Major Vault Protein Expression Research Articles

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

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.

Expression of major vault protein gene in osteosarcoma patients

Osteosarcoma (OS) is a primary malignant tumor of bone. Despite the successful use of multiple chemotherapeutic agents in the treatment of OS, more than 30% of OS tumors remain resistant to treatment. Elucidation of cellular resistance mechanisms may lead to better treatments for cancer patients. In this study, we used the low-density expression cDNA array, GEArray Q Series Human Cancer Drug Resistance and Metabolism Gene Array to screen genes related to drug resistance in 15 OS tumors. Expression patterns of the MPV gene were validated by real time PCR on 45 OS patient tumor samples and correlated with clinical and pathological data. Major vault protein (MVP) expression was present in 24 (53%) tumor samples and absent in 21 (47%). Samples from surgery showed correlation between the expression of MVP, metastatic disease at diagnosis and event free survival (EFS). The MVP gene expression correlates with metastatic disease at diagnosis after neoadjuvant chemotherapy (p=0.048), and is also associated with worse EFS (p=0.036). These findings suggest that MVP expression is involved in one of the mechanisms of drug resistance in OS and is induced by chemotherapy.

Cellular Functions of Vaults and their Involvement in Multidrug Resistance

Vaults are evolutionary highly conserved ribonucleoprotein (RNP) particles with a hollow barrel-like structure. They are 41 x 73 nm in size and are composed of multiple copies of three proteins and small untranslated RNA (vRNA). The main component of vaults represents the 110 kDa major vault protein (MVP), whereas the two minor vault proteins comprise the 193 kDa vault poly(ADP-ribose) polymerase (VPARP) and the 240 kDa telomerase-associated protein-1 (TEP1). Vaults are abundantly present in the cytoplasm of eukaryotic cells and they were found to be associated with cytoskeletal elements as well as occasionally with the nuclear envelope. Vaults and MVP have been associated with several cellular processes which are also involved in cancer development like cell motility and differentiation. Due to the over-expression of MVP (also termed lung resistance-related protein or LRP) in several P-glycoprotein (P-gp)-negative chemoresistant cancer cell lines, vaults have been linked to multidrug resistance (MDR). Accordingly, high levels of MVP were found in tissues chronically exposed to xenobiotics. In addition, the expression of MVP correlated with the degree of malignancy in certain cancer types, suggesting a direct involvement in tumor development and/or progression. Based on the finding that MVP binds several phosphatases and kinases including PTEN, SHP-2 as well as Erk, evidence is accumulating that MVP might be involved in the regulation of important cell signalling pathways including the PI3K/Akt and the MAPK pathways. In this review we summarize the current knowledge concerning the vault particle and discuss its possible cellular functions, focusing on the role of vaults in chemotherapy resistance.

The major vault protein is responsive to and interferes with interferon-γ-mediated STAT1 signals

The major vault protein (MVP) is the main component of vaults, large ribonucleoprotein particles implicated in the regulation of cellular signaling cascades and multidrug resistance. Here, we identify MVP as an interferon gamma (IFN-gamma)-inducible protein. Treatment with IFN-gamma resulted in a significant upregulation of MVP promoter activity as well as mRNA and protein levels. Activation of MVP expression by IFN-gamma involved transcriptional upregulation through the JAK/STAT pathway based on an interaction of STAT1 with an interferon-gamma-activated site (GAS) within the proximal MVP promoter. Mutation of this site distinctly reduced basal as well as IFN-gamma-stimulated MVP transcription. IFN-gamma also significantly enhanced the translation rate of MVP. Ectopic MVP overexpression in the MVP-negative lung cancer cell model H65 led to a downregulation of three known IFN-gamma-regulated genes, namely ICAM-1, CD13 and CD36. Additionally, presence of MVP in H65 cells blocked both basal and IFN-gamma-induced ICAM-1 expression whereas downmodulation of endogenous MVP levels by shRNA enhanced IFN-gamma-induced ICAM-1 expression in U373 glioblastoma cells. MVP-mediated IFN-gamma insensitivity was accompanied by significantly reduced STAT1 phosphorylation at Y701 and diminished translocation of STAT1 into the nucleus. Summarizing, we identify MVP as an IFN-gamma-responsive gene interfering with IFN-gamma-activated JAK/STAT signals. These data further substantiate that the vault particle functions as a general interaction platform for cellular signaling cascades.

