PKC-δ inhibits anchorage-dependent and -independent growth, enhances differentiation, and increases...

PKC-� Inhibits Anchorage-Dependent and-Independent Growth, Enhances Differentiation, and IncreasesApoptosis in CaCo-2 Cells

SONIA R. CERDA, MARC BISSONNETTE, BETH SCAGLIONE–SEWELL, MATTHEW R. LYONS,SHARAD KHARE, REBA MUSTAFI, and THOMAS A. BRASITUSDivision of Gastroenterology, Department of Medicine, University of Chicago, Chicago, Illinois

See editorial on page 1868.

Background & Aims: Previous studies showed decreasedprotein kinase C (PKC)-� expression in azoxymethane-induced rat and sporadic human colonic tumors. Toelucidate the role of PKC-� on the neoplastic phenotypeof human colon cancer cells, we established stabletransfectants of this isoenzyme in CaCo-2 cells.Methods: Human PKC-� complementary DNA was sub-cloned into 2 distinct metallothionein-regulated expres-sion vectors. Polyclonal populations of PKC-� transfec-tants were characterized by Western blotting. PKC-�activity was measured in situ using a PKC-�–specificsubstrate. Proliferation was determined by Coultercounter, and cell cycle distribution was analyzed by flowcytometry. In vitro transformation was assessed bygrowth in soft agar and differentiation by changes inalkaline phosphatase and sucrase isomaltase. Apopto-sis was evaluated by 4�,6-diamidino-2-phenylindole di-hydrochloride and terminal deoxynucleotidyl trans-ferase–mediated deoxyuridine triphosphate nick-endlabeling staining. Results: In the presence of Zn2�, PKC-�transfectants expressed a 4-fold increase in the proteinand a 2-fold increase in activity of PKC-�. PKC-� trans-fectants exhibited a 30% decrease (P < 0.05) in cellgrowth and an enhanced differentiation phenotype. In-creased PKC-� expression induced a significant G0/G1arrest, inhibited anchorage-independent growth (50%,P < 0.05), and caused a 2-fold increase in apoptosis(P < 0.05). Conclusions: Our studies show that in-creased expression of PKC-� inhibits anchorage-depen-dent and -independent growth, while inducing cellulardifferentiation and limiting survival of this human coloncancer cell line.

The serine/threonine protein kinase C (PKC) familyconsists of a group of at least 12 isoenzymes that

participate in many signal transduction pathways toregulate important cellular processes including prolifer-ation, differentiation, and apoptosis.1–4 Members of this

family are classified into 3 subfamilies, distinguished bydifferences in sequence homology and cofactor require-ments for activation.5 Although all isoforms requireacidic phospholipids, the conventional PKCs (�, �I, �II,and �) are activated by calcium (Ca2�) and 1,2-diacyl-sn-glycerol (DAG), whereas the novel class of PKCs (�, �,�, and ) are Ca2�-independent, but DAG-dependent.Although independent of both Ca2� and DAG, thecofactor requirements for activation of the atypical sub-family of PKCs (, �, and �) have not been firmlyestablished.5 In addition to these structural and regula-tory differences, PKC isoenzymes exhibit distinct pat-terns of tissue expression and subcellular localization,supporting the hypothesis that these isoenzymes haveunique functions in mediating cellular responses.1,6

Colon cancer is one of the leading causes of cancer-related deaths in the United States.7 Several lines ofevidence have implicated alterations in the expression,biochemical activity, and/or subcellular distribution ofspecific isoforms of PKC in colonic malignant transfor-mation in both human and experimental models of can-cer.8–11 Normal human and rat colonocytes express PKCisotypes �, �I, �II, �, �, and �.11,12 In human coloncancer, decreases in total PKC biochemical activity, aswell as in isoform-specific expression of PKC-�, �, and �,have been reported.13–17 Similarly, in experimental mod-els of colon cancer, our laboratory and others have shownsignificant decreases in the protein expression and/oractivities of several isoforms, including PKC-�, -�, and-�, and significant increases in PKC-�.18,19 Although

Abbreviations used in this paper: cDNA, complementary DNA; DAG,1,2-diacyl-sn-glycerol; DAPI, 4�,6-diamidino-2-phenylindole dihydro-chloride; DMEM, Dulbecco minimum essential medium; EV, emptyvector; HBSS, Hank’s balanced salt solution; PKC, protein kinase C;SDS-PAGE, sodium dodecyl sulfate-PAGE; TPA, 12-O-tetradecanoyl-phorbol 13-acetate; TUNEL, terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling.

© 2001 by the American Gastroenterological Association0016-5085/01/$35.00

doi:10.1053/gast.2001.24843

GASTROENTEROLOGY 2001;120:1700–1712

there were decreases in expression of specific PKC iso-forms accompanying malignant transformation in vivo,overexpression of PKC-� and PKC-�I in vitro causedgrowth inhibition and decreased tumorigenicity in sev-eral human colon cancer cell lines.20–22

In vitro overexpression of PKC-� has been associatedwith inhibition of proliferation, cell cycle arrest, en-hanced differentiation, and/or accelerated apoptosis inseveral noncolonic cell lines.23–34 Consistent with thispotential tumor suppressor role for PKC-�, inhibitorytyrosine phosphorylations of PKC-� were found to ac-company cell transformation induced by several receptorand nonreceptor tyrosine kinases.35–38 Additionally,transformation by stable transfection of the human ker-atinocyte line, HaCaT, with mutated cellular Ha-rasresulted in a selective loss of PKC-�.39 These studiessuggested that transformation by several proto-onco-genes may be mediated, at least in part, by decreases inthe activity and/or expression of PKC-�. Conversely,previous studies in src-overexpressing rat colonic epithe-lial cells have shown that PKC-� overexpression reversedthe src-induced transformed phenotype in these cells,causing an arrest in cellular proliferation.40 To date,however, no studies have examined the phenotypic con-sequences of increased PKC-� expression in transformedcolonocytes derived from human colonic epithelium. Inprevious studies, we have successfully assessed the directrole of another PKC isoform, PKC-�, in regulating cellproliferation and differentiation.20 Specifically, by trans-fecting sense complementary DNA (cDNA) for PKC-�,we determined that increased expression of this isoen-zyme in CaCo-2 cells, derived from a human coloncancer, caused growth inhibition, increased differentia-tion, and decreased tumorigenicity. As in the case ofPKC-�, PKC-� is down-regulated in human colon can-cers. Using a similar transfection strategy, we have nowdirectly examined the phenotypic characteristics inducedby increasing the expression of PKC-� in CaCo-2 cells.These cells, like their colon cancer counterparts, expresslow levels of PKC-�.

In the present studies, using CaCo-2 cells, we estab-lished stable transfectants with cDNA coding for fulllength PKC-�. This human colon cancer–derived cellline has been extensively used by investigators to studyintestinal epithelial growth and differentiation.51 Fur-thermore, Caco-2 cells do not contain mutations in c-K-ras or c-src, which are upstream regulators of PKC thatmight otherwise complicate mechanistic interpretationsof effects observed after PKC-� overexpression. We ex-amined the biological consequences of alterations in theexpression of PKC-� on the neoplastic phenotype of these

cells using 2 different Zn2�-inducible metallothioneineukaryotic expression vectors.42,43 This allowed us tooverexpress PKC-� during experiments but not in theclonal selection, expansion, and maintenance phases. Wechose these inducible systems because in previous studiesin rat intestinal epithelial cells, constitutive (noninduc-ible) overexpression of this isoform greatly inhibited cellgrowth.40 Mixed clones were selected to avoid unin-tended differences related to gene insertion, rather thanto PKC-� expression.

Our studies showed that increased expression ofPKC-� decreased anchorage-dependent growth associ-ated with an arrest in the G0/G1 phase of the cell cycle.Furthermore, overexpression of this isoenzyme inhibitedanchorage-independent growth while enhancing cellulardifferentiation and limiting the survival of these cells.Based on these findings, we suggest that the alterationsin expression of PKC-� observed in sporadic humancolon cancer are likely involved in several importantphenotypic characteristics that occur in malignant co-lonic cells.

