[Possible mechanism of the selective action of the inhibitors of glycolysis in the endothelial cells and the human carcinoma cells in the culture].

It is known that the production of energy and synthesis of macromolecules in cancer cells depend on the glucose metabolism to a greater extent than in non-tumor. In this paper we carry out a comparative study of the effectiveness of the two modifiers glycolysis 2 - D-deoxyglucose (2-DG) and dichloroacetate (DCA) in the induction of the cell death, changes in the cell cycle progression and in the alteration of the intracellular ROS levels in endothelial cells (line ECV304) and human carcinoma cells (line HeLa G-63) in order to identify cause-effect relations between these events. It has been shown that inhibition of the various stages of the glycolysis result in blocking cells in C2/M phase of the cell cycle and the induction of the cell death. This effect was record for HeLa G-63 cells only. DCA is inhibitor of the pyruvate dehydrogenase kinase and 2-DG is inhibitor of the glucose transport and glycosylation induced selective dose-dependent cytotoxic effect in HeLa G-63 cells. The increase of intracellular levels of the oxygen radicals induced by DCA in the cells HeLa G-63 suggests that the cytotoxic effect of the DCA is mediated by activation of the mitochondrial functions. The cytotoxic effect of 2-DG depend on the level of glucose in the culture medium, therefore we suggest that not only the oxidative stress, but and the energy depletion involved in selective response of the cancer cells on the actions of the inhibitors of glycolysis.

[Assessing of the role superoxide, nitric oxide and redox metals in cytotoxic effect of the H2O2 and amyloid-beta-protein].

On the cells of neuroendocrine tumor of rats (line PC12) in culture was estimating of the governing mechanisms of the cytotoxicity of the oxidative stress and the role of the amyloids in increasing this stress. Using flowcytometric assessment of the cytotoxicity H2O2 and fragment beta-amyloid (Abeta) peptide (25-35) has been shown the dose-dependent increasing of the quote of the cells with DNA content < 2c. Isoeffective consentrations were 1 MM H2O2 and 5 MkappaM Abeta. The cytotoxicity H2O2 and A were accompanig with the increasing of the intracellular level of O2-. The treatment of the cells GSNO (donor of NO) and o-phenantrolin (chelators of Fe ions) significantly decreased the intracellular level of O2- as well as the cytotoxicity H2O2 and Abeta. Thus, in direct experiments has been shown of the part amyloids in the increasing of the oxidative stress and participation of the reactive oxide radicals in the cytotoxic effect of the Abeta. The addition argument which confirmed contribution of the oxidative stress in the cytotoxic effect of the Abeta was the similarity of the cellular response on the action of the oxidative agent - H2O2 and Abeta.

Changes in nitric oxide and superoxide levels in human endotheliocytes and carcinoma cells after exposure to low-dose ionizing radiation.

The exposure of HeLa G63 and ECV-304 cells to γ-rays of (137)Cs as well as ß-particles of (3)H(2)O and (3)H-thymidine induced changes in redox status of not only irradiated cells, but also their progeny. Increased intracellular levels of nitric oxide (NO) were observed only in HeLa G63 cells and persisted over three cell generations; ß-particles from (3)H(2)O were most efficient. Intracellular superoxide (O(2)(-)) level had similar dynamics in both cell lines. Intracellular O(2)(-) level decreased immediately after irradiation, but then increased and significantly surpassed the control level. These changes in the intracellular level of O(2)(-) were accompanied by decondensation of nuclear chromatin. Increased level of free radicals in the progeny of irradiated cells and changes in chromatin conformation and the absence of correlation between radiation-induced structural damage to chromosomes and intracellular level of free radicals suggest participation of epigenetic mechanisms of inheritance.

[Immediate and delayed effects of low doses of beta-radiation in mammalian cells in culture].

We have carried out the comparative examination into the efficacy of induction of NO and superoxide anion by incorporated and unincorporated sources of ionizing radiation in endotheliocytes (line ECV 304) and carcinoma cells (line HeLa G63) expressing various forms of NO-synthases. The increased intracellular nitric oxide levels were observed after exposure of the cells to beta-particles of 3H-thymidine and 3H2O, as well as to gamma-rays of 137Cs in HeLa G63 cells expressing the inducible forms of NO-synthases. A higher incidence of the intracellular NO level was observed after exposure to beta-particles of 3H2O than to beta-particles of 3H-thymidine or gamma-rays of 137Cs even though 3H-thymidine and gamma-rays elicited more chromosomal damages. Modification of the intracellular superoxide level was shown to have a similar dynamics of the changes in time for the both cellular lines. Shortly after irradiation, the intracellular superoxide level was lower than in non-irradiated cells, and then it became higher than the control level. The increased intracellular superoxide and NO levels were observed after exposure of the cells to beta-particles of 3H-thymidine and 3H2O, as well as to gamma-rays of 137Cs in the progeny of irradiated cells. Modification of the intracellular superoxide level was accompanied by decondensation of the cellular chromatin. A higher intracellular free radical level in the progeny of irradiated cells along with decondensation of cellular chromatin, as well as the absence of correlation between a radiation-induced structural damage of chromosomes and intracellular free radical level allow us to speculate in favor of the participation of epigenetic inheritance mechanisms.

