From the British Columbia Cancer Research Centre, Vancouver, British Columbia V5Z 1L3 Canada
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ABSTRACT |
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Mouse transporter protein (MTP) is a highly
conserved polytopic membrane protein present in mammalian lysosomes and
endosomes. The role of MTP in regulating the in vivo
subcellular distribution of numerous structurally distinct small
molecules has been examined in this study by its expression in a
drug-sensitive strain of the yeast Saccharomyces
cerevisiae. Surprisingly, the expression of MTP in membranes of
an intracellular compartment resulted in a cellular resistance or
hypersensitivity to a range of drugs that included nucleoside and
nucleobase analogs, antibiotics, anthracyclines, ionophores, and
steroid hormones. The intracellular bioavailability of steroid hormones
was altered by MTP, as determined using an in vivo
glucocorticoid receptor-driven reporter assay in yeast, suggesting that
the MTP-regulated drug sensitivity arose due to a change in the
subcellular compartmentalization of steroid hormones and other drugs.
MTP-regulated drug sensitivity in yeast was blocked to varying degrees
by compounds that inhibit lysosomal function, interfere with
intracellular cholesterol transport, or modulate the multidrug
resistance phenotype of mammalian cells. These results indicate that
MTP is involved in the subcellular compartmentalization of diverse
hydrophobic small molecules and contributes to the inherent drug
sensitivity or resistance of the mammalian cell.
The mammalian cell is exquisitely compartmentalized into
organelles, distinct vesicle populations, cytoplasm, and the
extracellular space. Metabolic and catabolic processes present within
each of these compartments require the presence of one or more
different types of small molecules. Therefore, the transport and/or
retention of these small molecules between these subcellular
compartments must be tightly regulated to maintain the integrity of
these cellular processes. The lipid membrane architecture of cellular
organelles, vesicles, and the plasma membrane dictates that the
compartmentalization of small hydrophilic molecules occur by
protein-mediated transport processes. Many transporters of hydrophilic
molecules, such as amino acids (1), hexoses (2), and nucleosides (3),
have been identified in the plasma membrane. Membrane proteins that mediate the flux of nucleotide, nucleotide sugars, and nucleotide sulfate into the Golgi and endoplasmic reticulum (4), amino acid
neurotransmitters and biogenic amines into storage vesicles (1, 5), and
substrates across the inner mitochondrial membrane (6) have also been
identified and characterized.
In contrast, mechanisms or transport processes responsible for
regulating a subcellular compartmentalization of small hydrophobic molecules, which can readily permeate lipid membranes, are not as well
understood. Plasma membrane transporters of this type include the
multidrug efflux pump P-glycoprotein (7), the multidrug resistance-associated protein (8), the phosphocholine translocase MDR2
(9), bile acid transporters (10, 11), and fatty acid transporters (12).
P-glycoprotein and multidrug resistance-associated protein found to be
present within intracellular compartments presumably provide a similar
function to their plasma membrane counterparts (8, 13). However, little
else is known of proteins that mediate the intracellular transport of
small hydrophobic molecules.
Mouse transporter protein
(MTP)1 is a murine
4-transmembrane spanning protein present in lysosomes and late
endosomes (14),2 and its
transcript (or that of its mammalian homologs) is expressed in most, if
not all, tissues and cell types
(15).3 MTP exhibits 98%
identity with its human homolog and thus represents a highly conserved
mammalian protein. It is 233 amino acids in length and possesses a
hydrophilic C terminus containing several tyrosine-based sorting motifs
(YXX*, where * represents a bulky hydrophobic residue) that
have been demonstrated, in other membrane proteins, to be responsible
for localization to intracellular compartments (16). A phenotypic
selection assay using Saccharomyces cerevisiae originally
identified a truncated MTP that could mediate a cellular uptake of
thymidine by its inappropriate expression at the plasma membrane (15).
Based upon this observation, MTP was predicted to be involved in the
subcellular compartmentalization of nucleosides.