Materials and MethodsMaterials

Human colonic specimens were obtained from theDepartment of Surgical Pathology of the University of ChicagoHospitals. These included paired normal colonic mucosa dis-sected from the underlying layers and colon cancer samples.Specimens were flash frozen in liquid nitrogen within 1 hourof resection. Tissue and bacterial culture reagents were ob-tained from Fisher Scientific (Springfield, NJ), and mediasupplements were from GIBCO BRL–Life Technologies(Gaithersburg, MD). The mouse monoclonal antibody to hu-man PKC-� used for Western blotting was from TransductionLaboratories (Lexington, KY). Rabbit polyclonal antibodies tohuman PKC-�, -�I, -�II, -�, and -� were from Santa CruzBiotechnology (Santa Cruz, CA). The mouse monoclonal an-tibody to human �-actin was from Sigma Chemical (St. Louis,MO). Immobilon-P (polyvinylidene difluoride) membraneswere purchased from Millipore (Bedford, MA). The peroxi-dase-conjugated sheep anti-mouse and donkey anti-rabbit an-tibodies, enhanced chemiluminescence Western blotting pro-tein detection kit, and [�32P] adenosine triphosphate weresupplied by Amersham (Arlington Heights, IL). WhatmanP81 phosphocellulose chromatography paper was from FisherScientific. Instruments and reagents used for immunoblottingand gel electrophoresis, as well as the ion exchange resin,Chelex 100, were from Bio-Rad Laboratories (Richmond, CA).Kodak (Rochester, NY) supplied the X-OMAT AR film. Thephorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA),was obtained from Sigma. A PKC-�–specific peptide substratewas obtained from Calbiochem (San Diego, CA). The sequenceof this peptide corresponds to residues 422–443 of the murine

June 2001 PKC-� INHIBITS THE MALIGNANT PHENOTYPE OF CaCo-2 CELLS 1701

elongation factor eEF-1�, and contains Thr431 that is highlyspecific for PKC-� phosphorylation.44 All other reagents usedin this study were from Sigma Chemical or Fisher Scientific,unless otherwise indicated, and were of the highest purityavailable.

Construction of Human PKC-� ExpressionVectors

The cDNA encoding full length human PKC-�, sub-cloned into a Baculovirus transfer vector pBlueBac/PKC-�,45

was kindly provided by Dr. David J. Burns (Abbott Labora-tories, Abbott Park, IL). Full length PKC-� cDNA was ob-tained by PCR amplification from pBluBac/PKC-� and puri-fied by gel electrophoresis in 1% agarose. Forward and reverseprimers used for PCR amplification were purchased fromGIBCO-BRL. These primers were synthesized with sequencescomplimentary to the 5 and 3 ends of the published humanPKC-� coding sequence (Accession number L07860 [26-merscontaining 15 base pairs of overlap with creation of BamHIsites at each end]). PKC-� cDNA was sequenced to confirm itsidentity using an ABI Prism 377 DNA sequencer by theUniversity of Chicago Digestive Diseases Research Core Center(DDRCC) DNA Sequencing Facility. PKC-� was subclonedinto 2 Zn2�-inducible metallothionein mammalian expressionvectors, designated MRE �neo and MRE pMet, kindly pro-vided by Drs. R. D. Palmiter (University of Washington,Seattle, WA) and B. Thimmapaya (Northwestern UniversityCancer Center, Chicago, IL), respectively. The vector,pcDNA3.1/HisC, carrying the neomycin resistance gene wasfrom In Vitrogen (San Diego, CA). To generate PKC-�1 andPKC-�2 expression vectors, PKC-� cDNA was inserted 3 tothe metallothionein promoter in MRE �neo42 and MREpMet,43 respectively, per standard cloning procedures. PlasmidDNA was amplified by transformation into XL1-Blue MRsupercompetent cells (Stratagene, La Jolla, CA) and preparedusing the plasmid Mega Prep Kit from Quiagen (Valencia,CA).

Cell Culture

CaCo-2 cells, derived from a human colonic adenocar-cinoma (ATCC no. HTB37; Rockville, MD), were provided asa subclone relatively devoid of goblet cells by Dr. HugoDeJonge. Low passage cells (passages 21–31) were cultured at37°C in a humidified atmosphere of 5% CO2–95% air, aspreviously described by our laboratory.20 Cells were main-tained in standard Dulbecco minimum essential medium(DMEM) containing 4.5 g/L glucose, 10 mmol/L HEPES (pH7.4), 2 mmol/L L-glutamine, 50 U/mL penicillin G, 50 �g/mLstreptomycin, 2 �g/mL gentamicin, 1% essential and nones-sential amino acids, and 20% fetal bovine serum.

Preparation of Zinc-Depleted Medium

Zinc-depleted medium was prepared by a modificationof the method by Palmiter.42 Briefly, Chelex 100 was used todeplete zinc from heat-treated fetal calf serum. Chelex (5 g)was washed 3 times with Ca2�/Mg2�-free phosphate-buffered

saline (PBS) to activate the resin, which was then added to 50mL of serum and stirred at 4°C for 30 minutes. Zn2�-depletedserum was separated from the Chelex by centrifugation andfiltered with 450 mL of serum-free DMEM using a 0.22-micron cellulose acetate filter system (Corning, New York,NY).

Preparation of CaCo-2 Cell StableTransfectants With Human PKC-�

Low passage CaCo-2 cells were seeded at a density of5 � 105 cells in Falcon 60-mm tissue culture dishes. Togenerate MRE � stable transfectants, adherent cells (40%–50% confluent) were transfected with 10 �g of either theempty vector (EV) alone, MRE �neo (EV-1), or MRE�–PKC-� cDNA (PKC-�1), using Superfect Transfection Re-agent (Quiagen), following the protocol suggested by themanufacturer. To prepare MRE pMet stable transfectants, cellswere similarly transfected using 1 �g of pcDNA 3.1/His,containing the neomycin resistance gene, plus 10 �g of eitherMRE pMet vector alone (EV-2), or MRE pMet-PKC-� cDNA(PKC-�2). Cells transfected with EV-1 and EV-2 were used tocontrol for potential nonspecific effects of plasmid transfection.After a 3-hour transfection period, cells were washed 5 timesin Hank’s balanced salt solution (HBSS) and allowed to recoverfor 48 hours in fresh medium. Cells were then passaged at 1:10to 1:15 (vol/vol) and cultured in zinc-depleted medium sup-plemented with the antibiotic, geneticin (G418), at a concen-tration of 800 �g/mL for 4 weeks. For each transfection, afterselection, a polyclonal population of resistant clones waspooled and expanded for further study. Stable transfectantswere maintained in zinc-depleted medium containing 400�g/mL G418.

Western Blotting

Total cell lysates were prepared from EV- and PKC-�cDNA–transfected CaCo-2 cells grown in Chelex-treatedDMEM supplemented with 175 �mol/L Zn2�, a nontoxicconcentration of zinc that activates the metallothionein pro-moter (see Results). Briefly, medium was removed and cellswere boiled for 3 minutes in 2� Laemmli SDS buffer. Proteinwas precipitated with 20% trichloroacetic acid and assayed bythe bicinchoninic acid method using bovine serum albumin asthe standard (Bio-Rad). Equal amounts of protein (15 �g/lane)were subjected to sodium dodecyl sulfate–polyacrylamide gelelectrophoresis (SDS-PAGE) on a 10% resolving gel and trans-ferred to Immobilon-P membranes. To verify equal loading oflanes, blots were routinely stained with 0.1% India ink inTBST buffer, containing 50 mmol/L Tris-HCl (pH 7.4), 150mmol/L NaCl, and 0.05% Tween-20. Nonspecific binding ofantibodies was blocked by incubating blots at room tempera-ture for 3 hours with 5% non-fat dry milk in TBST. Afterblocking, blots were processed for immunoblotting by over-night incubation at 4°C with PKC isoform–specific antibodiesat final concentrations of 0.1 �g/mL in TBST. After incuba-tion, blots were rinsed 4 times for 5 minutes each in TBST andincubated for 1 hour with peroxidase-conjugated secondary

1702 CERDA ET AL. GASTROENTEROLOGY Vol. 120, No. 7

antibodies at 1:3000 final dilution in TBST. PKC isoformswere detected using an enhanced chemiluminescence systemfollowed by xerography on X-OMAT AR film, as recom-mended by the manufacturer, with exposure times adjusted toensure linear responses. The xerograms were scanned with aJX-3F6 scanner (Sharp Electronics, Mahwah, NJ) and quanti-fied using IP laboratory gel software (Signal Analytics, Vienna,VA). Under these immunologic detection conditions, thechemiluminescence assay was linear between 5 and 40 �g oftotal protein.

