND METHODS
Abbreviations: LDH, lactate dehydrogenase; PDBu, phorbol dibutyratc; PKC, protein kinase C; STAU, staurosporine; TPA, 12-0tctradecanoylphorbol-13-acetate; TdR, thymidine.
Chemicals and cell growth TPA and other reagents were purchased from Sigma o r Gibco (Poole, UK). STAU was a gift from Dr. H. Nakano
Kcceived: November 12, 1991 and in revised form December 17, 1991.
STAUROSPORINE ALTERS PHORBOL-ESTER-INDUCED EFFECTS
immunoglobulin was included as positive control. Proteins were electroblotted onto nitrocellulose as described by Towbin et al. (1979), using a Transblot apparatus (Bio-Rad). Blots were kept, first overnight at 4”C, then for 1 hr at room temperature, in 10 mM Tris-buffered saline (pH 7.4) with 5% (w/v) skimmed milk and 0.1% or 0.5% Tween 20 to block non-specific antibody binding. The nitrocellulose was incubated for 2 hr at 4°C with an antibody specific against murine PKC-o(,P (Amersham). Immunodetection was completed using the Amersham blotting detection kit for mouse antibodies. Band intensity was measured with a LKB 2202 Ultrascan laser densitometer. Values given under “Results” are the mean ? SD; numbers in parentheses refer to the number of observations.
loo
145
t k
RESULTS
Effects of STAU on PKC activity, growth and TPA-induced growth inhibition Figure 1shows that STAU is a potent inhibitor of the activity of PKC obtained from A549 cells, with an ICsOof 6.1 ? 1.4 nM (n = 15). This value is similar to the ICsOobserved for the inhibition of rat brain y P K C (2.7 nM) in the original report on the PKC-inhibitory effect of STAU (Tamaoki et al., 1986). STAU also inhibited the proliferation of A549 cells effectively. Measurement of cell growth following exposure for 96 hr furnished an apparent ICs0of 0.65 ? 0.05 nM (n = 6) (Fig 2). The cytotoxicity of STAU was measured after incubation for 96 hr, by determination of LDH release into the cellular medium, which gave an apparent ICsO of 18.4 -+ 1.1 nM (n = 3). This value was obtained after seeding 5 x lo4 cells, and it fluctuated considerably with differences in initial cell density and in incubation time. Surprisingly, STAU diminished DNA synthesis as determined by incorporation of 3H-TdR into A549 cells only following incubation for periods beyond 8 hr (Fig. 3). At earlier time points, thymidine incorporation was hardly affected. In contrast, a significant inhibitory effect of TPA or bryostatin 1 on DNA synthesis in these cells was observed already within 1 hr of incubation (Fig. 3). This difference between STAU and PKC activators in the rate at which growth arrest was initiated made it possible to assess the effect of STAU on the initial inhibition of DNA synthesis caused by TPA (Fig. 3a) or bryostatin 1 (Fig. 3b). On co-incubation with TPA or bryostatin 1 (10 nM), STAU (100 nM) partially cancelled the inhibition of DNA synthesis caused by these agents.
STAU concentration (M)
FIGURE2-Effect of STAU on the growth of A549 cells following incubation for 96 hr. Values are the mean k SD of 3 experiments, each conducted in triplicate.
Effects of STAU on TPA-induced PKC redistribution and PKC downregulation It is considered that redistribution of PKC to the membrane is a prerequisite for, and PKC downregulation a consequence of, TPA-induced enzyme activation. Therefore we tested the hypothesis that these processes are repressed when PKCmediated processes and PKC activity are inhibited by STAU. To that end, we incubated A549 cells with TPA (10 nM) and/or STAU (100 nM) for 1 or 24 hr. PKC was detected by Western blot analysis in both the cytosolic and particulate cellular fractions using a PKC-a,P-specific antiserum (Fig. 4). Quantitation of band intensity in the Western blot demonstrated that, after a I-hr period of incubation with TPA, 43 ? 7% (n = 3) of total immunodetectable PKC was left in the cytosol, while the rest had become firmly associated with the particulate fraction (Fig. 5a). STAU did not inhibit TPAinduced PKC redistribution but increased it, so that only 18 -+ 7% (n = 3) of PKC remained in the cytosol. An almost identical result was obtained when cytosolic phorbol ester receptor binding was determined: disappearance of phorbol ester receptors from the cellular cytosol caused by incubation with TPA for 1hr was increased significantly in the presence of STAU (Fig. 5b). STAU alone did not cause decreased cytosolic phorbol ester receptor binding or PKC redistribution. Results very similar to those shown in Figure 5a were obtained when cells were analysed for PKC using a 2-hr incubation period. On incubation with TPA for 24 hr, PKC was markedly downregulated. In the presence of STAU, TPA-induced PKC downregulation was not diminished (Fig. 4,6). Quantitation of immunoblot band intensity (Fig. 6b) suggests that TPAinduced downregulation of PKC in the presence of STAU resulted in the retention of twice as much PKC in the membrane as in the absence of STAU. DISCUSSION
STAU concentration (nM)
FIGURE1-Concentration dependence of effect of STAU on activity of PKC obtained by semi-purification from the cytosol of A549 cells. PKC activity was determined as described under “Methods”. Values are the mean ? SD from 5 experiments, each
conducted in triplicate.
