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Carcinogenesis Advance Access originally published online on June 13, 2006
Carcinogenesis 2006 27(12):2497-2510; doi:10.1093/carcin/bgl090
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Ductal origin of pancreatic adenocarcinomas induced by conditional activation of a human Ha-ras oncogene in rat pancreas

Shinobu Ueda1,2,3, Katsumi Fukamachi1, Yoichiro Matsuoka2,5, Nobuo Takasuka2,6, Fumitaka Takeshita3, Akihiro Naito2, Masaaki Iigo2,6, David B. Alexander1, Malcolm A. Moore1, Izumu Saito4, Takahiro Ochiya3 and Hiroyuki Tsuda1,2,*

1 Department of Molecular Toxicology, Nagoya City University Graduate School of Medical Sciences 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan
2 Experimental Pathology and Chemotherapy Division, National Cancer Center Research Institute, 5-1-1 Tsukiji Chuo-ku, Tokyo 104-0045, Japan
3 Section for Studies on Metastasis, National Cancer Center Research Institute, 5-1-1 Tsukiji Chuo-ku, Tokyo 104-0045, Japan
4 Laboratory of Molecular Genetics, Institute of Medical Science, University of Tokyo, 4-6-1 Shiroganedai Minato-ku, Tokyo 108-8639, Japan
5 Present address: Second Department of Pathology, Kansai Medical University, 10–15 Fumizono-cho, Moriguchi Osaka, Japan
6 Cancer Prevention Basic Research Project, National Cancer Center Research Institute Tokyo, Japan

*To whom correspondence and requests for reprints should be addressed. Tel: +81 52 853 8991; Fax: +81 52 853 8996; Email: htsuda{at}med.nagoya-cu.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results and discussion
 Summary
 Supplementary material
 References
 
Pancreatic ductal adenocarcinoma is one of the most debilitating malignancies in humans. Currently, radiation and chemotherapy are ineffective, with median survival times after treatment of <12 months. Animal models that reflect the human condition and can be used to explore screening and therapeutic approaches are clearly desirable. One feature of human pancreatic adenocarcinoma is an exceedingly high frequency of K-ras mutation. The present study was conducted to determine if targeted activation of a human oncogenic-ras transgene in rat pancreas would induce carcinomas correspondent to human pancreatic ductal adenocarcinomas. We established transgenic (Hras250) rats in which expression of a human Ha-rasG12V oncogene is regulated by the Cre/lox system. Targeted pancreatic activation of the transgene was accomplished by injection of Cre-carrying adenovirus into the pancreatic ducts and acini through the common bile duct. Adenoviral infection of injected animals was exclusive to the pancreas; infected cells could be identified in duct, intercalated duct, centroacinar and, less frequently, acinar cells, but not in endocrine islet cells. Four weeks after injection, proliferative lesions in the duct epithelium, intercalated ducts and centroacinar cells, but not acinar cells, were widespread. Tumorigenesis in other tissues was not observed. Most lesions, including atypical duct proliferative lesions, PanIN-like lesions and carcinomas, were positive for cytokeratins 19 and 7, cyclooxygenase 2 and MMP-7 but negative for amylase and chymotrypsin. Many adenocarcinoma lesions were positive for EGF and EGFR. Duct epithelial and atypical duct proliferative lesions and carcinoma lesions were all positive for transduced Ha-rasG12V oncogene expression. The cytogenesis of pancreatic ductal type carcinoma was depicted. This model exhibits important similarities to the human disease and promises to advance our understanding of the behavior of pancreas adenocarcinomas and expedite screening and therapy.

Abbreviations: AB, alcian blue; CK, cytokeratin; COX2, cyclooxygenase 2; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; GFP, green fluorescent protein; MMP, matrix metalloproteinase; PAS, periodic acid-Schiff's; PCNA, proliferating cell nuclear antigen; Rb, retinoblastoma; RT–PCR, reverse transcriptase polymerase chain reaction


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results and discussion
 Summary
 Supplementary material
 References
 
Cancers of the pancreas are diagnosed in ~1 in 10 000 people/year in the United States and are the fifth leading cause of cancer death. Most patients die within 1 year of diagnosis (1), and the 5 year survival rate is <5% (2,3). Ductal adenocarcinomas are diagnosed in ≥95% of the patients with pancreatic cancer. Of these patients, only ~10–15% can undergo potentially curative resection. Even surgical resection, however, offers a very low cure rate; the 5 year survival rate is <10%. Radiation and chemotherapy have so far proven ineffective (1,4). Clearly, our current understanding of this disease is far from sufficient.

Animal models are used extensively in the study of human disease. In the Syrian hamster, pancreatic ductal neoplasms similar to those in man can be induced by propylnitrosamines (5). These tumors are characterized by a high rate of K-ras activation (6), a characteristic that is believed to be of particular importance in human pancreatic cancers: while activated Ras is associated with ~40% of all human cancers, the figure for pancreatic adenocarcinomas is >90% (7–9). The hamster model (10–12), however, suffers from a number of disadvantages. These include the necessity of using a potent non-specific carcinogen, the small size of the animal and the inability to raise hamsters in pathogen-free conditions.

Attention has now become concentrated on the use of activated oncogenes for the generation of pancreatic lesions in transgenic animals. In most mouse models reported to date, however, pancreatic lesions are derived from acinar cells. For example, transgenic mice carrying a K-rasG12D gene with an elastase promoter (13) or an H-rasG12V gene with an elastase promoter (14) both lead to the development of pancreatic ductal lesions, which are probably derived from acinar cells. In humans, the cytogenesis of human pancreatic ductal adenocarcinomas is unclear; whether they are derived directly from duct cells has not yet been unequivocally established. However, it is important to consider the possibility that rodent models in which pancreatic tumors are derived from elements that do not give rise to human pancreatic tumors may not appropriately model the human condition.

