Induction of Heat Shock Protein 70 as a Predictive Marker of the Tumor Cell Radiosensitization with Inhibitors of the Heat Shock Protein 90 Activity.

Inhibitors of the heat shock protein 90 (HSP90) activity are considered as potential radiosensitizers of tumors with a perspective of their application in radiotherapy. However, there are tumors and tumor cell lines whose radioresistance is not decreased after treatment with the HSP90 activity inhibitors; therefore, a predictive marker is needed, which would allow one to predict the response of target cells. As such a marker, herein it is proposed to use induction of the heat shock protein 70 (HSP70) that is an early cellular response to the HSP90 dysfunction and can easily be immunodetected. It follows from the data obtained that the radiosensitization of HSP90 inhibitor-treated cells occurs only when this treatment causes the prominent induction of HSP70 in them. Determination of this marker enables one: 1) to predict a possibility of radiosensitization of any cells by means of the HSP90 activity inhibitors, 2) to design the inhibitor concentration range upon which the radiosensitizing effect seems likely to occur, 3) to find whether this radiosensitization will be selective towards cancer cells.

[Inhibitors of the heat shock protein 90 activity: a novel class of tumor radiosensitizers].

The 90 kDa heat shock protein (HSP90) is one of major chaperones of eukaryotes which catalyzes maturation and activation of its client proteins. Among the identified client proteins there are oncogene products, hormone or growth factor receptors and key components of signaling pathways responsible for the malignant growth of tumors or their resistance to chemotherapy and radiotherapy. In the case of inhibition of the HSP90 chaperone function, such proteins are inactivated and degraded soon that leads to simultaneous blocking several pathways essential for proliferation and survival of malignant cells; therefore, pharmacological inhibitors of the HSP90 chaperone activity could be used in anticancer therapy. At present, several HSP90 inhibitors are in preclinical testing or I-III Phase clinical trials as mono-agents or in combinations with other anticancer drugs or radiation. In the present review, all the data are summarized which characterize HSP90 inhibitors as effective radiosensitizers of tumor cells. Molecular mechanisms and selectivity of the radiosensitizing action of HSP90 inhibitors are here discussed as well as a possibility of their application to improve the outcome of radiotherapy.

[Preirradiation heat shock protein induction increases cellular radioresistance].

It was studied how does the transcriptional stress response and the heat shock protein (HSP) overexpression affect cellular radioresistance. For this purpose, normal murine fibroblasts and fibroblasts devoid of HSF1-gene (HSF1 is a transcriptional factor initiating stress-responsive HSP expression) were compared. Some cell samples were infected with specific vectors for expression of the constitutively active (mutant) HSF1 or individual HSP (HSP70, HSP56, HSP27). It was found that heat stress (43 degrees C, 30 min) increased the HSP level in normal fibroblasts and improved their survival following exposure to gamma-radiation, with both the effects being suppressed by quercetin (an inhibitor of HSF1-mediated HSP induction). In the HSF1-deprived cells, heat stress caused neither the up-regulation of HSP levels nor the enhancement of radioresistance, although both the effects were well manifested following the active HSF1 expression in those cells. The vector-induced over-expression of HSP70 or/and HSP27 equally enhanced the radioresistance in both cell cultures infected.

Pharmacological attenuation of apoptosis in reoxygenated endothelial cells.

BRX-235 (Iroxanadine), a novel drug developed by Biorex (Hungary), was previously characterized as a vasculoprotector against atherosclerosis, an activator of p38 kinase, and an enhancer of stress-responsive heat shock protein (Hsp) expression. The present data demonstrate that BRX-235 may improve survival of vascular endothelial cells (ECs) following ischemia/reperfusion stress. ECs cultured from human umbilical veins were exposed to hypoxia/reoxygenation to mimic ischemia/reperfusion. Caspase activation and apoptosis were monitored in the reoxygenated cells. Addition of BRX-235 (0.1-1 microM) to culture medium prior to hypoxia or at start of reoxygenation significantly reduced the caspase-dependent apoptosis. The cytoprotection conferred by the pre-hypoxic drug administration was sensitive to quercetin and seems to be based on enhanced Hsp accumulation in stressed ECs. In the case of post-hypoxic drug administration, the cytoprotection was strongly inhibited by SB202190 and SB203580 and appears to be associated with enhanced p38 kinase activation in reoxygenated ECs.

