The interphase death of dividing cells. The effect of oxygen and the pH of the medium on the interphase death of Ehrlich ascites carcinoma cells under the action of ionizing radiation

Interphase death of in vitro irradiated (200 Gy) Ehrlich ascites tumor cells was studied as a function of oxygenation level and medium pH. The presence of oxygen both during and after irradiation as well as the increase in pH from 7.4 to 8.1 were shown to increase interphase death rate. The pronounced threshold dependence of interphase death of cells upon their concentration may be attributed to hypoxia occurring in a pericellular medium when cells concentration exceeds the threshold.     

Effect of the sulfhydryl reagent thimerosal on cytosolic free Ca2+ and membrane potential of thymocytes.

The sulfhydryl reagent thimerosal at concentrations 5-100 microM has been found to induce a variety of changes in ion transport in rat thymocytes. In particular, [Ca2+]i increases about 10-fold from the basal level. The [Ca2+]i response to thimerosal displays a two-stage time course, with the main [Ca2+]i rise during the second stage. Evidence has been obtained for the depletion of intracellular Ca2+ pools in thimerosal-treated cells, however, Ca2+ mobilization from intracellular stores does not contribute significantly into [Ca2+]i rise. Thimerosal elicits permeability not only for Ca2+, but also for Mn2+ and Ni2+, which is Ca(2+)-dependent. We failed to get any evidence on thimerosal-induced inhibition of the plasma membrane Ca(2+)-ATPase. The induction of Ca2+ influx, rather than inhibition of Ca(2+)-ATPase, accounts for the disturbance of [Ca2+]i homeostasis in thimerosal-treated cells. Thimerosal also elicits changes in monovalent ion fluxes resulting in marked depolarization. The latter seems unrelated to the changes in [Ca2+]i and is suggested to be mediated both by increased permeability for Na+ and a decreased one for K+. Thimerosal significantly stimulates AA release from thymocytes. Evidence has been presented that AA metabolite(s), probably, LO product(s), may mediate the changes in the transport of mono- and divalent cations elicited by the sulfhydryl reagent. Prolonged treatment of thymocytes with thimerosal resulted in cell death.     

A study of the role of lipoxygenases in radiation-induced apoptosis of thymocytes. Inhibitory analysis

The radiation-induced apoptosis of thymocytes is suppressed by the common inhibitor of lipoxygenases nordihydroguaiaretic acid but not the inhibitors of cyclooxygenases or cytochrome P-450, which indicates the key role of lipoxygenases in apoptosis. However, the specific inhibitors of 5-lipoxygenase (AA861) and of 12-lipoxygenase (baicalein) do not suppress apoptosis and even enhance it. This effect can be explained by an increase in the yield of the 15-lipoxygenase product upon inhibition of 5- and 12-lipoxygenases. Indeed, the addition of 15-hydroxyecosotetraenic acid, a product of 15-lipoxygenase, into the incubation medium induces apoptosis in thymocytes. The results obtained suggest that 15-lipoxygenase is one of the enzymes involved in radiation-induced apoptosis of thymocytes.     

Apoptosis in P388 Leukemia Cells Induced by Specific Inhibitors of 5- and 12-Lipoxygenase and the Product of Cyclooxygenase, Prostaglandin E2

We studied the effect of specific inhibitors of 5- and 12-lipoxygenases as well as the product of cyclooxygenase activity, prostaglandin E₂, on proliferation and death of P388 leukemia cells. Inhibition of 5- and 12-lipoxygenases in the cells inhibits proliferation and induces apoptosis. The concentrations of baicalein, an inhibitor of 12-lipoxygenase, and AA861, an inhibitor of 5-lipoxygenase, causing a 50% death rate (LC₅₀) proved to be the same, 50 M. Excessive prostaglandin also inhibited proliferation of the cells and induced apoptosis. The LC₅₀ for prostaglandin E₂ was 4 M. The obtained data suggest that apoptosis in P388 cells after lipoxygenase inhibition can be induced by both deficiency of lipoxygenase products and excess of prostaglandins in the cell.     