In another model system, using transgenic mice with a K-rasG12V transgene under the control of the cytokeratin 19 (CK19) promoter, there was some hyperplasia of ductal epithelial cells, but no discernable morphological changes in acinar or islet cells (15). Moreover, the hyperplastic lesions did not progress into tumors. Recently, human-like pancreatic premalignant lesions have successfully been generated in mice in which oncogenic K-rasG12D protein is expressed at endogenous K-ras levels in pancreatic progenitor cells (16), and when this oncoprotein is expressed in Ink4a/Arf null progenitor cells the resulting tumors become highly invasive and metastatic (17). While these two animal models are strikingly similar to human pancreatic intraepithelial neoplasias and ductal carcinomas, they do not specify the cellular compartment from which the lesions arise and tumor induction is independent of exogenous control.

We have been focusing on the advantages of rat models (18) and initially established a transgenic rat carrying a human c-Ha-ras proto-oncogene. These animals are highly susceptible to carcinogens, developing mammary, skin, esophagus and bladder tumors depending on the carcinogen and route of exposure (18–21). Interestingly, in these tumors there is an extremely high incidence of mutation of the human c-Ha-ras transgene. Therefore, we proceeded to establish a transgenic rat carrying a human Ha-rasG12V oncogene in which expression is regulated by the Cre/lox system (22). Targeted expression of Cre recombinase specifies the tissues or cell types in which the Ha-rasG12V oncoprotein is expressed.

The present study was conducted to determine if transgenic rats carrying a conditionally expressed human Ha-rasG12V oncogene would be susceptible to induction of ductal pancreatic adenocarcinomas when Ha-rasG12V protein expression was induced in pancreatic tissue. Targeted activation of Ha-rasG12V was accomplished by injection of a Cre-carrying adenovirus into the pancreatic ducts through the common bile duct. Preneoplastic lesions derived from ductal and centroacinar cells that developed into adenocarcinomas were indeed induced. This rat model is a promising experimental system to provide a better understanding of pancreatic ductal adenocarcinoma cell origin and behavior and expedite experimental screening and therapies of one of the most lethal human carcinomas.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results and discussion
 Summary
 Supplementary material
 References
 
Recombinant DNA constructs and conditional transgenic rats
The CALNLHras (CAG promoter-loxP sequence–neo-resistant gene-loxP sequence–Ha-rasG12V) switching unit was constructed following the original protocol of Kanegae (22). Briefly, total RNA was extracted from cultured T24 cells (Cell Resource Center for Biomedical Research Institute of Development, Aging and Cancer, Tohoku University, Sendai, Japan) bearing an activated c-Ha-ras gene (23,24) using ISOGEN (NIPPON GENE, Toyama, Japan). The RNA was treated with DNase I (amplification grade; Life Technologies, Gaithersburg, MD), and reverse transcription polymerase chain reaction (RT–PCR) was performed with SuperScript II Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA) using oligo(dT)12–18 to prime first-strand DNA synthesis. The primers 5'-atgacggaatataagctggtggtggtg-3' and 5'-tcaggagagcacacacttgcagc-3' were used to amplify Ha-rasG12V. The resultant 570-bp amplicon was cloned into pCR2.1-TOPO using the TOPO TA Cloning system (Invitrogen), and the sequence was confirmed with a Thermo Sequenase Cy 5.5 Terminator cycle sequencing kit (Amersham Biosciences, Piscataway, NJ). The complete Ha-rasG12V sequence was subcloned into the SwaI site of pCALNLw (Riken Bioresources Center DNA Bank) to produce pCALNLHras. pCALNLHras was digested with SalI and HindIII, run out on an agarose gel, and the CALNLHras cassette was cut out and purified using a QIAquick Gel Extraction kit (QIAGEN). A total of 1 to 2 pl of the purified cassette at a concentration of 3.0 ng/µl was injected into the pronuclei of Sprague–Dawley rats (CLEA Japan, Tokyo, Japan). Techniques used for the generation of transgenic rats were the same as those reported previously (25). Transgenic founder rats were mated with Sprague–Dawley rats, and offspring were screened for the presence of the transgene by PCR and Southern blot analysis of genomic DNA isolated from tail biopsies at the age of 3 weeks.

Preparation of adenovirus vectors
Recombinant adenovirus vectors carrying the Cre gene (AxCANCre) or the GFP gene (AxCAGFP) and a control empty adenovirus vector (AxCAwt) were prepared as described previously (22). The virus stock was concentrated and purified at 1.0 x 1010 pfu/ml as reported previously (26).

Tumor induction and pathological examination
Twenty-eight 7-week-old male and female Hras250 rats were divided into two groups. One group received AxCANCre (21 rats) and the other group received AxCAwt (7 rats). To determine the site of infection, an adenovirus carrying the GFP reporter gene (AxCAGFP) was injected. All injections were into the pancreatic duct through the common duct according to the method of Taniguchi et al. (27). Animals were injected with 150 µl adenovirus solution (4 x 109 pfu/ml in DMEM) with a 31-G needle. A vital tracking dye (Indigo carmine, 2 mg/ml) was added to the vector suspension for visualization (27). Animals were killed by exsanguination from the inferior vena cava under deep ethyl ether anesthesia. The pancreas was excised and fixed in phosphate-buffered 10% formalin or acetone and processed for paraffin embedding for histological observation. All experiments were conducted according to the ‘Guidelines for Animal Experiments of the Nagoya City University Graduate School of Medical Sciences’ and the ‘Guidelines for Animal Experiments of the National Cancer Center’ of the Committee for Ethics of Animal Experimentation of the respective institutes.

