RESUMEN
MACC1 (metastasis associated in colon cancer 1) is a prognostic biomarker for tumor progression, metastasis and survival of a variety of solid cancers including colorectal cancer (CRC). Here we aimed to identify the MACC1-induced transcriptome and key players mediating the MACC1-induced effects in CRC. We performed microarray analyses using CRC cells ectopically overexpressing MACC1. We identified more than 1300 genes at least twofold differentially expressed, including the gene SPON2 (Spondin 2) as 90-fold upregulated transcriptional target of MACC1. MACC1-dependent SPON2 expression regulation was validated on mRNA and protein levels in MACC1 high (endogenously or ectopically) and low (endogenously or by knockdown) expressing cells. Chromatin immunoprecipitation analysis demonstrated the binding of MACC1 to the gene promoter of SPON2. In cell culture, ectopic SPON2 overexpression induced cell viability, migration, invasion and colony formation in endogenously MACC1 and SPON2 low expressing cells, whereas SPON2 knockdown reduced proliferative, migratory and invasive abilities in CRC cells with high endogenous MACC1 and SPON2 expression. In intrasplenically transplanted NOD/SCID mice, metastasis induction was analyzed with control or SPON2-overexpressing CRC cells. Tumors with SPON2 overexpression induced liver metastasis (vs control animals without any metastases, P=0.0036). In CRC patients, SPON2 expression was determined in primary tumors (stages I-III), and survival time was analyzed by Kaplan-Meier method. CRC patients with high SPON2 expressing primary tumors demonstrated 8 months shorter metastasis-free survival (MFS) compared with patients with low SPON2 levels (P=0.053). Combining high levels of SPON2 and MACC1 improved the identification of high-risk patients with a 20-month shorter MFS vs patients with low biomarker expression. In summary, SPON2 is a transcriptional target of the metastasis gene MACC1. SPON2 induces cell motility in vitro and CRC metastasis in mice. In patients, SPON2 serves as prognostic indicator for CRC metastasis and survival, and might represent a promising target for therapeutic approaches.
Asunto(s)
Biomarcadores de Tumor , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Proteínas de la Matriz Extracelular/genética , Proteínas de Neoplasias/genética , Factores de Transcripción/metabolismo , Animales , Movimiento Celular/genética , Proliferación Celular , Supervivencia Celular/genética , Análisis por Conglomerados , Neoplasias Colorrectales/mortalidad , Neoplasias Colorrectales/patología , Biología Computacional/métodos , Modelos Animales de Enfermedad , Proteínas de la Matriz Extracelular/metabolismo , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Ontología de Genes , Xenoinjertos , Humanos , Ratones , Metástasis de la Neoplasia , Proteínas de Neoplasias/metabolismo , Estadificación de Neoplasias , Pronóstico , Unión Proteica , Curva ROC , TransactivadoresRESUMEN
Bacterial toxins are known to be effective for cancer therapy. Clostridium perfringens enterotoxin (CPE) is produced by the bacterial Clostridium type A strain. The transmembrane proteins claudin-3 and -4, often overexpressed in numerous human epithelial tumors (for example, colon, breast, pancreas, prostate and ovarian), are the targeted receptors for CPE. CPE binding to them triggers formation of membrane pore complexes leading to rapid cell death. In this study, we aimed at selective tumor cell killing by CPE gene transfer. We generated expression vectors bearing the bacterial wild-type CPE cDNA (wtCPE) or translation-optimized CPE (optCPE) cDNA for in vitro and in vivo gene therapy of claudin-3- and -4-overexpressing tumors. The CPE expression analysis at messenger RNA and protein level revealed more efficient expression of optCPE compared with wtCPE. Expression of optCPE showed rapid cytotoxic activity, hightened by CPE release as bystander effect. Cytotoxicity of up to 100% was observed 72 h after gene transfer and is restricted to claudin-3-and -4-expressing tumor lines. MCF-7 and HCT116 cells with high claudin-4 expression showed dramatic sensitivity toward CPE toxicity. The claudin-negative melanoma line SKMel-5, however, was insensitive toward CPE gene transfer. The non-viral intratumoral in vivo gene transfer of optCPE led to reduced tumor growth in MCF-7 and HCT116 tumor-bearing mice compared with the vector-transfected control groups. This novel approach demonstrates that CPE gene transfer can be employed for a targeted suicide gene therapy of claudin-3- and -4-overexpressing tumors, leading to the rapid and efficient tumor cell killing in vitro and in vivo.
