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.

Intracellular ratio between monovalent cations (Na+/K+), and transcriptional activity of genes in tumour cells.

The effects of the intracellular Na+/K+ ratio on transcriptional activity of ribosomal, c-fos, beta-actin and histone genes of Ehrlich ascites tumour cells and P-388 leukaemia cells have been investigated. Optimum values of the Na+/K+ ratio for selective transcription of genes are shown, they are in accordance with the Na+/K+ ratio and mRNA content of respective genes during the cell cycle. Differential activation and repression of these genes do not correlate with the total synthesis rate of RNA during the cell cycle. The data indicate a selective nature of the action of monovalent cations on gene expression during the cell growth.

[Effects of intracellular Na+/K+ ratio on expression of c-fos oncogene and beta-actin gene in ascitic cells of leukemia P-388 and Ehrlich tumor]. / Vliianie vnutrikletochnogo sootnosheniia Na+/K+ na ékspressiiu onkogena c-fos i gena beta-aktina v astsitnykh kletkakh leikoza P-388 i raka Erlikha.

The intracellular Na+/K+ ratio was studied for its effect on expression gene beta-actin and oncogene c-fos and activity of these genes during the mitotic cycle in ascites cells of leukemia P-388 and Ehrlich tumour. It was established that gene beta-actin was activated at high and low ratios of Na+/K+, while c-fos only at high ones. The expression of these genes during the mitotic cell cycle was due to changes in the intracellular Na+/K+ ratio. The obtained data showed an important role of intracellular homeostasis of monovalent cations in regulation of gene expression during the mitotic cell cycle.

[Effects of intracellular Na+/K+ ratio on histone gene expression in ascitic cells of leukemia P-388 and Ehrlich cancer]. / Vliianie vnutrikletochnogo sootnosheniia Na+/K+ na ékspressiiu gistonovykh genov v astsitnykh kletkakh leikoza P-388 i raka Erlikha.

The intracellular Na+/K+ ratio was studied for its effect on histone genes expression in Ehrlich carcinoma and leukemia P-388 ascites cells. After equilibration with certain Na+/K+ ratio buffers at 0-4 degrees C the cells were treated during 1 h at 37 degrees C with ouabain added to the corresponding medium. Then total RNAs were extracted and analyzed by the reaction of blot-hybridization with histone genes cluster in plasmid p604. The maximal level of histone genes expression in leukemia P-388 cells was observed under conditions when intracellular Na+/K+ ratio corresponded to those at S-phase of the mitotic cycle. In Ehrlich carcinoma ascites cells the expression of histone genes was not dependent on the Na+/K+ ratio. A conclusion is drawn that the intracellular ratio of monovalent cations is one of the possible mechanisms which regulates the gene expression during the mitotic cycle.

[The effect of inhibitors of ionic passive transport on the structure of a cell population of Ehrlich's ascites carcinoma]. / Vliianie ingibitorov passivnogo transporta ionov na strukturu kletochnoi populiatsii astsitnoi kartsinomy Erlikha.

Triampur is a drug, containing triamterene and dichlotiazide, which inhibits passive influx of Na+ through cell membrane. Triampur was injected intraperitoneally to mice with the Ehrlich ascite carcinoma. The drug was injected in the exponential phase of tumor growth with 2 hour intervals within 24 hours. A subsequent sedimentation and fractionation of cells according to the cell cycle position displayed an additional peak in sedimentogram, that was absent in the control sedimentogram. This peak is due to cell blocking in G1-period; the pool of blocked cells contains 20-25% of the total cell population. If the drug was injected during 3 days and nights with 8-hour intervals, the pool of blocked cells amounted up to 40%. This effect is reversible: when the drug is cancelled, the peak of G1-blocked cells is reduced. Estimation of intracellular Na(+) and K(+) concentration and Na+, K(+) pump indicates a significant decrease in Na+ permeability in the blocked cells due to Triampur effect. The obtained data well compare with an idea of the importance of increasing a passive influx of Na+ for G1-S transition, and indicate a possibility to affect the structure of tumor cell population via the system of intracellular ionic homeostasis.

[Use of the method of nucleus sedimentation fractionation for studying the dynamics of cell populations in the regenerating liver]. / Ispol'zovanie metoda sedimentatsionnogo fraktsionirovaniia iader dlia issledovaniia dinamiki kletochnykh populiatsii regeneriruiushchei pecheni.

In different periods after partial hepatectomy (0, 11, 18, 24, 48 and 72 hours) isolated liver cell nuclei were separated according to their sizes using free sedimentation at 1 g in the exponential 0.1-1.0 M concentration gradient of sucrose at 4 degrees C. A sedimentogram of the intact liver cell nuclei displays three peaks corresponding to the nuclei of stromal cells, and diploid and tetraploid nuclei of hepatocytes. At 18 and 24 hours after the operation, but not at 0 or 11 hours, a curve of 3H-thymidine incorporation is represented by three maxima suggesting the influx of hepatocytes of different ploidy in the S-period. Then, 48 and 72 hours after the partial hepatectomy two additional peaks appear corresponding to 8- and 16-ploid nuclei, and a decrease in the portion of diploid nuclei is seen up to the full absence of the corresponding peak. Thus, the method of sedimentation at unit gravity of isolated nuclei using radioisotope labeling allows to obtain the information on the structure of the heterogeneous proliferating cell population.