Flavonoid dihydroquercetin, unlike quercetin, fails to inhibit expression of heat shock proteins under conditions of cellular stress.

Modification by natural flavonoids quercetin and dihydroquercetin of the in vitro cell response to hyperthermal and chemical stress was studied. Quercetin completely inhibited the synthesis and intracellular accumulation of 70-kD heat shock protein (HSP70) in response to hyperthermia or to treatment with sodium arsenite, whereas dihydroquercetin in the same or higher doses had no such effect. Stress exposures under conditions of the quercetin-inhibited HSP70 expression significantly increased the percentage of dead and damaged cells compared to the same exposures in the absence of quercetin. On the contrary, dihydroquercetin virtually failed to increase the damage and death of the stress-exposed cells which displayed typical induction of HSP70. The findings suggest a new strategy for pharmacological use of these flavonoids with similar structure.

Phosphatase inhibitors prevent HSP27 dephosphorylation, destruction of stress fibrils, and morphological changes in endothelial cells during ATP depletion.

Pretreatment with phosphatase inhibitors did not affect the decrease in ATP content in endothelial cells, but inhibited HSP27 dephosphorylation and redistribution, damages to actin cytoskeleton, and morphological changes in cells. Our results suggest that inhibition of stress-induced HSP27 dephosphorylation protects cells from ischemia-induced damages.

[The protective effect of thermal treatment before irradiation on CFUs from bone marrow of mice: a potential involvement of heat shock proteins]. / Zashchitnyi éffekt predradiatsionnoi termicheskoi obrabotki myshei na KOEs ikh kostnogo mozga: vozmozhnoe uchastie belkov teplovogo shoka.

It was studied on mice how prior whole body hyperthemia affects a colony-forming ability of bone marrow after gamma-irradiation. It was found that heating of the animals (42 degrees C, 10 min) 18-22 h before their total irradiation (4 Gy) increases 2-fold the level of CFUs8 and CFUs12 determined in the spleen exotest. The induced radioresistance correlated with accumulation of heat shock proteins, HSP70 and HSP25, in tissues of preheated mice. Injection of quercetin (a selective inhibitor of the heat shock protein synthesis) 0.5 h before the heating fully abolished both the subsequent heat shock protein accumulation and the rise in CFUs populations as compared with control. It is suggested that heat shock proteins, whose expression increases in response to hyperthermia, can play a role of endogenous radioprotectors. Possible mechanisms of their protective action under irradiation are discussed.

Modulation of in vivo HSP70 chaperone activity by Hip and Bag-1.

The chaperone activity of Hsp70 is influenced by the activities of both positive and negative regulatory proteins. In this study, we provide first time evidence for the stimulating effect of the Hsp70-interacting protein Hip on the chaperone activity in the mammalian cytosol. Overexpressing Hip enhances the refolding of the heat-inactivated reporter enzyme luciferase expressed in hamster lung fibroblasts. Also, it protects luciferase from irreversible denaturation under conditions of ATP depletion. We demonstrate that these stimulating actions depend on both the presence of the central Hsp70-binding site and the amino-terminal homo-oligomerization domain of Hip. The carboxyl terminus (amino acids 257-368) comprising the 7 GGMP repeats (Hsc70-like domain) and the Sti1p-like domain are dispensable for the Hip-mediated stimulation of the cellular chaperone activity. Bag-1, which inhibits the Hsp70 chaperone activity both in vitro and in vivo, was found to compete with the stimulatory action of Hip. In cells overexpressing both Hip and Bag-1, the inhibitory effects of Bag-1 were found to be dominant. Our results reveal that in vivo a complex level of regulation of the cellular chaperone activity exists that not only depends on the concentration of Hsp70 but also on the concentration, affinity, and intracellular localization of positive and negative coregulators. As the Hsp70 chaperone machine is also protective in the absence of ATP, our data also demonstrate that cycling between an ATP/ADP-bound state is not absolutely required for the Hsp70 chaperone machine to be active in vivo.

Effect of natural antiphospholipid antibodies on antithrombotic activity of vascular wall.

