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Tumor Biology |
Has the Potential to Advance the Recurrence of Human Prostate Cancer1
Department of Anatomy and Cell Biology, Brody School of Medicine, East Carolina University, Greenville, North Carolina 27858 [D. W., T. L. F., M. A. M., G. G. W., and D. M. T.], and Departments of Pediatrics [C. W. G.], Surgery (Division of Urology) [J. L. M.], Pathology and Laboratory Medicine [J. L. M.], and the Lineberger Comprehensive Cancer Center [J. L. M., O. H. F., R. F. A.], The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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(PKC), an oncogenic protein capable of promoting autocrine cell-signaling events. Gene transfer experiments demonstrated that PKC overexpression was sufficient to transform androgen-dependent LNCaP cells into an androgen-independent variant that rapidly initiated tumor growth in vivo in both intact and castrated male nude mice. This transformation was associated with an accelerated rate of androgen-independent LNCaP cell proliferation, resistance to apoptosis, hyperphosphorylation of the mitogen-activated protein kinase extracellular signal-regulated kinase and transcriptional repressor protein retinoblastoma, and increased expression of E2F-1 and other 5'-cap-dependent mRNAs, including the G1 cyclins, c-myc, and caveolin-1. Coimmunoprecipitation experiments indicated that PKC was associated with members of the extracellular signal-regulated kinase signaling cascade and the scaffolding protein caveolin-1. Caveolin-1, produced by LNCaP cells overexpressing PKC, was released into the medium, possibly through a Golgi-independent route, and significant growth inhibition was observed when these cells were cultured in the presence of an anti-caveolin-1 antiserum. Finally, antisense experiments established that endogenous PKC plays an important role in regulating the growth and survival of androgen-independent prostate cancer cells. This study provides several independent lines of evidence supporting the hypothesis that PKC expression may be sufficient to maintain prostate cancer growth and survival after androgen ablation. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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PKC is a member of the AGC family of Ser/Thr protein kinases that is known to have oncogenic potential (5)
and to be associated with the progression of many cancers (6, 7, 8, 9)
. Although there is recent evidence that PKC expression is elevated in tissue biopsies collected from patients with organ-confined CaP (10)
, the role of this isozyme in the progression to androgen independence has not been investigated. The activation of PKC
and PKC
induces apoptosis in LNCaP cells, an intensively studied androgen-sensitive CaP cell line, but not AI (DU145 and PC3) CaP cell lines (11, 12, 13)
. This finding indicates that at least some members of this gene family are capable of differentially regulating the growth and survival of CaP cells. Given the reciprocal functions of PKC isozymes in various cell types (5)
and the oncogenic activity of PKC, we hypothesized that this isozyme may oppose the proapoptotic influence of PKC and PKC in CaP.
There is evidence that caveolae might represent an important locus for PKC action (14 , 15) , and a positive correlation between caveolin-1 expression and the progression of human CaP has been described recently (16 , 17) . Caveolin-1 and -2 form homo- and hetero-oligomers on the inner membrane surface of caveolae and may serve as a scaffold for the assembly of multimeric signaling complexes that often include multiple components of the ERK cascade and certain members of the PKC family (14 , 18) . Exactly how these caveolin-associated signaling complexes function is unknown, although the overexpression of caveolin-1 alone is not sufficient to stimulate the AI proliferation of LNCaP cells (19) . We also do not understand the mechanisms that control the intracellular trafficking of caveolin-1 or how this membrane-type V protein gets rerouted into the secretory pathway of human CaP cells. However, aberrant transport is necessary for caveolin-1 to function as an autocrine/paracrine factor, now known to contribute to CaP metastasis and cell survival after androgen ablation (17) .
