Effect of Promising Antitumor Phenolic Antioxidant Anphen Sodium on the BCL-2 Family Proteins.

The proapoptotic effect of anphen (the effect on the level of the antiapoptotic protein Bcl-2) was investigated by immunoblotting. Incubation of Lewis carcinoma cell suspension with anphen at a concentration of 10-6 M for 0-3 h caused a 80% reduction in the level of the Bcl-2 protein and its homodimer. In vivo, when administered for 4 days to outbred mice, anphen (10-4 M) induced a decrease in the level of the Bcl-2 homodimer in the spleen cells by 20% and an increase in the content of the Bad protein (apoptosis activator) and the Bcl-XL protein. The antitumor effect of anphen may be due to blocking the hydrophobic pocket of the Bcl-2 protein.

Decrease in Bcl-2 Protein Level during the Development of Lewis Carcinosarcome.

We studied the development of Lewis carcinoma and possible antitumor effect of preliminary administered antioxidant anphen. The tumor was intramuscularly transplanted to C57Bl×DBA mice (7×106 cells per mouse). According to immunoblotting results, the content of anti-apoptotic Bcl-2 protein steadily decreased starting from post-transplantation day 11. In few days, its content decreased by 15-20% and soon the animals died. After administration of anphen, the content of Bcl-2 decreased more rapidly than in the control. Atomic force microscopy revealed a decrease in the mean volume of erythrocytes and then increase in this parameter at the terminal stage of tumor growth. These findings suggest that anphen does not affect the tumor growth rate and mouse lifespan, but enhances apoptosis of blood cells of animals with Lewis carcinoma at the terminal stages of tumor growth.

[Effects of smoking and tumor process on the contents of key proteins of apoptosis and activity of antioxidant enzymes in blood].

The effects of smoking on the contents of the apoptosis markers Bcl-2 and p53 proteins in blood plasma; the activity of the antioxidant (AO) enzymes Cu, Zn-superoxide dismutase (SOD), glutathione peroxidase (GP), glutathione reductase (GR), glutathione S-transferase (GST), and catalase; and the content of malondialdehyde (MDA) in erythrocytes from healthy donors and cancer patients were studied. Two groups of donors were revealed among healthy smokers: one with high SOD and GP activities and high Bcl-2 protein levels and the other with lower Bcl-2 levels compared with those found in nonsmokers. In the group of cancer patients (both smokers and nonsmokers), significantly increased p53 protein levels and increased activity of GST were found. A negative correlation between MDA and GST in the group of smoking healthy donors and a positive correlation between MDA and p53 in cancer patients were found. The results suggest a relationship between the components of enzymatic defence and lipid peroxidation and the content of apoptosis regulator proteins in healthy smokers and cancer patients.

[Impact of the antioxidant Phenozan and low dose radiation on the level of p53 and BCL-2 proteins of mice different lines].

The influence of the antioxidant Phenozan and 1.2 cGy gamma-radiation on the level of apoptotic proteins was determined for normal mice of F1 (CBA x C57BL) and leucosis AKR mice that are more sensitive to the irradiation. The constitutional level of p53 proteins in serum leucosis AKR mice was higher, than those in F1 (CBA x C57BL) mice, possible from accumulation of mutant p53 protein and viral infection of AKR mice. It was determined that injection of Phenozan in 10(-14) mol/kg and in 10(-4) mol/kg led to rising a p53 protein and bcl-2 protein level in serum and spleen in these line of mice, and lowering the number of double-strand breaks DNA spleen shown earlier. Common action Phenozan and 1.2 cGy gamma-radiation led to higher rising of p53 protein level than Phenozan only in F1 (CBA x C57BL) mice, stimulate possibly different pathway of p53 regulation. We assume that Phenozan can activate the reparation processes but not the processes of apoptosis in the cell and has a radioprotective properties.

[The effects of low doses of low-level gamma-radiation and phenozan injection on structural characteristics of mice spleen DNA].

It is well known that AKR mice with spontaneous leucosis are more sensitive to ionizing irradiation as compared to normal F1 (CBA x C57BL) mice. A study on changes of the structural characteristics of spleen DNA and level of protein p53 in the blood serum under the action of low-level gamma-irradiation in a dose of 1.2 cGy and injections of 10(-14) or 10(-4) mol/kg phenozan was performed. The changes in the structural characteristics of DNA (the adsorption on nitrocellulose filters and number of double-strand breaks) and p53 content were observed for each line of mice under gamma-irradiation and each phenozan concentration. Both factors showed long-time post-effects, and structural changes in AKR DNA were consistent with the life span of these mice. Phenozan in the above doses has abolished the induction of double-strand breaks in case of irradiation of F1 mice in a dose of 1.2 cGy and showed long-time post-irradiation effect. These facts suggest a radioprotection property of phenozan.

[Comparison of blood-serum p53 concentrations in patients with advanced breast tumors before and after chemotherapy].

