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The effects of thiols, ascorbic acid and thermal stress on the basal (steady-state) levels of oxidative DNA base modifications were studied. In various types of untreated cultured mammalian cells, the levels of total glutathione were found to be inversely correlated with the levels of DNA base modifications sensitive to the repair endonuclease Fpg protein, which include 8-hydroxyguanine (8-oxoG). A depletion of glutathione by treatment with buthionine sulphoximine increased the steady-state level in AS52 Chinese hamster cells by 50%. However, additional thiols in the culture medium did not reduce the level of Fpg-sensitive base modifications: 0-10 mM N-acetylcysteine had no effect, whereas cysteine ethylester even increased the oxidative DNA damage at concentrations gt;0.1 mM. Similarly, ascorbic acid (0-20 mM) failed to reduce the steady-state levels. When AS52 cells were grown at elevated temperature (41°C), the steady-state level of the oxidative DNA modifications increased by 40%, in spite of a concomitant 1.6-fold increase of the cellular level of total glutathione. Depletion of glutathione at 41°C nearly doubled the already elevated level of oxidative damage. A constitutive expression of the heat-shock protein Hsp27 in L929 mouse fibrosarcoma cells at 37°C increased the glutathione level by 60%, but had little effect on the level of oxidative DNA damage. [ABSTRACT FROM PUBLISHER]

Human small heat shock proteins are molecular chaperones that regulate fundamental cellular processes in normal and pathological cells. Here, we have reviewed the role played by HspB1, HspB4 and HspB5 in the context of Cystic Fibrosis (CF), a severe monogenic autosomal recessive disease linked to mutations in Cystic Fibrosis Transmembrane conductance Regulator protein (CFTR) some of which trigger its misfolding and rapid degradation, particularly the most frequent one, F508del-CFTR. While HspB1 and HspB4 favor the degradation of CFTR mutants, HspB5 and particularly one of its phosphorylated forms positively enhance the transport at the plasma membrane, stability and function of the CFTR mutant. Moreover, HspB5 molecules stimulate the cellular efficiency of currently used CF therapeutic molecules. Different strategies are suggested to modulate the level of expression or the activity of these small heat shock proteins in view of potential in vivo therapeutic approaches. We then conclude with other small heat shock proteins that should be tested or further studied to improve our knowledge of CFTR processing.

The Sonic Hedgehog (SHH) pathway plays a key role in cancer. Alterations of SHH canonical signaling, causally linked to tumor progression, have become rational targets for cancer therapy. However, Smoothened (SMO) inhibitors have failed to show clinical benefit in patients with cancers displaying SHH autocrine/paracrine expression. We reported earlier that the SHH receptor Patched (PTCH) is a dependence receptor that triggers apoptosis in the absence of SHH through a pathway that differs from the canonical one, thus generating a state of dependence on SHH for survival. Here, we propose a dual function for SHH: its binding to PTCH not only activates the SHH canonical pathway but also blocks PTCH-induced apoptosis. Eighty percent, 64%, and 8% of human colon, pancreatic, and lung cancer cells, respectively, overexpressed SHH at transcriptional and protein levels. In addition, SHH-overexpressing cells expressed all the effectors of the PTCH-induced apoptotic pathway. Although the canonical pathway remained unchanged, autocrine SHH interference in colon, pancreatic, and lung cell lines triggered cell death through PTCH proapoptotic signaling. In vivo , SHH interference in colon cancer cell lines decreased primary tumor growth and metastasis. Therefore, the antitumor effect associated to SHH deprivation, usually thought to be a consequence of the inactivation of the canonical SHH pathway, is, at least in part, because of the engagement of PTCH proapoptotic activity. Together, these data strongly suggest that therapeutic strategies based on the disruption of SHH/PTCH interaction in SHH-overexpressing cancers should be explored. SIGNIFICANCE: Sonic Hedgehog-overexpressing tumors express PTCH-induced cell death effectors, suggesting that this death signaling could be activated as an antitumor strategy., (©2020 American Association for Cancer Research.)

Human HspB1 (also denoted as Hsp27) belongs to the family of small (or stress) proteins (sHsps). The family, which contains ten members including αA,B-crystallin polypeptides, is characterized by a conserved C-terminal α-crystallin domain and molecular weights ranging from 20 to 40 kDa. Here, procedures are described for analyzing the dynamic oligomerization and phosphorylation patterns of HspB1 in cells exposed to different environments. Changes in the structural organization of HspB1 can reprogram its interaction with specific partner/client polypeptides. Methods are presented to analyze these interactions using tissue culture cells genetically modified to express different levels of this protein. In addition, the laboratory approaches presented here could be used to test the nine other human sHsp members as well as sHsps from other species.

Constitutively expressed small heat shock protein HspB1 regulates many fundamental cellular processes and plays major roles in many human pathological diseases. In that regard, this chaperone has a huge number of apparently unrelated functions that appear linked to its ability to recognize many client polypeptides that are subsequently modified in their activity and/or half-life. A major parameter to understand how HspB1 is dedicated to interact with particular clients in defined cellular conditions relates to its complex oligomerization and phosphorylation properties. Indeed, HspB1 structural organization displays dynamic and complex rearrangements in response to changes in the cellular environment or when the cell physiology is modified. These structural modifications probably reflect the formation of structural platforms aimed at recognizing specific client polypeptides. Here, I have reviewed data from the literature and re-analyzed my own studies to describe and discuss these fascinating changes in HspB1 structural organization.

