Vaults are 13-MDa ribonucleoprotein particles
composed largely of a 104-kDa protein, termed major vault protein or
MVP, and a small vault RNA, vRNA. While MVP levels have been found to
increase up to 15-fold in non-P-glycoprotein multidrug-resistant cell
lines, the levels of vault particles have not been investigated. As
both the function of vault particles and the mechanism of drug
resistance in non-P-glycoprotein cells are unknown, we decided to
determine whether vault synthesis was coupled to MDR. By cloning the
human gene for vRNA and careful quantitation of the MVP and vRNA levels in MDR cells, we find that vRNA is in considerable excess to MVP. Sedimentation measurements of vault particles in multidrug resistance cells have indeed revealed up to a 15-fold increase in vault synthesis, coupled with a comparable shift of associated vRNA, demonstrating that
vault formation is limited by expression of MVP or the minor vault
proteins. The observation that vault synthesis is linked directly
to multidrug resistance supports a direct role for vaults in drug
resistance.
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INTRODUCTION |
Multidrug resistance
(MDR)1 is the major cause of
chemotherapy failure in cancer treatment. Several mechanisms are
responsible for mediating MDR, including the overexpression of
transmembrane transporter molecules, such as P-glycoprotein (Pgp) and
multidrug resistance-associated protein (MRP), both of which act as
drug efflux pumps (1-4). However, several MDR cell lines have been described that do not overexpress either Pgp or MRP, indicating that
additional mechanisms are functioning (5). Recently a 110-kDa protein
termed lung resistance-related protein (LRP) has been found to be
overexpressed in several non-Pgp MDR tumor cell lines (6) and is
reported to be a possible candidate marker for in vitro and
in vivo prediction of chemotherapy success (7, 8). The
sequence of the LRP cDNA revealed that it was the human homologue
of the major vault protein (MVP; to avoid confusion we will hereafter
refer to LRP as MVP) (9).
Vaults are large, oval-shaped, cytoplasmic ribonucleoprotein particles
originally identified in preparations of clathrin coated vesicles
(10-12). Purified vaults are isolated from the microsomal fraction
(100,000 × g pellet; P100) by a series of sucrose
gradients followed by electrophoresis on nonsieving agarose gels (10, 13). Vaults purified from rat liver consist of three protein species
(210, 192, and 104 kDa) and a small RNA, vRNA. The vRNA constitutes 5%
of the rat vault particle by mass and is not a structural component, as
ribonuclease digestion does not alter particle morphology (14). vRNAs
isolated from different species share a similar predicted secondary
structure despite their differences in length, indicating that vRNA
association with vaults is not fortuitous (15). This suggests that it
has a fundamental role in the function of the vault particle possibly
through RNA-RNA or RNA-protein interactions. The 104-kDa MVP
constitutes >70% of the total protein from rat vaults and is the main
structural component of the particle. Although the size of the MVP
varies slightly among species, it retains immunological
cross-reactivity (16). Vaults are widely distributed throughout
eukaryotes, and their morphology is highly conserved among these
species. The structure of the vault particle has been extensively
studied by electron microscopy (14). Its dimensions have been
determined to be ~55 × 30 nm, with a molecular mass of about 13 MDa (three times the size of a ribosome). The intact vault particle has
2-fold symmetry, with each half vault capable of opening into a
flower-like structure containing eight petals surrounding a central
ring. The remarkable 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. Although vault function is undetermined, a portion of vaults have been
localized to the cytoplasmic face of the nuclear membrane at or near
nuclear pore complexes (17). Moreover, vault particle mass and symmetry
are strikingly similar to the predicted mass of the putative central
plug of the nuclear pore complexes, leading us to propose that vaults
participate in nuclear-cytoplasmic transport. This hypothesis has
increased in significance in view of the finding that MVP is
up-regulated in non-Pgp MDR cell lines.
Here we describe the cloning of the human vRNA genes and show by
subcellular fractionation of vault particles that both MVP and
vault-associated vRNA levels are similarly increased in various non-Pgp
drug-resistant cell lines. These results support the conclusion that
vaults are up-regulated in certain drug-resistant cell lines.
