[Changes in the type and frequency of chromosome associations in human peripheral blood lymphocytes after treatment with colcemid]. / Izmenenie kharaktera i chastoty khromosomnykh assotsiatsii v limfotsitakh perifericheskoi krovi cheloveka pri deistvii koltsemida.

Changes in the character and frequency of chromosome associations in metaphase cells of human lymphocyte cultures during colcemid treatment were studied. As the time of colcemid treatment was extended from 0 to 24 h, the number of both cells with chromosome associations and of chromosomes involved in this process was seen to increase. After a 1 h incubation of cells with colcemid the number of nucleolar-organizing chromosome associations increased considerably. In 3 h, non-nucleolar-organizing chromosomes were also involved in associations: among these most often were noticed chromosomes 1, 2 and 3, and less often chromosome 11. Following 24 h of colcemid influence, the number of telomeric associations of chromosomes increased, with most frequent involvement of chromosomes 1, 4, 6 and 16. Results of this investigation allow to propose that the step-by-step involvement of metaphase chromosomes in associations, as the time of colcemid treatment increases, may reflect the remoteness of the respective chromosomes from the nucleolus. Thus, in human blood lymphocytes chromosomes 1, 2, 3 and 11 are very likely localized near the nucleolus, or some regions of these chromosomes may contact with the nucleolus.

Karyotypic evolution of cells in culture: a new concept.

The Chapter summarizes peculiarities of karyotypic variability during establishment and long-term cultivation of permanent cell lines. A new concept on pathways of karyotypic evolution of cells in culture is put forward. A detailed description is presented of the author's original approach of cytogenetic analysis of cell lines provided for a principally new characteristic of the cell line: its generalized reconstructed karyotype (GRK). Its use as a criterion to evaluate authenticity, purity, and stability of cell lines is discussed. Based on analysis of the GRK, two stages of karyotype evolution of cell lines are revealed: establishment and stabilization, different in karyotypic variability of the cell population and in peculiarities of clone selection. Comparison of peculiarities of karyotypic variability of leukemic and tumor cells both in vitro and in vivo was made, and general regularities of their karyotypic evolution have been established, such as nonrandom changes in the number and structure of chromosomes and deletion of one of the sex chromosomes, as well as regularities characteristic only of cells in culture in most human and animal cell lines (at least 85%) of disomy on all autosomes. The rest of the cell lines, 15%, are characterized by either partial or total monosomies on certain autosomes during long-term cultivation. Three main compensatory mechanisms of maintaining viability of cell lines that have lost genetic material are discussed: polyploidization of the initial cell clone, amplification of oncogenes (predominantly of mys family), and extracopying of whole autosomes or of their fragments.

[The patterns of the karyotypic evolution of cells in culture]. / Zakonomernosti kariotipicheskoi évoliutsii kletok v kul'ture.

Numerous personal and literary data on the karyotypic variation of cell lines during their establishing and long-term culturing have been reviewed. A new notion about karyotypic evolutionary pathways of cells in culture is presented. A detailed original approach to cytogenetic study of permanent cell lines is given, which allows, via karyotype reconstruction, to obtain finally a new karyotypic characteristics-the generalized reconstructed karyotype (GRK). Application of the cytogenetic approach as a criterion for the control of authenticity, purity and stability of cell lines is discussed. The cell line analysis by means of GRK elicited that the cell line evolution in vitro passes through two stages: a stage of establishment, and a stage of stabilization, both differing in karyotypic variability of cell populations and clonal selection in culture. The data indicate that it is important in experiments to utilize cell lines being in the stage of stabilization and to characterize chromosomally the cell line at the same passage when it is used. Above all, a comparison of karyotypic variations of tumor and leukemic cells in vitro and in vivo has revealed their common karyotypic evolution regularities (a nonrandom character of numerical and structural chromosome changes and the loss of one of the sex chromosomes) and the karyotypic evolutionary regularities characteristic solely of cells in culture. The main of these being a balanced chromosome set in the cell population as a whole and obligatory retention of diploidy in all chromosomes of the normal set by the majority of human and animal cell lines. It has been revealed that no less than two homologs of each autosome are present in cells of at least 85% of examined lines. Other cell lines (at least 15%) of a generally neurogenic origin are shown to be notable by keeping partial or complete monosomies on autosomes throughout the long-term culturing. Peculiarities of the karyotypic evolution of the latter are regarded in detail in addition to the data on the expression of oncogenes and other growth-associated genes in their cells. It is suggested that there are three main compensatory mechanisms through which cell lines with autosomal monosomies may maintain the vitality in culture: polyploidization of initial cell clones, oncogene amplification, mainly of the myc-family oncogenes, and fragmentary or complete extracopying of several autosomes. In summary, perspective of cell line cytogenetics as a field of biology of the cell in culture is discussed.

