ARTICLE |
Correspondence to: Monique Doré, Département de Pathologie et Microbiologie, Faculté de Médecine Vétérinaire, Université de Montréal, CP 5000, St-Hyacinthe, Québec, Canada J2S 7C6. E-mail: doremo@medvet.umontreal.ca
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Summary |
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Squamous cell carcinoma is one of the most common cancers in humans and is also a frequently diagnosed neoplasm in dogs. Induction of cyclo-oxygenase-2 (COX-2), a key rate-limiting enzyme in prostaglandin biosynthesis, has been implicated in the oncogenesis of various cancers in humans, including squamous cell carcinomas. However, expression of COX-2 has not been reported in spontaneous squamous cell carcinomas of non-human species. Canine squamous cell carcinomas share several similarities with the human disease. Therefore, the objective of this study was to determine whether COX isoenzymes were expressed in naturally occurring cases of squamous cell carcinomas in dogs. Canine normal skin (n=4) and squamous cell carcinomas (n=40) were studied by immunohistochemistry and immunoblotting analysis using polyclonal antibodies selective for COX-1 or COX-2. COX-2 was strongly expressed by neoplastic keratinocytes in all cases of squamous cell carcinomas, whereas no COX-2 was detected in normal skin and in the non-neoplastic skin and oral mucosa included in the tumor tissue samples (p<0.01). Immunoblotting analysis confirmed the restricted expression of COX-2 (72,00074,000 molecular weight doublet) in squamous cell carcinomas only. In contrast, faint COX-1 staining was found in normal skin and in squamous cell carcinomas. This study demonstrates for the first time that COX-2 is induced in canine squamous cell carcinomas, and provides a new model to investigate the role and regulation of COX-2 gene expression in naturally occurring squamous cell carcinomas. (J Histochem Cytochem 49:867875, 2001)
Key Words: prostaglandin G/H synthase-2, cyclo-oxygenase-2, prostaglandins, keratinocytes, dogs, squamous cell carcinomas
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Introduction |
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SQUAMOUS CELL CARCINOMA is one of the most common malignant tumors in humans and can originate from different sites lined by a squamous epithelium (
Prostaglandins are believed to play a role in the development and progression of some forms of cancer (
Expression of COX-2 has been demonstrated in various human neoplasms, including colorectal, lung, gastric, pancreatic, prostate, breast, and hepatocellular adenocarcinomas, transitional cell bladder carcinomas, and adenocarcinomas and squamous cell carcinomas of the esophagus (
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Materials and Methods |
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Materials
Diethyldithiocarbamic acid (DEDTC), octyl ß-D-glucopyronoside (octyl glucoside), and diaminobenzidine tetrahydrochloride (DAB) were purchased from Sigma (St Louis, MO), [125I]-protein A and Biotrans nylon membranes (0.2 µm) from ICN Pharmaceuticals (Montreal, PQ, Canada), nitrocellulose membranes (0.45 µm) from Schleicher & Schuell (Keene, NH), rainbow molecular weight markers from Amersham (Arlington Heights, IL), Vectastain ABC kit from Vector Laboratories (Burlingame, CA), Kodak film X-OMAT AR from Eastman Kodak (Rochester, NY), PBS from Gibco (Grand Island, NY), and Bio-Rad Protein Assay and electrophoretic reagents from Bio-Rad Laboratories (Richmond, CA).
Tissue Samples and Platelet Isolation
Forty cases of canine squamous cell carcinomas submitted to the Departement of Pathology and Microbiology of the Faculty of Veterinary Medicine (Université de Montréal) were included in the study. All cases were confirmed as squamous cell carcinomas by examination of hematoxylineosinsaffran-stained (HES) sections by a veterinary pathologist. The malignant nature of the squamous cell carcinomas was defined using criteria such as anaplasia (anisocytosis, pleomorphism, anisokaryosis), loss of normal polarity, anarchic growth, atypical mitotic figures, and local or vascular invasion. The histological classification used was the World Health Organization International Histological Classification of Tumors of Domestic Animals (
Anti-COX Antibodies
Two anti-COX antibodies (antibodies 8223 and MF243) were used. Affinity-purified polyclonal antibody 8223 was raised in rabbits against ovine COX-1 and was shown to be selective for COX-1 in various species (
Immunohistochemistry
Immunohistochemical staining was performed using the Vectastain ABC kit as previously described (
Solubilized Cell Extracts and Immunoblotting Analysis
Solubilized cell extracts were prepared as previously described (
Statistical Analysis
The Fisher's exact test was used to compare the frequency of COX-2 expression between normal skin and skin squamous cell carcinomas. Statistical analyses were performed using the JMP Software (SAS Institute; Cary, NC).
