RESUMO
BACKGROUND/AIMS: To investigate the effect of gene therapy for hepatocellular carcinoma based on inhibition of cellular IGF-I expression, the technique of IGF-I triple helix was investigated in mice developing programmed hepatoma. METHODOLOGY: mhAT1F1 mouse hepatoma cell line was transfected in vitro with IGF-I triple helix expression vector (pMT-AG-TH) or with IGF-I antisense expression vector (pMT-Anti-IGF-I). 10 x 10(6) transfected cells of either triple helix or antisense type were inoculated intraperitonealy into transgenic ATIIITB6 mice developing genetically programmed hepatoma (mice die between the age of 6 and 7 months). In parallel, human cell cultures established from surgically removed hepatomas were investigated. RESULTS: mhAT1F1 and human primary cell cultures, transfected with pMT-AG-TH or pMT-Anti-IGF-I vectors resulted in total inhibition of IGF-I demonstrated by immunocytochemical and Northern blot techniques. Transfected cells changed their phenotype and recovered major histocompatibility complex I expression showed by fluorescence-activated cell sorting analysis and Western blot. Moreover, two phenomena were observed in IGF-I "antisense" or "triple helix" transfected cells: 1) the apoptosis, demonstrated by TUNEL technique; 2) the presence of IL-6 simultaneously with disappearance of tumor necrosis factor-alpha and IL-10, investigated by reverse transcriptase-polymerase chain reaction technique. In in vivo experiments, injection of murine transfected cells into mice in terminal-phase prolonged their survival 3-4 months in 100% of cases, as well in "antisense" group (8/8) as in "triple helix" group (10/10). CONCLUSIONS: Injection of hepatoma cells transfected with IGF-I triple helix expression vector, and showing immunogenic and apoptotic characteristics, can constitute an effective cellular therapy against hepatocellular carcinoma.
Assuntos
Fator de Crescimento Insulin-Like I/antagonistas & inibidores , Neoplasias Hepáticas Experimentais/terapia , Animais , Apoptose/genética , Humanos , Fator de Crescimento Insulin-Like I/genética , Interleucina-6/metabolismo , Neoplasias Hepáticas Experimentais/genética , Neoplasias Hepáticas Experimentais/patologia , Camundongos , Camundongos Transgênicos , Ratos , Transfecção , Células Tumorais Cultivadas/patologiaRESUMO
IGF-I antisense gene therapy has been applied successfully to animal models of glioma, hepatoma and teratocarcinoma. The antisense strategy has shown that tumor cells transfected with vectors encoding IGF-I antisense RNA lose tumorigenicity, become immunogenic and are associated with tumor specific immune response involving CD8+ lymphocytes. An IGF-I triple helix approach to gene therapy for glioma was recently described. The approach we have taken is to establish parameters of change using the IGF-I triple helix strategy. PCC-3 embryonal carcinoma cells derived from murine teratocarcinoma which express IGF-I were used as a model. The cells were transfected with vector which encodes an oligoribonucleotide that forms RNA-IGF-I DNA triple-helix structure. The triple-helix stops the production of IGF-I. Cells transfected in this manner underwent changes in phenotype and an increase in MHC-I and B-7 cell surface molecules. They also showed enhancement in the production of apoptotic cells (60-70%). The "triple helix" transfected cells lost the ability to induce tumor when injected subcutaneously in syngeneic 129 Sv mice. When co-transfected in vitro with expression vectors encoding both MHC-I and B-7 cDNA in antisense orientation, the "triple-helix" transfected cells were down-regulated in expression of MHC-I and B-7 and the number of apoptotic cells was significantly decreased. Injection of the doubly co-transfected cells into 129 Sv mice was associated with induction of teratocarcinoma. Comparison between antisense and triple-helix transfected cells strategies showed similar immunogenic and apoptotic changes. The findings suggest that triple-helix technology may offer a new clinical approach to treatement of tumors expressing IGF-I.
