Phorbol Diester-induced Alterations in the Expression of Protein ...

T H EJOURNAL OF BIOLOGICAL CHEMISTRY 1991 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 266, No. 23, Issue of August 15, pp. 15135-15143,1991 Printed in U.S.A.

(c)

Phorbol Diester-induced Alterationsin theExpression of Protein Kinase C Isozymes and TheirmRNAs ANALYSISINWILD-TYPEANDPHORBOLDIESTER-RESISTANTHL-60CELLCLONES* (Received for publication, April 24, 1991)

Rebecca L. McSwine-Kennick$, Evelyn M. McKeegang, Mark D. Johnson, andMichael J. Morinll From the Departmentsof Pharmacology and Pathology and The Cancer Center, Northwestern UniversityMedical School, Chicago, Illinois 6061 1

In an HL-60 cell subline (PR-17) which wasgreater protein kinase C genes in an isozyme-specific manner. than 100-fold resistant to the differentiating andcy- Comparable PMA-induced alterations inmRNA levels tostatic activities of phorbol 12-myristate 13-acetate were not observed in PMA-resistant cells, even under (PMA), the protein kinase C phenotype was found to conditions of significantactivationandsubsequent be nearly identical to thatof wild-type HL-60 cells. A down-regulation of protein kinase C protein. Taken measurable decrease (30%)in thespecific activities of together, these data suggest that activation and downcrude preparationsof PR-17 cell protein kinase C was regulation of the isozymes of protein kinaseC may not observed whenthe enzyme was measured with histonerepresent absolute determinants of the PMA-induced as the phosphate acceptor substrate, but other aspects differentiation of HL-60 cells, but that specific alterof the protein kinase C phenotype (intracellular con- ations in the levelsof the mRNA for thef3 isozymes of protein kinase C, or of other genes which maybe centrations and binding affinities of phorbol diester isozymes, are imporreceptors, translocation of activated enzyme fromcy- regulated by the activated kinase tant to the induction of leukemia cell differentiation tosolic toparticulatesubcellularfractions,relative expression of the a and 6 isozyme proteins) were equiv-by PMA. alent in both PMA-resistant PR-17 cells and inwildtype HL-60 cells. Direct analysisof the behavior of the a and B isozymes after the exposure of each cell type to 100 nM PMA for 1 2 h revealed that the activities and Protein kinase C exists as a family of isozymes coded for intracellularconcentrations of bothisozymes were by multiple genes (for reviews, see Refs. 1 and 2). There is downregulated to an equivalent extent in both wild- significant but indirectevidence to suggest that one or more of the individual kinase isozymes may play a major role in type and PMA-resistant cells. These results suggest cells. For that the cellular basis for the resistance to the effects differentiationanddevelopmentinmammalian of PMA was present “down-stream” from the activa- example, each of the isozymes may be developmentally regution and down-regulationof protein kinaseC and was lated, some are expressed in atissue-specific manner, and perhaps a nuclear component. Among the genes which many of the isozymes exhibit a significant degree of individual were likely to be differentially regulated when each of heterogeneity at a functional level (e.g.activation by free fatty the two cell lines were treated with PMA were those acids, dependence on Ca2+, substratespecificity, and suscepfor the protein kinase C isozymes themselves. In wild- tibilities to down-regulation by proteases) (3-8). Other data type HL-60 cells, the intracellular concentrations of indicate that alterations in one ormore of the isozymes can mRNA for each of the isozymes were increased (up modify the sensitivity of tumor cells tochemotherapeutic to 5-fold) 48 h after the initiation of PMA treatment; agents (9, 10) or induce transformed phenotypes in cells (11, further studies indicate that an activator of protein kinase C could influence the expressionof HL-60 cell 12). Additional studies also demonstrate that the induction of differentiation in various tumor cell lines can result in alterations in the intracellular concentration of the specific * Supported by Department of Health and HumanServices Grant isozymes of protein kinase C (13-16). CA-44589 from the National Cancer Institute, National Institutes of The role of protein kinase C in hematopoietic cell function Health. The costsof publication of this article were defrayed in part has also been widely examined inleukemia cell lines. Of those by the payment of page charges. This article must therefore hereby be marked “advertisement” in accordance with 18 U.S.C. Section 1734 agents which induce monocyte-like differentiation in the husolely to indicate thisfact. manHL-60 promyelocyticleukemia cell line, the phorbol The nucleotide sequence(s) reported in this paperhas been submitted esters are among the most potent efficacious and (for a review, to theGenBankTM/EMBLDataBankwith accession number(s) see Ref. 17). Exposure to nanomolar concentrations of PMA,’ M22199, M18254, M18.255, and M13977. which can activate protein kinase C both i n vitro and in vivo j: Submitted in partial fulfillment of the requirementsfor the Ph.D. (18), results in the rapid loss of proliferative potential, aswell degree in the Department of Pharmacology, NorthwesternUniversity. of characteristics related to the monRecipient of a Predoctoral Fellowship from The Lucille P. Markey as increased expression Charitable Trust. § Submitted in partial fulfillment of the requirements for the Ph.D. degree from the Program of Tumor Cell Biology, Northwestern University. Supported by a Predoctoral Training Program in Carcinogenesis (T32 CA-09560) from the National Cancer Institute, National Institutes of Health. ll To whom correspondence should be addressed Pfizer Central Research, Eastern Point Rd., Groton, C T 06340. Tel.: 203-441-5476; Fax: 203-441-4998.

The abbreviations used are: PMA, phorbol myristate acetate; PS, phosphatidyl serine; EGTA, ethylene glycol (bis aminoethyl ether) N,N,N’,N’-tetraacetic acid; PDBu, phorbol 12,13-dibutyrate; SDS, sodium dodecyl sulfate; D-PBS, Dulbecco’s cation free phosphatebuffered saline; HA-FPLC, hydroxylapatite fast protein liquid chromatography;EGR-1,early growth response gene 1; HPLC, high performance liquid chromatography; kb, kilobase(s); HEPES, 4-(2hydroxyethy1)-1-piperazineethanesulfonicacid; IL, interleukin.

