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5‐Flurouracil disrupts nuclear transport during apoptosis in a calcium dependent manner
Author(s) -
Koper Jarrett Abram,
Higby Kelly,
Foltz Lauren,
Resendes Karen
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.772.11
Subject(s) - nuclear transport , camptothecin , chemistry , apoptosis , microbiology and biotechnology , ran , calcium , cell nucleus , biology , nucleus , biochemistry , organic chemistry
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 derivative 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. The effects of these chemotherapies on the NPC and nuclear transport was analyzed using immunofluorescence of Ran, a key component of nuclear transport events and FG nucleoporins at the NPC. We demonstrate that 5‐FU, but not topotecan, disrupts Ran‐mediated nuclear transport before the disruption of the NPC. This Ran disruption correlates with increased NPC permeability, determined using the RGG2.2 cell line, which expresses a cytoplasmically restricted 70kD GFP tagged protein that only diffuses into the nucleus with an increase in NPC permeability limit above its 40kD baseline. Immunoblotting indicates that this change in the NPC occurs before caspase activation. Furthermore, we show that the increased NPC permeability is dependent on increased cellular calcium, as the Ca2+ chelator BAPTA‐AM, abolished the effects of 5‐FU on the NPC. Increased calcium alone induced by thapasigargin, which inhibits the uptake of calcium in the cell, was sufficient to disrupt the Ran gradient, and addition of BAPTA‐AM helped to reset the Ran gradient. In sum, our results indicate that 5‐FU disrupts nuclear transport early in apoptosis in a calcium dependent manner, a new mechanism of action for this therapeutic during apoptosis. This result opens the door to other potential combination chemotherapies that employ 5‐FU as a calcium mediated inhibitor of nuclear export, particularly in Crm1 overexpressing cells, where the excess nuclear export of several pro‐apoptotic factors has been identified as a potential trigger for cancerous transformation, as well as a mechanism for chemotherapy resistance.