RESUMEN
A majority of malignant melanomas harbor an oncogenic mutation in either BRAF or NRAS. If BRAF and NRAS transform melanoma cells by a similar mechanism, then additional genetic aberrations would be similar (or random). Alternatively, distinct mutation-associated changes would suggest the existence of unique cooperating requirements for each mutation group. We first analyzed a panel of 52 melanoma cell lines (n = 35, 11, 6 for BRAF*, NRAS*, and BRAF/NRAS(wt/wt), respectively) by array-based comparative genomic hybridization for unique alterations that associate with each mutation subgroup. Subsequently, those DNA copy number changes that correlated with a mutation subgroup were used to predict the mutation status of an independent panel of 43 tumors (n = 17, 13, 13 for BRAF*, NRAS*, and BRAF/NRAS(wt/wt), respectively). BRAF mutant tumors were classified with a high rate of success (74.4%, P = 0.002), whereas NRAS mutants were not significantly distinguished from wild types (26/43, P = 0.12). Copy number gains of 7q32.1-36.3, 5p15.31, 8q21.11, and 8q24.11 were most strongly associated with BRAF* tumors and cell lines, as were losses of 11q24.2-24.3. BRAF* melanomas appear to be associated with a specific profile of DNA copy number aberrations that is distinct from those found in NRAS* and BRAF/NRAS(wt/wt) tumors. These findings suggest that although both BRAF and NRAS appear to function along the same signal transduction pathway, each may have different requirements for cooperating oncogenic events. The genetic loci that make up this profile may harbor therapeutic targets specific for tumors with BRAF mutations.
Asunto(s)
Aberraciones Cromosómicas , Dosificación de Gen , Genes ras , Melanoma/genética , Proteínas Proto-Oncogénicas B-raf/genética , Línea Celular Tumoral , Distribución de Chi-Cuadrado , Humanos , Mutación , Hibridación de Ácido Nucleico , Análisis de Secuencia por Matrices de Oligonucleótidos , Transducción de SeñalRESUMEN
In murine macrophages, the anti-tumor agent, paclitaxel, induces expression of a wide variety of inflammatory and anti-inflammatory genes, and causes cytokine secretion via signaling pathways that overlap with those engaged by lipopolysaccharide (LPS), the endotoxic component of Gram-negative bacteria. Using semi-quantitative RT-PCR for detection of gene expression, coupled with ELISA for the detection of secreted gene products, we analyzed the responsiveness of an extensive panel of cytokine and non-cytokine genes to induction by paclitaxel and LPS in the murine DA-3 breast cancer line. A subset of the genes examined (e.g., G-CSF, MIP-2, iNOS, and IL-1 beta, and GM-CSF) was upregulated >3-20-fold by both LPS and paclitaxel in the DA-3 cell line, while IP-10 mRNA was induced by paclitaxel, but not by LPS. In the human MDA-MB-231 breast cancer cell line, LPS also increased mRNA levels for both GM-CSF and IP-10 significantly, while, paclitaxel increased IP-10 mRNA levels with delayed kinetics and failed to induce GM-CSF mRNA. Co-cultures of murine breast cancer cells and macrophages, stimulated with IFN-gamma plus either paclitaxel or LPS, resulted in augmented release of nitric oxide. As both GM-CSF and IP-10 have been implicated in tumor rejection in vivo through either indirect actions on the host immune system or by inhibiting tumor angiogenesis, our data strengthen the hypothesis that tumor cell-derived inflammatory mediators may, in part, underlie the anti-tumor efficacy of paclitaxel in breast cancer.
Asunto(s)
Neoplasias de la Mama/metabolismo , Quimiocinas/biosíntesis , Lipopolisacáridos/metabolismo , Neoplasias Mamarias Animales/metabolismo , Paclitaxel/farmacología , Animales , Quimiocina CXCL10 , Quimiocina CXCL2 , Quimiocinas CXC/biosíntesis , Técnicas de Cocultivo , Relación Dosis-Respuesta a Droga , Ensayo de Inmunoadsorción Enzimática , Factor Estimulante de Colonias de Granulocitos/biosíntesis , Factor Estimulante de Colonias de Granulocitos y Macrófagos/biosíntesis , Humanos , Interleucina-1/biosíntesis , Cinética , Macrófagos/metabolismo , Ratones , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa/biosíntesis , Óxido Nítrico Sintasa de Tipo II , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Factores de Tiempo , Células Tumorales Cultivadas , Regulación hacia ArribaRESUMEN
Activation of T cells through the T-cell receptor (TCR) induces the expression of Fas Ligand (CD95L). In turn, CD95L binds to the Fas receptor (CD95) and rapidly induces apoptosis in cycling cells. This interaction is involved in the elimination of reactive lymphocytes during an immune response. However, TCR activation cannot always trigger apoptosis because an effective immune response would then be compromised. Here we show that a short (2 to 3 h) activation of T cells through the TCR simultaneously induces an increase in CD95L mRNA and a dramatic decrease in caspase-8 mRNA levels and proteolytic activity in human CD8(+) T cells. In addition, there is a small reduction in CD95 mRNA and CD95 levels on the cell surface. We found that preactivation of T cells protected them from apoptosis induced by either religation of the TCR or direct exposure to CD95L. These results suggest a mechanism by which cycling CD95-sensitive peripheral T cells, become protected from CD95 mediated deletion when actively engaged in the specific recognition of target cells.
