[Immortalized cell lines from murine embryos are characterized by progressive destabilization of their karyotype structure]. / Immortalizovannye linii kletok, poluchennye iz transgennykh émbrionov myshei, kharakterizuiutsia progressiruiushzchei destabilizatsiei struktury kariotipa.

The karyotype structure was studied for three cell lines obtained from cells of transgenic murine embryos at early stages of their establishment. The first line was obtained from a transgenic embryonic explantant containing oncogen v-sis under promotor MMTV, two other lines originated from cells of transgenic embryos containing oncogen k51. The karyotypic analysis of G-banded metaphase chromosomes revealed deviations from the normal mouse karyotype as early as by the third passage of cultivation of independent embryonic cell lines that contained a foreign oncogene in their genome. The repeated analysis that involved 15-22 passages revealed similar abnormalities: variability and progression in chromosome number with the appearance of hyperpolyploid combinations, and a large number of rearranged chromosomes, both marker and unique ones. It is concluded that introduction of a foreign oncogene into murine cell genome leads to its enhanced and progressive non-specific destabilization. Oncogen v-sis produces a more valuable karyotype destabilization than oncogen k51.

Resistance to adriamycin in human chronic promyeloleukemia line K562 correlates with directed genome destabilization--amplification of MDR1 gene and nonrandom changes in karyotype structure. / Ustoichivost' kletok khronicheskogo promieloleikoza cheloveka linii K562 k adriamitsinu korreliruet s napravlennoi destabilizatsiei genoma--amplifikatsiei gena MDR1 i ne sluchainymi izmeneniiami v strukture genotipa.

Three independent subclones (B2, B3 and C9) of human myelogenous leukemia cell line K562 selected with adriamycin (ADM) were analysed. Cross-resistance of these ADM-resistant cells was examined for a resistance to the number of drugs including colchicine, actinomycin D and ethidium bromide. MDR 1 gene amplification in B3 and C9 subclones was detected using Southern-hybridization with specific probe. Additional genetical material was found in genomes of resistant cells by analysis of G-banded metaphase chromosomes. An extraordinarily long marker chromosome was observed in every C9 metaphase plate. The character of this chromosome G-banding suggests that it may be a derivative of chromosome 5 containing a large homogeneously staining region (HSR) in locus 5q15. Both B2 and B3 subclones expressed double minute chromosomes (DMs) in 5% of cells. In the course of a prolonged cultivation (about 2 years) of C9 cells in the presence of ADM a progressive karyotype destabilization was observed: the frequency of new markers formation in C9 cells increased, cells having additional copies of marker chromosome with HSR appeared, the length of HSR varied, coexistence of HSR and DMs being found in several C9 cells. These karyotypical changes may be regarded as patterns of genome destabilization due to the multidrug resistance of K562/ADM cells.

Accessibility of T-tubule vacuoles to extracellular dextran and DNA: mechanism and potential application of vacuolation.

In addition to its function in excitation-contraction coupling, the ability of the T-system of skeletal muscle fibres to undergo reversible vacuolation indicates that it may play a role in volume regulation. The mechanism of reversible vacuolation has been investigated by confocal microscopy using fluorescein dextran to probe the accessibility of T-tubules to the extracellular environment. Vacuolation was induced by loading the fibres with 60-100 nM glycerol for 30 minutes and then returning them to glycerol-free medium. Devacuolation was subsequently induced by the reentry of glycerol. During their formation from T-tubules, the vacuoles filled with fluorescent dextran from the extracellular medium. The inaccessibility of the vacuoles to extracellular ferritin observed in a previous study raised the possibility that the vacuoles may be detached from the surface membrane after their formation. However, it is apparent from the present work that, although the tubules of the treated fibres are constricted, the vacuoles maintain a open connection with the external medium for smaller macromolecules. In the light of these experiments, it is proposed that vacuolation is caused by water moving into T-tubules from the cytoplasm faster than it can exit to the extracellular space during a decrease in fibre volume. Since T-tubules have been implicated in the transfection of skeletal muscle by direct injection, the accessibility of plasmid DNA to T-tubules has also been investigated. DNA penetrated into the vacuoles from the extracellular medium less well than dextran, but many vacuoles containing fluorescent DNA were observed in the superficial layers of vacuolated fibres, and it is suggested that T-tubule vacuolation might be used to improve the efficiency of the transfection of skeletal muscle by direct injection.

[The growth-stimulating and immunomodulating activity of culture media conditioned by transformed fibroblasts from serum-free lines]. / Roststimuliruiushchaia i immunomoduliruiushchaia aktivnost' kul'tural'nykh sred, konditsionirovannykh transformirovannymi fibroblastami bessyvorotochnykh linii.

Five cell lines of transformed rodent fibroblasts capable of unlimited growth without exogenous proteins were studied. It has been demonstrated that the media conditioned by these cells (CMs), the protein preparations obtained from CMs, or the feeder cell cultures of the serum-free cell lines are mitogenic to mouse hybridoma cells as well as to primary cell cultures of the rat bone marrow. The addition of CMs proteins into mixtures of mouse splenocytes and transformed target cells resulted in stimulation or suppression of the splenocyte cytotoxicity when YAC-1 [correction of JAC-1] or K562 cell targets were used. A 18-20 h splenocyte preincubation with proteins, released by transformed rat fibroblasts of the serum-free cell line LRec-1sf, resulted in a 1.5-4-fold increase in K562 cell lysis by murine natural killer cells. By contrast, the target cell preincubation with the same proteins resulted in a 1.5-2-fold decrease in K562 cell sensitivity to cytotoxic effect of natural killer cells. Taken together our data show that the transformed rodent fibroblasts, growing without exogenous proteins, produce and release into the culture medium some growth stimulating, immunomodulating and immunoprotective factors.

