Short communication expression of glial fibrillary acidic protein in some tumors of dogs

Transkript

1 Short communication expression of glial fibrillary acidic protein in some tumors of dogs Fehmi ARIKAN*, Hamdi UYSAL** Abstract: Glial Fibrillary Acidic Protein (GFAP) is an intermediate filament protein in astrocytes of the Central Nervous System that has a molecular weight of approximately 50 kda. GFAP, as an antigen in nervous tissue identification, is used as a reliable immunohistochemical marker for glial neoplastic cells and benign astrocytoma cells. In veterinary medicine breast cancers, skin neoplasms and lymphatic tumors are frequent. In scientific literatures it has been made clear that GFAP is a remarkable marker for the normal and neoplastic glial cells nevertheless there is no information about GFAP as a marker for the tumors not generally considered to be of glial origin. Therefore, in this study to determine GFAP in the several dog tumors except glial neoplasms was intended. The results of Sodium Dodecyl Sulfate Polyacrylamide Gel Electroforesis (SDS-PAGE) and Semi-Dry Blotting analysis using antibody for GFAP (immunoblotting) showed that GFAP is not being expressed by the 14 tumors which we studied. It has been observed that GFAP expression is in enough amount in the brain tissues. The fact that GFAP has not been identified in the tumor tissues we analyzed points out that this protein is specific to nervous system tissues. Key Words: Glial fibrillary acidic protein, tumor, dogs, tumor markers Bazı Köpek Tümörlerinde Glial Fibriler Asidik Protein Ekspresyonu Öz: Glial Fibriler Asidik Protein (GFAP), molekül ağırlığı 50 kda olan merkezi sinir sistemine ait astrositlerin intermediyer filament bir proteinidir. Sinir dokusunun tanımlanmasında bir antijen olarak kullanılan GFAP, merkezi sinir sisteminde glial orijinli neoplastik hücrelerin ve iyi huylu astrositom hücrelerinin lokalize edilmesinde immunhistokimyasal bir belirteç olarak kullanılır. Veteriner hekimliği alanında köpek tümörlerine özellikle deri, meme, kemik ve lenf dokularında sıklıkla rastlanır. Bilimsel literatürlerde GFAP in glial orijinli normal ve neoplastik hücreler için güvenilir bir belirteç olduğu belirtilmektedir. Ancak merkezi sinir sisteminin dışındaki dokularda GFAP in bir belirteç olabileceği hakkında herhangi bir bilgi yoktur. Bu nedenle bu çalışmada köpeklerden elde edilen ve çeşitli dokulardan orijin alan tümörlerde GFAP in varlığı ve ayırıcı teşhisteki değerinin saptanması amaçlanmıştır. Elde edilen 14 adet tümörlü ve sağlıklı dokunun Sodyum Dodesil Sülfat Poliakrilamid Jel Elektroforezi (SDS-PAGE) ile yapılan protein analizleri ve GFAP antikoru ile yapılan immun blot analiz sonuçları gösterdi ki kullandığımız tümörlü dokularda GFAP in sentezi mümkün görünmemektedir. Beyin dokusunda GFAP in yeter miktarda bulunduğu gözlenmiştir. GFAP in elde ettiğimiz tümör dokularında tespit edilememiş olması bu proteinin genellikle sinir sistemi dokularına spesifik olduğuna işaret etmektedir. Anahtar Kelimeler: Glial fibriler asidik protein, tümör, köpek, tümör belirteçleri Glial Fibrillary Acidic Protein (GFAP) is an intermediate filament protein in astrocytes of the Central Nervous System that has a molecular weight of approximately 50 kda. Nearly 15 years ago, it has been acknowledged that GFAP can be used as a prototype antigen for nervous tissue identification and it has a standard marker value in basic and applied researches. As a member of cell skeleton proteins family, GFAP is thought to *Vet. Med, Department of Biochemistry, Faculty of Veterinary Medicine, University of Ankara, Ankara, Turkey ** Prof. Dr., Department of Biochemistry, Faculty of Veterinary Medicine, University of Ankara, Ankara, Turkey 23

