[Features of rat cells karyotypic abnormalities in rat cells in their transformation in vitro].

Neoplastic transformation of cells is characterized by karyotypic abnormalities involving aneuploidy, quantitative changes, as well as multiple clonal rearrangements of the number and structure of chromosomes. It is probable that chromosomes are one of the mechanisms of cells immortalization and transformation. Despite many years of study of chromosomal rearrangements, data on primary chromosomal rearrangements in early stages of transformation is still insufficient. We examined karyotypic abnormalities in embryonic rat fibroblasts in both the spontaneous transformation and transformation by oncogenes at different passages in vitro. Literature data and results of our cytogenetical analysis of rat cells lines established by different methods of transformation of cells of different tissue origin in vitro have shown that cell karyotype at early passages may either be normal or acquire diverse clonal chromosomal abnormalities. At later passages, other chromosomes of karyotype are involved in new rearrangements. Despite this, some of the lines do not acquire the malignant phenotype and remain to be immortalized. The role of instable chromosomes and their loci in immortalization and transformation of cells is discussed.

[On the issue of non-mutagenic non-targeted effects in low renewable tissues. Analysis of low dose radiation effects on the rat renal tubule epithelium].

Irradiation of rats with γ-quanta at relatively low doses induces a sustainable dose-independent increase in the occurrence of lethal cytoplasmic disorders in the renal tubules epithelium together with sustainable and as well dose-independent subcelluar compensation and restorative processes. Over the period of research (6 months) these processes led to no population recovery. The detected alterations are referred to the category of non-targeted non-mutagenic effects and they are of interest because they address the issue of the sensitivity of low renewable tissues to radiation.

[Special mechanisms for reducing life span of cells and organisms, initiated by some weak external signals].

The study presents the results of many-years research conducted using biological objects of different organization level. It demonstrates special species-nonspecific form of weak external signals negative effect to cells life expectancy reduction caused by program damage of cells populations. This effect is detected after weak radiation, radio-chemical and thermal influences. It leads to faster extinction of postmitotic populations which can be a reason for life expectancy reduction of multicellular organisms. A possibility of such effect inheritance in the asexual and sexual reproduction is shown. Epigenetic mechanisms of this phenomenon are assumed.

[Characteristics of the spontaneously transformed human endothelial cell line ECV304. I. Multiple chromosomal rearrangements in endothelial cells ECV304].

Karyotype of endothelial line ECV304 cells obtained from human umbilicus vein endothelial cells was studied using G-banding chromosome staining. It has been revealed that the cells have a polyploidy karyotype with 96-112 chromosomes and multiple numerical and structural clonal rearrangements. Almost all the chromosomes of the karyotype are involved in structural rearrangements. There are several double chromosome rearrangements revealed including del(9)(p21) as well as two derivatives of chromosome 3 with the breakpoint in the locus p25 - der(3)t(3;12)(3p25;12q11- 12q24.?1) and der(3)t(3;?)(3p25). The role of these rearrangements in the immortalization of endothelial cells and sighs of transformation are discussed. In connection with the information received about the fact that the cells of ECV304 line are not endothelial cells but T24, urinary bladder cancer cells (which karyotype was studied by Hurst et al., 2000), the comparative analysis of the karyotypes of these two lines was carried out. It has been revealed that these two lines differ by all cytogenetic characteristics. Neither identical structural chromosomal rearrangements nor cell characteristic of urinary bladder cancer cells were detected. Our line ECV304 is not identical to the line T24.

[Characteristics of the spontaneously transformed human endothelial cell line ECV304. II. Functional responses of the ECV304 cells].

