Diacylglycerol (DAG)-lactones, a New Class of Protein Kinase C(PKC) Agonists, Induce Apoptosis in LNCaP Prostate Cancer Cellsby Selective Activation of PKC�*

Received for publication, August 9, 2001, and in revised form, September 18, 2001Published, JBC Papers in Press, October 2, 2001, DOI 10.1074/jbc.M107639200

Maria Laura Garcia-Bermejo‡§, Federico Coluccio Leskow‡, Teruhiko Fujii‡, Qiming Wang¶,Peter M. Blumberg¶, Motoi Ohba�, Toshio Kuroki�, Kee-Chung Han**, Jeewoo Lee**,Victor E. Marquez‡‡, and Marcelo G. Kazanietz‡§§

From the ‡Center for Experimental Therapeutics and Department of Pharmacology, University of Pennsylvania School ofMedicine, Philadelphia, Pennsylvania 19104-6160, the ¶Laboratory of Cellular Carcinogenesis and Tumor Promotion,Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892, the �Department ofMicrobiology, School of Pharmaceutical Sciences, Showa University, Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan,the **Laboratory of Medicinal Chemistry, College of Pharmacy, Seoul National University, Shinlin-Dong, Kwanak-ku,Seoul 151-742, South Korea, and the ‡‡Laboratory of Medicinal Chemistry, Center for Cancer Research, NCI, NationalInstitutes of Health, Frederick, Maryland 21701

Phorbol esters, the archetypical (PKC) activators, induceapoptosis in androgen-sensitive LNCaP prostate cancercells. In this study we evaluate the effect of a novel class ofPKC ligands, the diacylglycerol (DAG)-lactones, as induc-ers of apoptosis in LNCaP cells. These unique ligandswere designed using novel pharmacophore- and receptor-guided approaches to achieve highly potent DAG surro-gates. Two of these compounds, HK434 and HK654, in-duced apoptosis in LNCaP cells with much higherpotency than oleoyl-acetyl-glycerol or phorbol 12,13-dibutyrate. Moreover, different PKC isozymes were foundto mediate the apoptotic effect of phorbol 12-myristate13-acetate (PMA) and HK654 in LNCaP cells. Using PKCinhibitors and dominant negative PKC isoforms, wefound that both PKC� and PKC� mediated the apo-ptotic effect of PMA, whereas only PKC� was involvedin the effect of the DAG-lactone. The PKC� selectivityof HK654 in LNCaP cells contrasts with similar potenciesin vitro for binding and activation of PKC� and PKC�.Consistent with the differences in isoform dependence inintact cells, PMA and HK654 show marked differences intheir abilities to translocate PKC isozymes. Both PMA andHK654 induce a marked redistribution of PKC� to theplasma membrane. On the other hand, unlike PMA,HK654 translocates PKC� predominantly to the nuclearmembrane. Thus, DAG-lactones have a unique profile ofactivation of PKC isozymes for inducing apoptosis inLNCaP cells and represent the first example of a selectiveactivator of a classical PKC in cellular models. An attrac-tive hypothesis is that selective activation of PKCisozymes by pharmacological agents in cells can beachieved by differential intracellular targeting of eachPKC.

The phorbol esters and related diterpenes are natural com-pounds that have been for many years the preferred pharma-cological tools for studying protein kinase C (PKC),1 a keyfamily of kinases implicated in growth factor- and G-protein-coupled receptor signaling. These compounds mimic the actionof the lipid second messenger diacylglycerol (DAG), a relativelysimple and highly flexible molecule generated by cellular phos-pholipases. The higher potency of phorbol esters and theirgreater stability compared with the second messenger DAGmakes these agents the preferred activators of PKC (1, 2).Phorbol esters regulate a variety of cellular functions, includ-ing cell cycle progression, differentiation, cytoskeleton remod-eling, and malignant transformation. Although phorbol esterspromote mitogenesis in several cell types, accumulating dataindicate that activation of PKC leads to inhibition of cellgrowth in many cells (3–6). Interestingly, phorbol esters induceapoptosis in several cell lines, including thymocytes, breastcancer cells, and prostate cancer cells (7–12).

The heterogeneity of effects of the phorbol esters is related tothe presence of multiple phorbol ester/DAG receptors, includ-ing PKC isozymes and “nonkinase” PKC receptors. The PKCfamily comprises at least 10 related kinases with differentialexpression, subcellular distribution and biochemical regula-tion. PKC isozymes have been classified into three subclassesaccording to their structure and regulation: “classical” orcalcium-dependent (“cPKCs” �, �I, �II, and �), “novel” or cal-cium-independent (“nPKCs” �, �, �, and �), and “atypical”(“aPKCs” � and /). A related enzyme, PKC� or protein kinaseD, is a distant relative of the PKC isozymes. Although aPKCsare insensitive to phorbol esters and DAG, cPKCs and nPKCsbind phorbol esters with high affinity in the presence of phos-pholipids as cofactors. PKC isozymes are subject to exquisiteregulatory mechanisms and can have either overlapping oropposite biological functions (13–16).

It is well established that activation of PKC by phorbol esterstriggers an apoptotic response in androgen-dependent prostate

* This work was supported in part by United States Department ofDefense Prostate Cancer Research Program Grant DAMD17-00-1-0531and National Institutes of Health Grant R01 CA89202-01A1 (toM. G. K.). The costs of publication of this article were defrayed in partby the payment of page charges. This article must therefore be herebymarked “advertisement” in accordance with 18 U.S.C. Section 1734solely to indicate this fact.

§ Recipient of a postdoctoral fellowship from Fundacion RamonAreces (Spain).

§§ To whom correspondence and reprint requests should be addressed:Center for Experimental Therapeutics, University of Pennsylvania School ofMedicine, 816 Biomedical Research Bldg. II/III, 421 Curie Blvd., Philadel-phia, PA 19104-6160. E-mail: marcelo@spirit.gcrc.upenn.edu.

1 The abbreviations used are: PKC, protein kinase C; DAG, diacyl-glycerol; PMA, phorbol 12-myristate 13-acetate; PDBu, phorbol 12,13-dibutyrate; OAG, 1-oleoyl-2-acetyl-sn-glycerol; DAPI, 4�,6-diamidino-2-phenylindole; cPKC, classical PKC; nPKC, novel PKC; aPKC, atypicalPKC; AdV, adenovirus; m.o.i., multiplicity of infection; pfu, plaque-forming unit(s); DN, dominant negative; FBS, fetal bovine serum; PBS,phosphate-buffered saline; GFP, green fluorescent protein; CHO, Chi-nese hamster ovary.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 1, Issue of January 4, pp. 645–655, 2002Printed in U.S.A.

