mdl-73811 and 1-methylspermidine

The regulation of polyamine transport and the roles of polyamine transport and synthesis in cell growth were investigated using cultured Chinese hamster ovary (CHO) cells and CHOMG cells which are mutants lacking polyamine-transport activity. Metabolically stable methylated polyamine analogues were used to measure polyamine accumulation, and the irreversible S-adenosyl-L-methionine decarboxylase inhibitor, 5'-([(Z)-4-amino-2-butenyl]methylamino)-5'-deoxyadenosine (AbeAdo), was used to inhibit synthesis. Exposure to AbeAdo lead to a dose-dependent decrease in growth for both cell lines, although CHOMG cells were more sensitive. Intracellular putrescine levels were greatly increased in AbeAdo-treated CHO cells and to a lesser extent in CHOMG cells, whereas intracellular spermidine and spermine levels were substantially reduced in both. Treatment with AbeAdo increased putrescine content in the culture medium to a much greater extent in CHOMG cultures indicating that a portion of the excess putrescine synthesized in response to AbeAdo treatment is excreted, but that CHO cells salvage this putrescine whereas it is lost to CHOMG cells which cannot take up polyamines. AbeAdo treatment increased polyamine transport into CHO cells despite high intracellular putrescine, suggesting that spermidine and/or spermine, and not putrescine, are the major factors regulating transport activity. The accumulation of either 1-methylspermidine or 1,12-dimethylspermine was significantly increased by AbeAdo treatment. Accumulation was increased even further when protein synthesis was blocked by cycloheximide, indicating that a short-lived protein is involved in the regulation of polyamine uptake. In the presence of cycloheximide and AbeAdo or alpha-difluoromethylornithine, methylated polyamine derivatives accumulated to very high levels leading to cell death. These results show that the polyamine-transport system plays an important role in retaining intracellular polyamines and that down-regulation of the transport system in response to increased intracellular polyamine content is necessary to prevent accumulation of toxic levels of polyamines.

Topics: Adenosylmethionine Decarboxylase; Animals; Biological Transport; Cell Division; CHO Cells; Cricetinae; Cycloheximide; Deoxyadenosines; Eflornithine; Kinetics; Polyamines; Putrescine; Spermidine; Spermine

The abilities of the natural polyamines, spermidine and spermine, and of the synthetic analogues, 1-methylspermidine and 1,12-dimethylspermine, to reverse the effects of the S-adenosyl-L-methionine decarboxylase inhibitor 5'-([(Z)-4-aminobut-2-enyl]methylamino)-5'-deoxyadenosine (AbeAdo) on L1210-cell growth were studied. L1210 cells were exposed to AbeAdo for 12 days to induce cytostasis and then exposed to spermidine, spermine, 1-methylspermidine or 1,12-dimethylspermine in the continued presence of AbeAdo. AbeAdo-induced cytostasis was overcome by the natural polyamines, spermidine and spermine. The cytostasis was also reversed by 1-methylspermidine. 1,12-Dimethylspermine had no effect on the AbeAdo-induced cytostasis of chronically treated cells, although it was active in permitting growth of cells treated with the ornithine decarboxylase inhibitor, alpha-difluoromethylornithine. The initial 12-day exposure to AbeAdo elevated intracellular putrescine levels, depleted intracellular spermidine and spermine, and resulted in the accumulation of unmodified eukaryotic translation initiation factor 5A (eIF-5A). Exposure of these cells to exogenous spermidine, which is the natural substrate for deoxyhypusine synthase, resulted in a decrease in the unmodified eIF-5A content. 1-Methylspermidine, which was found to be a substrate of deoxyhypusine synthase in vitro, also decreased the levels of unmodified eIF-5A in the AbeAdo-treated cells. Although spermine is not a substrate of deoxyhypusine synthase, spermine was converted into spermidine in the L1210 cells, and spermine addition to AbeAdo-treated cells resulted in the appearance of both intracellular spermine and spermidine and in the decrease in unmodified eIF-5A. Exogenous 1,12-dimethylspermine, which was not metabolized to spermine or to 1-methylspermidine and was not a substrate of deoxyhypusine synthase in vitro, did not decrease levels of unmodified eIF-5A. The finding that AbeAdo-induced cytostasis was only reversed by polyamines and polyamine analogues that result in the formation of hypusine or an analogue in eIF-5A is consistent with the hypothesis [Byers, Wiest, Wechter and Pegg (1993) Biochem. J. 290, 115-121] that AbeAdo-induced cytostasis is due to the depletion of the hypusine-containing form of eIF-5A, which is secondary to the depletion of spermidine by inhibition of S-adenosyl-L-methionine decarboxylase.

Topics: Adenosylmethionine Decarboxylase; Animals; Cell Division; CHO Cells; Chromatography, High Pressure Liquid; Cricetinae; Deoxyadenosines; Eflornithine; Eukaryotic Translation Initiation Factor 5A; Leukemia L1210; Lysine; Oxidoreductases Acting on CH-NH Group Donors; Peptide Initiation Factors; RNA-Binding Proteins; Spermidine; Spermine; Substrate Specificity; Tumor Cells, Cultured