[CANCER RESEARCH 60, 1104 –1110, February 15, 2000] The M r 193,000 Vault Protein Is Up-Regulated in Multidrug-resistant Cancer Cell Lines 1 Anouk B. Schroeijers, Amara C. Siva, George L. Scheffer, Mariska C. de Jong, Sophie C. E. Bolick, Danny F. Dukers, Jerry W. Slootstra, Rob H. Meloen, Erik Wiemer, Valerie A. Kickhoefer, Leonard H. Rome, and Rik J. Scheper 2 Department of Pathology, Academic Hospital Vrije Universiteit, 1081 HV Amsterdam, the Netherlands [A. B. S., G. L. S., M. C. d. J., S. E. B., D. F. D., R. J. S.]; Department of Biological Chemistry, University of California at Los Angeles School of Medicine, Los Angeles, California 90095 [A. C. S., V. A. K., L. H. R.]; Department of Molecular Recognition, Institute for Animal Science and Health (ID-DLO), 8219 PH Lelystad, the Netherlands [J. W. S., R. H. M.]; and Institute for Hematology, Erasmus University Rotterdam, 3000 DR Rotterdam, the Netherlands [E. W.] ABSTRACT Vaults are 13 megadalton ribonucleoprotein particles composed largely of the major vault protein (MVP) and two high molecular weight proteins, p240 and p193, and a small vault RNA (vRNA). Increased levels of MVP expression, vault-associated vRNA, and vaults have been linked directly to multidrug resistance (MDR). To further define the putative role of vaults in MDR, we produced monoclonal antibodies against the M r 193,000 vault protein and studied its expression levels in various multidrug-resistant cell lines. We find that, like MVP, p193 mRNA and protein levels are in- creased in various multidrug-resistant cell lines. Subcellular fractionation of vault particles revealed that vault-associated p193 levels are increased in multidrug-resistant cells as compared with the parental, drug-sensitive cells. Furthermore, protein analysis of postnuclear supernatants and co- immunoprecipitation studies show that drug-sensitive MVP-transfected tumor cells lack this up-regulation in vault-associated p193. Our obser- vations indicate that vault formation is limited not only by the expression of the MVP but also by the expression or assembly of at least one of the other vault proteins. INTRODUCTION Resistance to a broad range of cytostatic drugs (MDR 3 ) can be mediated by Pgp or the MRP1. Pgp and MRP1 function as efflux pumps, decreasing intracellular drug accumulation (1). In clinical drug resistance, however, other mechanisms may play a role, e.g., involving drug sequestration into exocytotic vesicles. Evidence has been obtained that subcellular particles named vaults may play a critical role in such a mechanism (2, 3). Vaults are 13 megadalton ribonucleoprotein particles containing three proteins of M r 240,000, 193,000, and 110,000, respectively, and a small untranslated vRNA. A vault interacting protein of M r 54,000 is occasionally observed in rat liver vault preparations (2). Vaults are widely distributed throughout eukaryotes, and their morphology is highly conserved among various species. The remarkable structural conservation and broad distribution of vaults suggest that their func- tion is essential to eukaryotic organisms and that the structure of the particle must be important for its function (4 – 6). Although vault function is undetermined, it has been proposed that vaults may me- diate transport of various substrates (7, 8). A role for vaults in intracellular traffic might be mediated by binding to cellular or- ganelles through direct interaction with its targets (9). Recently, an interaction of vaults with intracellular steroid hormone receptors has been reported (10). Although the majority of vault particles are distributed throughout the cytosol, a portion of vaults has been local- ized to the nuclear membrane at or near the nuclear pore complex. Furthermore, the recent three-dimensional reconstruction of the vault particle reveals a hollow interior, which may prove important in the transport/sequestration of large substrates (11). On the basis of striking similarities between vault particle mass and symmetry and the predictive mass of the putative central plug of the nuclear pore complexes, a role of vaults in nucleocytoplasmic exchange has been proposed (12). The discovery of a key role of VR-MDR in clinical drug resistance depended on the molecular identification of the LRP as the human MVP (3). LRP had been first identified in a non-small cell lung cancer cell line, selected in vitro for DOX resistance. The protein was subsequently found to be overexpressed in many human tumor cell lines characterized by their MDR phenotype, in the absence of drug accumulation defects such as mediated by Pgp (13). Moreover, LRP expression closely reflected known chemoresistance characteristics in broad panels of unselected tumor cell lines and untreated clinical cancers of different histogenetic origins (14, 15). Results from several, but not all, clinicopathological studies showed that LRP expression at diagnosis, rather than Pgp or MRP1 expression, is a strong and independent prognostic factor for poor response to chemotherapy and/or outcome, e.g., in ovarian carcinoma and leukemias (16). Most importantly, Kitazono et al. (17) demonstrated recently, using a LRP induction system and LRP-specific ribozymes, that LRP is involved in resistance to Adriamycin, vincristine, VP-16, Taxol, and gramicidine D and has an important role in the transport of Adriamycin between the nucleus and the cytoplasm in the SW-620 human colon carcinoma cell line. To avoid confusion, we will hereafter refer to LRP as MVP. Studies on the role of vaults in MDR, including the cloning of the MVP cDNA, have thus far been based on polyclonal antisera and two mAbs, LRP-56 and LMR-5 (13, 18), directed against the MVP. To further define the role of vaults in MDR, the other components must be characterized. The human vRNA genes have been cloned, and within tumor cells, not all of the vRNA was found to be vault- associated. Sedimentation measurements of vault components in VR- MDR cells have revealed up to a 15-fold increase in vault copy number, coupled with a comparable shift of vRNA to the 100,000  g pellet, demonstrating that vault formation is limited by expression of MVP and/or one of the other vault proteins (19). Because MVP-trans- fected cells did not show a drug-resistant phenotype (3), the other vault components are thought to be essential for vaults to play a role in MDR. Here we describe the production of the first mAbs against the M r 193,000 vault protein. The p193 was recently identified by its inter- action with the MVP in a yeast two-hybrid screen, and its identity was confirmed by peptide sequence analysis (20). Results from protein analysis of postnuclear supernatants and subcellular fractions, North- ern analysis, immunocytochemical and coimmunoprecipitation stud- ies show that: (a) p193 and MVP are both increased in various MDR cell lines and; and (b) vault-associated p193 levels are up-regulated in these MDR cell lines but not in a drug-sensitive, MVP full-length, cDNA-transfected cell line, supporting the conclusion that functional Received 8/30/99; accepted 12/16/99. 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. 1 Supported by Dutch Cancer Society Grant VU 95-923 (to R. J. S.), USPHS Grant GM38097, and a grant from the Margaret E. Early Foundation (to L. H. R.). 2 To whom requests for reprints should be addressed, at Department of Pathology, Academic Hospital Vrije Universiteit, De Boelelaan 1117, 1081 HV Amsterdam, the Netherlands. Phone: 31-20-444-4031; Fax: 31-20-444-2964; E-mail: rj.scheper@azvu.nl. 3 The abbreviations used are: MDR, multidrug resistance; MRP1, MDR protein 1; Pgp, P-glycoprotein; vRNA, vault RNA; LRP, lung resistance-related protein; VR-MDR, vault-related MDR; MVP, major vault protein; DOX, doxorubicin; mAb, monoclonal antibody. 1104 Research. on October 5, 2015. © 2000 American Association for Cancer cancerres.aacrjournals.org Downloaded from