From the Medical Research Council Clinical
Sciences Centre, Imperial College Faculty of Medicine, Hammersmith
Hospital Campus, Du Cane Road, London W12 0NN and § Cancer
Research UK, Paterson Institute for Cancer Research Cell Regulation
Laboratory, Manchester M20 4BX, United Kingdom
Received for publication, October 30, 2002, and in revised form, December 16, 2002
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ABSTRACT |
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Multidrug resistance in acute myeloid
leukemia is often conferred by overexpression of P-glycoprotein,
encoded by the MDR1 gene. We have characterized the key
regulatory steps in the development of multidrug resistance in K562
myelogenous leukemic cells. Unexpectedly, up-regulation of
MDR1 levels was not due to transcriptional activation but
was achieved at two distinct post-transcriptional steps, mRNA turnover and translational regulation. The short-lived (half-life 1 h) MDR1 mRNA of naïve cells (not exposed
to drugs) was stabilized (half-life greater than 10 h) following
short-term drug exposure. However, this stabilized mRNA was not
associated with translating polyribosomes and did not direct
P-glycoprotein synthesis. Selection for drug resistance, by long-term
exposure to drug, led to resistant lines in which the translational
block was overcome such that the stabilized mRNA was translated and
P-glycoprotein expressed. The absence of a correlation between
steady-state MDR1 mRNA and P-glycoprotein levels was
not restricted to K562 cells but was found in other lymphoid cell
lines. These findings have implications for the avoidance or reversal
of multidrug resistance in the clinic.
MDR1 is the most common
impediment to successful chemotherapy for a variety of cancers (1). The
most frequent form of drug resistance in relapsed acute leukemia is
overexpression of P-glycoprotein (2, 3). P-glycoprotein is a member of
the ATP-binding cassette superfamily of active transporters and
functions as an energy-dependent efflux pump that reduces
the intracellular concentration of cytotoxic compounds and, hence,
their toxicity. P-glycoprotein has a broad substrate specificity and
can confer resistance to a wide range of different cytotoxic compounds
(4).
Most pre-clinical and clinical efforts to overcome MDR aim to modulate
P-glycoprotein activity. However, clinical trials of compounds that
inhibit P-glycoprotein activity have had limited success and led to
adverse pharmacokinetic side effects (1). It may, therefore, be more
appropriate to target MDR1 expression. Indeed,
MDR1 transcription has been targeted with Ecteinascidin 743 in pre-clinical studies (5) and more recently by modulation of the
nuclear receptor SXR (6). Strategies involving antisense and
transcriptional decoy (7) and the use of anti-MDR1 mRNA hammerhead ribozymes have also been suggested (8).
Stresses such as short-term exposure to cytotoxic drugs results in the
up-regulation of MDR1 mRNA levels in many cell lines (9-13) and in human metastatic sarcomas in vivo (14). This
is frequently due to transcriptional activation of the MDR1
gene and has been reported in many cell lines after different physical and chemical stimulations and in cells selected for resistance to a
variety of cytotoxic drugs (5, 9, 10, 15, 16). In cell lines selected
for drug resistance, increased MDR1 gene expression is also
the result of amplification of the MDR1 locus and the
appearance of self-replicating episomes (4). Gene rearrangements that
constitutively activate MDR1 transcription have also been associated with refractory acute lymphocytic leukemia (17, 18).
Although regulation due to changes in the MDR1 mRNA
stability (19), P-glycoprotein turnover (20), or trafficking (21) have
been suggested, transcriptional regulation is widely considered to be
the key step accounting for the complex spatio-temporal pattern of
expression in vivo (22, 23). It has also generally been
assumed that up-regulation of MDR1 mRNA leads to an
increase in P-glycoprotein. For example, human renal carcinoma (16) and rat liver cells (24) up-regulate both MDR1 mRNA and
P-glycoprotein following different stresses and are consequently
transiently resistant to vinblastine.
In this study, we show that in K562 leukemic cells the levels of
MDR1 mRNA increase in a dose- and
time-dependent manner upon transient exposure to a variety
of cytotoxic drugs. However, in contrast to the general prevailing
models, we show that this is due to stabilization of mRNA and not
because of transcriptional activation. Furthermore, the newly
stabilized mRNA is not translated and so does not result in
expression of P-glycoprotein and drug efflux. Only on subsequent
long-term selection for drug resistance does the stabilized mRNA
associate with polyribosomes, permitting translation of P-glycoprotein
and drug efflux. The finding that drug resistance is a two-step
post-transcriptional process, mediated by changes in both
MDR1 mRNA stability and translation, suggests new
possibilities for treatment regimes to circumvent MDR in leukemia.
