[Characteristic of tumors developed after transplantation of transgenic GFP-positive C57BL/6 mice bone-marrow mesenchymal stem cells to mdx mice muscle].

The purpose of the study was the morphological and histochemical characteristics of differentiation of tumors developed after transplantation of GFP-positive mesenchymal bone-marrow stem cells (MSC) of transgenic mice C57BL/6 into M. quadriceps femoris of mdx mice. The tumors occurred only after transplantation of MSCs of 43-45th passages and did not arise after transplantation of MSCs of the 15th passage. No tumors developed also after transplantation of MSCs of 43-45th passages into muscle of C57BL/6 mice. The average weight of tumors appeared in 4 mdx mice studied was 1.3 +/- 0.5 g. All four tumors were classified as mesenchymomas because they originated from mesenchymal stem cells. Most of the periphery of the tumors was classified as fibrosarcomas with mitotic index 0.9 +/- 0.1%. The central parts of tumors had areas with epithelial like morphology of cells. Such cells showed positive reactivity for alcyan blue staining at pH 2.5, which indicated chondrocyte nature of the cells. No mitosis was observed in epithelial like cells. In the tumors, there were also areas with bone trabeculae containing megacaryocytes and foci of myeloid and erythrocyte hematopoiesis. There were also areas with neuronal and glial cells, and accumulations of adipocytes. One of the tumors was classified as a round cells sarcoma. The observed types of tumor cell differentiation in vivo were in accordance with described in literature types of MSCs differentiation after induction in vitro with special inductors. The spectrum of in vivo differentiation of transgenic GFP-positive MSCs after transplantation to mdx mice was broader than the spectrum of in vivo differentiation of transfected or transformed in vitro adult MSCs after transplantation to immunodeficient mice and mdx mice.

[Bio-objects and biological methods of space radiation effects evaluation].

The unique conditions of space experiments place austere requirements to bio-objects and biological methods of radiation effects evaluation. The paper discusses suitability of a number of bio-objects varying in stage of evolution and metabolism for space researches aimed to state common patterns of the radiation damage caused by heavy ions (HI), and character of HI-cell interaction. Physical detectors in space experiments of the BIOBLOCK series make it possible to identify bio-objects hit by space HI and to set correlation between HI track topography and biological effect. The paper provides an all-round description of the bio-objects chosen for two BIOBLOCK experiments (population of hydrophyte Wolffia arrhiza (fam. duckweed) and Lactuca sativa seeds) and the method of evaluating effects from single space radiation HI. Direct effects of heavy ions on cells can be determined by the criteria of chromosomal aberrations and delayed morphologic abnormalities. The evaluation results are compared with the data about human blood lymphocytes. Consideration is being given to the procedures of test-objects' treatment and investigation.

[Handling and maintenance of gerbils during the spacecraft Foton-M3 mission].

Pressurized low-sized module Kontur with an independent life support system (LSS) was developed by the Institute of Biomedical Problems cooperatively with the Special Design Bureau of Experimental Equipment to house gerbils (Meriones unguiculatus) aboard robotic technology-purpose spacecraft. Design of the module precludes pollution of the environment The fully equipped module weighs 69 kg; average daily power consumption is 62 watts. The environmental parameters for 12 animals flown on Foton-M3 in the period of September 14-26, 2007 were controlled within the following ranges: pO2 - 143-156 mm Hg, (mean 150 mm Hg), pCO2 - 0.76 mm Hg maximum (mean 0.64 mm Hg), temperature - 23-28 degrees C (mean 26.7 degrees C), relative humidity - 29% and 57% at the beginning and end of the flight, respectively (mean 39%). The animals consumed the palletized food prepared of natural products with a moisture content of approx. 20%. The day-night periods were 12 hrs. long. The daytime video recording of the animals went on continuously in the throughout the flight. The experiment showed that the module meets the requirements of experiments with mammals aboard returnable robotic spacecraft and piloted space stations. At the moment, the model is being redesigned for a 30-day BION-M1 mission.