In Situ Assay of PKC-� Biochemical Activity

EV- and PKC-� cDNA–transfected CaCo-2 cells wereplated in 24-multiwell plates at a density of 2 � 104 cells/welland grown in zinc-depleted DMEM for 2 days. Preconfluentcells were treated for a period of 48 hours in Chelex-treatedDMEM supplemented with 175 �mol/L Zn2�. After treat-ment, medium was aspirated and PKC activity was assayed insitu.46 Briefly, PKC-� activity was assayed by addition of 100�L of permeabilization-kinase buffer to each well. The buffercontained 137 mmol/L NaCl, 5.4 mmol/L KCl, 10 mmol/LMgCl2 , 5 mmol/L ethylene glycol-bis(�-aminoethyl ether)-N,N,N ,N -tetraacetic acid, 0.3 mmol/L Na2HPO4, 0.4mmol/L K2HPO4, 20 mmol/L HEPES (pH 7.2), 50 �g/mLdigitonin, 25 mmol/L �-glycerophosphate, 100 �mol/L [�32P]adenosine triphosphate (500 cpm/pmol), and 40 �mol/L PKC-�–specific substrate peptide derived from eEF-1� (Calbio-chem). After a 10-minute incubation, 25% (wt/vol) trichloro-acetic acid (10 �L) was added to each well to stop the reaction,and an aliquot (20 �L) was spotted onto a P81 phosphocellu-lose disk and washed with 1% (vol/vol) concentrated phospho-ric acid in water. Radioactivity was assessed by scintillationcounting.

Assay of Anchorage-Dependent Growth

EV- and PKC-� cDNA–transfected CaCo-2 cells wereseeded in 6-well tissue culture dishes at a density of 0.5 � 105

cells/well in zinc-depleted medium and allowed to attachovernight. PKC-� was induced by replacing medium withDMEM containing 175 �mol/L Zn2�. Fresh medium contain-ing Zn2� was replaced every 2 days and cultures were grownfor a period of 14 days. Cells from triplicate wells werecollected every other day in HBSS containing trypsin-EDTAand resuspended in an equal volume of medium containingserum to inhibit the trypsinization. Cell numbers were deter-mined using a Coulter counter (Coulter Electronics, Miami,FL). The number of cells per well is reported as the average �SD at the indicated days after plating.

Cell Cycle Analysis by Flow Cytometry

EV- and PKC-� cDNA–transfected CaCo-2 cells wereseeded in 6-well tissue culture dishes at a density of 1 � 105

cells/well in zinc-depleted medium and allowed to attachovernight. PKC-� was induced by replacing medium in trip-licate wells with Chelex-treated DMEM containing 175

�mol/L Zn2�, and cultures were grown for a period of 5–7days. We found that PKC-� activity levels were comparableduring this treatment period (data not shown). The distribu-tion of cells in the cell cycle was assessed according to themethod of Darzynkiewicz.47 Briefly, cells were fixed in 70%ethanol and stained for 30 minutes at room temperature with10 �g/mL propidium iodide in PBS containing 20 U/mL ofdeoxyribonuclease-free ribonuclease A. Cells (12,000 per sam-ple) were analyzed using CellQuest software in a FacScan flowcytometer (Becton Dickinson Immunocytometry Systems, SanJose, CA). The distribution of DNA in the cell cycle wasdetermined using Modfit LT software (Verity Software House,Topsham, ME).

Assay of Anchorage-Independent Growth

P10 (100 mm) tissue culture dishes (Corning) wereprecoated with 5 mL Chelex-treated media supplementedwith 175 �mol/L Zn2� and 0.7% Bactoagar (Difco, Detroit,MI). The agar layer was allowed to harden and 8 mL ofChelex-treated media supplemented with 175 �mol/LZn2�, 0.35 % Bactoagar, and 1 � 105 CaCo-2 cells, trans-fected with EV or PKC-� cDNA, were overlaid on theagar-coated dishes. All dishes were incubated at 37°C in ahumidified atmosphere of 5% CO2–95% air, and freshZn2�-free medium was added every 3 days for a period of 4weeks. (This addition of media every 3 days replaced evap-orative losses, but did not include Zn2�. The Zn2� was notlost from the agar. In preliminary studies, if repeatedlyadded with fresh media, Zn2� was found to accumulate totoxic concentrations.) Colonies were stained with 0.1%Giemsa and those larger than 0.2 mm were quantified aspreviously described by our laboratory.20

Alkaline Phosphatase Assay

EV- and PKC-� cDNA–transfected CaCo-2 cells wereseeded at a density of 0.5 � 105 cells/well in 6-well plates andallowed to attach overnight before initiating treatment withzinc (175 �mol/L). Cells were cultured for the indicated times,with replacement of fresh media containing Zn2� every 2 days,and cultures were grown for a period of up to 14 days. Cellsfrom triplicate wells were collected for alkaline phosphataseactivity. Briefly, cells were rinsed in cold PBS buffer andscraped into 300 �L of cold 2.0 mmol/L Tris–50 mmol/Lmannitol buffer (pH 7.4) and stored at �80°C until furtheranalysis. To determine alkaline phosphatase activity, cells weresonicated 3 times for 15 seconds each at an output setting of3 using a Model 250 Branson Sonifier (Branson Instruments,Danbury, CT). Total cell lysates (100 �L) were assayed foralkaline phosphatase activity using a Sigma Diagnostics alka-line phosphatase kit that uses p-nitrophenyl phosphate assubstrate. Units of alkaline phosphatase activity were deter-mined using a p-nitrophenol standard curve. Specific activitywas expressed as units of alkaline phosphatase per milligramsof protein per minute.


Sucrase Isomaltase Assay

EV- and PKC-� cDNA–transfected CaCo-2 cells wereseeded at a density of 0.5 � 105 cells/well in 6-well plates andallowed to attach overnight before initiating treatment withzinc (175 �mol/L). Cells were cultured for the indicated times,with replacement of fresh media containing Zn2� every 2 days,and cultures were grown for a period of up to 14 days. Cellsfrom triplicate wells were collected for sucrase activity. Cellswere rinsed in cold PBS buffer and scraped into 300 �L of cold0.1 mol/L sodium maleate buffer (pH 6.0) and stored at�80°C until further analysis. Sucrase activity was measured bythe ultramicro method of Messer and Dahlqvist.48 Briefly,samples were sonicated 3 times for 15 seconds each at anoutput setting of 3 using a Model 250 Branson Sonifier(Branson Instruments), and total cell lysates (100 �L) wereincubated with 50 mmol/L sucrose at 37°C for 30 minutes.The glucose generated from sucrose by sucrase-isomaltase wasmeasured using a Sigma Diagnostics Glucose Oxidase (GO)assay kit. Sucrase activity was expressed as nanomoles of glu-cose generated per minute per milligram of protein.

Assessment of Apoptosis

Apoptosis was determined by 2 independent methods.In the first method, apoptotic cells were detected by nuclearmorphologic changes using 4 ,6-diamidino-2-phenylindoledihydrochloride (DAPI), a fluorescent groove-binding probefor DNA. Briefly, EV- and PKC-� cDNA–transfected CaCo-2cells were seeded at a density of 1.5 � 105 cells in Nuncpolystyrene slideflasks (Nunc, Naperville, IL) and grown inzinc-depleted medium for 2 days. Preconfluent cells were thentreated for a period of 48 hours in Chelex-treated DMEMsupplemented with 175 �mol/L Zn2� in the presence ofvehicle (DMSO, 0.05%) or 10 nmol/L TPA. Cells were washedtwice with HBSS and fixed for 10 minutes at room tempera-ture with 4% paraformaldehyde in PBS. Fixative was removedby aspiration and the monolayer washed twice in PBS. DNAwas stained for 1 minute with 2 �g/mL DAPI (MolecularProbes, Eugene, OR) at room temperature. Excess DAPI stainwas removed, and the monolayer was extensively washed withwater and a coverslip mounted with Vectashield mountingmedium (Vector Laboratories, Burlingame, CA). Stained nu-clei were viewed at 200� using an Olympus BH-2 fluores-cence microscope (Olympus Optical Co., Ltd., Japan) with theUV cube in position. Representative fields of view were pho-tographed at 200� magnification using Kodak Ektachrome400 film. As a second assessment of apoptosis, programmedcell death was identified in situ via specific labeling of nuclearDNA fragmentation, using the terminal deoxynucleotidyltransferase–mediated deoxyuridine triphosphate nick-end la-beling (TUNEL) assay.49 Briefly, DNA strand breaks were endconjugated with digoxigenin-labeled nucleotide triphosphatesby terminal deoxynucleotidyl transferase, using the ApoTagperoxidase in situ apoptosis detection kit from Intergen (Pur-chase, NY), following the protocol suggested by the manufac-turer. Labeled DNA fragments, bound to peroxidase-conju-

gated antidigoxigenin antibodies, were detected using thechromogenic substrate DAB (3,3 diaminobenzidine). TUNELstained nuclei were viewed by light microscopy. Apoptoticcells, as assessed by nuclear condensation or DNA fragmenta-tion, were quantified in 1000 random cells in duplicate plat-ings per sample for each experiment. Slides were coded beforecounting and results confirmed by 2 independent observers.Rates of apoptosis were expressed as a mean percentage � SD(Apoptotic Nuclei Observed/Total Nuclei Counted �100).

