Correlation of the increase in DNA methylation and antioxidant activity of mouse liver nuclear lipids after administration of antioxidant and in Ehrlich ascites carcinoma

The content of 5'-methylcytosine in total DNA of mouse liver increases 2--2,5-fold 3 hrs after a single intraperitoneal injection of antioxidant (4-methyl-2,6-ditretbutylphenol) (20 or 60 mg per 1 kg of body weight) and makes up to 2--2.4 mol.%. The methylation of liver DNA is also increased more than 2-fold in Ehrlich ascite carcinoma. The DNA isolated from mouse liver after administration of antioxidant or during cancer growth markedly differs from liver DNA of intact animals in its CH3-accepting ability under in vitro methylation by the methylase complex from Enterobacter cloacea. The changes in DNA methylation in mouse liver under the effects of antioxidant and in Ehrlich ascite carcinoma are correlated with the changes in the antioxidant activity of liver nuclear lipids.     

Regulation of lipid oxidation and structural state of the nuclear membrane after the irradiation of tumor-bearing animals

Changes of antioxidative activity (AOA), lipid composition and microviscosity of different membrane regions in tumor cell nuclei and in the liver of tumor-host with Ehrlich ascite carcinoma (EAC) after irradiation were studied. On the basis of the obtained data the analysis of the control system of lipid oxidation in the membrane was carried out. This control system involves a relationship between AOA changes, lipid composition, their oxidative ability and the nuclear membrane structure. It was shown that after irradiation the control system in the nuclei of tumor cells had the same state as before irradiation and was different from the normal one. The control system in the nuclei of tumor-host liver after irradiation starts to work in a regime which is characteristic of irradiated cells. It was shown that the principle difference in the control system functioning in tumor and tumor-host nuclei disappeared after irradiation.     

Damage of cell membranes by linoleic acid hydroperoxide

It has been shown on Ehrlich ascite carcinoma cells that under the effect of linoleic acid hydroperoxides in vitro ionic permeability and membrane capacity of the cells sharply decrease after some threshold concentration of hydroperoxides (greater than 10(-5) M), while the threshold value decreases with the increase of the time of cell incubation in the presence of hydroperoxides. Interrelationship between the development of induced POL processes in the cell membranes and disturbance of their functional-structural state in the living cell is discussed.     

Use of liposomes for incorporation of alkylating derivatives of mono- and oligonucleotides into mammalian cells]

It was shown that within the liposomes mono- and oligonucleotides and their alkylating derivatives penetrate the cells of Ehrlich ascite carcinoma and peritoneal exudate of the mice. Inside the cells the alkylating reagents are mainly utilized for modification of proteins (42--76%), RNA (5--16%) and DNA (3--9%). Presumably DNA modification is largely dependent on the penetration of the reagents into the nuclei. No significant differences in alkylation of the cell components by oligoadenylate derivatives, capable of complementary interactions with nucleic acids and mononucleotide derivatives, incapable of such interactions, were observed.     

Changes in the ultraviolet fluorescence and the size of Ehrlich ascites carcinoma cells in the 1st hours after X-ray irradiation

Using the Ehrlich ascite carcinoma cells, a decrease in ultraviolet fluorescence (UVF) was demonstrated one hour following a 10 Gy X-irradiation, and an increase in UVF 5 hours following the same irradiation. The same changes were demonstrated for cell turbidity. Association between the optical changes and those in cell radius and membrane protein state is discussed.     

Neutral glycosphingolipids in Ehrlich ascites carcinoma cells

The structure of the neutral glycosphingolipids of the Ehrlich ascite carcinoma (EAC) cells was studied. The main four components were identified as glycosylceramide, lastosylceramide, N-acetylgalactosyllactosylceramide and galactosyl-N-acetyllactosylceramide (asialo-GM1). The neutral glycolipid pattern of the cells was found to depend on their density. Dilution of the cell suspension resulted in an increased content of asia-lo-GM1, whereas the content of the other neutral glycolipids remained unchanged. The possible connection between these changes and the earlier disclosed cell density dependence of the gangliosides in EAC cells is discussed.     

