[The reentrant binomial model of nuclear anomalies growth in rhabdomyosarcoma RA-23 cell populations under increasing doze of rare ionizing radiation].

Distributions of nuclear morphology anomalies in transplantable rabdomiosarcoma RA-23 cell populations were investigated under effect of ionizing radiation from 0 to 45 Gy. Internuclear bridges, nuclear protrusions and dumbbell-shaped nuclei were accepted for morphological anomalies. Empirical distributions of the number of anomalies per 100 nuclei were used. The adequate model of reentrant binomial distribution has been found. The sum of binomial random variables with binomial number of summands has such distribution. Averages of these random variables were named, accordingly, internal and external average reentrant components. Their maximum likelihood estimations were received. Statistical properties of these estimations were investigated by means of statistical modeling. It has been received that at equally significant correlation between the radiation dose and the average of nuclear anomalies in cell populations after two-three cellular cycles from the moment of irradiation in vivo the irradiation doze significantly correlates with internal average reentrant component, and in remote descendants of cell transplants irradiated in vitro - with external one.

[Sarcolysine sensitivity of the progeny of the clonogenic cells of rat rhabdomyosarcoma RA-2]. / Chuvstvitel'nost' k sarkolizinu potomstva klonogennykh kletok rabdomiosarkomy RA-2 krys.

Cells of rat rhabdomyosarcoma Ra-2 inoculated intravenously were trapped only in lungs and formed progressively growing monoclonal metastases. The population of clones was sensitive to the sarcolysin treatment (10-30 mg/kg) at all stages of clone formations (from 1 to 14 days). The drug acted by two ways: by the reduction of the number of clones and by the clone growth rate inhibition. Independent variability of both types of the clone sensitivity resulted in the highly heterogeneous response of clone populations to the sarcolysin treatment.

[Selection for a decrease in metastatic potential in a cell population of transplantable rat rhabdomyosarcoma RA-2]. / Otbor na snizhenie metastaticheskogo potentsiala v populiatsii kletok perevivnoi rabdomiosarkomy PA-2 krys.

The method of individual cloning was used for checking the reduction of metastatic potential (MP) in cell population of highly malignant rhabdomyosarcoma RA-2. In the course of 15 selection cycles MP was lowered by 10-15 times. These data enabled us to consider the MP value as a quantitative character of malignant cells with low heritability (the mean value of realized heritability coefficient is equal to 0.18).

[Isozymes of NADP-dependent isocitrate dehydrogenase in normal and tumor tissues of various mouse strains and their hybrids]. / Izozimy NADF-zavisimoi izotsitratdegidrogenazy v normal'nykh i opukholevykh tkaniakh myshei razlichnykh linii i ikh gibridov.

Normal tissues of DBA, CBA, CC57W, C3H, Balb/c, SHR mice and F1 hybrids CC57W/DBA appeared to differ in the ratios of mitochondrial and supernatant NADP-dependent isocitrate dehydrogenase (IDH). Tested inbred mice strains CC57W, C3H, SHR, Balb/c contain allelic form Idh-1a of supernatant IDH gene Idh-1, whereas allelic form Idh-1b is characteristic of mice strains DBA and CBA. In tumors IDH isozymes have the same mobility as do isozymes of homologous normal tissues; but their activity is lower. A high variability of each isozyme activity in the isozyme spectrum is revealed in various tissues of F1 hybrids CC57W/DBA. Allelic forms of gene Idh-1 were used as markers of normal and tumor cells for the experimental model: transplantation of sarcoma 37 (Idh-1a/Idh-1a) to subcutaneous tissue of the mouse strain DBA (Idh-1b/Idh-1b). It enables us to reveal isozymes of stromal cell in tumor IDH isozyme spectrum. The results indicate that the relation of normal and tumor isozymes vary in different tumors.

The karyotypic structure of cell populations in vitro as an integral system.

