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Tumor Biology |
Department of Pathology, Academic Hospital Vrije Universiteit, 1081 HV Amsterdam, the Netherlands [A. B. S., G. L. S., M. C. d. J., S. E. B., D. F. D., R. J. S.]; Department of Biological Chemistry, University of California at Los Angeles School of Medicine, Los Angeles, California 90095 [A. C. S., V. A. K., L. H. R.]; Department of Molecular Recognition, Institute for Animal Science and Health (ID-DLO), 8219 PH Lelystad, the Netherlands [J. W. S., R. H. M.]; and Institute for Hematology, Erasmus University Rotterdam, 3000 DR Rotterdam, the Netherlands [E. W.]
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Vaults are 13 megadalton ribonucleoprotein particles containing three proteins of Mr 240,000, 193,000, and 110,000, respectively, and a small untranslated vRNA. A vault interacting protein of Mr 54,000 is occasionally observed in rat liver vault preparations (2) . Vaults are widely distributed throughout eukaryotes, and their morphology is highly conserved among various species. The remarkable structural conservation and broad distribution of vaults suggest that their function is essential to eukaryotic organisms and that the structure of the particle must be important for its function (4, 5, 6) . Although vault function is undetermined, it has been proposed that vaults may mediate transport of various substrates (7 , 8) . A role for vaults in intracellular traffic might be mediated by binding to cellular organelles through direct interaction with its targets (9) . Recently, an interaction of vaults with intracellular steroid hormone receptors has been reported (10) . Although the majority of vault particles are distributed throughout the cytosol, a portion of vaults has been localized to the nuclear membrane at or near the nuclear pore complex. Furthermore, the recent three-dimensional reconstruction of the vault particle reveals a hollow interior, which may prove important in the transport/sequestration of large substrates (11) . On the basis of striking similarities between vault particle mass and symmetry and the predictive mass of the putative central plug of the nuclear pore complexes, a role of vaults in nucleocytoplasmic exchange has been proposed (12) .
The discovery of a key role of VR-MDR in clinical drug resistance depended on the molecular identification of the LRP as the human MVP (3) . LRP had been first identified in a non-small cell lung cancer cell line, selected in vitro for DOX resistance. The protein was subsequently found to be overexpressed in many human tumor cell lines characterized by their MDR phenotype, in the absence of drug accumulation defects such as mediated by Pgp (13) . Moreover, LRP expression closely reflected known chemoresistance characteristics in broad panels of unselected tumor cell lines and untreated clinical cancers of different histogenetic origins (14 , 15) . Results from several, but not all, clinicopathological studies showed that LRP expression at diagnosis, rather than Pgp or MRP1 expression, is a strong and independent prognostic factor for poor response to chemotherapy and/or outcome, e.g., in ovarian carcinoma and leukemias (16) . Most importantly, Kitazono et al. (17) demonstrated recently, using a LRP induction system and LRP-specific ribozymes, that LRP is involved in resistance to Adriamycin, vincristine, VP-16, Taxol, and gramicidine D and has an important role in the transport of Adriamycin between the nucleus and the cytoplasm in the SW-620 human colon carcinoma cell line. To avoid confusion, we will hereafter refer to LRP as MVP.
Studies on the role of vaults in MDR, including the cloning of the MVP cDNA, have thus far been based on polyclonal antisera and two mAbs, LRP-56 and LMR-5 (13 , 18) , directed against the MVP. To further define the role of vaults in MDR, the other components must be characterized. The human vRNA genes have been cloned, and within tumor cells, not all of the vRNA was found to be vault- associated. Sedimentation measurements of vault components in VR-MDR cells have revealed up to a 15-fold increase in vault copy number, coupled with a comparable shift of vRNA to the 100,000 x g pellet, demonstrating that vault formation is limited by expression of MVP and/or one of the other vault proteins (19) . Because MVP-transfected cells did not show a drug-resistant phenotype (3) , the other vault components are thought to be essential for vaults to play a role in MDR.
