Detection of the M r 110,000 Lung Resistance-related Protein LRP/MVP with Monoclonal Antibodies

Abstract

The M_r 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.

Keywords

lung resistance-related protein (LRP)major vault protein (MVP)vault multidrug resistance (MDR)monoclonal antibody (MAb)

MULTIDRUG RESISTANCE (MDR) is the major cause of chemotherapeutic failure in cancer treatment (Lehnert 1996). Several mechanisms are responsible for mediating MDR, including the overexpression of transmembrane transporter molecules, such as P-glycoprotein (reviewed in Germann 1996), multidrug resistance protein (MRP) 1, MRP2, MRP3 (reviewed in Borst et al. 1999), and breast cancer resistance protein (reviewed in Doyle et al. 1998), which act as drug efflux pumps, decreasing intracellular drug accumulation. In clinical drug resistance, however, other mechanisms may play a role, e.g., drug sequestration into exocytotic vesicles. Evidence has been obtained that subcellular particles called vaults may play a critical role in such a mechanism (Kedersha and Rome 1986; Scheffer et al. 1995). Vaults are evolutionarily highly conserved large ribonucleoprotein particles (Kedersha et al. 1990). The particles represent multimeric RNA-protein complexes with one predominant member, the major vault protein (MVP). The bulk of vaults is present in the cytoplasm. In addition, based on MVP immunolocalization in cells and tissues, a small fraction of vaults appear to be localized at or near the nuclear membrane and the nuclear pore complex (Chugani et al. 1993). Although the cellular role of the vault particle has remained elusive, several findings support the notion that vaults have a transport function, acting as a carrier, mediating bidirectional nucleo-cytoplasmic exchange, and vesicular transport of compounds, including cytostatic drugs (Rome et al. 1991; Chugani et al. 1993; Herrmann et al. 1996, 1999; Hamill and Suprenant 1997; Abbondanza et al. 1998; Kong et al. 1999). Recently, Kitazono et al. (1999) demonstrated, using an LRP-induction system and LRP-specific ribozymes, that LRP is involved in resistance to doxorubicin, vincristine, VP-16, taxol, and gramicidine-D, and has an important role in the transport of doxorubicin between the nucleus and the cytoplasm in the SW-620 human colon carcinoma cell line.

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 (Scheffer et al. 1995). LRP/MVP had been first identified in a non-small-cell lung cancer cell line, selected in vitro for doxorubicin resistance. Overexpression of the protein as well as elevated levels of vault particles were subsequently found in many human tumor cell lines characterized by their MDR phenotype, in the absence of drug accumulation defects (Scheper et al. 1993; Kickhoefer et al. 1998). Moreover, LRP/MVP expression closely reflected known chemoresistance characteristics in broad panels of unselected tumor cell lines and untreated clinical cancers of different histogenetic origins (Izquierdo et al. 1996a,b). Results from several, but not all, clinico-pathological studies showed that LRP/MVP expression at diagnosis, rather than P-glycoprotein or MRP1 expression, is a strong and independent prognostic factor for poor response to chemotherapy and/or outcome (Izquierdo et al. 1998).

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 (Scheper et al. 1993; Scheffer et al. 1995) and for functional studies to interfere with vault function (Kitazono et al. 1999; Schroeijers et al. 2000a). Furthermore, they have been used to analyze the tissue distribution and subcellular localization of LRP/MVP and to evaluate its presence in tumors (Scheper et al. 1993; Izquierdo et al. 1996b; reviewed in Izquierdo et al. 1998). The aim of this study was a comprehensive evaluation of previously as well as newly isolated MAbs detecting LRP/MVP immunoblotting analysis, on cytospins, and on frozen and formalin-fixed, paraffin-embedded tissues.