Sumoylation, a post-translational modification discovered over a decade ago, turns out to be a very important regulatory mechanism mediating multiple cellular processes. Recent studies from our laboratory and others also revealed that it plays a crucial role in regulating both differentiation and pathogenesis of the ocular lens. This review will summarize these progresses., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Our recent study has shown that αA-crystallin appears to act as a tumor suppressor in pancreas. Here, we analyzed expression patterns of αA-crystallin in the pancreatic tumor tissue and the neighbor normal tissue from 74 pancreatic cancer patients and also pancreatic cancer cell lines. Immunocytochemistry revealed that αA-crystallin was highly expressed in the normal tissue from 56 patients, but barely detectable in the pancreatic tumor tissue. Moreover, a low level of αA-crystallin predicts poor prognosis for patients with pancreatic duct adenocarcinoma (PDAC). In the 12 pancreatic cell lines analyzed, except for Capan-1 and Miapaca-2 where the level of αA-crystallin was about 80% and 65% of that in the control cell line, HPNE, the remaining pancreatic cancer cells have much lower αA-crystallin levels. Overexpression of αA-crystallin in MiaPaca-1 cells lacking endogenous αA-crystallin significantly decreased its tumorigenicity ability as shown in the colony formation and wound healing assays. In contrast, knockdown of αA-crystallin in the Capan-1 cells significantly increased its tumorigenicity ability as demonstrated in the above assays. Together, our results further demonstrate that αA-crystallin negatively regulates pancreatic tumorigenesis and appears to be a prognosis biomarker for PDAC.

During cell life, proteins often misfold, depending on particular mutations or environmental changes, which may lead to protein aggregates that are toxic for the cell. Such protein aggregates are the root cause of numerous diseases called "protein conformational diseases," such as myofibrillar myopathy and familial amyotrophic lateral sclerosis. To fight against aggregates, cells are equipped with protein quality control mechanisms. Here we report that NFκB transcription factor is activated by misincorporation of amino acid analogues into proteins, inhibition of proteasomal activity, expression of the R120G mutated form of HspB5 (associated with myofibrillar myopathy), or expression of the G985R and G93A mutated forms of superoxide dismutase 1 (linked to familial amyotrophic lateral sclerosis). This noncanonical stimulation of NFκB triggers the up-regulation of BAG3 and HspB8 expression, two activators of selective autophagy, which relocalize to protein aggregates. Then NFκB-dependent autophagy allows the clearance of protein aggregates. Thus NFκB appears as a central and major regulator of protein aggregate clearance by modulating autophagic activity. In this context, the pharmacological stimulation of this quality control pathway might represent a valuable strategy for therapies against protein conformational diseases., (© 2016 Nivon et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Human small heat shock proteins are molecular chaperones that regulate fundamental cellular processes in normal unstressed cells as well as in many cancer cells where they are over-expressed. These proteins are characterized by cell physiology dependent changes in their oligomerization and phosphorylation status. These structural changes allow them to interact with many different client proteins that subsequently display modified activity and/or half-life. Nowdays, the protein interactomes of small Hsps are under intense investigations and will represent, when completed, key parameters to elaborate therapeutic strategies aimed at modulating the functions of these chaperones. Here, we have analyzed the potential pro-cancerous roles of several client proteins that have been described so far to interact with HspB1 (Hsp27) and its close members HspB5 (αB-crystallin) and HspB4 (αA-crystallin).

Several human small heat shock proteins (sHsps) are phosphorylated oligomeric chaperones that enhance stress resistance. They are characterized by their ability to interact and form polydispersed hetero-oligomeric complexes. We have analyzed the cellular consequences of the stable expression of either wild type HspB5 or its cataracts and myopathies inducing R120G mutant in growing and oxidative stress treated HeLa cells that originally express only HspB1. Here, we describe that wild type and mutant HspB5 induce drastic and opposite effects on cell morphology and oxidative stress resistance. The cellular distribution and phosphorylation of these polypeptides as well as the oligomerization profile of the resulting hetero-oligomeric complexes formed by HspB1 with the two types of exogenous polypeptides revealed the dominant effects induced by HspB5 polypeptides towards HspB1. The R120G mutation enhanced the native size and salt resistance of HspB1-HspB5 complex. However, in oxidative conditions the interaction between HspB1 and mutant HspB5 was drastically modified resulting in the aggregation of both partners. The mutation also induced the redistribution of HspB1 phosphorylated at serine 15, originally observed at the level of the small oligomers that do not interact with wild type HspB5, to the large oligomeric complex formed with mutant HspB5. This phosphorylation stabilized the interaction of HspB1 with mutant HspB5. A dominant negative effect towards HspB1 appears therefore as an important event in the cellular sensitivity to oxidative stress mediated by mutated HspB5 expression. These observations provide novel data that describe how a mutated sHsp can alter the protective activity of another member of this family of chaperones.

Purpose: The recent discoveries in the field of human small heat shock proteins (sHSPs) clearly point to the important roles played by these adenosine triphosphate (ATP)-independent chaperones in the regulation of a large spectrum of vital cellular processes and in pathological diseases. These proteins are therefore considered as very attractive therapeutic targets., Aims: To understand the functions of the stress-inducible members of the sHSP family, HspB1, HspB5 and HspB8, and be able to therapeutically modulate their activities, researchers are faced with the complex oligomerisation and phosphorylation properties of these proteins and with their ability to interact with each other and with specific protein targets. Here, we have integrated, in a functionally orientated way, the up-to-date literature data concerning HspB1, HspB5 and HspB8 protein interactions which reflect their numerous crucial cellular functions. We also present data supporting the idea that specific phospho-oligomeric domains of HspB1 are involved in the interaction with particular client proteins., Conclusions: More information concerning the interactions between client protein targets and sHSPs or the multiple combinatorial chimeric oligomeric complexes formed by different sHSPs are urgently required to elaborate a comprehensive sHSPs protein interactome and propose efficient and pathology-specific therapeutic approaches.

