Thermoinduced structural changes in the membranes of animal organs following administration of antioxidants and during growth of malignancies

By means of weak-bound spin probes--stable iminoxyl free radicals differing in the level of hydrophobity there were studied thermoinduced structural transitions in the membranes of cell organells of animal liver after intraperitoneal injection of antioxidants and in the course of malignant growth, and in the nuclear membranes of Ehrlich ascite carcinoma as well. It has been found that during the growth of Ehrlich ascite carcinoma changes in rotationary mobility of probes in cell nuclei isolated from the liver of tumour-carrying animal are similar to the changes observed after antiocidants injection. A different pattern is observed in tumour cells. The membranes of ascite cell nuclei are characterized by a weak dependence of tau c on temperature for both probes. Within the temperature range studied no characteristic structural transitions proceeding in the nuclei of intact animals are observed.     

Karyotypic variability of a tumor cell population in the process of prolonged growth in the body]

An ascite variant of spontaneous mammary gland tumor of C3H/Sn mice was transplanted subcutaneously into syngenic mice. During the tumor growth (150 days) some alterations occur in the genetical structure of the tumor population: a change in the stem line, the increase and a further decrease of nearoctaploid cells.     

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.     

The role of endogenous glutathione in the action of sulfur-containing radio-protectors

In experiments on Ehrlich ascite tumor cells, the dependence of the radioprotective effect of beta-mercaptoethylamine and beta-mercaptopropionylglycine on the initial level of endogenous glutathione was studied. A varying degree of N-ethylmaleimide induced decrease of SH-glutathione content in the cells led to either easing or elimination of the radioprotective effect of the agents under study.     

In vivo action of valinomycin on Ehrlich ascites tumor cells. Inhibition of the proliferative activity of the tumor cells under the action of valinomycin]

It was shown that repeated administrations of valinomycin in doses of 1.0 and 0.01 gamma/gm to mice with Ehrlich ascitic tumors inhibited the ascite development. The radioautographic study using 3H-thimidine showed that 24 hours after the administration of valinomycin in doses of 0.1 and 0.01 gamma/gm transference of the cells into the phase of DNA synthesis was inhibited--inhibition of the tumor cell mitotic activity took place.     

Cytotoxic action of polymorphonuclear leukocytes on tumor and normal cells during ascite tumor development in vitro and in vivo

A vast number of studies, including the authors' own research, support the important role polymorphonuclear leukocytes (PMNL) in the development of ascite tumors. The method of luminol-dependent chemiluminescence (CL) was used to show the presence of two functionally different PMNL pools in a tumor-bearing organism: 1) "primed" PMNL, which circulate in the blood stream, and 2) "activated" PMNL, which are accumulated in the tumor zone and are capable of spontaneous CL. The purpose of the present investigation was to compare cytotoxic effects of primed and activated PMNL on tumor cells (ascite Ehrlich carcinoma (AEC), ascite Zajdel hepatoma) upon co-cultivation, as well as on normal cells of the organism, erythrocytes in vitro and in vivo. Upon stimulation with phorbol myristate acetate (PMA), PMNL effectively damaged AEC cells within the first 24 h until PMNL apoptosis occurred. Upon further co-cultivation, the tumor cells grew in number, which suggest the participation of PMNL in tumor protection. When stimulated with PMNL, pools suppressed tumor growth in vitro, since in this case the cytotoxicity was due to both reactive oxygen species and proteolytic enzymes. As it has been shown earlier by the authors, the functional potential of PMNL increases many times during tumor growth, and we suggested that not only tumor but also normal cells could be damaged. In this connection, we have studied the cytotoxic effect of primed and activated PMNL on rat erythrocytes in vitro on their co-cultivation. On stimulation with PMA, the rate of lysis of erythrocytes by primed PMNL increase many times compared to the norm. The fMLP-stimulated cytotoxity was 1.5-2.0 times higher than in the norm. Activated PMNL without stimulation are capable of producing only a partial lysis of erythrocytes (5-7 %). In order to assess the cytotoxic action of PMNL on erythrocytes in vivo, the hemoglobin content in erythrocytes and blood plasm of rats was measured in the course of tumor growth. The hemoglobin content in erythocytes during growth tumor decreased from 135 +/- 10 to 85 +/- 5 g/l, whereas in the blood plasm the hemoglobin content gradually increased by almost two times. The results enable us to suggest that one of death causes of tumor-bearing organisms may be the cytotoxic action of PMNL on normal cells of the organism caused by hyperproduction of ROS.     

