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Immunolocalization and Cell Expression of Lung Resistance-related Protein (LRP) in Normal and Tumoral Human Respiratory Cells

Lamiaâ Bouhamyia, Sandra Chantot-Bastaraud, Sakina Zaidi, Patricia Roynard, Claudie Prengel, Jean-François Bernaudin and Jocelyne Fleury-Feith

Service d'Histologie et Biologie Tumorale (LB,SZ,PR,CP,J-FB,JF-F) and Service d'Histologie-Cytogénétique (SC-B), Hôpital Tenon (AP-HP), Paris, France, and UPRES EA3499, Université Pierre et Marie Curie, Paris, France (LB,SZ,PR,CP,J-FB,JF-F)

Correspondence to: Dr. Jocelyne Fleury-Feith, Service d'Histologie et Biologie Tumorale, Hôpital Tenon, 4 rue de la chine, 75020, Paris, France. E-mail: jocelyne.fleury{at}tnn.aphp.fr

	Summary

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Lung resistance-related protein (LRP) is an integral part of the multidrug resistance (MDR) phenotype involved in cell resistance toward xenobiotics or chemotherapy. The aim of this study was to compare the intracellular localization and cell expression of LRP in normal bronchial cells and their tumoral counterparts from non-small cell lung cancer (NSCLC). LRP expression was also investigated concurrently with DNA ploidy and chromosome 16 (lrp gene locus) aberrations. Confocal microscopy showed that LRP localization was exclusively intracytoplasmic regardless of the cell type and was never observed in the nuclear pore complex. Flow cytometry demonstrated a similar level of LRP expression in normal bronchial cells and in cancer cells from NSCLC samples. FISH analysis, performed to evaluate the number of chromosome 16 and lrp loci, demonstrated a significant gain of chromosome 16 in DNA aneuploid tumors. Furthermore, we did not find any link between LRP expression and DNA ploidy status or chromosome 16 number. These results suggest that LRP expression observed in NSCLC, maintained through the carcinogenesis process of respiratory cells, is not altered by the increased number of copies of chromosome 16 and probably controlled by mechanisms different from those of MRP1 expression, whereas both proteins are associated with the MDR phenotype. (J Histochem Cytochem 55:773–782, 2007)

Key Words: lung resistance-related protein • bronchial cells • non-small cell lung cancer • DNA ploidy • flow cytometry • chromosome 16 • FISH

	Introduction

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MULTIDRUG RESISTANCE (MDR) phenotype is one major type of cell resistance toward xenobiotics for normal cells or chemotherapy for cancer cells. The MDR phenomenon is associated with a decrease in intracellular xenobiotics/drug accumulation and overexpression of transmembrane transporters such as P-gp170 (ABCB1/MDR1) and multidrug resistance-associated protein 1 (ABCC1/MRP1), both members of the ATP-binding cassette (ABC) protein transporter superfamily, acting as drug and/or xenobiotic efflux pumps (Cole et al. 1992; Lehnert 1996). However, as demonstrated in several MDR cell lines not overexpressing either Pgp or MRP1, additional mechanisms (Takeda et al. 1994) such as the lung resistance-related protein (LRP), which does not belong to the ABC transporter superfamily (Scheper et al. 1993), are also an integral part of the MDR phenotype.

LRP, initially described in non-small cell lung cancer (NSCLC) cell lines lacking P-gp170 (Kuiper et al. 1990; Scheper et al. 1993), is a 110-kDa protein encoded by the lrp gene localized on locus 16p11.2, proximal to the mrp1 gene on the short arm of chromosome 16 (Slovak et al. 1995). LRP has been found to be identical to the human major vault protein (MVP), which contributes to 70% of the mass of vault particles (Kedersha and Rome 1986; Scheffer et al. 1995). Vault particles are complex ribonucleoprotein particles that, in addition to the MVP, also contain two minor 193- and 240-kDa vault proteins and a small RNA, vRNA. Vault particles are arranged into hollow barrel flower-like structures measuring 35 x 65 nm with a nuclear mass of 13 MDa, with 8-fold symmetry, an invaginated waist, and two protruding caps (Kedersha et al. 1991). It has been calculated that each petal contains six copies of LRP and one vRNA molecule, for a total of 96 and 16 molecules, respectively, in each vault particle (Kedersha et al. 1991). Vaults are mainly cytoplasmic organelles, but some authors have reported that a small number of vaults (5%) are localized in the nuclear membrane and in the nuclear pore complex (NPC), supporting the hypothesis that vaults could act as shuttles between the nucleus and the cytoplasm (Chugani et al. 1993).

Respiratory airway cells continuously exposed to airborne pollutants, particularly genotoxic xenobiotics such as tobacco smoke, like all epithelial cells localized at the interface with the external environment are known to possess well-developed cell detoxification mechanisms, particularly the MDR phenotype (Dingemans et al. 1996). We, like others, have already explored the localization of two ABC transporters in airway tissues: P-gp170, a product of the mdr1 gene expressed in normal airways at the apical cell membrane of ciliated cells (Lechapt-Zalcman et al. 1997; Wioland et al. 2000), whereas MRP1 encoded by the mrp gene is expressed both at the basolateral cell membrane of ciliated and basal cells and at the intracellular level (Brechot et al. 1998; Wioland et al. 2000). However, few studies have precisely investigated the intracellular localization of LRP in normal respiratory cells (Berger et al. 2000).