Investigations of the role of p53 in tumorigenesis and growth, implementation of antitumor effect of cytostatics as well as emergence of tumor resistance have generally received great emphasis. Since most research was mostly concerned with use of tumor tissues, its dynamic aspects were ignored. Our study was concerned with p53 assay of blood serum from 10 patients with advanced breast tumors who underwent tests before and after a second cycle of chemotherapy. Due to immunoblotting technique, p53 was identified in all patients. Its concentration varied significantly and was twice as high in some as compared with the others. Prior to treatment, distinct differences were recorded in content as well as and in the nature of its age-dependent variation after chemotherapy. The highest levels were recorded in the age group over 60 yrs. In most patients (5 out of 6) under 55, post-treatment concentrations rose, on the average, by 13% while in all 4 cases of more than 60, they dropped by an average of 18%.

[P53 under low-intensity inluence of physical and chemical nature (ionizing radiation and antioxidant)].

It was found that low-intensity ionizing radiation and the antioxidant fenozan at a low concentration (10(-14) M) produce opposite effects on the content of protein p53 in the blood serum of mice. Thus, low-intensity gamma-irradiation of AKR mice with a dose of 1.2 cGy (0.6 cGy per day) led to a decrease in the content of p53 and acceleration of leukosis, whereas fenozan, which has membranolytic and radioprotective properties, when injected intramuscularly to F1 mice (CBA + c57 black) increased the content of p53. However, the dynamics of the activation of protein p53 depended on the concentration of fenozan (10(-4) or 10(-14)), which may be due to the difference in its binding to the membrane and the changes in its antioxidative properties depending on concentration.

[Changes in levels of p53 protein, immunoglobulin L-chains, and iron complexes in mice of leukosis strain AKR following low dose irradiation]. / Izmenenie soderzhaniia belka p53, L-tsepei immunoglobulinov i kompleksov zheleza pri obluchenii malymi dozami myshei leikoznoi linii AKR.

Changes in the content of protein p53 (regulator of the cell cycle) L-chains of immunoglobulins, and iron complexes (Fe2+) during the development of spontaneous leukosis in AKR mice and upon irradiation of animals with a dose of 1.2 cGy were studied by ESR spectroscopy, electrophoresis, and immunoblotting. It was found that irradiation leads to an increase in the incidence of leukoses in males by 7% and a decrease in life duration of females. A decrease in the content of protein p53 and L-chains in immunoglobulins in males and females was observed; however, in females, the decreases was less pronounced because the content of these proteins in females is naturally decreased. In mice irradiated with low doses at the age of three- to four months, a decrease in the amount of iron complexes at a later age (seven- to eight months) was registered. These data suggest that there is a relationship between the induction of protein p53 and the content of immunoglobulin L-chains in the blood serum of animals.

[Changes in the EPR spectra of the nitrosyl complexes of blood proteins in the low-intensity whole-body irradiation of mice]. / Izmenenie spektrov EPR nitrozil'nykh kompleksov gemoproteinov krovi pri nizkointensivnom total'nom obluchenii myshei.

After NO adding to mice blood and isolated erythrocytes ESR signal of nitrozyl complex HbNO (g = 2.07, g = 1.98) and NO-induced MetNg (g = 6.0) were registered. It was shown that the intensity of ESR spectra of these complexes increased after radiation of mice with a dose of 0.06, 0.6 and 5.4 cGy. Low-dose irradiation (0.6 and 0.06 cGy) caused the change in the form of ESR spectra of HbNO (g = 2.07), which is indicative of the shift from T-structure to R-structure and of the preferred formation of R-conformations of oxyhemoglobin in blood. It was found that dependence of NO-induced MetHb signal on irradiation dose is bimodal that may be connected with nonlinear response of the cells to irradiation and retarded adaptive response after radiation with low doses.

[EPR-spectroscopy of human blood preparations. Remote sequelae of the Chernobyl accident]. / EPR-spektroskopiia preparatov krovi cheloveka. Otdalennye posledstviia Chernobyl'skoi avarii.

It was shown, that as a result of Chernobyl accident the intensity of the EPR-signal of hemoproteins and ferroporphyrins of people blood increased, that most probably connected with disturbing of ferroprotein metabolism. Maximum intensity values had people, irradiated with low doses (to 2 cGy), that corroborate the theory of danger these doses for the people organism.

[Inhibition of endogenous oxidation of DNA by spin traps]. / Ingibirovanie éndogennogo okisleniia DNK spinovymi lovushkami.

It was shown that spin trap feeding resulted in a decrease of oxidation of rat DNA. This is an evidence of free radical mechanism of endogenous DNA oxidation.

[The effect of structure of benzimidazoles on the character of forming intramolecular cross-links in DNA and chromatin]. / Vliianie struktury preparatov klassa benzimidazola na kharakter obrazovaniia vnutrimolekuliarnykh sshivok v DNK i khromatine.