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cancer cells, such as LNCaP cells (10–12, 17, 18). Elegant workfrom Powell and co-workers (11, 18) revealed that phorbolester-induced apoptosis in LNCaP prostate cancer cells corre-lates with a persistent translocation of PKC� to membranes.We have recently demonstrated that overexpression of PKC� inLNCaP cells markedly potentiates phorbol 12-myristate 13-acetate (PMA)-induced apoptosis, suggesting a role for PKC� inthe phorbol ester effect. Likewise, expression of a dominantnegative (kinase-inactive) form of PKC� partially inhibitsPMA-induced apoptosis (17). Therefore, multiple PKCisozymes may contribute to the apoptotic effect of phorbol es-ters in LNCaP cells.

An interesting emerging concept is that PKC activators havevaried, unique pharmacological profiles and exert in manycases discrete cellular responses. Differential responses to PKCactivators have been observed for analogs such as bryostatin 1,mezerein, and 12-deoxyphorbol esters (1, 2, 5). In this paper wefocus on a novel class of synthetic analogs, the DAG-lactones,which bind with high potency to the phorbol ester/DAG bindingsite in PKC (the C1 domain). These novel C1 domain ligandswere designed through a pharmacophore-guided approachbased on the crystal coordinates of the C1b domain of PKC� incomplex with phorbol-13-acetate. Remarkably, the DAG-lac-tones show 3–4 orders of magnitude higher affinity for PKCisozymes than natural DAGs. To generate such potent DAGs,the glycerol backbone was constrained into a rigid structure (alactone ring), resulting in a reduced entropic penalty associatedwith DAG binding to the receptor (19–23). Like phorbol estersand the natural DAGs in an “open conformation,” the DAG-lactones bind to PKC and induce its activation in vitro. More-over, these simple DAG analogs have phorbol ester-like effectsin cells, such as inhibition of epidermal growth factor binding,with potencies similar to that of PDBu. Some of these com-pounds have displayed important antitumor activity in the invitro cell line screen of the NCI (National Institutes of Health),but their mechanism of action is largely unknown (22). Noinformation is available on the specificity of the DAG-lactonesfor individual PKC isozymes.

In this paper we show that DAG-lactones, like phorbol es-ters, induce apoptosis in LNCaP prostate cancer cells. A re-markable finding is that DAG-lactones and PMA exert theirapoptotic effect through the activation of a different subset ofPKC isozymes, as revealed by a series of pharmacological andmolecular approaches. Thus, DAG-lactones represent a noveltemplate for the design of potent selective PKC agonists, whichmay be useful tools to dissect PKC isozyme-specific functions incells.

EXPERIMENTAL PROCEDURES

Materials—PMA was obtained from LC Laboratories (Woburn, MA).GF109203X, Go6976, and rottlerin were purchased from Alexis Corp.(San Diego, CA). The pan-caspase inhibitor z-VAD was obtained fromCalbiochem (San Diego, CA). 4�,6-Diamidino-2-phenylindole (DAPI)was purchased from Sigma. Cell culture reagents and media werepurchased from Life Technologies, Inc.

Cell Culture—The human prostate cancer cell line LNCaP was ob-tained from the American Type Culture Collection (Rockville, MD).LNCaP cells were cultured in RPMI 1640 medium supplemented with10% fetal bovine serum (FBS), 100 units/ml penicillin, and 100 �g/mlstreptomycin at 37 °C in a humidified 5% CO2 atmosphere. LNCaP cellsoverexpressing Bcl-2 (LNCaP-Neo/Bcl-2) and the corresponding controlcells transfected with empty vector (LNCaP-Neo) (24) were a gift of Dr.L. Lothstein (University of Tennessee, Memphis, TN).

Infection of LNCaP Cells with PKC Adenovirus—In this study weused replication-deficient adenoviruses (AdV) for overexpression of in-dividual PKC isozymes. Generation of the PKC�AdV and PKC�AdV hasbeen described elsewhere (17, 25–27). Kinase-inactive PKCs were gen-erated by an Arg to Lys substitution at the ATP-binding site of thecatalytic domain, and the corresponding AdVs were then generated asdescribed in Ref. 26. AdVs were amplified in HEK 293 cells using

standard techniques (28). Titers of viral stocks were normally higherthan 1 � 109 pfu/cell. The absence of wild type AdV was confirmed byPCR using primers for the E1 region. An AdV for the LacZ gene(LacZAdV) was used as a control (17).

Subconfluent LNCaP cells in 6- or 12-well plates were infected withAdVs for 14 h at multiplicities of infection (m.o.i.) ranging from 1 to 30pfu/cell (in RPMI 1640 medium supplemented with 2% FBS). Followinginfection, the medium was replaced with fresh RPMI 1640 mediumsupplemented with 10% FBS and the cells were grown for an additional24 h. Maximum expression of PKC isozymes after AdV infection wasachieved with this protocol. Expression of recombinant protein re-mained stable for several days (17).

Synthetic DAG-lactones, PMA, or vehicle (ethanol) were added for1 h at different concentrations to either noninfected cells or to cellsinfected with different AdVs. After treatment, cells were grown inRPMI 1640 supplemented with 10% FBS for 24 h. PKC inhibitors (GF109203X, Go6976, or rottlerin) or the pan-caspase inhibitor z-VAD wereadded 1 h before and during DAG-lactone or PMA treatment.

Western Blot Analysis—Cells were harvested and lysed in a buffercontaining 50 mM Tris-HCl, pH 6.8, 10% glycerol, 2% SDS, and 5%�-mercaptoethanol. Equal amounts of protein (10 �g) were subjected toSDS-PAGE and transferred to nitrocellulose membranes. Membraneswere blocked with 5% nonfat milk and 0.1% Tween 20 in phosphate-buffered saline (PBS) and then incubated with a monoclonal anti-PKC�antibody (1:3000, Upstate Biotechnology, Inc., Lake Placid, NY) or apolyclonal anti-PKC� antibody (1:1000, Santa Cruz Biotechnology,Santa Cruz, CA). Membranes were washed three times with 0.2%Tween 20, PBS and incubated with anti-mouse or anti-rabbit secondaryantibody conjugated to horseradish peroxidase (1:10,000, Bio-Rad).Bands were visualized by the enhanced chemiluminescence (ECL)Western blotting detection system (Amersham Pharmacia Biotech).

Visualization by Confocal Microscopy of GFP-PKC Translocation—LNCaP cells were transfected with vectors encoding GFP fusion pro-teins for PKC� and PKC� (29) using LipofectAMINE Plus (LifeTechnologies, Inc.) according to the manufacturer’s instructions. Exper-iments were performed 3 days after transfection. Prior to observation,transiently transfected LNCaP cells were washed twice with standardmedium (Dulbecco’s modified Eagle’s medium without phenol red sup-plemented with 1% FBS) prewarmed to 37 °C. All PKC activators werediluted to specified concentrations in the same medium, and the finalconcentration of solvent (ethanol) was always less than 0.01%.

