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.

[Postradiation recovery of hematopoiesis in mice affected by Neogen (IEW)]. / Postradiatsionnoe vosstanovlenie krovetvoreniia u myshei pod vliianiem neogena (IEW).

The effect of the synthetic peptide IEW (Neogen) with immunomodulating properties on postradiation recovery of haemopoiesis was investigated. We have shown that Neogen is a potential stimulator of haemopoiesis. The administration of Neogen after irradiation shortened duration of period of the recovery of the compartment of CFU-S-8 and the amount of bone marrow cells. The comparision of the effects of Neogen and GM-CSF (Leucomax) and G-CSF (Granocyte 34) have shown that the targets for these agents are probably different: polypotent CFU-S-for Neogen, and CFU-GM-for GM-CFS. Based on the results, we suggested the mechanism of Neogen effects on heamopoiesis.

[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.

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.

[Lack of serum causes apoptosis of thymocytes not requiring protein synthesis and ATP generation]. / Lishenie syvorotki vyzyvaet apoptoz timotsitov, ne trebuiushchii sinteaz belka i generatsii ATP.

Incubation of rat thymocytes in serum-free media was found to result in their apoptotic death characterized by internucleosomal DNA fragmentation, nuclear pyknosis and subsequent irreversible plasma membrane damage. As in the case of glucocorticoid (hydrocortisone)-induced apoptosis, DNA fragmentation under serum withdrawal was suppressed by endonuclease inhibitors (Zn2+ and spermine). At the same time, protein synthesis inhibitors (cycloheximide and puromycin) failed to block the apoptosis induced by serum withdrawal but inhibited the hydrocortisone-induced apoptosis. Various inhibitors of oxidative phosphorylation (uncoupler, rotenone, oligomycin), causing sharp decrease in cellular ATP did not suppress DNA fragmentation, whereas thymocyte plasma membrane damage accelerated under their effect. The results obtained indicate that intact thymocytes contain all the components of the apoptotic system; however, in the absence of apoptotic stimuli (e.g., hydrocortisone) the system is blocked by some growth factors of serum origin. Serum withdrawal is sufficient by itself to induce apoptosis and does not require the synthesis of special proteins.

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.