2 be important in regulating astrocyte motility. In dulloblastomas, gangliogliomas, schwannomas, case of a trauma, disease or a genetic disorder in glial elemented teratomas, peripheral nerve sheath central nervous system of vertebrates, astrocytes tumors, neuroectodermal tumors and ependymomas as well as cerebellar abiotrophies. Non-glial react in a way called Astrogliosis. Astrogliosis is characterised by rapid GFAP synthesis and identified by increased levels of this protein (6). A recent are negative for GFAP extraction (1, 2, 3, 7, 8, 12). neoplasms, carcinomas, melanomas and sarcomas study in dogs revealed the In fact In In scientific that In there scientific is literatures a link literatures has been it has made been clear made that clear GFAP that is GFAP is is a a a remarkable is a remarkabl marke In scientific literatures it has been made clear between GFAP expression and age. GFAP level in the the normal the normal and neoplastic and neoplastic glial that cells GFAP glial nevertheless cells is a remarkable nevertheless there marker is is there is no no no information is the no normal information about GFAP abou the dentate gyrus decreases and the morphology of and neoplastic glial cells nevertheless there is no astrocytes changes by marker years for marker (9). for for the the for tumors the tumors not generally not information generally considered about considered to GFAP to to be be be of of as to of glial a be marker of origin. glial for origin. Therefore, the tu- Therefore, the the expre th Cell surface antigens of of of GFAP as well of in GFAP in as in the monoclonal the several in the several dog and tumors dog tumors not except generally except glial considered neoplasms glial neoplasms to was be of aimed glial was to origin. to aimed to study. to study. polyclonal antibodies produced for cell skeleton Therefore, the expression of GFAP in the several proteins have a potential value in tumor diagnosis 14 tumor and control of and tu- control dog tissue tumors samples tissue except samples were glial analysed were neoplasms analysed in in in was this aimed in study. this to study. The samples The s mors (11). GFAP is a reliable histopathologically marker for diagnosed normal diagnosed study. by by by Ankara by Ankara University, University, Faculty Faculty of of of Veterinary of Veterinar Medi and glial associated neoplastic cells (2). Studies 14 tumor and control tissue samples were proved that the level Department of GFAP Department of is of of closely Pathology. of related Pathology. All used All samples used samples were biopsy were biopsy material material which which was obtained was o analysed in this study. The samples were histopathologically tissues as tissues as as diagnosed well as as well age, by as sex Ankara age, and sex race University, and of of of race the the dogs of the are are dogs show GFAP is extracted not only from a number of cells with the malignity of the astrocytoma cells (4). pathology pathology department. department. Diagnosed Diagnosed Faculty of Veterinary Medicine, Department of in central nervous system Table 1. but 1. Table 1. also 1. from some tumor tissues that are considered non-glial (5). Pathology. All used samples were biopsy material which was obtained from pathology department. Extraction of GFAP has been determined in Diagnosed tissues as well as age, sex and race of astrocytomas, glioblastomas, Table 1: 1: Table 1: Neoplastic gliosarcomas, 1: Neoplastic tissue me- samples tissue the dogs samples of of of are dogs shown of used dogs in in Table in used this 1. research in this research