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Polyamine-dependent growth and calmodulin-regulated induction of ornithine decarboxylase.
D D Ginty; E R Seidel (Profiled Author: David Ginty)
Department of Physiology, School of Medicine, East Carolina University, Greenville, North Carolina 27858.
The American journal of physiology 1989;256(2 Pt 1):G342-8.
The proliferation of cultured gastrointestinal crypt epithelial cells (IEC-6) and the role of calcium in polyamine biosynthesis were examined after serum stimulation of quiescent cells. Ornithine decarboxylase (ODC) activity was high when cells grew in 5% fetal calf serum (FCS) and dropped to nearly nondetectable levels when cells reached contact inhibition of growth. Polyamines appeared to be necessary for the proliferation of these cells, as growth was completely inhibited by the addition of 5 mM difluoromethylornithine, a specific inhibitor of ODC, to the media. This effect was reversed by 10 microM putrescine. Serum deprivation of preconfluent cells resulted in a fall in ODC activity. Readdition of serum led to an increase in ODC activity, which peaked at 4 h after addition and preceded both putrescine accumulation and [3H]thymidine incorporation into acid-precipitable material. Furthermore, readdition of serum to serum-deprived cells resulted in an approximately twofold increase in the level of free, ionized, intracellular Ca2+ as measured spectrophotometrically by monitoring fura-2 fluorescence. Inhibition of calmodulin-mediated processes with N-(6-aminohexyl)-5-chloro-1-naphthalelesulfonamide (W-7), a calmodulin antagonist, inhibited the serum-stimulated induction of ODC in a dose-dependent manner, with an IC50 of approximately 10 microM. Similar results were obtained with trifluoperazine. Lastly, 30 microM W-7 completely inhibited serum-stimulated [3H]thymidine incorporation into acid-precipitable material. These data demonstrate that polyamine biosynthesis and subsequent DNA synthesis after serum refeeding of cells is regulated by a Ca2+ activated, calmodulin-dependent process. Furthermore, the production of polyamines is essential for normal proliferation of these epithelial cells in culture.
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