Analysis of MVP and VPARP promoters indicates a role for chromatin remodeling in the regulation of MVP

Multi-drug-resistant cancer cells frequently express elevated levels of ribonucleoprotein complexes termed vaults. The increased expression of vault proteins and their mRNAs has led to the suggestion that vaults may play a direct role in preventing drug toxicity. To further understand vault component up-regulation, the three proteins that comprise the vault, the major vault protein (MVP), vault poly(ADP-ribose) polymerase (VPARP), and telomerase-associated protein-1 (TEP1), were examined with respect to gene amplification and drug-induced chromatin remodeling. Gene amplification was not responsible for increased vault component levels in multi-drug-resistant cancer cell lines. The TATA-less murine MVP and human VPARP promoters were identified and functionally characterized. There was no significant activation of either the MVP or VPARP promoters in drug-resistant cell lines in comparison to their parental, drug-sensitive counterparts. Treatment of various cell lines with sodium butyrate, an inhibitor of histone deacetylase (HDAC), led to an increase in vault component protein levels. Furthermore, treatment with trichostatin A (TSA), a more specific inhibitor of HDAC, caused an increase in MVP protein, mRNA, and promoter activity. These results suggest that up-regulation of MVP in multi-drug resistance (MDR) may involve chromatin remodeling.     

Detection of the Mr 110,000 lung resistance-related protein LRP/MVP with monoclonal antibodies.

The Mr 110,000 lung resistance-related protein (LRP), also termed the major vault protein (MVP), constitutes >70% of subcellular ribonucleoprotein particles called vaults. Overexpression of LRP/MVP and vaults has been linked directly to MDR in cancer cells. Clinically, LRP/MVP expression can be of value to predict response to chemotherapy and prognosis. Monoclonal antibodies (MAbs) against LRP/MVP have played a critical role in determining the relevance of this protein in clinical drug resistance. We compared the applicability of the previously described MAbs LRP-56, LMR-5, LRP, 1027, 1032, and newly isolated MAbs MVP-9, MVP-16, MVP-18, and MVP-37 for the immunodetection of LRP/MVP by immunoblotting analysis and by immunocyto- and histochemistry. The availability of a broader panel of reagents for the specific and sensitive immunodetection of LRP/MVP should greatly facilitate biological and clinical studies of vault-related MDR.     

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.     

Cloning, expression, and purification of a highly immunogenic recombinant gonadotropin-releasing hormone (GnRH) chimeric peptide

To design an anti-gonadotropin-releasing hormone (GnRH) vaccine capable of eliciting strong immunogenicity, a gene fragment encoding a chimeric peptide was constructed using polymerase chain reaction and ligated into a novel expression vector for recombinant expression in a T7 RNA polymerase-based expression system. The chimeric peptide called GnRH3-hinge-MVP contained three linear repeats of GnRH (GnRH3), a fragment of the human IgG1 hinge region, and a T-cell epitope of measles virus protein (MVP). The expression plasmid contained the GnRH3-hinge-MVP construct ligated to its fusion partner (AnsB-C) via an unique acid labile Asp-Pro linker. The recombinant fusion protein was expressed in an inclusion body in Escherichia coli under IPTG or lactose induction and the target peptide was easily purified using washing of urea and ethanol precipitation. The target chimeric peptide was isolated from the fusion partner following acid hydrolysis and purified using DEAE-Sephacel chromatography. The purified GnRH3-hinge-MVP was determined to be highly homogeneous by IEF analysis and the N-terminal sequencing. Further, immunization of female mice with the recombinant chimeric peptide resulted in generation of high-titer antibodies specific for GnRH. The results showed that GnRH3-hinge-MVP could be considered as a candidate anti-GnRH vaccine.     

Downregulation of PGY1/MDR1 mRNA level in human KB cells by antisense oligonucleotide conjugates. RNA accessibility in vitro and intracellular antisense activity

Inhibition of PGY1/MDR1 (multidrug resistance gene 1) mRNA expression in multidrug resistant KB-8-5 cells by 5'-bis-pyrenyl-3'-aminohexyl oligodeoxyribonucleotide conjugates targeted to four sites of this mRNA has been investigated. Three of the tested oligonucleotide conjugates specifically inhibited the expression of PGY1/MDR1 mRNA as monitored by the RT-PCR assay. The oligonucleotide conjugate targeted to the region (+178; +194) of the PGY1/MDR1 mRNA decreased level of this mRNA to 10% compared to the control. Nuclease-resistant analogs of oligonucleotide, complementary to this MDR1 mRNA region therefore, might be considered as a prototype compounds for development of gene-targeted therapeutic agents for overcoming the MDR phenotype caused by the overexpression of the PGY1/MDR1 gene.     