This review describes regularities of karyotypic variability maintaining karyotypic stabilization of continuous cell lines. Statistical analysis of individual karyotypes of "marker" and "markerless" cell lines show that survival of cell population in vitro is maintained by a certain ratio of cells with different structural variants of karyotype (SVK). Characteristic feature of karyotypic variability in the "markerless" cell lines during long-term cultivation under various conditions is dicentric formation due to telomeric associations. These dicentrics seem to form genetical structures providing adaptation to conditions in vitro of the cell population as an autonomous system. Correlations between the numerical variability reflecting in SVK, and structural variability (dicentric formation) are manifestations of an integral cell-populational function. Experimental data allow to suggest that integrity of the karyotypic structure of cell populations is maintained not only by selection of random variations, but also by programmed (adaptive) changes of karyotype. As a whole, in the cell population the state is realized that can be called karyotypic homeostasis; the observed phenomena characterize processes maintaining such homeostasis.

[Changes in the LDH isoenzyme spectrum in transplantable rhabdomyosarcoma clones transplanted into the anterior chamber of the eye]. / Izmenenie spektra izozimov LDG v klonakh perevivnykh rabdomiosarkom, transplantirovannykh v peridniuiu kameru glaza.

Results of gel electrophoresis of transplantable rat (RA-2) and murine (MC-53) rhabdomyosarcomas show that the LDH isoenzyme spectrum of tumors growing subcutaneously differ from that of normal muscle tissue, LDH-1 and LDH-2 isoenzyme being absent in zymograms. Using the lung colony formation technique, clones of RA-2 and MC-53 were obtained and their zymograms were investigated. Clone populations of both the tumors were polymorphic for their LDH spectrum, in some clones of RA-2 normal LDH isoenzyme spectrum was found. After a 16-18 day cultivation in rat and mouse eye anterior chamber, LDH spectra of RA-2 and MC-53 clones changed sharply, LDH-1 and LDH-2 appearing. As a result, LDH isoenzyme spectra of some clones that grew in the eye anterior chamber became equal to those of the normal muscle tissue. The observed changes are discussed as biochemical signs of cytodifferentiation of tumor cells.

[Independence of tumor cell affinity for lung tissue from its interaction with unfixed macrophages]. / Nezavisimost' affiniteta opukholevykh kletok k legochnoi tkani ot ikh vzaimodeistviia s nefiksirovannymi makrofagami.

The selection of tumor cells for their affinity to pulmonary tissue significantly enhances their ability to invade and proliferate. The intraveneous inoculation of these cells into mice increases the number of animals bearing tumour clones and the number of clones per animal. The adduction of unfixed macrophages by means of intramuscular injection of BGG, which is known to increase the implanted tumour growth, did not increase either the percentage of animals bearing tumor cell clones or the number of clones per a lung, in our experiments. The affinity of tumor cells to pulmonary tissue obviously does not depend significantly on the interaction with unfixed macrophages. We assume that the selection of tumor cells for their affinity to pulmonary tissue is not necessarily connected with changes in their antigenous properties for recipient animals.

[Selection for affinity to lung tissue in cell populations of experimental neoplasms]. / Otbor na affinitet k legochnoi tkani v kletochnykh populiatsiiakh éksperimental'nykh opukholei.

Cell populations of eight experimental tumors (murine and rat rhabdomyosarcomas induced with 20-methylcholantrene, transplantable murine rhabdomyosarcoma A-7, and transplantable rat lymphosarcoma) have been selected for the affinity of their cells to lung tissue. The level of the affinity was measured as the number of lung nodules per 100 000 tumor cells injected intravenously. A 3-4-fold selection appeared to be non-effective, whereas 10-fold or more prolonged selections resulted in a gradual enhancement of the affinity of tumor cells to lung tissue. Thus, the transplantable murine and rat rhabdomyosarcomas were obtained with an increased capacity of their cells of yielding lung nodules. The affinity of rat rhabdomyosarcoma was 200-300 times higher after 40 steps of selection compared to the initial tumor affinity. With the rhabdomyosarcoma of CC57W mice, the affinity increased by 5 times after 20 steps of selection. Using our technique of selection (without an in vitro cultivation), the capacity of cells of persisting in lung tissue and yielding lung nodules looks likely as a quantitative character with a rather low heritability. It has been concluded that in cell populations of tumors examined there are only a few genetic population variations in cell capacity of making non-random metastases.

