Vladimir L. Gabai

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 heath 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.

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°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.

Association of blebbing with assembly of cytoskeletal proteins in ATP-depleted EL-4 ascites tumour cells

ATP depletion in EL-4 ascites tumour cells rapidly induced the changes in cell morphology (blebbing), cytoskeletal protein assembly and finally resulted in cell death. After 1 hr of incubation with 2 microM rotenone (inhibitor of respiration) in glucose-free medium, when ATP level was 4% of the initial level, there were increases in triton-insoluble actin and vinculin levels (2.5-fold and 2.8-fold, respectively) and 44% of cells showed blebs; such treatment damaged cells irreversibly. Ca2+ removal did not diminish the effect of ATP depletion on cytoskeleton, blebbing and cell death, although the elevation of free intracellular Ca2+ in rotenone-treated cells was prevented. The role of ATP in maintaining cytoskeleton and cell shape is discussed.

Inhibition of uncoupled respiration in tumor cells : a possible role of mitochondrial Ca2+ efflux

Uncouplers CCCP (2–4 μM) or DNP (200–400 μM) when added to EL‐4 thymoma or Ehrlich carcinoma ascites cells initially stimulated endogenous respiration about 2‐fold but then inhibited it to a first‐order rate 20–25% of controls. This inhibition was accelerated by intracellular acidification or by A23187, a Ca2+/H+‐antiporter (i.e. when mitochondrial Ca2+ efflux was stimulated) whereas Ruthenium red, an inhibitor of uniporter‐driven Ca2+ efflux, significantly slowed down the effect of uncouplers. The respiratory inhibition was associated with NAD(P)H oxidation and was partially reversed by exogenous substrates (glutamine or glucose). In the permeabilized cells, endogenous and glutamine‐supported respiration was inhibited by EGTA, while succinate‐supported respiration was Ca2+ independent. It is suggested that mitochondrial Ca2+ is necessary for NADH‐dependent respiration of tumor cells, and uncouplers inhibit it by activation of mitochondrial Ca2+ efflux.

Natural hidden antibodies reacting with DNA or cardiolipin bind to thymocytes and evoke their death

Both free and hidden natural antibodies to DNA or cardiolipin were obtained from immunoglobulins of a normal donor. The free antibodies reacting with DNA or cardiolipin were isolated by means of affinity chromatography. Antibodies occurring in an hidden state were disengaged from the depleted immunoglobulins by ion‐exchange chromatography and were then affinity‐isolated on DNA or cardiolipin sorbents. We used flow cytometry to study the ability of free and hidden antibodies to bind to rat thymocytes. Simultaneously, plasma membrane integrity was tested by propidium iodide (PI) exclusion. The hidden antibodies reacted with 65.2±10.9% of the thymocytes and caused a fast plasma membrane disruption. Cells (28.7±7.1%) were stained with PI after incubation with the hidden antibodies for 1 h. The free antibodies bound to a very small fraction of the thymocytes and did not evoke death as compared to control without antibodies. The possible reason for the observed effects is difference in reactivity of the free and hidden antibodies to phospholipids. While free antibodies reacted preferentially with phosphotidylcholine, hidden antibodies reacted with cardiolipin and phosphotidylserine.

Mitochondrial ATP hydrolysis and ATP depletion in thymocytes and Ehrlich ascites carcinoma cells

When Ehrlich ascites carcinoma (EAC) cells and thymocytes were treated with uncoupler or rotenone in glucose‐free medium, rapid ATP depletion was observed in both types of the cells. Oligomycin slowed down ATP loss in thymocytes, but not in EAC cells. Thus, mitochondrial ATP hydrolysis appears to be significant in deenergized thymocytes in contrast to EAC cells, in which other ATP consuming reactions were prevailing. Complete deenergization of mitochondria by uncoupler or rotenone in these cells resulted in inactivation of mitochondrial ATPase by 65–75%. The effect was observed after complete and rapid (20–30 s) disruption of the cells with detergent, Lubrol WX. ATPase was blocked by the specific inhibitor protein (IF1) as it was shown by the studies on reactivation of this enzyme. When respiration is blocked but ATP content is supported by glycolysis, mitochondrial ATPase is not suppressed by IF1, and maintains the energization of mitochondria. It is concluded that under complete de‐energization of mitochondria IF1, significantly inhibits mitochondrial ATP hydrolysis and may slow down ATP loss in thymocytes and EAC cells.

Glucose decreases respiratory control ratio in EL-4 tumor cells.

EL‐4 ascites thymoma cells are shown to have high aerobic glycolysis and decreased Pasteur effect. At the same time, glucose produces a much smaller inhibitory effect on cell respiration (Crabtree effect) than in Ehrlich ascites carcinoma (EAC) cells. In intact EL‐4 cells, the respiratory control ratio (RCR) was found to be 6.2 with endogenous substrates and 8.0 with glutamine. Glucose decreased the RCR to 3.2, by stimulating, the state 4 respiration. In rat thymocytes and EAC cells, such an effect of glucose was absent (RCR of 7.0 and 7.2, respectively). It is suggested that in EL‐4 tumor cells, the high aerobic glycolysis and small Crabtree effect may be due to glucose‐induced ‘uncoupling’ of oxidation and phosphorylation.

Heat Shock Proteins and the Regulation of Heat Shock Gene Expression in Eukaryotes

Our first chapter is devoted to the general description of stress proteins and peculiarities of their expression in eukaryotic cells. We also introduce readers to the modern views of the problem of “negative regulation” of heat shock gene transcription to facilitate an understanding of the subsequent sections of this book.

“Proteotoxicity” of ATP Depletion: Disruption of the Cytoskeleton, Protein Aggregation and Involvement of Molecular Chaperones

The majority of cell-stressing exposures affect proteins. Typical “proteotoxic” exposures such as heating, ultra-violet irradiation, de crease in pH and treatment with oxidants or heavy metals are able to damage various proteins both in vitro (in a solution) and in vivo (in a cell). Lack of ATP in artificially prepared protein solutions does not seem to be very critical for the stability of the soluted proteins; in contrast, the depletion of intracellular ATP destroys the cytoskeletal framework and evokes aggregation (or insolubility) of many cellular proteins including HSPs. Although this proteotoxic component is only one of the many harmful effects of ATP depletion on mammalian cells, we consider it the most crucial event coupling the mechanisms of cell injury and cell adaptation under metabolic (or ischemic) stress. That is why this phenomenon is considered substantially here.

Involvement of Heat Shock Proteins in Protection of Various Normal and Tumor Cells from Ischemic Insult

The heart is the first organ where the participation of HSPs in protecting against ischemia has been demonstrated directly by overexpressing HSP70 in transgenic animals (see the previous chapter). For other organs and tissues, adequate experimental evidence is absent. However, there is are numerous data indicating the possible involvement of HSPs in anti-ischemic protection. Below we discuss the main results obtained on this issue to date.