1 Department of Cancer Biology and 2 Department of Comparative Medicine, Comprehensive Cancer Center, Wake Forest University School of Medicine, Winston-Salem, NC 27157, USA, 3 Cell Signaling Technology, Beverly, MA 01915, USA, 4 GlaxoSmithKline, Research Triangle Park, NC 27709, USA, 5 Department of Environmental Health Sciences, Division of Toxicology, University of Cincinnati College of Medicine, Cincinnati, OH 45267, USA and 6 Division of Pulmonary Biology, Cincinnati Children's Hospital Medical Center, University of Cincinnati College of Medicine, Cincinnati, OH 45339, USA
* To whom correspondence should be addressed. Tel: +1 336 716 0795; Fax: +1 336 716 0255; Email: msmiller{at}wfubmc.edu
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Abstract |
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Abbreviations: AC, adenocarcinoma; AD, adenoma; CCSP, Clara cell secretory protein; DOX, doxycycline; Erk, extracellular regulated kinase; JNK, Jun kinase; MAPK, mitogen activated protein kinase; Mek, mitogen activated Erk kinase; RBD, Ras binding domain; RBP, Ral binding protein; rtTA, reverse tetracycline trans-activator; SP-C, surfactant protein C; tet, tetracycline
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Introduction |
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Numerous mouse lung tumor models have been utilized to try to mimic the effects of oncogenic Ki-ras in human lung tumors with either the use of chemical carcinogens or transgenes. Until recently, alterations in the Ki-ras oncogene and p16Ink4a and Retinoblastoma tumor suppressor genes were the most widely studied genes between human and mouse lung tumors (17). Technological advances have allowed the extension of these studies into the analysis of gene expression across the entire genome in both human and rodent tumors. Recently, Sweet-Cordero et al. (18) demonstrated that mouse lung tumors initiated in mice by a mutant Ki-ras allele could be used to classify human samples as either normal or tumor with 97% accuracy using upregulated or downregulated gene sets obtained from gene-expression profiling. Another group, also using gene-expression analysis, showed a similarity between chemically-induced mouse lung tumors and human lung tumors (19). These results suggest that mouse models are a useful tool in the study of lung tumorigenesis.
A previous mouse model from our laboratory demonstrated that, following in utero exposure to the polycyclic aromatic hydrocarbon, 3-methylcholanthrene, lung tumors induced in the offspring exhibited a high incidence of mutations (84%) in the Ki-ras gene (8,20). A high incidence of Ki-ras mutations were found in hyperplastic lung tissue (60%) as well, suggesting that activation of Ki-ras may be an early event in lung cancer formation. One of the most interesting findings from this (8) and a subsequent study (21) was that the type of mutation present in the lesions appeared to influence tumor development. Lesions harboring VAL12, ASP12 or ARG13 mutations were more likely to progress to ACs than were lesions with CYS12 mutations (8,21), suggesting that different mutant Ras alleles may have different oncogenic potentials. These results and others have demonstrated that mutation to the Ki-ras gene locus is a critical target in the initiation and progression of lung tumor development.
Although mutations of Ki-ras are considered an early event in the pathogenesis of lung tumors, direct evidence for this hypothesis has only recently been obtained. Both human and chemically-induced rodent tumors contain a number of genetic lesions in addition to mutated Ki-ras, making it difficult to identify the role for mutant Ki-ras in lung tumor initiation. Mouse lung tumorigenesis protocols usually involve treatment with chemical carcinogens, which can alter any one of a number of gene loci in addition to Ki-ras. Koera et al. (22) demonstrated that expression of the Ki-ras gene is critical for normal development in mouse embryos, as deletion of the Ki- , but not the Ha- or N-ras genes, resulted in embryonic lethality. A number of investigators have confirmed the importance of Ki-ras for normal lung development and shown that expression of Ki-ras in the lung increases during gestation, reaching peak levels in the adult, suggesting that Ki-ras plays a role in normal lung morphogenesis (23,24).
Recent studies by several laboratories have utilized inducible transgenic mouse systems to express various mutant alleles of Ki-ras in a timed and/or tissue-specific manner to more directly determine the effects of Ki-ras on cancer initiation and progression (6,2529). In these studies, expression of mutant Ki-ras was shown to be a potent oncogenic stimulus for lung epithelial cells, as expression of a mouse or human Ki-ras transgene resulted in a high tumor burden and formation of progressive ACs within 2 to 10 months.