We are interested in defining further the functional role of MTP in the
subcellular compartmentalization of various types of small molecules
within the mammalian cell. However, it is exceedingly difficult to
examine the in vivo distribution of small molecules in
mammalian cells, the exception being those molecules that are intrinsically fluorescent, such as anthracyclines (17). The yeast
S. cerevisiae has previously been used as a heterologous expression system to study the function of MTP (15) and other mammalian
membrane proteins, including the anion exchanger AE1 protein (18),
Golgi nucleotide-galactose transporters (19), the cystic fibrosis
transmembrane conductance regulator (20), and a vesicular copper
transporter (21). On this basis, we have utilized a sensitive in
vivo reporter system that employs a drug-sensitive strain of
S. cerevisiae to study the physiological role of MTP in
regulating the subcellular distribution of small molecules (22). The
S. cerevisiae protein data base contains no proteins that
exhibit any significant identity with MTP; therefore, interference by a
yeast ortholog of MTP would not be expected.3
The drug-sensitive yeast strain lacks the plasma membrane proteins Pdr5
and Snq2, which are ATP-binding cassette transporters that mediate a
cellular resistance to a wide variety of structurally diverse drugs by
acting as drug efflux pumps (23-28). In essence, these transporters
act to provide an extracellular compartmentalization of drugs. The
absence of these two transporters allows for the accumulation of toxic
concentrations of drug within the cell that ultimately leads to cell
death. Should the expression of MTP in this yeast regulate the
distribution of these toxic drugs between subcellular compartments, a
subcellular sequestration and altered cytotoxicity of the drug may
arise. Evidence to support a functional role for MTP in regulation of
the subcellular distribution of several structurally distinct small
molecules (or their metabolites) is presented.
MTP Expression Vectors--
The open reading frame of MTP was
amplified from pMTP (15) by the polymerase chain reaction method using
oligonucleotides that corresponded to the 5'
(5'-CGCATCCATGGTGTCCATGACTTTC-3', NcoI site
underlined) and 3' (5'-GGCATGGTACCTCAGGCAGGCAGGTAAGGA-3', KpnI site underlined) termini. The polymerase chain reaction
product was subjected to Taq DyeDeoxy terminator cycle
sequencing using an Applied Biosystems model 310 DNA sequencer. The
open reading frame of MTP was subcloned into the yeast expression
vector pYPGE15 to generate pYP-MTP. The pYPGE15 vector, a gift from Joe
Brunelli (Washington State University), contains a URA3 auxotrophic
marker, 2 µm origin, a constitutive phosphoglycerate kinase promotor, and a CYC1 transcriptional terminator.
Cell Culture--
The yeast strains YYM4 (MATa
ura3-52 leu2- Cellular Fractionation of Yeast--
Yeast was grown in
selective medium to an A600 of 1.0 and diluted
5-fold into 500 ml of YPD. The culture was grown to an
A600 of 1.0, whereupon sodium azide was added
(final concentration, 10 mM) and culture was chilled in ice
water. Cells were collected by centrifugation (4000 × g for 10 min) and suspended in 10 ml of SB-Zymolyase (1.4 M sorbitol, 50 mM potassium phosphate, pH 7.5, 10 mM sodium azide, 40 mM Drug Sensitivity Assays--
The sensitivity of yeast to
different chemical compounds was initially screened using a paper disc
assay. Yeast was grown in appropriate selective medium and plated (0.15 A600 units) in top agar (0.7% agarose in
selective medium) onto selective medium-agar plates. A 5-µl aliquot
of drug dissolved in either ethanol or Me2SO was spotted
onto a paper disc (Whatman no. 5, 7 mm diameter), which was then laid
on the top agar. Plates were incubated at 30 °C for 3 days and
examined for cleared zones of growth inhibition. Ethanol or
Me2SO alone spotted onto paper discs did not inhibit growth.
A quantitative drug sensitivity assay was performed using a method
similar to that of Egner et al. (22). Various concentrations of drug prepared in 55 °C selective medium-agar were plated in triplicate into 24-well plates. Actively growing cultures of cells were
diluted to A600 values of 0.12, 0.012, and
0.0012, and then 10-µl aliquots of each dilution were spotted in
triplicate onto drug-containing medium. Plates were incubated at
30 °C for 3 days. A quantitative measure of the drugs effect was
determined based upon the concentration of drug that inhibited colony
formation by 50% (IC50 value). Modulator studies were
performed as above except using a single concentration of drug in the
absence or presence of modulating drug.
