Amplification of sodium- and potassium-activated adenosinetriphosphatase in HeLa cells by ouabain step selection.

Ash, John F.; Fineman, Robert M.; Kalka, T; Morgan, Michael; Wire, B

doi:10.1083/jcb.99.3.971

Cited by 33 publications

(17 citation statements)

References 45 publications

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“…The increase in Na,K-ATPase content of C+ cells was correlated with the appearance of minute chromosomes, a hallmark of gene amplification (15). The reversion of C+ cells to ouabain sensitivity, coupled with a decrease in Na,K-ATPase and a loss of minute chromosomes (1,12), suggested that Na,K-ATPase overproduction in C+ cells was associated with gene amplification.…”

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confidence: 93%

“…One such cell line, C+, was found to express a 10-fold-higher level of Na,K-ATPase than parental HeLa cells (1,12). The increase in Na,K-ATPase content of C+ cells was correlated with the appearance of minute chromosomes, a hallmark of gene amplification (15).…”

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confidence: 99%

“…Cell lines resistant to levels of ouabain which are lethal to wild-type cells have been isolated in several laboratories (1,2,11,13,18). One such cell line, C+, was found to express a 10-fold-higher level of Na,K-ATPase than parental HeLa cells (1,12).…”

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confidence: 99%

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Amplification of DNA sequences coding for the Na,K-ATPase alpha-subunit in ouabain-resistant C+ cells.

Emanuel¹,

Garetz²,

Schneider³

et al. 1986

Mol. Cell. Biol.

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We have studied the mechanism of cellular resistance to cardiac glycosides in C+ cells. C+ cells were resistant to ouabain and overproduced plasma membrane-bound Na,K-ATPase relative to parental HeLa cells. Overexpression of Na,K-ATPase in C+ cells correlated with increased ATPase mRNA levels and amplification (approximately 100 times) of the ATPase gene. Growth of C+ cells in ouabain-free medium resulted in a marked decline in ATPase mRNA and DNA levels. However, when cells were reexposed to ouabain, they proliferated and ATPase mRNA and DNA sequences were reamplified. Restriction analysis of C+ and other human DNA samples revealed the occurrence of rearrangements in the region of the Na,K-ATPase gene in C+ cells. Furthermore, C+ cells expressed an ATPase mRNA species not found in HeLa cells. These results suggest that amplification of the gene coding for Na,K-ATPase results in overproduction of Na,K-ATPase polypeptides. Amplification of the ATPase gene or the expression of new ATPase mRNA sequences or both may also be responsible for acquisition of the ouabain-resistant phenotype.

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Amplification of DNA sequences coding for the Na,K-ATPase alpha-subunit in ouabain-resistant C+ cells.

Emanuel¹,

Garetz²,

Schneider³

et al. 1986

Mol. Cell. Biol.

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“…Ouabain-binding measurement and calculation of the number of ouabain-binding sites were performed as previously described (1).…”

Section: Methodsmentioning

confidence: 99%

“…A variant of the HeLa S3 cell line which expressed 10-to 12-fold more ouabain-binding sites was recently selected with the sodium-and potassium-activated ATPase (Na+,K+-ATPase) inhibitor ouabain (1). The unstable ouabainresistant phenotype was correlated with the presence of minute chromosomes, and amplification of the gene for Na+,K+-ATPase was proposed as the mechanism leading to drug resistance.…”

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

Pauw¹,

Johnson²,

Moore³

et al. 1986

Mol. Cell. Biol.

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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.

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Graded amplification of the Na, K‐ATPase across a subclonal series: Effects on membrane physiology

Pauw

Ash

1987

Journal Cellular Physiology

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We have generated a series of clonally related cell lines which differ in the level of amplified expression of the Na,K-ATPase. These lines, originally derived from the ouabain resistant HeLa variant C+, expressed different numbers of binding sites for the Na,K-ATPase inhibitor ouabain, ranging from 2.9 X 10(6)/cell to 11.8 X 10(6)/cell. Amplification of the genes for both subunits of the enzyme was also seen but was not strictly correlated with level of expression. The influxes of histidine and tetraphenylphosphonium were measured across a series, including HeLa S3 and revertants, expressing from 0.74 X 10(6) to 10.5 X 10(6) ouabain-binding sites per cell. Tetraphenylphosphonium influx rate, presumed to be a function of membrane potential, varied linearly with ouabain binding site number, while histidine influx varied with the log of ouabain binding site number. Our results suggest that membrane potential increases in a simple fashion across our series of amplified lines. However, histidine influx was unaffected by treatments which cause membrane depolarization and a decrease in tetraphenylphosphonium influx rate. We propose that increasing histidine influx rates across our amplified series reflects exchange acceleration of L system transport due to increased intracellular pools of L system reactive amino acids. The Na,K-ATPase is ultimately responsible for most active transport across the plasma membrane. The consistent, graded physiological alterations seen across this series of closely related lines, chosen for graded enzyme expression, demonstrate the value of this novel genetic approach to the study of the energization of membrane transport.

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Amplification of sodium- and potassium-activated adenosinetriphosphatase in HeLa cells by ouabain step selection.

Cited by 33 publications

References 45 publications

Amplification of DNA sequences coding for the Na,K-ATPase alpha-subunit in ouabain-resistant C+ cells.

Amplification of DNA sequences coding for the Na,K-ATPase alpha-subunit in ouabain-resistant C+ cells.

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

Graded amplification of the Na, K‐ATPase across a subclonal series: Effects on membrane physiology

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