Therefore, in an effort to understand the effects of activated Ki-rasG12C in lung tumorigenesis, we have utilized a bitransgenic mouse model that expresses the mutant human Ki-rasG12C allele in a lung-specific and tetracycline (tet)-inducible manner. In contrast to previous murine lung tumor models expressing mutant Ki-ras transgenes, expression of the CYS12 mutant allele allowed survival to an age of at least 12 months and resulted primarily in proliferative pulmonary lesions morphologically diagnosed as bronchioalveolar hyperplasias and adenomas (ADs). Activation of Ras downstream effector pathways, including the Raf/Mek/MAPK, p38, and Ral pathways was observed. The present study provides evidence for a novel lung tumor mouse model exhibiting little progression past the AD stage.
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Materials and methods |
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The Ki-ras transgene was constructed using standard molecular biological methods. Twenty µg of the pCR2.1-ras plasmid was linearized by digestion with Spe1 and blunt-ended with T4 DNA polymerase. The fragment was gel-purified and cut with Not1 to release the Ki-ras insert. The tetO7/CMV/Zeor expression vector was double digested with PmeI, which created a blunt end, and Not1 and then treated with shrimp alkaline phosphatase for 1 h at 37°C to prevent self-annealing. The linearized vector and the Ki-ras insert were then directionally cloned and ligated with 24 U of T4 DNA ligase, transformed into XL Blue cells, streaked onto an agar plate containing ampicillin and X-gal, and grown overnight at 37°C. Colonies were picked and expanded, then tested for the presence and orientation of the insertion by restriction enzyme digestion. The orientation of the inserted sequence relative to the CMV promoter was confirmed by gene sequencing.
The Ki-ras monotransgenic mouse was constructed at the Transgenic Mouse Facility at the University of Cincinnati, as described previously (3032). The tetO-CMV-Ki-ras insert was excised from the plasmid by digestion with SpeI and PvuII and purified by CsCl gradient centrifugation. The DNA was then microinjected into donor eggs obtained from superovulated FVB/N mice. The eggs were implanted into pseudopregnant mice and founder mice containing the transgene were established. These are referred to as monotransgenic Ki-rasG12C mice.
Genotyping
DNA was extracted from mouse tails using the Wizard Genomic DNA Purification Kit (Promega) according to the manufacturer's protocol. Genotyping was performed by PCR with Amplitaq Gold (Applied Biosystems, Foster City, CA). To identify Ki-rasG12C mice, an upstream primer in the CMV minimal promoter (5'-CCATCCACGCTGTTTTGACCTC-3') and a downstream primer in the human Ki-ras coding sequence (5'-TACTCCTCTTGACCTGCTGTGTCG-3') were used. Primers for genotyping mice for the presence of the CCSP-rtTA construct were 5'-ACTGCCCATTGCCCAAACAC-3' (forward) and 5'-AAAATCTTGCCAGCTTTCCCC-3' (reverse), and for the SP-C-rtTA construct 5'-GACACATATAAGACCCTGGTCA-3' (forward) and 5'-AAAATCTTGCCAGCTTTCCCC-3' (reverse). Samples were amplified following denaturation at 94°C for 2 min by 40 cycles of denaturation at 94°C for 30 s, annealing at 57°C (Ras) or 61°C (CCSP and SP-C) for 1 min, and extension at 72°C for 1 min, with a final extension for 7 min at 72°C (Bio-Rad iCycler, Hercules, VA).
Tumor studies
Eight week old CCSP/Ki-ras and SP-C/Ki-ras bitransgenic mice, as well as wild-type FVB/N and monotransgenic Ki-ras mice, were either untreated or given 500 µg/ml doxycycline (DOX) (Sigma, St Louis, MO) in the drinking water for 12 days, 1.5, 3, 6, 9 or 12 months. Mice were killed by CO2 asphyxiation/exsanguination, and macroscopic lung lesions were counted and measured prior to fixation. The lungs were then inflated and fixed in 10% phosphate-buffered formalin. At the 12-month time point, liver, spleen, kidney, pancreas, heart and either testis and prostate or ovaries were examined macroscopically and then removed and placed in 10% phosphate-buffered formalin. After 24 h, the formalin solution was replaced with 70% ethanol to avoid excessive aldehyde cross-linking for immunohistochemistry. Tissues were embedded in paraffin and 5 µm sections prepared by microtomy. The cut slides were stained with hematoxylin and eosin and examined by veterinary pathologists (J.E. and N.D.K.). Proliferative lesions were evaluated using established morphological criteria for mice (33).