Steroid Hormone Response Assays--
Assays were performed using
the yeast strain YNK410, which harbors the genomically integrated
reporter gene GT3Z (22), which had been transformed with the
glucocorticoid receptor expression plasmid pRS314-GN795 (30) and either
pYPGE15 or pYP-MTP. Cell cultures were grown overnight at 30 °C in
selective media, diluted 1:10 into fresh selective media that contained
dexamethasone, progesterone, or ethanol (carrier), and then grown for
14 h. Hormone-induced expression of Immunological Methods--
Yeast protein samples were
denatured in 4 M urea prior to being subjected to
electrophoretic separation by SDS-polyacrylamide gel electrophoresis
and electroblotted onto polyvinylidene fluoride membranes. Membranes
were sequentially incubated with primary antibodies and
peroxidase-conjugated anti-rabbit IgG (Jackson Immunologicals) as
described previously (15) and visualized using the enhanced
chemiluminescence method (Amersham Pharmacia Biotech). Rabbit antiserum
used was specific for DPAP A (Tom Stevens, University of Oregon),
Pep12p (Scott Emr, University of California, San Diego, CA), Vma4p
(Daniel Klionsky, University of California, Davis, CA), Pma1(Andre
Goffeau, Universite Catholique de Louvain), Sec61p (Randy Schekman,
University of California, Berkeley, CA), and MTP (15).
Expression of MTP in YYM4 Yeast--
The open reading frame of the
MTP cDNA was cloned into a high copy yeast expression vector, and
its expression in YYM4 cells was examined by immunoblotting analysis.
MTP was present as a low abundance protein in the membranes of cells
transformed with the MTP-containing vector, but was not detected in
cells transformed with the parental pYPGE15 vector (Fig.
1A). The cellular location of
MTP in yeast was assessed by immunoblot analysis of subcellular fractions isolated by means of discontinous sucrose gradient
centrifugation (Fig. 1B). MTP appeared to colocalize to
fractions most tightly associated with antibody markers for Golgi (DPAP
A) and prevacuolar vesicles (Pep12p), but not with those for plasma
membrane (Pma1), endoplasmic reticulum (Sec61p), and vacuole (Vma4p).
These results indicated that the localization of MTP was restricted to
intracellular membranes, most probably those of Golgi or Golgi-derived
vesicles and/or the endosome-like prevacuolar vesicles (31).
Drug Sensitivity of MTP-expressing Yeast--
MTP-expressing
yeast were initially screened for sensitivity to a variety of compounds
using a paper disc assay. Drug sensitivity, relative to control YYM4
yeast, was assigned based on the diameter of the zone of growth
inhibition (Fig. 2). The expression of
MTP in YYM4 yeast conferred increased resistance or a hypersensitivity to a range of compounds that included antibiotics, anthracyclines, neutral and carboxylic ionophores, nucleoside and nucleobase analogs, steriod hormones, dihydropyridines, phenothiazines, and cationic lipophilic molecules (Table I).
MTP-expressing yeast did not exhibit altered sensitivity toward
cyclohexamide, vancomycin, tamoxifen, or cisplatin, as compared with
control yeast. The parental strain, YPH499, exhibited none or little
sensitivity to any of the compounds examined (not shown).
The degree of cellular drug resistance or sensitivity conferred by the
expression of MTP toward a number of compounds was quantitated using a
colony formation assay, an example of which is presented in Fig.
3. The concentrations required to inhibit colony formation by 50% (IC50) determined for control and
MTP-expressing yeast are shown in Table
II. MTP conferred an increased resistance of approximately 1.5-15-fold to drugs that included carbonyl cyanide m-chlorophenyl-hydrazone (CCCP), nicardipine, erythromycin,
progesterone, and rhodamine 123. Conversely, MTP-expressing yeast was
2-50-fold more sensitive than control cells to trifluoperazine, TPP
bromide, 5-fluorouracil, and 5-fluorouridine. The expression of MTP
within the membranes of the Golgi and/or prevacuolar compartments could significantly modify the cytotoxicity of many structurally distinct drugs, suggesting that this effect occurred via a change in the subcellular distribution and subsequent intracellular bioavailability of these drugs.