Natural latent human antibodies cross-reacting with DNA and cardiolipin, interact with human endothelial cells, decrease antiaggregation activity of rat aortic wall, and increase fibrinolytic activity of the wall of the inferior vena cava. It is assumed that natural antiphospholipid antibodies present in immunoglobulin preparations in a latent state modify antithrombogenic activity of the vascular wall and are a potential cause of antiphospholipid syndrome.

Early and delayed tolerance to simulated ischemia in heat-preconditioned endothelial cells: a role for HSP27.

An ischemia-mimicking metabolic stress in cultured endothelial cells from the human aorta or umbilical vein caused ATP depletion, a rise in cytosolic free Ca2+, fragmentation and aggregation of actin microfilaments, retraction of the cytoplasm, and disintegration of cell monolayer. Simultaneously, the constitutive heat shock protein 27 (HSP27) underwent dephosphorylation and formed granules inside cell nuclei. Prior heat shock (45 degreesC, 10 min) in confluent cultures conferred two phases (early and delayed) of tolerance to simulated ischemia. Although heat preconditioning did not retard the ATP drop and the free Ca2+ overload within ischemia-stressed cells, each phase of the tolerance was manifested in longer preservation of normal cell morphology during the stress. Cells exhibiting the early tolerance within 3 h after heating altered the F-actin response to ischemic stress; no microfilament debris but, instead, translocation of F-actin to the tight submembranous layer was observed. In contrast, the delayed cytoprotection preserved the preexisting F-actin bundles under simulated ischemia; this happened only after 12- to 14-h post-heat shock recovery, elevating the intracellular HSP content, and was sensitive to blockers of HSP synthesis, cycloheximide and quercetin. The dephosphorylation and intranuclear granulation of HSP27 were markedly suppressed in both phases of the heat-induced tolerance. Without heat pretreatment, similar attenuation of the HSP27 dephosphorylation/granulation and the actin cytoskeleton stability during simulated ischemia were achieved by treating cells with the protein phosphatase inhibitors cantharidin or sodium orthovanadate. We suggest that prior heat shock ameliorates the F-actin response to ischemic stress by suppressing the HSP27 dephosphorylation/granulation; this prolongs a sojourn in the cytosol of phosphorylated HSP27, which protects microfilaments from the disruption and aggregation.

Protein phosphatase inhibitors and heat preconditioning prevent Hsp27 dephosphorylation, F-actin disruption and deterioration of morphology in ATP-depleted endothelial cells.

The vascular endothelium response to ischemic depletion of ATP was studied in vitro. Endothelial cells (EC) cultured from human aorta or umbilical vein were incubated in a glucose-free medium containing CCCP or rotenone. Such blockade of energy metabolism caused a drop in ATP, destruction of actin filaments, morphological changes, and eventually disintegration of EC monolayer within 2-2.5 h. While ATP fell and F-actin collapsed, the 27-kDa heat shock protein (Hsp27) lost basal phosphorylation and became Triton X-100-insoluble forming granules inside the cell nuclei. Protein phosphatase (PP) inhibitors (okadaic acid, cantharidin, sodium orthovanadate) did not delay the ATP decrease in energy-deprived EC but arrested both the alterations in the Hsp27 status and the changes for the worse in F-actin and cell morphology. Similarly, the Hsp27 dephosphorylation/insolubilization/granulation and the cytoskeletal and morphological disturbances resulting from lack of ATP were suppressed in heat-preconditioned (thermotolerant) cultures, this effect being sensitive to quercetin, a blocker of Hsp induction. The longer preservation of the cytosolic pool of phosphorylated Hsp27 during ATP depletion in the PP inhibitor-treated or thermotolerant EC correlated with the acquired resistance of F-actin and morphology. These data suggest that PP inhibitors as well as heat-inducible Hsp(s) can protect ischemia-stressed cells by preventing the ATP loss-provoked protein dephosphorylation and breakdown of the actin cytoskeleton.

Distinct effects of heat shock and ATP depletion on distribution and isoform patterns of human Hsp27 in endothelial cells.