[Localization of the CD4 receptor gene in the chromosomes of clone cells of the monocytoid line U-937 characterized by different sensitivities to HIV]. / Lokalizatsiia gena retseptora CD4 v khromosomakh kletok klonov monotsitoidnoi linii U-937, kharakterizuiushchikhsia razlichnoi chuvstvitel'nost'iu k VICh.

Karyotypes of two clones of U-937 line, with high and low sensitivity to HIV-1, were studied. The CD4-receptor gene-cellular receptor of HIV-1 was mapped. CD4-receptor gene was located according to in situ hybridization method, in locus 12 p11-p12, both in cells of high-sensitive clone U-937/16, and in cells of low-sensitive clone U-937/4. It is determined that in both the clones chromosomes 12 are presented in two copies and are not affected by rearrangements. That allows to conclude that the sensitivity of cells U-937 to HIV-1 does not depend on the dose of this gene, or on its transference in chromosomes.

[The interrelation between the morphological characteristics of the nucleolar apparatus in cultured human embryonic fibroblasts and the proliferative activity of the cells]. / Vzaimosviaz' mezhdu morfologicheskimi kharakteristikami iadryshkovogo apparata kul'tiviruemykh émbrional'nykh fibroblastov cheloveka i proliferativnoi aktivnost'iu kletok.

A relationship between the number of AgNOR, nucleolar area, the number of nucleoli per nuclei, the number of nucleoli touching nucleolar membrane and the proliferating activity of human embryonal fibroblast culture was investigated. Cell proliferation was arrested by serum deprivation. In 10 days, initiation of proliferation was performed by cessation of serum deprivation. The strongest correlation (r = 0.96) was found between the index of labeling (IL) and the number of nucleoli touching nucleolar membrane. A strong correlation was also observed between AgNOR count, nucleolar area and IL. The number of silver grains in the interphase nucleus decreased slowly and increased quickly, with quick decrease and increase of cell proliferation occurring, respectively. No correlation was found between the number of nucleoli and IL.

[The cytogenetic characteristics of human B-lymphoblastoid cell lines]. / Tsitogeneticheskaia kharakteristika B-limfoblastoidnykh kletochnykh linii cheloveka.

Permanent suspension B-cell lines HSL-1, HSL-2, HSL-3, HSL-4, HSL-5, HSL-8, HSL-9 have been obtained spontaneously from lymphocytes and cells of hematopoietic tissues of patients with various forms of leukemia and tumours. All cell lines contained Epstein-Barr virus (EBV). At early stages of cultivation (6-39th passages) normal karyotypes were established for all the tested lines which testified their origin from normal B-lymphocytes. In the process of cultivation of HSL-1 cell line, which (up to the 63rd passage) contained primarily the normal karyotypes, a substitution for tetraploid ones (92, XX, YY) by the 132nd passage was established. In line HSL-4, a pathological clone of cells with structural chromosome rearrangements (46, XY, -2, -12, der (12) t (2; 12) (p12, p13); del2 (p12), was revealed at the 26th passage. During a prolonged cultivation (up to the 130th passage) this abnormal clone forced out completely the clone with normal karyotype. Substitution of normal clone cells by cells with pathological karyotypes may point to their selective advantage.

[The cytogenetic characteristics of B-lymphoid lines from Papio hamadryas baboons and Macaca arctoides brown macaques]. / Tsitogeneticheskaia kharakteristika B-limfoidnykh linii pavianov gamadrilov Papio hamadryas i makakov burykh Macaca arctoides.

A cytogenetic analysis of 9 permanent suspension cell lines was performed. Cell lines were obtained from Papio hamadryas and Macaca arctoides cells by different methods. Five cell lines originated from non-malignant parental cells. Lines SPG-5 and SPG-6 were analysed at early stages of cultivation, with normal karyotypes being identified. In cell lines PL-11, MAL-1 and MAL-4 pathological clones with structural and numerical chromosomal rearrangements appeared only following a long-term cultivation. This phenomenon may be due presumably to the influence of cultivation on karyotype variability. In cell line LUG-4 chromosome marker 17p+ was found at early stages of cultivation. In sublines E9-1, E1-1 and E5-1, established from line LUG-4 by cloning, 17p+ chromosomal marker was found persisting. The presence of the marker 17p+ in all the cells at early passages testifies that the cell material from Papio with lymphoma, which was used for this line, might have the same chromosomal damage in its initial karyotype.

[The variability of the pattern of human G-band chromosomes in the late prophase-early metaphase]. / Izmenchivost' risunka G-okrashennykh khromosom cheloveka v pozdnei profaze--rannei metafaze.

