[Bcl-2 apoptosis regulating proteins in mouse tissues after exposure to 20 cGy of gamma-radiation]. / Belki-reguliatory apoptoza is semeistva bcl2 v tkaniakh myshei posle vozdeistviia gamma-izlucheniia.v doze 20 sGr.

A pronounced increase of Bcl-2 content was observed in thymus of mice exposed to 20 cGy of gamma-radiation 1 and 24 hours after the exposure; six days after the exposure the bcl-2 content lowered to the control one. No changes in bcl-2 level were revealed in mice liver during the period of observation. A level of Bax protein was stable in mice thymus during 60 days of observation.

Radiation-induced apoptosis in human myeloma cell line increases BCL-2/BAX dimer formation and does not result in BAX/BAX homodimerization.

A popular model of BCL-2 and BAX involvement in apoptosis suggests that upon apoptosis induction cytosolic BAX translocates to the mitochondria, where it displays the pro-apoptotic function, which involves its homodimerization. BCL-2 exerts anti-apoptotic function by forming heterodimers with BAX, thus neutralizing the pro-apoptotic activity of the latter. We have shown that irradiation of the human myeloma cell line RPMI-8226 induced apoptosis as determined by DNA degradation, cytochrome c release into cytoplasm and BCL-2 caspase-mediated cleavage. BCL-2 protein was present only in the membrane fraction, whereas BAX was found both in cytosol and membranes isolated from non-irradiated cells. Radiation induced moderate redistribution of BAX from cytosol to membranes with a concomitant increase in BCL-2/BAX heterodimer formation. Rapid and transient BCL-2 phosphorylation in membrane fractions of irradiated cells did not affect BCL-2/BAX heterodimerization. We failed to detect any BAX/BAX homodimers in apoptotic cells. Our findings show that in irradiated RPMI-8226 cells the formation of BCL-2/BAX heterodimers correlates with apoptosis. We conclude that BCL-2/BAX heterodimers are negative regulators of death protection, and our data agree with those who propose that BCL-2 does not require BAX to exert its survival function.

Radiation-induced apoptosis in human tumor cell lines: adaptive response and split-dose effect.

Irradiation of human ovarian carcinoma cells (OVCAR 3) and myeloma cells (RPMI 8226) with graded doses of 137Cs-gamma-rays led to a 35-40% increase in time-dependent apoptosis 72 hr after 6-8 Gy irradiation. Large individual variations in sensitivity to radiation-induced apoptosis were noted in human lymphocytes obtained from 5 donors. Pretreatment of OVCAR 3 and RPMI 8226 cells with 0.01 Gy increased their resistance to apoptosis after subsequent 6 Gy irradiation several hours or 48 and 72 hr later. A dose of 4 or 8 Gy given in 2 equal fractions at an interval of a few hours produced a low level of apoptosis compared to that resulting from a single administration of the same total dose. Adaptive response was demonstrated in 2 out of 3 samples of human lymphocytes isolated from different donors, and no split-dose effect for apoptosis was noted in 2 other donors. In split-dose experiments, there was no correlation between the sensitivity of cells to apoptosis and their position in the cell cycle, after the first half-dose. No G1 block was observed in irradiated cell lines. Adaptive response and split-dose effect were prevented by 3-aminobenzamide and okadaic acid which inhibit poly(ADP-ribose)polymerase and protein phosphatase, respectively. These results imply a common mechanism for acquired resistance to radiation-induced apoptosis in adaptive response and the split-dose effect.

Radiation-induced apoptosis in human ovarian carcinoma cells growing as a monolayer and as multicell spheroids.

Response to external gamma irradiation was studied in a human ovarian carcinoma cell line (OVCAR 3) growing as a monolayer and as multicell spheroids. Necrosis and apoptosis were documented using Trypan-blue uptake and acridine-orange staining, respectively, and apoptosis was quantified using a terminal deoxynucleotidyl transferase assay. Exposure of OVCAR 3 cells growing as a monolayer to 137Cs gamma radiation at a dose of 10 Gy produced 30-40% apoptosis 72 hr after irradiation. Cell-cycle analysis of irradiated cells showed an accumulation of cells in G2/M phase 24 hr after irradiation and then a decline at 48 hr in conjunction with apoptosis onset. The loss of G0/G1 cells in irradiated cultures suggested a preferential entry into apoptosis. No increase in apoptotic cell number was observed in OVCAR 3 spheroids after irradiation, and the cells probably died as a result of necrosis. When spheroids were disrupted immediately after irradiation to obtain a cell suspension, minor apoptosis was observed in association with a marked increase in TB-positive cell number after 96 hr of incubation following irradiation. Thus, a relationship was found between radiation-induced apoptosis and the cell cycle. Results with spheroids suggested the possible involvement of cell-to-cell interactions in apoptosis regulation.