Primary lung cancers, which arise from airway epithelial cells, are currently the leading cause of cancer deaths worldwide. They are classified into two main histological subtypes: small cell lung cancer (SCLC) and NSCLC, constituting 85% of all lung cancers. Tumor resistance in patients receiving chemotherapy alone or combined with radiotherapy represents a major problem in treatment of NSCLC (Rosell et al. 2002). As it can be hypothesized that intrinsic resistance of NSCLC could be derived from the cell detoxification mechanisms present in normal respiratory epithelial cells, comparative studies between normal respiratory cells and NSCLC cells would be worthwhile. Moreover, although LRP has been detected in lung cancer (Izquierdo et al. 1996a), its role in NSCLC is still a subject of controversy (Berger et al. 2000; Harada et al. 2003).

The present study was therefore designed first to precisely describe and compare the intracellular localization of LRP expression in normal bronchial cells and lung cancer cells from NSCLC samples and second to quantitatively compare the level of expression of LRP in these cells. Moreover, as a recent study from our laboratory showed that DNA aneuploid carcinomatous cells from NSCLC with an increased chromosome 16 number, harboring the mrp1 locus (16p13.1) close to the lrp locus (16p11.2), exhibited higher MRP1 expression than DNA diploid cells (Doubre et al. 2005), we therefore hypothesized that MRP1 and LRP could have a coordinate expression. We also investigated the potential role of the number of lrp gene copies on the level of LRP expression in NSCLC cancer cells.

	Materials and Methods

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Bronchial Epithelial Cells
Normal bronchial epithelial cells were recovered by bronchial brushings performed for diagnosis in patients suspected of having lung cancer. Bronchial samples (n=29) containing only cytologically normal epithelial cells were frozen in DMSO and stored at –80C until flow cytometry (seven samples), immunocytochemistry (four samples), confocal microscopy (11 samples), and FISH (seven samples). Samples were collected according to French legislation and the ethical rules of our institution at the time of experimentation.

Tumor Samples
NSCLC specimens, collected according to French legislation and the ethical rules of our institution at time of the experiment, were obtained from 14 patients after surgery performed in the Department of Thoracic Surgery (Dr. Bazelly; Hôpital Tenon, Paris, France). Tumor specimens were snap frozen in liquid nitrogen and stored at –80C. No patient had received chemotherapy before surgery.

Before tissue dissociation for flow cytometry, at least four sets of touch prints were systematically prepared, one stained by the May Grünwald Giemsa method for identification of carcinomatous cells, two others air dried overnight at room temperature and stored at –20C for immunocytochemistry and confocal microscopy, and one fixed with 3:1 methanol/acetic acid for 30 min and stored at –20C for FISH analysis. For flow cytometry, cells were mechanically dissociated from tissues by scraping of defrosted samples in 5% PBS–fetal calf serum (FCS). After PBS washes, aliquots of 4 x 10⁶ cells were stained for flow cytometry analysis.

A549 Cell Line
The NSCLC A549 cell line (American Type Culture Collection; Rockville, MD), known to spontaneously strongly express the LRP protein, was used for controls (Trussardi et al. 1998). A549 cells were expanded in DMEM (Invitrogen; Cergy Pontoise, France) supplemented with 10% (v/v) FCS (Invitrogen), 5 mM glutamine (Sigma; Saint Quentin Falavier, France), 50 U/ml penicillin–streptomycin (Sigma), and 20 µM Hepes (Invitrogen) at 37C in a humidified atmosphere containing 5% CO₂.

Antibodies and Reagents
Intracytoplasmic LRP was detected after incubation with monoclonal antibody (MAb) LRP-56 (Tebu-Bio; Le Perray en Yvelines, France) recognizing an intracellular epitope (Scheper et al. 1993) at different final working concentrations according to the method used. Incubation with a mouse IgG2b (Immunotech; Marseille, France) at the same protein concentration as MAb LRP-56 was used as the isotypic control. Secondary antibodies were a biotinylated rabbit anti-mouse F(ab')₂ (Dako; Trappes, France) for immunocytochemistry and a FITC-conjugated F(ab')₂ goat anti-mouse IgG (H&L; Immunotech) for confocal microscopy and flow cytometry.

To check the MAb LRP-56 specificity, immunoprecipitation was performed in five distinct experiments on cell lysates from A549 and two mesothelioma cell lines (BLA and CORO; kindly provided by Dr. M.C. Jaurand, INSERM U674, Paris, France). Approximately 10⁷ cells were processed as previously described (Meschini et al. 2002). Supernatants supplemented with 1% non-fat milk powder, precleared with 100 µl Protein G Sepharose (diluted 1:1 in PBS; Amersham Bioscience, Orsay, France) were spun at 10,000 x g for 10 min at 4C and incubated with 4 µg of undiluted MAb LRP-56 (100 µg/ml) or an equivalent amount of an irrelevant mouse IgG2b immunoglobulin for 2 hr at 4C. Immune complexes were precipitated by incubation with Protein G Sepharose (Amersham Bioscience), washed twice with lysis buffer containing 2% BSA and four times with PBS, taken up in 50 µl of sample buffer (200 mM Tris–HCl, pH 6.8, 1% 2-mercaptoethanol, 8% SDS, 10% glycerol, and 0.05% bromophenol blue), and microfuged. Supernatants were analyzed on 10% PAGE (125 V, 600 mA, 2 hr 30 min) after staining with Coomassie Brilliant Blue G (Sigma). After drying, gels were scanned with a model GS 800 (Bio-Rad; Marnes la Coquette, France). Molecular mass standards were obtained from Invitrogen.

