Indolyl- and Pyrrolylazine Derivatives Cause the Accumulation of Heat Shock Protein Hsp70 in Sh-Sy5Y Human Neuroblastoma Cells.

The heat shock protein Hsp70 is involved in cell defense from various types of stress, including the proteotoxic stress, which occurs during the development of many neurodegenerative diseases. This work presents data on the detection of small molecules, derivatives of indolyl- and pyrrolylazines, which can activate the synthesis of Hsp70 and cause its accumulation in the cell. The toxicity level of the new Hsp70 synthesis inducers was evaluated, and the safety of these compounds was demonstrated in experiments on SH-SY5Y neuroblastoma cell line. Derivatives of indolyl- and pyrrolylazines presented in this work can be potential therapeutic agents in models of neurodegenerative diseases that should be studied in more detail.

[Biodistribution of the recombinant heat shock protein rhHsp70 in intracranial C6 glioma models in Wistar rats and subcutaneos B16/F10 melanoma in C57BL/6 mice].

For the first time, the biodistribution of recombinant heat shock protein in rhHsp70 rats with grafted intracranial C6 glioma was evaluated. It was assessed using the fluorescent antibody accumulation chaperone rhHsp70 conjugated with fluorochrome Alexa Fluor 555 in tumor cells by intratumoral or intravenous administration. Assessment of the distribution and accumulation of labeled protein was carried out on the model of subcutaneous B16/F10 melanoma in C57BL/6 mice with the use of single-photon emission computer tomography. After 60 minutes after intravenous administration rhHsp70-I123 (20 MBq, 5 mg chaperone) accumulation of the drug mainly in the liver and tumor tissue was showed. The coefficient of the differential accumulation of the labeled protein KDN(tumor/background) was 3.14. It was turned out that comparing the level of fixation of rhHsp70-I123 in the liver and the tumor KDN(tumor/ liver) = 0.76. After 24 hours from the time of injection of rhHsp70-I123 it was observed increase the level of fixation of the labeled protein in the liver and melanoma: KDN(tumor/background) = 3.43; KDN(tumor/liver = 0.78.

Factors Affecting Aggregate Formation in Cell Models of Huntington's Disease and Amyotrophic Lateral Sclerosis.

Most neurodegenerative pathologies stem from the formation of aggregates of mutant proteins, causing dysfunction and ultimately neuronal death. This study was aimed at elucidating the role of the protein factors that promote aggregate formation or prevent the process, respectively, glyceraldehyde-3-dehydrogenase (GAPDH) and tissue transglutaminase (tTG) and Hsp70 molecular chaperone. The siRNA technology was used to show that the inhibition of GAPDH expression leads to a 45-50% reduction in the aggregation of mutant huntingtin, with a repeat of 103 glutamine residues in a model of Huntington's disease (HD). Similarly, the blockage of GAPDH synthesis was found for the first time to reduce the degree of aggregation of mutant superoxide dismutase 1 (G93A) in a model of amyotrophic lateral sclerosis (ALS). The treatment of cells that imitate HD and ALS with a pharmacological GAPDH inhibitor, hydroxynonenal, was also shown to reduce the amount of the aggregating material in both disease models. Tissue transglutaminase is another factor that promotes the aggregation of mutant proteins; the inhibition of its activity with cystamine was found to prevent aggregate formation of mutant huntingtin and SOD1. In order to explore the protective function of Hsp70 in the control of the aggregation of mutant huntingtin, a cell model with inducible expression of the chaperone was used. The amount and size of polyglutamine aggregates were reduced by increasing the intracellular content of Hsp70. Thus, pharmacological regulation of the function of three proteins, GAPDH, tTG, and Hsp70, can affect the pathogenesis of two significant neurodegenerative diseases.

[Chaperone therapy in the rat model of intracranial glioblastoma].