Our results suggest that in A549 cells both activation and inhibition of PKC elicit cessation of growth. The cells were more sensitive to the growth-inhibitory potential of STAU than many other cells in culture, exemplified by an IC50value of 0.65 nM, as compared to 29 nM in T-24 human bladder carcinoma and 130 nM in HL-60 human promyelocytic leukae-
146
BRADSHAW E T A L .
A
r
T
time of exposure (hr)
Fn
T
J ~
01
I
I
I
0
2
4
I I I 6 8 10 time of exposure (hr)
I
. + * -+ I -
1 Z T 1 8 - 2 4
FIGURE3-Influence of TPA (10 nM) (a) or bryostatin 1 (10 nM) (b) (O), STAU (100 nM) ( 0 )or of STAU together with either TPA (a) or bryostatin 1 (b) (0)on incorporation of ”-TdR into AS49 cells. Values for the combinations (0)are significantly different from those observed with PKC activators alone ( O ) ,in the case of TPA (a) at 1-8 hr @J < 0.001), and for bryostatin 1 (b) at the 1-, 4-, 8-hr 0, < 0.001) and the 3- and 6-hr points (p < 0.01). Values are the mean SD of 3 experiments, each conducted in triplicate.
*
mia cells, for example (Meyer et al., 1989). Cytotoxicity as measured by the release of LDH from A549 cells into the medium required concentrations of STAU which were at least 30 times higher than the ICjo for growth inhibition. These findings suggest that, as in the case of exposure of AS49 cells to PKC activators (Gescher and Reed, 1985), low concentrations of STAU cause cytostasis rather than cytotoxicity. Whereas, in the case of TPA or bryostatin, cessation of growth was reversible (Gescher and Reed, 1985; Dale and Gescher, 1989), the antiproliferative effect of STAU was irreversible (data not shown). In view of the relatively non-selective inhibition of kinases by STAU, it is impossible to judge whether the STAU-induced cytostasis is mediated mainly or exclusively by inhibition of PKC activity. STAU was undoubtedly a potent inhibitor of the PKC obtained from AS49 cells, which consists mainly of the a-isozyme (Hirai et aL, 1989). Growth inhibition occurred at a STAU concentration which was only a tenth of the ICsofor inhibition of A549 cell PKC caused by STAU. This suggests that STAU-induced growth arrest is probably mediated by a high-affinity interaction separate from PKC, perhaps with another enzyme. In some cells, such as mouse keratinocytes (Dlugosz and Yuspa, 1991), STAU functions primarily as a “PKC agonist”, and we have shown here that in A549 cells
FIGURE 4 - Influence of TPA (10 nM), STAU (100 nM) or a combination of both on PKC in the cytosolic (C) and particulate (M) fractions of AS49 cells. Western-blot analysis was performed with a PKC-a$-specific antiserum following incubation of cells for 1 or 24 hr. Arrow indicates position of the 80-kDa PKC protein. The blot shown is representative of 3 different experiments.
STAU similarly mimics the growth-inhibitory effect of PKC activators. On incubation of STAU together with TPA or bryostatin 1 for up to 8 hr, the inhibition of DNA synthesis caused by the PKC activators was partially reversed. This result supports the contention that the cytostasis caused by PKC activators in A549 cells is indeed mediated, at least in its initial stages, by enzyme activation. STAU did not diminish or abolish TPA-induced PKC redistribution or downregulation, but interfered potently with both the activity of semi-purified A549 PKC and TPA-induced cellular DNA synthesis, at concentrations in the M range. Therefore, it appears likely that STAU at 100 nM-the concentration used in the study of its effect on TPA-induced PKC redistribution and downregulation-was capable of repressing TPA-induced PKC activity effectively in incubates with intact A549 cells. Our results suggest that PKC redistribution as a consequence of exposure to PKC activators is probably not a prelude to, or corollary of, enzyme activation. This conclusion is incompatible with the original conjecture concerning the close link between PKC activation and redistribution (Kraft et al., 1982). However, it is consistent with the finding that, in 1321N1 cells, PKC redistribution to the membrane in response to muscarinic receptor stimulation did not correlate, either in duration or extent, with PKC activity as determined by substrate phosphorylation (Trilivas et al., 1991). We show here that in A549 cells STAU not only failed to abolish TPA-induced PKC redistribution to the membrane, but in fact increased it. We are currently investigating whether the amplification of the PKC-redistributory effect of TPA occurs also with PKC inhibitors other than STAU. Preliminary results suggest that calphostin C which, unlike STAU, inhibits PKC at the regulatory site (Bruns et al., 1991), does not elicit such an effect. On the basis of these results it could be argued that the PKC-activator complex acquires particular affinity for the cell membrane when another ligand, which occupies and thus inhibits the catalytic site of the enzyme, is attached to it. Such an interpretation is consistent with the finding of Wolf and Baggiolini (1988) that STAU in combination with PDBu induced the association of purified PKC with inside-out