Immunocytochemistry and Confocal Microscopy Analysis
Cytospin preparations of normal bronchial cells and A549 cells were prepared for immunocytochemistry, whereas touch prints were used for tumor cells from NSCLC samples, as previously described (Doubre et al. 2005). Slides were air dried overnight at room temperature, rehydrated with PBS for 5 min, and fixed by paraformaldehyde (PFA, 0.5% in PBS, pH 7.4, 25 min at room temperature). After two 10-min washes in PBS, cells were permeabilized with Triton X-100 0.3% in PBS, 30 sec at room temperature (Sigma), washed twice in PBS for 10 min, and incubated with the PBS/30% human AB serum saturation buffer (Abcys; Paris, France) for 30 min at room temperature before incubation with MAb LRP-56 or IgG2b.

For immunocytochemistry and light microscopy, cell preparations were incubated for 1 hr at room temperature with MAb LRP-56 (1/20; 5 µg/ml) or an equivalent amount of mouse IgG2b diluted in PBS/10% human AB serum and subsequently with biotinylated rabbit anti-mouse F(ab')₂ fragments (8.4 µg/ml) for 30 min at room temperature, followed by three 15-min washes in PBS. The conjugate was revealed using peroxidase-conjugated streptavidin (1/500, 30 min at room temperature) followed by three washes in PBS for 15 min (all reagents were obtained from Dako). Peroxidase activity was revealed with DAB (Dako), nuclei were counterstained with Harris hematoxylin (Bayer; Leverkusen, Germany), and the preparations were mounted with Eukitt (Kindler GmbH; Freiburg, Germany) and observed with a microscope (Zeiss Axioplan, Carl Zeiss, Jena, Germany) equipped with a color video camera (JVC KyF50).

For confocal microscopy, cell preparations were incubated for 1 hr with either MAb LRP-56 (1/5; 20 µg/ml) or the mouse IgG2b isotypic control (20 µg/ml). The slides were then incubated with a FITC-conjugated F(ab')₂ goat anti-mouse IgG (H&L) (0.014 mg/ml) for 30 min in the dark. Incubations with primary and secondary antibodies were performed at room temperature in PBS containing 10% human AB serum. Nuclei were counterstained with DRAQ-5 (1/200 in PBS; Biostatus, Leicestershire, UK) for 3 min at 37C. Centrifuge preparations were mounted with fluorescent mounting medium (Dako) and examined under a confocal laser-scanning microscope (Leica; Wetzlar, Germany). Cells were viewed in the X-Y or Y-Z plane, and the images were recorded.

For colocalization of LRP and actin, cells grown on Lab-Tek chamber slide system (ATGC; Marne la Vallee, France) with an 90% confluence were used. Cells were fixed for 15 min by PFA (1% in PBS, pH 7.4, 25 min at room temperature). After a PBS wash, cells were permeabilized with 0.3% Triton X-100 in PBS for 30 sec and washed three times for 10 min each with PBS. Cells were then incubated with tetramethylrhodamine B isothiocyanate-labeled phalloidin (1/200; Sigma) for 40 min. After three 10-min washes in PBS, cells were then incubated with either MAb LRP-56 (20 µg/ml) or the mouse IgG2b isotypic control (20 µg/ml). Further LRP staining procedures were performed as described above.

Flow Cytometry Analyses
Cells were double stained with MAb LRP-56 and propidium iodide (PI) to concomitantly evaluate the intracellular expression of LRP and DNA ploidy status. Cells were washed three times with PBS, and the pellet (5 x 10⁵ cells in 100 µl PBS) was fixed in 0.5% PFA in PBS for 10 min at room temperature. After centrifugation for 5 min at 1400 x g at 4C and blotting of residual PFA, cells were permeabilized by adding saponin (Intraprep kit 100 µl; Immunotech) for 5 min at room temperature. Cells were then incubated overnight at 4C either with MAb LRP-56 (4 µg/ml) or the isotypic control, a mouse IgG2b (4 µg/ml). After PBS washes and centrifugation, the cell pellet was incubated with a FITC-conjugated F(ab')₂ goat anti-mouse IgG (H&L) (0.028 mg/ml) for 30 min in the dark at room temperature. After one PBS wash, the cell pellet was suspended in 1 ml PI (50 ng/ml) containing RNase (DNA prep kit; Beckman Coulter, Villepinte, France) and kept in the dark at 37C for 30 min prior to flow cytometry analysis.

For flow cytometry analysis, samples were run on a Coulter EPICS Elite flow cytometer (Beckman Coulter) equipped with a single argon ion laser. Excitation was performed at 488 nm, and the emission filters used were 635 nm (red, PI) and 525 nm (green, FITC). At least 2 x 10⁴ cells per sample were analyzed, and data were stored in list mode files. LRP-positive cells were determined by reference to the negative level of the isotypic control and gated on dual parameter dot-plot LRP staining (green fluorescence) against side scatter (excluding cells with a high level of autofluorescence, such as macrophages).

Mean fluorescence index (MFI) was defined as the ratio of the mean fluorescence intensity obtained after incubation with MAb LRP-56 and after incubation with the isotypic control. This index was used for semiquantitative evaluation of LRP cell expression in cell types and to compare the levels of LRP expression in DNA diploid vs DNA aneuploid cells, as previously described (Doubre et al. 2005). DNA ploidy and S-phase data were analyzed using Wincycle software (Phoenix; San Diego, CA). DNA ploidy was analyzed according to the guidelines of the 1992 Cytometry Consensus Conference (Shankey et al. 1993). DNA ploidy was classified using a DNA index (DI), i.e., the ratio of the channel peaks between the studied cell population and the diploid cells. A sample was defined as DNA diploid when all the cell population has a DI between 0.95 and 1.05 and DNA aneuploid when at least one cell population with DI 0.95 or 1.05 was observed in at least 5% of the cells (Doubre et al. 2005).