Mechanism of differential effect of low dose adaptogens on the functional activity of normal and transformed cellular elements in vitro]

Influence of water solutions of chemically pure adaptogen--synthetic analog of Rhodiola Rosea extract phenol composition (SAR) on functional activity of hemopoietic and tumor cells of mice with Ehrlich ascite cancer was studied in vitro. The periodical character of SAR effects was shown to be different for both types of cells, and at 1 x 10(-2) and 1 x 10(-26) M concentrations simultaneous stimulation of blood marrow cells colony-forming activity and inhibition of the latter in tumor elements was revealed. Essential changes of reactions of both cell types after adding the DNA-dependent RNA polymerase blocker Actinomycin D permit to suggest SAR effects to be connected with drug influence on the membrane RNA of the target cells.     

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.     

Glutathione--an essential component of radio-modifying action of serotonin

5-HT applied prior to, immediately or 5 min after irradiation decreased equally the radiation damage to Ehrlich ascite tumor cells. A decrease in the endogenous glutathione content by N-ethylmaleimide (NEM) led to reduced the 5-HT radio-modifying effect. Its radio-modifying effect decreased irrespective of whether NEM was applied prior to or after irradiation.     

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]

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.     

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.     

Studies on the photosensitizing properties of angelicin, an angular furocoumarin forming only monofunctional adducts with the pyrimidine bases of DNA.

The bioligical photosensitizing properties of furocoumarins are due to the formation of adducts with the pyrimidine bases of DNA under irradiation with long wavelength ultraviolet light. The greatest importance is attributed to the difunctional adducts, which form cross-linkings between the 2 strands of DNA. As angelicin, photoreacting with DNA, forms only monofunctional adducts, and therefore no cross-linkings, its photosensitizing properties have been studied in order to evaluate the ability of monofunctional adducts to produce biological effects. The results obtained studying the inhibition of DNA, RNA and protein synthesis in Ehrlich ascite tumor cells after irradiation in the presence of angelicin and psoralen (for a comparison), and the inhibition of the ability of identically treated cells to transmit the tumor showed a remarkable ability of monofunctional adducts to produce biological effects.     

Dynamic changes in the pH and potassium concentration in a suspension of ascitic tumor cells under glucose load

A single addition of glucose to 10% (6 v/v) suspension of the Ehrlich ascite tumor cells results in biphasic changes of pH and potassium ion concentration in the extracellular medium during 120 minutes. Phases of pH and potassium changing do not coincide. Increasing glucose amount from 100 to 400 mumol/10 ml suspension causes no marked modification of ion dynamics. Possible mechanisms of the above phenomena and their relation to cell power are discussed.     

A simple method for isolation of DNA fragments associated with the nuclear lamina in vivo

We describe a simple method for the purification of DNA fragments associated with the nuclear lamina in vivo. Ehrlich ascite tumor cells are first u.v.-irradiated to crosslink DNA to proteins. The nuclear lamina is then isolated and purified by low-speed centrifugation through a cushion of 40% sucrose. The material sedimenting through the created density barrier represents nuclear lamina of a very high purity, free from any DNA fragments except those which were in a crosslinking distance to it in vivo.     

Cytochemical study of cells of primary and disseminated ascite Yoshida tumor cells

The different distribution of cytochemically demonstrable enzymes: lactate dehydrogenase (LDH, 1.1.1.27), succinate dehydrogenase (SDH, 1.3.99.1), dihydrofolate reductase (DHFR, 1.5.1.3), acid phosphatase (AcP, 3.1.3.2) and alkaline phosphatase (ALP, 3.1.3.1), has been documented in Yoshida ascites hepatoma cells in vivo or stored at 80 degrees C. The dehydrogenase activities (LDH, SDH, DHFR) show a strong reaction in all samples. An increased level of these enzyme activities has been observed in the malignant cells spreading through the organs of tumor bearing rats. On the contrary, in the same samples, acid and alkaline phosphatase activities are very low. The strong dehydrogenase activities observed in Yoshida ascite cells stress the rapid turnover of tumor cells. Our results indicate that the histochemical method may be a useful tool to detect the scattered tumor cells. Furthermore, the cytochemical methods allow the characterization of the metabolic pathways employed by the primary and disseminated tumor cells.     

