Sulphoxythiocarbamates modify cysteine residues in HSP90 causing degradation of client proteins and inhibition of cancer cell proliferation.

BACKGROUND: Heat shock protein 90 (HSP90) has a key role in the maintenance of the cellular proteostasis. However, HSP90 is also involved in stabilisation of oncogenic client proteins and facilitates oncogene addiction and cancer cell survival. The development of HSP90 inhibitors for cancer treatment is an area of growing interest as such agents can affect multiple pathways that are linked to all hallmarks of cancer. This study aimed to test the hypothesis that targeting cysteine residues of HSP90 will lead to degradation of client proteins and inhibition of cancer cell proliferation. METHODS: Combining chemical synthesis, biological evaluation, and structure-activity relationship analysis, we identified a new class of HSP90 inhibitors. Click chemistry and protease-mass spectrometry established the sites of modification of the chaperone. RESULTS: The mildly electrophilic sulphoxythiocarbamate alkyne (STCA) selectively targets cysteine residues of HSP90, forming stable thiocarbamate adducts. Without interfering with the ATP-binding ability of the chaperone, STCA destabilises the client proteins RAF1, HER2, CDK1, CHK1, and mutant p53, and decreases proliferation of breast cancer cells. Addition of a phenyl or a tert-butyl group in tandem with the benzyl substituent at nitrogen increased the potency. A new compound, S-4, was identified as the most robust HSP90 inhibitor within a series of 19 derivatives. CONCLUSION: By virtue of their cysteine reactivity, sulphoxythiocarbamates target HSP90, causing destabilisation of its client oncoproteins and inhibiting cell proliferation.

Human heat shock protein 70 (hsp70) protects murine cells from injury during metabolic stress.

Expression of heat shock protein 70 (hsp70) is stimulated during ischemia, but its proposed cytoprotective function during metabolic stress has remained conjectural. We introduced a human hsp70 gene into mouse 10T1/2 cells and assessed the susceptibility of these cells to injury in response to conditions that mimic ischemia. Transiently transfected cells, in the absence of stress, expressed human hsp70 to levels equal to or greater than those induced by heat shock, as assessed by RNAse protection, immunoblot, and immunohistochemical analyses. By comparison to cells transfected with a control plasmid, cells expressing the human hsp70 transgene were resistant to injury induced by glucose deprivation and inhibition of mitochondrial respiration. These results provide direct evidence for a cytoprotective function of hsp70 during metabolic stress.

Induction of stress proteins in cultured myogenic cells. Molecular signals for the activation of heat shock transcription factor during ischemia.

Expression of major stress proteins is induced rapidly in ischemic tissues, a response that may protect cells from ischemic injury. We have shown previously that transcriptional induction of heat-shock protein 70 by hypoxia results from activation of DNA binding of a preexisting, but inactive, pool of heat shock factor (HSF). To determine the intracellular signals generated in hypoxic or ischemic cells that trigger HSF activation, we examined the effects of glucose deprivation and the metabolic inhibitor rotenone on DNA-binding activity of HSF in cultured C2 myogenic cells grown under normoxic conditions. Whole-cell extracts were examined in gel mobility shift assays using a 39-bp synthetic oligonucleotide containing a consensus heat-shock element as probe. ATP pools were determined by high-pressure liquid chromatography and intracellular pH (pHi) was measured using a fluorescent indicator. Glucose deprivation alone reduced the cellular ATP pool to 50% of control levels but failed to activate HSF. However, 2 x 10(-4) M rotenone induced DNA binding of HSF within 30 min, in association with a fall in ATP to 30% of control levels, and a fall in pHi from 7.3 to 6.9. Maneuvers (sodium propionate and amiloride) that lowered pHi to 6.7 without ATP depletion failed to activate HSF. Conversely, in studies that lowered ATP stores at normal pH (high K+/nigericin) we found induction of HSF-DNA binding activity. Our data indicate that the effects of ATP depletion alone are sufficient to induce the DNA binding of HSF when oxidative metabolism is impaired, and are consistent with a model proposed recently for transcriptional regulation of stress protein genes during ischemia.

Disruption of heat shock factor 1 reveals an essential role in the ubiquitin proteolytic pathway.