Accumulation of cAMP in gamma-irradiated thymocytes and internucleosomal DNA fragmentation.

Ionizing radiation, glucocorticosteroids and chemical inducers of differentiation (CID) are cytotoxic to thymocytes, and induce internucleosomal DNA fragmentation. Tissue cAMP levels in thymi of irradiated mice were significantly elevated as early as 30 min post-irradiation. In contrast, cAMP content in the liver was not changed significantly up to 1 h post-irradiation, and then some decrease occurred. Irradiation of isolated thymocytes gave essentially the same results as after irradiation of animals, and the elevation in cAMP 30 min after the irradiation, DNA fragmentation and cell death were linearly related to the dose up to 2.5 Gy. The maximal induction of cAMP level occurs in the fractions of radiosensitive cortical thymocytes. In thymocytes all CID tested also induced the increase in cAMP level with concomitant DNA fragmentation. Unlike ionizing radiation, UVC light did not induce cAMP accumulation and DNA fragmentation in thymocytes. Treatment of UV-irradiated cells with But2 cAMP did not result in an increase in DNA fragmentation. Ionizing radiation induced DNA fragmentation and cell death can be prevented by adding the protein kinases inhibitor H-7. Theophylline was shown to reduce the cAMP response, DNA fragmentation and cell death in gamma-irradiated thymocytes, suggesting that the accumulation of cAMP may be partly related to adenosine receptor sites.

[The dopamine system in rat brain areas in the early period following irradiation with superhigh doses]. / Sistema dofamina v otdelakh golovnogo mozga u krys v rannie sroki posle oblucheniia v sverkhvysokikh dozakh.

In studying the main indices that characterize the neurochemical system of biosynthesis and degradation of a dopamine neuromediator, tyrosine hydroxylase-dopamine-monoamine oxidase, in different brain regions 5-6 min, 1 and 18 h after whole-body irradiation with high energy electrons (100 Gy) the authors have revealed a 25-40% inhibition of tyrosine hydroxylase and monoamine oxidase activity, and a 40% increase in the dopamine content of basal ganglia of the brain that control behavioural reactions of the organism. The neurochemical disturbances revealed are involved in the mechanisms of early transient incapacity after irradiation with superhigh doses.

[General similarities and differences in the postradiation death of various animal species and of man]. / Obshchie zakonomernosti i razlichiia postradiatsionnoi gibeli limfotsitov raznykh vidov zhivotnykh i cheloveka.

It was found in various animal species and man that an ordered internucleosome fragmentation of DNA is characteristic of lymphoid cells dying in the interphase. Both in vivo and in vitro, the postirradiation DNA degradation in thymocytes of rodents and piglets preceded the increase in the permeability of their plasma membrane. The in vivo kinetics of death of lymphoid cells from the thymus and spleen is similar in rodents and piglets. Rat thymocytes died in vitro earlier than thymocytes of piglets, calves and man which was evidently associated with a worse adaptive capacity of the latter to cultivation conditions.

Effect of the inducers of cellular differentiation and of ionizing radiation on thymus lymphocytes: chromatin degradation and programmed cell death.

The cytotoxic response of thymocytes to chemical inducers of differentiation does not represent a non-specific toxic action of these drugs. The death of thymocytes treated with the inducers or with gamma-rays is associated with internucleosomal chromatin fragmentation. All treatments are more effective on the most radiosensitive sub-population of these cells. This subpopulation is characterized by the maximal level of spontaneous DNA lesions. Incubation of thymocytes with the inducers of differentiation raises the level of these lesions. It is suggested that the processes of thymocyte death after the inducer treatment or irradiation and of cellular differentiation have features in common, and the capacity of thymocytes to limit or reverse the potentially lethal effects of these treatments is determined by the level of pre-existing spontaneous DNA lesions.

