The K-Ras 4A isoform promotes apoptosis but does not affect either lifespan or spontaneous tumor incidence in aging mice

Experimental Cell Research 312 (2006) 16 – 26 www.elsevier.com/locate/yexcr

Research Article

The K-Ras 4A isoform promotes apoptosis but does not affect either lifespan or spontaneous tumor incidence in aging mice Sarah J. Plowman a,1, Mark J. Arends b, David G. Brownstein c, Feijun Luo b, Paul S. Devenney a,2, Lorraine Rose a, Ann-Marie Ritchie a, Rachel L. Berry a,2, David J. Harrison a, Martin L. Hooper a, Charles E. Patek a,* a

Sir Alastair Currie Cancer Research UK Laboratories, Molecular Medicine Centre, The University of Edinburgh, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, UK b Department of Pathology, The University of Cambridge, Addenbrooke’s Hospital, Hills Road, Cambridge CB2 2QQ, UK c Research Animal Pathology Core Laboratory, Queen’s Medical Research Institute, The University of Edinburgh, 47 Little France Crescent, Edinburgh EH16 4TJ, UK Received 16 June 2005, revised version received 6 October 2005, accepted 7 October 2005

Abstract Ras proteins function as molecular switches in signal transduction pathways, and, here, we examined the effects of the K-ras4A and 4B splice variants on cell function by comparing wild-type embryonic stem (ES) cells with K-ras tmD4A/tmD4A (exon 4A knock-out) ES cells which express K-ras4B only and K-ras / (exons 1 – 3 knock-out) ES cells which express neither splice variant, and intestinal epithelium from wild-type and K-ras tmD4A/tmD4A mice. RT-qPCR analysis found that K-ras4B expression was reduced in K-ras tmD4A/tmD4A ES cells but unaffected in small intestine. K-Ras deficiency did not affect ES cell growth, and K-Ras4A deficiency did not affect intestinal epithelial proliferation. K-ras tmD4A/tmD4A and K-ras / ES cells showed a reduced capacity for differentiation following LIF withdrawal, and K-ras / cells were least differentiated. K-Ras4A deficiency inhibited etoposide-induced apoptosis in ES cells and intestinal epithelial cells. However, K-ras tmD4A/tmD4A ES cells were more resistant to etoposide-induced apoptosis than K-ras / cells. The results indicate that (1) K-Ras4A promotes apoptosis while K-Ras4B inhibits it, and (2) K-Ras4B, and possibly K-Ras4A, promotes differentiation. The findings raise the possibility that alteration of the K-Ras4A4B isoform ratio modulates tumorigenesis by differentially affecting stem cell survival and/or differentiation. However, K-Ras4A deficiency did not affect life expectancy or spontaneous overall tumor incidence in aging mice. D 2005 Elsevier Inc. All rights reserved. Keywords: Alternative splicing; Apoptosis; Differentiation; Intestine; Isoform; Proliferation; K-ras; Stem cell

Introduction The three classical mammalian ras genes, K-, N- and Hras, encode 21 kDa proteins that are members of the guanine nucleotide binding protein superfamily (reviewed in [1]). Ras proteins function as plasma membrane-bound

* Corresponding author. Fax: +44 131 651 1072. E-mail address: [email protected] (C.E. Patek). 1 Current address: Institute for Molecular Biosciences, University of Queensland, Brisbane, QLD 4072, Australia. 2 Current address: MRC Human Genetics Unit, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, UK. 0014-4827/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2005.10.004

guanine nucleotide binding proteins with intrinsic GTPase activity and act as molecular switches by regulating signal transduction pathways for hormones, growth factors and cytokine receptors, including the Raf/MEK/ERK (MAPK) and PI3-K/Akt kinase cascades, and so affect diverse cellular functions, including cell proliferation, differentiation, migration and apoptosis [2 – 5]. Activating point mutations that lead to constitutive activation of Ras proteins by stabilizing the active GTP-bound configuration are commonly found in human cancers, with K-ras mutations particularly prevalent in lung (30%), colon (40 –50%) and pancreatic (90%) cancers (reviewed in [6]). The presence of K-ras activating mutations in precancerous lesions, includ-

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ing aberrant crypt foci (reviewed in [7]), implies that they act during the early stage of tumorigenesis, and, indeed, expression of endogenous oncogenic K-ras promotes lung and pancreatic cancer and is sufficient to initiate transformation of embryonic fibroblasts (see [5,8]). However, understanding the roles of Ras in tumorigenesis has become more complex in recent years with evidence that the presence, or over-expression, of ras proto-oncogenes can either enhance or suppress the malignant phenotype (see [9]). Recent studies indicate that the K-ras proto-oncogene exhibits tumor suppressor activity since over-expression of K-ras reduces the incidence of MNU-induced mammary tumors in rats [10], and the wild-type K-ras allele protects against the progression of lung tumorigenesis in mice that harbor activating K-ras mutations induced by MNU or urethane [11]. The latter study also found that reexpression of wild-type K-ras in fibroblast and lung tumor cell lines that express oncogenic K-ras inhibits their growth in vitro and capacity for tumor formation in vivo. In agreement with this, human lung cancer progression is linked with loss of the wild-type K-ras allele in the presence of the oncogenic allele [12]. Furthermore, we reported that teratomas derived from K-ras / embryonic stem (ES) cells are larger than those that express wild-type K-ras, contain a higher proportion of undifferentiated embryonal carcinoma-like cells and show significantly increased mitotic activity, indicating that K-ras, like N-ras [13], can exhibit tumor suppressor activity in the absence of its oncogenic allele [4]. Comparisons between wild-type and K-ras / ES cells also found that K-Ras plays a crucial role in promoting apoptosis and differentiation in vitro [3,4]. Thus, since tumors can derive from tissue stem cells and may harbor Fcancer stem cells_ (reviewed in [14]), we postulated that absence of expression of the K-ras protooncogene might contribute to neoplastic progression by inhibiting apoptosis and promoting stem cell self-renewal rather than differentiation [4]. Understanding the role of K-ras in tumorigenesis is complicated further by the presence of two splice variants, K-ras 4A and K-ras 4B, which are generated by alternative splicing of the fourth coding exon. The isoforms differ at their C-terminus, which is termed the hypervariable region (HVR). Post-translational modifications of residues within this region target Ras proteins to the plasma membrane and are essential for their function. Both isoforms are farnesylated on their C-terminal cysteine residues, but, whereas KRas 4A is palmitoylated at additional upstream cysteine residues, K-Ras 4B contains a polybasic domain essential for its plasma membrane localization (reviewed in [15]). These differences are presumed to confer functional differences, and, indeed, oncogenic K-RasG12V isoforms differ in their ability to activate Raf-1 and induce transformed foci; only K-Ras 4A promotes anchorage-independent growth and only K-Ras 4B, cell migration [2]. Importantly, since Kras activating mutations usually occur at codons 12, 13 or 61 and therefore affect both isoforms, it was proposed that their cooperative actions could account for the high

