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Tumor Biology |
Laboratori d’Investigació Gastrointestinal, Institut de Recerca, Hospital de Sant Pau, 08025 Barcelona [S. G., I. C., L. F., R. M.]; Departament de Bioquímica i Biologia Molecular, Facultat de Químiques, Universitat de Barcelona 08028 [A. M.]; and Laboratori de Recerca Traslacional, Institut Català d’Oncologia, 08907 Barcelona [G. C.], Spain
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Several lines of evidence suggest that the malignant potential of tumor cells may be influenced not only by the presence or absence of ras mutations, but by its molecular nature (7, 8, 9) : (a) a reduced transforming capacity of the codon 13 mutation as compared with codon 12 mutation in in vitro and in vivo experimental systems (10, 11, 12) has been shown; (b) in human colorectal tumors, K-ras codon 12 mutations are much more frequent in carcinomas than in adenomas (7) and in metastatic than in nonmetastatic lesions (8 , 9) ; and (c) K-ras codon 13 mutations are exclusively present in tumors that show no local invasion, and they are never present in metastatic lesions (8) . These clinical and experimental findings suggest that tumor clones carrying K-ras codon 13 mutations are less aggressive than those with codon 12 mutations. In addition, colorectal tumors carrying K-ras codon 12 mutations show lower levels of apoptosis than tumors lacking this mutation (13) . This argues for a role of K-ras because K-ras mutation occurs at the adenoma stage, and resistance to apoptosis, in this tumor type, appears to be acquired during the adenoma-to-carcinoma transition (14 , 15) .
Here, we tested whether K-ras codon 12 mutation would confer upon the cell a more oncogenic phenotype than a K-ras codon 13 mutation or than the overexpression of the K-ras proto-oncogene. To this end, we transfected NIH3T3 cells with a plasmid containing K-ras with point mutations at codon 12 (K12) or at codon 13 (K13), or containing the K-ras proto-oncogene Kwt-oe; selected stable transfectants; and evaluated the possible changes in different functions contributing to transformation. In addition, we studied the alteration in expression and/or activation of proteins that participate in signal transduction downstream of Ras (JNK, MAPK, AKT, and RhoA), and of proteins involved in the regulation of apoptosis (bax and bcl-2), cell-cell (E-cadherin and ß-catenin) and cell-substrate (FAK) interactions. We observed that all transformants (K12, K13, and Kwt-oe) shared several characteristics of the malignant phenotype: (a) increased proliferation rates; (b) diminished growth dependence on serum; and (c) anchorage-independent growth (induced by plating on poly-heme). In contrast, K-ras codon 13 transformants showed confluency-dependent apoptosis. These changes were associated with JNK1 activation and some degree of E-cadherin, ß-catenin, and FAK overexpression. In contrast, K-ras 12 transformants did not enter apoptosis, were the only ones to show spontaneous anchorage-independent growth, and showed higher cell densities than K13 or Kwt-oe transformants, which already grew at higher densities than control cells. The changes in K12 transformants were associated with significantly higher AKT activation, bcl-2 overexpression, and RhoA underexpression, and even higher levels of E-cadherin, ß-catenin, and FAK overexpression.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A:T mutation at the first position of codon 12 (pMLK12), a
G:CA:T mutation at the second position of codon 13 (pMLK13), or the
wt K-ras gene sequence (pMLKwt), and a control
plasmid containing only vector and no K-ras coding sequences
(pMLneo). pMLK12, pMLK13, and pMLKwt plasmids were a gift of Dr. Manuel
Perucho of the Burham Institute at La Jolla, CA. All these plasmids are
derived from pBR and contain a neo-resistance selectable gene under the
thymidine kinase gene promoter and an ampicillin resistance
gene. The K-ras minigene contains all four coding exons (4B
is the fourth exon) separated by intronic regions, under the promoter
of K-ras, and a polyadenilation signal. Dulbecco’s
MEM (25 mM HEPES), FBS, glutamine, fungizone,
penicillin/streptomicin, ampicillin, acrylamide, and bis-acrylamide
were obtained from Life Technologies, Inc. Hoescht and poly-heme
(2-hydroxy-metacrylate), Tris-HCl, Triton X-100, SDS, glycerol,
benzamidine, phenylmethylsulfonyl fluoride, leupeptine, sucrose, Na
Acetate, NaCl EDTA, EGTA, bromphenol blue, DTT, glycine, Na
ß-glycerophosphate, Na fluoride, and PPi were
obtained from Sigma. Gentamicine sulfate was obtained from
BioWhittaker. The In Situ Cell Death Detection Kit,
peroxidase (TUNEL method) was from Roche, trypan blue was
obtained from Serva. APS, TEMED, and 2-ß-mercaptoethanol were
from Fluka. Na ortovanadate was from Panreac. Bactotryptone, yeast
extract, and bacto-agar were from Difco. Molecular weight markers were
from Bio-Rad.
