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Rapid Development of Salivary Gland Carcinomas upon Conditional Expression of K-ras Driven by the Cytokeratin 5 Promoter

2006; Elsevier BV; Volume: 168; Issue: 5 Linguagem: Inglês

10.2353/ajpath.2006.050847

1525-2191

Ana R. Raimondi, Lynn Vitale‐Cross, Panomwat Amornphimoltham, J. Silvio Gutkind, Alfredo Molinolo,

Cancer and Skin Lesions

We have used a recently described model in which a ras oncogene is expressed in cytokeratin 5 (K5)-expressing cells on doxycycline administration to explore the effects of this oncogene in salivary glands of adult mice. Inducible expression of a mutated K-ras gene under the control of the K5 promoter led to the development of hyperplastic and dysplastic epithelial lesions and carcinomas, with an incidence of 100% and a minimum latency of a week. All major salivary glands were affected, as well as a set of previously undescribed buccal accessory salivary glands located on the apex of the masseter muscle, close to the oral angle. The tumors appear to arise from the cytokeratin 5-positive basal cell compartment. Myoepithelial cells participated in the hyperplasias but not in carcinomas, because the tumors are negative for smooth muscle actin. Carcinomas did not accumulate immunoreactive p53 but are positive for p63, as assayed by immunohistochemistry using an antibody against the N terminus of ΔN p63, a splice variant of p63 that can inhibit p53 transcriptional activity. In this study, we provide evidence that the ras oncogene, targeted to a specifically sensitive cell compartment within the salivary glands, can trigger a series of event that are sufficient for full carcinogenesis. We have used a recently described model in which a ras oncogene is expressed in cytokeratin 5 (K5)-expressing cells on doxycycline administration to explore the effects of this oncogene in salivary glands of adult mice. Inducible expression of a mutated K-ras gene under the control of the K5 promoter led to the development of hyperplastic and dysplastic epithelial lesions and carcinomas, with an incidence of 100% and a minimum latency of a week. All major salivary glands were affected, as well as a set of previously undescribed buccal accessory salivary glands located on the apex of the masseter muscle, close to the oral angle. The tumors appear to arise from the cytokeratin 5-positive basal cell compartment. Myoepithelial cells participated in the hyperplasias but not in carcinomas, because the tumors are negative for smooth muscle actin. Carcinomas did not accumulate immunoreactive p53 but are positive for p63, as assayed by immunohistochemistry using an antibody against the N terminus of ΔN p63, a splice variant of p63 that can inhibit p53 transcriptional activity. In this study, we provide evidence that the ras oncogene, targeted to a specifically sensitive cell compartment within the salivary glands, can trigger a series of event that are sufficient for full carcinogenesis. Malignant tumors of the salivary glands are a relatively rare oral neoplasia, comprising <0.5% of all malignancies combined and ∼5% of malignancies of the head and neck region.1Speight PM Barrett AW Salivary gland tumours.Oral Dis. 2002; 8: 229-240Crossref PubMed Scopus (426) Google Scholar They have a special status in human neoplasia, because they exhibit the most complex histopathology of any tumor type, and for this reason they represent a morphologically diverse group of tumors.2Dardick I Color Atlas/Text of Salivary Gland Tumor Pathology. Igaku-Shoin Medical Publisher, Inc., New York1996Google Scholar This complexity is reflected in the current classification schemes that are mostly based in histological parameters,3Histological Typing of Salivary Gland Tumours World Health Organization International histological classification of tumours. TELOS, Springer-Verlag, New York1991Google Scholar although efforts have been made to correlate this growing list of tumors with their biological behavior.4Ellis GL Auclair PL Atlas of tumor pathology. Armed Forces Institute of Pathology Tumors of the salivary glands, Washington, DC1996: 31Google Scholar This histological approach to classification, although useful for other tumor types, has failed to satisfy the