No abstract

The translocation t(8;21)(q22;q22) affecting AML1 and ETO genes is known to be one of the frequent chromosome translocations in acute myeloid leukemia. But no data have been available up to date concerning mutual positioning of these particular genes in the nucleus of a living cell as well as the mechanism of their rapprochement and realignment. Here we show that there is no proximity between these two genes in the primary nuclei of normal human male fibroblasts and moreover that these genes are located in different nuclear layers. But we further show that treatment of cells with VP-16 (etoposide), an inhibitor of DNA topoisomerase II widely used in anticancer chemotherapy, causes the ETO gene repositioning which allows AML1 and ETO genes to be localized in the same nuclear layer. Inhibitor studies demonstrate that such an effect is likely to be connected with the formation of stalled cleavable complexes on DNA. Finally, inhibition of ETO gene repositioning by 2,3-butanedione monoxime (BDM) suggests that this process depends on nuclear myosin. Together, our data corroborate the so called "breakage first" model of the origins of recurrent reciprocal translocation.

Intercellular, nonartifactual variability of nucleolar organizer region (NOR)-Ag-staining was studied in cultured human peripheral blood lymphocytes, skin and embryonic fibroblasts. No differences in number and character of variable NORs and intensity of their staining were observed between lymphocytes stimulated to proliferate with phytohemagglutinin and pokeweed mitogen, as well as lymphocytes of first- and second division. The number of NOR associations per cell and the number of associated chromosomes per association were also similar. In a given individual these criteria were similar in lymphocytes and fibroblasts. In all nine clones derived from three independent parental fibroblast cultures the intercellular NOR-Ag-variability was similar to that observed in a given parental cell line. A significant decrease in the number of metaphases containing NOR associations was observed in second-division lymphocytes compared with first-division ones, as well as in skin fibroblasts compared with lymphocytes.

Clones of telomerized fibroblasts of adult human skin have earlier been obtained. It was shown that despite their fast growth in mass cultures, these cells poorly form colonies. Conditioned medium, antioxidants, and reduced partial oxygen pressure enhanced their colony formation, but not to the level characteristic of the initial cells. The conditioned medium of telomerized cells enhanced colony formation to a much greater extent than that of the initial cells. A study of proteome of the telomerized fibroblasts has revealed changes in the activities of tens of genes. A general trend consists in weakening and increased lability of the cytoskeleton and in activation of the mechanisms controlling protein degradation. However, these changes are not very pronounced. During the formation of immortal telomerized cells, selection takes place, which appears to determine changes in the expression of some genes. It was proposed that a decrease in the capacity of telomerized cells for colony formation is due to increased requirements of these cells to cell-cell contacts. The rate of cell growth reached that characteristic of mass cultures only in the largest colonies. In this respect, the telomerized fibroblasts resembled stem cells: they are capable of self-maintenance, but "escape" to differentiation in the absence of the corresponding microenvironment (niche), which is represented by other fibroblasts. Nondividing cells in the test of colony formation should be regarded as differentiated cells, since they have no features of degradation, preserve their viability, actively move, grow, phagocytize debris, etc. It was also shown that telomerization did not prevent differentiation of myoblasts and human neural stem cells. Thus, the results obtained suggest the existence of normal mechanisms underlying the regulation of proliferation in the telomerized cells, which opens possibilities of their use in cell therapy, especially in the case of auto-transplantation to senior people, when the cell proliferative potential is markedly reduced and accessibility of stem cells is significantly restricted.

Cultures of human and mammalian cells presenting 4 types of differentiation (normal human fibroblasts and myoblasts, human and Syrian hamster hepatoma cells, and mouse/mouse hybridoma cells) were used in a panel biotest system. This system allowed to evaluate the cytotoxic and stimulatory effect of bioactive compounds by determining the dose-effect relationships and some quantitative parameters including LD(50). Examination of some biolactive compounds of different nature (sangviritrin, escin, deltostim, cycloheximide, dexamethasone) confirmed high efficacy of this biotest system.