Differential regulation of NF-kappaB activation and function by topoisomerase II inhibitors

Abstract

Background: While many common chemotherapeutic drugs and other inducers of DNA-damage result in both NF-kappaB nuclear translocation and DNA-binding, we have previously observed that, depending on the precise stimulus, there is great diversity of the function of NF-kappaB. In particular, we found that treatment of U-2 OS osteosarcoma cells with the anthracycine daunorubicin or with ultraviolet (UV-C) light resulted in a form of NF-kappaB that repressed rather than induced NF-kappaB reporter plasmids and the expression of specific anti-apoptotic genes. Anthracyclines such as daunorubicin can induce DNA-damage though inhibiting topoisomerase II, intercalating with DNA and undergoing redox cycling to produce oxygen free radicals. In this study we have investigated other anthracyclines, doxorubicin and aclarubicin, as well as the anthracenedione mitoxantrone together with the topoisomerase II inhibitor ICRF-193, which all possess differing characteristics, to determine which of these features is specifically required to induce both NF-kappaB DNA-binding and transcriptional repression in U-2 OS cells.

Results: The use of mitoxantrone, which does not undergo redox cycling, and the reducing agent epigallocatechingallate (EGCG) demonstrated that oxygen free radical production is not required for induction of NF-kappaB DNA-binding and transcriptional repression by these agents and UV-C. In addition, the use of aclarubicin, which does not directly inhibit topoisomerase II and ICRF-193, which inhibits topoisomerase II but does not intercalate into DNA, demonstrated that topoisomerase II inhibition is not sufficient to induce the repressor form of NF-kappaB.

Conclusion: Induction of NF-kappaB DNA-binding and transcriptional repression by topoisomerase II inhibitors was found to correlate with an ability to intercalate into DNA. Although data from our and other laboratories indicates that topoisomerase II inhibition and oxygen free radicals do regulate NF-kappaB, they are not required for the particular ability of NF-kappaB to repress rather than activate transcription. Together with our previous data, these results demonstrate that the nature of the NF-kappaB response is context dependent. In a clinical setting such effects could profoundly influence the response to chemotherapy and suggest that new methods of analyzing NF-kappaB function could have both diagnostic and prognostic value.

Figure 1

Induction NF-κB DNA binding by topoisomerase II inhibitors and DNA-intercalators. U-2 OS cells were stimulated with 1 μM aclarubicin, 1 μM daunorubicin, 1 μM doxorubicin, 2.5 μM mitoxantrone and 4 μM ICRF-193, for the times indicated. Nuclear protein extracts were prepared and 5 μg was subjected to EMSA analysis using a ³²P labelled HIV-1 κB oligonucleotide.

Figure 2

Oxygen free radical generation is not required for induction of NF-κB DNA-binding. U-2 OS cells were stimulated with (A) 1 μM daunorubicin, (B) 2.5 μM mitoxantrone (C) 40 J/m² UV-C, or for the indicated times with, or without 1 hour pre-incubation with 20 μM EGCG or 10 mM NAC as indicated.

Figure 3

Mitoxantrone and aclarubicin but not ICRF-193, repress NF-κB reporter plasmid activity. U-2 OS cells were transfected with 2 μg of 3 × κB ConA luciferase reporter plasmid and 36 hours later stimulated with 4 μM ICRF-193, 1 μM aclarubicin or 2.5 μM mitoxantrone for 8 hours. Results are normalised such that no change in luciferase activity has a value of 0.

Figure 4

Oxygen free radical generation is not required for NF-κB dependent repression of transcription. (A) U-2 OS cells were transfected with 2 μg of 3 × κB ConA luc and 36 hours after transfection cells were unstimulated (control) or stimulated for 8 hours with 1 μM daunorubicin or 40 J/m² UV-C, with, or without, 1 hour pre-stimulation with 20 μM EGCG, as indicated. (B) U-2 OS cells were unstimulated (control) or stimulated for 6 hours with 1 μM daunorubicin or 40 J/m² UV-C, with, or without, 1 hour pre-stimulation with 20 μM EGCG, as indicated. Total RNA was prepared and semi-quantitative PCR analysis with primers specific to human Bcl-xL and GAPDH. (C) Mitoxantrone inhibits Bcl-xL and XIAP mRNA levels. U-2 OS cells were stimulated for 6 hours with 4 μM ICRF-193, 2.5 μM mitoxantrone and 1 μM doxorubicin. Total RNA was prepared and semi-quantitative PCR analysis performed using primers to human Bcl-xL, XIAP and GAPDH control. (D) Quantitative RT-PCR analysis. The effects of danuorubicin, doxorubicin and mitoxantrone on Bcl-xL and XIAP mRNA levels was confirmed by quantitative real-time PCR analysis.