RESUMO
In this study, a novel approach to antitumor therapy was devised by generating a chimeric tumor-targeted killer protein, referred to as immunocasp-3, that comprises a single-chain anti-erbB2/HER2 antibody with a NH(2)-terminal signal sequence, a Pseudomonas exotoxin A translocation domain, and a constitutively active caspase-3 molecule. In principle, cells transfected with the immunocasp-3 gene would express and secrete the chimeric protein, which then binds to HER2-overexpressing tumor cells. Subsequent cleavage of the constitutively active capase-3 domain from the immunocasp-3 molecule and its release from internalized vesicles would lead to apoptotic tumor cell death. To test this strategy, we transduced human lymphoma Jurkat cells with a chimeric immunocasp-3 gene expression vector and showed that they not only expressed and secreted the fusion protein but also selectively killed tumor cells overexpressing HER2 in vitro. i.v. injection of the transduced Jurkat cells led to tumor regression in a mouse xenograft model because of continuous secretion of immunocasp-3 by the transduced cells. The growth of HER2-positive tumor cells in this model was inhibited by i.m. as well as intratumor injection of immunocasp-3 expression plasmid DNA, indicating that the immunocasp-3 molecules secreted by transfected cells have systematic antitumor activity. We conclude that the immunocasp-3 molecule, combining the properties of a tumor-specific antibody with the proapoptotic activity of a caspase, has potent and selective antitumor activity, either as cell-based therapy or as a DNA vaccine. These findings provide a compelling rationale for therapeutic protocols designed for erbB2/HER2-positive tumors.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Anticorpos Antineoplásicos/uso terapêutico , Antígenos de Neoplasias/imunologia , Apoptose/efeitos dos fármacos , Caspases/uso terapêutico , Imunoconjugados/uso terapêutico , Região Variável de Imunoglobulina/uso terapêutico , Proteínas de Neoplasias/imunologia , Receptor ErbB-2/imunologia , Proteínas Recombinantes de Fusão/uso terapêutico , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Antineoplásicos/farmacologia , Caspase 3 , Caspases/farmacologia , Ativação Enzimática , Indução Enzimática , Feminino , Células HeLa , Humanos , Imunoconjugados/farmacologia , Região Variável de Imunoglobulina/farmacologia , Células Jurkat/metabolismo , Células Jurkat/transplante , Camundongos , Dados de Sequência Molecular , Neoplasias Ovarianas/patologia , Proteínas Recombinantes de Fusão/metabolismo , Transfecção , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
OBJECTIVE: To express human osteoprotegerin (OPG) in E. Coli and analyze its bioactivity in vitro. METHODS: Synthetic oligonucleotides were used to amplify human OPG gene by RT-PCR from total RNA of human osteosarcoma cell line MG63. The OPG cDNA coding for 380 amino acid residues was inserted into prokaryotic expression vector pRSET-A, transformed into competent E. Coli BL21, and induced by 0.1 mmol/l IPTG. SDS-PAGE and Western blot were performed to identify OPG-6His fusion protein. After purified by affinity chromatography, 1,000 microg/L or 1,500 microg/L of OPG-6His were added into the mouse bone marrow cells culture medium. The number of tartrate-resistant acid phophatase (TRAP)-positive multinucleated cells and resorption pits were counted to assess the bioactivity of expression products. RESULTS: The sequence of OPG mature peptide encoding cDNA obtained in this experiment was as same as reported. SDS-PAGE showed 24% of total bacterial protein was of OPG-6His fusion protein. Western blot assay demonstrated that the molecular weight of recombinant protein was about 46 KD and could react specifically with human anti-OPG antibody. The mouse bone marrow cells were induced by 1alpha, 25-dihydroxyvitaminD3 (10(-8) mol/L) and Dexamethasone (10(-7) mol/L) to form osteoclastic-like multinucleated cells. 1,500 microg/L of purified OPG-6His protein could decrease the number of resorption pits and TRAP-positive multinucleated cells in vitro (P < 0.05), but it didn't show the same effects when the concentration of OPG-6His fusion protein was of 1,000 microg/L. CONCLUSIONS: Human OPG-6His fusion protein is expressed and purified in E. Coli. The expression products have moderate inhibitory effects on osteoclast differentiation and bone resorption in vitro only when excessive amount of proteins are added into the culture medium, indicating that prokaryotic expression of fuctionalal OPG protein awaits further investigation.
