Development of a specific assay for cardiac glycoside-like compounds based on cross-resistance of human cell mutants

Abstract

The cross-resistance patterns of single- and two-step mutants ofHeLa cells resistant to SC4453 (a digoxin analog) and digoxin, which involve specific alteration in Na +, K +-ATPase towards numerous other compounds, have been examined. The mutants exhibited increased resistance to all of the steroidal compounds known to elicit a digitalis-like positive inotropic response (viz. various cardiac glycosides and their genins, erythrophleum alkaloid cassaine), but they showed no cross-resistance to any of a large number of other compounds which do not show cardiac glycoside (CG)-like biological activity. Based on the above characteristics of the mutants, a new cross-resistance assay for identifying compounds that show CG-like activity has been developed. In this assay, a sample is considered to possess CG-like activity if, in comparison to the parental HeLa cells, the CG R mutants exhibit increased resistance to it. From the known D 10value (drug concentration which reduces cloning efficiency of cells to 10%) of the drug for HeLa cells and the sample dilution necessary to produce equivalent cytotoxicity, the concentration of CGs in a given sample can be estimated. In blind studies the assay correctly identified all of the samples containing CGs; none of the other samples which lacked such activity tested positive. In the blind studies the assay also provided a good estimate of the concentration of CGs (±50% of the actual concentration) that was not affected by the presence of either serum components or a 20-fold excess of various steroidal compounds known to interfere in other assays. In view of the high specificity of the present assay for CG-like compounds, it should prove very useful in establishing/characterizing the presence of such activity in various biological (namely endogenous digitalis-like substances) and other samples.