Screening of deoxyribozyme with high reversal efficiency against multidrug resistance in breast carcinoma cells

Specific inhibition of P-glycoprotein (Pgp) expression, which is encoded by multidrug resistance gene-1 (MDR1), is considered a well-respected strategy to overcome multidrug resistance (MDR). Deoxyribozymes (DRz) are catalytic nucleic acids that could cleave a target RNA in sequence-specific manner. However, it is difficult to select an effective target site for DRz in living cells. In this study, target sites of DRz were screened according to MDR1 mRNA secondary structure by RNA structure analysis software. Twelve target sites on the surface of MDR1 mRNA were selected. Accordingly, 12 DRzs were synthesized and their suppression effect on the MDR phenotype in breast cancer cells was confirmed. The results showed that 4 (DRz 2, 3, 4, 9) of the 12 DRzs could, in a dose-dependent response, significantly suppress MDR1 mRNA expression and restore chemosensitivity in breast cancer cells with MDR phenotype. This was especially true of DRz 3, which targets the 141 site purine-pyrimidine dinucleotide. Compared with antisense oligonucleotide or anti-miR-27a inhibitor, DRz 3 was more efficient in suppressing MDR1 mRNA and Pgp protein expression or inhibiting Pgp function. The chemosensitivity assay also proved DRz 3 to be the best one to reverse the MDR phenotype. The present study suggests that screening targets of DRzs according to MDR1 mRNA secondary structure could be a useful method to obtain workable ones. We provide evidence that DRzs (DRz 2, 3, 4, 9) are highly efficient at reversing the MDR phenotype in breast carcinoma cells and restoring chemosensitivity.     

Hyperosmotic stress up-regulates the expression of major vault protein in SW620 human colon cancer cells

The major vault protein (MVP) is the major constituent of the vault particle, the largest ribonuclear protein complex described to date and is identical to lung resistance-related protein (LRP). Although MVP is also expressed in several normal tissues, little is known about its physiological role. MVP played a protective role against some xenobiotics and other stresses. We thus investigated the effect of osmotic stress on MVP expression by treating human colon cancer SW620 cells with sucrose or NaCl. The expression level of both MVP protein and MVP mRNA was increased by the osmostress. Sucrose or sodium chloride could also enhance MVP promoter activity. Inhibition of p38 MAPK in SW620 cells by SB203580 inhibited the expression of MVP under hyperosmotic stress. These findings suggested that osmotic stress up-regulated the MVP expression through p38 MAPK pathway. Down-regulation of MVP expression by MVP interfering RNA (RNAi) in SW620 cells increased the sensitivity of the cells to hyperosmotic stress and enhanced apoptosis. Furthermore, MVP RNAi prevented the osmotic stress-induced, time-dependent increase in phosphorylated Akt. These findings suggest that the PI3K/Akt pathway might be implicated in the cytoprotective effect of MVP.Our data demonstrate that exposure of cells to hyperosmotic stress induces MVP that might play an important role in the protection of the cells from the adverse effects of osmotic stress.     

Relationship of LRP-human major vault protein toin vitro and clinical resistance to anticancer drugs

Multidrug resistance (MDR) has been related to two members of the ABC-superfamily of transporters, P-glycoprotein (Pgp) and Multidrug Resistance-associated Protein (MRP). We have described a 110 kD protein termed the Lung Resistance-related Protein (LRP) that is overexpressed in several non-Pgp MDR cell lines of different histogenetic origin. Reversal of MDR parallels a decrease in LRP expression. In a panel of 61 cancer cell lines which have not been subjected to laboratory drug selection, LRP was a superior predictor forin vitro resistance to MDR-related drugs when compared to Pgp and MRP, and LRP's predictive value extended to MDR unrelated drugs, such as platinum compounds. LRP is widely distributed in clinical cancer specimens, but the frequency of LRP expression inversely correlates with the known chemosensitivity of different tumour types. Furthermore, LRP expression at diagnosis has been shown to be a strong and independent prognostic factor for response to chemotherapy and outcome in acute myeloid leukemia and ovarian carcinoma (platinum-based treatment) patients. Recently, LRP has been identified as the human major protein. Vaults are novel cellular organelles broadly distributed and highly conserved among diverse eukaryotic cells, suggesting that they play a role in fundamental cell processes. Vaults localise to nuclear pore complexes and may be the central plug of the nuclear pore complexes. Vaults structure and localisation support a transport function for this particle which could involve a variety of substrates. Vaults may therefore play a role in drug resistance by regulating the nucleocytoplasmic transport of drugs.Abbreviations LRP Lung Resistance-related Protein - MVP Major Vault Protein - MDR Multidrug resistance - MRP Multidrug resistance-associated Protein - NPC Nuclear Pore Complex - Pgp P-glycoprotein     