Modification of intracellular level of free radicals and apoptosis in cultured human endotheliocytes and carcinoma cells.

The intracellular levels of superoxide O(2)(-)and nitric oxide NO were directly measured under intravital conditions in cultured human endotheliocytes ECV 304 and carcinoma cells HeLa G-63. Comparative analysis of changes in the intracellular levels of superoxide and NO induced by ascorbic acid revealed a negative correlation between NO and O(2)(-)levels, whose strength depended on concentration of the acid. In pharmacological concentrations, ascorbic acid induced apoptotic death of carcinoma cells, but did not trigger apoptosis of endotheliocytes. The study demonstrated a possible way of cytotoxic action of ascorbic acid in pharmacological concentrations.

Dynamics of intracellular superoxide and NO content in human endotheliocytes and carcinoma cells after treatment with NO synthase inhibitors.

The dynamics of intracellular levels of superoxide and NO after cell treatment with NO synthase inhibitors were studied in human cells expressing various NOS isoforms: endotheliocytes and ECV-304 (eNOS) and carcinoma cells and HeLa-G63 (iNOS). Cytometric analysis of changes in the cell fluorescence intensity was carried out using superoxide and NO fluorescent indicators (dihydroethidene and DAF-2-DA, respectively). Intracellular levels of superoxide decreased in HeLa-G63 and ECV-304 cells after their incubation in medium with aminoguanidine, L-NAME, and D-NAME. Intracellular NO level decreased only in HeLa-G63 cells after incubation in medium with aminoguanidine and L-NAME, but not D-NAME. The level of NO returned to normal after 7-h culturing in inhibitor-free medium, while the level of superoxide increased and remained high throughout 3 generations. Incubation of cells with D-NAME did not increase the intracellular level of superoxide. Presumably, high prolonged generation of superoxide is a delayed result of inhibition of NO synthesis in HeLa-G63 cells.

[Characteristics of the spontaneously transformed human endothelial cell line ECV304. II. Functional responses of the ECV304 cells].

Functional responses of the spontaneously transformed human endothelial cell line ECV304 were studied in order to asses its applicability as an endothelial cell model for studying angiogenesis and signal transduction. The dependence of proliferation activity of this line on the presence of growth factor was shown. The absent serum in culture medium resulted in blocking of cells in G1-phase of a cell cycle which is not typical for tumor cell lines. Low doses of beta particles emitted during [3H]thymidine decay resulted in blocking the proliferation of these cells in G2M-phase in a dose-dependent manner. Incubation of the cells with another source of beta particles, 3H2O, under condition of equal specific activities of tritium resulted in preferable accumulation of the cells in S-phase. The different efficiency of beta particles of tritium as a part of 3H2O molecule or thymidine demonstrates that various mechanisms are responsible for various check points. The check point of G1/S is absent and that complies with the presence of deletion of chromosome 9 in locus p21. The level of NO produced by constitutive form of NO-synthase in ECV304 cells was relatively low and not modified by inducible NO-synthase inhibitors. The data obtained suggest that ECV304 line cells retained the properties of the initial spontaneously transformed cell line obtained from human umbilical vein (HUVEC) as well as they can be used as a model system for further studies of the properties of vascular endothelial.

Intracellular NO concentration and its changes in carcinoma cells and cultured human endotheliocytes under the influence of inhibitors and inductors of nitric oxide synthases.

We carried out cytophotometric evaluation of NO content in cells expressing various types of NO synthases. Endogenous production of NO can be modified by NO synthase inductors and inhibitors.

[The blocking of the proliferation of the human endothelial cells in culture by beta-irradiation from internal and external sources of the radiation. S-block is induced by the 3H2O beta-particles].