Immunostaining
Tissues were fixed in 4% formaldehyde fixative and embedded in paraffin. Primary antibodies against green fluorescent protein (GFP; CLONTECH Laboratories, CA) diluted 1 : 100; CK19 (Abcam, Cambridge, UK), diluted 1 : 100; cytokeratin 7 (CK7) (DAKO, Carpinteria, CA), undiluted; cyclooxygenase 2 (COX2) (IBL, Takasaki, Japan), diluted 1 : 20; epithelial growth factor (EGF; Biomedical, Stoughton, MA), diluted 1 : 100; EGF-receptor (EGFR; UBI, Lake Placid, NY), diluted 1 : 25; matrix metalloproteinase-7 (MMP-7; Santa Cruz, CA), diluted 1 : 50; proliferating cell nuclear antigen (PCNA; DAKO), diluted 1 : 50; anti-{alpha}-amylase (Sigma, St Louis, MO), diluted 1 : 200; and chymotrypsin (Biogenesis, Poole BH177DA England, UK), diluted 1 : 100 were incubated with slides for 2 h at room temperature to overnight at 4°C, and staining was performed using the biotin peroxidase complex (ABC) method (Vectastain ABC kits, Vector Laboratories, Burlingame, CA). For PCNA staining, section slides were autoclaved for 15 min in a 10 mM citrate buffer (pH 6.0) and then allowed to cool for 30 min before incubation with antibodies. For visualization of the transfer of GFP by adenovirus AxCAGFP in vivo, on Day 1 after injection of AxCAGFP the pancreas, liver, duodenum and spleen were examined. For mucin staining, periodic acid-Sciff's (PAS) and alcian blue (AB) staining were performed. Immunostaining of mucins using human antibodies such as MUC1 and MUC5a/c was negative throughout the normal pancreas tissue and neoplastic lesions including those of PanIN morphology, possibly owing to lack of cross-reactivity (rat antibodies are not currently available).

Detection of recombination and expression of the transgene in vivo
Pancreatic tissues were isolated 4 and 5 weeks after injection of AxCANCre or AxCAwt and genomic DNA and proteins were extracted using standard methods (28). Genomic DNA was used as the template for PCR reactions for detecting transgene recombination. The primers (Figure 1, arrows) were CAGp-f: 5'-cgtgctggttgttgtgctgtct-3' (in the CAG promoter region) and Hras3-4r: 5'-cctccactccctgccgggtc-3' (in the Ha-rasG12V coding region). PCR amplification was performed with TaKaRa Ex Taq TM (TAKARA SHUZO). Samples were denatured at 95°C for 5 min and then amplified for 33 cycles at 95°C for 40 s and 72°C for 2 min. A final extension at 72°C for 10 min was performed after the last cycle. Anti-pan Ras antibodies (Upstate, Charlottesville, VA) and goat anti-mouse IgG-HRP (Southern Biotechnology Associates, Birmingham, AL) were used to detect total Ras protein, and a Ras activation kit (Upstate) was used to detect activated Ras protein. All samples were subjected to SDS–PAGE in a 4–20% gradient gel (Daiichi Pure Chemicals, Tokyo, Japan) and electroblotted onto Immobilon-P membranes (Millipore, MA). Before incubation with antibodies, the blots were blocked with 5% skim milk in TPBS [0.1% Tween-20 in phosphate-buffered saline (PBS)]. Blots were incubated with antibodies according to the manufacturer's instructions. Visualization was carried out with the ECL+plus Western Blotting Detection System (Amersham Biosciences).


Figure 1
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  		Fig. 1 (A) The CALNLHrasG12V transgene is comprised of a hybrid CMV enhancer/chicken beta-actin promoter (CAG), a cassette for the neomycin resistance gene flanked by loxP sites, and a sequence containing human Ha- rasG12V. Infection with the Cre recombinase-carrying adenovirus AxCANCre results in Cre-mediated recombination of the transgene and removal of the neo-coding region and its associated mRNA polyadenylation signal, generating a functional Ha-rasG12V gene expression unit. pA, SV40 early poly(A) site; GpA, rabbit-ß-globin poly(A) site. Arrows, primers for the detection of recombination of the transgene. (B) Localization of adenovirus infection in the pancreas. At Day 1 after injection of the GFP-carrying adenovirus AxCAGFP into the pancreata of transgenic Hras250 rats, GFP is clearly shown in epithelial cells of a duct (panel a, arrows), intercalated ducts (panels b and d, arrows), centroacinar cells (panels b and c, arrowheads) and acinar cells (panels a–d, open arrowheads) (x132).

 
Detection of recombination of the transgene by laser capture microdissection (LCM)
Microdissection was performed using a PixCell IIe Laser Capture Microdissection Instrument (Arcturus Engineering, Mountain View, CA). DNAs were isolated from the captured samples and were used as templates for PCR reactions for detecting transgene recombination. The primers (Figure 1, arrows) were CAGp-f: 5'-cgtgctggttgttgtgctgtct-3' (in the CAG promoter region) and Hras2-3r: 5'-gatctgctccctgtactggtgg-3' (in the Ha-rasG12V coding region). PCR amplification was performed with TaKaRa Ex Taq TM (TAKARA SHUZO). Samples were denatured at 95°C for 5 min and then amplified for 40 cycles at 95°C for 40 s, 60°C for 30 s and 72°C for 1 min. A final extension at 72°C for 10 min was performed after the last cycle.