Plasmid Growth and Production of the K-ras
Transformants.
The different plasmids were grown and purified, after bacterial
transformation using the One Shot competent Escherichia coli
Kit (Invitrogen). Bacterial colonies were selected in plates containing
10-4g/ml ampicillin in Luria-Bertani medium
(10-2g/ml bactotryptone, 5 x 10-3g/ml yeast extract, 15 x 10-4g/ml bacto-agar,
10-2g/ml NaCl), picked up individually and grown
in Luria-Bertani medium at 37°C, shaking until they reached an
absorbance of 1.0 at 600 nm. Plasmids were then extracted with the
QIAprep Spin miniprep Kit (Qiagen) and used to transfect NIH3T3 cells.
3T3 cells were routinely maintained in 10% FBS in DMEM (25
mM HEPES), supplemented with 1% glutamine (Life
Technologies, Inc.), 0.5 µg/ml fungizone (Life Technologies, Inc.),
100 units/ml penicillin/streptomicin (Life Technologies, Inc.), and 5
µg/ml gentamicine sulfate (BioWhittaker) and incubated in a
humidified atmosphere containing 5% CO2 at
37°C. 3T3 cells, grown to 60% confluency in 60-mm plates, were
transfected with the pMLK12, pMLK13, pMLKwt, and pMLneo plasmids using
the calcium phosphate method. After overnight incubation, cells
were grown for 24 h in neomycin-free medium, and then transfected
clones were selected under 800 µg/ml G418 (Geneticin; Life
Technologies, Inc.) for 10–14 days.
Characterization of the Transformants.
A minimum of five different clones for each transfected construct, K12
(expressing the transfected plasmid pMLK12), K13 (expressing pMLK13),
Kwt-oe (overexpressing pMLKwt) and 3T3-neo (expressing pMLneo), and the
3T3wt (wt NIH3T3); were randomly identified,
coded, and analyzed for the presence of the transfected plasmid and
expression of K-Ras protein. Of these, we selected three clones per
construct in which expression of the foreign gene was confirmed for
further functional and molecular characterization. Presence of the
respective transfected constructs was evaluated by extracting DNA for
each of the selected clones and detecting the specific K-ras
point mutation by single-strand conformational polymorphism as
described (16)
. In addition, we used the PC-Image analysis
software (Foster Findlay Associates, Ltd.), to densitometrically
measure the levels of expression of the K-Ras protein in the three
selected clones per transfectant type after Western blot in whole-cell
lysates. Results were then expressed relative to the K-Ras level in
3T3wt cells. One-way ANOVA was performed to evaluate the possible
statistical significance of the differences in K-Ras expression among
groups (transfectant type) using the Statistical Package for Social
Sciences version 6.0 statistical package.
Morphological and Functional Analyses.
The three different clones for each transfected construct were analyzed
for morphological appearance (size, refringency, and presence of
filopodia, lamellopodia, and multiple nuclei) under a phase-contrast
microscope. Those clones were also functionally analyzed for several
characteristics: (a) proliferative capacity, by measuring
their replication time. Cells (5 x 104) were plated, in triplicate, and the number
of viable cells (stained with trypan blue) measured in a Neubauer
chamber at 0, 24, 48, 72, and 96 h; (b) changes in cell
growth patterns once confluency was reached. If cell death was observed
first, a time course of this effect was performed. To study a possible
apoptotic induction at 0, 8, 24, and 48 h after reaching
confluency, cells were fixed in 3.7% p-formaldehyde in PBS (pH 7.4)
for 10 min at -20°C. After rinsing three times with PBS, cells were
permeabilized, incubating with 0.5% Triton X-100 in PBS (pH 7.4) for 5
min at room temperature, and then rinsed again twice in PBS. Finally,
cells were stained with Hoescht (1:10,000 in PBS) for 10–30 min,
rinsed with water, suspended in PBS, mounted on a slide, and observed
on a fluorescent microscope at 334 nm absorption and 365 nm emission.