147
STAUROSPORINE ALTERS PI-IORBOL-ESTER-INDUCEDEFFECTS
-0
A
B
h
*
A Cytosol
B Membrane
7
I
T
FIGURE 5 - Effect of STAU on TPA-induced removal of immunodetectable PKC (a) or of phorbol ester receptor sites (b) from the cytosolic fraction of A549 cells. Cells were incubated with 10 nM TPA and/or 100 nM STAU for 1 hr. Cellular cytosol was separated from the particulate fraction and Western-blot analysis (a) was conducted with both fractions as described under “Methods”, using an antibody which recognizes the cleavage site of the PKC a and p isozymes. Band intensity was measured by laser densitometry. Values in (a) are expressed as a percentage of the sum of absorbance intensities of bands for the cytosolic and particulate fractions under each of the experimental conditions. The band intensities observed in the particulate fraction (not shown here) were therefore 100% minus the cytosolic PKC value shown above. The sum of band intensities for cytosolic and particulate fraction following incubation with TPA and/or STAU did not differ more than 5% from the control value, which indicates that appreciable PKC downregulation did not occur during 1hr of incubation with these agents. Phorbol ester receptor binding (b) was measured using the mixed micelle assay and PDBu as receptor ligand, as described under “Methods”. Values are the mean k SD of 3 experiments with measurement of absorbance intensity at 3 different band locations in each blot (a), or of 3 experiments (b), each conducted in triplicate. The asterisk indicates that values for the combination are significantly different from those for TPA alone (p < 0.005 in a, p < 0.002 in b).
vesicles from erythrocyte membranes. Of course, it is not possible to discount more complicated mechanisms which might involve secondary effects elicited by STAU, for example via inhibition of kinases other than PKC, or via modulation of intracellular Ca2+ levels or metabolism of diacylglycerols or phospholipids. The observed lack of effect of STAU on TPA-induced PKC downregulation is compatible with reports suggesting that PKC downregulation is independent of its phosphorylating activity (Freisewinkel et aZ., 1991; Pears and Parker, 1991; Lindner et al., 1991). In the presence of STAU, more immunodetectable PKC remained attached to the particulate fraction after TPA-induced enzyme downregulation than in its absence. This finding is congenial with an increased affinity for the cell membrane of the PKC-activator complex after association with STAU. In addition, it is consistent with the proposi-
FIGURE6 - Effect of incubation for 24 hr with TPA (10 nM), STAU (100 nM) or a combination of both on PKC levels in the cytosolic (a) or particulate (b) fractions of A549 cells. Detection was by Western-blot analysis using an anti-PKC-a,P antibody. Band intensity was measured by laser densitometry at 3 different positions per band. Values (mean t SD), expressed as percentages of band intensity in control cells, were obtained from 3 separate blots. Asterisks indicate that the difference between values for the combination and for TPA alone is significant (**p < 0.001; *p < 0.006 but only when blots were compared in each individual experiment).
tion that maximal proteolysis of membrane-bound PKC requires fully activated enzyme (Kishimoto et d., 1983). In conclusion, we have shown that the proliferative potential of A549 cells is very sensitive not only to exposure to PKC activators but also towards the PKC inhibitor STAU. The connections between cytostasis and activation, inhibition, redistribution and downregulation of PKC appears to be complex. Investigation of protein phosphorylation patterns elicited in cells by TPA and/or STAU should help to clarify them. Drug-induced changes in cellular localization or levels of PKC without preceding enzyme activation might ultimately be exploited in the design of novel therapeutic strategies. Now it appears possible that pharmacological intervention with proliferative diseases based on PKC modulation will eventually be successful with novel agents which possess higher specificity for particular tissues or PKC isozymes than the “traditional” PKC modulators. ACKNOWLEDGEMENTS
This study was supported by programme grant SP1.518 from the Cancer Research Campaign and a postgraduate studentship to T.D.B. from the Medical Research Council of Great Britain. The isolation of bryostatin 1was funded by PHS grant CA 16049-12, Outstanding Investigator Grant CA-44344-01A1 from the NCI, DHHS, the Fannie E. Rippel Foundation, the Arizona Disease Control Research Commission and the Robert B. Dalton Endowment Fund. W e thank Dr. P.J. Hanson (Aston University) for help with the Western-blot analysis, Mr. G.H. Smith (Aston University) for drawing the Figures, and Dr. J. Lord (University of Birmingham) for helpful discussions.
