Identification of growth inhibited cells by retention of a lipophilic fluorescent dye.
Cellular proliferation is regulated in both positive and negative ways. However, direct selection for growth inhibitory control elements is limited by the difficulty in identifying a growth inhibited cell against a background of cells which are proliferating. This study describes a positive selection technique for growth inhibited cells. This method is based on the retention of a lipophilic fluorescent dye which nonspecifically labels plasma membranes and distributes between daughter cells with membrane lipid as cells proliferate. Characterization of this assay is described using an epithelial cell line which is growth inhibited in response to transforming growth factor beta (TGF-beta) and dexamethasone and several mutant clones of that line which lack responsiveness to TGF-beta. Retention of dye in response to the growth inhibitors is proportional to the inhibition of thymidine incorporation of those cells. Mixing experiments were also carried out in which G418 resistant TGF-beta responsive epithelial cells were mixed with TGF-beta nonresponsive mutants. The mixture was labeled with PKH-2 and exposed to TGF-beta for 3 days. Subsequently, consecutive fractions of cells sorted on the basis of fluorescence intensity were selected in G418, and the TGF-beta responsive epithelial cells were found predominantly in the most fluorescent cells in the population. This method provides a positive selection for growth inhibited cells which may, in combination with classical gene transfer techniques, provide a way to select for growth inhibitory genes in a manner analogous to the focus forming assay selection for oncogenes.
Boyd
F T
FT
Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis 55455.
D000818:Animals; D002455:Cell Division / Q000187:drug effects*; D002460:Cell Line; D002469:Cell Separation; D003907:Dexamethasone / Q000494:pharmacology; D004395:Dye Dilution Technique; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D006131:Growth Inhibitors / Q000494:pharmacology*; D008055:Lipids / Q000737:chemistry*; D009930:Organic Chemicals; D012995:Solubility; D016212:Transforming Growth Factor beta / Q000494:pharmacology
D005456:Fluorescent Dyes; D006131:Growth Inhibitors; D008055:Lipids; D009930:Organic Chemicals; C068241:PKH-2; D016212:Transforming Growth Factor beta; D003907:Dexamethasone