In the present study, investigation of human CaP cell lines indicated a relationship between PKC expression and androgen independence. To better understand whether the expression of PKC could be of functional importance in CaP progression, we stably transfected LNCaP cells with a retroviral vector containing PKC cDNA. This analysis revealed that PKC overexpression was sufficient to transform LNCaP cells into an AI variant that rapidly initiated tumor growth, in the absence of Matrigel, in both intact and castrated male nude mice. This transformation of LNCaP growth was accompanied by changes in the expression of key cell cycle regulatory proteins, hyperphosphorylation of protein kinases in the ERK mitogenic signaling cascade, derepression of biosynthetic processes, increased production and expulsion of caveolin-1, and resistance to apoptosis. Finally, when antisense PKC ODNs were used to specifically block the expression of endogenous PKC in DU145 and PC3 AI CaP cells, we observed a significant inhibition of Raf-1 and ERK phosphorylation, caveolin-1 expression, and their AI proliferation. This study provides data from gene transfer and antisense experiments demonstrating that PKC expression may contribute to recurrent tumor growth in the absence of testicular androgens.
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MATERIALS AND METHODS |
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Expression Plasmid and Transfection into LNCaP Cells.
LNCaP cells were infected with pLXSN recombinant retrovirus (LNCaP/v) or pLXSN harboring the gene for p3/PKC (LNCaP/) as described previously (20)
. Stably expressing cells were selected and subcloned by limiting dilution in 500 µg/ml G418, and resultant subclones were then screened for PKC protein expression and in vitro kinase activity as described previously (20)
.
Assessment of in Vivo Tumor Growth.
Intact and surgically castrated nude male mice (NU/NU-nuBR) were purchased from Charles River Laboratories (Wilmington, MA) and inoculated s.c., into the dorsal flanks left and right of the midline, with 1 x 106 cells suspended in 250 µl of PBS/site and routinely inspected for tumor growth and morbidity for up to 10 weeks. Cell cultures used in these studies were free of Mycoplasma contamination. Solid tumor volumes were calculated by the formula: length x width x depth x 0.5236.
Immunoblot Analyses.
Immunoblot analyses were performed as described previously (20)
. Antibodies purchased from Santa Cruz Biotechnology, Inc. were raised against caveolin-1 (clone N-20), cyclin D1 (C-20), cyclin E (M-20), ERK1 (K-23), c-Myc (C-19), PKC (C-15), PKC (C-15), c-Raf-1 (C-12), and upstream binding factor-1 (H-300). Antibodies purchased from BD Transduction Laboratories (Lexington, KY) were raised against cyclin D3 (C28620), phospho-ERK1/2 (12D4), and the retinoblastoma protein (RB; clone 2). Anti-phospho-Raf-1 (Ser259; 9421) and anti-phospho-RB (Ser807/Ser811; 9308S) antisera were from Cell Signaling Technology (Beverly, MA). Anti-E2F-1 antibody (KH129) was purchased from Geneka Biotechnology (Montreal, Quebec) and anti-ß-actin antibody (JLA20) was purchased from Oncogene Research Products (Boston, MA).
RT-PCR Analyses.
The SuperScript One-Step RT-PCR System (Invitrogen) was used to analyze LNCaP variants for changes in the steady-state concentrations of PKC and ß-actin mRNA. The PKC sense and antisense primers were 5'-AGC CGG CTT CTG GAA ACT CCC-3' and 5'-AGC TGC CTT TGC CTA ACA CCT TGA T-3', respectively. The human ß-actin primers were purchased from Invitrogen. The following cycling conditions were used in a GeneAmp Thermal Cycler 2400 (Perkin-Elmer): cDNA synthesis and predenaturation in 1 cycle of 50°C for 30 min and 94°C for 2 min followed by 40 cycles of amplification at 94, 58, and 72°C for 1 min each. The final extension was performed in a single cycle at 72°C for 10 min, and RT-PCR products were analyzed using 5% PAGE. The only products visible on these gels are shown in Fig. 5A
and corresponded to either a 353-bp (ß-actin) or 380-bp (PKC) fragment.
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Apoptosis Detection.
