Abstract
Nuclear factor (NF)-YB, a subunit of the transcription factor nuclear factor Y (NF-Y) complex, binds and activates CCAAT-containing promoters. Our previous work suggested that NF-YB may be a mediator of topoisomerase IIα (Top2α), working through the Top2α promoter. DNA topoisomerase II (Top2) is an essential nuclear enzyme and the primary target for several clinically important anticancer drugs. Our teniposide-resistant human lymphoblastic leukemia CEM cells (CEM/VM-1-5) express reduced Top2α protein compared with parental CEM cells. To study the regulation of Top2α during the development of drug resistance, we found that NF-YB protein expression is increased in CEM/VM-1-5 cells compared with parental CEM cells. This further suggests that increased NF-YB may be a negative regulator of Top2α in CEM/VM-1-5 cells. We asked what causes the up-regulation of NF-YB in CEM/VM-1-5 cells. We found by microRNA profiling that hsa-miR-485-3p is lower in CEM/VM-1-5 cells compared with CEM cells. MicroRNA target prediction programs revealed that the 3′-untranslated region (3′-UTR) of NF-YB harbors a putative hsa-miR-485-3p binding site. We thus hypothesized that hsa-miR-485-3p mediates drug responsiveness by decreasing NF-YB expression, which in turn negatively regulates Top2α expression. To test this, we overexpressed miR-485-3p in CEM/VM-1-5 cells and found that this led to reduced expression of NF-YB, a corresponding up-regulation of Top2α, and increased sensitivity to the Top2 inhibitors. Results in CEM cells were replicated in drug-sensitive and -resistant human rhabdomyosarcoma Rh30 cells, suggesting that our findings represent a general phenomenon. Ours is the first study to show that miR-485-3p mediates Top2α down-regulation in part by altered regulation of NF-YB.
Footnotes
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The online version of this article (available at http://dmd.aspetjournals.org) contains supplemental material.
This work was supported by the National Institutes of Health National Cancer Institute [Grant CA40570]; the University of Illinois at Chicago; and the National Institutes of Health National Center for Research Resources [Grant C06-RR15482].
Details of the microRNA profiling experiments have been provided previously: He X, Arslan AD, Ho T-T, and Beck WT (2009) MicroRNA profiling in drug-resistant leukemic cells, in Proceedings of the 100th Annual Meeting of the American Association for Cancer Research; 2009 Apr 18–22; Denver, CO. Abstract 5514. American Association for Cancer Research, Philadelphia.
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
doi:10.1124/mol.110.069633.
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ABBREVIATIONS:
- NF-Y
- nuclear factor Y
- 3′-UTR
- 3′-untranslated region
- hsa-miR-485-3p
- human microRNA-485-3p
- ICE
- inverted CCAAT element
- MTT
- 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
- PCNA
- proliferating cell nuclear antigen
- PCR
- polymerase chain reaction
- RT
- reverse transcriptase
- Top2
- DNA topoisomerase II
- Top2α
- topoisomerase IIα
- HEK
- human embryonic kidney
- DMEM
- Dulbecco's modified Eagle's medium
- Ct
- cycle threshold.
- Received October 27, 2010.
- Accepted January 20, 2011.
- U.S. Government work not protected by U.S. copyright
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