Apoptotic induction was also studied, measuring the formation of DNA
strand breaks by TUNEL assay and following the recommendations of the
manufacturer (Roche); (c) capacity to spontaneously form
spheroids (tridimensional cell aggregates) either before or after
reaching confluency, provided that cells were maintained in culture for
1 week with fresh media; (d) changes in colony size and cell
density within the colonies, under phase contrast microscope;
(e) ability to grow in low (0.5% FBS) serum concentrations
for a week; and (f) capacity of forced anchorage-independent
growth (plating the cells on poly-heme, a reagent that avoids cell
anchorage to the plastic plates). To do so, 10-mm plates were covered
with a 4-ml solution of 10 mg/ml of poly-heme in 95% ethanol two
times, allowing them to dry under a sterile hood. Then,
105 cells were plated and their growth observed
for a 20-day period.
Molecular Analyses of the Transformants.
Western blots for the assessment of the expression of ß-actin, FAK,
ß-catenin, E-Cadherin, RhoA, GAPDH, bcl-2, and bax proteins and
activation of MAPK (Erk1 and Erk-2), JNK (JNK-1 and JNK-2) and
PKB/AKT and the processing of PARP was done in three different
clones for each transfected construct and for the wt NIH3T3 cells. To
study phosphorylated (activated forms) of some of the proteins, whole
cell lysates were prepared using a buffer containing 2 x 10-2 M Tris/Acetate (pH
7.5), 0.27 M sucrose, 10-3
M EDTA (pH 8.0), 10-3 M
EGTA (pH 8.0), 10-3 M Na
ortovanadate (pH 10.0), 10-2 M Na
ß-glycerophosphate, 5 x 10-2
M Na fluoride, 5 x 10-3 M PPi,
1% Triton X-100 en Tris/acetate-sucrose (210-2
M/0.27 M), 0.1% de 2-ß-mercaptoethanol,
10-3 M benzamidine, 2 x 10-4 M PMSF, 5 x 10-6g/ml leupeptine. The amount of
protein was quantitated by the Bradford method using the Bio-Rad
protein assay dye. Polyacrylamide gels were prepared with stacking
[2.7% acrylamide, 0.06% Bis-acrylamide, 0.08 M Tris-HCl (pH 6.8),
0.1% SDS, 0.1% APS, 0.07% TEMED] and separating [15% acrylamide,
0.003% bis-acrylamide, 0.375 M Tris-HCl (pH 8.8),
0.1%SDS, 0.1% APS, 0.07% de TEMED] gels. Samples were denatured at
100°C for 3 min, and after loading 75 µg of total protein, diluted
with x3 loading buffer [0.15 M Tris-HCl (pH 6.8), 6% SDS, 0.15%
bromphenol blue, 30% glycerol, 0.3 M DTT],
electrophoresis were run at 30–40 mAmp in Laemmli buffer [25 x 10-3 M Tris, 0.25 [scap]m
glycine (pH 8.3), 0.1% SDS] with molecular weight markers.
After the electrophoresis, samples were transferred at 200 mAmp overnight in transfer buffer (39 x 10-3 M glycine, 48 x 10-3 M Tris base, 0.037% SDS, 20% methanol) to nitrocellulose membranes. To control for protein loading, membranes were incubated for 10 min in 2 g/liter PonceauS (Sigma) in 3% acetic acid and rinsed with water. Afterward they were blocked in TBS-T buffer [0.132 M NaCl, 0.02 M Tris (pH7.5), 0.1% Tween 20(Sigma)] containing 5 g/100 ml of nonfat milk, and shaken at room temp for 1.5 h. Membranes were then incubated with the respective primary antibody at the indicated dilution (in TBS-T buffer with 1 g/liter BSA), shaken for 1 h at room temperature and then with the corresponding secondary antibody at the indicated dilution. Dilutions for primary antibodies were as follows: anti-K-Ras (Calbiochem), 1:2,000; anti-RhoA (Santa Cruz Biotech), 1:400; anti-active MAPK (Promega), 1:20,000; anti-active JNK (Promega), 1:10,000; anti-phospho-AKT (New England Biolabs), 1:10,000; anti-ß-actin (Santa Cruz Biotech), 1:2,000; anti-bcl-2 (Calbiochem), 1:2,500; anti-bax (Santa Cruz), 1:1,000; anti-GAPDH (Chemicon), 1:10,000; anti-PARP (Boehringer-Mannheim), 1:10,000; anti-ß-catenin (Santa Cruz), 1:400; anti-FAK (Santa Cruz), 1:4,000; and anti-E-cadherin (Santa Cruz), 1:400. Secondary antibodies were POD-conjugated goat antimouse, donkey antigoat, and goat antirabbit (all from Boehringer-Mannheim) and were all diluted 1:20,000. Protein bands were detected by chemiluminescence using Supersignal (Pierce).