Subconfluent LNCaP cells and their derivatives (3.5 x 104 cells/well in 24-well plates) were seeded and cultured in complete medium for 24 h at 37°C. Adherent cells were then incubated for an additional 24 h in fresh medium, with or without PMA (100 nM). Medium containing anoikis was transferred to microcentrifuge tubes and sedimented, whereas adherent cells were removed from tissue culture plates by trypsinization and transferred to tubes containing the corresponding anoikis. After centrifugation, cell pellets were washed with PBS, resedimented, and suspended in 10 µl of PBS containing a 2-µl aliquot of a dye solution containing 100 µg/ml acridine orange (Sigma Chemical Co.) and 100 µg/ml ethidium bromide (Sigma Chemical Co.) in PBS. Cells were examined by epifluorescence microscopy (Nikon Microphot-FX; excitation, 450–490 nm; barrier, 520 nm). The nuclei of apoptotic cells contained uniformly stained condensed or fragmented chromatin. One hundred cells were scored in triplicate for each cell line and treatment condition. Three independent experiments were conducted, and data are expressed as the percentage of apoptotic cells. Caspase-3 proteolytic activity was measured using the PharMingen (San Diego, CA) assay kit, according to the manufacturer’s specifications. Data are expressed as the percentage change in proteolytic activity measured in PMA-treated versus untreated cell cultures after a 6-h exposure to PMA.
Assays of Protein Synthesis.
The kinetics of total protein synthesis were analyzed by measuring the rate of L-[4,5-3H]leucine (153 Ci/mmol; Amersham) incorporation into the specified LNCaP subline after growing for 3 days in serum-free medium. Briefly, subconfluent cultures were washed with PBS and cultured for 12 h in leucine-free RPMI 1640 (Invitrogen) before the addition of radiolabel (4 µCi/ml) to the medium. Where indicated, test compounds were added to the leucine-free medium prior to the radiolabel. Cells were then incubated at 37°C for the specified time, washed with excess PBS, and lysed in 100 µl of MPER protein extraction reagent (Pierce Corp., Rockford, IL). Results are expressed as cpm/mg protein. Under these conditions, >95% of the radioactivity incorporated by LNCaP cells was inhibited by 10 µg/ml cycloheximide (data not shown).
Coimmunoprecipitation Assays.
Cells were grown in CDT for 3 days before lysis in a buffer containing 50 mM HEPES (pH 7.5), 150 mM NaCl, 10% glycerol (w/v), 1% Triton X-100 (w/v), 1 mM EGTA, 1.5 mM MgCl2, 0.4 mM phenylmethylsulfonyl fluoride, 2 µM pepstatin, 0.1 mg/ml aprotinin, and 1 mg/ml leupeptin. Cellular debris was sedimented by centrifugation (10 min at 12,500 x g), and the resulting supernatants were precleared by adding 60-µl aliquots of protein agarose A (Invitrogen) to each sample, mixing for 30 min at 4°C, and sedimentation. Precleared lysates were transferred to sterile microcentrifuge tubes containing either a mouse IgG (1.5 µg, control), anti-PKC, or a GST-anti-Raf-1 agarose conjugate (Upstate Biotechnology, Lake Placid, NY) and mixed overnight by rotation at 4°C. Protein agarose A (75 µl) was added and incubated for 1 h at 4°C before centrifugation (5 min at 12,500 x g). Supernatants were discarded, and immunoprecipitates were washed three times using excess lysis buffer before solubilizing the final pellets in 60 µl of a standard SDS-PAGE sample buffer. Masking of the ERK 1/2 bands (Mr 42,000–44,000) by the monomeric IgG heavy-chains (Mr 
50,000) was avoided by solubilizing the immune complexes at room temperature in SDS-PAGE buffer, rather than boiling, and allowing the IgG heavy-chains to dimerize (Mr 100,000) under this condition.
Antisense PKC ODN.