RT–PCR analysis
Total RNA from gross tumors isolated 4 and 5 weeks after adenovirus injection was prepared using the ISOGEN method (NipponGene) according to the manufacturer's protocol. Total RNA derived from AxCAwt-injected rat pancreas (control) was isolated as follows: pancreas samples were frozen using liquid nitrogen, the frozen sample was ground to a powder under liquid nitrogen, RNA was extracted using ISOGEN and the extracted RNA was spun (12 h at 41 000x g at 4°C) through a 5.7 M cesium chloride cushion containing 25 mM acetate (pH 6.0) and 1 mM EDTA and resuspended in RNA Secure (Ambion, Austin, TX). A total of 500 ng of total RNA (from control pancreas and tumor samples) was reverse-transcribed using Superscript III Reverse Transcriptase (Invitrogen) according to the manufacturer's instructions. Reverse-transcription reaction mixtures were diluted 1 : 5 and 1 µl was used for PCR. Real-time PCR was performed in a SmartCycler II (Cepheid, Sunnyvale, CA) using TaKaRa Ex Taq HS (TAKARA SHUZO) with SYBR Green in the reaction mixture according to the manufacturer's instructions. The initial amplification of nested PCR (Ink4a and Arf) was also performed in a SmartCycler II using TaKaRa Ex Taq HS, but without SYBR Green in the reaction mixture. The second amplification of nested PCR was performed in an iCycler (Bio-Rad, Hercules, CA) using 0.5 µl of initial PCR product in a 20 µl reaction with TaKaRa Ex Taq HS according to the manufacturer's instructions. The following primers were used: initial amplification of p16-Ink4a, 5'-gcgggcactgctggaa-3' and 5'-cacctgggcgtgcttga-3'; nested amplification of p16-Ink4a, 5'-ggggcttcaccaaacgc-3' and 5'cgttcccagcggaggaga-3'; initial amplification of p19-Arf, 5'-tcgtggccttggtgttga-3' and 5'- cacctgggcgtgcttga -3'; nested amplification of p19-Arf, 5'-agggccgcagccacat-3' and 5'- ccgcaaataccgcacgac-3'; Madh4, 5'-tcagcaccacccgcctat-3' and 5'-ctgccgcaaatcaaagacc-3'; p53, 5'-gatgcccgtgctgccgaggagt-3' and 5'-tgggccaggaaccagtttgcataga-3'; Mdm2, 5'-gtggcccagatgctcctgtca-3' and 5'-tggctcgatggcgttcagaga-3'; Rb, 5'-ataggcggtatttaaggtagcgtcag-3' and 5'-agcccttgacctaaacccaactaac-3'; p21-WafI/CipI, 5'-gtggccttgtcgctgtcttg-3' and 5'-tgggcacttcagggctttct-3'; cyclin D1, 5'-ggggattcaggacgactctta-3' and 5'-agcggcggcaagaatgt-3'; cyclin D2, 5'-cgatgccctgacggagctg-3' and 5'-ctcttgccgcccgaatgg-3'; cyclin D3, 5'-cttcagaaatcccgatagacgc-3' and 5'-gccaccagcccaaacctt-3'; ß-actin, as an internal control, 5'-ccgtaaagacctctatgccaaca-3' and 5'-cggactcatcgtactcctgctt-3'. Nested PCR parameters were as follows: initial amplification of p16-Ink4a, denature at 95°C for 3 min, amplify for 25 cycles at 95°C for 15 s and 60.5°C for 64 s; nested amplification of p16-Ink4a, denature at 95°C for 3 min, amplify for 20 cycles at 95°C for 20 s, 60°C for 20 s and 72°C for 30 s; initial amplification of p19-Arf, denature at 95°C for 3 min, amplify for 25 cycles at 95°C for 15 s and 61°C for 64 s; nested amplification of p19-Arf, denature at 95°C for 3 min, amplify for 20 cycles at 95°C for 20 s, 60°C for 20 s and 72°C for 27 s. For real-time PCR, samples were denatured for 3 min at 95°C and amplified for 35 cycles. Cycling parameters were as follows: Madh4, 95°C for 15 s and 61°C for 77 s; p53, 95°C for 15 s and 72°C for 30 s; Mdm2, 95°C for 15 s and 67.5°C for 27 s; Rb, 95°C for 15 s and 66°C for 30 s; p21-WafI/CipI, 95°C for 15 s and 64°C for 37 s; cyclin D1, 95°C for 15 s and 62°C for 60 s; cyclin D2, 95°C for 15 s and 70°C for 22 s; cyclin D3, 95°C for 15 s and 61°C for 80 s; ß-actin, 95°C for 15 s and 63°C for 29 s. PCR amplicons that were run out on agarose gels were generated using the same conditions as for real-time PCR except that reactions were stopped when the amplifications were in the log linear phase: Madh4, 28 cycles; p53, 30 cycles; Mdm2, 29 cycles; Rb, 30 cycles p21-Waf1/Cip, 31 cycles; cyclin D1, 27 cycles; cyclin D2, 27 cycles; cyclin D3, 33 cycles; and ß-actin, 24 cycles.


