Daunorubicin-induced apoptosis in rat cardiac myocytes is inhibited by dexrazoxane.
-The clinical efficacy of anthracycline antineoplastic agents is limited by a high incidence of severe and usually irreversible cardiac toxicity, the cause of which remains controversial. In primary cultures of neonatal and adult rat ventricular myocytes, we found that daunorubicin, at concentrations /=10 micromol/L induced necrotic cell death within 24 hours, with no changes characteristic of apoptosis. To determine whether reactive oxygen species play a role in daunorubicin-mediated apoptosis, we monitored the generation of hydrogen peroxide with dichlorofluorescein (DCF). However, daunorubicin (1 micromol/L) did not increase DCF fluorescence, nor were the antioxidants N-acetylcysteine or the combination of alpha-tocopherol and ascorbic acid able to prevent apoptosis. In contrast, dexrazoxane (10 micromol/L), known clinically to limit anthracycline cardiac toxicity, prevented daunorubicin-induced myocyte apoptosis, but not necrosis induced by higher anthracycline concentrations (>/=10 micromol/L). The antiapoptotic action of dexrazoxane was mimicked by the superoxide-dismutase mimetic porphyrin manganese(II/III)tetrakis(1-methyl-4-peridyl)porphyrin (50 micromol/L). The recognition that anthracycline-induced cardiac myocyte apoptosis, perhaps mediated by superoxide anion generation, occurs at concentrations well below those that result in myocyte necrosis, may aid in the design of new therapeutic strategies to limit the toxicity of these drugs. (+info)
Base excision repair of oxidative DNA damage activated by XPG protein.
Oxidized pyrimidines in DNA are removed by a distinct base excision repair pathway initiated by the DNA glycosylase--AP lyase hNth1 in human cells. We have reconstituted this single-residue replacement pathway with recombinant proteins, including the AP endonuclease HAP1/APE, DNA polymerase beta, and DNA ligase III-XRCC1 heterodimer. With these proteins, the nucleotide excision repair enzyme XPG serves as a cofactor for the efficient function of hNth1. XPG protein promotes binding of hNth1 to damaged DNA. The stimulation of hNth1 activity is retained in XPG catalytic site mutants inactive in nucleotide excision repair. The data support the model that development of Cockayne syndrome in XP-G patients is related to inefficient excision of endogenous oxidative DNA damage. (+info)
Chaperone activity with a redox switch.
Hsp33, a member of a newly discovered heat shock protein family, was found to be a very potent molecular chaperone. Hsp33 is distinguished from all other known molecular chaperones by its mode of functional regulation. Its activity is redox regulated. Hsp33 is a cytoplasmically localized protein with highly reactive cysteines that respond quickly to changes in the redox environment. Oxidizing conditions like H2O2 cause disulfide bonds to form in Hsp33, a process that leads to the activation of its chaperone function. In vitro and in vivo experiments suggest that Hsp33 protects cells from oxidants, leading us to conclude that we have found a protein family that plays an important role in the bacterial defense system toward oxidative stress. (+info)
The Golgi apparatus plays a significant role in the maintenance of Ca2+ homeostasis in the vps33Delta vacuolar biogenesis mutant of Saccharomyces cerevisiae.
The vacuole is the major site of intracellular Ca2+ storage in yeast and functions to maintain cytosolic Ca2+ levels within a narrow physiological range. In this study, we examined how cellular Ca2+ homeostasis is maintained in a vps33Delta vacuolar biogenesis mutant. We found that growth of the vps33Delta strain was sensitive to high or low extracellular Ca2+. This strain could not properly regulate cytosolic Ca2+ levels and was able to retain only a small fraction of its total cellular Ca2+ in a nonexchangeable intracellular pool. Surprisingly, the vps33Delta strain contained more total cellular Ca2+ than the wild type strain. Because most cellular Ca2+ is normally found within the vacuole, this suggested that other intracellular compartments compensated for the reduced capacity to store Ca2+ within the vacuole of this strain. To test this hypothesis, we examined the contribution of the Golgi-localized Ca2+ ATPase Pmr1p in the maintenance of cellular Ca2+ homeostasis. We found that a vps33Delta/pmr1Delta strain was hypersensitive to high extracellular Ca2+. In addition, certain combinations of mutations effecting both vacuolar and Golgi Ca2+ transport resulted in synthetic lethality. These results indicate that the Golgi apparatus plays a significant role in maintaining Ca2+ homeostasis when vacuolar biogenesis is compromised. (+info)
Characterization of transgenic mice with targeted disruption of the catalytic domain of the double-stranded RNA-dependent protein kinase, PKR.
