A new cell-based reporter system for sensitive screening of nuclear export inhibitors

Abstract

Nucleocytoplasmic transport of proteins across the nuclear pore complex (NPC), mediated by the nuclear localization signal (NLS) and the nuclear export signal (NES), is a vital homeostatic process in eukaryotic cells and also in mitogen-activated protein kinase (MEK) signaling molecule in tumor cell proliferation. Some viruses, including the influenza virus and HIV-1, also employ this nuclear export mechanism during their life cycle. Hence, drugs that control nucleocytoplasmic transport of proteins are putative candidate antivirals or anti-cancer agents. Thus, we previously developed a GFP/NES-MDCK reporter cell system for screening novel nuclear export inhibitors. NES signal-conjugated GFP accumulates in the nucleus in the presence of the nuclear export inhibitor leptomycin B (LMB). In this study, a stable GFP/NLS/NES fusion protein-expressing cell line was established, and its potential as a reporter was evaluated. The GFP/NLS/NES-MDCK cell line demonstrates improved nuclear accumulation of GFP in a time-course treatment with LMB. In addition, the dose-response data demonstrated superior sensitivity of GFP/NLS/NES-MDCK over GFP/NES-MDCK cells. As low as 0.01 ng/mL LMB is sufficient to cause accumulation of the GFP fusion protein in the nucleus in GFP/NLS/NES-MDCK cells, while at least 1 ng/mL of LMB is needed for the accumulation of GFP fusion protein in the nucleus of GFP/NES-MDCK cells. These results indicate that the newly established GFP/NLS/NES-MDCK cell line is a potentially powerful tool to screen for novel nuclear export inhibitors.