Cell Lines and Culture--
K562, CCRF-CEM, and MANN
cells were cultured in RPMI 1640 medium (Invitrogen) with 10%
fetal calf serum and 2 mM L-glutamine; KB-V1 cells (25) were cultured in Dulbecco's modified Eagle's medium
(Invitrogen) with 20% fetal calf serum and 110 µM
vinblastine (Sigma). All drugs were obtained from Sigma. For transient
drug treatments (inductions), exponentially growing cells were seeded at 1 × 106 cells/ml and incubated with drug for the
indicated times. The drug concentrations used were determined
previously to cause macroscopic changes in cell morphology, indicative
of cytotoxic stress, such as swelling, and changes in shape and
granularity, in greater than 50% of cells. Unless otherwise stated,
drug inductions were for 3 days using 3.4 nM doxorubicin,
22 µM vinblastine, 2.5 nM colchicine, 1.34 µM colcemid, or 100 µM cytarabine.
Drug-resistant K562 sublines were obtained in a one-step selection by
exposure to concentrations of the following cytotoxic drugs, which had been shown to result in 99.9% cell death after 14 days in culture in
preliminary experiments: 40 pM colchicine, 30 pM doxorubicin, or 25 µM
1- Determination of mRNA Expression by Semi-quantitative and
Real-time RT-PCR--
RNA was prepared from cells by RNAzol extraction
(Biogenesys, Poole, United Kingdom), reverse-transcribed (Roche
Molecular Biochemicals), and amplified by PCR. Semi-quantitative
RT-PCR estimation of MDR1 mRNA levels was performed as
described (26). Real-time quantitative PCR (Taqman; PerkinElmer Life
Sciences) used the following primers within the MDR1
coding sequence: sense, 5'-TTGTTCAGGTGGCTCTGGAT-3'; antisense,
5'-CTGTAGACAAACGATGAGCTATCACA-3'; and probe,
5'-AGGCCAGAAAAGGTCGGACCACCA-3'. Taqman Universal PCR Master Mix and
control amplimers for GAPDH and 18 S ribosomal RNAs were used as recommended by the supplier (PerkinElmer Life Sciences). Results were collected and analyzed with an ABI Prism 7700 sequence detection system (PerkinElmer Life Sciences) as follows: the
PCR cycle number that generated the first fluorescence signal above a
threshold (threshold cycle, CT; 10 standard deviations above the mean fluorescence generated during the baseline cycles) was
determined, and a comparative CT method was then used to
measure relative gene expression. The following formula was used to
calculate the relative amount of the transcript in the sample and
normalized to an endogenous reference (GAPDH or 18 S rRNA): 2 Determination of RNA Half-lives--
K562, KC40, and KD30 cells
were incubated with 12.5 µg/ml actinomycin D to inhibit
transcription. This concentration of actinomycin D was determined
empirically in a series of pilot experiments as that which inhibited
greater than 95% of [3H]uridine incorporation in both
the naïve and drug-resistant K562 cell lines, and thus nascent
transcription, within 1 h (27). Cells were harvested at different
times after actinomycin D addition, and total RNA was isolated as
above. Total RNA, or the poly(A)+ fraction isolated by
using an Oligotex kit (Qiagen), was used in real-time Taqman RT-PCR
assays. In addition, Northern blots (28) of 10 µg of total RNA from
the above samples were hybridized with probes derived from
gene-specific sequences from the MDR1, GAPDH,
Id2, or RAR- Genomic DNA and Southern Blotting--
Genomic DNA was isolated
from cells by proteinase K digestion and phenol/chloroform extraction,
digested with EcoRI, Southern blotted, and hybridized to
nick-translated radioactive probes by standard procedures (28). Probes
were derived from the MDR1 5'-end region (975-bp
PstI fragment comprising the transcription start point,
5'-untranslated region, and first intron) (31) and the Nuclear Run-on and Luciferase Transcription Assays--
Nuclei
were prepared from naïve, drug-induced, or drug-resistant K562
sublines as described (27). Nuclear run-on transcription and
hybridization methods have been described elsewhere (33). For the
MDR1 promoter-luciferase transcriptional assays, K562, KC40,
and KD30 cells were transiently transfected with
pMDR1( Measurement of Cell Surface P-glycoprotein by Flow
Cytometry--
Analysis of surface P-glycoprotein expression was by
flow cytometry using the fluorescently labeled monoclonal antibody UIC2 (Immunotech, Marseille, France), essentially as described (36), using a
Becton Dickinson Flow Cytometer (BD Biosciences). Live cells were
detected by exclusion of propidium iodide. Drug-induced cells were
monitored 1, 2, 3, and 4 days after drug addition. Where indicated,
single cells were sorted into 96-well plates after UIC2 staining at a
density of one cell per well by a FACS Vantage (BD Biosciences) and
clonally expanded.
