Chronic administration of phenytoin and pleomorphic adenoma: A case report and review of literature.

Adverse drug effects that are uncommon or appear only on chronic administration of a drug may not be detected in clinical trials. This explains the need of strict post-marketing vigilance on drug use. Phenytoin administration has been shown in the literature to be associated with development of neoplasia (benign/malignant). In our knowledge current work represents the first case of pleomorphic-adenoma of sub-mandibular salivary gland developed following chronic phenytoin use. A 40 year old male having a history of head trauma twenty years back, had been on tablet phenytoin 100 mg thrice daily since then. One year back he noticed a small swelling in left sub-mandibular region and gradually increasing in size. FNAC and CECT revealed the diagnosis of pleomorphic-adenoma of sub-mandibular salivary gland. Other causes were ruled out. Surgical excision was performed successfully and continuing follow-up with no recurrence at the end of 6 months. Histo-pathogical examination of the tissue did not show any malignant changes.

Therapy-related acute leukemia with mixed phenotype and t(9;22)(q32;q11.2): a case report and review of the literature.

Therapy-related acute leukemia showing mixed phenotype is extremely rare. We report a 49-year-old woman who presented with palpable masses in her neck and back. She had received systemic chemotherapy (adriamycin and cisplatin) and radiotherapy for endometrial adenocarcinoma 7 years before. Her peripheral blood and bone marrow showed increased blasts, which coexpressed myeloid (CD13, CD33, and myeloperoxidase) and B-lymphoid antigens (CD19 and CD79a). Cytogenetic analysis showed a karyotype of 46,XX,dup(1)(q21q32),add(5)(q33),t(9;22)(q34;q11.2)[12]/47,idem,+der(22)t(9;22)[8], and BCR/ABL1 rearrangement was detected. Leukemic infiltration was also confirmed in her back mass. After induction chemotherapy with idarubicin, cytarabine, and imatinib, she achieved complete remission. Only 2 cases of therapy-related acute leukemia with mixed phenotype have been reported so far: one with hyperploidy and the other with t(1;21)(p36;q22). To the best of our knowledge, this is the first case of therapy-related acute leukemia with mixed phenotype and t(9;22) as well as extramedullary leukemic infiltrations.

Graph models for phenotype and genotype association between oral mucosa and submandibular gland tumorigenesis in rat.

BACKGROUND: In recent years, success of statistics in field of genetics has been the identification of genes that affect the process of disease. Experimental models using animals enable early stages of tumor development to be studied. The aim of this study was to apply graph models to assess the association between the observed phenotypic changes in rat oral mucosa and induced tumorigenesis in the submandibular gland (SMG). MATERIALS AND METHODS: We studied changes in oncogenes TP53 and bcl-2, histopathological and immunomarker variables in samples of oral mucosa and SMG of Wistar male rats, 60 days old and 180 g in weight, in which tumorigenesis was induced in their SMG by a 0.5% solution of 9,10-dimethyl-1,2-benzanthracene in acetone. A set of linear structural equations were defined, with each formula indicating the response variables and the direct influences. In graph models, saliva was considered as a latent variable. The association was analyzed using Graphical Gaussian Markov models and odd ratios. RESULTS: About 40% of animals treated with 9, 10-dimethyl-1, 2-benzanthracene showed histological alterations in the epithelial basal strata of their oral mucosa only at 150 days. Statistical models indicated a relationship between gene alteration in gene bcl-2 in the SMG and histological changes observed in the oral mucosa (P = 0.04). CONCLUSION: Graph statistical model with one latent variable allows to conclude that these results associated with other clinical parameters may be useful in detecting early changes in SMG tumorigenesis. Furthermore, the design of randomized sampling of oral mucosa allows to validate these results and establish a reliable methodology for presumptive diagnosis or screening in the future.

Mucoepidermoid carcinoma of the submandibular gland after chemotherapy in a child.

Second malignant neoplasms (SMNs) have become a concern in survivors of childhood malignancy. Although there are many reports describing SMN in patients treated for childhood cancer, salivary gland tumors rarely appear in these reports. Radiotherapy is a well-known risk factor for the development of secondary salivary gland malignancies after the treatment of childhood cancer. However, it is not well known whether chemotherapy itself treatment increases the risk of salivary gland malignancies. We report a child case with mucoepidermoid carcinoma of the submandibular gland as a SMN after chemotherapy alone for acute myeloid leukemia.

