Polyamine oxidase activity and growth in human cancer cells

Abstract

lnterconversion of the polyamines, spermine + spermidine + putrescine, requires the N 1 -spermidine/spermine acetyltransferase (N1-SAT) and polyamine oxidase (PA01 enzymes. This process is used, in conjunction with biosynthesis and transport, to maintain the polyamine balance of the cell. N1-SAT is the rate-limiting step in the pathway and provides N1 -acetylpolyamine derivatives, the prefered substrate for PA0 [ll. PA0 contains a tightly bound FAD prosthetic group and acts on the secondary amine group of spermidine and spermine yielding spermidine and putrescine, respectively, along with 3-acetamidopropanal and hydrogen peroxide 121. Polyamine concentrations in cancerous tissue are high in comparison to normal tissue of the same type. High polyamine concentrations are also associated with rapidly dividing cells, suggesting alterations in either polyamine transport and/or polyamine metabolism are strongly associated with cell growth 131. As well as being substrates for PA0 acetylpolyamines have been suggested to be involved in excretion of polyamines 141. In this study we examined PA0 activity in a number of human tumour cell types and its relationship to cell growth. T47-D (breast carcinoma), G402 (renal leiomyoblastoma), and H T l l 5 (colonic carcinoma) cells were grown as monolayer cultures at a seeding density of 2 .5~104 celllcm2 (T47-D and G402) and 2.8~104 cell/cm2 (HT115). MOLT4-B (T leukaemia) cells were grown in suspension at 3x1 05 cells/ml. Culture medium was DMEM supplemented with 10% HS (HT1151, 10% FCS (G4021, 5% FCS & 1 % insulin (T47-D), or in RPMl with 10% FCS (MOLT4-B). Medium was changed every 48h where required. Cells were harvested at the appropriate growth condition for each cell type and the cells homogenised in Tris-HCI pH7.4 + 0.1 % Triton-X-l OO. PA0 activity was determined by a modification of the method of Table 1. PA0 activity in human tumour cells