RESUMO
Notch-1 inhibits apoptosis in some transformed cells through incompletely understood mechanisms. Notch-1 can increase nuclear factor-kappa B (NF-kappaB) activity through a variety of mechanisms. Overexpression of cleaved Notch-1 in T-cell acute lymphoblastic leukemia cells activates NF-kappaB via interaction with the I kappa B kinase (IKK) signalosome. Concomitant activation of the Notch and NF-kappaB pathways has been described in a large series of cervical cancer specimens. Here, we show that wild-type, spontaneously expressed Notch-1 stimulates NF-kappaB activity in CaSki cervical cancer cells by associating with the IKK signalosome through IKKalpha. A significant fraction of tumor necrosis factor (TNF)-alpha-stimulated IkappaB kinase activity in CaSki cells is Notch-1-dependent. In addition, Notch-1 is found in the nucleus in association with IKKalpha at IKKalpha-stimulated promoters and is required for association of IKKalpha with these promoters under basal and TNF-alpha-stimulated conditions. Notch-1-IKKalpha complexes are found in normal human keratinocytes as well, suggesting that IKK regulation is a physiological function of Notch-1. Both Notch-1 and IKKalpha knockdown sensitize CaSki cells to cisplatin-induced apoptosis to equivalent extents. Our data indicate that Notch-1 regulates NF-kappaB in cervical cancer cells at least in part via cytoplasmic and nuclear IKK-mediated pathways.
Assuntos
Quinase I-kappa B/metabolismo , Receptor Notch1/metabolismo , Neoplasias do Colo do Útero/metabolismo , Antineoplásicos/farmacologia , Apoptose , Linhagem Celular Tumoral , Cromatina/metabolismo , Cisplatino/farmacologia , Feminino , Humanos , Quinase I-kappa B/genética , NF-kappa B/metabolismo , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/metabolismo , Receptor Notch1/genética , Receptor Notch2/metabolismo , Receptor Notch4 , Receptores Notch/metabolismoRESUMO
In human papillomavirus (HPV) infected cervical epithelial cells the synthetic steroid dexamethasone inhibits radiation-induced apoptosis and increases the transcription of HPV E6/E7, enhancing p53 degradation. The aim of this study was to determine if suppression of apoptosis was mechanistically linked to changes in p53. HPV 16 E6 or E6/E7 expression vectors were transiently transfected into C4-1 HPV 18-positive cervical carcinoma cells to mimic the enhanced transcription following steroid treatment. After irradiation, apoptosis was suppressed in these cells comparable to the effect observed after steroid treatment alone. To confirm whether loss of p53 was responsible for the inhibition of apoptosis, residual p53 in C4-1 cells was targeted by stable transfection with a dominant-negative p53 mutant. While radiation-induced apoptosis increased after mutant transfection, inhibition of programmed cell death by steroid treatment was either eliminated or substantially reduced. Steroid-dependent inhibition of radiation-induced apoptosis in carcinoma of the cervix involves E6 modulation of p53 expression and may adversely affect treatment.
Assuntos
Anti-Inflamatórios/farmacologia , Antineoplásicos Hormonais/farmacologia , Apoptose/efeitos dos fármacos , Dexametasona/farmacologia , Proteína Supressora de Tumor p53/fisiologia , Neoplasias do Colo do Útero/patologia , Apoptose/efeitos da radiação , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Fragmentação do DNA , Feminino , Humanos , Proteínas de Neoplasias/fisiologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/efeitos da radiação , Papillomaviridae/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção , Células Tumorais Cultivadas , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/virologiaRESUMO
Vaccination with a peptide representing a CTL epitope from the human papillomavirus (HPV)16 E7 protein induces a specific CTL response that prevents the outgrowth of HPV16 E7-expressing tumors. In contrast, vaccination with a peptide encoding an adenovirus type 5 (Ad5) E1A CTL epitope results in CTL tolerance and enhanced growth of an Ad5 E1A-expressing tumor. It is unclear why these peptides induce such opposite effects. To determine whether a difference in pharmacokinetics can explain the functional contrasts, tritiated Ad5 E1A and HPV16 E7 peptides were injected into mice. Results show that the tolerizing peptide spread through the body 16 times faster than the activating peptide and was cleared at least 2 times faster. The HPV16 E7 peptide kinetics correlated with the kinetics of HPV16 E7-specific CTL induction. In contrast, Ad5 E1A peptide injection resulted in physical deletion of preexisting Ad5 E1A-specific CTLs within 24 h after injection. This tolerization occurred at the time when the peptide reached its maximum peptide concentration in the organs. These data suggest that ubiquitous expression of the tolerizing Ad5 E1A peptide within a short period of time causes activation-induced cell death of Ad5 E1A-specific CTLs. Therefore, information on the pharmacokinetics of peptides is vital for the safety and efficacy of peptide-based vaccines.
