Discrete localization of different DNA topoisomerases in HeLa and K562 cell nuclei and subnuclear fractions.
Monoclonal antibodies raised against DNA topoisomerase I and against topoisomerase II alpha and beta isoforms, which have been previously demonstrated to be highly specific and capable of detecting cell cycle-related variations of the topoisomerase II isoforms (Negri et al., 1992, Exp. Cell Res. 200, 452-459), have been utilized for a fine subcellular localization. Immunocytochemistry by confocal and electron microscopy have been used for a topological and quantitative evaluation of the fine distribution of the different topoisomerases in HeLa and K562 cells. Topoisomerase I and topoisomerase II alpha are present both in the nucleoplasm and in the nucleolus, though at different relative ratios, while topoisomerase II beta is exclusively present at the nucleolar level. This is further confirmed by immunoblotting and immunocytochemical quantitative evaluations performed on purified nuclear matrix fractions obtained from K562 cells. In fact, the amount of topoisomerase I and topoisomerase II alpha present in the whole cell nuclei is partly lost in isolated nuclei but, while topoisomerase I is further significantly reduced in nuclear matrix preparations, the topoisomerase II alpha content is only slightly decreased. On the other hand, the great majority of topoisomerase II beta is retained in the nuclear matrix and can be detected exclusively in association with the nucleolar remnant. These results are consistent with specific functional roles hypothesized for the different topoisomerase types.
Zini
N
N
Istituto di Citomorfologia Normale e Patologica, CNR, Bologna, Italy.
Santi
S
S
Ognibene
A
A
Bavelloni
A
A
Neri
L M
LM
Valmori
A
A
Mariani
E
E
Negri
C
C
Astaldi-Ricotti
G C
GC
Maraldi
N M
NM
D000911:Antibodies, Monoclonal; D002460:Cell Line; D002467:Cell Nucleus / Q000201:enzymology* / Q000648:ultrastructure; D004264:DNA Topoisomerases, Type I / Q000032:analysis*; D004250:DNA Topoisomerases, Type II / Q000032:analysis*; D006367:HeLa Cells; D006801:Humans; D007150:Immunohistochemistry; D007527:Isoenzymes / Q000032:analysis; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive; D016253:Microscopy, Immunoelectron; D015530:Nuclear Matrix / Q000201:enzymology* / Q000648:ultrastructure; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D007527:Isoenzymes; D004264:DNA Topoisomerases, Type I; D004250:DNA Topoisomerases, Type II