Transmembrane transfer of sodium ions in cell membranes of normal and tumor cells

Changes of sodium transport in tumor cells and their normal analogs were studied. For the first time we used a direct method of investigation with sodium-selective electrodes which permitted the study of ion transport in dynamics. As a result of investigation of sodium transport on malignant transplanted cultures of fibroblasts (7L strain) and on their normal prototypes--embryonic fibroblasts of hamsters, and also on the cells of Ehrlich ascite tumor--it was shown that when transferred the tumor cells in 15% hypertonic solution significant active transport of ions from the cells into the external medium was recorded. This phenomenon was not found in the normal cells. The experiments showed that in the process of malignancy significant changes began in the cellular membranes connected with the disturbance of activation of some enzymes, particularly Na,K-ATPase.     

DNA degradation and changes in the permeability and form of Ehrlich ascites tumor cells when incubated in an anaerobic medium without glucose

After a 3-hour incubation of the Ehrlich ascite tumor cells in buffered Hanks solution, without glucose and oxygen, the extensive cell injuries were observed. The time-course of appearance of these injuries was as follows: cell blebbing, staining of the cells with trypan blue, and then their staining with ethidium bromide. The DNA degradation registered with fluorometric method coincided in time with cell staining with trypan blue. All injuries (except DNA degradation) were delayed at pH 6.0 compared with those at pH 7.3. Glucose added to the cell suspension greatly protected the cells from these injuries, although DNA degradation at pH 6.0 in these conditions was a little higher than that at pH 7.3.     

Localization of antigens in the chromatin of normal rat liver cells, not detectable in hepatoma chromatin

Antibodies against rat liver chromatin interact with homologous chromatin as well as with chromatin of Zajdela ascite hepatoma and solid hepatoma 27, but not with the nuclear matrix isolated from these hepatomas. Rat liver chromatin regions hypersensitive to DNAase I and endogenous Mg2+-dependent nuclease are enriched with immunogenic nonhistone proteins. Using antiliver IgG pretreated with chromatin of Zajdela ascite hepatoma and solid hepatoma 27, it was shown that liver chromatin antigens that are not detectable in hepatoma cells are localized in hypersensitive to nucleases chromatin regions buy not in actively transcribed ones.     

An inhibitor of translation in cellular mRNA containing the 100S structure from Krebs II ascites carcinoma cells infected with encephalomyocarditis virus

As shown previously, the bulk of cellular mRNA in Krebs II ascite carcinoma cells infected with encephalomyocarditis virus during active virus-specific synthesis is bound to ribosomes within the 100S structure which is inactive in protein synthesis. In order to elucidate the reasons for the translation repression of cellular mRNA within the 100S structure, a fraction of loosely bound proteins which are liberated by treatment of the 100S structure with 0.5 M KCl an which contain sum translation factors, was obtained. This fraction was shown to contain an inhibitor which non-specifically represses the translation of endogenous viral and cellular mRNA within the composition of polyribosomes and of exogenous poly(A)-containing cellular mRNAs from ascite carcinoma cells.     

Studies of interaction of intracellular signalling and metabolic pathways under inhibition of mitochondrial aconitase with fluoroacetate

Mitochondrial aconitase has been shown to be inactivated by a spectrum of substances or critical states. Fluoroacetate (FA) is the most known toxic agent inhibiting aconitase. The biochemistry of toxic action of FA is rather well understood, though no effective therapy has been proposed for the past six decades. In order to reveal novel approaches for possible antidotes to be developed, experiments were performed with rat liver mitochondria, Ehrlich ascite tumor cells and cardiomyocytes, exposed to FA or fluorocitrate in vitro. The effect of FA developed at much higher concentrations in comparison with fluorocitrate and was dependent upon respiratory substrates in experiments with mitochondria: with pyruvate, FA induced a slow oxidation and/or leak of pyridine nucleotides and inhibition of respiration. Oxidation of pyridine nucleotides was prevented by incubation of mitochondria with cyclosporin A. Studies of the pyridine nucleotides level and calcium response generated in Ehrlich ascite tumor cells under activation with ATP also revealed a loss of pyridine nucleotides from mitochondria resulting in a shift in the balance of mitochondrial and cytosolic NAD(P)H under exposure to FA. An increase of cytosolic [Ca2+] was observed in the cell lines exposed to FA and is explained by activation of plasma membrane calcium channels; this mechanism, could have an impact on amplitude and rate of Ca2+ waves in cardiomyocytes. Highlighting the reciprocal relationship between intracellular pyridine nucleotides and calcium balance, we discuss metabolic pathway modulation in the context of probable development of an effective therapy for FA poisoning and other inhibitors of aconitase.