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Abstract: Topoisomerase II α (TopoIIα) and Topoisomerase II β (TopoIIβ) isoforms are different gene products having conserved catalytic activities. The α isoform is present in proliferating cell, while β isoform is predominantly present in non-proliferating cells namely neurons suggesting its role in non-replicating functions of DNA. The functions of TopoIIα and TopoIIβ isoforms are analyzed in peroxide-mediated DNA damage and double strand breaks (DSBs) repair in neuroblastoma and astrocytoma cells. The results show a strong correlation of TopoIIα level with the progression of DNA damage, while the TopoIIβ expression is correlated with the DNA DSBs repair activity of cells in Ku70, Werner’s helicase and pol-β dependent pathways. The functional roles of TopoIIα and TopoIIβ are assessed using siRNA mediated TopoIIα and TopoIIβ knockdown in cells. The results show that TopoIIα−TopoIIβ+ cells are resistant to peroxide-mediated DNA damage, while TopoIIα+TopoIIβ− cells are 2-fold more sensitive to peroxide and TopoIIβ deficiency lead to cellular apoptosis. These results are correlated with cell survival from peroxide-mediated insult. The result of this study that TopoIIα accelerates peroxide-mediated DNA damage, while TopoIIβ promotes DNA DSBs repair activity should provide new directions toward understanding of normalytic ageing processes in human brain. [Copyright &y& Elsevier]

Abstract: Human Topoisomerase II is present in two isoforms, 170KDa α and 180KDa β. Both the isoforms play a crucial role in maintenance of topological changes during DNA replication and recombination. It has been shown that Topoisomerase II activity is required for HIV-1 replication and the enzyme is phosphorylated during early time points of HIV-1 replication. In the present study, we have studied the molecular action of Topoisomerase II inhibitors, azalactone ferrocene (AzaFecp), Thiomorpholide amido methyl ferrocene (ThioFecp), and Ruthenium benzene amino pyridine (Ru(ben)Apy) on cell proliferation and also on various events of HIV-1 replication cycle. The Topoisomerase II β over-expressing neuroblastoma cell line shows a higher sensitivity to these compounds compared to the Sup-T1 cell line. All the three Topoisomerase II inhibitors show significant anti-HIV activity at nanomolar concentrations against an Indian isolate of HIV-193IN101 in Sup-T1 cell line. An analysis of action of these compounds on proviral DNA synthesis at 5h of post-infection shows that they inhibit proviral DNA synthesis as well as the formation of pre-integration complexes completely. Further analysis, using polymerase chain reaction and western blot, showed that both the Topoisomerase II α and β isoforms are present in the pre-integration complexes, suggesting their significant role in HIV-1 replication. [Copyright &y& Elsevier]

Abstract: Human topoisomerase II plays a crucial role in DNA replication and repair. It exists in two isoforms: topoisomerase II alpha (α) and topoisomerase II beta (β). The α isoform is localized predominantly in the nucleus, while the β isoform exhibits a reticular pattern of distribution both in the cytosol and in the nucleus. We show that both isoforms of topoisomerase II are phosphorylated in HIV infected cells and also by purified viral lysate. An analysis of the phosphorylation of topoisomerase II isoforms showed that extracts of HIV infected cells at 8 and 32h. post-infection (p.i.) contain maximal phosphorylated topoisomerase II α, whereas infected cell extracts at 4 and 64h p.i. contain maximum levels of phosphorylated topoisomerase II β. In concurrent to phosphorylated topoisomerase II isoforms, we have also observed increased topoisomerase II α kinase activity after 8h p.i and topoisomerase β kinase activity at 4 and 64h p.i. These findings suggest that both topoisomerase II α and β kinase activities play an important role in early as well as late stages of HIV-1 replication. Further analysis of purified virus showed that HIV-1 virion contained topoisomerase II isoform-specific kinase activities, which were partially isolated. One of the kinase activities of higher hydrophobicity can phosphorylate both topoisomerase II α and β, while lower hydrophobic kinase could predominantly phosphorylate topoisomerase II α. The phosphorylation status was correlated with catalytic activity of the enzyme. Western blot analysis using phosphoamino-specific antibodies shows that both the kinase activities catalyze the phosphorylation at serine residues of topoisomerase II α and β. The catalytic inhibitions by serine kinase inhibitors further suggest that the α and β kinase activities associated with virus are distinctly different. [Copyright &y& Elsevier]