Assuntos
Apoptose , Carcinoma Embrionário/imunologia , DNA , Fator de Crescimento Insulin-Like I/genética , Animais , Antígeno B7-1/genética , Antígeno B7-1/imunologia , Sequência de Bases , Carcinoma Embrionário/genética , Carcinoma Embrionário/patologia , Carcinoma Embrionário/terapia , Citometria de Fluxo , Técnica Indireta de Fluorescência para Anticorpo , Expressão Gênica , Terapia Genética , Vetores Genéticos , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Marcação In Situ das Extremidades Cortadas , Fator de Crescimento Insulin-Like I/metabolismo , Camundongos , Dados de Sequência Molecular , Transplante de Neoplasias , RNA Antissenso/genética , Transfecção , Células Tumorais CultivadasRESUMO
The transcriptional coactivator CBP displays an intrinsic histone acetyl transferase (HAT) activity which seems to participate in transcriptional activation through the destabilization of nucleosome structure. CBP is involved in the activity of several transcription factors that are nuclear endpoints of intracellular signal transduction pathways. In some instances, the transcription factors are phosphorylated upon cell activation, which induces their interaction with CBP. CBP itself is a phosphoprotein and can be phosphorylated by cycle-dependent kinases or by MAP kinases. Here we show that CBP phosphorylation by p44 MAP kinase/ERK1 results in the stimulation of its HAT enzymatic activity. The p44 MAP kinase/ERK1 phosphorylation sites are located in the C-terminal part of the protein, outside of the HAT domain. These sites are required for enzymatic stimulation, suggesting that phosphorylation by p44 MAP kinase/ERK1 induces a conformational change of the CBP molecule. Our data suggest that, in some instances, CBP itself might be a target for signal transduction pathways.
Assuntos
Acetiltransferases/metabolismo , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Proteínas Quinases Ativadas por Mitógeno , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae , Transativadores/metabolismo , Células 3T3 , Acetiltransferases/química , Trifosfato de Adenosina/metabolismo , Animais , Proteína de Ligação a CREB , Ativação Enzimática , Histona Acetiltransferases , Histonas/metabolismo , Camundongos , Proteína Quinase 3 Ativada por Mitógeno , Modelos Genéticos , Proteínas Nucleares/química , Nucleossomos/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação , Transdução de Sinais , Fatores de Tempo , Transativadores/químicaAssuntos
Carcinoma Hepatocelular/terapia , DNA Antissenso , Terapia Genética , Fator de Crescimento Insulin-Like I/genética , Neoplasias Hepáticas Experimentais/terapia , Neoplasias Hepáticas/terapia , Animais , Antígenos Virais de Tumores/genética , Antitrombina III/genética , Antitrombina III/fisiologia , Apoptose , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Humanos , Marcação In Situ das Extremidades Cortadas , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas Experimentais/genética , Complexo Principal de Histocompatibilidade , Camundongos , Camundongos Transgênicos , Regiões Promotoras Genéticas , Vírus 40 dos Símios/genética , Células Tumorais CultivadasRESUMO
Tumor cells frequently express the insulin-like growth factor I (IGF-I). Recently we demonstrated that rat glioma cells when transfected with a vector encoding antisense IGF-I cDNA lost tumorigenicity and induced a tumor specific immune response involving CD8+ lymphocytes. Here we show that the cultured transfected cell lines, rat C-6 glioma, human primary glioma and mouse teratocarcinoma, expressed an increased level of MHC-I and of co-signaling B-7 molecules. This increased expression of MHC-I and B-7, demonstrated by 51Cr release complement dependent cytoxicity assay and by immunostaining flow cytometry analysis, could contribute to the final immune recognition of glioma immunogenicity.
Assuntos
Antígenos CD/análise , Antígeno B7-1/análise , Antígenos de Histocompatibilidade Classe I/análise , Fator de Crescimento Insulin-Like I/genética , Glicoproteínas de Membrana/análise , Animais , Antígeno B7-2 , Citometria de Fluxo , Expressão Gênica/fisiologia , Glioma , Humanos , Fator de Crescimento Insulin-Like II/genética , Camundongos , Neuroblastoma , RNA Antissenso , RNA Mensageiro/metabolismo , Ratos , Teratocarcinoma , Transfecção , Células Tumorais Cultivadas/química , Células Tumorais Cultivadas/fisiologiaRESUMO
The replication of plasmid pSC101 requires the plasmid-encoded protein RepA. This protein has a double role: it binds to three directly repeated sequences in the pSC101 origin and promotes replication of the plasmid; it binds to two inversely repeated sequences in its promoter region and regulates its own transcription. A series of RepA protein derivatives carrying deletions of the C-terminal region were assayed for specific binding. We found that the last third of the protein is not needed for binding to the various specific sites. Truncated proteins that still bind can also form heterodimers with a wild-type protein. Analysis of band retardation assays conducted with wild-type and truncated proteins indicates that RepA binds to directly repeated sequences as a monomer and to inversely repeated sequences as a dimer.