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Redox Regulation of the G₁ to S Phase Transition in the Mouse Embryo Fibroblast Cell Cycle¹

Free Radical and Radiation Biology Program, Department of Radiation Oncology, University of Iowa, Iowa City, Iowa 52242 [S. G. M., E. H. S., D. R. S., M. S., H. Z., P. C. G., and Department of Radiation Oncology, Washington University Medical School, St. Louis, Missouri [R. H.]

The hypothesis that intracellular oxidation/reduction (redox) reactions regulate the G₀-G₁ to S-phase transition in the mouse embryonic fibroblast cell cycle was investigated. Intracellular redox state was modulated with a thiol-antioxidant, N-acetyl-L-cysteine (NAC), and cell cycle progression was measured using BrdUrd pulse-chase and flow cytometric analysis. Treatment with NAC for 12 h resulted in an 6-fold increase in intracellular low-molecular-weight thiols and a decrease in the MFI of an oxidation-sensitive probe, dihydrofluorescein diacetate, indicating a shift in the intracellular redox state toward a more reducing environment. NAC-induced alterations in redox state caused selective delays in progression from G₀-G₁ to S phase in serum-starved cells that were serum stimulated to reenter the cell cycle as well as to inhibit progression from G₁ to S phase in asynchronous cultures with no significant alterations in S phase, and G₂+M transits. NAC treatment also showed a 70% decrease in cyclin D1 protein levels and a 3–4-fold increase in p27 protein levels, which correlated with decreased retinoblastoma protein phosphorylation. Cells released from the NAC treatment showed a transient increase in dihydrofluorescein fluorescence and oxidized glutathione content between 0 and 8 h after release, indicating a shift in intracellular redox state to a more oxidizing environment. These changes in redox state were followed by an increase in cyclin D1, a decrease in p27, retinoblastoma protein hyperphosphorylation and subsequent entry into S phase by 8–12 h after the removal of NAC. These results support the hypothesis that a redox cycle within the mammalian cell cycle might provide a mechanistic link between the metabolic processes early in G₁ and the activation of G₁-regulatory proteins in preparation for the entry of cells into S phase.

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