K-ras Codon 12 Mutation Induces Higher Level of Resistance to Apoptosis and Predisposition to Anchorage-independent Growth Than Codon 13 Mutation or Proto-Oncogene Overexpression¹

The ras genes encode small GTPase proteins involved in signal transduction of extracellular signals. They become oncogenic by single point mutations, mainly at codons 12 or 13, which constitutively activate their protein products (1). Of the three ras genes, K-ras is the most frequently mutated in human tumors (2). Transformation by oncogenic Ras is associated with changes in morphology, increased proliferation, and inhibition of apoptosis (3, 4). In recent years, different Ras effectors have been identified (e.g., Raf,PI3K,³MEKK, Ras-GAP), and the study of their association with distinct functions contributing to transformation by Ras is being actively pursued (5, 6). Raf transduces proliferative signals through MAPK; PI3K inhibits apoptosis through PKB/AKT; MEKK associates with apoptotic induction through JNK; and RasGAP transduces signals through RhoA, which regulates apoptosis and alters morphology(6).

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-rasproto-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 β-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, β-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, β-catenin, and FAK overexpression.

NIH3T3 cells were obtained from the American Type Culture Collection. We used plasmids containing a K-ras minigene with a G:C→A:T mutation at the first position of codon 12 (pMLK12), a G:C→A: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, Naβ-glycerophosphate, Na fluoride, and PP_i 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-β-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.

The different plasmids were grown and purified, after bacterial transformation using the One Shot competent Escherichia coliKit (Invitrogen). Bacterial colonies were selected in plates containing 10⁻⁴g/ml ampicillin in Luria-Bertani medium(10⁻²g/ml bactotryptone, 5 × 10⁻³g/ml yeast extract, 15 × 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 5μg/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.

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-raspoint 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.

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 × 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.

Western blots for the assessment of the expression of β-actin, FAK,β-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 × 10⁻² m Tris/Acetate (pH 7.5), 0.27 m sucrose, 10⁻³m EDTA (pH 8.0), 10⁻³ mEGTA (pH 8.0), 10⁻³ m Na ortovanadate (pH 10.0), 10⁻² m Naβ-glycerophosphate, 5 × 10⁻²m Na fluoride, 5 × 10⁻³ m PP_i,1% Triton X-100 en Tris/acetate-sucrose (210⁻²m/0.27 m), 0.1% de 2-β-mercaptoethanol,10⁻³ m benzamidine, 2 × 10⁻⁴ m PMSF, 5 × 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 ×3 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 × 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 × 10⁻³ m glycine, 48 × 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 mTris (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-β-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-β-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).

Before studying the functional and molecular changes associated with transformation by the different K-ras constructs, we proceeded to characterize the transformants for the presence and expression of the respective transfected plasmids. The analysis of the mutations for each of the selected clones (three per construct) yielded the expected mutation. Moreover, all K-ras transformants expressed significantly (P < 0.001) higher levels of K-Ras protein than control 3T3-neo (0.85 ± 0.4) or than wt 3T3 cells. In addition, the differences in the level of K-Ras expression among K12 (3.8 ± 0.3), K13(3.3 ± 0.3), and Kwt-oe (3.9 ± 0.5)transformants were not statistically significant(P = 0.14).

All K-ras transformants [K-ras codon 12 mutation(K12), K-ras codon 13 mutation (K13), and K-raswt 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. 1,b), and showed increased filopodia and lamellopodia formation. In all of them, a percentage of polinucleated cells was observed (Fig. 1,c). In contrast, 3T3wt and 3T3-neo cells showed none of these changes (Fig. 1,a). 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. 1,d), 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).

Whereas all transformants showed some degree of cell-cell contact and cell-adhesion deregulation, K12 showed clearly different phenotypes than K13 or Kwt-oe transformants. Thus, K13 and Kwt-oe transformants showed forced (by plating on poly-heme) anchorage-independent growth. However, K12 transformants not only showed anchorage-independent growth when plated on poly-heme but when plated on plastic as well. In this setting they formed colonies (Fig. 3 b) that, with time, increased in size and cell density (Fig. 3,c) and eventually led to the spontaneous formation of spheroids or multicellular aggregates (Fig. 3,d). 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. 3 a) 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.

Phenotypic differences were also observed among transformants regarding their cell-cell contact interactions. Both K13 and Kwt-oe transfectants formed more dense colonies and achieved higher saturation densities once they reached confluency as compared with 3T3wt cells or the 3T3-neo transfectants. On the other hand, in the same culture conditions, K12 transformant cells grew in bigger colonies with much higher cell densities (Fig. 3,c) than K13 or Kwt-oe transformants. Moreover, because spheroid formation (Fig. 3 d) 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. 4,b) and by the TUNEL assay (Fig. 4,d). 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.

The functional differences observed between K12 and K13 or Kwt-oe transformants led us to analyze some of the more relevant pathways downstream of Ras, including those which regulate proliferation and apoptosis, as well as the expression of proteins playing significant roles in the regulation of cell-substrate and cell-adhesion interactions.

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 β-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.

Second, we evaluated the possible alterations in levels of several proteins known to play key roles in the regulation of cell-cell(E-cadherin and β-catenin) and cell-substrate interactions (FAK; Fig. 5) 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 β-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-M_r JNK form) pathway, whereas the level of JNK2 activation(54,000-M_r 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). β-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.

Finally, when apoptosis was induced in K13 or Kwt-oe transformants,both PARP and AKT (which are caspase substrates) became processed (Fig. 7). 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. β-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.

We tested the hypothesis that K-ras codon 12 mutations would confer upon the transfected cell a more aggressive transforming phenotype than mutations at codon 13 or the overexpression of the K-ras proto-oncogene. We used NIH3T3 fibroblast as the recipient cell because it is a reliable model to study transformation by ras and other oncogenes (17). We selected stable transfectants, expressing constructs with these mutations (K12, K13, Kwt-oe) and performed an evaluation of some of the functional and molecular alterations associated with transformation. The lack of statistically significant differences in K-Ras expression among K12, K13, and Kwt-oe transfectants indicated that all of the observed functional and molecular differences were attributable to the absence or presence of the different mutation types rather than to variations in expression.

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 β-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 andβ-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/β-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 versuscodon 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-rasalterations 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/β-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.

We thank Dr. Manuel Perucho for kindly supplying the pMLK12,pMLK13, and pMLKwt plasmids. We acknowledge Dr. Pere Puig and Ester Civit for technical support.