Protein Kinase C-alpha Activity Inversely Modulates Invasion and Growth of Intestinal Cells*

Eduard BatlleDagger , Javier Verdú§, David Domínguez, Maria del Mont Llosas, Víctor Díaz§, Noureddine Loukiliparallel , Rosanna Paciucci, Francesc Alameda, and Antonio García de Herreros**

From the Unitat de Biologia Cel.lular i Molecular, Institut Municipal d'Investigació Mèdica, Calle Dr. Aiguader 80, 08003 Barcelona, Spain

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

The phorbol ester phorbol 12-myristate 13-acetate induces remarkable phenotypic changes in intestinal HT-29 M6 cells; these changes consist of loss of homotypic adhesion and inactivation of E-cadherin. In parallel, cell growth is retarded. We have transfected HT-29 M6 cells with an activated form of the conventional protein kinase Calpha (cPK-Calpha ). Expression of this isoform induced the acquisition of a scattered phenotype, similar to that adopted by cells after addition of phorbol 12-myristate 13-acetate, with very low cell-to-cell aggregation and undetectable levels of functional E-cadherin. These cell clones were highly motile and rapidly invaded embryonic chick heart fragments. Furthermore, cells expressing activated-cPK-Calpha showed decreased proliferation in comparison to control clones. We have also studied how these two apparently antagonistic changes affect the tumorigenic ability of HT-29 M6 cells. When the different cell clones were xenografted into athymic mice, the effect on cell growth seemed to predominate. Expression of activated-cPK-Calpha significantly reduced the size of the tumors; the cells with the highest level of expression did not even form subcutaneous tumors. Besides their smaller size, the morphology of these tumors was clearly different from those originated by HT-29 M6 cells, and they could be defined as infiltrative on anatomo-pathological basis. These results indicate that cPK-Calpha controls both cell-to-cell adhesion and proliferation of intestinal cells.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Unveiling the mechanisms that control establishment and maintenance of intercellular contacts in the epithelium of the intestine is essential to understanding colon carcinogenesis. There is considerable evidence that protein kinase C (PK-C)1 isoforms are involved in this process; for example, addition of the phorbol ester phorbol 12-myristate-13-acetate (PMA) has been shown to reduce cell-to-cell contacts in epithelial cells (1). This compound has been widely used to activate PK-C in cells because it is membrane-permeable and causes a maintained stimulation of most members of this extended family (cPK-C and nPK-C) (2, 3). Addition of PMA to intestinal cell subpopulations derived from HT-29 cells, like its addition to many other epithelial cell lines, causes a striking alteration in their morphology and induces scattering of cell colonies (4). This process is characterized by the acquisition of a more fibroblastic phenotype, with lower cell-to-cell adhesion and inactivated E-cadherin (4, 5). In parallel, addition of this compound retards cell growth, an effect that has also been observed in other intestinal cells (6). Previous studies from our group have shown that cell scattering can also be induced by thymeleatoxin, a specific activator of conventional PK-Cs (cPK-Cs) (7). The goal of this study was to identify the PK-C isoform involved in these events and determine whether activation of this isoform in intestinal cells lead to altered tumorigenic properties of HT-29 M6 cells (HT-29 cell subpopulation isolated using 10-6 methotrexate) in vivo.

    EXPERIMENTAL PROCEDURES
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Introduction
Procedures
Results
Discussion
References

Reagents-- PMA, leupeptin and phenylmethylsulfonyl fluoride were supplied by Sigma. Gelatin and plasminogen were from Merck and Boehringer Mannheim, respectively. PK-C inhibitors GF109203X (Gf) and Go 6976 (Go) were purchased from Calbiochem (San Diego, CA); these products were dissolved in dimethyl sulfoxide (Me2SO) and stored protected from light at -40 °C. [gamma -32P]ATP was purchased from Amersham Pharmacia Biotech. Monoclonal antibodies against cPK-Calpha and E-cadherin (HECD-1) were obtained from Transduction Laboratories (Lexington, KY) and Zymed Laboratories Inc. (San Francisco, CA), respectively. Prestained SDS-polyacrylamide gel electrophoresis molecular weight markers were from Bio-Rad (Richmond, CA). All of the oligonucleotides used in our assays were synthesized by Amersham Pharmacia Biotech.

Cell Culture-- The properties of the cell line used in this study HT-29 M6 (M6), originally characterized with the name of HT-29 (10-6 methotrexate), have been extensively described (8, 9). Cells were seeded at a density of 2 × 104 cells/cm2 and cultured in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal bovine serum (Life Technologies, Inc.) as described previously (5). Experiments of scattering were performed using cells 4-5 days after plating, when they were 40-50% confluent.

