Stable gene amplification and overexpression of sodium- and potassium-activated ATPase in HeLa cells.

Abstract

Cell lines stably resistant to ouabain were isolated from an unstably resistant HeLa line after growth in nonselective medium. Stable resistant lines bound ouabain at levels 10-fold higher than did HeLa cells and at similar levels to those bound by the unstable C+ line previously described (J. F. Ash, R. M. Fineman, T. Kalka, M. Morgan, and B. Wire, J. Cell Biol. 99: 971-983). Expression and synthesis of the Na+, K+ -ATPase alpha chain showed a similar amplification over that for HeLa cells by Western blots and [35S]methionine pulse-labeling. In addition, a glycoprotein labeled with [3H]fucose and comigrating with the Na+, K+ -ATPase beta chain was eight- to ninefold amplified in stably resistant lines. Dot blots with a cDNA clone specific for Na+, K+ -ATPase alpha chain gene sequences confirmed the amplification of this gene. Karyotyping suggested that the amplification is associated with an expanded, abnormal banded region on the long (q) arm of one chromosome 17.

Structure/function analysis of Na+-K+-ATPase central isoform-specific region: involvement in PKC regulation

“…Resistance to sublethal concentrations of ouabain is occasionally achieved, for instance, by significant overexpression of pumps rather than the introduction of a resistant enzyme (15). Values are means Ϯ SE (n ϭ 6-11) of flux activations induced by PMA exposure, expressed as the percentage of their paired controls (same transfection group, same passage, same day), and were compared using paired 2-tailed Student's t-tests.…”

DNA amplification is rare in normal human cells.

“…In selections with MTX the frequency of colonies that exhibited some growth after isolation was 9 X 10-7. These frequencies are relative to starting cell numbers and (40)(41)(42)(43)(44), for amplification of the gene encoding the a-subunit of the Na, K-ATPase with oubain (45,46), for amplification of an adenine deaminase gene with adenine nucleosides (47), and for amplification of the UMP synthase gene with pyrazofurin (48 (25), ouabain (49,50), azaguanine (51), 6-thioguanine (50), and diphtheria toxin (52) and selection of keratinocytes with 6-thioguanine (53 (7,54) but not in one other (55). The …”

Three differentially expressed Na,K-ATPase alpha subunit isoforms: structural and functional implications.

“…In addition to al, ouabain resistance may also involve expression of other a subunits isoform(s), as well as different levels of expression of these isoforms. Moreover, the ouabain resistance conferred by intrinsic differences in the a subunit isoforms must be distinguished from other phenomena observed in acquired ouabain resistance, such as gene amplification (Emanuel et al, 1986;Pauw et al, 1986), and the expression of an ouabain resistance gene (Levenson et al, 1984).…”