Recently we shown that low doses (0.12-0.46 Gy) of (methyl-3H)-thymidine incorporated into human endothelial cells induce the accumulation cells in G2-phase of the cell cycle. The temperate doses of (1-6 Gy) gamma-rays 137Cs were less effective in the induction of the G2-block estimated by flow cytometry analysis of DNA content and in the induction of the chromosome aberrations (bridges and fragments in anaphase). The aim of this study was the comparative investigation of efficiency of beta-rays emitted 3H from 3H-thymidine and 3H2O by several of the cellular parameters. Here we shown that at the equal conditions of the incubation of the cells in medium with 3H2O induced the accumulation cells in S-phase without decreasing of the mitotic activity and without increasing of the chromosome aberrations level. Unlike from 3H2O the incubation of the cells with 3H-thymidine induced the accumulation cells in G2-phase with decrease of the mitotic activity and with increase of the chromosome aberrations level. Concurrent treatment cells with 3H-thymidine and thymidine abrogate these cellular effects of the 3H-thymidine. Inhibitor ATM-kinase caffeine abrogate as G2-block as S-phase block. These results suggest that the low-dose beta-radiation activates S-phase and G2-phase checkpoints requiring ATM-mediated signal transduction pathway. The factors, which impact on the efficiency of the internal and of the external sources of the irradiation, depend on theirs disposition in relation to radiosensitive target--DNA was discussed.

[Internal and external sources of the radiation induce the blocking of the proliferation of the human endothelial cells in culture. G2-block is induced by beta-particle 3H-thymidine and gamma-rays 137Cs].

We found that low doses (0.12-0.46Gy) of (methyl-) 3H-thymidine incorporated into human endothelial cells induce the accumulation cells in G2-phase of the cell cycle. Temperate doses of (1-6 Gy) gamma-rays 137Cs were less effective in the G2-block estimated by flow cytometry analysis of DNA content. Furthermore, the induced the high level of the chromosome aberrations (bridges and fragments in anaphases). 1Gy of gamma-ray 137Cs and 0.005 Gy of beta-rays induced the same per cent of the aberrant anaphases. Apparently, that the damages of the cellular hereditary structures are responsible for the blocking of the cellular proliferation in G2-phase. We suggest, that the disposition 3H-thymidine into radiosensitive target (DNA) defines the high cytotoxic of the beta-rays.

[Modification of cellular radiosensitivity by NO-synthase inhibitors].

We recently reported that the treatment of V-79 and HeLa cells with nitric oxide synthase (NOS) inhibitor N-nitro-L-arginine methyl ester (L-NAME) significantly reduced the level of the radiation-induced unstable chromosome aberrations. The stereoisomer D-NAME had no effect. We suggest that the radioprotective effect of L-NAME resulted from the action on the generation reactive radicals due to the inhibition of the NOS-activity. We tested this suggestion on the NO-resistant (ECV-304) and NO-sensitive (HeLa) cells, which were treated with L-NAME or aminoguanidine or D-NAME or cysteamine before gamma-irradiation. There are no significantly differences in radiosensitivity between these cells estimated after exposure by gamma-rays with different doses. However, the radioprotective effect of the NOS-inhibitors manifested only for HeLa. D-NAME had no radioprotective effect neither HeLa nor ECV-304. In contrast NOS-inhibitors, cysteamine treatment EVC-304 reduced the radiation-induced level chromosome aberrations almost twofold. The different mechanisms of the modification of cellular radiosensitivity are discussed.

Mutagenic effects of gamma-rays and incorporated 8-3H-purines on extracellular lambda phage: influence of mutY and mutM host mutations.

The lethal and mutagenic effects on phage lambdacI857 of 60Co gamma-rays and of decay of 3H incorporated into phage DNA both as 8-3H-deoxyadenosine and 8-3H-deoxyguanosine (using 8-3H-adenine as a labelled DNA precursor) were studied on four isogenic Escherichia coli strains: AB1157 M(+)Y(+) (wild type, mutM(+) mutY(+)), AB1157 M(-)Y(+) (mutM::kan mutY(+) mutant deficient in the formamidopyrimidine-DNA glycosylase MutM), AB1157 M(+)Y(-) (mutM(+) mutY mutant deficient in the A:G mismatch DNA glycosylase MutY), and AB1157 M(-)Y(-) (mutM::kan mutY double mutant deficient in both DNA glycosylases). The main products of transmutation component of 3H decay in position 8 of purine residues are 8-oxo-7, 8-dihydroadenine (8-oxoA) and 8-oxo-7,8-dihydroguanine (8-oxoG), the latter being responsible for the most part of the mutagenic effect. The lethal effects of both gamma-rays and tritium decay virtually did not depend on the repair phenotypes of the host strains used. Therefore, the MutM and MutY glycosylases are not involved in the repair of lethal DNA damages induced by ionizing radiation or by the transmutation component of 3H decay in purine residues of phage DNA. The efficiencies of mutagenic action of 3H-purines E(m) (frequencies of c-mutations per one 3H decay in phage genome) were 2.4-, 3.8- and 55-fold higher in the M(-)Y(+), M(+)Y(-) and M(-)Y(-) mutants, respectively, in comparison to the wild-type host. The mutagenic efficiencies E(m) for gamma-rays were nearly identical in the M(+)Y(+) and M(-)Y(+) hosts, but were increased 1.8- and 8.3-fold, respectively, in the M(+)Y(-) and M(-)Y(-) mutants. These data suggest that: (1) the MutY and MutM DNA glycosylases are important for prevention of mutations caused not only by spontaneous oxidation of guanine residues, but also by ionizing radiation or by decay of 3H incorporated into purine bases of DNA; (2) the MutY and MutM enzymes functionally cooperate in elimination of mutagenic damages induced by these agents.

