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Research Articles: Therapeutics, Targets, and Development

Depletion of major vault protein increases doxorubicin sensitivity and nuclear accumulation and disrupts its sequestration in lysosomes

Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, Virginia

Requests for reprints: Dan Theodorescu, Department of Molecular Physiology and Biological Physics, University of Virginia Health Sciences Center, Box 800422, Charlottesville, VA 22908. Phone: 434-924-0042; Fax: 434-982-3652. E-mail: dt9d{at}virginia.edu

Abstract

The major vault protein (MVP) is the major constituent of the vault particle, the largest known ribonuclear protein complex. To date, vaults have no clear function, although their low expression levels in de novo chemosensitive and curable tumors, such as testicular cancer, make them attractive candidates as contributors to intrinsic drug resistance. Here, we show that MVP knockdown in human bladder cancer cells via small interfering RNA results in sensitization toward doxorubicin in two distinct exposure protocols. The drug was detected in the nucleus immediately following addition and was subsequently sequestered to lysosomes, predominantly located adjacent to the nucleus. MVP knockdown leads to increased sensitivity toward doxorubicin and an enhanced nuclear accumulation of the drug as well as a loss of its perinuclear sequestration. Not only doxorubicin subcellular distribution was perturbed by MVP knockdown but lysosomal markers, such as pH-sensitive LysoSensor, pinocytosed dextran conjugates after 24-h chase period, and the lysosomal specific antigen Lamp-1, also showed a markedly different staining compared with controls. Lysosomes appeared dispersed through the cytoplasm without a clear organization adjacent to the nucleus. Microtubules, however, appeared unperturbed in cells with reduced MVP expression. Based on these data, we hypothesize that MVP and, by extension, vault complexes are important for lysosomal function and may influence cellular drug resistance by virtue of this role. [Mol Cancer Ther 2007;6(6):1804–13]

Grant support: NIH grant CA075115 (D. Theodorescu).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 6/26/06; revised 1/26/07; accepted 4/20/07.