Ras and Ral activation
CCSP/Ki-ras and SP-C/Ki-ras bitransgenic mice were either untreated or given 500 µg/ml DOX in the drinking water for 72 h. Lungs were removed and protein was extracted using modified RIPA buffer (50 mM TrisHCl (pH 7.4), 150 mM NaCl (pH 7.4), 1 mM EDTA, 1 mM PMSF, 1 µg/ml of aprotinin, leupeptin, pepstatin and 1 mM NaF) without detergents. Whole lung was homogenized using a Polytron homogenizer (Kinematica, Crystal Lake, IL) on setting 6 for 2 pulses of 30 s duration. Protein concentration was determined with the Bio-Rad protein assay kit according to the manufacturer's instructions (Bio-Rad, Hercules, CA). Activation of Ras and Ral was determined from the protein lysates using kits from Upstate Technologies (Charlottesville, VA). For each assay, 15 µl of agarose beads conjugated to either the Ras binding domain (RBD) of Raf or Ral binding protein (RBP) were added to 10 mg of lysate. The samples were rocked for 1 h at 4°C. The beads were washed and denatured with 50 µl loading dye containing 2-mercaptoethanol and boiled for 5 min. An aliquot of 20 µl of each sample was loaded onto a 10% SDSPAGE gel and transferred onto a nitrocellulose membrane. The membranes were incubated with either 1 µg/ml anti-Ras (clone Ras10) monoclonal antibody diluted in phosphate buffered saline containing 5% dry milk and 0.05% Tween-20 or 1 µg/ml anti-Ral A monoclonal antibody diluted in Tris buffered saline containing 5% dry milk and 0.05% Tween-20. Membranes were then incubated with a goat anti-mouse horseradish peroxidase-conjugated secondary antibody diluted 1:1000 in either phosphate-buffered saline or Tris buffered saline containing 5% dry milk and 0.05% Tween-20. Protein bands were visualized with Bio-Rad's Immun-Star Substrate detection kit by autoradiography.
Immunohistochemisry (Ras effectors)
Formalin fixed, paraffin embedded mouse lungs sectioned 5 µm thick were placed on Super Frost slides and air-dried. Slides were baked for 30 min at 58°C prior to deparaffinization. Baked slides were deparaffinized in xylene 3x 5 min, rehydrated in 100% ethanol 2x 10 min, 95% ethanol 2x 10 min, and water for 2x 5 min. Antigen retrieval was performed by heating slides to 100°C for 15 min in 10 mM sodium citrate (pH 6.0), slides were then cooled to room temperature before 2x water rinse. Endogenous peroxidase activity was quenched by a 10 min incubation in 3% peroxide followed by 2 water rinses of 5 min each. Slides were blocked with TBST [25 mM TrisHCl (pH 7.2), 100 mM NaCl and 0.1% Tween 20] containing 5% goat serum (Sigma) for 1 h. Slides were then incubated overnight at 4°C with the following primary antibodies (Cell Signaling Technology, Beverly, MA) diluted in blocking buffer: anti-phospho Erk (THR202/TYR204) rabbit monoclonal 1:300; anti-phospho p38 (THR180/TYR182) rabbit monoclonal 12F8 IHC specific 1:100; anti-phospho MAPKAPK-2 (THR319) rabbit polyclonal 1:100; anti-phospho p90Rsk (THR359/SER363) rabbit polyclonal 1:100; anti-phospho S6 (SER235/236) polyclonal 1:100; anti-phospho JNK (THR183/TYR185) rabbit polyclonal 1:50; anti-phospho Akt (SER473) rabbit polyclonal 1:50. Slides were washed 3x in TBST for 5 min then incubated with biotinylated goat anti-rabbit antibody (Vector Laboratories, Burlingame, CA) 1:1000 in TBST for 30 min, washed 3x 5 min with TBST, then incubated with avidinbiotin complex (Vector Laboratories) for 1 h. Negative controls consisting of tissue and secondary antibody only were performed to account for non-specific binding. Nova Red (Vector Laboratories) was used as per the manufacturer's instructions for 1 min before quenching in water. Slides were dehydrated in 2x 95% ethanol followed by 2x 100% ethanol for 10 s, then rinsed 3x for 10 s with xylene prior to mounting with Vectamount (Vector Laboratories). Slides were imaged with a Nikon Eclipse TE300 microscope fitted with an AxioCam digital camera.