   Results and discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
Summary
 Supplementary material
 References
 
Generation of rats with conditional regulation of activated c-Ha-ras expression
We obtained three founder rats carrying the CALNLHrasG12V transgene (Figure 1) transmittable to descendant generations (Hras218, 246 and 250). In these rats, Cre recombinase excises the stuffer DNA between the CAG promoter and the Ha-rasG12V gene, thereby initiating gene expression (Figure 1A). Southern blotting analysis indicated that Hras218 and 250 had one copy, and Hras246 had three copies of the transgene. Hras250 rats were used for these studies.

Adenovirus-mediated AxCAGFP expression
For detection of adenovirus-mediated gene transfer into adult rat pancreatic cells, a GFP-carrying adenovirus (AxCAGFP) was injected into the pancreatic ducts through the common bile duct. Immunostaining for GFP after injection indicated a clear localization of gene transfer into pancreatic duct cells (Figure 1B, panel a, arrows), intercalated duct cells (Figure. 1B, panels b and d, arrows) and centroacinar cells (Figure 1B, panels b and c, closed arrowheads). A small number of acinar cells were also positive for GFP (Figure 1B panels a–d, open arrowheads). Islet cells were negative for GPF. Liver, spleen and intestines were also examined for infection; these organs were negative (not shown).

Detection of transgene activation
For transgene activation, Hras250 rats were injected with an adenovirus carrying the Cre gene (AxCANCre); injection of an adenovirus without an exogenous insert (AxCAwt) served as a negative control. Four weeks after injection, genomic DNA was isolated from the pancreata of AxCANCre and AxCAwt animals and subjected to PCR. A 1784-bp band corresponding to the unmodified transgene was detected in the pancreata of both animals (Figure 2A, left panel). In addition, a 556-bp band corresponding to the recombinant transgene was generated by PCR in the AxCANCre group (Figure 2A, left panel). Activation of the transgene was assessed by western blot analysis. An extremely high level of active Ras was detected in the pancreata of the AxCANCre animals compared with AxCAwt animals (Figure 2A, right panel), indicating AxCANCre-mediated activation of the transgene.


Figure 2
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  		Fig. 2 (A) Induction of recombination of the CALNLHrasG12V transgene and activation of Ras expression four weeks after injection of AxCANCre into the pancreatic duct. A 556-bp band is apparent in pancreas tissue of Hras250 rats injected with AxCANCre, but not in rats injected with the empty control adenovirus AxCAwt, indicating recombination of the transgene (left panel). Activated Ras is clearly detected in the pancreas of AxCANCre-injected rats by western blot; very little activated Ras was detected in AxCAwt-injected animals (right panel). (B) Macroscopic appearance of pancreatic tumors induced by human activated c-Ha-ras at Week 4. Many nodules ~1–3 mm in diameter were observed in the pancreata of the AxCANCre group at Week 4 (left). No pancreatic lesions were induced in rats injected with AxCAwt (right). (C) (x33) Most of the nodular lesions were diagnosed as adenocarcinoma (carcinoma) with obvious invasion of the surrounding acinar structure (acini).

 
Gross and histological observation of pancreatic duct lesions
Animals were killed 4, 5 and 9 weeks after injection of AxCANCre or AxCAwt. At Week 4, many grossly visible whitish nodules of ~1–3 mm in diameter were observed throughout the pancreas in the AxCANCre group (Figure 2B, left photograph), but not in the AxCAwt group (Figure 2B, right photograph). Histological examination determined that these nodules were adenocarcinomas (Figure 2C). Incidence of tumors in the AxCANCre group was 100% (21 out of 21 animals). One animal died at Week 4, presumably as a result of tumor-associated pancreatic malfunction. By Week 9, a total of seven of the AxCANCre animals had died, all with severely damaged pancreas. No neoplastic lesions were evident in organs other than the pancreas (data not shown). None of the AxCAwt group displayed any pancreatic lesions (or lesions in other tissues) even after 9 weeks (not shown).

Histological characterization of the lesions
Early proliferative lesions that developed in AxCANCre-treated animals were largely divided into three types: intraductal epithelial lesions, centroacinar cell lesions and ductular (intercalated duct) lesions. The epithelial cells in the intraductal lesions tended to be tall columnar and the lesions were quite similar to those of human intraductal PanIN-1 (Figure 3) or PanIN-2/3 (Figure 4) (29,30). The cells were positive for CK19 and CK7. These cells were also positive for COX2 and EGF. Compared with the PanIN-1 lesions, the more advanced PanIN-2/3 lesions exhibited a more dramatic increase in COX2. The lesions also began displaying increased EGFR in the cell membranes and MMP-7 in the cytoplasm near the apical border. PanIN-2/3 lesions with obvious intraluminal growth and papillary projection showed some irregular localization of ß-catenin. The lesions were partly positive for mucopolysaccharides, as demonstrated by PAS and AB. Lesion cells were negative for amylase and chymotrypsin (Figure 4, amylase data not shown). The presence of COX2 in the cytoplasm indicates similarity to human pancreatic carcinomas. Co-expression of EGF and EGFR indicates that tumor growth may be controlled in an autocrine manner, and the presence of MMP-7, another feature of human pancreatic carcinomas (31–34), indicates progression toward malignancy.


Figure 3
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  		Fig. 3 (x66) Semi-serial sections through an early intraductal proliferative lesion in a pancreatic duct. Lesion cells are slightly tall columnar with some cellular and nuclear dysplasia resembling a human PanIN-1 lesion. The lesion is positive for CK19 and CK7 with increases in COX2 and EGF.