To study the cytoprotective capacity of Hsp27 under various cellular stresses, we compared the effects of heating and energy deprivation on its distribution and isoform composition. Cultured endothelial cells from human aorta or umbilical vein were subjected to heat shock (45 degrees C) and ATP-depleting metabolic stress (CCCP or rotenone in a glucose-free medium). Both exposures led to the translocation of Hsp27 into the Triton X-100-insoluble cellular fraction, whereas the immunofluorescent Hsp27 pattern was characteristic for each stress employed. Heating (5-30 min) caused unexpected association of Hsp27 with thick bundles of actin microfilaments (stress fibers). ATP depletion within 30-120 min resulted in the appearance of Hsp27-containing compact granules in the nucleus. The insolubilization and relocalization of Hsp27 were reversible in both cases. The stress-induced shifts in the Hsp27 isoform spectrum indicate an increase in phosphorylation of Hsp27 in heat-shocked cells and its dephosphorylation in ATP-depleted cells. We suggest that these stresses diversely affect the phosphorylation status of endothelial Hsp27, thus altering its localization, supramolecular organization and functional activity toward actin.

Resistance of Ehrlich tumor cells to apoptosis can be due to accumulation of heat shock proteins.

Previously we have found that stationary Ehrlich ascites carcinoma (EAC) cells in vivo accumulated heat shock proteins (HSPs) and became resistant to necrotic death induced by prolonged energy deprivation of hyperthermia. Here we report that apoptotic death induced by nutrient starvation, transient ATP depletion, heat shock and a microtubule-disrupting drug, vinblastine, was also suppressed in stationary EAC cells comparing with exponential cells. When exponential (sensitive) cells were subjected to short-term heating with recovery to accumulate inducible form of HSP70, they also became resistant to all of the employed apoptosis-inducing exposures, and an inhibitor of cytosolic protein synthesis, cycloheximide, prevented acquisition of the resistance. It is suggested that in vivo accumulation of HSPs in stationary tumor cells can be endogenous protective device against apoptotic death induced by starvation or some anticancer treatments.

Adaptation of Ehrlich ascites carcinoma cells to energy deprivation in vivo can be associated with heat shock protein accumulation.

Tumor adaptation to chronic energy starvation in vivo was studied on Ehrlich ascites carcinoma (EAC) cells. EAC cells were isolated from mice and incubated in a glucose-free medium containing blocators of mitochondrial ATP generation (rotenone, 2,4-dinitrophenol, or oligomycin). ATP level in the treated cells decreased to 3-4% of the initial during 30 min of the incubation. The aggregation of cytoskeletal proteins, blebbing, and necrotic death within 2-3 h were observed in ATP-depleted EAC which were isolated and treated in the exponential phase of growth (5 days after inoculation), whereas stationary EAC (8 days after inoculation) were considerably more resistant to ATP depletion, and actin aggregation as well as bleb formation were suppressed in these cells despite the ATP loss. In contrast to the exponentially growing cells, thermotolerance and unexpected expression of inducible HSP68 and HSP27 as well as an elevated level of HSP90 were found in stationary EAC. Since the stationary cells had decreased content of ATP, ATP/ADP ratio, and energy charge, we suggest that this energy dysbalance may be conducive to HSP induction within the ascites tumor in vivo, and, at the same time, EAC cells with elevated content of HSPs acquire resistance to chronic energy starvation occurring in late stages of the tumor growth.

Heat shock-induced accumulation of 70-kDa stress protein (HSP70) can protect ATP-depleted tumor cells from necrosis.

The phenomenon of cell resistance to prolonged energy deprivation after mild thermal stress was studied in vitro. Murine P3O1 myeloma and Ehrlich ascites carcinoma cells were treated with rotenone (an inhibitor of respiration) in glucose-free medium to block ATP generation. ATP rapidly decreased in these cells to 3-6% of the initial level that resulted in powerful aggregation of cytoskeletal proteins, blebbing, and necrotic death of 60-70% cells within 2 h. Prior heat shock (43 degrees C for 10 min) with a subsequent 3-h recovery in a rich medium considerably suppressed the rotenone-induced actin aggregation and rate of necrosis in the energy-deprived cells without effecting the ATP drop in them. Using [14C]leucine labeling, gel electrophoresis, and fluorography, stimulation of the heat-shock protein (HSP) synthesis and total suppression of any other translation were revealed in the cells during recovery after the heat pretreatment. Significantly elevated levels of HSP70 but not HSP90 and HSP27 were found by means of immunoblotting in both cell cultures rendered resistant to necrosis under ATP-depleting conditions. Inhibition of the thermo-induced HSP synthesis by cycloheximide fully prevented development of the tolerance to energy deprivation. A novel function of HSP70 consisting of protection of ATP-deprived cells from "lethal" aggregation of cytoskeletal proteins is suggested.