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expression of PMA-responsive genes were ocytic phenotype. Included among theseare adhesion of tate alterations in the treated cells to plastic growth surfaces, increased activity of obtained from a commercial source (American Type Culture CollecRockville, MD), exceptfor that for the EGR-1probe, which was lysosomal enzymes, increased phagocytosis, and the generation, a generous gift from Dr. Vikas P. Sukhatme, Universityof Chicago. tion of tumoricidal effector cells (17). Taken together with Cell Growth and Maturation in Wild-type and PMA-resistant HLthe observation that the cellular receptor for phorbol esters 60 Cell Clones-Rat-6 fibroblast cells and retrovirally transfected copurifies with protein kinase C activity (19,20), these results Rat-6 cells which stably overexpressed the rat protein kinase C 01 have led to the hypothesis that the biochemical basis for the isozyme were grown as described previously (11, 28). Early passage differentiating effects of PMA in intact myeloid leukemia HL-60 cells were originally obtained from Dr. Robert C. Gallo of the National Cancer Institute andgrown in RPMI 1640 medium supplecells lies principally in the activation of this protein kinase mented with 20% heat-inactivated, iron-supplemented bovine calf (17). Consistent with these observations are data indicating serum. HL-60 cell cultures, which were maintained in logarithmic that many cell lines which are resistant to the cytostatic and growth in a humidified atmosphere of 9.25% air and 7.5% CO, at differentiating effects of phorbol diesters have measurable 37 "C, exhibited a doubling time of 22 h. PMA-resistant clones were by growing low-passage wild-type cells defects in the activity or behavior of protein kinase C (21, generated without mutagenesis in increasing concentrations (10 nM to 3 p ~ of) PMA for several 22). For example, Homma and coworkers (21) have observed weeks (half-log increases in concentration each week). Cells which that sublines of PMA-resistant HL-60 cells exhibited both were capable of growth a t 3 ~ L MPMA were subcloned by limiting altered membrane-association after activation and modified dilution in the absence of phorbol diester. Each clonal subline was expanded until sufficient numbersof cells (lo6)were available for the affinities for well defined phosphate acceptor substrates. There are other data (23-27) which suggest, however, that evaluation of resistance to PMA. Clones which were found to maingrown in the absence of the the differentiation response in HL-60 cells is complex, and tain stable resistance to PMA when phorbol diesterfor several weekswere cryopreserved. Of the 30 clones that biochemical activation of protein kinase C is alone in- so isolated,several were subsequentlyfoundtoexhibit defective sufficient to account for the cellular effects of PMA. We have proteinkinase Cactivity, includingthesublinePR-20,in which noted similarities in the biochemical effects produced when translocation of the kinase after activation may have been defective cells were exposed to either PMA or to cell-permeable diacyl- (see "Results").Cell numbers were assessed using a model ZBI Coulter Diagnostics, Hialeah, FL). glycerols, such as 1,2-dioctanoylglycerol.Among these were particle counter and Channelizer (Coulter of myeloid an equivalent activation of protein kinase C in vitro and in Cellular adhesion to plastic and other quantitative aspects cell differentiation were assessed as described earlier (23, 24). uiuo, an increased phosphorylation of several identical proMeasurement of PKC Catalytic Actiuity-The activity of protein teins in intact cells, and anincreased incorporation of choline kinase C, in both crude fractions and in fractions partially purified into phospholipids (23, 24). However, significantly dissimilar by HA-FPLC, was assessed essentially as described previously (23, cellular effects were observed with the two different classes 24), with minormodifications. Aliquots of cells (2 X 10') were washed of activators of protein kinase C, in that monocytic differen- in D-PBS, resuspended in1.0 ml of lysis buffer (20 mM Tris-HCI, 0.1 EGTA, 10% sucrose, 50 mM 2-mercaptoethanol at pH 7.5), and tiation was not detected in populations of diacylglycerol- mM disrupted by sonication.Kinaseactivity was extractedfromthe treated cells, even when the diacylglycerols were replete in sonicates on ice for 15 min with lysis buffer containing 0.3% Triton the media of HL-60 cell cultures at bihourly intervals (23- X-100 and DE-52 cellulose (50% v:v in lysis buffer). The resin was washed twice with the same lysis buffer, and the enzyme was eluted 25). In this report, alterations in the activities and expression with lysisbuffer containing 300 mM NaC1. The PS- and Ca2+111s of protein kinase C isozymes inPMA-sensitive wild-type HL- dependentincorporation of [y-"PIATPintohistonefraction was used to assay the enzyme. Aliquots of partially purified kinase 60 cells have been compared to alterations which occur in a (250 pl) were incubated a t 30 "C in the presenceof 150 pg of PS and unique PMA-resistant subline. PR-17 cells were found to be 50 pg of 1,2-dioctanoylglycerol, 27 mM Tris-HC1, 34 g M EGTA, 17 stably resistant to PMA and to have minimal defects in their mM 2-mercaptoethanol, 3.4% sucrose, 10 mM MgCI2, 100 pM ATP, protein kinase C phenotype. The results of the present studies 100 pg/ml histone fraction IIIS, and 0.13 mM Ca2+at pH 7.5. The with this clonal subline are consistent with previous obser- reaction was terminated a t 4 "C by dilution with a cold solution of Nonidet P-40 (O.ll%), followed by rapid precipitation with cold 30% vations that biochemical activation of protein kinase C is trichloroacetic acid.Acid-precipitable phosphoproteins were realone insufficient to account for the differentiation response, covered by centrifugation, hydrolyzed in NaOH, and "PO4 incorpoand that defects in other aspects of the pathway, "down- rated into protein was quantitated by liquid scintillation spectromewere deterstream" from the activated kinase, can account for the resist- try. Protein concentrations in the kinase preparations ance of PR-17 cells to differentiation by PMA. Among the mined by a dye binding technique (32) with bovine serum albumin potential targets may be proteins which regulate the expres- used as the protein standard.All measurements were obtained under conditions of linear reaction kinetics (protein phosphorylation uersus sion of genes for the protein kinase C /3 isozymes (or other time). In some experiments, the translocation, or change in abunintermediate-to-late protein kinase C-responsive genes), dance of kinase activity ina soluble uersus a particulate fraction, was which were found to be transcribed at a greater rate in wild- quantitated after PMA treatment of HL-60 cells, essentially as detype HL-60 cells than in PMA-resistant cells during exposure scribed earlier (24). Cells were treated with 100 nM PMA for l h, washed in D-PBS, and then disrupted at 4 "C with 15 strokes of a to PMA.

