RESUMO
PURPOSE: Measles virus (MV) causes the regression of human lymphoma xenografts. The purpose of this study was to determine if canine lymphoid cells could be infected in vitro with MV or canine distemper virus (CDV, the canine Morbillivirus equivalent of MV) and determine if in vitro viral infection leads to apoptotic cell death. EXPERIMENTAL DESIGN: Reverse transcriptase-PCR was used to examine the expression of both signal lymphocyte activation molecule (CD150) and membrane cofactor molecule (CD46) mRNA. An attenuated CDV expressing enhanced green fluorescent protein was used to infect canine cells in vitro. Both flow cytometry and reverse transcriptase-PCR was used to document CDV infection. Cell death was examined using a propidium iodide staining assay and Annexin V binding. RESULTS: Canine lymphoid cell lines and neoplastic B and T lymphocytes collected from dogs with spontaneous lymphoma expressed the Morbillivirus receptor CD150 mRNA. In contrast, only neoplastic lymphocytes expressed detectable levels of CD46 mRNA. Although MV did not infect canine cells, CDV efficiently infected between 40% and 70% of all three canine lymphoid lines tested. More importantly, CDV infected 50% to 90% of neoplastic lymphocytes isolated from dogs with both B and T cell lymphoma. Apoptosis of CDV-infected cell lines was documented. CONCLUSIONS: Attenuated CDV may be a useful treatment for canine lymphoma. As such, dogs with lymphoma may represent a biologically relevant large animal model to investigate the feasibility, safety, and efficacy of Morbillivirus therapy in a clinical setting with findings that may have direct applicability in the treatment of human non-Hodgkin's lymphoma.
Assuntos
Vírus da Cinomose Canina/crescimento & desenvolvimento , Linfócitos/virologia , Animais , Antígenos CD/genética , Apoptose , Linhagem Celular Tumoral , Chlorocebus aethiops , Vírus da Cinomose Canina/genética , Cães , Citometria de Fluxo , Expressão Gênica , Glicoproteínas/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Imunoglobulinas/genética , Células Jurkat , Linfócitos/metabolismo , Linfoma/terapia , Linfoma/virologia , Proteína Cofatora de Membrana , Glicoproteínas de Membrana/genética , Plasmídeos/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Superfície Celular , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Membro 1 da Família de Moléculas de Sinalização da Ativação Linfocitária , Transfecção , Células VeroRESUMO
OBJECTIVE: To evaluate canine histiocytic sarcoma cell lines and tumor samples for dysregulation of the Kit/stem-cell factor (SCF), Flt3/Flt3 ligand (Flt3L), and Met/hepatocyte growth factor (HGF) receptor tyrosine kinase signaling pathways, as these are known to contribute to the differentiation and survival of normal dendritic cells as well as malignant transformation of dendritic cells in mouse models. SAMPLE POPULATION: 4 histiocytic sarcoma tumor cell lines and 35 formalin-fixed histiocytic sarcoma specimens obtained from dogs. PROCEDURE: Histiocytic sarcoma cell lines were evaluated for expression of Kit/SCF, Flt3/Flt3L, and Met/HGF by use of reverse transcriptase-PCR procedures. Histiocytic sarcoma cell lines and tumor samples were evaluated for mutations in Kit, Flt3, and Met by use of PCR analysis of genomic DNA, followed by both sequencing and fluorescent PAGE for deletions or internal tandem duplications. The ability of the multi-targeted split-kinase inhibitor SU11654 to block proliferation and induce apoptosis of histiocytic sarcoma cell lines was also evaluated. RESULTS: No mutations in Kit, Flt3, and Met were identified in any of the cell lines or tumor samples evaluated. Furthermore, SU11654 did not induce cell-cycle arrest or apoptosis of histiocytic sarcoma lines, even at supratherapeutic doses. CONCLUSIONS AND CLINICAL RELEVANCE: These data suggest that dysregulation of Kit/SCF, Flt3/Flt3L, and Met/HGF signaling pathways is unlikely to occur in histiocytic sarcomas of dogs and that inhibitors of the Kit, Flt3, and Met pathways are unlikely to provide clinical benefit to dogs with histiocytic sarcomas.
Assuntos
Doenças do Cão/genética , Transtornos Histiocíticos Malignos/veterinária , Proteínas de Membrana/genética , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas c-met/genética , Sarcoma/veterinária , Tirosina Quinase 3 Semelhante a fms/genética , Animais , Sequência de Bases , Linhagem Celular Tumoral , Clonagem Molecular , Análise Mutacional de DNA , Primers do DNA , Cães , Éxons , Transtornos Histiocíticos Malignos/genética , Reação em Cadeia da Polimerase , Sarcoma/genética , Fator de Células-Tronco/genéticaRESUMO
An activating mutation in codon 599 of BRAF has been identified in approximately 60% of human cutaneous nevi and melanomas, but not melanomas of mucosal origin. The purpose of this study was to determine if BRAF mutations occur in canine oral malignant melanomas. The canine BRAF gene was first cloned from normal canine testicular cDNA, and a novel previously unreported splice variant involving exon 5 was identified during this process. To screen canine melanoma samples for BRAF mutation in codon 599, cDNA and genomic DNA were isolated from canine malignant melanoma cell lines and primary tumor samples respectively, all from cases seen at the Veterinary Medical Teaching Hospital at the University of California, Davis. Polymerase chain reaction (PCR) was performed for exon 15 using primers based at the 5' end of exon 15 and the 5' end of intron 15 and the resultant products were directly sequenced. No mutations in codon 599 or exon 15 were identified in any of the 17 samples evaluated. However, all of the melanoma cell lines expressed BRAF and demonstrated high levels of basal ERK phosphorylation suggesting that dysregulation of this pathway is present. Therefore, similar to the case with human mucosal melanomas, canine oral malignant melanomas do not possess codon 599 BRAF mutations commonly identified in human cutaneous melanomas. This finding supports the notion that melanomas arising from non-sun-exposed sites exhibit distinct mechanisms of molecular transformation.