Materials and Methods

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) (Zijlstra et al. 1987); the non-small-cell lung carcinoma cell line SW-1573 (low LRP/MVP expression) and its doxorubicin-selected MDR variants SW-1573/2R120 (selected with 120 nM doxorubicin; high, heterogeneous LRP/MVP expression); and SW1573/2R160 (160 nM; ~97% LRP/MVP-negative/~3% high LRP/MVP expression) (Kuiper et al. 1990). All cell lines were propagated in Dulbecco's modified Eagle's medium (Bio-Whittaker; Verviers, Belgium) supplemented with 10% fetal calf serum (Integro; Zaandam, The Netherlands), penicillin (50 U/ml), and streptomycin (50 μg/ml) at 37C in a humidified atmosphere containing 5% CO₂. Cells were routinely tested to ensure the absence of Mycoplasma. The drug-selected cell lines were cultured in the presence of drugs until 3–10 days before the experiments.

Immunization and Generation of Hybridomas

Male Balb/c mice (n = 3) received footpad injections of 5 μg of full-length recombinant human LRP/MVP (Schroeijers et al. 2000b) emulsified in Freund's complete adjuvant (Difco; Detroit, MI). A booster injection (approximately 2.5 μg of antigen without adjuvant) was given after 9 days. Four days before fusion, a second booster injection (2.5 μg of antigen) was administered. Lymphocytes were isolated from the draining popliteal lymph nodes of the mice. Lymphocytes were mixed with mouse myeloma Sp2/0 cells in a ratio of 6:1 and fused using polyethylene glycol (M_r 1450) (Kodak Chemicals; Weesp, The Netherlands). Cultures were fed with RPMI 1640 (Bio-Whittaker) containing 100 μM hypoxanthine, 0.4 μM aminopterin, 16 μM thymidine, 20% heat-inactivated high clone fetal calf serum (Hyclone; Logan, UT), penicillin (50 U/ml), and streptomycin (50 μg/ml).

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; Schroeijers et al. 2000b) and two LRP/MVP-specific murine MAbs previously isolated in our laboratory: mouse MAb LRP-56 and rat MAb LMR-5, which were raised by immunization of mice with the drug resistant tumor cell line SW-1573/2R120 (Scheper et al. 1993; Flens et al. 1997). Furthermore, the following anti-LRP/MVP mouse MAbs were used: LRP (Transduction Laboratories; Lexington, KY), 1027, and 1032 (Abbondanza et al. 1998). MAbs 1027 and 1032 were kindly provided by Dr. C. Abbondanza and Dr. B. Moncharmont. Negative controls included both omission of the first antibody step and substitution of the primary antibodies by irrelevant antibodies of the same species.

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 streptavidin-HRP (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 (Peranen et al. 1993; immunocytochemical staining procedure involving GdnHCl treatment was described before in Schroeijers et al. 2000b); 0.5% (v/v) trypsin (Gibco; Grand Island, NY) in Tris-buffer, pH 7.6 for 30 min at 37C; citrate buffer (0.01 mol/liter citric acid, pH 6.0) at 100C three times for 5 min; 1 mM ethylene-diaminetetraacetate (Merck; Darmstadt, Germany) three times for 5 min at 100C. A microwave was used to raise the temperature to 100C.

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 phenyl-methylsulfonyl fluoride. All subsequent steps were performed at 4C. Samples were vortexed, incubated on ice for 5 min, and centrifuged at 9000 × 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. Antibody-antigen complexes were recovered by incubation with 14% (w/v) protein A-Sepharose CL-4B (Pharmacia Biotech; Woerden, The Netherlands). Precipitated proteins were shown by immunoblotting (as described above).

Results

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 IgG_2b.

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 M_r 110,000 LRP/MVP protein (Figure 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.

As reported earlier (Scheper et al. 1993; Flens et al. 1997) both anti-LRP/MVP MAbs LRP-56 and LMR-5 are unsuitable for immunoblotting analysis. To create a better understanding of the applicability of the available anti-LRP/MVP MAbs (specifications listed in Table 1), we decided to compare the performance of this panel of MAbs in immunoblotting analysis, on cytospins, and on frozen and formalin-fixed, paraffin-embedded tissues. The results of these experiments are described below and summarized in Table 2; a rating system is used for performance.