[The role of extracellular chaperone Hsp70 in creating antitumor immunity in rat rhabdomyosarcoma RA-2 model].

Immunization of experimental animals with extract or membranes of rat rhabdomyosarcoma RA-2 in combination with pure Hsp70 did not offer any significant effect of protection from subsequent RA-2 cells-stimulated tumor growth. By contrast, immunization with preparations of pure Hsp70 led to a significant decrease in number and size of tumors as well as elevation of concentrations of antibodies against RA-2 cells. Also, enhanced blood levels of Hsp70 involved delayed tumor growth. In vitro tests Hsp70 incubation with RA-2 cells was followed by a 30-35% rise in cytotoxic lymphocytes levels. An ability of pure Hsp70 preparations to stimulate humoral and antitumor response was demonstrated. Hence, they may be used in developing vaccine formulas.

[Structural changes of the nucleolar organizing regions in rhabdomyosarcoma RA-23 tumor cells induced by okadaic acid]. / Strukturnye preobrazovaniia interfaznykh IaOR v opukholevykh kletkakh rabdomiosarkomy PA-23, indutsirovannye okalaikovoi kislotoi.

It has been recently shown that okadaic acid (OA), an inhibitor of dephosphorilation, is capable of inducing changes in the nucleolar organizer regions (AgNORs) of some mammalian cells. Our work was focused on studying the structural changes in AgNORs of tumour cells of rat rhabdomyosarcoma RA-23 by their exposure to 100 nM OA. A standard silver staining procedure of interphase AgNORs in tumour cells was used. We measured no less than 100 tumour cells in each clone. In the examined tumour cell populations, the index of interphase AgNORs varied from 1.54 to 4.35. A clear structure and form of AgNORs was not observed in 30% okadaic acid-treated tumour cells, as opposed to 10% of the control ones. AgNORs in these cells looked like a mixture of thin threads encompassing some dark dots lying, mostly, separately. Additional procedures of tumour cell staining with Giemsa and fluorescent dye Hoechst 33,258, respectively, revealed that such structures were not chromosomes. Meanwhile, the frequency of cells at the stage of prophase exceeded 3%, as opposed to the control, where the frequency of cells at this stage was less than 0.5%. Thus, we can conclude that we have detected specific changes in AgNORs and chromatin structure of okadaic acid-treated tumour cells.

[Survivability and morphologic anomalies in higher plants wolffia arrhiza following exposure to heavy ions of the galactic space radiation]. / Vyzhivaemost' i morfologicheskie anomalii u vysshikh rastenii (vol'fii beskornevoi) posle vozdeistviia tiazhelykh ionov galakticheskogo kosmicheskogo izlucheniia.

Experimental data on the effects of spaceflight factors, space radiation in particular, on higher plant Wolffia arrhiza firstly exposed in the "Bioblock" assembly and measurements made by physical track detectors of heavy ions (HI) are presented. Death of individual Wolffia plants and morphologic anomalies were the basic evaluation criteria. The peculiar feature of this biological object consists in the possibility to reveal delayed effects after 1-2 months since space flight as Wolffia has a high rate of vegetative reproduction. German investigators through microscopic examination of track detectors performed identification of individual plants affected by HI. With specially developed software and a coordinate system of supposition of biolayers and track detectors with the accuracy of 1 micron, tracks and even separate sections of individual HI tracks were determined in biological objects. Thereafter each Wolffia plant hit by HI was examined and data were compared with other variants. As a result, correlation between Wolffia death rate and morphologic anomalies were determined at different times post flight and topography of HI tracks was found. It is hypothesized that morphological anomalies in Walffia were caused by direct hits of plant germs by heavy ions or close passage of particles.

[Effect of cycloferon on dissemination of Lewis lung carcinoma in mice and rhabdomyosarcoma ra-23 in rats]. / Vliianie tsikloferona na metastazirovanie kartsinomy l]egkikh Lewis u myshei i rabdomiosarkomy ra-23 u kryc.