Modulation of MTP-mediated Drug Sensitivity--
The profile
of drug resistance mediated by MTP exhibited a striking similarity to
that exhibited by multidrug-resistant (MDR) mammalian cells that
overexpress the P-glycoprotein (also termed MDR1). P-glycoprotein is a
ATP-binding cassette transporter that mediates resistance to
rhodamines, anthracyclines, steroid hormones, and carboxylic ionophores
(7, 32). Cells that overexpress P-glycoprotein also exhibit a
collateral sensitivity or hypersensitivity toward various hydrophobic
molecules (33); however, P-glycoprotein does not mediate resistance or
hypersensitivity toward 5-fluorouracil or 5-fluorouridine (34). The MDR
phenotype in mammalian cells can be blocked by the presence of one of
many different modulators, which are hydrophobic molecules that
generally possess a tertiary nitrogen (35).
We assessed the ability of several MDR modulators to block the drug
hypersensitivity or resistance mediated by the expression of MTP in
YYM4 yeast through the use of the quantitative plating assay, an
example of which is shown in Fig. 4. The
modulators, which included amiodarone, dipyridamole, reserpine, and
verapamil, did not exhibit toxicity to YYM4 yeast at the concentrations
utilized (not shown). All modulators completely failed to reverse the
hypersensitivity to 5-fluorouridine or the resistance to nicardipine
(Table III). Conversely, all modulators
completely blocked resistance to progesterone. Resistance to CCCP,
erythromycin, and rhodamine 123 was blocked to various degrees by each
modulator. These results indicated that these MDR modulators were, to
varying extents, able to block MTP-mediated
resistance/hypersensitivity. This modulation effect was dependent upon
drug-modulator combinations present, a characteristic that is shared
with the interaction of these modulators with P-glycoprotein (32,
35).
Regulation of Intracellular Steroid Bioavailability by
MTP--
The mechanism by which steroid hormones exert their
cytotoxicity in yeast is not known but is thought to be related to
their interference in the pathways of biosynthesis or cellular
transport of sterols. The yeast ATP-binding cassette transporter Pdr5
can reduce the cellular accumulation of steroid hormones by
transporting these molecules into the extracellular space, thereby
circumventing their toxic effects (22, 25, 27). Decreased cellular
accumulation of steroid hormones mediated by Pdr5 has been directly
demonstrated by an in vivo reporter assay in the YNK410
yeast strain (22, 25). YNK410 yeast harbors (i) a genomically
integrated cassette composed of glucocorticoid response elements
upstream of a
MTP was able to mediate a cellular resistance to steroid hormone
toxicity; therefore, we used the YNK410
The correlation of steroid resistance with an increased intracellular
bioavailability in MTP-expressing cells suggests that MTP does not
provide a cellular resistance to steroid hormone toxicity by tightly
binding and sequestering these molecules, but rather by mediating a
change in the subcellular localization of steroid hormones that
prevents their interference in pathways of sterol transport or biosynthesis.
MTP is a member of the mammalian 4-transmembrane spanning
protein superfamily, which includes the "tetraspanin" family (37), the PMP22 family (38), the "synaptic vesicle" family (39, 40), the
"gap/tight junction" family (41, 42), TWIK-1 (43), and micosomal
epoxide hydrolase (10). Members of the synaptic vesicle and gap/tight
junction families participate in the formation of pores, TWIK-1
functions as a K+ channel, and microsomal epoxide hydrolase
can transport bile acids. However, the physiological functions of the
majority of these four-transmembrane-spanning proteins have not yet
been defined, and most are postulated to be involved in roles ranging
from pore formation to cell proliferation and differentiation. The
present study, which employed in vivo yeast reporter
systems, has demonstrated that MTP expressed in intracellular
organelles can function to regulate the cytotoxicity and subcellular
localization of molecules that include cationic amphiphilic drugs,
hydrophobic anions and cations, hydrophobic steroids, and the more
hydrophilic charged nucleobases/nucleoside analogs.
Transport processes that mediate the flux of small hydrophilic
molecules across the membranes of lysosomes and endosomes in mammalian
cells are relatively well documented (44, 45). In contrast, the
mechanisms of lysosomal and endosomal compartmentalization and/or
retention of small hydrophobic molecules are not as well understood.
