Commentary: locating the restriction point.

Attempts to map the Restriction Point in the mammalian cell cycle typically involve stimulating quiescent cells with mitogens for increasing intervals, removing the stimulus and then determining the proportion of cells that reach S phase at some point later. This "fixed point" estimate assumes that further cell cycle commitment ceases as soon as the stimulus is removed. In fact, kinetic analysis shows that the probability of cell cycle commitment does not fall back to its initial low value, immediately after a pulse of mitogens, but may instead remain slightly elevated for some while afterwards, compared to the starting quiescent population. Thus, cells entering S phase after a brief exposure to mitogens are not those that pass the Restriction Point early. Rather, they represent cells that continue on to S phase as a result of this residual, low probability of cell cycle commitment. Instead, the mitogen-regulated process(es) affecting the probability of cell cycle commitment are much closer to the start of S phase itself. Since the acquisition of (apparent) mitogen independence is such a poor indicator of the timing of cell cycle commitment, it is argued that a better measure is the point of insensitivity to CDK4,6 inhibitors such as palbociclib, which indicates when hyperphosphorylation of the Retinoblastoma Protein, RB, ceases to be dependent on mitogen-signalling pathways regulating CDK4,6/cyclin D activity.

Cell Cycle Commitment and the Origins of Cell Cycle Variability.

Exit of cells from quiescence following mitogenic stimulation is highly asynchronous, and there is a great deal of heterogeneity in the response. Even in a single, clonal population, some cells re-enter the cell cycle after a sub-optimal mitogenic signal while other, seemingly identical cells, do not, though they remain capable of responding to a higher level of stimulus. This review will consider the origins of this variability and heterogeneity, both in cells re-entering the cycle from quiescence and in the context of commitment decisions in continuously cycling populations. Particular attention will be paid to the role of two interacting molecular networks, namely the RB-E2F and APC/CCDH1 "switches." These networks have the property of bistability and it seems likely that they are responsible for dynamic behavior previously described kinetically by Transition Probability models of the cell cycle. The relationship between these switches and the so-called Restriction Point of the cell cycle will also be considered.

Cell cycle arrest in replicative senescence is not an immediate consequence of telomere dysfunction.

In replicative senescence, cells with critically-short telomeres activate a DNA-damage response leading to cell-cycle arrest, while those without telomere dysfunction would be expected to cycle normally. However, population growth declines more gradually than such a simple binary switch between cycling and non-cycling states would predict. We show here that late-passage cultures of human fibroblasts are not a simple mixture of cycling and non-cycling cells. Rather, although some cells had short cycle times comparable to those of younger cells, others continued to divide but with greatly extended cycle times, indicating a more-gradual approach to permanent arrest. Remarkably, in late passage cells, the majority showed prominent DNA-damage foci positive for 53BP1, yet many continued to divide. Evidently, the DNA-damage-response elicited by critically-short telomeres is not initially strong enough for complete cell-cycle arrest. A similar continuation of the cell cycle in the face of an active DNA-damage response was also seen in cells treated with a low dose of doxorubicin sufficient to produce multiple 53BP1 foci in all nuclei. Cell cycle checkpoint engagement in response to DNA damage is thus weaker than generally supposed, explaining why an accumulation of dysfunctional telomeres is needed before marked cell cycle elongation or permanent arrest is achieved.