REFERENCES
BRADFORD, M.M., A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteindye binding.Anal. Biochem., 72,248-254 (1976). BRADSHAW, T.D., GESCHER, A. and PETIT, G.R., The effect of foetal calf serum on growth arrest caused by activators of protein kinase C. Znt. . I Cancer, . 47,929-932 (1991). BRUNS,R.F., MILLER, F.D., MERRIMAN, R.L., HOWBERT, J.J., HEATH, W.F., KOBAYASHI, E., TAKAHASHI, I., TAMAOKI, T. and NAKANO, H.,
Inhibition of protein kinase C by calphostin C is light-dependent. Biochem. biophys. Res. Comm., 176,288-293 (1991). DALE,I.L., BRADSHAW, T.D., GESCHER, A. and PETIT, G.R., Comparison of effectsofbqostatin 1 and 2 and 12-O-tetradecanoylphorbol-13acetate on protein kinase activity in A549 human lung carcinoma cells. Cancer Rex, 49,3242-3245 (1989). DALE,I.L. and GESCHER, A., Effects of activators of protein kindse C
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including bryostatin 1 and 2 on the growth of A549 human lung carcinoma cells. Int. J. Cancer, 43, 158-163 (1989). DLUGOSZ,A.A. and YUSPA,S.H., Staurosporine induces protein kinase C agonist effects and maturation of normal and neoplastic mouse keratinocytes in vifro. Cancer Rex, 51,4677-4684 (1991). FREISEWINKEL, I., RIETHMACHER, D. and STABEL, S., Downregulation of protein kinase C-7 is independent of a functional kinase domain. FEBS Left.,280,262-266 (1991). GESCHER, A. and REED,D.J., Characterization of the growth inhibition induced by tumor-promoting phorbol esters and their receptor binding in A549 human lung carcinoma cells. Cancer Rex, 45, 4315-4321 (1985). HANNUN, Y.A. and BELL,R.M., Phorbol ester binding and activation of protein kinase C on Triton X-100 mixed micelles containing phosphatidylserine. J. biol. Chem., 261,9341-9347 (1986). HIRAI,M., GAMOU, S., KOBAYASHI, M. and SHIMIZU, N., Lung cancer cells often express high levels of protein kinase C activity. Jap. J. CancerRes., 80,204-208 (1989). KISHIMOTO, A,, KAJIKAWA, N., SHIOIA,M. and NISHIZUKA, Y., Proteolytic activation of calcium-activated, phospholipid-dependent protein kinase b calcium dependent neutral protease. J. bid. Chem., 258,1156-1164 8983). KRAFT, AS., ANDERSON,W.B., COOPER,H.L. and SANDO,J.J., Decrease in cytosolic calciurniphospholipid-dependent protein kinase activity following phorbol ester treatment of EL4 thymoma cells. J. bid. Chem., 257,13193-13196 (1982). LAEMMLI, U.K., Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (Lond.), 227,680-685 (1970). LEATHWOOD, P.D. and PLUMMER, D.T., Enzymes in rat urine. 1. A metabolism cage for the complete separation of urine and faeces. Enzymologia, 37,240-250 (1969). LINDNER, D., GSCHWENDT, M. and MARKS,F., Down-regulation of
protein kinase C in Swiss 3T3 fibroblasts is independent of its phosphorylating activity. Biochem. biophys. Res. Comm., 176, 12271231 (1991). MEYER,T., REGENASS, U., FABBRO,D., ALMKI,E., ROSEL,J., MULLER, M., CARAVATTI,G. and MATTER,A,, A derivative of staurosporine (CGP 41 251) shows selectivity for protein kinase C inhibition and in vitro anti-proliferative as well as in vivo anti-tumor activity. Znt. J. Cancer, 43,851-856 (1989). PEAKS,C. and PARKER, P.J., Down-regulation of a kinase defective PKC-a. FEBSLetf., 284,120-122 (1991). PEr-ril, G.R., HERALD, C.L., DOUBEK, S.L., HERALD,D.L., ARNOLD, E. and CLARDY,J., Isolation and structure of bryostatin 1. J. Amer. chem. Soc., 104,6846-6848 (1982). RODRIGUEZ-PENA, A. and ROZENGURT, E., Disappearance of Ca2+sensitive, phospholipid-dependent protein kinase activity in phorbolester-treated 3T3 cells. Biochem. biophys. Res. Comm., 120,1053-1059 (1984). TAMAOKI, T., NOMUrO, H., TAKAHASHI, l., KATO, Y., MORIMOTO, M. and TOMITA,F., Staurosporine, a potent inhibitor of phospholipid/ Ca2+-dependentprotein kinase. Biochem. biophys. Res. Comm., 135, 397-402 (1986). TOWBIN, H., STAEHELIN, T. and GORDON, J., Electrophoretic transfer of proteins from poyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. nut. Acad. Sci. (Wash.), 76, 43504354 (1979). TRILIVAS, I., MCDONOUGH, P.M. and BROWN,J.H., Dissociation of protein kinase C redistribution from the phosphorylation of its substrates. J. bid. Chem., 266,8431-8438 (1991). WOLF,M. and BAGGIOLINI, M., The protein kinase inhibitor staurosporine, like phorbol esters, induces the association of protein kinase C with membranes. Biochem. biophys. Res. Comm., 154, 1273-1279 (1988).