Asunto(s)
Apoptosis/inmunología , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/metabolismo , Receptores de Antígenos de Linfocitos T/fisiología , Receptor fas/fisiología , Apoptosis/genética , Linfocitos T CD8-positivos/inmunología , Caspasa 8 , Caspasa 9 , Inhibidores de Caspasas , Caspasas/genética , Caspasas/metabolismo , Muerte Celular/inmunología , Citotoxicidad Inmunológica , Regulación hacia Abajo/inmunología , Humanos , ARN Mensajero/antagonistas & inhibidores , Transducción de Señal/inmunología , Células Tumorales Cultivadas , Receptor fas/genéticaRESUMEN
Previous studies have found conflicting associations between susceptibility to activation-induced cell death and the cell cycle in T cells. However, most of the studies used potentially toxic pharmacological agents for cell cycle synchronization. A panel of human melanoma tumor-reactive T cell lines, a CD8+ HER-2/neu-reactive T cell clone, and the leukemic T cell line Jurkat were separated by centrifugal elutriation. Fractions enriched for the G0-G1, S, and G2-M phases of the cell cycle were assayed for T cell receptor-mediated activation as measured by intracellular Ca(2+) flux, cytolytic recognition of tumor targets, and induction of Fas ligand mRNA. Susceptibility to apoptosis induced by recombinant Fas ligand and activation-induced cell death were also studied. None of the parameters studied was specific to a certain phase of the cell cycle, leading us to conclude that in nontransformed human T cells, both activation and apoptosis through T cell receptor activation can occur in all phases of the cell cycle.
Asunto(s)
Apoptosis/fisiología , Activación de Linfocitos/fisiología , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/metabolismo , Anticuerpos Monoclonales , Antígenos de Superficie/inmunología , Antígenos de Superficie/metabolismo , Complejo CD3/inmunología , Complejo CD3/metabolismo , Calcio/metabolismo , Ciclo Celular/fisiología , Proteína Ligando Fas , Humanos , Glicoproteínas de Membrana/inmunología , Glicoproteínas de Membrana/metabolismo , Proteínas Recombinantes/metabolismo , Células Tumorales CultivadasRESUMEN
'Naked' nucleic acid vaccines are potentially useful candidates for the treatment of patients with cancer, but their clinical efficacy has yet to be demonstrated. We sought to enhance the immunogenicity of a nucleic acid vaccine by making it 'self-replicating'. We accomplished this by using a gene encoding an RNA replicase polyprotein derived from the Semliki forest virus, in combination with a model antigen. A single intramuscular injection of a self-replicating RNA immunogen elicited antigen-specific antibody and CD8+ T-cell responses at doses as low as 0.1 microg. Pre-immunization with a self-replicating RNA vector protected mice from tumor challenge, and therapeutic immunization prolonged the survival of mice with established tumors. The self-replicating RNA vectors did not mediate the production of substantially more model antigen than a conventional DNA vaccine did in vitro. However, the enhanced efficacy in vivo correlated with a caspase-dependent apoptotic death in transfected cells. This death facilitated the uptake of apoptotic cells by dendritic cells, providing a potential mechanism for enhanced immunogenicity. Naked, non-infectious, self-replicating RNA may be an excellent candidate for the development of new cancer vaccines.