This analysis was performed on bronchial cells (seven samples) and on tumor cells obtained from NSCLC (14 samples), as well as on A549 cell line (from three different experiments) for control.

FISH
Dual-color FISH analysis on interphasic nuclei was performed with a mixture of spectrum green-labeled lrp gene probe and a rhodamine-labeled chromosome 16 -satellite centromere probe (Amplitech; Compiegne, France), which allowed simultaneous determination of a number of lrp gene copies and chromosome 16. The bacterial artificial chromosome (BAC clone RP11-279M12 from the RP11 human BAC clone library mapping to 16p11.2 at lrp locus; Wellcome Trust Sanger Institute, Cambridge, UK) was used for lrp gene hybridization. Isolation and purification of DNA were performed using the Qiagen Hispeed Plasmid Maxi Kit (Qiagen; Courtaboeuf, France) according to the manufacturer's instructions. BAC DNA was labeled with spectrum green dUTP by nick translation (Vysis; Maurence Scopont, France). This probe was purified by ethanol precipitation and dissolved in 1/10 TE buffer, pH 8.

Dual-color FISH was performed on cytospin preparations of normal bronchial cells (seven samples) and A549 tumor cell line and on touch imprints from 14 NSCLC samples. Cells were fixed and incubated with the mixture of directly labeled lrp gene probe (spectrum green) and a directly labeled chromosome 16 centromeric probe (rhodamine) [1 µl of chromosome 16 -satellite centromere probe and 5 µl of BAC DNA (50 ng/µl) in 4 µl of LSI hybridization buffer (Vysis)].

Briefly, slides were treated in a pepsin solution (50 ng/ml, 0.01 N HCl; Sigma) for 15 min at 37C; dehydrated in a chilled alcohol series of 70%, 80%, and 100% for 2 min each; and hybridized with 10 µl of the hybridization mixture applied onto the slide. Denaturation was then performed at 72C for 2 min. Hybridization was performed overnight in a dark moist chamber at 37C. Slides were subsequently washed in 0.4X SSC at 73C for 2 min and in 2X SSC, 1% Tween 20, pH 7, at room temperature for 2 min. Nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI) in Vectashield (Abcys).

Slides were examined with a DM400B Leica epifluorescence microscope equipped with filter sets optimized for DAPI, spectrum green, and rhodamine. Images were captured with a Zeiss microscope and a cooled CCD camera using Genikon Imaging System (Alphelys; Plaisir, France). Same-colored signals were counted as two if they were separated by at least one signal diameter and had the same intensity, size, and shape. Hybridization signals were scored in at least 100 intact non-overlapping nuclei.

Statistical Analysis
Results are expressed as mean ± SD. Statistical analysis was performed with Statview Software (Abacus Concept; Berkeley, CA). Non-parametric tests were applied for the various data comparisons: a paired test (Wilcoxon) to compare values from individual specimens and a non-paired test (Mann-Whitney). Statistical significance was estimated when p value 0.05.

	Results

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Immunoprecipitation With MAb LRP-56
Immunoprecipitation results of cell lysates from cell lines with MAb LRP-56 are shown in Figure 1 . A band at the expected size of LRP, i.e., 110 kDa (Scheper et al. 1993; Izquierdo et al. 1996a) was clearly demonstrated after precipitation with MAb LRP-56 and was absent after precipitation with the mouse IgG2b immunoglobulin. These results confirmed the specificity of MAb LRP-56 to detect human LRP, as already reported (Scheper et al. 1993; Izquierdo et al. 1996a).

View larger version (21K): [in this window] [in a new window]   Figure 1 Immunoprecipitation with monoclonal antibody (MAb) LRP-56. Immunoprecipitation of BLA, CORO, and A549 cell lysates using MAb lung resistance-related protein (LRP)-56. Lysates were immunoprecipitated as described in Materials and Methods with LRP-56 or a mouse IgG2b. A 110-kDa band is observed in the cell lysates from the three cell lines. No band was detected after precipitation with the mouse IgG2b.

View larger version (130K): [in this window] [in a new window]   Figure 2 Immunodetection of LRP by light microscopy. Immunostaining of LRP with MAb LRP-56 (5 µg/ml) using immunoperoxidase in normal bronchial epithelial cells (A), A549 cell line (B), and tumor cells from NSCLC (C). Left column: isotypic controls; right column: MAb LRP-56. Cells were counterstained with hematoxylin. Note that the brown staining indicating LRP inside the cytoplasm has a granular pattern in the various cell types. Bar = 40 µm.

View larger version (101K): [in this window] [in a new window]   Figure 3 Confocal microscopy of LRP in normal bronchial epithelial cells, non-small cell lung cancer (NSCLC), and A549 cells. Illustrations of indirect immunofluorescence analyzed on horizontal (x-y axis) optical sections after incubation with MAb LRP-56. (A) Normal bronchial epithelial cells. (B) DNA aneuploid NSCLC. LRP (in green) is present in the cytoplasm (middle column), nuclei are stained with DRAQ-5 (left column), and overlays are shown in the right column. No perinuclear reinforcement or nuclear pore complex staining was observed. (C) A549 cells cultured on chamber slides. Actin microfilaments are labeled by TRITC phalloidin (red), nuclei by DRAQ-5 (blue), and overlay is shown in the right column. Double staining of LRP and actin confirmed the cytoplasmic distribution of LRP not colocalized with actin microfilaments. Bars: A,B = 20 µm; C = 40 µm; Inset in A = 40 µm.