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frequency of K-ras mutations detected in human cancers. K-Ras 4A might activate effector pathways that transform cells and enable anchorage-independent growth, whereas KRas 4B could promote cell motility and thereby facilitate angiogenesis, invasion and, ultimately, metastasis. Recent studies show that K-Ras proto-oncoproteins also have different functions: only K-Ras 4B promotes expression of matrix metalloproteinase 2 [16] and has a role in cardiovascular homeostasis [17]. Indeed, a difference in functions is consistent with earlier reports that K-ras 4A and 4B are expressed differentially during mouse development and in adult tissues [18 – 20] and that K-ras / mice, which express neither splice variant, die in utero [21,22], whereas K-ras tmD4A/tmD4A and KrasKI mice (generated by knock-out and knock-in gene targeting strategies respectively) which only express the K-ras 4B splice variant develop normally [17,20]. However, no specific role or normal cellular function has been ascribed to K-Ras 4A to date. To address this issue, we examined its effect on cell proliferation, apoptosis and differentiation by comparing intestinal crypt epithelial cells from wild-type and K-ras tmD4A/tmD4A mice and wild-type and K-ras tmD4A/tmD4A ES cells. Additionally, K-ras / and K-ras tmD4A/tmD4A ES cells were compared to gain insight as to whether K-Ras 4B also regulates stem cell function. Previously, we reported that K-ras tmD4A/tmD4A mice are healthy at 3 months [20]. Since K-ras exhibits tumor suppressor activity in the absence of its oncogenic allele [4], we also examined the long-term consequence of K-Ras 4A deficiency to determine whether it affects life expectancy and the spontaneous tumor incidence in aging mice.

Materials and methods Mice K-ras tmD4A/tmD4A mice were generated as described previously [20]. ES cells The culture conditions and the production of K-ras / HM1-derived ES cells (which harbor a homozygous deletion of exons 1– 3) and K-ras tmD4A/+ ES cells (where exon 4A of one allele is replaced by a neomycin resistance cassette) have been reported previously [3,20]. K-ras tmD4A/ tmD4A ES cells were derived from two K-ras tmD4A/+ clones isolated in separate targeting experiments using HM1 and E14 ES cell lines by culture in a high concentration of geneticin (G418, 2 mg ml 1, Invitrogen) (see [23]). ES cell clones harboring a homozygous deletion of exon 4A were identified initially by PCR using the primer pair Px4AS (5VCATTGGTGAGAGAGATCCGACAGTAC-3V) and Px4AA (5V-TCACACAGCCAGGAGTCTTTTCTTC-3V) positioned in K-ras exon 4A that generate a 72 bp product in wild-type

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and K-ras tmD4A/+ ES cells but give no product in Kras tmD4A/tmD4A cells. For control purposes, to test for the presence of DNA of PCR quality, the primer pair Px3S (5VGACTCTGAAGATGTGCCTATGGTCC-3V) and Px3A (5VGCTGAGGTCTCAATGAACGGAATCC-3V) positioned in K-ras exon 3 was used to generate a 125 bp product. In both reactions, DNA was denatured for 4 min at 94-C and amplified at 94-C for 30 s and 58-C for 30 s for 30 cycles. The genotype of K-ras tmD4A/tmD4A ES cells was confirmed by Southern blotting using internal and external probes as described previously [20]. Reverse transcription-PCR (RT-PCR) analysis K-ras 4A and 4B expression was determined by RT-PCR as reported previously using primers in exons 1 and 4B that co-amplify both splice variants in the same reaction [18]. RNA was extracted from tissues using the Trizol reagent as per the manufacturer’s protocol (Invitrogen). First-strand cDNA synthesis was performed using the SuperScripti preamplification system (Invitrogen) using 1 –5 Ag of RNA. For each sample, minus RT controls were included and in all cases proved negative. Analysis of K-ras 4B transcript expression levels by quantitative RT-PCR

Measurement of apoptosis in vitro Etoposide (Sigma) was dissolved in DMSO to a stock concentration of 100 mM before diluting into growth medium at the indicated concentrations, and all dishes received DMSO to a final concentration of 0.02% at the time of etoposide addition, 24 h after seeding. Cells were harvested and stained with propidium iodide [24]. Analysis was performed on a Coulter EPICS-XL flow cytometer and the proportion of apoptotic cells taken as the proportion of nuclei with DNA content in the sub-G1/G0 (or hypodiploid) peak [3,25]. Hoechst 33342 staining and fluorescent microscopy was used to confirm nuclear morphological changes typical of apoptosis [26]. Measurement of apoptosis in vivo Mice were administered etoposide, [1 mg/kg (Sigma)] dissolved in DMSO and diluted in 0.25 ml saline, by intraperitoneal injection. Three mice from each genotype were culled at each time point, and the small intestines were fixed overnight in methacarn (4 volumes methanol, 2 volumes chloroform and 1 volume glacial acetic acid). Apoptosis was scored in histological sections as described previously [27]. Measurement of cell proliferation in vitro

Total RNA (100 ng) from ES cells or tissues was reverse transcribed in 25 Al volume using the iTaq SYBR Green RTPCR kit (Bio-Rad) following the manufacturer’s instructions. All real-time quantitative reverse transcription polymerase chain reactions (RT-qPCR) were amplified starting with denaturation at 95-C for 3 min then 45 cycles of 95-C for 15 s and 60-C for 1 min. The following exon-spanning primers were used: mouse h-actin upstream primer (5V-AAGCTGTGCTATGTTGCTCTAGACT-3V) and downstream primer (5V-CACTTCATGATGGAATTGAATGTAG-3V); mouse Kras 4B upstream primer (5V-GAGTAAAGGACTCTGAAGATGTGCC-3V) located in K-ras exon 3 and downstream primer (5V-CATCGTCAACACCCTGTCTTGTCTT-3V) spanned the junction of K-ras exon 3 and 4B (specific for the mouse K-ras 4B transcript as this sequence is absent from the K-ras 4A transcript). The PCR product sizes derived from K-ras 4B and h-actin transcripts were 158 bp and 148 bp respectively. The specificities of the PCR reactions were confirmed by dissociation curve analysis and 2% agarose gel electrophoresis. All PCR products were analyzed when in the exponential phase of PCR amplification. Quantitation of the relative expression levels of Kras 4B was obtained using standard curves with normalization against those of h-actin transcripts from the same sample. The fold difference in relative expression levels was the ratio of the value of each sample normalized against h-actin. All RT-qPCR reactions were performed in triplicate, and the average relative expression level was calculated.

