ABSTRACT
The overexpression of proteins P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP1), mutant p53, and the enzyme glutathione-S-transferase (GSTpi) are related to resistance to chemotherapy in neoplasms. This study evaluated the expression of these markers by immunohistochemistry in two groups of canine TVT, without history of prior chemotherapy (TVT1, n=9) and in TVTs presented unsatisfactory clinical response to vincristine sulfate (TVT2, n=5). The percentage of specimens positively stained for P-gp, MRP1, GSTpi and p53 were, respectively 88.8%, 0%, 44.5% and 22.2% in TVT1 and 80%, 0%, 80% and 0% in TVT2. In TVT1, one specimen presented positive expression for three markers and four specimens for two markers. In TVT2, three specimens expressed P-gp and GSTpi. In conclusion, the canine TVTs studied expressed the four markers evaluated, but just P-gp and GSTpi were significantly expressed, mainly at cytoplasm and cytoplasm and nuclei, respectively, either before chemotherapy as after vincristine sulfate exposure. Future studies are needed to demonstrate the function of these two markers in conferring multidrug resistance (MDR) or predict the response to chemotherapy in canine TVT.
A superexpressão das proteínas glicoproteína-P (Gp-P), proteína associada à resistência à múltiplas drogas 1 (MRP1) e p53 mutante e a enzima glutationa-S-transferase pi (GSTpi) está relacionada com resistência à quimioterapia em neoplasias humanas e caninas. Este estudo avaliou a expressão, por meio da imuno-histoquímica desses marcadores em espécimes de TVT caninos sem histórico de quimioterapia prévia (TVT1, n=9) e em TVT caninos que apresentaram resposta clínica insatisfatória ao sulfato de vincristina (TVT2, n=5). A porcentagem de espécimes positivos para Gp-P, MRP1, GSTpi e p53 foram, respectivamente 88,8%, 0%, 44,5% e 22,2% no grupo TVT1 e 80%, 0%, 80% e 0% no grupo TVT2. No TVT1, um espécime apresentou expressão positiva para três marcadores e quatro para dois marcadores. No TVT2, três espécimes expressaram a Gp-P e GSTpi. Em conclusão, os TVTs caninos estudados expressaram os quatro marcadores avaliados, no entanto apenas a Gp-P e GSTpi foram significativamente expressas, principalmente no citoplasmas e no citoplasma e no núcleo, respectivamente, tanto antes da quimioterapia quanto após à exposição ao sulfato de vincristina. Estudos futuros são necessários para demonstrar a função desses dois marcadores em conferir resistência à multiplas drogas (RMD) ou predizer a resposta a quimioterapia no TVT canino.
Subject(s)
Animals , Dogs , Dogs/metabolism , Drug Resistance, Neoplasm , Venereal Tumors, Veterinary/chemistry , Vincristine/administration & dosage , Glutathione , Immunohistochemistry/veterinary , ATP Binding Cassette Transporter, Subfamily BABSTRACT
El objetivo del presente estudio fue visualizar y caracterizar las Regiones Organizadoras Nucleolares Argénticas (AgNORs) en células neoplásicas del tumor venéreo transmisible (TVT) de caninos a través de la impregnación con nitrato de plata. Se trabajó con una muestra tomada al azar de tejido parafinado de 30 caninos diagnosticados histológicamente (con coloración de Hematoxilina y Eosina) como TVT canino entre el 2000 al 2006. En 100 células por muestra se visualizó la presencia y ubicación de los AgNORs en las células neoplásicas. Se encontró un número medio de AgNORs por célula de 1.53, por núcleo de 0.91 y por nucléolo de 0.62. La distribución de los AgNORs en el total de células evaluadas fue de 60.7% (1821/3000) en el núcleo, 34.3% (1029/3000) en el nucléolo y 5% (150/3000) en ambas estructuras.
The objective of the present study was to visualize and characterize the Nucleolar Organizer Regions (AgNORs) in Cells of Transmissible Venereal Tumor (TVT) in thecanine through the impregnation with silver nitrate. One sample was collected at random from paraffin tissue of 30 canine tissue samples that were histological diagnosed (using Hematoxylin and Eosin stain) as TVT during the period of 2000-2006. The AgNORs were visualized and localized in 100 cells per sample. The average number of AgNORs per cellwas 1.53, per cell nucleus was 90.91 and per nucleolus was 0.62. The distribution ofAgNORs in the total number of cells was 60.7% (1821/ 3000) in the nucleus, 34.3% (1029/3000) in the nucleolus, and 5% (150/3000) in both structures.
Subject(s)
Animals , Dogs , Biomarkers, Tumor , Neoplasms , Nucleolus Organizer Region , Venereal Tumors, Veterinary/chemistryABSTRACT
LINEs (long interspersed nuclear elements or long interspersed repeated DNA elements) contains two open reading frames (ORFs), ORF1 and ORF2. We analysed the ORF2 located in the 5' region to the first exon of oncogene c-myc in canine transmissible venereal tumor (TVT) cell. We also showed the transcription activation was induced by this TVT-LINE sequence using CAT assay. To identify the mutation of tumor suppressor gene, sequence analysis of p53 from TVT cell was performed. We identified the point mutation of 964 nucleotide (T-C) resulting in the change of amino acid (Phe-Ser) of p53 tumor suppressor protein.
