Functional Study of the Regulation of DNA Topoisomerase II and its Cellular Role in Drug Responsiveness

Resistance to chemotherapeutic agents often occurs in many cancers and is a barrier to effective treatment. Such resistance is multifactoral and one is associated with the altered expression of drug’s target. DNA topoisomerase II (Top2) is an essential cellular enzyme involved in DNA metabolism and the target of important chemotherapeutic agents. In mammalian cells, there are two isoforms of DNA topoisomease II, designated Top2a and Top2b. We first determined the role of Top2a in Top2 poison-mediated responsiveness. A reduced expression of Top2a resulted in drug-resistance in cancer cells. Our previous work suggested that the transcription factor NF-YB is a negative regulator of Top2a, working through the Top2a promoter (Morgan and Beck, Mol.Pharmacol 59:203,2001). Our data suggest that the increased NF-YB may be related to or be the cause of reduced Top2a in drug-resistant cells. Furthermore, recent studies indicate that microRNAs are often aberrantly expressed in cancer and may mediate drug responsiveness. MicroRNAs function through base pairing with protein coding mRNA 3’-untranslated regions (3’-UTRs) for mRNA degradation or translational repression. We have found by microRNA profiling that one microRNA, hsa-miR-485-3p, is consistently expressed at substantially lower levels in drug resistant CEM/VM-1-5 cells and microRNA target-predicting algorithms revealed that hsa-miR-485-3p has a potential target to 3’-UTR of NF-YB. We further validated the binding of hsa-miR-485-3p to 3’-UTR of NF-YB. Moreover, ectopic expression of hsa-miR-485-3p repressed NF-YB expression and rescued the expression of Top2a. On the other hand, we constructed tetracycline inducible system to turn-on and -off the expression of Top2a shRNA with and without the adding of antibiotic doxycycline (Doxy), respectively. However, we observed the cytotoxicity effect of doxycycline in CEM cells and all other cancer cell lines tested. The inhibitory effect of doxycycline on cancer cell lines was partly due to apoptosis of the cancer cells. Furthermore, we observed the downregulation of Top2a and upregulation of Top2b in Doxy-treated cells. Our results presented herein suggest that the upregulation of Top2b in Doxy-treated cells is not mediated by differentiation of the cells.