Original modification of the harvest procedure, hypotonic treatment and slide-making techniques were used to obtain prometaphase spreads of good quality fitted to G-banding or to FISH. Human blood cultures were synchronized with a methotrexate block during synthesis, and following this with a thymidine release. Cells were then proceeded into prometaphase and early metaphase and were quickly harvested without exposure to colcemid. Following incubation in a mixture of 0.56% KCl and 1% sodium citrate (1:1) for 20-23 min at room temperature, cells were fixed in 4 changes of fixative made of 3 parts of absolute methanol and 1 part of glacial acetic acid. Three or four 100-105 microliters volume of a suspension of fixed cells were blew with a strength through pasteur pipette tip from a distance 5-6 cm onto chilled superclean slides wetted by water. These slides were then quickly flame dried. Prior to staining, prometaphase bearing spreads were held in thermostat overnight at 37 degrees C. Prometaphases were G-banded with the aid of a modified technique elaborated in the Department of Clinical Genetics, Lund University Hospital (Sweden). The slides were incubated for 1-4 min at room temperature in 0.01 versene solution, containing 0.011% w/v trypsin (Difco), 0.4 mg/ml D-glucose, 0.17 mg/ml KCl, 1.7 mg/ml NaCl. Following a 15 sec incubation in a solution containing 1 mg/ml D-glucose, 0.4 mg/ml KCl, 8 mg/ml NaCl, G-banding was completed by staining the slides with 2% Giemsa (Merck) in pH 6.8 phosphate buffer for 4-5 min.(ABSTRACT TRUNCATED AT 250 WORDS)

[The dependence of the quantity of silver granules detectable in interphase nuclei after the silver nitrate staining of the chromosomal nucleolus-organizer regions on the conditions for the hypotonic treatment of the cells and the effect on this index of cell treatment by a pharmacological agent causing dissociation of the nucleolar components]. / Zavisimost' kolichestva granul serebra, vyiavliaemykh v interfaznykh iadrakh posle okrashivaniia iadryshkoobrazuiushchikh raionov khromosom nitratom serebra, ot uslovii gipotonicheskoi obrabotki kletok i vliianie na étot pokazatel' obrabotki kletok farmakologicheskim agentom, vyzyvaiushchim dissotsiatsiiu komponentov iadryshka.

We investigated effects of hypotonic treatment of cultured HL-60 cells and human fibroblasts, and of DRB on the quantity of AgNORs revealed following AgNO3 staining. The average number of grains increases after KCl hypotonic treatment. The lower the concentration of KCl in the solution, the higher the number of silver grains revealed in the nucleoli due to their dissociation into substructures. The use of adenosine analogue (DRB) before the hypotonic treatment of cells facilitates the following count of silver deposits. A maximum increase in the mean number of silver grains was observed after a 1.5 h incubation.

[The structure and function of the chromosomal nucleolus organizer regions: the molecular, cytological and clinical aspects]. / Struktura i funktsiia iadryshkoobrazuiushchikh raionov khromosom: molekuliarnye, tsitologicheskie i klinicheskie aspekty.

Numerous data concerning nucleolar organizer region (NOR) structure and function in normal and pathological human cells are analysed. The contents of argentophilic nucleolar proteins vary in the cells tested. The silver-stained test is closely connected with cellular proliferative potentials and differentiation status of the cells. This test also depends on the level of tumor cell malignancy and seemed to be very useful for megakaryocyte and cardiomyocyte studies. In summary, the NOR silver-staining approach is of great value for both fundamental cytological investigations and wide medical practice.

[The use of DNA hybridization in situ for identifying chromosomal rearrangements in the karyotyping of cell lines]. / Ispol'zovanie gibridizatsii DNK in situ dlia identifikatsii khromosomnykh perestroek pri kariotipirovanii kletochnykh linii.

A complex karyological analysis of three human cell lines (PLC-PRF-5, MT-4 and U937) was carried out that involved traditional methods of G-, C-banding and silver staining, in addition to the in situ hybridization technique using 4 alpha-satellite DNA probes: DNA specific for centromeric regions of chromosome 11, 6, 13, and 21, and 14 and 22. The application of this additional method allowed to identify, prove or detalize the structure of 13 markers in PLC-PRF-5 cells, 1 marker in MT-4 cells, and 3 markers in U937 cells. The results show that the in situ hybridization method would be successful in cell line karyotyping for a more objective identification of some markers difficult for analysis.

Karyological approach to the identification of true cell lines susceptible to the human immunodeficiency virus (HIV).

