[Disruption of plasma cell membrane permeability for Ca2+ ions in radiation-induced apoptosis of thymocytes].
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Biomedical subjects
V L Gabaĭ
Publications and source records attributed to V L Gabaĭ.
[Effect of protein inhibitors of mitochondrial ATPase in intact rat thymocytes and Ehrlich ascites carcinoma cells].
It has been found that inhibition of mitochondrial ATPase in living thymocytes and Ehrlich ascites carcinoma (EAC) cells after incubation of cells with uncoupler, rotenone or cumene hydroperoxide, depends, in a large measure, on the inhibitor protein (IF1) action. Maximum inhibition (up to 70% of the oligomycin-sensitive ATPase activity) was found in the presence of the uncoupler in the incubation medium. Even when IF1 action was maximum, the residual ATPase activity caused marked ATP depletion in thymocytes, while in EAC cells other ATP-consuming processes prevailed. No inactive ATPase-IF1 complexes were found in intact thymocytes and EAC cells. The extent of inhibition of mitochondrial ATPase under oxidative stress was higher in thymocytes than in EAC cells and depended on cumene hydroperoxide concentration and duration of cell cellular ATP depletion. It is suggested that under certain experimental conditions IF1 can prevent cell death by slowing down the hydrolysis of cellular ATP.
Adenosine Triphosphate↗
[Lack of serum causes apoptosis of thymocytes not requiring protein synthesis and ATP generation].
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.
Adenosine Triphosphate↗
[Damage to and interphase death of Ehrlich ascites carcinoma tumor cells at different growth stages during energy starvation and heat shock].
The reaction of the Ehrlich ascite carcinoma cells, being at different phases of their growth, to the energy deprivation (rotenone in glucose-free medium) and heat shock (HS) was investigated. The criteria of this reaction were interphase death (according to Trypan blue staining) and structural changes (appearance of big blebs). It was found that proliferating cells (from log phase), judging from the two criteria, were more sensitive to a separate action of both energy deprivation and HS, than the resting ones (from stationary phase). Under combined actions (energy deprivation plus HS), when cell damage is much accelerated, the difference in their sensitivity was revealed only in relation to structural damages. Under the action of starvation and HS, changes in ATP content in the cells of both ages were similar; the reaction of cells to both the agents, after removing the calcium from medium (by chelator), was not changed. It means that specificity of the reaction of cells being at different growth phases to the damaging agents is not determined by disturbance in their energetic and calcium homeostases. In the proliferating cells, the cytoskeletal protein aggregation under energetic deprivation proceeded faster than that in the resting ones. It is proposed that the reaction of cells being on different growth phases depends on the stability of cytoskeletal proteins and on the content of stress proteins.
Adenosine Triphosphate↗
[Fragmentation of Ehrlich ascites carcinoma DNA during influences causing aggregation of cytoskeletal proteins].
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.
Animals↗
[Change in energy metabolism of ascites cancer cells with a decrease in pH].
In Hank's balanced salt solution EL-4 ascites thymoma cells possessed endogenous respiration which was sufficient for the maintenance of their ATP level: pH decrease down to 6.0 had no effect either on endogenous respiration or the ATP level. Glucose had no influence on the respiration of EL-4 cells but inhibited that of Ehrlich ascites carcinoma (EAC) cells by 40% (Crabtree effect); respiration of the both cell lines was strongly (4-fold) inhibited after simultaneous addition of glucose, lactate and pH decrease. EL-4 cells had no endogenous glycolysis; EAC cells showed a low level of glycolysis only after pH decrease. Glucose addition led to activation of glycolysis (both inhibited 2-fold after a decrease of pH down to 6.0. The respiration inhibition at pH 7.3 and 6.0 caused no decrease of ATP depletion when glucose was present in the medium; this result may be due to suppression of ATP consumption. Incubation of EL-4 cells under respiration and glycolysis deficiency conditions resulted in a sharp ATP depletion; pH decrease delayed this depletion.