Dounce homogenizer. Particulate-associated andsoluble enzymefractions were prepared by subjecting the cell homogenates to centrifugation in anAirFuge (30 p.s.i.; Beckman Instruments; PaloAlto, CA) Materials-PS and 1,2-dioctanoylglycerol were purchasedfrom for 5 min, after which each fraction was extracted in detergent and Avanti Polar Lipids (Birmingham, AL), and PMA was obtained from eluted batch-wise from DE-52, as described above. LC Services (Woburn, MA). Aprotinin and histone fraction 111s were Binding of Radiolabeled Phorbol Diester-Cell lysates were prepurchased from Sigma.Antibodies to the a, p, and y isozymes of pared as described above and utilized to assess the binding of ["HI protein kinase C were purchased from SeikagakuAmerica (Rockville, PDBu (Du Pont-New England Nuclear). Phorbol diester binding was MD). Iron-supplemented bovine calf serum was obtained from Hyassessed essentially as described by others (29, 30) with minor modClone (Logan, UT), and RPMI 1640 culture medium and D-PBS ifications. Aliquots of cell lysates, prepared as described above (7.5 were purchased from GIBCO. 1,25-Dihydroxyvitamin Da was a gen- mg protein/ml), were incubated for 15 min a t 30 "Cinreaction erous gift from Dr. Milan R.Uskokovic, Hoffmann-La Roche (Nutley, mixtures containing 0-120 nM ["HIPDBu (10.2 Ci/mmol), 0.1% boNJ). [."H]PDBu and ["'PIATP were purchased from Du Pont-New vine serum albumin, 0.13 mM Ca2+,300 pg/ml PS, 32 mM Tris-HC1, England Nuclear. Cognate antisense DNAoligonucleotides (40-mers) 10 mM MgCI2,60 p~ EGTA, and6.2% sucrose at pH7.5. Nonspecific for the kinase isozymes were synthesized by phosphoramidite chem- binding was determined in parallel experiments run in the presence istry and repurified by HPLC (Northwestern University Biotechnol- of a100-fold excess (12 p ~of)unlabeled PDBu. The binding reactions ogy Facility, Evanston, IL). Eachof the other probesused to quanti- were terminated by the addition of ice-cold polyethylene glycol (lo%, EXPERIMENTAL PROCEDURES