Mutagenesis of cPK-Calpha and Transfection to M6 Cells-- Human cPK-Calpha was obtained from ATCC. Mutation of the alanine situated in position 25 to glutamic acid (A25E) was carried out according to the method of Kunkel et al. (10), using the oligo 5'-CCCGCAAAGGGGAGCTCAGGCAGAAG-3' (corresponding to nucleotides 89-114 of the sequence of human cPK-Calpha ). In addition to the change C to A (position 102, in bold), required to replace Ala with Glu, the G present in position 106 was mutated to a C (underlined); this second silent mutation generates a restriction site for SacI, which is useful to recognize the mutated cDNA. The presence of the mutations in the final construction was verified by sequencing using Sequenase (U. S. Biochemical Corp.). Mutated cPK-Calpha , denominated cPK-Calpha (+), was inserted in the NotI site of pOPRSV expression plasmid (Stratagene, La Jolla, CA) and transfected to M6 cells using LipofectAMINE (Life Technologies, Inc.) under the conditions indicated by the manufacturer. After 21 days of selection in Dulbecco's modified Eagle's medium supplemented with G-418 (0.6 mg/ml) (Sigma), resistant clones were isolated. As a control, transfection of HT-29 M6 with pOPRSV plasmid without insert was performed; two clones resistant to G-418 were isolated and analyzed.

Analysis of the Transfectants-- RNA was obtained from the different clones using the guanidium thiocyanate method (11), treated with DNase I, and reverse transcribed into cDNA using random hexamers. This cDNA was first analyzed by PCR with oligos A1 (5'-TGGACAAACTACCTACAGAGATT-3') and A2 (5'-TCCCAAACCCCCAGATGAAGTCG-3'); oligo A1 corresponds to nucleotides 2638-2660 of pOPRSV, a sequence located downstream from the transcription start point; oligo A2 corresponds to nucleotides 211-189 of cPK-Calpha . To verify the specificity of the amplification, the 275-base pair product of amplification was subjected to electrophoresis, transferred to a Nylon-membrane (Amersham Pharmacia Biotech) and blotted with oligo B1 (5'-GGACCATGGCTGACGTTTTC-3', nucleotides 23-42 of cPK-Calpha ) labeled with [gamma -32P]ATP by T4 polynucleotide kinase. In order to compare the relative expression of the mutated and wild-type forms of cPK-Calpha , the cDNA was amplified quantitatively with oligos B1 and B2 (5'-ACAGCAAACTTGGACTGGAA-3', nucleotides 239-220), which flank the mutation. The product of the PCR was digested with SacI and analyzed as above using oligo A2 labeled with 32P. After treatment with SacI, wild-type cPK-Calpha generated a fragment of 217 base pairs; the mutated cDNA generated a fragment of 142 base pairs. As a control, the cDNA was subjected to 20 cycles of amplification using two oligonucleotides specific for human actin (sequences 10-30 and 358-338).

Invasion into Embryonic Chick Heart Fragments-- Invasiveness into embryonic chick heart fragments was assayed following the method of Mareel et al. (12), with minor modifications. Briefly, heart fragments of 9-day-old chick embryos were dissected and incubated for 2 days in an orbital shaker set at 100 rpm at 37 °C in an atmosphere of 95% air, 5% CO2. Precultured heart fragments with a diameter of 0.5 mm were selected under a stereomicroscope. Cell suspensions were brought into contact with precultured heart fragments placed on semisolid agar medium. After incubation for 8 h at 37 °C to allow the cells to attach to the external fibroblastic layer of precultured heart fragments, individual confrontations were transferred to 5-ml Erlenmeyer flasks for further incubation in an orbital shaker (120 rpm) at 37 °C. After 1-4 days, confrontations were fixed and processed for transmission electron microscopy.

Semithin and Ultrathin Sections-- Cell confrontations were fixed with 2.5% glutaraldehyde, treated with 2% OsO4, and dehydrated washing in ascending series of graded ethanol (once in 50%, once in 70%, twice in 95%, and five times in 100% ethanol). Cells were then embedded in Spur resin (TAAB Laboratories, Aldermaston, United Kingdom). For light microscopy, semithin sections were cut with a LKB ultramicrotome and stained with toluidine blue. For transmission electron microscopy, ultrathin sections of 500-800 Å were placed on uncoated 300-mesh copper grids prior to staining with uranyl acetate (5% in absolute ethanol) and lead citrate and viewed at original magnifications from 4500 to 15,000 in a Hitachi H700 transmission electron microscope operated at 75 kV.

Xenografting-- Cells were trypsinized, washed, and resuspended in sterile phosphate-buffered saline solution at a concentration of 1 × 107 cells/ml. Tumorigenic ability was determined by inoculation of 1 × 106 cells in the subcutis of 5-week-old athymic female nude mice (Criffa, Barcelona, Spain). Viability of the uninjected cells, determined at the end of the process, always exceeded 95%. Tumors were followed externally. After 6 weeks, mice were sacrificed by cervical dislocation, and the site of the injection, as well as the liver, lungs, and lymph nodes, was examined. Tissues were fixed, embedded in paraffin, and analyzed under the microscope after hematoxylin-eosin staining.