Phosphorothioate ODNs were obtained from Invitrogen. Sequences for the antisense PKC ODN and corresponding scrambled control ODN were exactly as specified (21)
. Subconfluent (70–80%) DU145 and PC3 cultures were washed with Opti-MEM 1 (Invitrogen) before introducing a mixture of ODN (1 µM) and lipofectin (2 µg/ml; Invitrogen). After 6 h at 37°C, the cells were washed twice with serum-free medium and incubated 18 h in lipofectin-free medium containing ODN and fresh CDT. Medium was replenished, and the CaP cells were incubated for an additional 3 days in CDT before harvesting using trypsinization.
Data Analysis.
Values shown are representative of three or more experiments, unless otherwise specified, and treatment effects were evaluated using a two-sided Student’s t test. Errors are SEs of averaged results, and values of P < 0.05 were taken as a significant difference between means.
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RESULTS AND DISCUSSION |
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Causes Androgen-independent Growth and Tumorigenicity. expression compared with control benign tissues (10)
. To determine whether signals transduced through PKC had the potential to contribute to the AI progression of CaP, LNCaP cells overexpressing PKC were established using the pLXSN retroviral vector. The pooled population of PKC overexpressing LNCaP cells (LNCaP/) were selected for by their collective resistance to G418, and a representative subclone (LNCaP/3) was isolated from this pool of transfectants by limiting dilution and maintained in culture. The pLXSN vector control line was called LNCaP/v. LNCaP/ and LNCaP/3 cells expressed equivalent levels of PKC (Fig. 2C)
and, compared with parental and vector controls, the catalytic activity of PKC was increased, in the absence of an exogenous PKC activator, by 4.3- and 5.8-fold in adherent LNCaP/ and LNCaP/3 sublines, respectively (not shown). LNCaP cells are PTEN-/- (22)
and, because PTEN plays a dominant role in suppressing PKC activity (23)
, this may account for the increased basal activity of PKC in the LNCaP/ and LNCaP/3 sublines.
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and gradually became arrested in G1 upon androgen removal (Ref. 24
and Fig. 1B
). FACS analysis of PI-stained LNCaP cells indicated that >90% of these parental cells were in G1 after a 96-h incubation in CDT (data not shown). The forced expression of PKC dramatically altered this phenotypic response to androgen withdrawal and enabled LNCaP cells to proliferate in the absence of androgens (Fig. 1B)
or in serum-free medium (not shown). LNCaP cells overexpressing PKC (LNCaP/) remained responsive to the growth-promoting effects of DHT (Fig. 1A)
, but their expression of androgen receptor and prostate-specific antigen proteins was substantially reduced and could only be detected after prolonged film exposures (not shown). The conservation of androgen responsiveness in the presence of a declining steady-state concentration of androgen receptor protein suggested that the overexpression of PKC in LNCaP cells could be associated with androgen hypersensitivity. These results prompted investigation into the effects of PKC overexpression on the tumorigenicity of LNCaP cells in the absence and presence of testicular androgens.
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3 cells (1 x 106/site) were injected s.c. into intact and castrated male nude mice. Matrigel was not used as an adjunct in any of these experiments, and all cells were injected alone as a suspension in PBS. Within 3–4 weeks, tumors appeared with a take rate of 100%, and the onset of tumor growth was more rapid in castrated animals (Fig. 1C)
. A similar result was obtained using the heterogeneous LNCaP/ subline in castrated mice (Fig. 1C)
. No tumors formed during a 10-week observation period when an equal number of LNCaP or LNCaP/v cells were injected without Matrigel into intact male nude mice. The finding that LNCaP cells overexpressing PKC grew tumors in vivo more rapidly in castrated than intact male mice is similar to that of LNCaP variants that have been clonally selected in CDT medium for AI growth, e.g., LNCaP 104-S (25)
and CL-1 (26)
. Forced overexpression of the HER-2 receptor tyrosine kinase also enables LNCaP cells to form xenograft tumors in castrated nude mice but only if mixed with Matrigel (27)
, a potent inducer of LNCaP cell growth. These studies indicate that PKC has the potential to advance the progression of CaP and may be sufficient to initiate recurrent tumor growth in the absence of testicular androgens.