Regulation of PKC Gene Expression and Activity

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w:v), and ["HIPDBu specifically associated with thepolyethyleneglyspun-column chromatography. St,andard amounts of total RNA (20 col precipitates was quantitated by liquid scintillation spectrometry. pg) or poly(A+) RNA (3 pg) were separated by electrophoresis in 1% Data from the saturation binding isotherms (specific binding versus formaldehyde agarose gels, and the RNA was then transferred to PDBu concentration)were replotted by the methodof Scatchard (31), nitrocellulose (38). Standard amounts of RNA (0.1-0.8 pg) were also and values for k d and cellular PDBu receptor densities were derived applied to nitrocellulose using a slot blot apparatus (Schleicher and from the slopes and n intercepts of the plots obtained after least Schuell). In both cases, the RNA was baked on to the nitrocellulose squares analysis with commercially available data plotting software at 80 "C in a vacuum oven for 1 h. The blots were prehybridized for (Jandel Scientific, Sausalito, CA). 1 h at 42 "C in 0.04 ml/cm2 prehybridization buffer (5 X Denhardt's Separation of Protein Kinase Isozymes by HA-FPLC-Soluble cell solution, 0.1% SDS, 100 pg/ml denatured salmon sperm DNA, 5 X extracts were prepared as described above with the exception that SSC, 50mM sodium phosphate,p H 6.5). Radiolabeled antisense DNA the partially purified enzyme was eluted from DE-52 cellulose with probes(5ng/ml) were addedtofresh hybridizationbuffer(40% lysis buffer containing 300 mM NaCl plus 10 pg/ml aprotinin. The deionized formamide, 5 X Denhardt's solution, 0.1% SDS, 100 pg/ml enzyme extracts were then concentrated using a Centricon-30 filter denatured salmon sperm DNA, 5 X SSC, 50 mM sodium phosphate, (Amicon, Danvers, MA) and resuspended in 0.3 ml of lysis buffer p H 6.5) and the blots were hybridized at 42 "C for 18 h. Following containing 10 pg/ml aprotinin. The isozymes were separated at 4 "C hybridization, the blotswere washed twice a t 42 "C for 15 min in 0.1 by FPLC (Pharmacia, LKBBiotechnology Inc.) on a hydroxylapatite X SSC, 0.1% SDS. The blots were air-dried and exposed to Kodak column (100 X 7.8 mm, Bio-Jel HPHT, Bio-Rad), and eluted with X-Omat AR film a t -70 "C for 24-72 h. In parallel experiments, the lysis buffer containing linearly increasing concentrations of potas- expression of a putative housekeeping gene, glyceraldehydephosphate sium phosphate (0-300 mM) (33, 34). The column flow rates were dehydrogenase, was assessed to normalize the RNA data obtained maintained at0.5 ml/min, and up to 60 separate fractions (2were ml) from different treatment groups (39). The resultant autoradiographs collected and analyzedfor protein kinase C activity as described were analyzed with a scanning laser densitometer, and quantitated above. The results shown (see Fig. 1) represent the Ca'+- and lipid- on the basis of micrograms of RNA applied uers'sus relative radiodependent kinase activity(specific activity) for each fraction. graphic signal. The specific contents (slopes of the plots of hybridiWestern Blotting of Isozyme Proteins-For Western blot transfer zation signal uersus microgramsof RNA) of the totalRNA and mRNA of protein kinase C isozyme proteins to nitrocellulose membranes, for the PKC isozymes were determined for the treatment conditions isozyme fractions were obtained as described above, and preparations described above and normalized to the expression of glyceraldehyde of total cellular protein were obtained as described previously. The phosphate dehydrogenase. proteins were resolved by SDS-polyacrylamide gel electrophoresis as Nuclear Run-on Analysis of Transcriptional Rates-Nuclear rundescribed previously (23); nitrocellulose membranes (0.20 pm, on analysis of relative transcriptional rates was carried out by the Schleicher and Schuell) and the SDS gels were equilibrated separately method of Groudine and coworkers (40), with minor modifications. in transfer buffer (25 mM Tris-HC1, 192 mM glycine, 20% methanol) Plasmids (1 pg) containing cDNA for the a, 6 , and y isozymes of rat for 30 min. Blotting was conducted in a Hoeffer Transphor System protein kinase C (American Type CultureCollection, Rockville, MD), (Hoeffer, San Francisco,CA) at 100 v for 2 h in transfer buffer. The and control plasmid, were linearized by sonication, denatured with nitrocellulose membranes were removed and incubated in blocking sodium hydroxide, neutralized, and applied to nitrocellulose filters buffer (50 mM Tris-HC1,200 mM NaCl, 0.05% Tween-20, and 5% dry using a slot blot apparatus. The filters were baked in a vacuum oven milk, pH 7.5) for 2 h a t room temperature. Nitrocellulose membranes a t 80"C for2 h, and prehybridized in abuffer (containing50% were incubated with each antibody (2 pg/ml anti$, 5 pg/ml anti-a, formamide, 6 X SSC, 10 X Denhardt's solution, 0.2% SDS, and 50 or 5 pg/ml anti-y) in separate, sealed plastic bags for 12 h at room pg/ml tRNA) for 4 hat 42 "C. Aliquots of cells (5 X lo7)from cultures temperature, at which point the membranes were then washed three which were untreated or exposed to PMA for various intervals were mM Tris-HCl,200 mM NaC1,0.05% time for 5 min in TBS-Tween (50 washed in PBS and then incubated on ice in lysis buffer (10 mM Tween, pH 7.5). The secondary antibody, an anti-mouseIgG mono- NaCl, 3 mM MgCl,, 10 mM Tris, and 0.5% Nonidet P-40, pH 7.4). clonal antibody conjugated to horseradish peroxidase (Bio-Rad) was Nuclei were pelleted by centrifugation, resuspended in storagebuffer used a t a 1:lOO dilution in blocking buffer. The blots were incubated (40% glycerol, 50 mM Tris, 5 mM MgC12, 0.1 mM EDTA, pH8.0) and for 2 h a t room temperature and thenwashed three times inblocking immediately frozen in an ethanol/dryice bath. Prior to theiruse, the buffer. Protein kinase C isozyme bands were developed by incubating nuclei were thawed on ice and then incubated a t room temperature theblots for 30 min inTris-bufferedsalinecontaining 0.06% 4for 30 min in reaction buffer (25 mM HEPES.KOH, 2.5 mM MgCI2, chloro-1-napthol and0.02% H202. 2.5 mM dithiothreitol, 75 mM KC1, 5% glycerol, pH 7.4) containing RNA Extraction and Preparation of Poly(A+) RNA-RNA was 0.4 p~ U T P plus 0.2 mCi of [32P]UTP. 350 p M ATP, GTP, CTP, and extracted from HL-60 cells by a guanidinium CsCl technique (35). (20 units) of DNase I (Worthington Biochemical, Freehold, An aliquot. The cells were disrupted by sonication, extracted in guanidinium, and the extract was applied over 5.7 M CsCl and centrifuged for 18 h a t NJ) was then added and incubateda t 37 "C for 20 min. The reaction 105,000 X g. The resultant pellets were washed twicewith 70% ethanol was terminated by incubation at 45 "C for 60 min with 600 pl of stop and then resuspended in 10 mM EDTA. The RNA was extracted once buffer (2% SDS, 7 M urea, 350 mM LiC1, 1 mM EDTA, 10 mM Tris with pheno1:chloroform:isoamyl alcohol (1:24:1, v/v), once with chlo- pH 8.0) containing 80 pg of proteinase K and 100 pg of tRNA. The roform:isoamyl alcohol (24:1, v/v), and then precipitated in ethanol. RNA was precipitated on ice for 20 min with the additionof trichloThe precipitates were resuspended in diethylpyrocarbonate-treated roacetic acid to a final concentration of 10%. The precipitates were water and stored a t -70 "C. Poly(A+) RNA was purified by choro- recovered by centrifugation a t 15,000 X g for 15 min, washed in 95% as describedby Aviv and Leder (36). ethanol, resuspended ina buffer containing 10 mM Tris, 1 mM EDTA, matographyonoligo(dT) Oligo(dT)cellulose (type 3, Collaborative Research Inc., Bedford, MA) and 0.5% SDS, pH 8.0, and transferred to tubes containing hybridization buffer and the nitrocellulose filters blotted with the kinase was packed in 1-ml columns, RNA was diluted to 400 rg/ml with elution buffer, denatured at 75 "C for 5 min and then placed on ice. cDNAs. The blots were allowed to hybridize to 42 "C for 72-96 h and The salt concentrations in the samples were adjusted to500 mM with were washed twice for 30 min a t 65 "C in buffers with increasing stringency:6 X SSC, 0.2% SDS; 2 X SSC, 0.2% SDS; 0.2 X SSC, 4 M NaCl, and the RNAwas applied to the columns(flow rate of 0.5 ml/min). The flow-through fractions (poly(A-) RNA) were collected 0.2% SDS. The blots were air-dried and analyzed autoradiographiand reapplied to the columns. The columns were then washed with cally (96-h exposures) asdescribed above. 30 ml of high salt binding buffer (10 mM Tris, p H 7.5, containing 1 RESULTS mM EDTA, 500 mM NaCl, and 0.2% SDS) followed by 10 ml of low salt binding buffer (50 mM NaCl). Poly(A+) RNA was collected in PMA-resistant HL-60 cell clones were selected in an atwarmed (45 "C) elution buffer (binding buffer without NaCl) and tempt toidentify alterations in the protein kinaseC pathway precipitated in ethanol. Preparation of Labeled Antisense DNA Probes; Northern and Slot which were functionallylinked to the commitment of the Blot Analyses-Maximally divergent sequences for the a, PI, P2, and leukemiccells to differentiation.Twoclones, which were y isozymes of humanproteinkinase Cwere obtainedfromthe selected on the basis of stable resistance to PMA,were found GenBank database, and cognate antisense DNA oligonucleotides (40to have growth rates which were similar to those in wild-type mers) were synthesized by phosphoramidite chemistry andrepurified by HPLC (Northwestern University Biotechnology Facility, Evans- HL-60 cells. Unlike wild-type cells, when the clones were ton, IL). The DNA probeswere end-labeled with [y-32P]ATPby the treated for 72 h with concentrations of PMA as high as 2 p ~ , T-4 kinase method (37), and unincorporated ATP was removed by they continued toproliferate and did not become adherent to