Secondary structure and methylation of DNA in reinoculated tumors and cultures of embryonic cells transformed by oncogenic viruses]

Using the kinetic formaldehyde method the concentration of secondary structure defects (SSD) in the DNAs of ascite leukosis L 1210 cells and cultures of hamster embryonic cells transformed by virus SV-40 and polyoma was evaluated. It was found that this concentration was considerably higher than in the DNAs from normal liver and primary culture of mouse embryonic cells. The occurrence of the defects in malignant cell DNAs is not due to enzymatic degradation of DNA. Using thin-layer chromatography the content of m5C in the DNAs from 17 sources (transformed cell cultures, experimental tumours and liver cells of mouses with Ehrlich ascite carcinoma) were determined. The methylation level for all these DNAs was higher than for normal mouse and rat liver DNAs. No correlation between the SSD concentration and m5C content in the DNAs studied was observed.     

Sensitivity of intranuclear glucocorticoid complex from normal rat liver and Zajdela ascites hepatoma to ionic strength and nuclease treatment

After 30 min of intraperitoneal injection of dexamethasone to adrenectomized rats about 15% of the label are incorporated into liver homogenate and only 1% of the cytosol-bound hormone is detected in the cell nuclei. The binding of the "in vitro" injected hormone by the nuclei does not obey the second-order reactions (the Scatchard plots). This is probably due to the existence of various ancillary mechanisms, which control the translocation of the hormone complex into cell nuclei at the level of cytoplasm and nuclear membranes. DNAase I, micrococcal nuclease and endogenous nucleolysis markedly increase the part of the nuclear hormone complex resistant to 0.4 M NaCl. In hepatocyte nuclei obtained by the collagenase method, the content of the 0.4 M NaCl-resistant receptor complex is also increased. The resistance of 0.4 M NaCl was also found in 80% of the glucocorticoid-insensitive nuclear complex from Zajdela ascite hepatoma cells. The changes in interaction of the hormone-receptor complex with nuclear acceptor sites eventually resulting in impaired sensitivity of host tissues to hormonal control can be due to the damage of chromatin structure induced by different influences and tumour growth.     

Elevated polyamine levels in erythrocytes of SLC:ICR mice inoculated with ehrlich ascites carcinoma cells

Ehrlich ascites carcinoma cells (4×10⁵ cells/mouse) were inoculated intraperitoneally in 7-week-old SLC:ICR mice, and polyamine levels in peripheral erythrocytes and in ascites cells were determined periodically. Polyamine levels in peripheral erythrocytes increased linearly until 10 days after cell inoculation, while ascites cells showed exponential growth.The effect of carbazilquinone on cellular growth and polyamine levels in erythrocytes was also studied. When 1 or 2mg/kg of carbazilquinone was injected intraperitoneally on day 4 or on day 7, cellular growth was suppressed and the survival time of the mice was lengthened. The polyamine levels in erythrocytes were also markedly decreased 3 days after the carbazilquinone injection.These results suggest that the polyamine levels in peripheral erythrocytes are closely related to the cellular growth of Ehrlich ascites carcinoma cells.     

Proteinase activity of nuclei from various tissues towards endogenous histones]

The proteinase activities of nuclei isolated from tissues differing in their mitotic activities (brain, thymus, liver, ascite lymphoma) towards the histones and non-histone acid -- extractable proteins were studied. The sensitivity of different histone fractions to nuclear proteinase depends on temperature and time of nuclei incubation under conditions providing for complete dissociation of chromatin proteins from DNA (2 M NaCl--5 M urea). The proteinase activity in the brain and thymus nuclei is revealed only under prolonged (43 hrs) incubation of the nuclei at 25 degrees C, which is accompanied by partial proteolysis of histone H1. Histone H4 from brain nuclei and histone H2a from thymus nuclei are preferably degraded. In the nuclei isolated from the mice ascite cell lymphoma NK/ly and from rat liver the enzyme activity is revealed mainly towards the arginine-enriched histones H3 and H4. The proteolysis of the arginine-enriched histones in tumour cell nuclei is more complete. A high sensitivity to proteolysis was observed for non-histone acid-extractable proteins with low electrophoretic mobility, which were found in brain and tumour cell nuclei.     

