Cellular oxidation and reduction (redox) environment is influenced by the production and removal of reactive oxygen species (ROS). In recent years, several reports support the hypothesis that cellular ROS-levels could function as “second messengers” regulating numerous cellular processes, including proliferation. Periodic oscillations in the cellular redox environment, a redox cycle, regulate cell cycle progression from quiescence (G0) to proliferation (G1, S, G2, and M) and back to quiescence. A loss in the redox control of the cell cycle could lead to aberrant proliferation, a hallmark of various human pathologies. This review discusses the literature which supports the concept of a redox cycle controlling the mammalian cell cycle with an emphasis on how this control relates to proliferative disorders including cancer, wound healing, fibrosis, cardiovascular diseases, diabetes, and neurodegenerative diseases. We hypothesize that reestablishing the redox control of the cell cycle by manipulating the cellular redox environment could assuage many aspects of the proliferative disorders.

SummaryIn recent years, the intracellular reactive oxygen species (ROS) levels have gained increasing attention as a critical regulator of cellular proliferation. We investigated the hypothesis that manganese superoxide dismutase (MnSOD) activity regulates proliferative and quiescent growth by modulating cellular ROS levels. Decreasing MnSOD activity favored proliferation in mouse embryonic fibroblasts (MEF), while increasing MnSOD activity facilitated proliferating cells' transitions into quiescence. MnSOD (+ + + + /-) and (-/-) MEFs demonstrated increased superoxide steady-state levels; these fibroblasts failed to exit from the proliferative cycle, and showed increasing cyclin D1 and cyclin B1 protein levels. MnSOD (+ + + + /-) MEFs exhibited an increase in the percentage of G 2 cells compared to MnSOD (+ + + + /+ + + + ) MEFs. Overexpression of MnSOD in MnSOD (+ + + + /-) MEFs suppressed superoxide levels and G 2 accumulation, decreased cyclin B1 protein levels, and facilitated cells' transit into quiescence. While ROS are known to regulate differentiation and cell death pathways, both of which are irreversible processes, our results show MnSOD activity and, therefore, mitochondria-derived ROS levels regulate cellular proliferation and quiescence, which are reversible processes essential to prevent aberrant proliferation and subsequent exhaustion of normal cell proliferative capacity. These results support the hypothesis that MnSOD activity regulates a mitochondrial 'ROS-switch' favoring a superoxide-signaling regulating proliferation and a hydrogen peroxide-signaling supporting quiescence.

We tested the hypothesis that manganese superoxide dismutase (MnSOD), an antioxidant enzyme, regulates the proliferative potential of confluent human fibroblasts. Normal human skin (AG01522) and lung (WI38, CCL-75) fibroblasts kept in confluence (>95% G 0 /G 1 ) showed a significant decrease in their capacity to reenter the proliferation cycle after 40 -60 days. The inhibition of re-entry was accompanied with the agedependent increase of p16 protein levels in the confluent culture. Adenoviral mediated overexpression of MnSOD during confluent growth suppressed p16, enhanced p21 protein accumulation, and protected fibroblasts against the loss of proliferation potential. Increases in p21 protein levels in MnSOD overexpressing confluent fibroblasts were independent of p53 protein levels. p53 protein levels did not change in control, replication-defective adenovirus containing an insertless vector (AdBgl II), or AdMnSOD-infected confluent cells cultured for 20 and 60 days. In addition, MnSOD-induced protection of the proliferation capacity of confluent fibroblasts was independent of their telomerase activity. However, telomerase-transformed fibroblasts showed increased MnSOD expression in confluent growth, maintaining their capacity to re-enter the proliferation cycle. Although inactivation of the retinoblastoma protein in cells subcultured from the 60-day confluent control, AdBgl II-, and AdMnSOD-infected fibroblasts was identical, only MnSOD-overexpressing cells showed a higher percentage of S-phase. These results support the hypothesis that a redox-sensitive checkpoint regulated the progression of fibroblasts from G 0 /G 1 to S-phase.In mammalian cells, intracellular antioxidant enzymes include superoxide dismutase, catalase, and glutathione peroxidase. There are two intracellular forms of superoxide dismutase as follows: CuZnSOD, 1 found in the cytoplasm and nucleus, and MnSOD, found in mitochondria (1, 2). Different isozymes of glutathione peroxidase are found in most subcellular compartments, whereas catalase is found primarily in peroxisomes and cytoplasm (1). Antioxidant enzymes neutralize reactive oxygen species (ROS) generated from the univalent reduction of oxygen by mitochondrial electron transport chains as well as biochemical reactions of oxygen-metabolizing enzymes (3-5). ROS, including superoxide, hydrogen peroxide, hydroxyl radical, singlet molecular oxygen, and organic hydroperoxides, are oxygen-containing molecules that have higher chemical reactivity than ground state molecular oxygen. ROS have traditionally been thought of as unwanted and toxic by-products of living in an aerobic environment (6, 7). In recent years, several studies suggest metabolic production of ROS is tightly regulated and serves a physiological function during mitogenic stimulation of cultured cells (6 -11). It has been suggested that ROS operate as a key signaling process in the cascade of events leading to cell proliferation following stimulation with platelet-derived growth factor (9), epidermal growth factor (10), cytokine...

Polychlorinated biphenyls (PCBs) are environmental chemical contaminants believed to adversely affect cellular processes. We investigated the hypothesis that PCB-induced changes in the levels of cellular reactive oxygen species (ROS) induce DNA damage resulting in cytotoxicity. Exponentially growing cultures of human non-malignant breast epithelial cells (MCF10A) were incubated with PCBs for 3 days and assayed for cell number, ROS levels, DNA damage, and cytotoxicity. Exposure to 2,2',4,4',5,5'-hexachlorobiphenyl (PCB153) or 2-(4-chlorophenyl)benzo-1,4-quinone (4-Cl-BQ), a metabolite of 4-chlorobiphenyl (PCB3) significantly decreased cell number, MTS reduction, and increased the percentage of cells with sub G 1 DNA content. Results from electron paramagnetic resonance (EPR) spectroscopy showed a 4-fold increase in the steady-state levels of ROS, which was suppressed in cells pre-treated with catalase. EPR measurements in cells treated with 4-Cl-BQ detected the presence of a semiquinone radical, suggesting that the increased levels of ROS could be due to the redox-cycling of 4-Cl-BQ. A dose-dependent increase in micronuclei frequency was observed in PCB-treated cells, consistent with an increase in histone 2AX-phosphorylation. Treatment of cells with catalase blunted the PCB-induced increase in micronuclei frequency and H2AX phosphorylation that was consistent with an increase in cell survival. Our results demonstrate a PCB-induced increase in cellular levels of ROS causing DNA damage, resulting in cell killing.

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]