Phosphatidylcholine-Specific Phospholipase C Activation in Epithelial Ovarian Cancer Cells

Phosphatidylcholine-Specific Phospholipase C Activation in

Epithelial Ovarian Cancer Cells

Francesca Spadaro,1Carlo Ramoni,

1Delia Mezzanzanica,

2Silvia Miotti,

2Paola Alberti,

2

Serena Cecchetti,1Egidio Iorio,

1Vincenza Dolo,

3Silvana Canevari,

2and Franca Podo

1

1Section of Molecular and Cellular Imaging, Department of Cell Biology and Neurosciences, Istituto Superiore di Sanita, Rome, Italy;2Unit of Molecular Therapies, Department of Experimental Oncology and Laboratories, Fondazione IRCCS Istituto Nazionaledei Tumori, Milan, Italy; and 3Department of Experimental Medicine, University of L’Aquila, L’Aquila, Italy

Abstract

Elucidation of the mechanisms responsible for aberrantphosphatidylcholine (PC) metabolism in cancer cells mayallow identification of novel biomarkers of tumor progressionand design of new targeted anticancer therapies. We recentlyreported up-regulation of PC-specific phospholipases in epi-thelial ovarian cancer cells (EOC) compared with nontumoral(normal or immortalized) counterparts (EONT). In the pre-sent study, we focused, in the same cell systems, on levels,subcellular localization, and activity of PC-specific phospho-lipase C (PC-PLC), for which a key role in cell prolifera-tion, differentiation, and apoptosis has been shown in severalmammalian cells. A 66-kDa PC-PLC isoform, detected innuclear and cytoplasmic compartments of both EOC andEONT cells, accumulated on the external plasma membraneof cancer cells only, where it colocalized with B1 integrin,in nonraft membrane domains. PC-PLC activity was 3-foldhigher in total cell lysates and 5-fold higher in membrane-enriched fractions of EOC compared with EONT cells. Serumdeprivation induced in EOC, but not in EONT, cells a 3-folddecrease in PC-PLC activity, associated with a 40% drop inS-phase fraction. The recovery of both variables to theiroriginal levels in serum-restimulated (or lysophosphatidicacid–restimulated) EOC cells was strongly delayed, for atleast 24 h, in the presence of the PC-PLC inhibitor tricyclo-decan-9-yl-potassium xanthate (D609). The S-phase of serum-restimulated EONT cells was not sensitive to D609. Thesefindings warrant further investigations on the role of PC-PLCand on the effects of its inhibition on the pathways respon-sible for constitutive EOC cell stimulation and cell proliferation.[Cancer Res 2008;68(16):6541–9]

Introduction

Epithelial ovarian cancer (EOC), the most common cause ofdeath from gynecologic malignancy in the Western world, stillpresents a high case-fatality ratio with an overall 5-year survivallower than 45%, mainly due to asymptomatic disease progressionat early stage, high recurrence rate, and frequent onset of chemo-

resistance after first line treatment (1, 2). The existing gap in theknowledge of molecular mechanisms responsible for EOC progres-sion leads to current limitations of therapy regimens, still mainlyrestricted to surgery followed by chemotherapy with cytotoxicdrugs. Only limited attention has been, thus far, devoted to altera-tions of metabolic fluxes and enzyme activities associated in thesetumors with gene-driven deregulation of cell signaling pathwaysresponsible for uncontrolled cell proliferation, cell invasiveness,and escape from default apoptotic programs.An aberrant phosphatidylcholine (PC) metabolism is reported in

cancer cells, in which substantial changes in the steady-state levelsof PC cycle derivatives are commonly detected by in vitro andin vivo nuclear magnetic resonance signals (3–5). Among these, anincrease in phosphocholine (PCho) content is a common feature ofdifferent cancer cells, beyond their otherwise wide phenotypicvariability (5). Elucidation of the underlying molecular mechanismsmay characterize novel biomarkers of tumor progression (6–8) andallow identification of metabolic targets for newly designedselective antitumor therapies (9–11).We recently reported (8) a 3-fold to 8-fold increase in the PCho

content in EOC cell lines compared with epithelial ovariannontumoral (normal or immortalized) cells (EONT). Enzymaticassays showed that PCho accumulation in EOC cells wasassociated with activation of choline kinase, a biosynthetic enzymewell known for its implication in human carcinogenesis (12–14),and PC-specific phospholipases of the C and D type (PC-PLC andPC-PLD). These findings prompted our interest on investigatingthe role of PC-PLC in ovary tumor progression, also in view of ourprevious reports on ectopic localization and/or activation of thisenzyme in oncogene-transformed and mitogen-stimulated fibro-blasts (15, 16).A growing body of evidence has pointed, since the 1990s, to the