Publication of the International Union Against Cancer Publication de I Union Internationale Contre le Cancer
MODULATION BY STAUROSPORINE OF PHORBOL-ESTER-INDUCED EFFECTS ON GROWTH AND PROTEIN KINASE C LOCALIZATION IN A549 HUMAN LUNG-CARCINOMA CELLS Tracey D. BRADSHAW', Andreas G ~ S C H F Rand ' George R. PFTTIT* Cancer Research Campaign Expenmental Chemotherapy Group, Pharmaceutical Sciences Institute, Aston University, Birmingham B47ET, U E and 2Cancer Research Institute, Anzona State University, Tempe, AZ, USA. I 2-0-tetradecanoylphorbol- I 3-acetate (TPA) and bryostatin I are activators of protein kinase C (PKC). TPA is a potent inhibitor of the growth of A549 cells, while bryostatin I exerts a weak antiproliferative effect upon this cell line. We tested the hypothesis that the PKC inhibitor staurosporine (STAU) can interfere with the effects of TPA or bryostatin I on A549 cells. STAU alone arrested A549 cell growth effectively with an ICs0of 0.65 nM as determined by cell counting after incubation for 96 hr. It also caused the release of lactate dehydrogenase from cells with an lCsoof 18.4 nM. On incubation with cells for up to 8 hr, STAU (I 00 nM) alone did not reduce thymidine incorporation into cells. However, it partially abrogated the inhibition of DNA synthesis caused by TPA or bryostatin I (I 0 nM). The IC,, for inhibition by STAU of the activity of PKC purified from A549 cells was 6. I nM. Localizationand levels of PKC were studied by Western blot and phorbol ester receptor binding analyses. STAU (I 00 nM) did not prevent the TPA-induced rapid redistribution of PKC to the cell membrane, but instead increased it by 25%. The PKC downregulation caused by TPA was not reduced in the presence of STAU. The results suggest that (i) PKC activation is involved in growth inhibition caused by TPA or bryostatin I in A549 cells, and (ii) subcellular localization or levels of PKC can be pharmacologically manipulated even under conditions of inhibited kinase function.
Activators of PKC, such as TPA (Gescher and Reed, 1985) or bryostatin 1 (Dale and Gescher, 1989), interfere with the growth of human-derived A549 adenocarcinoma cells at exquisitely low, non-toxic concentrations. The mechanism of this growth arrest, and the precise role which PKC activation plays in this mechanism, are unclear. The study described here was designed to help elucidate the mechanisms by which PKC activators disturb cell proliferation. Activation of PKC in cells is thought to involve a tight association with membranes of soluble PKC or of enzyme which is loosely connected with the membrane (Kraft et al., 1982). Furthermore, chronic exposure to PKC activators generally results in the downregulation of cellular enzyme activity (Rodriguez-Pena and Rozengurt. 1984). Both TPA and bryostatin 1 are very similar in their ability to cause the redistribution and downregulation of PKC in A549 cells (Dale et al., 1989). However, considerable differences exist between TPA and bryostatin 1 in their effects on cell growth (Dale and Gescher, 1989). Bryostatin 1 exerts growth arrest only at concentrations within the narrow range of to lo-' M, and interference with proliferation is temporary, lasting for only 18 hr (Dale and Gescher, 1989), whereas TPA inhibits cell growth at concentrations of > lo-'" M for up to 6 days (Gescher and Reed, 1985). The aim of the work described here is to gain a better understanding of the link between growth inhibition induced by PKC activators and PKC redistribution and downregulation. To that end we tested the hypothesis that effects caused by TPA or bryostatin 1 can be abrogated by STAU. STAU is a potent, but not particularly selective, inhibitor of PKC, mediating its effect by interacting with the catalytic site of the enzyme (Tamaoki et al., 3986).
(Kyowa Hakko, Tokyo). Bryostatin 1was isolated and purified as described previously (Pettit et al., 1982). Stock solutions of TPA, STAU or bryostatin 1 were prepared in DMSO and stored at -20°C. The final concentration of DMSO in the medium did not exceed 0.5%, which alone did not affect cell growth. A549 human lung adenocarcinoma cells, which originated from the ATCC (Rockville, MD) were cultured in Ham's F-12 medium supplemented with 10% fetal calf serum; these conditions ensure optimal growth (Gescher and Reed, 1985). Cells (5 x lo4) were seeded in Nunclon 6-well multidishes (3.5 cm diameter, 3 ml medium), and became attached to the plastic within 4 hr. Medium was aspirated and replenished every other day. Cells were counted with a Coulter Counter (model ZM) (Luton, UK) after incubation for 4 days. Incorporation of 3H-TdR into cells was measured as described previously (Dale and Gescher, 1989). Cytotoxicity was assessed by determination of LDH released into the medium, according to Leathwood and Plummer (1969).
Measurement of PKC activity and cytosolic phorbol ester receptor binding The cytosolic fraction was prepared by disruption of cells via sonication and centrifugation for 30 min at 100,000 g. The particulate (membrane) fraction was obtained by suspending the pellet in H8 buffer containing Nonidet P40 (1%) and recentrifugation (l00,000g, 30 min) (Dale et al., 1989). Protein concentration of the fractions was determined by the assay of Bradford (1976). PKC activity was measured using a commercially available kit (Amersham, Aylesbury, UK), where the TPA-induced Ca2+ and phospholipid-dependent transfer of the y-phosphate group of 32P-ATPto a PKC-specific peptide was measured. Formation of mixed micelles from a semi-purified extract of the cytosolic fraction and measurement of phorbol ester receptor binding were performed as described by Hannun and Bell (1986) with some specifications as outlined by Bradshaw et al. (1991) using 3H-PDBu (New England Nuclear, Stevenage, UK) as ligand. Non-specific binding was < l o % of total binding. Westem-blot analysis For Western-blot analyses, proteins in the cytosolic or particulate fractions from approximately 4 x los cells were separated by SDS-PAGE on gels containing 8% (wiv) polyacrylamide, by the method of Laemmli (1970) in a Mini-Protean apparatus (Bio-Rad, Watford, UK). Rainbow markers (molecular weight range 14.3-20.0 kDa) were used and mouse
'To whom correspondence and reprint requests should be sent. Fax: 021-333 3172.