[Changes in the antigenic phenotype of mouse thymocytes after exposure to chemical inducers of differentiation and ionizing radiation]. / Izmenenie antigennogo fenotipa timotsitov myshei pri deistvii khimicheskikh induktorov differentsirovki i ioniziruiushchei radiatsii.

Irradiation of a mouse thymocyte fraction enriched by T-lymphocyte precursors changes the antigenic phenotype of cells toward the increase of their highly differentiated forms. Similar changes in membrane marker antigens are produced by chemical inductors of differentiation and thymotropin. The changes in the cell phenotype induced by the above agents are associated with both membrane and intragenome rearrangements. The results of the experiments on preventing the expression of some antigens by puromycin and the data on the level of spontaneous genome lesions in thymocyte fractions have prompted an assumption that destabilization of the genome upon irradiation increases DNA injury above some critical level which may serve a stimulus for "sorting out" the most radiosensitive thymocyte fraction.

[Degradation of chromatin in thymus lymphocytes during inhibition of repair of spontaneous DNA damage]. / Degradatsiia khromatina v limfotsitakh timusa pri ingibirovanii reparatsii spontannykh povrezhdenii DNK.

Chromatin degradation and cell death were observed after 6-7 h incubation of mouse thymus lymphocytes with 1-beta-D-arabinofuranosylcytosine and hydroxyurea. The time dynamics of both processes was similar. In this case, just as after gamma-irradiation, nucleosomes and their oligomeres were the products of degradation. Puromycin and cycloheximide prevented the toxic action of DNA synthesis inhibitors on thymocytes. It is suggested that the accretion of unrepaired DNA damages to some critical level triggers the process of the internucleosome degradation of chromatin, i.e. implements the program of lymphocyte death.

[The role of spontaneous DNA damage in the radiosensitivity of thymus lymphocytes]. / Rol' spontannykh povrezhdenii DNK v radiochuvstvitel'nosti limfotsitov timusa.

The most radiosensitive thymus cortical lymphocytes are highly sensitive to inhibitors of repair of spontaneous DNA lesions known by endonucleases: this is indicative of different level of injury to cells. The preincubation of cells with chemical inductors of differentiation increases the number of spontaneous lesions in them. The preincubation of thymocytes with concanavalin A removes almost completely the differences in the level of injury.

[Nature of the death of thymus lymphocytes as affected by different kinds of harmful factors]. / Kharakter gibeli limfotsitov timusa pri deistvii povrezhdaiushchikh faktorov razlichnoi prirody.

It was shown that thymus lymphocyte death caused by the effect of dithiobisdinitrobenzoic acid, hyperthermia, and osmotic shock is not accompanied by ordered chromatin degradation. Protein synthesis inhibitors do not prevent thymocyte death under these effects. The authors discuss the kinds of lymphoid cell death which depend upon the nature of the damaging factor and differ both in morphology and the character of nuclear material degradation.

[Role of hypoxanthine in the radiation death of thymocytes in vitro]. / Rol' gipoksantina v radiatsionnoi gibeli timotsitov in vitro.

Hypoxanthine in vitro causes death of thymocytes and concurrent intranucleosome degradation of chromatin. This process is more manifest in a more radiosensitive thymocyte fraction and prevented by protein synthesis inhibitors. The increase in the yield of hypoxanthine after the effect of lympholytic agents of different nature is not the result of cell death. It is assumed that hypoxanthine, formed in the exposed cells, may be an additional cytotoxic factor on reaching a subliminal concentration.

[Chromatin degradation during the exposure of thymus lymphocytes in vitro to differentiation inducers and gamma irradiation]. / Degradatsiia khromatina pri deistvii induktorov differentsirovki i gamma-oblucheniia na limfotsity timusa in vitro.

Chemical inductors of differentiation were shown to cause chromatin degradation in thymus lymphocytes. This process was prevented by the protein synthesis inhibitors. The fragments formed after the effect of chemical differentiation inductors on thymocytes were fully identical to chromatin internucleosome degradation products formed in the exposed cells. Chromatin degradation under the effect of chemical differentiation inductors was most pronounced in a more radiosensitive thymocyte fraction.

