Chinese hamster ovary cell line DXB-11: chromosomal instability and karyotype heterogeneity.

BACKGROUND: Chinese hamster ovary cell lines, also known as CHO cells, represent a large family of related, yet quite different, cell lines which are metabolic mutants derived from the original cell line, CHO-ori. Dihydrofolate reductase-deficient DXB-11 cell line, one of the first CHO derivatives, serves as the host cell line for the production of therapeutic proteins. It is generally assumed that DXB-11 is identical to DUKX or CHO-DUK cell lines, but, to our knowledge, DXB-11 karyotype has not been described yet. RESULTS: Using differential staining approaches (G-, C-banding and Ag-staining), we presented DXB-11 karyotype and revealed that karyotypes of DXB-11 and CHO-DUK cells have a number of differences. Although the number of chromosomes is equal-20 in each cell line-DXB-11 has normal chromosomes of the 1st and 5th pairs as well as an intact chromosome 8. Besides, in DXB-11 line, chromosome der(Z9) includes the material of chromosomes X and 6, whereas in CHO-DUK it results from the translocation of chromosomes 1 and 6. Ag-positive nucleolar organizer regions were revealed in the long arms of chromosome del(4)(q11q12) and both chromosome 5 homologues, as well as in the short arms of chromosomes 8 and add(8)(q11). Only 19 from 112 (16.96%) DXB-11 cells display identical chromosome complement accepted as the main structural variant of karyotype. The karyotype heterogeneity of all the rest of cells (93, 83.04%) occurs due to clonal and nonclonal additional structural rearrangements of chromosomes. Estimation of the frequency of chromosome involvement in these rearrangements allowed us to reveal that chromosomes 9, der(X)t(X;3;4), del(2)(p21p23), del(2)(q11q22) /Z2, der(4) /Z7, add(6)(p11) /Z8 are the most stable, whereas mar2, probably der(10), is the most unstable chromosome. A comparative analysis of our own and literary data on CHO karyotypes allowed to designate conservative chromosomes, both normal and rearranged, that remain unchanged in different CHO cell lines, as well as variable chromosomes that determine the individuality of karyotypes of CHO derivatives. CONCLUSION: DXB-11and CHO-DUK cell lines differ in karyotypes. The revealed differential instability of DXB-11 chromosomes is likely not incidental and results in karyotype heterogeneity of cell population.

Fluorometric Na+ Evaluation in Single Cells Using Flow Cytometry: Comparison with Flame Emission Assay.

BACKGROUND/AIMS: Sodium is a key player in the fundamental cell functions. Fluorescent probes are indispensable tools for monitoring intracellular sodium levels in single living cells. Since the fluorescence of sodium-sensitive dyes in cells is significantly different from that in an aqueous solution, the fluorescence signal is calibrated in situ indirectly using ionophores for equalizing external and intracellular ion concentration. Attempts to compare data obtained using fluorescent probes and by direct flame emission analysis are sparse and results are inaccurate. METHODS: We determined the intracellular sodium concentration in U937 cells by flow cytometry using the Na+-sensitive probe Asante Natrium Green-2 (ANG), and by standard flame emission photometry combined with the cellular water determination by cell density in Percoll gradient. The intracellular Na+ concentrations was modified using known ionophores or, alternatively, by blocking the sodium pump with ouabain or by causing cell apoptosis with staurosporine. RESULTS: It is revealed that both methods are comparable when intracellular sodium concentration was modified by ouabain-mediated blockage of the sodium pump or staurosporine-induced apoptosis. The ANG fluorescence of cells treated with ionophores is approximately two times lower than that in cells with the same Na+ concentration but not treated with ionophores. Although the mechanism is still unknown, this effect should be taken into account when a quantitative assessment of the concentration of intracellular sodium is required. CONCLUSION: The sodium sensitive dye ANG-2 is a sensitive and useful probe for determination changes in Na+ content and concentration both in single cells and subcellular microparticles. The ANG fluorescence determined in the studied cells in the absence of ionophores, cannot be used as a measure of the real intracellular concentration of Na+ if calibration was carried out in the presence of ionophores.

Pump and channel K (Rb+) fluxes in apoptosis of human lymphoid cell line U937.

Ouabain-sensitive (OS) and -resistant (OR) Rb(+) influx was examined in three sublines of U937 cells to compare alterations of K(+) channel permeability and the Na(+),K(+)-ATPase pump leading to the shift in ion and water balance during apoptosis induced by 0.2 and 1microM staurosporine (STS) for 4-5 h. Cell K(+), Rb(+), Na(+) and Cl(-) content was determined by flame photometry and (36)Cl distribution. Changes in cell water content were monitored by measurement of buoyant cell density and distribution of [(3)H]-glycerol or 3-O-methyl-D-[(3)H]glucose. Apoptosis was detected by DNA flow cytometry and light microscopy of the native cells stained with acridine orange. Treatment with 0.2 microM STS for 5 hours led to mild apoptosis with 10-13 % cell dehydration and either moderate increase of channel mediated Rb(+) influx without significant changes in the pump activity or moderate decrease of pump Rb(+) influx without significant change of channel influx, depending on the cell line used. Treatment with 1 microM STS was followed by 18-23 % cell dehydration, a decrease of the pump activity and a small or insignificant increase in the OR Rb(+) influx in all studied sublines. It is concluded that moderate apoptotic cell shrinkage may be associated with both an increase in K(+) channel permeability and inhibition of the pump whereas more remarkable shrinkage occurs presumably due to inhibition of the pump.