ES cells were seeded at 7.5  105 cells/90 mm tissue culture dish and the medium replenished after 24 and 72 h. Duplicate dishes were harvested at the indicated time points. The cell pellets were resuspended in medium, diluted in Isoton II balanced electrolyte solution (Beckman-Coulter) and counted in a Coulter Particle Count and Size Analyser. DNA content and cell cycle phase distribution were determined by flow cytometry of nuclei isolated by the detergent-trypsin method [24], and analysis using MultiCycle AV for Windows software (Phoenix Flow Systems). Measurement of cell proliferation in vivo Mice were administered bromodeoxyuridine [BrdU, 50 mg/kg (Sigma)] by intra-peritoneal injection and culled after 2 h. The small intestines were fixed overnight in methacarn, and wax sections (5 Am) were dewaxed and rehydrated. Following antigen retrieval (1 M HCl at 60-C for 10 min), endogenous peroxidase activity was quenched using 1.5% H2O2 for 20 min. Sections were incubated with 1% bovine serum albumin (BSA) for 20 min to block non-specific binding sites, then incubated with an HRP-conjugated mouse monoclonal anti-BrdU antibody [Roche, 1:50 in 1% BSA in phosphate buffered saline (PBS)] for 3 h. Signal was developed using 3,3V-Diaminobenzidine (Vector), and the sections counter-stained with hematoxylin. Negative controls consisted of substitution of the primary antibody with 1% BSA in PBS, and these sections gave no signal.

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BrdU-positive nuclei were counted in 25 full crypts from two age-matched mice of each genotype. Stem cell self-renewal/differentiation assay ES cells were seeded in triplicate at 103 cells per well of a gelatinized 6-well plate and cultured for 5 days in medium without leukemia inhibitory factor (LIF). Colonies were fixed and stained for the ES cell marker alkaline phosphatase (alkaline phosphatase leukocyte staining kit, Sigma Diagnostics) as described previously [28].

Results Generation of K-ras tmD4A/tmD4A ES cells K-ras tmD4A/+ ES cells were generated previously by homologous recombination using a targeting vector that replaced exon 4A by a neomycin resistance cassette [20]. K-ras tmD4A/tmD4A ES cells were isolated from two independent K-ras tmD4A/+ clones, derived from HM1 and E14 ES cells, by culture in a high concentration of geneticin (see [23]). K-ras tmD4A/tmD4A cells were identified initially by PCR analysis using primers within exon 4A which showed the absence of a 72 bp product (Fig. 1A, upper panel). The presence of PCR quality DNA was established by the amplification of a PCR product from exon 3 (Fig. 1A, lower panel). The genotype of the K-ras tmD4A/tmD4A clones was subsequently confirmed by Southern blotting using internal and external probes (Fig. 1B), and by RTPCR which found that wild-type ES cells express both

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splice variants, albeit with K-ras 4A transcripts at a low level, whereas K-ras tmD4A/tmD4A clones express K-ras 4B only (Fig. 2). The latter is consistent with our earlier finding that colon from K-ras tmD4A/tmD4A mice no longer expresses K-ras 4A [20]. Importantly, the finding that ES cells normally express both K-ras splice variants, as does mouse intestinal epithelium where they are expressed at comparable levels [18,20], enabled examination of the role of the K-Ras isoforms on cellular function, including effects on cell proliferation, differentiation and apoptosis. While examination of the role of K-Ras 4B is possible by comparison of K-ras tmD4A/tmD4A and K-ras / ES cells, examination of the role of K-Ras 4A by comparing wildtype and K-ras tmD4A/tmD4A genotypes is more complex since the phenotype could reflect either loss of K-Ras 4A, a consequent change in level of K-Ras 4B or a combination of both. To address this issue, K-ras 4B expression was examined in wild-type and K-ras tmD4A/tmD4A tissues/ES cells by real-time RT-qPCR (Fig. 3). It was found that tissues which normally express low/moderate levels of Kras 4A, including small intestine, kidney, liver and lung [18 – 20], show no change in the level of K-ras 4B expressed ( P = 0.17, P = 0.27, P = 0.65 and P = 0.83 respectively, Student’s t test), whereas tissues that normally express high levels of K-ras 4A, including colon and caecum [18 – 20], expressed increased levels of K-ras 4B ( P = 0.0011 and P = 0.012, respectively, Student’s t test). This is consistent with a recent report on Smad2 alternative splicing which found that deletion of the splice variant that is expressed at the highest level results in increased expression of the remaining splice variant [29]. In contrast, compared with wild-type ES cells which normally express only trace amounts of K-ras 4A (Fig. 2), the K-ras tmD4A/tmD4A ES cells expressed less K-ras 4B (Fig. 3; P = 0.030, combined value for HM1- and E14derived ES cells, Fisher’s method for combination of probabilities, ref. [30] pp. 794 –797). This does not fall into the pattern displayed by the mouse tissues and may reflect in vitro culture. K-Ras 4B promotes ES cell differentiation

Fig. 1. Genotyping of K-ras tmD4A/tmD4A ES cells. (A) Upper panel, primers located in exon 4A generate a 72 bp product in wild-type (+/+) and Kras tmD4A/+ (+/ ) ES cell clones but give no product in K-ras tmD4A/tmD4A clones (nos. 8, 12, 16, 18 and 24). Lower panel, primers within exon 3 generate a 125 bp product in all ES cell clones. ( ) minus DNA control; (*) 25 bp ladder, the major band is 125 bp. (B) Southern analysis using a 3V internal probe (upper panel) detecting 2 kb wild-type and 6 kb targeted bands following digestion with HindIII and a 5V external probe (lower panel) detecting 5 kb wild-type and 4 kb targeted bands following PvuII digestion (see [20]).

Previously, we reported that K-ras is implicated in stem cell differentiation since K-ras / ES cells show a reduced capacity to differentiate (as judged by high levels of alkaline phosphatase activity—a marker of undifferentiated ES cells) when cultured in the absence of LIF [4]. To examine the role of the K-Ras isoforms in differentiation, we compared wild-type, K-ras / and K-ras tmD4A/tmD4A HM1-derived ES cells and wild-type and K-ras tmD4A/tmD4A E14-derived ES cells in eight separate experiments involving several different K-ras mutant clones. Following LIF withdrawal, wild-type ES cells showed very highly significantly greater levels of differentiation than Kras tmD4A/tmD4A cells ( P = 8.62  10 10 for HM1 cells, P = 2.18  10 15 for E14 cells, Student’s t test), and both

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Fig. 2. RT-PCR analysis of K-ras tmD4A/tmD4A ES cells. Primers located in exons 1 and 4B co-amplify K-ras 4A (687 bp) and K-ras 4B (565 bp) in wild-type (+/+) ES cells, but K-ras tmD4A/tmD4A ( / ) ES cells express Kras 4B only. St, mouse stomach. *100 bp ladder, the major band is 600 bp.