[Lethal and mutagenic effects of tritium incorporated into position 8 of the purines in phage lambda DNA and the role of the Fpg protein]. / Letal'noe i mutagennoe deistvie tritiia, inkorporirovannogo v 8-e polozhenie purinov v DNK faga liambda, i rol' belka Fpg.

The lethal and mutagenic effects of the decay of 3H incorporated in phage lambda DNA as 8-3H-adenosine and 8-3H-guanosine were studied, using the DNA of 8-3H-adenine as a labeled DNA precursor. A transmutation component of 3H decay is involved in formation of 8-oxoguanine (8-oxo-G) and 8-oxoadenine (8-oxo-A) residues in phage DNA. The efficiency of phage inactivation (the number of lethal lesions per one tritium decay in the phage genome) for 3H decay in position 8 of purines was the same as that measured in positions 5 and 6 of pyrimidines (alpha = 0.14 +/- 0.01) and virtually did not depend on the fpg-1::kan mutation in the host gene encoding the Fpg protein (formamidepyrimidine-DNA-glycosylase). The efficiency of the mutagenic effect of 3H-purines Em (frequency of c mutations per one 3H decay in the phage genome) was (2.9 +/- 0.3) x 10(-5) in the fpg+ host and (4.6 +/- 0.4) x 10(-5) in the fpg-host. This means that the Fpg protein excised approximately 40% of premutational DNA lesions (probably, 8-oxo-G residues). Induction of the mutagenic SOS system by UV light caused a 1.5-fold increase in the frequency of c mutations induced by 8-3H-purines in fpg+ cells over that in fpg-cells. This suggests that apurinic AP sites produced after the excision of 8-oxo-G by the Fpg protein are substrates for mutagenic SOS repair.

[Characterization of the adaptive response to the action of gamma-rays, induced by low doses of 14C in Chinese hamster fibroblasts]. / Kharakteristika adaptivnogo otveta k deistviiu gamma-luchei, indutsirovannogo malymi dozami 14C v fibroblastakh kitaiskogo khomiachka.

It has been studied the correlation of the mitotic activity of the chromosome aberrations and apoptosis, in the V-79 cells pre-exposure to an adapting dose of ionizing radiation from 14C-thymidine prior to an acute challenge dose of gamma-rays. In spite of that the incubation of the cells with isotope increased of the yield of the chromosome aberrations, but the cells became more resistant to following gamma-irradiation. Increasing the adaptive dose of the 14C on degree didn't influence on the present of the adaptive response. However, using concentrations of the 14C damaged metaphase/anaphase transition and cells blocked in this check-point by apoptotic death. The results suggest, that the cellular selection has been involved in 14C-induced adaptive response, estimated by level of asymmetric chromosome aberrations in V-79 cells.

[The lethal and mutagenic action of 3H incorporated into the 2' position of the deoxyribose in the DNA of the extracellular phage lambda]. / Letal'noe i mutagennoe deistvie 3H, inkorporirovannogo v polozhenie 2' dezoksiribozy v DNK vnekletochnogo faga lambda.

The lethal and mutagenic effects of 3H decay in 2' position of deoxyribose residues in DNA of extracellular lambda phage were studied, [2'-3H]-deoxyadenosine (3H-dA) or [2'-3H]-thymidine (3H-dT) being used as labelled DNA precursors. As estimated by the efficiency of the lethal and mutagenic actions of 3H decay in position 2' was significantly lower than that of the decay in the incorporated 3H-pyrimidines. The genetic effects of 3H decay in 2' position may be attributed to the radiation effect of beta-particles on DNA. In UV-irradiated E. coli cells, with the induced SOS repair, the mutagenic effect of 3H-dA in phage lambda is significantly higher than that of 3H-dT. This is perhaps related to the formation in DNA of AP-sites, resulting from 3H-decay in 2' position, and to the predominant incorporation of adenosine residues opposite to AP-sites during SOS repair.

