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PubMedDate not supplied

Lung resistance-related protein gene expression and drug sensitivity in human gastric and lung cancer cells.

Lung resistance-related protein (LRP) is the human major vault transporter protein and is suggested to confer anticancer drug resistance. We quantitated the level of LRP mRNA expression in 10 gastric and 14 lung cancer cell lines by RT-PCR, and examined the relationship between its level in these cells and their sensitivities to anticancer drugs. HT1080 fibrosarcoma cells were used as positive controls for LRP. LRP mRNA was expressed in all gastric and lung cancer lines, and its level in each cell type was less than two-fold that of HT1080 cells, except for two lung cancer lines. The correlation between the level of LRP mRNA expression and cisplatin sensitivity was significant in lung cancer lines (r = 0.762, P = 0.028), and borderline in gastric cancer lines (r = 0.631, P = 0.129). There was no correlation between the level of LRP mRNA expression and etoposide, doxorubicin, vincristine, or SN-38. Our results suggest that LRP is commonly expressed in gastric and lung cancers, and may confer their resistance to cisplatin.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMedDate not supplied

Altered expression of resistance associated genes in hepatoblastoma xenografts incorporated into mice following treatment with adriamycin or cisplatin.

To study the development of drug resistance in childhood hepatoblastoma (HB) using a model closely related to in vivo conditions, we cultivated HB xenografts from 3 patients into mice and treated these tumors with either adriamycin (ADR) or cisplatin (CIS). Determination of the relative expression levels of various resistance associated genes by a cDNA-PCR approach showed: a) Significantly enhanced MDR1 gene expression levels after treatment with ADR in each case. b) Significantly enhanced glutathione S-transferase mu (GST mu) gene expression levels after treatment with CIS in 2/3 xenografts. c) Significantly decreased levels of topoisomerase IIa (TOPO IIa) in tumors of the same two patients after treatment with either ADR or CIS. These findings give evidence that the MDR1-, GST mu- and TOPO IIa-gene products may contribute to drug resistance in HB.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1998

Activation of the LRP (lung resistance-related protein) gene by short-term exposure of human leukemia cells to phorbol ester and cytarabine.

Treatment-induced secondary drug resistance of tumor cells is a major cause of relapsed disease and therapeutic failure in cancer patients. It has been shown that the expression of the multidrug resistance MDR1/P-glycoprotein gene could be induced by short-term in vitro exposure of cells to protein kinase C (PKC) agonists or different chemotherapeutic drugs. We studied whether other genes involved in drug resistance are regulated by similar signaling pathways. Transient (up to 24 h) treatment of HL-60 or K562 leukemia cells with phorbol 12-myristate 13-acetate (TPA) resulted in increased steady-state level of LRP (lung resistance-related protein) mRNA and protein. Among conventional chemotherapeutic drugs tested, only cytarabine (Ara C) induced the LRP mRNA expression though no increase in LRP protein was detected. LRP gene activation was not detectable in either H9 T-cell leukemia or in solid carcinoma cell lines (BT-20, ZR-75-1, and SW 1573). None of the agents influenced the levels of MRP (multidrug resistance-associated protein) mRNA in any cell line tested. In HL-60 cells, the LRP activation by TPA or Ara C was sustained for at least 23 days after withdrawal of inducing agents. bis-Indolylmaleimide I, a potent PKC inhibitor, attenuated TPA-induced LRP activation. In contrast, the inhibitor had no effect on the LRP induction by Ara C. These data indicate that the LRP gene can be activated by different mechanisms, some of which involve PKC.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMedDate not supplied

Expression of lung-resistance protein gene is not associated with platinum drug exposure in lung cancer.

BACKGROUND: Platinum drug resistance is an important problem in lung cancer chemotherapy. In this study, we examined lung-resistance protein (LRP) gene expression in vivo and in vitro in relation to platinum drug exposure. MATERIALS AND METHODS: The expression levels of the LRP gene were assessed by the reverse transcription polymerase chain reaction, in 80 autopsy samples (40 lung tumors and 40 corresponding normal lung tissues), two lung cancer cell lines and in peripheral mononuclear cells collected from 8 lung cancer patients before and after platinum drug administration. RESULTS: The LRP gene expression levels of autopsy specimens exposed antemortem to platinum drugs were not significantly different to those of specimens without platinum drug exposure, for both lung tumors and normal lung tissues. Our results also demonstrate that LRP gene expression was not induced by platinum drugs either in vitro or in vivo. CONCLUSIONS: The present results indicate that LRP gene expression is not associated with platinum drug exposure in lung cancer.

