Cytotoxic effects induced by oxidative stress in cultured mammalian cells and protection provided by Hsp27 expression

There is currently great interest in the development of methods to analyze intracellular redox state and the cellular damages generated by oxidative stress. General methods for analyzing reactive oxygen species and glutathione level are presented together with more recently developed protocols to analyze the consequences of oxidative stress on the oxidation of macromolecules. Finally, techniques to study modalities of constitutive expression of Hsp27 in mammalian cells are considered as well as methods used to determine the protective activity of this small heat shock protein against the deleterious effects induced by oxidative stress.     

Mitochondrial biogenesis and mitochondrial DNA maintenance of mammalian cells under oxidative stress

Mitochondrial biogenesis and mitochondrial DNA (mtDNA) maintenance depend on coordinated expression of genes in the nucleus and mitochondria. A variety of intracellular and extracellular signals transmitted by hormones and second messengers have to be integrated to provide mammalian cells with a suitable abundance of mitochondria and mtDNA to meet their energy demand. It has been proposed that reactive oxygen species (ROS) and free radicals generated from respiratory chain are involved in the signaling from mitochondria to the nucleus. Increased oxidative stress may contribute to alterations in the abundance of mitochondria as well as the copy number and integrity of mtDNA in human cells in pathological conditions and in aging process. Within a certain level, ROS may induce stress responses by altering expression of specific nuclear genes to uphold the energy metabolism to rescue the cell. Once beyond the threshold, ROS may cause oxidative damage to mtDNA and other components of the affected cells and to elicit apoptosis by induction of mitochondrial membrane permeability transition and release of pro-apoptotic proteins such as cytochrome c. On the basis of recent findings gathered from this and other laboratories, we review the alterations in the abundance of mitochondria and mtDNA copy number of mammalian cells in response to oxidative stress and the signaling pathways that are involved.     

Proteolytic response to oxidative stress in mammalian cells

Oxidative stress in mammalian cells is an inevitable consequence of their aerobic metabolism. The production of reactive oxygen and nitric oxide species causes oxidative modifications of proteins often combined with a loss of their biological function. Like most partially denatured proteins, moderately oxidized proteins are more sensitive to proteolytic attack by proteases. The diverse cellular proteolytic systems are an important secondary defense against oxidative stress by degrading oxidized and damaged proteins, thereby preventing their intracellular accumulation. In mammalian cells, a range of proteases exists which are distributed throughout the cell. In this review we summarize the function of the cytosolic (proteasome and calpains), the lysosomal, the mitochondrial and the nuclear proteolytic pathways in response to oxidative stress. Particular emphasis is given to the proteasomal system, since this pathway appears to be the most important proteolytic system involved in the removal of oxidatively modified or damaged proteins.     

The small heat shock protein Hsp22 of Drosophila melanogaster is a mitochondrial protein displaying oligomeric organization

Drosophila melanogaster has four main small heat shock proteins (Hsps), D. melanogaster Hsp22 (DmHsp22), Hsp23 (DmHsp23), Hsp26 (DmHsp26), and Hsp27 (DmHsp27). These proteins, although they have high sequence homology, show distinct developmental expression patterns. The function(s) of each small heat shock protein is unknown. DmHsp22 is shown to localize in mitochondria both in D. melanogaster S2 cells and after heterologous expression in mammalian cells. Fractionation of mitochondria indicates that DmHsp22 resides in the mitochondrial matrix, where it is found in oligomeric complexes, as shown by sedimentation and gel filtration analysis and by cross-linking experiments. Deletion analysis using a DmHsp22-EGFP construct reveals that residues 1-17 and an unknown number of residues between 17-28 are necessary for import. Site-directed mutagenesis within a putative mitochondrial motif (WRMAEE) at positions 8-13 shows that the first four residues are necessary for mitochondrial localization. Immunoprecipitation results indicate that there is no interaction between DmHsp22 and the other small heat shock proteins. The mitochondrial localization of this small Hsp22 of Drosophila and its high level of expression in aging suggests a role for this small heat shock protein in protection against oxidative stress.     