[Lethal and mutagenic effect of incorporated 6-3H-pyrimidines on extracellular phage lambda]. / Letal'noe i mutagennoe deistvie inkorporirovannykh 6-3H-pirimidinov na vnekletochnyi fag lambda.

A study was made of lethal and mutagenic effects of 6-3H-thymidine and 6-3H-cytosine, incorporated into DNA, on extracellular phage lambda. The lethal effects of both 6-3H-pyrimidines (the number of lethal hits per 3H decay) do not differ from those of [3H-methyl]thymidine and 5-3H-cytosine. The mutagenic effects (at equal survival rates) are as follows: 6-3H-thymidine approximately 3H2O less than [3H-methyl]thymidine less than 6-3H-cytosine less than 5-3H-cytosine. UV-irradiation of host cells induces a more pronounced W-mutagenesis with 6-3H-cytosine than with 6-3H-thymidine.

[W-mutagenesis in the bisulfite-treated lambda phage]. / W-mutagenez u faga liambda, obrabotannogo bisul'fitom.

Survival of phage lambda cI857 inactivated by bisulfite (pH 5.6, 37 degrees C) is higher (the dose modification factor approx. 1.2) and frequency of bisulfite-induced c-mutations 2-4-fold lower on the lawn of the wild-type strain ung+, as compared to ung-1 mutant deficient in uracil-DNA glycosylase. Irradiation of host cells by a moderate UV dose inducing SOS repair system enhances the frequency of bisulfite-induced c-mutations 2-3-fold in the wild-type (ung+) host, but not in the ung-1 mutant. It is suggested that W-mutagenesis in bisulfite-treated lambda phage in the ung+ cells is due to SOS repair of apyrimidinic sites which are produced during excision of uracil residues, the products of cytosine deamination.

[Lethal and mutagenic action of incorporated 5-3H-cytosine on extracellular phage lambda]. / Letal'noe i mutagennoe deistvie inkorporirovannogo 5-3H-tsitozina na vnekletochnyi fag lamda.

A study was made of the lethal and mutagenic effects on extracellular phage gamma of 5-3H-cytosine incorporated into DNA. The efficiencies of inactivation by incorporated 3H were equal for 5-3H-cytosine and [3H-methyl]-thymidine, but the yield of c-mutations for the former was 14 times higher. The lethal and mutagenic effects of incorporated 5-3H-cytosine did not depend on ung mutation of host cells which caused a deficiency in uracil-DNA-glycosylase. The mutagenic effect was not enhanced when SOS-repair system was induced by UV-radiation. The mutagenic effect of 5-3H-cytosine was associated with the modified mispairing bases but not with uracil residues.

[Lethal and mutagenic activity of tritiated water and incorporated [3H-methyl]-thymidine on extracellular phage lambda]. / Letal'noe i mutagennoe deistvie tritievoi vody i inkorporirovannogo [3H-metil]-timidina na vnekletochnyi fag liambda.

A study was made of lethal and mutagenic effects on extracellular phage lambda of beta-particles from tritiated water and of [3H-methyl]-thymidine incorporated into DNA. It was shown that the mean lethal dose D10 and the yield of c-mutations per unit of the dose absorbed during external beta-irradiation in 3H2O or during the decay of incorporated [3H-methyl]-thymidine were equivalent to those obtained during 60Co-gamma-irradiation in 4% nutrient broth or at a dried state respectively.

[Low transfecting efficiency of phage lambda ring chromosomes and their fragments formed by membrane nucleases]. / Nizkaia éffektivnost' kol'tsevykh khromosom faga lambda i ikh fragmentov, obrazovannykh obolochechnymi nukleazami, pri transfektsii.

Transfection efficiency of a number of lambda DNA samples differing in ring to linear molecules ratio was determined. Graphic extrapolation to the zero content of linear molecules showed that efficiency of ring molecules did not exceed 5% of that of linear molecules. Probably, this difference is caused by more fast penetration of linear molecules into the cell and, therefore, by lower probability of their degradation by cell wall nucleases. Fragments of both ring and linear molecules formed by cell wall nucleases proved to be inactive in marker rescue experiments.