For live cell imaging, the Bioptechs Focht Chamber System (FCS2)was inverted and attached to the microscope stage with a custom stageadapter. The cells cultured on a 40-mm round coverslip were introducedinto the chamber system, which was connected to a temperature con-troller set at 37 °C, and medium was perfused through the chamberwith a model P720 microperfusion pump (Instech, Plymouth Meeting,PA). As indicated, the perfusate to the chamber was changed to thatcontaining the specified ligand for PKC and sequential images of thesame cell were then collected at 1-min intervals using LaserSharpsoftware through a Bio-Rad MRC 1024 confocal scan head mounted ona Nikon Optiphot microscope with a 60� planapochromat lens. Excita-tion at 488 nm was provided by a krypton-argon gas laser with a 522/32emission filter for green fluorescence.

Apoptosis Assays—To assess morphological changes in chromatinstructure, cells were stained with DAPI. Cells were then trypsinized,mounted on glass slides, fixed in 70% ethanol, and stained for 20 minwith 1 mg/ml DAPI. Apoptosis was characterized by chromatin conden-sation and fragmentation when examined by fluorescence microscopy.The incidence of apoptosis in each preparation was analyzed by count-ing 500 cells and determining the percentage of apoptotic cells (17).DNA laddering was measured using the Apoptotic DNA-Ladder kitfrom Roche Molecular Biochemicals. For flow cytometry analysis, cellswere fixed in 70% ethanol and resuspended in PBS containing pro-pidium iodide (1 mg/ml) and RNase (40 �g/ml). Cell cycle progressionand apoptosis were analyzed in an Epics XL flow cytometer (CoulterCorp, Hialeah, FL). For each treatment 7,500 events were recorded.

[3H]PDBu Binding—[3H]PDBu binding to PKC isozymes was meas-ured using the polyethylene glycol precipitation assay. A detailed de-scription of the methodology is presented in Ref. 30. To measure com-petition of [3H]PDBu binding by different analogs, we used a fixedconcentration of [3H]PDBu (3 nM) and increasing concentrations (intriplicate) of the competing nonradioactive ligand. ID50 values weredetermined from the competition curve, and the Ki for the competingligand was calculated from the ID50 by using the relationship Ki �ID50/(1 � L/Kd), where Kd is the dissociation constant for [3H]PDBu andL is the concentration of free [3H]PDBu at the ID50. PKC� and PKC�

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used in these assays were generated by baculovirus infection ofSf9 insect cells and subsequent purification, as described pre-viously (30).

PKC Activity—PKC activity was assayed by measuring the incorpo-ration of 32P from [�-32P]ATP into a specific PKC substrate (�-pseudosubstrate peptide), as described previously (30), using 100 �g/mlphospholipid vesicles (20% phosphatidylserine, 80% phosphatidylcho-line) prepared by sonication. The reaction was carried out at 30 °C for10 min. Kinase activity was linear with time over this incubationperiod.

Protein Determination—Protein determinations were made with theMicro BCA Protein Assay from Pierce, using bovine serum albumin asa standard.

RESULTS

Induction of Apoptosis by Synthetic DAG-lactones—DAGspossess substantially lower potency for binding to PKCs andreduced metabolic stability compared with the correspondingphorbol ester analogs. An approach that was used to success-fully generate potent DAG analogs involved constraining theglycerol backbone into a five-member ring (DAG-lactone). Withthe combined use of pharmacophore- and receptor-guided ap-proaches facilitated by the crystal structure of the PKC� C1bdomain, it was possible to rationally design structurally simpleDAG-lactone ligands that displayed binding affinities for PKC�in the low nanomolar range (19–23). However, very limitedinformation on the biological activity of these DAG-lactones isavailable other than their in vitro binding affinity for PKC� asa receptor.

A group of 29 DAG-lactones that exhibited high bindingaffinities for PKC� (in the low nanomolar range) were selectedto generate a structurally diverse set of compounds displayingvarious combinations of R1 and R2 groups (general structure,Fig. 1). Their affinities for PKC� expressed in term of Ki valuescovered a wide range: 2–6 nM (10 compounds), 6–9 nM (3compounds), 9–13 nM (6 compounds), 12–20 nM (4 compounds),20–40 nM (2 compounds), and �� 40 nM (4 compounds). Thelipophilicity range (log P) spanned between 3.5 and 6.5. Be-cause phorbol esters exhibit pro-apoptotic activity in LNCaPprostate cancer cells, we decided to evaluate these 29 com-pounds for their apoptotic effect in these cells. From this group,four compounds were selected for their unique structure-activ-ity relationship in terms of their apoptotic inducing potency.The structures of these compounds are shown together with thecode names used in the text (Fig. 1). The complete set of 29compounds, as well as the description of their syntheses and

characterization, including the selected compounds for thiswork, will be described elsewhere.2

LNCaP cells were treated with different DAG-lactones at asingle concentration (10 �M) for 1 h, and apoptosis was assessed24 h later by counting the number of apoptotic cells after DAPIstaining. After such time, a maximum apoptotic response isnormally observed following PKC activation (17). Both HK434and HK654 induced �30% of apoptosis, which equals the max-imum response observed with PMA under the experimentalconditions used in our studies. On the other hand, DAG-lac-tones HK204 and HK602 showed only a modest response, aswas also observed with 1-oleoyl-2-acetylglycerol (OAG), a DAGthat is commonly used to activate PKC in cellular models (Fig.2A). A representative field with apoptotic cells under the fluo-rescent microscope is shown in Fig. 2B. As shown in the figure,a large number of cells with morphological changes distinctiveof apoptosis, including nuclear fragmentation and cell shrink-age, were observed after treatment with PMA or DAG-lactones.HK434, HK654, and PMA were able to induce a characteristicpattern of DNA fragmentation, visualized as DNA laddering inagarose gels (Fig. 2C). A concentration-dependence analysisshowed that HK434 and HK654 were more potent than OAG orPDBu, a phorbol ester commonly used to activate PKC. Asexpected, the inactive phorbol ester 4�-PMA was totally inef-fective in inducing apoptosis in LNCaP cells. The presence ofapoptotic cells after treatment with DAG-lactones was alsodetected by flow cytometry analysis (see Fig. 4).

