Our knowledge of the pathways that regulate p21 is extensive. A variety of mechanisms operate at the transcriptional, post-transcriptional and post-translational levels in different cell types and under different conditions to dictate the level of p21. In this manuscript, we demonstrate that hdm2-dependent p21 protein turnover can control p21 accumulation in S and G2 phase and prevent the elaboration of a p53-induced, p21-dependent cell cycle arrest in response to DNA damage at that time.
Using selective synchrony methods based on either cell volume (elutriation) or DNA content (cell sorting), we enriched populations of cells in each phase of the cell cycle and showed that p21 accumulation was curtailed in S phase in transformed, immortalized and normal cells following exposure to ionizing radiation. Ionizing radiation induces double-strand DNA breaks, leading to activation of chk2 and a number of downstream checkpoints dependent on p53, cdc25A, BRCA1 or Nbs1.1
Our finding that p53-induced p21 accumulation was dependent on the phase of the cell cycle is consistent with the prior observation of Deptala and colleagues,16,20
who examined p53 and p21 accumulation in MCF7 cells treated with camptothecin. However, our results extend their finding by showing that p53 was in the nucleus and transcriptionally active in S-phase cells, but a proteolytic barrier prevented p21 protein accumulation.
The effect of cell cycle phase on p53 activation had been studied by a number of groups. Gottifredi et al. reported that a transcriptional blockade prevented p53-induced-accumulation of p21 but not other p53 targets in S-phase RKO cells.21
These investigators also reported that a proteolytic mechanism could contribute in other cell types,13,14
but the molecular nature of this mechanism was not addressed. The translocation of p53 to the nucleus may also be regulated in some cells.22
p21 protein turnover is regulated by a number of pathways, with both ubiquitin-dependent and ubiquitin-independent mechanisms contributing to its regulation in S-phase cells. These include a WISp39-associated chaperone pathway,23
and an hdm2-dependent pathway.27,28
Overwhelming cellular, biochemical and genetic evidence indicates that p27 is a bona fide substrate for SCFskp2
in S and G2
and, although it is generally accepted that skp2 can regulate p21 in cycling cells as well, the evidence for this is largely drawn from the observation that p21 levels rise in quiescent serum-deprived, skp2-deficient mouse embryo fibroblasts induced to re-enter the cell cycle.24
Our inability to define a similar S-phase role for skp2 may reflect cell type- or species-specific differences or, because we can see a strong G2
-phase role for skp2 and a more modest one for G1
-phase, could reflect the purity of the populations that were studied by Bornstein and colleagues. For example, the degree of synchronization obtained in mouse embryo fibroblasts by serum starvation/release protocols is not sufficient to eliminate G1
cell contamination, when skp2 promotes p21 turnover.
Hdm2 can regulate p21 accumulation in two ways. Indirectly, hdm2 ubiquitinates p53, targeting it for degradation; reducing hdm2 can increase p53-dependent transcription of p21. More directly, hdm2 can bind p21 and target it to the proteasome for degradation.27,28
We noted that neither p53-dependent transcription of p21 nor the accumulation of hdm2 was affected by cell cycle phase; however, in S-phase cells, unlike G1
cells, the p53-dependent accumulation of p21 was limited by hdm2. This raises an interesting question as to what cell cycle phase-dependent events are controlling the ability of hdm2 to promote p21 turnover.
Are their additional factors missing in G1
cells? Are there factors that are present in G1
cells preventing turnover? To address such questions in the future, we are attempting to develop an in vitro hdm2-p21 turnover system that will allow the biochemical identification of such factors (or modifications) and validate their effect in cellular systems. Additionally, given recent reports that the mTOR signaling environment could affect the outcome of p53 induction vis a vis reversible growth arrest or irreversible senescence,35,36
it is reasonable to speculate that cell cycle phase-specific differences in the signaling environment might also impact mdm2-dependent turnover of p21. Consistent with this, we note that LY294002 and PD98059 were able to inhibit G1
accumulation of p21 protein, although the accumulation of mdm2 mRNA and protein were unaffected (Bhakta R and Koff A, unpublished data). In contrast, neither of these compounds, nor rapamycin, induced S-phase accumulation of p21 in treated cells. This suggests that the activity of PI3-kinase and MEK pathways may be required to control hdm2-p21 turnover events.
Having such an S- and G2-phase dependent proteolytic mechanism is consistent with the findings that persistent blockade of cdk activity in S phase can lead to apoptosis (Shapiro 2006). Thus, the presence of the hdm2-dependent regulatory mechanism to eliminate p21 may allow for the dominance of cdc25-dependent mechanisms to inhibit kinases, a process which is more easily reversible. Thus, unraveling the pathways that prevent p21 turnover in S phase may allow us to consider therapeutic strategies that would drive a cell toward apoptosis through p21-mediated inhibition of cdk activity in S phase.