Here we describe the production of the first mAbs against the Mr 193,000 vault protein. The p193 was recently identified by its interaction with the MVP in a yeast two-hybrid screen, and its identity was confirmed by peptide sequence analysis (20) . Results from protein analysis of postnuclear supernatants and subcellular fractions, Northern analysis, immunocytochemical and coimmunoprecipitation studies show that: (a) p193 and MVP are both increased in various MDR cell lines and; and (b) vault-associated p193 levels are up-regulated in these MDR cell lines but not in a drug-sensitive, MVP full-length, cDNA-transfected cell line, supporting the conclusion that functional vault formation is limited by expression of MVP and expression of at least one of the other vault components, p193.
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MATERIALS AND METHODS |
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Immunization and Generation of Hybridomas.
Escherichia coli BL21 (DE3) bacteria were
transformed with the pET-28a(+) expression vector (Novagen, Madison,
WI) containing a segment of p193 cDNA corresponding to amino acids
408–611 (20)
. The vector construct was induced to produce
the His-tagged recombinant protein using
isopropyl-ß-D-thiogalactoside (0.33
mM; 4–6 h; 37°C). Total bacterial lysate was
used as immunization antigen. Female BALB/c mice
(n = 4) received footpad injections of 25
µg of antigen emulsified in Freund’s incomplete adjuvant (Difco,
Detroit, MI). Two booster injections (
18 µg of antigen without
adjuvant) were given at 4- and 2-week intervals, respectively. Four
days before fusion, a third booster injection (10 µg of antigen) was
administered. Lymphocytes were isolated from the draining popliteal
lymph nodes of the mice, mixed with mouse myeloma Sp2/0 cells in a
ratio of 6:1, and fused using polyethylene glycol
(Mr 1450; Kodak Chemicals, Weesp, the
Netherlands). Cultures were fed with RPMI 1640 containing 100
µM hypoxanthine, 0.4 µm aminopterin, 16
µM thymidine, 20% heat-inactivated fetal
bovine serum (Hyclone, Logan, UT), 50 units/ml penicillin, and 50
µg/ml streptomycin.
Screening, Cloning, and Isotyping.
After 9 days of growth in selective medium, the hybridoma
supernatants were tested for the presence of antibodies of interest by
ELISA. Plates (96-well) were coated with 5 µg/ml of the immunization
antigen or, as a control, 5 µg/ml of pET-28a(+) transformed E.
coli BL21 (DE3) bacterial lysate induced to produce a His-tagged,
ß-galactosidase recombinant protein. Hybridomas secreting antibodies
of interest were subcloned three times by limiting dilution.
Immunoglobulin subtypes of the selected mAbs were determined using an
isotype reagent kit (Boehringer Mannheim, Indianapolis, IN).
PEPSCAN.
All overlapping dodecapeptides (12-mers) covering amino acids
408–611 of the p193 protein (beginning with the 12-mers 408–419,
409–420, and so forth) were synthesized and screened using the
minipepscan method as described previously (24
, 25)
. In
credit card format of mini-PEPSCAN cards (455 peptides/card), the
binding of the anti-p193 mAbs to each peptide was tested in a
PEPSCAN-based ELISA. The 455-well credit card format polyethylene
cards, containing the covalently linked peptides, were incubated with
mAbs p193-4, p193-6, and p193-10 (8, 15, and 10 µg/ml, respectively).
After washing, the cards were incubated with rabbit antimouse
peroxidase (Dako, Glostrup, Denmark; 1 h at 25°C), and
subsequently, the peroxidase substrate
2,2'-azino-di-3-ethylbenzthiazoline sulfonate and 2 µl/ml 3%
H2O2, was added. The color
development of the ELISA was measured after 1 h and quantified
with a CCD camera and an image processing system. The setup consisted
of a CCD camera and a 55-mm lens (Sony CCD Video Camera XC-77RR; Nikon
Micro-Nikkor 55-mm f/2.8 lens), a camera adaptor (Sony Camera adaptor
DC-77RR), and the Image Processing Software package TIM, version 3.36
(Difa Measuring Systems, Breda, the Netherlands).