needs of those directly involved in the treatment of salivary gland tumors,1Speight PM Barrett AW Salivary gland tumours.Oral Dis. 2002; 8: 229-240Crossref PubMed Scopus (426) Google Scholar who often fail to see in these complex diagnoses a useful guide for treatment standardization. This problem, largely reflecting our lack of understanding of the possible correlation between histological patterns and tumor prognosis, has been the focus of extensive review.2Dardick I Color Atlas/Text of Salivary Gland Tumor Pathology. Igaku-Shoin Medical Publisher, Inc., New York1996Google Scholar Several approaches, including the use of chemicals,5Ide F Suka N Kitada M Sakashita H Kusama K Ishikawa T Skin and salivary gland carcinogenicity of 7,12-dimethylbenz[a]anthracene is equivalent in the presence or absence of aryl hydrocarbon receptor.Cancer Lett. 2004; 214: 35-41Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar viruses,6Azuma M Tamatani T Kasai Y Sato M Immortalization of normal human salivary gland cells with duct-, myoepithelial-, acinar-, or squamous phenotype by transfection with SV40 ori- mutant deoxyribonucleic acid.Lab Invest. 1993; 69: 24-42PubMed Google Scholar radioactive isotopes,7Espinal EG Ubios AM Cabrini RL Salivary gland tumors induced by 32P.J Oral Pathol. 1984; 13: 686-691Crossref PubMed Scopus (5) Google Scholar and genetically engineered animals (reviewed by Dardick8Dardick I Ho J Paulus M Mellon PL Mirels L Submandibular gland adenocarcinoma of intercalated duct origin in Smgb-Tag mice.Lab Invest. 2000; 80: 1657-1670Crossref PubMed Scopus (9) Google Scholar), have been used to developed experimental models that may help understand the carcinogenic process in salivary glands. Compared to other approaches, transgenic mouse models have the advantage of allowing a refined molecular dissection of each of the steps involved in tumor development and progression, because the variability within a given model is generally low and the carcinogenic events proceed through homogeneous steps and similar histological changes. Several of the available models have taken advantage of the mouse mammary tumor virus (MMTV) promoter to target the expression of different oncogenes to salivary and mammary glands. However, the resulting salivary gland phenotypes are not homogeneous across different models and have been less characterized than, for example, mammary gland tumors, the induction of which has been in most studies the original purpose of the experimental design. In this regard, a wide spectrum of premalignant and malignant changes have been described in the salivary glands of transgenic mice in which the MMTV promoter drives the expression of different oncogenes, though with a variable incidence rate and usually a prolonged latency. For example very few transgenic mice in which an int-1 allele resembling those found in virus-induced tumors is expressed from the MMTV long terminal repeat developed salivary adenocarcinomas.9Tsukamoto AS Grosschedl R Guzman RC Parslow T Varmus HE Expression of the int-1 gene in transgenic mice is associated with mammary gland hyperplasia and adenocarcinomas in male and female mice.Cell. 1988; 55: 619-625Abstract Full Text PDF PubMed Scopus (588) Google Scholar Incomplete differentiation and dysplastic changes in all glands, as well as poorly differentiated adenocarcinomas apparently arising from the parotid gland, have been reported in the MMTV-int-3 mouse,10Jhappan C Gallahan D Stahle C Chu E Smith GH Merlino G Callahan R Expression of an activated Notch-related int-3 transgene interferes with cell differentiation and induces neoplastic transformation in mammary and salivary glands.Genes Dev. 1992; 6: 345-355Crossref PubMed Scopus (299) Google Scholar and well circumscribed salivary gland adenocarcinomas developed with a low incidence (3 out of 62 mice) in the MMTV-Fgf8 transgenic mouse.11Daphna-Iken D Shankar DB Lawshe Ornitz DM Shackleford GM MacArthur CA MMTV-Fgf8 transgenic mice develop mammary and salivary gland neoplasia and ovarian stromal hyperplasia.Oncogene. 