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plastic (Table I). In separate studies, it was determined that the cells did retain the capacity to differentiate in response to 1,25-dihydroxyvitaminD3(data not shown), indicating that the lack of response to PMA was not ageneral differentiation resistance phenomenon in these clones (21, 22). Analysis of the levels of total protein kinase C activity in the wild-type and resistant cell lines revealed a reproducible decrease (30%) in the specific activity of the kinase in each of the resistant clones when histone was used asthe exogenous acceptor substrate (Table I). Scatchard analysis of [3H]PDBu binding isotherms revealed that this decrease in activity in the resistant clones was probably not due to decreased content of the kinase or an altered binding affinity for activating ligands such as phorbol diesters (Table I). Furtheranalysis of possible functional alterations demonstratedthat, inone of the clones (PR-20), translocation of protein kinase C from cytosolic to particulate subcellular fractions following treatment with PMA was defective, in that theassociation of the kinase with the particulate fraction was nearly equivalent in untreated and in PMA-treated PR-20 cells. These results were consistent with those of Homma and coworkers (21), who had observed that sublines of PMA-resistant HL-60 cells exhibited both altered membrane-association after activation and modified affinities for well defined, phosphate-acceptor substrates. To evaluate possible defects in resistant cells which were distinct from alterations in the behavior of the kinase, subsequent studies were focused on the PR-17 cell line, in which the translocation of protein kinase C was equivalent to that in wild-type cells (Table I). To determine if changes in isozyme expression in the resistant clones were responsible for the alterationsin the

sensitivity to PMA, the profiles of protein kinase C isozyme activities eluted from both untreated and PMA-treated HL60 cells and PR-17cells were obtained (Fig. 1).The two major activities present in both cell types eluted from hydroxyapatite at 110-135 mM Po4and 150-175 mM Po4,respectively (Fig. 1, A and D ) . These chromatographic behaviors were consistent with those of the P and a isozymes of a number of cell types from a number of different species (1, 2, 15,33, 34). The identities of the major peaks were further characterized in parallel experiments in which kinase activities extracted from fibroblasts overexpressing the Pi isoform (11, 28) were chromatographed on the same column (Fig. lA),and by Western blots of the recovered HL-60 cell isozyme fractions, in which the eluted proteins were identified with isozymespecific antibodies (Fig. 2). When the activity profiles of PMA-sensitive and -resistant cells were compared, it was apparent that they were similar, but a minor decrease in the ratio of p:a isozyme activity was detectable in PR-17 cells. Treatment with 100 nM PMA for 1 2 h resulted in a significant down-regulation of protein kinase C activity in each cell type (Fig. 1, B and E ) . Removal of PMA for 24 h after a 12-h exposure resulted in measurable recovery of the two major activity peaks in both cell lines (Fig. 1, C, and F ) . Taken together, these results suggested that alterations in the activation, turnover, and recovery of the a and P isoforms of protein kinase C could not account for the resistance of PR17 cells to differentiation by phorbol diesters. To begin to characterize the putatively altered determinants which were “down-stream” from the biochemical activation of protein kinase C isozymes, the expression of three major kinase isozymes ( a , PI, and p2) was measured in untreated and PMA-treated HL-60 and PR-17 cells. OligonucleTABLE I otide (40-mer) probes for the maximally divergent sequences Characterization of wild-type and PMA-resistant HL-60 cells: cellular within the coding regions of each of the protein kinase C response to phorbol esters and the protein kinaseC phenotype isozyme genes (Fig. 3A) were synthesized, labeled, and used HL-60 PR-17 PR-20 to quantitate steady-statelevels of isozyme mRNA.The specDoubling (h) time 21.820.9 21.7 ificities of the probes were demonstrated by Northern analysis of mRNA from normal rat fibroblasts and fibroblasts transPMA fected with the gene for PI isozyme (11,28). The a oligonucle>2000 >2000 ICso (nM) growth inhibi- 20 f 6 tion“ otide probe detected equivalent amounts of a isozyme mRNA >2000 >2000 Echo(nM) adhesion’ 60 f 11 in normal andtransfected cells (Fig. 3B).Northern blots probed with the f12 and y 40-mers showed expression of neither Protein kinase C 4.94 f 0.40 3.58 0.52 3.57 f 0.31 Specific activity‘ of these isoforms in fibroblasts; subsequent studies in the 22.2 f 3.4 21.0 f 3.521.6 f 1.7 Molecules/cell ( X 10-4)d appropriate cell types demonstrated that the p2 probe (Fig. 10 f 2 9 f1 2O f 2 & (nM) PDBu bindingd 3B) and the y probe (in Lan-5 neuroblastoma cells, data not 3.6 1.9 3.4 f 1.21.6 f 0.7 Subcellular distribution‘ shown) were also specific for their cognate genes. The p1 Inhibition of cell growth measured under conditions of a single oligonucleotide probe specifically detected the overexpression treatment with PMA and assessment of cell numbers 72 h after the of that isoform in the transfected fibroblasts (Fig. 3B). In addition of the phorbol diester. * Cell adhesion was measured as described previously (23, 24) after Northern blots of HL-60 cell mRNA (Fig. 3C), the a , PI, and a single treatment with PMA. ECSo value represents concentrations P2 probes were used to specifically detect transcripts with the of PMA yielding 50% of the maximal (generally 45-65% of the total) expected sizes (a:9.7, 8.1, and 2.2 kb; PI and p 2 : 8.6 and 3.4 fraction of adherent cells in the population. kb). ‘ Proteinkinase C activity(pmol of PO, mg-’ min” X lo-’) The isozyme-specific probes were utilized to evaluate altermeasured as described under “Experimental Procedures.” Values for ations in the steady-state levels of protein kinase C isozyme the PR-17 and PR-20cell lines were significantly different at confimRNA in wild-type andPMA-resistant HL-60 cells after dence levels of p < 0.005 as determined by the Student’s t test. Receptor densities and Kd values for [“HIPDBu binding detertreatment with PMA. These results, summarized inFig.4, mined by Scatchard analysis. Values were not statistically different revealed an isozyme-specific regulation of mRNA levels after for the wild-type and PMA-resistantcell lines (identicala t confidence the addition of PMA. In wild-type cells (Fig. 4A), the levels levels of p < 0.01 as determined by the Student’s t test). “ Distribution of protein kinase C activities between particulate of isozyme mRNA were essentially invariant immediately and cytosolic fractions was determined as described previously (24) after PMA treatment;there was a measurable but minor and under “Experimental Procedures.” Values shown represent the decrease in the steady-state levelof the mRNA for the a ratio P,:C,/P,:C,, where P , and Ct represent specific activity values for isozyme after 48 h of exposure to PMA. Conversely, the the particulate and cytosolic fractions in PMA-treated cells, and PC relative expression of mRNA for each of the isozymes was and C, represent the same values in untreated cells. Values for the PR-17 cell line were not significantly different from those for the significantly elevated (up to5-fold) 12-48 h after the addition wild-type cells, while the values for the PR-20 cell line were different of PMA (Fig. 4A). This response may have been specifically related to cellular commitment to differentiation and not to (0.1 < p < 0.05).