Oxidative stress, antioxidant defenses, and damage removal, repair, and replacement systems

Oxidative stress is an unavoidable consequence of life in an oxygen-rich atmosphere. Oxygen radicals and other activated oxygen species are generated as by-products of aerobic metabolism and exposure to various natural and synthetic toxicants. The "Oxygen Paradox" is that oxygen is dangerous to the very life-forms for which it has become an essential component of energy production. The first defense against oxygen toxicity is the sharp gradient of oxygen tension, seen in all mammals, from the environmental level of 20% to a tissue concentration of only 3-4% oxygen. These relatively low tissue levels of oxygen prevent most oxidative damage from ever occurring. Cells, tissues, organs, and organisms utilize multiple layers of antioxidant defenses and damage removal, and replacement or repair systems in order to cope with the remaining stress and damage that oxygen engenders. The enzymes comprising many of these protective systems are inducible under conditions of oxidative stress adaptation, in which the expression of over 40 mammalian genes is upregulated. Mitotic cells have the additional defensive ability of entering a transient growth-arrested state (in the first stages of adaptation) in which DNA is protected by histone proteins, energy is conserved by diminished expression of nonessential genes, and the expression of shock and stress proteins is greatly increased. Failure to fully cope with an oxidative stress can switch mitotic cells into a permanent growth-arrested, senescence-like state in which they may survive for long periods. Faced with even more severe oxidative stress, or the declining protective enzymes and adaptive capacity associated with aging, cells may "sacrifice themselves" by apoptosis, which protects surrounding healthy tissue from further damage. Only under the most severe oxidative stress conditions will cells undergo a necrotic death, which exposes surrounding tissues to the further vicissitudes of an inflammatory immune response. This remarkable array of systems for defense; damage removal, replacement, and repair; adaptation; growth modulation; and apoptosis make it possible for us to enjoy life in an oxygen-rich environment.     

Sir2 is induced by oxidative stress in a yeast model of Huntington disease and its activation reduces protein aggregation

Huntington disease (HD) is a neurodegenerative disorder caused by expansion of CAG trinucleotide repeats, leading to an elongated polyglutamine sequence (polyQ) in the huntingtin protein. Misfolding of mutant polyQ proteins with expanded tracts results in aggregation, causing cytotoxicity. Oxidative stress in HD has been documented in humans as important to disease progression. Using yeast cells as a model of HD, we report that when grown at high glucose concentration, cells expressing mutant polyQ do not show apparent oxidative stress. At higher cell densities, when glucose becomes limiting and cells are metabolically shifting from fermentation to respiration, protein oxidation and catalase activity increases in relation to the length of the polyQ tract. Oxidative stress, either endogenous as a result of mutant polyQ expression or exogenously generated, increases Sir2 levels. Δ sir2 cells expressing expanded polyQ lengths show signs of oxidative stress even at the early exponential phase. In a wild-type background, isonicotinamide, a Sir2 activator, decreases mutant polyQ aggregation and the stress generated by expanded polyQ. Taken together, these results describe mutant polyQ proteins as being more toxic in respiring cells, causing oxidative stress and an increase in Sir2 levels. Activation of Sir2 would play a protective role against this toxicity.     

Impairing the microRNA biogenesis pathway induces proteome modifications characterized by size bias and enrichment in antioxidant proteins

Perturbation of individual microRNAs, or of the microRNA pathway, plays a role in carcinogenesis. In certain cancer cells, inhibition of the microRNA biogenesis pathway leads to a growth arrest state (CoGAM for Colony Growth Arrest induced by Microprocessor inhibition), which can be rescued by re-expression of individual microRNAs such as miR-20a. We now report that inhibition of the microRNA biogenesis pathway induced proteome changes characterized by a size bias in differentially expressed proteins, with induction of small proteins and inhibition of large ones. This size bias was observed in cells undergoing CoGAM, as well as in CoGAM-resistant cells, and in CoGAM-sensitive cells rescued by miR-20a. In this case, GO analysis of induced proteins identified by mass spectrometry revealed a significant enrichment in proteins involved in resistance to oxidative stress. In addition, H(2) O(2) treatment of Saccharomyces cerevisiae or mammalian cells led to similarly size-biased proteome modifications. Our results point to size bias as a relevant readout of proteome modifications, in particular in conditions of stress such as inhibition of the microRNA biogenesis pathway or oxidative stress. They also suggest research avenues to study the role of the microRNA pathway in proteostasis.     