[Analysis of the statistical characteristsics of the distribution of NK/Ly lymphoma cells obtained by laser flow cytometry]. / Analiz statisticheskikh kharakteristik raspredelenii kletok limfomy NK/Ly, poluchennykh metodom lazernoi protochnoi tsitometrii.

Distributions in volumes of NK/Ly ascite lymphoma cells in different periods after inoculation were studied by laser cytometry. With the age of tumour the primary peak of distribution shifts to an area corresponding to large cells, but from the sixth day there appears an additional peak of distribution due to an accumulation of a pool of nonproliferating quiscent G0(R1) cells. The obtained data are in good agreement with results of sedimentation fractionation of NK/Ly lymphoma cells. Discrepancies in statistical characteristics of distributions, found by the both methods, are not more than 20%.

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.

[Isolation and physico-chemical properties of P-9 protein, marker for the resting cells of lymphoma NK/Ly and Gerene carcinoma]. / Vydelenie i fiziko-khimicheskie svoistva belka P-9, markiruiushchego pokoiashchiesia kletki limfomy NK/Ly i kartsinomy Gerena.

A fraction of tumour cells in the state of proliferative rest was prepared by sedimentation from aged Ehrlich tumor and lymphoma NK/Ly. These cells do not incorporate [3H]-thymidine and contain fewer proteins and RNAs than the proliferating cells. The incorporation of [3H]-uridine by these cells makes up to 3.5-10% of the maximal incorporation by proliferating cells at the S-phase. The resting cells have a low rate of glycolysis and a high rate of respiration. The activity of lactate dehydrogenase, glucose-6-phosphate dehydrogenase and malate dehydrogenase in these cells is considerably reduced. The resting cell nuclei were found to contain a low molecular weight protein with molecular weight of 9000 +/- 500 (protein P-9) absent in proliferating cell nuclei. Using acetone fractionation and a subsequent preparative electrophoresis in a phenol solution, protein P-9 was isolated from the nuclei of lymphoma NK/Ly and Gerene carcinoma in a pure state. The physico-chemical properties of the protein from various sources were found to be similar. Thus, protein P-9 can be attributed to low molecular weight non-histone proteins with pI at the weakly alkaline region of pH.

[Cell population structure and nuclear macromolecular synthesis at different stages of the cell cycle in Guerin's carcinoma according to the data from the sedimentation fractionation of isolated nuclei]. / Struktura kletochnoi populiatsii i sintez iadernykh makromolekul na razlichnykh étapakh kletochnogo tsikla kartsinomy Gerena po dannym sedimentatsionnogo fraktsionirovaniia izolirovannykh iader.

For separating isolated nuclei of the Geren carcinoma, according to phases of the cell cycle, the sedimentation fractionation technique in steep gradients of sucrose at 1 g was used. Four peaks were displayed on a sedimentogram, corresponding to the four bands of sedimentating nuclei. From the results of DNA determination and of thymidine-3H incorporation in "pulse chase" experiments, the three peaks of the sedimentogram are identified as those corresponding to nuclei being in G1- and G2-periods of cycles of diploid and tetraploid tumour subpopulations, and ranges between the peaks are identified as nuclear fractions corresponding to S-periods of cycles of both the subpopulations. Data on synthesis of nuclear macromolecules allow us to suppose, that one of the peaks on the sedimentogram may correspond to nuclei of diploid cells being in the late G1-period of the cell cycle.

[Lactate dehydrogenase and malate dehydrogenase isoenzymes at different stages of the mitotic cycle in Ehrlich tumor cells]. / Izozimy laktatdegidrogenazy y malatdegidrogenazy na razlichnykh étapakh mitoticheskogo tsikla kletok opukholi Erlikha.

LDH is represented in the tumor almost exclusively by an extremely slow migrating isozyme LDH-5. There is actually no LDH-1, LDH-2, LDH-3 in the spectrum, while LDH-4 is in small amounts. MDH is represented by cytoplasmic isozyme (S-MDH) and a mitochondrial fraction (m-MDH). The activity of LDH-5 and S-MDH is altered in a synchronous way: it shows a very sharp increase at the very beginning of the mitotic cycle and reaches the maximum value already by the mid-S-phase. The m-MDH activity through the cycle is enhanced twice: at the beginning of the cycle and following the termination of S-phase. There was found a direct correlation between the activity of m-MDH and the rate of anaerobic glycolysis through the mitotic cycle of Ehrlich tumor cells.