A new bioassay using transient transfection for invasion-related gene analysis.
To understand the mechanisms of tumor invasion and metastasis, model systems are required that isolate the individual steps of these complicated, multifaceted processes. We propose a new procedure to identify genes involved in cell invasion and/or motility that features the combined advantages of transient gene transfection and Matrigel invasion assays. Cancer cells were transiently cotransfected with two vectors expressing the gene of interest and luciferase, as a marker of transfected cells, and then assayed for Matrigel invasion. Luciferase cotransfection appeared to be a sensitive semiquantitative assay for transfected cells and was maximal throughout the invasion assay. The proposed transfection procedure, using calcium phosphate precipitation, did not affect cell invasiveness and allowed cellular coexpression of both genes. When applying this method, we found that transient expression of the unliganded and liganded human estrogen receptor alpha prevented invasiveness of MDA-MB-231 breast cancer cells. In conclusion, we propose rapid and versatile in vitro procedure for studying the effects of individual cloned genes on cellular processes, such as invasion and motility.
Platet
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Institut National de la Santé et de la Recherche Médicale, Unité Hormones et Cancer (U148) and Université de Montpellier I, Montpellier, France.
Garcia
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D001485:Basement Membrane / Q000473:pathology; D001681:Biological Assay; D001943:Breast Neoplasms / Q000235:genetics / Q000473:pathology; D002465:Cell Movement; D003094:Collagen; D004338:Drug Combinations; D004958:Estradiol / Q000494:pharmacology; D047628:Estrogen Receptor alpha; D005260:Female; D020869:Gene Expression Profiling; D017930:Genes, Reporter; D006801:Humans; D007797:Laminin; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics; D009361:Neoplasm Invasiveness / Q000235:genetics*; D016066:Pleural Effusion, Malignant / Q000235:genetics / Q000473:pathology; D011509:Proteoglycans; D011960:Receptors, Estrogen / Q000235:genetics / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014162:Transfection; D014407:Tumor Cells, Cultured
D004338:Drug Combinations; D047628:Estrogen Receptor alpha; D007797:Laminin; D011509:Proteoglycans; D011960:Receptors, Estrogen; D011993:Recombinant Fusion Proteins; C059485:matrigel; D004958:Estradiol; D003094:Collagen; D008156:Luciferases