ARTICLE |
Correspondence to: Rik J. Scheper, Academic Hospital Vrije Universiteit, Dept. of Pathology, PO Box 7057, 1007 MB Amsterdam, The Netherlands. E-mail: rj.scheper@vumc.nl
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Summary |
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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.
(J Histochem Cytochem 49:13791385, 2001)
Key Words: lung resistance-related protein, (LRP), major vault protein (MVP), vault, multidrug resistance (MDR), monoclonal antibody (MAb)
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Introduction |
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MULTIDRUG RESISTANCE (MDR) is the major cause of chemotherapeutic failure in cancer treatment (
The discovery of a key role of vault-related MDR in clinical drug resistance depended on the molecular identification of the lung resistance-related protein (LRP) as the human MVP (
Antibodies that specifically recognize LRP/MVP are important in the fundamental and clinical analysis of vault-related MDR. They have been used for the characterization of the LRP/MVP protein (
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Materials and Methods |
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Cells and Cell Culture
The following cell lines were used in this study: the small-cell lung carcinoma cell line GLC4 (low LRP/MVP expression) and its doxorubicin-selected partner GLC4/ADR (1152 nM; high LRP/MVP expression) (97% LRP/MVP-negative/
3% high LRP/ MVP expression) (
Immunization and Generation of Hybridomas
Male Balb/c mice (n=3) received footpad injections of 5 µg of full-length recombinant human LRP/MVP (
Screening, Cloning, and Isotyping
After 14 days of growth in selective medium, hybridoma supernatants were tested for the presence of antibodies of interest by ELISA. Plates (96-well) were coated with 5 µg/ml of recombinant LRP/MVP or, as a control, 2 µg/ml of E. coli bacterial proteins (DH10B). Four hybridomas secreting antibodies of interest were subcloned three times by limiting dilution. Immunoglobulin subtypes of the MAbs produced by the stable hybridoma clones obtained were determined using an isotype reagent kit (Boehringer Mannheim; Indianapolis, IN). For large-scale antibody production, hybridomas were cultured in 1.5 liters of growth medium containing 1% (v/v) Nutridoma serum replacement (Boehringer Mannheim; Mannheim, Germany) in 750-cm3 tissue culture flasks. After 10 days, supernatants were harvested and concentrated in ST25 capillary flow dialyzers (Travenol AG; Baxter, Germany).
Monoclonal Antibodies
LRP/MVP expression was studied with the four newly isolated MAbs (see above;
Immunocyto- and Histochemistry
Cytocentrifuge preparations of tumor cell lines were air-dried, fixed in acetone for 5 min, -20C methanol for 10 min, or 4% paraformaldehyde for 10 min. To study reactivity of normal human, rat, mouse, and guinea pig tissues with the LRP/MVP MAbs, cryostat sections of the tissue samples were cut at 5 µm thick and air-dried directly before staining. Sections of normal human tissues 5 µm thick were also obtained from tissue blocks fixed in 4% neutral formalin and embedded in paraffin. Histologically normal adult tissues (lung, salivary gland, kidney, and colon) derived from autopsy specimens were obtained from our tissue bank. The cytocentrifuge preparations and tissue sections were incubated with primary antibodies (60 min at room temperature or overnight at 4C; 30 min at 37C in combination with guanidine hydrochloride pretreatment; see below), followed by incubation with biotinylated rabbit anti-mouse F[ab']2 fragments (Dako, Copenhagen, Denmark; 1:500, 60 min) or rabbit anti-rat immunoglobulins (1:100; Dako), and streptavidinHRP (Zymed Laboratories; San Francisco, CA; 1:500, 30 min). Bound peroxidase was visualized with 4 mg (w/v) 3-amino-9-ethylcarbazole and 0.02% (v/v) H2O2 in 0.1 M NaAc (pH 5.0) or 4 mg (w/v) 3,3'-diaminobenzidine tetrahydrochloride and 0.02% (v/v) H2O2 in PBS, nuclei were counterstained with hematoxylin, and the tissue preparations were mounted.