Assuntos
Proteínas E1A de Adenovirus/imunologia , Proteínas E1A de Adenovirus/farmacocinética , Tolerância Imunológica/imunologia , Ativação Linfocitária/imunologia , Proteínas Oncogênicas Virais/imunologia , Proteínas Oncogênicas Virais/farmacocinética , Papillomaviridae/imunologia , Linfócitos T Citotóxicos/imunologia , Proteínas E1A de Adenovirus/administração & dosagem , Animais , Deleção Clonal , Difusão , Epitopos de Linfócito T/imunologia , Epitopos de Linfócito T/metabolismo , Feminino , Injeções Subcutâneas , Cinética , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Oncogênicas Virais/administração & dosagem , Especificidade de Órgãos/imunologia , Proteínas E7 de Papillomavirus , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/farmacocinética , Ligação Proteica/imunologia , Linfócitos T Citotóxicos/metabolismo , Fatores de Tempo , Trítio/metabolismo , Vacinas Virais/administração & dosagem , Vacinas Virais/imunologia , Vacinas Virais/farmacocinéticaRESUMO
Loss of immunogenic epitopes by tumors has urged the development of vaccines against multiple epitopes. Recombinant DNA technologies have opened the possibility to develop multiepitope vaccines in a relatively rapid and efficient way. We have constructed four naked DNA-based multiepitope vaccines, containing CTL, Th cell, and B cell epitopes of the human papillomavirus type 16. Here we show that gene gun-mediated vaccination with an epitope-based DNA vaccine protects 100% of the vaccinated mice against a lethal tumor challenge. The addition of spacers between the epitopes was crucial for the epitope-induced tumor protection, as the same DNA construct without spacers was significantly less effective and only protected 50% of the mice. When tested for therapeutic potential, only the epitope construct with defined spacers significantly reduced the size of established tumors, but failed to induce tumor regression. Only after targeting the vaccine-encoded protein to the protein degradation pathway by linking it to ubiquitin, the vaccine-induced T cell-mediated eradication of 100% of 7-day established tumors in mice. The finding that defined flanking sequences around epitopes and protein targeting dramatically increased the efficacy of epitope string DNA vaccines against established tumors will be of importance for the further development of multiepitope DNA vaccines toward clinical application.