Immunohistochemistry (CCSP, SP-C, Ki-67 and cleaved caspase 3)
Slides were deparaffinized with 3x 5 min washes with xylene. The slides were hydrated through graded alcohols 3x 1 min each. They were then placed in distilled water for 5 min. Antigen retrieval was performed using 10 mM citrate buffer (pH 6.0) for 35 min using a steamer. The slides were cooled for 20 min and placed in distilled water for 5 min. Primary antibodies were added: anti-CCSP 1:1000 (34); anti-SP-C 1:2000 (3538); anti-Ki-67 1:25 (Abcam, Cambridge, MA), and anti-cleaved caspase 3 1:50 (Cell Signaling Technology). Samples as well as negative controls were incubated overnight at 4°C. Slides were then washed 5x with 1x Tris buffer (Biomeda, Foster City, CA) containing 0.5% casein. A secondary biotinylated anti-rabbit antibody (BioGenex, San Ramon, CA) was added at a dilution of 1:20 for 30 min at 32°C. Slides were washed 5x in Tris buffer followed by a 1:20 dilution of Streptavidin-alkaline phosphatase conjugate (BioGenex) for 30 min at 32°C. Slides were washed 5x in Tris buffer and Vector Red substrate (Vector Laboratories) was added for 5 min. Slides were then washed 2x in 0.1 M TrisHCl buffer (pH 8.28.4) and 5x with distilled water. Slides were counterstained with Mayer's hematoxylin for 5 min, then washed under running water for 5 min. Slides were dehydrated through graded alcohol and cleared through several changes of p-xylene.
Expression of transgenic and endogenous Ki-ras RNA by real-time fluorescent PCR
Eight week old CCSP/Ki-ras and SP-C/Ki-ras bitransgenic mice, as well as monotransgenic Ki-ras mice, were either untreated or given 500 µg/ml DOX in the drinking water for 24 h, 48 h, 7 or 14 days. Thirty mg of whole lung tissue was homogenized with a Polytron homogenizer in RLT lysis buffer supplied in the RNeasy Mini Kit at speed 6. Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Valencia, CA). In addition, RNA was also isolated from the liver, spleen, thymus, pancreas, kidneys, heart, intestines and either testis and prostate or ovaries at 14 days of treatment. For RTPCR, cDNA was initially generated from 1 µg of RNA using the iScript cDNA Synthesis Kit (Bio-Rad). One tenth of the cDNA (2 µl) was used to amplify the Ki-ras transgene, the endogenous murine Ki-ras gene, and GAPDH using the iCycler (Bio-Rad) for 40 cycles with SYBR Green Supermix (Bio-Rad).
To generate a standard curve, each PCR product (Ki-ras transgene, endogenous Ki-ras, and GAPDH) was first cloned into a pCR2.1 TA cloning vector. The plasmids were then utilized to generate the standard curves for each of the three genes using the same primers to be used for the unknown samples in order to quantify the copy number of each gene by the relative standard curve method (3941). Both the Ki-ras transgene copy number and endogenous Ki-ras copy number were normalized to GAPDH copy number. PCR efficiencies and R2 values for the standard curves ranged from 99 to 103% and 0.9910.998, respectively.
The primers used to amplify the Ki-rasG12C transgene were 5'-CTGCAGAATTCGCCCTTATGACTGA-3' (forward) and 5'-TAGCTGTATCGTCAAGGCACTCTTGC-3' (reverse); for the endogenous murine Ki-ras gene were 5'-GCAGGGTTGGGCCTTACAT-3' (forward) and 5'-ATGCGTCGCCACATTGAAT-3' (reverse); and for GAPDH were 5'-TCTCCCTCACAATTTCCATCCCAG-3' (forward) and 5'-GGGTGCAGCGAACTTTATTGATGG-3' (reverse). Cycling conditions included an initial denaturation step at 94°C for 3 min followed by 40 cycles of denaturation at 94°C for 30 s, 66.5°C (transgene and GAPDH) or 57°C (endogenous Ki-ras) for 1 min, and 72°C for 1 min. A melt curve was determined following each real-time PCR run to ensure the presence of a single product.
For values reported in Table II, the iCycler software determined a copy number for the Ki-ras transgene, endogenous Ki-ras, and GAPDH for each unknown sample based on the gene-specific standard curve. Both transgene and endogenous Ki-ras copy numbers were normalized to GAPDH by dividing each sample by their respective GAPDH copy numbers. These normalized values were averaged and standard deviations calculated. The numbers in parentheses were determined by dividing all of the normalized averages by the monotransgenic normalized endogenous Ki-ras value.
