Progressive increase in polyamine levels in 9L cells in vitro during the cell cycle: comparison between cells isolated by centrifugal elutriation and cells grown in synchrony.

Abstract

Centrifugal elutriation was used to separate 9L rat brain tumour cells into fractions enriched in the G1, S, or G2/M phases of the cell cycle. Cells enriched in early G1 phase were recultured, grown in synchrony, and harvested periodically for analysis of their DNA distribution and polyamine content. Mathematical analysis of the DNA distributions indicated that excellent synchrony was obtained with low dispersion throughout the cell cycle. Polyamine accumulation began at the time of seeding, and intracellular levels of putrescine, spermidine, and spermine increased continuously during the cell cycle. In cells in the G2/M phase of the cell cycle, putrescine and spermidine levels were twice as high as in cells in the G1 phase. DNA distribution and polyamine levels were also analysed in cells taken directly from the various elutriation fractions enriched in G1, S, or G2/M. Because we did not obtain pure S or G2/M populations by elutriation or by harvesting synchronized cells, a mathematical procedure--which assumed that the measured polyamine levels for any population were linearly related to the fraction of cells in the G1, S, and G2/M phases times the polyamine levels in these phases and that polyamine levels did not vary within these phases--was used to estimate 'true' phase-specific polyamine levels (levels to be expected if perfect synchrony were achieved). Estimated 'true' phase-specific polyamine levels calculated from the data obtained from cells either sorted by elutriation or obtained from synchronously growing cultures were very similar.