Effects of exposure of different skin areas to low-power laser light

The effect of He-Ne laser light of extremely low power (632.8 nm, 0.2 mW/cm²) on the immune status of mice bearing solid tumors was studied. The state of tumor-bearing animals was assessed taking into account the number of immunocompetent cells, concentration of cytokines (tumor necrosis factor and interleukin-2), production of nitric oxide, expression of heat shock proteins 70 and 90, and the activity of natural killer cells. The model of a solid tumor was formed by subcutaneous transplantation of Ehrlich ascite carcinoma cells to mice; the average lifespan of animals was approximately 55 days. Different areas of skin of tumor-bearing mice were irradiated with laser light either singly (1 min; dose, 0.012 J/cm²) or repeatedly (1 min every 3 days over 30 days; total dose, 0.1 J/cm²). It was established that long-term chronic exposure of mice bearing Ehrlich ascite carcinoma cells to low-power laser light in the thymus projection area and especially in the tumor projection area leads to a decrease in the natural antitumor potential, which is manifested in acceleration of tumor growth and a tendency to decrease in the lifespan of tumor-bearing mice. Conversely, stimulation of antitumor immunity was observed over several days after a single exposure to low-power laser radiation. The results suggest that it is expedient to continue studies of the immunomodulating effects of low-power laser light and demonstrate the necessity of monitoring the immune system in the course of laser therapy.     

Action of embryonic thymus and liver cells on the development of the ascitic form of Ehrlich's carcinoma

The influence of the cells of embryonic thymus and liver on the development of Ehrlich carcinoma was studied. The intraperitoneal injection of the embryonic cells in the adult mice infested by the Ehrlich carcinoma resulted in a marked lengthening of the life time of animals and an increase of the survival percentage. The embryonic cells of thymus and liver inhibited sharply the growth of carcinoma cells in the diffusion chambers as well. In contrast to this, the thymus and bone marrow cells of adult animals, taken in the same concentrations as the embryonic cells, exhibited only a slight inhibiting effect on the growth of tumour cells. On the basis of these data a suggestion is put forward to the effect that the embryonic immunocompetent cells determine the stronger inhibition of tumour growth in the embryos as compared with the adult animals.     

A high-resolution 31P-NMR study of the disorders in the energy metabolic system of Ehrlich ascites cancer cells exposed to antitumor preparations]

The disturbance in energetic metabolism of Ehrlich ascite carcinoma cells during antitumor drug treatment was examined using high-resolution 31P-NMR. The value of antitumor drug effect was shown to be characterized by the kinetics of ATP and KF level alteration in tumor cells. The results correlated with the indexes of therapeutical activity of studied drugs.     

Membrane-bound Ca2+ in the mitochondria of Ehrlich ascitic tumor cells

Ca2+ transport in mitochondria of Ehrlich ascite tumour cells and in liver mitochondria has been compared. It has been shown that in tumour cell mitochondria unlike liver ones even small amounts of Ca2+ caused marked increase in membrane-bound Ca2+ level. Therefore, a decrease in the electro-neutral Ca2+ efflux, stabilization of mitochondria membranes and inhibition of phosphorylated respiration were observed. It has been proposed that high content of membrane-bound Ca2+ is predetermined by a higher affinity of membrane phospholipids to Ca2+.     

The in vivo arrest of Ehrlich ascitic carcinoma cells in the G2 phase of the cell cycle

Evidence for arrested G2 cell subpopulation in the Ehrlich ascite carcinoma has been provided using flow cytometry stathmokinetic and bromdesoxyuridine--Hoechst 33258 techniques. After exposure in DME--F-12 media G2-arrested cells synchronically enter mitosis and then move trough G1 period. Durations have been measured for G1. G2 phases and G2-G1 transition.     