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FIG. 1. Elution profiles (HA-FPLC) of Ca2+-and phospholipid-dependent protein kinase C activities in HL-60 cells (A-C) and PR-17 cells (D-F). Solid lines indicate the PO, gradients applied to the columns, and the open triangles in A represent an elution profile from fibroblasts transfected with the PI isozyme of the kinase ( 1 1).A and D represent untreated cells; B and E represent cells treated for 12 h with 100 nM PMA: C and F represent cells treated with 100 nM PMA for 12 h, and then washed extensively and grown in the absence of PMA for an additional 24 h. The profiles shown are given as specific activities (lipid- and Ca'+-dependent kinase activity), and arerepresentative of data obtained in three or more separate experiments.

the loss of proliferative capacity in maturing cells, in that in the PMA-resistant subline, PR-17. More striking effects mRNA levels for each isozyme fell to approximately 50% of were observed, however, when nuclearrun-onassays were for the individual control valueswhen the cells were renderedquiescent by employed t o assess the transcriptional rates serum deprivation for 48 h (fraction of expression relative to isozymes in PMA-treated cells (Fig. 5). For up to24 h followwas little or no that in log-growing cells: a, 0.53; PI, 0.50; p2, 0.43). In PR-17 ing exposure to the phorbol diester, there cells, the steady-state levels of protein kinase C mRNA were change in the transcription of the a isozyme, but the trannot modified by exposure to PMA(Fig. 4B). Taken together, scription of the p isozyme was increased approximately 3the resultsof these experimentsrevealed that the mRNAs for fold. Taken together with an increase in the stability of the protein kinaseC, a cellularreceptor for PMA,were modulated mRNAs for the ,6 kinase isozymes, this transcriptional rein an isozyme-specific manner in the presence of PMA, and sponse could account for the significant isozyme-specific inlevels of protein kinase C ,6 mRNA that the capacity to increase the expression of the p isozymes crease in the steady-state during chronic treatment with PMA was defective in PMA- observed in PMA-treated wild-type HL-60 cells. resistant PR-17 cells. These datawere consistent with the concept that, in PMAPreviously published studies indicate that the treatment of resistant cell lines which retain a wild-type protein kinase C cells with phorbol diesters resulted in significant alterations phenotype, determinants "down-stream'' from the activated in the half-lives of specific mRNA (41). The possible stabili- enzyme could be defective or deficient. One such targetcould zation of p isozyme mRNA in PMA-treated HL-60 cells was be the AP-1 or fos-jun transcription factor complexes, which examined to determineif this could account for the observed are known to modulate the expression of genes under the increases in steady-state mRNA levels. The results of these control of PMA-responsive elements(42). It isnow clear that experiments indicated that the half-lives of the mRNA for the regulation of AP-1 activity is complex, and is itself modboth and p2 were increased somewhat (from 5.7 to 8.3 h for ulated by the presence of occupied receptors for vitamins A PI;from 4.1 to 8.4 h for p2) in PMA-treated cells. Consistent and D (43), each of which induce differentiation in HL-60 with eachof the previous results, thiseffect was not observed cells. The activity of AP-1 is also regulated by the expression


15140

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FIG.2. Western blots of protein kinase C fractions before and after chromatography on HA-FPLC. Aliquots of the unfractionated injectate (inJ),and HA-FPLC fractions7-12 and 16-20 were concentrated, separated by SDS-polyacrylamide gel electrophoresis, blotted, and reacted with antibodies against the 01 (upper panel)and (9 (lowerpanel) isoforms of protein kinase C. The blots were visualized as described under “Experimental Procedures.” Arrows indicate the positions of molecular weight markers (M,= 97,000 and 66,000 respectively). of jun-B, a negative regulator of AP-1 function (44) which is itself induced during HL-60 cell differentiation (45). Moreover, while the upstream regulatory sequences of the @ isozymes are not yet known, those for the y isozyme have been reported (46). This regulatory domain contains the cognate DNA sequences for the binding of not only AP-1, but also those for AP-2, CAMP-responsive element binding proteins, Sp-1, and others. To obtain a more general picture of the multiple nuclear alterations which could be operative in the PR-17 cell line, we chose to examine PMA-inducedalterations in theexpression of “immediate-early” genes (many of which are regulated by AP-I), intermediate genes, and genes which are expressed relatively late in the differentiation response to PMA (45). The results of these studies indicated that the “immediate-early” response to PMA in genes such as EGR-1 and tumor necrosis factor was not impaired in the PR-17cell line (data not shown). This result suggested that the lack of differentiation response in PR-17cells treated with PMA was not due to a gross defect in or deletion of AP-1 activity. A lack of responsiveness was seen at later time points in terms of the level of the expression of tissue inhibitor of metalloproteinase (47, 48) and the 94-kDa collagenase (49) (data not shown). These studies were suggestive of an alteration or alterations in molecular determinants of differentiation in PR-17 cells which were “down-stream” from the activated protein kinase C isozymes and unrelated togross alterations in the“immediate-early’’transcriptional activity of one of the putative nuclear substrates for protein kinase C, AP-1. Given the isozyme-specific increase in protein kinase C p mRNA in the PMA-responsive wild-type cells, a more complete analysis of the impact of long-term PMA treatment on the kinase protein in sensitive and resistant cells, and of the role of accumulated mRNA on the synthesis of new @ isozyme protein, was carried out. The results of Western blot studies demonstrated that, for both thewild-type and PMA-resistant cell lines, turnover of the kinase protein in the presence of PMA (for up to 48 h) was the dominant event, even in cells (HL-60 wild-type) which had accumulated significant levels

C.

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FIG.3. A , a schematic representation of the protein kinase gene family (adapted from Nishizuka, Ref. 1)showing the locations of the maximally divergent regions chosen to generate the isozyme-specific probes. B, specificity of the probes demonstrated by Northern RNA analysis of rat fibroblasts which were sham-transfected ( N )or transfected (T)with protein kinase C isozyme PI uia a retroviral vector. The largest transcripts for each of the isoform RNAs detected by the probes are shown and were found to be of the appropriate size (approximately 8.7 kb). C, Northernblotanalysis of HL-60 cell protein kinase C isozymes mRNA using the oligonucleotide probes. Arrows indicate positions of molecular weight markers, and thesizes of the detectable mRNA species for the isozyme bands are 9.7, 8.1, and 2.2 kb for 01; 8.6 and 3.4 kb for both PI and P 2 . of p isozyme mRNA (data not shown). In Fig. 6, data are shown from Western blot experiments in which cells were treated with PMA for 36 h, and were then washed and allowed to recover in media without PMA. These results revealed that the levels of the @ isozyme protein recovered to control levels within 1 h in wild-type cells which had accumulated mRNA for the @ isozyme. In the resistant PR-17 cells, the recovery was more gradual and was not completed as rapidly in the absence of PMA, despite the fact that these cells, unlike HL60 cells, were growing at their maximal rate. The results of this setof experiments were consistent with the concept that a physiological role for the accumulation of kinase mRNA may have been a more rapid repletion of p isozyme protein immediately after theremoval of an activator, thereby providing for amore efficient and rapid recovery from the dominant down-regulation of the activated kinase protein. DISCUSSION