implication of PC-PLC in proliferation, differentiation, and apop-tosis of mammalian cells. In fact, this enzyme has been implicatedin cellular processes crucial for cell signaling, such as (a) mitogen-driven and oncogene-driven extracellular signal-regulated kinasephosphorylation and activation of gene transcription factors infibroblasts (16, 17), (b) agonist stimulation of cells transfected withreceptor proteins (18), (c) neuronal and endothelial cell differen-tiation (19–21), (d) programmed cell death (22, 23), and (e) acti-vation of cells of the immune system (24–30).Currently, the mammalian PC-PLC has not been cloned and its

sequence is unknown. Only scanty knowledge has been accumu-lated on expression, subcellular distribution, and activity of PC-PLCin cancer cells. Nevertheless, indirect evidence on increased PC-PLC activity was reported in breast tumor cells (31). Moreover, thepresent unavailability of specific monoclonal antibodies (mAb) canbe partly compensated by the use of rabbit polyclonal antibodiesraised against bacterial (Bacillus cereus) PC-PLC, possessing proved

Note: Supplementary data for this article are available at Cancer Research Online(http://cancerres.aacrjournals.org/).

F. Spadaro and C. Ramoni contributed equally to this work.Requests for reprints: Franca Podo, Department of Cell Biology and

Neurosciences, Istituto Superiore di Sanita, Viale Regina Elena 299, 00161 Rome,Italy. Phone: 39-06-49902686; Fax: 39-06-49387144; E-mail: [email protected] or SilvanaCanevari, Department of Experimental Oncology, Fondazione IRCCS IstitutoNazionale dei Tumori, Via Venezian 1, 20133 Milan, Italy. Phone: 39-02-23902567;Fax: 39-02-23903073; E-mail: [email protected].

I2008 American Association for Cancer Research.doi:10.1158/0008-5472.CAN-07-6763

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selective cross-reactivity against mammalian PC-PLC (32). Thisapproach allowed identification of PC-PLC isoforms in variousnormal and tumor cells of different origin (15, 16, 25–27, 32, 33).This article reports evidence on subcellular localization and acti-

vity of PC-PLC in EOC cell lines compared with epithelial ovariannormal cells or immortalized cell variants. The possible role of thisenzyme in cell proliferation was investigated by measuring PC-PLCactivity in relation to alterations in the S-phase fraction induced inEOC cells by serum deprivation and subsequent restimulation bygrowth factors, in the presence or absence of a PC-PLC inhibitor.

Materials and Methods

Antibodies and reagents. Rabbit polyclonal anti–PC-PLC antibodyraised against bacterial (B. cereus) PC-PLC and selectively cross-reacting

with mammalian PC-PLC was obtained according to the procedure de-

scribed by Clark and colleagues (32) and modified as reported in ref. 15.Immunochemical characterization and specificity of these antibodies for

proteins with PC-PLC activity have already been described (15, 16, 25). In

particular, specific binding inhibition tests showed that this antibody was

directed against the catalytic site of a mammalian 66-kDa PC-PLC isoform(16). Anti–h-actin mAb was supplied by Sigma-Aldrich. Anti–folate receptor(FR) MOV18 and MOV19 (34), available through Alexis Life Sciences, Inc.,

and anti–h1 integrin MAR4 (35) mAbs were produced at the Fondazione

IRCCS Istituto Nazionale dei Tumori, Milan. Rabbit anti–caveolin-1 (Cav-1)antibody was from Santa Cruz Biotechnology. Alexa Fluor-488 and Alexa

Fluor-594 F(ab)2 fragment of goat anti-rabbit IgG (H + L) and Alexa Fluor-

594 F(ab)2 fragment of goat anti-mouse IgG (H + L), purchased from

Molecular Probes, Inc., were used as secondary antibodies.Epidermal growth factor (EGF) was supplied by Calbiochem. Lysophos-

phatidic acid (LPA) was purchased from Avanti Polar Lipids, Inc. Triton

X-100, propidium iodide, tricyclodecan-9-yl-potassium xanthate (D609),5-bromo-2¶-deoxyuridine (BrdUrd), and all other chemicals and bioche-

micals were from Sigma-Aldrich.

Normal ovarian epithelial cells, immortalized cell variants, andcancer cells. Normal ovarian surface epithelial (OSE) cells (six independentpreparations), stably immortalized OSE cell variants obtained by OSE

transfection with SV40 large T antigen (IOSE) and by further IOSE

transfection with cDNA of the catalytic subunit of human telomerase

reverse transcriptase (hTERT), and EOC cell lines (IGROV1, OVCAR3,SKOV3, and CABA I) were obtained and cultured as reported (8). Ascitic

fluids were collected during surgical procedures from four advanced stage

ovarian cancer patients undergoing debulking surgery at the FondazioneIRCCS Istituto Nazionale dei Tumori at the first diagnosis and not

previously treated with chemotherapeutic regimens. Histopathologic

diagnosis for all patients was poorly differentiated serous papillary ovarian

cancer. All clinical specimens used for this study were obtained withInstitutional Review Board approval from patients who gave informed

consent to use leftover biological material for investigative purposes.