Assuntos
Escherichia coli/genética , Glicoproteínas/genética , Receptores Citoplasmáticos e Nucleares/genética , Proteínas Recombinantes de Fusão/biossíntese , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Clonagem Molecular , Glicoproteínas/biossíntese , Humanos , Osteoclastos/efeitos dos fármacos , Osteoclastos/fisiologia , Osteoprotegerina , Receptores Citoplasmáticos e Nucleares/biossíntese , Receptores do Fator de Necrose Tumoral , Proteínas Recombinantes de Fusão/farmacologiaRESUMO
AIM: To express and purify two alternative splicing mouse Era proteins and detect whether anti-human Era antibody can be used in the study of mouse Era proteins. METHODS: Two fusion protein expression vectors, pMAL-meraW and pMAL-meraS, were constructed, then the MBP-mEra proteins were expressed in E. coli. The target proteins were purified by amylose affinity chromatography. The specificity of rabbit anti-human Era antibody to the proteins was identified by Western blot. RESULTS: The expressed MBP-mEraW and MBP-mEraS proteins constituted approximately 17% and 19% of the total bacterial proteins. The purity of the fused proteins was 67% and 61% respectively after amylose affinity chromatography. Rabbit anti-human Era antibody had high specificity to these two kinds of splicing mouse Era proteins. CONCLUSION: Two fusion mera genes could be expressed in E. coli by using gene recombination technique. The high specificity of rabbit anti-human Era antibody to the two splicing mouse ERA proteins indicates that this antibody can be used to study the function of these two kinds of splicing mouse Era.
Assuntos
Processamento Alternativo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/isolamento & purificação , Animais , Western Blotting , Eletroforese em Gel de Ágar , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Proteínas de Ligação ao GTP , Expressão Gênica , Engenharia Genética , Humanos , Camundongos , Plasmídeos/genética , Reação em Cadeia da Polimerase , Proteínas de Ligação a RNA/biossíntese , Proteínas de Ligação a RNA/química , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificaçãoRESUMO
OBJECTIVE: To review the current concepts of gene therapy approaches mediated by adenovirus vectors for bone trauma and bone disease. METHODS: The recent literature concerned gene therapy mediated by adenovirus vectors was reviewed, which provides new insights into the treatments of bone trauma and bone disease. RESULTS: Adenovirus vectors was efficient, achieved high expression after transduction, and could transfer genes to both replicating and nonreplicating cells, such as osteoblasts, osteoclasts, fibroblasts, chondrocytes, bone marrow stromal cells, etc. Gene therapy mediated by adenovirus vectors achieved affirmative results in enhancing bone union and in curing bone diseases, such as osteoporosis and rheumatoid arthritis. CONCLUSION: Gene therapy mediated by adenovirus offers an exciting avenue for treatment of bone trauma and bone diseases.
Assuntos
Adenoviridae/genética , Doenças Ósseas/terapia , Terapia Genética/métodos , Vetores Genéticos , Fraturas Ósseas/terapia , Técnicas de Transferência de GenesRESUMO
OBJECTIVE: To construct a co-expressing vector of human bone morphogenetic protein 2 (BMP-2) and osteoprotegerin (OPG) and to determine the expression of BMP-2 and OPG in myoblast C2C12. METHODS: Using the isolated total RNA from osteosacoma cell line MG63 as a template, the cDNA encoding region of human OPG was amplified by reverse transcription-polymerase chain reaction (RT-PCT) method and cloned into sites EcoR 1 and BamH I of mammalian expressing vector pIRES2-EGFP, and the cDNA encoding region of human BMP-2 was cloned into endonucleases site BstX I. Then the recombinant plasmid pIRES2-BMP-2-OPG was transformed into C2C12 cell line, the expression of OPG and BMP-2 were determined by Western blot assay. RESULTS: The sequence of OPG cDNA obtained was the same as that reported, recombinant plasmid pIRES2-BMP-2-OPG was constructed successfully. Human OPG and BMP-2 co-expression cell line C2C12 was selected and confirmed by Western blot analysis. CONCLUSION: The co-expressing vector of OPG and BMP-2 is constructed and can expressed stably in myoblast C2C12. The co-expression of human OPG and BMP-2 may be logical approach for treatment of osteoporosis and bone metastasis.
