CHARACTERIZATION OF TOPOTECAN-MEDIATED REDISTRIBUTION OF DNA TOPOISOMERASE-I BY DIGITAL IMAGING MICROSCOPY

Topographical image measures have been used to characterize the subnuc lear distribution of DNA topoisomerase I in human tumor cell lines. Th is topographical analysis allowed a mathematical description of staini ng patterns to be produced that did not depend on subjective grading. The redistribution of topoisomerase I in response to increasing concen trations of topotecan was then monitored by this method. The cell line s were stained for topoisomerase I by indirect immunofluorescence meth ods. Digital imaging microscopy and image analysis were used to extrac t the nucleus from each cell, and nine parameters describing the topog raphy of the distribution of topoisomerase I within the nucleus were c omputed for each. Use of multivariate analysis of variance enabled thi s nine-parameter set to be reduced to a single canonical variable, rep resenting 60-90% of the observed internuclear variance. Plotting the c anonical variable vs drug concentration resulted in dose-response curv es that could be fitted well by a simple E-max model. From these curve fits, EC50 and E-max values for drug-induced redistribution of topois omerase I were determined. Our results indicate that neither the maxim um extent of topoisomerase I redistribution (E-max) nor the EC50 for d rug-induced redistribution correlated well with the growth inhibition produced by continuous exposure to topotecan in these cell lines. Howe ver, the EC50 determined for the l-h high-concentration exposure did r eflect the growth inhibition produced in cells exposed to the drug for 1 h. The methodology described may also be generally applied to any a ntigen of interest. (C) 1998 Academic Press.