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Results |
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Characteristics of Dogs with Squamous Cell Carcinomas
The age of dogs with squamous cell carcinomas ranged from 3.5 to 12.8 years (mean 8.4 years). Eighty-five percent of dogs (34/40 cases) were more than 6 years old at the time of cancer diagnosis, which is more than 40 years old when expressed in equivalent human years (
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COX Expression in Normal Canine Skin and Oral Mucosa
To determine whether COX-1 and/or COX-2 was expressed under physiological conditions, immunohistochemical staining was performed on normal canine skin (n=4). Results showed that no COX-1 was present in the epidermis or in the follicle walls (Fig 1A), whereas some COX-1 staining was found in smooth muscle cells of arrector pili muscles (Fig 1C). However, no COX-2 expression was detected in normal canine skin (Fig 1B). Moreover, no COX-2 immunostaining was found in the non-neoplastic skin included in the tumor tissue samples (26/33 cases) and in the non-neoplastic oral mucosa included in the tumor samples (5/7 cases) (Fig 1D).
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Induction of COX-2 in Canine Squamous Cell Carcinomas
In contrast to normal skin and oral mucosa in which no COX-2 expression was found, all cases of squamous cell carcinomas (100%) displayed immunoreactivity for COX-2. The extent and intensity of the COX-2 staining varied among the tumors (Table 1). There was no correlation between site, grade, and/or COX-2 intensity. COX-2 immunostaining was predominantly localized in the cytoplasm of neoplastic keratinocytes (Fig 2A2C), often being concentrated around the nuclear membrane (Fig 2C). In a small number of tumors (n=3/40), fusiform fibroblast-like cells located in the stroma were COX-2-positive (Fig 2D). In the majority of carcinomas (n=35/40), neoplastic keratinocytes also displayed very weak positive staining for COX-1 (Fig 2E).
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Immunoblotting of COX Isoforms in Canine Tissues
To confirm the specificity of each antibody and to characterize each COX isoform, solubilized cell extracts were prepared from normal skin, a squamous cell carcinoma, and canine platelets, and proteins were analyzed by Western blotting. When a selective anti-ovine COX-1 antibody was used, a very faint 69,000 Mr band was detected in the squamous cell carcinoma (Fig 3A). A band of identical molecular weight was detected in canine platelets (Fig 3A) and therefore corresponded to canine COX-1. When a selective anti-ovine COX-2 antibody was used, no signal was detected in normal skin but strong COX immunoreactivity was observed in the squamous cell carcinoma (Fig 3B). Canine COX-2 appeared as a 72,00074,000 Mr doublet and a small 62,000 Mr band (Fig 3B) believed to correspond to a proteolytic fragment, as previously observed in other species (
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Discussion |
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This study is the first to document the expression of COX-2 in naturally occurring squamous cell carcinomas in animals. Interestingly, whereas no or very low expression of the enzyme was observed in normal skin, all cases of canine squamous cell carcinomas examined expressed COX-2. COX-2 was abundantly present in neoplastic keratinocytes and in stromal cells within some tumors. Tumors originated from different anatomic locations (i.e. mouth, feet, or skin), with no predilection of COX-2 expression for a specific site because all cases contained immunoreactive cells. In humans, squamous cell carcinomas from different anatomic sites, including the skin, head, and neck, the lung, and the esophagus have also been shown to overexpress COX-2 (
Among the etiologic factors contributing to skin cancer, ultraviolet (UV) irradiation plays a central role in the development of cutaneous squamous cell carcinomas in humans, in part through its ability to induce prostaglandin synthesis (
Overexpression of COX-2 has also been reported in various experimental models of skin carcinogenesis (, transforming growth factor-
, or 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in the induction of COX-2 mRNA or protein (
was ineffective in inducing COX-2 expression in squamous cell carcinoma cell lines, suggesting alterations in the IFN-
response pathway in cancerous keratinocytes (
The precise mechanisms by which COX-2 expression contribute to carcinogenesis are beginning to unravel. These include effects on cell proliferation, apoptosis, cell invasion, and immune response (
Evidence for the role of COX-2 in tumor progression in humans also comes from promising results obtained with trials of therapy aimed at the specific inhibition of COX-2 expression in colon, bladder, and skin cancer (
In summary, squamous cell carcinomas share many similarities between humans and dogs, including an increased prevalence with advancing age, an absence of breed or race predilection, and the presence of tumors arising from a variety of anatomic sites. The present study further strengthens the parallel between the two species by establishing that, as in humans, induction of COX-2 occurs in most if not all squamous cell carcinomas in dogs. It should also be considered that, as companion animals, dogs share with humans a similar environment and may be exposed to the same external factors that contribute to cancer. In view of all these similarities, we propose the dog as a valuable model to study the role and regulation of COX-2 gene expression in naturally occurring cases of squamous cell carcinoma.
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Acknowledgments |
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Supported by grants from the Morris Animal Foundation (MD and JS), the Natural Sciences and Engineering Council of Canada (MD), and the Canadian Institutes for Health Research (CIHR) (JS). JS is the recipient of a CIHR Investigator Award.
We thank Drs Jilly F. Evans and Stacia Kargman (Merck Frosst Centre for Therapeutic Research; Pointe-Claire-Dorval, Québec) for kindly providing antibody MF243.
Received for publication November 3, 2000; accepted January 31, 2001.
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