Inhibition of proteasome-mediated protein degradation machinery is a potent stress stimulus that causes accumulation of ubiquitinated proteins and increased expression of heat shock proteins (Hsps). Hsps play pivotal roles in homeostasis and protection in a cell, through their well-recognized properties as molecular chaperones. The inducible Hsp expression is regulated by the heat shock transcription factors (HSFs). Among mammalian HSFs, HSF1 has been shown to be important for regulation of the heat-induced stress gene expression, whereas the function of HSF2 in stress response is unclear. Recent reports have suggested that both HSF1 and HSF2 are affected during down-regulation of ubiquitin-proteasome pathway (Y. Kawazoe et al., Eur. J. Biochem. 255:356-362, 1998; A. Mathew et al., Mol. Cell. Biol. 18:5091-5098, 1998; D. Kim et al., Biochem. Biophys. Res. Commun. 254:264-268, 1999). To date, however, no unambiguous evidence has been presented as to whether a single specific HSF or multiple members of the HSF family are required for transcriptional induction of heat shock genes when proteasome activity is down-regulated. Therefore, by using loss-of-function and gain-of-function strategies, we investigated the specific roles of mammalian HSFs in regulation of the ubiquitin-proteasome-mediated stress response. Here we demonstrate that HSF1, but not HSF2, is essential and sufficient for up-regulation of Hsp70 expression during down-regulation of the ubiquitin proteolytic pathway. We propose that specificity of HSF1 could be an important therapeutic target during disease pathogenesis associated with abnormal ubiquitin-dependent proteasome function.

Heat shock response: lessons from mouse knockouts.

Organisms are endowed with integrated regulatory networks that transduce and amplify incoming signals into effective responses, ultimately imparting cell death and/or survival pathways. As a conserved cytoprotective mechanism from bacteria to humans, the heat shock response has been established as a paradigm for inducible gene expression, stimulating the interests of biologists and clinicians alike to tackle fundamental questions related to the molecular switches, lineage-specific requirements, unique and/or redundant roles, and even efforts to harness the response therapeutically. Gene targeting studies in mice confirm HSF1 as a master regulator required for cell growth, embryonic development, and reproduction. For example, sterility of Hsf1-null female but not null male mice established strict requirements for maternal HSF1 expression in the oocyte. Yet Hsf2 knockouts by three independent laboratories have not fully clarified the role of mammalian HSF2 for normal development, fertility, and postnatal neuronal function. In contrast, Hsf4 knockouts have provided a consistent demonstration for HSF4's critical role during lens formation. In the future, molecular analysis of HSF knockout mice will bring new insights to HSF interactions, foster better understanding of gene regulation at the genome level, lead to a better integration of the HSF pathway in life beyond heat shock, the classical laboratory challenge.

Induction of a heat shock factor 1-dependent stress response alters the cytotoxic activity of hsp90-binding agents.

In addition to its classic role in the cellular stress response, heat shock protein 90 (Hsp90) plays a critical but less well appreciated role in regulating signal transduction pathways that control cell growth and survival under basal, nonstress conditions. Over the past 5 years, the antitumor antibiotics geldanamycin and radicicol have become recognized as selective Hsp90-binding agents (HBA) with a novel ability to alter the activity of many of the receptors, kinases, and transcription factors involved in these cancer-associated pathways. As a consequence of their interaction with Hsp90, however, these agents also induce a marked cellular heat shock response. To study the mechanism of this response and assess its relevance to the anticancer action of the HBA, we verified that the compounds could activate a reporter construct containing consensus binding sites for heat shock factor 1 (HSF1), the major transcriptional regulator of the vertebrate heat shock response. We then used transformed fibroblasts derived from HSF1 knock-out mice to show that unlike conventional chemotherapeutics, HBA increased the synthesis and cellular levels of heat shock proteins in an HSF1-dependent manner. Compared with transformed fibroblasts derived from wild-type mice, HSF1 knock-out cells were significantly more sensitive to the cytotoxic effects of HBA but not to doxorubicin or cisplatin. Consistent with these in vitro data, we found that systemic administration of an HBA led to marked increases in the level of Hsp72 in both normal mouse tissues and human tumor xenografts. We conclude that HBA are useful probes for studying molecular mechanisms regulating the heat shock response both in cells and in whole animals. Moreover, induction of the heat shock response by HBA will be an important consideration in the clinical application of these drugs, both in terms of modulating their cytotoxic activity as well as monitoring their biological activity in individual patients.

beta Adrenergic receptor desensitisation may serve a cardioprotective role.

