Abstract
Nucleostemin (NS) is a nucleolar-nucleoplasmic shuttle protein that regulates cell proliferation, binds p53 and Mdm2, and is highly expressed in tumor cells. We have identified NS as a target of oxidative regulation in transformed hematopoietic cells. NS oligomerization occurs in HL-60 leukemic cells and Raji B lymphoblasts that express high levels of c-Myc and have high intrinsic levels of reactive oxygen species (ROS); reducing agents dissociate NS into monomers and dimers. Exposure of U2OS osteosarcoma cells with low levels of intrinsic ROS to hydrogen peroxide (H(2)O(2)) induces thiol-reversible disulfide bond-mediated oligomerization of NS. Increased exposure to H(2)O(2) impairs NS degradation, immobilizes the protein within the nucleolus, and results in detergent-insoluble NS. The regulation of NS by ROS was validated in a murine lymphoma tumor model in which c-Myc is overexpressed and in CD34+ cells from patients with chronic myelogenous leukemia in blast crisis. In both instances, increased ROS levels were associated with markedly increased expression of NS protein and thiol-reversible oligomerization. Site-directed mutagenesis of critical cysteine-containing regions of nucleostemin altered both its intracellular localization and its stability. MG132, a potent proteasome inhibitor and activator of ROS, markedly decreased degradation and increased nucleolar retention of NS mutants, whereas N-acetyl-L-cysteine largely prevented the effects of MG132. These results indicate that NS is a highly redox-sensitive protein. Increased intracellular ROS levels, such as those that result from oncogenic transformation in hematopoietic malignancies, regulate the ability of NS to oligomerize, prevent its degradation, and may alter its ability to regulate cell proliferation.
Highlights
Olus to the nucleoplasm in response to a variety of cellular stressors, including inhibition of RNA synthesis [5]
Reactive oxygen species (ROS)-dependent Oligomerization of NS—Having first observed during purification from Raji cells that NS existed as a were preincubated with NAC (10 mM or 50 mM) for 16 h and exposed to 10 mM H2O2 for 30 min, followed by separation on SDS-PAGE and analysis for NS using goat antibody against full-length NS under non-reducing (B, left panel) or reducing conditions
Moderate increases in ROS may act to promote cell proliferation, signal transduction, and possibly genomic instability [38, 39], whereas higher levels of ROS can induce extensive oxidation of essential cellular proteins, leading to the accumulation of unfolded or misfolded aggregates and protein inactivation [18, 40, 41]. Accumulation of such aggregated proteins is known to play an important role in oxidative damage-mediated cell death [41]
Summary
Olus to the nucleoplasm in response to a variety of cellular stressors, including inhibition of RNA synthesis [5]. ROS Regulate Oligomerization of Nucleostemin expression of the Myc oncogene because of amplification, mutation, increased transcription, or translocation results in increased ROS levels that appear to promote genomic instability in transformed cells [19, 24]. Further understanding of the oxidant-dependent signaling pathways induced during transformation should be useful in identifying previously unrecognized redox-sensitive targets that may play a role in the progression of diseases such as CML or those resulting from increased c-Myc expression. Cell lines with Myc amplification express an oligomerized form of NS that is reduced after incubation with NAC and other reducing agents This observation led us to examine the role of intracellular ROS as a potentially important regulator of NS stability and function
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