[The cultivation features, growth characteristics and oncogenicity of "serum-free" populations of transformed fibroblasts]. / Osobennosti kul'tivirovaniia, kharakteristiki rosta i onkogennost' "bessyvorotochnykh" populiatsii transformirovannykh fibroblastov.

The authors report about five previously selected cell lines of transformed mouse and rat fibroblasts, capable of growing in the medium lacking exogenous proteins. In contrast to cells of the parental lines, the cells under investigation have the ability to semi-suspension growth pattern and demonstrate an increasing sensitivity to damaging action of trypsin. The lowering of the autological conditioned medium (CM) concentration below 10-15%, as well as the reducing of the initial population density to 0.2 x 10(4)-0.4 x 10(5) cells per 1 cm2, resulted in inhibition of the serum-free cell proliferation. The feeder cultures of the serum-free cells immobilized by X-ray are able to stimulate proliferation of the intact autological cells in medium with semi-liquid agar in the presence of serum. CMs of the serum-free cell cultures stimulated the DNA synthesis in stationary cultures of NIH 3T3 cells in the absence of serum. The loss of dependence on serum was accompanied by a considerable broadening of the metionine-containing protein spectrum in the culture medium of the transformed rat embryo fibroblasts. In addition, it was demonstrated that selection for independence on exogenous proteins was accompanied by the loss of oncogenicity in both sensitive and resistant to cytotoxic effect of ethidium bromide serum-free cell lines, derived from L929 cell line. On the contrary, the serum-free variants from spontaneously transformed rat embryo fibroblasts as well as from C3H 10T1/2 cell line exhibit an enhanced oncogenicity as compared with the parental cells.(ABSTRACT TRUNCATED AT 250 WORDS)

[Electron microscopic study of the minichromosomes in murine fibroblast cells fixed in situ]. / Elektronno-mikroskopicheskoe izuchenie minikhromosom v kletkakh myshinykh fibroblastov, fiksirovannykh in situ.

The ultrastructure and differential staining of minute chromosomes (MC) from mouse fibroblasts line C3H10T1/2 (LS9/0.1) was studied. MC in mitotic cells of this line contain R-positive material and are not stained according to the G- and C-methods. Reconstruction of ultrathin serial sections made it possible to identify MC in situ. It is shown that the basic structural unit of MC is a DNP fibril 20-30 nm in diameter. The density of packing of the MC material corresponds to that of normal chromosomal arms. In some cases the fibrillar material is present between MC and normal chromosomes. It is suggested therefore that some MC may be structurally linked to a normal chromosome arm. The MC structure is discussed in terms of the ultrastructural properties of R- and C-positive segments.

[Karyotype characteristics of C3H10T1/2-strain mouse fibroblasts undergoing long-term selection for their capacity to multiply in the presence of ethidium bromide]. / Osobennosti kariotipa fibroblastov myshi linii C3H10T1/2, proshedshikh dlitel'nuiu selektsiiu na sposobnost' razmnozhat'sia v prisutstvii bromistogo étidiia.

Variants Br-0.5 and Br-1 of minimally transformed mouse fibroblasts of C3H10T1/2 line were selected for their ability to proliferate in the medium with 0.5 and 1 mkg/ml of ethidium bromide (EB) toxic for cells of the parent line. Karyological analysis of metaphase chromosomes, stained by Giemsa for G-bands, revealed the number of significant changes in the karyotype of cells resistant to EB. In cells of the resistant sublines the variability of chromosomes was higher than in those of the sensitive population. Two groups of cells are distinguished in the Br-0.5 subline: those with near-diploid and tetraploid chromosome numbers, respectively. The number of polyploid cells in the EB-resistant sublines increases up to 38%, compared to 2% in the parent population. The marker chromosomes in resistant cells originated from translocations, deletions and inversions, with preferential involvement of the material from chromosomes 1.4 and 6. The pericentromeric region of chromosome 4 and the distal region of chromosome I (region 1H1-1H6) were characterized by the increased variability and preferential involvement in rearrangements. In cells of both resistant sublines double mini-chromosomes (1-5 copies per cell) were found. The relation between the revealed chromosomal rearrangements and the mechanism of EB-resistance is discussed.

[Control of cell multiplication in vitro. IV. Isolation and characteristics of C3H10T1/2-line cells with decreased dependence for multiplication on blood serum]. / Kontrol' razmonozheniia kletok in vitro. IV. Poluchenie i kharakteristika kletok linii C3H10T1/2 so snizhennoi zavisimost'iu razmnozheniia ot syvorotki krovi.

Mouse embryo fibroblasts of C3H10T1/2 cell line were selected in the medium with a low serum content. The frequency of clone occurrence was about 1.10(-5) (for 0.5% serum). The treatment of cells by N-methyl-N'-nitrosoguanidine significantly increased the frequency of occurrence of these clones. The obtained spontaneous variants were assayed for stability of growth characteristics. The phenotypic analysis of clones with low serum growth dependence discovered at least two cell types differing in some morphological and growth characteristics. The former did not differ from the parental line C3H10T1/2 in any phenotype characteristics but their serum growth dependence; whereas, the latter were poorly spread over the substrate and showed an enhanced saturation density and a decreased population doubling time. These cells also differed in their growth dependence on the homologous conditioned medium.