Yumin Chen

ROS and p53: A versatile partnership

The tumor suppressor protein p53 is a redox-active transcription factor that organizes and directs cellular responses in the face of a variety of stresses that lead to genomic instability. One of the most important questions in the study of p53 is how selective transactivation of certain p53 target genes is achieved. Reactive oxygen species (ROS), generated by cells as products or by-products, can function either as signaling molecules or as cellular toxicants. Cellular generation of ROS is central to redox signaling. Recent studies have revealed that each cellular concentration and distribution of p53 has a distinct cellular function and that ROS act as both an upstream signal that triggers p53 activation and a downstream factor that mediates apoptosis. Here, we examine the newly discovered role of p53 in regulating cellular ROS generation and how ROS modulate selective transactivation of p53 target genes. The focus is on interlinks between ROS and p53.

A neuronal model of Alzheimer's disease: an insight into the mechanisms of oxidative stress-mediated mitochondrial injury.

Alzheimers disease (AD) is associated with beta-amyloid accumulation, oxidative stress and mitochondrial dysfunction. However, the effects of genetic mutation of AD on oxidative status and mitochondrial manganese superoxide dismutase (MnSOD) production during neuronal development are unclear. To investigate the consequences of genetic mutation of AD on oxidative damages and production of MnSOD during neuronal development, we used primary neurons from new born wild-type (WT/WT) and amyloid precursor protein (APP) (NLh/NLh) and presenilin 1 (PS1) (P264L) knock-in mice (APP/PS1) which incorporated humanized mutations in the genome. Increasing levels of oxidative damages, including protein carbonyl, 4-hydroxynonenal (4-HNE) and 3-nitrotyrosine (3-NT), were accompanied by a reduction in mitochondrial membrane potential in both developing and mature APP/PS1 neurons compared with WT/WT neurons suggesting mitochondrial dysfunction under oxidative stress. Interestingly, developing APP/PS1 neurons were significantly more resistant to beta-amyloid 1-42 treatment, whereas mature APP/PS1 neurons were more vulnerable than WT/WT neurons of the same age. Consistent with the protective function of MnSOD, developing APP/PS1 neurons have increased MnSOD protein and activity, indicating an adaptive response to oxidative stress in developing neurons. In contrast, mature APP/PS1 neurons exhibited lower MnSOD levels compared with mature WT/WT neurons indicating that mature APP/PS1 neurons lost the adaptive response. Moreover, mature APP/PS1 neurons had more co-localization of MnSOD with nitrotyrosine indicating a greater inhibition of MnSOD by nitrotyrosine. Overexpression of MnSOD or addition of MnTE-2-PyP(5+) (SOD mimetic) protected against beta-amyloid-induced neuronal death and improved mitochondrial respiratory function. Together, the results demonstrate that compensatory induction of MnSOD in response to an early increase in oxidative stress protects developing neurons against beta-amyloid toxicity. However, continuing development of neurons under oxidative damage conditions may suppress the expression of MnSOD and enhance cell death in mature neurons.

Specificity Protein 1-dependent p53-mediated Suppression of Human Manganese Superoxide Dismutase Gene Expression

Manganese superoxide dismutase (MnSOD) is a primary antioxidant enzyme necessary for the survival of aerobic life. Previously, we demonstrated that specificity protein 1 (Sp1) is essential for the basal transcription of the MnSOD gene. We also identified nucleophosmin (NPM), an RNA-binding protein, as an important co-activator of NF-κB in the induction of MnSOD by cytokine and tumor promoter. Here, using chromatin immunoprecipitation (ChIP) analysis, we demonstrate that Sp1 and NPM interact in vivo to enhance NF-κB-mediated MnSOD induction. Interaction between NPM and Sp1 or NF-κB at the promoter and enhancer of the MnSOD gene in vivo were verified by the presence of the PCR products from the promoter and enhancer elements in the ChIP assay. Unexpectedly, we also found p53, another transcription factor, to be a component of the complex detected by ChIP assay. The presence of p53 in this transcription complex was verified by immunoprecipitation of p53 proteins with antibody to Sp1 in nuclear extracts. Using a vector expressing full-length p53 cDNA, we demonstrated that p53 overexpression suppresses MnSOD mRNA and protein levels. Consistent with the negative role of p53 in the expression of the MnSOD gene, expression of small interfering RNA for p53 leads to an increase of MnSOD mRNA and protein levels. Using ChIP assays and immunoprecipitation, we further demonstrated that p53 interacts with Sp1 to suppress both the constitutive and 12-O-tetradecanoylphorbol-13-acetate-stimulated expression of the MnSOD gene. Inhibition of the MnSOD gene by p53 was abolished when Sp1 sites on the MnSOD promoter were mutated or when the Sp1 protein was reduced by siRNA approaches. Because expression of MnSOD protects against cell death, our findings reveal a previously unrecognized mechanism of p53-mediated cell death and demonstrate an intricate relationship between the positive and negative control of MnSOD expression.