Plasminogen Activator Activity-- Activity of plasminogen activators was determined by zymography. Conditioned medium from cells cultured for 24 h in the absence of fetal bovine serum (10%) was centrifuged at 13,000 × g for 15 min at 4 °C. Sample volumes were adjusted on the basis of protein concentration in the corresponding cell lysate, and proteins were separated by SDS-polyacrylamide gel electrophoresis in plasminogen and gelatin-containing gels, as described elsewhere (13). Protease activity was revealed by incubating the gels in 2.5% Triton X-100 for 1 h followed by incubation in 0.1 M glycine, pH 8.3, overnight at 37 °C. After fixing of proteins with methanol/acetic acid/water (30:10:60), gels were stained with 0.1% Amido Black and destained.

Other Methods-- To determine cell dissociation, the assay developed by Nagafuchi et al. (14) was used, with the modifications described previously (7). The extent of dissociation was represented by the index NTC/NTE, where NTC and NTE are the total particle number after treatment of cells with trypsin in the presence or absence of calcium, respectively. E-cadherin association to the cytoskeleton was determined analyzing by Western blot the presence of this protein in Triton-soluble and -insoluble fractions, prepared as described by Nelson and co-workers (15). Autoradiograms were quantified by scanning densitometry (Hoefer GS-300 Scanning Densitometer).

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Inhibitors of cPK-C Isoforms Block Scattering of M6 Cells Induced by PMA-- We have previously reported that PMA induces remarkable morphological changes in M6 cultures (4). After addition of this phorbol ester, M6 colonies scatter, and cells acquire a fibroblastic aspect. These effects are observed shortly after the addition of this phorbol ester at concentrations as low as 20 nM. Among the properties altered by PMA, cells lose homotypic adhesion, and concomitantly, E-cadherin is inactivated (5). These effects of PMA are prevented if the cells are simultaneously incubated with the PK-C inhibitor Gf (2 µM) (7). Gf is a bisindolymaleimide derivative that selectively inhibits PK-C isoforms; in vitro, it shows a ranked order of potency of cPK-Cs > nPK-Cs > atypical PK-Cs (16). As shown in Table I, the action of PMA on cell scattering, homotypic adhesion, or E-cadherin inactivation was blocked by similar concentrations of Gf. The minimal dose of Gf required to prevent morphological scattering induced by PMA was 0.5 µM; in the presence of this concentration, cells incubated with PMA presented a similar phenotype to untreated controls (not shown). The effect on this inhibitor on the loss of homotypic adhesion was also estimated; IC50 was obtained with approximately 70 nM Gf, a concentration lower than the IC50 for nPK-Cs delta  or epsilon  activities (210 and 130 nM, respectively), determined by in vitro protein kinase assays. Concomitantly with the effect on homotypic aggregation, low doses of Gf (0.5 µM) PMA also blocks the decrease in E-cadherin-associated to cytoskeleton caused by PMA (Table I).

                              
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Table I
Inhibition by Gf and Go of the effects of PMA on M6 cells
M6 cells, grown in complete medium (DMEM plus 10% fetal bovine serum) for 5 days after seeding were incubated in the presence of PMA (100 nM) and inhibitors.

Recently, the preparation of a selective inhibitor of cPK-Cs with respect to nPK-C or atypical PK-C has been described (16). In vitro, this compound, Go, displays high activity against cPK-Cs alpha  and beta  (with IC50 values of 2.5 and 6.2 nM, respectively) whereas no effect was observed on nPK-Cs epsilon  or delta  or or atypical PK-Czeta even at micromolar concentrations (16). In M6 cells, Go blocked the action of PMA on the three parameters studied (Table I), although the doses required were considerably higher than those of Gf. This difference could be explained by a lower ability to penetrate the cells; although the activity of Go and Gf have been studied in vitro, their relative potency in whole cells has not been characterized. However, even the highest concentration of Go used in our assays did not inhibit nPK-Cepsilon activity in vitro (data not shown).

Expression of an Activated Form of cPK-Calpha Causes Scattering of M6 Cells-- The use of these inhibitors suggests that a cPK-C is responsible for triggering the process of scattering and loss of cell-to-cell adhesion. We have previously described that the only cPK-C expressed in HT-29 M6 cells is cPK-Calpha (7). The presence of cPK-Calpha , and not of cPK-Cs beta  or gamma , was demonstrated both by Western blot with specific monoclonal antibodies and by reverse transcription-PCR analysis, using two oligos corresponding to consensus sequences of these three isoforms. Therefore, to prove the role of cPK-Calpha in cell scattering, we have overexpressed this isoform in M6 cells.

