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MDM2 and CDK4 expression in canine liposarcoma
68
ESVP/ECVP Proceedings 2012
152:1, 2015
MDM2 AND CDK4 EXPRESSION IN CANINE LIPOSARCOMA G. Avallone *, B. Brunetti *, C. Bernardini *, P. Roccabianca y, M. Forni *, E. Lepri z, L. Crippa x, A. Olandese * and G. Sarli* *Department of Veterinary Medical Sciences, University of Bologna, y Dipartimento di Scienze Veterinarie e Sanita Pubblica, University of Milano, z Dipartimento di Medicina Veterinaria, University of Perugia and xIstovet, Besana Brianza (MB), Italy Introduction: Canine liposarcoma (LP) is an uncommon neoplasm usually arising in the subcutis and classified as well-differentiated (WDLP), myxoid (MLP) and pleomorphic (PLP). In man, LP is classified as WDLP/atypical lipomatous tumour (ATL), dedifferentiated (DDLP), MLP and PLP. WDLP/ALT and DDLP are considered different morphological presentations of the same entity, bearing the amplification of genes encoding for MDM2 and CDK4, and overexpressing these proteins. The aim of this study was to assess the expression of MDM2 and CDK4 in canine LP by immunohistochemistry. Materials and Methods: Selected cases were classified according to the WHO classification of tumours of domestic animals. When this was not possible, the human classification was applied. Cross-reactivity of antibodies specific for anti-mdm2 and CDK4 was assessed by western blot analysis. Immunohistochemistry was performed on formalin-fixed tissue with heat-induced antigen retrieval. Cases were considered positive when O10% of neoplastic cells had nuclear labelling. Results: Forty-seven LP, were collected: 19 WDLP, 18 PLP and seven MLP. Three cases were consistent with DDLP. Fifteen cases expressed MDM2 and 37 CDK4. MDM2 and CDK4 were respectively expressed in 12/19 and 17/19 cases of WDLP, 1/7 and 5/7 of MLP, 0/ 18 and 12/18 of PLP, and 2/3 and 3/3 of DDLP. Taken together, WDLPs and DDLPs expressed MDM2 in 14/22 (63.6%) and CDK4 in 20/22 cases (90.9%). Conclusions: These results suggest that WDLP/DDLP may represent a biological entity characterized by MDM2 and CDK4 overexpression, paralleling LP in man, and suggesting the presence of similar gene amplifications.
THE COMPARISON OF N-CADHERIN EXPRESSION IN CANINE AND HUMAN OSTEOSARCOMA CELL LINES AND IN SPONTANEOUS OSTEOSARCOMAS R. Ciaputa *, M. Kandefer-Gola *, I. Janus *, M. Nowak *, nskaJ.A. Madej *, I. Janus *, D. Poradowski y, B. Obmi orzy nskaz Mrukowicz y and E. G *Department of Pathology, Division of Pathomorphology and Veterinary Forensics, yDepartment of Biochemistry, Pharmacology and Toxicology, Division of Pharmacology and Toxicology, University of Environmental and Life Sciences, Wroclaw and zHistopathological Laboratory ‘Hist-Med’, Wroclaw, Poland Introduction: N-cadherin plays an important role in intercellular adhesion. The expression level of this protein in tumours may be useful in assessing the ability of the tumour to metastasize. The aim of this study was to compare the expression of N-cadherin in canine and human spontaneous osteosarcoma with the expression of this protein in the cells derived from the established canine (D-17) and human (U-2 OS) osteosarcoma cell lines. Materials and Methods: Ten samples of canine osteosarcoma and five samples of human osteosarcoma were used. The cell lines were grown on 10-well hydrophobic slides. Immunohistochemistry was performed using N-cadherin antibodies (Clone 6G11, DAKO). Results: Expression of N-cadherin, according to the Remmel scale, in the cells of spontaneous canine osteosarcoma was assessed at 4 points and for human osteosarcoma at 3 points, while in the canine osteosarcoma cell line it was at 12 points and in the human cell line at 8 points. Conclusions: Decreased expression of N-cadherin in the spontaneous osteosarcomas compared with their equivalent species cell lines may suggest a relatively high metastatic potential. The stronger expression of the tested protein in the cell lines may be due to the fact that these cells might have stronger adhesion when compared with spontaneously arising tumours. Decreased expression of N-cadherin in human osteosarcoma, when compared with canine osteosarcoma, may indicate a greater potential for metastasis in the human tumour.