Molecular chaperons can effectively activate innate and adaptive anti-tumor immune response. In the model of intracranial glioma C6 in Wistar rats we assessed immunomodulatory activity of recombinant protein Hsp70 in case of local, intratumoral injection. Single intratumoral infusion of chaperone had led to dramatic delay in tumor volume growth (on MRI of rat brain), which was accompanied by increase in survival rates. Incubation of rat spleenocytes with C6 cells elevated the levels of INF-gamma, that shows an immunologically specific T-cell response. With immunohistochemical assay we observed a marked infiltration of the tumor by T-lymphocytes and NK-cells. Thus, purified Hsp70 can efficiently induce innate and adaptive anti-tumor response and could be used as adjuvant in treatment of malignant brain tumors of central nervous system.

[Content of chaperon Hsp70 in dopaminergic neurons of the black substance increases in proteasome dysfunstion].

In Wistar rats, specific inhibitor UPS lactacystin induced degeneration of 24% of the dopaminergic neurons in the black substance. The work shows that a moderate weakening of the UPS function is characterized by an enhanced activity of the shaperon system. This process seems to restore and maintain population of the dopaminergic neurons.

Kinetics of chaperone activity of proteins Hsp70 and Hdj1 in human leukemia u-937 cells after preconditioning with thermal shock or compound u-133.

Kinetics of the chaperone activity of proteins Hsp70 and Hdj1 were analyzed in human U-937 promonocytes during their response to heat shock or to treatment with the echinochrome triacetyl glucoside derivative U-133. To measure the chaperone activity of both proteins, a special test was developed for their recognition and binding of a denatured protein. Using this test, the chaperone activity could be concurrently estimated in large numbers of cellular or tissue extracts. We also estimated the contents of both chaperones in cells by immunoblotting. The values for contents of Hsp70 and Hdj1 obtained by two independent test systems coincided, and this suggested that the substrate-binding activity could change proportionally to the chaperone content in the protein mixture. Therefore, the test developed by us can be employed for high throughput screening of drugs activating cellular chaperones. The analysis of quantity and activity of two cellular chaperones during the cell response to heat stress or to the drug-like substance U-133 showed that both factors caused the accumulation of chaperones with similar kinetics. We conclude that the efficiency of drug preconditioning could be close to the efficiency of hyperthermia and that the high activity of chaperones could be retained in human cells for no less than 1.5 days.

[Novel compounds increasing chaperone Hsp70 expression and their biological activity].

Hsp70 possesses chaperonic activity, the property associated with the protective function that was demonstrated in experiments on a great number of cell and animal models. Therefore, it seems important to search for the substances able to innocuously elevate the chaperone concentration in an organism cells and tissues. In our work, we screened of more that 60 compounds and found two chemicals, derivatives of shikonin and echinochrome that able to increase the chaperone level in a variety of human cells. It was shown that in human erythroleukemia K562 cells treated with the both substances concomitantly with elevation of Hsp70 level the absolute chaperonic activity was also increased; this can indicate mobilization of the whole cellular chaperonic machinery by above mentioned compounds. Estimating biological activity of the two substances, we demonstrated that treatments of cells by them prior to hard heat stress, hydrogen peroxide or staurosporine reduced cell mortality by 20-50 % depending on a cytotoxic factor. The results show that after simple chemical modifications these compounds might be taken as a basis of pharmaceuticals for therapy of wide range of disorders.

[Dual role of chaperones in the response of a cell and of a whole organism to stress].

Chaperones constitute a class of proteins able to recognize newly synthesized and(or) damaged polypeptides and to transport these to the sites of their allocation or promote the degradation of irreversibly spoiled ones. The members of Hsp70 family can be classified as the first-discovered chaperones, and to date the chaperonic mechanism based on the proteins is well understood. Using this mechanism Hsp70 executes the dual role: it corrects the structure of nascent and damaged polypeptides or promotes degradation of incorrigible polypeptides. Chaperonic activity appears to form key functions of Hsp70, protective and adjuvant ones. The former is proved in a large amount of experiments both in vitro and in vivo. From the beginning of the 2000th, it has become clear that Hsp70 can be released by cells treated by a number of stressful factors, and exogenous chaperone can influence the cells of innate immunity system proving that the protein uses its protective power already at a level of the whole organism. One of the goals of this review is to describe the functions performed by Hsp70 inside and outside a cell. In a view of its properties Hsp70 can be of significant interest for creation of novel therapeutic technologies. This direction to the practical application of the chaperone is also considered in the review.