Figure 1

Western analysis of LRP/MVP using the newly isolated MAbs MVP-9, MVP-16, MVP-18, and MVP-37 in LRP/MVP immunoprecipitates (IP; using the MAb LRP-56) and control immunoprecipitates and the supernatants (sup) of these immunoprecipitates. The precipitations were carried out on the postnuclear supernatant of GLC4/ADR cells.

Table 1

Specifications of MAbs detecting LRP/MVP

Monoclonal antibody
Name	Species	Isotype	Reference/source
LRP-56	Mouse	IgG_2b	Scheper et al. (1993)
LMR-5	Rat	IgG_2a	Flens et al. (1997)
MVP-9	Mouse	IgG₁	This study
MVP-16	Mouse	IgG_2b	This study
MVP-18	Mouse	IgG₁	This study
MVP-37	Mouse	IgG_2b	This study
LRP	Mouse	IgG₁	Transduction Laboratories
1027	Mouse	IgM	Abbondanza et al. (1998)
1032	Mouse	IgG₁	Abbondanza et al. (1998)

Protein Blotting Analysis

Figure 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 Abbondanza et al. (1998), showed weak immunoreactivity, whereas other MAbs from this series (1011 and 1014; data not shown) were unreactive in our hands.

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.

Table 2

Performance of MAbs detecting LRP/MVP a

	Reactivity
Mab	Western blotting	Cytospins	Frozen sections	Paraffin sections
LRP-56	-	++ b	++ b	++ c
LMR-5	-	++ b	++ b	++ c
MVP-9	++	++	+	-
MVP-16	+	++	+	++
MVP-18	++	++	++	++
MVP-37	++	++	+	++
LRP	++	+	+	++
1027	±	+	±	±
1032	±	++	±	++

-, no reactivity; ±, weak reactivity; +, good reactivity; ++, very good reactivity.

Applicable without GdnHCl pretreatment.

Overnight incubation of primary MAb required (see Table 3).

Figure 2

Western analysis of LRP/MVP with all available MAbs in a postnuclear supernatant of multidrug-resistant GLC4/ADR cells, showing specific detection of the LRP/MVP protein.

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 Izquierdo et al. 1998). After acetone fixation, we observed clear immunoreactivity with LRP-56 and LMR-5, as reported previously in LRP/MVP-expressing cells (Scheper et al. 1993; Flens et al. 1997). The overall quality (intensity, specificity, and morphology) did not benefit from other fixation or pretreatment methods. On the contrary, MVP-9, MVP-16, MVP-18, MVP-37, LRP, 1027, and 1032 showed the best staining results using paraformaldehyde fixation and subsequent GdnHCl denaturation. MAb 1027 showed only weak reactivity with the cells compared with the other MAbs, despite varying fixation, pretreatment, and primary antibody incubation conditions. The composite Figure 3a-3f illustrates the results of LRP/MVP detection in cytospin preparations using LRP-56, MVP-18, MVP37, and 1027. Labeling of GLC4/ADR and SW-1573/2R120 was uniform, whereas the SW-1573/2R160 cells were mainly negative, with only a few positive-staining cells (1–3%) for LRP/MVP, consistent with results published previously (Scheper et al. 1993). As mentioned, for all MAbs LRP/MVP staining was granular, throughout the cytoplasma. After paraformaldehyde fixation and GdnHCl pretreatment, a densely stained spot appeared in the GLC4/ADR cells. Most likely, the cross-linking and/or denaturation influences the staining pattern.

Table 3

Optimal staining conditions of anti-LRP/MVP MAbs on cytospins, frozen, and formalin-fixed, paraffin-embedded tissue sections a