Functional responses of the spontaneously transformed human endothelial cell line ECV304 were studied in order to asses its applicability as an endothelial cell model for studying angiogenesis and signal transduction. The dependence of proliferation activity of this line on the presence of growth factor was shown. The absent serum in culture medium resulted in blocking of cells in G1-phase of a cell cycle which is not typical for tumor cell lines. Low doses of beta particles emitted during [3H]thymidine decay resulted in blocking the proliferation of these cells in G2M-phase in a dose-dependent manner. Incubation of the cells with another source of beta particles, 3H2O, under condition of equal specific activities of tritium resulted in preferable accumulation of the cells in S-phase. The different efficiency of beta particles of tritium as a part of 3H2O molecule or thymidine demonstrates that various mechanisms are responsible for various check points. The check point of G1/S is absent and that complies with the presence of deletion of chromosome 9 in locus p21. The level of NO produced by constitutive form of NO-synthase in ECV304 cells was relatively low and not modified by inducible NO-synthase inhibitors. The data obtained suggest that ECV304 line cells retained the properties of the initial spontaneously transformed cell line obtained from human umbilical vein (HUVEC) as well as they can be used as a model system for further studies of the properties of vascular endothelial.

[The study of DNA integrity in somatic cells and spermatozoa by single cell gel electrophoresis with silver staining].

In present work we studied DNA damage in human and bovine lymphocytes and spermatozoa by means of single cell gel electrophoresis followed by silver staining. The spontaneous frequency of DNA damage estimated manually in spermatozoa from healthy donors did not exceed 9% (on average -- 4.8 +/- 1.2%). The frequency of DNA damages in bull sperm after short (less than a year) and long period (more than 20 years) of cryopreservation was assessed as 3.1 +/- 0.9 and 4.3 +/- 0.5%, correspondingly. The comparative estimation of DNA damages in lymphocytes followed by silver staining is a valuable tool to estimate DNA damage in populations of somatic and reproductive cells.

[Chromosomal rearrangements and their effects in spontaneous immortalization and transformation of rat embryo cells in vitro].

G-banding analysis of LRec-1 and LRec-3, spontaneously immortalized cell lines from rat embryo fibroblast, revealed diploid karyotypes with specific clonal structural rearrangements of chromosomes 7 and 19 - del(7)(q11.2q22.1), t(7;19)(q11.1;q12) in malignant stage. Both clonal abnormalities of chromosomes 7 and 19 were also revealed in LRec-1k clone and LRec-1 sf cell line. Previous study of LRec-1 and LRec-3 cells showed the presence of karyotypes with pseudodiploid modal chromosome number, partial trisomy of chromosome 7 and same clonal structural rearrangements of chromosomes 7 and 19 in immortalized stage. In malignant stage, the trisomy 6 and new clonal structural rearrangements of chromosomes 1, 2, 11, 15, 18, 19 and of chromosomes 10, 20 were also found in LRec-1 sf and LRec-1 cells, accordingly. There were no new clonal structural chromosome rearrangements in LRec-1 k and LRec-3 cells. We compared locies of chromosomes involved in rearrangements with mapped genes on these chromosomes according to RATMAP. Supposedly these genes are involved in spontaneous immortalization of rat embryo fibroblast and malignant transformation of LRec-1 and LRec-3 cells and rearrangements of chromosomes 1, 2, 11, 15 and 18 facilitate expression of growth factors of LRec-1 sf cells.

[NO-dependent pathway of the modifying of the cellular sensitivity to gamma- and neutron irradiation]. / NO-zavisimyi put' modifikatsii kletochnoi chuvstvel'nosti k deistviiu gamma- i neitronnogo izlucheniia.

The modifying effect of L-NAME, the NO-synthase inhibitor and D-NAME, the inactive enantiomer was investigated in human carcinoma cells (HeLa) and Chinese hamster fibroblasts (V-79) exposed to different doses of gamma-rays and 0.85 MeV neutrons. We estimated the level of the chromosome aberrations manifested as the bridges and fragments in anaphases. Radioprotective effect of L-NAME showed the inverse dependence on the exposure dose and at low doses (1 Gy) it was higher in the V-79 cells, than in the HeLa cells. However, at high doses (3, 4, 6 Gy) the efficiency of L-NAME for these cellular lines was almost equal (DFR = 2). The modifying effect of L-NAME was almost equal for gamma-irradiation and neutrons, although the exposure of V-79 cells to neutrons induced more the asymmetric chromosome aberrations (RBE = 4). The D-NAME had no effect on the level of the radiation-induced chromosome aberrations, although D-NAME treatment of cells increased the chromatin condensation, as well as L-NAME. The counteractive condensation does not play the major role in the radioprotective effect of L-NAME. We suggest that the radioprotective effect of L-NAME resulted from the action on the generation reactive radicals due to the inhibition of the inducible NO-synthase.