OBJECTIVE: The aim was to test the hypothesis that beta adrenoceptor desensitisation is a form of cardioprotection whereby the myocardium is protected from the injurious effects of excessive adrenergic stimulation by isoprenaline. METHODS: A new sensitive and specific method of identifying cardiac myocyte necrosis with monoclonal antimyosin was employed. Antibody labelled necrotic cardiomyocytes from male Sprague-Dawley rats (190-300 g) were identified by immunofluorescence. Homologous beta adrenoceptor desensitisation was induced by 9 d pretreatment with isoprenaline infusion (20 mg.kg-1.d-1), and heterologous desensitisation by treatment with 0.15% propylthiouracil in the diet for six weeks. The effects of isoprenaline induced cardiomyocyte injury in these animals were compared with those in control rats. RESULTS: In euthyroid control rats, treatment with the beta adrenergic agonist, isoprenaline, produced prolonged tachycardia and hypotension, and a significant amount of cardiac myocyte necrosis subendocardially. When the same isoprenaline challenge was given to rats pretreated for 9 d with an isoprenaline infusion, the haemodynamic response was markedly attenuated and very little myocyte necrosis was noted. In the model of heterologous desensitisation occurring in hypothyroidism, isoprenaline challenge produced markedly diminished haemodynamic response and little cardiac myocyte necrosis. CONCLUSIONS: In both homologous and heterologous models of beta adrenoceptor desensitisation, the susceptibility of the myocardium to isoprenaline induced cytotoxicity is markedly reduced.

Heat shock factor 1-mediated thermotolerance prevents cell death and results in G2/M cell cycle arrest.

Mammalian cells respond to environmental stress by activating heat shock transcription factors (eg, Hsf1) that regulate increased synthesis of heat shock proteins (Hsps). Hsps prevent the disruption of normal cellular mitosis, meiosis, or differentiation by environmental stressors. To further characterize this stress response, transformed wild-type Hsf1+/+ and mutant Hsf1-/- mouse embryonic fibroblasts (MEFs) were exposed to (1) lethal heat (45 degrees C, 60 minutes), (2) conditioning heat (43 degrees C, 30 minutes), or (3) conditioning followed by lethal heat. Western blot analysis demonstrated that only Hsf1+/+ MEFs expressed inducible Hsp70s and Hsp25 following conditioning or conditioning and lethal heat. Exposure of either Hsf1+/+ or Hsf1-/- MEFs to lethal heat resulted in cell death. However, if conditioning heat was applied 6 hours before lethal heat, more than 85% of Hsf1+/+ MEFs survived, and cells in G2/M transiently increased 3-fold. In contrast, conditioned Hsf1-/- MEFs neither survived lethal heat nor exhibited this G2/M accumulation. Coinfection with adenoviral Hsp70 and Hsp25 constructs did not fully recreate thermotolerance in either Hsf1+/+ or Hsf1-/- MEFs, indicating other Hsf1-mediated gene expression is required for complete thermotolerance. These results demonstrate the necessity of Hsf1-mediated gene expression for thermotolerance and the involvement of cell cycle regulation, particularly the G2/M transition, in this thermotolerant response.

Matrix metalloproteinases: from biology to therapeutic strategies in cardiovascular disease.

