Redox regulation of the G1 to S phase transition in the mouse embryo fibroblast cell cycle.
The hypothesis that intracellular oxidation/reduction (redox) reactions regulate the G(0)-G(1) 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 approximately 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 G(0)-G(1) to S phase in serum-starved cells that were serum stimulated to reenter the cell cycle as well as to inhibit progression from G(1) to S phase in asynchronous cultures with no significant alterations in S phase, and G(2)+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 G(1) and the activation of G(1)-regulatory proteins in preparation for the entry of cells into S phase.
Free Radical and Radiation Biology Program, Department of Radiation Oncology, University of Iowa, Iowa City 52242, USA.
Sarita G
SG
Menon
Ehab H
EH
Sarsour
Douglas R
DR
Spitz
Ryuji
R
Higashikubo
Mary
M
Sturm
Hannah
H
Zhang
Prabhat C
PC
Goswami
D000111:Acetylcysteine / Q000494:pharmacology; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D004622:Embryo, Mammalian; D005347:Fibroblasts / Q000166:cytology* / Q000378:metabolism; D005434:Flow Cytometry; D016193:G1 Phase / Q000187:drug effects / Q000502:physiology*; D051379:Mice; D010084:Oxidation-Reduction; D016196:S Phase / Q000187:drug effects / Q000502:physiology*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D016159:Tumor Suppressor Protein p53 / Q000502:physiology
D000975:Antioxidants; D018797:Cell Cycle Proteins; D016159:Tumor Suppressor Protein p53; D000111:Acetylcysteine