 

Figure 4
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  		Fig. 4 (x66) Semi-serial sections through an intraductal proliferative lesion in a pancreatic duct. The lesion is positive for CK19 and CK7 and shows increases in EGF, EGFR, COX2 and MMP-7 (insets are x5 higher magnification). The cellular arrangement is irregular, as can be seen with ß-catenin staining, partly exhibiting papillary formation resembling PanIN-3 lesions. Some epithelial cells have mucinous material positive for PAS and AB staining. Chymotrypsin is negative (inset, acini on the same slide).

 
Early centroacinar cell lesions (Figure 5A, arrowheads) and ductular proliferative lesions (Figure 5A, arrows) exhibited a transitional figuration to an incomplete duct-like arrangement with nuclear atypia and increased labeling for PCNA (Figure 5A, right panel). No PCNA labeling was observed in acinar cells. These ductular lesions also showed expansive growth into the surrounding acinar structure (Figure 5B). Most of the cells comprising the lesions were positive for CK19, CK7, COX2 (not shown), EGF, EGFR (not shown) and MMP-7; were weakly positive for AB in the cytoplasm and at the apical boarder; and were negative for chymotrypsin and amylase (Figure 5B and C).


Figure 5
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  		Fig. 5 (A) (x132) Proliferation of intercalated duct (arrows) and centroacinar cell lesions (arrowheads) (left). Cells exhibit incomplete duct-like arrangements with slight nuclear atypia. An obvious increase in PCNA labeling was observed (right). (B) (x66) These atypical ductular proliferative lesions exhibit compression of the surrounding acinar structure. The cells in the lesions were variably positive for CK19, CK7, EGF, MMP-7 and PCNA. (C) (x66) Another view of an atypical ductular proliferative lesion. The higher magnification (x264) panel clearly shows that AB is weakly positive in the cytoplasm in some cells. Amylase and chymotrypsin are negative.

 
Advanced lesions, including adenocarconomas of moderately differentiated morphology, exhibited expansive growth into the surrounding acinar structure (Figures 6 and 7). These lesions shared several common characteristics with early proliferative epithelial and ductular lesions: many of the component cells were positive for CK19, CK7, EGF, EGFR, COX2 and MMP-7; weakly positive for mucopolysaccharides; and negative for chymotrypsin and amylase. Carcinoma tissue occasionally showed invasion of liver capsular tissue with accompanying fibrous reaction (not shown). Proliferative lesions in the acinar component were not observed. Comparison of cellular characteristics comprising the different lesions are summarized in Table I.


Figure 6
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  		Fig. 6 In larger lesions, cells with moderate nuclear atypia exhibit a fusiform arrangement. (A) (x33) Many of the component cells were positive for CK19, CK7, EGF, EGFR, COX2 and MMP-7. Middle top (x123) is a higher magnification view of left top. In contrast to acinar cells, although the cytoplasm is slightly eosinophilic, no granular material was observed. ß-catenin is localized primarily in the cell membrane, but is also seen in the cytoplasm. Insets (x66) are from the same lesion as the top middle panel. (B) (x33) Similar lesion as in A. The apical borders of the tumor cells are slightly positive for PAS and AB staining (inset x66), but entirely negative for chymotrypsin and amylase [insets are intact acini from the respective slides (x66)].

 

Figure 7
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  		Fig. 7 (A) (x33) Moderately differentiated adenocarcinoma, exhibiting characteristics similar to those in Figure 6. Fusiform glands are irregularly arranged and surrounded by fibrous tissue resembling a desmoplastic morphology. Many of the component cells were positive for CK19, COX2, EGF (x33 and x99), EGFR (x33 and x99) and MMP-7. ß-catenin is localized primarily in the cell membrane. (B) (x66) Similar lesion as in A. Carcinoma cells are negative for both chymotrypsin and amylase. Inset (x66) are acini from the respective slides.

 

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  		Table I Summary of cellular characteristics

 
Thus, the histopathological appearance of these adenocarcinomas closely resembles that described in humans (29,30). Most human pancreatic neoplasms have a ductal morphology (29). The exact cellular origin of these lesions, however, is not certain. For example, rats treated with the non-specific carcinogen 7,12-dimethylbenz[a]anthracene (DMBA) develop pancreatic tumors that exhibit ductal marker expression in the early stages of tumorigenesis (35), but the possibility that these tumors could arise through dedifferentiation of acinar cells has attracted a great deal of attention (36). In vivo experiments have provided evidence that suggests that injured or carcinogen-treated acinar cells may assume a ductal phenotype in the rat (36) and in the hamster (37). Also, transdifferentiation of neoplastic acinar cells to give rise to duct-like structures in the rat has been described previously (38). Moreover, several models for the induction of duct-like tumors in transgenic mice appear to involve acinar cell precursors (13,14). In vitro studies of acinar cell preparations have also shown transition of acinar cells to duct-like cells (39–41).

Our results clearly indicate that the pancreatic lesions induced in the Hras250 rat express duct characteristics but do not express an acinar phenotype. Thus, our findings suggest that centroacinar and intercalated duct epithelial cells were the precursors of adenocarcinomas, and that de-differentiation of acinar cells was not a contributing factor. Our results agree with the observation made in the Pten knockout mouse that centroacinar cell proliferation may be a cause of pancreatic ductal carcinomas (42). A schematic diagram of Hras250 rat pancreatic carcinogenesis is summarized in Figure 8. Precursor lesions are hyperplastic and dysplastic proliferation of centroacinar cells, intercalated ducts and duct epithelium exhibiting PanIN-like lesions, but not of acinar cells. Irrespective of cell origin the eventual morphology is adenocarcinoma with a ductular phenotype.