A mutation in the pseudosubstrate domain of cPK-Calpha was performed in order to obtain a higher basal activity of this enzyme in the absence of external activators. This mutation consists of the replacement of Ala (residue 25) present in the central position of the pseudosubstrate by a charged Glu residue. This substitution, A25E, activates cPK-Calpha because it reduces the affinity of the autoinhibitory pseudosubstrate peptide for the catalytic site (17). Although considerably more active in the absence of effectors than the wild-type enzyme, this mutated cPK-Calpha , denominated cPK-Calpha (+), can be further activated by the addition of PMA and phospholipids (17). Four different cPK-Calpha (+) transfectant clones were selected and analyzed in detail; these four clones summarized the different phenotypes obtained in our experiments. One of the clones (A1) presented a phenotype identical to control M6 (Fig. 1) and a similar sensitivity to PMA (not shown). Clone A2 showed a phenotype slightly less compact than control cells; the two other clones (A3 and A4) presented the phenotype previously defined as "scattered" to different extents. A3 cells formed colonies composed of flattened cells, with a low number of cell-to-cell contacts, whereas the phenotype of A4 cells was almost identical to that displayed by M6 chronically treated with PMA. In parallel, a transfection with a control plasmid that did not contain insert was performed; all of the clones observed presented a morphology identical to HT-29 M6 cells (not shown), and two of these clones (C1 and C2) were isolated and used as negative controls in our studies. Addition of the PK-C inhibitor Gf to clone A2 or A3 induced the formation of colonies identical to control cells; reversion by Gf, although important, was not complete in M6 cells chronically treated with PMA or in A4 cells (Fig. 1). Because the mutant cPK-Calpha is not fully activated by the mutation and can be further stimulated by addition of PMA, we analyzed the sensitivity of the clones to the phorbol ester. Clones A2, A3, and A4 were sensitive to doses of PMA that did not induce scattering of any of the control clones; for instance, A2 cell colonies dispersed in response to 5-10 nM phorbol ester, a 4-fold lower dose than that required with control cells (Fig. 1, bottom row). In A4 and A3 cells, the addition of PMA induced the acquisition of an extremely stretched phenotype, characterized by long cellular extensions (Fig. 1, bottom row).


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Fig. 1.   Morphology of cPK-Calpha (+) transfectants. Cells were grown in complete medium for 6 days; when indicated, PMA (100 nM) was added at the time of seeding, and Gf (1 µM) was added during the last 2 days of incubation. In the experiment shown in the bottom row, cells were incubated with PMA (10 nM) for 4 h. Pictures were taken under a phase-contrast microscope at × 200 magnification, except in the case of A4 cells incubated with PMA, which was taken at × 350. No morphological differences respect to the control were observed in C1 and C2 clones, in cells incubated with Gf, or with PMA and Gf added simultaneously (results not shown).

The expression of the mutant cPK-Calpha was analyzed by RT-PCR; only clones A2, A3, and A4, but not A1 or the controls, showed expression of the mutant cPK-Calpha (+) transcript (Fig. 2). Although the amplification was only semiquantitative, the rank of expression could be established as A4 > A3 > A2, demonstrating a correlation between expression of cPK-Calpha (+) and cell scattering. Taking advantage of the new restriction site created with the mutation, the relative amounts of the wild-type and mutant forms were determined; only A4 presented levels of cPK-Calpha (+) transcript higher than those of the wild-type form.


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Fig. 2.   PCR analysis of the transfectants. cDNA was obtained from the different clones and analyzed by PCR using oligos A1 and A2 (PCR A), B1 and B2 (PCR B), or actin-specific (Actin) as described under "Experimental Procedures." A scheme of the construction with the position of the oligos is shown. The product of PCR A was transferred to a nylon membrane and analyzed with oligo B1 labeled with 32P; the product of PCR B was digested with SacI and analyzed with oligo A2. The figure shows representative results of PCRs A and B (autoradiograms) and of the amplification of the actin fragment used as control (ethidium bromide staining). The three amplifications were performed at low number of cycles (20-25 cycles) to avoid saturation of the reaction. In PCR B, a control of digestion of the plasmid with SacI was included; although it is not shown, the bottom fragment was not observed in M6 or A1 cells.

Because activation of cPK-Calpha leads, in many cases, to down-regulation of this enzyme, determinations of total levels of this protein were not informative. However, the levels of other PMA-responsive PK-C isoforms were analyzed in transfectant clones by Western blot, to verify whether the higher sensitivity to PMA was due to increased synthesis of PK-Cs other than PK-Calpha . We did not detect any increase in the levels or change in localization of the nPK-Cs epsilon  and eta  in the cPK-Calpha (+)-transfectant clones, nor was cPK-Cbeta , absent in M6, induced in these cells (data not shown).