Spontaneous overexpression of heat-shock proteins in Ehrlich ascites carcinoma cells during in vivo growth.

Ehrlich carcinoma (EC) cells isolated from mice at different phases of ascites growth were exposed to hyperthermia (44 degrees C), or oxidative stress (hydrogen peroxide or vikasol), or ATP depletion induced by rotenone. These exposures caused protein aggregation and rapid necrotic death in exponentially growing EC cells. On the contrary, the same cell culture at stationary phase of growth became considerably more resistant to all the above cytotoxic treatments, and the level of aggregated protein was significantly lower in stressed stationary EC cells than that in exponential ones. Comparative immunoblotting has revealed the unexpected expression of inducible 70 kDa heat-shock protein form (HSP68), as well as accumulation of HSP27 and HSP90 in the thermo- and drug-resistant stationary EC cells. It is suggested that the in vivo occurring HSP overexpression in stationary EC cells is an adaptive modulation of the tumor cell phenotype to maintain the viability of ascites EC cells under chronic deficiency of oxygen and nutrients.

Heat-shock proteins maintain the viability of ATP-deprived cells: what is the mechanism?

ATP depletion causes necrosis in mammalian cells. However, a previous heat shock can protect cells from the effects of energy deprivation, probably as a result of the synthesis and accumulation of heat-shock proteins (hsps). We propose that hsps protect ATP-depleted cells from rapid necrotic death by inhibiting the aggregation of cytoskeletal proteins that occurs when ATP synthesis is blocked.

[Fragmentation of Ehrlich ascites carcinoma DNA during influences causing aggregation of cytoskeletal proteins]. / Fragmentatsiia DNK astsitnoi kartsinomy Erlikha pri vozdeistviiakh, vyzyvaiushchikh agregatsiiu belkov tsitoskleta.

Upon exposures of Ehrlich ascites carcinoma cells to heat shock (44 degrees, 1 hr), oxidative stress or energy deprivation, their DNA undergoes fragmentation (35-45% after 5 hrs of incubation) which is considered as a hallmark of apoptosis. Prior to DNA fragmentation the cells exhibited blebbing (55-90% after 1 hr), thus being suggestive of cytoskeletal damage and a 1.5-2-fold increase in the Triton-insoluble protein concentration (protein aggregation) after 3 hrs. Rapid cell death (75% after 4 hrs) occurred only under oxidative stress. Electrophoresis of the Triton-insoluble protein fraction revealed that the common feature of all stress exposures used in this study was a dramatic increase in the aggregation of cytoskeletal proteins--actin and the 57 kDa protein. No dependence of DNA fragmentation on intracellular Ca2+ increase was found. Both DNA fragmentation and protein aggregation were suppressed by glucose, whereas Zn2+, an endonuclease inhibitor, suppressed only DNA fragmentation without any effect on protein aggregation. It is suggested that cytoskeletal damage may trigger tumor cell apoptosis.

Induction of heat-shock protein synthesis and thermotolerance in EL-4 ascites tumor cells by transient ATP depletion after ischemic stress.

The effect of a short-term energy deprivation (ischemia) on thermoresistance and heat-shock protein (HSP) synthesis in murine ascites EL-4 thymoma cells was studied in vitro. The incubation of the cells in glucose-free medium with rotenone (respiratory inhibitor) for 10 min caused rapid ATP depletion (to 9% of the initial level). After recovery, the synthesis of HSP70 and HSP90 was stimulated in the cells and they became greatly more resistant to hyperthermia than the control cells. The simultaneous rotenone and thermal treatment significantly decreased cell viability. The transition of HSP70 to Triton X-100-insoluble cell fraction was found in the ATP-depleted cells as well as in the heat-shocked cells, and 1 mM ATP fully reversed such insolubilization when it was added in Triton extraction buffer. The data obtained reveal that transient ATP depletion per se is sufficient to result in the HSP70 insolubilization, thus being conducive to induction of HSP synthesis and thermotolerance in the cells which recovered after energy deprivation. A novel mechanism of protein aggregation in ATP-deficient cells and a possible role of transient ischemia in development of tumor thermotolerance in vivo are discussed.