Western Blot Analysis--
Crude cell membrane fractions were
prepared from 1 × 108 cells essentially as described
(37), with minor modifications. Briefly, cells were lysed with a
hypotonic buffer (50 mM mannitol, 50 mM Tris-HCl, pH 7.4, 2 mM EGTA) and centrifuged at a low speed
(500 × g) to pellet nuclei and associated membranes
such as endoplasmic reticulum and Golgi apparatus (plasma
membrane-depleted fraction). The supernatant from the low speed
fractionation was further centrifuged at 100,000 × g
to obtain the plasma membrane-enriched fraction. Plasma
membrane-enriched and -depleted fractions (200 µg) were separated by
SDS-PAGE, and proteins were transferred electrophoretically to
Immobilon membranes (Millipore, Watford, United Kingdom). Filters were
incubated overnight at 4 °C with 0.1 µg/ml of the
anti-P-glycoprotein monoclonal C219 (Cis-Bio International,
Gif-Sur-Yvette, France) or 1:1000
anti-Na+/K+-ATPase De Novo Protein Synthesis--
Incorporation of
[35S]Met in nascent proteins was determined by
trichloroacetic acid precipitable counts, following standard procedures
(38).
Sucrose Gradient Density Centrifugation and Detection of RNA
across Polysome Profiles--
Extracts of drug-induced K562, KC40, and
MANN cells, sucrose gradient centrifugation, and RNA extraction
followed standard procedures (39). Where indicated, buffers contained
20 mM EDTA. Isolated RNA was precipitated with 3 M LiCl and resuspended in 10 µl of water. Detection of
MDR1 mRNA was by RT-PCR as described above.
MDR1 mRNA Is Up-regulated in Drug-induced and Drug-resistant
K562 Cells--
The myeloid leukemia-derived cell line K562 expresses
very low levels of MDR1 mRNA, barely detectable by
standard RT-PCR assays (Fig.
1A), although readily
determined by a more sensitive method like real-time RT-PCR using
poly(A)+ mRNA (data not shown). K562 cells responded to
short-term exposure (drug induction) to several different cytotoxic
drugs (doxorubicin, colchicine, colcemid, vinblastine, and cytarabine)
by up-regulating MDR1 mRNA levels (Fig. 1A).
Real-time RT-PCR showed a 30- to 100-fold increase in MDR1
mRNA levels in drug-induced cells compared with naïve (not
exposed to drug) K562 cells (data not shown). This effect was due to
the cytotoxic drug, because the increase in MDR1 mRNA
was both time- and dose-dependent (Fig. 1B).
To generate K562 sublines resistant to low levels of drugs, three
cytotoxic drugs with different modes of action were used, colchicine
(which binds tubulin and prevents mitosis), doxorubicin (a DNA
intercalating agent), and cytarabine (a pyrimidine analogue). A
one-step drug selection resulted in the generation of resistant pools
of clones (see "Experimental Procedures"). These lines were called
KC40, KD30, and KA25 (colchicine-, doxorubicin-, and cytarabine (araC)-resistant, respectively). MDR1 mRNA was
substantially up-regulated in each of these lines (Fig. 1A),
shown by real-time RT-PCR to be between 2- and 5-fold greater than the
levels found in the 3-day drug-induced cells (data not shown). Thus,
both drug induction and selection for drug resistance result in
substantial increases in steady-state MDR1 mRNA levels,
independent of the mode of action of the drug.
Up-regulation of MDR1 mRNA Is Not Due to Gene Amplification,
Gene Rearrangement, or Transcriptional Activation--
Gene
amplification is a common mechanism for the up-regulation of
MDR1 mRNA in cell lines (4) and is normally accompanied by rearrangements and deletions in the amplified locus. Genomic DNA
from drug-resistant (KC40 and KD30) cells had the same MDR1 gene copy number as the parental line K562, thus no detectable amplification or rearrangements of the locus had occurred (Fig. 2A).
To determine whether MDR1 mRNA up-regulation was due to
transcriptional activation (40), we used two different approaches. We
initially used a gene reporter assay in which an MDR1
promoter fragment was placed upstream of the luciferase reporter gene
(34). This reporter plasmid has been used previously to demonstrate transcriptional activation of MDR1 in, among others, human
colon carcinoma SW620 cells (34). Luciferase expression was equivalent in naïve (not drug-treated) and all of the drug-resistant K562 lines (Fig. 2B), suggesting that there is no promoter
up-regulation. As AP-1 plays a role in the transcriptional activation
of MDR1 (41), and because K562 cells resistant to etoposide
(another P-glycoprotein substrate) have up-regulated levels of
c-jun and c-fos mRNAs and increased AP-1
binding activity (42, 43), it was necessary to exclude the possibility
that the AP-1 pathway had been inactivated in these cells. To do this
we used a reporter plasmid, pAP1-luc, in which the
luciferase gene expression was driven by four human collagenase
TPA-responsive elements and the minimal rat prolactin promoter (35).
This TPA-responsive element has been used in numerous studies because
of its high affinity for the AP-1 complex, and, as expected, transient
transfection in Jurkat T-cells results in an increase of luciferase
activity upon TPA activation (35), which increases MDR1 gene
expression in several cell lines, including K562 cells (44).