A karyological analysis of twenty-two variants of eight cell lines, which differed in their susceptibility to the human immunodeficiency virus (HIV) and which had been obtained from different sources, was carried out by means of differential chromosome staining for G and C bands. The karyotypes of eight T-lymphoblastoid cell lines were identified, including five (MT-4, Molt-3, CEM, H-9, and Hut-78) not previously studied by cytogenetic methods. Karyotyping confirmed the identity of seventeen variants of the eight cell lines, and five variants of four lines were found to be misidentified. Comparative analysis of the cytogenetic characteristics of the three CEM-line variants demonstrates the need for karyotype evaluation in the course of in vitro cell cultivation. Fourteen identical marker chromosomes were revealed in H-9 and Hut-78 cell karyotypes, confirming the common origin of these two lines. It was found that the cells of the HIV-susceptible lines had a tendency to undergo polyploidisation both during the initial stages after isolation and in the course of cultivation.

[Differences in the localization of Alu-repeats in various chromosomes of peripheral blood cells from normal donors and bone marrow cells from patients with acute leukemia]. / Razlichiia v lokalizatsii povtorov Alu-semeistva v nekotorykh khromosomakh kletok perifericheskoi krovi normal'nykh donorov i kletok kostnogo mozga bol'nykh ostrym leikozom.

The dispersion of the Alu-family DNA repeats in phytohemagglutinin-stimulated lymphocytes from peripheral blood of normal donors as well as in nonstimulated bone marrow cells of four patients suffering from acute leukemia was studied by hybridization on metaphase chromosomes in situ. DNA of bacteriophage lambda CAR42 clone containing the insertion of at least 8 copies of Alu-family DNA-repeats and labelled with tritium was used as a probe in hybridization. All patients with acute leukemia had the same pattern of changes in hybridization of the bone marrow cells. It consists of silver grains clustering over 3q26, 8p12, 14q24. The pattern may reflect amplification transposition of Alu-family DNA repeats in the human genome connected with cellular differentiation or malignant transformation of blood cells.

Combined trisomy 1q and monosomy 17p due to translocation t(1;17) in a patient with myelodysplastic syndrome.

A nonconstitutional translocation, t(1;17)(q21;p11), has been observed in bone marrow cells from a young patient with myelodysplastic syndrome during a 4-year period. Cytogenetic findings and some clinical aspects are briefly discussed.

[Cytogenetic and virological characteristics of the initial line and clones of RH cells with various degrees of sensitivity to the tick-borne encephalitis virus]. / Tsitogeneticheskaia i virusologicheskaia kharakteristika iskhodnoi linii i klonov kletok RH s razlichnoi chuvstvitel'nost'iu k virusu kleshchevogo éntsefalita.

Virological and cytogenetic characterization of line RH and its two clones (RHk-20 and RHk-13/6), having different sensitivity to the tick-borne encephalitis virus (TBE), has been made. The RHk-13/6-cells are 10,000 times more sensitive to cytopathic effect of the tick-borne encephalitis virus in comparison with the RHk-20-cells. By its sensitivity to the virus the base line is in the intermediate position. Cytogenetic analysis revealed the variability interval decrease as to the chromosome number at cloning, and showed the structural stability of the line and clone karyotypes. The line RH and its clone K-20 have modal number of chromosomes equal to 65; the cell population with the chromosome number equal to 66 dominated in RHk-13/6. The application of methods of chromosome differential (G, C) staining and these for the detection of active nucleolar organizers (NO) permitted identifying the marker chromosomes and determining the difference between the clones and the line as associated with the presence of an additional homologue of chromosome 8 in the sensitive clone.

[Detection of viral particles in the blood lymphocytes of a patient with T-cell acute lymphoblastic leukemia]. / Vyiavlenie virusnykh chastits v limfotsitakh krovi bol'nogo T-kletochnym ostrym limfoblastnym leikozom.

The electron microscopic examinations of the short-term cultures of mononuclear cells from patients with T-cell acute lymphoblastic leukemia were conducted for the detection of viral particles. The viral particles which correspond in their morphology to C type retroviruses are observed in the intercellular space of the culture.

The activity of nucleolar organizer regions of human bone marrow cells studied with silver staining. II. Acute leukemia.

The activity of nucleolar organizer regions (NOR) in chromosomes and interphase nuclei of bone marrow cells from 11 adult patients with acute lymphoblastic leukemia (ALL), 35 patients with acute nonlymphoblastic leukemia (ANLL), and eight healthy donors has been studied with silver nitrate staining. PHA-stimulated lymphocytes of the same individuals were used as standards of the maximum silver-staining patterns for each person. In 90% of patients with acute leukemia the average number of Ag+NOR in metaphases was lower when compared with that of PHA-stimulated lymphocytes. A variable expression of NOR was observed within the cell population and between individual patients. The populations tested showed high heterogeneity in relation to the content of Ag-negative mitoses. Ag+NOR per metaphase and the content of Ag-negative mitoses in bone marrow did not differ between patients with ALL and ANLL. Differences in the staining pattern in leukemic cells are discussed.