[Heritability of cell size in experimental rhabdomyosarcomas]. / Nasleduemost' razmerov kletok éksperimental'nykh rabdomiosarkom.

Cell diameter and cell value variations were studied in murine and rat rhabdomyosarcomas, both in clones obtained in lung tissue, and in subcutaneous tumors. Coefficients of heritability (h2) of these characters were calculated on the basis of their variances. They were equal to 0.15--0.31 for the diameters of cells, and 0.13--0.87 for the cell volumes. The problem of investigation of heritability of quantitative characters in somatic cell populations is discussed.

[Cytophotometric study of the progeny of clonogenic mouse rhabdomyosarcoma cells in lung tissue]. / Tsitofotometricheskoe izuchenie potomstv klonogennykh kletok rabdomiosarkomy myshi v legochnoi tkani.

50 clones of murine rhabdomyosarcoma were grown in lungs, DNA contents of interphase nuclei of these clones being measured cytophotometrically. Significant differences in the mean DNA contents of clones obtained were observed, reflecting karyotypic differences of the initial clonogenic cells. The progenies of clonogenic cells had very high genome mutation rates (17.4 +/- 0.3.10(-2), and cells with increased numbers of chromosomes arose more frequently than hypoploid cells (12.5 +/- 0.3 and 2.9 +/- 0.2.10(-2), respectively). The mutability of tumor cells was non-equal in different clones, and positive correlations were observed between the mean DNA contents and mutability of the clones, whereas negative correlations were noticed between mutability and rates of cell proliferations.

[Variation in the number of C-heterochromatin blocks in the interphase nuclei of the cells of a transplantable murine rhabdomyosarcoma]. / Var'irovanie chisla blokov C-geterokhromatina v interfaznykh iadrakh kletok perevivnoi rabdomiosarkomy myshi.

By means of staining procedure which reveals the constitutive heterochromatin (CH), the number of CH-blocks was detected in the nuclei of interphase cells obtained from the subcutaneously growing rhabdomyosarcoma and from its 10 lung colonies. The range of variability of the number of CH-blocks in tumor cell populations was greater than in the populations of normal lung cells and lymphocytes. The mean numbers of CH-blocks varied from 17.8 +/- 0.9 to 28.0 +/- 1.2 in cells of different clones, the mean number of CH-blocks in clones being the same as in the subcutaneously growing uncloned tumor. The coefficient of heritability h2 of character "the number of CH-blocks", calculated on the basis of population and interclonal variances, was equal to 0.15, and that calculated by one-factor dispersional analysis was actually the same (0.16). It is concluded that the high heterogeneity of tumor cells may be conditioned not only by their high phenotypic instability, but also by a high frequency of mutations.

[Changes in primary heat sensitivity of rhabdomyosarcoma PA-2 cells in rats during selection for heat resistance]. / Izmenenie pervichnoi termoustoichivosti kletok rabdomiosarkomy PA-2 krys v protsesse selektsii na termorezistentnost'.

Using the technique of multistep selection, a thermoresistant substrain (TR RA-2T45) was obtained from a rhabdomyosarcoma RA-2 strain. The cells of this substrain retain the ability to form lung metastasis after a 20 minute treatment at 45 degrees C in vitro. The selection of cells for their ability to retain their reproduction activity is accompanied by the increase in their primary thermoresistance. The increased reproductive thermoresistance and primary thermoresistance of the selected cells is hereditary stabilized. Cells of both original strain and its substrain are capable of thermal tolerance, which makes perspective the selection for further increase in thermoresistance in RA-2T4t cell population.

[Internuclear chromosomal bridges and nuclei with protrusions in cellular populations of rat PA-23 rhabdomyosarcoma]. / Mezh''iadernye khromosomnye mosty i iadra s protruziiami v kletochnykh populiatsiiakh rabdomiosarkomy PA-23 krys.