Early phenotypic and genotypic alterations in submandibular gland oncogenesis in rats.

The present study evaluates the phenotypic and genotypic changes that take place during early oncogenesis. The submandibular glands of male rats were injected with a 0.5% solution of 9,10-dimethyl-1,2-benzanthracene (DMBA) in acetone. Gland samples were taken at 0, 7, 30 and 150 days post-injection and submitted to histological, biochemical, immunocytochemical and PCR evaluation. Histopathological analysis was performed on hematoxylin-eosin stained slides. Total protein content was assessed by Lowry's method and the protein profile was analyzed by 12% SDS-PAGE. Bcl-2 was demonstrated by silver-enhanced gold immunolabeling. p53 immunolabeling was performed using the streptavidin-biotin system. All the treated animals developed carcinoma-like lesions at 30 and 150 days. Total protein concentration rose significantly (p < 0.05) above control values at 7, 30 and 150 days. The treated glands exhibited positive immunolabeling for p53 in the nuclei of neoplastic cells at 30 and 150 days. Treated glands also showed positive cytoplasmic immunolabeling for Bcl-2, exhibiting statistically significant differences between 7, 30 and 150 days (p = 0.0015), and with controls (p < 0.0001). No p53 mutations were observed whereas a point mutation, C-to-A, of the Bcl-2 gene was detected at 7, 30 and 150 days by PCR amplification. This mutation led to a single aminoacid change (thre --> asn) in the protein molecule. Our results suggest that the early histopathological changes correspond to quantitative and qualitative protein changes. The histopathological, biochemical, immunocytochemical and genetic alterations observed during the course of experimental carcinogenesis in the submandibular gland of the rat could constitute reproducible indices of malignant transformation applicable to human oncogenesis, given the high degree of homology between the oncogenes of mice, rats and human beings.

Effects of soy oil on murine salivary tumorigenesis.

Dietary fat influences dimethylbenzanthracene (DMBA)-induced tumorigenesis of several organs, including the salivary glands. There is not enough evidence to suggest that soy oil could also affect growth of salivary tumors. The main purpose of this work therefore was to study the effects of dietary soy oil on macroscopic parameters of chemically induced murine salivary gland tumors. Eighty BALB/c male mice were assigned to four groups: soy oil (SO), corn oil (CO, control), fish oil (FO) and olein (O). Two weeks later, tumors were induced by 9,10-dimethyl-1,2-benzanthracene (DMBA). At the 13th post-injection week, the animals were sacrificed. In vivo tumor diameter, gland volume (total resected mass), tumor volume (microscopically measured), tumor remission and tumor histopathology were analyzed. The initial in vivo tumor diameter, gland and tumor volume were significantly greater in soy oil than in fish oil group. 26.7% of animals on the soy oil diet showed tumor remission. Sarcomas were more often found in the SO group, carcinomas in FO and the mixed-type tumors both in SO and CO groups. This study shows that the soy oil treatment resulted in larger tumors, some of which later became undetectable. It is necessary to further investigate these divergent results.

Reduction of p53 dosage renders mice hypersensitive to 7, 12-dimethylbenz(alpha) anthracene-induced salivary gland tumorigenesis.

BACKGROUND: The role of p53 during the evolutionary steps of experimental salivary gland tumorigenesis has not been fully elucidated. MATERIALS AND METHODS: Each genotype group of p53-deficient mice received a single intrasubmandibular gland injection of 1 mg of 7, 12-dimethylbenz(a)anthracene. In addition to the routine histopathological examination, immunohistochemical detection of p53 protein and molecular biological analysis of wild-type p53 allele status in the p53+/- tumors developed were performed. RESULTS: Fourteen weeks following injection, submandibular gland tumors developed in 100% of p53-/- and 70% of p53+/- mice, whereas only 10% of p53+/+ mice yielded tumors. Salivary gland tumors in p53-deficient mice were predominantly sarcomas (70%). In 64% of p53+/- tumors, overexpression of a mutant version of p53 protein was evident. Loss of the wild-type allele of p53 could not be detected in all p53+/- tumors. CONCLUSION: The earlier tumor development in p53-deficient mice provides the in vivo evidence that reduction of p53 dosage or possibly p53 haploinsufficiency might be sufficient to promote salivary gland tumorigenesis.

Enhancing effects of fibroblast growth factor on the proliferation of salivary gland carcinoma cells and salivary gland carcinogenesis.