Naïve and drug-resistant cells transiently transfected with
pAP1-luc produced similar levels of luciferase activity,
both before and after TPA activation (Fig. 2C). Thus, the
pathway responsible of activating AP-1 is equally functional in both
naïve and drug-resistant K562 cells, and the lack of
transcriptional activation of the MDR1 promoter in
drug-resistant cells cannot be due to a lack of AP-1 functionality.
To confirm that transcriptional activation of the MDR1
promoter is not responsible for MDR1 mRNA up-regulation
we also studied the MDR1 promoter in its native chromosomal
context by nuclear run-on experiments. Despite the increase in
steady-state MDR1 mRNA levels, transcription initiated
from the MDR1 promoter was low compared with transcription
from other control genes (e.g. GAPDH,
Up-regulation of MDR1 Is Due to mRNA
Stabilization--
Because the increase in steady-state
MDR1 mRNA was not due to de novo mRNA
synthesis, we asked whether changes in the rates of mRNA decay
might be involved. To determine the half-lives of MDR1
mRNA in naive, drug-induced and drug-resistant cells, we treated
cells with actinomycin D to inhibit transcription (see "Experimental
Procedures"). MDR1 mRNA from naïve K562 cells
had a very short half-life (approximately 1 h) determined by
real-time RT-PCR (Fig. 3A). In
contrast, MDR1 mRNA half-life values for doxorubicin-
and colchicine-resistant K562 cells were 12-16 h (Fig. 3A).
This was confirmed by Northern analysis (Fig. 3B) and is in
good agreement with the 14-h half-life reported for another independently derived doxorubicin-resistant K562 line (K562/ADR) (45).
Drug-induced K562 cells also had a long MDR1 mRNA
half-life (Fig. 3A). The half-lives of other short-lived
messages, such as Id2 and RAR- Drug Induction of MDR1 mRNA Does Not Lead to P-glycoprotein
Expression--
Transient exposure to cytotoxic drugs (drug induction)
led to an increase in MDR1 mRNA levels through mRNA
stability. This observation led us to ask whether MDR1
mRNA up-regulation was accompanied by an increase in P-glycoprotein
expression. Cell surface expression of P-glycoprotein was measured by
flow cytometry using the P-glycoprotein-specific monoclonal antibody
UIC2 (36). There was no significant increase in UIC2-positive cells
following drug induction at any stage during the 4-day incubation
period compared with naïve K562 cells. In contrast, cells
selected for resistance to the P-glycoprotein substrates colchicine
(KC40 cells) or doxorubicin (KD30 cells) showed a large (10- to
100-fold) increase in UIC2 reactivity, indicating the presence of
active P-glycoprotein on the cell surface. Cells selected for
resistance to cytarabine (KA25 cells), which is not transported by
P-glycoprotein, showed much lower levels of surface P-glycoprotein
expression than the KC40 and KD30 cells, despite having similar
MDR1 mRNA levels. Only 40% of cytarabine-resistant
(KA25) cells expressed surface P-glycoprotein, consistent with the fact
that resistance to cytarabine is known to be mediated by other
P-glycoprotein-independent mechanisms (11) (Fig.
4A).
The Absence of P-glycoprotein at the Cell Surface Is Not Due to a
Defect in Protein Trafficking--
Because the assay above detects
only active P-glycoprotein in the plasma membrane, it was necessary to
exclude the possibility that P-glycoprotein was expressed in
drug-induced cells, but either inserted in the plasma membrane in an
inactive form or accumulated intracellularly. Cell membranes from
naïve, drug-induced, and drug-resistant K562 cells were
analyzed for P-glycoprotein expression by immunoblotting using the
monoclonal antibody C219. A band of ~190 kDa, corresponding to mature
P-glycoprotein, was detected in the plasma membrane-enriched fraction
from drug-resistant lines but not from the naïve or
drug-induced cells (Fig. 4B). P-glycoprotein was also absent
from plasma membrane-depleted fractions (i.e. nuclear and
rough endoplasmic reticulum) of these cells, indicating that the
protein was not retained intracellularly (Fig. 4B). The intensity of the C219-specific band was consistent with the level of
UIC2 binding (Fig. 4A). Thus, although drug-induced and
drug-resistant cells express MDR1 mRNA, only
drug-resistant cells are able to express P-glycoprotein from
MDR1 mRNA.
MDR1 mRNA-positive Cells That Cycle Normally Can Lack
P-glycoprotein--
Because transient treatment with cytotoxic drugs
led to cell cycle arrest, it was necessary to exclude the possibility
that inhibition of cell cycle progression was responsible for the
failure to express P-glycoprotein. The colchicine analogue colcemid was used, because its microtubule depolymerizing activity can be relieved following extensive washes of the cells (46). K562 cells were induced
for 4 days with colcemid, which completely arrested the cell cycle and
up-regulated MDR1 mRNA (Fig.