Statistical Analysis

Numerical data is expressed as means � SD. Statisticalsignificance of the differences between samples was determinedby the unpaired Student t test. Data was considered signifi-cantly different at the level of P � 0.05.

ResultsInducible Overexpression of PKC-� inCaCo-2 Cells

Previous studies have shown a loss of PKC-�expression in human and rodent colonic tumors com-pared with normal colonic mucosa. In agreement withthese studies, as shown in Figure 1, the expression ofPKC-� protein in human colonic carcinomas was de-creased to 60% of that of normal human colonic mucosa(Figure 1, lanes 6 and 7 vs. 4 and 5). In addition,expression of PKC-� was further decreased in humancolon cancer–derived CaCo-2 cells, with only 30% ofthat of normal colonic epithelium (Figure 1, lanes 2 and3 vs. 4 and 5). There were no differences in proteinexpression for �-actin, indicating equal amounts of pro-tein sample loaded in each lane (Figure 1). To investigatethe functional consequences of this loss of PKC-�, wetransfected full length human PKC-� cDNA into these

Figure 1. Western blot analysis of PKC-� expression in CaCo-2 cellsand in human colonic normal mucosa and colon tumor samples.Whole cell lysates were prepared from rat brain control, CaCo-2 cells,and human normal (N) and tumor (T) paired colon tissues. Proteins(20 �g/lane) were resolved by SDS-PAGE on 10% polyacrylamide gels,transferred to nitrocellulose membranes, and immunoblotted withantihuman PKC-� and �-actin monoclonal antibodies. Upper panel:PKC-� expression. PKC-� protein comigrates with rat brain PKC-� withMR � 78 kilodaltons. Lower panel: �-actin expression.


cells and selected stable polyclonal populations. Withrespect to PKC-� expression, these transfectants would,therefore, more closely mimic their nontransformedcolonocyte counterparts. For our expression system, weused a Zn2�-inducible metallothionein promoter,42,43

thereby avoiding constitutively increased levels ofPKC-� protein that might potentially be toxic to thesecells.40 We isolated 2 polyclonal populations, PKC-�1(MRE p�neo-�) and PKC-�2 (MRE pMet-�), which inresponse to Zn2� overexpressed full length PKC-� inCaCo-2 cells. EV-transfected polyclonal CaCo-2 cell pop-ulations, EV-1 (MRE p�neo) and EV-2 (MRE pMet),served as controls for PKC-�1 and PKC-�2, respectively,in these experiments. Except where indicated, transfec-tants were maintained in zinc-depleted DMEM.

In initial titration studies, we found that CaCo-2 cellsproliferated normally in the presence of 175 �mol/LZn2� in DMEM that contained 20% Chelex-treatedserum, but not at higher Zn2� concentrations, whichwere toxic to these cells (data not shown). Therefore, allsubsequent experiments were carried out using Chelex-treated media supplemented with 175 �mol/L Zn2�. Toanalyze the expression of PKC-� in these clones, wholecell lysates were prepared from EV- and PKC-�–trans-fected CaCo-2 cells cultured in Chelex-treated DMEMsupplemented with 175 �mol/L Zn2�. Maximal induc-tion of this gene was achieved by 24 hours. PKC-�migrated as a 78-kilodalton protein in rat brain control(Figure 2A and B, lane 1). Western blot analysis of wholecell extracts showed that expression of PKC-� was in-creased in PKC-�1– and PKC-�2–transfected cells byapproximately 4.5- and 3.8-fold, respectively, comparedwith EV controls (Figure 2A and B, lane 3 vs. 2). Levelsof nontargeted PKC isoforms -�, -�I, -�II, -�, and -�,which are also expressed in CaCo-2 cells,20 were analyzedin these clones. In contrast to PKC-� expression, bothEV- and PKC-�–transfected CaCo-2 cells showed mini-mal changes in the protein expression levels of the non-targeted isoforms, PKC-�, -�I, -�II, -�, and -�, com-pared with the nontransfected parental CaCo-2 cells (datanot shown).

To further characterize the overexpression of PKC-� inthese CaCo-2 transfectants, we measured the biochemicalactivity of this isoenzyme in these clones. Activity wasassayed in situ in digitonin permeabilized cells,46 using aPKC-�–specific peptide substrate that contains residues422–443 of the murine elongation factor, eEF-1�.44

Basal levels of PKC-� kinase activity were increased 2.0-and 3.0-fold in PKC-�1 and PKC-�2 clones, respec-tively, compared with EV controls, EV-1 and EV-2(Figure 3).

Overexpression of PKC-� InhibitsAnchorage-Dependent and -IndependentGrowth of CaCo-2 Cells

PKC-� has previously been shown to alter theproliferation of several noncolonic cell types.41 We,therefore, hypothesized that overexpression of PKC-� inCaCo-2 cells might result in altered growth rates in thesecells. Figure 4A and B represent growth curves for EV-and PKC-�–transfected CaCo-2 cells cultured for a pe-riod of up to 14 days in DMEM containing 175 �mol/LZn2�. The proliferative rates of parental- and EV-trans-fected CaCo-2 cells were not affected by this concentra-tion of zinc (data not shown). As shown in Figure 4,PKC-� overexpression caused significant decreases in cellnumbers, as early as day 3 (PKC-�1; Figure 4A ) and day7 (PKC-�2; Figure 4B) after plating, relative to EVcontrol cells (P � 0.01). This PKC-� antiproliferativeeffect remained significant through day 14 for bothtransfected clones. By day 14, there were 20% and 30%less PKC-�1 and PKC-�2 cells, respectively, comparedwith their EV controls (P � 0.01).

Figure 2. Western blot analysis of PKC-� expression in EV- and PKC-�–transfected CaCo-2 cells. Whole cell lysates were prepared from ratbrain control and EV- and PKC-�–transfected cells. Proteins (15 �g/lane) were resolved by SDS-PAGE on 10% polyacrylamide gels, trans-ferred to nitrocellulose membranes, and immunoblotted with PKC-�monoclonal antibodies. (A) PKC-� expression in PKC-�1 and EV-1stable transfectants. (B) PKC-� expression in PKC-�2 and EV-2 stabletransfectants. Human PKC-� comigrates with rat brain PKC-�; MR � 78kilodaltons. (B) PKC-� may migrate as a doublet because of phosphor-ylation.


Because growth inhibition may reflect alterations inthe cell cycle, we investigated the effects of PKC-� oncell cycle distributions by flow cytometry. Cell cycledistributions for EV cells were similar for EV-1 andEV-2. Therefore, data was pooled for EV-1 and EV-2 andexpressed as EV. Cell cycle analysis showed 55.8% �0.7% and 30.5% � 0.7% of EV cells were present in theG0/G1 and S phases, respectively (Figure 5). In contrast,PKC-� overexpression increased the number of cells inG0/G1 to 67.0% � 2.2%, with a reciprocal decrease inthe S phase for PKC-�2 cells (P � 0.05, n � 3). Therewere no statistical differences in the percent of cells inG2/M between PKC-�2 and EV-2. Cell cycle distribu-tions for PKC-�1 cells were similar to PKC-�2 (data notshown). These data indicate that PKC-� suppresses cellgrowth, at least in part, by inhibiting the G13S tran-sition in the cell cycle.

To assess anchorage-independent proliferation, and asan in vitro assay of cellular transformation, EV- andPKC-�–transfected CaCo-2 cells were analyzed for theirability to form colonies in soft agar. Consistently, in vitrotransformation characteristics were inhibited in thePKC-�–transfected clones, which displayed an attenu-ated capacity for anchorage-independent growth (Figure6). Specifically, PKC-�1 and PKC-�2 transfectants

showed a significant decrease of 46.0% � 3.1% and 68%� 4.7%, respectively, in the number of colonies formedin soft agar, compared with EV control cells (P � 0.05).