View larger version (16K): [in this window] [in a new window]   Figure 4 Mean fluorescence index (MFI) of LRP in various cells analyzed by flow cytometry. (Left panel) MFI of LRP (mean ± SD) evaluated on seven normal bronchial cells samples, 14 tumor cells from NSCLC, and the A549 tumor cell line. No significant differences (NS) in the levels of LRP protein expression were detected between normal bronchial cells, the A549 tumor cell line, and tumor cells from NSCLC. (Right panel) LRP MFI (mean ± SD) evaluated on cells obtained from three DNA diploid and 11 DNA aneuploid tumor samples. Columns on the right show results restricted to the DNA diploid and DNA aneuploid cell subpopulations from DNA aneuploid tumors. No significant difference (NS) in LRP protein expression was detected between DNA diploid tumors and DNA aneuploid tumors or between DNA diploid and DNA aneuploid cell subpopulations from DNA aneuploid tumors.

FISH Analysis
FISH analysis of chromosome 16 and lrp gene loci performed on normal bronchial cells, A549 cell line, and tumor imprints was correlated with DNA ploidy (Figure 5 ; Table 1). After dual-color FISH of interphasic nuclei with chromosome 16 -satellite centromere probe and BAC DNA clone RP11-279M12, the number of copies of lrp gene loci and chromosome 16 centromere per interphasic nucleus was always similar, regardless of the cell type studied.

View larger version (52K): [in this window] [in a new window]   Figure 5 Examples of FISH results after cohybridization of interphasic nuclei with a chromosome 16 centromeric probe (red signals) and BAC DNA clone RP11-279M12 (lrp gene on 16p11.2, green signals). (A) Normal bronchial cells. (B) A549 cells. Normal bronchial cells were disomic for chromosome 16 (two red spots), but A549 cells were trisomic (three red spots). (C) DNA diploid tumors (the majority of cells were chromosome 16 disomic, two red spots). (D) DNA aneuploid tumors (chromosome 16 aneusomic, three red spots). In all samples analyzed, the number of lrp gene locus (green spots) copies is the same as the number of chromosomes 16 (red spots). Bar = 20 µm.

Among the 14 samples from NSCLC, 3 tumors were DNA diploid and 11 tumors DNA aneuploid. In DNA diploid tumors, an average of 75.7 ± 38.2% of tumor cells exhibited two signals for chromosome 16. In contrast, intracellular variations in the chromosome 16 copy number were observed in DNA aneuploid tumors with an average of 68 ± 24.1% of tumor cells with more than two copies of chromosome 16 (Figure 5 and Figure 6 ; Table 1). The most frequent anomalies were three or more than three copies of chromosome 16.

View larger version (12K): [in this window] [in a new window]   Figure 6 Distribution of the chromosome 16 and lrp gene copy numbers per 100 cells (mean ± SD) observed after FISH analysis in relation to DNA ploidy status in seven samples of normal bronchial cells, three samples of A549 cells, and in three DNA diploid and 11 DNA aneuploid tumor samples.

	Discussion

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The second step of this study was to use a flow cytometry method for semiquantitative evaluation of LRP expression. Most of the studies concerning LRP expression in solid tumors were based on immunohistochemistry using a score evaluation (Dingemans et al. 1996; Volm et al. 1997) or reverse transcription–polymerase chain reaction (RT-PCR) evaluating LRP mRNA (Ohno et al. 2001), whereas the majority of studies on hematological malignancies used flow cytometry for LRP detection (Damiani et al. 2004). Moreover, this method has not been previously used to compare LRP expression in normal and carcinomatous cells from the same tissues. We previously reported the use of such a method for evaluation of MRP1 expression in normal bronchial and NSCLC cells (Doubre et al. 2005). LRP expression was found in 100% of NSCLC cells, in line with the results reported by Berger et al. (2000) who evaluated LRP expression by mRNA detection. However, the rate of LRP-positive cells evaluated by flow cytometry is higher than that previously reported for LRP expression evaluated by immunohistochemistry (Dingemans et al. 1996; Volm et al. 1997; Harada et al. 2003; Raybarova et al. 2004). This difference can probably be explained by a higher sensitivity of flow cytometry analysis compared with immunohistochemistry (Leers et al. 2002). Similarly, LRP was detected in bronchial epithelial cells in our study as in other studies (Nooter et al. 1995), suggesting a role of LRP in the cell detoxification process for cells localized at the air–tissue interface (Dingemans et al. 1996; Izquierdo et al. 1996a) where detoxification mechanisms are particularly well developed. A similar level of LRP expression was observed in bronchial cells and carcinomatous cells from NSCLC samples, similar to the A549 cell line known to spontaneously have a high level of LRP expression (Trussardi et al. 1998). This is at variance with observations in the colon, where LRP is expressed in the surface epithelium and upper part of the crypts of normal colorectal epithelium (Dingemans et al. 1996; Meijer et al. 1999), but a definitive and abrupt increase of LRP levels is observed early in the process of colorectal carcinogenesis, from normal epithelium to adenoma and from adenoma to carcinoma (Meijer et al. 1999). Some types of carcinomatous cells have been reported to express significantly higher levels of LRP than their normal counterparts (e.g., thyroid carcinoma), whereas LRP expression appears to be downregulated during malignant transformation in other tumors (e.g., germ-cell tumors) (Izquierdo et al. 1996a). These results suggest that a similar level of LRP expression was maintained in epithelial respiratory cells throughout the carcinogenesis process.

