RESUMO
The antiapoptotic Livin/ML-IAP gene has recently gained much attention as a potential new target for cancer therapy. Reports indicating that livin is expressed almost exclusively in tumours, but not in the corresponding normal tissue, suggested that the targeted inhibition of livin may present a novel tumour-specific therapeutic strategy. Here, we compared the expression of livin in renal cell carcinoma and in non-tumorous adult kidney tissue by quantitative real-time reverse transcription-PCR, immunoblotting, and immunohistochemistry. We found that livin expression was significantly increased in tumours (P=0.0077), but was also clearly detectable in non-tumorous adult kidney. Transcripts encoding Livin isoforms alpha and beta were found in both renal cell carcinoma and normal tissue, without obvious qualitative differences. Livin protein in renal cell carcinoma samples exhibited cytoplasmic and/or nuclear staining. In non-tumorous kidney tissue, Livin protein expression was only detectable in specific cell types and restricted to the cytoplasm. Thus, whereas the relative overexpression of livin in renal cell carcinoma indicates that it may still represent a therapeutic target to increase the apoptotic sensitivity of kidney cancer cells, this strategy is likely to be not tumour-specific.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Apoptose , Biomarcadores Tumorais/genética , Carcinoma de Células Renais/genética , Regulação Neoplásica da Expressão Gênica , Proteínas Inibidoras de Apoptose/genética , Neoplasias Renais/genética , Rim/metabolismo , Proteínas de Neoplasias/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Biomarcadores Tumorais/metabolismo , Carcinoma de Células Renais/metabolismo , Humanos , Técnicas Imunoenzimáticas , Proteínas Inibidoras de Apoptose/metabolismo , Neoplasias Renais/metabolismo , Proteínas de Neoplasias/metabolismo , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Cancer cells are typically characterized by apoptosis deficiency. In order to investigate a possible role for the anti-apoptotic livin gene in renal cell cancer (RCC), we analyzed its expression in tumor tissue samples and in RCC-derived cell lines. In addition, we studied the contribution of livin to the apoptotic resistance of RCC cells by RNA interference (RNAi). Livin gene expression was detected in a significant portion of RCC tumor tissue specimens (13/14, 92.9%) and tumor-derived cell lines (12/15, 80.0%). Moreover, targeted inhibition of livin by RNAi markedly sensitized RCC cells towards proapoptotic stimuli, such as UV irradiation or the chemotherapeutic drugs etoposide, 5-fluorouracil, and vinblastine. These effects were specific for livin expressing tumor cells. We conclude that livin can contribute significantly to the apoptosis resistance of RCC cells. Targeted inhibition of livin could represent a novel therapeutic strategy to increase the sensitivity of renal cancers towards pro-apoptotic agents.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Apoptose/fisiologia , Carcinoma de Células Renais/patologia , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Neoplasias Renais/patologia , Proteínas de Neoplasias/antagonistas & inibidores , Carcinoma de Células Renais/fisiopatologia , Linhagem Celular Tumoral , Inativação Gênica , Humanos , Neoplasias Renais/fisiopatologia , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/isolamento & purificação , Interferência de RNA , RNA Neoplásico/genética , RNA Neoplásico/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
A substantial proportion of the worldwide liver cancer incidence is associated with chronic hepatitis B virus (HBV) infection. The therapeutic management of HBV infections is still problematic and novel antiviral strategies are urgently required. Using the peptide aptamer screening system, we aimed to isolate new molecules, which can block viral replication by interfering with capsid formation. Eight peptide aptamers were isolated from a randomized expression library, which specifically bound to the HBV core protein under intracellular conditions. One of them, named C1-1, efficiently inhibited viral capsid formation and, consequently, HBV replication and virion production. Hence, C1-1 is a novel model compound for inhibiting HBV replication by blocking capsid formation and provides a new basis for the development of therapeutic molecules with specific antiviral potential against HBV infections.