Major Vault Protein Does Not Play a Role in Chemoresistance or Drug Localization in a Non-Small Cell Lung Cancer Cell Line

The human major vault protein (MVP) is the primary component of the 13 MDa vault complex. MVP has been implicated in the development of non-P-glycoprotein-mediated drug resistance in cancer cells. Here we present several lines of evidence that dispute this assertion. siRNAs capable of specifically and efficiently knocking down expression of MVP do not alter the ability of resistant cells to remove doxorubicin from the nucleus and do not increase sensitivity to the drug. Conversely, upregulation of MVP in chemosensitive cells does not confer increased drug resistance. In multi-drug resistant (MDR) lung carcinoma cells, fluorescence microscopy reveals that doxorubicin enters the nucleus and is then removed, inconsistent with suggestions that vaults either act to prevent the drug from entering the nucleus or are involved as a nuclear efflux pump. These data suggest that vaults play no direct role in the MDR phenotype in non-small cell lung carcinoma cells and that their cellular function remains unknown. These results also have important implications concerning the value of MVP as a drug target and as a prognostic marker for chemotherapy failure. Our results suggest the need for further investigation into the link between upregulation of vaults and malignancy, the mechanism behind non-P-gp-mediated drug resistance, and the role of vaults in human cells.

Intrinsic chemotherapy resistance to the tubulin‐binding antimitotic agents in renal cell carcinoma

Renal cancer is one of the most chemoresistant tumor types. Using a panel of 10 established renal cancer cell lines that have not been subjected to prior drug selection, the range of functional resistance phenotypes to the tubulin-binding agents paclitaxel, vinblastine, vincristine and patupilone (epothilone B, EPO906) was determined, together with expression of P-glycoprotein (PgP), multidrug resistance associated protein-2 (MRP2) and major vault protein (MVP) proteins. The IC(50) values for vincristine correlated positively with PgP expression (r = 0.73; p = 0.031), with values for paclitaxel and vinblastine just failing to reach significance. A significant positive correlation was observed for sensitivity to paclitaxel and MRP2 expression only (r = 0.8; p = 0.013). MVP expression did not correlate with sensitivity to any of the drugs examined. All cell lines exhibited much greater sensitivity to patupilone, demonstrating for the first time the potential use of patupilone in this cancer. In tissue samples from chemotherapy-naive renal cell carcinoma (RCC) patients, marked downregulation or absence of PgP in many tumor cells with expression levels more similar to sensitive cell lines rather than the resistant lines was seen. Similarly, MRP2 was absent or only weakly present in tumor cells, whereas MVP was very strongly upregulated in most tumor samples. This study illustrating discrepancies between results exclusively based on studies in cell lines and findings in vivo suggests that the role of PgP and MRP2 in intrinsic resistance in RCC in vivo may be less than expected from the in vitro findings and supports a potential role for MVP on the basis of in vivo expression studies.

Expression and Cellular Distribution of Major Vault Protein: A Putative Marker for Pharmacoresistance in a Rat Model for Temporal Lobe Epilepsy

Because drug transporters might play a role in the development of multidrug resistance (MDR), we investigated the expression of a vesicular drug transporter, the major vault protein (MVP), in a rat model for temporal lobe epilepsy. By using real-time polymerase chain reaction (PCR) analysis and immunocytochemistry, we quantified MVP mRNA and protein from the dentate gyrus (DG) and parahippocampal cortex (PHC) taken from EEG-monitored rats at 1 week after electrically induced status epilepticus (SE) and at 5-9 months after SE, when rats exhibit spontaneous seizures. Within 1 week after SE, MVP mRNA levels increased in both DG and PHC compared with those in controls. In chronic epileptic rats, MVP mRNA was still significantly upregulated in the PHC, whereas in the DG, the expression returned to control levels. MVP protein increased within 1 day after SE in reactive microglial cells within most limbic regions; the hippocampus showed the highest expression at 1 week after SE. In chronic epileptic rats, MVP protein expression was largely decreased in most brain regions, but it was still high, especially in the piriform cortex. The occurrence of SE was a prerequisite for increased MVP expression, because no increase was found in electrically stimulated rats that did not exhibit SE. MVP expression is upregulated in chronic epileptic rats and may contribute to the development of pharmacoresistance.

