Recruitment of the Major Vault Protein by InlK: A Listeria monocytogenes Strategy to Avoid Autophagy

L. monocytogenes is a facultative intracellular bacterium responsible for listeriosis. It is able to invade, survive and replicate in phagocytic and non-phagocytic cells. The infectious process at the cellular level has been extensively studied and many virulence factors have been identified. Yet, the role of InlK, a member of the internalin family specific to L. monocytogenes, remains unknown. Here, we first show using deletion analysis and in vivo infection, that InlK is a bona fide virulence factor, poorly expressed in vitro and well expressed in vivo, and that it is anchored to the bacterial surface by sortase A. We then demonstrate by a yeast two hybrid screen using InlK as a bait, validated by pulldown experiments and immunofluorescence analysis that intracytosolic bacteria via an interaction with the protein InlK interact with the Major Vault Protein (MVP), the main component of cytoplasmic ribonucleoproteic particules named vaults. Although vaults have been implicated in several cellular processes, their role has remained elusive. Our analysis demonstrates that MVP recruitment disguises intracytosolic bacteria from autophagic recognition, leading to an increased survival rate of InlK over-expressing bacteria compared to InlK− bacteria. Together these results reveal that MVP is hijacked by L. monocytogenes in order to counteract the autophagy process, a finding that could have major implications in deciphering the cellular role of vault particles

CD34-related coexpression of MDR1 and BCRP indicates a clinically resistant phenotype in patients with acute myeloid leukemia (AML) of older age

Clinical resistance to chemotherapy in acute myeloid leukemia (AML) is associated with the expression of the multidrug resistance (MDR) proteins P-glycoprotein, encoded by the MDR1/ABCB1 gene, multidrug resistant-related protein (MRP/ABCC1), the lung resistance-related protein (LRP), or major vault protein (MVP), and the breast cancer resistance protein (BCRP/ABCG2). The clinical value of MDR1, MRP1, LRP/MVP, and BCRP messenger RNA (mRNA) expression was prospectively studied in 154 newly diagnosed AML patients ≥60 years who were treated in a multicenter, randomized phase 3 trial. Expression of MDR1 and BCRP showed a negative whereas MRP1 and LRP showed a positive correlation with high white blood cell count (respectively, p < 0.05, p < 0.001, p < 0.001 and p < 0.001). Higher BCRP mRNA was associated with secondary AML (p < 0.05). MDR1 and BCRP mRNA were highly significantly associated (p < 0.001), as were MRP1 and LRP mRNA (p < 0.001) expression. Univariate regression analyses revealed that CD34 expression, increasing MDR1 mRNA as well as MDR1/BCRP coexpression, were associated with a lower complete response (CR) rate and with worse event-free survival and overall survival. When adjusted for other prognostic actors, only CD34-related MDR1/BCRP coexpression remained significantly associated with a lower CR rate (p = 0.03), thereby identifying a clinically resistant subgroup of elderly AML patients

Role of drug transporters and drug accumulation in the temporal acquisition of drug resistance

<p>Abstract</p> <p>Background</p> <p>Anthracyclines and taxanes are commonly used in the treatment of breast cancer. However, tumor resistance to these drugs often develops, possibly due to overexpression of drug transporters. It remains unclear whether drug resistance <it>in vitro </it>occurs at clinically relevant doses of chemotherapy drugs and whether both the onset and magnitude of drug resistance can be temporally and causally correlated with the enhanced expression and activity of specific drug transporters. To address these issues, MCF-7 cells were selected for survival in increasing concentrations of doxorubicin (MCF-7_DOX-2), epirubicin (MCF-7_EPI), paclitaxel (MCF-7_TAX-2), or docetaxel (MCF-7_TXT). During selection cells were assessed for drug sensitivity, drug uptake, and the expression of various drug transporters.</p> <p>Results</p> <p>In all cases, resistance was only achieved when selection reached a specific threshold dose, which was well within the clinical range. A reduction in drug uptake was temporally correlated with the acquisition of drug resistance for all cell lines, but further increases in drug resistance at doses above threshold were unrelated to changes in cellular drug uptake. Elevated expression of one or more drug transporters was seen at or above the threshold dose, but the identity, number, and temporal pattern of drug transporter induction varied with the drug used as selection agent. The pan drug transporter inhibitor cyclosporin A was able to partially or completely restore drug accumulation in the drug-resistant cell lines, but had only partial to no effect on drug sensitivity. The inability of cyclosporin A to restore drug sensitivity suggests the presence of additional mechanisms of drug resistance.</p> <p>Conclusion</p> <p>This study indicates that drug resistance is achieved in breast tumour cells only upon exposure to concentrations of drug at or above a specific selection dose. While changes in drug accumulation and the expression of drug transporters does occur at the threshold dose, the magnitude of resistance cannot be attributed solely to changes in drug accumulation or the activity of drug transporters. The identities of these additional drug-transporter-independent mechanisms are discussed, including their likely clinical relevance.</p