Action of avermectins on lymphoid leukemia P-388 cells in vitro]

Avermectins are final products in the fermentation process with Streptomyces avermitilis. They have parasitocidic activity and are used as the main substances of insectoacaronematocides. The study of the activity of the natural avermectin complex (aversectin C) and separate avermectins A1, A2, B1 and B2 in the cell culture of lymphoid leukemia P-388 showed that within the concentrations of 0.1 to 1.0 microgram/ml aversectin C inhibited the growth of the tumor cells and induced their death. The inhibition was due to blocking the cell mitosis. The cell death was accompanied by internucleosomal degradation of the DNA nuclei i.e. the death was of the apoptosis type. The sensitivity of the cells to aversectin C was directly proportional to their initial proliferative activity. As for the separate avermectins only avermectin A1 had the cytotoxic activity within the concentrations used, avermectin A2 had the cytostatic activity and avermectins B1 showed no activity under the experimental conditions.     

Change of multidrug resistance and sensitivity of leukemic cells to oxidative stress in large tumors

The possibility of modification of vincristine-resistant strain of leukemic cells upon long-term tumor transplantation was studied. DBA2 mice were inoculated with cells P388VR, a vincristine-resistant strain of leukemic cells P388 either from small 5-day-old tumors or from large 7 to 8-day-old tumors. Multidrug resistance (MDR) of the cells was evaluated by cell sensitivity to vincristine and from the transport of calcein ester out of cells. Sensitivity of cells to oxidative stress was determined by cell viability and the production of reactive oxygen species in cells, by dichlorofluorescein assay. It has been shown that properties of the P388VR strain did not change in mice inoculated with the cells from small tumors. However, if the cells from large tumors were used for inoculation, the strain underwent the following modifications: tumors became contaminated with erythrocytes; resistance to vincristine increased; transport of calcein ester into and out of the cells accelerated, and sensitivity to oxidative stress raised. These changes in the properties of the P388VR strain persisted upon consequent propagation of the tumor and could be caused by a constitutive activation of the HIF system. The possibility of the alterations of the leukemic cells upon tumor transplantation from large tumors has to be taken into account in the experimental work with tumor cell lines.     

The effect of post-radiation hypoxia on potential cell injury

The effect is described of increasing the reproductive death of pro- and eucaryotes by the action of a short-term (approximately 60 min) acute hypoxia after a single irradiation thereof in normal culture medium at the logarithmic phase of growth. The effect observed is probably associated with the fact that potential damages are poorly repaired and are fixed under hypoxic conditions.     

The effect of melittin on proliferation and death of thymocytes

The effect of melittin, an activator of phospholipase A₂, on proliferation and death of rat thymocytes in a broad concentration range was studied. Cell proliferation was estimated by the accumulation of colchicin metaphases, necrotic death was determined from lysis and staining of cells with trypan blue, and apoptosis was assessed from the type of DNA fragmentation, the amount of fragmented DNA, and the percentage of cells with subdiploid DNA. It was shown that low melittin concentrations (below 5 μg/ml) stimulate thymocyte proliferation. At high melittin concentrations, thymocytes die by the primary necrosis type. Throughout the concentration range studied, melittin does not produce apoptosis in thymocytes. Conversely, high melittin concentrations even inhibit thymocyte apoptosis in the control and after irradiation. An inhibitor of RNA synthesis actinomycin D does not affect thymocyte death in the presence of melittin. It is concluded that the activation of phospholipase A₂ can induce necrosis but not apoptosis and thus is not a necessary step in the signaling cascade that initiates apoptosis in thymocytes.     

The interphase death of dividing cells. The relation of interphase death to cell energy requirements

In experiments with irradiated cells of Chinese hamster and Ehrlich ascites tumor a study was made of the influence of energy provision on their interphase death rate. The presence of the uncoupler of respiration and oxidative phosphorylation--carbonyl cyanide-3-chlorophenylhydrazone--in a medium without glucose was shown to drastically increase the interphase death rate of cells of both types, whereas this effect was not observed in a medium with glucose.