Expression and functions of heat shock proteins in the normal and pathological mammalian eye

Heat shock proteins (Hsps) are expressed in mammalian embryonic, adult and aging lens, cornea and retina. These proteins, particularly those belonging to the family of small Hsps, such as αA-crystallin (HspB4) and αB-crystallin (HspB5), play important roles in the differentiation of lens cells and are essential for the maintenance and protection of the supraorganization of proteins in differentiated corneal and lens fiber cells. Hsps are molecular chaperones characterized by their protective activity against different types of stress. They also have anti-apoptotic and anti-oxidant functions that help lens and corneal cells to better cope with the oxidative conditions that result from light induced injuries. They are also effective to protect the retina against the high rate of oxidative metabolism observed in this tissue. The goal of this review is to highlight recent works describing the expression and function(s) of the different Hsps as an attempt to better understand their roles in the normal and pathological eye. Particular emphasis is given to the α-crystallin polypeptides which, in addition to their protective functions, are key structural polypeptides that are essential for the refractive and light focusing properties of the lens, a property demonstrated by the caractogenic potential of their mutation.     

Human hsp27, Drosophila hsp27 and human alphaB-crystallin expression-mediated increase in glutathione is essential for the protective activity of these proteins against TNFalpha-induced cell death.

Expression of small stress proteins (shsp) enhances the survival of mammalian cells exposed to heat or oxidative injuries. Recently, we have shown that the expression of shsp from different species, such as human hsp27, Drosophila hsp27 or human alphaB-crystallin protected murine L929 cells against cell death induced by tumor necrosis factor (TNFalpha), hydrogen peroxide or menadione. Here, we report that, in growing L929 cell lines, the presence of these shsp decreased the intracellular level of reactive oxygen species (ROS). shsp expression also abolished the burst of intracellular ROS induced by TNFalpha. Several downstream effects resulting from the TNFalpha-mediated ROS increment, such as NF-kappaB activation, lipid peroxidation and protein oxidation, were inhibited by shsp expression. We also report that the expression of these different shsp raised the total glutathione level in both L929 cell lines and transiently transfected NIH 3T3-ras cells. This phenomenon was essential for the shsp-mediated decrease in ROS and resistance against TNFalpha. Our results therefore suggest that the protective activity shared by human hsp27, Drosophila hsp27 and human alphaB-crystallin against TNFalpha-mediated cell death and probably other types of oxidative stress results from their conserved ability to raise the intracellular concentration of glutathione.     

LEDGF binds to heat shock and stress-related element to activate the expression of stress-related genes

We have investigated the mechanism by which LEDGF protects cells against environmental stress. Our earlier report showed that a low level of LEDGF was present in the nucleus of most cell types and significant elevation of LEDGF level was induced by heat and oxidative stress. The cells overexpressing LEDGF-activated expression of heat shock proteins and enhanced survival of many cell types. Here we show that LEDGF binds to heat shock element (HSE) and stress-related regulatory element (STRE) to activate the expression of stress-related genes (Hsp27 and alphaB-crystallin). Apparently, HSE and STRE are present in promoters of many stress-related genes. Elevation of many stress-related proteins (STRPs) induced by LEDGF may protect cells against environmental stress. In yeast, it has been demonstrated that a single stress can activate the expression of multiple STRPs. This is known as "cross-protection," and now similar mechanism has been found in mammalian cells and LEDGF plays a vital role in it.     

Overexpression of calreticulin fails to abolish its induction by perturbation of normal ER function.

