PURPOSE: To investigate the involvement of polyamines (putrescine, spermidine, and spermine) in the proliferation of retinal pigment epithelium (RPE) using cultured bovine RPE cells. METHODS: The polyamine content and the activities of rate-limiting enzymes in polyamine biosynthesis (ornithine decarboxylase [ODC] and S-adenosylmethionine decarboxylase [SAMDC]) and in polyamine biodegradation (spermidine spermine N1-acetyltransferase [SAT]) were measured after proliferative stimulation by 10% fetal calf serum (FCS). DNA synthesis was determined by [3H]thymidine incorporation into the acid-insoluble fraction after the addition of an inhibitor of ODC (alpha-difluoromethylornithine [DFMO]) or SAMDC (methylglyoxal bis[guanylhydrazone] [MGBG]). The effects of exogenous polyamines on DNA synthesis after the additions of inhibitors also were determined. RESULTS: ODC and SAMDC activities were elevated after stimulation by FCS and reached their peaks 16 hours and 4 hours, respectively, after the addition of FCS. SAT activity was not increased. Polyamine content was increased significantly after stimulation by FCS. DFMO did not inhibit DNA synthesis induced by FCS, and only putrescine content was decreased significantly among polyamines. However, MGBG inhibited DNA synthesis in a dose-dependent manner, and the amounts of spermidine and spermine were decreased significantly. Exogenous polyamines, especially spermine, restored MGBG-induced inhibition of DNA synthesis. CONCLUSIONS: Polyamines are essential for the proliferation of cultured bovine RPE cells. These data suggest that, of the polyamines, spermine has the greatest effect on DNA synthesis although other polyamines can substitute for spermine at higher concentrations with similar results. As for polyamine metabolism in RPE proliferation, it is possible that SAMDC is the key enzyme rather than ODC.
PURPOSE: To investigate the involvement of polyamines (putrescine, spermidine, and spermine) in the proliferation of retinal pigment epithelium (RPE) using cultured bovine RPE cells. METHODS: The polyamine content and the activities of rate-limiting enzymes in polyamine biosynthesis (ornithine decarboxylase [ODC] and S-adenosylmethionine decarboxylase [SAMDC]) and in polyamine biodegradation (spermidinespermine N1-acetyltransferase [SAT]) were measured after proliferative stimulation by 10% fetal calf serum (FCS). DNA synthesis was determined by [3H]thymidine incorporation into the acid-insoluble fraction after the addition of an inhibitor of ODC (alpha-difluoromethylornithine [DFMO]) or SAMDC (methylglyoxal bis[guanylhydrazone] [MGBG]). The effects of exogenous polyamines on DNA synthesis after the additions of inhibitors also were determined. RESULTS:ODC and SAMDC activities were elevated after stimulation by FCS and reached their peaks 16 hours and 4 hours, respectively, after the addition of FCS. SAT activity was not increased. Polyamine content was increased significantly after stimulation by FCS. DFMO did not inhibit DNA synthesis induced by FCS, and only putrescine content was decreased significantly among polyamines. However, MGBG inhibited DNA synthesis in a dose-dependent manner, and the amounts of spermidine and spermine were decreased significantly. Exogenous polyamines, especially spermine, restored MGBG-induced inhibition of DNA synthesis. CONCLUSIONS:Polyamines are essential for the proliferation of cultured bovine RPE cells. These data suggest that, of the polyamines, spermine has the greatest effect on DNA synthesis although other polyamines can substitute for spermine at higher concentrations with similar results. As for polyamine metabolism in RPE proliferation, it is possible that SAMDC is the key enzyme rather than ODC.
Authors: Hyunju Lee; Hyewon Chung; Hilal Arnouk; Folami Lamoke; Richard C Hunt; William J M Hrushesky; Patricia A Wood; Sung Haeng Lee; Wan Jin Jahng Journal: Int J Biol Macromol Date: 2010-05-25 Impact factor: 6.953