Characterization of MVP and VPARP assembly into vault ribonucleoprotein complexes

Vaults are barrel-shaped cytoplasmic ribonucleoprotein particles composed of three proteins: the major vault protein (MVP), the vault poly(ADP-ribose)polymerase (VPARP), and the telomerase-associated protein 1, together with one or more small untranslated RNAs. To date, little is known about the process of vault assembly or about the stability of vault components. In this study, we analyzed the biosynthesis of MVP and VPARP, and their half-lives within the vault particle in human ACHN renal carcinoma cells. Using an immunoprecipitation assay, we found that it took more than 4 h for newly synthesized MVPs to be incorporated into vault particles but that biosynthesized VPARPs were completely incorporated into vaults within 1.5 h. Once incorporated into the vault complex, both MVP and VPARP were very stable. Expression of human MVP alone in Escherichia coli resulted in the formation of particles that had a distinct vault morphology. The C-terminal region of VPARP that lacks poly(ADP-ribose)polymerase activity co-sedimented with MVP particles. This suggests that the activity of VPARP is not essential for interaction with MVP-self-assembled vault-like particles. In conclusion, our findings provide an insight into potential mechanisms of physiological vault assembly.

SP-transcription factors are involved in basal MVP promoter activity and its stimulation by HDAC inhibitors

The major vault protein (MVP) has been implicated in multidrug resistance, cellular transport, and malignant transformation. In this study we aimed to identify crucial MVP promoter elements that regulate MVP expression. By mutation as well as deletion analysis a conserved proximal GC-box element was demonstrated to be essential for basal human MVP promoter transactivation. Binding of Sp-family transcription factors but not AP2 to this element in vitro and in vivo was shown by EMSA and ChIP assays, respectively. Inhibition of GC-box binding by a dominant-negative Sp1-variant and by mithramycin A distinctly attenuated MVP promoter activity. In Sp-null Drosophila cells, the silent human MVP promoter was transactivated by several human Sp-family members. In human cells the MVP promoter was potently stimulated by the histone deacetylase (HDAC) inhibitors butyrate (NaB) and trichostatin A (TSA), resulting in enhanced MVP expression. This stimulation was substantially decreased by mutation of the single GC-box and by application of mithramycin A. Treatment with HDAC inhibitors led to a distinct decrease of Sp1 but increase of Sp3 binding in vivo to the respective promoter sequence as demonstrated by ChIP assays. Summarising, this study identifies variations in Sp-transcription factor binding to a single proximal GC-box element as critical for basal MVP promoter activation and its stimulation by HDAC inhibitors.

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.

Overexpression of the Human Major Vault Protein in Gangliogliomas

Recent evidence has been obtained that the major vault protein (MVP) may play a role in multidrug resistance (MDR). We investigated the expression and cellular localization of MVP in gangliogliomas (GGs), which are increasingly recognized causes of chronic pharmacoresistant epilepsy. Surgical tumor specimens (n = 30), as well as peritumoral and control brain tissues, were examined for the cellular distribution pattern of MVP with immunocytochemistry. Western blot analysis showed a consistent increase in MVP expression in GGs compared with that in control cortex. In normal brain, MVP expression was below detection in glial and neuronal cells, and only low immunoreactivity (IR) levels were detected in blood vessels. MVP expression was observed in the neuronal component of 30 of 30 GGs and in a population of tumor glial cells. In the majority of the tumors, strong MVP IR was found in lesional vessels. Perilesional regions did not show increased staining in vessels or in neuronal and glial cells compared with normal cortex. However, expression of MVP was detected in the hippocampus in cases with dual pathology. The increased expression of MVP in GGs is another example of an MDR-related protein that is upregulated in patients with refractory epilepsy. Further research is necessary to investigate whether it could play role in the mechanisms underlying drug resistance in chronic human epilepsy.

Expression of multidrug resistance genes MVP, MDR1, and MRP1 determined sequentially before, during, and after hyperthermic isolated limb perfusion of soft tissue sarcoma and melanoma patients.

