Intrinsic checkpoint deficiency during cell cycle re-entry from quiescence

Jacob Peter Matson; Amy M. House; Gavin D. Grant; Huaitong Wu; Joanna Perez; Jeanette Gowen Cook

Many cells in vivo switch between periods of active proliferation and periods of G0, repeatedly re-entering the cell cycle to proceed through this presumably underlicensed first cell cycle. We considered the possibility that repeated cell cycle exit and re-entry would lead to additional replication stress sensitivity over time. To measure accumulation of replication stress during repeated cell cycle re-entry, we synchronized RPE1 cells in G0 by contact inhibition for 48 hours and released them into the cell cycle for about 48 hours (~2-3 cycles) and then they became contact inhibited again. We reiterated this procedure for a total of 3 rounds of G0 arrest and re-entry. In the third release, we treated with gemcitabine and measured the induction of γH2AX as before (Fig. S2B, C) . Cells re-entering S phase from three repeated arrests in G0 ("3xG0") were more likely to induce γH2AX cells than cells that been arrested only once ("1xG0"), and these cells were also more intensely γH2AX positive per cell (Fig. S2D , E). The increased replication stress sensitivity after three rounds of G0 varied somewhat due to inherent variations in the G0 synchronization over multiple rounds Wang et al., 2017) . This variability was reflected by unpaired t test p values of ~p=0.15 for both the number of γH2AX positive cells and the γH2AX intensity Fig. S2D , E. Nevertheless, we observed a clear trend that repeated cell cycle re-entry from G0 increased sensitivity to replication stress.