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5-Flurouracil disrupts nuclear export and nuclear pore permeability in a calcium dependent manner

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Abstract

Regulation of nuclear transport is an essential component of apoptosis. As chemotherapy induced cell death progresses, nuclear transport and the nuclear pore complex (NPC) are slowly disrupted and dismantled. 5-Fluorouracil (5-FU) and the camptothecin derivatives irinotecan and topotecan, are linked to altered nuclear transport of specific proteins; however, their general effects on the NPC and transport during apoptosis have not been characterized. We demonstrate that 5-FU, but not topotecan, increases NPC permeability, and disrupts Ran-mediated nuclear transport before the disruption of the NPC. This increased permeability is dependent on increased cellular calcium, as the Ca2+ chelator BAPTA-AM, abolishes the effect. Furthermore, increased calcium alone was sufficient to disrupt the Ran gradient. Combination treatments of 5-FU with topotecan or irinotecan, similarly disrupted nuclear transport before disassembly of the NPC. In both single and combination treatments nuclear transport was disrupted before caspase 9 activation, indicating that 5-FU induces an early caspase-independent increase in NPC permeability and alteration of nuclear transport. Because Crm1-mediated nuclear export of tumor suppressors is linked to drug resistance we also examined the effect of 5-FU on the nuclear export of a specific target, topoisomerase. 5-FU treatment led to accumulation of topoisomerase in the nucleus and recovered the loss nuclear topoisomerase induced by irinotecan or topotecan, a known cause of drug resistance. Furthermore, 5-FU retains its ability to cause nuclear accumulation of p53 in the presence of irinotecan or topotecan. Our results reveal a new mechanism of action for these therapeutics during apoptosis, opening the door to other potential combination chemotherapies that employ 5-FU as a calcium mediated inhibitor of Crm1-induced nuclear export of tumor suppressors.

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Acknowledgements

This work was supported in part by the Westminster College Drinko Center for Undergraduate Research and the Westminster College Dietz-Sullivan Biology Research Experience Award. The authors thank Raymond Lewis for critically reviewing the manuscript.

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Author notes

  1. Kelly J. Higby

    Present address: Dana-Farber Cancer Institute, 450 Brookline Ave, Boston, MA, 02215, USA

  2. Melissa M. Bischak

    Present address: Ohio Valley Hospital, 25 Heckel Road, Pittsburgh, PA, 15136, USA

  3. Christina A. Campbell

    Present address: Cook MyoSite Inc., 105 Delta Drive, Pittsburgh, PA, 15238, USA

  4. Rebecca G. Anderson

    Present address: Department of Cancer Biology, Wake Forest School of Medicine, Winston-Salem, NC, 27157, USA

  5. Sarah A. Broskin

    Present address: MCBG Program, Drexel University College of Medicine, Philadelphia, PA, 19102, USA

  6. Lauren E. Foltz

    Present address: Division of Biological Sciences, University of Montana, Missoula, MT, 59812-4824, USA

Authors and Affiliations

  1. Department of Biology Hoyt Science Center 222, Westminster College, 319 S Market Street, New Wilmington, PA, 16172, USA

    Kelly J. Higby, Melissa M. Bischak, Christina A. Campbell, Rebecca G. Anderson, Sarah A. Broskin, Lauren E. Foltz, Jarrett A. Koper, Audrey C. Nickle & Karen K. Resendes

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Electronic supplementary material

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10495_2016_1338_MOESM1_ESM.eps

Supplemental Fig. 1 5-FU and topotecan propidium iodide timeline. HeLa cells were treated with a 5-FU (100μM) or b topotecan (20μM) for the time points indicated. In each of three replicate experiments, 500 cells were characterized as having no propidium iodide staining or positive nuclear propidium iodide staining. The average percentage of cells with positive nuclear propidium iodide staining is shown for each time point (EPS 724 KB)

10495_2016_1338_MOESM2_ESM.eps

Supplemental Fig. 2 Ionomycin and Thapsigargin propidium iodide timeline. HeLa cells were treated with a ionomcyin (5µg/mL) or b thapsigargin (0.25µM) for the time points indicated. In each of three replicate experiments, 500 cells were characterized as having no propidium iodide staining or positive nuclear propidium iodide staining. The average percentage of cells with positive nuclear propidium iodide staining is shown for each time point (EPS 732 KB)

10495_2016_1338_MOESM3_ESM.eps

Supplemental Fig. 3 Combination treatment propidium iodide timeline. HeLa cells were treated with 5-FU (100μM) and topotecan (20μM) or irinotecan (100μM) for the time points indicated. In each of three replicate experiments, 500 cells were characterized as having no propidium iodide staining or positive nuclear propidium iodide staining. The average percentage of cells with positive nuclear propidium iodide staining is shown for each time point (EPS 580 KB)

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Higby, K.J., Bischak, M.M., Campbell, C.A. et al. 5-Flurouracil disrupts nuclear export and nuclear pore permeability in a calcium dependent manner. Apoptosis 22, 393–405 (2017). https://doi.org/10.1007/s10495-016-1338-y

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