In this comprehensive review of matrix remodeling, one central theme that bears re-emphasis is the extensivecross-talk and dynamic interactions that exist between terminally differentiated, postmitotic cells, proliferative cells, and the ECM of the cardiovascular system. The activities of MMPs and TIMPs constitute a well-orchestrated contest to maintain tissue integrity and homeostasis. Overexpression of MMPs tilts the balance in favor of irreversible tissue destruction of joints (eg, as in rheumatic disease), and efforts to curtail such errant pathways are ongoing (123). Thrombolytic therapy and percutaneous transluminal coronary angioplasty represent effective strategies for restoring antegrade flow in occluded vessels, but multiple factors preclude most patients with AMI from receiving either of these treatments. Tissue healing and remodeling is a process in which the biology of MMPs becomes universally applicable. Basic lessons from the biochemistry and enzymology of MMPs, combined with the mechanisms of gene expression, will undoubtedly impact the development of future therapies involving MMPs and their endogenous inhibitors. In addition, formidable challenges, ranging from bioavailability to tissue penetration and toxicity in animal models, face investigators using existing pharmacotherapeutics. For congenital diseases, such as Marfan syndrome, which primarily affects the connective tissue, future therapies may be targeted to the underlying pathobiology involving MMPs. Strategies aimed at correction of the genetic defect may be complemented by those to prevent or ameliorate fundamental imbalances in matrix turnover and deposition. The future challenge for cardiovascular medicine is to appropriately shift the pendulum, not to the exclusion of, but to the recognition of the dynamic interaction that exists between myocyte and nonmyocyte populations, which clearly affect the pathogenesis of many acquired and genetic disorders.

Stress proteins and cardiovascular disease.

Understanding the molecular basis by which cells of the heart and blood vessels adapt to physiological stress conditions is an important goal for cardiovascular investigators. The ubiquitous heat shock response provides a model for cellular adaptations to metabolic stresses that are encountered in cardiac disease. Stress-induced synthesis of a family of highly conserved proteins serves to protect cells from injury. In addition, members of this family have essential roles in protein processing and assembly of macromolecular complexes, and in regulation of gene expression, even in unstressed cells. Research concerning the regulation and function of stress proteins potentially is pertinent to the pathophysiology of myocardial hypertrophy, remodeling, and failure, to age-related changes in the cardiovascular system, as well as to ischemic heart disease.

Stress (heat shock) proteins: molecular chaperones in cardiovascular biology and disease.

How a cell responds to stress is a central problem in cardiovascular biology. Diverse physiological stresses (eg, heat, hemodynamics, mutant proteins, and oxidative injury) produce multiple changes in a cell that ultimately affect protein structures and function. Cells from different phyla initiate a cascade of events that engage essential proteins, the molecular chaperones, in decisions to repair or degrade damaged proteins as a defense strategy to ensure survival. Accumulative evidence indicates that molecular chaperones such as the heat shock family of stress proteins (HSPs) actively participate in an array of cellular processes, including cytoprotection. The versatility of the ubiquitous HSP family is further enhanced by stress-inducible regulatory networks, both at the transcriptional and posttranscriptional levels. In the present review, we discuss the regulation and function of HSP chaperones and their clinical significance in conditions such as cardiac hypertrophy, vascular wall injury, cardiac surgery, ischemic preconditioning, aging, and, conceivably, mutations in genes encoding contractile proteins and ion channels.

Differential expression of B-crystallin and Hsp27 in skeletal muscle during continuous contractile activity. Relationship to myogenic regulatory factors.

AlphaB-crystallin (alphaBC) is a major structural protein (22 kDa) of the ocular lens as well as a bona fide heat shock protein in non-lens tissue. The alphaBC gene is abundantly expressed in tissues with high oxidative capacity, including the heart and type I skeletal muscle fibers, and is regulated by the MyoD family of basic helix-loop-helix transcription factors during myogenesis. To test the hypothesis that alphaBC expression may be directly regulated by the demand for oxidative metabolism, we examined the expression of alphaBC and the ancestral-related Hsp27 in rabbit tibialis anterior muscle subjected to continuous low frequency motor nerve stimulation (3 V, 10 Hz). alphaBC mRNA and protein increased within the 1st day of continuous contractile activity (5- and 2.5-fold, respectively) and achieved maximum levels (>20-and 4-fold, respectively) after 21 d of stimulation. Hsp27 mRNA and protein levels also increased with stimulation, but with a less specific and dramatic induction pattern. In agreement with the Northern analysis, in situ hybridization performed on cross sections from tibialis anterior muscle revealed progressively increasing alphaBC transcript signal, localized in a ringlet pattern, from 1 through 21 days of stimulation. Serial sections subjected to myosin immunohistochemistry revealed that alphaBC expression was confined to slow-twitch type I and a subpopulation of fast twitch type II fibers after 1 day but present in nearly all fibers after 21 days of stimulation. Transcript levels of all four myogenic regulatory factors (MyoD, myogenin, myf-5, and MRF4) also increased with stimulation in a pattern temporally similar with alphaBC, suggesting that expression of alphaBC in response to stimulation may, in part, be regulated through myogenic regulatory factor(s) interaction with the canonical E-box element located within the alphaBC promotor. These data demonstrate that expression of the small heat shock protein, alphaBC, is rapidly induced independent of the ancestrally related Hsp27 in a fiber type specific pattern in skeletal muscle subjected to the oxidative stress imposed by continuous contractile activity.

