Abstract
Cultured cells require the actions of growth factors to enter the cell cycle, but how individual members of a population respond to the same stimulus remains unknown. Here we have employed continuous monitoring by live cell imaging in a dual-reporter cell model to investigate the regulation of short-term growth factor signaling (protein kinase B (PKB/Akt) activity) and longer-term progression through the cell cycle (cyclin-dependent kinase 2 activity). In the total population, insulin-like growth factor-I (IGF-I)-enhanced cell cycle entry by >5-fold compared with serum-free medium (from 13.5 to 78%), but at the single cell level we observed a broad distribution in the timing of G1 exit (4-24 h, mean ∼12 h) that did not vary with either the amount or duration of IGF-I treatment. Cells that failed to re-enter the cell cycle exhibited similar responses to IGF-I in terms of integrated Akt activity and migration distance compared with those that did. We made similar observations with EGF, PDGF-AA, and PDGF-BB. As potential thresholds of growth factor-mediated cell cycle progression appeared to be heterogeneous within the population, the longer-term proliferative outcomes of individual cells to growth factor stimulation could not be predicted based solely on acute Akt signaling responses, no matter how robust these might be. Thus, although we could define a relationship at the population level between growth factor-induced Akt signaling dynamics and cell cycle progression, we could not predict the fate of individual cells.
Highlights
Movement through the cell cycle is controlled at multiple stages
Analyzing Akt and cyclin-dependent kinase 2 (Cdk2) Signaling in Individual Cells— Growth factors play a key role in cell proliferation, but their actions have been found to be temporally limited [1,2,3, 15]
Before reaching the restriction point in the G1 phase of the cell cycle, growth factors are required; after it, cell cycle movement to S phase is growth factor independent. It is unclear if individual cells show behaviors similar to the entire population with regard to responsiveness to growth factor signaling and cell cycle progression
Summary
Cyclin-dependent kinase 2; IGF-I, insulinlike growth factor-I; pRb, retinoblastoma protein; HDHB, human DNA helicase B; SFM, serum-free medium. We have developed an experimental cell system that allows simultaneous tracking in real time of initial and longerterm responses to different growth factors, which we have applied to assess cell cycle progression in C3H10T1/2 fibroblasts. We find that at the population level there are clear doseand time-thresholds for individual growth factors to promote the G1 to S phase transition, but that in single cells these thresholds are variable. Growth factor signaling activity correlates with cell cycle progression in the overall population, it does not in individual cells. An acute signaling response cannot be used to determine if a cell will commit to S phase. Our studies reveal how growth factor signaling is encoded into cell cycle progression at the population level but not in single cells
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