MATERIAL AND METHODS
Abbreviations: LDH, lactate dehydrogenase; PDBu, phorbol dibutyratc; PKC, protein kinase C; STAU, staurosporine; TPA, 12-0tctradecanoylphorbol-13-acetate; TdR, thymidine.
Chemicals and cell growth TPA and other reagents were purchased from Sigma o r Gibco (Poole, UK). STAU was a gift from Dr. H. Nakano
Kcceived: November 12, 1991 and in revised form December 17, 1991.
STAUROSPORINE ALTERS PHORBOL-ESTER-INDUCED EFFECTS
immunoglobulin was included as positive control. Proteins were electroblotted onto nitrocellulose as described by Towbin et al. (1979), using a Transblot apparatus (Bio-Rad). Blots were kept, first overnight at 4”C, then for 1 hr at room temperature, in 10 mM Tris-buffered saline (pH 7.4) with 5% (w/v) skimmed milk and 0.1% or 0.5% Tween 20 to block non-specific antibody binding. The nitrocellulose was incubated for 2 hr at 4°C with an antibody specific against murine PKC-o(,P (Amersham). Immunodetection was completed using the Amersham blotting detection kit for mouse antibodies. Band intensity was measured with a LKB 2202 Ultrascan laser densitometer. Values given under “Results” are the mean ? SD; numbers in parentheses refer to the number of observations.
loo
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t k
RESULTS
Effects of STAU on PKC activity, growth and TPA-induced growth inhibition Figure 1shows that STAU is a potent inhibitor of the activity of PKC obtained from A549 cells, with an ICsOof 6.1 ? 1.4 nM (n = 15). This value is similar to the ICsOobserved for the inhibition of rat brain y P K C (2.7 nM) in the original report on the PKC-inhibitory effect of STAU (Tamaoki et al., 1986). STAU also inhibited the proliferation of A549 cells effectively. Measurement of cell growth following exposure for 96 hr furnished an apparent ICs0of 0.65 ? 0.05 nM (n = 6) (Fig 2). The cytotoxicity of STAU was measured after incubation for 96 hr, by determination of LDH release into the cellular medium, which gave an apparent ICsO of 18.4 -+ 1.1 nM (n = 3). This value was obtained after seeding 5 x lo4 cells, and it fluctuated considerably with differences in initial cell density and in incubation time. Surprisingly, STAU diminished DNA synthesis as determined by incorporation of 3H-TdR into A549 cells only following incubation for periods beyond 8 hr (Fig. 3). At earlier time points, thymidine incorporation was hardly affected. In contrast, a significant inhibitory effect of TPA or bryostatin 1 on DNA synthesis in these cells was observed already within 1 hr of incubation (Fig. 3). This difference between STAU and PKC activators in the rate at which growth arrest was initiated made it possible to assess the effect of STAU on the initial inhibition of DNA synthesis caused by TPA (Fig. 3a) or bryostatin 1 (Fig. 3b). On co-incubation with TPA or bryostatin 1 (10 nM), STAU (100 nM) partially cancelled the inhibition of DNA synthesis caused by these agents.
STAU concentration (M)
FIGURE2-Effect of STAU on the growth of A549 cells following incubation for 96 hr. Values are the mean k SD of 3 experiments, each conducted in triplicate.
Effects of STAU on TPA-induced PKC redistribution and PKC downregulation It is considered that redistribution of PKC to the membrane is a prerequisite for, and PKC downregulation a consequence of, TPA-induced enzyme activation. Therefore we tested the hypothesis that these processes are repressed when PKCmediated processes and PKC activity are inhibited by STAU. To that end, we incubated A549 cells with TPA (10 nM) and/or STAU (100 nM) for 1 or 24 hr. PKC was detected by Western blot analysis in both the cytosolic and particulate cellular fractions using a PKC-a,P-specific antiserum (Fig. 4). Quantitation of band intensity in the Western blot demonstrated that, after a I-hr period of incubation with TPA, 43 ? 7% (n = 3) of total immunodetectable PKC was left in the cytosol, while the rest had become firmly associated with the particulate fraction (Fig. 5a). STAU did not inhibit TPAinduced PKC redistribution but increased it, so that only 18 -+ 7% (n = 3) of PKC remained in the cytosol. An almost identical result was obtained when cytosolic phorbol ester receptor binding was determined: disappearance of phorbol ester receptors from the cellular cytosol caused by incubation with TPA for 1hr was increased significantly in the presence of STAU (Fig. 5b). STAU alone did not cause decreased cytosolic phorbol ester receptor binding or PKC redistribution. Results very similar to those shown in Figure 5a were obtained when cells were analysed for PKC using a 2-hr incubation period. On incubation with TPA for 24 hr, PKC was markedly downregulated. In the presence of STAU, TPA-induced PKC downregulation was not diminished (Fig. 4,6). Quantitation of immunoblot band intensity (Fig. 6b) suggests that TPAinduced downregulation of PKC in the presence of STAU resulted in the retention of twice as much PKC in the membrane as in the absence of STAU. DISCUSSION
STAU concentration (nM)
FIGURE1-Concentration dependence of effect of STAU on activity of PKC obtained by semi-purification from the cytosol of A549 cells. PKC activity was determined as described under “Methods”. Values are the mean ? SD from 5 experiments, each
conducted in triplicate.
