From the Unitat de Biologia Cel.lular i Molecular, Institut Municipal d'Investigació Mèdica, Calle Dr. Aiguader 80, 08003 Barcelona, Spain
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ABSTRACT |
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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 C (cPK-C
). 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-C
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-C
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-C
controls both cell-to-cell adhesion and proliferation of intestinal
cells.
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INTRODUCTION |
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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 106
methotrexate) in vivo.
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EXPERIMENTAL PROCEDURES |
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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. [
-32P]ATP
was purchased from Amersham Pharmacia Biotech. Monoclonal antibodies
against cPK-C
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
(106 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-C and Transfection to M6 Cells--
Human
cPK-C
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-C
). 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-C
, denominated cPK-C
(+), 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-C. 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-C
) labeled with [
-32P]ATP
by T4 polynucleotide kinase. In order to compare the relative expression of the mutated and wild-type forms of cPK-C
, 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-C
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).
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RESULTS |
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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 or
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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Expression of an Activated Form of cPK-C 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-C
(7). The presence of cPK-C
,
and not of cPK-Cs
or
, 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-C
in cell
scattering, we have overexpressed this isoform in M6 cells.
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Expression of cPK-C(+) 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-C
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-C
(+) 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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Cell Growth Is Retarded by the Activation of
cPK-C(+)--
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-C
(+)-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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Expression of cPK-C(+) Stimulates Invasion of Chick Embryo Heart
Fragments--
The results obtained so far have indicated that
activation of cPK-C
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-C
(+) 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-C
(+). 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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Growth and Characteristics of Tumors Originated by M6 Cells Are
Affected by Expression of cPK-C(+)--
We have demonstrated that
activation of cPK-C
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-C
(+), the different clones and controls were
xenografted into the subcutis of nude mice. Expression of cPK-C
(+)
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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DISCUSSION |
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Several conclusions can be drawn from our results. First, it is
evident that activation of cPK-C 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-C
is sufficient to induce the acquisition of the
scattered phenotype and to alterate the functionality of both
E-cadherin and uPA.
Expression of cPK-C(+) 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-C
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-C
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-C
.
Based on these data and the localization of this enzyme along the
crypt-villus axis, a role for cPK-C
in the negative control of cell
growth in intestinal epithelium has been suggested (6, 26, 27).
Association of cPK-C
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-C, 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-C in
colon carcinogenesis. This enzyme might exert a dual action: 1) a
moderate activation of cPK-C
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-C
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-C
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-C 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-C
in these cells. Although overexpression of
a dominant negative mutant does not demonstrate involvement of cPK-C
in cell scattering induced by PMA, the results presented by these
authors are totally consistent with those described in this work.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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* 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.
Predoctoral fellowship from CIRIT (Generalitat de Catalunya).
§ Supported by funds from La Marató de TV3.
¶ Predoctoral fellowship from Ministerio de Educación.
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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REFERENCES |
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