Internuclear chromosome bridges (CB) and nuclear protrusions (NP) were investigated in cell populations of RA-23 rat rhabdomyosarcoma. A morphological classification of different types of CB and NP has been offered. The obtained data suggest a morphological similarity between CB and NP. It is likely that NP could arise from CB after its break. So, NPs may be regarded as remains of broken CB.

[Variability in the DNA content in the progeny of the clonogenic cells from transplantable rat rhabdomyosarcoma]. / Izmenchivost' soderzhaniia DNK v potomstvakh klonogennykh kletok perevivnoi rabdomiosarkomy krys.

Transplantable rat rhabdomyosarcoma RA-2 selected for affinity to lung tissue was cloned in vivo, and DNA contents of interphase nuclei was cytophotometrically measured in progenies of 121 clonogenic cells implanted in the lungs. Clone populations have shown a high karyotypic heterogeneity, karyotypic structure being unchanged from the 16th to the 28th selection cycles investigated. Karyotypic heterogeneity is reproduced due to the high rate of genome mutations in the progenies of clonogenic cells (11.10-2-14.10-2 per cell per generation). Cells with increased numbers of chromosomes arose 3 fold more frequently than cells with decreased chromosome numbers, tumor cell mutability being non-equal in different clones.

[Frequency of genome mutations in progeny of colony-forming bone marrow cells in BALB/c mice]. / Chastota genomnykh mutatsii v potomstvakh kolonieobrazuiushchikh kletok kostnogo mozga myshei BALB/c.

Spleen colonies of syngenic bone marrow cells were grown in sublethally irradiated BALB/c mice. DNA contents of interphase nuclei of the colony cells have been measured using the Feulgen cytophotometry. The rates of appearance of cells with variations in chromosome number were calculated according to the following equation: (Formula: see text), where No - the total number of colonies, Nm - the number of mutant colonies, and n- the number of cells examined. The rate of genome mutations was found to be 7.7 +/- 0.6X X10(-2) per cell per generation. Binominal distribution of clones according to the number of mutant cells has been noticed. Cells with decreased numbers of chromosomes (hypodiploid mutants) were of a more frequent occurrence than cells with increased chromosome numbers (hyperdiploid mutants). It is concluded that the propagation of colony-forming bone marrow cells in irradiated animals is accompanied by almost the same increase in frequency of genome mutations as in the case of the cultivation of cells in monolayer cultures.

[Genome mutation frequency in the cells of spleen colonies of different origins in NIH mice]. / Chastota genomnykh mutatsii v kletkakh selezenochnykh kolonii razlichnogo proiskhozhdeniia u myshei NIH.

Genome mutation frequencies (GMF) were determined in cells of endogenous (from bone marrow) and exogenous (from bone marrow, spleen and embryonic liver) spleen colonies on the basis of variations in DNA contents of interphase nuclei. In cells of the former GMF varied from 1.1 X 10(-2) to 10.8 X 10(-2), and in the latter these were equal to 8.9 X 10(-2). In the cells of exogenous colonies derived from X-irradiated precursors (1.8 and 5.9 Gy) GMF were 10.1 X 10(-2) and 11.9 X 10(-2), resp. The mode of transplantation influenced greatly on the GMF: after an additional short transplantation (4-6 days) the number of GMF increased by 1.5-2 times. It is concluded that the increased number of GMF may be responsible for the limited life-span of bone marrow stem cells in the course of their serial transplantations in the irradiated syngenic mice.

[Pathological mitoses in clones of the RA-2 subline of rat transplantable rhabdomyosarcoma selected for increased and decreased frequencies of the formation of spontaneous micronuclei]. / Patologicheskie mitozy v klonakh sublinii perevivnoi rabdomiosarkomy krys RA-2, otselektirovannykh na povyshennuiu i ponizhennuiu chastotu formirovaniia spontannykh mikroiader.