Isolated, hyperthermic limb perfusion (ILP) with recombinant human tumor necrosis factor alpha and melphalan is a highly effective treatment for advanced soft tissue sarcoma (STS) and locoregional metastatic malignant melanoma. Multidrug resistance (MDR)-associated genes are known to be inducible by heat and drugs; expression levels of the major vault protein (MVP), MDR1, and MDR-associated protein 1 (MRP1) were determined sequentially before, during, and after ILP of patients. Twenty-one STS or malignant melanoma patients were treated by ILP. Tumor tissue temperatures were recorded continuously and ranged from 33.4 degrees C initially to peak values of 40.4 degrees C during ILP. Serial true-cut biopsy specimens from tumor tissues were routinely microdissected. Expression analyses for MDR genes were performed by real-time reverse transcriptase polymerase chain reaction and immunohistochemistry. In 83% of the patients, MVP expression was induced during hyperthermic ILP. MVP-mRNA inductions often paralleled the increase in temperature during ILP. Increased MVP protein expressions either were observed simultaneously with the MVP-mRNA induction or were delayed until after the induction at the transcriptional level. Inductions of MDR1 and MRP1 were observed in only 13% and 27% of the specimens analyzed. Temperatures and drugs applied preferentially led to an induction of MVP and were not sufficient to induce MDR1 and MRP1 in the majority of tumors. This study is the first to analyze the expression of MDR-associated genes sequentially during ILP of patients and demonstrates that treatment might lead to increased levels of MVP, whereas enhanced levels of MDR1 and MRP1 remain rare events.

The genomic sequence of the murine major vault protein and its promoter

Vaults are ribonucleoproteins of unknown function, consisting of three different proteins and multiple copies of small untranslated RNA molecules. One of the protein subunits has been identified as TEP1, a protein that is also associated with the telomerase complex. Another protein appears to contain a functional PARP domain and is hence called VPARP. The third protein, major vault protein (MVP), is believed to make up 70% of the total mass of the vault complex and to be responsible for the typical barrel-shaped structure of vaults. We have isolated the murine MVP cDNA and compared the amino acid sequence with MVP from other species. Over 90% of sequence identity was found between mouse, human and rat, and a considerable degree of identity between mouse and MVPs from lower eukaryotes. We also found that the genomic structure of the murine MVP gene closely resembles the organization of the human MVP gene, both consisting of 15 exons of which most have exactly the same size. Finally we have isolated a genomic region upstream (and partially overlapping) the first untranslated exon, that displayed promoter activity in a luciferase reporter assay. Furthermore, we showed that the sequences from the first exon together with the 5′-end of the first intron enhance the promoter activity, implying the presence of essential promoter elements in this region. Alignment of the murine promoter region with the homologous sequences of the human gene revealed an identity of 58%. The apparent presence of conserved promoter elements suggests a similar regulation of human and murine MVP expression.

Assembly of Vault-like Particles in Insect Cells Expressing Only the Major Vault Protein

Vaults are the largest (13 megadalton) cytoplasmic ribonucleoprotein particles known to exist in eukaryotic cells. They have a unique barrel-shaped structure with 8-fold symmetry. Although the precise function of vaults is unknown, their wide distribution and highly conserved morphology in eukaryotes suggests that their function is essential and that their structure must be important for their function. The 100-kDa major vault protein (MVP) constitutes approximately 75% of the particle mass and is predicted to form the central barrel portion of the vault. To gain insight into the mechanisms for vault assembly, we have expressed rat MVP in the Sf9 insect cell line using a baculovirus vector. Our results show that the expression of the rat MVP alone can direct the formation of particles that have biochemical characteristics similar to endogenous rat vaults and display the distinct vault-like morphology when negatively stained and examined by electron microscopy. These particles are the first example of a single protein polymerizing into a non-spherically, non-cylindrically symmetrical structure. Understanding vault assembly will enable us to design agents that disrupt vault formation and hence aid in elucidating vault function in vivo.

A small upstream open reading frame causes inhibition of human major vault protein expression from a ubiquitous mRNA splice variant

Overexpression of the major vault protein (MVP) has been linked to a multidrug resistance (MDR) phenotype. We describe a ubiquitously expressed MVP mRNA splice variant (long (L)-MVP) differing from the regular isoform (short (S)-MVP) within the 5′-leader. Only L-MVP mRNA contains a small upstream open reading frame which was proven to inhibit in vitro and in vivo MVP expression in cis. L-MVP represented an almost constant portion of total MVP mRNA in diverse normal tissues, but was more variable in malignant cell types. MDR sublines with altered MVP expression displayed changed S-MVP/L-MVP ratios as compared to their drug-sensitive counterparts. Our results suggest alternative splicing as one mechanism for regulation of MVP expression.