Differential expression and nuclear localization of human topoisomerase IIIalpha during the cell cycle progression in PHA-activated peripheral blood lymphocytes and HL-60 leukemia cells.

摘要

Human topoisomerase IIIα (huTop IIIα) is a nuclear enzyme that is involved in resolving topological problems during DNA metabolism. However, its function remains unknown. This study was focused on elucidating the cellular function of huTop IIIα by examining the expression level and cellular localization of huTop IIIα during cell cycle progression, cell differentiation, and DNA damage in different types of cells and by testing the interaction between huTop IIIα and Werner's syndrome (WS)/Bloom's syndrome (BLM) protein.; In the Western blot analyses, huTop IIIα levels remained relatively constant during the cell cycle progression in HL-60 leukemia cells, but decreased as PHA-activated human peripheral blood lymphocytes entered S and G₂ /M phases. In addition, the indirect immunofluorescence staining indicated that huTop IIIα was predominantly localized in the nucleolus during the cell cycle progression in several different cell types. However, the expression pattern of huTop IIIα measured by immunocytochemistry and by laser scanning cytometry (LSC) revealed a cell cycle-independent pattern in PHA-activated peripheral blood lymphocytes, HeLa, and HL-60 cells. Further studies demonstrated that when PHA-activated lymphocytes progressed through S and G₂/M phases, huTop IIIα underwent proteolytic cleavage to generate low molecular weight derivatives, resulting in the disappearance of the intact huTop IIIα.; During the course of HL-60 cell differentiation, huTop IIIα levels decreased when HL-60 cells were induced to granulocytic maturation in the presence of 1.3% DMSO, but its levels remained unchanged when HL-60 cells were induced to monocytic maturation in the presence of 50 nM 1,25-dihydroxyvitamin D₃. However, in either case, huTop IIIα was found to be accentuated in the nucleolus. These data also suggest that the function of huTop IIIα is correlated with rDNA metabolism, especially with rRNA synthesis which is required for cell proliferation. In the analyses of DNA damage in Molt 4 cells induced by Methyl methanesulfonate (MMS), Mitomycin (MMC), phleomycin, or 4-nitroquinoline-1-oxide (4NQO), huTop IIIα was not involved in these types of DMA damage in that the nucleolar accentuation of huTop IIIα did not change and huTop IIIα level either remained the same or decreased.; In the experiments of testing the interaction between huTop IIIα and WS/BLM helicases, the recombinant huTop IIIα synthesized in the in vitro transcription/translation system did not coprecipitate the recombinant BLM protein, and the anti-WS antibody was also unable to coimmunoprecipitate huTop IIIα.; This study provides evidence that the expression level of huTop IIIα in PHA-activated lymphocytes is subjected to regulation at the post-translational level, which accounts for the differential expression of huTop IIIα between PHA-activated lymphocytes and HL-60 cells, and that huTop IIIα plays a role in ribosomal DNA (rDNA) metabolism, but perhaps not in DNA replication.