Digoxin up-regulates MDR1 in human colon carcinoma Caco-2 cells

Because MDR1 (P-glycoprotein) plays an important role in pharmacokinetics such as absorption and excretion of xenobiotics and multidrug resistance, an understanding of the factors regulating its function and expression is important. Here, the effects of digoxin on cell sensitivity to an anticancer drug, MDR1 function, and expression were examined by assessing the growth inhibition by paclitaxel, the transport characteristics of the MDR1 substrate Rhodamine123, and the level of MDR1 mRNA, respectively, using human colon carcinoma Caco-2 cells, which are widely used as a model of intestinal epithelial cells. The sensitivity to paclitaxel, an MDR1 substrate, in Caco-2 cells pretreated with digoxin was lower than that in non-treated cells. The accumulation of Rhodamine123 was reduced by pretreatment with digoxin and its efflux was enhanced. The level of MDR1 mRNA in Caco-2 cells was increased in a digoxin concentration-dependent manner. These results taken together suggested that digoxin up-regulates MDR1 in Caco-2 cells.     

Influence of IGF-I and cell density on MDR1 expression in the T-lymphoblastoid cell line CCRF-CEM

The debate about a direct or indirect effect of GH and IGF-I on the recurrence of malignancy, especially in the case of rhGH therapy in patients with leukemia, is still going on. Recent studies suggested that IGF-I plays a role in drug resistance during anticancer therapy. This resistance to diverse cytotoxic drugs, named multidrug-resistance (MDR), is mainly due to high levels of P-glycoprotein (P-gp). The gene encoding this membrane-associated transporter protein was named MDR1, and increased levels of P-gp are linked to enhanced MDR1 mRNA expression. Our aim was to investigate a possible effect of rhIGF-I on MDR1 gene expression in vitro. We cultured the T-lymphoblastoid cell line CCRF-CEM with different rhIGF-I concentrations (0, 5, 20 and 50 ng/ml) in serum-free medium for 3 days. CCRF-CEM cells are drug-sensitive and express MDR1 at low levels. MDR1 mRNA expression was measured by semiquantitative RT-PCR using a competitive assay with a heterologous DNA construct. In addition, GAPDH mRNA was amplified as an internal control for RNA integrity. P-gp activity was determined by a flow cytometric assay measuring rhodamine 123 accumulation. Furthermore, cell proliferation was monitored in all experiments. Our data do not support an effect of rhIGF-I on MDR1 mRNA expression, P-gp activity or cell proliferation in the CCRF-CEM cell line. MDR1 mRNA levels were inversely correlated to cell density with high significance (p < 0.0001). In conclusion, multidrug resistance linked to P-gp is not induced by IGF-I in CCRF-CEM cells. At high density, CCRF-CEM cells downregulate MDR1 gene expression. Our experimental model provides a very useful tool for monitoring the influence of growth factors on multidrug resistance in vitro.     

The AU-rich 3' untranslated region of human MDR1 mRNA is an inefficient mRNA destabilizer.

The human multidrug resistance gene MDR1 encodes a membrane-bound protein, referred to as P-glycoprotein, that acts as a pump to extrude toxins from cells. The 3' untranslated region (3'UTR) of the human MDR1 mRNA is very AU-rich (70%) and contains AU-rich sequences similar to those shown to confer rapid decay on c-myc, c-fos, and lymphokine mRNAs. We tested the ability of the MDR1 3'UTR to act as an mRNA destabilizing element in the human hepatoma cell line HepG2. The MDR1 mRNA has an intermediate half-life of 8 h in HepG2 cells compared to a half-life of 30 min for c-myc mRNA. The MDR1 mRNA half-life was prolonged to >20 h upon treatment with the protein synthesis inhibitor cycloheximide. We constructed expression vectors containing the human beta-globin coding region with the 3'UTR from either MDR1 or c-myc. The c-myc 3'UTR increased the decay of the chimeric mRNA, but the MDR1 3'UTR had no effect. We tested the ability of MDR1 3'UTR sequences to compete for interaction with AU-binding proteins in cell extracts; MDR1 RNA probes had a fivefold lower affinity for AU-binding proteins that interact with the c-myc AU-rich 3'UTR. Overall, our data suggest that the MDR1 3'UTR does not behave as an active destabilizing element in HepG2 cells.     