The proliferation of mouse submandibular gland carcinoma YT-12 cells was stimulated by endothelial cell growth factor (ECGF)/bovine brain-derived acidic fibroblast growth factor (aFGF) and recombinant human aFGF. To determine whether aFGF was capable of modifying salivary gland carcinogenesis, the effect of brain-derived aFGF was examined in vivo. Mice in Groups 1 and 2 were injected with 9,10-dimethyl-1,2-benzanthracene (DMBA) into the left submandibular gland, and then Group 1 mice received bovine brain-derived aFGF and Group 2 mice received vehicle subcutaneously for 10 weeks. Group 3 and 4 mice received either bovine brain-derived aFGF or vehicle only. Sixteen weeks after the start of the experiment, the incidence of submandibular gland carcinomas in Group 1 was significantly greater than that in Group 2. Immunohistochemical study indicated that ducts in the normal submandibular glands and carcinomas showed positive staining with anti-aFGF antibody. Immunoblot and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed the expression of aFGF in these tissues. FGF receptor (FGFR)-1 and FGFR-4 were detectable in the mouse submandibular glands and carcinomas. These findings suggest that bovine brain-derived aFGF stimulates the proliferation of submandibular gland carcinoma cells and promotes mouse submandibular gland carcinogenesis.

Induction of adenocarcinoma containing myoepithelial cells in rat submandibular gland by 7,12-dimethylbenz(a)anthracene.

In an attempt to induce adenocarcinoma containing myoepithelial cells (MECs) in the rat submandibular gland, we injected 7,12-dimethylbenz(a)anthracene (DMBA) dissolved in acetone into the glands of rat pups at the age of 10 days. In both male and female pups, the glands, including their developing terminal secretory units, contained far greater numbers of cells positive for proliferating cell nuclear antigen (PCNA) than did adult glands. A single administration of 1% DMBA (0.05 ml/130 g b.w.) did not produce adenocarcinoma, but did induce occasional sarcomas, such as rhabdomyosarcoma and fibrosarcoma, in 2 months. Most glands regenerated with minimal scar formation. Microscopically, these glands were atypical in that they contained increased numbers of PCNA-positive cells, underdeveloped granular ducts, and striated ducts surrounded by MECs positive for alpha smooth muscle actin (alphaSMA). Though these features were also observed in the regenerated glands after acetone injection, the number of PCNA-positive cells was relatively high in the glands of DMBA-treated females, especially in the terminal secretory unit. The second DMBA injection at 10 weeks of age produced adenocarcinoma made up of alphaSMA-positive MECs and keratin 19-positive duct cells. Such MEC-associated adenocarcinoma was induced in the glands of more than half the female but not the male animals. Replacement of either of the double DMBA treatments with acetone, or DMBA treatment, single or double, of adult glands did not produce adenocarcinoma, but did produce sarcoma and squamous cell carcinoma. These results suggest that (1) at least two genetic mutations are necessary for induction of adenocarcinoma with MECs in the rat submandibular gland, (2) the mutation is efficiently introduced to pup glands whose terminal secretory units exhibit extreme proliferative activity, and (3) the second mutation is difficult to introduce in male glands, whose proliferative activity is relatively low, and/or transformed cells need some female hormone after the mutation to propagate.

[Establishment of rat submandibular gland squamous cell carcinoma induced by DMBA].

OBJECTIVE: To study carcinogenesis and development of salivary gland tumor and establish an animal model of submandibular gland (SMG) tumor. METHODS: Histopathological study during carcinogenesis in rat SMGs using (9,10-dimethyl-1,2-benzanthracene (DMBA) was evaluated. A total of 50 male and female Sprague-Dawley (SD) rats of 8 weeks old and 180-200 g weight were obtained from the Animal Center of Henan Medical University. Under pentobarbital sodium anesthesia, the left SMGs were exposed by surgical procedure. A sponge pellet (1.0 mm x 1.0 mm x 1.0 mm, made by authors) was used as the carrier of the carcinogen. The sponge containing 2% DMBA (Fluka, Switzerland)/acetone solution was implanted into the glandular tissue of the left SMGs. Four rats (2 males and 2 females) were killed after every 2 weeks of the DMBA/sponge implantation. The same method of sponge implantation without DMBA was used at the right side of SMG as a control. All rats left were killed after 20 weeks. The SMGs were fixed in 10% formalin buffer solution for 24 hours, and embedded in paraffin, then 4 microns-thick sections were made for histopathological study. RESULTS: The earliest tumor occurred after 4 weeks of implantation of sponge, a total of 21 lateral tumors were induced (10 females, 11 males). There was no tumor found in the controlled SMG. The peak time of tumor genesis was after 8-12 weeks of implantation, all tumors induced were squamous cell carcinomas(SCCs). The induced tumors grew slowly below the mandibles of rats, which were present as nodular masses without capsules, and the borders were not clear. They were slightly hard when palpated. The process of carcinogenesis can be described as following: squamous metaplasia of cyst-like structures occurred, then SCCs were induced and invaded surrounding tissues. No metastasis was observed in regional lymph nodes and other organs. CONCLUSION: SCCs of SMG can be induced by implantation of DMBA. The present study supports the conclusion that all duct segments undergo squamous metaplasia, and therefore may participate in the genesis of neoplasia during experimental carcinogenesis.