PKC-� Overexpression Enhances theDifferentiation Phenotype of CaCo-2 Cells

The acquisition of a differentiation phenotype incolonic cells, including CaCo-2 cells, is associated withan increase in the activity of alkaline phosphatase, abrush-border membrane enzyme.50,51 In addition, onreaching confluence, this cell line spontaneously differ-entiates into an ileal-like cell with the development of anapical brush-border membrane containing high levels ofsucrase-isomaltase enzyme.51 We investigated whetheroverexpression of PKC-� in CaCo-2 cells would alter

Figure 3. PKC-� overexpression increases PKC-�–specific phosphor-ylating activity in situ. CaCo-2 cells, transfected with EV or full lengthhuman PKC-� (PKC-�1 and PKC-�2), were plated in 24-multiwellplates. PKC-� phosphorylating activity was measured in subconfluentcells in the presence of a permeabilizing phosphorylation reactionmixture containing digitonin, [�32P] ATP, and a PKC-�–specific sub-strate derived from eEF-1� as described in Materials and Methods.32P-labeled eEF-1� peptide was then measured by scintillation count-ing. Values are expressed as fold increases in PKC-� activity (mean �SD) in transfected cells (PKC-�1 and PKC-�2), compared with the EVcontrols (n � 3 independent experiments in triplicate). *P � 0.05,compared with their respective EV control.

Figure 4. PKC-� overexpression inhibits the anchorage-dependentgrowth of CaCo-2 cells. EV- (F) and PKC-�– (■) transfected cells(0.5 � 105 cells/well) were seeded in 6-well plates as described inMaterials and Methods. At the indicated days after plating, cells weretrypsinized, collected, and then resuspended and counted using aCoulter counter. Data represents the average � SD of 2 independentexperiments, each carried out in triplicate. Error bars not shown arecontained within the data points. (A) PKC-�1–transfected cells. (B)PKC-�2–transfected cells. *P � 0.05, compared with their respectiveEV controls.


these differentiation markers. PKC-�–transfected cellsexhibited enhanced differentiation, shown by signifi-cantly increased alkaline phosphatase– and sucrase-iso-maltase–specific activities by days 9 (Figure 7) and 14(Table 1), respectively, compared with EV-transfectedcontrol cells (P � 0.05). At subsequent later times,compared with EV controls, these PKC-�–overexpress-ing cells continued to have significantly increased alka-

line phosphatase and sucrase-isomaltase activities duringthe duration of these experiments.

Overexpression of PKC-� Increases theApoptotic Rate of CaCo-2 Cells

Based on previous studies that suggested a role forPKC-� in apoptosis in several noncolonic cancer celllines,41 it was of interest to determine whether overex-pression of PKC-� would increase the rate of apoptosis inCaCo-2 cells. Such an increase would also contribute tothe antiproliferative effects of PKC-� that we observed inthese clones. Preconfluent cells were incubated for aperiod of 48 hours in Chelex-treated DMEM supple-

Figure 5. PKC-� overexpression causes a G1 arrest in CaCo-2cells. Preconfluent CaCo-2 cells, transfected with EV (EV1 � EV2;data pooled and expressed as EV, �; or full length human PKC-�,PKC-�2, ■), were assessed for their distribution in the cell cycleas described in Materials and Methods. Cells were fixed andanalyzed using CellQuest software in a FacScan flow cytometer(Becton Dickinson Immunocytometry Systems, San Jose, CA).The distribution of DNA in the cell cycle was determined usingModfit LT software. Values are reported as mean of 3 sam-ples � SD. *P � 0.05, compared with EV control. As noted in thetext, cell cycle distributions for PKC-�1 were similar to those ofPKC-�2.

Figure 6. PKC-� overexpression inhibits anchorage-indepen-dent growth of CaCo-2 cells. CaCo-2 cells (1 � 105), trans-fected with EV (EV1 � EV2; data pooled and expressed as EV) orPKC-� (PKC-�1 or PKC-�2), were plated in 0.35% agar and analyzedfor their ability to form colonies in soft agar as described inMaterials and Methods. Data are the mean � SD of 2 indepen-dent experiments of 10 determinations each. *P � 0.05, com-pared with EV.

Figure 7. Effect of PKC-� overexpression on alkaline phosphatase–specific activity. EV- (F) and PKC-�– (■) transfected cells (0.5 � 105)were plated in 6-well plates. Cells from triplicate wells were collectedon the indicated days and assayed for alkaline phosphatase activityas described in Materials and Methods. Specific activity was ex-pressed as units of alkaline phosphatase per milligrams of protein perminute. (A) PKC-�–stable transfectants. (B) PKC-�2–stable transfec-tants. Data are means � SD of 2 separate experiments each carriedout in triplicate. *P � 0.05, compared with their respective EV control.


mented with 175 �mol/L Zn2� in the presence of vehicle(DMSO) or 10 nmol/L TPA, and apoptosis was quanti-fied by nuclear morphologic changes and DNA fragmen-tation, as assessed by DAPI and TUNEL staining, re-spectively. The rates of apoptosis of EV-transfected cells,EV-1 and EV-2, were similar to that of parental CaCo-2cells (data not shown). As assessed by DAPI and shownin Figure 8, PKC-�1– and PKC-�2–transfected cellsexhibited significantly increased nuclear condensation inthe presence of 175 �mol/L Zn2�, compared with EVcontrols (6.6% � 0.1% and 8.3% � 0.1%, respectively,compared with 4.8% � 0.5% for EV-1 cells; P � 0.05,n � 4). In a similar fashion, these transfectants exhibitedsignificantly increased DNA fragmentation by TUNELassay, compared with EV controls (4.4% � 0.3% and6.0% � 0.1% for PKC-�1 and PKC-�2, respectively,compared with 2.7% � 0.5% for EV-1 cells; P � 0.05,n � 4). Consistent with an activation of PKC by phorbolesters, compared with untreated transfectants, TPA fur-ther enhanced apoptosis in each of their respective clonesby DAPI (8.6% � 0.4% and 10.3% � 0.3% forPKC-�1 and PKC-�2, respectively, and 7.0% � 0.4%for EV cells, P � 0.05) and TUNEL assay (6.6% � 0.4%and 8.2% � 0.3% for PKC-�1 and PKC-�2, respec-tively, compared with 4.0% � 0.9% for EV-1 cells; P �0.05). These studies by 2 independent methods showincreased programmed cell death in PKC-� transfectants.

DiscussionThe normal architecture of the colonic crypt is

maintained by a balance between colonocyte proliferationat the base and apoptosis at the colonic luminal surface,with acquisition of differentiation features during theupward migration of these cells. In colonic neoplasms,however, the normal architecture of the colonic crypt iscompletely altered, with marked disturbances in prolif-eration, differentiation, and apoptosis characterizingthese malignancies.52 PKC isoenzymes are involved indiverse cellular functions, including regulation of cell

balance and maturation.1–4 Several lines of evidence havealso implicated aberrant alterations in PKC in the dys-regulation of these events that characterize colon can-cer.8–11,16 For example, changes in the expression, bio-chemical activity, and/or subcellular distribution ofspecific isoforms of PKC have been associated with co-lonic malignant transformation in both human and ex-perimental models of colon cancer. These findings sug-gest that PKC isoenzymes are involved not only in themaintenance of normal colonic cellular homeostasis, butalso contribute to the process of colonic carcinogenesis.In this regard, elucidation of the isoform-specific cellularfunctions of members of this family of kinases will becritical to understand their roles in colonic tumorigene-sis. PKC-�, in particular, has been implicated in controlof growth, differentiation, and cell death of many non-colonic cell types.41 In malignant colonocytes, previousstudies have shown a significant decrease in the expres-sion of this isoenzyme in both human and experimentalcolonic tumors.17-19 In the present study, compared withnormal mucosa, we have not only confirmed thesechanges in sporadic human colon cancers, but also ob-served that CaCo-2 cells have an even lower expression ofthis isoform.

