[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.

[Radioadaptive response in vitro of unstimulated rat lymphocytes according to metallothionein test]. / Radioadaptivnyi otvet in vitro nestimulirovannykh limfotsitov krys po metallotioneinovomu testu.

It was shown that 24 h after acute action of gamma-radiation in vitro on unstimulated rat lymphocytes the metallothionein (MT) level did not change for doses of 0.01; 0.5 and 1.0 Gy, while dosed of 1.75 and 2.5 Gy increased this parameter 2 and 2.6 times on the average. After exposure to 0.01 Gy of adaptive radiation 2 h before exposure to 1.75 Gy of the damaging dose reliably lower (1.5 times on the average) increase in MT content was found. The heterogeneity in display of the radioadaptive response by MT test for various lymphocyte preparations was found. Two possible mechanisms of the obtained changes were discussed. One of them is connected with the selection irradiation death of cells with the initially low MT level, and the other is caused by postradiation MT synthesis as a result of accumulation of the DNA damages and MT genes amplification.

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.

Validation of 213Bi-alpha radioimmunotherapy for multiple myeloma.

The efficacy of radioimmunotherapy (RIT) with beta emitters has been clinically demonstrated in the treatment of refractory forms of lymphoma. Alpha-emitting radionuclides with a short half-life are also good potential candidates for RIT directed at tumor targets easily accessible to radioimmunoconjugate molecules and small enough to benefit from the short range of alpha particles (<100 microm). The purpose of this study was to demonstrate the feasibility of ex vivo purging of multiple myeloma-invaded bone marrow. Tumor cells were targeted by a specific monoclonal antibody (B-B4) coupled to 213Bi by a chelating agent (pentaacetic triamine diethylene p-aminobenzyl acid). The efficacy of alpha-RIT was assessed in vitro by analysis of thymidine incorporation, cell mortality, apoptosis of myeloma cells, and the study of nonspecific irradiation of hematopoietic cell lines not recognized by B-B4-pentaacetic triamine diethylene p-aminobenzyl acid immunoconjugate. High dose-dependent cell mortality of myeloma cells was found with radiolabeled B-B4, and this mortality was total at 30 kBq/10(5) cells. Cells were found in apoptotic state at rates of up to 40% for a dose of 7.4 kBq/10(5) cells. Nonspecific mortality was low compared with specific mortality (up to 1%).

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.

Cell death induced by a 131I-labeled monoclonal antibody in ovarian cancer multicell spheroids.

Treatment of OVCAR-3 spheroids with 131I-OC125 monoclonal antibody produced a decrease in spheroid volume and a concomitant rise in necrotic cell number. No increase in apoptotic cell number was observed during incubation of spheroids with the labeled antibody. Necrosis began early, reaching a maximum after 3 Gy of accumulated dose delivered at a dose rate of 1.8 cGy/h. Higher accumulated doses induced necrosis for longer incubation times. Thus, dose rate and time are both determinants of ultimate radiation effects when spheroids are incubated with labeled antibodies, although dose rate is the most important factor.

[The radiobiology of metallothioneins]. / Radiobiologiia metallotioneinov.

The data about the increase of cell and tissue metallothioneins (MT) level after the irradiation by ionizing radiation and UV rays were present. The conclusion was made that increase was conditioned by DNA damage by free radicals with following MT-genes amplification. Some agents which increased endogenous MT content also exogenous MT protected biological object from the irradiation and agents which caused oxidative stress. The possible mechanisms of MT protection effect (principal is the MT antioxidative properties) and some aspects of practical application of MT were examined.

[Study of DNA damage and heterogeneity of cells by a gel microelectrophoresis method]. / Issledovanie povrezhdennosti DNK i geterogennosti kletok metodom gel'-mikroélektroforeza.

The influence of some cell parameters--cell size, cell DNA content, cell cycle phase--on migration of DNA from individual cell in constant electric field was studied on human peripheral lymphocytes and mouse lymphoblasts L5178Y by microelectrophoresis (neutral DNA-comet assay). It has been shown that the full comet length (l) did not depend on the cell parameters for intact and moderately irradiated cells (up to 25 Gy). It is proposed that migration capacity of DNA is related with damage to DNA. Then the heterogeneity of intact cells in the comet length can mirror the variation of background level of DNA lesions from cell to cell in the same population. Lymphoblasts of radiosensitive strain (LY-S cells) show higher average DNA-mobility and heterogeneity in DNA-comet length as compared to the cells of radioresistant strain (LY-R). This difference is suggested to be compatible with relatively higher background DNA-breakage in LY-S cells due to deficiency in repair of double-strand breaks in these cells. A subpopulation (about 20%) in LY-S cells with very migrating DNA was determined with neutral DNA-comet assay. It is reasonable to identify this subpopulation with cells of this line showing the lowest DNA repair rate. Thus neutral DNA-comet assay can be useful for evaluation of damage to and repair of DNA in individual cells and their heterogeneity.

[The characteristics of the structural organization and spontaneous DNA damage in the cells of 2 lymphoma L5178Y lines differing in their radiosensitivity]. / Osobennosti strukturnoi organizatsii i spontannaia povrezhdenoost' DNK v kletkakh dvukh linii limfomy L5178Y, razlichaiushchikhsia po radiochuvstvitel'nosti.