5, A and B). The
washed cells were transferred to drug-free medium, where they remained quiescent for a further 2-3 days before proliferating at the same rate
as untreated cells (Fig. 5A). The colcemid-induced increase in MDR1 mRNA persisted for at least 19 days after drug
withdrawal (Fig. 5B), yet this did not lead to the synthesis
of P-glycoprotein (Fig. 5C). Therefore, mitotic arrest is
not responsible for the lack of P-glycoprotein expression in
drug-induced cells.
This was further confirmed by studying single-cell-derived clones from
KA25 cells (selected as resistant to the non-P-glycoprotein substrate
cytarabine). Clones from both P-glycoprotein-expressing (UIC2-positive)
and non-expressing (UIC2-negative) subpopulations (Fig. 4A)
(data not shown) were obtained by flow cytometry sorting. RNA analysis
indicated that the MDR1 transcript was up-regulated in both
types of cells (data not shown). Thus, cell selected for resistance to
cytarabine and growing normally can also express MDR1
mRNA but still fail to express P-glycoprotein. Therefore, cells that cycle normally, either after drug withdrawal or after selection with a non-P-glycoprotein substrate, can up-regulate MDR1 mRNA but fail to express P-glycoprotein.
Increased Levels of MDR1 mRNA Do Not Correlate with
P-glycoprotein Levels in Other Cell Types--
To test whether the
absence of P-glycoprotein expression in MDR1
mRNA-positive cells was a phenomenon particular to K562 cells, we
studied the acute lymphocytic leukemia-derived cell line CCRF-CEM. In
contrast to K562 cells, naïve CCRF-CEM cells were found to have
detectable MDR1 mRNA levels, which increased upon drug
induction. No P-glycoprotein was detected in the induced cells (data
not shown). We also studied a non-leukemic-derived cell line, the EBV-transformed B cell line MANN, in which the naïve
untreated cells express significant levels of MDR1 mRNA
but no P-glycoprotein (binding to UIC2 monoclonal antibody; data not
shown). These cells are also unable to efflux rhodamine 123, a
fluorescent P-glycoprotein substrate (47). The lack of correlation
between steady-state MDR1 mRNA and P-glycoprotein levels
in these cell lines suggests that the mechanism regulating
P-glycoprotein expression is not restricted to K562 cells.
MDR1 mRNA Is Not Associated with Translating Polyribosomes in
Cells Failing to Express P-glycoprotein--
To test whether the
drug-induced MDR1 mRNA had undergone RNA editing or
processing that rendered it untranslatable, the full MDR1
cDNA from colcemid-induced cells was sequenced and compared with
the sequence deposited in the databases (GenBankTM
accession number M14758). When polymorphisms were found, they were
always confirmed to exist in the MDR1 cDNA sequence from
a drug-resistant K562 cell expressing P-glycoprotein (KC40). No
differences between the mRNA sequences were found, showing that the
MDR1 mRNA does not undergo editing or processing during
short-term drug exposition or drug selection, which could account for
lack of P-glycoprotein.
Because stress can have a general negative effect on protein synthesis
(48-50) we also measured the rate of incorporation of [35S]Met into nascent proteins in naïve and
drug-induced K562 cells. There was approximately an 8% decrease in the
rate of overall protein synthesis after 24 h in the presence of
cytotoxic drug, which decreased further to 40% after 72 h (data
not shown). Thus, upon treatment with cytotoxic drugs there is a
general decrease in the rate of protein synthesis. However, although
de novo translation is decreased by the drug treatment, this
effect is insufficient to account for the complete lack of expression
of P-glycoprotein. To confirm that the absence of P-glycoprotein
expression in drug-induced cells was due to a failure to translate
MDR1 mRNA, we examined MDR1 mRNA
association with polyribosomes. Cell lysates were fractionated by
sucrose gradient ultracentrifugation, and the amount of MDR1 mRNA in individual fractions was determined by semi-quantitative PCR (51). In drug-resistant cells, a substantial portion of MDR1 mRNA was associated with high molecular weight
polyribosomes (toward the bottom of the gradient) indicating that the
MDR1 mRNA was efficiently translated. When ribosomes
were dissociated by including EDTA in the extraction buffer and sucrose
gradients, the position of the MDR1 mRNA shifted toward
the lighter (non-polysome) fractions of the gradient, as expected. In
contrast, for drug-induced K562 cells MDR1 mRNA migrated
at approximately the same position as for EDTA-dissociated KC40 cells
(top of the gradient), indicating that in these cells MDR1
mRNA is not associated with polyribosomes and is not being
translated (Fig. 6). To exclude the
possibility that the absence of association between MDR1
mRNA and polyribosomes is due to a general reduction in the rate of
protein synthesis, caused by the cytotoxic drugs, the association of
MDR1 mRNA with ribosomes was also examined in
P-glycoprotein-negative EBV-transformed B lymphocytes (MANN cell line).