Cell substrains with increased and decreased spontaneous levels of micronuclei have been obtained by artificial selection. Clones of the substrains were investigated for mitotic division fidelity. About 200 mitoses were investigated in each clone. Frequencies of mitotic abnormalities such as ana- and telophases with bridge, chromosome and fragment delays at ana- and telophases, scattered chromosomes, chromosomes dislocated from spindle at metaphases and tripolar ana- and telophases were significantly higher in substrains with increased level of micronuclei. This finding indicates that reasons for arising of spontaneous micronuclei and alterations in mitotic division fidelity are the same or closely related. These reasons can be amplified as a result of the artificial selection.

[The dependence of the incidence of encountering cells with micronuclei in the experimental metastases of rat rhabdomyosarcoma on the duration of the growth and on the size of these metastases]. / Zavisimost' chastoty vstrechaemosti kletok s mikroiadrami v éksperimental'nykh metastazakh rabdomiosarkomy krys ot dlitel'nosti rosta i razmerov étikh metastazov.

Cell suspensions of rat transplantable rhabdomyosarcoma RA-2 were injected to white rats. The experimentally induced lung metastases (ELM) were estimated according to the frequency of micronucleated cells (MNC) that appeared 9-20 days after injection (a. i.). The frequency of MNC in ELM decreased from 2.7% (9 days a. i.) to 0.7% (20 days a. i.). The ELM which were of the same size but were examined at different time after injection had equal frequencies of MNC. At each investigated time after injection the least frequencies of MNC were observed in the ELV of the biggest size. The data provided allow to infer that the growth time and site of ELM may influence the spontaneous karyotype instability rates.

[Changes in the frequency of cells with micronuclei in cell populations of rat transplantable rhabdomyosarcoma RA-2 as a result of selection for an increased and a decreased frequency of such cells]. / Izmenenie chastoty kletok s mikroiadrami v kletochnykh populiatsiiakh perevivnoi rabdomiosarkomy RA-2 krys v resul'tate otbora na povyshennuiu i ponizhennuiu chastotu takikh kletok.

Artificial selection for increasing and decreasing frequencies of cells with micronuclei (FCM) was conducted in populations of cell clones of the rat transplantable RA-2 rabdomyosarcoma. The selection was effective in both the opposite directions. The average FCM increased from 3.3 +/- 0.2 to 6.9 +/- 0.6% and decreased from 3.3 +/- 0.2 to 1.6 +/- 0.2%. Heritability coefficient, h2, varied from 0.2 to 0.4. In the cell population with increased FCM, 37% of anaphases and early telophases had lagged chromosomes and fragments. In contrast, in the cell population with decreased FCM, lagging were observed in 11% of cases. The DNA-content in G1 cell varied greatly in cell population with increased FCM (P < 0.01). The selection for increased FCM has led to a significant decrease in metastatic capacities of cells.

[Clonal analysis of karyotypic instability and metastatic potential in tumor cell populations]. / Klonal'nyi analiz kaiotipicheskoi nestabil'nosti i metastaticheskogo potentiala v populiatsiiakh opukholevykh kletok,

Rat organospecific transplantable RA-2 rhabdomyosarcoma strains RA-2H and RA-2L were investigated using single cell cloning technique for "Micronuclear containing cell frequency" (MCF) and "Metastatic potential" (MP). The RA-2H was obtained from RA-2 during selection for increasing metastatic potential, whereas the RA-2L was the result of selection in opposite direction. MP was assessed by lung colony formation technique and in the case of RA-2H, it was one hundred times greater than in the case of RA-2L (2490 +/- 280 and 22 +/- 11 lung colonies i.v. after injection of 10 cells, respectively). Average MCE for RA-2H was 3 and for RA-2L 5.3%. Also, significant differences were observed between these substrains in percentage of anaphase and telophase cells with bridges and fragments (24 +/- 4 for RA-2H and 56 +/- 5% for RA-2L, P less than 0.05). The selection of RA-2H clones with high level of MCF was successful. In the population obtained MCF was increased up to 7.8% and MP decreased (79 +/- 44). So, the selection for decreasing of MP led to significant increase in genome instability characteristics and the selection for increasing of MCF--to the abrupt decline of MP, which proved the existence of negative correlation between these two characters in populations studied. It is concluded that correlation between malignancy and genome instability in tumor populations with different levels of malignancy is not, as a rule, positive.