Tumor cells were isolated by centrifugation on a discontinuous 75% to 100%

Ficoll gradient followed by separation from adherent cells, essentially asdescribed (36).

Confocal laser scanning microscopy and flow cytometry analyses.PC-PLC subcellular distribution in either unfixed or fixed and permeabilizedcells was characterized by indirect immunofluorescence and confocal laser

scanning microscopy (CLSM) analysis, as previously described (16, 25–27).

CLSM observations were performed on a Leica TCS SP2 AOBS apparatus,

using excitation spectral laser lines at 405, 488, and 594 nm and using theLeica Confocal Software (Leica Lasertechnik GmbH) and Adobe Photoshop

software programs (Adobe Systems).

Signals from different fluorescent probes were taken in parallel, and

colocalization was detected in yellow. Different fields of view (>200 cells)were analyzed on the microscope for each labeling condition, and

representative results are shown.

Flow cytometry analyses were performed on cells suspended in 0.2%

PBS-EDTA and stained as described (16).

Western blot analyses. Total cell lysates were obtained suspending cellpellets in ice-cold lysis buffer [150 mmol/L NaCl, 100 mmol/L Tris-HCl

(pH 8), 1% Triton X-100, 1 mmol/L MgCl2, and protease inhibitor cocktail]

for 20 min. Lysates were cleared of detergent insoluble materials by 10 min

centrifugation at 20,000 � g .Membrane fractions were prepared, and their purification was controlled

as described (37).

Protein concentration was determined by Bio-Rad protein assay

according to the manufacturer’s protocol (Bio-Rad Laboratories, Inc.) andtotal cell lysates or purified membranes (30 Ag proteins) were analyzed byWestern blotting, as previously described (27). Densitometry analyses of

protein bands were performed with a Bio-Rad apparatus (Bio-Rad

Laboratories Srl) using the Quantity One software.In vitro PC-PLC and PLD activity assays. Relative changes of PC-PLC

and PLD activities were determined in whole-cell lysates and enriched mem-

brane fractions using the Amplex Red assay kit (Molecular Probe, Inc.), asdescribed by the manufacturer and adapted by Spadaro and colleagues (26).

Separation of lipid rafts by sucrose gradient. Cells grown in T75 flaskswere washed in cold PBS, lysed in 1% Triton X-100 containing buffer

[25 mmol/L MES (pH 6.5), and 150 mmol/L NaCl], and the lysate was mixed1:1 with 80% sucrose solution. Gradient fractionation (5–30% sucrose) and

protein separation were carried out as previously described (38, 39). After

ultracentrifugation at 200,000 � g for 17 h in TH641 Sorvall rotor, twelve

1-mL fractions were collected from the top of the gradient, and the samevolume (90 AL) was taken from each fraction for analysis in SDS-PAGE in

parallel with that of total cell lysate (35 Ag total protein). To concentrateraft membranes, top fractions 3 to 6 were diluted 1:2 with 25 mmol/L MES(pH 6.5) and 150 mmol/L NaCl, and raft membranes were pelleted by

centrifugation at 13,000 rpm for 30 min. Pellet was resuspended in loading

buffer for SDS-PAGE analysis. Distribution of relevant proteins separated

by 10% SDS-PAGE was analyzed by Western blotting.Glycosyl-phosphatidyl inositol–anchored FR and Cav-1 were used as

markers of lipid rafts, whereas h1 integrin was considered as a nonraft

marker. To analyze FR and h1 integrin reactivity, samples were analyzed

under nonreducing conditions.Immunohistochemical detection of PC-PLC in EOC tissues. Analyses

were performed on a commercial tissue array slide constructed with

paraffin sections from 50 ovarian cancer specimens of different histo-types (SuperBioChips Laboratories). Antigen retrieval was performed in

10 mmol/L citrate buffer (pH 6), at 120jC for 2 min; endogenous peroxidasewas quenched with 3% H2O2 for 10 min, and slides were saturated in

blocking solution (30 min) and then incubated with rabbit anti–PC-PLCantibodies (1 h at room temperature) and revealed with biotin-conjugated

antirabbit secondary antibody followed by avidin-biotin complex (Vectas-

tain Universal Elite ABC kit, Vector Laboratories, Inc.). Peroxidase reaction

was developed with 3,3-diaminobenzidine, and sections were counter-stained with hematoxylin. Slides incubated with the secondary antibodies

alone provided negative controls; a control with hematoxylin/eosin was also

prepared. Staining was evaluated considering both staining intensity and

the proportion of cells showing positive reaction.Statistical analysis. Data were analyzed using GraphPad software

version 3.03. Statistical significance of differences was determined by one-

way ANOVA or by Student’s t test (as specified). Differences were consideredsignificant at P < 0.05.