Small heat shock proteins (sHsps) regulate a large number of fundamental cellular processes and are involved in many pathological diseases. They share complex oligomerization and phosphorylation properties allowing them to interact and modulate the activity of many client proteins. Here, the up-to date protein interactome of the ten human sHsps is presented as an illustration of their multiple cellular functions. In addition of forming homo-oligomers, some of these proteins interact whith each other and form hetero-oligomeric complexes that could bear new protein targets recognition abilities. Here, novel informations are presented on how the formation of HspB1/HspB5 complex can stimulate the activity of the oxidoresistance promoting enzyme glucose 6-phosphate dehydrogenase through its interaction with newly formed highly phosphorylated HspB1 homo-oligomers., (Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Human HSP27 (HSPB1) is a molecular chaperone sensor which, through dynamic changes in its phosphorylation and oligomerization, allows cells to adapt to changes in their physiology and/or mount a protective response to injuries. In pathological conditions, the high level of HSPB1 expression can either be beneficial, such as in diseases characterized by cellular degenerations, or be malignant in cancer cells where it promotes tumourigenesis, metastasis and anti-cancer drug resistance. Structural changes allow HSPB1 to interact with specific client protein partners in order to modulate their folding/activity and/or half-life. Therefore, the search is open for therapeutic compounds aimed at either down- or upregulating HSPB1 activity. In this respect, we have previously described two peptide aptamers (PA11 and PA50) that specifically interact with HSPB1 small oligomers and decrease its anti-apoptotic and tumourigenic activities. A novel analysis of the different HSPB1-interacting aptamers that were isolated earlier revealed that one aptamer (PA23) has the intriguing ability to stimulate the protective activity of HSPB1. We show here that this aptamer abolishes the dominant negative effect induced by the R120G mutant of αB-crystallin (HSPB5) by disrupting its interaction with HSPB1. Hence, developing structure-based interfering strategies could lead to the discovery of HSPB1-based therapeutic drugs.

Heat shock proteins (HSP) are a subset of the molecular chaperones, best known for their rapid and abundant induction by stress. HSP genes are activated at the transcriptional level by heat shock transcription factor 1 (HSF1). During the progression of many types of cancer, this heat shock transcriptional regulon becomes co-opted by mechanisms that are currently unclear, although evidently triggered in the emerging tumor cell. Concerted activation of HSF1 and the accumulation of HSPs then participate in many of the traits that permit the malignant phenotype. Thus, cancers of many histologies exhibit activated HSF1 and increased HSP levels that may help to deter tumor suppression and evade therapy in the clinic. We review here the extensive work that has been carried out and is still in progress aimed at (1) understanding the oncogenic mechanisms by which HSP genes are switched on, (2) determining the roles of HSF1/HSP in malignant transformation and (3) discovering approaches to therapy based on disrupting the influence of the HSF1-controlled transcriptome in cancer.

Human HspB1 (Hsp27), a molecular chaperone bearing tumorigenic and metastatic roles, is characterized by its dynamic phosphorylation and heterogenous oligomerization in response to changes in cell physiology. The phenomenon is particularly intense and specific when cells are exposed to different death inducers. This favors the hypothesis that the structural organization of HspB1 acts as a sensor which, through reversible modifications, allows cells to adapt and/or mount a protective response. A large number of HspB1 interacting partners have already been described in the literature. Specific changes in oligomerphosphorylation organization may therefore allow HspB1 to interact with the more appropriate polypeptides and to subsequently modulate their folding/activity and/or half-life. This could indirectly link HspB1 to multiple cellular functions and could explain the apparently unrelated effects associated to its over- or underexpression. In cancer, HspB1 is tumorigenic, stimulates metastasis and provide cancer cells with resistance to many anti-cancer drugs, so compounds aimed at disrupting HspB1 deleterious pro-cancer activity are actively looked for. One example, is brivudine that impairs HspB1 ability to recognize pathological protein substrates and appears as a promising anti-cancer drug. Similarly, we have observed that peptide aptamers that specifically interfere with HspB1 structural organization reduced its anti-apoptotic and tumorigenic activities. We propose that, in addition to RNA interference approaches, the tumorigenic activity of HspB1 could be inhibited by altering HspB1 structural organization and consequently its interaction with inappropriate procancerous polypeptide partners. Hence, developping HspB1 structure-based interfering strategies could lead to new anti-cancer drugs discovery.

Background: The small stress heat shock protein 27 (Hsp27) has recently turned as a promising target for cancer treatment. Hsp27 upregulation is associated with tumour growth and resistance to chemo- and radio-therapeutic treatments, and several ongoing drugs inhibiting Hsp27 expression are under clinical trial. Hsp27 is now well described to counteract apoptosis and its elevated expression is associated with increased aggressiveness of several primary tumours. However, its role in the later stage of tumour progression and, more specifically, in the later and most deadly stage of tumour metastasis is still unclear., Methods/results: In the present study, we showed by qRT-PCR that Hsp27 gene is overexpressed in a large fraction of the metastatic breast cancer area in 53 patients. We further analysed the role of this protein in mice during bone metastasis invasion and establishment by using Hsp27 genetically depleted MDA-MB231/B02 human breast cancer cell line as a model. We demonstrate that Hsp27 silencing led to reduced cell migration and invasion in vitro and that in vivo it correlated with a decreased ability of breast cancer cells to metastasise and grow in the skeleton., Conclusion: Altogether, these data characterised Hsp27 as a potent therapeutic target in breast cancer bone metastasis and skeletal tumour growth.