Asunto(s)
Claudinas/metabolismo , Enterotoxinas/genética , Genes Transgénicos Suicidas , Terapia Genética/métodos , Neoplasias/terapia , Animales , Efecto Espectador , Línea Celular Tumoral , Claudina-3 , Claudina-4 , Claudinas/genética , Células HCT116 , Humanos , Masculino , Ratones , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Dendritic cells (DCs) are the most potent antigen-presenting cells, because they are also able to induce native T cells. Thus they are crucial in the induction of antiviral immune responses. Several viral immune escape mechanisms have been described; here we concentrate on the interaction between DCs and herpes simplex virus type 1 (HSV-1). DCs can be infected by HSV-1; however, only immature DCs generate infectious viral particles, whereas mature DCs do not support virus production and only immediate-early and early viral transcripts are generated. To induce potent immune responses DCs must mature. Interestingly, HSV-1 interferes with this maturation process, thus inhibiting antiviral T cell stimulation. Furthermore, HSV-1 strongly interferes with DC-mediated T cell proliferation. A striking finding was the complete degradation of CD83, the best-known marker for mature DC, after HSV-1 infection in lysosomal compartments. This CD83 degradation coincided with a clearly reduced T cell stimulation representing an additional new escape strategy. The functional role and the importance of CD83 are discussed in detail.
Asunto(s)
Células Dendríticas/virología , Herpesvirus Humano 1/fisiología , Inmunoglobulinas/inmunología , Glicoproteínas de Membrana/inmunología , Linfocitos T/inmunología , Antígenos CD , Senescencia Celular , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Células Dendríticas/patología , Herpesvirus Humano 1/inmunología , Humanos , Activación de Linfocitos/inmunología , Antígeno CD83RESUMEN
The present study was conducted to characterise the centrally active analgesic drug tramadol hydrochloride [(1RS,2RS)-2-[(dimethyl-amino)-methyl]-1-(3-methoxyphenyl)-cyclohe xanol hydrochloride] and its metabolites M1, M2, M3, M4 and M5 at the cloned human mu-opioid receptor. Membranes from stably transfected Chinese hamster ovary (CHO) cells were used to determine the four parameters of the ligand-receptor interaction: the affinity of (+/-)-tramadol and its metabolites was determined by competitive inhibition of [3H]naloxone binding under high and low salt conditions. The agonist-induced stimulation of [35S]GTPgammaS binding permits the measurement of potency (EC50), efficacy (Emax = maximal stimulation) and relative intrinsic efficacy (effect as a function of receptor occupation). The metabolite (+)-M1 showed the highest affinity (Ki=3.4 nM) to the human mu-opioid receptor, followed by (+/-)-M5 (Ki=100 nM), (-)-M1 (Ki=240 nM) and (+/-)-tramadol (Ki=2.4 microM). The [35S]GTPgammaS binding assay revealed an agonistic activity for the metabolites (+)-M1, (-)-M1 and (+/-)-M5 with the following rank order of intrinsic efficacy: (+)-M1>(+/-)-M5>(-)-M1. The metabolites (+/-)-M2, (+/-)-M3 and (+/-)-M4 displayed only weak affinity (Ki> 10 microM) and had no stimulatory effect on GTPgammaS binding. These data indicate that the metabolite (+)-M1 is responsible for the mu-opioid-derived analgesic effect.
Asunto(s)
Analgésicos Opioides/farmacología , Receptores Opioides mu/efectos de los fármacos , Tramadol/farmacología , Analgésicos Opioides/metabolismo , Animales , Unión Competitiva/efectos de los fármacos , Células CHO , Clonación Molecular , Cricetinae , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Humanos , Naloxona/metabolismo , Antagonistas de Narcóticos/metabolismo , Tramadol/metabolismoRESUMEN
Phosphorylation of human p34(cdc2) at Thr 161 seems to be necessary for its catalytic activity. CAK (cdk activating kinase) containing p40(MO15) from Xenopus egg extracts phosphorylates and activates p34(cdc2) in a cyclin dependent manner at Thr 161. We describe the cDNA sequence coding for human MO15, which predicts a serine/threonine kinase of 346 aa. Despite the high homology of 91% between the human and Xenopus proteins we observed a rather different mRNA distribution in adult tissues: In contrast to ubiquitously expressed human MO15-transcripts MO15-mRNA expression in Xenopus is restricted to oocytes indicating a different cellular role in these two phylogenetically distant species. By virtue of the homology to members of the family of cell cycle kinase genes we examined MO15 mRNA expression for its correlation to the proliferative activity of cells. Stimulation of lymphocytes showed MO15 mRNA expression to be independent of mitotic activity.