Tablo 1: 1: Tablo 1: Bu Bu Bu araşt rmada 1: Bu araşt rmada kullan lan kullan lan neoplastik neoplastik doku örnekleri doku örnekleri Table 1: Neoplastic tissue samples of dogs used in this research Tablo 1: Bu araştırmada kullanılan neoplastik doku örnekleri Histopathologic Histopathologic Diagnose Diagnose Race Race Age Sex Age Sex Sex Eosinophilic Eosinophilic Granuloma Granuloma Terrier Terrier Female 6.5 Female Malign Mixed Malign Tumor Mixed Tumor Terrier Terrier Female 10 Female Malign Mixed Malign Tumor Mixed Tumor Terrier Terrier Female 16 Female Leiomyoma Leiomyoma Terrier Terrier 999 Female 9 Female Complex Complex Carcinoma Carcinoma Terrier Terrier Female 10 Female Sebaceous Sebaceous Gland Adenoma Gland Adenoma Collie Collie 555 Female 5 Female Mast Cell Mast Tumor Cell Tumor Boxer Boxer 555 Male 5 Male Lenfoma Lenfoma Pitbull Pitbull 888 Male 8 Male Adenocarcinoma Adenocarcinoma Cocker Cocker 999 Female 9 Female Adenocarcinoma Adenocarcinoma Terrier Terrier 999 Female 9 Female Rhabdomyosarcoma Rhabdomyosarcoma Kangal Kangal 777 Male 7 Male Lipoma Lipoma Kangal Kangal 777 Male 7 Male Malign Mixed Malign Tumor Mixed Tumor Terrier Terrier Female 12 Female Malign Mixed Malign Tumor Mixed Tumor Terrier Terrier Female 11 Female 24 For the the For analysis the analysis of of of GFAP of in GFAP in in in histopathologically diagnosed diagnosed neoplastic neoplastic samples samples by by by SDS-PA by μg μg μg tissue 20 μg extracts tissue extracts were loaded were on loaded on on the the on polyacrylamide the polyacrylamide gels. Protein gels. Protein analysis analysis of of of the the sam os

3 For the analysis of GFAP in histopathologically diagnosed neoplastic samples by SDS-PAGE, 20 μg tissue extracts were loaded on the polyacrylamide gels. Protein analysis of the samples was performed by using denaturing 10 % SDS-PAGE according to the method of Laemmli (10). SE Mighty Small II Slab Gel Electrophoresis Unit (Hoefer Scientific Instruments) was used for electrophoretic analysis. Proteins in the gel were transferred to nitrocellulose membrane by using semi-dry transfer unit (Hoefer Scientific Instruments) Figure Figure 1a: Determination 1a: Determination of protein profiles of protein in normal profiles and in normal and neoplastic tissues of dogs by SDSneoplastic tissues of dogs by SDS-PAGE analysis. Proteins PAGE analysis. Proteins of tumor tissues were seperated of tumor tissues on 10 were % polyacrylamide seperated on 10 % gel polyacrilamide and stained gel with and stained Coomassie with Coomassie Blue (G-250). Blue (G-250). Lane Lane 1; Sebaceous 1; Sebaceous Gland Gland Adenoma. Adenoma. Lane 2; Sebaceous Lane 2; Sebaceous Gland Adenoma Gland Control. Adenoma Lane 3; Control. Mast Cell Lane Tumor. 3; Lane Mast 4; Cell Mast Cell Tumor. Tumor Lane Control, 4; Mast Lane Cell 5; Leiomyoma. Tumor Control, Lane 6; Lane Leiomyoma 5; Leiomyoma. Control. Lane Lane 7; Rhabdomyosarcoma. 6; Leiomyoma Control. Lane 8; Lane 7; Rhabdomyosarcoma. Lane 8; Rhabdomyosarcoma Control. Lane 9; Lipoma. Lane 10; Li- Lipoma Control Şekil 1a: Köpeklerin normal ve neoplastik dokular nda SDS- Şekil 1a: Köpeklerin normal ve neoplastik do- Rhabdomyosarcoma Control. Lane 9; Lipoma. Lane 10; kularında PAGE analizi SDS-PAGE ile protein analizi profillerinin ile protein belirlenmesi. profillerinin doku belirlenmesi. proteinleri %10 luk Tümör poliakrilamid doku proteinleri jelde ayr şt r ld %10 luk ve poliakrilamid Coomassie mavisi jelde (G-250) ayrıştırıldı ile boyand. ve Coomassie mavi- Tümör si (G-250) ile boyandı. S ra 1; Sebaseöz Bez Adenomu. S ra 2; Sebaseöz Bez Adenomu Sıra 1; Kontrol. Sebaseöz S ra 3; Bez Mast Adenomu. Hücreli