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Translocation of nucleophosmin from nucleoli to nucleoplasm requires ATP.

ABSTRACT: The movement of nucleophosmin from nucleoli to nucleoplasm in HeLa cells induced by cytotoxic drugs and detected by immunofluorescence is inhibited by concomitant treatment with antimycin A in glucose-free medium. Incubation of HeLa cells with antimycin A (300 nM; 30 min) and glucose-free medium resulted in an approximately 90% decrease in cellular ATP pools. To study the biochemical events involved in nucleophosmin translocation, we used an in vitro system consisting of Triton-permeabilized HeLA cells. Incubation of permeabilized cells with ATP (0.5 mM; 1 h) resulted in the translocation of nucleophosmin from nucleoli to nucleoplasm and cytoplasm. Similarly to drug-induced nucleophosmin translocation in whole cultured cells, there is no reduction (measured by e.l.i.s.a.) or degradation of nucleophosmin or change in the ratio of the high-molecular-mass form to the monomeric form (ascertained by Western blotting) during ATP treatment of permeabilized cells. Together, these results indicate a requirement for ATP for redistribution of nucleophosmin from nucleoli to nucleoplasm. Because this permeabilized cell model is simple and efficient and works effectively with exogenous factors, it should provide a powerful tool for investigating the biochemical features of nucleophosmin translocation from nucleoli to nucleoplasm.

SUBMITTER: Wu MH

PROVIDER: S-EPMC1136355 | biostudies-other | 1995 Feb

REPOSITORIES: biostudies-other

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Project description:BackgroundCucurbitacin B, an oxygenated tetracyclic triterpenoid compound extracted from the Thai medicinal plant Trichosanthes cucumerina L., has been reported to have several biological activities including anti-inflammatory, antimicrobial and anticancer. Cucurbitacin B is great of interest because of its biological activity. This agent inhibits growth of various types of human cancer cells lines.MethodsIn this study, we explored the novel molecular response of cucurbitacin B in human breast cancer cells, MCF-7 and MDA-MB-231. The growth inhibitory effect of cucurbitacin B on breast cancer cells was assessed by MTT assay. The effects of cucurbitacin B on microtubules morphological structure and tubulin polymerization were analyzed using immunofluorescence technique and tubulin polymerization assay kit, respectively. Proteomic analysis was used to identify the target-specific proteins that involved in cucurbitacin B treatment. Some of the differentially expressed genes and protein products were validated by real-time RT-PCR and western blot analysis. Cell cycle distributions and apoptosis were investigated using flow cytometry.ResultsCucurbitacin B exhibited strong antiproliferative effects against breast cancer cells in a dose-dependent manner. We show that cucurbitacin B prominently alters the cytoskeletal network of breast cancer cells, inducing rapid morphologic changes and improper polymerization of the microtubule network. Moreover, the results of 2D-PAGE, real-time RT-PCR, and western blot analysis revealed that the expression of nucleophosmin/B23 and c-Myc decreased markedly after cucurbitacin B treatment. Immunofluorescence microscopy showed that cucurbitacin B induced translocation of nucleophosmin/B23 from the nucleolus to nucleoplasm. Treatment with cucurbitacin B resulted in cell cycle arrest at G2/M phase and the enhancement of apoptosis.ConclusionsOur findings suggest that cucurbitacin B may inhibit the proliferation of human breast cancer cells through disruption of the microtubule network and down-regulation of c-Myc and nucleophosmin/B23 as well as the perturbation in nucleophosmin/B23 trafficking from the nucleolus to nucleoplasm, resulting in G2/M arrest.

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Translocation of nucleophosmin from nucleoli to nucleoplasm requires ATP.

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