RESUMO
Cell lines 4A4 and 2C5 are the respective metastatic and non-metastatic variants of the human mammary carcinoma cell line MDA-MB-435 in the nude mouse system. We compared the transcriptional profile of approximately 5000 full-length genies using the Affymetrix HuGene FL Array technology. We have shown that the metastatic phenotype is mediated by different functional categories of genes, e.g. genes involved in immune response, genes responsible for tumor antigens, genes involved in migration and invasion, genes involved in mediating signal transduction, genes responsible for transcription factors, genes involved in phospholipid signaling, genes involved in modulation of extracellular matrix and cytoskeleton, genes with a cell-type specific mode of expression and genes which do not fit into the subclasses as defined above. Our results suggest an important role of Autocrine Motility Factor (AMF) as a mediator of metastasis in this system.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Metástase Neoplásica , Transcrição Gênica , Animais , Northern Blotting , Linhagem Celular , Regulação para Baixo , Feminino , Humanos , Camundongos , Camundongos Nus , Transplante de Neoplasias , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , RNA Mensageiro/metabolismo , Transdução de Sinais , Células Tumorais Cultivadas , Regulação para CimaRESUMO
In order to identify genes associated with metastasis of mammary carcinoma, we compared the transcriptional profile (Affymetrix chip technology) of two cell lines derived from primary mammary carcinoma, three cell lines derived from bone marrow micrometastasis, a cell line derived from a lymph node metastasis as well as a cell line derived from malignant ascites. We found that 11 genes (0.16%) were up-regulated in all five cell lines derived from metastasis and 32 genes (0.45%) were up-regulated in four of these cell lines. Sixteen genes (0.23%) were down-regulated in the five metastatic cell lines, while 24 genes (0.34%) were down-regulated in four of the metastatic cell lines. The usefulness of our system for the identification of genes associated with metastasis of mammary carcinoma is demonstrated by the identification of genes which have already been implicated in metastasis of mammary carcinoma. This suggests that further evaluation of identified de-regulated genes, which until now have not been seen in context with metastasis of mammary carcinoma, should be undertaken.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Neoplasias da Medula Óssea/genética , Neoplasias da Medula Óssea/secundário , Neoplasias da Mama/metabolismo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Metástase Linfática , Metástase Neoplásica , Transcrição Gênica , Células Tumorais Cultivadas , Regulação para CimaRESUMO
Identification of new genes involved in the pathogenesis of breast cancer opens new avenues for improved diagnostic markers and new molecular targets for improved treatment of this malignancy. In the following we review genes with proved involvement in invasion and metastasis of breast cancer as well as genes which exhibit an expression pattern that correlates with invasion and metastasis.
Assuntos
Neoplasias da Mama/genética , Metástase Neoplásica/genética , Proteínas de Neoplasias/genética , Sequência de Aminoácidos , Animais , Neoplasias da Mama/patologia , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/fisiologia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/fisiologia , Endopeptidases/genética , Endopeptidases/fisiologia , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/fisiologia , Feminino , Regulação Neoplásica da Expressão Gênica , Genes , Substâncias de Crescimento/genética , Substâncias de Crescimento/fisiologia , Humanos , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/patologia , Modelos Moleculares , Dados de Sequência Molecular , NAD(P)H Desidrogenase (Quinona)/genética , Invasividade Neoplásica/genética , Proteínas de Neoplasias/fisiologia , Inibidores de Proteases , Conformação Proteica , Receptores de Fatores de Crescimento/genética , Receptores de Fatores de Crescimento/fisiologia , Roedores , Transdução de Sinais/genéticaRESUMO
Differential display technique was applied to a pair of cell lines derived from human breast carcinoma cell line MDA-MB 435 with metastatic and non-metastatic properties in the nude mouse system, with the objective to isolate genes involved in metastasis. DRIM (Down-Regulated In Metastasis) was the only gene found to be differentially expressed in this system. DRIM encodes a protein comprising 2785 amino acids with significant homology to a protein in yeast and C. elegans. The protein contains a conserved positively charged tail and several HEAT repeats, designated after four functionally characterized proteins in which the repeat was detected. Most of the hydrophobic regions of DRIM can be assigned to HEAT repeats. Expression of DRIM at the RNA level was investigated in several normal tissues and tumor cell lines.
Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Regulação Neoplásica da Expressão Gênica , Biossíntese de Proteínas , Proteínas , Sequência de Aminoácidos , Animais , Sequência de Bases , Neoplasias da Mama/genética , Caenorhabditis elegans/genética , Sequência Conservada , Feminino , Humanos , Camundongos , Camundongos Nus , Dados de Sequência Molecular , Metástase Neoplásica , Proteínas Nucleares , Especificidade de Órgãos , Reação em Cadeia da Polimerase , RNA Mensageiro/biossíntese , RNA Neoplásico/biossíntese , Sequências Repetitivas de Ácido Nucleico , Ribonucleoproteínas , Saccharomyces cerevisiae/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transcrição GênicaRESUMO
As a model system for the identification of genes involved in the progression of human breast cancer, differential gene expression in cell lines MCF-7 and MCF-7ADR was investigated. The latter cell line is derived from the former. Cell line MCF-7 is estrogen receptor-positive, vimentin-negative and uninvasive in the Matrigel outgrowth assay and in the nude mouse, while MCF-7ADR is estrogen receptor-negative, hormone-resistant, vimentin-positive, invasive in the Matrigel outgrowth assay and in the nude mouse and resistant to adriamycin due to overexpression of glycoprotein gp170. We have shown that tumor progression in this model system is mediated by transcriptional regulation of mitochondria-related genes, proteases, transmembrane receptors and cell cycle-related gene proteins. Among the genes differentially regulated at the transcriptional level in the cell lines MCF-7 and MCF-7ADR are a new mitochondrial transcript, mitochondrial creatine kinase, matrix metalloproteinase-1, stromelysin-3, urokinase and its receptor, tissue factor, E-cadherin, epidermal growth factor receptor, transmembrane proteins Mat-8 and progression associated protein (PAP), cyclin E, cyclin-dependent kinase-2 and cell cycle inhibitory proteins p16, p21 and p27.
Assuntos
Neoplasias da Mama/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Mamárias Experimentais/genética , Animais , Sequência de Bases , Caderinas/genética , Caderinas/metabolismo , Clonagem Molecular , Ciclinas/genética , Ciclinas/metabolismo , DNA Mitocondrial , Modelos Animais de Doenças , Progressão da Doença , Endopeptidases/genética , Endopeptidases/metabolismo , Biblioteca Gênica , Camundongos , Camundongos Nus , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RNA Mensageiro/análise , Transcrição Gênica , Células Tumorais CultivadasRESUMO
Differential gene expression in mammary carcinoma cell lines MCF-7 and MCF-7ADR was investigated by Differential Display Technique. These two cell lines represent a model system for progression of mammary carcinoma: MCF-7 is estradiol-dependent for growth, estrogen-receptor positive, tamoxifen responsive, vimentin negative, Adriamycin sensitive and not invasive in vitro or in vivo while MCF-7ADR is estradiol-independent for growth, estrogen-receptor negative, tamoxifen resistant, vimentin positive, Adriamycin resistant and invasive in vitro and in vivo. Here we describe the identification of a new receptor expressed exclusively in MCF-7ADR. The receptor covers 157 amino acids with a predicted topology of four transmembrane and two extracellular domains. Additional members of the gene family are: peripheral membrane protein PMP22, epithelial membrane protein EMP-1 and squamous cell differentiation marker CL-20.