Tümör. Sıra S ra 2; 4; Sebaseöz Mast Hücreli Bez Adenomu Kontrol. Sıra 3; Mast Hücreli Tümör Kontrol. S ra 5; Leiomyom. S ra 6; Tümör. Sıra 4; Mast Hücreli Tümör Kontrol. Sıra Leiomyom Kontrol. S ra 7; Rhabdomyosarkom. S ra 8; 5; Leiomyom. Sıra 6; Leiomyom Kontrol. Sıra 7; Rhabdomyosarkom. Kontrol. Sıra S ra 8; 9; Lipom. Rhabdomyosarkom S ra 10; Lipom Kontrol. Sıra 9; Lipom. Sıra 10; Lipom Kontrol. Figure Figure 1b: Determination 1b: Determination of protein profiles of protein in normal profiles and in normal and neoplastic tissues of dogs by SDSneoplastic tissues of dogs by SDS-PAGE analysis. Proteins PAGE analysis. Proteins of tumor tissues were seperated of tumor tissues on 10 were % polyacrylamide seperated on 10 % gel polyacrilamide and stained gel with and stained Coomassie with Coomassie Blue (G-250). Blue (G-250). Lane Lane 1; Eosinophilic 1; Granuloma. Granuloma. Lane 2; Lane Eosinophilic 2; Eosinophilic Granuloma Control. Granuloma Lane Control. 3; Mammary Lane Glands 3; Mammary Control. Glands Lanes 4 Control. and 5; Malign Lanes Mixed 4 and Tumor. 5; Malign Lane Mixed 6; Complex Tumor. Lane 6; Complex Carcinoma. Lanes 7 and 8; Carcinoma. Lanes 7 and 8; Adenocarcinoma. Lanes 9 and Adenocarcinoma. Lanes 9 and 10; Malign Mixed Tumor. 10; Malign Lane Mixed 11; Tumor. Glandula Lane 11; mammaria Glandula mammaria Control. Lane Control. 12; Lane 12; Lenfoma. Lane 13; Lenfoma Control. Control. Lane Lane 14; Brain 14; Brain Tissue Tissue Control. Control. Lane 15; Lane Mammary 15; Mammary Glands Glands Control Control Şekil Şekil 1b: Köpeklerin 1b: Köpeklerin normal ve normal neoplastik ve dokular nda neoplastik SDS- dokularında SDS-PAGE analizi ile protein profillerinin belirlenmesi. Tümör doku proteinleri %10 luk PAGE analizi ile protein profillerinin belirlenmesi. Tümör poliakrilamid doku proteinleri jelde %10 luk ayrıştırıldı poliakrilamid ve Coomassie jelde ayr şt r ld mavisi Coomassie (G-250) mavisi ile boyandı. (G-250) ile boyand. ve S ra Sıra 1; 1; Eozinofilik Granüloma. S ra Sıra 2; Eozinofilik 2; Eozinofilik Granüloma Granüloma Kontrol. S ra Kontrol. 3; Meme Sıra Dokusu 3; Meme Kontrol. Dokusu S ra 4; Kontrol. Malin Miks Sıra Tümör. 4; Malin S ra 5; Miks Malin Tümör. Miks Sıra Tümör. 5; S ra Malin 6; Miks Tümör. Sıra 6; Kompleks Karsinom. Sıra 7; Kompleks Karsinom. S ra 7; Adenokarsinom. S ra 8; Adenokarsinom. Sıra 8; Sıra 9; Malin Adenokarsinom. Miks Tümör. S ra 9; Malin Sıra 10; Miks Malin Tümör. Miks S ra 10; Tümör. Malin Sıra Miks 11; Tümör. Meme S ra Dokusu 11; Meme Kontrol. Dokusu Sıra Kontrol. 12; Lenfoma. S ra 30 12; Sıra Lenfoma. 13; S ra Lenfoma 13; Lenfoma Kontrol. Kontrol. Sıra S ra 14; 14; Beyin Beyin Dokusu Kontrol. Sıra 15; Meme Dokusu Kontrol. Kontrol. S ra 15; Meme Dokusu Kontrol. 25

4 Rabbit-anti-GFAP, as primary antibody, and Goat anti-rabbit-gfap, as secondary antibody, were used in order to search antigens on the nitrocellulose membrane after semi-dry blotting (immunoblotting). Profiles of the Coomassie Blue (G-250) dyed proteins are shown in Figure 1a and 1b. As shown in the figures, control and tumor tissues have different numbers of protein bands which means that control and tumor tissues have different protein contents from each other. In dogs, GFAP activity is previously observed especially in fibrous astrocytes, Schwann cells, axons and peripheral ganglions (13). Therefore brain tissue was used as positive control in order to show if the immunostaining method we used is working with anti-gfap (Figure 2a and 2b) Figure 2a: Seperated tissue proteins by SDS- PAGE were transferred from gel to nitrocellulose membrane by semi-dry blotting and immunostained with GFAP antibody (immunoblotting). Figure 2a: Seperated tissue proteins by SDS-PAGE were transferred from gel to nitrocellulose membrane by semi-dry blotting and immunostained with GFAP antibody (immunoblotting). Lane 1; Brain Tissue Control. Lane 2; Mast Cell Tumor. Lane 3; Mast Cell Tumor Control. Lane 4; Leiomyoma. Lane 5; Leiomyoma Control. Lane 6; Rhabdomyosarcoma. Lane 7; Rhabdomyosarcoma Control. Lane 8; Lipoma. Lane 9; Lipoma Control. Lane 10; Brain Tissue Control Lane 1; Brain Tissue Control. Lane 2; Mast Cell Tumor. Lane 3; Mast Cell Tumor Control. Lane 4; Leiomyoma. Lane 5; Leiomyoma Control. Lane 6; Rhabdomyosarcoma. Lane 7; Rhabdomyosarcoma Control. Lane 8; Lipoma. Lane 9; Lipoma Şekil Control. 2a: SDS-PAGE Lane 10; Brain ile Tissue ayrıştırılan Control doku proteinleri yarı-kuru blot yöntemi ile jel den nitroselüloz membrana aktarılmış ve GFAP antikorları ile immun boyama yapılmıştır (İmmun blot yöntemi). Şekil 2a: SDS-PAGE ile ayr şt r lan doku proteinleri yar kuru blot yöntemi ile jel den nitroselüloz membrana aktar lm ş ve GFAP antikorlar ile immun boyama Sıra yap lm şt r 1; Beyin (İmmun Dokusu blot yöntemi). Kontrol. Sıra 2; Mast Hücreli Tümör. Sıra 3; Mast Hücreli Tümör Kontrol. S ra 1; Beyin Dokusu Kontrol. S ra 2; Mast Hücreli Tümör. Sıra 4; Leiomyom. Sıra 5; Leiomyom Kontrol. Sıra S ra 3; Mast Hücreli Tümör Kontrol. S ra 4; Leiomyom. 6; Rhabdomyosarkom. Sıra 7; Rhabdomyosarkom Kontrol. S ra 5; Leiomyom Sıra 8; Kontrol. Lipom. S ra Sıra 6; Rhabdomyosarkom. 9; Lipom Kontrol. Sıra S ra 7; 10; Rhabdomyosarkom Beyin Dokusu Kontrol. S ra 8; Lipom. S ra 9; Lipom Kontrol. S ra 10; Beyin Dokusu Kontrol. 26 Figure 2b: Seperated tissue proteins by SDS-PAGE were transferred from gel to nitrocellulose membrane by semi-dry blotting and immunostained with GFAP antibody (immunoblotting). Lane 1; Brain Tissue Control. Lane 2; Eosinophilic Granuloma. Lane 3; Eosinophilic Granuloma Control. Lane 4; Mammary Glands Control. Lanes 5-7; Malign Mixed Tumor. Lanes 8 and 9; Adenocarcinoma. Lane 10; Complex Carcinoma. Lane 11; Lenfoma. Lane 12; Lenfoma Control. Lane 13; Sebaceous Gland Adenoma. Lane 14; Sebaceous Gland Adenoma Control. Lane 15; Brain Tissue Control Şekil 2b: SDS-PAGE ile ayr şt r lan doku proteinleri yar kuru blot yöntemi ile jel den nitroselüloz membrana aktar lm ş ve GFAP antikorlar ile immun boyama yap lm şt r (İmmun blot yöntemi). Beyin Dokusu Kontrol. Figure 2b: Seperated tissue proteins by SDS- PAGE were transferred from gel to nitrocellulose membrane by semi-dry blotting and immunostained with GFAP antibody (immunoblotting). Lane 1; Brain Tissue Control. Lane 2; Eosinophilic Granuloma. Lane 3; Eosinophilic Granuloma Control. Lane 4; Mammary Glands Control. Lanes 5-7; Malign Mixed Tumor. Lanes 8 and 9; Adenocarcinoma. Lane 10; Complex Carcinoma. Lane 11; Lenfoma. Lane 12; Lenfoma Control. Lane 13; Sebaceous Gland Adenoma. Lane 14; Sebaceous Gland Adenoma Control. Lane 15; Brain Tissue Control Şekil 2b: SDS-PAGE ile ayrıştırılan doku proteinleri yarı-kuru blot yöntemi ile jel den nitroselüloz membrana aktarılmış ve GFAP antikorları ile immun boyama yapılmıştır (İmmun blot yöntemi). Sıra 1; Beyin Dokusu Kontrol. Sıra 2; Eozinofilik S ra 1; Granüloma. Beyin Dokusu Kontrol. Sıra 3; S ra Eozinofilik 2; Granüloma Kontrol. Granüloma. Sıra S ra 4; 3; Eozinofilik Meme Dokusu Granüloma Kontrol. Sıra S ra 5-7; 4; Malin Meme Dokusu Miks Kontrol. Tümör. S ra Sıra 5-7; 8 Malin ve 9; Miks Adenokarsinom. Tümör. S ra 8 Sıra ve 9; Adenokarsinom. 10; Kompleks S ra Karsinom. 10; Kompleks Sıra Karsinom. 11; Lenfoma. S ra 11; Sıra 12; Lenfoma Kontrol. Sıra 13; Sebaseöz Bez Lenfoma. S ra 12; Lenfoma Kontrol. S ra 13; Sebaseöz Bez Adenomu. Sıra 14; Sebaseöz Bez Adenomu Kontrol. Adenomu. Sıra S ra 15; 14; Beyin Sebaseöz Dokusu Bez Adenomu Kontrol. Kontrol. S ra 15; The results of SDS PAGE and semi-dry blotting analysis showed that GFAP is not