PKC Accelerates Transit through the G1 Restriction Point.
FACS analysis of exponentially multiplying LNCaP/3 cells revealed that PKC overexpression decreased the number of cells in the diploid compartment and increased the number of cells in S-phase, relative to their parental counterparts (Fig. 2A)
. In complete medium, LNCaP and LNCaP/v cells doubled in 55 h and had an average G1 phase duration of 42 h (76% in G1 x 55 h = 41.8 h). In contrast, the LNCaP/ and LNCaP/3 lines had an average doubling time of 19 h and traversed G1 in 8 h (43% in G1 x 19 h = 18.2 h). Thus, increased expression of PKC accelerated G1 to S transit of LNCaP cells.
PKC and ERK Signaling to RB.
The G1 arrest associated with androgen ablation in LNCaP cells is dependent on the growthrestraining activity of the RB family of transcriptional repressor proteins (28)
; androgen independence may be achieved through signals that disrupt the function of pocket proteins such as RB, p130, and p107. Collectively, the data in Fig. 2
provide strong support for this hypothesis. Raf-1 is an imminent, if not direct, target of PKC (29)
that is capable of inactivating RB (30)
. Coimmunoprecipitation experiments showed that PKC remained constitutively associated with phospho-Raf-1 and ERK 1/2 in both quiescent LNCaP cells and the AI-LNCaP/ cells that continued to proliferate after 3 days in CDT (Fig. 2
B, upper panel). The association of PKC with Raf-1 was confirmed by using a GST-anti-Raf-1 agarose conjugate to perform a reciprocal pull-down of endogenous Raf-1 from precleared LNCaP and LNCaP/ lysates. The lower panel in Fig. 2B
shows that Raf-1 immunoprecipitates from these two cell lines contained equivalent amounts of PKC protein. In contrast to LNCaP cells, however, PKC immunoprecipitates from LNCaP/ cells pulled down caveolin-1 (Fig. 2B
, upper panel). It has been reported that PKC remains physically associated with Raf-1 in a biochemically inactive signaling complex when fibroblasts are forced into quiescence by serum starvation (29)
and that the scaffolding domain of caveolin-1 inhibits the in vitro catalytic activity of PKC and PKC
(15)
. These observations suggested that the caveolin-based PKC:Raf-1:ERK 1/2 complex may not be actively signaling in LNCaP/ cells. However, immunoblot analyses of whole cell lysates indicated that the general levels of Raf-1 and ERK 1/2 phosphorylation remained elevated in both LNCaP/ and LNCaP/3 cells after 3 days in CDT, in comparison to either the parental or vector controls (Fig. 2C)
. These experiments implied that PKC was able to colocalize with caveolin-1 and Raf-1 while indirectly promoting ERK phosphorylation (activation). The fact that PKC remains biochemically active when linked to caveolin-1 has been attributed to the unique location of the caveolin-binding motif in this isozyme (i.e., subdomain IV of PKC versus subdomain IX of the PKC catalytic domain, see Ref. 15
). On the basis of these and other studies, we hypothesized that PKC has the potential to activate members of the ERK cascade that are capable of deregulating the cell cycle progression of LNCaP cells through hyperphosphorylation of RB (30)
.
In the absence of androgen and/or serum, LNCaP cells undergo a programmed response that is characterized by reduced levels of G1 cyclin-dependent kinase and RB activities (24)
. The data in Fig. 2D
show that the overexpression of PKC was sufficient to disrupt this inherent cellular response. Compared with the parental and vector controls, both LNCaP/ and LNCaP/3 cells maintained elevated levels of inactive/phosphorylated RB and continued to express elevated levels of cyclins D1, D3, and E and the E2F-1 transcription factor after 3 days in serum-free medium. Other cell cycle regulators and transcription factors were either unaltered in their expression (p21waf1/cip1 and upstream binding factor-1) or showed no consistent changes (p27kip1 and TATA binding protein-1).4
PKC overexpression also increased cellular levels of c-myc (Fig. 2D)
, which is an oncogene with functional E2F binding sites in the promoter DNA sequence that is up-regulated in expression during the progression to androgen independence in some CaPs (3)
. Taken together, the data point to PKC as an active regulator of the ERK to RB signaling pathway in LNCaP cells.