There are significant data tosuggest that protein kinase C plays a key role in the regulation of the response of hematopoietic cells in bothphysiological and nonphysiological inducers of functional mitogenesis and terminaldifferentiation. In T lymphoid cell lineswith mutatedor chemically downregulated protein kinase C activity, the mitogenic responses of the cells to T-cell receptor ligands, but not to IL-2, have been found to be significantly impaired (50-53). Moreover, the binding of IL-1 to T-cells, and the binding of IL-3 and


Gi

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HOURS AFTER PMA WASHOUT

FIG. 6. Kinetics of the recovery of immunodetectablej3 isozyme protein in HL-60 and PR-17 cells from which PMA has been removed. HL-60 cells (open bars) andPR-17 cells (crosshatched bars) were treated with 100 nM PMA for 36 h, at which point the cellswerewashedextensively in medium containing 20% calf serum. The cells were resuspended in medium and, at the indicated intervals, aliquotsof cells were recovered by centrifugation. Proteins were extracted for Westernblotting as described under“Experimental Procedures,” and the Western blots were quantitated by scanning laser densitometry. The data shown represent at least four separate determinations for each time point (mean & S.E.).

FIG. 4. Alterations in the specific activities of the indicated cytokine receptors (58, 59). The activated kinase may also isozyme mRNAs after exposure to PMA. HL-60 cells ( A ) and become associated with thenucleus, where it may function by PR-17 cells ( B ) were treated with 100 nM PMA, and poly(A+) RNA altering gene expression during the differentiation response fractions were prepared as described and applied to slot blots. “*P- (60-62). labeled oligonucleotide probes (V,a; O, PI; O, p2) were hybridized to There are also significant data to suggest that the cellular the blots which were then washed and autoradiographed.The linear response tothe phorbol diesters is complex and may be slopes of the slot blot data (signal versus pg of RNA) were used as dependent on a number of events which are separable from specific activity values, which were then normalized to the values obtained for a housekeeping gene, glyceraldehyde phosphate dehydro-the activation of protein kinase C. By comparing the phenogenase.Theresults shown are data from three or more separate typic responses of HL-60 cells to cell-permeable diacylglycexperiments (meanvalues k standard error of the mean). *, signifi- erols and PMA it was demonstrated that, even with compacantly lesser value than that in untreated cells ( p < 0.05). **, signif- rable degrees of kinaseactivationandsimilarpatterns of icantly greatervalue than that in untreated cells ( p < 0.01). protein phosphorylation in intact cells (23, 24), a disparity existed between the activation of protein kinase C and the differentiating activities of the two classes of compounds. A more direct analysis of the determinants of the cellular response to PMA was undertaken with the present studies, in which modifications in the expression and activation of specific isozymes of protein kinase C were assessed in wild-type and PMA-resistant HL-60 cells. A potentially important aspect of these studies was the selection of stably resistant clonalsublines which hadnotbeen exposed to PMA for several weeks prior to these experiments. In contrast to the sublines isolated and characterized in other laboratories(21, 10 15 20 25 22), the PR-17 clone was stably and significantly (100-fold) HOURS OF TREATMENT FIG. 5. Increased transcription of protein kinase C j3 iso- resistant to PMA and yet exhibited a nearly normal protein kinase C phenotype. The use of a cell line such as this may zyme mRNA in PMA-treated cells. Nucleiwereisolatedfrom have significant advantages in terms of the identification of untreated HL-60 cells and from cells treated with 100 nM PMA for the times indicated. Each group of nuclei was incubated with [“’PI elements which regulate cellularresponses andfunction UTP and the other ribonucleotide triphosphates, andradioactive “down-stream’’ from the activation of protein kinaseC. When RNA was recovered as described under “Experimental Procedures” the intracellular PMA responses of the kinaseswere measured and applied toslotblotsbearing cDNAfor the 0 ( 0 ) and a (0) cells, it became isozymes. The relative abundance of labeled (3 and CY isozyme RNA in sensitive HL-60 cells and in resistant PR-17 of kinase isozymes extracted from each set of nuclei were assessed from densitometric apparent that the activation and turnover of PMA was not defective inthePR-17 analysis of the resultant autoradiographs (96-h exposures), and ex- inthepresence pressed relative to the same values from untreated cells. The results subline; there was, however, a significant lag in the recovery shown are data from a single experiment, which was representative rate for fi isozyme protein after the removal of PMA in the of those obtained from three separate experiments. resistant cells, eventhough their growth rates were minimally effected by treatment with 100 nM PMA. This observation granulocyte macrophage-colony stimulating factor tomyeloid was discordant with theconceptthataltered expression, cells, results in the activation of protein kinase C uia the function, or turnover (63) of one or more of the isozymes generation of diacylglycerols, possibly from the turnover of could alone account for the lack of PMA responsiveness in (54phosphatidylcholineratherthanphosphatidylinositol resistant cell lines. 57). When activated by these ligands, protein kinaseC underA novel defect was characterized at the level of PMAgoes what has been termed translocation to the plasma mem-induced modulations of steady-state levels of kinase isozyme brane, where it mayregulate thesubsequentfunction of mRNAs. Wild-type HL-60 cells treated with PMAwere found