Asunto(s)
Linfocitos B/inmunología , Linfocitos T CD8-positivos/inmunología , Vacunas contra el Cáncer/uso terapéutico , Neoplasias del Colon/prevención & control , ARN Polimerasa Dependiente del ARN/genética , ARN Polimerasa Dependiente del ARN/uso terapéutico , Animales , Formación de Anticuerpos , Apoptosis , Neoplasias del Colon/inmunología , Células Dendríticas/inmunología , Elementos de Facilitación Genéticos , Inyecciones Intramusculares , Ratones , Ratones Endogámicos BALB C , Plásmidos , Regiones Promotoras Genéticas , ARN Polimerasa Dependiente del ARN/biosíntesis , Proteínas Recombinantes/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Virus de los Bosques Semliki/enzimología , Virus de los Bosques Semliki/genética , Transfección , Células Tumorales CultivadasRESUMEN
CD8+ T lymphocytes that specifically recognize tumor cells can be isolated and expanded ex vivo. While the lytic properties of these cells have been well described, their fate upon encounter with cognate tumor is not known. We performed reverse 51Cr release assays in which the lymphocyte effectors rather than the tumor cell targets were radioactively labeled. We found that melanoma tumor cells caused the apoptotic death of tumor-specific T cells only upon specific MHC class I-restricted recognition. This death was entirely blockable by the addition of an Ab directed against the Fas death receptor (APO-1, CD95). Contrary to the prevailing view that tumor cells cause the death of anti-tumor T cells by expressing Fas ligand (FasL), our data suggested that FasL was instead expressed by T lymphocytes upon activation. While the tumor cells did not express FasL by any measure (including RT-PCR), functional FasL (as well as FasL mRNA) was consistently found on activated anti-tumor T cells. We could successfully block the activation-induced cell death with z-VAD-fmk, a tripeptide inhibitor of IL-1 beta-converting enzyme homologues, or with anti-Fas mAbs. Most importantly, these interventions did not inhibit T cell recognition as measured by IFN-gamma release, nor did they adversely affect the specific lysis of tumor cell targets. These results imply that Fas-mediated activation-induced cell death could be a limiting factor in the in vivo efficacy of adoptive transfer of class I-restricted CD8+ T cells and provide a means of potentially enhancing their growth in vitro as well as their function in vivo.
Asunto(s)
Apoptosis/inmunología , Inhibidores de Caspasas , Activación de Linfocitos/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/patología , Melanoma/inmunología , Receptor fas/fisiología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/patología , Inhibidores de Cisteína Proteinasa/farmacología , Pruebas Inmunológicas de Citotoxicidad , Citotoxicidad Inmunológica , Epítopos de Linfocito T/biosíntesis , Proteína Ligando Fas , Humanos , Linfocitos Infiltrantes de Tumor/enzimología , Melanoma/enzimología , Melanoma/metabolismo , Glicoproteínas de Membrana/fisiología , Receptores de Antígenos de Linfocitos T/metabolismo , Células Tumorales Cultivadas , Receptor fas/biosíntesisRESUMEN
A recent report described the expression of Fas ligand (FasL) by melanoma cells as an important mechanism involved in the immune evasion by tumors [M. Hahne et al., Science (Washington DC), 274: 1363-1366, 1996]. To investigate the expression of FasL by melanomas, we screened a panel of early-passage cell lines by functional assay and reverse transcriptase-PCR. Using conditions designed to replicate those in the original report, we did not find functional FasL on any of the 19 human melanoma lines established at the National Cancer Institute. Furthermore, we additionally evaluated our melanoma lines using reverse transcriptase-PCR and found that 0 of the 26 human melanoma cell lines expressed FasL mRNA. FasL mRNA was abundantly expressed by anti-melanoma T-cell lines after activation. These data do not support a role for FasL expression in the escape of melanoma cells from immune destruction.
Asunto(s)
Melanoma/inmunología , Glicoproteínas de Membrana/biosíntesis , Proteína Ligando Fas , Regulación Neoplásica de la Expresión Génica , Humanos , Melanoma/patología , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/inmunología , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Células Tumorales CultivadasRESUMEN
The oncogene HER-2/neu is genetically amplified and overexpressed in a large number of human adenocarcinomas and has been implicated in the tumorigenic phenotype. Although it is a nonmutated self-protein, it is barely detectable in adult tissues, and immune responses toward it have been described in a number of patients. It is, thus, an attractive candidate antigen for the immunotherapy of cancer patients. HLA-A2+ patients with metastatic breast, ovarian, or colorectal adenocarcinomas that overexpressed HER-2/neu were immunized with the HLA-A2-binding epitope p369-377 (p369). Patients were treated by repeated immunization with 1 mg of p369 in Freund's incomplete adjuvant every 3 weeks. Peripheral blood mononuclear cells were collected prior to immunization and following two and four immunizations and were stimulated in vitro with peptide and assayed for peptide and tumor recognition. In three of four patients, peptide-specific CTLs were detected in post- but not preimmunization blood. These CTLs recognized peptide-pulsed target cells at peptide concentrations of > or =1 ng/ml yet failed to react with a panel of HLA-A2+ HER-2/neu+ tumor lines. In addition, infecting HLA-A2+ cells with recombinant vaccinia virus encoding HER-2/neu or up-regulating HLA-A2 with IFN-gamma in HER-2/neu+ cells also failed to confer reactivity by p369-reactive T-cells. A T-cell response to the HLA-A2 binding epitope p369 can be easily generated by immunizing patients with peptide in Freund's incomplete adjuvant. However, the CTLs failed to react with HER-2/neu+ tumor cells. Further studies are needed to determine whether and how HER-2 might serve as an antigen for tumor immunotherapy.