To compare transgene expression of Ki-rasG12C to Ki-rasG12D, RNA samples from the CCSP/Ki-rasG12D mice were obtained from the University of Cincinnati Transgenic Mouse Facility for each of the following treatment groups, no DOX treatment, 24 h DOX treatment, and 7 days DOX treatment. Each treatment group was provided in triplicate. A 2 months DOX-treated lung sample was also provided by Dr Harold Varmus from the Memorial Sloan-Kettering Cancer Center. An additional time point of 2 months was included for CCSP/Ki-rasG12C mice as a comparison with this CCSP/Ki-rasG12D sample. Expression of the murine Ki-rasG12D transgene, endogenous Ki-ras, and GAPDH genes were determined by real-time PCR as described above, except that the primers for the mouse transgene used in their construct (26) were: 5'-CAAGGACAAGGTGTACAGTTATGTGACT-3' (forward) and 5'-GGCATCTGCTCCTGCTTTTG-3' (reverse).
Statistical methods
Continuous variables were compared among groups with t-tests. Logistic regression adjusting for sex, time and type of experiment was used to estimate adjusted means. 2-test was used to evaluate adjusted means and generated P-values.
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Results |
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To generate mice containing the Ki-rasG12C transgene, the insert was prepared as described in the Materials and methods section and microinjected into donor eggs obtained from superovulated FVB/N mice. The eggs were implanted into pseudopregnant mice and founder mice containing the transgene were established. To screen the pups for the presence of the transgene, genomic DNA was isolated from a small piece of the tail and then probed for the presence of the tetO and Ki-ras sequence using PCR, producing a 264 bp product that extended from the 3' region of the tetO7CMV expression vector through to exon 2 of the Ki-ras gene.
Initially, five Ki-rasG12C founders were identified by PCR and bred to non-transgenic littermates to establish germ line transmission. One founder did not transmit the transgene to offspring. Animals from each of the remaining four lines were bred to CCSP-rtTA or SP-C-rtTA activator mice to obtain bitransgenic mice that were then treated with DOX for 7 days to induce transgene expression. By RTPCR analysis, one line had robust transgene expression, two had low expression, and one was not induced; however, expression was not quantified. The uninduced line was not characterized further. Bitransgenic mice from the two lines expressing low levels of Ki-ras RNA were treated with DOX from 6 weeks to 6 months. Upon killing, analysis of lung histology from these animals revealed no proliferative lesions and these lines were terminated. The remaining line developed lung adenomas upon induction of oncogenic Ki-ras expression and was used for our studies. Twenty five µg of liver DNA from theKi-rasG12C mouse line was restricted with EcoRI and analysed by Southern blotting with a 73 bp exon 1 probe to determine the copy number of the inserted transgene. By densitometric analysis, the ratio of the 430 bp transgenic fragment to the 700 bp endogenous fragment demonstrated that there were 22 copies of the transgene present in the mouse genome.
Treatment with DOX induces morphological changes in the lung
Bitransgenic CCSP/Ki-ras and SP-C/Ki-ras, monotransgenic Ki-ras, and control wild-type FVB/N mice were either untreated or treated with 500 µg/ml of DOX in the drinking water for 12 days, 1.5, 3, 6, 9 and 12 months. At each time point, mice were killed, macroscopic lesions counted, and the lung tissues were evaluated microscopically for the presence of proliferative changes. DOX-treated bitransgenic mice on either the CCSP or SP-C background exhibited small, hyperplastic lung foci after only 12 days of DOX treatment (Figure 1B). By 5 weeks of treatment, extensive epithelial hyperplasia of the alveolar region of the lung tissue could be seen. Lung morphology in untreated bitransgenic mice (Figure 1A) or DOX-treated single transgenic or control mice was normal.
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Ki-ras is required for tumor maintenance
Recent studies by Fisher et al. (26), utilizing a similar bitransgenic mouse system, reported that, following induction of mutant murine Ki-rasG12D expression, removal of DOX resulted in regression of the lung tumors. In order to demonstrate that the Ki-rasG12C allele is similarly important for tumor maintenance, bitransgenic CCSP/Ki-ras and SP-C/Ki-ras mice were treated with 500 µg/ml of DOX for 9 months and then withdrawn from DOX treatment for up to 1 month. Two weeks after DOX withdrawal, there were only four lung tumors still visible on the surface of the lungs in both CCSP/Ki-ras and SP-C/Ki-ras mice. By 1 month of withdrawal, 4 tumors were still visible on the surface of the lung of CCSP/Ki-ras mice, whereas no lesions were seen in SP-C/Ki-ras mice. Unfortunately, the macroscopic lesions were too small for further analysis; however, the persistent lesions could possibly be attributed to a second genetic alteration inhibiting tumor regression. Microscopically, both SP-C/Ki-ras and CCSP/Ki-ras mice exhibited few lesions after 1 month of DOX withdrawal, with only minimal hyperplastic foci generally present (Figure 1L). The remaining foci following 2 weeks of DOX withdrawal showed scattered positive nuclear staining for Ki-67, while the foci remaining following 1 month of DOX withdrawal were negative. None of the lesions were positive for cleaved caspase 3, indicating that these lesions were not regressing through an apoptotic mechanism. These data indicate that expression of Ki-rasG12C is necessary for maintenance of lung lesions and regression occurs through a mechanism alternative to apoptosis.