Inhibition of Apoptosis by a Pan-caspase Inhibitor and Bcl-2Overexpression—Caspases are essential molecules for the exe-cution of apoptosis (31). This family of Asp-directed cysteine-proteases can be specifically inhibited by cell-permeable pep-tides. We treated LNCaP cells with the pan-caspase peptidez-VAD (50 �M) and evaluated the apoptotic effect of the DAG-lactone HK654 and PMA. As shown in Fig. 3A, z-VAD mark-edly reduced the number of apoptotic cells after HK654 or PMAtreatment.

The role of Bcl-2 as a key anti-apoptotic molecule is wellestablished in many cell types, including prostate cancer cells(31, 32). We investigated the effect of DAG-lactones in LNCaPcells transfected with a mammalian expression vector thatencodes for Bcl-2. These cells (LNCaP-Neo/Bcl-2) expressedhigh levels of Bcl-2 compared with vector-transfected cells(LNCaP-Neo) (24). Cells overexpressing Bcl-2 showed a markedresistance to apoptosis when stimulated with either HK654 orPMA. The apoptotic responses to PMA and HK654 in Bcl-2-overexpressing cells are only 41 and 32%, respectively, of thoseobserved in vector-transfected cells (Fig. 3B).

Effect of PKC Inhibitors—To confirm that the apoptotic effectof DAG-lactones in LNCaP cells is mediated by activation ofPKC, we assessed the effect of these compounds in the presenceof PKC inhibitors. This is important because phorbol esters andDAG have other targets in addition to PKC isozymes (2, 5, 16,33). We first used the PKC inhibitor GF 109203X, an inhibitorof cPKCs and nPKCs capable of blocking the apoptotic effect ofPMA in LNCaP cells (17). GF 109203X (5 �M) also blocked theapoptotic effect of HK434 and HK654 almost completely, asjudged by counting of apoptotic cells after DAPI staining (Fig.4A). The incidence of apoptosis after treatment of LNCaP cellswith GF 109203X in the absence of PKC activators was lessthan 2% (see Ref. 17). The inhibitory effect of GF 109203Xcould also be ascertained by flow cytometry analysis. The DNAhistograms shown in Fig. 4B revealed a large number of cellswith sub-G0/G1 DNA content after HK434, HK654, or PMA

2 J. Lee, K.-C. Han, J.-H. Kang, N. E. Lewin, S. Yan, S. Benzaria,M. C. Nicklaus, P. M. Blumberg, and V. E. Marquez, submitted forpublication.

FIG. 1. Structure of DAG-lactones.

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treatment, which is consistent with the presence of apoptoticcells. In all cases the effect was abolished by treatment with GF109203X. Treatment with GF 109203X alone did not produceany noticeable increase in the population of cells with sub-G0/G1 DNA content.

LNCaP cells express the classical PKC�, the novel PKC�,and the phorbol ester/DAG-unresponsive PKC� and PKC.Very low levels of PKC� and PKC� were also detected (17).These results agree with those reported previously by Powelland co-workers (11, 18). Previous work from the Powell labo-ratory has established that PKC� is a mediator of PMA-in-duced apoptosis in LNCaP cells (11, 18). Our recent workestablished that PKC� is also a pro-apoptotic isozyme inLNCaP cells (17), suggesting the existence of overlapping rolesfor both PKC� and PKC� in this cellular model. To evaluate theinvolvement of PKC� and PKC� as mediators of the pro-apo-ptotic effect of DAG-lactones, we first used a pharmacologicalapproach. Two PKC inhibitors known to have selectivity forPKC isozymes were used: Go6976, an inhibitor of the cPKCs(34), and rottlerin, which preferentially inhibits PKC� (35).Because the only cPKC present in LNCaP cells is PKC�,Go6976 represents a PKC�-selective inhibitor in our model.Fig. 5A shows that Go6976 inhibits the apoptotic effect of

HK654 or PMA in a dose-dependent fashion. Go6976 did notaffect the basal levels of apoptosis in LNCaP cells. Interest-ingly, Go6976 did not fully inhibit the apoptotic effect of PMA.In fact, the inhibitory effect of Go6976 on PMA-induced apo-ptosis seems to plateau at �50% at 10–30 �M, whereas furtherinhibition by Go6976 was observed for HK654. This partialinhibition of PMA-induced apoptosis suggests that PKC� is notthe only PKC isozyme that mediates the phorbol ester effect.Remarkably, the PKC� inhibitor rottlerin partially blockedPMA-induced apoptosis but did not affect the apoptotic effect ofHK654 (Fig. 5B). Treatment of rottlerin alone induces �5% ofapoptosis at the highest concentration tested. These experi-ments using PKC inhibitors suggest that, although PMA in-duces apoptosis through activation of PKC� and PKC�, onlyPKC� is involved in the pro-apoptotic effect of theDAG-lactones.

Effects of Overexpression of PKC� or PKC� on HK654- andPMA-induced Apoptosis—The differential sensitivity of PMAand DAG-lactones to PKC inhibitors prompted us to explorethe issue of isozyme selectivity in further detail. An approachthat has been extensively used to assess PKC isozyme selectiv-ity is the overexpression of individual PKCs. We have success-fully used adenoviral delivery of PKC� to LNCaP cells as a

FIG. 2. DAG-lactones induce apoptosis in LNCaP cells. LNCaP cells were treated with different compounds for 1 h. Apoptosis wasdetermined 24 h later. Panel A, apoptosis induced by 100 nM PMA, 10 �M OAG, or 10 �M DAG-lactones. Panel B, LNCaP cells were stained withDAPI and nuclear morphology was assessed by fluorescence microscopy. Apoptotic cells are indicated by arrows. Cc, vehicle (control). Panel C, DNAfragmentation visualized in 2% agarose gels after staining with ethidium bromide. Lane 1, untreated cells; lane 2, HK434 (10 �M); lane 3, 10 �M

HK654; lane 4, 100 nM PMA. Ms, molecular size standards. Panel D, concentration-dependence analysis of apoptosis induced by phorbol esters(PMA, 4�-PMA, and PDBu), OAG, and DAG-lactones. In panels A and D, apoptosis was determined after DAPI staining. The incidence of apoptosisin each preparation was analyzed by counting 500 cells and determining the percentage of apoptotic cells. Results are the mean � S.E. of threeindependent experiments.