In order to measure the endogenous activity of cPK-Calpha , we employed an indirect assay: the analysis of expression of a gene containing phorbol ester-response elements. This method has been used previously by other authors with this goal (17, 18). The gene used in these studies was the urokinase-type plasminogen activator (uPA); the promoter of this gene contains several phorbol-ester responsive elements, and its expression is greatly stimulated by activation of several PK-C isoforms (19, 20). Therefore, in order to analyze endogenous cPK-Calpha activity in the M6 cells and in the clones transfected with cPK-Calpha (+), expression of uPA was determined by zymography. This method estimates the activity of uPA, a parameter that correlates with its expression. M6 or control clones (C1 and C2) presented very low levels of plasminogen activators. High levels of a protease activity with a molecular mass of 54 kDa, corresponding to uPA, were observed in the supernatant of these cells chronically treated with PMA (Fig. 3). A1 or A2 cells did not show significant levels of uPA; the presence of this activity was detected in A3 cells and, to a much greater extent, in A4 cells, where it was a level similar to that seen in M6 cells incubated with PMA (Fig. 3). These data allow us to rank the cells in terms of cPK-Calpha activity as A4 >>  A3 > A2 = controls.


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Fig. 3.   uPA activity in cPK-Calpha (+) transfectants. Media conditioned for 24 h from the different cell clones or M6 cells were treated when indicated with PMA because seeding were subjected to SDS-polyacrylamide gel electrophoresis in plasminogen and gelatin-containing gels, and protease activity was measured as described under "Experimental Procedures." In addition of a protease of 55 kDa, corresponding to uPA, a minor band of lower molecular mass (probably consequence of the processing of uPA) was also detected in A4 cells.

Expression of cPK-Calpha (+) Modifies Other Cellular Properties Associated with Scattering: Transfectants Show Higher Mobility, Lower Cell-to-Cell Adhesion, and Decreased Levels of E-cadherin Compared to Controls-- Scattering is associated with an increase in the mobility of cells. To determine whether expression of cPK-Calpha enhanced mobility, a wound was inflicted in the epithelial monolayer, and the ability of the cells to fill the gap was determined. After 24 h, control M6 cells had not moved into the denuded area (Fig. 4). Incubation with 100 nM PMA but not with 10 nM PMA induced the cells to significantly fill the free space. Expression of cPK-Calpha (+) correlated with the extent of the healing; A4 cells showed higher mobility than A3, and these in turn showed higher mobility than A2. A2 and A3 were sensitive to low doses of PMA (10 nM) that did not induce the movement of M6 cells (Fig. 4).


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Fig. 4.   cPK-Calpha (+) expression increases mobility of M6 cells. Cell mobility was determined using an assay of wound healing. Confluent cell clones were wounded with two cross-shaped scratches; PMA was added when indicated, and the ability of the cells to refill the gaps was examined after 24 h. The × 50 magnification micrographs show the results of one representative experiment of three performed.

Besides the modification of other cellular properties, scattering requires the loss of homotypic aggregation. In M6 cells, the decrease in this parameter is concomitant with the inactivation of E-cadherin (5). Therefore, as expected, clones A3 and A4 showed dissociation indexes much higher than M6 cells or the control clones analyzed (A1, C1, and C2) (Table II). These indexes were very similar to that calculated for HT-29 M6 cells incubated in the presence of PMA. Clone A2 presented an index not significantly different from control cells, but these cells were much more sensitive to the addition of PMA (Table II). Very similar results were obtained when the function of E-cadherin was studied. Inactivation of this protein (loss of the association to the cytoskeleton) is reflected by an increase in its solubility in Triton X-100 (15). In untreated M6 cells and in the control clones, approximately 35% of total E-cadherin was insoluble in Triton X-100 (Fig. 5). However, in long-term PMA-treated cells or in A3 and A4 cells, the level of E-cadherin present in this fraction, which corresponds to the functional E-cadherin, was barely detectable (Fig. 5). The total levels of this protein were also lower, especially in M6 cells treated with the phorbol ester and in A4 cells (not shown).

                              
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Table II
Expression of cPK-Calpha (+) decreases homotypic aggregation
NTC/NTE index was determined from cells 50% confluent as described. When indicated, cell medium was supplemented with PMA for 24 h. The mean ± S.D. of three independent experiments is shown.


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Fig. 5.   cPK-Calpha (+) transfectants present lower levels of E-cadherin associated to the cytoskeleton. Fractions soluble in Triton X-100 or only in SDS were prepared from different clones or M6 cells treated, when indicated, with PMA (100 nM) since seeding. Equivalent amounts of both fractions were analyzed by Western blot with an anti-E-cadherin monoclonal antibody; only a band of 120 kDa, corresponding to this protein, was detected. The figure shows a representative experiment of three performed.