Activation of the heat shock transcription factor by hypoxia in mammalian cells.

Members of the stress protein family such as HSP70 are induced in ischemic tissues and may contribute to the ability of cells to survive episodes of transient circulatory insufficiency. However, the biochemical events that lead to this induction, and their degree of similarity with pathways triggered by heat stress, have not been defined. In this study, we demonstrate that transient exposure of cultured C2C12 mouse myogenic cells to a hypoxic atmosphere stimulates DNA binding activity of the heat shock transcription factor through mechanisms that are independent of new protein synthesis. Activation of heat shock transcription factor in hypoxic cells is temporally associated with induction of endogenous HSP70 gene transcription and with induction of a heterologous reporter gene controlled by the human HSP70 promoter. Furthermore, induction of the human HSP70 promoter by hypoxia requires an intact heat shock element, indicating that other cis-acting transcriptional control elements contained within this complex promoter are not sufficient to transduce signals generated within hypoxic cells. These findings provide strong evidence that hypoxia and heat shock induce expression of the HSP70 gene by similar, if not identical, mechanisms.

Cardiac hypertrophy in idiopathic dilated congestive cardiomyopathy: a clinicopathologic study.

Although clinical studies indicate that patients with idiopathic dilated congestive cardiomyopathy who develop electrocardiographic or angiographic signs of left ventricular (LV) hypertrophy may survive longer, there is little morphologic evidence for such anatomic favorable of unfavorable prognostic groups. We studied 30 autopsied patients who died of dilated cardiomyopathy; of these, 15 died within 1 year of the first symptom of their disease (short-term survivors) and 15 patients died 1-14 years after initial symptoms (long-term survivors). There were no significant differences in sex, race, clinical presentation or cause of death between the groups, but there were significant morphologic differences. In the short-term survivors, average heart weight was 540 g and LV wall thickness was 1.0 cm, whereas in the long-term survivors, the average heart weight was 759 g and LV wall thickness was 1.3 cm (p less than 0.001). LV cavity dilatation as measured by maximal transverse diameter from the postmortem angiograms did not differ between the two groups. These patients were compared with 10 autopsied patients with normal hearts and no clinical cardiac disease and 10 autopsied patients with volume overload secondary to valvular regurgitation. An LV hypertrophy/dilatation index (thickness/diameter) was 0.17 +/- 0.07 for the short-term survivors, 0.21 +/- 0.07 for the long-term survivors, 0.38 +/- 0.07 for volume overload patients, and 0.48 +/- 0.19 for normal subjects (F = 20.24, p less than 0.001). Thus, in patients with hypertrophy due to volume overload, wall thickening increased with dilatation, returning the ratio of wall thickness to cavity size toward normal. In contrast, among the idiopathic congestive cardiomyopathies, dilatation was disproportionate to hypertrophy and the difference was most marked for short-term survivors.

Temporospatial expression of the small HSP/alpha B-crystallin in cardiac and skeletal muscle during mouse development.

Although the small (22 Kd) heat shock protein/alpha B-crystallin functions as a major structural protein and molecular chaperone in the vertebrate lens, little is known about the protein's role in nonlenticular tissues such as the heart and skeletal muscle. Recent studies have demonstrated that alpha B-crystallin expression is uniquely regulated during myogenesis in vitro. We report here for the first time that the temporal and spatial expression of alpha B-crystallin is similarly regulated in vivo during mouse embryogenesis. Expression of alpha B-crystallin mRNA was detected by in situ hybridization in the primitive heart at 8.5 days postconception (p.c.) and in the myotome of the somites at 10.5 days p.c. This tissue-restricted pattern was corroborated by immunohistochemical studies. alpha B-crystallin mRNA and protein expression were uniform in the developing atria and ventricles without regional differences or gradients. alpha B-crystallin expression was absent in the endocardial cushion, pulmonary trunk, aorta, and endothelium. Examination of muscle precursors revealed expression throughout the dorsoventral aspect of the myotomes and in developing skeletal muscle. Our findings suggest that alpha B-crystallin may serve pivotal roles as a structural protein and a molecular chaperone in myofiber stabilization of metabolically active tissues during early embryogenesis. Thus, early alpha B-crystallin expression in myogenic lineages supports the hypothesis that the putative functions of alpha B-crystallin are coupled to the activation of genetic programs responsible for myogenic differentiation and cardiac morphogenesis.

