A simple assay for examining the effect of transiently expressed genes on programmed cell death.
Programmed cell death (PCD) has been observed in a wide variety of cell types in response to physiologic signals or types of stress. How these stimuli trigger PCD, and whether there is a common PCD signal transduction pathway, is not clear. As more genes are described that may participate in or regulate PCD, an assay system in which gene products can easily be introduced and/or modulated would be of great value. To avoid the generation and screening of multiple individual stable cell transfectants, a simple transient transfection death assay has been developed. 2B4.11, a murine T cell hybridoma, was transfected by electroporation with a constitutively active beta-galactosidase reporter gene and the cells were incubated in culture medium or with a PCD-inducing stimulus. The amount of beta-galactosidase activity remaining in the intact cells at the end of the culture period represented only viable transfected cells. Bcl-2 was chosen to examine whether this system would be useful to study the effect of transiently transfected genes since it blocks PCD in a number of experimental systems. Consistent with data obtained using stable transfectants, transient expression of Bcl-2 in 2B4.11 completely protected cells from glucocorticoid- and cytotoxic agent-induced PCD. This protection from death was confirmed at the individual cell level by the transient co-expression of a class I Ld surface antigen and flow cytometric analysis. Some of the advantages of the transient transfection death assay described here are; (1) the simple and sensitive beta-galactosidase assay, (2) the rapidity of the assay, (3) the ability to perform conventional viability assays to monitor treatment-induced cytotoxicity, (4) multiple gene products can be tested alone, and in combination, (5) antisense or dominant negative approaches can be used, and (6) the adaptability of this assay system to other cell types, transfection techniques, or reporter and expression vectors. The transient transfection death assay should make it easier to identify and order important steps in the PCD signal transduction pathways.
Memon
S A
SA
Laboratory of Immune Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-1152.
Petrak
D
D
Moreno
M B
MB
Zacharchuk
C M
CM
D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D002460:Cell Line; D002478:Cells, Cultured; D005434:Flow Cytometry; D015870:Gene Expression; D017930:Genes, Reporter; D006825:Hybridomas; D051379:Mice; D010957:Plasmids; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000502:physiology*; D019253:Proto-Oncogene Proteins c-bcl-2; D015398:Signal Transduction; D013601:T-Lymphocytes / Q000502:physiology; D014162:Transfection / Q000235:genetics; D001616:beta-Galactosidase / Q000032:analysis* / Q000235:genetics
D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D001616:beta-Galactosidase