HM1- and E14-derived K-ras tmD4A/tmD4A cells showed very highly significantly greater levels of differentiation than Kras / cells ( P = 6.50  10 23 and P = 6.72  10 20, respectively, Student’s t test; Fig. 4). The results indicate that K-Ras 4B can inhibit ES cell self-renewal and promote differentiation. While the findings that Kras tmD4A/tmD4A ES cells show lower levels of differentiation than wild-type cells, and higher levels of differentiation than K-ras / cells suggest that K-Ras 4A, like K-Ras 4B, may also promote differentiation, interpretation of the data is complicated by the fact that K-ras tmD4A/tmD4A ES cells express a reduced level of K-ras 4B (see Fig. 3). K-Ras 4A deficiency does not affect proliferation of ES cells or intestinal epithelial cells Expression of endogenous oncogenic K-ras promotes proliferation of embryonic fibroblasts in vitro and induces pre-neoplastic epithelial hyperplasias in lung and colon (see [5,8]). Here, we examined whether expression of endogenous K-Ras 4A and 4B proto-oncoproteins affects cell growth by comparing wild-type, K-ras tmD4A/tmD4A and K-ras / HM1-derived ES cells and intestinal epithelial cells from wild-type and K-ras tmD4A/tmD4A mice. Wild-type E14 and HM1-derived ES cells were compared and showed similar doubling times, 22.4 h and 22.8 h respectively, and the mean estimated doubling times of the mutant ES cells determined from three independent

Fig. 4. Undifferentiated ES cells. Mean percentage T SEM of alkaline phosphatase positive colonies from eight experiments involving E14- and HM1-derived wild-type and K-ras tmD4A/tmD4A ES cells (two E14-derived and three HM1-derived mutant clones) and HM1-derived K-ras / ES cells (three clones) plated in the absence of LIF expressed as a percentage of the total number of colonies scored. For all the HM1 genotypes and both E14 genotypes, the percentage of alkaline phosphatase-positive colonies in the presence of LIF averaged at least 93%. Wild-type (filled columns); Kras tmD4A/tmD4A (open columns); K-ras / (gray column).

experiments were not significantly different from wildtype (Fig. 5A; wild-type 20.4 T 1.6 h, K-ras tmD4A/tmD4A 18.9 T 0.8 h and K-ras / 19.1 T 0.8 h; P = 0.074 and P = 0.13 respectively, Student’s t test). The lack of effect of K-Ras isoforms on cell growth was confirmed by DNA flow cytometric analysis of ES cells from all three genotypes which showed very similar cell cycle parameters (Fig. 5B). The effect of K-Ras 4A on cell proliferation in vivo was examined by comparing BrdU incorporation in intestinal crypt epithelial cells between wild-type and K-ras tmD4A/tmD4A mice. Two mice from each genotype were examined, and the number of cells undergoing DNA synthesis was similar in both cases (wild-type, 267/ 1100 and 331/1100; K-ras tmD4A/tmD4A , 300/1100 and 325/ 1100; P = 0.307, Student’s t test). K-ras 4A promotes etoposide-induced apoptosis in ES cells and intestinal epithelial cells

Fig. 3. Real-time RT-qPCR analysis showing relative expression of K-ras 4B by E14- and HM1-derived wild-type and K-ras tmD4A/tmD4A ES cells and tissues from age-matched wild-type and K-ras tmD4A/tmD4A mice (mean of at least 4 mice T SEM). Wild-type (filled columns); K-ras tmD4A/tmD4A (open columns); SI, small intestine. Data for HM1-derived mutant cells are the mean of four clones and, for E14-derived mutant cells, the mean of two clones.

Since K-ras expression is important for the response of ES cells to genotoxic damage [3], the role of the individual isoforms in apoptosis was investigated. Wild-type, Kras tmD4A/tmD4A and K-ras / cells were treated with different doses of etoposide and apoptosis quantified by DNA flow cytometry [3,25]. Wild-type ES cells from the E14 and HM1 parental cell lines showed similar levels of apoptosis following treatment with etoposide (Fig. 6A). In contrast, the dose response revealed clear differences in the response of K-ras mutant ES cells to etoposide treatment at the higher doses used. The order of sensitivity was as follows: K-ras tmD4A/tmD4A < K-ras / < wild-type, with Kras tmD4A/tmD4A cells derived from both parental cell lines showing the least apoptosis (Figs. 6B, C). Time-course

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Fig. 5. ES cell proliferation. (A) Growth curve showing the mean number of cells for duplicate dishes of wild-type (diamond), K-ras tmD4A/tmD4A (square) and K-ras / (triangle) HM1-derived ES cells (TSEM). The graph is representative of three independent experiments. (B) Flow cytometric DNA profile analysis showing the mean number of cells detected in each cell cycle phase (TSEM). Wild-type (filled column), K-ras tmD4A/tmD4A (open column) and K-ras / (gray column) HM1-derived ES cells.

analysis with E14-derived K-ras tmD4A/tmD4A ES cells found that the apoptotic response to 5 AM etoposide was delayed compared to wild-type and K-ras / cells (Fig. 6D). When all the data from 24 h etoposide treatment (Figs. 6B– D) were analyzed using Fisher’s method for combining probabilities ([30], p. 794– 797) and statistical significance assessed by sequential Bonferroni tests ([30], p. 240 – 242), it was found that K-ras tmD4A/tmD4A ES cells showed very highly significantly lower levels of apoptosis than wild-type (v 122 = 77.26, P = 1.37  10 11) and K-ras / ES cells (v 122 = 36.83, P = 0.000238). Furthermore, K-ras / cells showed significantly less apoptosis than wild-type ES cells (v 122 = 25.09, P = 0.0144). Hoechst 33342 staining confirmed that cell death induced in ES cells by etoposide was due to apoptosis as judged by characteristic chromatin condensation and nuclear fragmentation [26]. Overall, 3 studies were performed using HM1-derived wild-type and K-ras / ES cells, and K-ras tmD4A/tmD4A ES cells derived from both E14 and HM1 lines, and, for each genotype, 250 cells were scored 18 h following treatment with etoposide (5 AM). In agreement with the hypodiploid data, the combined data from wild-type ES cells showed the highest level of

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apoptosis (30% T 3.7%) followed by K-ras / ES cells (20.3% T 5.4%), with K-ras tmD4A/tmD4A ES cells showing a significantly lower level of apoptosis (8% T 1.7%) than wild-type ES cells ( P = 0.001, Student’s t test). The finding that K-ras tmD4A/tmD4A ES cells (which express the K-ras 4B splice variant only) show lower levels of apoptosis than K-ras / cells (which express neither splice variant) implies that K-Ras 4B is anti-apoptotic. However, while the finding that K-ras tmD4A/tmD4A ES cells show less apoptosis than wild-type cells suggests that K-Ras 4A is proapoptotic, interpretation of the data is complicated by the fact that K-ras tmD4A/tmD4A ES cells express less K-ras 4B than wild-type cells (see Fig. 3). To resolve the issue as to whether K-Ras 4A is indeed pro-apoptotic, we examined etoposideinduced apoptosis in wild-type and K-ras tmD4A/tmD4A small intestines since they express similar levels of K-ras 4B (see Fig. 3). Intestinal crypt epithelial cells from wild-type and Kras tmD4A/tmD4A mice showed similar basal levels of apoptosis, 0.9 T 0.6% and 1.1 T 0.4% respectively. In contrast, epithelium from K-ras tmD4A/tmD4A mice contained fewer apoptotic cells up to 12 h after etoposide treatment, and the timing of the apoptotic peak was delayed (Fig. 7). The finding that apoptosis is reduced in K-ras tmD4A/tmD4A small intestine which expresses K-ras 4B at wild-type levels indicates that it reflects loss of K-ras 4A expression. Furthermore, the finding that K-ras tmD4A/tmD4A ES cells have a more severe apoptotic phenotype than K-ras / cells which do not express K-ras 4B shows that the lower apoptosis in K-ras tmD4A/tmD4A ES cells is not the consequence of the reduced K-ras 4B levels. Overall, the results indicate that K-Ras 4A promotes etoposide-induced apoptosis, and this applies to both stem cells in vitro and somatic cells in vivo. K-ras 4A deficiency does not affect life expectancy or the spontaneous tumor incidence in aging mice Previous studies indicate that K-Ras 4A-deficient mice remain healthy at 3 months [17,20]. However, reports that K-ras can exhibit tumor suppressor activity [4,11], together with the present findings that the K-Ras 4A protooncoprotein exhibits actions consistent with tumor suppressor activity (i.e. promotes apoptosis and possibly differentiation), prompted an examination of the long-term consequence of K-Ras 4A deficiency in mice. Survival curves were compared using the Mantel –Haenszel test (see [31]). No significant difference in survival was found between K-ras tmD4A/tmD4A and wild-type inbred mice (strain 129/Ola) either for males (v 12 = 0.307, P = 0.580) or females (v 12 = 0.091, P = 0.763; Fig. 8). Similar results were obtained for mice with a crossbred (129/OlaC57BL/6) genetic background (for aggregate data for both sexes from 11 wild-type and 24 K-ras tmD4A/tmD4A mice v 12 = 0.056, P = 0.813). Postmortem histopathological examination after sacrifice of surviving overtly healthy inbred (strain 129/ Ola) wild-type and K-ras tmD4A/tmD4A mice, aged between