Immunohistochemical study of fibroblast growth factor-2 (FGF-2) and fibroblast growth factor receptor (FGF-R) in experimental squamous cell carcinoma of rat submandibular gland.

Fibroblast growth factor-2 (FGF-2) is a member of the heparin-binding growth factor family and has mitogenic activity. Immunohistochemical expression of FGF-2 and its receptor (FGFR) was evaluated in experimentally induced squamous cell carcinoma as well as transforming cells of rat submandibular gland (SMG) induced by 9,10-dimethyl-1,2-benzanthracene (DMBA)/sponge implantation. Proliferating cells detected by proliferating cell nuclear antigen (PCNA) staining during carcinogenesis were also compared with FGF-2 and FGFR stainings. In the normal SMG, FGF-2 and FGFR were present in the excretory, striated and intercalated duct cells. Pillar and transition cells of granular convoluted tubule (GCT) showed FGF-2 staining, PCNA-labeled cells in normal SMG were rarely observed. In 2-3 weeks after carcinogen implantation, the reactions of FGF-2 and FGFR were expressed in epithelial islands, duct-like structures and affected ductal segments. PCNA-positive cells were developed in these epithelial structures. In 4-8 weeks after carcinogen implantation squamous epithelium appeared surrounding DMBA/sponge and gradually transforming with high PCNA labeling in the based cells and strong staining of FGF-2 and FGFR. Squamous cell carcinoma arose within about 12 weeks of the experiment. In squamous cell carcinoma, there was an intense immunohistochemical expression of FGF-2 and FGFR. Basal and parabasal layers of the squamous cell carcinoma showed high PCNA labeling. FGF-2-positive cells were found in the connective tissue stroma and in inflammatory cells around the proliferating duct-like structure. Coexpression of FGF-2 and FGFR was indicated in transforming cells during carcinogenic processes and in experimental squamous cell carcinoma of rat SMG. These findings suggested that FGF may play an important role for squamous metaplasia and carcinogenesis in rat SMG as an autocrine factor and FGF-positive stromal cells may also act to stimulate epithelial proliferation.

Effect of epidermal growth factor administration on the development of mouse salivary gland carcinomas.

This study investigated whether epidermal growth factor (EGF) administration was capable of modifying salivary gland carcinogenesis. Two groups of mice were given 1 mg of 9,10-dimethyl-1,2-benzanthracene (DMBA) into the left submandibular gland, and then Group 1 mice received 2 microg of EGF and Group 2 mice received vehicle subcutaneously for 8 weeks. Mice in two other groups, 3 and 4, received either EGF or vehicle alone. Twelve weeks after the start of the experiment, the incidences of submandibular gland carcinomas in Groups 1 and 2 were 39% and 58%, respectively, although this difference was not statistically significant. Duct- and cyst-like structures and carcinomas in the left submandibular glands were weakly stained by anti-EGF receptor (EGFR) antibody. Immunoblot and reverse transcriptase polymerase chain reaction (RT-PCR) analyses revealed the expression of EGFR in the submandibular glands and carcinomas. However, EGFR was undetectable in YT cells that were derived from a submandibular gland undifferentiated carcinoma of a Group 2 mouse. These findings indicate that EGF does not promote tumor induction in mouse salivary gland carcinogenesis. This may be ascribed in part to the low expression level of EGFR in tumor cells.

[Rat submandibular gland tumor induced by 3-methylcholanthrene].