An investigation of a number of benzimidazole class preparations, being distinguished by a position of aminomethyl substitutes, has been carried out. It has been shown, that the non-substituted preparation BIO-10 does not form UV-cross-links in DNA and chromatine; BIO-40, having one substitute in the position 2, causes the formation of inter-molecular cross-links DNA-DNA. The preparation BIO-50, having 2 aminomethyl groups in the imidazole nucleus positions 2 and 6, forms cross-links DNA-DNA and DNA-protein in chromatine. The generation of radicals by the preparations BIO-10 and BIO-50 has been studied by the EPR-method by use of spin trap. It has been demonstrated, that BIO-10, unlike BIO-50, actively generates superoxide. A supposition has been made, that an UV-formation of superoxide-radical in the presence of BIO-10 might be a reason of DNA-macromolecule destruction.

[Titration of SH-groups of histone H3 in a DNP preparation of normal and neoplastic animal cells with a mercury-containing spin marker and with a DTNB preparation]. / Titrovanie SH-grupp gistona N3 v DNP normal'nykh i opukholevykh kletok zhivotnykh rtut'soderzhashchei spinovoi metkoi i preparatom DTNB.

DNP samples isolated from the cells of calf thymus and Ehrlich ascite carcinoma of mice were examined. SH-groups of histone H3 of chromatin from these cells were titrated with mercury-containing spin label and with DTNB under joint action of different salt and sarcosyl concentrations on DNP. The results revealed differences in accessibility and titration of histone H3 SH-groups in DNP of normal and tumor cells with DTNB, as well as in molecular dynamics of the mercury-containing spin label introduced to these SH-groups.

[The action of benzimidazole derivative preparations on the formation of DNA-protein cross-links in the UV irradiation of chromatin]. / Deistvie preparatov proizvodnykh benzimidazola na obrazovanie sshivok DNK--belok pri UF-obluchenii khromatina.

Effect of benzimidazole-derivatives on the DNA-protein binding formation was studied after UV-radiation of chromatin. These derivatives were shown to protect chromatin from UV-induced DNA-protein binding formation. Structural analog contained two aminomethyl residuals sensibilized additional binding formation in chromatin. Results suggested, that benzimidazole interacted with DNA, while aminomethyl groups interacted with protein and sensibilized binding of DNA with histone H1.

[Comparison of DNA interaction with sphingomyelin, spermine and magnesium ions using a spin label method]. / Sravnenie vzaimodeistviia DNK so sfingomielinom, sperminom i ionami magniia metodom spinovykh zondov.

Identical interactions of liposomes from sphingomyelin, spermine and magnesium ions with DNA was shown by spin probe method using spin-labeled 9-aminoacridine. The interactions proceed in the phosphate groups at the expense of hydrophobic part of phospholipid. The functional role of sphingomyelin--DNA interaction in matrix biosyntheses is discussed.

[Local homology of amino acid sequences of histones H3, H4 and the protein repressor lambda Cro. Possible conformation of homologous histone sites in DNP]. / Lokal'naia gomologiia aminokislotnykh posledovatel'nostei gistonov H3, H4 i belka repressora lambda Cro. Vozmozhnaia konformatsiia gomologichnykh uchastkov gistonov v DNP.

A mathematical analysis of amino acid sequences was carried out with a view of detecting possible homology between histones H3 and H4 and repressor-activator proteins of prokaryotes according to the A. I. criterion which reflects the similarity of their primary structure. It was found that the sites of eukaryotic histones H3 (102-123) and H4 (68-85) and site alpha 3 (24-25) of the prokaryotic repressor protein lambda Cro, i. e., the site of protein interaction with DNA, reveal a statistically significant homology. The A. I. value for the H3 site of lambda Cro is 3.37, that for the H4 site of calf thymus and sea horse is 3.28. The amino acid sequences of these proteins in the alpha 2-alpha 3 site, i. e., the site in which the homology between amino acid sequences of histones and DNA-binding proteins had been established previously, with regard to similarity of their secondary structure of the helix-turn-helix type, were analyzed. A pairwise comparison of H3 and protein lambda Cro showed that the A. I. value for histones H3 from various sources is approximately 2.7; however, the homology of the alpha 2 site is lower than that of site alpha 3. It is concluded that there exists an evolutionary relationship between homologous segments of histones H3 and H4 and protein lambda Cro, which can be preserved in order to maintain a definite secondary structure, presumably for binding to DNA.

[Characteristics of sulfhydryl groups of histone H3 from the calf thymus using mercury-containing nitroxyl radicals]. / Kharakteristika sul'fgidril'nykh grupp gistona H3 iz timusa telenka s pomoshch'iu rtut'soderzhashchikh nitroksil'nykh radikalov.

The interaction between total histone and deoxyribonucleoprotein (DNP) preparations from calf thymus with mercury-containing nitroxyl radicals in low ionic strength solutions, 2 M NaCl and urea was investigated. It was found that the label is rapidly incorporated into the SH-groups of histone H3 to produce characteristic EPR signals. Titration of SH-groups within DNP demonstrated that in low ionic strength solutions only one SH-group (presumably, the SH-group of the cysteine residue in position 110) is accessible to the reagents. After dissociation by 2 M NaCl, two SH-groups become titrable; however, the EPR spectra point to differences in the conformational state of these two groups. In 4 M urea, these differences are compensated for by structural disintegration. The spin labels may be used for the analysis of SH-groups under different conditions and at different functional states of nucleoproteins.