Results

Increased levels of PC-PLC localization and activity on themembrane of EOC compared with EONT cells. CLSM analyseson unfixed cells showed massive (although different) levels of PC-PLC localization on the outer membrane surface of a number ofEOC cell lines, as well as on that of cancer cells isolated fromascitic exudates (examples in Fig. 1A). This characteristic PC-PLCdistribution was much less evident in hTERT and only barelydetectable in OSE and IOSE cells. Flow cytometry analyses onunfixed cells showed a 5.0 F 0.9 (SD)–fold increase (ANOVA

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Cancer Res 2008; 68: (16). August 15, 2008 6542 www.aacrjournals.org



P < 0.0001) in the median value of fluorescence intensity ofOVCAR3 and IGROV1 compared with OSE and IOSE cells (Fig. 1B).No significant difference was found between hTERT and the otherEONT cells (P = 0.09). Moreover, it is worth noting that, whereasonly 25.0 F 3.8% (SD) of unfixed OSE and IOSE cells were positiveto PC-PLC immunostaining, this fraction increased to 53.7 F 9.5%in hTERT (P = 0.003) and reached values as high as 84.6 F 5.7% inEOC cell preparations (P < 0.0001).Both EOC and EONT cells exhibited, however, similar PC-PLC

intracellular distribution patterns, as shown by CLSM analyses onfixed and permeabilized cell preparations (examples in Supple-mentary Fig. S1). PC-PLC–positive granules were clearly detected inboth cytoplasmic and nuclear regions, as well as in the filamentousstructures protruding from cell periphery.Immunohistochemical examinations of a commercial tissue

array slide of ovarian tumor tissues, including 31 epithelial ovariancancer sections, revealed high levels of PC-PLC in 27 samples (87%)with variable cellular localization (examples in SupplementaryFig. S2). In the PC-PLC–positive epithelial tumors, staining waslocalized to the cytoplasm. Among these cases, six also showed

staining at the membrane level and the other six at the nuclearlevel (Table 1). PC-PLC localization in tumor specimens was ingeneral agreement with the results obtained in EOC cell lines byCLSM analysis. In fact, as in CLSM examinations on fixed cells(Supplementary Fig. S1), the majority of the staining was mainlylocalized to the cytoplasm also in formalin-fixed paraffin-embedded tissue sections (Supplementary Fig. S2).Immunoblotting analyses allowed detection of a distinct PC-PLC

isoform with apparent molecular weight Mr = 66 kDa in fresh, totalcell lysates of all investigated EONT and EOC cells (Fig. 2A), inagreement with previous reports in other mammalian cells (15, 16,25–27, 33). By densitometric analysis, the intensity of the corre-sponding protein band in most of the investigated cancer cell lines(OVCAR3, SKOV3, and IGROV1) was 1.9F 0.5 times higher than thatobserved in nontumoral cells. Moreover, the intensity of the PC-PLCband at 66 kDa was 2.6 F 0.1–fold higher in membrane-enrichedfractions of OVCAR3 compared with immortalized cells (Fig. 2B).Amplex Red assays performed on total cell lysates showed no

significant differences (P = 0.62) in the relative PC-PLC activityvalues of nontumoral immortalized cells (IOSE and hTERT); data

Figure 1. CLSM and flow cytometry analyses of PC-PLC distribution on the plasma membrane of EOC cells compared with EONT cells. A, PC-PLC detection byCLSM analysis (three-dimensional reconstruction) on the outer membrane surface of unfixed, subconfluent EOC cell lines (SKOV3, CABAI, IGROV1, and OVCAR3),normal OSE, and nontumoral immortalized cells (IOSE, hTERT) cultured to early confluence. An example of observations on cancer cells isolated from patientexudate is also reported. Offset and gain values of the photomultiplier channel were regulated with respect to the setup selected for OSE cell analysis to makefluorescence intensity comparable across experiments on different samples. Scale bar, 20 Am. B, examples of flow cytometry profiles of unfixed EOC and EONT cells(three independent series of experiments for each analyzed cell line). Left, representative cytofluorimetric histograms (CTR, control cells, IOSE, or OVCAR3, incubatedwith rabbit IgG plus secondary antibody); center, median (F SD) values of fluorescence intensity; right , percentages of PC-PLC–positive cells (mean F SD).

PC-PLC in Ovarian Cancer




were therefore pooled together, and their mean value wasnormalized to 1.0. In this arbitrary unit scale, the PC-PLC activityof EOC cell lines (Fig. 2C) in repeated experiments was 2.9 F 0.7(FSD)–fold higher than that of normal cells (ANOVA P < 0.0001).Assays on enriched membrane fractions (Fig. 2D) showed that the

PC-PLC activity of OVCAR3 cells was 5-fold higher than that ofEONT cells, and a similar result was obtained from ascitic cancercells isolated from patient exudate.These results altogether indicated that EOC differed from EONT

cells in a substantial increase in the PC-PLC level on outer cell

Table 1. Immunohistochimical detection of PC-PLC in an ovarian cancer tissue array slide

Samples No. sections No/weak staining* Immunohistochemical staining

Strong staining* localized to

Cytoplasm Cytoplasm/membrane Cytoplasm/nucleus

Epithelial tumors: 31 4 15 6 6Mucinous 1 1

Serous 17 2 11 1 3

Mullerian 2 2

Endometrioid 5 1 2 2Clear cell 5 1 2 1 1

Brenner tumor 1 1

*Immunohistochemical score was obtained by adding the value of staining intensity (0, no staining; 1, faint staining; 2, moderate staining; 3, intensestaining) to the estimated percentage of positive cells (0, no reactive cells; 1, 1–10% reacting cells; 2, 11–25% reacting cells; 3, 26–50% reacting cells;

4, over 50% reacting cells). Weak staining, score V 3.