Adenocarcinoma↗
PubMed2002

Disruption of the murine major vault protein (MVP/LRP) gene does not induce hypersensitivity to cytostatics.

Vaults are ribonucleoprotein particles with a distinct structure and a high degree of conservation between species. Although no function has been assigned to the complex yet, there is some evidence for a role of vaults in multidrug resistance. To confirm a direct relation between vaults and multidrug resistance, and to investigate other possible functions of vaults, we have generated a major vault protein (MVP/lung resistance-related protein) knockout mouse model. The MVP(-/-) mice are viable, healthy, and show no obvious abnormalities. We investigated the sensitivity of MVP(-/-) embryonic stem cells and bone marrow cells derived from the MVP-deficient mice to various cytostatic agents with different mechanisms of action. Neither the MVP(-/-) embryonic stem cells nor the MVP(-/-) bone marrow cells showed an increased sensitivity to any of the drugs examined, as compared with wild-type cells. Furthermore, the activities of the ABC-transporters P-glycoprotein, multidrug resistance-associated protein and breast cancer resistance protein were unaltered on MVP deletion in these cells. In addition, MVP wild-type and deficient mice were treated with the anthracycline doxorubicin. Both groups of mice responded similarly to the doxorubicin treatment. Our results suggest that MVP/vaults are not directly involved in the resistance to cytostatic agents.

Animals↗
PubMed2005

Solution structure of a two-repeat fragment of major vault protein.

Major vault protein (MVP) is the main constituent of vaults, large ribonucleoprotein particles implicated in resistance to cancer therapy and correlated with poor survival prognosis. Here, we report the structure of the main repeat element in human MVP. The approximately 55 amino acid residue MVP domain has a unique, novel fold that consists of a three-stranded antiparallel beta-sheet. The solution NMR structure of a two-domain fragment reveals the interdomain contacts and relative orientations of the two MVP domains. We use these results to model the assembly of 672 MVP domains from 96 MVP molecules into the ribs of the 13MDa vault structure. The unique features include a thin, skin-like structure with polar residues on both the cytoplasmic and internal surface, and a pole-to-pole arrangement of MVP molecules. These studies provide a starting point for understanding the self-assembly of MVP into vaults and their interactions with other proteins. Chemical shift perturbation studies identified the binding site of vault poly(ADP-ribose) polymerase, another component of vault particles, indicating that MVP domains form a new class of interaction-mediating modules.

Amino Acid Sequence↗
PubMed1999

The 193-kD vault protein, VPARP, is a novel poly(ADP-ribose) polymerase.

Mammalian vaults are ribonucleoprotein (RNP) complexes, composed of a small ribonucleic acid and three proteins of 100, 193, and 240 kD in size. The 100-kD major vault protein (MVP) accounts for >70% of the particle mass. We have identified the 193-kD vault protein by its interaction with the MVP in a yeast two-hybrid screen and confirmed its identity by peptide sequence analysis. Analysis of the protein sequence revealed a region of approximately 350 amino acids that shares 28% identity with the catalytic domain of poly(ADP-ribose) polymerase (PARP). PARP is a nuclear protein that catalyzes the formation of ADP-ribose polymers in response to DNA damage. The catalytic domain of p193 was expressed and purified from bacterial extracts. Like PARP, this domain is capable of catalyzing a poly(ADP-ribosyl)ation reaction; thus, the 193-kD protein is a new PARP. Purified vaults also contain the poly(ADP-ribosyl)ation activity, indicating that the assembled particle retains enzymatic activity. Furthermore, we show that one substrate for this vault-associated PARP activity is the MVP. Immunofluorescence and biochemical data reveal that p193 protein is not entirely associated with the vault particle, suggesting that it may interact with other protein(s). A portion of p193 is nuclear and localizes to the mitotic spindle.

Alpha-Globulins↗
PubMed2006

The major vault protein is responsive to and interferes with interferon-gamma-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.

Active Transport, Cell Nucleus↗
PubMed2000

Expression of the major vault protein LRP in human non-small-cell lung cancer cells: activation by short-term exposure to antineoplastic drugs.