To gain further insight into the roles of PKC-� as aregulator of CaCo-2 cell proliferation, differentiation,and apoptosis, we constructed eukaryotic expression vec-tors that code for full length human PKC-�. Becauseconstitutive PKC-� overexpression severely impaired cellviability in rat colonic epithelial cells,40 we used 2 dis-tinct Zn2�-inducible metallothionein expression sys-tems.42,43 This allowed us to select and expand trans-fected clones in the basal state, thereby limitingconstitutive overexpression of this isoform during thesestages, to prevent a growth-related selection bias. Weestablished 2 polyclonal populations of PKC-� transfec-tants in CaCo-2 cells, PKC-�1 and PKC-�2. In thepresence of Zn2�, these clones overproduced PKC-� pro-tein �4-fold and showed an �2-fold increase in basal

Table 1. Effect of PKC-� Overexpression on the Sucrase-Isomaltase Activity of CaCo-2 Cells

Time (days after plating)

Sucrase-isomaltase activity (nmol/mg/min)

EV-1 PKC-�1 EV-2 PKC-�2

6 2.02 � 0.5 2.65 � 0.4 1.5 � 0.3 1.66 � 0.212 9.5 � 1.0 11.1 � 0.5 6.7 � 1.7 10.35 � 0.514 13.7 � 0.9 19.8 � 0.9a 14.4 � 2.4 22.6 � 0.7a

16 13.4 � 1.7 27.41 � 1.6a 16.8 � 1.2 27.56 � 2.8a

NOTE. EV- and PKC-�– (PKC-�1 and PKC-�2) transfected cells (0.5 � 105) were plated in 6-well plates. Cells from triplicate wells were collectedon the indicated days and assayed for sucrase-isomaltase activity as described in Materials and Methods. Sucrase-isomaltase activity wasexpressed as nanomoles of glucose-released per milligram of protein per minute. Values are reported as means � SD of 3 samples.aP � 0.05, compared with their respective EV control.


levels of PKC-�–specific biochemical activity. Overex-pression of PKC-� significantly decreased the growth ofCaCo-2 cells, as assessed by cell numbers. These findingsare consistent with previous studies in noncolonic celllines. For example, PKC-� overexpression inhibited cellproliferation in NIH 3T3 fibroblasts,23 CHO epithelialcells,24 and capillary endothelial cells.26 In agreementwith our findings, recent studies in src-overexpressing ratcolonic epithelial cells have shown that overexpression ofPKC-� reversed the src-induced transformed phenotype,causing an arrest of cellular proliferation.40 There areoccasional exceptions to this growth inhibitory effect ofPKC-�, emphasizing the importance of cell context inthe growth changes induced by this isoform.53 Takentogether, however, the preponderance of prior results, as

well as the findings in this study, strongly support theinvolvement of PKC-� as a growth suppressor of trans-formed colonic epithelial cells.

Although the downstream targets involved in thetumor-suppressing activity of this isoenzyme are un-known, we speculated that the antiproliferative effectsinduced by PKC-� overexpression might be mediated byalterations in the cell cycle. In recent years, evidence hassuggested a role for PKC-� in the regulation of cell cycleprogression in the G1 and G2/M phases of the cell cycle.3

Overexpression of this PKC isozyme, for example, de-layed G13S progression in endothelial cells,3,26 mousekeratinocytes, and rat vascular smooth muscle cells,3

while causing a cell cycle arrest in G2/M in CHO cells.24

These differences in the effects of PKC-� on the cell cycle

Figure 8. PKC-� overexpression increases the apoptosis ofCaCo-2 cells. Preconfluent CaCo-2 cells, transfected with EV or fulllength human PKC-� cDNA (PKC-�1 and PKC-�2), were treated witheither 10 nmol/L TPA (�TPA) or vehicle DMSO (�TPA) as de-scribed in Materials and Methods. Treated cells were fixed, andnuclei or fragmented DNA were stained by DAPI or TUNEL assay,respectively, and visualized by microscopy. Representative fieldsof view were photographed at 200� magnification. (A) DAPI-stained cells from the indicated clones. Examples of apoptoticcells are indicated with arrows. (B) Apoptotic rates. Rates ofapoptosis, expressed as a percentage of apoptotic nuclei, wereassessed in DAPI- (■) and TUNEL- (�) stained cells by counting1000 random cells in duplicate platings per experiment. Resultsare the mean � SD of 2 independent experiments each in dupli-cate. a,bP � 0.05, compared with EV control group. c,dP � 0.05,compared with their respective DMSO-treated clone (TPA control).Because the rates of apoptosis among parental- and EV-trans-fected cells (EV-1, EV-2) were similar, only EV-1 is shown for clarity.


may reflect the restricted availability of tissue-specificPKC-� activators or substrates, including cell cycle reg-ulatory elements. In this study, overexpression of PKC-�in CaCo-2 cells caused a G0/G1 cell cycle arrest with adecrease in the number of cells in S phase, withoutaffecting the proportion of cells in the G2/M phase. Inrat 3Y1 fibroblasts and vascular smooth muscle cells,PKC-� regulates the G13S progression by inhibitingE2F activity and decreasing the expression of cyclins D1and E, respectively.3 In future studies, elucidation of themechanisms by which this isoform influences cell cycleregulatory proteins in CaCo-2 cells will help to identifythe links between PKC-� signal transduction pathwaysand cellular proliferation in these human colon cancercells.

In this study, increased expression of PKC-� alsoinhibited CaCo-2 cell anchorage–independent growth,an in vitro measure of cellular transformation. Hence,loss of PKC-�, as occurs in colon cancer, might therebyenhance the malignant phenotype of CaCo-2 cells. Sev-eral studies have investigated the role of alterations in theexpression of this isoenzyme in malignant transforma-tion.41 In this regard, overexpression of a constitutivelyactive PKC-� inhibited colony formation of both wildtype– and c-Ha-ras–transformed NIH 3T3 cells.54

PKC-� overexpression was also found to reverse thetransformed phenotype of c-src–overexpressing rat co-lonic epithelial cells.40 In contrast, depletion of PKC-�,by expression of a dominant negative mutant, enhancedthe transformed phenotype of c-src overexpressing ratfibroblasts.36 As in the case of proliferation, there areoccasional exceptions to this inhibition of growth in softagar by PKC-�.53,55 Again, our studies on anchorage-independent growth, as well as the majority of publishedreports in other cell types, support a role for PKC-� as atumor suppressor, suggesting that decreases in its expres-sion may be involved in critical steps in the multistageprocess of colonic carcinogenesis, including increasedcellular proliferation and acquisition of anchorage-inde-pendent growth.

We have also shown that increasing PKC-� expressionresulted in a more differentiated phenotype, as assessedby increases in alkaline phosphatase and sucrase-isomal-tase activities, markers of CaCo-2 cell differentiation.51

Several groups have investigated the effects of PKC-� oncellular differentiation, showing that overexpression ofthis isoenzyme enhanced phorbol ester–induced macro-phage differentiation25,27 and caused maturation of nor-mal human keratinocytes.56 Although a preponderance ofstudies support a role for this isoenzyme as an inducer ofdifferentiation, down-regulation of PKC-� was found to

accelerate differentiation of murine erythroleukemiacells.57 These differences, as in the case of growth, mostlikely reflect cell-specific effects of PKC-� on differenti-ation. Although in normal mouse and rat colonocytes,PKC-� levels in situ were low and predominantly cyto-solic in proliferating colonocytes at the base of the crypt,increasing levels of expression were found to coincidewith progressive colonocyte differentiation in postmi-totic cells.11 This was accompanied by an increased as-sociation of PKC-� in the membrane/cytoskeletal frac-tion. In contrast, in colonic malignancy, loss ofdifferentiation markers are associated with decreases inPKC-� expression.17 Taken together, these findings sug-gest that increased expression of PKC-� is involved inthe acquisition of differentiation characteristics in thecolonic epithelium. Furthermore, they suggest that lossof PKC-�, observed in colonic tumors in vivo, maycontribute to the block in differentiation associated withcolonic neoplasia. To our knowledge, this is the firstdirect demonstration that increased PKC-� expressioninduces a differentiation phenotype in a human coloncancer–derived cell line.

Besides the involvement of PKC-� in the regulation ofCaCo-2 cell growth and differentiation, our studiesshowed that overexpression of this isoenzyme also en-hanced the rate of apoptosis in these cells. These findingsare consistent with recent studies showing the involve-ment of PKC-� in phorbol ester–induced apoptosis ofprostate cancer cells.30 Apoptosis mediated by PKC-� isthought to be associated with proteolysis of this isoen-zyme, generating a 40-kilodalton catalytically activefragment that phosphorylates DNA-dependent proteinkinase.58 This, in turn, may contribute to DNA damage-induced apoptosis.58 Furthermore, caspase-dependentpathways have been implicated in this proteolytic acti-vation of PKC-� in some cell types,29,59 but not others.30

Recent studies in SNU-C1 and SNU-C4 human coloncancer cells have shown that concentrations of phorbolesters inducing apoptosis caused the translocation ofPKC-�, also suggesting the involvement of this isoen-zyme in regulating programmed cell death in thesecells.60 Our study showed the involvement of PKC-� inthe apoptosis of CaCo-2 cells. Increased expression of thisPKC isoform enhanced the rate of apoptosis in thesecells, which was further augmented by phorbol estertreatment. Future studies will be required to elucidatethe specific proapoptotic pathways that are abrogated bythe loss of PKC-� in colon cancer, thereby enhancingtumorigenesis.