Enzymic Digestion and Detergent Treatment
Sections were treated with the following digestion and detergent treatments: 6 N guanidine hydrochloride (GdnHCl) in 50 mM Tris-HCl, pH 7.5, for 10 min (
Protein Blot Analysis
Extracts were prepared from GLC4/ADR cells by the following procedure. Cells were harvested, and resuspended in cold Buffer A [50 mM Tris-Cl (pH 7.4), 1.5 mM MgCl2, 75 mM NaCl] containing 0.5% Nonidet P-40 and 1 mM phenylmethylsulfonyl fluoride. All subsequent steps were performed at 4C. Samples were vortexed, incubated on ice for 5 min, and centrifuged at 9000 x g for 20 min. The resulting supernatant was designated the postnuclear supernatant. Protein concentration was determined with a Bio-Rad protein assay (Bio-Rad; Richmond, CA). Postnuclear supernatant samples containing 40 µg of protein were fractionated by SDS/10% PAGE and transferred to a nitrocellulose filter by electroblotting. After blotting, the filters were blocked for at least 2 hr in block buffer [PBS containing 1% (w/v) bovine serum albumin, 1% (w/v) milk powder, and 0.05% (v/v) Tween 20], followed by a 2 h incubation with the primary antibodies in block buffer. Immunoreactivity was visualized with peroxidase-conjugated rabbit anti-mouse or anti-rat immunoglobulins (Dako) in block buffer, followed by staining with 0.05% 4-chloro-1-naphthol and 0.03% H2O2 in PBS.
Immunoprecipitation
GLC4/ADR cells were used in the immunoprecipitation assays. Aliquots of the postnuclear supernatant (prepared as described above) containing 750 µg of protein were brought up to 500 µl and incubated for at least 2 hr at 4C with 8 µg of MAb LRP56 or irrelevant MAb. Antibodyantigen complexes were recovered by incubation with 14% (w/v) protein ASepharose CL-4B (Pharmacia Biotech; Woerden, The Netherlands). Precipitated proteins were shown by immunoblotting (as described above).
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Results |
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Monoclonal Antibody Production
Using lymph nodes from mice immunized with full-length human recombinant LRP/MVP, murine hybridomas were generated and screened for their ability to detect the immunization antigen in ELISA. In the end, four stable cloned hybridoma cell lines, designated MVP-9, MVP-16, MVP-18, and MVP-37, were obtained. MAbs MVP-9 and MVP-18 were determined to be of the IgG1 subclass; MAbs MVP-16 and MVP-37 were both IgG2b.
To confirm the LRP/MVP specificity of these MAbs, immunoblotting analyses on immunoprecipitated LRP/MVP were carried out. The LRP/MVP protein was immunoprecipitated from the small-cell lung cancer cell line GLC4/ADR with MAb LRP-56, using protein A to bind immune complexes. MAbs MVP-9, MVP-16, MVP-18, and MVP-37 all detected the precipitated Mr 110,000 LRP/MVP protein (Fig 1). No immunoreactivity was observed when the immunoprecipitation was carried out with a control MAb. In those instances, as expected, the LRP/MVP protein was detected in the corresponding supernatants.
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As reported earlier (
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Protein Blotting Analysis
Fig 2 shows the immunoreactivity of all MAbs with postnuclear supernatant from LRP/MVP-overexpressing GLC4/ADR cells. Indeed, no immunoreactivity was found in Western blots, whereas LRP/MVP was easily detectable using the newly isolated MAbs MVP-9, MVP-16, MVP-18, and MVP-37. In addition, the MAb LRP from Transduction Laboratories clearly detects the LRP/MVP in accordance with the accompanying production sheet. The two MAbs 1027 and 1032, selected on protein blotting immunoreactivity by
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Immunoblotting methods offer the assurance of specificity but are difficult to apply to all clinical samples because they are time-consuming and require large samples. To be widely useful for the detection of LRP/MVP in many experimental applications and in the analysis of clinical samples, it is important that LRP/MVP-specific MAbs are able to recognize LRP/MVP epitopes in fixed cells and tissues. For this reason, labeling of tumor cell lines was examined by immunocytochemistry. In addition, immunohistochemical studies of certain frozen and formalin-fixed, paraffin-embedded tissues were performed.