[The role of extracellular chaperone Hsp70 in creating antitumor immunity in rat rhabdomyosarcoma RA-2 model].

Immunization of experimental animals with extract or membranes of rat rhabdomyosarcoma RA-2 in combination with pure Hsp70 did not offer any significant effect of protection from subsequent RA-2 cells-stimulated tumor growth. By contrast, immunization with preparations of pure Hsp70 led to a significant decrease in number and size of tumors as well as elevation of concentrations of antibodies against RA-2 cells. Also, enhanced blood levels of Hsp70 involved delayed tumor growth. In vitro tests Hsp70 incubation with RA-2 cells was followed by a 30-35% rise in cytotoxic lymphocytes levels. An ability of pure Hsp70 preparations to stimulate humoral and antitumor response was demonstrated. Hence, they may be used in developing vaccine formulas.

[Molecular chaperone Hsp70 protects neuroblastoma SK-N-SH cells from hypoxic stress].

HIF-1alpha is synthesized constutively, however under normoxia it is specifically degraded. Hypoxia blocks the factor degradation, and it activates the transcription of genes whose products control multiple cellular processes. Hsp70 molecular chaperone is known to protect neural cells from the deleterious effects of hypoxic stress, though the mechanism of this action remains elusive. To understand how Hsp70 protects cells affected by hard hypoxia the model cell line was constructed based on human neuroblastoma SK-N-SH cells and over-expressing the chaperone when treated by zinc salt. The cells were shown to be resistant to the treatment by CoCl2 imitating in the experiments the reaction to hypoxia. Life span of HIF-1alpha was elevated in these cells as compared with parental line due to the fact that Hsp70 formed long-time complex with HIF-1alpha. The data show that Hsp70 interferes with signaling pathways of cellular response to hypoxic stress at the level of regulation of HIF-1alpha stability.

Production of heat shock proteins, cytokines, and nitric oxide in toxic stress.

Expression of heat shock proteins Hsp27, Hsp90, and Hsp70 and production of tumor necrosis factors (TNF-alpha, TNF-beta), interferon-gamma (IFN-gamma), interleukin-2, -3, -6, and nitric oxide (NO) were studied under conditions of acute and chronic intoxication of animals with lipopolysaccharides. Injection of endotoxin increased expression of heat shock proteins Hsp70 and Hsp90-alpha in mouse cells. Acute toxic stress also provoked a sharp increase in the production of TNF-alpha, TNF-beta, and NO in mouse cells. The production of other cytokines (interleukins and IFN-gamma) was changed insignificantly. In the model of chronic toxic stress, changes in the production of Hsp70, Hsp90, TNF, and NO were followed during 11 days after the beginning of the toxin injections. The expression of Hsp70 and Hsp90 in acute stress was significantly higher than at the final stage of the chronic exposure. The changes in the TNF and NO productions, on one hand, and the production of heat shock proteins, on the other hand, were synchronous. The findings indicate that repeated injections of increasing endotoxin doses result in a decreased ability of the body cells to respond to stress by overproduction of heat shock proteins, TNF, and NO.

[Hsp70 chaperone and the prospects of its application in anticancer therapy].

Major stress protein Hsp70 is known to possess two important properties: ATP-dependent activity and protective activity; these two are thought to play a significant role in anticancer therapy. Many malignant tumors contain high amounts of intracellular Hsp70. Moreover, many anticancer drugs themselves are able to elevate Hsp70 expression in tumor cells. Since Hsp70 was found to disturb many signal pathways of apoptosis in many points, the high chaperone expression may lead to an increased resistance of tumor cells to anticancer drugs. On the other hand, when overexpressed by a certain mechanism, Hsp70 is able to emerge at the cell surface by itself or together with tumor antigens and present these to immune cells T-lymphocytes and natural killers, in such a manner that makes cancer cell recognized and abolished. These properties make Hsp70 very promising instrument in designing some novel anticancer vaccines.

