Hamster fibroblasts defective in thrombin-induced mitogenesis. A selection for mutants in phosphatidylinositol metabolism and other functions.
Growth of Chinese hamster lung fibroblasts (CCL39) on thrombin as sole mitogen is dependent on phosphatidylinositol (PI) metabolism and activation of the Na+/H+ antiporter. By modifying a H+ suicide selection developed for the isolation of antiporter mutants in these cells, we enriched for and isolated CCL39 variants deficient in the thrombin mitogenic response (thrombin nongrowers). These mutants retain alternate mitogenic mechanisms and, hence, grow well on media containing serum. When challenged with thrombin, the mutants show decreased, increased, or unchanged levels of inositol phosphates produced as compared with wild type cells. One of the mutants (D1-6b) has decreased inositol phosphates production not only with thrombin but also with serotonin (5-hydroxytryptamine) and AlF4-, suggesting a defect distal to the thrombin receptors. Extracts of this mutant reveal marked decreased phospholipase C activity toward PI. From the different phenotypes of the thrombin nongrowers, it is clear that the selection is general and that mutants with various biochemical defects should lead to a better understanding of the PI cycle as well as of functions essential to mitogenesis.
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110.
H M
HM
Rath
G A
GA
Doyle
D F
DF
Silbert
D000818:Animals; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D005020:Ethyl Methanesulfonate / Q000494:pharmacology; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects; D007700:Kinetics; D008168:Lung; D009154:Mutation; D010716:Phosphatidylinositols / Q000378:metabolism*; D012701:Serotonin / Q000494:pharmacology; D012969:Sodium Fluoride / Q000494:pharmacology; D013917:Thrombin / Q000494:pharmacology*
D010716:Phosphatidylinositols; D012701:Serotonin; D012969:Sodium Fluoride; D005020:Ethyl Methanesulfonate; D013917:Thrombin