We previously found that the NF-κB transcription factor is activated during the recovery period after heat shock; moreover, we demonstrated that NF-κB is essential for cell survival after heat shock by activating autophagy, a mechanism that probably helps the cell to cope with hyperthermic stress through clearance of damaged proteins. In this study, we analyze the involvement of NF-κB in basal and heat-stress-induced protein quality control, by comparing the level of multiubiquitylated and/or aggregated proteins, and proteasome and autophagic activity in NF-κB-competent and NF-κB-incompetent cells. We show that NF-κB has only a minor role in basal protein quality control, where it modulates autophagosome maturation. By contrast, NF-κB is shown to be a key player in protein quality control after hyperthermia. Indeed, NF-κB-incompetent cells show highly increased levels of multiubiquitylated and/or aggregated proteins and aggresome clearance defects; a phenotype that disappears when NF-κB activity is restored to normal. We demonstrate that during heat shock recovery NF-κB activates selective removal of misfolded or aggregated proteins--a process also called 'aggrephagy'--by controlling the expression of BAG3 and HSPB8 and by modulating the level of the BAG3-HspB8 complex. Thus NF-κB-mediated increase in the level of the BAG3-HspB8 complex leads to upregulation of aggrephagy and clearance of irreversibly damaged proteins and might increase cell survival in conditions of hyperthermia.

Hsp27 belongs to the heat shock protein family and displays chaperone properties in stress conditions by holding unfolded polypeptides, hence avoiding their inclination to aggregate. Hsp27 is often referenced as an anti-cancer therapeutic target, but apart from its well-described ability to interfere with different stresses and apoptotic processes, its role in non-stressed conditions is still not well defined. In the present study we report that three polypeptides (histone deacetylase HDAC6, transcription factor STAT2 and procaspase-3) were degraded in human cancerous cells displaying genetically decreased levels of Hsp27. In addition, these proteins interacted with Hsp27 complexes of different native size. Altogether, these findings suggest that HDAC6, STAT2 and procaspase-3 are client proteins of Hsp27. Hence, in non stressed cancerous cells, the structural organization of Hsp27 appears to be a key parameter in the regulation by this chaperone of the level of specific polypeptides through client-chaperone type of interactions.

Small heat shock proteins (small Hsps) are stress-induced molecular chaperones that act as holdases towards polypeptides that have lost their folding in stress conditions or consequently of mutations in their coding sequence. A cellular protection against the deleterious effects mediated by damaged proteins is thus provided to cells. These chaperones are also highly expressed in response to protein conformational and inflammatory diseases and cancer pathologies. Through specific and reversible modifications in their phospho-oligomeric organization, small Hsps can chaperone appropriate client proteins in order to provide cells with resistance to different types of injuries or pathological conditions. By helping cells to better cope with their pathological status, their expression can be either beneficial, such as in diseases characterized by pathological cell degeneration, or deleterious when they are required for tumor cell survival. Moreover, small Hsps are actively released by cells and can act as immunogenic molecules that have dual effects depending on the pathology. The cellular consequences linked to their expression levels and relationships with other Hsps as well as therapeutic strategies are discussed in view of their dynamic structural organization required to interact with specific client polypeptides.

Human heat shock protein 27 (Hsp27, HspB1) is an anti-apoptotic protein characterized for its tumorigenic and metastatic properties, and now referenced as a major therapeutic target in many types of cancer. Hsp27 biochemical properties rely on a structural oligomeric and dynamic organization. Downregulation by small interfering RNA or inhibition with dominant-negative mutant have proven their efficiency to counteract the anti-apoptotic and protective properties of Hsp27. In this study, we report the isolation and characterization of Hsp27-targeted molecules interfering with its structural organization. Using the peptide aptamer (PA) strategy, we isolated PAs that specifically interact with Hsp27 and not with the other members of the small heat shock protein family. In mammalian cell cultures, PAs expression perturbed the dimerization and oligomerization of Hsp27, and acted as negative regulators of the anti-apoptotic and cytoprotective activities of this protein. Further studies analyzing SQ20B cell xenografts in immunocompromised mice showed that PAs strongly reduced tumor development through cell cycle arrest. Our data suggest that PAs could provide a potential tool to develop strategies for the discovery of Hsp27 chemical inhibitors.

Human HspB1 (also denoted Hsp27) is a well-known member, together with alphaB-crystallin, of the small heat-shock (or stress) proteins (sHsps) (20-40 kDa). In this chapter, I describe procedures for testing the oligomeric and phosphorylation patterns of HspB1 as well as its interaction with specific partner/client polypeptides using tissue culture cells genetically modified to express different levels of this protein. The procedures have been developed in my laboratory and could be used in any well-established cellular laboratory. In addition, the different procedures presented here could be extended to test the nine other human sHsp members as well as sHsps from other species.

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 HspB1 (also denoted Hsp27) is an oligomeric anti-apoptotic protein that has tumorigenic and metastatic roles. To approach the structural organizations of HspB1 that are active in response to apoptosis inducers acting through different pathways, we have analyzed the relative protective efficiency induced by this protein as well its localization, oligomerization and phosphorylation. HeLa cells, that constitutively express high levels of HspB1 were treated with either etoposide, Fas agonist antibody, staurosporine or cytochalasin D. Variability in HspB1 efficiency to interfere with the different apoptotic transduction pathways induced by these agents were detected. Moreover, inducer-specific dynamic changes in HspB1 localization, native size and phosphorylation were observed, that differed from those observed after heat shock. Etoposide and Fas treatments gradually shifted HspB1 towards large but differently phosphorylated oligomeric structures. In contrast, staurosporine and cytochalasin D induced the rapid but transient formation of small oligomers before large structures were formed. These events correlated with inducer-specific phosphorylations of HspB1. Of interest, the formation of small oligomers in response to staurosporine and cytochalasin D was time correlated with the rapid disruption of F-actin. The subsequent, or gradual in the case of etoposide and Fas, formation of large oligomeric structures was a later event concomitant with the early phase of caspase activation. These observations support the hypothesis that HspB1 has the ability, through specific changes in its structural organization, to adapt and interfere at several levels with challenges triggered by different signal transduction pathways upstream of the execution phase of apoptosis., (Copyright 2010 Elsevier Inc. All rights reserved.)