Targeted disruption of heat shock transcription factor 1 abolishes thermotolerance and protection against heat-inducible apoptosis.

Heat shock transcription factor 1 (HSF1) is a member of the vertebrate HSF family that regulates stress-inducible synthesis of heat shock proteins (HSPs). Although the synthesis of the constitutively expressed and inducible members of the heat shock family of stress proteins correlates with increased cellular protection, their relative contributions in acquired cellular resistance or "thermotolerance" in mammalian cells is presently unknown. We report here that constitutive expression of multiple HSPs in cultured embryonic cells was unaffected by disruption of the murine HSF1 gene. In contrast, thermotolerance was not attainable in hsf1(-/-) cells, and this response was required for protection against heat-induced apoptosis. We conclude that 1) constitutive and inducibly expressed HSPs exhibit distinct physiological functions for cellular maintenance and adaptation, respectively, and 2) other mammalian HSFs or distinct evolutionarily conserved stress response pathways do not compensate for HSF1 in the physiological response to heat shock.

Isoproterenol-induced myocardial fibrosis in relation to myocyte necrosis.

Treatment of rats with the beta-adrenergic agonist isoproterenol results in cardiac hypertrophy, myocyte necrosis, and interstitial cell fibrosis. Our objectives in this study have been to examine whether hypertrophy and fibrosis occur in a compensatory and reparative response to myocyte loss or whether either process may be occurring independently of myocyte loss and thus be a reactive response to adrenergic hormone stimulation. We have examined this question by evaluating each of these responses in rats treated with different doses and forms of isoproterenol administration. Myocyte necrosis was evaluated using in vivo labeling with monoclonal antimyosin for identification of myocytes with permeable sarcolemma, which was indicative of irreversible injury. Myocardial fibrosis was evaluated by morphometric point counting of Gomori-stained tissue sections and by assessment of the stimulation of fibroblast proliferation by determination of increased levels of DNA synthesis. Stimulation of fibroblast DNA synthesis was determined from DNA specific radioactivities and radioautography after pulse labeling with [3H]thymidine. The evidence provided by this study suggests that the degree and timing of myocardial hypertrophy does not follow the course of myocyte loss and, thus, appears to be either a response to altered cardiac loading or a reactive response to beta-adrenergic hormone stimulation rather than a compensation for myocyte loss. Myocardial fibrosis, on the other hand, appears to be more closely related to myocyte necrosis with respect to collagen accumulation in the same areas of the heart, its dose-response relation to the amount of isoproterenol administered, and the timing of increased DNA synthesis, or fibroblast proliferation, after myocyte loss.

[Effects of hsf1 genotype on the constitutive expression of alphaB-crystallin in mice myocardium].

AIM: Try to clarify the effects of HSF1 gene on the constitutively expressed alphaBC. METHODS: To investigate the levels of constitutively expressed alphaB-Crystallin (alphaBC) in hsf1 knockout (hsf1 -/-) and hsf1 wild type (hsf1 +/+) mice myocardium by Western blot and immunohistochemistry. RESULTS: The alphaBC levels in hsf1 -/- and hsf1 +/+ were 68.42% +/- 4.16%, 100% +/- 7.58%, respectively (P < 0.05, cytosolic fraction), and 20.53% +/- 1.01%, 37.55% +/- 1.91%, respectively (P < 0.05, pellet fraction). The alphaBC signals decreased significantly in hsf1 -/- myocardium compared with hsf1 +/+ myocardium stained with fluorescence immunohistochemistry. CONCLUSION: hsf1 is the important, but not the only factor, which mediates the constitutively expressed alphaBC.