RESUMEN
The aim of this study was to investigate the impact of increased mRNA levels encoding GIRK1 in breast tumours on GIRK protein expression. mRNA levels encoding hGIRK1 and hGIRK4 in the MCF7, MCF10A and MDA-MB-453 breast cancer cell lines were assessed and the corresponding proteins detected using Western blots. cDNAs encoding for four hGIRK1 splice variants (hGIRK1a, 1c, 1d and 1e) were cloned from the MCF7 cell line. Subcellular localisation of fluorescence labelled hGIRK1a-e and hGIRK4 and of endogenous GIRK1 and GIRK4 subunits was monitored in the MCF7 cell line. All hGIRK1 splice variants and hGIRK4 were predominantly located within the endoplasmic reticulum. Heterologous expression in Xenopus laevis oocytes and two electrode voltage clamp experiments together with confocal microscopy were performed. Only the hGIRK1a subunit was able to form functional GIRK channels in connection with hGIRK4. The other splice variants are expressed, but exert a dominant negative effect on heterooligomeric channel function. Hence, alternative splicing of the KCNJ3 gene transcript in the MCF7 cell line leads to a family of mRNA's, encoding truncated versions of the hGIRK1 protein. The very high abundance of mRNA's encoding GIRK1 together with the presence of GIRK1 protein suggests a pathophysiological role in breast cancer.
Asunto(s)
Neoplasias de la Mama/genética , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/genética , Edición de ARN/genética , Animales , Secuencia de Bases , Western Blotting , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Células Clonales , Femenino , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/metabolismo , Humanos , Inmunohistoquímica , Microscopía Confocal , Datos de Secuencia Molecular , Técnicas de Placa-Clamp , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Mensajero/análisis , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Xenopus laevisRESUMEN
More and more members of the scientific community interested in 3R related topics of animal experiments are using Internet-Homepages as a low-budget forum to present either their scientific results, or opinions on specific subjects or to announce publications. Supra/inter/national authorities/universities/laboratories are also offering information, documents, access to databases and much more to the interested public. Therefore the Internet is an advantageous new medium for 3R related subjects.
RESUMEN
An increasing variety of in vitro test systems is used in cancer research to detect mutagenic or carcinogenic factors, to improve diagnosis and therapy and to study cancer cell functions and metastasis. This short review indicates that these in vitro techniques are not only superior to some animal experiments but often are the leading techniques. Improved 3D cell culture techniques, studies of membrane of organelle functions as well as molecular biology and genetic techniques will be discussed in the light of the cancer research strategic recommendations of the European Community (Europe against cancer). The multistep cascade of events during metastasis can be studied by in vitro techniques with outstanding precision, making many painful animal experiments avoidable. Metabolically competent human cells are superior tools to uncover carcinogenic compounds and mutagenic factors.