A study of polyamine acetylation and excretion in cultured human cancer cells

• Main Author: Coleman, Catherine S.
• Published: University of Aberdeen 1990
• Subjects: 572; Biochemistry
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.315072

HT29/219 and HT115 human colon cancer cells were adapted to grow in medium supplemented with serum lacking amine oxidase activity. In HepG2 human hepatoma cells grown in the presence of foetal calf serum, aminoguanidine was added to inhibit extracellular polyamine oxidation. All three cell lines contained spermine as their major polyamine > spermidine > > putrescine. No acetylpolyamines were detected in HT29/219 or HT115 cells although HepG2 cells did contain N¹-acetylspermidine. The total intracellular polyamine content of all cells were depleted by treatment with growth inhibitory concentrations of certain antimetabolites including the polyamine inhibitors, DFMO and MGBG. The growth inhibitory effects of DFMO were reversed by putrescine. Pretreatment of HT29/219 cells with DFMO increased the intracellular accumulation of MGBG to levels which were toxic to the cells. MGBG caused structural damage to HT29219 cell mitochondria although the cells were able to recover slowly from its growth inhibitory effects - exogenous spermidine enhanced the rate of recovery. Three spermidine acetyltransferase (SAT) activities were distinguished using an improved assay coupled with identification of products. N⁸-acetylspermidine was formed by nuclear and cytosolic enzymes. MGBG was the only growth inhibitor treatment to increase significantly a cytosolic spermidine N¹-acetyltransferase (N¹-SAT). HepG2 cells contained the highest inducible N¹-SAT activity > HT115 > HT29/219 cells. Analysis of the substrate specificity of crude cytosolic extracts from HepG2 and HT29/219 cells showed that MGBG treatment caused a specific increase in the acetylation of substrates containing aminopropyl moieties. N¹- and N⁸-acetylspermidine were identified in the extracellular medium of HT29/219 and HT115 cells. These were the major excretory products of HT115 cells but accounted for < 1% of polyamines excreted from HT29/219 cells: putrescine > > spermidine were the main polyamines excreted from HT29/219 cells although no spermine was detected. Growth inhibitory treatments increased polyamine excretion from cells mainly in the form of putrescine and spermidine. In contrast, HepG2 cells excreted spermine > N¹-acetylspermidine > spermidine but no putrescine was detected. Putrescine was however excreted from HepG2 cells after treatment with MGBG. These results support the direct excretion of preformed polyamines from growing cells, a process which is increased in response to growth inhibition. While acetylation and excretion may be linked, acetylation may not be a prerequisite to excretion in all cell types.