Overexpression of Human DNA Topoisomerase II α by Fusion to Enhanced Green Fluorescent Protein

DNA topoisomerase (topo) IIα is a major target for many anticancer agents. However, progress towards understanding how these agents interact with this enzyme in human cells and how resistance to these agents arises is greatly impeded by difficulties in expressing this gene. Here, we report on achieving a high level of expression of a full-length human topo IIα gene in human cells. We started with the topo IIα cDNA driven by a strong cytomegalovirus promoter and transiently transfected HeLa cells. Although topo IIα mRNA was consistently detected in transfected cells, no exogenous topo IIα protein was detected. By contrast, when the same cDNA was fused to an enhanced green fluorescent protein (EGFP), we detected a high level of expression at both mRNA and protein levels. The exogenous topo IIα was localized to cell nuclei as expected, indicating that the fusion protein is properly folded. Furthermore, overexpression of the EGFP-topo IIα fusion protein increased the sensitivity of the transfected cells to teniposide, suggesting that it functions as the endogenous counterpart. Thus, in addition to being used as a gene tag, the GFP fusion approach may be generally applicable for expressing genes, such as topo IIα, that are difficult to express by conventional methods. INTRODUCTION DNA topoisomerase (topo) II is an essential enzyme that regulates DNA topology for DNA replication and transcription during cell growth and cell cycle progression (8). In mammalian cells, although two types of topo II exist, α and β (5,10), topo IIα seems to be the predominant isoenzyme in most tissues. Its clinical importance comes from findings that this enzyme is a major target for many anticancer drugs (7). Although much information about the physiological and biochemical aspects of the enzyme has been accumulated from studies of the yeast and Drosophila enzyme, little is known about regulation and expression of the gene for human topo IIα in mammalian systems. This unfamiliarity has been attributed to the difficulty in expressing the topo IIα gene in mammalian systems. For example, several mutations in human topo IIα have been identified in drugresistant cell lines (9), but testing whether any of these mutations confer drug resistance has not been possible in mammalian systems. Human topo IIα is 170-kDa nuclear protein consisting of 1530 amino acids (10). Although some success has been reported in yeast systems (4), as with many other large proteins, expression of this gene in mammalian cells has been a great challenge. While the expression of human topo IIα in mammalian cells has been reported (1), the results were difficult to interpret because the exogenous gene product was indistinguishable from the endogenous counterpart. To express human topo IIα in human cells, we have tried a variety of approaches, including use of constitutive strong, weak or inducible promoters (e.g., cytomegalovirus [CMV], Rous sarcoma virus [RSV] and tetracycline, respectively) without success. All these efforts only led to expression of this gene at the transcriptional level, and no exogenous topo IIα was detected by Western blot using antibody specific to the exogenous protein (Y.-Y. Mo and W.T. Beck, unpublished results). However, while we were examining expression of the C-terminal region of topo IIα for its subcellular localization, we fused the C terminus of the gene to the enhanced green fluorescent protein (EGFP) and found very high expression of the fusion protein. We subsequently examined the possibility of expressing the full-length human topo IIα. By using the EGFP fusion, we were able to detect high levels of exogenous topo II expression at both the mRNA and protein levels in mammalian cells. This protein localized to the cell nucleus and appeared to be catalytically active, because it conferred sensitivity to the specific inhibitor of topoisomerase II, teniposide (VM-26). MATERIALS AND METHODS Construction of Expression Plasmids To label human topo IIα, we introduced the RGS-6×His sequence from a QIAexpress pQE high level bacterial 1052 BioTechniques Vol. 25, No. 6 (1998) Cancer Research Techniques Overexpression of Human DNA Topoisomerase IIα by Fusion to Enhanced Green Fluorescent