THE COMPARISON OF SURVIVIN EXPRESSION IN CANINE AND HUMAN SPONTANEOUS OSTEOSARCOMA AND ESTABLISHED OSTEOSARCOMA CELL LINES D. Poradowski *, B. Obmi nska-Mrukowicz *, R. Ciaputa y, orzy nskaz M. Kandefer-Gola y, M. Nowak y, I. Janus y and E. G *Department of Biochemistry, Pharmacology and Toxicology, Division of Pharmacology and Toxicology, yDepartment of Pathology, Division of Pathomorphology and Veterinary Forensics, University of Environmental and Life Sciences, Wroclaw and zHistopathological Laboratory ‘Hist-Med’, Wroclaw, Poland Introduction: Survivin is a protein belonging to the family of inhibitors of apoptosis (IAP). This protein is expressed by some normal cell types and its overexpression occurs in many types of cancers. The aim of this study was to compare the expression of survivin in canine and human spontaneous osteosarcoma with expression in cells derived from the established canine (D-17) and human (U-2 OS) osteosarcoma cell lines. Materials and Methods: Paraffin wax blocks from archival collections (10 samples of canine and five samples of human osteosarcoma) were sectioned at 4 mm. Cells from human and canine osteosarcoma cell lines were grown on 10-well hydrophobic slides (Thermo Scientific). Immunohistochemistry was performed using survivin antibodies (clone 12C4, DAKO). Results: The strength of survivin expression in canine and human spontaneous osteosarcoma was at the level of + +, while in the case of the canine osteosarcoma cell line at the level of ++ and the human osteosarcoma cell line at the level of +. Conclusions: Expression of survivin was comparable in both spontaneous osteosarcomas and the canine cell line, while weaker in the human cell line. This may indicate that the studied tumours and cell lines demonstrate a comparable level of resistance to apoptosisinducing agents. In-vitro studies may be useful in refining a suitable animal model that can be used in research on human oncogenesis.
THE INFLUENCE OF RISEDRONATE SODIUM ON CANINE (D-17) AND HUMAN (U-2 OS) OSTEOSARCOMA CELL LINES D. Poradowski *, B. Obmi nska-Mrukowicz *, R. Ciaputa y, y M. Kandefer-Gola , M. Nowak y and I. Janusy *Department of Biochemistry, Pharmacology and Toxicology, Division of Pharmacology and Toxicology and yDepartment of Pathology, Division of Pathomorphology and Veterinary Forensics, University of Environmental and Life Sciences, Wroclaw, Poland Introduction: Risedronate sodium is a new generation bisphosphonate; a drug used in human and veterinary medicine in the treatment of diseases such as osteoporosis, Paget’s disease and multiple myeloma. Bisphosphonates also have cytotoxic activity and are used as premedication in the treatment of osteosarcoma. The aim of this study was to evaluate the viability of canine and human osteosarcoma cell lines treated with risedronate sodium dependent on the concentrations applied. Materials and Methods: The cell lines were exposed to risedronate sodium for a period of 72 h. The drug was tested in the following concentrations: 300 150 100, 30, 15, 10, 3, 1.5, 1, 0.3, 0.15 mg/ml. The viability of the cells was evaluated using the MTT assay. Four independent repetitions were performed then, for each, the EC50 (half maximal effective concentration) was calculated and the results given as the average of these values. Results: The EC50 value of risedronate sodium in the canine osteosarcoma line was 144.83 6.22 mg/ml and in the human osteosarcoma line 98.1 5.4 mg/ml. For the concentration of 300 mg/ml, the percentage of live cells in the case of the canine osteosarcoma cell line was 29.1 2.17%, while in the case of the human osteosarcoma cell line this was 14.84 2.36%. Conclusions: Canine osteosarcoma cells are more resistant to risedronate sodium than human osteosarcoma cells. This suggests that risedronate sodium may be a useful adjunct to standard therapy of osteosarcoma in both man and animals.