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Fig. 6. Apoptosis in ES cells. Mean number of hypodiploid nuclei detected in the pre-G0/G1 peak after treatment with etoposide. Untreated cultures received no DMSO, and those labeled as 0 – 40 AM etoposide also received 0.02% DMSO. (A) Wild-type E14 (filled columns) and HM1 (open columns) ES cells, 24 h treatment (Thalf-range of 2 observations). (B) HM1-derived wild-type (filled columns), K-ras tmD4A/tmD4A (open columns) and K-ras / (gray columns) ES cells, 24 h treatment (TSEM of 3 observations). (C) Similar experiment to that shown in panel (B) except that open columns represent E14-derived Kras tmD4A/tmD4A ES cells. The study was performed once. (D) ES cells were treated with etoposide (5 AM) and harvested at the indicated times. Two separate experiments are shown, one with HM1-derived wild-type (filled diamond), K-ras tmD4A/tmD4A (filled square) and K-ras / (filled triangle) ES cells (Thalfrange of 2 observations), and the other comparing HM1-derived wild-type (open diamond) and K-ras / (open triangle) ES cells with E14-derived Kras tmD4A/tmD4A (open square) ES cells (TSEM of 3 observations).

596 and 698 days, revealed a similar overall spontaneous tumor incidence, with a total of 6 tumors from 5 wild-type mice and 11 tumors from 11 K-ras tmD4A/tmD4A mice (Table

1). With the exception of Harderian gland adenomas, the tumor profiles differed between the genotypes, but, in view of the small numbers, this may have occurred simply by

Fig. 7. Apoptosis in intestinal crypt epithelial cells. Mean percentage of apoptotic nuclei induced by etoposide in 25 full small intestinal crypts (1100 cells) from three age-matched wild-type (filled triangle) and Kras tmD4A/tmD4A (open square) mice (strain 129/Ola) at each time point (TSEM).

Fig. 8. Lifespan of inbred 129/Ola mice, limited by sacrifice at the first sign of ill-health, as a function of sex and K-ras genotype. Light continuous line, wild-type males (n = 5); heavy continuous line, K-ras tmD4A/tmD4A males (n = 13); light broken line, wild-type females (n = 10); heavy broken line, K-ras tmD4A/tmD4A females (n = 13).

S.J. Plowman et al. / Experimental Cell Research 312 (2006) 16 – 26

23

Table 1 Incidence of spontaneous neoplasms in aging (596 – 698 days) K-Ras 4A-deficient (K-ras tmD4A/tmD4A ) and wild-type (+/+) control strain 129/Ola mice Neoplasm

Harderian gland: adenoma Uterus: sarcoma, endometrial stromal Uterus: polyp, endometrial Ovary: sarcoma Pancreas: carcinoma, islet cell Urinary bladder: papilloma Liver: carcinoma, hepatocellular Lung: adenoma, bronchiolo-alveolar Lung: carcinoma, bronchiolo-alveolar Sternum: neuroendocrine tumor Total neoplasms

Males

Females

+/+ [n = 1]

K-ras

1 – – – – – – – – 1 2

2 – – – – – – – – – 2

chance, and no overall pattern was evident as regards the tissues/organs affected.

Discussion Since tumors can derive from tissue stem cells and may harbor Fcancer stem cells_ (reviewed in [14]), a greater understanding of the mechanisms that regulate ES cell function can provide valuable insights into cancer biology. ES cells and tissue stem cells share many characteristics with cancer cells in that they are capable of either differentiation or self-renewal and are subject to a common nucleolar mechanism regulating cell cycle progression [32]. Moreover, ES cells and tissue stem cells show similar transcriptional profiles, including enrichment of K-ras expression [33], and studies with K-ras / ES cells indicate that K-Ras plays a crucial role in regulating stem cell apoptosis and self-renewal [3,4]. Here, we addressed whether K-Ras 4A contributes to the tumor suppressor activity of the K-ras proto-oncogene [4,11] by examination of cell proliferation, differentiation and apoptosis using Kras tmD4A/tmD4A ES cells and intestinal crypt epithelial cells from K-ras tmD4A/tmD4A mice. Since ES cells deficient only in K-ras 4B expression are not available, K-ras / and Kras tmD4A/tmD4A ES cells were also compared to determine whether K-Ras 4B affects these processes and whether the isoforms have overlapping or different biological functions. Since the K-ras mutant genotypes were derived by gene targeting, this approach enables a comparison of stem cells that express both isoforms, neither isoform or K-Ras 4B only, on an otherwise normal genetic background. Importantly, the phenotype of K-ras / ES cells reflects the genotype since reexpression of wild-type K-ras in K-ras / ES cells restored their capacity for apoptosis and differentiation to wild-type levels [3,4]. The finding here that Kras tmD4A/tmD4A ES cells from two different parental lines (HM1 and E14) show identical phenotypes is consistent with this view. The present study found that either absence of expression of endogenous proto-oncogenic K-ras 4A and 4B, or

tmD4A/tmD4A

[n = 2]

+/+ [n = 4]

K-ras tmD4A/tmD4A [n = 9]