OBJECTIVE: To study the genesis and the development of salivary gland tumor (SGT). METHODS: SGT animal models were established by injection of 3-methylcholanthrene (3-MCA) oil solution into 40 submandibular glands of Sprague-Dawley (SD) rats, then histopathology and ultrastructure of induced tumors were observed under microscope, and immunohistochemical detection of keratin and actin was carried out. RESULTS: 32 models of submandibular gland tumors were induced successfully, with 4 rats of squamous cell carcinoma and 28 leiomyosarcoma, and the carcinogenesis of 3-MCA and tissue genesis of induced tumors were discussed. CONCLUSION: Submandibular gland tumor can be induced by 3-MCA, and sarcoma was of high incidence.

The effect of streptozotocin and a high-fat diet on BOP-induced tumors in the pancreas and in the submandibular gland of hamsters bearing transplants of homologous islets.

The effect of a high-fat diet (HF) and streptozotocin (STZ) was investigated in the rapid cancer induction model developed in our laboratories. Syrian golden hamsters bearing homologous islets transplanted in their right submandibular gland (SMG) received a HF or a low-fat diet (LF). Half of the animals from each dietary group received STZ (HF-STZ and LF-STZ groups) and the other half did not (HF and LF groups). One week later, all hamsters were treated with N-nitrosobis(2-oxopropyl)amine (BOP) weekly for 3 weeks and the experiment was terminated 12 weeks after the last BOP injection. Pancreatic lesions were found in many hamsters, with a lower incidence in the LF-STZ group (13%) than in other groups (35-45%). A HF diet counteracted the inhibitory effect of STZ on pancreatic tumor induction by yet unknown mechanisms. SMG tumors, all ductal-type adenocarcinomas, developed in all groups and the incidence was lowest in the HF group (6%) compared to the LF group (15%), LF-STZ group (17%) and HF-STZ group (18%). However, the difference was not statistically significant. It was concluded that a HF diet counteracts the inhibitory effect of STZ on BOP-induced pancreatic lesions but has no effect on the induction of tumors in the SMG. STZ pretreatment does not influence tumor induction of the SMG of these hamster.

Growth pattern of experimental squamous cell carcinoma in rat submandibular glands--an immunohistochemical evaluation.

Histopathological and immunohistochemical studies during carcinogenesis in rat submandibular glands (SMGs) using a carcinogen (9,10-dimethyl-1,2-benzanthracene: DMBA) were evaluated. For carcinogenesis, the carcinogen-containing sponge was surgically inserted into the gland. Histopathological features during carcinogenesis were as follows; dilatation of ductal segments, the presence of duct-like structures and cystic lesion around the sponge were observed within 3 weeks of the experiment, squamous metaplasia in duct-like structures and lining epithelium of the cystic structures around the sponge were observed at 4-6 weeks of the experiment, and finally well differentiated squamous cell carcinomas (SCCs) were observed after 8 weeks of the experiment. The immunoreactivity of K8.12 keration (K8.12), S-100 protein (S-100), epidermal growth factor (EGF), laminin, and proliferating cell nuclear antigen (PCNA) were evaluated. In the normal SMG, EGF was confined to the granular cells and S-100 to the pillar cells of granular convoluted tubules (GCTs). K8.12 was found in striated (SD) and excretory duct (ED) cells and laminin showed linear staining of the basement membrane around the ducts, acini and blood vessels. PCNA-positive nuclei were rarely observed in the normal glandular parenchyma. During carcinogenesis, during the first stage, EGF in granular cells and S-100 in pillar cells of GCT segments disappeared, and cytokeration K8.12 was observed in duct-like structures and cystic epithelium around the DMBA sponge. PCNA-positive nuclei in the first stage were mainly confined to basal cells of morphologically altered ducts. During the second stage, squamous metaplastic cells showed an intense K8.12 reaction. During the third stage, the well differentiated SCC showed strong reaction for K8.12, and the linear staining for laminin staining had disappeared at the invading fronts. The PCNA index was nearly 40% in the tumour cell component. The stem cells or the progenitor cells during experimental carcinoma were most likely to be the ductal basal cells, and carcinogenesis was initiated with an increase of proliferating activity in small cell clusters surrounding a necrotic area, basal cells of dilated excretory ducts and duct-like structures. Thus, all ductal segments undergoing squamous metaplasia may participate in the genesis of neoplasia during experimental carcinogenesis.

Induction of adenocarcinomas in the submandibular salivary glands of female Wistar rats treated with 7,12-dimethylbenz(a)anthracene.