Expression of Ki-ras transgene
Real-time PCR was used to quantitate expression of the human Ki-rasG12C transgene in the lungs following 24 and 48 h, and 7 and 14 days of DOX treatment. Liver, spleen, pancreas, intestines, kidneys, heart, thymus and either testis and prostate or ovaries were screened for transgene expression after 14 days of antibiotic treatment. In the lung, transgene expression was shown to increase by at least 3 logs following 24 h of DOX treatment, and increased an additional 2-fold by 7 days in both lines of bitransgenic mice (Table II). Expression of the transgene in CCSP/Ki-ras mice was 2-fold higher at every time point than in SP-C/Ki-ras mice, although this difference was not statistically significant (P < 0.9). This difference could be due to the fact that in CCSP/Ki-ras mice, both Clara cells and alveolar type II cells express the Ki-ras transgene, whereas in SP-C/Ki-ras mice only alveolar type II cells express the transgene. Except in testis and prostate, there was no detectable level of transgene expression in any of the other organs examined (Figure 3A). Since the rtTA protein should not be expressed in these two tissues, we tested the monotransgenic Ki-ras mice for transgene expression and found that the level of expression in the testis was
6.3-fold higher than in the DOX-treated, bitransgenic lungs of SP-C/Ki-ras mice (Figure 3B). Interestingly, the testis exhibited no morphological changes even after 12 months of DOX treatment, consistent with the fact that testicular cancers do not have a high incidence of mutations in any of the Ras genes (50). The expression of the transgene in the prostate was
8-fold higher than the lung in the same monotransgenic mice and, similar to the testis, displayed no alterations in normal histology. These results suggest that the transgene may have integrated downstream of a region of the chromosome that is regulated by male-specific hormones.
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The Ki-rasG12C mice used in this study demonstrated a much less severe phenotype than reported by several other laboratories utilizing transgenic mouse lines (6,2629). In particular, the mice described by Fisher et al. (26) utilized the same vector constructs, tetO7CMV and CCSPrtTA, and expressed the murine Ki-rasG12D mutant allele in the same genetic background (FVB/N). Our previous data had suggested that the Ki-rasG12D mutation would be highly oncogenic, while the Ki-rasG12C mutation used in this study would be less oncogenic (8,21). To compare the levels of transgene expression, we obtained RNA from Ki-rasG12D mice either not treated or treated for 24 h and 7 days with DOX. In addition, we also isolated RNA from a sample of lung tumor tissue fromKi-rasG12D mice (a generous gift from Dr Varmus' laboratory). In an individual experiment with newly generated standard curves, we compared these samples with RNA isolated from lung tissue in Ki-rasG12C mice following exposure to DOX for each time point. The results showed that murine Ki-rasG12D transgene expression for 24 h and 7 days was 4.31 x 104 ± 2.11 x 104 and 1.46 x 103 ± 1.24 x 103 compared with 6.83 x 104 ± 6.83 x 104 and 8.90 x 104 ± 1.25 x 103, respectively, in Ki-rasG12C mice, neither time point displaying any statistically significant difference between the two lines of mice (P = 0.5758 and 0.6022). There was a large difference in transgene expression between mice, accounting for the large standard deviations. However, this variation was not seen in our housekeeping gene, GAPDH, indicating that induction of transgene expression varied between mice. At 2 months, expression of the Ki-rasG12D transgene was 2.5 times higher than the Ki-rasG12C allele in CCSP/Ki-ras mice. The tumor mass and lung weights were also two times higher in the Ki-rasG12D mice (26), which accounts for this difference in the RNA levels observed at 2 months between the two transgenic mouse lines. These results suggest that differences in phenotype between Ki-rasG12D and Ki-rasG12C mice were not due to differences in expression of their respective transgenes.