In these cells, which had not been exposed to cytotoxic drugs,
MDR1 mRNA was not associated with polyribosomes (Fig.
6). The translational block is, therefore, not due to the effect of
cytotoxic drugs and represents a novel mechanism for regulating
P-glycoprotein expression.
Expression of MDR1/P-glycoprotein in blasts from
leukemia patients has been adversely correlated with the clinical
response to chemotherapy (2, 3, 52). We have used a K562 leukemic cell
model system to study the mechanisms regulating MDR1 gene expression involved in the acquisition of MDR. Unexpectedly, we found
that in these cells, up-regulation of P-glycoprotein expression was a
two-step process, mRNA stabilization and relief from a
translational block.
Three drug-resistant K562 cell lines were developed, resistant to the
P-glycoprotein substrates, doxorubicin and colchicine, or to the
non-P-glycoprotein substrate, cytarabine. In contrast to many previous
studies, we developed these lines by just a single-step selection to
very low doses of cytotoxic drug, rather than the traditional stepwise
selection at increasing drug concentrations. This enabled us to work at
drug concentrations and levels of drug resistance closer to those found
in the clinic and avoided amplification and rearrangements of the
MDR1 locus frequently found in highly resistant cell lines
but never documented in vivo (Fig. 2A) (1).
As expected, drug-resistant K562 lines, as well as naïve K562
cells transiently exposed to a range of cytotoxic agents, up-regulated steady-state MDR1 mRNA levels (Fig. 1A). This
effect was independent of the mode of action of the drug. Similarly, it
has been reported that heat shock, UV radiation, arsenate, or sodium
butyrate also induces MDR1 gene expression in a variety of
systems (40). Transcriptional activation is generally assumed to be the
principal mechanism for up-regulating MDR1 gene expression,
and several regulatory elements (AP-1, heat shock element, Sp1, Y-box,
CAAT box) have been identified and characterized in the MDR1
promoter region (40). In this study we found that in K562 leukemic
cells transiently exposed to drugs or further selected for drug
resistance the increased MDR1 mRNA levels could not be
accounted for by transcriptional activation of the MDR1
promoter (Fig. 2B). Furthermore, the
AP-1-dependent activation of the MDR1 promoter,
observed in other cell lines, was not apparent in K562 cells despite
the fact that the AP-1 activation pathway by protein kinase C was shown
to be functional in both naïve and drug-resistant K562 cells
(Fig. 2C). Because K562 cells do not express the tumor
suppressor gene p53 (53), the absence of transcriptional
activation in the cells could, at least in part, be due to a lack of
functional p53, which is known to activate the MDR1 promoter
(54).
In contrast to transcriptional activation, we found that up-regulation
of MDR1 mRNA in K562 cells is due to an increase in mRNA stability (Fig. 3, A and B). This was
specific to the MDR1 mRNA as the stability of other
short-lived mRNA species remained unaffected (Fig. 3C).
Interestingly, in naïve K562 cells, MDR1 mRNA
has a very short half-life (around 1 h), which, in addition to the
low rate of transcription from the MDR1 gene, can account for the low steady-state levels found in naïve cells (Fig.
1A). The cis-determinants involved in mRNA
stabilization are usually, but not exclusively, located in the
3'-untranslated region of the transcripts (55). The MDR1
3'-untranslated region is short (378 nucleotides) and contains several
potential AU-binding protein recognition sites that could regulate
rapid mRNA decay (55). The short half-life of MDR1
mRNA in naïve K562 cells contrasts with the few studies
that report a longer half-life, i.e. 8 h in the human
hepatoma cell line HepG2 (56). The MDR1 mRNA half-life increased 10-fold upon transient exposure of cells to cytotoxic drugs
and in the drug-resistant lines (KD30 and KC40) (Fig. 3). Similar long
half-lives have also been described in a K562 line resistant to
doxorubicin (45). The stabilized MDR1 mRNA from drug-induced K562 cells persisted for several days after the recovery from the cytotoxic stress but eventually returned to the basal level of
naïve K562 cells (Fig. 5B), consistent with dilution of a trans-acting stabilizing factor upon cell division to
levels below a functional concentration. One interpretation of the data is that this putative factor(s) is activated upon drug exposure.
In contrast to other studies using different systems (16, 24),
following transient drug exposure (drug induction) the increase in
steady-state MDR1 mRNA was not accompanied by a parallel increase in P-glycoprotein expression in K562 cells (Fig. 4). This
effect could, in part, be due to a decrease in the rate of global
protein synthesis due to cytotoxic stress caused by the drug (48-50).