The hypothesized role of LRP in resistance to chemotherapy is still a subject of controversy. However, the role of LRP as a prognosis factor in cancer is probably not restricted to the MDR phenotype, MVP being associated with several cellular processes also involved in cancer development like cell proliferation, motility, differentiation, or cell signaling (Steiner et al. 2006). Clinically, LRP overexpression has been associated with poor responses to chemotherapy in different malignancies such as NSCLC (Izquierdo et al. 1995; List et al. 1996; Volm et al. 1997; Raaijmakers et al. 1998). Similarly, LRP has been shown to be overexpressed in a subgroup of cisplatin-resistant NSCLC cell lines (Berger et al. 2000), and LRP has recently been reported to be a predictive marker for treatment response in NSCLC and testicular germ-cell tumors (Harada et al. 2003; Mandoky et al. 2004). However, other studies failed to detect such an association (Dingemans et al. 1996), and another study showed that LRP expression in NSCLC was not enhanced in patients treated with chemotherapy (Berger et al. 2005). However, it has been shown that the other lung cancer subtype, SCLC, which is initially very sensitive to chemotherapy, displays very low LRP expression (Dingemans et al. 1996).

We recently showed that DNA aneuploid NSCLC carcinomatous cells with an increase in chromosome 16 number exhibited a higher MRP1 expression than DNA diploid cells (Doubre et al. 2005). As the lrp gene is localized on the short arm of chromosome 16 (16p11.2), 27 centimorgan or an estimated 18 megabases proximal to the mrp1 gene (16p13.1), we tested the hypothesis that LRP and MRP1 could both be concomitantly overexpressed in the case of chromosome 16 polysomy. However, we found a similar level of LRP expression in DNA diploid and DNA aneuploid NSCLC cells as in normal bronchial epithelial cells, regardless of the chromosome 16 number. This dissociation between LRP and MRP1 expression is at variance with the results described in ovarian carcinoma (Yakirevich et al. 2006). However, several studies in leukemia and unselected human cancer cell lines (Izquierdo et al. 1996b; Michieli et al. 1997) have provided evidence that MRP and LRP expression are not coregulated. In our study, the increase of chromosome 16 copies was associated with DNA aneuploidy but not with an increase in the level of LRP protein expression. Similarly, differences in the expression of LRP mRNA and protein have been described in other studies (Laurencot et al. 1997), suggesting a strong posttranscriptional regulation of LRP expression. Our results do not support the gene dosage hypothesis for LRP expression control. Moreover, no lrp overexpression was observed in NSCLC cells, in line with the results reported by Berger et al. (2005). These results suggest that the LRP expression observed in NSCLC is controlled by mechanisms different from those involved in MRP1 expression, whereas both proteins are associated with the MDR phenotype.

In conclusion, in the present study LRP expression, which is associated with a ubiquitous role of vaults in cell detoxification processes, was found in NSCLC carcinomatous cells from tumor samples similar to that observed in normal bronchial epithelial cells. No alteration of the LRP intracellular localization and/or level of expression was observed when considering the normal and carcinomatous subsets of cells from the same tissue, i.e., respiratory airways. Moreover, on the basis of these results, we can therefore hypothesize that the level of LRP expression observed in NSCLC is maintained through the carcinogenesis process of respiratory cells and not altered by the increased number of copies of chromosome 16 catalyzed by cell aneuploidy.

	Acknowledgments

 
This study was supported by "Legs POIX" 2005 and Amis des Centres des Tumeurs de Tenon.

We thank the Department of Thoracic Surgery, particularly Dr. B. Bazelly, and the Department of Pathology (Dr. M. Antoine) for the generous gifts of the NSCLC specimens, and the Department of Respiratory Medicine (Prof. C. Mayaud) for normal bronchial epithelial cells. We are grateful to Prof. J.P. Siffroi, A. Leneveu, S. Giacuzzo, and S. Lucchini for their technical assistance in cytogenetics.

	Footnotes

 
Received for publication January 1, 2007; accepted March 22, 2007

	Literature Cited

Top Summary Introduction Materials and Methods Results Discussion Literature Cited

 

Berger W, Elbling L, Micksche M (2000) Expression of the major vault protein LRP in human non-small-cell lung cancer cells: activation by short-term exposure to antineoplastic drugs. Int J Cancer 88:293–300[CrossRef][Medline]

Berger W, Setinek U, Hollaus P, Zidek T, Steiner E, Elbling L, Cantonati H, et al. (2005) Multidrug resistance markers P-glycoprotein, multidrug resistance protein 1, and lung resistance protein in non-small cell lung cancer: prognostic implications. J Cancer Res Clin Oncol 131:355–363[CrossRef][Medline]

Brechot JM, Hurbain I, Fajac A, Daty N, Bernaudin JF (1998) Different pattern of MRP localization in ciliated and basal cells from human bronchial epithelium. J Histochem Cytochem 46:513–517[Abstract/Free Full Text]

Chugani DC, Rome LH, Kedersha NL (1993) Evidence that vault ribonucleoprotein particles localize to the nuclear pore complex. J Cell Sci 106:23–29[Abstract]