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All K-ras transformants [K-ras codon 12 mutation
(K12), K-ras codon 13 mutation (K13), and K-ras
wt overexpressors (Kwt-oe)] showed similar morphological changes when
growing attached to the plate (they became smaller, more rounded and
more refringent), grew forming colonies (Fig. 1b)
, and showed increased filopodia and lamellopodia
formation. In all of them, a percentage of polinucleated cells was
observed (Fig. 1c)
. In contrast, 3T3wt and 3T3-neo cells
showed none of these changes (Fig. 1a)
. Moreover, all
K-ras transformants grew in 0.5% FBS, showed forced
anchorage-independent growth as defined by their ability to grow in
poly-heme, forming spheroids (Fig. 1d)
, whereas 3T3wt and
3T3-neo did not have these capacities. In addition, all
K-ras transformants showed reduced doubling times
(20.05 ± 0.35 h for K12, 20.55 ± 1.06 h for K13, and 20.08 ± 1.36 h for
Kwt-oe), as compared with the 3T3wt (27.06 ± 0.33 h) or the 3T3-neo (27.27 ± 0.32 h) clones (Fig. 2)
. These results suggest that distinct types of ras mutations
and an increased dosage of the wt allele induce some of the
characteristics associated with the transformed phenotype, including
increased proliferative rates, similar morphological changes,
diminished growth dependence on serum, and forced anchorage-independent
growth (by plating on poly-heme).
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b) that, with time, increased in size and cell density (Fig. 3c)
and eventually led to the spontaneous formation
of spheroids or multicellular aggregates (Fig. 3d)
. This was
associated with changes of cell morphology typical of the growth in
suspension. No spontaneous spheroid formation was observed in K13 or
Kwt-oe transformants, which only showed small colonies attached to the
plate (Fig. 3a)
even 1 week after culturing under
logarithmic growth. As expected, neither forced nor spontaneous
anchorage-independent growth was observed in 3T3wt or 3T3-neo cells.
Therefore, we observed some level of cell adhesion deregulation in all
K-ras transformants, which was more intense in those
carrying codon 12 mutation.
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than K13 or Kwt-oe
transformants. Moreover, because spheroid formation (Fig. 3d)
occurred in K12 transformants before confluency was
reached, a monolayer of adhered and confluent K12 cells was never
obtained. As expected, 3T3wt and 3T3-neo transfectants did not form
colonies nor undergo growth arrest when they reached confluency.
Therefore, there was a higher level of cell-cell contact deregulation
in K12 than in K13 or Kwt-oe transformants.
The most striking difference among K-ras transformants
occurred in apoptotic induction. K13 and Kwt-oe transformants showed
confluency-dependent apoptosis. Thus, 24–48 h after reaching
confluency, all cells in these transformants died (showing changes in
morphology and becoming detached). This death occurred synchronously
during a short (3–4 h) period and was apoptotic, as assessed by
nuclear condensation (Fig. 4b)
and by the TUNEL assay (Fig. 4d)
.
Confluency-dependent apoptosis did not occur in 3T3wt or 3T3-neo cells.
K12 transformants showed no sign of cell death (Fig. 4, a and c)
when similarly plated and cultured under the same
conditions for as long as 1 week, provided that fresh media was
supplied. Accordingly, no apoptotic induction was observed in K12
transformants. Thus, we observed an increased resistance to apoptosis
in K12 as compared with K13 or Kwt-oe transformants.
