In recent years, the intracellular oxidation-reduction (redox) state has gained increasing attention as a critical mediator of cell signaling, gene expression changes and proliferation. This review discusses the evidence for a redox cycle (i.e., fluctuation in the cellular redox state) regulating the cell cycle. The presence of redox-sensitive motifs (cysteine residues, metal co-factors in kinases and phosphatases) in several cell cycle regulatory proteins indicate periodic oscillations in intracellular redox state could play a central role in regulating progression from G 0 /G 1 to S to G 2 and M cell cycle phases. Fluctuations in the intracellular redox state during cell cycle progression could represent a fundamental mechanism linking oxidative metabolic processes to cell cycle regulatory processes. Proliferative disorders are central to a variety of human pathophysiological conditions thought to involve oxidative stress. Therefore, a more complete understanding of redox control of the cell cycle could provide a biochemical rationale for manipulating aberrant cell proliferation.

Arginine methylation is a widespread post-translational modification of proteins catalyzed by a family of protein arginine methyltransferases (PRMTs). The ancient protozoan parasite, Trypanosoma brucei, possesses five putative PRMTs, a relatively large number for a single-celled eukaryote. Trypanosomatids lack gene regulation at the level of transcription, instead relying on post-transcriptional control mechanisms that act at the levels of RNA turnover, translation, and editing, all processes that likely involve multiple RNA-binding proteins, which are common targets of arginine methylation. Here, we report the characterization of a trypanosome PRMT, TbPRMT7, which is homologous to human PRMT7. Interestingly, trypanosomatids are the only single-celled eukaryotes known to harbor a PRMT7 homologue. TbPRMT7 differs dramatically from all known metazoan PRMT7 homologues in lacking the second AdoMet binding-like domain that is required for activity of the human enzyme. Nevertheless, bacterially expressed TbPRMT7 exhibits robust methyltransferase activity toward multiple targets in vitro. High resolution ion exchange chromatography analysis of methylated substrates reveals that TbPRMT7 is a type III PRMT, catalyzing the formation of only monomethylarginine, thereby representing the only exclusively type III PRMT identified to date. TbPRMT7 is expressed in both mammalian and insect stage T. brucei and is apparently dispensable for growth in both life cycle stages. The enzyme is cytoplasmically localized and is a component of several higher order complexes in vivo. Together, our studies indicate that TbPRMT7 is a Type III PRMT, and its robust activity and presence in numerous complexes suggest it plays multiple roles during the complex T. brucei life cycle.

Thiol antioxidants, including N-acetyl-L-cysteine (NAC), are widely used as modulators of the intracellular redox state. We investigated the hypothesis that NAC-induced reactive oxygen species (ROS) signaling perturbs cellular proliferation by regulating the cell cycle regulatory protein cyclin D1 and the ROS scavenging enzyme Mn-superoxide dismutase (MnSOD). When cultured in media containing NAC, mouse fibroblasts showed G 1 arrest with decreased cyclin D1 protein levels. The absence of a NAC-induced G 1 arrest in fibroblasts overexpressing cyclin D1 (or a nondegradable mutant of cyclin D1-T286A) indicates that cyclin D1 regulates this G 1 arrest. A delayed response to NAC exposure was an increase in both MnSOD protein and activity. NAC-induced G 1 arrest is exacerbated in MnSOD heterozygous fibroblasts. Results from electron spin resonance spectroscopy and flow cytometry measurements of dihydroethidine fluorescence showed an approximately 2-fold to 3-fold increase in the steady-state levels of superoxide (O 2˙À ) in NAC-treated cells compared with control. Scavenging of O 2˙À with Tiron reversed the NAC-induced G 1 arrest. These results show that an O 2˙À signaling pathway regulates NACinduced G 1 arrest by decreasing cyclin D1 protein levels and increasing MnSOD activity. [Cancer Res 2007;67(13):6392-9]

RhoA is overexpressed in human cancer and contributes to aberrant cell motility and metastatic progression; however, regulatory mechanisms controlling RhoA activity in cancer are poorly understood. Neuroepithelial transforming gene 1 (Net1) is a RhoA guanine nucleotide exchange factor that is overexpressed in human cancer. It encodes two isoforms, Net1 and Net1A, which cycle between the nucleus and plasma membrane. Net1 proteins must leave the nucleus to activate RhoA, but mechanisms controlling the extranuclear localization of Net1 isoforms have not been described. Here, we show that Rac1 activation causes relocalization of Net1 isoforms outside the nucleus and stimulates Net1A catalytic activity. These effects do not require Net1A catalytic activity, its pleckstrin homology domain, or its regulatory C terminus. We also show that Rac1 activation protects Net1A from proteasome-mediated degradation. Replating cells on collagen stimulates endogenous Rac1 to relocalize Net1A, and inhibition of proteasome activity extends the duration and magnitude of Net1A relocalization. Importantly, we demonstrate that Net1A, but not Net1, is required for cell spreading on collagen, myosin light chain phosphorylation, and focal adhesion maturation. These data identify the first physiological mechanism controlling the extranuclear localization of Net1 isoforms. They also demonstrate a previously unrecognized role for Net1A in regulating cell adhesion.

Reactive oxygen species (ROS) and ROS signaling have been implicated in a variety of human pathophysiological conditions that involve aberrant cellular proliferation, particularly cancer. We hypothesize that intracellular redox state differentially affects cell-cycle progression in nonmalignant versus malignant cells. The thiol antioxidant, N-acetyl-L-cysteine (NAC), was used to alter intracellular redox state in nonmalignant human breast epithelial (MCF-10A) and breast cancer cells (MCF-7 and MDA-MB-231). Treatment of cells with NAC resulted in significant augmentation of intracellular small-molecular-weight thiols, glutathione and cysteine. In addition, NAC treatment decreased oxidation of a prooxidant-sensitive dye in MCF-10A cells, but not in MDA-MB-231 and MCF-7 cells. NAC-induced shifts in intracellular redox state toward a more reducing environment caused G(1) delays in MCF-10A cells without causing any significant changes in MCF-7 and MDA-MB-231 cell-cycle progression. NAC treatment of MCF-10A (but not MCF-7 and MDA-MB-231) was accompanied by a decrease in cyclin D1 and an increase in p27 protein levels, which correlated with increased retinoblastoma protein hypophosphorylation. These results show differential redox control of progression from G(1) to S in nonmalignant versus malignant cells and support the hypothesis that loss of a redox control of the cell cycle could contribute to aberrant proliferation seen in cancer cells.