[Increase of probability of cell loss in the endothelium of blood vessel capillaries in progeny of rats after a low-dose irradiation of one of the parents (electron microscopy study)]. / Povyshenie veroiatnosti gibeli kletok v éndotelii krovenosnykh kapilliarov u potomkov krys posle oblucheniia odnogo iz roditelei v malykh dozakh (élektronno-mikroskopicheskoe issledovanie).

The total gamma-irradiation of Wistar rats at a dose of 0.25 Gy as well as at higher doses (0.5, 2 and 4.5 Gy) produces in the capillary endothelial cells of myocardium and lung a pronounced, dose-independent increase of the yield of necrotized cells. Similar changes were revealed in the animals, of which one of the parents (a male one day, a female seven days prior to copulation) was irradiated at doses of 0.25 and 0.5 Gy. This effect was observed in all studied descendants. The massive induction of the changes already by low radiation actions and their dose-independence allow considering the revealed effects as a manifestation of peculiar cellular reactions that presumably have epigenetic nature.

[Specific long-term cellular changes under effects of low doses of radiation]. / Osobye dolgovremennye izmeneniia kletok pri vozdeistvii radiatsii v malykh dozakh.

We examined the peculiar form of a tissue postirradiative reaction characterizing by massive, dose-independent transition of cell populations to the steady state modification with the essential raise of cell damage and cell loss probability as compared with the probability level of the same alterations in controls. We described some other signs of such type of cellular transformation. It was found that the indicated cellular condition occurred both in active and slowly proliferating tissues. The reaction occurred at relatively low doses of irradiation. Some nonmutagenic factors also may evoke such effects. Our experimental data allow us to suppose the epigenetic mechanizms taking part in the induction and preservation of such alterations. The discovered form of cellular reaction manifestating in different biological objects may be considered as some general biological tendency. The importance of the studied reaction in the pathogenesis of late consequences of low dose irradiation is discussed.

[Atypical transformation of cell populations after low irradiation and other actions]. / Neobychnaia transformatsiia kletochnykh populiatsii posle slabykh radiatsionnykh i nekotorykh drugikh vozdeistvii.

Ionizing irradiation at low doses and some weak non-irradiational (including non-mutagenic) actions can produce a peculiar cell reaction, such as a massive, dose-independent transition to a new regime of existence. The changes appear saltationally. They are maintained for a very long time by both rarely and actively dividing cells. The most characteristic phenotype of the studied transformation is a stable increase of probability of cell damage and death. Some other manifestations of this transformation have been also revealed. This unusual response is found upon observation of totally different biological objects (unicellular organisms, cells of various mammalian tissues) to be regarded as a general biological regularity. The role of such radiational alterations in pathogenesis of remote consequences of low-dose irradiation is discussed.

[Nuclei with protrusions--"tailed" nuclei--and radiation cytogenetic markers in a lymphocyte culture after x-ray irradiation]. / Iadra s protruziiami--"khvostatye" iadra i radiatsionnye tsitogeneticheskie markery v kul'ture limfotsitov posle rentgenovskogo oblucheniia.

Frequency of the appearance of binuclear cells with nuclei having outgrowth into the cytoplasmic space and arise after first mitosis in human lymphocyte culture is linear-square dependent on the X-irradiation at doses from 0.0 to 4.0 Gy. Positive correlation between frequency of cells with "tailed" nuclei and frequency of metaphases of first mitosis having dicentrics and rings was established. Apparently, formation such "tailed" nuclei is connected with dicentrics and rings.