1998; 17: 2711-2717Crossref PubMed Scopus (55) Google Scholar On the other hand, 22% of MMTV-v-Ha-ras animals developed acinic cell carcinomas only in the parotid gland at 73 to 150 days of age.12Dardick I Burford-Mason AP Garlick DS Carney WP The pathobiology of salivary gland. II. Morphological evaluation of acinic cell carcinomas in the parotid gland of male transgenic (MMTV/v-Ha-ras) mice as a model for human tumours.Virchows Arch A Pathol Anat Histopathol. 1992; 421: 105-113Crossref PubMed Scopus (20) Google Scholar This limited effect of ras is surprising because expression of the activated form of genes for the Ras family of GTPases, H-Ras, K-Ras, and N-Ras has been associated with the pathogenesis of a wide variety of human tumors, including salivary gland neoplasm.13Rodenhuis S ras and human tumors.Semin Cancer Biol. 1992; 3: 241-247PubMed Google Scholar, 14Milasin J Pujic N Dedovic N Gavric M Vranik V Petrovic V Minic A H-ras gene mutations in salivary gland pleomorphic adenomas.Int J Oral Maxillofac Surg. 1993; 22: 359-361Abstract Full Text PDF PubMed Scopus (24) Google Scholar, 15Stenman G Sandros J Mark J Nordkvist A High p21RAS expression levels correlate with chromosome 8 rearrangements in benign human mixed salivary gland tumors.Genes Chromosomes Cancer. 1989; 1: 59-66Crossref PubMed Scopus (19) Google Scholar, 16Zachos G Spandidos DA Expression of ras proto-oncogenes: regulation and implications in the development of human tumors.Crit Rev Oncol Hematol. 1997; 26: 65-75Abstract Full Text PDF PubMed Scopus (46) Google Scholar Indeed, the H-ras gene has been found mutated in 35% of the salivary gland pleomorphic adenomas14Milasin J Pujic N Dedovic N Gavric M Vranik V Petrovic V Minic A H-ras gene mutations in salivary gland pleomorphic adenomas.Int J Oral Maxillofac Surg. 1993; 22: 359-361Abstract Full Text PDF PubMed Scopus (24) Google Scholar and in adenocarcinomas of the parotid glands, albeit with a slight lower incidence (23%).17van Halteren HK Top B Mooi WJ Balm AJ Rodenhuis S Association of H-ras mutations with adenocarcinomas of the parotid gland.Int J Cancer. 1994; 57: 362-364Crossref PubMed Scopus (10) Google Scholar Leaky transgenic expression of oncogenes in salivary gland tissues when using a variety of promoters can also result in a low incidence of salivary gland tumors. For example, animals expressing the human H-ras oncogene from the murine whey acidic protein promoter develop salivary gland adenocarcinomas in male animals after several months.18Andres AC Schonenberger CA Groner B Hennighausen L LeMeur M Gerlinger P Ha-ras oncogene expression directed by a milk protein gene promoter: tissue specificity, hormonal regulation, and tumor induction in transgenic mice.Proc Natl Acad Sci USA. 1987; 84: 1299-1303Crossref PubMed Scopus (152) Google Scholar Expression of a similar H-ras oncogene driven by the human prostate-specific antigen promoter leads to the development of salivary gland tumors of possible ductal origin, ranging from well differentiated adenocarcinomas to anaplastic carcinomas, in <50% of the animals older than 11 months.19Schaffner DL Barrios R Shaker MR Rajagopalan S Huang SL Tindall DJ Young CY Overbeek PA Lebovitz RM Lieberman MW Transgenic mice carrying a PSArasT24 hybrid gene develop salivary gland and gastrointestinal tract neoplasms.Lab Invest. 1995; 72: 283-290PubMed Google Scholar If instead a promoter from the neonatal submandibular gland secretory protein b gene is used to express the SV40 large T antigen specifically in salivary glands, at 1 month of age animals develop duct luminal cell hyperplasias that progress to dysplasia within 3 to 4 months and severe dysplasia and in situ carcinoma within 4 to 6 months. Submandibular gland adenocarcinomas originating from the intercalated ducts also develop in about half of the male mice by 12 months of age.8Dardick I Ho J Paulus M Mellon PL Mirels L Submandibular gland adenocarcinoma of intercalated duct origin in Smgb-Tag mice.Lab Invest. 