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EXPERIMENTAL PROCEDURES |
Cell Line Maintenance--
MDR cell lines, SW1573/2R120
(non-small cell lung cancer) (18), and GLC4/ADR (small cell lung
cancer) (19, 20), were cultured in the presence of 115 and 1156 nM doxorubicin, respectively, once every week. MCF-7/MR
(breast cancer cells) (21) and 8226/MR4, 8226/MR20 (myeloma cells) (22)
were cultured in the presence of 80, 40, and 200 nM
mitoxanthrone, respectively, twice weekly. Drug-sensitive SW1573,
GLC4/S, MCF-7, and 8226/S cells were grown in RPMI 1640 medium,
supplemented with 10% fetal bovine serum and antibiotics.
Human vRNA Cloning--
To isolate the genes encoding the human
vault RNAs we screened a Lambda FIX II (Stratagene) human genomic DNA
library that was constructed by Kathy Kampf (UCLA MRRC Molecular
Biology Core Facility). A total of 6.25 × 105
recombinants were screened as described previously (15) using a random
primed partial human vRNA gene. Comparisons between the rat and
bullfrog vRNA sequences revealed that bases 11-27 and 110-129 (based
on the rat vRNA sequence) were conserved (15). Primers to these
conserved regions were synthesized on an Applied Biosystems DNA
synthesizer and used to amplify partial human vRNA genes by polymerase
chain reaction from genomic DNA. The purified polymerase chain reaction
products were subcloned into pBluescript SK+ (Stratagene) and sequenced
by the dideoxy method using Sequenase (U. S. Biochemical Corp.). These
partial human vRNA genes were used as hybridization probes. Two clones
were identified and plaque-purified (1 and 4). Based on Southern blot
analysis a 350-base SacI fragment was subcloned into
pBluescript SK+ (HVG1) and sequenced. Subcloning and
sequence analysis revealed that the second clone (no. 4) contained two
vRNA genes (HVG2 and HVG3) within about 7 kilobase pairs of each other. The sequences have been submitted to
GenBankTM (accession nos. AF045143, AF045144, and AF045145;
HVG1 through HVG3, respectively).
Subcellular Fractionation--
Extracts were prepared from
various drug-sensitive, -resistant, and -revertant cell lines by the
following procedure. Cells (108) were harvested, counted,
and resuspended in 5 ml of cold buffer A (50 mM Tris-Cl (pH
7.4), 1.5 mM MgCl2, 75 mM NaCl)
containing 0.5% Nonidet P-40, 1 mM phenylmethylsulfonyl
fluoride, and protease inhibitor mixture (2 µg/ml aprotinin, 0.5 mM benzamidine, 2 µg/ml chymostatin, 5 µM
leupeptin, 5 µM pepstatin). All subsequent steps were
performed at 4 °C. Cells were vortexed, incubated on ice for 5 min,
and centrifuged at 20,000 × g for 20 min. The
postnuclear supernatant fraction was centrifuged at 100,000 × g for 1 h. The resulting supernatant was designated the
S100 fraction. The nuclear and 100,000 × g pellets
(P100) were resuspended by Dounce homogenization in buffer A containing
10% glycerol, 1 mM phenylmethylsulfonyl fluoride, and
protease inhibitors in the original volume. Equal volume amounts of
fractions were analyzed for protein and RNA content. The GLC4/ADR P100
fraction was then applied to a 20/30/40/45/50/60% sucrose step
gradient in buffer A (containing 1/2/2/2/2/1 ml in each layer,
respectively) and centrifuged at 28,000 rpm in a Beckman SW-41 rotor
for 16 h. Under these conditions, intact vault particles localize
to the 40/45% sucrose layers (10). Gradient fractions for each layer
were collected, diluted 4-fold with buffer A, and centrifuged for
3 h at 100,000 × g. Pellets were resuspended in buffer A and analyzed for both protein and RNA content.