1 – – 1 – – – 1 1 – 4

3 2 1 – 1 1 1 – – – 9

absence of K-ras 4A alone, does not affect proliferation of ES cells in vitro and that K-Ras 4A deficiency does not alter intestinal epithelial proliferation in vivo. The latter finding is consistent with our earlier study which found no histological abnormalities in 3-month-old K-ras tmD4A/tmD4A mouse tissues that normally express K-ras 4A, including lung, intestine, stomach, kidney, liver and pancreas [20]. We previously reported that absence of K-ras expression causes reduced differentiation of ES cells when grown in the absence of LIF as evident by cell morphology and high levels of alkaline phosphatase activity [4]. Here, we found that K-Ras 4B promotes stem cell differentiation and that this may also apply to K-Ras 4A (given that K-ras tmD4A/tmD4A ES cells not only show lower levels of differentiation than wildtype cells but also differentiate more readily than K-ras / cells). Dysregulation of stem cell self-renewal, which may increase the chance that a mutant cell acquires additional DNA damage, is linked with cancer development since several genes identified initially for their role in carcinogenesis have been implicated in normal stem cell self-renewal decisions and include genes in the Bmi-1, Notch, Wnt and Sonic hedgehog signaling pathways (reviewed in [14]). Like K-ras, the Wnt pathway is mutated at an early stage in colorectal tumorigenesis, and it has been reported that ES cells which harbor mutant Apc alleles (that allow h-catenin accumulation) have only a limited capacity for differentiation in vitro and in teratomas in vivo [34]. Likewise, Apc mutant intestinal cells exhibit an immature Fcrypt progenitorlike_ phenotype [35]. Previously, we reported that K-ras G12V and K-ras / ES cells show a reduced capacity to differentiate in vitro and in teratomas in vivo and, in accordance with the Fstem cell model_ for cancer formation, proposed that K-ras activating mutations or loss of K-Ras function (either directly, by homozygous deletions and/or inactivating mutations of K-ras, or indirectly, by inactivating mutations of downstream genes in the Ras signal transduction pathways) may support neoplastic progression by promoting stem cell self-renewal rather than differentiation [4]. Although we found that K-Ras 4B promotes differentiation, we were unable to reach a definitive conclusion about whether K-Ras 4A also does so. Different effects of the two

24

S.J. Plowman et al. / Experimental Cell Research 312 (2006) 16 – 26

isoforms are conceivable since the Raf/MAP kinase pathway regulates stem cell differentiation (see [36]), and the K-Ras 4A and 4B oncoproteins (G12V) differ in their ability to activate Raf-1 [2]. Thus, a change in their ratio may also perturb stem cell self-renewal and differentiation. The effect of Ras proteins on regulation of apoptosis is complex as it varies between family members and with their mutational status [3,37 – 39]. Using two independent methods to measure apoptosis, we found that K-ras tmD4A/tmD4A ES cells and intestinal crypt epithelial cells were more resistant to etoposide-induced apoptosis than wild-type cells, indicating that K-Ras 4A exerts a pro-apoptotic effect in vivo and in vitro. However, while we confirmed that absence of K-ras expression in ES cells suppresses etoposide-induced apoptosis [3], comparisons between K-ras / and K-ras tmD4A/tmD4A ES cells which, respectively, express neither isoform and K-Ras 4B only found that the latter were more resistant, with delayed apoptosis. These findings suggest that the isoforms may exert antagonistic actions, with K-Ras 4A and 4B exhibiting pro- and anti-apoptotic actions respectively. This possibility is not without precedent since K-Ras 4A and 4B oncoproteins affect Raf-1 differentially, indicating that they may affect different effector pathways [2], and, in this respect, the effect of Ras on apoptosis depends on the signaling pathway activated: PI3-K/AKT/Rac and NF-nB signaling are antiapoptotic, RASSF1/Nore1/Mst1 signaling is pro-apoptotic, whereas Raf/MEK/ERK signaling can be either (reviewed in [37]). Thus, the effect of K-ras on apoptosis may depend ultimately on the overall K-Ras isoform balance and, consequently, the effector pathway(s) activated. Indeed, different isoforms of other cancer-related genes, including tumor suppressor genes, exhibit either pro- or anti-apoptotic actions, and their isoform balance is altered in some tumors in favor of the anti-apoptotic isoform (reviewed in [40,41]). In this respect, it has recently been reported that the K-ras 4A/4B splice variant ratio is reduced in the SW480 human colon cancer cell line compared with normal colon [42]. Therefore, in view of the present findings that the K-Ras 4A and 4B protooncoproteins promote and inhibit apoptosis respectively, it is conceivable that the altered ratio in favor of K-Ras 4B may contribute to neoplastic change by enabling the survival of cells with DNA damage. Overall, the study shows that while the K-Ras protooncoproteins do not influence cell proliferation they may have both overlapping and antagonistic actions as regards their effects on stem cell differentiation and apoptosis respectively. While the results suggest they jointly regulate stem cell function and that the tumor suppressor activity of the K-ras proto-oncogene may depend on their cooperative actions, further studies are required to determine the full repertoire of their actions and how they are affected by the corresponding mutationally activated K-Ras 4A and 4B oncoproteins. While K-ras activating mutations play a key role in neoplastic progression [5,8], our earlier finding that

K-ras can exhibit tumor suppressor activity in the absence of its oncogenic allele [4] suggests that absence of K-ras expression or, indeed, inactivation of downstream effector pathways might also promote tumorigenesis. However, the present findings that the K-Ras proto-oncoproteins affect stem cell self-renewal and apoptosis suggest that the involvement of K-Ras in tumorigenesis could be even more complex since they raise the possibility that alterations in their ratio could also modulate neoplastic change by inappropriate promotion of stem cell survival and/or selfrenewal. Indeed, the K-ras 4A/4B ratio is altered in the SW480 colorectal cancer cell line [42]. Therefore, since many tissues express both K-ras splice variants, including colon, pancreas and lung [18 – 20] where tumors with K-rasactivating mutations arise, it will be of interest to determine whether the K-Ras isoform ratio is affected in these tumors and, importantly, whether this also applies to tumor types that normally lack K-ras activating mutations. Ras proteins regulate signal transduction pathways for hormones, growth factors and cytokine receptors, yet Hras / , N-ras / , H-ras / /N-ras / and K-Ras 4Adeficient mice are viable and remain healthy in early adulthood [17,20,43 – 45]. However, no studies have addressed in any detail the long-term consequence of Ras deficiency in aging mice. Here, we found that Kras tmD4A/tmD4A mice have a normal lifespan on both inbred and crossbred genetic backgrounds, and inbred mice show no difference in overall spontaneous tumor incidence at 20– 23 months compared with wild-type mice. While the results indicate that K-Ras 4A proto-oncoprotein deficiency, in the absence of challenge by xenobiotic agents, does not affect longevity, it remains to be determined, given widespread expression of K-ras 4A in adult tissues [18 – 20], whether K-ras tmD4A/tmD4A mice harbor a more subtle physiological phenotype. The finding that wild-type and K-ras tmD4A/tmD4A mice show a similar spontaneous tumor incidence is possibly unexpected given that K-ras can exhibit tumor suppressor activity in the absence of its oncogenic allele [4], and K-ras 4A is expressed widely, including the colon, pancreas and lung where tumors with K-ras activating mutations usually arise [18 –20], and exhibits functions consistent with tumor suppressor activity i.e. is pro-apoptotic and may inhibit stem cell self-renewal (present study). While N-Ras, like K-Ras 4A, is not essential for mouse development, it does play a crucial role in carcinogen-induced tumorigenesis [44]. Interestingly, evidence suggests that this might also apply to K-Ras 4A: (1) exogenous K-ras 4AG12V transforms cells with greater efficiency than K-ras 4BG12V and promotes anchorageindependent growth in vitro [2], (2) mutationally activated K-ras 4A is expressed by a wide range of cancer cell lines, including lung, bladder, pharangeal, neuroblastoma and colon cancer cells [18,19,42,46], (3) K-ras 4A expression is down-regulated in colon cancer cells [42] and (4) the level of K-ras 4A expression in the lung correlates with lung tumor susceptibility among inbred strains of mice