Figure 2. Expression and activity ofPC-PLC in cell lysates of nontumoral (OSE,IOSE, hTERT) and EOC cell lines at earlyconfluence. Western blotting of total celllysates (A ) and membrane fractions (B).h-Actin and h1 integrin were used as loadingcontrol for densitometric analyses in Aand B , respectively. Representative setsof experiments [of three (A ) or two (B )performed] are reported. PC-PLC activity(C and D ) was measured from the relativeincrease in Resorufin fluorescence in AmplexRed assay in cancer cells compared withIOSE and hTERT cells. C, relative increasein PC-PLC activity in whole-cell lysates(mean F SD of five experiments; P < 0.0001by ANOVA in EOC with respect to IOSE plushTERT cell samples); D , relative increase inPC-PLC activity in cell membrane preparations(mean F SD of three experiments; P < 0.0002by Student’s t test). The PC-PLC activity ofascitic cells from peritoneal exudates wasmeasured in triplicate (variability F 10%).

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membrane, in a higher activity of this enzyme in total cell lysates,including membrane fragments and in an even higher activity inmembrane-enriched fractions.

PC-PLC localizes on EOC cells plasma membrane in nonraftdomains. The granular PC-PLC distribution pattern on plasmamembrane of cancer cells suggested that the enzyme could local-ize in selected membrane domains. To investigate the nature ofthese domains, a sucrose gradient separation of lipid rafts wasperformed on OVCAR3 cell lysates (see Materials and Methods).The distribution of PC-PLC was monitored by Western blotand compared with the distribution of nonraft (h1 integrin) andraft (FR and Cav-1) markers. At variance with the raft markersFR and Cav-1, PC-PLC was below detection level in raft frac-tions (3–6), not only when equal sample volumes were analyzed(not shown), but also when raft membranes were pelleted bycentrifugation and the resulting 18-fold–concentrated sampleswere used for comparative analyses, to enhance the sensitivityof the detection method (Fig. 3A). A distinct protein band at66 kDa was instead clearly detected by anti–PC-PLC antibodieseven in unconcentrated h1 integrin–containing samples ( frac-tions 9–12).CLSM analyses on unfixed OVCAR3 cells showed a substantial

colocalization of PC-PLC with h1 integrin (Fig. 3B), but not with FR(not shown), a pattern which was even clearer in cancer asciticcells (Fig. 3C). A very low level of colocalization of PC-PLC and h1integrin could instead be detected on the membrane of unfixed

hTERT cells (Fig. 3D), in which the phospholipase was present toa much lower level (in agreement with Fig. 1A).Interestingly, the extent of colocalization of PC-PLC with h1

integrin substantially decreased on the plasma membrane ofserum-deprived OVCAR3 cells and progressively returned to thepattern observed in subconfluent control cells after EGF stimula-tion (data reported in Supplementary Fig. S3).Overall, these results showed that PC-PLC localization on the

plasma membrane of EOC cells occurs in discrete nonraft domainsand suggest a spatial (and perhaps functional) relationshipbetween this phospholipase and the h1 integrin adhesion protein.

Changes of PC-PLC activity in OVCAR3 cells after serumdeprivation and cell restimulation in the presence or absenceof D609. Amplex Red analyses showed that the mean value ofPC-PLC activity decreased f3-fold in OVCAR3 cells cultured for48 h in FCS-deprived medium compared with control cells grownto subconfluence in complete medium (Fig. 4A). Under theseconditions, the PC-PLC activity of EOC cells became comparablewith that of EONT cells grown in complete medium. When serum-deprived cells were restimulated by FCS, the PC-PLC activityreturned almost to the level measured in control cells within1 h. Similar results were obtained when cell restimulation wasinduced by LPA alone [20 Amol/L in unsupplemented RPMI], apotent mitogen for EOCs (40). In fact, 1-hour LPA stimulationinduced a 3.4 F 1.1–fold increase in PC-PLC activity of serum-deprived cells.