2000; 80: 1657-1670Crossref PubMed Scopus (9) Google Scholar In this study, we took advantage of the observation that salivary glands express cytokeratin 5 (K5) in ductal epithelia and the availability of a recently described inducible animal model in which a ras oncogene is expressed in K5-expressing cells on doxycycline administration to explore the biological consequences of the conditional expression of this oncogene in salivary glands in adult mice. We found that, when a mutated K-ras gene was conditionally expressed in the salivary glands under the control of the K5 promoter, it induces hyperplastic and dysplastic lesions and carcinomas, with an incidence of 100% of the animals and a minimum latency as short as 1 week. Indeed, we provide evidence that the ras oncogene, when targeted to a specifically sensitive cell compartment within the salivary glands, can trigger a series of event that are sufficient for full carcinogenesis. The K5-rTA (K5-tet-on), tet-ras (K-ras4bG12D), and tetO-LacZ transgenic mice have been previously described.20Vitale-Cross L Amornphimoltham P Fisher G Molinolo AA Gutkind JS Conditional expression of K-ras in an epithelial compartment that includes the stem cells is sufficient to promote squamous cell carcinogenesis.Cancer Res. 2004; 64: 8804-8807Crossref PubMed Scopus (108) Google Scholar Doxycycline was administered via the drinking water at a concentration of 1 g/L to a total of 24 animals of 21 days of age. A similar number of wild-type animals as well as transgenic mice not receiving doxycycline treatment were used as controls. All mice were examined daily. K5-tet-on/tet-o-ras mice as well as control animals were injected with 5-bromo-2′-deoxyuridine (BrdU; Sigma-Aldrich Corp., St. Louis, MO) and euthanized with CO2 after 2 hours. All major salivary glands were carefully dissected. To better assess the location and microscopic features of the accessory buccal glands, samples, including skin and buccal mucosa from the oral angle, were obtained from several wild-type animals. All tissues were fixed overnight in buffered 4% paraformaldehyde, transferred to 95% ethanol, and embedded in paraffin. Five-micron sections were cut and stained with hematoxylin and eosin or used for immunocytochemical studies. Alcian blue staining was used in selected buccal salivary gland sections to provide a better contrast for illustration purposes. To ascertain the expression of the promoter, double transgenic mice expressing the β-galactosidase gene under the control of the K5-tet-on system were euthanized with CO2. Cryostat sections were obtained from frozen salivary gland samples and fixed in 4% buffered paraformaldehyde 1 hour at 4°C. For immunocytochemical studies, the following antibodies were used: rabbit polyclonal antibody against mouse keratin 5 (Covance, Denver, PA) at 1:500 dilution; mouse p63monoclonal antibody (4A4, sc-8431 Santa Cruz Biotechnology Inc., Santa Cruz, CA), raised against amino acids 1 to 205 mapping at the N terminus of ΔN p63of human origin, at 1:100 dilution; rabbit p53 monoclonal antibody (Cell Signaling Technology, Beverly, MA) at 1:40 dilution; rabbit polyclonal antibody against smooth muscle actin (RB-9010; Lab Vision, NeoMarkers and Lab Vision Corporation, Fremont, CA) at dilution 1:100; and rat anti-BrdU monoclonal antibody (Accurate Chemical, Westbury, NY) at 1:10 dilution. Secondary antibodies were conjugated with biotin (Vector, Burlingame, CA) and diluted 1:400. All antibodies were diluted in 2.5% bovine serum albumin (BSA, Sigma, St. Louis, MO) in phosphate-buffered saline (PBS). Cryostat sections were washed with PBS and processed for β-galactosidase staining.21Ramirez A Bravo A Jorcano JL Vidal M Sequences 5′ of the bovine keratin 5 gene direct tissue- and cell-type-specific expression of a lacZ gene in the adult and during development.Differentiation. 1994; 58: 53-64PubMed Google Scholar The tissues slides were dewaxed in SafeClear II (Fisher), hydrated through graded alcohols, and immersed in 3% hydrogen peroxide in PBS for 30 minutes to quench the endogenous peroxidase. After washing in distilled water, antigen retrieval was performed with 10 mmol/L citric acid in a microwave for 20 minutes (2 minutes at 100% power and 18 minutes at 10% power). Slides were allowed to cool for 30 minutes at room temperature, rinsed thoroughly with distilled water and PBS, and incubated in blocking solution (2.5% BSA in PBS) for 30 minutes at room temperature. Excess solution was discarded, and the sections were incubated with the primary antibody diluted in blocking solution at 4°C overnight. After washing with PBS, the slides were sequentially incubated with the biotinylated secondary antibody (1:400; Vector, Burlingame, CA) for 1 hour, followed by the avidin-biotin complex method (Vector Stain Elite, ABC kit; Vector) for 30 minutes at room temperature. For BrdU staining the slides were dewaxed as indicated, incubated with 2 N HCl at 37°C for 1 hour, and washed with water and PBS and then finally with 2.5% BSA in PBS (PBS-BSA). After discarding the PBS-BSA solution, the slides were incubated with the anti-BrdU antibody at 4°C overnight. The secondary antibody used was anti-rat immunoglobulins (Vector), diluted 1:400 in PBS-BSA at room temperature for 30–45 minutes. The slides were washed and developed in 3,3′-diaminobenzidine (FASTDAB tablet; Sigma) under microscopic control. The reaction was stopped in tap water, and the tissues were counterstained with Mayer's hematoxylin, dehydrated, and mounted. Briefly the sections were rinsed in PBS and washed for 15 minutes in rinse buffer (21). They were then incu-bated overnight at 37°C in X-gal staining solution (1 mg/ml 5-bromo-4-chloro-3-indolyl-β-galactopyranoside, 10 mmol/L K3Fe(CN)6, 10 mmol/L K4Fe(CN)6 and 1.2 mmol/L MgCl2 in PBS). After the reaction was fully developed, the sections were postfixed in 4% paraformaldehyde and counterstained with Nuclear Fast Red. All stained slides were scanned at 200× using an Aperio T3 Scanscope (Aperio Technologies, Inc., Vista, CA) to produce high definition images. For selected samples, several partially overlapping high definition images were taken with a total magnification of 160× using a real-time Retiga 1300 digital camera with the QImaging Pro software (MVIA, Inc., Monaca, PA). The images were then stitched together using the PanaVue Image Assembler Software (PanaVue Co., Quebec City, QC, Canada). Dysplasias were defined as any intraepithelial, noninvasive morphological change carrying any atypical cytological markers that are widely accepted to precede invasive carcinomas.22Robbins & Cotran Pathologic Basis of Disease. W. B. Saunders, Philadelphia2004Google Scholar Mice expressing the K-ras oncogene under the control of the K5-tet-on-inducible system developed a wide range of proliferative lesions in the skin and other squamous epithelia.20Vitale-Cross L Amornphimoltham P Fisher G Molinolo AA Gutkind JS Conditional expression of K-ras in an epithelial compartment that includes the stem cells is sufficient to promote squamous cell carcinogenesis.Cancer Res. 2004; 64: 8804-8807Crossref PubMed Scopus (108) Google Scholar Of note, these mice also exhibited a complex salivary gland phenotype, presenting visible lesions in the major and accessory buccal salivary glands (Figure 1) after only 1 week of doxycycline treatment (Table 1). A swollen, irregular neck area could be easily observed in live animals; at palpation, the glands felt hard and irregular. At necropsy the salivary glands looked enlarged and distorted, frequently weighing up to three times their normal weight (Figure 1, A–C). Bilateral tumor-looking structures were also seen on the masseter muscles ranging from 1 to 3 mm in diameter (Figure 1A). These tumors seem to have originated in small, microscopic mixed accessory salivary glands located close to the superficial muscular layer, which is close to the buccal mucosa, in the oral angle, and close to the border between the skin and the buccal mucosa (Figure 1D). These glands differed in histology and location from the extraorbital lacrimal glands, which were not affected by the carcinogenic process, and from the sebaceous glands located in the angles of the mouth that open directly to the mucosal surface.23The Staff of the Jackson Laboratory Biology of the Laboratory Mouse. Dover Publications, Inc., New York1981Google Scholar No gross alterations were evident in untreated transgenic or wild-type animals.Table 1Salivary Glands Phenotype of K5-tet-on/tet-o-K-rasMiceMouse no.SexDoxycycline treatment (days)DiagnosticSMSLPAccessory glands1F6HyperplasiaYes––nd2F7AdenosisYesYesYesnd3F7SCCYes––nd4M7HyperplasiaYesYes–nd5M7SCCYes––nd6M7Hyperplasia/SCCYesYes–nd7M7HyperplasiaYes––nd8M7Hyperplasia/SCCYes––nd9M7Hyperplasia/fibrosis/SCCYes––nd10F7Hyperplasia/fibrosisYes––nd11F7HyperplasiaYesYesYesYes12F10Hyperplasia/SCCYesYes–Yes13M11Hyperplasia/SCCYesYes––14M14HyperplasiaYesYesYes–15M15HyperplasiaYesYes–nd16M15Hyperplasia/SCCYes––nd17M15Adenosis/hyerplasiaYesYesYes–18M15Adenosis/hyperplasiaYesYes––19M23HyperplasiaYesYesYes–20F28HyperplasiaYesYesYes–21M29HyperplasiaYesYes–Yes22M30Hyperplasia/SCCYesYes–Yes23M30Hyperplasia/SCC–––Yes24F32Adenosis/hyperplasiaYes–––Doxycycline (RPI) was administrated via the drinking water at a concentration of 1g/L to 24 animals of 21 days of age. nd, not done. SM, submaxillary; SL, sublingual; SCC, squamous cell carcinoma; P, parotid. Open table in a new tab Doxycycline (RPI) was administrated via the drinking water at a concentration of 1g/L to 24 animals of 21 days of age. nd, not done. SM, submaxillary; SL, sublingual; SCC, squamous cell carcinoma; P, parotid. Submaxillary glands were affected in all cases, displaying the wider range of pathological alterations. Epithelial proliferation ranged from hyperplasia of the small secretory ducts, in the form of adenosis (Figure 3, lower inset), to solid hyperplasia and squamous metaplasia of the larger ducts. The acini did not seem to be involved in the proliferative lesions. Sublingual and parotid glands were less affected, and the lesions progressed from ductal, intralobular, and extralobular structures (Figure 2, Figure 3, Figure 4).Figure 2Dysplastic changes with solid squamous metaplastic proliferation (A) and adenosis (B) are evident in the submaxillary gland. The parenchyma of the sublingual gland seems unaffected, but the extralobular ducts show focal intraductal cribiform proliferation (inset). Magnifications: ×40 and ×100 (inset).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4A: This multifocal squamous cell carcinoma arises in an otherwise unaffected submaxillary gland; no signs of hyperplasia or dysplasia are observed. The inset shows a distinct limit between neoplasia and normal tissue (arrow). B: Multifocal spindle-cell carcinomas in a sublingual gland arising from ductal structures; the inset depicts the border between the carcinoma and the normal tissue; a fibrous pseudocapsule with inflammatory mononuclear cells is seen (arrow). C: Moderately differentiated multifocal squamous cell carcinoma arising in a submaxillary gland; the tumors form solid masses that infiltrate the surrounding stroma. Distorted mucous acini are seen in the periphery. D: Infiltrating spindle-shaped carcinoma, with a desmoplastic stroma. E: Early carcinoma arising in a dysplastic sublingual gland. Note that, although the lesion is small, all of the cells display malignant features; a rapid transition to invasion characterized these tumors, with no evident dysplasias. F: Metastatic squamous cell carcinoma in a cervical lymph node; the arrows indicate remnants of the lymph node parenchyma. Magnifications: ×20 (A); ×30 (B); ×45 (C–F); and ×100 (insets).View Large Image Figure ViewerDownload Hi-res image Download (PPT) During the development of the phenotype, the excretory interlobular ducts of both submaxillary and sublingual glands showed immature squamous metaplasia in the early lesions, with cytological features of dysplasia, but these lesions did not tend to progress. The proliferation seems to arise from a basal cell compartment, and the dysplastic features were present from the very beginning. Within the lobular compartment the early stages of the phenotype were characterized only by epithelial proliferation; in later stages, particularly the submaxillary glands, there was a complex phenotype in which hyperplasia, squamous metaplasia, and dysplasia of the intercalated and secretory ducts were associated with fibroblastic proliferation and fibrosis (Figure 2, Figure 3). In certain areas, the general structure of the gland showed a fibroadenomatous-like picture (Figure 3, upper inset). Adenosis, with proliferation of small tubular structures surrounded by loose connective tissue, was also seen (Figure 3B, lower inset). Some of the metaplastic/dysplastic ducts showed early progression into invasive carcinomas, and within a few days, overt moderately to poorly differentiated squamous carcinomas developed that infiltrated the gland and surrounding tissues (Figure 4). In submaxillary and sublingual glands, as well as in the accessory buccal glands, invasive carcinomas lesions were frequently multifocal (Figure 4, B and C). Sublingual