Assuntos
Antivirais/farmacologia , Antígenos do Núcleo do Vírus da Hepatite B/metabolismo , Hepatite B/tratamento farmacológico , Neoplasias Hepáticas/tratamento farmacológico , Peptídeos/farmacologia , Sequência de Aminoácidos , Antivirais/metabolismo , Aptâmeros de Peptídeos , Capsídeo/efeitos dos fármacos , Vírus da Hepatite B/efeitos dos fármacos , Humanos , Neoplasias Hepáticas/virologia , Dados de Sequência Molecular , Biblioteca de Peptídeos , Peptídeos/química , Peptídeos/metabolismo , Células Tumorais Cultivadas , Técnicas do Sistema de Duplo-Híbrido , Vírion/efeitos dos fármacos , Replicação Viral/efeitos dos fármacosRESUMO
The ability to specifically interfere with the function of proteins of pathological significance has been a goal for molecular medicine for many years. Peptide aptamers comprise a new class of molecules, with a peptide moiety of randomized sequence, which are selected for their ability to bind to a given target protein under intracellular conditions. They have the potential to inhibit the biochemical activities of a target protein, can delineate the interactions of the target protein in regulatory networks, and identify novel therapeutic targets. Peptide aptamers represent a new basis for drug design and protein therapy, with implications for basic and applied research, for a broad variety of different types of diseases.
Assuntos
Quinases relacionadas a CDC2 e CDC28 , Proteínas de Ciclo Celular , Proteínas de Ligação a DNA , Peptídeos/farmacologia , Ligação Proteica/efeitos dos fármacos , Antivirais/farmacologia , Quinase 2 Dependente de Ciclina , Quinases Ciclina-Dependentes/antagonistas & inibidores , Desenho de Fármacos , Fatores de Transcrição E2F , Proteínas Oncogênicas Virais/antagonistas & inibidores , Peptídeos/uso terapêutico , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Fatores de Transcrição/antagonistas & inibidores , Proteínas do Core Viral/antagonistas & inibidoresRESUMO
Cytomegaloviruses encode numerous functions that inhibit antigen presentation in the major histocompatibility complex (MHC) class I pathway in vitro. One example is the mouse cytomegalovirus (MCMV) glycoprotein gp40, encoded by the m152 gene, which selectively retains murine but not human MHC class I complexes in the endoplasmic reticulum-Golgi intermediate compartment/cis-Golgi compartment (Ziegler, H., R. Thäle, P. Lucin, W. Muranyi, T. Flohr, H. Hengel, H. Farrell, W. Rawlinson, and U.H. Koszinowski. 1997. Immunity. 6:57-66). To investigate the in vivo significance of this gene function during MCMV infection of the natural host, we constructed recombinants of MCMV in which the m152 gene was deleted, as were the corresponding virus revertants. We report on the following findings: Deletion of the m152 gene has no effect on virus replication in cell culture, whereas after infection of mice, the m152-deficient virus replicates to significantly lower virus titers. This attenuating effect is lifted by reinsertion of the gene into the mutant. Mutants and revertants grow to the same titer in animals deprived of the function targeted by the viral gene function, namely in mice deficient in beta2-microglobulin, mice deficient in the CD8 molecule, and mice depleted of T cells. Upon adoptive transfer of naive lymphocytes into infected mice, the absence of the m152 gene function sensitizes the virus to primary lymphocyte control. These results prove that MHC-reactive functions protect CMVs against attack by CD8(+) T lymphocytes in vivo.
Assuntos
Glicoproteínas de Membrana/genética , Linfócitos T/metabolismo , Células 3T3 , Animais , Apresentação de Antígeno/imunologia , Linfócitos T CD8-Positivos/imunologia , Testes Imunológicos de Citotoxicidade , Deleção de Genes , Genes MHC Classe I/imunologia , Imunidade , Glicoproteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos , Mutação , Proteínas Virais/imunologia , Virulência , Replicação ViralRESUMO
The murine cytomegalovirus (MCMV) fcr-1 gene codes for a glycoprotein located at the surface of infected cells which strongly binds the Fc fragment of murine immunoglobulin G. To determine the biological significance of the fcr-1 gene during viral infection, we constructed MCMV fcr-1 deletion mutants and revertants. The fcr-1 gene was disrupted by insertion of the Escherichia coli lacZ gene. In another mutant, the marker gene was also deleted, by recombinase cre. As expected for its hypothetical role in immunoevasion, the infection of mice with fcr-1 deletion mutants resulted in significantly restricted replication in comparison with wild-type MCMV and revertant virus. In mutant mice lacking antibodies, however, the fcr-1 deletion mutants also replicated poorly. This demonstrated that the cell surface-expressed viral glycoprotein with FcR activity strongly modulates the virus-host interaction but that this biological function is not caused by the immunoglobulin binding property.