Cell Growth Is Retarded by the Activation of cPK-Calpha (+)-- Another of the features that appear in M6 cells treated with PMA is a retardation in cell growth (4). As observed in Fig. 6, this effect of the phorbol ester requires concentrations significantly higher than those required to induce scattering. For instance, addition of 20 nM PMA, the lowest concentration that induced scattering, did not have any effect on the proliferation of HT-29 M6 cells. Growth of the different clones was measured either in the absence or in the presence of 20 and 100 nM PMA. Control cells (C1 and C2) grew with times of duplication similar to those of M6 cells and showed similar sensitivity to PMA. Minor differences were found between control and A2 cells either in the absence of PMA or in the presence of the two different concentrations of this compound. As shown in Fig. 6, growth of A3 cells in absence of phorbol ester was similar to control. However, these cells were much more sensitive to the phorbol ester; 20 nM PMA inhibited proliferation of A3 by 50% versus less than 5% to M6 or C1 cells. The highest cPK-Calpha (+)-expressing clone, A4, proliferated at a lower rate and responded to low doses of PMA with a total block in proliferation (Fig. 6).


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Fig. 6.   Expression of cPK-Calpha (+) retards cell growth. Proliferation of cells was measured during the exponential phase of growth directly by cell counting in the absence or in the presence of the indicated concentrations of PMA. The figure shows the mean of three different of experiments, which did not differ by more than 5%. The number in parentheses corresponds to the duplication time of the cells in the different conditions studied.

Expression of cPK-Calpha (+) Stimulates Invasion of Chick Embryo Heart Fragments-- The results obtained so far have indicated that activation of cPK-Calpha induced the acquisition of a fibroblastic phenotype, with low functionality of E-cadherin and enhanced secretion of uPA, two alterations that have been related to a more invasive phenotype of epithelial cells (21, 22). Therefore, we analyzed whether transfection of cPK-Calpha (+) had indeed enhanced the invasive properties of M6 cells, using the assay of invasion of embryonic chick heart fragments. Single-cell suspensions were added to the precultured heart fragments, and samples were processed after different periods of incubation. Histological analysis of cultures revealed striking differences between control cells and cells expressing cPK-Calpha (+). M6 cells, or clone C1 cells, which behaved identically, gave rise to compact colonies that progressively occupied the peripheral parts of the heart fragment without invading the myocardial tissue (Fig. 7, G and H). Only after long times of confrontation (6 days) were small clusters of cells seen inside the heart tissue (not shown). In contrast, A4 cells were highly invasive; spindle-shaped A4 cells could be observed penetrating the heart tissue as early as 8 h after the initiation of the assay (Fig. 7, A and B). After 24 h, A4 cells had invaded the heart fragment and replaced extensive areas of this tissue (Fig. 7, C and D). These cells did not survive well in the co-culture; at longer periods of time (3-6 days), most of the A4 cells showed symptoms of cell damage, such as clumping of chromatin and shrinkage of cytoplasm (not shown). The invasive capability of A3 cells was also determined; these cells invaded more slowly than A4 cells, although an extensive colonization of the fragment was observed by 24 h (Fig. 7, E and F).


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Fig. 7.   Expression of cPK-Calpha (+) stimulates invasion of chick heart fragments by M6 cells. The figure shows light micrographs of semithin sections from confrontations between precultured embryo heart fragments (marked H) and clones A4 (A-D), A3 (E and F), or M6 cells (G and H) fixed after 8 h (A and B), 24 h (C-F), or 3 days (G and H). T, M6 cells; arrowheads in A and B: A4 cells; asterisk in H: fibroblast showing signs of cell damage. Magnifications: A, C, E, and G: × 150; B, D, F, and H, × 600.

Ultrastructural sections of cultures revealed an active progression of A4 cells through the myocardial tissue, with a marked hypertrophy of their rough endoplasmic reticulum and numerous cell extensions (Fig. 8, A and B). In contrast, M6 cells were localized in the periphery of the fragment and showed a polarized morphology with many mucous granules near the apical membrane and a microvilli (Fig. 8C). M6 cells lying in close apposition to the heart tissue showed a flattened morphology with no cytoplasmic processes.


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Fig. 8.   Alterations in cell ultrastructure in cells expressing cPK-Calpha (+) after invasion of EHFs. The figure shows representative transmission electron micrographs from some of the samples analyzed in Fig. 7. A, two A4 cells (T) actively progressing inside the heart tissue. H, myoblasts (× 4500). B, detail from A showing an A4 cell (T) with a marked hypertrophy of rough endoplasmic reticulum cisternae (asterisk). Myofilaments (M) can be distinguished in neighboring myoblasts (H). Arrowheads, fuzzy material; arrow, Z-line (× 15,000). C, M6 cells did not invade the heart tissue (H) after 3 days of culture. Arrow, mucosecretory granules; arrowheads, microvilli (× 5000).