Assuntos
Proteínas Morfogenéticas Ósseas/biossíntese , Glicoproteínas/biossíntese , Mioblastos/metabolismo , Receptores Citoplasmáticos e Nucleares/biossíntese , Fator de Crescimento Transformador beta , Animais , Proteína Morfogenética Óssea 2 , Proteínas Morfogenéticas Ósseas/genética , Diferenciação Celular , Células Cultivadas , Escherichia coli , Expressão Gênica , Vetores Genéticos , Glicoproteínas/genética , Humanos , Mioblastos/citologia , Osteoprotegerina , Receptores Citoplasmáticos e Nucleares/genética , Receptores do Fator de Necrose Tumoral , Proteínas Recombinantes , TransfecçãoRESUMO
Functional analysis of new genes is playing a central role in postgenomic era. Here we reviewed several main strategies including bioinformatics, gene transduction, antisense technology, certain gene silence induced by RNA interference (RNAi), transgene and gene knockout and artificial chromosome transduction.
Assuntos
Biologia Computacional/métodos , Genes/fisiologia , Transdução Genética/métodos , Animais , HumanosRESUMO
The OPG/RANKL/RANK system plays an important role in osteoclastogenesis and represents a great progress in bone biology. RANKL, which expresses on the surface of osteoblast/stromal cells and activated T cells, binds to RANK on the osteoclastic precursors or mature osteoclasts, and promotes osteoclastogenesis and bone resorption. While osteoprotegerin (OPG), which is expressed by osteoblasts/stromal cells, strongly inhibits bone resorption by binding to its ligand RANKL and thereby blocks the interaction between BANKL and RANK. A number of cytokines and hormones exert their effects on bone metabolism by regulating the OPG/RANKL ratio in the bone marrow microenvironment. RANK is also expressed on mammary epithelial cells and RANKL expression in these cells is induced by pregnancy hormones, RANKL and RANK are essential for the formation of the lactating mammary gland and the transmission of maternal calcium to neonates in mammalian species. Modulation of these systems provides a unique opportunity to develop novel therapeutics to inhibit bone loss in osteoporosis, rheumatoid arthritis, and bone metastasis of cancer. Further research should be focused on the cooperation of OPG/RANKL/RANK system with other signal pathways and the interactions among bone remodeling, immune system and endocrinology system. Currently, the development of OPG analogues or compounds which may stimulate OPG expression is becoming an attractive industry which may be profitable to both patients and manufacturers.
Assuntos
Reabsorção Óssea/imunologia , Reabsorção Óssea/metabolismo , Osteoprotegerina/metabolismo , Ligante RANK/metabolismo , Receptor Ativador de Fator Nuclear kappa-B/metabolismo , Animais , Humanos , Osteoclastos/citologia , Osteoclastos/metabolismo , Osteoclastos/patologia , Osteogênese/efeitos dos fármacos , Osteogênese/genética , Osteogênese/imunologia , Osteoprotegerina/fisiologia , Ligante RANK/fisiologia , Receptor Ativador de Fator Nuclear kappa-B/farmacologia , Receptor Ativador de Fator Nuclear kappa-B/fisiologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologiaRESUMO
AIM: To clone the PID domain of human DOC-2 (nDOC-2) and express it in E.coli DH5alpha. METHODS: The cDNA fragment encoding the PID domain of nDOC-2 was amplified by RT-PCR from normal human ovarian tissue and cloned to pUC19. The DNA fragment from the pUC19-nDOC-2 digested with BamH I and EcoR I was ligated to the BamH I/EcoR I digested prokaryotic expression vector pGEX-4T-1.The expression of fusion protein was induced with IPTG and the expressed product was identified by SDS-PAGE. RESULTS: (1)The sequencing and endonucleases digestion analysis showed that the fragment of nDOC-2 gene was insert into vectors pUC19 and pGEX-4T-1;(2)SDS-PAGE showed the nDOC-2 gene had been expressed in E.coli DH5alpha. CONCLUSION: The PID domain of nDOC-2 was expressed successfully in prokaryote, which makes preparation for further researching the function of DOC-2 and preparing antibodies to DOC-2 protein.
Assuntos
Expressão Gênica , Plasmídeos , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Vetores Genéticos , HumanosRESUMO
Using the isolated total RNA from osteosacoma cell line MG63, the cDNA encoding human OPG was amplified by RT-PCR. A recombinant adenoviral vector carrying cDNA of OPG was constructed and OPG expression in mouse myoblast C2C12 cells was confirmed by Western blot and ELISA. The secreted expression of OPG protein persisted more than 6 weeks in vitro, and the growth of C2C12 cells infected by recombinant adenoviral were in good state. Osteoclasts derived from mouse bone marrow cells infected with recombinant adenoviral made less number of TRAP positive cells and resorption pits formed on dentine slices.