S.J. Plowman et al. / Experimental Cell Research 312 (2006) 16 – 26

[19]. Since K-ras mutations jointly affect both isoforms, a greater understanding of the role(s) each oncoprotein plays in malignant transformation, including the signal transduction pathways affected, is crucial in the development of therapeutic approaches in cancer treatment, which include the use of drugs that target isoform-specific post-translational modifications [6] and of antisense oligonucleotides to modulate alternative splicing [47]. Importantly, the finding that K-ras tmD4A/tmD4A mice exhibit normal life expectancy and an overall similar spontaneous tumor incidence as wild-type mice indicates the K-ras tmD4A/tmD4A mouse offers an excellent experimental model to examine the role of the K-Ras isoforms in carcinogenesis resulting from mutational activation of K-ras or the presence of mutations in other genes.

[11]

[12]

[13]

[14]

[15]

Acknowledgments The study was supported by a Medical Research Council studentship and grants from Cancer Research UK (CUK), The Melville Trust for the Care and Cure of Cancer and The Row Fogo Charitable Trust.

[16]

[17]

[18]

References [1] M. Malumbres, M. Barbacid, RAS oncogenes: the first years, Nat. Rev., Cancer 3 (2003) 459 – 465. [2] J.K. Voice, R.L. Klemke, A. Le, J.H. Jackson, Four human ras homologs differ in their abilities to activate Raf-1, induce transformation, and stimulate cell motility, J. Biol. Chem. 274 (1999) 17164 – 17170. [3] D.G. Brooks, R.M. James, C.E. Patek, J. Williamson, M.J. Arends, Mutant K-ras enhances apoptosis in embryonic stem cells in combination with DNA damage and is associated with increased levels of p19 (ARF), Oncogene 20 (2001) 2144 – 2152. [4] R.M. James, M.J. Arends, S.J. Plowman, D.G. Brooks, J.D. West, C.E. Patek, K-ras proto-oncogene exhibits tumour suppressor activity as its absence promotes tumourigenesis in murine teratomas, Mol. Cancer Res. 1 (2003) 820 – 825. [5] D.A. Tuveson, A.T. Shaw, N.A. Willis, D.P. Silver, E.L. Jackson, S. Chang, K.L. Mercer, R. Grochow, H. Hock, D. Crowley, S.R. Hingorani, T. Zaks, C. King, M.A. Jocobetz, L. Wang, R.T. Bronson, S.H. Orkin, R.A. DePinho, T. Jacks, Endogenous oncogenic K-ras (G12D) stimulates proliferation and widespread neoplastic and developmental defects, Cancer Cell 5 (2004) 375 – 387. [6] A.A. Adjei, Blocking oncogenic Ras signaling for cancer therapy, J. Natl. Cancer Inst. 93 (2001) 1062 – 1074. [7] L. Cheng, M.-F. Lai, Aberrant crypt foci as microscopic precursors of colorectal cancer, World J. Gastroenterol. 9 (2003) 2642 – 2649. [8] C. Guerra, N. Mijimolle, A. Dhawahir, P. Dubus, M. Barradas, M. Serrano, V. Campuzano, V.M. Barbacid, Tumor induction by an endogenous K-ras oncogene is highly dependent on cellular context, Cancer Cell 4 (2003) 111 – 120. [9] R. Diaz, L. Lopez-Barcons, D. Ahn, A. Garcia-Espana, A. Yoon, J. Matthews, R. Mangues, R. Perez-Soler, A. Pellicer, Complex effects of Ras proto-oncogenes in tumourigenesis, Carcinogenesis 25 (2004) 535 – 539. [10] T. Thompson, J.D. Haag, M.J. Lindstrom, D.E. Griep, J.K. Lohse, M.W. Gould, Decreased susceptibility to NMU-induced mammary

[19] [20]

[21]

[22]

[23]

[24]

[25]

[26] [27]

[28]

[29]

25

carcinogenesis in transgenic rats carrying multiple copies of a rat ras gene driven by the rat Harvey ras promoter, Oncogene 21 (2002) 2797 – 2804. Z. Zhang, Y. Wang, H.G. Vikis, L. Johnson, G. Lui, J. Li, M.W. Anderson, R.C. Sills, H.L. Hong, T.R. Devereux, T. Jacks, K.L. Guan, M. You, Wildtype Kras2 can inhibit lung carcinogenesis in mice, Nat. Genet. 29 (2001) 25 – 33. J. Li, Z. Zhang, Z. Dai, C. Plass, C. Morrison, Y. Wang, J.S. Wiest, M.W. Anderson, M. You, LOH of chromosome 12p correlates with Kras2 mutation in non-small cell lung cancer, Oncogene 22 (2003) 1243 – 1246. R.D. Diaz, D. Ahn, L. Lopez-Barcons, M. Malumbres, I.P. de Castro, J. Lue, N. Ferrer-Miralles, R. Mangues, J. Tsong, R. Garcia, R. PerezSoler, A. Pellicer, The N-ras proto-oncogene can suppress the malignant phenotype in the presence or absence of its oncogene, Cancer Res. 62 (2002) 4514 – 4518. M. Al-Hajj, M.W. Becker, M. Wicha, I. Weissman, M.F. Clarke, Therapeutic implications of cancer stem cells, Curr. Opin. Genet. Dev. 14 (2004) 43 – 47. J.F. Hancock, Ras proteins: different signals from different locations, Nat. Rev., Mol. Cell Biol. 4 (2003) 373 – 384. J. Liao, J.C. Wolfman, A. Wolfman, K-Ras regulates the steady-state expression of matrix metalloproteinase 2 in fibroblasts, J. Biol. Chem. 278 (2003) 31871 – 31878. N. Potenza, C. Vecchione, A. Notte, A. De Rienzo, A. Rosica, L. Bauer, A. Affuso, M. De Felice, T. Russo, R. Poulet, G. Cifelli, G. De Vita, G. Limbo, R. Di Lauro, Replacement of K-Ras with H-Ras supports normal embryonic development despite inducing cardiovascular pathology in adult mice, EMBO Rep. 6 (2005) 432 – 437. S. Pells, M. Divjak, P. Romanowski, H. Impey, N.J. Hawkins, A.R. Clarke, M.L. Hooper, D.J. Williamson, Developmentallyregulated expression of murine K-ras isoforms, Oncogene 15 (1997) 1781 – 1786. Y. Wang, M. You, Y. Wang, Alternative splicing of the K-ras gene in mouse tissues and cell lines, Exp. Lung Res. 27 (2001) 255 – 267. S.J. Plowman, D.J. Williamson, M.J. O’Sullivan, J. Doig, A.-M. Ritchie, D.J. Harrison, D.W. Melton, M.J. Arends, M.L. Hooper, C.E. Patek, While K-ras is essential for mouse development, expression of the K-ras 4A splice variant is dispensable, Mol. Cell. Biol. 23 (2003) 9245 – 9250. L. Johnson, D. Greenbaum, K. Cichowski, K. Mercer, E. Murphy, E. Schmitt, R.T. Bronson, H. Umanoff, W. Edelmann, R. Kucherlapati, T. Jacks, K-ras is an essential gene in the mouse with partial functional overlap with N-ras, Genes Dev. 11 (1997) 2468 – 2481. K. Koera, K. Nakamura, K. Nakao, J. Miyoshi, K. Toyoshima, T. Hatta, H. Otani, A. Aiba, M. Katsuki, K-ras is essential for the development of the mouse embryo, Oncogene 15 (1997) 1151 – 1159. R.M. Mortensen, D.A. Conner, S. Chao, A.A. Geisterfer-Lowrance, J.G. Seidman, Production of homozygous mutant ES cells with a single targeting construct, Mol. Cell. Biol. 12 (1992) 2391 – 2395. L.L. Vindelov, I.J. Christensen, N.I. Nissen, A detergent-trypsin method for the preparation of nuclei for flow cytometric DNA analysis, Cytometry 3 (1983) 323 – 327. I. Nicoletti, G. Migliorati, M.C. Pagliacci, F. Grignani, C. Riccardi, A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry, J. Immunol. Methods 139 (1991) 271 – 279. M.J. Arends, A.H. Wyllie, Apoptosis: mechanisms and roles in pathology, Int. Rev. Exp. Pathol. 32 (1991) 223 – 254. A.R. Clarke, S. Gledhill, M.L. Hooper, C.C. Bird, A.H. Wyllie, p53 dependence of early apoptotic and proliferative responses within the mouse intestinal epithelium following gamma-irradiation, Oncogene 9 (1994) 1767 – 1773. H. Niwa, T. Burdon, I. Chambers, A. Smith, Self-renewal of pluripotent embryonic stem cells is mediated via activation of STAT3, Genes Dev. 12 (1998) 2048 – 2060. N.R. Dunn, C.H. Koonce, D.C. Anderson, A. Islam, E.K. Bikoff, E.J.