ESVP/ECVP Proceedings 2012
152:1, 2015
MDM2 AND CDK4 EXPRESSION IN CANINE LIPOSARCOMA G. Avallone *, B. Brunetti *, C. Bernardini *, P. Roccabianca y, M. Forni *, E. Lepri z, L. Crippa x, A. Olandese * and G. Sarli* *Department of Veterinary Medical Sciences, University of Bologna, y Dipartimento di Scienze Veterinarie e Sanita Pubblica, University of Milano, z Dipartimento di Medicina Veterinaria, University of Perugia and xIstovet, Besana Brianza (MB), Italy Introduction: Canine liposarcoma (LP) is an uncommon neoplasm usually arising in the subcutis and classified as well-differentiated (WDLP), myxoid (MLP) and pleomorphic (PLP). In man, LP is classified as WDLP/atypical lipomatous tumour (ATL), dedifferentiated (DDLP), MLP and PLP. WDLP/ALT and DDLP are considered different morphological presentations of the same entity, bearing the amplification of genes encoding for MDM2 and CDK4, and overexpressing these proteins. The aim of this study was to assess the expression of MDM2 and CDK4 in canine LP by immunohistochemistry. Materials and Methods: Selected cases were classified according to the WHO classification of tumours of domestic animals. When this was not possible, the human classification was applied. Cross-reactivity of antibodies specific for anti-mdm2 and CDK4 was assessed by western blot analysis. Immunohistochemistry was performed on formalin-fixed tissue with heat-induced antigen retrieval. Cases were considered positive when O10% of neoplastic cells had nuclear labelling. Results: Forty-seven LP, were collected: 19 WDLP, 18 PLP and seven MLP. Three cases were consistent with DDLP. Fifteen cases expressed MDM2 and 37 CDK4. MDM2 and CDK4 were respectively expressed in 12/19 and 17/19 cases of WDLP, 1/7 and 5/7 of MLP, 0/ 18 and 12/18 of PLP, and 2/3 and 3/3 of DDLP. Taken together, WDLPs and DDLPs expressed MDM2 in 14/22 (63.6%) and CDK4 in 20/22 cases (90.9%). Conclusions: These results suggest that WDLP/DDLP may represent a biological entity characterized by MDM2 and CDK4 overexpression, paralleling LP in man, and suggesting the presence of similar gene amplifications.
THE COMPARISON OF N-CADHERIN EXPRESSION IN CANINE AND HUMAN OSTEOSARCOMA CELL LINES AND IN SPONTANEOUS OSTEOSARCOMAS R. Ciaputa *, M. Kandefer-Gola *, I. Janus *, M. Nowak *, nskaJ.A. Madej *, I. Janus *, D. Poradowski y, B. Obmi orzy nskaz Mrukowicz y and E. G *Department of Pathology, Division of Pathomorphology and Veterinary Forensics, yDepartment of Biochemistry, Pharmacology and Toxicology, Division of Pharmacology and Toxicology, University of Environmental and Life Sciences, Wroclaw and zHistopathological Laboratory ‘Hist-Med’, Wroclaw, Poland Introduction: N-cadherin plays an important role in intercellular adhesion. The expression level of this protein in tumours may be useful in assessing the ability of the tumour to metastasize. The aim of this study was to compare the expression of N-cadherin in canine and human spontaneous osteosarcoma with the expression of this protein in the cells derived from the established canine (D-17) and human (U-2 OS) osteosarcoma cell lines. Materials and Methods: Ten samples of canine osteosarcoma and five samples of human osteosarcoma were used. The cell lines were grown on 10-well hydrophobic slides. Immunohistochemistry was performed using N-cadherin antibodies (Clone 6G11, DAKO). Results: Expression of N-cadherin, according to the Remmel scale, in the cells of spontaneous canine osteosarcoma was assessed at 4 points and for human osteosarcoma at 3 points, while in the canine osteosarcoma cell line it was at 12 points and in the human cell line at 8 points. Conclusions: Decreased expression of N-cadherin in the spontaneous osteosarcomas compared with their equivalent species cell lines may suggest a relatively high metastatic potential. The stronger expression of the tested protein in the cell lines may be due to the fact that these cells might have stronger adhesion when compared with spontaneously arising tumours. Decreased expression of N-cadherin in human osteosarcoma, when compared with canine osteosarcoma, may indicate a greater potential for metastasis in the human tumour.