The heat shock response is a widely described defense mechanism during which the preferential expression of heat shock proteins (Hsps) helps the cell to recover from thermal damages such as protein denaturation/aggregation. We have previously reported that NFkappaB transcription factor is activated during the recovery period after heat shock. In this study, we analyze the consequences of NFkappaB activation during heat shock recovery, by comparing the heat shock response of NFkappaB competent and incompetent (p65/RelA-depleted) cells. We demonstrate for the first time that NFkappaB plays a major and crucial role during the heat shock response by activating autophagy, which increases survival of heat-treated cells. Indeed, we observed that autophagy is not activated during heat shock recovery and cell death is strongly increased in NFkappaB incompetent cells. Moreover, if autophagy is artificially induced in these cells, the cytotoxicity of heat shock is turned back to normal. We show that despite a post-heat shock increase of Beclin 1 level in NFkappaB competent cells, neither Beclin 1/class III PI3K complex, Bcl(2)/Bcl-X(L) nor mTOR kinase are NFkappaB targets whose modulation of expression could be responsible for NFkappaB activation of autophagy during heat shock recovery. In contrast, we demonstrate that aberrantly folded/aggregated proteins are prime events in the signaling pathway leading to NFkappaB mediated autophagy after heat shock. Hence, our findings demonstrate that NFkappaB-induced autophagy during heat shock recovery is an additional cell response to HS-induced protein denaturation/aggregation; this mechanism increases cell survival, probably through clearance of irreversibly damaged proteins.

In leukemia cells, hyperthermia enhances tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. The phenomenon is caspase-dependent and results in membrane changes leading to an increased recognition of TRAIL death receptors by TRAIL. Because either caspase-2 or an apical proteolytic event has been recently proposed to act as an initiator of the cell death mechanism induced by heat shock, we have investigated the hierarchy of caspase activation in cells exposed to the combined heat shock plus TRAIL treatment. We report here that caspases-2, -3, and -8 were the first caspases to be activated. As expected, caspase-8 is required and indispensable during the initiation of this death signaling. Caspase-2 may also participate in the phenomenon but, in contrast to caspase-8, its presence appears dispensable because its depletion by small interfering RNA is devoid of effects. Our observations also suggest a role of caspase-3 and of a particular cleaved form of this caspase during the early signals of heat shock plus TRAIL-induced apoptosis.

US11 protein, one of herpes simplex virus type 1 (HSV-1) true late gene products, plays a role in the virally induced post-transcriptional control of gene expression. In addition, US11 expression also interferes with the cellular response to HSV-1 infection that can lead to apoptosis. We have previously shown that US11 expression enhanced the recovery of cellular protein synthesis and increased cell survival in response to thermal stress. Since heat shock can activate apoptosis, we tested for a possible anti-apoptotic behavior of US11. Here, we show that, in HeLa cells, US11 expression strongly reduced heat induced apoptosis, a phenomenon independent of Hsp expression and characterized by a delayed cytochrome c efflux from mitochondria and reduced caspase 3 activation. Moreover, US11 expression also protected against staurosporine induced apoptosis. Hence, our results favor an anti-apoptotic activity of US11 polypeptide that appears to be located at the level of mitochondria or upstream signaling pathways.

We recently reported that a mild heat shock induces a long lasting stimulation of TRAIL-induced apoptosis of leukemic T-lymphocytes and myeloid cell lines, but not normal T-lymphocytes, which correlates with an enhanced ability of TRAIL to recognize its receptors. As shown here, this phenomenon could be inhibited by the xanthogenate agent D609, a sphingomyelin/ceramide pathway inhibitor. A caspase-dependent and D609-sensitive two-fold increase in ceramide level was elicited by heat shock plus TRAIL combined treatment. One day after heat shock, a similar increase in ceramide was induced by TRAIL. Sphingolipids/ceramides are known to regulate membrane integrity, and heat shock increases membrane fluidity. In this regard, the heat shock plus TRAIL combined treatment resulted in a D609-sensitive membrane fluidization which was far more intense than that induced by heat shock only. We also report that membrane fluidizers, that mimic the effect of heat shock, such benzyl alcohol and ethanol, potently stimulated TRAIL-induced apoptosis. As heat shock, these alcohols increased, in a D609-sensitive manner, membrane fluidity in the presence of TRAIL, the recognition of TRAIL death receptors, and ceramide levels. These results suggest that stress agents that trigger ceramide production and an overall increase in membrane fluidity are stimulators of TRAIL apoptosis.

Hsp27 and alphaB-crystallin are molecular chaperones that are constitutively expressed in several mammalian cells, particularly in pathological conditions. These proteins share functions as diverse as protection against toxicity mediated by aberrantly folded proteins or oxidative-inflammation conditions. In addition, these proteins share anti-apoptotic properties and are tumorigenic when expressed in cancer cells. This review summarizes the current knowledge about Hsp27 and alphaB-crystallin and the implications, either positive or deleterious, of these proteins in pathologies such as neurodegenerative diseases, myopathies, asthma, cataracts and cancers. Approaches towards therapeutic strategies aimed at modulating the expression and/or the activities of Hsp27 and alphaB-crystallin are presented.