Non-small-cell lung cancer (NSCLC) cells are characterised by resistance to the toxic impact of antineoplastic drugs both in vivo and in vitro. The lung resistance-related protein (LRP), identical with the human major vault protein, is over-expressed in a variety of tumour cells characterised by intrinsic or acquired chemoresistance. We investigated the expression and cellular localisation of LRP in 16 unselected NSCLC cell lines, immortalised bronchial epithelial cells and embryonic lung fibroblasts. All cell lines analysed expressed LRP mRNA, while protein expression ranged from undetectable up to high levels. Cell fractionation and immunofluorescence staining in selected cell lines localised LRP almost exclusively to the cytoplasm. LRP was contained in the 100, 000 g pellet and absent in the soluble, cytosolic fraction and nuclei. A small proportion of LRP, however, was shown to be loosely associated with the outside of the nuclei. Sucrose gradient equilibrium centrifugation revealed presence of LRP exclusively in the fraction known to accumulate purified vault particles. Short-term exposure (16 hr) to subtoxic daunomycin (DM)-, and bleomycin (BM)-concentrations significantly (up to 4-fold) enhanced LRP expression in 2/4 cell lines tested. Cisplatin (CDDP) had a minor effect while vinblastine (VBL) was ineffective. At cytotoxic conditions all drugs rather decreased than increased LRP expression. When basic LRP expression was compared with chemosensitivity, a significant correlation was detected for resistance to CDDP but not DM, doxorubicin (DOX), etoposide (VP-16), VBL and BM. Summing up, our data suggest a role of vaults both in basic CDDP resistance and, additionally, in an short-term defensive response of NSCLC cells against several other drugs.

Antineoplastic Agents↗
PubMed2001

The Telomerase/vault-associated protein TEP1 is required for vault RNA stability and its association with the vault particle.

Vaults and telomerase are ribonucleoprotein (RNP) particles that share a common protein subunit, TEP1. Although its role in either complex has not yet been defined, TEP1 has been shown to interact with the mouse telomerase RNA and with several of the human vault RNAs in a yeast three-hybrid assay. An mTep1(-/-) mouse was previously generated which resulted in no apparent change in telomere length or telomerase activity in six generations of mTep1-deficient mice. Here we show that the levels of the telomerase RNA and its association with the telomerase RNP are also unaffected in mTep1(-/-) mice. Although vaults purified from the livers of mTep1(-/-) mice appear structurally intact by both negative stain and cryoelectron microscopy, three-dimensional reconstruction of the mTep1(-/-) vault revealed less density in the cap than previously observed for the intact rat vault. Furthermore, the absence of TEP1 completely disrupted the stable association of the vault RNA with the purified vault particle and also resulted in a decrease in the levels and stability of the vault RNA. Therefore, we have uncovered a novel role for TEP1 in vivo as an integral vault protein important for the stabilization and recruitment of the vault RNA to the vault particle.

Animals↗
PubMed1997

Development and characterisation of novel human multidrug resistant mammary carcinoma lines in vitro and in vivo.

Clinical chemotherapy of breast carcinomas must be considered insufficient, mainly due to the appearance of drug resistance. The multidrug resistance (MDR) phenotype, either intrinsically occurring or acquired, e.g., against a panel of different antineoplastic drugs, is discussed in relation to several MDR-associated genes such as the MDR-gene mdr1 encoding the P-glycoprotein (PGP), the MRP gene (multidrug resistance protein) encoding an MDR-related protein or the LRP gene encoding the lung resistance protein. Numerous experimental and clinical approaches aiming at reversing resistance require well-characterised in vitro and in vivo models. The aim of our work was to develop multidrug resistant sublines from human xenotransplanted breast carcinomas, in addition to the broadly used line MCF-7 and its multidrug resistant subline MCF-7/AdrR. MDR was induced in vitro with increasing concentrations of Adriablastin (ADR) for several weeks, resulting in a 3.5- to 35-fold increase in IC50 values using the MTT-test. Cell lines were cross-resistant toward another MDR-related drug, vincristine, but remained sensitive to non-MDR-related compounds such as cisplatin and methotrexate. The resistance toward Adriamycin and vincristine was confirmed in vivo by a lack of tumour growth inhibition in the nude mouse system. Gene expression data for the mdr1/PGP, MRP/MRP and LRP/LRP on both the mRNA (RT-PCR) and the protein levels (immunoflow cytometry) demonstrated that induction of mdr1 gene expression was responsible for the acquired MDR phenotype. Rhodamine efflux data, indicated by PGP overexpression, underlined the development of this MDR mechanism in the newly established breast carcinoma lines MT-1/ADR, MT-3/ADR and MaTu/ADR.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2007

Role of chemotherapy resistance genes in outcome of neuroblastoma.

