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

To quantify the amount of loaded MCM at the G1/S transition, we analyzed the S phase cells from Fig. 1C , and defined very early S phase cells as EdU-positive with ~2C DNA content ( Fig. 1E black rectangles). Early S phase cells in the first cycle had a broad range of loaded MCM levels that included many cells with low MCM, whereas early S phase cells in the second cycle primarily had high loaded MCM levels. The second cell cycle was nearly indistinguishable from asynchronously proliferating cells that had not been arrested. We thus used the second cell cycle to define the normal licensing levels, and classified cells with less MCM loaded in early S as "underlicensed" (Fig. 1E dashed line) . To visualize and directly compare MCM loading distribution in different populations, we generated histograms of loaded MCM levels in early S phase cells (Fig. 1F ). On this plot the differences between the first and second S phases were also readily apparent (compare orange and grey lines). We quantified the differences as fold change between the mean MCM loaded in the first and second S phases. Cells in the first cycle after re-entry from G0 loaded only half as much MCM as cells in the second cell cycle (Fig. 1G) . We then used this same comparison to test if this underlicensed first cell cycle is common among different untransformed cell lines or methods of quiescence induction. We observed similar underlicensing after quiescence induction in RPE1-hTert by serum starvation and re-stimulation and in two normal fibroblast cell lines arrested by contact inhibition and release . We observed that not only was the mean MCM loaded by the first S phase half that of subsequent S phases, but also that the majority of first S phase cells were underlicensed (Fig. 1H) . Thus, re-entry from G0 is characterized by substantial underlicensing in the first cell cycle.