Activation of downstream signaling pathways
Mutations in Ki-ras result in constitutive activation of the Ras protein and increased signaling to downstream effectors. Therefore, the activation of Ras and one of its downstream effectors, Ral, was assayed. Ras and Ral activation were detected with pull down assays that detected binding of the activated protein to either the RBD of Raf or RBP, respectively. The activated proteins were then detected by western blot using antibodies for either the Ras or Ral proteins. In both bitransgenic mouse lines, Ras and Ral activation increased following treatment of the mice with DOX for 72 h. Ras activation was shown to be elevated 1.4- and 2.2-fold in DOX-treated SP-C/Ki-ras and CCSP/Ki-ras mice, respectively, compared with untreated mice (Figure 4A). Similarly, Ral activation was increased 1.4- and 1.8-fold in DOX-treated SP-C/Ki-ras and CCSP/Ki-ras mice, respectively, compared with untreated mice (Figure 4B). Thus, expression of theKi-rasG12C allele increased signaling to downstream effectors of Ras. It should be emphasized that lysates used in the binding assays were tissue preparations from whole lung after 72 h of DOX treatmentgiven the large number of cell types in the lung and the restricted expression of the transgene to Clara and/or alveolar type II cells, it is likely that the activation assays have probably underestimated the actual amount of enhanced activation.
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Discussion |
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There are three possible explanations for the phenotypic differences seen between the Ki-rasG12C and Ki-rasG12D mice. First, previous studies from this laboratory provided evidence that different Ki-ras mutations influenced tumor progression, with lesions harboring the more potent VAL12 and ARG12 mutations more likely to progress to a malignant phenotype than lesions containing wild-type sequences or a CYS12 mutation (8,21). Recent studies have suggested that the correlation of mutated Ki-ras with decreased patient survival may depend on the actual base substitution, with the VAL12 and ARG12 mutations showing a trend with poor patient outcome (7,13,16). Thus, the results presented in this study, combined with those cited above, may explain the severe phenotypic differences and could be the first to demonstrate in vivo that the type of mutation induced in Ki-ras may determine the rate of lung tumor progression and the severity of the lesion produced.
Second, the Ki-rasG12C allele is a human allele while theKi-rasG12D is a murine allele, both expressed in identical murine backgrounds. While the Ki-ras transcript and protein sequences are highly conserved between mouse and human, demonstrating 93% mRNA and 98% protein homology, there is the possibility that the human protein is signaling differently than the murine protein. Anytime there is cross-species expression, this must be considered.
Third, these transgenic mice were made through a random insertion event. Since the insertion site of neither transgene is known, there is the possibility that the phenotype is an effect of differential insertion. This problem can generally be addressed with multiple founder mice; however, we were unable to obtain a second founder line with comparable transgene expression. Further research needs to be done to distinguish between these potential mechanisms.
The benign phenotype of this model prompted us to further characterize the effects of the Ki-rasG12C transgene at the molecular level, thus we examined the activation of downstream Ras effectors. Activated Ras is thought to induce tumorigenicity by turning on multiple downstream effector pathways that increase cell proliferation and inhibit the induction of apoptosis (51). To determine effector activation, Ras and Ral activation assays as well as immunohistochemistry with phospho-specific antibodies to detect activation of downstream effectors were used. Both Ras and Ral activity were elevated 2-fold in Ki-rasG12C expressing mice relative to untreated controls. Immunohistochemical analysis revealed that Erk and its downstream effectors, p90Rsk and pS6, were highly phosphorylated in lung tumor cells expressing mutant Ki-ras compared with non-tumor tissue or untreated, DOX naive mice. However, while we detected no increase in the activity of the JNK or Akt pathways, increased phosphorylation of p38 and MAPKAPK-2 were detected. Interestingly, Tuveson et al. (25) failed to detect phosphorylated Erk1/2 by immunohistochemistry in murine lung hyperplasias induced by expression of the Ki-rasG12D allele, although they detected elevated levels of cyclin D1. The reason for these contrasting observations is not known, but could be due to differences in the Ras alleles used in the transgene constructs, differences in levels of expression of the different Ki-ras transgenes, or differences in the antibodies or staining methods used.