Tumor growth and proliferative activity of tumor cells were suppressed and the number of pulmonary metastases in C57B16 mice decreased 3.3-fold following seven injections of cycloferon (100 mg/kg body) to induce interferon production. Injections were carried out 1-16 days after subcutaneous transplantation of Lewis lung carcinoma. After mice were immunized with ovine red blood cells, cycloferon administration raised thymus-dependent humoral immune response. After eight injections of cycloferon (50 mg/kg body) into rats, from day of intravenous transplantation of rhabdomyosarcoma RA-23 until day 20, no significant effect on metastasizing into the lung was recorded. However, single injection of cyclophosphamide 50 mg/kg inhibited metastasis formation. The highest suppressor effect was registered with combination cycloferon-cyclophophamide treatment: mean weight of metastasis decreased by half, as compared with treatment with cyclophosphamide alone. Both drugs caused karyotypical abnormalities to occur in metastatic cells. Tumor growth and spreading suppression after cycloferon should be attributed to cytotoxic antitumor action, cell proliferation inhibition and immunomodulating effect.

[The possibility of performing artificial selection in vivo for a change in the mutability level in populations of mouse bone marrow stem cells]. / O vozmozhnosti provedeniia iskusstvennogo otbora in vivo na izmenenie urovnia mutabil'nosti v populiatsiiakh stvolovykh kostnomozgovykh kletok myshei.

Bone marrow cells of CBA males were transplanted to irradiated syngeneic females and after 13 days the mean frequency of blood erythrocytes with micronuclei, cloning efficiency of haemopoietic stem cells (HSC), and variability range of frequency of cells with micronuclei in spleen colonies were studied in 44 primary recipients. A wide range of variability has been shown for the indicators of mutability and cloning efficiency (0.1-1.8% for frequency of erythrocytes with micronuclei, 200-300-1500 for cloning efficiency, 0.1-0.5% for frequency of cells with micronuclei in spleen colonies of individual transplants). A weak negative correlation between the frequency of erythrocytes with micronuclei and the cloning efficiency was observed. We conclude that the difference in frequency of erythrocytes with micronuclei between HSC subpopulations is sufficiently high to carry out an artificial selection for high and low mutability in the course of transplantations. The cloning efficiency can be increased when spleen colonies are studied by micronuclear analysis. Data on the importance of such a selection in studying the behavior of "mutability" and "cloning efficiency" characters, immortalization, "aging", and death of HSC populations of CBA mice have been reported.

[Selection in vivo in cell populations of rat RA-23 rhabdomyosarcoma at the trait "frequency of cells with bridges"]. / Selektsiia in vivo v kletochnykh populiatsiiakh rabdomiosarkomy RA-23 krys po priznaku "chastota kletok s mostami".

Selection in vivo of cell clones of rat transplantable organospecific rhabdomyosarcoma RA-23 for increased and decreased frequencies of cells with chromosomal bridges (FCB) was performed. The initial average FCB in clones was 0.8% at a range of variation of from 0 to 3.0%. Selection for an increase, as well as for a decrease, in the FCB in RA-23 clones was effective. After one step of selection for an increase in the FCB, the average FCB increased to 3.0%. Thereafter, selection for an increase in the FCB was impracticable due to loss of transplantability in cell populations in which the FCB exceeded 5.0%. Over five steps of selection for a decrease in the FCB, the average FCB significantly decreased to 0.3% (P < 0.001). The heritability coefficient h2 of the trait FCB upon selection ranged from 0.25 to 0.30. The high and low FCBs attained by selection correlated with the index "frequency of cells with micronuclei." The population of RA-23 cells after selection for a decrease in the FCB differed from the population of cells of the original RA-23 strain by significantly lower karyotypic heterogeneity. The values of h2 obtained upon selection for the FCB and the effectiveness of selection for an increase, as well as for a decrease, in the FCB show that, with selection at the trait "frequency of cells with bridges," which characterizes the stability of the karyotype, existing cell strains can be subjected to karyotypic stabilization or destabilization.