Nucleotide particles, resulting from mild lysis procedure, obtained from two strains of murine L5178Y lymphoma cells, differing in radiosensitivity (L-R and LY-S) showed differences in the sedimentation rate and in the halo rewinding in the presence of 40 micrograms/ml Ethidium bromide (EB). Microgel electrophoresis of DNA after an exhaustive lysis of cells (in the presence of sodium laurylsarcosine and proteinase for 20-24 h at room temperature) disclosed not only heterogeneity in DNA migration length among LY-R cells (LR) and LY-S cells (LS), but also differences between two strains with respect to this parameter. L-value for LY-S cells exceeds that for LY-R strain by 20 per cent, and the ratio LS/LR remains constant regardless of the voltage applied. Thus, the cell distribution pattern according to L-value is the intrinsic parameter for both LY-R and LY-S cells, and DNA of LY-S cells is suggested to bear more background strand breaks as compared with the radioresistant LY-R cells. LY-R cells irradiated with 10 Gy repair their DNA completely after 60-90 min of incubation at 37 degrees C but DNA migration pattern for irradiated and repaired LY-S cells is characterized by the predominance of DNA with a higher L-value than that for intact cells. Hence, we suggest that the higher radiosensitivity of LY-S cells might be compatible with a relatively high background DNA breakage in these cells.

[The connection between the content of metallothioneins in the bone marrow and liver and the survivability of irradiated mice after cadmium chloride administration]. / Sviaz' mezhdu soderzhaniem metallotioneinov v kostnom mozge, pecheni i vyzhivaemost'iu obluchennykh myshei posle vvedeniia khloristogo kadmiia.

The survival rate of mice gamma-irradiated at different times after administration of Cd2+ (0.75 mg/kg) was shown to be tightly connected with the amount of metallothioneins induced by this heavy metal in the bone marrow (r = 0.96). The connection between the amount of metallothioneins in the liver and the survival rate was less pronounced.

[Lowering the acute toxicity of ethanol with a zinc-metallothionein compound]. / Snizhenie ostroi toksichnosti étanola preparatom tsink-metallotioneina.

The homogeneous drug of zinc-metallothionein (zinc-MT) which characterized according to the molecular weight and UV-absorption spectra was obtained from the liver of the rats preliminary injected with the zinc chloride. It was shown that zinc-MT (0.5-4 mg/kg of protein) injected to the mouse 10 min before the ethanol decrease the acute toxicity of the alcohol. The preliminary injection of the control mixture consisted the albumin, cysteine and zinc chloride in proportions and dose corresponding to the dose 3 mg/kg of zinc-MT did not change LD50 of ethanol. The possible mechanism of the effect is discussed. The most possible is the stabilization of the lipid peroxidation in the liver or the restoration of the thiol groups pool at the alcohol intoxication by the zinc-MT.

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.

Relationship of single-stranded DNA-binding proteins of Ehrlich ascites tumour to cell growth phase and DNA replications.

The possible involvement of SSB-proteins in DNA replication in Ehrlich ascites tumour (EAT) has been investigated. A direct relation (the computer-generated correlation coefficient was 0.9) between the SSB-proteins content in chromatin and intensity of the replicative synthesis of DNA in various preparation of EAT in vivo and in vitro is observed. Addition of exogenous SSB-proteins to the permeable EAT cells has been found to increase the replicative synthesis. Although eukaryotic SSB-proteins are not complete analogs of the prokaryotic SSB-proteins, they evidently participate in DNA replication in eukaryotic cells and possibly are intracellular regulators of proliferation.

[Use of benzoyl-naphthyl-DEAE-cellulose chromatography for determining the ability of proteins from Ehrlich ascites carcinoma, binding with single-stranded DNA (SSB-proteins), to destabilize the DNA double helix]. / Ispol'zovanie khromatografii na benzoil-naftil-DEAE-tselliuloze dlia opredeleniia sposobnosti belkov iz astsitnoi kartsinomy Erlikha, sviazyvaiushchikhsia s odnonitevoi DNK (SSB-belkov), destabilizirovat'dvoinuiu spiral' DNK.

Single-stranded DNA-binding proteins (SSB-proteins) isolated from Ehrlich ascites tumour (EAT) cells were incubated for 30 min at 5 mM NaCl with salmon sperm DNA or [3H]DNA from EAT at the SSB-protein/DNA ratio (w/w) of 0 to 4.5. After addition of sodium dodecyl sulfate up to a 0.05% concentration, the proteins were applied to columns with benzoylated naphthoylated DEAE-cellulose. Double-stranded DNA was eluted by 1 M NaCl; the DNA containing single-stranded regions was eluted by 50% dimethylformamide. There was a progressive lowering of the DNA content in the first eluate and a rise in the second eluate, as could be evidenced from the increase in the SSB-protein/DNA w/w ratio. This effect was more pronounced in the case of homologous DNA and was not coupled with the nuclease activity of SSB proteins. It was concluded that EAT SSB-proteins are "DNA-unwinding" proteins.