gland lesions developed later than those of the submaxillary gland and consisted of hyperplasias, squamous metaplasias/dysplasias, and invasive carcinomas. Adenosis of small ducts with the associated stromal proliferation and fibrosis, so frequently observed in submaxillary, were not seen in this gland. One of the distinctive features of these lesions was the early transformation; even relatively small groups of proliferating cells had already acquired the histological features of nonkeratinizing squamous cell carcinomas and were infiltrating the surrounding parenchyma (Figure 4). The majority of these carcinomas, frequently spindle-shaped, were moderately or poorly differentiated tumors (Figure 4, D and E), composed of densely packed chords of neoplastic cells with a high mitotic index and abnormal mitotic figures. Of the three major salivary glands, the parotids were the least frequently affected, usually starting late as compared with the other glands. These lesions were mainly characterized by scattered squamous metaplasia, hyperplasia, and dysplasia of the small intralobular ducts. Accordingly, progression into invasive carcinoma was also less frequent and a late event as well. An interesting feature of the general phenotype was the observation of lesions in a set of buccal accessory salivary glands located anatomically on the apex of the masseter muscle, close to the oral angle. Although these glands are not normally visible at necropsy, they became grossly evident when they developed proliferative lesions. Histologically they are mixed serous/mucinous glands in the submucosa of the mouth, seeming to arise from two independent components and draining into the oral cavity through a short and wide duct (Figure 1). Figure 5 depicts a low magnification image of the affected glands in which the whole spectrum of lesions, ranging from hyperplasia to squamous carcinoma, can be recognized. Most animals with accessory gland lesions also had lesions of the major salivary glands. Metastasis to regional cervical lymph nodes was observed in two cases (Figure 4F). It was not possible to determine from which gland they arose, but in both cases the submaxillary gland had the most advanced alterations. Untreated double transgenic K5-tet-on/tet-ras, as well as wild-type mice, were histologically normal. Persistence of salivary gland tumors as well as skin lesions, part of the general phenotype already described,20Vitale-Cross L Amornphimoltham P Fisher G Molinolo AA Gutkind JS Conditional expression of K-ras in an epithelial compartment that includes the stem cells is sufficient to promote squamous cell carcinogenesis.Cancer Res. 2004; 64: 8804-8807Crossref PubMed Scopus (108) Google Scholar were observed in four K5-tet-on/tet-o-ras animals necropsied a month after stopping the doxycycline treatment. These preliminary results of a set of ongoing experiments suggest that the malignant salivary phenotype continue to progress despite suspension of the doxycycline treatment. Double transgenic mice expressing the β-galactosidase gene under the control of the K5-tet-on system revealed β-galactosidase activity in the ducts of the salivary glands after administration of doxycycline, confirming the specific expression of the K5 promoter in the salivary glands (Figure 6A). Indeed, immunocytochemistry for K5 revealed staining of basal cells in all salivary gland ducts (Figure 6, B–D). The submaxillary gland (Figure 6D) showed a higher number of positive structures as compared with the sublingual gland (Figure 6C); the myoepithelial cells were also immunoreactive for K5 in both glands. Double β-galactosidase/K5 staining confirmed that all cells expressing the transgene were K5-positive (data not shown). Furthermore, immunostaining of hyperplastic and dysplastic lesions revealed that the hyperproliferating cells arose from the K5-positive cell compartment (Figure 6, Figure 7). In carcinomas, K5 expression varied on tumor differentiation. Almost all cells were positive in moderately differentiated carcinomas, whereas in spindle-shaped tumors, as well as in metastases, expression could be patchy (Figure 7C and inset). Immunostaining for smooth muscle actin showed the participation of myoepithelial cells in the stromal a