MVP Antibodies.
MVP expression was studied using the rabbit polyclonal
antibody Pab W. Pab W was raised as follows. A
NcoI-EcoRV fragment of 2631 bp (amino acids
1–871) was cloned between the NcoI and filled-in
HindIII sites of the pGEX-KG polylinker (26)
.
The resulting glutathione S-transferase MVP fusion protein
was synthesized in E. coli DH5
as described by Guan and
Dixon (26)
, except that the cells were lysed by
sonication. The largely soluble glutathione S-transferase
MVP fusion protein was bound to glutathione-Sepharose 4B beads
(Pharmacia Biotechnology, Uppsala, Sweden), after which the MVP part
was released by a thrombin digest. The fraction containing the
MVP was concentrated by freeze-drying and used to immunize a
rabbit. Also, MVP expression was studied with two MVP-specific murine
mAbs (both of the IgG2b subclass) obtained in our laboratory: LRP-56,
which was raised by immunization of mice with the MDR tumor cell line
SW-1573/2R120 (13)
; and MVP-37, raised in mice against the
above-described MVP construct.
Immunocytochemistry.
Cytocentrifuge preparations of tumor cell lines were
air-dried, fixed at room temperature in acetone for 10 min or 3% (w/v)
paraformaldehyde/0.4% (w/v) glucose in PBS for 10 min. The
paraformaldehyde-fixed cells were washed two times with PBS and then
incubated with 20 mM glycine (pH 7.5) in PBS for 10 min to
block unreacted aldehyde groups. This was followed by two washes in
PBS/0.2% (w/v) BSA. Denaturation of intracellular proteins was done by
applying 50 µl of 6 N guanidine hydrochloride in 50
mM Tris-HCl (pH 7.5) to the cytospin preparations for 10
min (27)
. The cells were then rinsed three times with
PBS/0.2% BSA. All antibody dilutions were made in PBS/1% BSA. Between
incubation steps, the acetone-fixed cytospin preparations were washed
(three times during 15 min) with PBS; paraformaldehyde-fixed
preparations were washed with PBS/0.2% BSA. Primary antibodies were
applied for 60 min at room temperature to the acetone-fixed cytospin
preparations and for 30 min at 37°C to the paraformaldehyde-fixed
preparations. Irrelevant mouse IgG1 (Cappel, Organon Teknika Aurora,
OH) was used as negative control. Subsequently, the preparations
were incubated with biotinylated rabbit antimouse
F(ab')2 fragments (Dako; 60 min at room
temperature), followed by peroxidase-conjugated streptavidin (Zymed,
San Francisco, CA; 30 min at room temperature). Bound peroxidase was
visualized with 4 mg (w/v) amino-ethyl-carbazole and 0.02% (v/v)
H2O2 in 0.1 M
NaAc (pH 5.0), nuclei were counterstained with hematoxylin, and the
cytospin preparations were mounted with Kaiser’s mounting medium.
Double Immunofluorescence.
For double-labeling immunofluorescence experiments, cytospin
preparations were fixed in paraformaldehyde and pretreated with glycine
and guanidine hydrochloride as described above (see
"Immunocytochemistry"). After a blocking step with 2% normal goat
serum and 2% normal rabbit serum (Dako) for 20 min at room
temperature, the cells were incubated simultaneously with anti-p193
(mAb p193-4) and anti-MVP (mAb MVP-37) for 30 min at 37°C.
Subsequently, p193-4 was detected using biotinylated goat antimouse
IgG1 (Southern Biotechnology Associates, Inc., Birmingham, AL) with the
addition of 10% human pooled serum and 10% normal goat serum,
followed by the detection of MVP-37 with peroxidase-conjugated rabbit
antimouse (Dako; 30 min/incubation). Biotinylated goat antimouse IgG1
was detected using streptavidin conjugated with
R-phycoerythrin (Dako) for 30 min, whereas rabbit antimouse
peroxidase was detected by the deposition of FITC-conjugated tyramine
(15 min; Refs. 28
and 29
). The cytospin
preparations were counterstained with 4',6-diamidino-2-phenylindole,
mounted with Prolong mounting medium (Molecular Probes, Eugene, OR),
and evaluated with a Leica DMRB fluorescence microscope. Negative
controls consisted of simultaneously processed slides with
isotype-matched control mAbs replacing either p193-4 or MVP-37 (mouse
IgG1, Cappel; mouse IgG2b, anti-chromogranin A, Dako).
