Levels of Pura, a protein implicated in control of both DNA replication and gene transcription,¯uctuate during the cell cycle, being lowest in early S phase and highest just after mitosis. Here we have employed a new video time-lapse technique enabling us to determine the cell cycle position of each cell in an asynchronous culture at a given time and to ask whether introduction of Pura protein at speci®c times can a ect cell cycle progression. Approximately 80% of all NIH3T3 cells injected with Pura were inhibited from passing through mitosis. Cells injected with Pura during S or G2 phases were e ciently blocked with a 4N (G2 phase) DNA level, as determined by quantitative DNA photometry of individual cells. Of the cells injected with Pura during G1 phase, 40% experienced a rapid cell death characterized by extreme cellular fragmentation. Of those G1 injected cells which remained viable, approximately equal numbers were arrested with either 2N or 4N DNA levels. Cells arrested by Pura in G2 phase grew to cover a large surface area. These results link¯uctuations in Pura levels to aspects of cell cycle control.
We conclude that the PNC2 plasmid selectively inhibits oncogenic ras-p21 and may therefore be highly effective in blocking proliferation of ras-induced cancer cells. Also, from the patterns of inhibition, by PNC2 and other ras- and raf-related peptides, of raf- and constitutively activated MEK-induced maturation, we conclude that PNC2 peptide inhibits oncogenic ras p21 downstream of raf.
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