15142


to have an increased expression of the mRNA for the p isozymes of protein kinase C. The observation that a phorbol diester agonist for the receptor kinase specifically modulated the expression of the gene for the kinase itself may be analogous to results of other studieswhich have demonstrated that specific alterations in receptor gene expression can be induced by the addition of an agonist (e.g. epidermal growth factor, IL-2) for that receptor (64, 51). Recent data from other laboratories suggest that alterations in the expression of protein kinase C isozyme genes may occur in a cell type-specific (64) and agonist-specific (15) manner. Isakov and coworkers (65) have demonstrated that after long-term PMA exposure of the JurkatT leukemia cell line, under conditions in which levels of immunoreactive /3 protein kinase C isozyme were unchanged, the steady-state levels of mRNA for each of the isozymes werealso unchanged (65). Obeid and coworkers (15) have recently demonstrated that treatment of HL-60 cells with 1,25-dihydroxyvitamin DB increased the expression of genes for both the a and p isozymes (15). The alterations in steady-state mRNAlevels observed in our studies (in the absence of cycloheximide)were relatively modest, and occurred while the cellular commitment to differentiation was ongoing in wild-type cells. However, three separate observations were each indicative of a functional relationship between this effect and the differentiation response: (a)the increase in /3 isozyme mRNA levels occurred in an isozyme-specific manner (steady-state levels of a isozyme mRNA were unchanged or decreased slightly after exposure to PMA), ( b ) comparable alterations were not observed in growth-arrested cells, and (c) similar results were not obtained under conditions of comparable activation and turnover of protein kinase C in differentiation-resistant PR-17 cells. The mechanisms through which the steady-state levels of p isozyme mRNAare specifically modulated during exposure to PMA have not yet been fully defined, but two components of this response appear to be a decrease in RNA turnover and a significant increase in the transcription of the /3 isozyme mRNAs in PMA-sensitive, but not in PMA-resistant, HL-60 cells. It was intriguing to speculate that factors which influence the transcription of these genes and otherkinase C-responsive genes, such as the immediate-early genes jun, fos, and EGR1 are altered or deficient in the PMA-resistant PR-17 cells. Recent results from Colburn’s laboratory (66) have demonstrated thata murine epidermal cell line which is resistant to the tumor promotion effects of PMA has a measurable defect in thefunction of AP-1or the fos-jun transcription complexes, which are themselves subject to regulation by activated protein kinase C (42,67,68). However, the regulation of AP-1 is complex, particularly during differentiation when both positive and negative factors are undergoing dramatic alterations in their intracellular concentrations and activities (43-45). Analysis of the steady-state levels of multiple mRNAs after the addition of PMA to wild-type and resistant HL-60 cells reveals that theearly increases in the expression of junp and EGR-1 areunimpaired in the resistant cells, that theincrease in the expression of tissue inhibitor of metalloproteinase is partially impaired, and that the increased expression of the differentiation-associated 94-kDa collagenase gene is abolished in PMA-treated PR-17cells. More direct analysis of the factors involved in the expression of intermediate and late genes and of their role in the altered transcription rates for the /3 isozyme mRNAs would be required before conclusions regarding the significance of these results could be drawn for this cell system. The functional basis for these changes also remains an open question. Analysis of the response of sensitive and resistant

cells at theisozyme protein level demonstrates that themajor isoforms of the protein kinase C activities in both cell types are responsive to PMA and can be reexpressed to an equivalent extent when the phorbol diester was removedfor at least 12 h. At earlier time points, the accumulation of p isozyme mRNA appeared to confer on wild-type cells the ability to recover more quickly an intracellular concentration of the j3 isozyme which was equivalent to that of untreated cells. In preliminary studies of the kinetics of changes in steady-state levels of kinase mRNAs, a significantly elevated rate of recovery in the levels of a isozyme mRNA after release from PMA exposure was noted (data notshown). It may be possible that anincrease in the expression of isozyme mRNA in cells chronically exposed to PMA, or another more physiological ligand which activates protein kinase C through the appropriate receptor, may equip the cell to more rapidly overcome the down-regulation of the kinase at the protein level. This would allow for a more efficient activation of protein kinase C-regulated pathways during a subsequent exposure to an agonist. More detailed studies to examine the kinetics of recovery of protein kinase C gene expression and enzymatic activity in chronically treated wild-type and resistant HL-60 cells are currently underway to examine this possibility. REFERENCES 1. Nishizuka, Y. (1988) Nature 334,661-665 2. Nishizuka, Y. (1989) Cancer 6 3 , 1892-1903 3. Yoshida, Y., Huang, F. L., Nakabayashi, H., and Huang, K.-P. (1988) J. Biol. Chem. 263,9868-9873 4. Hidaka, H., Tanaka, T., Onoda, K., Hagiwara, M., Watanabe, M., Ohta, H., Ito, Y., Tsurudome, M., and Yoshida, T. (1988) J. Biol. Chem. 263,4523-4526 5. Ohno, S., Kawasaki, H., Imajoh, S., Suzuki, K., Inagaki, M., Yokokura, H., Sakoh, T., and Hidaka, H. (1987) Nature 3 2 5 , 161-166 6. Sekiguchi, K., Tsukuda, M., Ogita, K., Kakkawa, U., and Nishizuka, Y. (1987) Biochem.Biophys. Res. Commun. 145, 797802 7. Ohno, S., Akita, Y., Konno, Y., Imajoh, S., and Suzuki, K. (1988) Cell 53,731-741 8. Ido, M., Sekiguchi, K., Kikkawa, U., and Nishizuka, Y. (1987) FEBS Lett. 219,215-218 9. Posada, J. A., McKeegan, E. M., Worthington, K. F., Morin, M. J., Jaken, S., and Tritton, T. R. (1989) CancerCommun. 1 , 285-292 10. Quino, A., Warren, B. S., Omichinski, J., Hartman, K. D., and Glazer, R. I. (1990) Biochem. Biophys. Res. Commun. 166, 723-728 11. Housey, G. M., Johnson, M. D., Hsiao, W. L. W., O’Brian, C. A., Murphy, J. P., Kirschmeier, P., and Weinstein, I. B. (1988) Cell 52,343-354 12. Megidish, T., and Mazurek, N. (1989) Nature 342,807-811 13. Melloni, E., Pontremoli, S., Michetti, M., Sacco, O., Cakiroglu, A. G., Jackson, J. F., Rifkind, R. A,, and Marks, P. A. (1987) Proc. Natl. Acad. Sci. U. S. A. 84,5282-5286 14. Makowske, M., Ballester, R., Cayre, Y., and Rosen, 0. M. (1988) J . Biol. Chem. 263,3402-3410 15. Obeid, L. M., Okazaki, T., Karolak, L. A., and Hannun, Y. A. (1990) J. Biol. Chem. 265, 2370-2374 16. Wada, H., Ohno, S., Kubo, K., Taya, C., Tsuji, S., Yonehara, S. and Suzuki, K. (1989) Biochem. Biophys. Res. Commun. 165, 533-538 17. Vandenbark, G. R., and Niedel, J. D. (1984) J.Natl. Cancer Inst. 73,1013-1019 18. Nishizuka, Y. (1984) Nature 308,693-698 19. Niedel, J. E., Kuhn, L., and Vandenbark, G.R.(1983) Proc. Natl. Acad. Sci. U. S. A. 80, 36-40 20. Ashendel, C. L., Staller, J. M., and Boutwell, R. K. (1983) Cancer Res. 43,4333-4337 21. Homma, Y., Gemmell, M. A., and Huberman, E. (1988) Cancer Res. 48,2744-2748 22. Gailani, D., Caldwell, F. J., O’Donnell, P. S., Hromas, R. A., and McFarlane, D. E. (1989) Cancer Res. 49,5329-5333

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