Northern Analysis.
Total RNA was isolated following the procedure of Chomczynski
and Sacchi (30)
. The RNA (20 µg) was fractionated on a
formaldehyde-agarose gel and transferred to Hybond-N membrane (Amersham
Corp., Little Chalfont, United Kingdom). Hybridization was carried out
according to the manufacturer’s recommendation with a randomly primed
p193 probe (bases 4515–5490). Hybridized bands were visualized on a
Phosphor- Imager screen (Molecular Dynamics).
Protein Analysis of Postnuclear Supernatant.
Extracts were prepared from various drug-sensitive, resistant,
and revertant cell lines by the following procedure. Cells were
harvested and resuspended in cold buffer A [50 mM Tris-Cl
(pH 7.4), 1.5 mM MgCl2, and 75
mM NaCl] containing 0.5% NP40 and 1 mM
phenylmethylsulfonyl fluoride. All subsequent steps were performed at
4°C. Samples were vortexed, incubated on ice for 5 min, and
centrifuged at 9000 x g for 20 min. The
resulting supernatant was designated as the postnuclear supernatant.
Protein concentration was determined with a Bio-Rad protein assay
(Bio-Rad, Richmond, CA). Protein samples were fractionated by SDS/6%
PAGE and subsequently transferred to nitrocellulose filter by
electroblotting. After blotting, the filters were blocked for at least
2 h in block buffer (PBS containing 1% BSA, 1% milk powder, and
0.05% Tween 20), followed by overnight incubation with the primary
antibodies in block buffer/10% FCS. Immunoreactivity was visualized
with peroxidase-conjugated swine antirabbit or rabbit antimouse
immunoglobulins (Dako) in block buffer/10% FCS, followed by staining
with 0.05% chloronaphtol and 0.03%
H2O2 in PBS. Protein levels
were determined by densitometric scanning (GelDoc; Bio-Rad) of the
filters. The density of the protein bands was analyzed using the
software of the manufacturer (Molecular Analyst; Bio-Rad). The values
obtained were expressed as absorbance (A) x mm2.
Subcellular Fractionation.
Nuclear, S100, and P100 extracts were prepared from various
drug-sensitive, resistant, and revertant cell lines as described
previously (19)
, except resuspended at a concentration of
4 x 107 cell/ml. Equal volume
amounts of fractions were analyzed for protein content. Protein samples
were solubilized in SDS sample loading buffer, fractionated on 7.5%
SDS-PAGE, and transferred to Hybond-C (Amersham Corp.) by
electroblotting. Western blots were performed using the mouse anti-p193
mAbs following established procedures. Reactive bands were detected
using the enhanced chemiluminescence system (Amersham Corp.).
Immunoprecipitation.
A2780, AC16, and GLC4/ADR cells were used in the
immunoprecipitation assays. Aliquots of postnuclear supernatants
containing 750 µg of protein (prepared as described in "Protein
Analysis of Postnuclear Supernatant") were brought up to 500 µl and
incubated for at least 2 h at 4°C with 8 µg of mAb.
Antibody-antigen complexes were recovered by incubation with 14% w/v
protein A-Sepharose CL-4B (Pharmacia Biotech BV, Woerden, the
Netherlands). Precipitated proteins were detected by immunoblotting (as
described above in "Protein Analysis of Postnuclear Supernatant").
Electron Microscopy.
Negative staining was performed by adsorbing
immunoprecipitates of the GLC4/ADR cells onto Formvar-coated 75-mesh
copper grids (Stork Veco, Eerbeek, the Netherlands) for 5 min, blotting
of the sample, and adding 1% uranyl acetate for 4–5 min. Excess stain
was than removed by blotting, and the specimens were air dried and
viewed on a Jeol 1200 EX electron microscope.