The vault complex.

Vaults are large ribonucleoprotein particles found in eukaryotic cells. They are composed of multiple copies of a Mr 100,000 major vault protein and two minor vault proteins of Mr 193,000 and 240,000, as well as small untranslated RNAs of 86-141 bases. The vault components are arranged into a highly characteristic hollow barrel-like structure of 35 x 65 nm in size. Vaults are predominantly localized in the cytoplasm where they may associate with cytoskeletal elements. A small fraction of vaults are found to be associated with the nucleus. As of yet, the precise cellular function of the vault complex is unknown. However, their distinct morphology and intracellular distribution suggest a role in intracellular transport processes. Here we review the current knowledge on the vault complex, its structure, components and possible functions

Vaults: a ribonucleoprotein particle involved in drug resistance?

Vaults are ribonucleoprotein particles found in the cytoplasm of eucaryotic cells. The 13 MDa particles are composed of multiple copies of three proteins: an M(r) 100 000 major vault protein (MVP) and two minor vault proteins of M(r) 193 000 (vault poly-(ADP-ribose) polymerase) and M(r) 240 000 (telomerase-associated protein 1), as well as small untranslated RNA molecules of approximately 100 bases. Although the existence of vaults was first reported in the mid-1980s no function has yet been attributed to this organelle. The notion that vaults might play a role in drug resistance was suggested by the molecular identification of the lung resistance-related (LRP) protein as the human MVP. MVP/LRP was found to be overexpressed in many chemoresistant cancer cell lines and primary tumor samples of different histogenetic origin. Several, but not all, clinico-pathological studies showed that MVP expression at diagnosis was an independent adverse prognostic factor for response to chemotherapy. The hollow barrel-shaped structure of the vault complex and its subcellular localization indicate a function in intracellular transport. It was therefore postulated that vaults contributed to drug resistance by transporting drugs away from their intracellular targets and/or the sequestration of drugs. Here, we review the current knowledge on the vault complex and critically discuss the evidence that links vaults to drug resistance

Efflux kinetics and intracellular distribution of daunorubicin are not affected by major vault protein/lung resistance-related protein (vault) expression.

Vaults may contribute to multidrug resistance by transporting drugs away from their subcellular targets. To study the involvement of vaults in the extrusion of anthracyclines from the nucleus, we investigated the handling of daunorubicin by drug-sensitive and drug-resistant non-small lung cancer cells, including a green fluorescent protein (GFP)-tagged major vault protein (MVP)-overexpressing transfectant (SW1573/MVP-GFP). Cells were exposed to 1 microm daunorubicin for 60 min, after which the cells were allowed to efflux the accumulated drug. No significant differences in daunorubicin efflux kinetics were observed between the sensitive SW1573 and SW1573/MVP-GFP transfectant, whereas the drug-resistant SW1573/2R120 cells clearly demonstrated an increased efflux rate. It was noted that the redistribution of daunorubicin from the nucleus into distinct vesicular structures in the cytoplasm was not accompanied by changes in the intracellular localization of vaults. Similar experiments were performed using mouse embryonic fibroblasts derived from wild-type and MVP knockout mice, which were previously shown to be devoid of vault particles. Both cell lines showed comparable drug efflux rates, and the intracellular distribution of daunorubicin in time was identical. Reintroduction of a human MVP tagged with GFP in the MVP(-/-) cells results in the formation of vault particles but did not give rise an altered daunorubicin handling compared with MVP(-/-) cells expressing GFP. Our results indicate that vaults are not directly involved in the sequestration of anthracyclines in vesicles nor in their efflux from the nucleus