[The heterogeneity of cell populations by the cytochemical characteristics of the cellular chromatin]. / Geterogennost' kletochnykh populiatsii po tsitokhimicheskim kharakteristikam khromatina kletok.

Cell heterogeneity of cytochemical characteristics of chromatin in the norm and after provocative factors in vitro (a heating at 45 degrees C, X-irradiation and joint actions of heating and X-ray irradiation) was investigated in the peripheral blood mononuclear cells of sheep and mouse, the lymphoid cell populations of the murine bone marrow, spleen and thymus. As a criterion of cytochemical heterogeneity were used the distinctions in the rate of staining of cell nuclei with DNA-specific dyes, that was registered by means of measurement of coefficient variation of the DNA-histogram basis peak, with a flow cytometry. Heterogeneity of cytochemical characteristics are dealt with showed a variability of spatial organization of interphase nuclei of single cells. The provocative factors bring about a decrease in the level of the cytochemical characteristics heterogeneity in the sheep mononuclear cells. In the murine cell populations studied no such equalizing of the marker of variability was revealed.

[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 characteristics of homologous DNA recombination in somatic cells. II. The characteristics of the extrachromosomal recombination of plasmid DNA in cultured cells differing in their tumor potential]. / Izuchenie osobennostei gomologichnoi rekombinatsii DNK v somaticheskikh kletkakh. II. Osobennosti ékstrakhromosomnoi rekombinatsii plazmidnykh DNK v kul'tiviruemykh kletkakh, razlichaiushchikhsia opukholevym potentsialom.

The frequencies of homologous extrachromosomal recombination, following plasmid transfection into cell lines differing in their tumour potential were measured. A 10-fold increase in extrachromosomal plasmid recombination has been reported in tumour cells (Ra-2, Jf-1) relative to immortal (Rat-2) and normal (REF). The differences between the normal and tumour cells are preserved in the in vitro recombination experiments (the recombination in the cell nuclei extracts). However, the recombination activity of nuclear extracts from the immortality transformed cells (Rat-2) is similar to that of nuclear extracts from the tumour cells.

[The characteristics of homologous DNA recombination in somatic cells. I. The construction of a DNA plasmid containing deletion variants of the aminoglycoside phosphoribosyltransferase gene]. / Izuchenie osobennostei gomologichnoi rekombinatsii DNK v somaticheskikh kletkakh. I. Konstruirovanie plazmidnykh DNK, soderzhashchikh deletsionnye varianty gena aminoglikozidfosforiboziltransferazy.

A plasmid DNA family containing deletion variants of Neo-gene has been constructed for investigation of somatic cell homologous recombination. Experimental data are presented provided by studies of extrachromosomal recombination, recombination following plasmid transfection into somatic cells, and recombination in the nuclear extracts. The opportunities of application of the plasmid constructions in the different pattern homologous recombination experiments performed on mammalian cells have been discussed.

[Interclonal and interpopulational differences in the frequency of spontaneous karyotypic changes in cell populations of transplantable rat rhabdomyosarcoma RA-2]. / Mezhklonal'nye i mezhpopuliatsionnye razlichiia po chastote spontannykh kariotipicheskikh izmenenii v kletochnykh populiatsiiakh perevivnoi rabdomiosarkomy RA-2 krys.

Rat organospecific transplantable RA-2 rhabdomyosarcoma substrains RA-2H (high metastatic potential), RA-2L (low metastatic potential) and RA-2T (thermal resistance) were investigated using single cell cloning technique for frequency of micronucleated cells (FMC). Significant interclonal and interpopulational differences were established. Average FMC for RA-2H was 2.96 +/- 0.13%; 5.94 +/- 0.24% for RA-2L and 1.89 +/- 0.12% for RA-2T. Clones showed 5-10 times differences in FMC in each substrain. FMC versus growth time and clone size was studied in RA-2H: average FMC dropped from 2.7% on day 9 to 0.5% on day 20 after administration and the lowest values were recorded in larger clones at each stage. Perspectives of FMC studies in tumor cell populations are discussed.

[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.