An investigation of the mechanism of polyamine efflux from human colorectal carcinoma cells.

Molecules, depending on their nature, can cross the lipid bilayer of the cell membrane by simple diffusion or by a protein carrier-mediated process. Camermediated transport occurs by facilitated diffusion through a channel or pore, or by the specific interaction with a 'mobile' transporter protein. The transport of polyamines into and out of the cell has been found to compliment de nova biosynthesis in regulating cellular pol yamine levels. The accumulation of polyamines into cells has been characterid in numerous cell types as being a timedependent, saturable process involving one or more protein transporter [reviewed in 11. Although movement of polyamines out of the cell has been shown to be important in altering their cellular levels [2], the transport process involved in efflux is unknown. In this study, the mechanism by which efflux occurs from monolayer cultures of a human colorectal cancer cell line was investigated. HTll5 cells were grown in Dulbecco's Modified Eagle's Medium supplemented with 10% horse serum at a seeding density of 2.8 x 104 celldcm2. Cultures were grown in the presence of [3H]putrescine for 36h to radiolabel cellular polyamines. The medium was then removed and fresh medium added. After a further 12h. the medium was changed and 1mM putrescine, spermidine or spermine was added to analytical cultures ; 0.9% saline was added to controls. After 24h, the medium was removed and analysed for radioactivity using liquid scintillation spectrometry. Polyamines were extracted from the medium and cells in 0.2M-perchloric acid, separated by h.p.1.c. and quantified either by fluorescenceor radiomatic detection. Protein determination was by the method of Lowry et al[3]. Addition of 'cold' polyamines to the culture medium resulted in apparent trans-acceleration of polyamine efflux from HTll5 cells, so that an increase in radioactive polyamines in the medium was observed. Fig 1. Effect of exogenous addition of 'cold' polyamines on emu of cellular polyamines from HTllS c e b Results are mean f SD for 3 independent experiments