Association of topoisomerase II with the hepatoma cell nuclear matrix: the role of intermolecular disulfide bond formation.
Previous studies have resulted in conflicting data regarding the recovery of the nuclear enzymes topoisomerase (topo) II and topo I in the nuclear matrix fraction. In the present study we have assessed the effect of systematically altering a single extraction procedure on the distribution of these enzymes during the subfractionation of nuclei from HTC hepatoma tissue culture cells. When nuclear monolayers (prepared by treating attached cells in situ with the neutral detergent Nonidet-P40 at 4 degrees C) were isolated in the presence of the irreversible sulfhydryl blocking reagent iodoacetamide, subsequent treatment with DNase I and RNase A followed by 1.6 M NaCl resulted in structures which were extensively depleted of intranuclear components as assessed by phase contrast microscopy and conventional transmission electron microscopy. These structures contained 12 +/- 4% of the total protein present in the original nuclear monolayers. The lamins and polypeptides with molecular weights comparable to those of actin and vimentin were the predominant polypeptides present on SDS-polyacrylamide gels. Western blotting revealed that less than 5% of the total nuclear topo II molecules were present in these structures. In contrast, when the sulfhydryl cross-linking reagent sodium tetrathionate (NaTT) was substituted for iodoacetamide, the same extraction procedure yielded structures containing components of the nucleolus and an extensive intranuclear network. These structures contained a wide variety of nonlamin, nonhistone nuclear polypeptides including 23 +/- 4% of the total nuclear topo II. SDS-polyacrylamide gel electrophoresis performed under nonreducing conditions revealed that topo II in these nuclear matrices was present as part of a large disulfide cross-linked complex. Treatment of these structures with reducing agents in 1.6 M NaCl released the topo II. In contrast, topo I did not form disulfide cross-linked oligomers and was not detectable in any of these nuclease- and salt-resistant structures prepared at 4 degrees C. To assess the effect of in vitro heat treatment on the distribution of the topoisomerases, nuclear monolayers (isolated in the absence of iodoacetamide and NaTT) were heated to 37 degrees C for 1 h prior to treatment with nucleases and 1.6 M NaCl. The resulting structures (which retained 26 +/- 5% of the total nuclear protein) were morphologically similar to the NaTT-stabilized nuclear matrices and contained 15 +/- 4% of the total nuclear topo II. High-molecular-weight disulfide cross-linked oligomers of topo II were again demonstrated. Attempts to demonstrate these disulfide cross-linked oligomers in intact cells were unsuccessful.
Kaufmann
S H
SH
Department of Pharmacology, Johns Hopkins University School of Medicine, Johns Hopkins Hospital, Baltimore, Maryland 21205.
Shaper
J H
JH
D006528:Carcinoma, Hepatocellular; D002458:Cell Fractionation / Q000379:methods; D003432:Cross-Linking Reagents; D004264:DNA Topoisomerases, Type I / Q000032:analysis; D004250:DNA Topoisomerases, Type II / Q000032:analysis*; D003850:Deoxyribonuclease I; D003902:Detergents; D004220:Disulfides / Q000378:metabolism; D006358:Hot Temperature; D007399:Interphase; D015530:Nuclear Matrix / Q000201:enzymology*; D017830:Octoxynol; D011092:Polyethylene Glycols; D012259:Ribonuclease, Pancreatic; D012965:Sodium Chloride; D012995:Solubility; D013776:Tetrathionic Acid; D014407:Tumor Cells, Cultured
D003432:Cross-Linking Reagents; D003902:Detergents; D004220:Disulfides; D011092:Polyethylene Glycols; D012965:Sodium Chloride; D013776:Tetrathionic Acid; D017830:Octoxynol; C010615:Nonidet P-40; D003850:Deoxyribonuclease I; D012259:Ribonuclease, Pancreatic; D004264:DNA Topoisomerases, Type I; D004250:DNA Topoisomerases, Type II