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RESULTS |
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subclass, mAb p193-6 of the
IgG2b subclass, and mAb p193-10 was an IgG2a. PEPSCAN analysis
showed that the smallest peptide sequence recognized by mAb p193-4 is
HPGE (amino acids 491–494), by mAb p193–6 FSKVEDY (amino acids
593–599), and by mAb p193-10 VALGK (amino acids 506–510; Fig. 1
).
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193,000 protein in the MDR cell lines GLC4/ADR (small cell lung
cancer cell line) and SW-1573/2R120 (non-small cell lung cancer cell
line; Fig. 2
). Similar results were obtained with mAb p193-6 and p193-10 (not
shown). The expression of the p193 protein in these cell lines, as well
as lower molecular weight bands, most likely reflecting breakdown
products, shows a similar trend in up-regulation as the MVP protein.
The parental drug-sensitive GLC4, SW-1573 and MCF-7 (breast carcinoma
cell line) and the multidrug resistant SW-1573/2R160 and MCF-7/D40 cell
lines analyzed with no or only low levels of MVP also lacked detectable
p193 protein (Fig. 2)
. Densitometric analysis confirmed the observed
coordinate expression of p193 and MVP in the panel of cell lines GLC4,
GLC4/ADR, SW-1573, SW-1573/2R120, SW-1573/2R160, MCF-7, and MCF-7/D40,
as indicated by the A x mm2 values of p193 (including lower protein
bands) and MVP bands: 0.4, 10.1, 1.5, 11.9, 1.2, 0.3, 0.6 and 0, 3.7,
1.1, 3.4, 0.2, 0, 1.4, respectively.
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. Thus, both p193 mRNA and protein expression are regulated according
to drug resistance level, concomitant with MVP mRNA and protein levels
(3
, 19
, 31)
.
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and SW-1573/2R120 cells (Fig. 4d)
is similar to
that observed by MVP staining (Fig. 4, f and h
,
respectively). No expression of p193 was detected in the parental
drug-sensitive GLC4/S (Fig. 4a)
and SW-1573 (Fig. 4c)
cell lines, which show no and only weak expression of
MVP (Fig. 4, e and g
, respectively).
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. Similarly, colocalization of p193 and MVP
distribution was found in the vault-positive SW-1573/2R120 cells (data
not shown). As described previously (13)
, only 1–3% of
the cells from the SW-1573/2R160 cell line stained positive for MVP
(Fig. 5e)
. These cells also showed p193 (Fig. 5d)
staining, which completely colocalized with the MVP (Fig. 5f)
. No R-phycoerythrin/p193 labeling was
observed when irrelevant mouse IgG1 (Cappel) replaced p193-4. No
FITC/MVP labeling was observed when irrelevant mouse IgG2b
(anti-chromogranin A; Dako) replaced MVP-37.
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. Unlike the MVP protein, the p193 protein was observed in all of the
fractions: N, S100, and P100; however, the greater part fractionated
with the P100. The detection of p193 in the S100 fraction indicates
that besides vault-associated p193 also non-vault-associated p193 is
present. Furthermore, a comparison of parental, resistant, and
revertant cells revealed that a higher amount of p193 fractionates with
the P100 of the MDR GLC4/ADR cell line. Although less obvious, the N
and S100 fractions of these drug-resistant cells also contained
increased levels of p193 protein as compared with the parental and
revertant cells. Analysis of additional MDR lines, including the
parental, drug-sensitive lines (SW-1573, SW-1573/2R120, MCF-7, and
MCF-7/MR; data not shown) confirmed our observation that in MDR cells
clearly a higher amount of the p193 is localized in the P100, the
fraction that reflects the level of assembled vault particles.