[The balance between Hsp70 and its cochaperones Hdj1 and Bag1 determines its substrate-binding activity].

Heat shock protein Hsp70 presents one of the most effective cell protective systems. Its protective activity is mostly due to the fact that Hsp70 is able to restore native conformation of newly synthesized or damaged proteins. Two other proteins. Hdj and Bag 1, are involved in the process, allowing Hsp70 to perform binding-release cyclec of target proteins. The aim of this study was to investigate interactions between cochaperones Hdj 1 and Bag 1, and the major cell chaperone Hsp in vitro. The accumulation of Hsp70 and Hdj 1 in human erythroleukemia K562 cells was stimulated by heat stress (43 degrees C, 60 min). Cells were collected at certain time periods after heat stress, and amounts of cell chaperones were measured using Western blotting and ELISA assay. The level of Hsp70 chaperone activity in cell extracts was estimated using original technique. The effects of exogenous cochaperones and of their parts on this activity were also investigated. The results of the study indicate that Hsp70 chaperone activity is regulated by the level of its cochaperones, especially Hdj 1. At the same time the amount of ATP appears to be critical for functional activity of Hsp70. Hdj 1 and Bag 1 peptides, which bind to Hsp70 with high affinity, are able to significally reduce its chaperone activity. This finding confirms the possibility of using peptide approach for regulation of Hsp70 function at the cellular and organismal levels.

[Dynamics of chaperone complex Hdj1-Hsp70-Bag1 as a response of erythroleukemia K562 cells to heat stress]. / Dinamika shaperonnykh kompleksov Hsp70 s belkami-pomoshchnikami Hdj1 i Bag1 v reaktsii kletok éritroleikemii cheloveka K562 na teplovoi stress.

Heat shock protein Hsp70 is known to play an important role in cell protection against a variety of harmful factors. This property, at least in part, is due to Hsp70 ability to restore the native conformation of newly synthetized or damaged proteins. In this activity Hsp70 is accompanied by two proteins, Hdj1 and Bag1, that enable Hsp70 to peform cycles of binding-release of target proteins. The aim of this study was to investigate interactions of Hdj1 and Bag1 co-chaperones with Hsp70 in vivo. The accumulation of Hsp70 was stimulated by heat stress, and later, at certain periods following the stress, cell probes were collected for biochemical and microscopic analysis. The data of Western blotting showed that within 24 h after heat shock amounts of Hsp70 and Hdj1 raised to remain at the elevated level for nearly 48 h. Several time points within this period were chosen for analysis of the complexes between Hsp70 and co-chaperones. The data of reciprocal immunoprecipitation/immunoblotting and confocal microscopy showed that Hsp70-Hdj1 complexes were detected primarily at early stage after heat shock, then Hsp70 was preferably bound to Bag1. The dynamics of chaperone complex formation and changes in their intracellular localization are discussed in terms of cell reaction to stress.

[The role of Hsp70 chaperone in the reaction of human leukemic cells to anticancer drugs]. / Rol' shaperona Hsp70 v reaktsii kletok leikemii cheloveka na protivoopukholevye preparaty.

Most of anti-tumor factors are designed to kill selectively cancer cells; in most cases this action is related to the ability of the above substances to induce apoptosis. One of potent anti-apoptotic mechanisms is based on Hsp70 protein. Since the level of this protein is often higher in malignant tumors than in normal tissues, the aim of this study was to establish whether the elevated Hsp70 content may influence the process of apoptosis induced by anti-tumor drugs in cancer cells population. The increase of intracellular content of Hsp70 in human leukemia U-937 cells was attained by a mild heat stress or by transfection of cells with the human hsp70 gene. The elevation of Hsp70 quantity, irrespective of the way it was performed, leads to the inhibition of apoptosis in cells treated with two substances, etoposide or adriamycin. The inhibition of apoptosis was accompanied with the reducing of the share of cells with fragmented nuclei and with the delay in caspase activation. It is suggested that in addition to the previously discovered targets, whose activity is suppressed by the Hsp70 chaperone, this protein can inhibit the activity of caspase-3 and -7; this delays the onset of apoptosis in part of a cancer cell population.