We recently reported that, in cultured leukemic T lymphocytes and promyelocytic cells, a mild heat shock treatment (1 h at 42 degrees C) induced a long lasting stimulation of the apoptosis induced by TNF-related apoptosis inducing ligand (TRAIL). On the opposite, no effects were recorded toward normal human T lymphocytes. The apoptogenic efficiency of TRAIL in leukemic lymphocytes is linked to the long lasting increased ability of TRAIL to recognize and bind DR4 and DR5 receptors during hyperthermia. Here, we have analyzed whether this new apoptotic co-treatment could be relevant toward primary cells from patients suffering of chronic lymphocytic leukemia. Analysis of samples from 24 patients with different ages, sex and disease stages revealed that half of them had lymphocytes that, once isolated and analyzed in vitro, positively responded (increase of cell death) to the heat shock plus TRAIL co-treatment. Analysis of the level of expression of various anti-apoptotic proteins in the cell samples revealed a great heterogeneity between patients and no clear relationships could be drawn. Nevertheless, most cell samples that were sensitive to TRAIL plus heat shock induced apoptosis displayed a higher level of cell surface DR4 and DR5 receptors than the non-sensitive counterparts. Hence, analysis of the level of TRAIL surface receptors is a prerequisite for future clinical applications based on this protocol.

Background: Improved chemical hazard management such as REACH policy objective as well as drug ADMETOX prediction, while limiting the extent of animal testing, requires the development of increasingly high throughput as well as highly pertinent in vitro toxicity assays., Methodology: This report describes a new in vitro method for toxicity testing, combining cell-based assays in nanodrop Cell-on-Chip format with the use of a genetically engineered stress sensitive hepatic cell line. We tested the behavior of a stress inducible fluorescent HepG2 model in which Heat Shock Protein promoters controlled Enhanced-Green Fluorescent Protein expression upon exposure to Cadmium Chloride (CdCl2), Sodium Arsenate (NaAsO2) and Paraquat. In agreement with previous studies based on a micro-well format, we could observe a chemical-specific response, identified through differences in dynamics and amplitude. We especially determined IC50 values for CdCl2 and NaAsO2, in agreement with published data. Individual cell identification via image-based screening allowed us to perform multiparametric analyses., Conclusions: Using pre/sub lethal cell stress instead of cell mortality, we highlighted the high significance and the superior sensitivity of both stress promoter activation reporting and cell morphology parameters in measuring the cell response to a toxicant. These results demonstrate the first generation of high-throughput and high-content assays, capable of assessing chemical hazards in vitro within the REACH policy framework.

Cells possess effective mechanisms to cope with chronic or acute disturbance of homeostasis. Key roles in maintaining or restoring homeostasis are played by the various heat shock or stress proteins (Hsps). Among the Hsps, the group of proteins characterized by low molecular masses (between 20 to 30 kDa) and homology to alpha-crystallin are called small stress proteins (denoted sHsps). The present chapter summarizes the actual knowledge of the protective mechanisms generated by the expression of mammalian Hsp27 (also denoted HspB1 in human) against the cytotoxicity induced by heat shock and oxidative stress. It also describes the anti-apoptotic properties of Hsp27 and their putative consequences in different pathological conditions.

Recently, we reported that the transient expression of huntingtin exon1 polypeptide containing polyglutamine tracts of various sizes (httEx1-polyQ) in cell models of Huntington disease generated an oxidative stress whose intensity was CAG repeat expansion-dependent. Here, we have analyzed the intracellular localization of the oxidative events generated by the httEx1-polyQ polypeptides. Analysis of live COS-7 cells as well as neuronal SK-N-SH and PC12 cells incubated with hydroethidine or dichlorofluorescein diacetate revealed oxidation of these probes at the level of the inclusion bodies formed by httEx1-polyQ polypeptides. The intensity and frequency of the oxidative events among the inclusions were CAG repeat expansion-dependent. Electron microscopic analysis of cell sections revealed the presence of oxidation-dependent morphologic alterations in the vicinity of httEx1-polyQ inclusion bodies. Moreover, a high level of oxidized proteins was recovered in partially purified inclusions. We also report that the iron chelator deferroxamine altered the structure, localization and oxidative potential of httEx1-polyQ inclusion bodies. Hence, despite the fact that the formation of inclusion bodies may represent a defense reaction of the cell to eliminate httEx1 mutant polypeptide, this phenomenon appears inherent to the generation of iron-dependent oxidative events that can be deleterious to the cell.

Detection of toxic substances interfering with endocrine system is one of the major preoccupations of the European community. A whole-cell bioassay for pollution detection based on stress induction has been designed. Well characterized toxicants, cadmium chloride and thiram (a dithiocarbamate fungicide), were used to optimize the detection conditions such as time-course conditions, cell line and reporter gene to be used. HeLa cells containing the firefly luciferase (luc) reporter gene under the control of the Drosophila melanogaster hsp22 promoter were compared to liver cells (HepG2) containing the same stress gene promoter fused either to the luc or the EGFP (Enhanced-Green Fluorescent Protein) gene. The sensitivity of the obtained bioassay was found to be enhanced by the concomitant use of liver cells and EGFP reporter gene. The detection limits of the toxicants were then lowered from 1 to 0.1 microM and from 1 to 0.01 microM for CdCl(2) and thiram, respectively.