Interactions between SIRT1 and AP-1 reveal a mechanistic insight into the growth promoting properties of alumina (Al2O3) nanoparticles in mouse skin epithelial cells

The physicochemical properties of nanomaterials differ from those of the bulk material of the same composition. However, little is known about the underlying effects of these particles in carcinogenesis. The purpose of this study was to determine the mechanisms involved in the carcinogenic properties of nanoparticles using aluminum oxide (Al(2)O(3)/alumina) nanoparticles as the prototype. Well-established mouse epithelial JB6 cells, sensitive to neoplastic transformation, were used as the experimental model. We demonstrate that alumina was internalized and maintained its physicochemical composition inside the cells. Alumina increased cell proliferation (53%), proliferating cell nuclear antigen (PCNA) levels, cell viability and growth in soft agar. The level of manganese superoxide dismutase, a key mitochondrial antioxidant enzyme, was elevated, suggesting a redox signaling event. In addition, the levels of reactive oxygen species and the activities of the redox sensitive transcription factor activator protein-1 (AP-1) and a longevity-related protein, sirtuin 1 (SIRT1), were increased. SIRT1 knockdown reduces DNA synthesis, cell viability, PCNA levels, AP-1 transcriptional activity and protein levels of its targets, JunD, c-Jun and BcL-xl, more than controls do. Immunoprecipitation studies revealed that SIRT1 interacts with the AP-1 components c-Jun and JunD but not with c-Fos. The results identify SIRT1 as an AP-1 modulator and suggest a novel mechanism by which alumina nanoparticles may function as a potential carcinogen.

Pharmacokinetics of the potent redox-modulating manganese porphyrin, MnTE-2-PyP5+, in plasma and major organs of B6C3F1 mice

Mn(III) tetrakis(N-ethylpyridinium-2-yl)porphyrin, MnTE-2-PyP(5+), a potent catalytic superoxide and peroxynitrite scavenger, has been beneficial in several oxidative stress-related diseases thus far examined. Pharmacokinetic studies are essential for the better assessment of the therapeutic potential of MnTE-2-PyP(5+) and similar compounds, as well as for the modulation of their bioavailability and toxicity. Despite high hydrophilicity, this drug entered mitochondria after a single 10 mg/kg intraperitoneal injection at levels high enough (5.1 muM; 2.95 ng/mg protein) to protect against superoxide/peroxynitrite damage. Utilizing the same analytical approach, which involves the reduction of MnTE-2-PyP(5+) followed by the exchange of Mn(2+) with Zn(2+) and HPLC/fluorescence detection of ZnTE-2-PyP(4+), we measured levels of MnTE-2-PyP(5+) in mouse plasma, liver, kidney, lung, heart, spleen, and brain over a period of 7 days after a single intraperitoneal injection of 10 mg/kg. Two B6C3F1 female mice per time point were used. The pharmacokinetic profile in plasma and organs was complex; thus a noncompartmental approach was utilized to calculate the area under the curve, c(max), t(max), and drug elimination half-time (t(1/2)). In terms of levels of MnTE-2-PyP(5+) found, the organs can be classified into three distinct groups: (1) high levels (kidney, liver, and spleen), (2) moderate levels (lung and heart), and (3) low levels (brain). The maximal levels in plasma, kidney, spleen, lung, and heart are reached within 45 min, whereas in the case of liver a prolonged absorption phase was observed, with the maximal concentration reached at 8 h. Moreover, accumulation of the drug in brain continued beyond the time of the experiment (7 days) and is likely to be driven by the presence of negatively charged phospholipids. For tissues other than brain, a slow elimination phase (single exponential decay, t(1/2)=60 to 135 h) was observed. The calculated pharmacokinetic parameters will be used to design optimal dosing regimens in future preclinical studies utilizing this and similar compounds.

Mrp1 Localization and Function in Cardiac Mitochondria after Doxorubicin

Multidrug resistance-associated protein 1 (Mrp1; Abcc1) is expressed in sarcolemma of murine heart, where it probably protects the cardiomyocyte by mediating efflux of endo- and xenobiotics. We used doxorubicin (DOX), a chemotherapeutic drug known to induce oxidative stress and thereby cardiac injury, as a model cardiotoxic compound and observed changes in the Mrp1 expression pattern in cardiac tissue of DOX-versus saline-treated mice. Confocal immunofluorescent and immunogold electron microscopy, together with subcellular fractionation followed by immunoblot analyses and transport measurements, localized functional Mrp1 to mitochondria after DOX. Expressions of Mrp1 in heart homogenate, sarcolemma, and submitochondrial particles (SMP) were increased 1.6-, 2-, and 3-fold, respectively, at 24 h after DOX. Mitochondrial Mrp1 expression was markedly increased 72 h after DOX, whereas transport of Mrp1 substrates in SMP was maximal at 24 h. ATP-dependent transport in SMP occurred into an osmotically sensitive space and was inhibited by the anti-MRP1 antibody QCRL3. Adduction of a 190-kDa protein with the reactive lipid peroxidation product 4-hydroxy-2-nonenal (HNE) was detected in SMP and was maximal at 72 h after DOX; immunoprecipitation confirmed Mrp1-HNE adduction. In vitro, HNE (10 μM) inhibited mitochondrial respiration and transport activity in SMP, suggesting that Mrp1 is adversely affected by oxidative stress. These data demonstrate that after DOX, functional Mrp1 is detected in mitochondria in addition to that in sarcolemma; however, adduction with HNE inhibits Mrp1 activity. Mrp1 may serve to protect the heart by mediating the efflux of toxic products of oxidative stress from mitochondria and cardiomyocytes.