Finally, it is important to emphasize that, althoughthe antiproliferative effects of PKC-� overexpression


were modest, albeit significant, their accumulated im-pact on tumor growth may be of considerable biologicalrelevance. Tumorigenesis is characterized by a net in-crease in proliferation over programmed cell death.Mathematical models of tumorigenicity have suggestedthat even modest decreases in proliferative rates, or in-creases in apoptosis, could result in significant inhibitionof tumor formation.61,62

In summary, our results provide the first direct evi-dence that alterations in PKC-� expression may play anessential role in the neoplastic phenotype of CaCo-2 cells.Reversing the constitutive low level of this isoform byoverexpression inhibited anchorage-dependent and -in-dependent growth, caused a G1 cell cycle arrest, in-creased differentiation, and enhanced the apoptosis ofCaCo-2 cells. These in vitro phenotypic alterations ofCaCo-2 cells induced by PKC-�, if applicable in vivo,have important implications with respect to the role thatloss of this isoenzyme may play in human colonic ma-lignant transformation. Furthermore, it suggests thatfuture interventions to selectively preserve colonicPKC-� expression may provide novel approaches to in-hibit the development or progression of this malignancy.

References1. Nishizuka Y. Protein kinase C and lipid signaling for sustained

cellular responses. FASEB J 1995;9:484–496.2. Liu WS, Heckman CA. The sevenfold way of PKC regulation. Cell

Signal 1998;10:529–542.3. Livneh E, Fishman DD. Linking protein kinase C to cell-cycle

control. Eur J Biochem 1997;248:1–9.4. Lavin MF, Watters D, Song Q. Role of protein kinase activity in

apoptosis. Experientia 1996;52:979–994.5. Newton AC. Regulation of protein kinase C. Curr Opin Cell Biol

1997;9:161–167.6. Wetsel WC, Khan WA, Merchenthaler I, Rivera H, Halpern AE,

Phung HM, Negro-Vilar A, Hannun YA. Tissue and cellular distri-bution of the extended family of protein kinase C isoenzymes.J Cell Biol 1992;117:121–133.

7. Greenlee RT, Murray T, Bolden S, Wingo PA. Cancer statistics,2000. CA Cancer J Clin 2000;50:7–33.

8. Weinstein IB. The roles of specific isoforms of protein kinase C ingrowth control and human colon cancer. Princess TakamatsuSymp 1991;22:277–283.

9. Kahl-Rainer P, Karner-Hanusch J, Weiss W, Marian B. Five of sixprotein kinase C isoenzymes present in normal mucosa showreduced protein levels during tumor development in the humancolon. Carcinogenesis 1994;15:779–782.

10. Brasitus TA, Bissonnette M. PKC isoforms: villains in colon can-cer? Gastroenterology 1998;115:225–227.

11. Verstovsek G, Byrd A, Frey MR, Petrelli NJ, Black JD. Colonocytedifferentiation is associated with increased expression and al-tered distribution of protein kinase C isozymes. Gastroenterology1998;115:75–85.

12. Davidson LA, Jiang YH, Derr JN, Aukema HM, Lupton JR, ChapkinRS. Protein kinase C isoforms in human and rat colonic mucosa.Arch Biochem Biophys 1994;312:547–553.

13. Guillem JG, O’Brian CA, Fitzer CJ, Forde KA, LoGerfo P, Treat M,Weinstein IB. Altered levels of protein kinase C and Ca2�-depen-

dent protein kinases in human colon carcinomas. Cancer Res1987;47:2036–2039.

14. Kopp R, Noelke B, Sauter G, Schildberg FW, Paumgartner G,Pfeiffer A. Altered protein kinase C activity in biopsies of humancolonic adenomas and carcinomas. Cancer Res 1991;51:205–210.

15. Levy MF, Pocsidio J, Guillem JG, Forde K, LoGerfo P, Weinstein IB.Decreased levels of protein kinase C enzyme activity and proteinkinase C mRNA in primary colon tumors. Dis Colon Rectum1993;36:913–921.

16. McGarrity TJ, Peiffer LP. Protein kinase C activity as a potentialmarker for colorectal neoplasia. Dig Dis Sci 1994;39:458–463.

17. Craven PA, DeRubertis FR. Loss of protein kinase C delta isozymeimmunoreactivity in human adenocarcinomas. Dig Dis Sci 1994;39:481–489.

18. Craven PA, DeRubertis FR. Alterations in protein kinase C in1,2-dimethylhydrazine induced colonic carcinogenesis. CancerRes 1992;52:2216–2221.

19. Wali RK, Frawley BP Jr, Hartmann S, Roy HK, Khare S, Scaglione-Sewell BA, Earnest DL, Sitrin MD, Brasitus TA, Bissonnette M.Mechanism of action of chemoprotective ursodeoxycholate in theazoxymethane model of rat colonic carcinogenesis: potentialroles of protein kinase C-�, -�II, and -�. Cancer Res 1995;55:5257–5264.

20. Scaglione-Sewell B, Abraham C, Bissonnette M, Skarosi SF, Da-vidson NO, Wali RK, Davis BH, Sitrin M, Brasitus TA. DecreasedPKC-� expression increases cellular proliferation, decreases dif-ferentiation, and enhances the transformed phenotype of CaCo-2cells. Cancer Res 1998;58:1074–1081.

21. Goldstein DR, Cacace AM, Weinstein IB. Overexpression of pro-tein kinase C �I in the SW480 colon cancer cell line causesgrowth suppression. Carcinogenesis 1995;16:1121–1126.

22. Choi PM, Tchou-Wong KM, Weinstein IB. Overexpression of pro-tein kinase C in HT29 colon cancer cells causes growth inhibitionand tumor suppression. Mol Cell Biol 1990;10:4650–4657.

23. Mischak H, Goodnight JA, Kolch W, Martiny-Baron G, SchaechtleC, Kazanietz MG, Blumberg PM, Pierce JH, Mushinski JF. Over-expression of protein kinase C-� and -� in NIH 3T3 cells inducesopposite effects on growth, morphology, anchorage dependence,and tumorigenicity. J Biol Chem 1993;268:6090–6096.

24. Watanabe T, Ono Y, Taniyama Y, Hazama K, Igarashi K, Ogita K,Kikkawa U, Nishizuka Y. Cell division arrest induced by phorbolester in CHO cells overexpressing protein kinase C-� subspecies.Proc Natl Acad Sci U S A 1992;89:10159–10163.

25. Mischak H, Pierce JH, Goodnight J, Kazanietz MG, Blumberg PM,Mushinski JF. Phorbol ester–induced myeloid differentiation ismediated by protein kinase C-� and -� and not by protein kinaseC-�II, -�, -�, and -. J Biol Chem 1993;268:20110–20115.

26. Harrington EO, Loffler J, Nelson PR, Kent KC, Simons M, Ware JA.Enhancement of migration by protein kinase C-� and inhibition ofproliferation and cell cycle progression by protein kinase C-� incapillary endothelial cells. J Biol Chem 1997;272:7390–7397.

27. Wang QJ, Acs P, Goodnight J, Giese T, Blumberg PM, Mischak H,Mushinski JF. The catalytic domain of protein kinase C-� in recip-rocal -� and -� chimeras mediates phorbol ester-induced macro-phage differentiation of mouse promyelocytes. J Biol Chem1997;272:76–82.

28. Reddig PJ, Dreckschmidt NE, Ahrens H, Simsiman R, Tseng CP,Zou J, Oberley TD, Verma AK. Transgenic mice overexpressingprotein kinase C-� in the epidermis are resistant to skin tumorpromotion by 12-O-tetradecanoylphorbol-13-acetate. Cancer Res1999;59:5710–5718.

29. Denning MF, Wang Y, Nickoloff BJ, Wrone-Smith T. Protein kinaseC-� is activated by caspase-dependent proteolysis during ultravi-olet radiation–induced apoptosis of human keratinocytes. J BiolChem 1998;273:29995–30002.

30. Fujii T, Garcia-Bermejo ML, Bernabo JL, Caamano J, Ohba M,


Kuroki T, Li L, Yuspa SH, Kazanietz MG. Involvement of proteinkinase C � (PKC �) in phorbol ester–induced apoptosis in LNCaPprostate cancer cells. Lack of proteolytic cleavage of PKC �. J BiolChem 2000;275:7574–7582.