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; Refs. 2
, 4, 5, 6
). The slight distortion of the vault
morphology is probably attributable to the presence of the bound
antibodies on the surface of the vault particle or disruption of the
particle from antibody interactions. No vault structures were detected
in control immunoprecipitates (not shown). Immunoprecipitation of the
MVP/vaults from postnuclear supernatant of the multidrug-resistant
GLC4/ADR cell line, followed by detection of MVP as well as p193 by
immunoblotting experiments, showed clear coimmunoprecipitation of the
p193 with the MVP (Fig. 8)
. Control immunoprecipitation with an isotypic mAb revealed neither MVP
nor coimmunoprecipitation of p193. Both proteins were localized in the
corresponding supernatant. In contrast with our fractionation data, no
soluble p193 was detected in the supernatant that remained after MVP
precipitation. This is not surprising because the supernatants that
were examined are much less concentrated than the immunoprecipitation
samples. As a result, the relatively low levels of non-vault-associated
p193 may remain below the level of detection.
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. Furthermore, immunoprecipitation of the MVP from the postnuclear
supernatant of the AC16, followed by SDS-PAGE analysis, showed only
detection of MVP (Fig. 8)
.
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DISCUSSION |
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Studies on the function and structure of vaults to date have been focused on the MVP and vRNA. In this study, we generated mAbs against the p193 and used these to further characterize the p193 in human tumor cell lines. A strong positive correlation was found between p193 and MVP expression by Western analysis, suggesting that besides MVP also p193 expression is indicative for VR-MDR. In addition, analysis of total RNA indicated that besides the p193 protein also p193 mRNA levels increase accordingly to an increase in the number of vaults. Immunocytochemical analysis showed that the distribution of p193 staining is compatible with the cytoplasmic location of MVP/vaults. Paraformaldehyde fixation followed by a guanidine hydrochloride denaturation pretreatment (27) is a prerequisite for exposing the epitopes recognized by the anti-p193 mAbs. This protocol could not retrieve the antigenic site for the anti-MVP mAb LRP-56, which gives a positive signal in cells fixed with acetone. This is not surprising because the anti-p193 mAbs were raised against a fusion protein and therefore are more likely to be unreactive with their antigens in a more native conformation than the anti-MVP mAb LRP-56, which was raised against tumor cell lysate.
Comparison of the distribution of the p193 with the MVP protein in tumor cells reported earlier to contain high amounts of vault particles (19) reveals an identical staining pattern in corresponding immunofluorescent images. Typical vault granules are present in the entire cytoplasm, which colocalize in the double-exposed image. The double staining results strongly suggest a high degree of association of the p193 with MVP/vaults. On the basis of MVP overexpression, vaults have been reported to be most abundant in epithelial cells (5 , 15) . Although the precise tissue distribution of the p193 is still under investigation, we found p193 to be present in normal human lung, with highest expression in the epithelial cells lining the respiratory tract. Thus, vault expression in normal human tissues also involves p193 expression.
Upon subcellular fractionation, the MVP is exclusively present in the large vault complex retained in a particulate fraction (2 , 5 , 19 , 32) . Western analysis of similar subcellular fractions of tumor cell lines demonstrates the presence of vault-associated as well as a relatively low level of non-vault-associated p193. In a previously reported study, (20) the presence of non-vault-associated p193 was also observed in the cytosol and nucleus by Western blot analysis upon subcellular fractionation. In contrast with the present findings, the nuclear localization of p193 was also shown in these studies by immunofluorescence using an anti-p193 polyclonal antibody. Probably the relatively low level of non-vault-associated p193 in the drug-resistant cell lines remains below the detection level in our immunofluorescence double-labeling experiments using anti-p193 mAbs, or the soluble p193 signal is masked by the abundance of MVP located in the cytoplasm. A comparison upon subcellular fractionation of resistant and parental tumor cell lines reveals a clear increase in vault-associated p193 levels in the resistant cells. Furthermore, in the revertant GLC4 cell line, which was isolated by culturing in the absence of drug but still is a drug-resistant cell line (albeit at a lower concentration of drug), vault-associated p193 protein levels decrease. Thus, an increase in the general pool of vaults (as in the drug resistant cells) results in an increase in vault-associated p193.