Our results suggest that in A549 cells both activation and inhibition of PKC elicit cessation of growth. The cells were more sensitive to the growth-inhibitory potential of STAU than many other cells in culture, exemplified by an IC50value of 0.65 nM, as compared to 29 nM in T-24 human bladder carcinoma and 130 nM in HL-60 human promyelocytic leukae-
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BRADSHAW E T A L .
A
r
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time of exposure (hr)
Fn
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I I I 6 8 10 time of exposure (hr)
I
. + * -+ I -
1 Z T 1 8 - 2 4
FIGURE3-Influence of TPA (10 nM) (a) or bryostatin 1 (10 nM) (b) (O), STAU (100 nM) ( 0 )or of STAU together with either TPA (a) or bryostatin 1 (b) (0)on incorporation of ”-TdR into AS49 cells. Values for the combinations (0)are significantly different from those observed with PKC activators alone ( O ) ,in the case of TPA (a) at 1-8 hr @J < 0.001), and for bryostatin 1 (b) at the 1-, 4-, 8-hr 0, < 0.001) and the 3- and 6-hr points (p < 0.01). Values are the mean SD of 3 experiments, each conducted in triplicate.
*
mia cells, for example (Meyer et al., 1989). Cytotoxicity as measured by the release of LDH from A549 cells into the medium required concentrations of STAU which were at least 30 times higher than the ICjo for growth inhibition. These findings suggest that, as in the case of exposure of AS49 cells to PKC activators (Gescher and Reed, 1985), low concentrations of STAU cause cytostasis rather than cytotoxicity. Whereas, in the case of TPA or bryostatin, cessation of growth was reversible (Gescher and Reed, 1985; Dale and Gescher, 1989), the antiproliferative effect of STAU was irreversible (data not shown). In view of the relatively non-selective inhibition of kinases by STAU, it is impossible to judge whether the STAU-induced cytostasis is mediated mainly or exclusively by inhibition of PKC activity. STAU was undoubtedly a potent inhibitor of the PKC obtained from AS49 cells, which consists mainly of the a-isozyme (Hirai et aL, 1989). Growth inhibition occurred at a STAU concentration which was only a tenth of the ICsofor inhibition of A549 cell PKC caused by STAU. This suggests that STAU-induced growth arrest is probably mediated by a high-affinity interaction separate from PKC, perhaps with another enzyme. In some cells, such as mouse keratinocytes (Dlugosz and Yuspa, 1991), STAU functions primarily as a “PKC agonist”, and we have shown here that in A549 cells
FIGURE 4 - Influence of TPA (10 nM), STAU (100 nM) or a combination of both on PKC in the cytosolic (C) and particulate (M) fractions of AS49 cells. Western-blot analysis was performed with a PKC-a$-specific antiserum following incubation of cells for 1 or 24 hr. Arrow indicates position of the 80-kDa PKC protein. The blot shown is representative of 3 different experiments.