Protein samples were solubilized in SDS sample loading buffer,
fractionated on 7.5% SDS-polyacrylamide gel electrophoresis, and
transferred to Hybond-C (Amersham Corp.) by electroblotting. Western
blots were performed using the anti-rat vault polyclonal antibody (N2)
following established procedures (10). Reactive bands were detected
using the enhanced chemiluminescence system (Amersham Corp.). RNA from
cellular fractions were purified by phenol/chloroform extraction and
ethanol precipitation. Total RNA was isolated by the guanidinium-phenol
method. Subsequently the RNAs were fractionated on 8 M
urea, 10% polyacrylamide gels, and electroblotted to Zeta GT membrane
(Bio-Rad). The membrane was hybridized with a randomly primed human
vRNA gene probe (HVG1, specific activity 1 × 109 cpm/µg). Hybridization was carried out according to
the manufacturer's recommendation. Hybridized bands were detected by
autoradiography. Quantitation of reactive bands (either protein or RNA)
was carried out by scanning with a Molecular Dynamics Personal
Densitometer SI using ImageQuant software. Fold changes in Table I were
calculated from multiple data sets, except for the GLC4 data set, which
matched previously determined values (9).
Copy Number--
Vault levels in the parental cell lines were
determined by comparison of protein levels in the high speed pellet
(P100) fraction from 4 × 105 cells per lane to
purified rat liver vaults (0.05, 0.10, 0.15, 0.20, and 0.25 µg).
Western analysis and quantitation was carried out as described above. A
vault standard curve was generated and linear regression analysis was
used to estimate the number of vault particles per cell in the parental
cell lines (see Table II). Values for the drug-resistant and -revertant
lines were calculated using the fold changes determined in Table I.
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RESULTS AND DISCUSSION |
MVP has been shown to be overexpressed in many non-Pgp MDR tumor
cell lines, including the SW1573/2R120 (non-small cell lung cancer)
(18), GLC4/ADR (small cell lung cancer) (19, 20), MCF-7/MR (breast
cancer) (21), and 8226/MR20 (myeloma) (22) cell lines (6). Furthermore,
revertant cell lines, which were isolated by culturing in the absence
of drug, down-regulate the expression of MVP (6). However, transfection
studies have shown that overexpression of the MVP cDNA alone is not
sufficient to confer a drug resistant phenotype (9). This is not
unexpected as the MVP comprises only 70% of the vault particle mass.
Therefore additional components of the vault particle (like the minor
vault proteins and vRNA) could also be required for drug resistance. Partial sequence conservation between rat and bullfrog vRNAs allowed us
to clone the human vRNA genes. Humans contain three vRNA homologues that share about 84% identity with each other: one is 96 bases in
length (human vRNA gene, HVG1) and the other two are 86 bases in length (HVG2 and HVG3; Fig.
1). The human vRNAs can be folded into
secondary structures similar to those for rat and bullfrog vRNAs (15)
(data not shown). Like other vRNA genes, the human vRNA genes contain
internal RNA polymerase III-type promoter elements and end with a
typical polymerase III termination signal of four Ts (Fig. 1). We have
previously shown, by subcellular fractionation, that vaults pellet at
100,000 × g (P100) and that all of the MVP is
associated with this fraction and is assembled into vaults (10). In
contrast, only a portion of the total cellular vRNA fractionates to the
P100 where it is associated with vaults. This non-vault-associated vRNA
fractionates in the soluble or S100 fraction (Fig.
2A). Although there are
multiple human vRNAs, we have determined that only one form (HVG1)
associates with the vault particle (as evidenced by pelleting at
100,000 × g, Fig. 2A). Surprisingly, the
86-bp encoded vRNAs are not present in all of the cell lines (Fig.
2B, lanes 1-3, 9, and 10).
However, PCR analysis of genomic DNA from these lines indicates the
genes are present (data not shown). These data indicate that, while the
86-bp RNAs (HVG2 and HVG3) are vRNA-related, they are apparently not
vault-associated RNAs.

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Fig. 1.
Human vRNA genes (HVG1-3) DNA
sequence alignment. Identical bases are indicated by
dashes, asterisks indicate a deletion. RNA
polymerase III internal promoter elements (termed A and B boxes) are
located at bases 11-20 and bases 65-75, respectively.
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Fig. 2.