Growth and Characteristics of Tumors Originated by M6 Cells Are Affected by Expression of cPK-Calpha (+)-- We have demonstrated that activation of cPK-Calpha induces two alterations that are presumably antagonistic in the process of tumorigenesis: enhanced cell invasion and retarded proliferation. In order to analyze the tumorigenic ability of cells expressing cPK-Calpha (+), the different clones and controls were xenografted into the subcutis of nude mice. Expression of cPK-Calpha (+) was associated to a decrease in the size of the tumors originated by M6 cells (Fig. 9). In contrast to control C1 or A1 cells, which originated large tumors after eight weeks, A4 cells did not form macroscopic tumors at this late time (Table III). Absence of tumors was confirmed by microscopic analysis of the area of implantation after hematoxylin-eosin staining. No evidence of tumor cells was observed when the analysis was performed at longer (10 weeks) or shorter (10 days) times after grafting. Implantation of these cells in the spleen gave similar results (not shown).


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Fig. 9.   Histological analysis of tumors. The figure shows representative tumors obtained from different xenografts stained with hematoxylin-eosin. Tumors A and B were obtained from animals injected with C1 cells, C and D from animals injected with with A2 cells, and E-G from animals injected with A3 cells. Details are shown to the right of the indicated tumors; they are labeled as the letter corresponding to the tumor followed by a number. Magnifications: A-G × 12.5; G1, × 40;, B1, D1, E1, and E2, × 100.

                              
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Table III
Expression of cPK-Calpha decreases tumorigenicity of M6 cells
Mice were injected subcutaneously in the flank with 106 cells; presence of tumors was analysed as described 8 weeks later. ND, tumor not detected.

The clones A2 and A3, with a lower expression of cPK-Calpha (+), showed intermediate results. In both cases, xenografting gave rise to the formation of tumors, which were smaller than in the case of control cells. This decrease in tumor size correlated with the expression of cPK-Calpha (+) (Table III).

The morphology of these A3-derived tumors, and in to a lesser extent A2 tumors, also showed differences with respect to the controls. Control tumors presented well-defined limits without any sign of infiltration of neighboring tissues. These tumors grew expansively and pressed the skin without invading the dermis (Fig. 9, A and B). In some cases, probably due to their larger size, tumors showed areas of necrosis (Fig. 9A). In contrast, all of the A3 tumors examined were characterized by an irregular contour (Fig. 9, E-G). Areas in which the tumor cells have invaded the surrounding tissues were easily detected and comprised most of the periphery of the tumor. Single cells infiltrating the neighboring tissue (Fig. 9, E1 and G1) and muscle fibers surrounded by tumor cells (Fig. 9E2) were two characteristics of these A3 tumors. This infiltrative phenotype was also observed to a lesser extent in the A2 tumors but only in a small part of the periphery of 1 of the 16 controls analyzed. A noteworthy feature found in the A2 tumors was their ability to invade the dermis of the mice. As shown in Fig. 9, control tumors grew expansively without altering the skin structure (Fig. 9B1); A2 tumors infiltrate dermis and, in some cases, epidermis as well (Fig. 9, C, D, and D1). This invasion of the skin was not found in A3 tumors, probably due to their small size.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

Several conclusions can be drawn from our results. First, it is evident that activation of cPK-Calpha in M6 cells induces the acquisition of a fibroblastic phenotype, denominated scattered. Cell scattering is a consequence of co-ordinated changes that decrease cell-to-cell interactions, increase the adhesion to the extracellular matrix, and stimulate the migration of the cells. The basis of these phenotypic changes is alteration in the function of several proteins; for instance, inactivation of E-cadherin and increased uPA activity have been related to loss of homotypic adhesion and enhanced mobility, respectively (22, 23). We show here that expression of an activated form of cPK-Calpha is sufficient to induce the acquisition of the scattered phenotype and to alterate the functionality of both E-cadherin and uPA.

Expression of cPK-Calpha (+) also increased the ability of M6 cells to invade chick heart embryo fragments. Similar data have been obtained with other epithelial cells: overexpression of cPK-Calpha in breast cells induced the acquisition of a fibroblastic phenotype and enhanced the proliferation rate and the tumorigenic and metastatic capabilities (24). However, differently to the results in breast cells, activation of cPK-Calpha in intestinal M6 cells decreased proliferation and tumorigenicity. Similar data were obtained by Weinstein and co-workers (25) in HT-29 cells after transfection of another cPK-C, cPK-Cbeta . Based on these data and the localization of this enzyme along the crypt-villus axis, a role for cPK-Calpha in the negative control of cell growth in intestinal epithelium has been suggested (6, 26, 27). Association of cPK-Calpha to the membrane has been detected by immunofluorescence in the mid-crypt region, where cells cease proliferation; this alteration has been suggested to reflect an increased activity of this enzyme, although this conclusion has not been definitively proved.