	Cytospins			Frozen sections			Paraffin sections
MAb	Concentration (μg/ml)	Fixation	Pretreatment	Concentration (μg/ml)	Fixation	Pretreatment	Concentration (μg/ml)
LRP-56	0.5–2	Acetone	-	0.5–2	Acetone	-	5–10 b
LMR-5	0.5–2	Acetone	-	0.5–2	Acetone	-	1–4 b
MVP-9	1.5–6	PAF	GdnHCl	CS	PAF	GdnHCl	CS
MVP-16	CS	PAF	GdnHCl	CS	PAF	GdnHCl	CS
MVP-18	CS	PAF	GdnHCl	CS	PAF	GdnHCl	CS
MVP-37	1.5–6	PAF	GdnHCl	CS	PAF	GdnHCl	1.5–6
LRP	1–4	PAF	GdnHCl	5–10	PAF	GdnHCl	1–4
1027	CS	PAF	GdnHCl	CS	PAF	GdnHCl	CS
1032	CS	PAF	GdnHCl	CS	PAF	GdnHCl	CS

PAF, paraformaldehyde; CS, hybridoma culture supernatant.

Overnight incubation of primary antibody.

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 pre-treatment 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 Figures 3g-3l. 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 (Izquierdo et al. 1996a,b).

Discussion

Although drug resistance can be mediated by a number of mechanisms (Lehnert 1996), the role of LRP/MVP expression has attracted great interest in recent years because it may be related to clinical MDR. For the analysis of LRP/MVP in clinical samples and the detection in many experimental applications, LRP/MVP-specific MAbs that are widely useful are important. In this study we have reported the production of four new anti-LRP/MVP MAbs (MVP-9, MVP-16, MVP-18, and MVP-37) and compared their immunoreactivity in a variety of methods (immunoblotting, immunocytochemistry, and immunohistochemistry) with two MAbs (LRP-56 and LMR-5) previously raised in our laboratory (Scheper et al. 1993; Flens et al. 1997), and three other MAbs (LRP, 1027 and 1032) (Abbondanza et al. 1998).

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 Abbondanza et al. (1998). Indeed, MAbs 1027 and 1032 recognized the LRP/MVP protein band, but the staining intensity of the protein blot signal was less compared to the other reactive MAbs under the same conditions.

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 (Peranen et al. 1993) is a prerequisite for optimal exposure of the epitopes recognized by these MAbs, revealing staining patterns similar to those using MAbs LRP-56 and LMR-5 (Scheper et al. 1993; Flens et al. 1997).

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.

Figure 3

Detection of LRP/MVP in cytocentrifuge preparations of human tumor cell lines (a-f) and in formalin-fixed, paraffin-embedded tissue sections (g-l). (a-f) Tumor cell line-specific expression patterns of LRP/MVP with MAb LRP-56 in GLC4/ADR cells (a), MAb MVP-18 in GLC4/ADR, SW-1573/2R120 and SW-1573/2R160 cells (b,c,e), MAb MVP-37 (d) and 1027 (f) in GLC4/ADR cells. (g-l) Immunohistochemical detection of LRP/MVP in formalin-fixed, paraffin-embedded tissue sections of human lung (g,h; 3-amino-9-ethylcarbazole) and salivary gland (i-l; 3,3-diaminobenzidine tetra-hydrochloride). Immunoreactivity was observed mainly in the bronchial epithelia and alveolar macrophages and in small striated ducts, respectively. Using a conventional staining procedure, good immunoreactivity was found with a 60-min incubation period for the MAbs (g) MVP-16, (h) MVP-18, and (i) MVP-37, in contrast to (j) LRP-56. (k) Using LRP-56, stronger staining intensity was obtained after overnight incubation at 4C. (l) Mouse IgG_2b (isotypic control for LRP-56).

Footnotes

Acknowledgments

Supported by the Dutch Cancer Society, grant VU 95–923.

We wish to thank Dr B. Moncharmont for useful comments on the manuscript.

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23.

Scheper

Broxterman

Scheffer

Kaaijk

Dalton

van Heijningen

van Kalken

Slovak

de Vries

van der Valk

Meijer

CJML

Pinedo

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Schroeijers

Reurs

Stam

AGM

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Wiemer

EAC

de Gruijl

Scheper

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Siva

Scheffer

de Jong

Bolick

Dukers

Slootstra

Meloen

Wiemer

Kickhoefer

Rome

Scheper

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26.

Zijlstra

de Vries

Mulder

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