BACKGROUND: Neuroblastoma is a heterogeneous pediatric disease. Most patients with localized disease usually have a favorable prognosis, but patients with advanced disease have a poor prognosis despite combination chemotherapy. Treatment failure may be attributable to resistance to cytotoxic drugs. PROCEDURE: Using quantitative RT-PCR, we investigated the clinical significance of the level of mRNA expression of multidrug resistance genes (MDR1, MRP1, MRP5, LRP) in a series of 29 advanced neuroblastoma samples. RESULTS: At the end of induction chemotherapy, 48% of patients achieved a clinical complete response, 28% achieved a partial response or stable disease, and 24% presented progressive disease. MDR1 mRNA overexpression (i.e., mRNA level >2 copies of MDR1 gene) was observed in 74% of samples, and MRP1, MRP5, LRP overexpression was observed less frequently (30, 33, and 33% of samples, respectively). None of these parameters were predictive of response, relapse, or survival. However, clinical response to treatment was highly predictive of relapse-free survival and overall survival. CONCLUSIONS: High expression of these multidrug resistance genes in advanced neuroblastoma is not the main parameter of response to cytotoxic drugs; clinical response to treatment remains the most important parameter in predicting the prognosis of patients with advanced neuroblastoma, until other relevant laboratory parameters have been identified.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2001

Mechanisms of taxotere-related drug resistance in pancreatic carcinoma.

BACKGROUND: Pancreatic adenocarcinoma (PAC) is generally refractory to most chemotherapeutic agents, including docetaxel (Taxotere; TXT). Specific mechanisms for TXT-related drug resistance in PAC have not been defined. The hypothesis of this study was that PAC resistance to TXT is primarily related to P-glycoprotein (P-gp), the expression product of multiple drug resistance (MDR)-1, as opposed to lung resistance protein (LRP) or multidrug resistance protein (MRP). MATERIALS AND METHODS: The sensitivity of the PAC cell line SUIT-2 and its sublines to TXT, doxorubicin (DOX) and 5-fluorouracil (5-FU) was evaluated with a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay. MDR1 (P-gp), MRP, LRP, and beta-tubulin isotype gene expressions were detected at the messenger RNA level by reverse transcription-polymerase chain reaction (RT-PCR). Verapamil and indomethacin (IMC) were used to test the functionality of P-gp and MRP, respectively. RESULTS: The SUIT-2 subline S-020 and the TXT-selected SUIT-2 cell line S2/TXT were significantly resistant to TXT. Both showed cross-resistance to DOX but no resistance to 5-FU. RT-PCR demonstrated strong expression of P-gp in S-020 and S2/TXT and weaker or no expression in other cells lines. MRP and LRP expression was found in most of these cell lines but had no relationship to the TXT resistance. TXT resistance in S2-020 and S2/TXT could be reversed by verapamil but not by IMC. Levels of beta-tubulin isotype II and III were increased in S2/TXT compared with S-020 and SUIT-2. CONCLUSIONS: Intrinsic and acquired TXT resistance is primarily mediated by P-gp, but not by MRP or LRP, and is markedly reversed by the P-gp modulator verapamil. Hence future related studies should focus on the use of agents that block the transporter action of P-gp.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2000

Expression of lung resistance protein in epithelioid sarcoma in vitro and in vivo.