26

[30] [31] [32]

[33]

[34]

[35]

[36]

[37] [38]

S.J. Plowman et al. / Experimental Cell Research 312 (2006) 16 – 26 Robertson, Mice exclusively expressing the short isoform of Smad2 develop normally and are fertile, Genes Dev. 19 (2005) 152 – 163. R.R. Sokal, F.J. Rohlf, Biometry, W.H. Freeman and Co., New York, 1995. E.T. Lee, Statistical Methods for Survival Data Analysis, 2nd ednR, Wiley, New York, 1992. R.Y.L. Tsai, R.D.G. McKay, A nucleolar mechanism controlling cell proliferation in stem cells and cancer cells, Genes Dev. 16 (2002) 2991 – 3003. M. Ramalho-Santos, S. Yoon, Y. Matsuzaki, R.C. Mulligan, D.A. Melton, ‘‘Stemness’’: transcriptional profiling of embryonic and adult stem cells, Science 298 (2002) 597 – 600. M.F. Kielman, M. Rindapaa, C. Gaspar, N. van Poppel, C. Breukel, S. van Leeuwen, M.M. Taketo, S. Roberts, R. Smits, R. Fodde, Apc modulates embryonic stem-cell differentiation by controlling the dosage of beta-catenin signalling, Nat. Genet. 32 (2002) 594 – 605. O.J. Sansom, K.R. Reed, A.J. Hayes, H. Ireland, H. Brinkman, I.P. Newton, E. Batlle, P. Simon-Assmann, H. Clevers, I.S. Nathke, A.R. Clarke, D.J. Winton, Loss of Apc in vivo immediately perturbs Wnt signaling, differentiation and migration, Genes Dev. 18 (2004) 1385 – 1390. T. Burdon, A. Smith, P. Savatier, Signalling, cell cycle and pluripotency in embryonic stem cells, Trends Cell Biol. 12 (2002) 432 – 438. A.D. Cox, C.J. Der, The dark side of Ras: regulation of apoptosis, Oncogene 22 (2003) 8999 – 9006. J.A. Choi, M.T. Park, C.M. Kang, H.D. Um, S. Bae, K.H. Lee, T.H. Kim, J.H. Kim, C.H. Cho, Y.S. Lee, H.Y. Chung, S.J. Lee, Opposite effects of Ha-Ras and Ki-Ras on radiation-induced apoptosis via differential activation of PI3K/Akt and Rac/p38 mitogen-activated protein kinase signaling pathways, Oncogene 23 (2004) 9 – 20.

[39] Y. Ninomiya, K. Kato, A. Takahashi, Y. Ueoka, T. Kamikihara, T. Arima, T. Matsuda, H. Kato, J. Nishida, N. Wake, K-Ras and N-Ras activation promote distinct consequences on endometrial cell survival, Cancer Res. 64 (2004) 2759 – 2765. [40] D. Mercatante, R. Kole, Modifications of alternative splicing pathways as a potential approach to chemotherapy, Pharmacol. Ther. 85 (2000) 237 – 243. [41] B.M.W. Brinkman, Splice variants as cancer biomarkers, Clin. Biochem. 37 (2004) 584 – 594. [42] J.A. Butz, K.G. Roberts, J.S. Edwards, Detecting changes in the relative expression of KRAS2 splice variants using polymerase colonies, Biotechol. Prog. 20 (2004) 1836 – 1839. [43] H. Umanoff, W. Edelmann, A. Pellicer R Kucherlapati, The murine Nras gene is not essential for growth and development, Proc. Natl. Acad. Sci. U. S. A. 92 (1995) 1709 – 1713. [44] K. Ise, K. Nakumura, K. Nakao, S. Shimizu, H. Harada, T. Ichise, J. Miyoshi, Y. Gondo, T. Ishikawa, A. Aiba, M. Katsuki, Targeted deletion of the H-ras gene decreases tumour formation in mouse skin carcinogenesis, Oncogene 19 (2000) 2951 – 2956. [45] L.M. Esteban, C. Vicario-Abejon, P. Fernandez-Salguero, A. Fernandez-Medarde, N. Swaminathan, K. Vieger, E. Lopez, M. Malumbres, R. McKay, J.M. Ward, A. Pellicer, E. Santos, Targeted disruption of H-ras and N-ras, individually or in combination, reveals the dispensability of both loci for mouse growth and development, Mol. Cell. Biol. 21 (2001) 1444 – 1452. [46] D.J. Capon, P.H. Seeburg, J.P. McGrath, J.S. Hayflick, U. Edman, A.D. Levinson, D.V. Goeddel, Activation of Ki-ras2 gene in human colon and lung carcinomas by two different point mutations, Nature 304 (1983) 507 – 513. [47] P. Sazani, R. Kole, Therapeutic potential of antisense oligonucleotides as modulators of alternative splicing, J. Clin. Invest. 122 (2003) 481 – 486.