RESUMO
Mantle cell lymphoma (MCL) is a moderately aggressive B-cell lymphoma that responds poorly to currently used therapeutic protocols. In order to identify tumour characteristics that improve the understanding of biology of MCL, analysis of oligonucleotide microarrays were used to define specific gene expression profiles. Biopsy samples of MCL cases were compared to reactive lymphoid tissue. Among genes differentially expressed in MCL were genes that are involved in the regulation of proliferation, cell signalling, adhesion and homing. Furthermore, some genes with previously unknown function, such as C11orf32, C2orf10, TBC1D9 and ABCA6 were found to be differentially expressed in MCL compared to reactive lymphoid tissue. Of special interest was the high expression of the cannabinoid receptor 1 (CB1) gene in all MCL cases analysed. These results were further confirmed at the cellular and protein level by immunocytochemical staining and immunoblotting of MCL cells. Furthermore, there was a reduced expression of a regulator of G protein signalling, RGS13 in all MCLs, with a complete absence in the majority of cases while present in control lymphoid tissue. These results were further confirmed by PCR. Sequencing of the RGS13 gene revealed changes suggesting polymorphisms, indicating that downregulation of the expression of RGS13 is not related to mutations, but may serve as a new specific marker for MCL. Moreover, comparison between individual cases of MCL, revealed that the CCND1 gene appears to be differently expressed in MCL cases with high vs low proliferative activity.
Assuntos
Transformação Celular Neoplásica/genética , Linfoma de Célula do Manto/genética , Proteínas de Neoplasias/genética , Proteínas RGS/genética , Receptores de Droga/genética , Adolescente , Adulto , Idoso , Linfoma de Burkitt/genética , Linfoma de Burkitt/metabolismo , Linfoma de Burkitt/patologia , Estudos de Casos e Controles , Divisão Celular , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Criança , Ciclina D1/genética , Ciclina D1/metabolismo , DNA de Neoplasias/análise , DNA de Neoplasias/genética , Regulação para Baixo , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Hibridização in Situ Fluorescente , Leucemia de Células B/genética , Leucemia de Células B/metabolismo , Leucemia de Células B/patologia , Linfoma de Célula do Manto/metabolismo , Linfoma de Célula do Manto/patologia , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas RGS/metabolismo , RNA Mensageiro/análise , RNA Neoplásico/genética , Receptores de Canabinoides , Receptores de Droga/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Human tumour xenografts maintained in nude mice are a valuable research tool. The passaging and maintenance of human tumour xenografts in immune-deficient animals are expensive and labour-intensive. This study presents a protocol that permits long-term cryopreservation of viable glioblastoma xenograft tissue pieces in liquid nitrogen. Twenty different human glioblastoma xenografts that have been successfully transplanted and repeatedly passaged in nude mice were cryopreserved to validate the method. Different passages were cryopreserved for up to 40 months. On retransplantation of the tumours, all cases except one grew successfully. In order to ensure that the individual tumours did grow (not induced mouse tumours) restriction fragment length polymorphism analysis with variable number of tandem repeats (VNTR) probes was carried out. The method permits the long-term storage of viable, retransplantable glioblastoma xenografts. It minimizes animal use for the maintenance of xenografts and permits cryo-back-up of valuable tumours, thus markedly reducing the cost and increasing the accessibility of human tumour xenografts as a research tool in biology and genetics.
Assuntos
Neoplasias Encefálicas/patologia , Criopreservação , Glioblastoma/patologia , Transplante de Neoplasias/métodos , Animais , Southern Blotting , Neoplasias Encefálicas/genética , Divisão Celular , Sobrevivência Celular , DNA de Neoplasias/análise , DNA de Neoplasias/isolamento & purificação , Glioblastoma/genética , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Transplante HeterólogoRESUMO
Amplification of the epidermal growth factor receptor (EGFR) occurs in about 40% of human glioblastomas. In half of these cases, rearrangements of the amplified gene result in aberrant transcripts and proteins. The most frequent rearrangement affects the external domain of the receptor and results in nonbinding of ligand and constitutive activity. Less frequent rearrangements involve changes resulting in the loss of cytoplasmic amino acid sequences necessary for downregulation of the receptor following ligand binding. Here we report the development and selection for a rearranged amplified EGF receptor, which lacks cytoplasmic amino acid sequences in a human glioblastoma xenograft. An identical aberration has previously been reported in glioblastoma tissue. The patient tumor material, as well as the first passages of the xenograft showed amplification of the EGFR gene, but no evidence of gene rearrangement or an aberrant transcript. Interphase FISH data show the amplified gene on double minutes. Between passages 3 and 16, the growth rate of the xenograft almost doubled, the rearranged amplicon became dominant, as did the aberrant transcript, indicating selection under these conditions.