[Expression of Hsp70 and Hdj1 chaperone proteins in human tumor cells]. / Analiz ékspressii belkov-shaperonov, Hsp70 i Hdj1, v opukholevykh kletkakh cheloveka.

Heat shock proteins (Hsp), or "stress proteins", play an important role in maintenance of cellular homeostasis both under normal conditions and during cellular stress. We tested malignant female urogenital and breast tumors for Hsp70 and Hdj1 (Hsp40) chaperones. Immunoenzyme procedure (based on Hsp70 and ATP interaction) was used to assay Hsp70 levels. Hsp70 and Hdj1 expression was higher in malignant cells than in benign ones. We were the first to demonstrate the feasibility of using Hdj1 expression as a novel prognostic factor for neoplastic disease.

[Induction and accumulation of HSP70 leads to formation of its complexes with other cell proteins]. / Induktsiia i nakoplenie BTSh70 privodit k formirovaniiu ego kompleksov s drugimi kletochnymi belkami.

The expression of a major stress protein Hsp70 may be induced by a great number of cytotoxic agents and also by factors known to cause process of cell proliferation or apoptosis. After induction the synthesis of Hsp70 is reduced to its basic level within first 2-6 hours after inducer application, while the high content of the protein may persist up to several days. The aim of this study was to understand the reason of such a long storage of Hsp70 in U-937 cells induced with three distinct factors. The data of immunoblotting showed that the mild heat shock at 43 degrees C for 60 min, phorbol ester and tumor necrosis factor (the two latter known to induce differentiation and apoptosis, respectively) caused Hsp70 accumulation, the protein level being high within 48 hours and then slowly declined to the basal level. According to the results of chromatography on anti-Hsp70 antibody-Sepharose gel, approximately 50% of the protein was retarded by the gel, while the other part was not recognized by immunoglobulins. Among proteins bound to the Hsp70, three polypeptides were identified as actin, p65 and c-Rel, two latter being constituents of NF-kappa B regulatory complex. The data demonstrate that besides its traditional target, i.e. newly synthesized or abnormal polypeptides, Hsp70 is able to bind mature, active proteins.

[Stress proteins in eukaryotic cells]. / Belki stressa v éukarioticheskoi kletke.

The review concerns properties and function of heat shock proteins (hsp) in the eukaryotic cell. The factors inducing Hsps' expression are considered with the emphasis on the fact that most of these agents are able to affect the function of peptide-synthesizing and protein-modifying machineries. The common outcome of this effect is accumulation of wrongly assembled protein structures which results in activation of heat shock transcription factors. The major property of proteins belonging to Hsp70 and Hsp90 is their chaperonic activity or the ability to bind newly synthesized or damaged polypeptides followed by restoration of the native structure of the latter. In this activity Hsp70 and Hsp90 are assisted by other proteins, and the work of such chaperonic systems is described. The function of the major stress protein Hsp70 in the eukaryotic cell was analysed, and on the base of a huge amount of data in has been resumed that proteins of this family constitute one of the most abundant systems of cell protection against cytotoxic factors. In the concluding part of the review we present some ways of application of our knowledge of Hsp in ecology, for analysing biological pollution, and in medicine, for increasing tissue or organismal resistance to injuring factors.

[Induction of apoptosis in murine myeloma cells NS0/1, transfected with the gene for the basic heat shock protein HSP70i]. / Induktsiia apoptoza v kletkakh myshinoi mielomy NS0/1, transfitsirovannoi genom osnovnogo belka teplovogo shoka BTSh70i.

Murine myelomas are rare cell variants deficient in inducible isoform of Hsp70 that protects cells from injury. In these cells Hsp70 is absent and is not induced under stress conditions. In this study myeloma cells NS0/1 were transfected with hsp70, and their susceptibility to apoptosis was challenged by serum deprivation or hydrogen peroxide. Expression of Hsp70 in NS0/1 cells made them more resistant to apoptosis in serum-free medium but did not affect their response to hydrogen peroxide. Hsp70 involvement in the protection of myeloma cells from apoptosis caused by different agents is discussed.