We recently reported that the transient expression of polyglutamine tracts of various size in exon 1 of the huntingtin polypeptide (httEx1) generated abnormally high levels of intracellular reactive oxygen species that directly contributed to cell death. Here, we compared the protection generated by heat shock proteins to that provided by the antioxidant agent N-acetyl-L-cysteine. In cells expressing httEx1 with 72 glutamine repeats (httEx1-72Q), the overexpression of Hsp27 or Hsp70 plus Hdj-1(Hsp40) or treatment of the cells with N-acetyl-L-cysteine inhibited not only mitochondrial membrane potential disruption but also the increase in reactive oxygen species, nitric oxide and protein oxidation. However, only heat shock proteins and not N-acetyl-L-cysteine reduced the size of the inclusion bodies formed by httEx1-72Q. In cells expressing httEx1 polypeptide with 103 glutamine repeats (httEx1-103Q), heat shock proteins neither decreased oxidative damage nor reduced the size of the inclusions. In contrast, N-acetyl-L-cysteine still efficiently decreased the oxidative damage induced by httEx1-103Q polypeptide without altering the inclusions. N-Acetyl-L-cysteine was inactive with regard to proteasome inhibition, whereas heat shock proteins partially restored the caspase-like activity of this protease. These observations suggest some relationships between the presence of inclusion bodies and the oxidative damage induced by httEx1-polyQ.

We report that a mild heat shock, that did not impair cell growth, stimulated TNF-related apoptosis inducing ligand (TRAIL)-mediated apoptosis of leukemic T lymphocytes and promyelocytic cells, but not normal human T lymphocytes. The death stimulation was maximal when the heat shock was performed at the beginning of the exposure to TRAIL. However, enhanced apoptosis was still observed when TRAIL was added one day after heat shock. The phenomenon was transcription and translation independent suggesting that newly made heat shock proteins were not involved. TRAIL-induced apoptosis after heat shock was dependent on caspases and FADD and an enhanced FlipL/S processing was noticed. However, since after the heat shock FlipL/S processing was transient, events upstream of caspase 8 and FADD may be responsible of the long lasting enhanced TRAIL apoptosis observed after heat shock. No heat-mediated alteration in the antibody recognition of cell surface DR4 and DR5 TRAIL receptors was observed. However, in the presence of TRAIL, a long lasting attenuation in the antibody detection of DR4 and DR5 was observed in heat shock-treated cells that correlated with the enhanced apoptogenic efficiency of TRAIL.

A whole-cell bioassay has been developed for the total toxicity testing of liquid samples. The method is based on the induction of the bioluminescent activity of genetically manipulated mammalian cells. For that purpose, transfection was used to introduce, in HeLa cells, a DNA sensing element that responds to chemical stress agents (heavy metals, genotoxic agents, and endocrine-disrupting chemicals). Such element was designed to direct the expression of a reporting gene (firefly luciferase) through the activation of Drosophila melanogaster hsp22 promoter. A molecular approach was conducted to optimize hsp22 promoter element in order to decrease the background expression level of the reporting gene and to increase the sensitivity of the bioassay for testing endocrine disruptors. As a result, in the presence of 20-100 microM cadmium chloride, a 6-fold increase in luciferase expression was obtained using a specially designed truncated hsp22 promoter construction. The following chemicals known to be found in the polluted samples were tested: CdCl2, Cd(NO3)2, NaAsO2, alachlore, fentine acetate, thiram, and maneb. The stressing effect of each of them was sensitively detected by the present bioassay in the 0.05-50 microM concentration range.

Heat shock proteins (Hsps) are highly conserved and under physiological conditions act as molecular chaperones and/or have anti-apoptotic activities. Expression in the brain of two heat shock proteins, the70 kDa Hsp (Hsp70) and the 27 kDa Hsp (Hsp27), is notable because both proteins are highly inducible in glial cells and neurons following a wide range of noxious stimuli including ischemia, epileptic seizure and hyperthermia. In the central nervous system, constitutive expression of Hsp27 is limited to many (but not all) sensory and motor neurons of the brain stem and spinal cord, while there is little or no constitutive expression of Hsp70. However, inducible expression of both Hsp70 and Hsp27 is present in many areas of the brain and retina and is associated with cellular resistance to a variety of insults. The potential for manipulating the expression levels of Hsps for therapeutic advantage in neurodegenerative diseases such as Alzheimer's disease, stroke and glaucoma will be explored.

Exposure to different conditions or agents that destabilize cell homeostasis often alters protein folding. Depending on stress intensity irreversible protein aggregation and cell death can occur. Cells have developed a conserved defense mechanism aimed at reducing the deleterious effects induced by protein folding alteration. This mechanism is characterized by the expression of a small number of genes encoding specific proteins, named Hsps. Several of these proteins act as molecular chaperones through their ability to refold polypeptides with an altered conformation. Moreover, constitutive Hsps homologues have been characterized that participate in the folding of newly made polypeptides, in the assembly of protein complexes in the endoplasmic reticulum, in the translocation of polypeptides through membranes or in masking mutations that alter protein folding. Neurodegeneratives and cancereous diseases are discussed as examples where high levels of Hsp expression can be either beneficial or deleterious to the cells.

Small stress proteins [small heat shock proteins (sHsps)] are molecular chaperones that modulate the ability of cells to respond to oxidative stress. The current knowledge concerning the protective mechanism generated by the expression of mammalian heat shock protein-27 (Hsp27) that allows cells to increase their resistance to oxidative stress is presented. We describe the effects mediated by Hsp27 expression toward crucial enzymes such as glucose-6-phosphate dehydrogenase and glutathione reductase that uphold glutathione in its reduced form. New data are presented showing that the expression of sHsps correlates with a drastic decrease in the intracellular level of iron, a catalyzer of hydroxyl radical (OH( . )) generation. A decreased ability of sHsps expressing cells to concentrate iron will therefore end up in a decreased level of oxidized proteins. In addition, we propose a role of Hsp27 in the presentation of oxidized proteins to the proteasome degradation machinery. We also present an analysis of several Hsp27 mutants that suggests that the C-terminal part of this stress protein is essential for its protective activity against oxidative stress.

