Investigation of p21-dependent population heterogeneity in cycling and quiescent cellular states [electronic resource]
Keywords: Chemical Engineering.

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. Cells with exactly the same genetic material and epigenetic markings can respond differently to the same stimulus. While stochastic fluctuations in gene expression can cause cell-to-cell variation, fluctuations alone cannot readily explain the coexistence of distinct phenotypic states within a population. However, stochasticity in the expression of genes involved in complex signaling and regulatory pathways governing non-linear behavior can lead to heterogeneity in cellular states. Using live-cell imaging, flow cytometry, and kinetic modeling, we demonstrated that two states—quiescence and cell-cycling—can coexist within an isogenic population of human cells. This population heterogeneity resulted from relatively low, basal expression levels of p21, a cyclin-dependent kinase inhibitor. While p21 is generally studied in the context of DNA damage repair pathways where p53 directly upregulates transcription of p21, we have investigated the role of constant, non-DNA damage dependent p21 expression in regulating cell-cycle activity. In unperturbed cells, Cyclin E and Cyclin-dependent kinase 2 (CDK2) form a complex that promotes progression through the G1/S transition and cell-cycle entry. p21 binds to this complex and inhibits its activity, thus arresting the cell cycle. However, p21 bound to CDK2 is regulated by the SCF/Skp2 E3 ubiquitin ligase complex that targets p21 for proteasomal degradation. Thus, these interactions form a double-negative feedback loop where p21 inhibits active CDK2, and active CDK2, through the action of SCF/Skp2, regulates p21 stability. Under the right conditions, double-negative feedback can generate bistability and heterogeneity in a system's response to a stimulus. We therefore attributed the observed heterogeneity in cell-cycle activity to this double-negative feedback regulation involving CDK2, SCF/Skp2, and p21. In support of this mechanism, both our experimental data and our kinetic modeling 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 cellular states. Cells with high p21 and low CDK2 activity remained quiescent while cells with low p21 and high CDK2 activity were actively cycling. This double-negative feedback regulatory mechanism between cell-cycle activators and inhibitors has been adopted widely, in mammalians as well as yeast. We believe that this is a general mechanism for maintaining heterogeneity in cell-cycle states in systems such as stem cell pools and tumors. While heterogeneity in cell-cycle states of stem cells can be beneficial in maintaining a pool of undifferentiated stem cells, tumor heterogeneity is less desirable and could lead to decreased efficacy of chemotherapy and radiation treatments. Decreasing the heterogeneity in cell-cycle states within a tumor could therefore be beneficial and possibly reduce the frequency of cancer relapse after remission.