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First, we analyzed the activation of the MAPKs proliferative pathways
(Fig. 5)
. All three tested K-ras transformants (Kwt-oe, Fig. 5
,
Lane B; K12, Lane C; and K13, Lane D)
showed similarly high levels of both activated isoforms Erk1 and Erk2,
as compared with the 3T3wt (Lane A) and 3T3-neo (Lane
E) cells. Nevertheless, the increase in Erk2 activation was much
more pronounced than the increase in Erk1 activation despite the fact
that the basal levels of Erk2 activation were already higher than those
of Erk1 activation in 3T3wt or 3T3-neo control cells (Fig. 5)
. In
addition, all K-ras transformants similarly increased the
expression of the metabolic protein GAPDH, which is associated with
increased proliferation. The levels of ß-actin expression were
similar in all K-ras transfectants and in control 3T3-neo
and 3T3wt cells (Fig. 5)
. These observations indicate that all
transformants have similarly activated the MAPK pathway and are in
agreement with the similar replication times observed for the different
transformants.
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) because we observed differences in cell density within the colonies
and in the level of deregulation of anchorage-dependent growth among
transfectants. Accordingly, under logarithmic growth, there was an
increase in the expression of ß-catenin and E-cadherin in all
K-ras transformants that was more intense in K12 (Fig. 5
,
Lane C) than in K13 (Lane D), or Kwt-oe
(Lane B) transformants. Similarly, there was an increase in
FAK expression in all transfectants that was much more important in K12
transformants (Lane C) than in K13 (Lane D) or in
Kwt-oe (Lane B) transformants. Therefore, the observed
differences in cell-cell contact inhibition and anchorage-independent
growth observed between the K12 and K13 or Kwt-oe transfectants were
associated with increasingly higher levels of proteins regulating these
interactions.
Third, we analyzed the possibility of a differential activation of
pathways previously related to the regulation of apoptosis (Fig. 6)
. In cells cultured under logarithmic growth, we observed an increased
activation of the AKT protein, and an overexpression of the
anti-apoptotic protein bcl-2, in the K12 (Fig. 6
, Lane C)
transformants when compared with K13 (Lane D) or Kwt-oe
(Lane B) transformants, which still showed an increase in
AKT activation and bcl-2 expression as compared with 3T3wt and 3T3-neo
clones (Fig. 6)
. In addition, K12 transformants showed RhoA
underexpression, whereas K13 and Kwt-oe maintained the level of RhoA
present in the control, 3T3wt, and 3T3-neo cells (Fig. 6)
. In contrast,
K13 and Kwt-oe transformants showed increased activation of the JNK1
(46,000-Mr JNK form) pathway, whereas the
level of JNK2 activation
(54,000-Mr JNK form) remained
unaltered in all transformants and similar to that observed in the
control 3T3wt and 3T3-neo cells (Fig. 6)
. The level of expression of
the pro-apoptotic protein, bax, in all K-ras transfectants
(K12, K13, and Kwt-oe) was similar to the endogenous level observed in
3T3-neo transfectants and 3T3wt cells (Fig. 6)
. ß-actin was similarly
expressed in all K-ras transfectants and in control 3T3-neo
and 3T3wt cells (Fig. 6)
. Therefore, specific proteins regulating the
apoptotic process are differently activated and/or expressed between
K12 and K13 or Kwt-oe transformants.
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. PARP processing started to become evident 
8 h after the cells
reached confluency (Fig. 7
, Kwt-oe, Lane E, and K13,
Lane I), increased in intensity at 24 h (Kwt-oe,
Lane F, and K13, Lane J), and became complete by
48 h (Kwt-oe, Lane G, and K13, Lane K). In
contrast, PARP processing was not observed in K12 transformants
(Lane C), in 3T3-neo transfectants (Lane B), nor
in 3T3wt cells (Lane A), cultured under the same conditions
and for the same time period. No PARP processing was observed in either
K13 (Lane H) or Kwt-oe (Lane D) if confluency was
avoided. Moreover, in K13 and Kwt-oe transformants, AKT processing
started 24 h after confluency (Fig. 7
, Kwt-oe, Lane F;
and K13, Lane J), and was completed by 48 h (Kwt-oe,
Lane G and K13, Lane K). No AKT processing was
observed in control 3T3 cells (Lane A), 3T3-neo
transfectants (Lane B), or in K12 transformants (Lane
C). Apoptotic induction also associated with increased activation
of the JNK1 and JNK2 pathways in K13 and Kwt-oe transformants (Fig. 7)
.