To further evaluate the association of p193 with MVP/vaults, we performed immunoisolation of vault particles from GLC4/ADR cells. Negative staining electron microscopy showed that vault particles were isolated as judged by the structure resemblance to vaults described previously (2 , 5 , 6) . Western analysis of the immunoisolates revealed coimmunoprecipitation of the p193 with the MVP, demonstrating the specificity of p193 association with MVP/vaults.
Consistently, vault formation is limited by the expression of MVP (19) . However, the previously constructed MVP transfectant tumor cell line AC16 shows no signs of drug resistance (3) . This is not unexpected because the MVP comprises only 70% of the vault particle. Therefore, additional components of the vault particle could also be required for vault function and drug resistance. Within tumor cells, vRNA was found to be in considerable excess to MVP (19) , suggesting that in the AC16 cells, this component is not limiting functional vault formation. Western analysis revealed that endogenous p193 was not overexpressed. Furthermore, no detectable coimmunoprecipitation of p193 with MVP was found in the immunoisolates of the MVP transfectant AC16. These findings support our view that MVP transfection did not result in an acquired VR-MDR phenotype, because the assembly of functional vault particles is impaired by the lack of overexpression of at least another vault constituent and subsequent vault association, leading to high numbers of incomplete dysfunctional vault particles.
Taken together, using newly developed mAbs against the p193, we have demonstrated that the MVP and p193 are co-up-regulated in various MDR cell lines. Furthermore, we present evidence that functional vault formation is not only limited by expression of the MVP but also by expression of the p193. Considering that the expression of two vault proteins is essential for functional vault formation, the contribution of the Mr 240,000 vault protein, which was recently identified as a component in the telomerase complex (telomerase-associated protein, TEP1; Ref. 33 ) needs to be clarified to complete the picture on vault function and the role of VR-MDR in clinical drug resistance.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by Dutch Cancer Society Grant VU
95-923 (to R. J. S.), USPHS Grant GM38097, and a grant from the
Margaret E. Early Foundation (to L. H. R.). 
2 To whom requests for reprints should be
addressed, at Department of Pathology, Academic Hospital Vrije
Universiteit, De Boelelaan 1117, 1081 HV Amsterdam, the Netherlands.
Phone: 31-20-444-4031; Fax: 31-20-444-2964; E-mail: rj.scheper{at}azvu.nl
3 The abbreviations used are: MDR, multidrug
resistance; MRP1, MDR protein 1; Pgp, P-glycoprotein; vRNA, vault RNA;
LRP, lung resistance-related protein; VR-MDR, vault-related MDR; MVP,
major vault protein; DOX, doxorubicin; mAb, monoclonal antibody.
Received 8/30/99. Accepted 12/16/99.
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 A. B. Schroeijers, G. L. Scheffer, A. W. Reurs, A. C.L.M. Pijnenborg, C. Abbondanza, E. A.C. Wiemer, and R. J. Scheper Detection of the Mr 110,000 Lung Resistance-related Protein LRP/MVP with Monoclonal Antibodies J. Histochem. Cytochem., November 1, 2001; 49(11): 1379 - 1386. [Abstract] [Full Text] [PDF]
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E. Perkins, D. Sun, A. Nguyen, S. Tulac, M. Francesco, H. Tavana, H. Nguyen, S. Tugendreich, P. Barthmaier, J. Couto, et al. Novel Inhibitors of Poly(ADP-ribose) Polymerase/PARP1 and PARP2 Identified Using a Cell-based Screen in Yeast Cancer Res., May 1, 2001; 61(10): 4175 - 4183. [Abstract] [Full Text]
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 A. van Zon, M. H. Mossink, M. Schoester, G. L. Scheffer, R. J. Scheper, P. Sonneveld, and E. A. C. Wiemer Multiple Human Vault RNAs. EXPRESSION AND ASSOCIATION WITH THE VAULT COMPLEX J. Biol. Chem., September 28, 2001; 276(40): 37715 - 37721. [Abstract] [Full Text] [PDF]
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