Global enhancement of nuclear localization‐dependent nuclear transport in transformed cells

Abstract

Fundamental to eukaryotic cell function, nucleocytoplasmic transport can be regulated at many levels, including through modulation of the importin/exportin (Imp/Exp) nuclear transport machinery itself. Although Imps/Exps are overexpressed in a number of transformed cell lines and patient tumor tissues, the efficiency of nucleocytoplasmic transport in transformed cell types compared with nontransformed cells has not been investigated. Here we use quantitative live cell imaging of 3 isogenic nontransformed/transformed cell pairs to show that nuclear accumulation of nuclear localization signal (NLS)-containing proteins, but not their NLS-mutated derivatives, is increased up to 7-fold in MCF10CA1h human epithelial breast carcinoma cells and in simian virus 40 (SV40)-transformed fibroblasts of human and monkey origin, compared with their nontransformed counterparts. The basis for this appears to be a significantly faster rate of nuclear import in transformed cell types, as revealed by analysis using fluorescence recovery after photobleaching for the human MCF10A/MCF10CA1h cell pair. Nuclear accumulation of NLS/nuclear export signal-containing (shuttling) proteins was also enhanced in transformed cell types, experiments using the nuclear export inhibitor leptomycin B demonstrating that efficient Exp-1-mediated nuclear export was not impaired in transformed compared with nontransformed cells. Enhanced nuclear import and export efficiencies were found to correlate with 2- to 4-fold higher expression of specific Imps/Exps in transformed cells, as indicated by quantitative Western blot analysis, with ectopic expression of Imps able to enhance NLS nuclear accumulation levels up to 5-fold in nontransformed MCF10A cells. The findings indicate that transformed cells possess altered nuclear transport properties, most likely due to the overexpression of Imps/Exps. The findings have important implications for the development of tumor-specific drug nanocarriers in anticancer therapy.

Principles of protein targeting to the nucleolus

“…This difficulty is further aggravated by the fact that validated and predicted NoLS depict high sequence diversity. 18,57 Our results show first that all poly-K and poly-R peptides ranging from 5-12 amino acids accumulate in the cell nucleus compared to the cytoplasmic level and, therefore, match the criteria for nuclear localization sequences. Similarly, all NLS bearing GFP versions tested show a nuclear accumulation, which is generally a feature of nuclear proteins bearing a NLS.…”

“…Similarly, all NLS bearing GFP versions tested show a nuclear accumulation, which is generally a feature of nuclear proteins bearing a NLS. 57 In line with this interpretation, the GFP protein without NLS shows a homogeneous distribution between nucleus and cytoplasm. Interestingly, the mean cytoplasmic fluorescence intensity of all L-peptides tested (0.61 to 0.76 fold) is much higher than for any of the NLS containing GFP versions (0.08 to 0.39) showing that passive nuclear pore leakage is less efficient and slower for larger molecules.…”

Targeting the Nuclear Import Receptor Kpnβ1 as an Anticancer Therapeutic

“…As the goal of cancer therapy is to promote the death of cancer cells without causing too much damage to normal cells this selectivity is advantageous and suggests that the development of anti-nuclear import drugs may have therapeutic potential. The selectivity may derive from the increased nuclear transport rates in cancer cells compared with normal cells, and thus increased reliance on the nuclear transport machinery (9). Alternatively, it is also possible that the selectivity toward cancer cells is due to the cancer cells being "primed" to undergo apoptosis (closer in proximity to the apoptotic threshold) compared with normal cells (49).…”

“…We previously showed that Kpnb1 mRNA and protein are expressed at elevated levels in cervical tumors and cervical cancer cell lines (6), and that the promoter is more highly active in cervical cancer cells due to its activation by the cell-cycle regulator, E2F (7). Smith and colleagues (2010) found that Kpnb1 mRNA was elevated in ovarian cancer cell lines and transformed ovarian cells (8) and Kuusisto and colleagues (2011) also described increased levels of Kpnb1 in several transformed cell lines (9). These lines of evidence suggest that the Kpnb1 protein is associated with cellular transformation and cancer.…”

Fatty Acid-binding Proteins 1 and 2 Differentially Modulate the Activation of Peroxisome Proliferator-activated Receptor α in a Ligand-selective Manner

“…Multipoint template FRAP experiments were performed by selecting regions of interest (ROIs) in multiple neighboring cells corresponding to either the cell nuclei or an area of the cytoplasm as described previously (25). The ROIs were photobleached (12 scans, 12.5 s/pixel, 100% laser power), and the fluorescence recovery was monitored (4% laser power, 10 s/pixel) at 20 s intervals for 500 s. The relative level of fluorescence in the bleached and unbleached regions was quantitated by image analysis using ImageJ.…”