Tailed nuclei and dicentric chromosomes in irradiated subjects.

A study of peripheral blood smears from irradiated Chernobyl liquidators and other subjects has shown nuclei of some lymphocytes to have a protrusion into the cytoplasm. Such abnormal nuclei are called "tailed" nuclei (TN). Sixteen main morphologic types, observed in human peripheral blood lymphocytes, are described. The frequency of appearance of lymphocytes with TN in a group of Chernobyl liquidators was significantly higher than in control groups (p < 0.001, in all cases). A positive correlation was found between the TN frequency in lymphocytes and dicentric chromosomes in lymphocytes cultured to metaphase (p < 0.001). Elevated frequencies of dicentrics, higher than 0.1%, were found in 17 out of 22 subjects in whom the frequency of lymphocytes with TN was 0.8% and more. Abnormalities of the TN type in lymphocytes are likely to result from breakdown of chromosome bridges formed by dicentrics. The TN can be considered as a possible marker of irradiation and, therefore, their detailed study is important.

[External signals can induce stable increase in frequency of cell death in populations]. / Vneshnie signaly mogut indutsirovat' ustoichivoe povyshenie chastoty gibeli kletok v populiatsiiakh.

Relatively weak radiation and some other external actions, producing no "forced" cell death, trigger some intracellular mechanisms in various unicellular organisms (amoebae, ciliates, yeasts) and in the studied mammalian cells (rat vascular endotheliocytes). These mechanisms provide spasmodic changes: massive transition of cell populations into a stationary alternative state which is characterized by an increased predisposition to cell death, in comparison with the initial level. This phenomenon is considered as a particular, widely spread in nature form of genetic control of cell death frequency populations.

[Morphological anomalies in "tailed" lymphocyte nuclei and their connection with dicentric chromosomes in irradiated patients]. / Morfologicheskie anomalii iader tipa "khvostov" v limfotsitakh i ikh sviaz' s ditsentricheskimi khormosomami u obluchennykh patsientov.

Lymphocyte nuclei with a narrow outgrowth into the cytoplasm ("tail") were detected on peripheral blood smears of patients exposed to ionizing radiation during the clean up after the Chernobyl meltdown (liquidators). On average, lymphocytes with tailed nuclei occurred at a frequency of 0.43% and 0.15% in exposed patients (n = 101) and healthy donors (n = 50), respectively; the difference between the samples was significant, P < 0.001. The coefficient of correlation between frequencies of lymphocytes with tailed nuclei and those with dicentric chromosomes was 0.74 (n = 27, P < 0.001). The frequency of lymphocytes with dicentric chromosomes was increased in 9 out of 10 patients, in which lymphocytes with tailed nuclei occurred at a frequency larger than 0.8%. A double-color FISH was used to localize centromeric (not more than two signals) and telomeric (not more than one signal) regions in nuclear tails. Abnormal tailed lymphocyte nuclei were assumed to result from breaking the chromosome bridges formed by the dicentric chromosomes.

[New immortalized cell lines from transgenic rat embryos. I. The isolation and characteristics of cell lines from rat embryos with an injected v-mos oncogene coupled with or without the gene for genecitin resistance]. / Novye linii immortalizovannykh kletok iz transgennykh émbrionov krysy. I. Poluchenie i kharakteristika linii kletok iz émbrionov krysy s in''etsirovannym onkogenom v-mos odnovremenno s genom ustoichivosti k genetitsinu ili bez nego.

