Spermine uptake is necessary to induce haemoglobin synthesis in murine erythroleukaemia cells

Abstract

To determine whether intracellular uptake of spermine is necessary to induce haemoglobin synthesis in murine erythroleukaemia (MEL) DS 19 cells, we used single-step selection for resistance to N1,N12-bis(ethyl)spermine (BESM), a cytotoxic spermine analogue, to isolate clones deficient in polyamine transport. The cells were approximately 500-fold more resistant to BESM than parental cells and were unable to accumulate BESM, putrescine, spermidine or spermine. Addition of spermine to the polyamine-transport-deficient cells failed to induce haemoglobin synthesis. Hexamethylene-1,6-bisacetamide, a well-known differentiating agent, induced haemoglobin synthesis in both parental and resistant cells. Polyamine-transport-deficient cells transfected with DNA purified from the parental cell line were further selected for their ability to grow in the presence of alpha-difluoromethylornithine and putrescine. The transfectants had an active transport system for polyamines, and spermine added to their culture medium accumulated inside the cells and induced haemoglobin production. These findings indicate that intracellular spermine uptake is required to induce haemoglobin production in MEL cells.

Synthesis and Biological Evaluation of N-(Anthracen-9-ylmethyl)triamines as Molecular Recognition Elements for the Polyamine Transporter

“…The K m value of spermidine transport was determined by Lineweaver-Burke analysis as described. 38 The ability of conjugates to compete for [ 14 C]-spermidine uptake was determined in L1210 cells by a 10 min uptake assay in the presence of increasing concentrations of competitor, using 1 µM [ 14 C]-spermidine as substrate. Ki values for inhibition of spermidine uptake were determined using the Cheng-Prusoff equation 39 from the IC50 value derived by iterative curve fitting of the sigmoidal equation describing the velocity of spermidine uptake in the presence of the respective competitor.…”

Molecular Requirements for Targeting the Polyamine Transport System. Synthesis and Biological Evaluation of Polyamine−Anthracene Conjugates

“…The K m value of spermidine transport was determined by the Lineweaver-Burke analysis as described. 48 The ability of conjugates to compete for [ 14 C]-spermidine uptake was determined in L1210 cells by a 10 min uptake assay in the presence of increasing concentrations of competitor, using 1 µM [ 14 C]-spermidine as substrate. Ki values for inhibition of spermidine uptake were determined using the Cheng-Prusoff equation 49 from the IC50 value derived by iterative curve fitting of the sigmoidal equation describing the velocity of spermidine uptake in the presence of the respective competitor.…”

“…The respective radioactivity of each sample was measured using a scintillation counter. The K m value of spermidine transport was determined by the Lineweaver−Burke analysis as described …”

Defining the Molecular Requirements for the Selective Delivery of Polyamine Conjugates into Cells Containing Active Polyamine Transporters

“…The Km value of spermidine transport was determined by Lineweaver-Burke analysis as described. 48 The ability of conjugates to compete for [ 14 C]spermidine uptake were determined in L1210 cells by a 10-min uptake assay in the presence of increasing concentrations of competitor, using 1 µM [ 14 C]spermidine as substrate. Ki values for inhibition of spermidine uptake were determined using the Cheng-Prusoff equation 41 from the IC50 value derived by iterative curve fitting of the sigmoidal equation describing the velocity of spermidine uptake in the presence of the respective competitor.…”