Transfection-mediated cell-cycle signaling: considerations for transient transfection-based cell-cycle studies.
Transient transfection of recombinant genes into cells is a commonly used approach for analyzing cell-cycle- and/or apoptotic-related activities of cell-cycle control proteins. In this approach, information regarding the functional consequence of expressing a recombinant protein transiently is garnered by comparing against results obtained from cells which are transfected with either a control expression plasmid and/or with mutant expression plasmids. In general however, little attention is paid to whether the transfection procedure itself influences these experiments. Using the calcium phosphate transfection method, we show that the introduction of DNA into cells induces signaling of the cell-cycle control machinery. In Hela cells, a transient increase in G0/G1 cells is observed 8 h after transfection. Furthermore, the introduction of DNA into several cell lines induces apoptosis. Transfection-mediated apoptosis can be elicited through a p53-independent mechanism, suggesting the possible extrapolation to many tumor cell lines. Last, we show that due to a likely cell-cycle-specific entry of marker genes into the nucleus, a highly biased cell-cycle distribution is observed in successfully transfected cells at early times following transfection. The importance of these issues in the interpretation as well as the design of transient transfection-based cell-cycle experiments is discussed.
Rodriguez
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Harvard University and Dana-Farber Cancer Institute, 44 Binney Street, Boston, Massachusetts 02115, USA.
Flemington
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D002448:Cell Adhesion; D002453:Cell Cycle; D002460:Cell Line; D053938:DNA Fragmentation; D005434:Flow Cytometry / Q000379:methods; D016193:G1 Phase; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics; D009707:Nucleosomes / Q000235:genetics; D010957:Plasmids; D016192:Resting Phase, Cell Cycle; D015398:Signal Transduction; D014162:Transfection / Q000379:methods*
D008164:Luminescent Proteins; D009707:Nucleosomes; D049452:Green Fluorescent Proteins