It is remarkable that activation of the same enzyme, cPK-Calpha , exerts two different actions on M6 cells: it increases invasion and retards cell growth, two effects that seem antagonistic in tumor development. The results presented in this report demonstrate that although both effects are triggered by activation of the same PK-C isoform, they are differentially sensitive to the extent of this activation. A moderate stimulation of the enzyme (for instance, as present in A3 cells) causes an increase in the ability of these cells to infiltrate surrounding tissues and a decrease in the growth of the tumor; on the other hand, when the activation of the enzyme is extensive (A4 cells), the growth inhibitory effect is predominant, and the tumor is unable to develop. These effects mimic what happens in M6 cells treated with PMA: cell scattering and loss of cell-to-cell contacts require low doses of this phorbol ester, whereas higher concentrations are required to affect cell growth. Similar results were obtained with other intestinal cell lines with respect to the sensitivity of these two parameters to PMA. In all of these cell lines (WiDr, Caco-2, HRT18, SW620, and SWCo15), maximal inhibition of growth (26-52%, depending on the cell lines) required concentrations of the phorbol ester about 100 nM, whereas scattering was observed at much lower doses.2

Several lines of evidence indicate that alterations in PK-C may be involved in malignant transformation of colon in humans. Colon adenocarcinomas show a reduction in PK-C activity compared with normal adjacent mucosa, indicating down-regulation of this enzyme in the tumors (28-30). Experimental models of colon carcinogenesis have provided evidence of changes in PK-C both in premalignant and malignant epithelial cells; these changes include translocation of PK-C and subsequent down-regulation of this enzyme (31). Alterations in the content of specific PK-C isozymes have been also described in colon tumors with respect to normal tissue either in human samples or in experimental animal models (32, 33); however, different laboratories have reported contrary results of these analyses (32, 34, 35). Therefore, at the present, it is not clear whether the initial activation or the later down-regulation are related to colon tumorigenesis, neither the role of specific isoforms in this process.

The results presented here can help to explain the role of cPK-Calpha in colon carcinogenesis. This enzyme might exert a dual action: 1) a moderate activation of cPK-Calpha would take place at the first stages of carcinogenesis, being involved in the loss of function of E-cadherin, and 2) a later down-regulation and inactivation of cPK-Calpha would result in an uncontrolled growth of the primary tumor or the metastasis. According to this model, studies performed with cell lines or tumors that have progressed beyond the first stage would not show any positive effect of PK-Calpha activation in tumor development. Experiments directed to the validation of this model using transgenic animals are currently in progress in our laboratory.

While this work was being written, the article by Rosson et al. (36) came to our notice; it describes the involvement of cPK-Calpha in the scattering of LLC-PK1 cells induced by PMA. Their conclusion was obtained by expression of the wild-type form and a negative mutant of cPK-Calpha in these cells. Although overexpression of a dominant negative mutant does not demonstrate involvement of cPK-Calpha in cell scattering induced by PMA, the results presented by these authors are totally consistent with those described in this work.

    ACKNOWLEDGEMENTS

We thank the members of the Unitat de Biologia Cellular i Molecular of the Institut Municipal d'Investigació Mèdica for their comments and help, especially Drs. C. Harvey and E. Sancho. The technical help of M. C. Torns and M. Garrido is greatly appreciated.

    FOOTNOTES

* This research was supported by Grants SAF94-1008 and SAF97-0080 from the Fundación Ramón Areces and Comisión Interministerial de Ciencia y Tecnología (to A. G. d. H.) and by Grant GRQ 93-9301 from Comissió Interdepartamental de Recerca i Tecnologia to the Unitat de Biologia Cellular i Molecular.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Predoctoral fellowship from CIRIT (Generalitat de Catalunya).

§ Supported by funds from La Marató de TV3.

Predoctoral fellowship from Ministerio de Educación.

parallel Supported by Fundación Ramón Areces.

** To whom correspondence should be addressed. Tel: 3-221-1009; FAX: 3-221-3237; E-mail: agarcia{at}imim.es.

1 The abbreviations used are: PK-C, protein kinase C; Gf, PK-C inhibitor GF109203X; Go, PK-C inhibitor Go 6976; cPK-C, conventional PK-C; nPK-C, atypical PK-C; PMA, phorbol 12-myristate 13-acetate; uPA, urokinase-type plasminogen activator.

2 E. Batlle and A. Garcia de Herreros, unpublished observations.

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Abstract
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Results
Discussion
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