THE COMPARISON OF SURVIVIN EXPRESSION IN CANINE AND HUMAN SPONTANEOUS OSTEOSARCOMA AND ESTABLISHED OSTEOSARCOMA CELL LINES D. Poradowski *, B. Obmi nska-Mrukowicz *, R. Ciaputa y, orzy nskaz M. Kandefer-Gola y, M. Nowak y, I. Janus y and E. G *Department of Biochemistry, Pharmacology and Toxicology, Division of Pharmacology and Toxicology, yDepartment of Pathology, Division of Pathomorphology and Veterinary Forensics, University of Environmental and Life Sciences, Wroclaw and zHistopathological Laboratory ‘Hist-Med’, Wroclaw, Poland Introduction: Survivin is a protein belonging to the family of inhibitors of apoptosis (IAP). This protein is expressed by some normal cell types and its overexpression occurs in many types of cancers. The aim of this study was to compare the expression of survivin in canine and human spontaneous osteosarcoma with expression in cells derived from the established canine (D-17) and human (U-2 OS) osteosarcoma cell lines. Materials and Methods: Paraffin wax blocks from archival collections (10 samples of canine and five samples of human osteosarcoma) were sectioned at 4 mm. Cells from human and canine osteosarcoma cell lines were grown on 10-well hydrophobic slides (Thermo Scientific). Immunohistochemistry was performed using survivin antibodies (clone 12C4, DAKO). Results: The strength of survivin expression in canine and human spontaneous osteosarcoma was at the level of + +, while in the case of the canine osteosarcoma cell line at the level of ++ and the human osteosarcoma cell line at the level of +. Conclusions: Expression of survivin was comparable in both spontaneous osteosarcomas and the canine cell line, while weaker in the human cell line. This may indicate that the studied tumours and cell lines demonstrate a comparable level of resistance to apoptosisinducing agents. In-vitro studies may be useful in refining a suitable animal model that can be used in research on human oncogenesis.
THE INFLUENCE OF RISEDRONATE SODIUM ON CANINE (D-17) AND HUMAN (U-2 OS) OSTEOSARCOMA CELL LINES D. Poradowski *, B. Obmi nska-Mrukowicz *, R. Ciaputa y, y M. Kandefer-Gola , M. Nowak y and I. Janusy *Department of Biochemistry, Pharmacology and Toxicology, Division of Pharmacology and Toxicology and yDepartment of Pathology, Division of Pathomorphology and Veterinary Forensics, University of Environmental and Life Sciences, Wroclaw, Poland Introduction: Risedronate sodium is a new generation bisphosphonate; a drug used in human and veterinary medicine in the treatment of diseases such as osteoporosis, Paget’s disease and multiple myeloma. Bisphosphonates also have cytotoxic activity and are used as premedication in the treatment of osteosarcoma. The aim of this study was to evaluate the viability of canine and human osteosarcoma cell lines treated with risedronate sodium dependent on the concentrations applied. Materials and Methods: The cell lines were exposed to risedronate sodium for a period of 72 h. The drug was tested in the following concentrations: 300 150 100, 30, 15, 10, 3, 1.5, 1, 0.3, 0.15 mg/ml. The viability of the cells was evaluated using the MTT assay. Four independent repetitions were performed then, for each, the EC50 (half maximal effective concentration) was calculated and the results given as the average of these values. Results: The EC50 value of risedronate sodium in the canine osteosarcoma line was 144.83 6.22 mg/ml and in the human osteosarcoma line 98.1 5.4 mg/ml. For the concentration of 300 mg/ml, the percentage of live cells in the case of the canine osteosarcoma cell line was 29.1 2.17%, while in the case of the human osteosarcoma cell line this was 14.84 2.36%. Conclusions: Canine osteosarcoma cells are more resistant to risedronate sodium than human osteosarcoma cells. This suggests that risedronate sodium may be a useful adjunct to standard therapy of osteosarcoma in both man and animals.