This activation started 8 h after confluency (Kwt-oe, Lane
D; K13, Lane H), peaked at 24 h (Kwt-oe,
Lane E; K13, Lane I), and returned to basal
levels by 48 h. ß-actin remained unprocessed in all cell types
(Lanes A-D, F, J and
H) except at 48 h after confluency in cells entering
apoptosis (Lanes G and K), because it is also a
caspase substrate. Therefore, and differently from K12 transformants,
K13 and Kwt-oe transformants underwent confluency-dependent apoptosis
associated with PARP and AKT processing and activation of the JNK
pathways.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Three main functional differences between K12 and K13 or Kwt-oe transformants were observed. K12 transformants: (a) formed colonies of increased cell density; (b) underwent spontaneous (not only forced) anchorage-independent growth; and (c) showed a reduced ability to enter apoptosis. The most striking of these differences involved the regulation of apoptosis. Whereas, K12 transformants did not undergo apoptosis, K13 and Kwt-oe transformants unexpectedly underwent confluency-dependent apoptosis rather than cell arrest, as observed in the control cells. The increased resistance (or reduced predisposition) to apoptosis of the K12 transformants was associated, in our model, with a significant increase in AKT activation and bcl-2 expression and a significant decrease in RhoA expression. In contrast, the reduced apoptotic threshold observed in Kwt-oe and K13 transformants was associated with a significant increase in JNK1 activation. The differences in apoptotic regulation were not associated with alterations in the expression of bax, because this protein was not regulated by transformation. These findings are consistent with AKT activation (18, 19, 20, 21) or bcl-2 overexpression (22 , 23) mediating the anti-apoptotic function of Ras. Our observations are also consistent with the pro-apoptotic role of RhoA overexpression (24 , 25) and with the association of JNK activation with apoptotic induction (26) .
The different transfectants also showed distinct phenotypes related with cell-cell contact and cell-substrate interaction. The overexpression of the wt K-ras allele or codon 13 mutations were sufficient to increase the expression of cell-cell contact (E-cadherin and ß-catenin) and cell adhesion (FAK) proteins to a level that deregulate cell-cell contact inhibition of growth or deregulate the cell-adhesion interaction. The former would increase saturation density and the latter would permit growth under forced anchorage independence conditions. In contrast, only the codon 12 mutation increased the expression of cell-cell contact (E-cadherin and ß-catenin) molecules to a significantly higher level, leading to higher cell-cell contact deregulation, which may be responsible for the ability to form bigger colonies with higher cell densities. Moreover, only K12 increased the expression of the cell adhesion protein FAK, leading to a degree of cell-adhesion deregulation, which may have induced spontaneous (not just forced) anchorage-independent growth. Therefore, our model helps explain the differences in transforming capacity of the different transformants, relating them, not only to apoptotic deregulation, but also to deregulation of cell-cell and cell-adhesion interactions. Our results are consistent with overexpression of functional E-cadherin/ß-catenin complexes inducing tight cell-cell contacts (27) and constitutive FAK expression rendering cells capable of anchorage-independent growth (28 , 29) .
In some of the analyzed phenotypes, all K-ras transformants behaved similarly. In all K-ras transformants (Kwt-oe, K13 and K12), we observed a similar activation of the Erk1 and Erk2 proliferative pathways, associated with a correspondingly similar reduction in doubling time (increase in proliferation rate), and in GAPDH expression, as compared with the 3T3wt and the 3T3-neo cells. These observations are consistent with the report of a reduction in the G1 phase period (30) and an increase in expression of the proliferation-dependent GAPDH expression (31) in Ras mediated-transformation.
The quantitative rather than qualitative (absence or presence of specific bands) differences in expression or activation of the studied molecules observed among these three transformant types suggest that their different functional outcomes may be the consequence of different levels of activation of the same Ras downstream targets, and not to their activation of different substrates. This agrees with the observation that mutations at codon 12 or 13 change the amino acid sequence in the guanine nucleotide binding region (32) , which renders Ras unresponsive to GAP, but leaves unaltered the overall structure of the molecule (33) , which makes qualitative changes in the affinity of interaction with effector targets unlikely (34) .
In our model, ras 12 mutant would condition a specific interaction pattern with PI3K, MEKK, Ras-GAP, or other Ras effectors that, directly or indirectly, would increase the activation of the AKT and the bcl-2 pathways and would decrease the RhoA pathway activity. This pattern would differ from that of the ras13 mutant or the overexpression of the wt protein, which activates the JNK1 pathway. In contrast, interaction with Raf or other targets that activate MAPK and bax pathways is not affected by the molecular nature of ras mutations, supporting the idea of K-Ras mutations changing its affinity for effectors other than Raf, which aggrees with the lack of significant changes in affinity of Ras for Raf among different Ras mutants (35 , 36) .