[Effect of irradiation and chemical differentiation inducers on the survival of thymus lymphocytes in mice]. / Vliianie oblucheniia i khimicheskikh induktorov differentsirovki na vyzhivaemost' limfotsitov timusa myshei.

Chemical inductors of differentiation were shown to produce a cytotoxic effect on thymus cells. The protein synthesis inhibitors prevented this effect. The toxic action of the inductors was more pronounced in a most radiosensitive thymocyte fraction. A combination of differentiation inductors and ionizing radiation did not produce the additive effect. This was observed after the effect of radiation and substances being not the differentiation inductors but toxic for cells.

[Radiobiology of differentiating mammalian cells]. / Radiobiologiia differentsiruiushchikhsia kletok mlekopitaiushchikh.

The data are reviewed in a comparative aspect concerning the influence of ionizing radiation and chemical inductors on mammalian cell differentiation. Molecular bases for the effects observed are discussed. Such an approach is shown to be adequate in studying the mechanism of radiation death of thymus lymphocytes.

[Molecular mechanisms of radiation death of lymphoid cells. Chromatin degradation and DNA replication in fractions of irradiated thymocytes]. / Issledovanie molekuliarnykh mekhanizmov radiatsionnoi gibeli limfoidnykh kletok. Degradatsiia khromatina i replikatsiia DNK vo fraktsiiakh obluchennykh timotssitov.

Chromatin of irradiated large and small thymocytes starts degrading 2 h following irradiation. However the yield of polydeoxynucleotides is more intensive in small thymocytes which are more radiosensitive. Immediately after irradiation small thymocytes exhibit activation of impulse incorporation of 3H-thymidine into a DNA fraction which does not exceed half the replicon by its molecular mass.

[Study of the nature of increased weight of lymphocyte superhelical DNA during cell incubation in vitro]. / Issledovanie prirody "utiazheleniia" superspiral'noi DNK limfotsitov v protsesse inkubatsii kletok in vitro.

The nature of the increased sedimentation of superhelical DNA (sDNA) during incubation of both intact and irradiated thymocytes has been studied. The "weighting" of sDNA was not consequent on the changes in superhelical density. No quantitative or qualitative changes in the composition of proteins connected with sDNA in the course of its "weighting" were found either. It is assumed that sDNA "weighting" seen during incubation of the cells exposed to non-physiological factors (irradiation, hyperthermia, incubation in a poor medium) reflects the processes of chromatin condensation followed by the apoptic death of lymphoid cells.

[Molecular mechanisms of radiation death of lymphoid cells. Protein poly-ADP-ribosylation in thymocyte fractions]. / Issledovanie molekuliarnykh mekhanizmov radiatsionnoi gibeli limfoidnykh kletok. Poli-ADF-ribozilirovanie belkov vo fraktsiiakh timotsitov.

A study was made of the process of poly-ADP-ribosylation of proteins in cells of radioresistant and radiosensitive fractions of mouse thymus lymphocytes after irradiation of isolated cells with a dose of 25 Gy. It was shown that activation of ADP-ribosylation of proteins occurred simultaneously with the formation of single-strand DNA breaks in thymocytes, and the degree of activation in radioresistant and radiosensitive fractions was nearly the same.

[Molecular mechanisms of radiation death of lymphoid cells. Radioprotective effect of cysteamine on a subpopulation of thymocytes differing in radiation sensitivity]. / Issledovanie molekuliarnykh mekhanizmov radiatsionnoi gibeli limfoidnykh kletok. Radiozashchitnoe deistvie tsisteamina na subpopuliatsii timotsitov, razlichaiushchiesia po radiochuvstvitel'nosti.

The radioprotective effect of cysteamine, incubated with thymocytes for 20 min prior to gamma-irradiation, was much more manifest with regard to subpopulations of radiosensitive small cortical cells (DMF = 1.8) than large thymocytes (DMF = 1.2). Radioprotective concentrations of cysteamine relaxed completely superhelical DNA of thymocytes. The relaxation was not due to the formation of single-strand breaks. A partial recovery of superhelical conformation of DNA of small thymocytes before irradiation reduced the radioprotective effect of cysteamine.