Assuntos
Adjuvantes Imunológicos/genética , Cisteína Endopeptidases/metabolismo , DNA Intergênico/imunologia , Epitopos/genética , Epitopos/imunologia , Complexos Multienzimáticos/metabolismo , Neoplasias Experimentais/prevenção & controle , Vacinas de DNA/administração & dosagem , Adjuvantes Imunológicos/administração & dosagem , Animais , Apresentação de Antígeno/genética , Linhagem Celular Transformada , Cisteína Endopeptidases/genética , Citotoxicidade Imunológica/genética , DNA Intergênico/administração & dosagem , DNA Intergênico/genética , Epitopos/metabolismo , Feminino , Vetores Genéticos/administração & dosagem , Vetores Genéticos/imunologia , Vetores Genéticos/metabolismo , Antígeno HLA-A2/genética , Antígeno HLA-A2/imunologia , Antígeno HLA-A2/metabolismo , Humanos , Hidrólise , Injeções Intradérmicas , Injeções Intraperitoneais , Injeções Subcutâneas , Camundongos , Camundongos Endogâmicos C57BL , Complexos Multienzimáticos/genética , Neoplasias Experimentais/enzimologia , Neoplasias Experimentais/imunologia , Complexo de Endopeptidases do Proteassoma , Linfócitos T Citotóxicos/imunologia , Células Tumorais Cultivadas , Ubiquitinas/genética , Ubiquitinas/imunologia , Vacinas de DNA/genética , Vacinas de DNA/imunologiaRESUMO
As a result of its transforming abilities, activated Ras is expressed in a great number of cancers. The ras mutation frequency varies between 95% in pancreatic cancer and 5% in breast cancer. In leukemia, the highest frequency (30%) is found in acute myeloid leukemia. The presence of ras mutations has been correlated with a poor prognosis and negative clinical outcome. This suggests that mutated Ras activates mechanisms, which favor tumor growth, enhance the metastatic capacity of tumors or modulate tumor-specific immune responses. Several new functions of Ras, such as downregulation of major histocompatibility complex molecules, upregulation of certain cytokines, growth factors and degradative enzymes have been uncovered in the last decade. Additionally, mutated Ras can also serve as a primary target for the development of immunotherapy or drug therapy. This review will discuss the mechanisms by which Ras expressing tumors are able to evade destruction by the immune system and enhance their growth and metastatic potential. It will further elaborate on the attempts to develop successful immunotherapy and drug therapy targeting Ras expressing tumors.
Assuntos
Genes ras , Sistema Imunitário/metabolismo , Proteínas de Neoplasias/fisiologia , Neoplasias/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/fisiologia , Alquil e Aril Transferases/antagonistas & inibidores , Animais , Apresentação de Antígeno , Antineoplásicos/química , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Moléculas de Adesão Celular/metabolismo , Transformação Celular Neoplásica/genética , Citocinas/metabolismo , Desenho de Fármacos , Endopeptidases/metabolismo , Ativação Enzimática , Farnesiltranstransferase , Proteínas Fúngicas/fisiologia , Proteínas de Fusão bcr-abl/fisiologia , Substâncias de Crescimento/metabolismo , Guanosina Trifosfato/fisiologia , Humanos , Imunoterapia , Leucemia/genética , Leucemia/metabolismo , Camundongos , Modelos Biológicos , Mutação , Metástase Neoplásica , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/terapia , Neurofibromina 1 , Oligonucleotídeos Antissenso/farmacologia , Proteínas/fisiologia , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Infecções por Reoviridae/fisiopatologia , Proteínas Repressoras/fisiologia , Proteína SOS1 , Transdução de Sinais , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismoRESUMO
An important role in the immune defense against deoxyribonucleic acid virus induced tumors is mediated by T-cells, as is evident from aetiological, animal model, and clinical data. In this review the most recent observations in this field are described for three prominent members of this family of viruses, namely human papillomavirus associated with human cervical cancer, human adenovirus associated with lung infections in humans and tumors in rodents, and simian virus 40 associated with rodent tumors and human mesothelioma, osteosarcoma and ependymoma.
Assuntos
Vírus de DNA Tumorais/imunologia , Imunoterapia , Infecções Tumorais por Vírus/imunologia , Infecções Tumorais por Vírus/terapia , Animais , Humanos , Imunidade Celular , Camundongos , Linfócitos T/imunologiaRESUMO
As the nature of the T cell immune response is defined by T cell receptor recognition of small protein fragments, referred to as peptides, the identification of peptides would lead us to understanding and directing the T-cell-mediated immune response. Immunogenic peptides might be used for vaccination and activation of the immune reaction against cancer- and virus-infected cells. Additionally, the knowledge of immunogenic peptides was expected to lead to blocking of allergic reactions and autoimmune diseases. Based on these assumptions, the search for immunogenic peptides was started in mice and man in the mid-1980s. After a decade of peptide identification and testing in vitro and in vivo, this may be a proper time to evaluate the results from the peptide-related work and determine the possible applications of this knowledge for the next decade. In this review we discuss the identification of peptides, their use in murine models, as well as clinical data from peptide vaccinations or therapies. Potential hazards and limitations of peptide use in immunotherapy and other possible applications for peptides or peptide motifs in immunotherapy are evaluated.