Cationic amphiphilic drugs, such as anthracyclines, nicardipine,
trifluoperazine, and erythromycin, are concentrated within lysosomes
and endosomes due to the passive diffusion of drug across the organelle
membrane and their subsequent protonation into a membrane impermeable
form (17, 46-48). Hydrophobic anions (i.e. nigericin and
monensin) and cations (i.e. TPP, CCCP, and ethidium) also
become concentrated inside endosomes and lysosomes (22, 49-53). The
mechanism responsible for localization of these molecules is apparently
driven by the activity of V-type H+-ATPases (54), but there
also appears to be a second, as yet unknown, mechanism for uptake
and/or binding of these types of molecules in lysosomes (46).
Progesterone and other steroid hormones can block the transport of
cholesterol from lysosomes by an unknown mechanism (55-57), implying
the presence of steroid hormones within membranes of these organelles.
These limited studies on the interaction of hydrophobic molecules with
lysosomes and endosomes suggest that their compartmentalization arises,
at least in part, through mediated transport processes. The ability of
MTP, a membrane protein localized to lysosomes and endosomes, to
regulate the distribution of these hydrophobic molecules in yeast
reporter systems suggests that MTP may be involved in regulating the
localization and/or retention of these hydrophobic molecules within
these organelles in mammalian cells.
In light of the apparent interaction of MTP with hydrophobic small
molecules, the ability of MTP to regulate the cytotoxicity of
6-aza-adenine, 5-fluorouracil, and 5-fluorouridine is intriguing. Nucleobases and nucleosides, including 5-fluorouracil and
5-fluorouridine, are rapidly metabolized into their respective
nucleotide forms upon uptake into mammalian cells (58, 59); therefore,
these predominant nucleotide forms should be the substrates for any subcellular compartmentalization event. The extent of distribution of
these nucleotides into lysosomes and endosomes is unclear, although
evidence for an energy-dependent vesicular nucleotide transporter exists (60). MTP has previously been shown to mediate an
increased membrane permeability toward nucleosides (15); however, these
studies were preformed under in vivo conditions that may
have allowed for conversion of nucleoside to its nucleotide form. In
addition, the nucleobases and nucleoside employed in the present study
differ from their physiological counterparts by the presence of a
positive or negative charge on their purine or pyrimidine moieties,
which in itself may be an important factor for regulation by MTP.
Clearly, further studies are required to define the interaction of MTP
with nucleotides, nucleosides, and nucleobases.
The MDR phenotype in mammalian cells that arises due to the
overexpression of P-glycoprotein can be blocked by MDR modulators, the
action of which has been attributed to their ability to bind to
P-glycoprotein and inhibit its function (32, 35). It also appears these
modulators can induce subtle changes in the inherent drug sensitivity
of non-drug-resistant or "normal" cells (61, 62), but their
mechanisms of action under these conditions have not been defined. It
is relevant that MDR modulators can block MTP-mediated resistance
to several different drugs in our yeast reporter system, because this
observation suggests that MDR modulators may be capable of altering the
inherent drug sensitivity in mammalian cells by interfering with the
function of MTP in lysosomes or endosomes.
Most MDR modulators can induce an in vivo inhibition of
lysosomal function (47, 63, 64), but there is clear evidence for a
specific interaction of MDR modulators in the transport of cholesterol
from lysosomes. Cholesterol is transported bidirectionally between the
lysosomes and the plasma membrane (65), and it has been suggested that
this transport occurs via mediated processes based upon the ability of
(i) steroid hormones and cationic amphiphilic MDR modulators to block
these transport processes and induce a lysosomal accumulation of
cholesterol (55-57, 65), and (ii) oxysterols to reverse this
cholesterol accumulation in a manner independent of lysosomal pH (65).
Proteins involved in this homostasis of intracellular cholesterol
transport would be expected highly conserved and expressed in all
tissues and cell types. One such protein is NPC1, a membrane protein
implicated in Niemann-Pick C disease (66). An absence of NPC1 leads to
the disruption of intracellular cholesterol transport; however, its
actual function and subcellular location are unknown (67). It is
interesting that there exists an interaction between MTP, a highly
conserved and widely expressed lysosomal/endosomal protein, and both
steroid hormones and MDR modulators. Our study suggests that MTP may be
involved in the homostasis of intracellular transport of steroid
hormones or cholesterol.