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strategy to demonstrate the involvement of this nPKC in PMA-induced apoptosis (17). In agreement with our previous studies(17), PKC� overexpression markedly potentiated the apoptoticeffect of PMA (Fig. 6A). The effect was proportional to the levelof expression of PKC�, as judged by Western blot (Fig. 6C) or byin vitro kinase assays (Ref. 17, and data not shown). Notsurprisingly, a similar potentiation of the PMA effect was ob-served after infection of LNCaP cells with PKC�AdV (m.o.i. �1–30 pfu/cell). In this later case, the potentiation of the PMAeffect also correlates with the expression levels of PKC�. Onthe other hand, a control LacZAdV was totally ineffective (Fig.6A). Interestingly, a different pattern was observed for theDAG-lactone HK654. As in the case of PMA, overexpression ofPKC� using the PKC�AdV markedly potentiated the apoptoticeffect of the HK654. However, infection of LNCaP cells withPKC�AdV did not produce any significant effect on HK654-induced apoptosis. As expected, infection with the LacZAdVwas also without effect (Fig. 6B). As shown previously (17),infection with PKC�AdV, PKC�AdV or LacZAdV (m.o.i. � 1–30pfu/cell) induces only �2% of apoptosis in the absence of a PKCactivator (data not shown). In agreement with the data usingspecific PKC inhibitors, these results suggest that PMA andDAG-lactones activate a different subset of PKC isozymes topromote apoptosis in LNCaP cells.

Effect of Dominant Negative PKC� (DN-PKC�) and Domi-nant Negative PKC� (DN-PKC�) Mutants on HK654- and PMA-induced Apoptosis—To further explore the role of PKC� andPKC� as mediators of apoptosis in LNCaP cells, we decided toevaluate the effect of the expression of kinase-deficient mu-

tants, which were shown to act as dominant negatives (DN)that interfere with PKC function. We used AdVs for PKC� andPKC� mutants in which an Arg to Lys mutation was introducedin the ATP-binding site (DN-PKC�AdV and DN-PKC�AdV)(26, 27). These mutants are kinase-inactive after expression in

FIG. 3. Inhibition of DAG-lactone-induced apoptosis by acaspase inhibitor and Bcl-2 overexpression. Panel A, LNCaP cellswere treated with 100 nM PMA (open bars), 10 �M HK654 (solid bars),or vehicle (hatched bars) in the absence or presence of the pan-caspaseinhibitor z-VAD (50 �M) added 1 h before and during treatment. PanelB, LNCaP cells overexpressing Bcl-2 (LNCaP-Neo/Bcl-2) or mock-trans-fected cells (LNCaP-Neo) were treated with 100 nM PMA (open bars), 10�M HK654 (solid bars), or vehicle (hatched bars). Cells were collected24 h later and stained with DAPI. The incidence of apoptosis in eachpreparation was analyzed by counting 500 cells and determining thepercentage of apoptotic cells. Results are the mean � S.E. of threeindependent experiments.

FIG. 4. Effect of the PKC inhibitor GF 109203X. LNCaP cellswere treated with vehicle, 100 nM PMA, 10 �M HK654, or 10 �M HK434,either in the absence or presence of 5 �M GF 109203X, which waspresent 1 h before and during the treatment with DAG-lactones orPMA. Panel A, cells were collected 24 h after treatment and stainedwith DAPI. The incidence of apoptosis in each preparation was ana-lyzed by counting 500 cells and determining the percentage of apoptoticcells. Results are the mean � S.E. of three independent experiments.Panel B, after staining with propidium iodide, DNA content was ana-lyzed by flow cytometry. A representative experiment is shown. Similarresults were observed in two additional experiments. Cc, control (vehi-cle); GF, GF 109203X.

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LNCaP cells (17). Expression of DN-PKCs after infection witheither DN-PKC�AdV or DN-PKC�AdV can be readily detectedby Western blot using specific anti-PKC� or anti-PKC� anti-bodies, respectively (Fig. 7C). The data in Fig. 7A show thatinfection of LNCaP cells with increasing m.o.i. values of DN-PKC�AdV inhibits the apoptotic effect of both PMA andHK654. On the other hand, whereas expression of DN-PKC�

inhibits the apoptotic effect of PMA by �30%, it does not affectthe apoptotic effect of HK654. Taken together, these findingsstrongly suggest that PKC�, but not PKC�, mediates the apo-ptotic effect of HK654 in LNCaP cells.

Effect of HK654 on [3H]PDBu Binding and PKC Activa-tion—We next evaluated the interaction of HK654 with PKCisozymes using binding assays. The issue we wished to test waswhether the differences in isozyme specificity observed inLNCaP cells could be explained by a differential pattern ofrecognition of the ligand. To test this hypothesis, we deter-mined the binding affinity of HK654 for PKC� expressed in Sf9insect cells (30). Binding was determined using a fixed concen-tration of the radioligand [3H]PDBu and increasing concentra-tions of HK654. Fig. 8 shows that HK654 very efficiently com-petes for [3H]PDBu binding to PKC�. The Ki for inhibition ofbinding to PKC�, determined from the ID50, was 1.6 � 0.1 nM

(n � 5). Under the same experimental conditions, the Ki forinhibition of binding to recombinant PKC� was 5.4 � 0.3 nM

(n � 3). Therefore, HK654 binds with high affinity to bothPKC� and PKC�.

We then compared the ability of HK654 to activate kinaseactivity in vitro using �-pseudosubstrate peptide, a substratethat can be efficiently phosphorylated both by PKC� and PKC�

(30). Fig. 9 shows that HK654 activates PKC� and PKC� withsimilar potency. The maximum activation observed withHK654 was similar to that observed with 1 �M PMA (a concen-tration that induces maximum activation in our experimental

FIG. 6. Effect of overexpression of PKC� or PKC� on HK654- and PMA-induced apoptosis. LNCaP cells were infected with PKC�AdV(open bars), PKC�AdV (solid bars), or LacZAdV (hatched bars) at different m.o.i. values (1–30 pfu/cell) for 14 h. Twenty four h later cells weretreated with either 3 nM PMA (panel A) or 1 �M HK654 (panel B) for 1 h. Cells were collected 24 h later and stained with DAPI. The incidence ofapoptosis in each preparation was analyzed by counting 500 cells and determining the percentage of apoptotic cells. Results are the mean � S.E.of three independent experiments. Panel C shows representative Western blots for the expression of PKC� or PKC� after infection with PKC�AdVor PKC�AdV, respectively, using the m.o.i. values indicated in the figure. Expression of the corresponding PKC isozyme in cell extracts wasevaluated using specific PKC� and PKC� antibodies, as described under “Experimental Procedures.”

FIG. 5. Effect of Go6976 and rottlerin. LNCaP were treated withincreasing concentrations of either Go6976 (panel A) or rottlerin (panelB), which was present 1 h before and during PMA or HK654 treatment.Twenty-four h later cells were stained with DAPI. The incidence ofapoptosis in each preparation was then assessed by counting 500 cellsand determining the percentage of apoptotic cells. Results are themean � S.E. of three independent experiments. Open bars, 100 nM

PMA; solid bars, 10 �M HK654; hatched bars, vehicle.