Adenosine Triphosphate↗
[The relationship of damage to and death of ascitic tumor cells during starvation to the ATP and free calcium content in the cells].
Ascite tumor cells EL-4 were incubated in conditions of energy starvation (Hanks salt solution with rothenone and without glucose) at 37 degrees C for 3 hours. Under these conditions, some structural cell damages appeared within the first hours: enlarging and flattening of the cells, blebbing, vacuolization of the cytoplasm, nuclear chromatin condensation. Later on, a share of cells with obvious damage decreased, whereas that of the cells stained with trypan blue (dead cells) much increased (up to 90% after a 3 hour incubation). The cellular ATP decreased abruptly (up to 10% of the control) during the first 10 minutes of starvation. Free Ca2+ concentration increased within 1 hour of incubation more than two-fold. The conditions promoting Ca2+ influx (ionophore A23187 + Ca2+ in medium) accelerated the damage and cell death. However, the increase in free Ca2+ concentration did not trigger any damage in the energy-starved cells, since in the Ca2(+)-depleted medium (no increase in free Ca2(+)-concentration) the development of damages was not prevented. The damage initiation was irreversible: the addition of glucose to cell suspensions after 0.5-1 hour of their incubation in energy-starved condition did not prevent the development of damage, while ATP content in these cells was much increased.
Adenosine Triphosphate↗
[DNA degradation and changes in the permeability and form of Ehrlich ascites tumor cells when incubated in an anaerobic medium without glucose].
After a 3-hour incubation of the Ehrlich ascite tumor cells in buffered Hanks solution, without glucose and oxygen, the extensive cell injuries were observed. The time-course of appearance of these injuries was as follows: cell blebbing, staining of the cells with trypan blue, and then their staining with ethidium bromide. The DNA degradation registered with fluorometric method coincided in time with cell staining with trypan blue. All injuries (except DNA degradation) were delayed at pH 6.0 compared with those at pH 7.3. Glucose added to the cell suspension greatly protected the cells from these injuries, although DNA degradation at pH 6.0 in these conditions was a little higher than that at pH 7.3.
Anaerobiosis↗
[Increasing the concentration of free calcium in thymocytes during gamma-irradiation may induce their death].
Free Ca2+ concentration in thymocytes increased 0.5-1.5h after gamma-irradiation (10 Gy) as was measured by Quin-2AM fluorescent probe. Cycloheximide, a protein synthesis inhibitor, suppressed Ca2+ increase and inhibited radiation-induced thymocyte death. EL-4 thymoma cells did not exhibit any changes in free Ca2+ concentration and interphase death after gamma-irradiation. It is believed that the radiation-induced increase of free Ca2+ concentration in thymocytes may induce their death.
Animals↗
[Increased plasma membrane permeability for Ca2+ in radiation-induced thymocyte apoptosis].
Parameters of the Ca2+ permeability (the 45Ca influx rate in the presence of orthovanadate blocking the Ca(2+)-ATPhase, and the initial rate of the 45Ca uptake) and the DNA fragmentation were determined in rat thymocytes after gamma-irradiation-induced (with a dose of 5 Gy) apoptosis. Within the first 30-90 min after irradiation, the 45Ca influx rate that is characteristic of the membrane passive permeability remained unchanged. The initial rate of the 45Ca uptake that is characteristic of the Ca2+ exchange almost doubled. In 90-180 min, the rate of the 45Ca influx in the irradiated cells increased 1.5 to 2.0 times. An increase in the DNA fragmentation was observed in 90-120 min after irradiation of thymocytes; in 3 h, it developed up to 50%. Thus, modification of the membrane Ca2+ permeability in the irradiated thymocytes precedes a stage of initiation of the DNA degradation. A degree of disturbance of the membrane passive permeability increases as the thymocyte apoptosis progresses. The obtained data suggest that disturbance of the passive Ca2+ permeability and the intracellular Ca2+ homeostasis are responsible for the apoptosis of lethally irradiated thymocytes.
Animals↗