Figure 3. PC-PLC localization in nonraft membrane domains and its colocalization with h1 integrin in epithelial ovarian cancer and immortalized cells. A, sucrosegradient separation of Triton X-100 lysate from OVCAR3 cells (see Materials and Methods). The distribution of PC-PLC in the recovered fractions (3–6 for raftsand 9-12 for nonrafts) and in total cell lysate (T ) was monitored by Western blot analysis and compared with that of nonraft (h1 integrin) and raft (FR and Cav-1)markers. Raft fractions were 18-fold concentrated to increase the sensitivity of PC-PLC detection. B–D, CLSM analyses (central optical sections) of unfixed cellsdouble-stained with anti–PC-PLC antibodies (red) and mAb against h1 integrin (green ). B , OVCAR3 cells; right, background labeling in absence of primary antibodies.C, ascitic cancer cells (from patient 4). D, hTERT cells. Colocalization in yellow. Nuclear staining by 4¶,6-diamidino-2-phenylindole (blue ). Scale bar, 20 Am.





However, when cell restimulation occurred in the presenceof the PC-PLC inhibitor D609 (50 Ag/mL), the PC-PLC activity inFCS-stimulated cells maintained, for at least 24 h, valuescomparable with those of serum-deprived cells (Fig. 4A). Cellincubation with D609 similarly prevented the recovery of PC-PLCactivity in LPA-stimulated cells. Parallel Amplex Red PLD assaysshowed that the activity of this enzyme was not significantlyaltered by D609 in OVCAR3 cells under any of the adoptedconditions of serum deprivation or serum resupplementation(Fig. 4B). It is also worth noting that the percentage of apop-totic cells, measured by flow cytometry on propidium iodide–

stained EOC cells, was below 3% both in the presence and absenceof D609.Experiments performed on hTERT cells showed that PC-PLC

activity remained practically unaltered (within F10%) in these cellsafter 48 hours of serum deprivation and during subsequent FCS re-stimulation, irrespectively of cell exposure to D609 (data not shown).These results suggest the existence of a fine regulation

mechanism controlling PC-PLC activity in ovarian cancer (butnot in nontumoral, immortalized epithelial ovarian cells) inresponse to exogenous growth factors.

Effect of D609 on the S-phase fraction of OVCAR3 andhTERT cells. To investigate possible relationships between PC-PLCactivity and cell proliferation in EOC and EONT cells, dual flowcytometry analyses were performed on OVCAR3 and hTERT cellsstained with BrdUrd and propidium iodide at the end of the serumdeprivation period and after cell restimulation by serum (FCS, for8 or 24 h) either in the presence or absence of D609. Cell cyclemeasurements in these samples were compared with those ofcontrol cells cultured in the presence of complete medium andharvested at early confluence (Fig. 5).The cell cycle distribution G0-G1/S/G2M was 47 F 6%: 30 F

2%: 16 F 2% in control OVCAR3 cells (mean of five independentexperiments), changing to 60 F 5%: 17 F 3%: 18 F 3% in serum-deprived cells. There were no substantial changes in the cell cycledistribution of cells deprived of serum for further 8 or 24 h after theinitial 48-h serum starvation (i.e., for total serum deprivationperiods of 56 and 72 h). The S-phase fraction almost returned tothe control value (S = 35 F 5%) in cells restimulated by FCS for8 h (or 24 h) after 48-h serum deprivation (Fig. 5, top). However,this recovery was totally inhibited in cells restimulated for 8 hin the presence of D609 (S = 16 F 2%) and was still partiallyinhibited (P = 0.004) after 24 h of cell reexposure to FCS.Similar results were obtained when cell restimulation was exerted

by LPA alone, instead of FCS. In particular, the recovery of the S-phase fraction in either FCS-restimulated or LPA-restimulated cellswas equally inhibited by D609 for at least 24 hours (data not shown).No apoptosis was induced by D609 under any of these experi-

mental conditions.Cell cycle analyses performed under the same conditions on

hTERT cells gave a G0-G1/S/G2M percentage distribution of 36 F4%: 45 F 10%: 13 F 4% in subconfluent control cells. These valuesindicated that the basal S-phase fraction of these nontumoral,immortalized cells was even higher than that of a highly aggressivecarcinoma cell line, such as OVCAR3. With respect to the latter,serum deprivation of hTERT cells induced smaller decreases inthe S-phase fraction (by 24% at 56 h and 36% at 72 h). Similarto OVCAR3 cells, FCS restimulation restored in 8 h the originalS-phase fraction in hTERT cells. However, different from thecarcinoma cell line, D609 did not prevent, at any incubation time,in these nontumoral cells the FCS-induced process of S-phaserecovery (Fig. 5, bottom).These data altogether indicate that cell exposure to the PC-PLC

inhibitor results in a long-lasting inhibition of serum growthfactor–induced proliferation of cancer cells, but does not inhibitproliferation of nontumoral immortalized cells.

