RESUMO
N-terminal methionine removal is an important cellular process required for proper biological activity, subcellular localization, and eventual degradation of many proteins. The enzymes that catalyze this reaction are called Methionine Aminopeptidases (MAPs). To date, only two MAP family members, MAP1A and MAP2, have been well characterized and studied in mammals. In our studies, we have cloned a full length MAP1D gene. Expression and purification of full length recombinant protein shows that the sequence encodes an enzyme with MAP activity. MAP1D is overexpressed in colon cancer cell lines and in colon tumors as compared to matched normal tissue samples. Downregulation of MAP1D expression by shRNA in HCT-116 colon carcinoma cells reduces anchorage-independant growth in soft agar. These data suggest that MAP1D is a potentially oncogenic, novel member of the MAP gene family that may play an important role in colon tumorigenesis.
Assuntos
Aminopeptidases/biossíntese , Aminopeptidases/genética , Neoplasias do Colo/enzimologia , Acetiltransferases/genética , Sequência de Aminoácidos , Aminopeptidases/fisiologia , Linhagem Celular Tumoral , Clonagem Molecular , Neoplasias do Colo/genética , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Humanos , Metionil Aminopeptidases , Proteínas Associadas aos Microtúbulos/genética , Dados de Sequência Molecular , Filogenia , Proteínas Recombinantes/químicaRESUMO
Abnormalities in cellular differentiation are frequent occurrences in human cancers. Treatment of human melanoma cells with recombinant fibroblast interferon (IFN-beta) and the protein kinase C activator mezerein (MEZ) results in an irreversible loss in growth potential, suppression of tumorigenic properties and induction of terminal cell differentiation. Subtraction hybridization identified melanoma differentiation associated gene-7 (mda-7), as a gene induced during these physiological changes in human melanoma cells. Ectopic expression of mda-7 by means of a replication defective adenovirus results in growth suppression and induction of apoptosis in a broad spectrum of additional cancers, including melanoma, glioblastoma multiforme, osteosarcoma and carcinomas of the breast, cervix, colon, lung, nasopharynx and prostate. In contrast, no apparent harmful effects occur when mda-7 is expressed in normal epithelial or fibroblast cells. Human clones of mda-7 were isolated and its organization resolved in terms of intron/exon structure and chromosomal localization. Hu-mda-7 encompasses seven exons and six introns and encodes a protein with a predicted size of 23.8 kDa, consisting of 206 amino acids. Hu-mda-7 mRNA is stably expressed in the thymus, spleen and peripheral blood leukocytes. De novo mda-7 mRNA expression is also detected in human melanocytes and expression is inducible in cells of melanocyte/melanoma lineage and in certain normal and cancer cell types following treatment with a combination of IFN-beta plus MEZ. Mda-7 expression is also induced during megakaryocyte differentiation induced in human hematopoietic cells by treatment with TPA (12-O-tetradecanoyl phorbol-13-acetate). In contrast, de novo expression of mda-7 is not detected nor is it inducible by IFN-beta+MEZ in a spectrum of additional normal and cancer cells. No correlation was observed between induction of mda-7 mRNA expression and growth suppression following treatment with IFN-beta+MEZ and induction of endogenous mda-7 mRNA by combination treatment did not result in significant intracellular MDA-7 protein. Radiation hybrid mapping assigned the mda-7 gene to human chromosome 1q, at 1q 32.2 to 1q41, an area containing a cluster of genes associated with the IL-10 family of cytokines. Mda-7 represents a differentiation, growth and apoptosis associated gene with potential utility for the gene-based therapy of diverse human cancers.
Assuntos
Antígenos de Neoplasias/genética , Apoptose/genética , Cromossomos Humanos Par 1/genética , Diterpenos , Genes , Substâncias de Crescimento/genética , Interleucinas , Proteínas de Neoplasias/genética , Neoplasias/genética , Antígenos de Neoplasias/biossíntese , Antígenos de Neoplasias/isolamento & purificação , Sequência de Bases , Carcinoma/patologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Divisão Celular/genética , Clonagem Molecular , Dimetil Sulfóxido/farmacologia , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genes Supressores de Tumor , Glioblastoma/patologia , Substâncias de Crescimento/biossíntese , Substâncias de Crescimento/isolamento & purificação , Células HL-60/metabolismo , Células HL-60/patologia , Humanos , Interferon Tipo I/farmacologia , Células K562/metabolismo , Células K562/patologia , Masculino , Melanócitos/metabolismo , Melanoma/química , Melanoma/genética , Melanoma/patologia , Dados de Sequência Molecular , Peso Molecular , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/isolamento & purificação , Especificidade de Órgãos , Osteossarcoma/patologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Neoplásico/biossíntese , RNA Neoplásico/genética , Proteínas Recombinantes de Fusão/fisiologia , Proteínas Recombinantes , Terpenos/farmacologia , Acetato de Tetradecanoilforbol/farmacologia , Transfecção , Células Tumorais Cultivadas/patologiaRESUMO
Current cancer therapies are highly toxic and often nonspecific. A potentially less toxic approach to treating this prevalent disease employs agents that modify cancer cell differentiation, termed 'differentiation therapy.' This approach is based on the tacit assumption that many neoplastic cell types exhibit reversible defects in differentiation, which upon appropriate treatment, results in tumor reprogramming and a concomitant loss in proliferative capacity and induction of terminal differentiation or apoptosis (programmed cell death). Laboratory studies that focus on elucidating mechanisms of action are demonstrating the effectiveness of 'differentiation therapy,' which is now beginning to show translational promise in the clinical setting.