Cole SP, Bhardwaj G, Gerlach JH, Mackie JE, Grant CE, Almquist KC, Stewart AJ, et al. (1992) Overexpression of a transporter gene in a multidrug- resistant human lung cancer cell line. Science 258:1650–1654[Abstract/Free Full Text]

Damiani D, Michieli M, Michelutti A, Candoni A, Stocchi R, Masolini P, Geromin A, et al. (2004) Antibody binding capacity for evaluation of MDR-related protein in acute promyelocytic leukemia: onset versus relapse expression. Cytometry B Clin Cytom 59:40–45[Medline]

Dingemans A, van Ark-Otte J, van der Valk P, Apolinario RM, Scheper RJ, Postmus PE, Giaccone G (1996) Expression of the human major vault protein LRP in human lung cancer samples and normal lung tissues. Ann Oncol 7:625–630[Abstract/Free Full Text]

Doubre H, Cesari D, Mairovitz A, Benac C, Chantot-Bastaraud S, Dagnon K, Antoine M, et al. (2005) Multidrug resistance-associated protein (MRP1) is overexpressed in DNA aneuploid carcinomatous cells in non-small cell lung cancer (NSCLC). Int J Cancer 113:568–574[CrossRef][Medline]

Hamill DR, Suprenant KA (1997) Characterisation of the sea urchin major vault protein: a possible role for the vault ribonucleoprotein particles in nucleocytoplasmic transport. Dev Biol 190:117–128[CrossRef][Medline]

Harada T, Ogura S, Yamazaki K, Kinoshita I, Itoh T, Isobe H, Yamashiro K, et al. (2003) Predictive value of expression of P53, Bcl2 and lung resistance-related protein for response to chemotherapy in non-small cell lung cancers. Cancer Sci 94:394–399[CrossRef][Medline]

Herrmann C, Golkaramnay E, Inman E, Rome L, Volknandt W (1999) Recombinant major vault protein is targeted to neuritic tips of PC12 cells. J Cell Biol 144:1163–1172[Abstract/Free Full Text]

Izquierdo MA, Scheffer GL, Flens MJ, Giaccone G, Broxterman HJ, Meijer CJ, van der Valk P, et al. (1996a) Broad distribution of the multidrug resistance-related vault lung protein in normal human tissues and tumors. Am J Pathol 148:877–887[Abstract]

Izquierdo MA, Shoemaker RH, Flens MJ, Scheffer GL, Wu L, Prather TR, Scheper RJ (1996b) Overlapping phenotypes of multidrug resistance among panels of human cancer-cell lines. Int J Cancer 65:230–237[CrossRef][Medline]

Izquierdo MA, van der Zee AG, Vermorken JB, van der Valk P, Beliën JA, Giaccone G, Scheffer GL, et al. (1995) Drug resistance-associated marker LRP for prediction of response to chemotherapy and prognoses in advanced ovarian cancer. J Natl Cancer Inst 87:1230–1237[Abstract/Free Full Text]

Kedersha NL, Heuser JE, Chugani DC, Rome LH (1991) Vaults. III. Vault ribonucleoprotein particles open into flower-like structures with octagonal symmetry. J Cell Biol 112:225–235[Abstract/Free Full Text]

Kedersha NL, Rome LH (1986) Isolation and characterization of a novel ribonucleoprotein particle: large structures contain a single species of small RNA. J Cell Biol 103:699–709[Abstract/Free Full Text]

Kuiper CM, Broxterman HJ, Baas F, Schuurhuis GJ, Haisma HJ, Scheffer GL, Lankelma J, et al. (1990) Drug transport variants without P-glycoprotein overexpression from a human squamous lung cancer cell line after selection with doxorubicin. J Cell Pharmacol 1:35–41

Laurencot CM, Scheffer GL, Scheper RJ, Shoemaker RH (1997) Increased LRP mRNA expression is associated with the MDR phenotype in intrinsically resistant human cancer cell lines. Int J Cancer 72:1021–1026[CrossRef][Medline]

Lechapt-Zalcman E, Hurbain I, Lacave R, Commo F, Urban T, Antoine M, Milleron B, et al. (1997) MDR1-Pgp 170 expression in human bronchus. Eur Respir J 10:1837–1843[Abstract]

Leers MP, Schoffelen RH, Hoop JG, Theunissen PH, Oosterhuis JW, vd Bijl H, Rahmy A, et al. (2002) Multiparameter flow cytometry as a tool for the detection of micrometastatic tumor cells in the sentinel lymph node procedure of patients with breast cancer. J Clin Pathol 55:359–366[Abstract/Free Full Text]

Lehnert M (1996) Clinical multidrug resistance in cancer: a multifactorial problem. Eur J Cancer 32A:912–920[CrossRef]

List AF, Spier CS, Grogan TM, Johnson C, Roe DJ, Greer JP, Wolff SN, et al. (1996) Overexpression of the major vault transporter protein lung-resistance protein predicts treatment outcome in acute myeloid leukemia. Blood 87:2464–2469[Abstract/Free Full Text]

Mandoky L, Geczi L, Doleschall Z, Bodrogi I, Csuka O, Kasler M, Brak M (2004) Expression and prognostic value of the lung resistance-related protein (LRP) in germ cell testicular tumors. Anticancer Res 24:1097–1104[Medline]

Meijer GA, Schroeijers AB, Flens MJ, Meuwissen SG, van der Valk P, Baak JP, Scheper RJ (1999) Increased expression of multidrug resistance related proteins Pgp, MRP1, and LRP/MVP occurs early in colorectal carcinogenesis. J Clin Pathol 52:450–454[Abstract]

