QUANTITATIVE IMMUNOFLUORESCENCE AND IMMUNOELECTRON MICROSCOPY OF THE TOPOISOMERASE II-ALPHA ASSOCIATED WITH NUCLEAR MATRICES FROM WILD-TYPEAND DRUG-RESISTANT CHINESE-HAMSTER OVARY CELL-LINES

Topo II alpha is considered an important constituent of the nuclear ma trix, serving as a fastener of DNA loops to the underlying filamentous scaffolding network, To further define a mechanism of drug resistance to topo II poisons, we studied the quantity of topo II alpha associat ed with the nuclear matrix in drug-resistant SMR16 and parental cells in the presence and absence of VP-16. Nuclear matrices were prepared f rom nuclei isolated in EDTA buffer, followed by nuclease digestion wit h DNase II in the absence of RNase treatment and extraction with 2 M N aCl. Whole-mount spreading of residual structures permits, by means of isoform-specific antibody and colloidal-gold secondary antibodies, an estimate of the amount of topo II alpha in individual nuclear matrice s. There are significant variations in Lope II alpha amounts between i ndividual nuclear matrices due to the cell cycle distribution. The par ental cell line contained eight to ten times more nuclear matrix-assoc iated topo II alpha than the resistant cell line matrices. Nuclear mat rix-associated lope II alpha from wild-type and resistant cell lines c orrelated well with the immunofluorescent staining of the enzyme in nu clei of intact cells. The amount of DNA associated with residual nucle ar structures was five times greater in the resistant cell line. This quantity of DNA was not proportional to the quantity of topo II alpha in the same matrix; in fact they were inversely related. In situ whole -mount nuclear matrix preparations were obtained from cells grown on g rids and confirmed the results from labeling of isolated residual stru ctures. (C) 1997 Wiley-Liss, Inc.