Semi-quantitative RT-PCR method to estimate full-length mRNA levels of the multidrug resistance gene

Expression levels of P-glycoprotein (P-gp), the transporter encoded by the human multidrug resistance gene (MDR1), may play an important role in drug disposition. The ability to quantitate full-length MDR1 mRNA levels may be predictive of P-gp expression and function. Therefore, a semi-quantitative RT-PCR assay was developed to assess full-length MDR1 mRNA levels. Levels offull-length 3.8-kb MDR1 mRNA were estimated by comparing PCR amplification of the RNA extract with that of an internal standard, deltaMDR1. The 2.9-kb deltaMDR1 competitor RNA standard was constructed by deleting 965 bpfrom the interior of MDR1 mRNA. The full-length MDR1 and deltaMDR1 share identical 5' and 3'primer binding sequences, allowing for their simultaneous amplification in the same RT-PCR. With this approach, MDR1 mRNA levels can be sensitively and reliably estimated with a detection limit of 2000 copies. Full-length MDR1 mRNA levels in various human cell lines and lymphocytes from leukemia patients varied over 100-fold, ranging from 0.3 to 36.5 x 10(5) copies/microg total RNA. The semi-quantitative full-length RT-PCR assay may be useful in estimating MDR1 mRNA levels to assess P-gp expression, which may be important in studying the role of P-gp in drug disposition and cancer chemotherapy efficacy.     

On the role of major vault protein in the resistance of senescent human diploid fibroblasts to apoptosis

Major vault protein (MVP), the main component of vault complex, is overexpressed in many multidrug-resistant cancer cell lines, suggesting a possible role for MVP in cell signaling and survival. In this study, we have found that MVP is markedly increased in senescent human diploid fibroblasts (HDFs) as well as in aged organs. We examined whether MVP expression might be affected by apoptotic stress in an aging-dependent manner. We treated young and senescent HDFs with apoptosis-inducing agents such as H(2)O(2), staurosporine and thapsigargin, and monitored MVP expression. We found that MVP expression is markedly reduced in young HDFs but not in senescent HDFs, in response to apoptotic stresses. Downregulation of MVP increased the sensitivity of senescent HDFs to apoptosis. Also, the level of antiapoptotic B-cell lymphoma protein-2 (Bcl-2) was significantly reduced and the accumulation of c-Jun increased in MVP knocked-down senescent HDFs. Moreover, treatment of MVP knocked-down senescent HDFs with SP600125, a specific c-Jun NH(2)-terminal kinase (JNK) inhibitor, restored the level of Bcl-2 protein. Taken together, these results suggest that MVP is important in the resistance of senescent HDFs to apoptosis by modulation of Bcl-2 expression by JNK pathway.     

Structure and expression of the human MDR (P-glycoprotein) gene family.

The human MDR (P-glycoprotein) gene family is known to include two members, MDR1 and MDR2. The product of the MDR1 gene, which is responsible for resistance to different cytotoxic drugs (multidrug resistance), appears to serve as an energy-dependent efflux pump for various lipophilic compounds. The function of the MDR2 gene remains unknown. We have examined the structure of the human MDR gene family by Southern hybridization of DNA from different multidrug-resistant cell lines with subfragments of MDR1 cDNA and by cloning and sequencing of genomic fragments. We have found no evidence for any other cross-hybridizing MDR genes. The sequence of two exons of the MDR2 gene was determined from genomic clones. Hybridization with single-exon probes showed that the human MDR1 gene is closely related to two genes in mouse and hamster DNA, whereas MDR2 corresponds to one rodent gene. The human MDR locus was mapped by field-inversion gel electrophoresis, and both MDR genes were found to be linked within 330 kilobases. The expression patterns of the human MDR genes were examined by enzymatic amplification of cDNA. In multidrug-resistant cell lines, increased expression of MDR1 mRNA was paralleled by a smaller increase in the levels of MDR2 mRNA. In normal human tissues, MDR2 was coexpressed with MDR1 in the liver, kidney, adrenal gland, and spleen. MDR1 expression was also detected in colon, lung, stomach, esophagus, muscle, breast, and bladder.     

Vault-related resistance to anticancer drugs determined by the expression of the major vault protein LRP

In this review we analyze the data supporting the notion that vault-related MDR, as reflected by LRP/MVP overexpression, represents a marker of drug resistance in vitro and in the clinic. Vaults, besides playing a fundamental biological role, may be involved in a novel mechanism of MDR. This revised version was published online in August 2006 with corrections to the Cover Date.