Meschini S, Marra M, Calcabrini A, Monti E, Gariboldi M, Dolfini E, Arancia G (2002) Role of the lung resistance-related protein (LRP) in the drug sensitivity of cultured tumor cells. Toxicol In Vitro 16:389–398[CrossRef][Medline]

Michieli M, Damiani D, Ermacora A, Raspadori D, Michelutti A, Grimaz S, Fanin R, et al. (1997) P-glycoprotein (PGP) and lung resistance-related protein (LRP) expression and function in leukaemic blast cells. Br J Haematol 96:356–365[CrossRef][Medline]

Nooter K, Westerman AM, Flens MJ, Zaman GJR, Scheper RJ, van Wingerden KE, Burger H, et al. (1995) Expression of the multidrug resistance-associated protein (MRP) gene in human cancers. Clin Cancer Res 1:1301–1310[Abstract]

Ohno N, Tani A, Uozumi K, Hanada S, Furukawa T, Akiba S, Sumizawa T, et al. (2001) Expression of functional lung resistance-related protein predicts poor outcome in adult T-cell leukemia. Blood 98:1160–1165[Abstract/Free Full Text]

Raaijmakers HG, Izquierdo MA, Lokhorst HM, de Leeuw C, Belien JA, Bloem AC, Dekker AW, et al. (1998) Lung resistance-related protein expression is a negative predictive factor for response to conventional low but not to intensified dose alkylating chemotherapy in multiple myeloma. Blood 91:1029–1036[Abstract/Free Full Text]

Raybarova S, Hajdukova M, Hodorova I, Kocisova M, Boor A, Brabencova E, Kasan P, et al. (2004) Expression of the multidrug resistance-associated protein 1 (MRP1) and the lung resistance-related protein (LRP) in human lung cancer. Neoplasma 51:169–174[Medline]

Rosell R, Monzo M, Alberola V, Taron M, Barnadas A, Sanchez JM, Manzano JL, et al. (2002) Determinants of response and resistance to cytotoxics. Semin Oncol 29:110–118[CrossRef][Medline]

Scheffer GL, Wijingaard PLJ, Flens MJ, Izquierdo MA, Slovak ML, Pinedo HM, Meijer CJ, et al. (1995) The drug resistance-related protein LRP is the human major vault protein. Nat Med 1:578–582[CrossRef][Medline]

Scheper RJ, Broxterman HJ, Scheffer GL, Kaaijk P, Dalton WS, van Heijningen TH, van Kalken CK, et al. (1993) Overexpression of a Mr 110,000 vesicular protein in-non-P-glycoprotein-mediated multidrug resistance. Cancer Res 53:1475–1479[Abstract/Free Full Text]

Shankey TV, Rabinovitch PS, Bagwell B, Bauer KD, Duque RE, Hedley DW, Mayall BH, et al. (1993) Guidelines for implementation of clinical DNA cytometry. International Society for Analytical Cytology. Cytometry14:472–477[CrossRef][Medline]

Slesina M, Inman EM, Rome LH, Volknandt W (2005) Nuclear localization of the major vault protein in U373 cells. Cell Tissue Res 321:97–104[CrossRef][Medline]

Slovak ML, Ho JP, Cole SP, Deeley RG, Greenberger L, de Vries EG, Broxterman HJ, et al. (1995) The LRP gene encoding a major vault protein associated with drug resistance maps proximal to MRP on chromosome 16: evidence that chromosome breakage plays a key role in MRP or LRP gene amplification. Cancer Res 55:4214–4219[Abstract/Free Full Text]

Steiner E, Holzmann K, Elbling L, Micksche M, Berger W (2006) Cellular functions of vaults and their involvement in multidrug resistance. Curr Drug Targets 7:923–934[CrossRef][Medline]

Takeda Y, Nishio K, Niitani H, Saijo N (1994) Reversal of multidrug resistance by tyrosine-kinase inhibitors in a non-P-glycoprotein-mediated multidrug-resistant cell line. Int J Cancer 57:229–239[CrossRef][Medline]

Trussardi A, Poitevin G, Gorisse MC, Faroux MJ, Bobichon H, Delvincourt C, Jardillier JC (1998) Sequential overexpression of LRP and MRP but not P-gp 170 in VP16-selected A549 adenocarcinoma cells. Int J Oncol 13:543–548[Medline]

van Zon A, Mossink MH, Houtsmuller AB, Schoester M, Scheffer GL, Scheper RJ, Sonneveld P, et al. (2006) Vault mobility depends in part on microtubules and vaults can be recruited to the nuclear envelope. Exp Cell Res 312:245–255[Medline]

Volm M, Mattern J, Koomagi R (1997) Expression of lung resistance-related protein (LRP) in non-small cell lung carcinomas of smokers and non-smokers and its predictive value for doxorubicin resistance. Anticancer Drugs 8:931–936[Medline]

Wioland MA, Fleury-Feith J, Corlieu P, Commo F, Monceaux G, Lacau-St-Guily J, Bernaudin JF (2000) CFTR, MDR1, and MRP1 immunolocalization in normal human nasal respiratory mucosa. J Histochem Cytochem 48:1215–1222[Abstract/Free Full Text]

Yakirevich E, Sabo E, Naroditsky I, Sova Y, Lavie O, Resnick MB (2006) Multidrug resistance-related phenotype and apoptosis-related protein expression in ovarian serous carcinomas. Gynecol Oncol 100:152–159[CrossRef][Medline]

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