MTP may regulate the intracellular compartmentalization of small
molecules in the yeast reporter systems and mammalian cells by one of
several mechanisms. The protein may be a transporter that interacts
directly with small molecules to facilitate their flux across the
membranes of lysosomes and endosomes. Conversely, the presence of MTP
may regulate the function of other transporters by direct
protein-protein interaction or by indirectly altering the character of
the membrane lipid, the transmembrane H+ gradient, or the
lumenal contents of the lysosome/endosome. Future studies will be
necessary to define the mechanistic nature of MTP, but whichever
mechanism is responsible, this study clearly demonstrates that MTP is
capable of regulating the subcellular compartmentalization of small
hydrophobic molecules.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 his3-
200 ade2-101oc
lys2-801amb
pdr5::TRP1
snq2::hisG) and YPH499 (MATa
ura3-52 leu2-
1 his3-
200 ade2-101oc
lys2-801amb trp1-
63) were kindly provided by Karl
Kuchler (University and Biocenter of Vienna). Yeast strain YNK410
(MATa ura3-52 leu2-
1 his3-
200
ade2-101oc lys2-801amb trp1-
63
pdr5::GT3Z) was obtained from Anastasia Kralli
(Biozentrum, University of Basel). Yeast strains were grown at 30 °C
on YPD or selective media that contained auxotrophic supplements for plasmid maintenance as described by Kaiser et al. (29).
-mercaptoethanol,
0.3 mg/ml Zymolyase 100T) and incubated at 30 °C for 40 min.
Spheroplasts were pellet by centrifugation (700 × g
for 10 min) and suspended in 5 ml of lysis buffer (0.8 M
sorbitol, 10 mM MOPS, pH 7.2, 1 mM EGTA) that contained Complete protease inhibitor mixture (Roche Molecular Biochemicals). Spheroplasts were lysed by Dounce homogenization, and
homogenate was subjected to centrifugation (2500 × g,
10 min, 4 °C) to pellet unbroken cells. The supernatant was
collected, and 1-ml portions of the homogenate were layered onto a
discontinuous gradient composed of 1 ml each of 12, 18, 24, 30, 36, 42, 48, 54, and 60% (w/v) sucrose in lysis buffer. Gradients were
centrifuged (100,000 × g for 16 h at 4 °C),
and 1-ml fractions were collected from the bottom of the gradient.
Total cellular membranes were collected by centrifugation (100,000 × g for 60 min at 4 °C) of the Dounce homogenate.
-galactosidase (
-gal) in
cell lysates was measured using a glass bead
lysis/o-nitrophenyl-
-D-galactosidase assay method (29). Reactions were terminated after identical periods of
incubation, and A420 was measured. Protein
content of cell lysates was determined, and specific activity of
-gal in cell lysate was expressed as nmol/min/mg of protein.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (27K):
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Fig. 1.
Expression and subcellular localization of
MTP in S. cerevisiae. YYM4 yeast transformed with
pYPGE15 or pYP-MTP were examined for the presence of MTP. A,
total membrane fractions were prepared, and equal quantities were
subjected to SDS-polyacrylamide gel electrophoresis and immunoblot
analysis with anti-MTP antibody. Arrow indicates
immunoreactive species. B, YYM4 yeast transformed with
pYP-MTP were lysed and subjected to subcellular fractionation by
centrifugation on a discontinous 12-60% (w/v) sucrose gradient. Equal
portions of gradient fractions were subjected to SDS-polyacrylamide gel
electrophoresis and immunoblot analysis using anti-MTP antibody or
antibodies specific for markers of the plasma membrane
(Pma1), Golgi (DPAP A), endoplasmic reticulum
(Sec61p), vacuole (Vma4p), and prevacuolar
(Pep12p) compartments.
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Fig. 2.
Assessment of drug sensitivity of
MTP-expressing yeast using the disc assay. YYM4 yeast containing
pYPGE15 vector (control) or pYP-MTP (MTP) were
plated in top agar and subjected to paper disc assays to determine
MTP-mediated drug resistance. In this example, 5-µl aliquots of the
following drug solutions (in Me2SO) were deposited onto
individual paper discs: nicardipine (194 mM),
trifluoperazine (312 mM), progesterone (636 mM), or ethidium bromide (220 mM). Zones of
growth inhibition were examined after 3 days of incubation at
30 °C.