[Stimulation of replicative DNA synthesis by eukaryotic proteins bound to single-stranded DNA]. / Stimuliatsiia replikativnogo sinteza DNK belkami éukariot, sviazyvaiushchimisia s odnonitevoi DNK.

The paper deals with the effect of the single-strand (ss) DNA-binding proteins (SSB-proteins) from the Ehrlich ascites tumor (EAT) cells and from the eggs of silkworm, as well as the mouse serum blood proteins, having preferential affinity to ss DNA, on the DNA replicative synthesis in the EAT cells permeable for the macromolecules, and, for the silkworm proteins and on the DNA replicative synthesis in the nuclei from the eggs of silkworm proteins and on the DNA replicative synthesis in the nuclei from the eggs of silkworm permeable for macromolecules. SSB-proteins of EAT to considerable extent stimulated the DNA synthesis. At the same time, the other proteins (from the silkworm and from the serum) activated the DNA synthesis in the permeable cells to the less extent. It was found that SSB-proteins from the silkworm had a 1.5-13 fold stimulating effect on the DNA replicative synthesis in the homologous system (in the permeable nuclei). If the permeability for the macromolecules of the cells and nuclei treatment with Triton X-100 may be different, it is supposed that the activation of the DNA synthesis by the exogenous proteins depends on the homologous system of the DNA replicative complex. It is possible that the effect of the serum proteins on the DNA synthesis is connected with the masking of the ss regions of DNA which inhibited DNA-polymerase alpha. Perhaps the mechanisms of the activation of the DNA replicative synthesis by the proteins in vitro with the purified DNA polymerase alpha and in vivo are of different nature and are conditioned by homology of the deoxyribonucleoproteins.

[The phenomenon of adaptive response in radiobiology]. / Fenomen adaptovnogo otveta kletok v radiobiologii

Consideration is given to various adaptive reactions to low-level radiation, their association with an absorbed dose, dose rate, radiation quality and time-interval between exposures, as well as with a cell cycle phase. Possible mechanisms of the adaptive response and the character and role of DNA damages, that can induce gene expression of the adaptive response, are discussed. The data on the influence of a preliminary long-term exposure to low-level radiation on the radiosensitivity of biological objects are analyzed with due regard for the adaptive cell response. It is concluded that the adaptive response of cells to ionizing radiation is a particular case of the phenomenon of cell adaptation to the effect of genotoxic factors of the environment.

[Antigenic properties of heterogeneous nuclear ribonucleoprotein particles weakly binding nonhistone proteins and proteins binding single-stranded DNA (SSB proteins) from Ehrlich ascites tumor]. / Antigennye svoistva belkov geterogennykh iadernykh ribonukleoproteidnykh chastits, labil'no sviazannykh negistonovykh belkov i belkov, sviazyvaiushchikhsia s odnonitevoi DNK (belkov SSB) iz astsitnoi kartsinomy Erlikha.

Antigenic properties of the proteins of heterogeneous nuclear ribonucleoprotein particles, (hnRNP), weakly bound nonhistone chromatin proteins (WB(N)P) and single-strand DNA-binding proteins (SSB proteins) from chromatin and extrachromatin fraction of the Ehrlich ascites tumor cells have been comparatively studied. The chromatin and extrachromatin SSB proteins displayed similar mobility in the tube and slab SDS/PAGE, had the same ssDNA-binding capacity and similarly stimulated the replicative synthesis in permeable cells. However, the chromatin SSB proteins contained 1.4 times higher phosphate amount than the extrachromatin ones (3.1 and 2. 2. moles phosphorus per 1 mole protein, respectively). The study of four protein groups with the use of a rabbit antiserum to/against extrachromatin SSB proteins (titer 1:13000 by enzyme immunoassay) showed that the chromatin and the extrachromatin SSB proteins have similar antigenic properties. One fraction of the hnRNP proteins was also reactive with the antiserum, whereas the WB(N)P displayed no cross-reactivity. The specificity of the ferm "SSB proteins" as applied to eukaryotic cells, their affinity with hnRNP proteins and differences from the HMG proteins are discussed.

[Chromatin degradation in the death of thymic lymphocytes induced by radiation or dexamethasone: the necessity for a stage of preliminary proteolysis]. / Degradatsiia khromatina pri gibeli limfotsitov timusa, indutsirovannoi radiatsiei ili deksametazonom: neobkhodimost' étapa predvaritel'nogo proteoliza.

Proteolytic activity in a protein fraction of a rat thymocyte nuclear matrix was found to increase 1-2 h after gamma-irradiation or administration of dexamethazone. Cycloheximide did not prevent the observed protease activation. Neither histons nor thymocyte nuclear matrix proteins were subjected to proteolysis after exposure to radiation or the hormone. Such proteolysis inhibitors as phenylmethylsulfonyl fluorine, trasilol, and partly leupeptine inhibited nuclear DNA degradation in irradiated and dexamethazone treated thymus lymphocytes. In all appearance, this effect was not due to Ca/Mg-dependent endonuclease inactivation. The same was observed in the system of autolytic chromatin degradation in isolated thymocyte nuclei.