Basal p21 controls population heterogeneity in cycling and quiescent cell cycle states

Abstract

Phenotypic heterogeneity within a population of genetically identical cells is emerging as a common theme in multiple biological systems, including human cell biology and cancer. Using live-cell imaging, flow cytometry, and kinetic modeling, we showed that two states--quiescence and cell cycling--can coexist within an isogenic population of human cells and resulted from low basal expression levels of p21, a Cyclin-dependent kinase (CDK) inhibitor (CKI). We attribute the p21-dependent heterogeneity in cell cycle activity to double-negative feedback regulation involving CDK2, p21, and E3 ubiquitin ligases. In support of this mechanism, analysis of cells at a point before cell cycle entry (i.e., before the G1/S transition) revealed a p21-CDK2 axis that determines quiescent and cycling cell states. Our findings suggest a mechanistic role for p21 in generating heterogeneity in both normal tissues and tumors.

Keywords: cell dormancy; nongenetic cell heterogeneity; positive feedback loop; synthetic uORF; tumor heterogeneity.

Fig. 1.

p21 causes population heterogeneity in cell cycle activity. (A) Single-cell tracking of cell cycle progression using a DHB-YFP reporter of CDK2 activity. (Left) CDK2-dependent translocation of DHB-YFP from the nucleus to the cytoplasm occurs during G1/S, S, and G2 phases. DHB-YFP returns to the nucleus after mitosis (M) and remains during early G1. H2B-mTurquoise (H2B-mTurq) is the nuclear marker. *During mitosis, after nuclear membrane collapse, the cytoplasmic-to-nuclear ratio (cyt/nuc) is not well-defined. (Right) CDK2 activity (cyt/nuc DHB-YFP) vs. time for cell in Left; arrows indicate depicted time points. (B) Single-cell traces of CDK2 activity in MCF10A WT and p21-deficient (^−/−) cycling (blue) and quiescent (red) cells. Traces for cycling cells were aligned to the second mitosis. B and D show results for populations grown under varying growth factor concentrations (EGF and serum). (C) Immunoblot of p21. The right three immunoblot lanes show WT and p21^−/− cells without ionizing irradiation (IR) and WT with 10 Gy IR. The left eight immunoblot lanes show expression of GFP-p21-ER in p21^−/− cells. Expression levels were tuned by varying translation initiation site bases preceding the GFP-p21-ER gene and/or synthetic uORFs. Slashes separate specified bases preceding each of one to three uORFs and bases preceding GFP-p21-ER. Bases without a slash precede GFP-p21-ER without any uORF. Expression of RFP-p21-ER in D was tuned with two uORFs (ACC/ACC/ACC and schematic) to approximate basal expression. (D) Traces for CDK2 activity and RFP-p21-ER expressed in p21^−/− cells.

Fig. 2.

p21 enables coexistence of cycling and quiescent cells over a range of growth factor stimulation. (A) Histograms for cell populations (y axes show numbers of cells); the cycling subpopulation is distinguished from the quiescent subpopulation by BrdU incorporation. (B) Percentage of quiescent cells from A. The shaded region indicates conditions at which 25–75% of WT cells remained quiescent. Data are represented as means ± SDs of triplicate samples.

Fig. 3.

Double-negative feedback regulation explains p21-dependent heterogeneity in cell cycle activity. (A) Schematic of (Left) interacting factors and (Right) double-negative feedback regulation. Ub, ubiquitin. (B) Steady-state balance plots generated by the model: steady-state p21 (green) and CDK2 (purple) activity levels, where intersections indicate stable quiescent (red) and cycling (blue) states at three growth factor (GF) concentrations. Open circles indicate an unstable steady state. At intermediate stimulation (GF = 1.75 relative units), two states (high p21/low CDK2 activity and low p21/high CDK2 activity) can stably coexist in a single population. (C) Live-cell imaging: RFP-p21-ER vs. CDK2 activity for cycling (blue) and quiescent (red) cells. Values for cycling cells were determined at a point in G1 3 h after mitosis and before cell cycle entry.

Fig. 4.

Negative regulation of p21 is dependent on SCF/Skp2 and p21 lysine residues targeted for ubiquitination. (A) GFP-p21-ER was expressed in p21^−/− cells. Population distributions for GFP-p21-ER were determined by flow cytometry, and GFP-low and -high populations were quantified. Cell cycle phase distributions were determined by PI staining of DNA content (2 N–4 N). (B) Inhibition of SCF/Skp2 by the cullin inhibitor MLN-4924. (C) Expression of GFP-p21K6R-ER, where ubiquitination by SCF/Skp2 has been reduced by mutating six lysine targets.

Fig. 5.

A p21–CDK2 axis governs p21-dependent population heterogeneity in quiescent and cycling cellular states. (A) Cells deficient in p21 respond to growth factor stimulation in a more switch-like manner, whereas cells expressing basal levels of p21 respond in a more graded manner. The shaded region represents growth factor levels that support p21-dependent heterogeneity in cell cycle states. (B) Cell states mapped onto a p21–CDK2 axis (shaded region). At a stage before cell cycle entry, cells with high p21 and low CDK2 activity are quiescent (red region), whereas cells with low p21 and high CDK2 activity are cycling (blue region).