Effect of expression of MTP on the drug sensitivity of YYM4 yeast
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Fig. 3.
Quantitation of drug sensitivity of
MTP-expressing yeast by plating assay. A quantitative measure of
the drug resistance or sensitivity was determined by measuring the
colony formation of serially diluted cultures of YYM4 yeast containing
pYPGE15 vector (control) or pYP-MTP (MTP) that
had been aliquoted onto selective media that contained either drug
carrier (Me2SO) or increasing concentrations of drug
solution. Shown are examples using drug carrier (Me2SO),
nicardipine (670 µM), trifluoperazine (440 µM), progesterone (500 µM), erythromycin
(1500 µM), and 5-fluorouridine (40 µM).
Quantitation of the MTP-mediated resistance/hypersensitivity in YYM4
yeast
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Fig. 4.
Modulation of drug sensitivity mediated by
MTP. The ability of MDR modulators to block MTP-mediated drug
resistance or sensitivity was assessed by the inclusion of modulator in
quantitative plating assays that employed a single drug concentration.
In this example, YYM4 yeast containing pYPGE15 vector
(control) or pYP-MTP (MTP) was aliquoted onto
selective media that contained progesterone (500 µM)
alone or in addition to verapamil (1570 µM), dipyridamole
(400 µM), reserpine (470 µM), or amiodarone
(650 µM).
Effect of modulators on MTP-mediated drug resistance/hypersensitivity
-gal reporter gene, and (ii) a disrupted
PDR5 gene (the absence of the Pdr5 transporter allows for an
intracellular accumulation of steroid hormones). Expression of a
glucocorticoid receptor in YNK410 yeast drives transcription of the
-gal reporter gene in a steroid hormone-dependent
fashion and therefore provides a direct measurement of the
intracellular bioavailability of steroid hormone.
-gal reporter system to
measure whether this resistance was associated with an alteration in
the intracellular bioavailability of steroid hormones. Exposure of
MTP-expressing cells to 10 µM dexamethasone or
progesterone induced approximately 15- and 25-fold greater levels of
-gal activity, respectively, than compared with control cells under identical conditions (Fig.
5A). This effect of
dexamethasone and progesterone was dose-dependent, because
greater levels of
-gal expression were observed in the presence of
MTP than in its absence over a 100-fold concentration range (Fig.
5B). The apparent saturation and decline of
-gal
expression that was observed at higher concentrations of dexamethasone
has previously been documented in this assay and the ability of
dexamethasone to induce greater overall levels of
-gal expression
than progesterone in both MTP-expressing and -nonexpressing cells
correlated with the different affinities of these steroid hormones for
the glucocorticoid receptor (36). These experiments revealed that the
expression of MTP in YNK410 cells resulted in an increased
intracellular bioavailability of progesterone and dexamethasone that
subsequently allowed for greater steroid hormone interaction with the
glucocorticoid receptor.
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Fig. 5.
Effect of MTP expression on steroid hormone
response. YNK410 cells harboring pRS314-GN795 and either not
expressing (solid) or expressing (hatched) MTP
were grown in steroid hormone-containing medium, and cell lysates were
prepared and assayed for -gal activity. A, activity in
cells grown in 10 µM dexamethasone or progesterone.
Presented are mean measurements for three independent assays. S.E.
values are indicated (bars) or were too small to be
observed. B, activity in cells grown in increasing
concentrations of dexamethasone or progesterone. Shown is a single
representative assay.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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FOOTNOTES |
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* This study was supported by the National Cancer Institute of Canada and the Alberta Heritage Foundation for Medical Research.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Recipient of an Alberta Heritage Foundation for Medical Research
Fellowship. To whom correspondence should be addressed: BC Cancer
Research Centre, 601 W. 10th Ave., Vancouver, British Columbia V5Z 1L3, Canada. Tel.: 604-877-6000; Fax: 604-877-6155; E-mail: dhouge{at}bccancer.bc.ca.
2 C. Cass, personal communication.
3 D. Hogue, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are:
MTP, mouse
transporter protein;
-gal,
-galactosidase;
CCCP, carbonyl cyanide
m-chlorophenyl-hydrazone;
MDR, multidrug-resistant;
TPP, tetraphenylphosphonium;
MOPS, 4-morpholinepropanesulfonic acid.
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