MDR1 mRNA, but no P-glycoprotein, was also demonstrated in K562 cells after colcemid withdrawal (Fig. 5B) and in
K562 cells resistant to the non-P-glycoprotein substrate cytarabine (data not shown). In addition, CCRF-CEM cells and EBV-transformed human
lymphocytes do not express P-glycoprotein despite being MDR1
mRNA-positive (data not shown). A lack of correlation between MDR1 expression and P-glycoprotein function has also been
reported previously in acute myeloid leukemia cell lines (57) and human bone marrow lymphoid cells (58). There is, therefore, a clear lack of
correlation between the presence of MDR1 mRNA and
P-glycoprotein in several lymphocyte cell models, showing it is not
specific to K562 cells or the drug treatment regime.
In this study we show that translational regulation plays an important
role in determining the levels of P-glycoprotein expression. Translational control most likely operates at the level of initiation, because MDR1 mRNA does not migrate with the polysome
fractions in P-glycoprotein-negative cells. As the sequence of
MDR1 mRNA from P-glycoprotein-expressing and
non-expressing cells is identical, regulation must involve
trans-acting factors (59, 60). Upon drug selection this
translational block is overcome, enabling the cells to grow in the
presence of the cytotoxic drug.
In summary, the data show that in K562 cells up-regulation of
MDR1 gene expression occurs at two distinctive steps,
mRNA stabilization and translational initiation. That these two
steps are distinct is also illustrated by data obtained with
cytarabine, a cytotoxic drug not transported by P-glycoprotein.
Cytarabine was as effective as the P-glycoprotein substrates in
inducing MDR1 mRNA stabilization, suggesting that this
is a general response to drug-induced stress. Upon selection for
cytarabine resistance, P-glycoprotein was expressed in a subpopulation
of cells, in contrast to selection for resistance to P-glycoprotein
substrates where all cells expressed P-glycoprotein (Fig.
2A). MDR1 mRNA was up-regulated in clones
derived from both the P-glycoprotein-positive and
P-glycoprotein-negative cells (data not shown) indicating that in these
cells the first step in the acquisition of MDR has occurred. The second
step, relief from the translational block, had also occurred in a
subpopulation of cells. As cytarabine induces apoptosis (61), and
caspase-dependent apoptotic pathways are inhibited in
P-glycoprotein-overexpressing K562 and CCRF-CEM cells (62), the
expression of P-glycoprotein in this subpopulation of
cytarabine-resistant cells may indicate that P-glycoprotein
confers a selective growth advantage, perhaps through inactivation of
caspase-dependent processes.
These findings have implications for attempts to circumvent or overcome
drug resistance in the clinic, at least for leukemia. First, the
demonstration that MDR1 mRNA levels do not necessarily correlate with P-glycoprotein expression show that measuring
MDR1 mRNA as a clinical surrogate for
P-glycoprotein-mediated drug resistance is inappropriate. Second, as
drugs themselves can induce stabilization of MDR1 mRNA,
the first necessary step in acquiring P-glycoprotein-mediated drug
resistance, drug treatment regimes should be developed to minimize this
occurrence. Finally, as induction of drug resistance involves two
specific steps, stabilization of MDR1 mRNA and
subsequent overcoming of a translational block, each of these steps
provides novel and potentially specific targets for circumventing drug
resistance in the clinic.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-arabinofuranosylcytosine (cytarabine). Lines
resistant to these concentrations of drug were designated KC40, KD30,
and KA25 lines, respectively.
CT, where
CT is the difference in CT between the gene
of interest and GAPDH or 18 S rRNA, and
CT for the sample =
CT of the
actual sample
CT of the lowest expressing sample.
genes (29, 30). Hybridization
signals were quantified with a PhosphorImager (Amersham Biosciences).
-globin locus
(3.0-kb EcoRI fragment containing DNase I hypersensitive
site V) (32).
-Globin to MDR1 ratios were obtained by
quantitation using a PhosphorImager.
1202), carrying the luciferase reporter gene from
pGL2B (Promega, Madison, WI) under the control of MDR1
promoter (34), together with pEFlacZ carrying the bacterial
-galactosidase gene under the control of the EF1
promoter. After 2 days, cells extracts were prepared, and luciferase
activity was determined with a luciferase assay system (Promega) and
normalized against
-galactosidase expression (28). The
AP-1-dependent luciferase reporter plasmid
(pAP1-luc), containing four tandem copies of the
TPA-responsive element consensus motif TGACTCA coupled to the
36 to
+37 rat prolactin minimal promoter (35), was cotransfected with
pEFlacZ into K562 and KD30 cells. When indicated, cells were
stimulated with 20 ng/ml phorbol 12-myristate 13-acetate (Sigma) during
the last 12 h, and luciferase and
-galactosidase expression
were measured as above. Transfections were by electroporation using a
Bio-Rad gene pulser (Bio-Rad) with 5 × 106 cells
essentially as described (28).