The incidence of epithelioid sarcoma among patients with malignant soft tissue tumors is small, but the rates of recurrence and metastasis of this type of sarcoma are high. To date, effective chemotherapy for advanced epithelioid sarcoma has not been established and, furthermore, epithelioid sarcoma is known to exhibit multidrug resistance (MDR). The chemosensitivities to anticancer agents of two cell lines established from epithelioid sarcoma were examined in this study. The results showed that the ES-OMC-MN and SFT-8606 cell lines were resistant to vincristine (IC50 1190 nM and 872 nM, respectively) and Adriamycin (IC50 921 nM and 650 nM, respectively), but sensitive to actinomycin D (IC50 < 10 nM). P-glycoprotein (p-Gp) and MDR-associated protein (MRP) were not expressed in these cell lines, but a high expression level of lung resistance protein (LRP) was observed. The original tumor tissues from which the two cell lines were established were also found to be LRP-positive but not to express p-Gp or MRP. Their chemosensitivities to Adriamycin were not significantly altered in the presence of 2.5 microg/ml anti-LRP antibody (LRP-56), but the IC50 of vincristine was much less (IC50 128 nM and 27 nM, respectively) than that for an untreated cell line. It is thus suggested that the vincristine resistance in the two cell lines is LRP-mediated. Since cyclosporin A, known to be a modifier of p-Gp, also induced reversal of vincristine resistance in the ES-OMC-MN and SFT-8606 cell lines (IC50 6.2 nM and 17 nM, respectively), it is suggested that cyclosporin A acts as a modifier of MDR mediated by LRP.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed2001

Recent findings in the modern RNA world.

It is assumed that modern life forms arose from a molecular ancestor in which RNA molecules both stored genetic information and catalyzed biochemical reactions. In modern cells, these functions are carried out, respectively, by DNA and proteins, but diverse cellular RNAs are also involved in key cellular functions. In this paper, we review the cellular RNAs that are ubiquitous and/or that perform essential biological functions, and we discuss the evolutionary relationships of such RNAs with a prebiotic RNA world. This unexpected biological diversity of cellular RNAs and the crucial functions they perform in cellular metabolism demonstrate the complexity of an RNA-driven metabolism in an ancient RNA world and in modern life. Cellular RNAs are involved in translation (tRNA and rRNA) but also in ribosome maturation (snoRNA) and more generally in RNA processing (snRNA and snoRNA), replication (telomerase RNA), editing, protein translocation (SRP RNA), cellular transport (vRNA) and translation quality control (tmRNA). In addition, the function of many other cellular RNAs has not yet been determined. Future investigations of RNA function will allow us to better understand not only early evolutionary biological processes but also the central metabolism of modern cells.

Biological Evolution↗
PubMed2003

C(H)OP refractory chronic lymphocytic leukemia patients in whom salvage chemotherapy chosen by evaluating multiple chemotherapeutic drug-resistant factors was remarkably effective.

It is well known that the expression of anticancer drug-resistant factors is elevated in patients with primary refractory or relapsed chronic lymphocytic leukemia (CLL) who have been treated with chemotherapy. We report here two C(H)OP refractory patients with CLL in whom salvage chemotherapy chosen by evaluating anticancer drug-resistant factors (glutathione-S-transferase-Pi [GST-Pi], glycoprotein [GP]-170, multidrug resistance-associated protein [MRP], and lung resistance protein [LRP]) was remarkably effective. A 71-year-old male patient was refractory to induction therapy with cyclophosphamide, vincristine, and prednisone (COP), and his leukemic cells at diagnosis displayed overexpression of GST-Pi and GP-170. A 74-year-old female patient's condition had been stable; she had received ten courses of COP over 9 years. However, because systemic lymphadenopathies recurred, she was treated with chemotherapy consisting of cyclophosphamide, adriamycin, vincristine, and prednisone (CHOP) or dexamethasone, etoposide, ifosphamide, and carboplatin (DeVIC). However, she did not respond at all, and her leukemic cells at recurrence displayed overexpression of GST-Pi. Therefore, we chose for these patients a salvage therapy consisting of dexamethasone and high-dose cytosine arabinoside (Ara C), to which neither GST-Pi nor GP-170 show any drug resistance. In both patients, this salvage therapy proved effective.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed2001

Intracellular P-gp contributes to functional drug efflux and resistance in acute myeloid leukaemia.

Drug compartmentalization as well as drug efflux can contribute to drug resistance. We demonstrate the presence of P-gp in intracellular vesicles in certain AML cell lines and show localization of DNR to a similar subcellular compartment(s) that can be altered in the presence of P-gp inhibitors. Analysis of leukaemic cell lines and 50 AML patient samples showed that the level of P-gp mRNA or total P-gp protein correlated better with drug efflux than surface P-gp protein, suggesting that intracellular P-gp may contribute to MDR in AML. Therefore, the level of total P-gp protein or mRNA may be a better indicator of MDR than surface P-gp protein. In addition, we provide evidence for a novel mechanism of drug sequestration in K562 myeloid leukaemic cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