Assuntos
Neoplasias da Mama/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteínas de Membrana/genética , Proteínas de Neoplasias/genética , Receptores de Superfície Celular , Sequência de Aminoácidos , Sequência de Bases , DNA Complementar/análise , Feminino , Humanos , Proteínas de Membrana/análise , Dados de Sequência Molecular , Proteínas da Mielina/fisiologia , RNA Mensageiro/análise , Células Tumorais CultivadasRESUMO
The general feasibility of chimerization of monoclonal antibodies (mAbs) has already been shown for a large number of them. In order to evaluate in vitro parameters relevant to immunosuppressive therapy, we have chimerized and synthesized two anti-CD4 mAbs recognizing two different epitopes on the human T-lymphocyte antigen, CD4. The chimerized mAbs are produced at levels corresponding to those of the original hybridoma cell lines. With respect to activation of human complement, the individual Abs are negative; however, when used in combination, complement activation was performed. When applied in combination, they were found to modulate the CD4 antigen, whereas the individual mAb do not display this property. Individually they mediate an up to 60% inhibition of the mixed lymphocyte reaction (MLR). However, by combination of an anti-CD4 mAb with one directed against the alpha-chain of the human IL2 receptor, nearly 100% inhibition of the MLR was achieved, even with reduced dosage of the mAbs. Our data suggest that the combination of an anti-CD4 mAb and an anti-IL2R alpha chain mAb is more effective with respect to immunosuppression than each mAb by itself, indicating that this mAb cocktail could be a new strategy for immunosuppressive therapy.
Assuntos
Anticorpos/química , Antígenos CD4/imunologia , Receptores de Interleucina-2/imunologia , Sequência de Aminoácidos , Animais , Reações Antígeno-Anticorpo , Modulação Antigênica , Sequência de Bases , Clonagem Molecular , Ativação do Complemento , Expressão Gênica , Humanos , Hibridomas , Ativação Linfocitária , Teste de Cultura Mista de Linfócitos , Camundongos , Dados de Sequência Molecular , RNA Mensageiro/genética , Proteínas Recombinantes de Fusão , TransfecçãoRESUMO
General aviation and air taxi approach phase accidents, which occurred during Visual and Instrument Flight Rules (VFR and IFR, respectively) over the last 25 years, were analyzed. The data suggest that there is a 204% higher risk during the approach and landing phase of VFR flights, than during similar IFR operations (14.82 vs. 7.27 accidents/100,000 approaches). Alarmingly, the night single pilot IFR (SPIFR) accident rate is almost 8 times the rate of day IFR, 35.43 vs. 4.47 accidents/100,000 approaches, and two and a half times that of day VFR approaches, 35.43 vs. 14.82 accidents/100,000 approaches. Surprisingly, the overall SPIFR accident rates are not much higher than dual-pilot IFR (DPIFR), 7.27 vs. 6.48 accidents/100,000 approaches. The generally static ratio of the statistics for SPIFR/DPIFR accident rates may be accounted for by little or no change in general aviation cockpit technology during the last 25 years, and because IFR operational flight task management training has not kept pace.
Assuntos
Acidentes Aeronáuticos/estatística & dados numéricos , Aeronaves/instrumentação , Apresentação de Dados/normas , Escuridão , Falha de Equipamento , Humanos , Fatores de Risco , Segurança , Percepção VisualRESUMO
We describe a new method for amplification, by polymerase chain reaction (PCR), of rearranged segments encoding the variable part of light and heavy chains of an antibody (Ab) from the chromosomal DNA of hybridoma cells for the chimerization of Abs. A fundamental prerequisite for this is the knowledge of the exact sequences in the 5'-untranslated region of light and heavy chain mRNA, and of the joining segment used for rearrangement. This allows the design of nondegenerated oligodeoxyribonucleotides for PCR. The primer design permits directional cloning of the amplified, promoterless fragments into cassette vectors, in which they will be linked to the appropriate human constant domains and immunoglobulin (Ig) promoter/enhancer elements. The method is illustrated for chimerization of an Ab directed against the human T-lymphocyte antigen, CD4. The chimerized Ab is secreted in abundant quantities after transfection of the engineered plasmids into non-Ig-producing myeloma cells.