A collection of established cell lines was made by means of their explanation into 15 day old transgenic rat embryos. Some of these cell lines were characterized by measuring the cultivated population redoubling time, the saturation density and oncogenicity. A cytogenetic analysis was also carried out. The phenotypical analysis and studies of reproduction permit to define these lines as transformed immortalized non-oncogenic lines capable of contact inhibition. Cytogenetic studies were performed only on Mos N3, N6 and Mos+Neo N1, N6 lines. The karyotypes of cells in Mos lines were normal, and the karyotypes of cells in Mos+Neo lines had chromosomal markers (2 and 3, resp.). These markers result from arrangements of chromosomes 6, 9, 14, 15 and 17. the "thru deletions" of region q1 2qter (line N1) and region q22qter (line N6) of chromosome 15 were revealed in Mos+Neo lines by the summarized reconstruction karyotype method. We propose that these deletions of chromosome 15 and other chromosomes rearrangements may play an important role in transformation of cells from transgenic embryos in vitro, because RB1 antioncogene was mapped on rat chromosome 15.

[A cytogenetic analysis of the cells of spontaneously transformed LREC rat cell lines. I. Chromosomal rearrangements detected in the early stages of rat embryonic cell transformation in vitro]. / Tsitogeneticheskii analiz kletok spontanno transformirovannykh kletochnykh linii krys LREC. I. Perestroiki khromosom, vyiavliaemye na rannikh stadiiakh transformatsii émbrional'nykh kletok krys in vitro.

Seven spontaneously transformed cell lines LREC of rat embryo fibroblasts were obtained by an originally elaborated cloning technique (method 2T7). Six lines (1-6) were obtained from Rattus norvegicus cells, and one line (LREC-7) from Wistar rats. Method 2T7 was based on a serial propagation of rat embryo cells, brought to a "crisis" stage under conditions of a higher cell density and followed by the appearance of actively proliferating cell clones. These lines were analysed cytogenetically at the earliest stages using the Giemsa G-banding technique. In the karyotypes of six LREC (1-6) lines two abnormal chromosomes 7 were revealed: one marker chromosome M1-t(7; 19) results from translocation between chromosomes 7 and 19, the other marker chromosome M2--del(7) is a result of deletion of the second homolog of chromosome 7 in the q11.2 q22.1 loci; besides an extra normal homolog of chromosome 7 was revealed. There are only two marker chromosomes M2 in the LREC-7 line karyotype. Cells of LREC (1-3) lines could be transformed from the immortalized stage to the malignant one by the 30-45th passages. The cells of LREC (4, 7) lines became malignant at the 10-8th passages, resp. The rearrangements of chromosome 7 are supposed to be specific for LREC lines obtained by our method. A hypothesis is put forward that the translocation of chromosome 7 may play an important role for the immortalization of the rat embryo cells. The deletion of chromosome 7 may be associated with a malignant transformation of cells, as it is possible that the deleted loci have a recessive oncogene. Method 2T7 allows to obtain constantly spontaneously transformed cell lines of rat embryo cells with the least abnormal karyotype.

[Cytogenetic changes in an explanted mouse rhabdomyosarcoma during prolonged cultivation]. / Tsitogeneticheskie izmeneniia éksplantirovannoi rabdomiosarkomy myshi pri dlitel'nom kul'tivirovanii.

Quantitative and qualitative chromosome rearrangements, dynamics of distribution of double-minute chromosomes (DMs), and morphological characteristics of tumor rhabdomyoblasts MH-82 during explantation and following in vitro cultivation are analysed. Cells of the 13th and 27th passages of cultivation were characterized by the epithelial type of growth, although their form and size varied. Chromosome analysis of tumor rhabdomyoblasts was carried out on passages 4, 14, 20, 25 and 30 of in vitro cultivation. The modal class with 53-55 chromosomes was established within 20 passages. Heterogeneity of cell population in concern to the chromosome number and content of hypotetraploid cells (72-78) diminished during cultivation. Chromosome rearrangements (marker chromosomes) in hyperdiploid and hypotetraploid cell subpopulations differed. The number of cells with DMs and the number of DMs per cell decreased till the full disappearance by the 30th passage. It is concluded that the establishment of the MH-82 cell line was completed up to the 30th passage of cultivation.