Discussion

In this study, we report accumulation of PC-PLC on the outersurface of the plasma membrane in EOC cell lines, as well as inEOC cells isolated from patient ascites compared with in vitro

Figure 4. Relative changes of PC-PLC (A ) and PLD (B) activities in OVCAR3cells after serum deprivation and subsequent restimulation in the presence orabsence of the PC-PLC inhibitor D609. Amplex Red assays were performed ontotal cell lysates. White columns, cells deprived of serum for 48 h and thenrestimulated by 10% FCS for the indicated time intervals. Black columns,cells exposed to D609 (50 Ag/mL) for 15 min at the end of the 48-h serumdeprivation period and maintained in the presence of the inhibitor during FCSrestimulation. Gray column , subconfluent control cells (CTR ) maintained inculture in complete medium. Relative fluorescence (mean F SD, five series ofindependent experiments) was normalized to that of serum-deprived cells(made equal to 1; experimental variability F 30%). Cell viability was 95% to98% under all conditions.

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cultured EONT cells. Cellular PC-PLC redistribution in EOC cellslines was consistent with immunohistochemical detection ofPC-PLC in a relevant subset of archival EOC tissue samples. Theectopic PC-PLC localization in EOC cells was associated withhighly significant enzyme activation with respect to EONT cells,corresponding to a PC-PLC activity increase of 3-fold and 5-foldin total EOC cell lysates and membrane-enriched fractions, re-spectively. The lower increase in enzyme activity measured in totalcell lysates suggests that the enzyme undergoes a much lower(if any) activation in nonmembranous cytoplasmic compartments.Furthermore, by exposing cells to either FCS or LPA in the pre-sence of the PC-PLC inhibitor D609, we provided the first directindication that PC-PLC activity in EOC cells is likely involved in thecontrol of cell proliferation, mainly affecting the S-phase fraction.

PC-PLC externalization on the plasma membrane of EOCcells. The massive localization of the enzyme on the membranesurface of EOC (but not EONT) cells is in general agreement withsimilar findings reported on ras -transformed and mitogen-stimulated fibroblasts (15, 16), as well as on cytokine-activatednatural killer (NK) cells (25–27). Among signaling-activated PC-specific phospholipases (41, 42) which are also known to

translocate to the plasma membrane, PC-PLC presents the pecu-liar feature of accumulating on the outer, instead of the inner,membrane surface of mammalian cells. Furthermore, differentfrom PC-PLD, whose localization is mainly restricted to caveolin-enriched microdomains (43), our study shows that, in EOC cells,PC-PLC is localized in nonraft domains in close proximity withh1 integrin.Further investigations are needed to elucidate whether protein

phosphorylation and/or interaction with specific cytoskeletoncomponents are involved in PC-PLC activation and accumulationon the plasma membrane under conditions of constitutive oragonist-mediated cell stimulation of EOC cells. Indirect evidenceon PC-PLC phosphorylation is inferred from a previous study (19),in which nerve growth factor–induced increase of an antiphos-photyrosine immunoreactive 66-kDa band in PC12 cells has beenreported to be inhibited by cell exposure to D609. Regarding thepossible involvement of cytoskeleton components in PC-PLCtranslocation to the membrane, we previously reported a kinesin-supported PC-PLC transport from the microtubule organizingcenter to the membrane and PC-PLC interaction with filamentousactin in interleukin 2–activated, but not in resting NK cells (25).

Figure 5. S-phase fraction in OVCAR3 and hTERT cellsafter serum deprivation and subsequent restimulation incomplete medium in the presence or absence of D609.White columns, percentage of the S phase measured incells deprived of serum for 48 h and then cultured for 8 or24 h in the presence or absence of 10% FCS, asindicated. Black columns, percentage of S-phase fractionmeasured in parallel sets of samples as above, exposed toD609 (50 Ag/mL) for 15 min at the end of the 48-h serumstarvation period and henceforth for 8 or 24 h in thepresence or absence of FCS. Gray column, CTR,subconfluent control cells maintained in culture in completemedium. Numbers of experiments performed are indicatedin brackets within each data column.





Regarding functional aspects, externalization of PC-PLC on thecell membrane may confer to this activated enzyme the capabilityof participating in membrane receptor-mediated cell signaling, byproducing diacyl glycerol (DG) second messengers from hydrolysisof PC, the major phospholipid component of the outer layer ofeukaryotic cells. In fact, a persistent production of PC-derived DGsis reputed to contribute to long-lasting (PKC-mediated or PKC-independent) signaling events ending with gene transcription andDNA synthesis (43, 44). In this context, it is interesting to note thateven exogenously added bacterial PC-PLC has been shown to elicita potent mitogenic response in fibroblasts by promoting DNAsynthesis and cell proliferation (45).Furthermore, the ectopic localization of activated PC-PLC on

cancer cell membrane may allow this enzyme to modulate theexpression of some membrane receptors. In fact, we recentlyreported evidence on the crucial role of ectopically localizedactivated PC-PLC in selectively regulating the expression of CD16,the FcIgG receptor on the membrane of human NK cells, withstriking effects on CD16-mediated cytotoxicity (27). The changesreported herein of PC-PLC and h1 integrin colocalization on themembrane of serum-deprived and EGF-restimulated OVCAR3 cells(Supplementary Fig. S3) may point to a similar role of PC-PLC inregulating the expression and signaling of this adhesion protein inEOC cells. A previous report on the mutual relationships betweenPC-PLC and h1 integrin in regulating cell-substratum adhesion invascular endothelial cells (46) seems to reinforce this hypothesis.Studies are in progress to further investigate and quantify possibleinteractions of PC-PLC with this adhesion molecule under differentconditions of EGF stimulation.