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conditions), suggesting that HK654 fully activates PKC� andPKC�. Therefore, the isozyme-specific effects of HK654 in LNCaPcells cannot be explained by a differential intrinsic ability to bindand/or activate discrete PKC isozymes.

Subcellular Distribution of PKC� and PKC� in LNCaPCells—Experiments using subcellular fractionation by ultra-centrifugation show that HK654, like PMA, translocates PKC�from soluble (cytosolic) to particulate (membrane) fraction inLNCaP cells (data not shown). An attractive hypothesis is thatthe differential activation of PKC isozymes by HK654 in LNCaPcells is the consequence of a differential pattern of translocationof PKCs. To explore this issue, we used GFP-tagged PKCs. Plas-mids encoding for GFP-PKC� and GFP-PKC� were transfectedinto LNCaP cells and the pattern of subcellular redistribution inresponse to PMA and HK654 evaluated by confocal microscopy.Fig. 10A shows that both PMA and HK654 redistribute GFP-

PKC� from the cytoplasm to the plasma membrane. A quantita-tive analysis of translocation revealed that translocation to theplasma membrane peaked at �10 min for 1 �M PMA and at 7 minfor 100 �M HK654 (Fig. 10, B and C). No translocation to thenuclear membrane was detected. However, a different pattern oftranslocation was observed for GFP-PKC� in LNCaP cells. Inagreement with a previous report in Chinese hamster ovary(CHO) K1 cells (29), PMA induced a rapid redistribution of GFP-PKC� to the plasma membrane, followed by a slower redistribu-tion to nuclear membrane (Fig. 10, D and E). On the other hand,HK654 induced a pronounced and rapid translocation of GFP-PKC� to the nuclear membrane. A patchy pattern of fluorescenceappearing throughout the cytoplasm was observed in some cases.Although slight plasma membrane staining was observed insome GFP-PKC�-transfected cells after HK654 treatment, a

FIG. 7. Effect of DN-PKC�AdV andDN-PKC�AdV on HK654- and PMA-induced apoptosis. LNCaP cells were in-fected with DN-PKC�AdV (panel A) andDN-PKC�AdV (panel B) for 14 h at them.o.i. values indicated in the figure. After24 h, 100 nM PMA (open bars) and 10 �M

HK654 (solid bars) were added for 1 h.Apoptosis was determined 24 h later afterDAPI staining. The incidence of apoptosisin each preparation was analyzed by count-ing 500 cells and determining the percent-age of apoptotic cells. Results are themean � S.E. of three independent experi-ments. Panel C, representative Westernblot of extracts of LNCaP cells infectedwith DN-PKC�AdV (panel A) and DN-PKC�AdV (panel B) using the m.o.i. valuesindicated in the figure. Expression wasevaluated with specific anti-PKC� and an-ti-PKC� antibodies, as described under“Experimental Procedures.”

FIG. 8. Competition of [3H]PDBu binding to PKC� by HK654.Binding to recombinant PKC� was performed using a fixed concentra-tion of [3H]PDBu (5 nM) in the presence of 100 �g/ml phosphatidyl-serine, and six increasing concentrations (in triplicate) of nonradioac-tive HK654. Each point represents the mean � S.E. of triplicatedeterminations in a single experiment. Four additional experimentsgave similar results.

FIG. 9. Activation of PKC� and PKC� by HK654. PKC� and PKC�activities were assayed in the presence of 100 �g/ml phospholipid ves-icles (20% phosphatidylserine, 80% phosphatidylcholine) and increas-ing concentration of HK654. Results are expressed as percentage ofmaximum activation observed with 1 �M PMA (dotted line) and repre-sent the mean � S.E. of triplicate determination in a single experiment.Two additional experiments gave similar results.

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careful quantitative analysis revealed that this effect was negli-gible if compared with the intensity of nuclear membrane trans-location (Fig. 10F). Therefore, significant differences in the pat-tern of translocation of PKC� in LNCaP cells were observed forHK654 and PMA.

DISCUSSION

In the present study we demonstrated that DAG-lactones, anovel class of synthetic DAG analogs, induce apoptosis in LNCaPprostate cancer cells. Unlike PMA, which mediates apoptosisthrough the activation of PKC� and PKC�, HK654 activates onlyPKC� in LNCaP cells. The design of the �-lactone templaterepresents an important step in the rational synthesis of novelPKC activators with biological activity. These molecules wereproduced by constraining the glycerol backbone of DAG into afive-membered ring, a modification that was able to reduce theentropic penalty associated with DAG binding. An extensive syn-thetic effort based on molecular docking experiments that uti-lized the PKC C1b domain of PKC� complexed with phorbol13-acetate led to the generation of a series of DAG mimetics within vitro affinities for PKC in the micromolar and nanomolarrange (19–21). An important step to achieve low nanomolarbinding affinities was the use of branched acyl or �-alkylidenechains in these minimal structures, a strategy that optimized theinteractions of these compounds through van der Waals contactswith highly conserved amino acids of the C1 domain and with themembrane (22, 23). Our results show that, in LNCaP prostatecancer cells, the DAG-lactones HK434 and HK654 were consid-erably more potent than the phorbol ester PDBu and the widelyused DAG analog OAG.

The heterogeneity in the cellular responses of PKC agonistshighlights the complexity in the regulation of PKC isozymes. Itappears that exquisite regulatory mechanisms and discreteactivation of signaling pathways by individual members of thePKC family may take place in different cell types, leading toeither overlapping or opposite biological functions. The datapresented in this paper reveal that both PKC� and PKC�, themost prominent cPKC and nPKC isozymes in LNCaP cells,have overlapping pro-apoptotic functions in this cell line. Wehave shown previously that PKC� promotes apoptosis in an-drogen-sensitive prostate cancer cells (17), an effect also de-scribed in hemopoietic cells, keratinocytes, and salivary glandacinar cells (40–43). A growth-inhibitory role for PKC� wasalso reported in numerous cell types. Early work from Mischaket al. (36) showed that overexpression of PKC� in NIH 3T3fibroblasts inhibits cell growth, in contrast to the effect ob-served with PKC�, which promotes cell growth and transfor-mation in this cell type. In other cell types, such as CHO orHL-60 cells, ectopic expression of PKC� leads G2/M arrest (37,38). On the other hand, PKC� enhances anchorage-indepen-dent growth and metastatic potential in mammary adenocar-cinoma cells (39). Different roles for PKC� in proliferation andapoptosis have also been described in several cell types. Earlywork using overexpression strategies suggested that PKC�promotes cell growth in fibroblasts (44). PKC� also has a pro-tective effect against apoptosis after interleukin-3 withdrawalin 32D myeloid progenitor cells (45). Furthermore, inhibition ofPKC� function is sufficient to trigger cell death in Ramos-BL Bcells (46), glioma cells (47), and COS cells (48). Anti-apoptotic

effects have also been ascribed to other PKC isozymes, includ-ing PKC� and the atypical PKCs (49, 50). Despite the growth-promoting effect described for PKC�, this cPKC inhibits prolif-eration or has pro-apoptotic properties in many cellular models(3, 6, 51). Given the growth inhibitory properties of PKCisozymes, an emerging theme is that PKC activation ratherthan PKC inhibition may have therapeutic value in appropri-ate systems, and indeed several PKC agonists are under eval-uation as anti-cancer agents in clinical studies (52–54).