Figure 8
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  		Fig. 8 Schematic presentation of cytogenesis of pancreatic ductal adenocarcinomas induced in human Ha- rasG12V transgenic rats. Precursor lesions are hyperplastic and dysplastic proliferation of centroacinar cells, intercalated ducts and duct epithelium (PanIN-like lesions), but not of acinar cells. Irrespective of cytogenesis, the eventual morphology is adenocarcinoma with ductular phenotype.

 
Detection of recombination of the transgene
To identify in which cellular compartment the transgene underwent recombination, samples were removed using LCM (Figure 9A), the DNA was extracted and PCR was performed. A 1624-bp band corresponding to the unmodified transgene was detected in all captured samples (Figure 9B). A 396-bp band corresponding to transgenes that had undergone recombination was generated by PCR in tumor tissue (Figure 9A, panel a and 9B, lanes 3 and 4), mixed populations of proliferating centroacinar cells and intercalated ducts (Figure 9A, panel b and 9B, lanes 5 and 6), a PanIN-2/3-like lesion (Figure 9A, panel c and 9B, lanes 7 and 8) and in normal-looking acinar cells surrounding mixed populations of proliferating centroacinar cells and intercalated ducts (Figure 9A, panel d and 9B, lanes 9–11). A recombinant transgene was not detected in acinar (Figure 9B, lane 1) or ductal cells (Figure 9B, lane 2) obtained from the pancreata of AxCAwt-treated animals. Pancreatic cancers exhibit multiple genetic and molecular alterations, several of which have been identified in the last few years (8,43–46). It is generally well accepted that K-ras gene mutation is a highly frequent and early event in pancreatic carcinogenesis (6–9,43–46) Similarly, in the Hras250 rat, the initiating signal in pancreatic ductal adenocarcinogenesis is expression of the Ha-rasG12V oncogene.


Figure 9
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  		Fig. 9 Detection of recombination of the transgene by LCM. (A) Tissue from a pancreas of an AxCANCre-treated animal captured by LCM. Panel a, adenocarcinoma; panel b, mixed population of proliferating centroacinar cells and intercalated ducts; panel c, PanIN-2/3-like lesion; panel d, normal looking acinar cells surrounding proliferating centroacinar cells and intercalated ducts. (B) PCR amplification of the transgene. The 1624-bp band corresponds to the unmodified transgene, and the 396-bp band corresponds to transgenes that have undergone recombination. 1, acinar tissue from an AxCAwt-treated pancreas; 2, duct from an AxCAwt-treated pancreas; 3 and 4, captured tumor tissue from panel A-a; 5 and 6, mixed population of proliferating centroacinar cells and intercalated ducts from panel A-b; 7 and 8, PanIN-2/3-like lesion from panel A-c; 9,10 and 11, normal looking acinar cells surrounding a mixed population of proliferating centroacinar cells and intercalated ducts from panel A-d.

 
Molecular analysis of pancreatic adenocarcinomas
We conducted a molecular analysis of the tumors to determine the status of pathways that are commonly altered in human pancreatic adenocarcinomas (8,43–46). We assessed the expression profile of Madh4, p21-Waf1/Cip1, p53, Mdm2, p19-Arf, Rb, p16-Ink4a, cyclin D1, cyclin D2 and cyclin D3 in pancreatic adenocarcinomas from AxCANCre-injected animals and pancreatic tissue from AxCAwt-injected animals (control pancreas) using PCR (Figure 10).


Figure 10
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  		Fig. 10 RT–PCR analysis of pancreatic tumors in Hras250 animals. Total RNA isolated from an AxCAwt-injected Hras250 rat pancreas (control) and five pancreatic tumor samples from AxCANCre-injected Hras250 rats were used to generate cDNA by reverse transcription. The cDNA was amplified using primers specific for Madh4, p21-waf1/cip1, p53, Mdm2, p19-Arf, Rb, p16-Ink4a and cyclin D1, D2 and D3. Amplification of ß-actin served as an internal control. (A) PCR was stopped during the log-linear phase of the reaction and amplicons were run out on an agarose gel. (B) Graphical results of semi-quantitative real-time PCR analysis. (Numerical data can be found in Supplemental material.) C, Control pancreas; 2, 4, 21, 23 and 24, pancreatic tumors from AxCANCre-treated Hras250 animals. *Amplicons were not generated for either p19-Arf or p16-Ink4a during real-time PCR. Therefore, nested PCR was used to generate amplicons for these cDNAs. Quantification was not attempted for the nested PCR products. # Amplicons were generated for cyclin D3 during real-time PCR only for the control tissue and not for any of the tumor tissues.

 
Madh4 (MAD homolog 4), also known as Smad4 and Dpc4, is an important regulator of the transforming growth factor-ß (TGF-ß) signaling pathway (47). In tumor tissue, expression of Madh4 was depressed. Decreased expression of Madh4 is expected to depress differentiation and anti-proliferative signaling by TGFß, resulting in enhanced growth.

p21-Cip1/Waf1 (cyclin-dependent kinase inhibitor 1A, Cdkn1a), an inhibitor of cyclin-dependent kinases (48,49), is another protein important in antiproliferative pathways (50,51). Like Madh4, expression of p21-Cip1/Waf1 was depressed in tumor tissue, and, as with Madh4, the expected consequence of decreased p21-Cip1/Waf1 expression is enhanced proliferation.

p53 signaling is believed to be disrupted in virtually all metastatic cancers (52–55). In contrast to Madh4 and p21- Cip1/Waf1, we could not detect any obvious downregulation of p53 in tumor tissue. Two key regulators of p53 are Mdm2 (56,57) and p19-Arf (57). Mdm2 promotes p53 protein degradation and p19-Arf stabilizes the p53 protein. Mdm2 and p19-Arf expression levels were unchanged in the tumor tissue. Therefore, in the pancreatic adenocarcinomas that developed in the Hras250 animals, the p53 signaling pathway is likely to be intact.