Along with other endoplasmic reticulum (ER) Ca2+-binding proteins, notably the glucose-response proteins grp78 and grp94, expression of calreticulin is induced in response to perturbation of normal ER function. It has yet to be clearly defined how this stress is signaled from the ER to the nucleus in mammalian cells, particularly with regard to its initiation. Using a GFP-calreticulin fusion protein, we have generated and selected stably transfected HeLa cells that overexpress calreticulin to investigate whether the protein might be involved in signaling its own induction. Basal levels of endogenous calreticulin mRNA and protein were unaffected in these cells, indicating that overexpression alone does not induce a stress response. ER stress induced calreticulin expression in response to either thapsigargin or tunicamycin was equivalent in these cells to that seen in control, nontransfected cells, leading us to conclude that calreticulin is unlikely be involved in its own induction. Levels of the mRNA encoding the fusion protein were also increased by tunicamycin, but not thapsigargin, suggesting that, in agreement with our previous observations, inhibition of N-linked glycosylation may increase the stability of calreticulin mRNA. This indicates that in mammalian cells, there is more than one signaling pathway for the ER stress response.     

Plant rac proteins induce superoxide production in mammalian cells

The small GTP-binding protein family including Rac proteins represents a paradigm for signaling molecules shared by animal and plants. In mammalian cells, Rac induces the activation of NADPH oxidase leading to superoxide production. In plants, evidence suggests that resistance to pathogens depends on superoxide that is generated via NADPH oxidase-like enzymes. We have identified four closely related Rho/Rac genes from Zea mays that exhibit a high degree of homology to the human Rac. We hypothesized that these plant Rac proteins could function as their mammalian counterpart and activate an enzymatic complex that leads to superoxide production. Here, we show that like human Rac1, activated Zea mays Rac genes can induce superoxide production, when expressed in a mammalian system: NIH 3T3 cells. Our results suggest that in plants, Rac proteins can function as activators of oxidative burst and indicate the remarkable functional and structural conservation of Rho/Rac proteins between plant and animal kingdoms during evolution.     

Involvement of the Leishmania donovani virulence factor A2 in protection against heat and oxidative stress

Leishmania is an obligate intracellular protozoan parasite that infects cells of the reticulo-endothelial system. Host defences against Leishmania include fever and oxidant production, and the parasite has developed a number of defence mechanisms to neutralize the host response. The Leishmania donovani A2 family of proteins has been shown to be essential for survival in mammalian visceral organs. Here we provide evidence that A2 proteins protect the parasite against host defences, namely heat stress (fever) and oxidative stress. A2 is however unable to protect the cells from endoplasmic reticulum stress induced by dithiothreitol. To downregulate A2 protein expression, L. donovani was transfected with an A2 antisense RNA expressing-vector, resulting in significant reduction of A2 levels. The resulting A2-deficient cells were more sensitive to heat shock and this was associated with increased production of internal oxidants during heat shock. Moreover, axenic amastigotes with downregulated A2 expression had increased internal oxidants and decreased viability following treatment with hydrogen peroxide or a nitric oxide donor when compared to control cells. Overall, these results suggest that A2 protects L. donovani from a variety of stresses, thereby allowing it to survive in the internal organs of the mammalian host and to cause visceral disease.     

Protein oxidation and proteolysis

One of the hallmarks of chronic or severe oxidative stress is the accumulation of oxidized proteins, which tend to form high-molecular-weight aggregates. The major proteolytic system responsible for the removal of oxidized cytosolic and nuclear proteins is the proteasome. This complicated proteolytic system contains a core proteasomal form (20S proteasome) and several regulators. All of these components are affected by oxidative stress to various degrees. The ATP-stimulated 26S proteasome is sensitive to oxidative stress, whereas the 20S form seems to be more resistant. The nuclear proteasome selectively degrades oxidatively damaged histones in the nuclei of mammalian cells, where it is activated and regulated by automodified PARP-1 after oxidative challenge. In this brief review we highlight the proteolysis and its regulatory effects during oxidative stress.     

