Quantitative immunofluorescence and immunoelectron microscopy of the topoisomerase II alpha associated with nuclear matrices from wild-type and drug-resistant chinese hamster ovary cell lines.
Topo II alpha is considered an important constituent of the nuclear matrix, 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 associated with the nuclear matrix in drug-resistant SMR16 and parental cells in the presence and absence of VP-16. Nuclear matrices were prepared from nuclei isolated in EDTA buffer, followed by nuclease digestion with DNase II in the absence of RNase treatment and extraction with 2 M NaCl. 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 matrices. There are significant variations in topo II alpha amounts between individual nuclear matrices due to the cell cycle distribution. The parental cell line contained eight to ten times more nuclear matrix-associated topo II alpha than the resistant cell line matrices. Nuclear matrix-associated topo II alpha from wild-type and resistant cell lines correlated well with the immunofluorescent staining of the enzyme in nuclei of intact cells. The amount of DNA associated with residual nuclear 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 grids and confirmed the results from labeling of isolated residual structures.
Valkov
N I
NI
H. Lee Moffitt Cancer Center & Research Institute, Department of Internal Medicine and Biochemistry, University of South Florida, Tampa 33612, USA.
Gump
J L
JL
Sullivan
D M
DM
D000818:Animals; D000951:Antigens, Neoplasm; D015153:Blotting, Western; D016466:CHO Cells; D006224:Cricetinae; D003593:Cytoplasm / Q000201:enzymology; D004250:DNA Topoisomerases, Type II / Q000032:analysis*; D004268:DNA-Binding Proteins; D003850:Deoxyribonuclease I; D004351:Drug Resistance / Q000502:physiology; D004706:Endodeoxyribonucleases; D004791:Enzyme Inhibitors; D005047:Etoposide / Q000494:pharmacology; D007527:Isoenzymes / Q000032:analysis* / Q000037:antagonists & inhibitors; D008856:Microscopy, Fluorescence / Q000379:methods; D016253:Microscopy, Immunoelectron / Q000379:methods; D015530:Nuclear Matrix / Q000201:enzymology*; D059005:Topoisomerase II Inhibitors
D000951:Antigens, Neoplasm; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D059005:Topoisomerase II Inhibitors; D005047:Etoposide; D004706:Endodeoxyribonucleases; D003850:Deoxyribonuclease I; C019329:deoxyribonuclease II; D004250:DNA Topoisomerases, Type II