The mechanisms underlying the pro-apoptotic effects of PKCisozymes are still poorly understood. Apoptosis upon UV radi-ation of hemopoietic cells and keratinocytes involves the gen-eration of an active 40-kDa fragment corresponding to thePKC� C-terminal catalytic domain. A caspase-3 cleavage site ispresent in the hinge region (V3 domain) of PKC�. Moreover, acaspase-3 inhibitor prevents the generation of the 40-kDa cat-alytic fragment, which suggests an important role for caspase-3in the activation of PKC� during apoptosis induced by UVradiation (40, 42). In LNCaP cells, however, we have shownthat phorbol ester-induced apoptosis is caspase-3-independent,as judged by the lack of effect of the caspase-3 inhibitor DEVDand lack of caspase-3 cleavage (17). We hypothesize that, inLNCaP cells, phorbol esters and DAG-lactones promote theallosteric activation of PKC isozymes, and that PKCs are pri-mary effectors or participate in a pathway that signals toapoptosis. Therefore, distinct mechanisms, namely proteolyticcleavage and allosteric activation, may be involved in apoptosistriggered by different stimuli. Whether each of these mecha-nisms involves the activation of different signaling pathways oroccurs at different phases of the apoptotic process is still un-known. Allosteric activation of PKC, rather than proteolyticcleavage, is probably the primary mechanism of activation byphorbol esters and DAG-lactones in LNCaP cells, a hypothesisthat is also supported by the fact that PKC� does not possess acaspase cleavage site in its structure, and that proteolyticcleavage of PKC� or PKC� was not observed upon activationwith phorbol esters or HK654 in LNCaP cells (Ref. 17, and datanot shown).

The subcellular redistribution or translocation of cPKCs andnPKCs from cytosol to plasma membrane is a hallmark of theiractivation. A striking observation in this paper is the contrast-ing pattern of translocation of PKC isozymes in LNCaP cells.Both PKC� and PKC� are translocated to the plasma mem-brane by PMA. PKC� is translocated to the plasma membraneafter HK654 treatment, but PKC� is primarily redistributed tothe nuclear membrane by the DAG-lactone. Although we haveobserved some PKC� localization at the plasma membraneafter HK654 treatment, our quantitative analysis revealedthat it was minimal when compared with nuclear transloca-tion. Because a substantial proportion of PKC� is normally asso-ciated to membranes in unstimulated cells (data not shown), wecannot rule out that this small pool of PKC� is already present inthe plasma membrane before stimulation. It may be possible thatthe apoptotic effect of PKC isozymes requires the phosphoryla-tion of a PKC substrate in the plasma membrane. It has beendemonstrated that PMA-induced apoptosis in LNCaP cells in-volves the persistent membrane translocation of PKC� and theactivation of the Raf-mitogen-activated protein kinase pathway

FIG. 10. Translocation of PKC isozymes by DAG-lactones. Representative fluorescent images of LNCaP cells expressing GFP-PKC� (panelA) and GFP-PKC� (panel D) treated with 100 nM PMA or 10 �M HK654 for the times indicated in the figure. Quantitative changes in the fluorescentdistribution of GFP-PKC� and GFP-PKC� at the plasma membrane (open symbols) and nuclear membrane (closed symbols) in response to differentdoses of PMA or HK654 are shown in panels B and C (for GFP-PKC�) and in panels E and F (for GFP-PKC�). Results are expressed as changesin the ratios of plasma membrane and nuclear membrane translocation as a function of time. Ratio of membrane translocations was calculated asthe ratio of (Im Icyto)/Icyto, where Im represents the mean fluorescent intensity on the plasma or nuclear membrane in a given area and Icyto isthe mean fluorescent intensity in a comparable area of the cytoplasm or the nucleoplasm, respectively. The quantitative values represent theaverage of at least three experiments with 2–3 cells evaluated in each experiment.

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(18). Recent findings from our laboratory suggest that PKC alsoregulates Akt phosphorylation in LNCaP cells.3 Li et al. (25) havereported that translocation of PKC� to mitochondria is an essen-tial step for apoptosis in keratinocytes. However, we did notobserve any co-localization of PKC isozymes with MitoTracker (amitochondrial marker) in LNCaP cells upon PMA or HK654treatment (data not shown), suggesting divergent mechanismsinvolved in translocation in different cell types.

An important lesson from these studies is that marked dis-crepancies exist between in vitro and cellular effects of PKCligands. It is remarkable that HK654 does not discriminatebetween PKC isozymes in ligand binding and kinase assays(Figs. 8 and 9) but shows PKC� selectivity in translocation andapoptosis in LNCaP cells. Therefore, in vitro assays may un-derestimate the potential selectivity of PKC ligands in cells.Differences in in vitro binding affinities of PKC ligands for PKCisozymes and other phorbol ester receptors are normally verysmall (30, 33). Although in some cases this may reflect the highdegree of homology between the C1 domains of individualPKCs, one cannot rule out that this may be because of thesaturated amount of phospholipid (phosphatidylserine) cofac-tor used in in vitro binding and kinase assays, a condition thatdoes not necessarily reflect the physiological interactions thatoccur in a membrane environment. In support of this concept,12-deoxyphorbol esters, a family of analogs with anti-tumorpromoter activity in the mouse skin, showed marked differ-ences in the pattern of translocation of cPKCs and nPKCs inmouse keratinocytes (55), despite the similar potency for bind-ing to PKC isozymes in in vitro assays (30). Likewise, we haveillustrated in several systems that the structure-activity rela-tions for ligand binding to PKC (and related receptors) is de-pendent upon the lipid environment. As expected from theprinciples of pharmacology, under phospholipid conditions dif-ferentially limiting for one receptor, apparent ligand affinity tothis receptor is differentially impaired (56, 57).