Several studies have shown that stimulation of Ras activity in primary cells induces both an increase in cell proliferation and also activates a defensive mechanism to enhance apoptosis or cell cycle arrest in response to inappropriate mitogenic signals (52,53). The tumors in this model demonstrated scattered positive staining for Ki-67 but were negative when stained with an antibody specific for the cleaved fragment of caspase 3. The lack of apoptosis in both the proliferative lung lesions and in the tissue undergoing regression following DOX withdrawal suggests that there may be another regulatory mechanism accounting for the benign phenotype in this tumor model. Whereas signaling through JNK and Akt have been suggested to inhibit apoptosis (54,55), the role of p38 in the regulation of cell growth, differentiation, and cell death remains somewhat controversial (56). While an increase in Ras-mediated p38 signaling has been shown by some groups to inhibit proliferation or increase apoptosis in a number of different cell types (5759), elevated levels of p38 have been observed in human lung tumor samples (60), suggesting that activation of p38 in some cell contexts could have a mitogenic effect. It is likely that the Ki-rasG12C allele activates a specific subset of downstream effector pathways relative to other Ki-ras mutants, and that this could determine how quickly tumors progress to more severe phenotypes.
Until recently, there has been much controversy regarding the putative progenitor cells of peripheral lung tumors. Many late-stage lung tumors express both SP-C and CCSP, making it difficult to distinguish the cell of origin. Recent transgenic mouse models have demonstrated that the majority of peripheral lung tumors stain positively for SP-C only whereas none of the lesions demonstrated staining for CCSP alone, and only a few lung tumors stain positively for both SP-C and CCSP (26,27,29). Recent studies by Kim et al. (49) have identified a potential common stem cell in the lung, which they have termed bronchioalveolar stem cells, that was identified in the bronchioalveolar junction. The authors provided evidence that these cell types may be the progenitor cells of both Clara and alveolar type II cells, and in addition may be the cells of origin for peripheral lung tumors. In the mouse model described in the present study, the Ki-rasG12C transgene is expressed in both Clara and alveolar type II cells when expressed in the CCSP-rtTA activator line, but immunohistochemical analysis demonstrated positive staining for SP-C and very little or no staining for CCSP in the proliferative cells. These data provide evidence that alveolar type II cells may serve as progenitors of murine lung tumors, but are also consistent with the possibility that tumors arise from a common progenitor cell for both Clara and alveolar type II cells.
One of the advantages of the Tet-on system is the ability to turn off transgene expression and thus determine the role of activated Ki-ras in tumor maintenance and progression. Results obtained in this study, as well as those reported by Fisher et al. (26), demonstrated that, following removal of DOX for 1 month, the number of proliferative lesions was reduced, consistent with regression of the tumors and the requirement for oncogenic Ki-ras expression for maintenance of the tumor phenotype. Using a nude mouse assay, Roth's group transfected antisense RNA constructs of Ki-ras into human tumor cell lines and demonstrated a strong inhibition of tumor cell growth in mice, demonstrating the importance of the mutated ras allele in stimulating tumor cell proliferation and maintenance of the transformed phenotype in human cells as well (61,62). Interestingly, although the tumors that developed in Ki-rasG12C mice were not as severe, 1 month following DOX withdrawal a small number of lesions were still present when transgene expression was driven from the CCSP promoter. The remaining lesions stained positive for Ki-67 following 2 weeks of DOX withdrawal and negative following 1 month of withdrawal. However, all of the lesions were negative for cleaved caspase 3, suggesting that once the proliferative stimulus of elevated mutant Ki-rasG12C expression is removed, either tissue remodeling or the initiation of a non-apoptotic cell death pathway (63,64) may mediate the reversion of the benign lesions to normal-looking pulmonary tissue. It is possible that the longer latency of tumor development in Ki-rasG12C mice allowed a second genetic mutation to occur in some lesions that interfered with the ability of some lesions to regress. However, we also cannot rule out the possibility that due to the leakiness of transgene expression (Table II), low levels of transgene expression may prevent tumor regression in a small number of lesions.
Our results support the development of a novel mouse model for lung tumorigenesis where the tumors do not progress past the adenoma stage, in contrast to other models using Ki-ras transgenes. This long latency will allow the tumors to be monitored for an extended time period without the complications encountered by a high tumor burden or early death in the mice. With an understanding of specific activation pathways of Ras effectors, more effective targeting of novel anti-cancer agents to the specific molecules activated by specific-mutant Ras alleles could be developed, allowing therapies to be tailored to the specific molecular lesions of the tumor. Thus, these results strongly confirm the current strategy of targeting Ras proteins or members of the Ras signaling pathway in the development of novel anti-neoplastic and chemopreventive agents.
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Acknowledgments |
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Conflict of Interest Statement: None declared.
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