The interferon-inducible, double-stranded RNA-dependent protein kinase PKR has been implicated in anti-viral, anti-tumor, and apoptotic responses. Others have attempted to examine the requirement of PKR in these roles by targeted disruption at the amino terminal-encoding region of the Pkr gene. By using a strategy that aims at disruption of the catalytic domain of PKR, we have generated mice that are genetically ablated for functional PKR. Similar to the other mouse model of Pkr disruption, we have observed no consequences of loss of PKR on tumor suppression. Anti-viral response to influenza and vaccinia also appeared to be normal in mice and in cells lacking PKR. Cytokine signaling in the type I interferon pathway is normal but may be compromised in the erythropoietin pathway in erythroid bone marrow precursors. Contrary to the amino-terminal targeted Pkr mouse, tumor necrosis factor alpha-induced apoptosis and the anti-viral apoptosis response to influenza is not impaired in catalytic domain-targeted Pkr-null cells. The observation of intact eukaryotic initiation factor-2alpha phosphorylation in these Pkr-null cells provides proof of rescue by another eukaryotic initiation factor-2alpha kinase(s). (+info)
Increased lipophilicity and subsequent cell partitioning decrease passive transcellular diffusion of novel, highly lipophilic antioxidants.
Oxidative stress is considered a cause or propagator of acute and chronic disorders of the central nervous system. Novel 2, 4-diamino-pyrrolo[2,3-d]pyrimidines are potent inhibitors of iron-dependent lipid peroxidation, are cytoprotective in cell culture models of oxidative injury, and are neuroprotective in brain injury and ischemia models. The selection of lead candidates from this series required that they reach target cells deep within brain tissue in efficacious amounts after oral dosing. A homologous series of 26 highly lipophilic pyrrolopyrimidines was examined using cultured cell monolayers to understand the structure-permeability relationship and to use this information to predict brain penetration and residence time. Pyrrolopyrimidines were shown to be a more permeable structural class of membrane-interactive antioxidants where transepithelial permeability was inversely related to lipophilicity or to cell partitioning. Pyrrole substitutions influence cell partitioning where bulky hydrophobic groups increased partitioning and decreased permeability and smaller hydrophobic groups and more hydrophilic groups, especially those capable of weak hydrogen bonding, decreased partitioning, and increased permeability. Transmonolayer diffusion for these membrane-interactive antioxidants was limited mostly by desorption from the receiver-side membrane into the buffer. Thus, in this case, these in vitro cell monolayer models do not adequately mimic the in vivo situation by underestimating in vivo bioavailability of highly lipophilic compounds unless acceptors, such as serum proteins, are added to the receiving buffer. (+info)
Inactivation of both RNA binding and aconitase activities of iron regulatory protein-1 by quinone-induced oxidative stress.
Iron regulatory protein-1 (IRP-1) controls the expression of several mRNAs by binding to iron-responsive elements (IREs) in their untranslated regions. In iron-replete cells, a 4Fe-4S cluster converts IRP-1 to cytoplasmic aconitase. IRE binding activity is restored by cluster loss in response to iron starvation, NO, or extracellular H2O2. Here, we study the effects of intracellular quinone-induced oxidative stress on IRP-1. Treatment of murine B6 fibroblasts with menadione sodium bisulfite (MSB), a redox cycling drug, causes a modest activation of IRP-1 to bind to IREs within 15-30 min. However, IRE binding drops to basal levels within 60 min. Surprisingly, a remarkable loss of both IRE binding and aconitase activities of IRP-1 follows treatment with MSB for 1-2 h. These effects do not result from alterations in IRP-1 half-life, can be antagonized by the antioxidant N-acetylcysteine, and regulate IRE-containing mRNAs; the capacity of iron-starved MSB-treated cells to increase transferrin receptor mRNA levels is inhibited, and MSB increases the translation of a human growth hormone indicator mRNA bearing an IRE in its 5'-untranslated region. Nonetheless, MSB inhibits ferritin synthesis. Thus, menadione-induced oxidative stress leads to post-translational inactivation of both genetic and enzymatic functions of IRP-1 by a mechanism that lies beyond the "classical" Fe-S cluster switch and exerts multiple effects on cellular iron metabolism. (+info)
Identification and functional characterization of a novel mitochondrial thioredoxin system in Saccharomyces cerevisiae.
The so-called thioredoxin system, thioredoxin (Trx), thioredoxin reductase (Trr), and NADPH, acts as a disulfide reductase system and can protect cells against oxidative stress. In Saccharomyces cerevisiae, two thioredoxins (Trx1 and Trx2) and one thioredoxin reductase (Trr1) have been characterized, all of them located in the cytoplasm. We have identified and characterized a novel thioredoxin system in S. cerevisiae. The TRX3 gene codes for a 14-kDa protein containing the characteristic thioredoxin active site (WCGPC). The TRR2 gene codes for a protein of 37 kDa with the active-site motif (CAVC) present in prokaryotic thioredoxin reductases and binding sites for NADPH and FAD. We cloned and expressed both proteins in Escherichia coli, and the recombinant Trx3 and Trr2 proteins were active in the insulin reduction assay. Trx3 and Trr2 proteins have N-terminal domain extensions with characteristics of signals for import into mitochondria. By immunoblotting analysis of Saccharomyces subcellular fractions, we provide evidence that these proteins are located in mitochondria. We have also constructed S. cerevisiae strains null in Trx3 and Trr2 proteins and tested them for sensitivity to hydrogen peroxide. The Deltatrr2 mutant was more sensitive to H2O2, whereas the Deltatrx3 mutant was as sensitive as the wild type. These results suggest an important role of the mitochondrial thioredoxin reductase in protection against oxidative stress in S. cerevisiae. (+info)