31. Savickiene J, Gineitis A, Stigbrand T. Modulation of apoptosis ofproliferating and differentiating HL-60 cells by protein kinaseinhibitors: suppression of PKC or PKA differently affects celldifferentiation and apoptosis. Cell Death Differ 1999;6:698–709.

32. Cross T, Griffiths G, Deacon E, Sallis R, Gough M, Watters D, LordJM. PKC-� is an apoptotic lamin kinase. Oncogene 2000;19:2331–2337.

33. Villalba M. A possible role for PKC-� in cerebellar granule cellsapoptosis. Neuroreport 1998;9:2381–2385.

34. Sumitomo M, Shen R, Goldberg JS, Dai J, Navarro D, Nanus DM.Neutral endopeptidase promotes phorbol ester–induced apopto-sis in prostate cancer cells by inhibiting neuropeptide-inducedprotein kinase C � degradation. Cancer Res 2000;60:6590–6596.

35. Zang Q, Lu Z, Curto M, Barile N, Shalloway D, Foster DA. Asso-ciation between v-Src and protein kinase C � in v-Src–transformedfibroblasts. J Biol Chem 1997;272:13275–13280.

36. Lu Z, Hornia A, Jiang YW, Zang Q, Ohno S, Foster DA. Tumorpromotion by depleting cells of protein kinase C �. Mol Cell Biol1997;17:3418–3428.

37. Li W, Michieli P, Alimandi M, Lorenzi MV, Wu Y, Wang LH, Heida-ran MA, Pierce JH. Expression of an ATP binding mutant of PKC-�inhibits Sis-induced transformation of NIH 3T3 cells. Oncogene1996;13:731–737.

38. Li W, Jiang YX, Zhang J, Soon L, Flechner L, Kapoor V, Pierce JH,Wang LH. Protein kinase C-� is an important signaling molecule ininsulin-like growth factor I receptor-mediated cell transformation.Mol Cell Biol 1998;18:5888–5898.

39. Geiges D, Marks F, Gschwendt M. Loss of protein kinase C � fromhuman HaCaT keratinocytes upon ras transfection is mediated byTGF �. Exp Cell Res 1995;219:299–303.

40. Perletti GP, Marras E, Concari P, Piccinini F, Tashjian AH Jr. PKC-�acts as a growth and tumor suppressor in rat colonic epithelialcells. Oncogene 1999;18:1251–1256.

41. Gschwendt M. Protein kinase C �. Eur J Biochem 1999;259:555–564.

42. Palmiter RD. Regulation of metallothionein genes by heavy met-als appears to be mediated by a zinc-sensitive inhibitor thatinteracts with a constitutively active transcription factor, MTF-1.Proc Natl Acad Sci U S A 1994;91:1219–1223.

43. Morosov A, Phelps WC, Raychaudhuri P. Activation of the c-fosgene by the HPV16 oncoproteins depends upon the cAMP-re-sponse element at �60. J Biol Chem 1994;269:18434–18440.

44. Kielbassa K, Muller H-J, Meyer HE, Marks F, Gschwendt M.Protein kinase C-�–specific phosphorylation of the elongationfactor eEF-1� and an eEF-1� peptide at threonine 431. J BiolChem 1995;271:6156–6162.

45. Aris P, Basta PV, Holmes WD, Ballas LM, Moomaw C, Rankl NB,Blobel G, Loomis CR, Burns DJ. Molecular and biochemical char-acterization of a recombinant human PKC-� family member. Bio-chim Biophys Acta 1993;1174:171–181.

46. Khare S, Bissonenette M, Scaglione-Sewell B, Wali RK, SitrinMD, Brasitus TA. 1,25-dihydroxyvitamin D3 and TPA activatephospholipase D in CaCo-2 cells: role of PKC-�. Am J Physiol1999;276:G993–G1004.

47. Darzynkiewicz Z. Cell cycle analysis by flow cytometry. In: Cellbiology: a laboratory handout. San Diego, CA, Academic, 1994:261–271.

48. Messer M, Dahlqvist A. A one-step ultramicro method for the

assay of intestinal disaccharidases. Anal Biochem 1966;14:376–392.

49. Gavrieli Y, Sherman Y, Ben-Sasson SA. Identification of pro-grammed cell death in situ via specific labeling of nuclear DNAfragmentation. J Cell Biol 1992;119:493–501.

50. Halline AG, Davidson NO, Skarosi SF, Sitrin MD, Tietze C, AlpersDH, Brasitus TA. Effects of 1,25-dihydroxyvitamin D3 on prolifer-ation and differentiation of Caco-2 cells. Endocrinology 1994;134:1710–1717.

51. Pinto M, Robine-Leon S, Appay M-D, Kedinger M, Triadou N,Dussaulx E, LaCroix B, Simon-Assmann P, Haffen K, Fogh J,Zweibaum A. Enterocyte-like differentiation and polarization ofthe human colon carcinoma cell line Caco-2 in culture. Biol Cell1983;47:323–330.

52. Lipkin M. Proliferation and differentiation of normal and neoplas-tic cells in the colon of man. Cancer 1971;28:38–40.

53. Kiley SC, Clark KJ, Duddy SK, Welch DR, Jaken S. Increasedprotein kinase C � in mammary tumor cells: relationship totransformtion and metastatic progression. Oncogene 1999;18:6748–6757.

54. Hirai S, Izumi Y, Higa K, Kaibuchi K, Mizuno K, Osada S, SuzukiK, Ohno S. Ras-dependent signal transduction is indispensablebut not sufficient for the activation of AP1/Jun by PKC �. EMBO J1994;13:2331–2340.

55. Kiley SC, Clark KJ, Goodnough M, Welch DR, Jaken S. Proteinkinase C � involvement in mammary tumor cell metastasis. Can-cer Res 1999;59:3230–3238.

56. Ohba M, Ishino K, Kashiwagi M, Kawabe S, Chida K, Huh NH,Kuroki T. Induction of differentiation in normal human keratino-cytes by adenovirus-mediated introduction of the and � iso-forms of protein kinase C. Mol Cell Biol 1998;18:5199–5207.

57. Pessino A, Passalacqua M, Sparatore B, Patrone M, Melloni E,Pontremoli S. Antisense oligodeoxynucleotide inhibition of � pro-tein kinase C expression accelerates induced differentiation ofmurine erythroleukaemia cells. Biochem J 1995;312:549–554.

58. Bharti A, Kraeft SK, Gounder M, Pandey P, Jin S, Yuan ZM,Lees-Miller SP, Weichselbaum R, Weaver D, Chen LB, Kufe D,Kharbanda S. Inactivation of DNA-dependent protein kinase byprotein kinase C�: implications for apoptosis. Mol Cell Biol 1998;18:6719–6728.

59. Pongracz J, Webb P, Wang K, Deacon E, Lunn OJ, Lord JM. Sponta-neous neutrophil apoptosis involves caspase 3–mediated activationof protein kinase C-�. J Biol Chem 1999;274:37329–37334.

60. Weller SG, Klein IK, Penington RC, Karnes WE Jr. Distinct proteinkinase C isozymes signal mitogenesis and apoptosis in humancolon cancer cells. Gastroenterology 1999;117:848–857.

61. Tomlinson IP, Bodmer WF. Failure of programmed cell death anddifferentiation as causes of tumors: some simple mathematicalmodels. Proc Nat Acad Sci U S A 1995;92:11130–11134.

62. Solyanik GI, Berezetskaya NM, Bulkiewicz RI, Kulik GI. Differentgrowth patterns of a cancer cell population as a function of itsstarting growth characteristics: analysis by mathematical model-ing. Cell Prolif 1995;28:263–278.

Received August 21, 2000. Accepted February 5, 2001.Address requests for reprints to: Marc Bissonnette, M.D., The Uni-

versity of Chicago Hospitals and Clinics, Department of Medicine, MC4076, 5841 South Maryland Avenue, Chicago, Illinois 60637. e-mail:[email protected]; fax: (773) 702-2182.

Supported in part by National Institutes of Health grants DK39573,CA36745 (to T.A.B. and M.B.), CA69532 (to M.B.), and P30DK42086(to T.A.B., Digestive Disease Research Core Center), as well as by theSamuel Freedman GI Cancer Laboratory at the University of Chicago.


PKC-δ inhibits anchorage-dependent and -independent growth, enhances differentiation, and increases...

Documents

Transcript of PKC-δ inhibits anchorage-dependent and -independent growth, enhances differentiation, and increases...