A, drug-resistant myeloma cells were
separated into nuclear (N), high speed supernatant
(S), and high speed pellet (P) fractions. RNA was
extracted and analyzed by Northern blotting. B, total RNA
(10 µg) from the indicated cell lines (lanes 1-10) were
examined by Northern analysis. Arrows indicate the positions
of the human vRNAs, HVG1, and HVG2,3.
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Previous studies of non-Pgp MDR cells have determined that MVP levels
are increased, but the levels of vault particles has not been
investigated (6, 9). Here we measured vault particle levels by
sedimentation analysis in conjunction with an analysis of the levels of
MVP and associated HVG1 vRNA in the small cell lung cancer line GLC4/S
(parental), and its derivative cell lines the drug resistant GLC4/ADR
and drug revertant GLC4/REV (Fig. 3,
Table I). A comparison of the resistant
and parental MVP revealed a 15-fold increase in MVP protein levels and
suggested that the increased protein was assembled into a
macromolecular form able to pellet at 100,000 × g
(Fig. 3A, lanes 3 and 6). This 15-fold increase in MVP protein levels is in agreement with the increased expression of MVP mRNA (9). In addition, Northern analysis of the
same fractions extracted for RNA revealed that vault-associated HVG1
vRNA increased about 15-fold (Fig. 3B, lanes 3 and 6). A concomitant shift of the vRNA from the S100 to the
P100 fraction in the drug resistant line (Fig. 3B,
lanes 5 and 6) suggests that a larger fraction of
vRNA is associated with vaults. Likewise in the revertant line, which
was isolated by culturing in the absence of drug, but is still a
drug-resistant cell line (albeit at a lower concentration of drug),
protein and vRNA levels decrease (Fig. 3A, lanes
6 and 9; Fig. 3B, lanes 6 and
9). Both the MVP and vRNA levels decrease to comparable
levels in the revertant line (Table I).

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Fig. 3.
A, MVP levels were determined by Western
blotting. Sample extracts for each cell line are in groups of three:
nuclear (N), high speed supernatant (S),
and high speed pellet (P) from the non-small cell lung cell
line GLC4, parental (GLC4/S), resistant (GLC4/ADR), and revertant
(GLC4/REV). B, Northern analysis of RNA extracted from
fractions (see "Experimental Procedures"). C, Northern
analysis of 10 µg of total RNA.
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Analysis of total RNA indicates that vRNA levels remain constant in the
different lines, and that increased association with vaults is not due
to an increase in transcription of the vRNA gene (Fig. 3C).
This is not surprising since we have determined that on average only
about 20% of the total vRNA is associated with the vault particle in
these MDR cancer cell lines (data not shown). These results support the
hypothesis that there is a dynamic relationship between the vRNA and
vaults and that there is a pool of vRNA from which a certain fraction
is associated with the vault particle at any particular time. Thus an
increase in the general pool of vaults (as in the drug resistant lines)
results in an increase in the fraction of the vRNA pool that is vault
associated (Fig. 3B, lanes 5 and 6).
Correspondingly, in the revertant lines, a shift of vRNA from the P100
back to the S100 pool is observed (Fig. 3B, lanes
8 and 9), supporting our view that the vRNA is a
dynamic component of the vault particle. Recent studies in our laboratory have demonstrated by UV cross-linking that the vRNA interacts primarily with the minor vault proteins and not the MVP.2 Further fractionation
of the GLC4/ADR P100 fraction on a sucrose equilibrium gradient
revealed that the majority of MVP and vRNA were present in the 45%
layer, coincident with the previously published behavior of purified
vault particles (Fig. 4). This data
verifies that the increases of MVP and vRNA seen in the P100 fraction
accurately reflect the level of assembled vault particles. From these
data we conclude that the co-ordinate changes in MVP and vRNA levels in
the P100 fraction indicate that vault particle levels vary depending on
the level of drug susceptibility in the cell lines.

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Fig. 4.