In 12 male and 12 female Wistar rats, 7-9 weeks of age, a solution of 0.05 ml of 1% 7,12-dimethylbenz(a)anthracene (DMBA) dissolved in acetone was injected directly into the submandibular glands biweekly, after the gland had been injured. In some rats the carcinogen was injected 1 week after the injury. Each rat received six to seven injections. Swelling was observed in the submandibular gland region as early as 4 weeks after the last injection. The animals were killed 4-8 weeks after the last injection and glands with tumour tissues were processed for light microscopy. The control rats of the same age that received a corresponding amount of acetone only (three male, three female), carcinogen injection alone (three male, five female), and injury only (four male, four female) were killed at the same time. Histological examination revealed adenocarcinomas of the submandibular gland in 12/12 (100%) female rats, six of which were associated with fibrosarcomas. The adenocarcinomas basically consisted of ductal and glandular structures. Sometimes tubular, cystic, papillary-cystic and cribriform-like structures were also observed. Male rats mainly developed fibrosarcomas, although there was one squamous-cell carcinoma. The reasons for the sex difference are not known. One adenocarcinoma developed in the submandibular gland of a female rat (1/5) that received carcinogen injections alone. Thus direct injections of DMBA into the submandibular gland produce adenocarcinomas in female Wistar rats.

Effects of testosterone on tumor induction and epidermal growth factor production in the mouse submandibular gland.

To determine whether testosterone administration was capable of modifying salivary gland carcinogenesis, female mice were given 1 mg of 9,10-dimethyl-1,2-benzanthracene (DMBA) into the left submandibular gland and then Group 1 mice received 5 mg of testosterone propionate and Group 2 mice received vehicle, olive oil, subcutaneously for 8 weeks. Twelve weeks after the start of the experiment, the weight of the left submandibular gland of the Group 2 mice was greater than that of the Group 1 mice. The incidences of submandibular gland carcinoma in Groups 1 and 2 were 41% (12/29) and 57% (17/30), respectively. Epidermal growth factor (EGF) levels of the left submandibular gland were significantly higher in Group 1 as compared with Group 2. These findings indicate that testosterone increases the production of EGF in the DMBA-injected submandibular gland, but does not promote the development of submandibular gland carcinoma.

Carcinogenesis of mouse submandibular salivary gland using DMBA implant.

Carcinogenesis of 35 ddy male mice submandibular salivary glands were attempted; using implants of a 1 mg (2 mm) prepared pellets of the potent chemical carcinogen 9, 10-dimethyl-1, 2-benzanthracene (DMBA) dry powder without a vehicle or carrier. This method appeared to be easy, fast and effective. Within a period ranging from 7-14 weeks; twenty animals developed epidermoid carcinoma (two of them developed squamous cell carcinoma of covering skin as well), and two animals developed mixed tumors (pleomorphic adenoma like tumor) but neither adenocystic carcinoma nor sarcoma were found, the results were adequately discussed.

Immunohistochemical evidence that tumors elicit the synthesis of estrogen receptors in the submandibular gland of female rats.

We have demonstrated the presence of estrogen receptor mRNA and the mature protein in the cytoplasm and nucleus, respectively, of a 9,10-dimethyl-1,2-benzathracene-induced submandibular gland tumor in female rats. We have previously shown that progesterone receptors are also present in human salivary gland tumors. These results suggest that endocrine therapy may be effective in treatment of submandibular gland tumors.

Immunohistochemical localization of estrogen receptors in the submandibular gland tumors of female rats.

The localization of estrogen receptors (EsR) in the tumor tissues of submandibular glands was examined in female rats, using the indirect immunoperoxidase method in combination with the in situ hybridization technique. Tumors were experimentally produced by 9,10-dimethyl-1,2-benzanthracene (DMBA), and the tumor tissues were fixed with formalin or paraformaldehyde and then embedded in paraffin. In the DMBA-induced submandibular gland tumors, immunoreactivity to EsR-peroxidase conjugate was found in nuclei of the tumor cells which occupied the peripheral rim of the tumor cell nests. In contrast, the reactivity in the normal submandibular glands without tumor was mostly confined to nuclei of the duct cells. When EsR mRNA expression was analyzed in the tumor tissue by in situ hybridization with a cDNA probe, its distribution was identical with that of immunoreactivity to EsR. These data suggest that the ductal cells of the submandibular gland are responsive to ovarial steroids, and that estrogens may play an important role in the maintenance of growth of the submandibular gland tumors.