Altogether, our observations suggest that K12 mutations confer a more aggressive tumor phenotype, not by changing cell morphology or proliferative capacity, but by altering the threshold of apoptotic induction. In contrast, K13 mutations or the overexpression of the K-ras wt protein reduce this threshold. To our knowledge, this is the first description of functional and molecular differences in apoptosis regulation between different ras mutants. Our results are in agreement with the higher transformation efficiency, using the focus formation and/or the nude mouse tumorigenicity assays, of K-ras transfectants carrying codon 12 versus codon 13 mutants (10) or K-ras wt overexpression (37) . They are also in agreement with the fact that tumors carrying K-ras codon 12 mutations show lower apoptotic indeces than tumors not bearing codon 12 mutations (13) . Our results suggest that cells carrying K-ras codon 13 mutations or overexpressing the wt allele, may have a reduced survival ability and could be selected against in the adenoma to carcinoma transition. In addition, K-ras alterations may also differently deregulate cell-cell and cell-adhesion interaction, affecting the predisposition of the tumor cells to local invasion and metastasis, which are important determinants of tumor aggressiveness, involving FAK (38) or E-cadherin/ß-catenin deregulation (39) .
In vivo tumors are likely to contain multiple mutational changes in addition to K-ras, and thus many additional molecular interactions are likely to occur in the real clinical setting, which may make these interactions complex. Nevertheless, even in this context, K12 is likely to predispose to a more aggressive tumor phenotype than K13 or Kwt-oe.
In summary, our results reinforce the notion that not only the presence of a ras mutation, but its molecular nature, may significantly influence the biological behavior of a given tumor cell. That is, the type of alteration in K-ras which occurs at the adenoma stage may differently predispose the transformed cells toward a more benign or a more aggressive tumor phenotype. Therefore, the spectrum of ras mutations may be critical when assessing the biological behavior of tumors in different clinical settings.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work has been supported in part by Grants
SAF98-0042, SAF99-0089, and 2FD97-2018-C02-02 from the Comisión
Interministerial de Ciencia y Tecnología. S. G. is a fellow of
the Spanish Ministry of Education and Science. L. F. is a fellow of
the Comissió Interdepartamental de Recerca i Innovació
Tecnològica. R. M. is a researcher of the Spanish National
Health System. 
2 To whom requests for reprints should be
addressed, at Laboratori d’Investigació Gastrointestinal,
Institut de Recerca, Hospital de Sant Pau, Avda. Sant Antoni M. Claret,
167, Barcelona, Spain. Phone: 34-93-291-9106; Fax: 34h93h291h9263;
E-mail: rmangues{at}santpau.es
3 The abbreviations used are: PI3K,
phosphatidylinositol 3'-kinase; TUNEL terminal deoxynucleotidyl
tranferase-mediated dUTP nick-end labeling; K12, K-ras
codon 12 transformants; K13, K-ras codon 13
transformants; Kwt-oe, transformants overexpressing the
K-ras proto-oncogene; poly-heme,
poly(2-hidroxyethylmethacrylate); FAK, focal adhesion kinase; wt, wild
type; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MAPK,
mitogen-activated protein kinase; JNK, c-Jun N-terminal kinase; PARP,
poly-(ADP-ribose)-polymerase; GAP, GTPase-activating protein; MEKK,
MAPK kinase kinase; TEMED,
N,N,N',N'-tetramethylethylenediamine;
APS, ammonium persulfate; FBS, fetal bovine serum; AKT/PKB, protein
kinase B.
Received 4/11/00. Accepted 9/27/00.
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radiation. Mol. Cell Biol., 10: 405-408, 1990.This article has been cited by other articles:
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), K13 (
), and Kwt-oe
(x), transformants, which show significantly reduced doubling times
(20.05 ± 0.35 h, 20.55 ± 1.06 h, and 20.08 ± 1.36 h, respectively), as compared
with the control cells [3T3wt (
) and 3T3-neo (
); 27.06 ± 0.33 h. and 27.27 ± 0.32 h.,
respectively]. The mean ± SD of the cell counts for
three independent experiments are depicted in a neperian logarithmic
scale of cell number versus time plot to obtain the
duplication times after adjusting by the minimum square method.
