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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.

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.

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., (Copyright 2001 Wiley-Liss, Inc.)

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%).

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.

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.

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 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.

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.

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 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.

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.

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.

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.

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.

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.

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 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.

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.

The in vitro gamma-irradiated mouse thymocytes were embedded in low melting agarose at 37 degrees C. After getting at 4 degrees C, the cells were lysed in neutral detergent solution containing proteinase K and ethidium bromide. Microscopic visualization of single lysed and stained cells showed the presence of the central "core" (nuclear matrix) surrounded with "halo" (relaxed nuclear DNA). During electrophoresis (2-5 V/sm, 5 min) this "halo" migrated towards the anode forming a "tail". The use of microdensitometric system provided measuring the size of the tail (L) and quantity of migrated DNA (S) for individual cells as well as obtaining the distribution of these parameters among the cells. The latter may be characteristic of heterogeneity of the cell population. It was shown that L and S increased linearly with the dose irradiation at least between 0.2 and and 5.0 Gy. In irradiated thymocyte (3 Gy) the DNA repair occurred within 10-20 min, but residual DNA damage could be observed even after 60 min of incubation. These damages may initiate the degradation of DNA in irradiated thymocytes that was observed after the repair of DNA.

Binding of SSB-protein from Ehrlich ascites tumor to ssDNA from M13 phage leads to its compactization. The structure of the complex at the protein/DNA ratios far from the saturation level looks like "beads-on the string". DNA that was fully saturated with protein forms collapsed globular structure. Binding of the protein to the dsDNA from phage lambda increases its flexibility and decreases the coil dimensions; no "beads-on the string" structure are seen. The protein possess slight destabilizing effect on hairpin helices of M13DNA. Competition studies demonstrate that the binding properties of protein with polyribonucleotide lattices and DNA's decrease in ranking as follows: poly(rG) greater than or equal to poly(rI) greater than or equal to ssDNA greater than dsDNA greater than poly(rA) congruent to approximately poly(rU). Thus SSB-protein from Ehrlich ascites tumor differs significantly from its presumed prokaryotic analogs.

Quick test for estimation of DNA-bound proteins (PBD) in mammalian blood serum involved filtration of blood serum through Wathman filters containing DNA. DNA was fixed in the Wathman filters by means of UV irradiation. Some physicochemical and chromatographic properties of PBD from mice blood serum were studied. The data obtained suggest that blood serum PBD constituted one of gamma-globulin fractions. Applicability of the PBD test for studies of tumoral diseases was shown using mice with Ehrlich ascites carcinoma.

Possible involvement of the single-strand DNA-binding protein (SSB-protein) in DNA replication in Ehrlich ascite tumour (EAT) cells was studied. There was a direct correlation between the content of SSB-protein in chromatin and the intensity of replicative synthesis of DNA in various preparations of EAT in vitro and in vivo (the computed value of the correlation coefficient was equal to 0.9). It was shown that the addition of exogenous SSB-protein to permeable EAT cells increased the replicative synthesis. It was concluded that although eukaryotic SSB-proteins are not complete analogs of prokaryotic ones, they may participate in DNA replication in eukaryotic cells and, possibly, are intracellular regulators of proliferation.

The lymphocyte nucleoids of mouse thymus contain about 40% of rapidly labelled nuclear RNA, about 9% of total intracellular protein and all nuclear DNA. Relaxation of superhelical DNA after thymocyte nucleoids treatment with pronase or RNAase suggests that non-histone proteins and/or RNAs are involved in conformational restrictions in the superhelical domains of cell DNAs. Thymocyte nucleoids proteins are represented by two groups of nonhistone proteins with molecular weights of 50 000-60 000 and 75 000-85 000. An essential role in the appearance of conformational restrictions in thymocyte superhelical DNA belongs to disulfide bonds.

Single-strand breaks (SB) in DNA of irradiated thymocytes are first repaired, at a half-recovery period of a few minutes, then the number of breaks rapidly increases indicating that in addition to chromatin endonucleolysis there is an imbalance between the repair enzymes that generate DNA breaks and eliminate them. The rate of SB accumulation in thymocyte fractions, upon incubation of the cells with 1-beta-D-arabinofuranosyl cytosine (AraC) and hydroxyurea (HU), is directly proportional to their radiosensitivity, whereas DNA ligase activity is higher in a fraction of highly radioresistant thymocytes. Chemical compounds that increase the survival rate of irradiated thymocytes decelerate SB accumulation in DNA and sensitivity of cells to AraC and HU. Ineffective substances have no such an effect.

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.

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.

Adenosine and a majority of adenine mononucleotides have a radioprotective action; adenine and 2'-deoxyadenosine have no radioprotective effect, and that of 3',5'-cAMP only approaches the detectable level. Ribo- and deoxyribonucleosides and nucleotides of guanine, uracil, thymine, and cytosine have no protective action. Dipyridamole increases and alkylxanthines block the radioprotective effect of adenosine. So it follows that the radioprotective effect is realized via A-receptors of the plasmatic membrane external surface.