PKC Stimulates Protein Synthesis and Cellular Proliferation.
Abnormal stimulation of the translation apparatus may constitute a major step toward tumor development (31)
, and our studies demonstrate that under the stress of serum starvation, the autocrine growth of LNCaP/ and LNCaP/3 cells was associated with a 7-fold increase in their rate of general protein synthesis, measured as the incorporation of [3H]leucine over time (Fig. 2E)
. Although the cellular content of DNA was increased 4-fold in LNCaP/3 cells (Fig. 2A)
, their rate of total RNA synthesis ([3H]uridine incorporation) did not change relative to the parental or vector controls (data not shown). PKC appears to stimulate translation upstream of the polypeptide elongation stage. Pseudomonas exotoxin A, an inhibitor of elongation factor-2, decreased protein synthesis with equal potency in all cell lines with a maximum inhibitory effect of 96% at 10 µg/ml (data not shown).
PKC Renders LNCaP Cells Resistant to the Apoptotic Effects of PMA.
PMA induces a massive apoptogenic response in LNCaP cells (13)
. Greater than 80% of parental or vector control cells cultured in the presence of PMA (100 nM) for 24 h tested positive for apoptosis using acridine orange staining of the cells (Fig. 3A)
. In contrast, apoptosis was rarely observed when LNCaP/ or LNCaP/3 cells were treated with equimolar concentrations of PMA. In LNCaP cells, the apoptosis induced by PMA was preceded by the stimulation of caspase 3 proteolytic activity, whereas no change in the activity of caspase 3 was detected in LNCaP/ or LNCaP/3 cells after a 6-h exposure to 100 nM PMA (Fig. 3B)
. It has been proposed recently that both dephosphorylation (activation) of RB and the repression of c-myc transcription are prerequisite events for PKC-mediated apoptosis in LNCaP cells (31)
. The present studies indicate that PKC overexpression renders LNCaP cells resistant to the apoptogenic effects of PMA (Fig. 3)
while also preventing the activation of RB or c-myc repression during androgen ablation (Fig. 2D)
. Additional studies of the response of LNCaP/ cells to PMA should reveal whether PKC signaling uncouples the apoptotic response of LNCaP cells to PMA by either preventing RB activation or c-myc repression.
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Enhances the Synthesis and Expulsion of Caveolin-1. overexpression significantly increased both the steady-state levels of cellular caveolin-1 and the "secretion" of this protein by LNCaP cells (Fig. 4A)
. Control (LNCaP and LNCaP/v) and PKC overexpressing LNCaP cells were grown in serum-free medium for 3 days in the absence of any exogenous secretagogues before cell lysates and medium were analyzed for caveolin-1 by immunoblotting. In a separate experiment, LNCaP/3 cells were cultured in serum-free medium for 1 day in the absence or presence of brefeldin A (10 µM) to determine whether caveolin-1 must enter the default secretory pathway via the lumen of the ER/Golgi apparatus to exit the cell. Exposure to brefeldin A significantly depleted the intracellular pool of caveolin-1 and increased the amount of this protein in conditioned medium (Fig. 4B)
. This finding supports the notion that caveolin-1 may accumulate into preexisting lipid droplets in the presence of brefeldin A (32)
and be released as a lipoprotein that remains associated with a fibrous protein(s) after dissolution of the lipid droplet by Triton X-100 (33)
. Our studies also confirm an earlier report that caveolin-1 has the potential to stimulate the survival of CaP cells (17)
. Medium preconditioned by LNCaP/3 cells (3 days) had a positive effect on the growth/survival of LNCaP cells held in serum-free medium (Fig. 4C)
. Treatment of the LNCaP/3 conditioned medium with anti-caveolin-1 antiserum completely blocked this growth-promoting response and significantly reduced the viability of LNCaP cells (P < 0.001) compared with the IgG-treated medium (Fig. 4C)
. These studies suggest that PKC had the potential to ensure the survival of CaP cells by promoting the expression and expulsion of caveolin-1.
