Control of cell cycle progression in human natural killer cells through redox regulation of expression and phosphorylation of retinoblastoma gene product protein.
Using thiol deprivation, we have previously shown that the response of natural killer (NK) cells to interleukin-2 (IL-2) is subject to redox regulation downstream of IL-2 binding and internalization. We have now used the IL-2-dependent cell line, NK3.3 to study redox regulation of NK cells further, and found that NK3.3 cells neither incorporated [3H]-thymidine nor completed the G1-S phase transition in medium lacking the thiol-related compounds, L-cystine, and glutathione, despite the presence of sufficient IL-2. Thiol deprivation did not alter the induction of DNA interferon-gamma activated sequence (GAS)-binding activity in response to IL-2. However, the retinoblastoma gene product (RB), a cyclin-dependent kinase (CDK) substrate, was phosphorylated within 24 hours after IL-2 stimulation in standard medium, but its expression and phosphorylation were reduced in thiol-depleted medium in both NK3.3 cells and freshly isolated NK cells. These reductions were not associated with an increased level of p27Kip1, an inhibitor of CDKs CDK6/2 in association with G1 cyclins. Reducing agents, N-acetylcysteine, reduced glutathione or 2-ME restored both RB phosphorylation and DNA synthesis in thiol-deprived NK3.3 cells. The in vitro kinase activities of CDK6 and CDK2 were prematurely increased by thiol deprivation. This enhancement was associated with CDK hyperphosphorylation and prolonged phosphorylation, and could be observed before and beyond IL-2 stimulation. The data suggest the possibility that the premature and prolonged enhancement of CDK activity in thiol-deprived NK cells is associated with, and therefore may contribute to, the reduced expression and phosphorylation of RB, and the associated cell cycle arrest.
Yamauchi
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Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892, USA.
Bloom
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D002453:Cell Cycle; D002460:Cell Line; D005786:Gene Expression Regulation; D006801:Humans; D007694:Killer Cells, Natural / Q000166:cytology* / Q000378:metabolism; D010084:Oxidation-Reduction; D010766:Phosphorylation; D016160:Retinoblastoma Protein / Q000378:metabolism*; D015398:Signal Transduction
D016160:Retinoblastoma Protein