Relationships between PC-PLC activity and S-phase fractionin EOC cells. Serum deprivation caused a 3-fold decrease in thePC-PLC activity of OVCAR3 cells, inducing a drop in the activityof this enzyme down to the level steadily measured in EONT cells.PC-PLC down-regulation was associated in EOC cells with a 40%decrease in the S-phase fraction of serum-deprived OVCAR3 cells.PC-PLC reactivation in FCS-stimulated or LPA-stimulated OVCAR3cells preceded a full recovery of the S-phase fraction to the valuemeasured before serum deprivation. Similarly, although quantita-tively different, changes in PC-PLC activity (5-fold decrease) andS-phase fraction (f100% drop) were reported in serum-deprivedfibroblasts, where subsequent cell restimulation by platelet-derivedgrowth factor (PDGF) promptly restored PC-PLC activity and theoriginal proliferative status (16). The higher residual percentage ofPC-PLC activity maintained in serum-deprived EOC cells comparedwith the residual PC-PLC activity reported in serum-deprivednormal fibroblasts may be attributed to the well-known reduceddependence of cancer cells upon exogenous growth factors fortheir survival and proliferation (47). The long-lasting ( for at least24 h) inhibition on the recovery of PC-PLC activity induced byD609 in serum-restimulated or LPA-restimulated OVCAR3 cells isin general agreement with a similar effect observed in PDGF-stimulated fibroblasts (16), suggesting that altered PC-PLC activity

and changes in the S-phase fraction may both be linked to commonmitogen-dependent pathways. Further investigations are requiredto elucidate the nature of the underlying molecular mechanisms.The present study also shows that the suggested relationships

between PC-PLC activity and S-phase fraction in OVCAR3 do nothold true for the nontumoral immortalized hTERT cells, in whichsubcellular PC-PLC localization was mainly confined to innercellular compartments. In spite of their higher S-phase fraction atsubconfluence (probably due to telomerase transfection), thesecells maintained a 3-fold lower PC-PLC activity compared withOVCAR3 cells and no substantial change was observed in PC-PLCactivity in serum-deprived and FCS-restimulated cells, either in thepresence or in the absence of D609. Noteworthy, only scantyalterations were observed in the S-phase fraction of hTERT cellsexposed to D609.Further studies are needed to elucidate whether other enzymes,

besides PC-PLC, may also be directly or indirectly involved in D609-induced effects on EOC cell proliferation. In fact, it has been shownthat PC-PLC inhibition by D609 may also interfere with cellsignaling, by blocking, in some cell systems, mitogen-inducedprotein kinase activation (48, 49) or by inhibiting other enzymes,such as sphingomyelin synthase (50).In overall, the results reported in the present work support the

view that PC-PLC is directly involved in the complex machinery ofpathways responsible for EOC cell proliferation. Further evidenceon these mechanisms is expected from PC-PLC sequencing andcloning, which would allow a better elucidation of the role of thisenzyme in the multiple pathways controlling the development ofaberrant phenotypes typical of these (and probably other)malignant cells, together with the possible identification of noveltargets for selective antitumor therapies. At the present stage, thedifferent involvement of PC-PLC in EONT and EOC cellproliferation suggests that the use of a PC-PLC inhibitor (possiblyone even more effective and specific than D609) might provide apowerful method to selectively interfere, likely in combination withantitumor drugs, with some of the aberrant pathways responsiblefor EOC proliferation and invasiveness.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

Received 12/20/2007; revised 5/28/2008; accepted 6/6/2008.Grant support: Cassa di Risparmio delle Provincie Lombarde Foundation

CARIPLO grant 2003.1740/10.4890 (S. Canevari and F. Podo) and Associazione Italianaper la Ricerca sul Cancro grant 2007 (F. Podo).

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Dr. C. Pini and Dr. B. Barletta, Istituto Superiore di Sanita, for preparationand purification of anti–PC-PLC antibodies, the clinical staffs of the Gynecological andPathologic Units of Fondazione IRCCS Istituto Nazionale dei Tumori for providingpatients’ materials, and Marco Sabatini for excellent graphical assistance.

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2008;68:6541-6549. Cancer Res Francesca Spadaro, Carlo Ramoni, Delia Mezzanzanica, et al. Epithelial Ovarian Cancer CellsPhosphatidylcholine-Specific Phospholipase C Activation in

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Phosphatidylcholine-Specific Phospholipase C Activation in Epithelial Ovarian Cancer Cells

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