Reversible accumulation of plant suspension cell cultures in G1 phase and subsequent synchronous traverse of the cell cycle

Abstract

The induction of DNA synthesis in Datura innoxia Mill, cell cultures was determined by flow cytometry. A large fraction of the total population of cells traversed the cell cycle in synchrony when exposed to fresh medium. One hour after transfer to fresh medium, 37% of the cells were found in the process of DNA synthesis. After 24 hours of culture, 66% of the cells had accumulated in G2 phase, and underwent cell division simultaneously. Only 10% of the cells remained in G0 or G1. Transfer of cells into a medium, 80% (v/v) of which was conditioned by a sister culture for 2 days, was adequate to inhibit this simultaneous traverse of the cell cycle. A large proportion of dividing cells could be arrested at the G0 + G1/S boundary by exposure to 10 millimolar hydroxyurea (HU) for 12 to 24 hours. Inhibition of DNA synthesis by HU was reversible, and when resuspended into fresh culture medium synchronized cells resumed the cell cycle. Consequently, a large fraction of the cell population could be obtained in the G2 phase. However, reversal of G1 arrested cells was not complete and a fraction of cells did not initiate DNA synthesis. Seventy- four percent of the cells simultaneously reached 4C DNA content whereas the frequency of cells which remained in G0 + G1 phase was approximately 17%. Incorporation of radioactive precursors into DNA and proteins identified a population of nondividing cells which represents the fraction of cells in G0. The frequency of cells entering G0 was 11% at each generation. Our results indicate that almost 100% of the population of dividing cells synchronously traversed the cell cycle following suspension in fresh medium.