The effect of a single-stranded DNA-binding protein (SSB-protein) form Ehrlich ascites tumour cells (EAT) on the activity of homologous purified DNA-polymerases alpha and beta, DNA-replicase, primase and DNA-polymerases from phage T4 and Bacillus stearothermophillus was studied. It was shown that the SSB-protein caused a 1.5-2.5-fold stimulation of the DNA-polymerase alpha activity on different templates (e.g., denaturated and activated DNA, poly(dA). The degree of stimulation depended on the template type, protein/template ratio and purity of DNA-polymerase alpha. The activity of DNA-polymerase was inhibited by the SSB-protein, when the activated DNA was used as a matrix and was unchanged on the denaturated DNA. The activity of some prokaryotic DNA-polymerases was increased under the influence of the SSB-protein. The protein enhanced the processivity of T4 DNA-polymerase and strongly inhibited the activity of replicase and primase. A conclusion about the complex effect of the SSB-protein on the activity of replicative and repair enzymes is drawn.

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.

A decrease in the endogenous NAD content that occurs immediately after gamma-irradiation of thymus lymphocytes is attributed to activation of poly (ADP-ribosylation) and not to changes in the activity of NAD-glycohydrolase and/or to the release of NAD from cells. The addition of benzamide 60 min before irradiation prevents the postirradiation drop of the NAD level and produces a radioprotective effect. At the same time, benzamide inhibits nuclear superhelix DNA repair and causes an average of 40 per cent decrease in the activity of DNA ligases I and II. The authors discuss the idea that the content of intracellular NAD in thymocytes is a critical factor responsible for the vitality of these cells.

The decrease in the average DNA size in thymocytes starts soon after in vivo irradiation and at approximately 45 min reaches a plateau, thereafter showing only minor changes up to 3 h. This fall in extent of chromatin cleavage coincides with the accumulation of 1.0-1.5 kb DNA fragments. Double-strand breaks generated by endonucleases are not randomly distributed along DNA but clustered in such a way that they give rise to fragments of 1-5 nucleosomes in size. Cycloheximide treatment partially inhibits nuclease activity in nuclear extracts isolated from thymus of irradiated mice. This suggest that DNA fragmentation is an early event in programmed death of thymocytes mediated by irradiation. The data indicate that it requires protein synthesis and that it precedes release of polydeoxyribonucleotides.

It was established that the postirradiation changes in incorporation of 14C-adenine into acid-soluble and acid-insoluble fractions of thymocytes reflected cell death. When added after irradiation, nalidixic and oxolinic acids exerted a radioprotective action. The effect was absent after single washing of thymocytes incubated with these compounds for 60 min before or after irradiation. Both substances inhibited utilization of 14C-adenine and incorporation thereof into the acid-insoluble fraction of nonirradiated thymocytes and did not influence the viability of cells.

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.

The effect of different chemical compounds on the cAMP/cGMP ratio in the bone marrow of mice and radioresistance of animals has been studied. It has been shown that all compounds possessing radioprotective properties give rise to the cAMP/cGMP ratio in the bone marrow of mice. No changes in cAMP and cGMP level were noted after the administration of nonradioprotective substances. The maximal radioprotective effect coincide in time with the largest increase of the cAMP/cGMP ratio. The injection of radioprotectors at different doses demonstrate clearly that only at radioprotective doses the increase in the cAMP/cGMP ratio takes place. The administration of some substances 2, 15 and 60 min after the irradiation of mice shows that the radioprotective effect, though modest, was evident only in one case of elevated cAMP/cGMP ratio (the injection of 2-Mercaptoethylamine 2 min after the irradiation). Evident radioprotective effect occurs at the cAMP/cGMP ratio of about 170-200%; the ratio of about 130-140% corresponds to small radioprotection.

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.

The diminution of NAD level in mouse thymus lymphocytes precedes their death under the effect of various genotoxic agents and manifests itself by the time of the onset of chromatin degradation. At the same time, in vitro, NAD does not influence the activity of micrococcus nuclease of Ca2+,Mg2+-dependent endonuclease from human spleen. Stimulation of protein poly(ADP-ribosylation) by exogenous NAD does not change the sensitivity of chromatin to micrococcus nuclease. In contrast to hepatocytes, in the thymus, no inhibition of Ca2+,Mg2+-endonuclease, resulting from ADP-ribosylation, occurs which may be due to low activity of ADP-ribosyl transferase in thymocytes. Incubation of thymus lymphocytes with benzamide prior to irradiation does not inhibit chromatin degradation. It is suggested that the decrease in the NAD level is one of the indications of the injury to thymocytes which is not related to the induction of their death. In contrast to thymocytes, the pretreatment of Ehrlich ascites tumor cells with benzamide produces a radiosensitizing effect.

Single-stranded DNA-binding protein (SSB-protein) has been purified and characterized from Ehrlich ascite tumour (EAT) cells. The purification procedure was performed in analytical and preparative variants. It was shown that in the analytical variant of the purification procedure can be used to determine protein concentration in the cell. The molecular mass of the SSB-protein as determined by SDS polyacrylamide gel electrophoresis is 36 and 43 kD; that determined by gel filtration is 27, 28, 43 and 44 kD; pI is 7.4. The use of nitrocellulose filters showed that the SSB-protein binds preferentially to ss-DNA. The protein contains no admixtures of DNA-polymerases, endo- or exonucleases, DNA-dependent ATPase, lactate dehydrogenase and HMG-proteins. The SSB-protein stimulates 1.5-2-fold the activity of DNA-polymerase alpha from EAT, it does not activate DNA-polymerase beta from EAT and strongly inhibits the activity of exonuclease (snake venom phosphodiesterase). The specificity of the term "SSB-protein" which makes it different from other non-histone proteins of chromatin is discussed.