5 The results of SDS PAGE and semi-dry blotting analysis showed that GFAP is not being expressed by the 14 tumors which we studied (See Figure 2a and Figure 2b). It has been observed that GFAP expression was seen in enough amounts as expected in the brain tissues. The fact that GFAP has not been identified in the tumor tissues we analysed points out that this protein is quite specific to nervous system tissues. In conclusion, GFAP is not being expressed in neoplastic tissue samples of dogs with eosinophilic granuloma, malign mixed tumor, leiomyoma, complex carcinoma, sebaceous gland adenoma, mast cell tumor, lenfoma, adenocarcinoma, rhabdomyosarcoma and lipoma. These results suggest that GFAP cannot be used as a marker for nonglial tumor tissues in dogs. Obtained results can be considered as an original data in terms of non-glial origin tumors since GFAP is a specific marker protein to healthy and neoplastic glial cells. Acknowledgement This research has been financially supported by The Scientific and Technological Research Council of Turkey (TUBITAK). References 1. Bergmann W, Burgener IA, Roccabianca P, Rytz U, and Welle M (2009): Primary Splenic Peripheral Nerve Sheath Tumour in a Dog. Journal of Comparative Pathology, 141, Bignami A, Dahl D, Rueger DC (1980): Glial fibrillary acidic protein in normal neural cells and in pathological conditions. Adv Cellul Neurobiol, 1, Budka H (1986): Non-glial specificities of immunocytochemistry for the glial fibrillary acid protein. Acta Neuropathology, 72, Chen MH, Yang WK, Whang-peng J, Lee LS, Huang TS (1998): Differential inducibilities of GFAP expression, cytostasis and apoptosis in primary cultures of human astrocyctic tumours. Apoptosis, 3, Curran A, White DK, Damm DD, Murrah V (2001): Polymorphous low-grade adenocarcinoma versus pleomorphic adenoma of minor salivary glands. Resolution of a diagnostic dilemma by immunohistochemical analysis with glial fibrillary acidic protein. Oral Surgery Oral Medicine Oral Pathology, 91, Eng LF, Ghirnikar RS, Lee YI (2000): Glial Fibrillary Acidic Protein: GFAP-Thirty-One Years ( ). Neurochemical Research, 25, Gumber S, Cho DY, Morgan TW (2010): Late onset of cerebellar abiotrophy in a boxer dog. Veterinary Medicine International, 2010, 8. Headley SA, Koljonen M, Gomes LA, and Sukura A (2009): Central Primitive Neuroectodermal tumour with ependymal differentiation in a dog. Journal of Comparative Pathology, 140, Hwang IK, Choi JH, Li H, Yoo KY, Kim DW, Lee CH, Sun Shin S, Sung JK, Lee IS, Yoon YS, Won MH (2008): Changes in Glial Fibrillary Acidic Protein Immunoreactivity in the Dentate Gyrus and Hippocampus Proper of Adult and Aged Dogs. J. Vet. Med. Sci., 70, Laemmli UK (1970): Cleavage of structural proteins during the assembly of the head of bacteriophage. Nature, 227, Moulton J (1990): Tumors in Domestic Animals. University of California Press, Berkeley. 12. Ottinger T, Lindberg R, Ekman S (2009): Malignant Acoustic Schwannoma in a Dog. Journal of Veterinary Diagnostic Investigation, 21, Vos JH, Van den I, Misdorp W, Ramaekers FCS, Van Mil FN, And Neijs M (1989): An immunohistochemical study of canine tissues with vimentin, demsin, glial fibrillary acidic protein and neurofilament antisera. Journal of Veterinary Medicine, 36, Geliş Tarihi: / Kabul Tarihi: Corresponding Author: Prof. Dr. Hamdi Uysal Department of Biochemistry, Faculty of Veterinary Medicine, University of Ankara, 06110, Ankara, Turkey huysal@veterinary.ankara.edu.tr 27