Effect of adrenaline on the mitosis-inhibiting action of a chalone-containing preparation in Ehrlich ascites tumor

The effect of adrenaline and Ehrlich ascite carcinoma (EAC) chalone on cell division was studied. It has been established that EAC chalone inhibited cell proliferation. The action of adrenaline was also accompanied by a decrease in mitotic index, but the inhibitory effect of the hormone was weaker than that of chalone, it occurred later and its duration was less. A combined effect of adrenaline and chalone depended on the time interval between the administration of the substances. It has been found that chalone administration 1 h after adrenaline administration prolonged mitotic inhibitory effect by 4 h and its synchronous action on cell division in EAC was weak during the experiment. Combined effect of adrenaline and chalone did not differ from the effect of chalone alone if chalone was administered 3 h after adrenaline administration.     

Dependence of the nature of the action of metabolic inhibitors on ribosomal RNA synthesis in Ehrlich ascites carcinoma cells on cell integrity

Ribosomal RNA (rRNA) synthesis in the intact Ehrlich ascite carcinoma cells is selectively inhibited by papaverin (ED50 = 0.01 mM), 2,4-dinitrophenol (DPN; ED50 = 5 microM), and actinomycin D (ED50 = 0.1 microgram/ml). The inhibition of rRNA synthesis is not connected with a direct action of these agents on the rRNA synthesis apparatus, and they had no effect on isolated cell nuclei. The rRNA synthesis in cells permeabilized with triton X-100 (0.05%) becomes insensible to the action of papaverine and DPN, but is inhibited by actinomycin D in low doses. In cells permeabilized with digitonin (0.01%) the rRNA synthesis shows no sensibility to the action of low doses of actinomycin D. The results suggest that the action of these agents on the rRNA synthesis may depend on cell integrity and on the permeabilization method employed.     

Role of microviscosity of the lipid phase of microsomal membranes in regulation of enzymatic activity of glucose-6-phosphatase under microsomal modifications in vivo and in vitro

The changes in microviscosity of the lipid phase of microsomal membranes under microsomal modification in vivo and in vitro were studied. It was shown that in intact microsome lipids there occur five thermo-induced structural transitions within the temperature range of 5--50 degrees. Delipidation of microsomes results in a shift in structural transitions temperature. Based on the literary and own data it was assumed that the breaks on the Arrhenius plots for glucose-6-phosphatase (EC 3.1.3.9) activity are due to phase-structural changes of microsomal lipids.     

Study of the effect of tegalide and bithionol on biological membranes using fluorescent probes

Fluorescent probes were used to study structural changes in different membranes affected by gamma-radiation, protonophores and radioprotective agents, tegalide and bithionol. The preparations of the defined concentrations decreased the microviscosity of membranes, lowered the peaks and changed the temperature of phase transitions in liposomes from dipalmitoyl lecithin, and induced the output of Ca2+ from mitochondria. The effects depended on the radiation dose, the structure, concentration and lipophilicity of the preparation; protonophores produced a specific effect.     

The effect of phorbol ester in a wide range of concentrations on the lipid structure of microsome membranes of tumor cells in vitro

The action of 12-O-tetradecanoyl-13-acetate (TPA) in vitro in a wide range of concentration from 10(-3) mol/l down to ultra-low doses 10(-23) mol/l and dilution 10(-24) mol/l on the microsome membranes isolated from tumor--Ehrlich ascite carcinoma (EAC) has been studied by ESR-method using two spin probes: 5- and 16-doxyl stearates (5- and 16-DS) localized in the different regions of lipid bilayer. From the ESR spectra obtained it was calculated the following parameters: an order of the long axis 5-DS (S) related to order of the fatty acids chains in the lipid bilayer; two rotation correlation times (Tc1 and Tc2) of 16-DC to estimate a microviscosity value and structural-sensitive ones. It was found the stage changes of all these parameters (increase and decrease) as compared with control level (the membranes untreated by TPA) depending on TPA concentration into the range of 10(-3)-10(-24) mol/l; in particular, the most significant shape changes of structural-sensitive parameters have been observed at TPA doses below 10(-16) mol/l. It is concluded that tumor membranes are very sensitive to TPA action in vitro in a wide range of concentration included ultra-low doses.