Oxidative stress-alleviating strategies to improve recombinant protein production in CHO cells

Large scale biopharmaceutical production of biologics relies on the overexpression of foreign proteins by cells cultivated in stirred tank bioreactors. It is well recognized and documented fact that protein overexpression may impact host cell metabolism and that factors associated with large scale culture, such as the hydrodynamic forces and inhomogeneities within the bioreactors, may promote cellular stress. The metabolic adaptations required to support the high-level expression of recombinant proteins include increased energy production and improved secretory capacity, which, in turn, can lead to a rise of reactive oxygen species (ROS) generated through the respiration metabolism and the interaction with media components. Oxidative stress is defined as the imbalance between the production of free radicals and the antioxidant response within the cells. Accumulation of intracellular ROS can interfere with the cellular activities and exert cytotoxic effects via the alternation of cellular components. In this context, strategies aiming to alleviate oxidative stress generated during the culture have been developed to improve cell growth, productivity, and reduce product microheterogeneity. In this review, we present a summary of the different approaches used to decrease the oxidative stress in Chinese hamster ovary cells and highlight media development and cell engineering as the main pathways through which ROS levels may be kept under control.     

Oxygen sensing and redox signaling: the role of thioredoxin in embryonic development and cardiac diseases

It is important to regulate the oxygen concentration and scavenge oxygen radicals throughout the life of animals. In mammalian embryos, proper oxygen concentration gradually increases in utero and excessive oxygen is rather toxic during early embryonic development. Reactive oxygen species (ROS) are generated as by-products in the respiratory system and increased under inflammatory conditions. In the pathogenesis of a variety of adult human diseases such as cancer and cardiovascular disorders, ROS cause an enhancement of tissue injuries. ROS promote not only the development of atherosclerosis but also tissue injury during the reperfusion process. The thioredoxin (TRX) system is one of the most important mechanisms for regulating the redox balance. TRX is a small redox active protein distributed ubiquitously in various mammalian tissues and cells. TRX acts as not only an antioxidant but also an anti-inflammatory and an antiapoptotic protein. TRX is induced by oxidative stress and released from cells in response to oxidative stress. In various human diseases, the serum/plasma level of TRX is a well-recognized biomarker of oxidative stress. Here we discuss the roles of TRX on oxygen stress and redox regulation from different perspectives, in embryogenesis and in adult diseases focusing on cardiac disorders.     

A leader sequence capable of enhancing RNA expression and protein synthesis in mammalian cells

Many applications in biotechnology require human proteins generated from human cells. Stable cell lines commonly used for this purpose are difficult to develop, and scaling to large numbers of proteins can be problematic. Transient expression can circumvent this problem, but protein yields are generally too low for most applications. Here we report a novel 37‐nucleotide leader sequence that promotes rapid and high transgene expression in mammalian cells. This sequence was identified by in vitro selection and functions in a transient vaccinia‐based cytoplasmic expression system. Vectors containing this sequence produce microgram levels of protein in just 6 h from a small‐scale expression in 10⁶ cells. This level of protein synthesis is ideal for high throughput production of human proteins, and could be scaled to generate milligram quantities of protein. The technology is compatible with a broad range of cell lines, accepts plasmid and linear DNA, and functions with viruses that are approved for use under BSL1 conditions. We suggest that these advantages provide a powerful method for generating human protein in mammalian cells.     

Genotoxic potential of copper oxide nanoparticles in human lung epithelial cells

Copper oxide nanoparticles (CuO NPs) are increasingly used in various applications. Recent studies suggest that oxidative stress may be the cause of the cytotoxicity of CuO NPs in mammalian cells. However, little is known about the genotoxicity of CuO NPs following exposure to human cells. This study was undertaken to investigate CuO NPs induced genotoxic response through p53 pathway in human pulmonary epithelial cells (A549). In addition, cytotoxicity and oxidative stress markers were also assessed. Results showed that cell viability was reduced by CuO NPs and degree of reduction was dose dependent. CuO NPs were also found to induce oxidative stress in dose-dependent manner indicated by depletion of glutathione and induction of lipid peroxidation, catalase and superoxide dismutase. The expression of Hsp70, the first tier biomarker of cellular damage was induced by CuO NPs. Further, CuO NPs up-regulated the cell cycle checkpoint protein p53 and DNA damage repair proteins Rad51 and MSH2 expression. These results demonstrate that CuO NPs possess a genotoxic potential in A549 cells which may be mediated through oxidative stress. Our short-term exposure study of high level induction of genotoxic response of CuO NPs will need to be further investigated to determine whether long-term exposure consequences may exist for CuO NPs application.