Murine small stress protein [heat shock protein 25 (Hsp25)] expression confers thermotolerance and protection against oxidative stress. Hsp25 is an oligomeric ATP-independent phospho-chaperone that can generate a glutathione-dependent pro-reducing state in cells that are normally devoid of small stress protein constitutive expression. Hsp25 contains only one cysteine residue (position 141) that is highly susceptible to oxidation. We have explored the significance of this reactive residue by generating a mutant in which cysteine-141 was substituted by an alanine residue (C141A mutant). We report here that the C141A mutant did not form dimers when expressed in either murine L929 or human HeLa cells, hence, demonstrating that cysteine-141 regulates Hsp25 dimer formation. The C141A mutant also interfered with the dimerization of human Hsp27, a constitutively expressed small stress protein in HeLa cells. The mutated polypeptide showed a decreased ability to multimerize, but its expression was still able to induce cellular protection against oxidative stress. The C141A mutant was, however, less efficient than the wild-type protein in counteracting staurosporine-induced apoptosis, and it showed no in vivo chaperone activity. Hence, the cellular protection mediated against different stressors may require specific structural organizations of Hsp25 that are differently altered by the mutation. Of interest, when expressed concomitantly with wild-type Hsp25, the C141A polypeptide induced a dominant-negative effect, a phenomenon that may result from the ability of small stress proteins to interact and multimerize with each other.

Many differentiation programs are accompanied by an increase in small heat shock proteins (sHsps) level. Most of the time transient, this accumulation takes place during the early phase of the process and is correlated with the growth arrest that precedes the differentiation. Important biochemical modifications of sHsps occur, such as changes in phosphorylation and oligomerization. The fact that these proteins are induced independently of the signal that triggers differentiation, of the differentiation type, and of the cell type strongly suggests their involvement in fundamental mechanisms of cellular differentiation. Moreover, impairment of sHsps accumulation leads to abortion of the differentiation program and, subsequently, to a massive commitment to cell death. Recent advances in this field of research are presented as well as the hypothesis that should be tested to unravel the mode of action of these proteins during cellular differentiation., (2004 Wiley-Liss, Inc.)

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.

Heat shock (HS) proteins (Hsps) function in tissue protection through their chaperone activity and by interacting with cell signaling pathways to suppress apoptosis. Here, we investigated the effect of HS treatment on the nuclear factor (NF)-kappaB signaling pathway in the angiotensin II (ANG II) model of inflammation. Male Sprague-Dawley rats were divided into sham and HS-, ANG II-, and HS + ANG II-treated groups. HS treatment was administered 24 h before the initiation of ANG II infusion. HS treatment (42 degrees C for 15 min) decreased 7-day ANG II-induced hypertension from 191 +/- 4 to 147 +/- 3 mmHg (P < 0.01). Histological staining of hearts showed that HS treatment reduced ANG II-induced leukocyte infiltration, perivascular and interstitial inflammation, and fibrosis. Heart NF-kappaB nuclear translocation and activity, examined by Western blot analysis and electrophoretic mobility shift assay, was suppressed by HS treatment. HS treatment depleted IkappaB kinase-alpha (IKK-alpha) and phosphorylated IKK-alpha and suppressed the depletion of IkappaB-alpha and the accumulation of phosphorylated IkappaB-alpha. HS treatment blocked ANG II induced expression of IL-6 and ICAM-1 in the heart. ANG II and HS treatment induced high-level expression of Hsp27 and Hsp70 and their phosphorylation. Phosphorylated isoforms of Hsp27 and Hsp70 may play an important role in protecting the heart against ANG II-induced inflammation.

We report that in Jurkat T cells or freshly isolated T lymphocytes, physiological concentrations of high-molecular weight sulfated polysaccharides such as heparin, heparan sulfate, and dextran sulfate significantly increased the percentage of cell death induced by Fas IgM agonistic antibody. The phenomenon was caspase dependent and P53 independent and correlated with an increased accessibility of cell surface Fas receptors. We also observed that the Fas IgM agonistic antibody-dependent formation of sodium dodecyl sulfate (SDS)-resistant large structures containing Fas receptor was decreased in the presence of heparin-like agents. In contrast, the different agents had no effect when cell death was triggered by FasL, the natural ligand of Fas that does not generate SDS-resistant forms of Fas. Interestingly, the synergistic effect of heparin-like agents toward Fas IgM agonistic antibody-mediated cell death abolished Hsp27 antiapoptotic activity but did not alter much the protection generated by Bcl-2 expression.

ATP- and ubiquitin-independent proteolysis by the 20S proteasome is responsible for the selective degradation of oxidized proteins. In vitro, the 20S proteasome shows an increased proteolytic activity toward oxidized polypeptides and the suc-LLVY-MCA peptide specific for its chymotrypsin-like activity. We have analyzed the effect of the intracellular redox status on the chymotrypsin-like activity of the 20S proteasome in human T47D cells overexpressing the detoxifiant enzyme seleno-glutathione peroxidase-1 (GPx-1). We report a 30% decreased activity of the chymotrypsin-like activity in cells overexpressing GPx-1. This phenomenon correlated with a 2-fold increase in IkappaB alpha half-life, a protein whose basal turnover is 20S proteasome-dependent. Following exposure to H2O2, these cells showed a seleno-dependently decreased accumulation of intracellular reactive oxygen species and 20S proteasome chymotrypsin-like activity. Similar results were obtained in HeLa cells transiently overexpressing human GPx-1. Moreover, exposure of HeLa cells to antioxidant compounds reduced the proteasome 20S chymotrypsin-like activity. In contrast, no effects were observed when HeLa cell extracts used to determine proteasome activity were incubated with either reduced or oxidized glutathione. These results suggest that GPx-1 activity or pro-reducing conditions can downregulate basal 20S proteasome activity. Hence, the intracellular redox status, probably through the level of oxidized proteins, is an important element that can either activate or down-regulate the 20S proteasome chymotrypsin-like activity in living cells.