[Positive and negative regulation of replication in hybrid cells]. / Pozitivnaia i negativnaia reguliatsiia replikatsii v gibridnykh kletkakh.

DNA replication blockage in various differentiated cells was investigated on the model of heterokaryons. Two distinct types of DNA synthesis regulation in heterokaryons "differentiated cell + proliferating cell" were revealed: I. Neutrophils and nucleated erythrocytes efficiently prevented the entry of non-malignant proliferating cells nuclei into the S-period but usually failed to substantially inhibit the replication in malignant cells nuclei. Both "mortal" and immortalized proliferating cells activated the DNA synthesis in neutrophil and chicken erythrocyte nuclei. II. Macrophages did not influence the DNA synthesis in the nuclei of non-malignant cells in heterokaryons but drastically inhibited that in the nuclei of malignant cells. Only immortalized cells reactivated DNA synthesis in the nuclei of macrophages. These data show that the mechanisms maintaining differentiated cells in non-proliferating state are not uniform. Nucleated erythrocytes were shown to suppress the duplication of centrioles in partner cells. The possibility of the blockage of DNA replication upon the fusion of two proliferating cells (fibroblast + leukemia cell) was demonstrated for the first time in the present work. The influence of various oncogenes upon the regulation of DNA synthesis in heterokaryons was investigated in detail. New modifications of the methods of cell fusion, enucleation and heterokaryon identification were proposed.

[Proliferative senescence of embryo fibroblasts of Japanese senescence accelerated mice (SAM) is accompanied by parallel decreasing of response to various growth factors]. / Proliferativnoe starenie émbrional'nykh fibroblastov iaponskikh uskorenno stareiushchikh myshei (SAM) soprovozhdaetsia parallel'nym oslableniem otveta na razlichnye rostovye faktory.

The Japanese senescence accelerated mice (SAM) are a group of the low-longevity mouse lines and represent a new convenient model for studying the senescence process. We studied the proliferation of embryo fibroblasts of SAMP1 and SAMR1 mouse lines. It was shown that fibroblasts of the shortest longevity line SAMP1 have a markedly decreased proliferative potential of the mean 8.7 population doublings, whereas fibroblasts of a relatively high-longevity line SAMR1 have an average proliferative potential of 12.3 doublings. The fibroblast senescence in both lines is accompanied by a simultaneous lowering of the cell proliferative response to the blood serum, epidermal, fibroblast, and platelet-derived growth factors. At initial stages of the cell culture growth, lines SAMP1 and SAMR1 exhibit the same reactions to growth factors, but already beginning from the fifth doubling, the SAMP1 cell response is sharply decreased as compared with SAMR1. Lowering the proliferative reaction is accompanied by a decreased phosphorylation of tyrosine in the cell proteins responsible for mitogenic reaction. Thus, the parallel decrease of proliferative response to different growth factors during fibroblast senescence is most likely due the emergence of a regulatory block at common stages of the mitogenic signal transduction.

[Effect of enucleation on the structural and functional state of the cytoplasm in L cells]. / Vliianie énukleatsii na strukturno-funktsional'noe sostoianie tsitoplazmy L-kletok.

The viability of cytochalasin-enucleated L-cells (cytoplasts) was investigated using autoradiography in addition to fluorescence and electron microscopy. L-cells were enucleated according to a modified Prescott's method (thermostabilized centrifugation, low centrifuge acceleration). Relatively mild conditions of enucleation permitted us to prolong the life-span of cytoplasts up to 24 hours. Soon after enucleation, the disaggregation of polyribosomes in cytoplasts takes place, reflecting, apparently, m-RNA degradation. The cytoplasts are shown to be able to live for 1--2 days with a completely stopped protein synthesis. Acridin orange staining and ultrastructural investigation showed lysosomal hypertrophy in senescent cytoplasts. The death of cytoplasts is suggested to be due to disruption of lysosomes.

[DNA synthesis in heterokaryons obtained by the fusion of polymorphonuclear leukocytes and cultured cells possessing different proliferative potentials]. / Sintez DNK v geterokarionakh, poluchennykh pri sliianii segmentoiadernykh leikotsitov i kul'tiviruemykh kletok, obladaiushchikh razlichnym proliferativnym potentsialom.

Heterokaryons between terminally differentiated polymorphonuclear leukocytes (PL) and culture cells of different proliferative potentials: mouse and rat embryo fibroblasts (EFM, EFR); immortal cells NIH 3T3 and E2; malignant cells NCC2, L929, He239 and SV 3T3,--were obtained by means of electrofusion. Radioautographic study of 3H-thymidine incorporation in the nuclei of heterokaryons showed that all the cells taken for fusion were able to induce reactivation of DNA synthesis in PL nuclei, however, with different rates: 7-37% for EFM and NIH 3T3 and 20-40% for malignant cells. The presence of oncogenes Elan in E2 cells and ras in NCC2 cells increased the rate of PL reactivation approximately twice as compared with the cells of original lines (EFR and NIH 3T3, correspondingly). In parallel to reactivation of DNA synthesis in PL nuclei inhibition of the synthesis in culture cell nuclei in the same heterokaryons was found. The rate of inhibition was about 70% for non-malignant and 23, 40 and 18% for NCC2, L and SV 3T3 cells, respectively. He239 cells, transformed by a temperature-dependent mutant of virus SV40 showed at permissive temperature the increased capacity of inducing reactivation of PL nuclei, though He239 cells susceptibility to inhibitory action of PL nuclei did not change with temperature. According to the behaviour in heterokaryons PL were found to be similar to chick erythrocytes, but differing from them by a pronounced inhibiting effect upon DNA synthesis in the nuclei of malignant cells.

[Use of cell enucleation in studying the stability of cytoplasmic organelles and cytoplasm organization]. / Primenenie énukleatsii kletok dlia izucheniia stabil'nosti tsitoplazmaticheskikh organell i organizatsii tsitoplazmy.

A study was made of the viability and ultrastructure of cytoplasts produced by enucleation of cytochalasin-induced A9 cells in suspension. These cytoplasts are in general as viable as cells enucleated in the monolayer. The organization of the cytoplasm, i.e. a specific distribution of cytoplasmic organelles, is conserved for at least 24 hours in the absence of the nucleus. This fact may reflect a high degree of autonomy of the cytoskeleton.

[The nature of a proliferation block in differentiated cells with heterokaryons as a model: various types of absence of proliferation in cells in terminal differentiation]. / Issledovanie prirody bloka proliferatsii v differentsirovannykh kletkakh na modeli geterokarionov: razlichnye tipy otsutstviia proliferatsii v kletkakh pri terminal'noi differentsirovke.

Heterokaryons obtained by fusion of proliferating and terminally differentiated cells were studied. The data obtained suggest that mechanisms of proliferation arrest are different in macrophages on one hand and nucleate erythrocytes and polymorph leukocytes on the other. Macrophages appeared to be devoid of factors preventing replication in nontransformed and spontaneously immortalized cells. Inhibition of proliferation was probably due to certain modifications of macrophage genome which arise during differentiation and can be compensated by the effect of "immortalizing" oncogenes. On the contrary, nucleate erythrocytes and polymorphs evidently contain some factors mediating negative control of proliferation. For reactivation of DNA synthesis in these cell types after fusion with other cells the latter did not have to be immortalized. After cell fusion macrophages specifically inhibit DNA synthesis in cells containing active oncogenes.