Antitumor resistance formation in animals in early neonatal period.

Immunization of new-born mice during the first three days after birth enhances immunity in adult life, and it remains high throughout the entire life course. The antitumor resistance is raised so high in order to produce cancerogenesis blockade caused by 20-methylcholanthrene injection to adult animals even one year after vaccination. The dynamics of growth of new-borns shows that immediately after birth the animal growth proceeds very quickly, following an exponential law. The same occurs in rats and even in human beings. Vaccination of animals during fast growth phase ensures an antitumor effect, while vaccination after fast growth phase leads to antitumor immunity breakdown. It was assumed fetal lymphocytes do not attain full sexual maturity and their cytotoxic properties start developing only after birth. This hypothesis is in full agreement with our results. Vaccination of new-borns during fast growth phase leads to a significant increase of immature lymphocytes of those clones which are sensitive to an injected antigen. Upon vaccination termination, these lymphocytes attain complete maturation and remain in tissues, thereby ensuring a prolonged and high immunity. Our earlier experiments clearly demonstrated the increase in lymphocytes number in animals which had been exposed to early postnatal immunization. The kinetic analysis showed that full tumor destruction would be only possible if the rate of tumor cells destruction by cytotoxic lymphocytes is greater than the rate of tumor cells reproduction, as the tumor is incapable to make up for loss of its own cells. Immunization during fast growth phase does allow maximum increase in the number of cytotoxic lymphocytes and, thereby, enhance an entire cell immunity - unattainable after full lymphocyte maturation.

Antitumor resistance activation in mice: can the immunological memory cells enhance resistance?

Immunization of adult animals with the Ehrlich ascytic cancer cells homogenate three months prior to an experiment, did not affect either tumor transplantation or the progress of cancerogenesis induced by injection of 20-methylcholanthrene oil solution into the femoral muscle. All consequences of adult animal vaccination disappeared in 30-40 days following antigen administration. Quite different consequences were observed after immunization of the newborn mice. The same antigen (Ehrlich cancer cells homogenate) injected to newborn mice on days 1 and 3 after birth in a dose that failed to develop tolerance not only significantly increased the ascytic tumor transplantation threshold (by nearly 200 times for sarcoma 37 cells and by nearly 400 times for Ehrlich cancer cells) in adult animals but also led to almost 50% inhibition of cancerogenesis (induced by injection of 20-methylcholanthrene oil solution in the femoral muscle of mature mouse) after three and even after 12 months following immunization. The MTT-analysis did not reveal any noticeable differences in the number and activity of the cytotoxic lymphocytes in populations of splenocytes obtained from the intact mice (control) and from the adult animals which had been exposed to postnatal immunization (experiment).However, after a new vaccination such differences were found. In the populations of splenocytes obtained from control animals, the cytotoxic activity measured on day 10 after vaccination had increased 2.86-fold mainly at the expense of an increased number of effector cells. In the populations of splenocytes obtained from the experimental group of animals the activation was much greater (25.8-fold), being accomplished not only at the expense of an increased number of the effector cells, as observed in the control group, but also at the expense of their higher activity. The kinetic analysis of a mechanism of effector cells/target cells interaction has led to derive equations for estimation of the limiting rates of such interaction and of the equilibrium constants for interacting cells. Analysis of a generally accepted mechanism of the cytotoxic lymphocytes formation, with an account of the kinetic analysis data, has shown that a major reason of low antitumor resistance of animal organism is the negligible population of resting cells--the precursors of antitumor cytotoxic lymphocytes. Newborn mice vaccination does not produce any increase in the number of resting cells of the necessary type. This circumstance explains both, increase of the ascytic tumor transplantation threshold and increase of the resistance to 20-methylcholanthrene action in adulthood. Adult animal immunization does not possess such action. Analysis of the problem leads to the conclusion that the system of organism's antitumor resistance becomes effective only in those cases when, owing to antigen activation of resting cells, the concentration of cytotoxic lymphocytes rises to such an extent that the rate of tumor cell destruction becomes greater than the rate of target cells reproduction.

Morphometric patterns and functional activity of the ascites tumour cells at different cell cycle stages.