The studies of Wang et al. (29, 58) both provide a markedparallel with the current findings and suggest a mechanisticbasis for them. These authors showed that the pattern of trans-location of PKC� in CHO-K1 cells differed markedly with thespecific ligand: phorbol esters, bryostatin 1, or DAG-lactones.In that study, as here, the DAG-lactones failed to induce trans-location of PKC� to the plasma membrane. In contrast, PKC�in CHO-K1 cells translocated to the plasma membrane in re-sponse to phorbol esters, bryostatin 1 or DAG-lactones,4 againsimilar to the results reported here for the LNCaP cells anddistinct DAG-lactones. We know for PKC� that the pattern oftranslocation depends, among other variables, on the lipophi-licity of the ligand (58). Under conditions of reduced lipophilic-ity, translocation to the nuclear membrane is favored. Whymay that be? From other studies, we know that PKC� differsfrom PKC� in its interaction with phospholipid membranes invitro.4 Under conditions of sufficient calcium, PKC� binds moreweakly to phospholipids than does PKC�. Under limiting con-ditions, PKC� would thus require a greater hydrophobic con-tribution from the ligand to achieve insertion to the plasmamembrane.

Although the full understanding of the structure-activityrelations for PKC� localization remains to be determined, anunambiguous finding from our studies is that PKC� can betargeted pharmacologically to different intracellular compart-ments and that this differential targeting may be consequen-tial. An attractive hypothesis is that “mislocalization” of a PKCisozyme may also lead to functional antagonism. Indeed, bryo-

statin 1 and 12-deoxyphorbol 13-phenylacetate, which translo-cate PKC� predominantly to nuclear membrane rather thanthe plasma membrane (29), antagonize PMA-mediated re-sponses. This hypothesis still needs to be tested withDAG-lactones.

In summary, our results provide strong evidence that DAG-lactones induce apoptosis in LNCaP prostate cancer cells byselective activation of PKC�, the only cPKC present in thismodel. It would be important to evaluate whether this isozymeselectivity occurs in cell types other than LNCaP cells. Like-wise, although PKC� is the only cPKC expressed in numerouscell types, it will be necessary to evaluate whether the DAG-lactones retain selectivity for PKC� in cells expressing othercPKCs. Because of the simplicity of their structures, DAG-lactones represent novel templates for the rational synthesis ofpotent selective agonists through pharmacophore and receptor-guided approaches. DAG-lactones are useful tools for studyingPKC isozyme selectivity and therefore help to overcome thelimitations that exist in the study of isozyme-specific functionsin cellular models.

Acknowledgment—We thank Dr. Patricia Lorenzo (NCI, NationalInstitutes of Health, Bethesda, MD) for GFP-PKC� plasmid.

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DAG-lactones Induce Apoptosis in LNCaP Cells 655

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Marquez and Marcelo G. KazanietzPeter M. Blumberg, Motoi Ohba, Toshio Kuroki, Kee-Chung Han, Jeewoo Lee, Victor E. Maria Laura Garcia-Bermejo, Federico Coluccio Leskow, Teruhiko Fujii, Qiming Wang,

αInduce Apoptosis in LNCaP Prostate Cancer Cells by Selective Activation of PKCDiacylglycerol (DAG)-lactones, a New Class of Protein Kinase C (PKC) Agonists,

doi: 10.1074/jbc.M107639200 originally published online October 2, 20012002, 277:645-655.J. Biol. Chem. 

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Additions and Corrections

Vol. 279 (2004) 5565–5572

Identification of cathepsin B as a mediator of neuronaldeath induced by A�-activated microglial cells using afunctional genomics approach.

Li Gan, Shiming Ye, Alan Chu, Kristin Anton, Saili Yi,Valerie A. Vincent, David von Schack, Daniel Chin, JosephMurray, Scott Lohr, Laszlo Patthy, Mirella Gonzalez-Zulueta,Karoly Nikolich, and Roman Urfer

Page 5571, Fig. 4C: The first value shown at bottom of Fig. 4Cshould be 0.5 �M. A corrected figure is shown at right.

Also, in the Fig. 4 legend, second line from the last, within theparentheses “[em],” should be deleted. It should read “(p �0.05) . . . .”

FIG. 4

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 22, Issue of May 28, pp. 23845–23846, 2004© 2004 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

We suggest that subscribers photocopy these corrections and insert the photocopies at the appropriate places where the article to becorrected originally appeared. Authors are urged to introduce these corrections into any reprints they distribute. Secondary (abstract)services are urged to carry notice of these corrections as prominently as they carried the original abstracts.

23845

Vol. 277 (2002) 645–655

Diacylglycerol (DAG)-lactones, a new class of proteinkinase C (PKC) agonists, induce apoptosis in LNCaPprostate cancer cells by selective activation of PKC�.

Maria Laura Garcia-Bermejo, Federico Coluccio Leskow,Teruhiko Fujii, Qiming Wang, Peter M. Blumberg, Motoi Ohba,Toshio Kuroki, Kee-Chung Han, Jeewoo Lee, Victor E. Marquez,and Marcelo G. Kazanietz

The structures of compounds HK654 and HK602 studied in thispaper were incorrectly reported as N-hydroxylamides in Lee etal. (Lee, J., Han, K.-C., Kang, J.-H., Pearce, L. L., Lewin, N. E.,Yan, S., Benzaria, S., Nicklaus, M. C., Blumberg, P. M., andMarquez, V. E. (2001) J. Med. Chem. 44, 4309–4312; Correc-tion (2003) J. Med. Chem. 46, 2794). The compounds corre-spond instead to esters HK434 and HK204. The reader shouldbe aware that the biological properties described for HK654correspond instead to HK434. When taking into considerationthe small difference in molecular weight (HK654, Mr � 397.55,and HK434, Mr � 382.54), the values reported remain virtuallyunchanged. The slightly greater potency for the alleged HK654in Fig. 2 is due to this difference, which resulted in testing aslightly more concentrated solution of HK434. Since discover-ing the problem, authentic samples of HK654 and HK602 havebeen synthesized and tested. They showed a nearly 1000-foldreduction in binding affinity towards PKC�. The correct struc-tures appeared in Choi et al. (Choi, Y., Kang, J. H., Lewin, N.E., Blumberg, P. M., Lee, J., and Marquez, V. E. (2003) J. Med.Chem. 46, 2790–2793). HK434 has the attributes of isozymespecificity and apoptotic-inducing activity originally associatedwith the N-hydroxylamide, and therefore the main conclusionsof our paper remain unchanged.

Vol. 279 (2004) 1176–1183

Diacylglycerols containing omega 3 and omega 6 fattyacids bind to RasGRP and modulate MAP kinaseactivation.

Sihem Madani, Aziz Hichami, Mustapha Cherkaoui-Malki,and Naim A. Khan

Page 1176: Dr. Cherkaoui-Malki’s name was misspelled in thisarticle. The correct spelling is shown above.

Additions and Corrections23846