Immunocytochemistry
The immunoreactivity of the anti-LRP/MVP MAbs was tested on cytocentrifuge preparations of a small panel of human parental, sensitive, and drug-selected MDR cell lines that express the LRP/MVP protein at different levels, varying from negative/low (SW-1573/2R160, GLC4) to high (SW-1573/2R120, GLC4/ADR) LRP/MVP expression. To detect LRP/MVP with the best sensitivity, several modalities of conditional circumstances such as fixation and pretreatment methods, primary antibody concentration, incubation time, and temperature were evaluated. The optimal staining conditions, i.e., resulting in the best overall quality (intensity, specificity, morphology) of the staining results with the anti-LRP/MVP MAbs on cytospin preparations, are summarized in Table 3. Typically, LRP/MVP staining in tumor cells as well as in normal tissues (see below) was in the cytoplasm, in a granular fashion, compatible with the primarily cytoplasmic location of vaults (reviewed in
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Immunohistochemistry
In frozen sections of normal human lung, clear LRP/MVP expression in the epithelial cells lining the bronchioles was detected with LRP-56 and LMR-5 after acetone fixation. Similar to what was found for the staining of cytocentrifuge preparations, MVP-9, MVP-16, MVP-18, MVP-37, LRP, 1027, and 1032 showed optimal staining results using paraformaldehyde fixation and the GdnHCl protocol. MAbs 1027 and 1032 showed only weak reactivity with the frozen tissue sections, despite varying fixation, pretreatment, and primary antibody incubation conditions. The optimal staining conditions on frozen tissue sections are summarized in Table 3. In addition, the applicability of MVP-16, MVP-18, MVP-37, LRP-56, and LMR-5 on frozen tissue sections of mouse, rat, and guinea pig was tested. No immunoreactivity was observed.
Previously, we reported that in formalin-fixed, paraffin-embedded tissues optimal staining results for LRP-56 and LMR-5 are found using overnight incubation at 4C of these primary MAbs. In contrast, the newly isolated MAbs MVP-16, MVP-18, and MVP-37, and MAbs LRP, 1027, and 1032 performed very well on this material, using merely a 60-min incubation. MVP-9 showed no staining despite varying pretreatment and primary antibody incubation conditions. The optimal staining conditions on paraffin sections are summarized in Table 3. Examples of immunohistochemical staining results in salivary gland and lung are shown in Fig 3g3l. Although minor differences in staining intensity on paraffin vs frozen material were occasionally observed, we conclude that the results of LRP/MVP found with the anti-LRP/MVP MAb panel show high concordancy on both materials, confirming the tissue distribution reported earlier in lung, salivary gland, kidney, and colon (
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Discussion |
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Although drug resistance can be mediated by a number of mechanisms (
Among the various techniques, differences in reactivity and suitability were noted (Table 2). Epitope conformation is known to depend on the way the antigen is treated. In general, MAbs selected with linear (poly) peptides in ELISA systems perform well in protein blotting techniques, in which the antigen is fully linearized. MAbs selected with more native proteins (e.g., as present on viable cells or cell membranes) are more likely to detect non-linear epitopes and usually are less suitable for immunoblotting. Typically, LRP-56 and LMR-5, which were selected on LRP/MVP-overexpressing tumor cell cytospin preparations, are unreactive in protein blots. However, the newly isolated MAbs MVP-9, MVP-16, MVP-18, and MVP-37, which were selected with linear full-length human LRP/MVP in ELISA, perform well in immunoblotting techniques. In addition, the MAb LRP from Transduction Laboratories clearly detects LRP/MVP by immunoblot analysis. MAbs 1027 and 1032 were selected on immunoblot reactivity by
Because in formalin-fixed tissues the antigens are present in a more denatured form, it is not surprising that (with the exception of MAb MVP-9) MAbs MVP-16, MVP-18, MVP-37, LRP, 1027, and 1032 perform well using a standard, straightforward immunohistochemical staining technique. In contrast, on frozen tissue sections and cytospin preparations, paraformaldehyde fixation followed by a GdnHCl denaturation pretreatment (
The practical implications of the present findings include the following: (a) LRP/MVP expression can be studied by protein blotting analysis using MVP-9, MVP-16, MVP-18, MVP-37, LRP, 1027, and 1032; (b) evaluation of LRP/MVP expression on frozen tissue specimens and cytospin preparations is easiest using LRP-56 or LMR-5; and (c) the second generation of anti-LRP/MVP MAbs, reported here, and the three other MAbs tested (LRP, 1027, 1032) are most appropriate on paraffin tissue specimens. The availability of this panel of MAbs suitable for a variety of experimental applications should greatly expedite studies on the putative role of vaults in clinical drug resistance.
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Acknowledgments |
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Supported by the Dutch Cancer Society, grant VU 95-923.
We wish to thank Dr B. Moncharmont for useful comments on the manuscript.
Received for publication December 21, 2000; accepted June 12, 2001.
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