Cellular and regulatory roles of human DNA topoisomerase IIβ

Abstract

DNA topoisomerase IIβ is an enzyme that manipulates DNA topology. Its mechanism of action involves introduction of transient breaks in the DNA phosphodiester backbone enabling the passage of another DNA strand through the break. TOP2β has been suggested to be implicated in chromosomal translocations associated with development of secondary malignancy induced by anti-cancer drugs that targeting TOP2 enzymes. TOP2β also plays a role in transcriptional regulation, possibly via the introduction of transient DNA breaks at promoters enabling local remodelling of chromatin. To investigate the isoform-specific contribution of TOP2β in cytotoxicity and genotoxicity of TOP2 targeting drugs, a Nalm6 cell line previously knocked out for TOP2β and its parent cell line were utilised in this study. Their response to drugs idarubicin or daunorubicin alone or in combination with Ara-C has been investigated. Gene expression was investigated by RNA-seq of the Nalm6 cell lines. Analysis showed that expression of a number of genes was altered and this was confirmed by RT-qPCR on the same cell line. To be sure which genes changed a new Nalm6 TOP2β knockout was generated by CRISPR cas9. In the newly created knockout, RT-qPCR was carried out to confirm which genes still had altered gene expression. Strikingly, CBR1, a gene implicated in doxorubicin-induced cardiotoxicity, was found to be down regulated in both Nalm6 TOP2β knockout lines. Chip followed by RT-qPCR confirmed that TOP2β was involved in regulating CBR1 transcription. I also generated several TOP2β knockout clones in further two cell lines (K562 and SH-SY5Y) using CRISPR. The response of wild type and three knockout SH-SY5Y clones to retinoic acid was compared in a number of ways including neurite outgrowth and RT-qPCR. Differential gene expression pattern was investigated using RNA-seq followed by IPA analysis. Results showed reduced level of ATRA-induced differentiation in the knockout cells compared with wild type. RNA-seq analysis of showed downregulation of key neuronal genes involved in neuronal functions.