Subject(s)
Dog Diseases/genetics , Long Interspersed Nucleotide Elements/genetics , Tumor Suppressor Protein p53/genetics , Venereal Tumors, Veterinary/genetics , Amino Acid Sequence , Animals , Base Sequence , Cells, Cultured , Cricetinae , DNA, Neoplasm/chemistry , DNA, Neoplasm/genetics , Dogs , Molecular Sequence Data , Point Mutation , Polymerase Chain Reaction , Sequence Alignment , Transcription, Genetic , Tumor Suppressor Protein p53/chemistry , Venereal Tumors, Veterinary/chemistryABSTRACT
LINEs (long interspersed nuclear elements or long interspersed repeated DNA elements) contains two open reading frames (ORFs), ORF1 and ORF2. We analysed the ORF2 located in the 5' region to the first exon of oncogene c-myc in canine transmissible venereal tumor (TVT) cell. We also showed the transcription activation was induced by this TVT-LINE sequence using CAT assay. To identify the mutation of tumor suppressor gene, sequence analysis of p53 from TVT cell was performed. We identified the point mutation of 964 nucleotide (T-->C) resulting in the change of amino acid (Phe-->Ser) of p53 tumor suppressor protein.
Subject(s)
Animals , Cricetinae , Dogs , Amino Acid Sequence , Base Sequence , Cells, Cultured , DNA, Neoplasm/chemistry , Dog Diseases/genetics , Long Interspersed Nucleotide Elements/genetics , Molecular Sequence Data , Point Mutation , Polymerase Chain Reaction , Sequence Alignment , Transcription, Genetic , Tumor Suppressor Protein p53/chemistry , Venereal Tumors, Veterinary/chemistryABSTRACT
The collective immunohistochemical expression of human lysozyme, human alpha-1-antitrypsin, human CD3 antigen, calf vimentin, human keratins, human lambda light chains,canine immunoglobulins IgG, IgM, and bovine protein S-100 has been analyzed on formalin-fixed, paraffin-embedded tissue sections of 25 spontaneous canine transmissible venereal tumors (CTVT) from both genital and extragenital locations using the avidin-biotin-peroxidase complex technique. Lysozyme immunoreactivity was detected in 10/25 CTVT, alpha-1-antitrypsin in 14/25 CTVT, and vimentin in 25/25 CTVT. All CTVT cells were negative to keratins 5 + 8 of the Moll catalogue (RCK-102), S-100 protein, lambda light-chain immunoglobulins, IgG, IgM, and CD3 antigen. The intratumoral T-and B-lymphocyte infiltrate was differentiated using CD3 antigen, lambda light-chain immunoglobulins, IgG, and IgM, and this technique could be useful to evaluate the regressive or progressive growth stage of venereal tumors. Our findings support the hypothesis of a histiocytic immunophenotype for CTVT, and these staining techniques could be used in the differential diagnosis with lymphomas.
Subject(s)
Dog Diseases/pathology , Venereal Tumors, Veterinary/chemistry , Venereal Tumors, Veterinary/pathology , Animals , CD3 Complex/analysis , CD3 Complex/metabolism , Cattle , Diagnosis, Differential , Dog Diseases/metabolism , Dogs , Female , Humans , Immunoglobulin G/analysis , Immunoglobulin G/metabolism , Immunoglobulin M/analysis , Immunoglobulin M/metabolism , Immunohistochemistry/methods , Keratins/analysis , Keratins/metabolism , Male , Muramidase/analysis , Muramidase/metabolism , S100 Proteins/analysis , S100 Proteins/metabolism , Venereal Tumors, Veterinary/metabolism , Vimentin/analysis , Vimentin/metabolism , alpha 1-Antitrypsin/analysis , alpha 1-Antitrypsin/metabolismABSTRACT
Ten canine transmissible venereal tumour (TVT) cases were studied by digital image analysis quantification on sections stained with silver to demonstrate nucleolar organizer regions (Ag-NORs). In each animal, 100 neoplastic cells were randomly selected for evaluation. The following parameters were measured or calculated: area of nucleus, area of Ag-NOR dot, the mean number of Ag-NOR dots per nucleus, the mean area of Ag-NOR dots per nucleus and the ratio of mean nuclear dot area to nuclear area. All 10 cases were treated with vincristine at a dose of 0.6 mg/m2 intravenously once a week. Two, which showed malignant characteristics (i.e. uncontrolled growth, local invasion or metastasis), did not respond to multiple (12) treatments and had a fatal outcome. Of the remaining eight cases, seven responded to two to six treatments and one required 12 treatments. The average number of Ag-NORs per nucleus and the area of Ag-NORs per nucleus were lower in the seven cases that responded to two to six treatments than in the other three cases, but the difference was not significant. However, there was a significant difference in the ratios of Ag-NOR area to nuclear area between the two groups of cases. Thus, poor prognosis was (1) possibly correlated with an increase in the mean number of Ag-NORs per nucleus and an increase in the mean area of Ag-NORs per nucleus, and (2) definitely correlated with an increase in the mean ratio of Ag-NOR area to nuclear area.