STAU similarly mimics the growth-inhibitory effect of PKC activators. On incubation of STAU together with TPA or bryostatin 1 for up to 8 hr, the inhibition of DNA synthesis caused by the PKC activators was partially reversed. This result supports the contention that the cytostasis caused by PKC activators in A549 cells is indeed mediated, at least in its initial stages, by enzyme activation. STAU did not diminish or abolish TPA-induced PKC redistribution or downregulation, but interfered potently with both the activity of semi-purified A549 PKC and TPA-induced cellular DNA synthesis, at concentrations in the M range. Therefore, it appears likely that STAU at 100 nM-the concentration used in the study of its effect on TPA-induced PKC redistribution and downregulation-was capable of repressing TPA-induced PKC activity effectively in incubates with intact A549 cells. Our results suggest that PKC redistribution as a consequence of exposure to PKC activators is probably not a prelude to, or corollary of, enzyme activation. This conclusion is incompatible with the original conjecture concerning the close link between PKC activation and redistribution (Kraft et al., 1982). However, it is consistent with the finding that, in 1321N1 cells, PKC redistribution to the membrane in response to muscarinic receptor stimulation did not correlate, either in duration or extent, with PKC activity as determined by substrate phosphorylation (Trilivas et al., 1991). We show here that in A549 cells STAU not only failed to abolish TPA-induced PKC redistribution to the membrane, but in fact increased it. We are currently investigating whether the amplification of the PKC-redistributory effect of TPA occurs also with PKC inhibitors other than STAU. Preliminary results suggest that calphostin C which, unlike STAU, inhibits PKC at the regulatory site (Bruns et al., 1991), does not elicit such an effect. On the basis of these results it could be argued that the PKC-activator complex acquires particular affinity for the cell membrane when another ligand, which occupies and thus inhibits the catalytic site of the enzyme, is attached to it. Such an interpretation is consistent with the finding of Wolf and Baggiolini (1988) that STAU in combination with PDBu induced the association of purified PKC with inside-out
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STAUROSPORINE ALTERS PI-IORBOL-ESTER-INDUCEDEFFECTS
-0
A
B
h
*
A Cytosol
B Membrane
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FIGURE 5 - Effect of STAU on TPA-induced removal of immunodetectable PKC (a) or of phorbol ester receptor sites (b) from the cytosolic fraction of A549 cells. Cells were incubated with 10 nM TPA and/or 100 nM STAU for 1 hr. Cellular cytosol was separated from the particulate fraction and Western-blot analysis (a) was conducted with both fractions as described under “Methods”, using an antibody which recognizes the cleavage site of the PKC a and p isozymes. Band intensity was measured by laser densitometry. Values in (a) are expressed as a percentage of the sum of absorbance intensities of bands for the cytosolic and particulate fractions under each of the experimental conditions. The band intensities observed in the particulate fraction (not shown here) were therefore 100% minus the cytosolic PKC value shown above. The sum of band intensities for cytosolic and particulate fraction following incubation with TPA and/or STAU did not differ more than 5% from the control value, which indicates that appreciable PKC downregulation did not occur during 1hr of incubation with these agents. Phorbol ester receptor binding (b) was measured using the mixed micelle assay and PDBu as receptor ligand, as described under “Methods”. Values are the mean k SD of 3 experiments with measurement of absorbance intensity at 3 different band locations in each blot (a), or of 3 experiments (b), each conducted in triplicate. The asterisk indicates that values for the combination are significantly different from those for TPA alone (p < 0.005 in a, p < 0.002 in b).
vesicles from erythrocyte membranes. Of course, it is not possible to discount more complicated mechanisms which might involve secondary effects elicited by STAU, for example via inhibition of kinases other than PKC, or via modulation of intracellular Ca2+ levels or metabolism of diacylglycerols or phospholipids. The observed lack of effect of STAU on TPA-induced PKC downregulation is compatible with reports suggesting that PKC downregulation is independent of its phosphorylating activity (Freisewinkel et aZ., 1991; Pears and Parker, 1991; Lindner et al., 1991). In the presence of STAU, more immunodetectable PKC remained attached to the particulate fraction after TPA-induced enzyme downregulation than in its absence. This finding is congenial with an increased affinity for the cell membrane of the PKC-activator complex after association with STAU. In addition, it is consistent with the proposi-
FIGURE6 - Effect of incubation for 24 hr with TPA (10 nM), STAU (100 nM) or a combination of both on PKC levels in the cytosolic (a) or particulate (b) fractions of A549 cells. Detection was by Western-blot analysis using an anti-PKC-a,P antibody. Band intensity was measured by laser densitometry at 3 different positions per band. Values (mean t SD), expressed as percentages of band intensity in control cells, were obtained from 3 separate blots. Asterisks indicate that the difference between values for the combination and for TPA alone is significant (**p < 0.001; *p < 0.006 but only when blots were compared in each individual experiment).
tion that maximal proteolysis of membrane-bound PKC requires fully activated enzyme (Kishimoto et d., 1983). In conclusion, we have shown that the proliferative potential of A549 cells is very sensitive not only to exposure to PKC activators but also towards the PKC inhibitor STAU. The connections between cytostasis and activation, inhibition, redistribution and downregulation of PKC appears to be complex. Investigation of protein phosphorylation patterns elicited in cells by TPA and/or STAU should help to clarify them. Drug-induced changes in cellular localization or levels of PKC without preceding enzyme activation might ultimately be exploited in the design of novel therapeutic strategies. Now it appears possible that pharmacological intervention with proliferative diseases based on PKC modulation will eventually be successful with novel agents which possess higher specificity for particular tissues or PKC isozymes than the “traditional” PKC modulators. ACKNOWLEDGEMENTS
This study was supported by programme grant SP1.518 from the Cancer Research Campaign and a postgraduate studentship to T.D.B. from the Medical Research Council of Great Britain. The isolation of bryostatin 1was funded by PHS grant CA 16049-12, Outstanding Investigator Grant CA-44344-01A1 from the NCI, DHHS, the Fannie E. Rippel Foundation, the Arizona Disease Control Research Commission and the Robert B. Dalton Endowment Fund. W e thank Dr. P.J. Hanson (Aston University) for help with the Western-blot analysis, Mr. G.H. Smith (Aston University) for drawing the Figures, and Dr. J. Lord (University of Birmingham) for helpful discussions.
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