A, Western analysis of MVP levels in a
sucrose equilibrium gradient. Fractions correspond to the load 20, 30, 40, 45, 50, and 60% layers (lanes 1-6, respectively);
lane 7, GLC4/ADR P100; lane M is the Novagen
Perfect Protein Marker. B, Northern analysis of RNA
extracted from gradient fractions (lanes 1-6) and a total
RNA (SW1573) standard (lane 7).
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We extended this analysis to four additional MDR lines including the
parental and when available to revertant line(s). The results are
summarized in Table I which shows the fold change in the relative
levels of P100 MVP and vRNA compared with their parental cell lines.
This data supports the conclusion that vault particle levels are also
up-regulated in these MDR cell lines. Analysis of Table I revealed that
as little as 1.3-fold up-regulation was sufficient to confer the drug
resistant phenotype in one cell line compared with a high of 15-fold
seen in another. We reasoned that this variability might be explained
by a difference in the number of vault particles present in the
parental cell lines, perhaps a higher induction would be necessary if
the parent cell started with a lower level of endogenous vaults. Vault
particle levels were determined by analysis of protein levels in the
P100 fraction compared with increasing amounts of purified vaults (Fig. 5). As summarized in Table
II, vault levels per cell vary
considerably among the different cell lines examined and, consistent
with our hypothesis, lines with lower levels of endogenous vault
particles (GLC4/S and MCF7/S) showed the highest fold induction seen in Table I. Among the lines described in Table II, two (GLC4 and SW1573)
were selected with doxorubicin (18-21), the others were selected with
mitoxanthrone (21, 22). The very high number of vaults in the GLC4 drug
resistant line (245,000 vaults per cell) might reflect the high
concentration of doxorubicin to which this line is resistant (1 µM), this level of doxorubicin is ten times greater that
the level used to select the SW1573 resistant lines 0.1 µM (6) which display only one third the number of vaults
per cell. The GLC4 revertant has a reduction in vault number that is
still over 4-fold higher than the parent line. However, this line,
although now sensitive to killing by 1 µM doxorubicin, is
still resistant to 0.1 µM doxorubicin and therefore is
more similar to the SW1573 resistant line with regard to drug
sensitivity than to the SW 1573 revertant. Interestingly the vault
levels in these lines (71,000 versus 85,000) are also quite
similar. This suggests that absolute vault levels may directly dictate the extent of drug resistance. Another factor which may influence the
level of vault induction may be related to the tissue specific distribution of vaults and the cancer type. Thus cells with higher endogenous vault levels, might be primed to become drug resistant and
can do so with a relatively modest induction of vault levels. This
finding is consistent with the distribution of vaults in tissues with
the highest level of exposure to xenobiotics where greater levels of
endogenous vaults could predispose these tissues to drug resistance
(23). Interestingly, the lowest level of vaults was found in the MCF7/S
cell line (10,633 vaults/cell). This line was derived from a breast
carcinoma, and its drug-resistant derivative MCF7/MR (39,000 vaults/cell), has a level of vaults comparable to the drug sensitive
SW1573/S and 8226/S cell lines and might reflect a different required
level of vaults to achieve drug resistance in this tissue type.

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Fig. 5.
Determination of vault copy number in
parental cell lines. P100 fractions representing 2 × 105 or 4 × 105 cells per lane from the parental cell lines (lanes 1, 2 and 3, 4 respectively) and purified rat liver vaults
(0.05-0.25 µg per lane as indicated, lanes 5-9) were
analyzed by Western blotting.
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The mechanism of vault up-regulation is unknown. Although the
overexpression of vaults in MDR cells is intriguing, this does not
prove that vaults are responsible for drug resistance. We hypothesize
that vault overexpression is a critical component of a pathway involved
in non-Pgp MDR and that the mechanism of vault-mediated MDR may be
through vault binding directly to drugs or possibly through vault
interaction with a protein or RNA that binds drugs.
We thank E. G. E de Vries and H. J. Broxterman for kindly providing the MDR cell lines derived from GLC4
and SW1573 lung carcinomas for these studies. We thank Nancy Kedersha,
Michael Carey and members of the Rome laboratory for comments and
discussion of the manuscript.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF045143, AF045144, and AF045145, for HVG1, HVG2, and HVG3