Comparison of the morphometric patterns obtained by electron microscopy with data from the biochemical analysis of metabolism in Ehrlich ascites tumour cells and P-388 leukemia cells, in each phase of the cell cycle, shows a distinct correlation between structure and function which is demonstrated by the fact that stage duration in the structure changes coincides with metabolism alteration. Cell volume and total protein content increase simultaneously in all cell cycle stages. Decrease in the heterochromatin fraction in the nuclei occurs against the sharp DNA reduplication and transcription background. Throughout a cell cycle, the cell respiration intensity gradually increases. Simultaneously the number and functional activity of mitochondria increase.

Energy supply of the mitotic cell cycle and the Na+/H+-antiport in ascites tumors.

The activation of Na+ transport is due to the exchange of protons formed via glucose conversion into lactate for Na+, i.e., to the stimulation of the Na+/H+-antiport. Experimental results and theoretical calculations suggest that in glucose-containing medium the Na+ transport increases from 0.75 to 1.78 pmol/hour per cell. The permeability of plasma membranes for K+ increases 2.75 fold, while the passive flux of Na+ diminishes. The intensity of O2 adsorption by ascites tumor cells does not practically depend on the monovalent cation concentration gradient between the cells and the culture medium, whereas the rate of glycolysis decreases simultaneously with the diminution of the concentration gradient. In synchronized cultures at the beginning of the mitotic cycle, the bulk of ATP resynthesized via glycolysis is utilized for the synthesis of biopolymers, whereas that at the end of the S-phase and in the G2-phase is utilized for cation transport across plasma membranes. From 35 to 100% of the whole amount of ATP resynthesized via glycolysis is utilized for transport purposes. It is concluded that the observed increase in the Na+/K+ ratio in ascites tumor cells is connected with their enhanced ability to synthesize lactic acid. Presumably, glycolysis is one of the regulatory mechanisms of intracellular ratios of monovalent cations.

Ascitic tumor cell cycle metabolism and energetics.

Data are presented on Ehrlich ascitic tumor cells energetic metabolism, activities of the glycolytic enzymes and the pentose phosphate pathway enzymes, contents and synthesis rates of the macromolecules at various cell cycle stages. An attempt was made to correct the direct measurement by taking into consideration a systematic error introduced in the experiment by incomplete cells synchronization. Cell metabolism activation sharply increased at two mitotic cycle stages. At the first stage (end of G1-period, beginning of S-period) the processes associated with the preparation to reduplication and DNA synthesis were activated. The second activation wave (end of S-period, G2-period) was connected with cell preparation of mitosis. The coordination of cell metabolism variations during the cell cycle is shown.

Isolation of low molecular protein P-9 from nuclei of NK/Ly lymphoma and Guerin carcinoma.

Protein with molecular weight 9000 (protein-9) was found in nuclei of resting cells of NK/Ly lymphoma and Guerin carcinoma by electrophoresis in polyacrylamide gel. Method of preparative isolation P-9 concluding in fractionation of nuclei from phenol extract by acetone at different pH and following electrophoresis in polyacrylamide gel was made. Electrophoretically homogeneous preparation of protein P-9 from nuclei of NK/Ly lymphoma and Guerin carcinoma was obtained.

The influence of glucose, succinate, pH of the medium and higher temperature on the cytotoxic activity of the preparation Ukrain.

Cells of ascitic forms of Ehrlich's carcinoma and sarcoma 37 transplanted into C57BI/6 mice were used to study the influence of glucose, succinate, and pH of the medium on the cytotoxic activity of the preparation Ukrain. Viability of cells was estimated by the method of intravital staining with tripan blue after the incubation of cell suspension in the presence of the preparation (1.05 x 10(-5) mol and 1.69 x 10(-4) mol). It was established that glucose (5 mmol) reduces the cytotoxic activity of Ukrain, while succinate (5 mmol) increases it. The activity of the preparation was practically absent at pH 6.1-6.7 of the incubation medium and was at a maximum at pH 7.3-8.0. A temperature of 41.5 degrees C has no influence on the effectiveness of Ukrain.