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could play a dominant role in the clonal expansion of prostatic epithelial cells that naturally overexpress this oncoprotein in recurrent CaP. At the same time, however, we remained circumspect and questioned whether the proliferative autonomy of LNCaP/ cells could have arisen as an artifact because of the forced overexpression of this oncoprotein rather than the activation of a normal program for oncogenesis. In this regard, it is important to note that essentially the same phenotypic changes were observed in two independently transfected and heterogeneous pools of LNCaP/ cells (selected in G418 but not subcloned). Nevertheless, it was evident that alternative experimental strategies would be required to critically examine the importance of PKC in the proliferation and survival of CaP cells. For this reason, we extended our analysis of PKC expression to include additional models of the progression to androgen independence in CaP and used complimentary antisense strategies to determine whether AI CaP cells are dependent for their proliferation or survival on the expression of endogenous PKC.
Increased Steady-State Concentrations of PKC in AI CaP Cell Lines.
Immunoblot analysis revealed that the levels of endogenous PKC were increased in AI DU145 and AI PC3 cell lines in comparison with the androgen-dependent LNCaP cell line (Fig. 5A)
. PKC protein was not detected in immunoblots of whole cell lysates prepared from subconfluent cultures of LNCaP cells, although the protein could be detected in PKC immunoprecipitates (not shown). Compared with the LNCaP cell line, AI DU145 and AI PC3 cells expressed elevated levels of PKC, Raf-1, phospho-ERK 1/2, and caveolin-1 (Fig. 5A)
. In DU145 and PC3 cells, the increased level of PKC protein was associated with an increase in the amount of a RT-PCR product encoding PKC (Fig. 5A)
, implying that the PKC gene may be transcriptionally up-regulated in some AI CaP cells. Alternatively, the AI DU145 and AI PC3 cell lines may have been derived from metastases that had grown to be relatively homogeneous for PKC-positive cells in vivo, possibly via a selection process favoring the survival of these cells.
Antiproliferative Effects of Antisense PKC ODNs in AI CaP Cells.
To investigate the role of endogenous PKC in the growth of AI CaP cells, an antisense PKC ODN was used for selective down-regulation of expression of this isoenzyme in DU145 (PTEN+/+) and PC3 (PTEN-/-) CaP cells. DU145 and PC3 cells were preincubated with either the scrambled or antisense PKC ODN (1 µM) for 3 days, and lysates were analyzed by immunoblotting for changes in the steady-state concentration of PKC, PKC, phospho-Raf-1, phospho-ERK 1/2, caveolin-1, and ERK 1/2. Translation of PKC mRNA was selectively and effectively down-regulated by the antisense PKC ODN, whereas the steady-state concentrations of PKC and ERK 1/2 remained unaltered (Fig. 5B)
. Under identical conditions, neither lipofectin alone nor lipofectin plus the scrambled PKC ODN inhibited PKC, PKC, caveolin-1, or ERK1/2 synthesis in these two cell lines (Fig. 5B)
. The decreased expression of PKC induced by the antisense PKC ODN was associated with sequence-specific reductions in the phosphorylation of Raf-1 and ERK 1/2 and the expression of caveolin-1 in both the DU145 and PC3 cell lines (Fig. 5B)
. In addition, after a 3-day incubation, 1 µM antisense PKC ODN inhibited the growth of DU145 and PC3 cells in complete medium by 45%, relative to the scrambled PKC ODN control. The growth-inhibitory effects of this treatment were augmented when these same cells were grown in CDT. When DU145 and PC3 cells were cultured for 3 days in CDT, 1 µM antisense PKC ODN inhibited the AI growth of DU145 and PC3 cells by 75 and 54%, respectively (Fig. 5C)
. These results are consistent with the suggestion that PKC may function upstream of Raf-1 in the ERK signaling cascade, where it plays an important role in sustaining the expression of caveolin-1 and the AI growth and proliferation in both PTEN+/+ (DU145) and PTEN-/- (PC3) CaP cells.