Inactivation of the retinoblastoma (Rb) signaling pathway is also thought to be a central theme in tumorigenesis (52–55). As with p53, there was no consistent change in Rb expression in tumor tissue compared with control pancreas. Important components of the Rb signaling pathway are the D-type cyclins and their associated kinase subunits, CDK4 and CDK6, and p16-Ink4a (cyclin-dependent kinase inhibitor 2A; Cdkn2a). Cyclin D-CDK4/6 complexes phosphorylate Rb and promote movement through the cell cycle (58), while p16-Ink4a binds to CDK4 and CDK6, inhibiting their activation by D-type cyclins, and represses movement through the cell cycle (59). Defects in Ink4a/Arf signaling have been shown to be important in pancreatic carcinogenesis: mutations in the Ink4a/Arf locus resulting in a null phenotype are frequently observed in human pancreatic cancers and are connected with a worse prognosis (43–46,60), and expression of K-rasG12D in Ink4a/Arf null pancreatic progenitor cells results in induction of highly metastatic ductal carcinomas (17). Expression of p16-Ink4a, a Ras inducible gene (59), was clearly elevated in Hras250 rat pancreatic tumor tissue, indicating that transcription of p16-Ink4a can still be activated in the tumor cells. In agreement with the finding that the tumor tissue had more PCNA positive cells than the surrounding tissue, cyclin D1 expression was also induced. These results suggest that the cells comprising the pancreatic adenocarcinomas are actively proliferating, but that the Rb pathway, which plays an important role in coordinating movement through the cell cycle, is probably intact.

Finally, expression of both cyclin D2 (Ccnd2) and cyclin D3 (Ccnd3) was reduced compared with control pancreatic tissue. This is consistent with a clonal origin of the tumors.

Taken together, Hras250 rat pancreatic carcinogenesis is initiated by expression of the Ha-rasG12V oncogene. Proliferation is further stimulated by an EGF–EGFR autocrine loop and decreased expression of inhibitory proteins such as Madh4 and p21-Cip1/Waf1. The resultant increase in proliferative capacity of the cells is demonstrated at the cellular level by increased expression of cyclin D1 and PCNA and at the tissue level by tumor formation. However, while the cells comprising the tumors exhibit abnormally high proliferation, p53 and Rb signaling pathways appear to be intact, suggesting essentially normal movement through each cell cycle.


   Summary
Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Summary
Supplementary material
 References
 
Few animal models exist that recapitulate human pancreatic ductal carcinogenesis. In this report, we document the establishment of a rat line, Hras250, carrying a human Ha-rasG12V oncogene under the control of the Cre/lox system. Injection of Cre-carrying adenovirus induces tubular adenocarcinomas of ductal and ductular origin in the pancreas with high penetrance and short latency. These adenocarcinomas arise from centroacinar cells, intercalated ducts and duct epithelium, but not acinar cells (Figure 8). Also, a preliminary survey suggests that Ha-rasG12V–initiated carcinogenesis may occur through proliferative mechanisms without the disruption of the p53 or Rb signaling pathways. These results, taken in conjunction with the morphology of the lesions, suggest that this animal model exhibits characteristics of premetastatic pancreatic carcinomas that are progressing towards frank malignancy. The Hras250 rat carrying a Cre recombinase-regulated human Ha-rasG12V gene promises to be an excellent tool for the analysis of pancreatic tumor histogenesis, screening and therapeutics. The ease of tumor induction; the similarity to human pancreatic tumors; the high penetrance, rapidity and multiplicity of tumor induction; and the large size of the rat pancreas are all factors that will facilitate research into activated ras-associated pancreatic neoplasia using this animal model.


   Supplementary material
Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Summary
 Supplementary material
References
 
Supplementary data are available at carcinogenesis online.


   Acknowledgments
 
We gratefully thank Dr Junichi Miyazaki of Osaka University (Osaka, Japan) for his kind provision of the CAG promoter. We also thank Drs M. Tsutsumi and Y. Konishi of Nara Medical University (Nara, Japan) and M. Tatematsu of Aichi Center Research Institute (Nagoya, Japan) for histological evaluation of induced lesions, Dr Takao Sekiya of Mitsubishi Kagaku Institute of Life Sciences (Machida, Tokyo) for gene construction and Dr Tomoyuki Shirai of Nagoya City University Graduate School of Medical Sciences (Nagoya, Japan) for kind advice and assistance in preparation of histological slides. This study was supported by the following grants: a Grant-in-Aid for Scientific Research (KAKENHI) on Priority Areas from the Ministry of Education, Science, Sports and Culture of Japan; a Grant-in-Aid for the Long-range Research Initiative from the Japan Chemical Industry Association (CC05-01); a Grant-in-Aid for the Second-Term Comprehensive 10-Year Strategy for Cancer Control and a Grant-in-Aid for Cancer Research from the Ministry of Health, Labour and Welfare, Japan; a Grant-in-Aid from the Surveillance Study for Assessment of Refined Petroleum Products from the Ministry of Economy, Trade and Industry.

Conflict of Interest Statement: None declared.


   References
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Summary
 Supplementary material
 References
 

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Received December 15, 2005; revised May 6, 2006; accepted May 8, 2006.


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