The major finding of the present study was that PKC is an oncogenic protein with the potential to induce AI growth of LNCaP tumors in castrated animals. Now that there is direct evidence that PKC is capable of functioning as a complete oncogene in the LNCaP tumor model, the signaling mechanism(s) that confer this potential should be rigorously investigated. Our gene transfer experiments demonstrate that PKC overexpression transforms LNCaP cells into AI tumor cells that recapitulate many hallmark features of recurrent CaP. The overexpression of PKC leads to an uncontrolled and accelerated proliferation of LNCaP cells associated with the constitutive activation of the ERK signaling cascade, hyperphosphorylation of the transcriptional repressor RB, and the increased expression of E2F-1 and other m7 GTP cap-dependent mRNAs, including the G1 cyclins, c-myc, and caveolin-1. Although there is no question that Raf-1 is a downstream target of PKC, Ras/Raf induction alone is insufficient to promote the AI proliferation of LNCaP cells (34
, 35)
. Therefore, PKC must signal to additional downstream targets, possibly within the caveolin-1 signaling complex, to overcome the growth-regulatory signals that normally control the cell cycle progression of LNCaP cells. Caveolin-1 differentially influences the biochemical activity of its binding partners, up-regulating ligand-dependent androgen receptor signaling (36)
while inhibiting the activity of proapoptotic (PKC and PKC) but not oncogenic (PKC) isozymes of PKC (14
, 15)
. It is of note that multiple oncoproteins, and PKC isozymes, have been overexpressed in LNCaP cells without producing the phenotype of LNCaP/ cells. Although many important details need to be further investigated, this study demonstrates that PKC may play an important role in the progression to androgen independence in some human prostate cancers.
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FOOTNOTES |
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1 Supported by NIH Grants ES8397 (to D. M. T.), P01 CA77739 (to J. L. M.), and U54 HD35041 (Tissue Culture Core), and the Department of the Army Contract Nos. DAMD17-02-1-0110 (to C. W. G.) and DAMD17-02-1-0053 (to D. M. T.). 
2 To whom requests for reprints should be addressed, at Department of Anatomy and Cell Biology, Brody School of Medicine, East Carolina University, 600 Moye Boulevard, Greenville, NC 27858. Phone: (252) 816-3247; Fax: (252) 816-2850; E-mail: terriand{at}mail.ecu.edu
3 The abbreviations used are: CaP, prostate cancer; AI, androgen independent; ERK, extracellular signal-regulated protein kinase; FBS, fetal bovine serum; CDT, charcoal dextran-treated serum; RT-PCR, reverse transcription-PCR; DHT, 5-dihydrotestosterone; ODN, oligodeoxynucleotide; PKC, protein kinase C; PI, propidium iodide; PMA, phorbol 12-myristate 13-acetate; RB, retinoblastoma protein.
4 D. Wu and D. M. Terrian, unpublished data.
Received 8/ 8/01. Accepted 2/13/02.
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), LNCaP/v (
), and LNCaP/
) cells after growing for 3 days in serum-free medium. Data are the means of triplicate determinations in three independent experiments; bars, SE.
) cells incubated at 37°C for 3 days in CDT. Cells were serum starved overnight before the introduction of DHT, and cell proliferation was measured by counting the total number of viable cells/plate by trypan blue exclusion. Data are the means of triplicate determinations in three independent experiments; bars, SE. B, growth of LNCaP (