2 subunit,
clone IID8, (Affinity Bioreagents Inc., Golden, CO) in 5 g/100 ml
skimmed milk phosphate-buffered saline containing 0.1 g/100 ml
Tween 20. Protein was visualized by enhanced chemiluminescence (Amersham Biosciences) after incubating with and horseradish
peroxidase-conjugated goat and mouse IgG secondary antibody (1/1000)
(Dako, Ely, United Kingdom).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (47K):
[in a new window]
Fig. 1.
MDR1 mRNA is up-regulated in
drug-induced and drug-resistant K562 cells. MDR1 and
GAPDH transcripts were amplified by RT-PCR and detected by
Southern blotting and hybridization with specific internal
oligonucleotide probes. A, left hand lane shows
naïve K562 cells (not treated with drug), middle
lanes show K562 cells transiently induced with doxorubicin
(D), cytarabine (A), colchicine (C),
colcemid (CD), or vinblastine (V) for 3 days;
right hand lanes show K562 cells selected for resistance to
doxorubicin (KD30, KD), cytarabine (KA25, KA), or
colchicine (KC40, KC). KBV1, human KB epidermoid
carcinoma line. B, dose-time response for induction of
MDR1 mRNA by colcemid using concentrations of 0.1, 1.0, and 10.0 µM.
View larger version (25K):
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Fig. 2.
Increased MDR1 mRNA
levels are not due to gene amplification or transcriptional
activation. A, Southern blot analysis of the
MDR1 and -globin loci of naïve
(untreated) and drug-resistant (KC40 and KD30) K562 cells. The KB-V1
cell line was used as a positive control for amplification and
rearrangement of the MDR1 locus; arrows show the
positions of rearranged MDR1 fragments in this cell line.
B, transient expression of a luciferase reporter gene driven
by the MDR1 promoter was measured after cotransformation of
naïve and drug-resistant (KC40 and KD30) cells with
pMDR1(
1202) and a bacterial
-galactosidase reporter
gene driven by the constitutive EF1
promoter
(pEFlacZ). Luciferase activity was normalized to
-galactosidase activity, and the data are shown as transcription
relative to naïve K562 cells 2 days after transfection. Data
show the average and S.D. from three independent experiments.
C, AP-1 is not involved in the up-regulation of
MDR1 mRNA in drug-resistant (KD30) cells. The
AP-1-dependent luciferase reporter plasmid
(pAP1-luc), containing four tandem copies of the
TPA-responsive element coupled to the rat prolactin minimal promoter,
was cotransfected with pEFlacZ into K562 and KD30 cells
(clear bars). Cells were stimulated with 20 ng/ml phorbol
12-myristate 13-acetate during the last 12 h, and luciferase and
-galactosidase expression were measured as above (filled
bars).
-actin, or
-globin) and did not increase
significantly in drug-resistant or drug-induced cells (data not shown).
Thus, transcriptional activation does not appear to be responsible for the up-regulation of MDR1 mRNA in K562 cells following
drug induction or upon selection for drug resistance.
, were confirmed
to be 1-2 h as reported previously (29, 30) in both naïve and
drug-resistant K562 cells (Fig. 3C). Similarly, the
half-life of a long-lived message (GAPDH) was also unchanged
in drug-resistant cells (20-24 h in both naïve K562 and KD30
cells) (Fig. 3B). Thus, the stabilization of the MDR1 mRNA is specific and not a general phenomenon
affecting other short-lived mRNAs. In conclusion, up-regulation of
MDR1 mRNA levels following exposure to drugs, after
either a transient induction or drug selection, is primarily due to a
specific increase in mRNA stability.
View larger version (18K):
[in a new window]
Fig. 3.
Increased MDR1 mRNA
levels are due to an increase in mRNA half-life. A,
MDR1 mRNA decay pattern after actinomycin D addition to
naïve (white circle), drug-induced (black
square), and drug-resistant (white triangle, KC40;
black triangle, KD30) K562 cells obtained by RT and
real-time quantitative PCR. A representative drug induction (with
vinblastine) is shown. MDR1 mRNA half-lives were 1 h for the naïve and greater than 10 h for drug-induced and
-resistant cells. B, mRNA decay pattern of K562
(white symbol) and KD30 (black symbols) cells was
followed by Northern analysis (using ten µg of total RNA) and
hybridization with GAPDH (circles)- and
MDR1 (triangle)-specific probes. The GAPDH
mRNA half-life was 20-24 h in both naïve and
drug-resistant cells, and the half-life of MDR1 mRNA was
15-20 h in KD30 cells. Similar half-lives were obtained for
colchicine-resistant KC40 cells (data not shown). MDR1
message was undetectable by Northern hybridization in naïve
K562 cells. C, mRNA decay analyzed by Northern analysis
and hybridization to probes for other short-lived messages
(circles, Id2; triangles,
RAR-a) in naïve K562 (white symbols) and
doxorubicin-resistant KD30 (black symbols) cells. The
half-lives of the transcripts (1-2 h) were similar in each cell type.
Results from KC40 cells were essentially the same (data not
shown).
View larger version (45K):
[in a new window]
Fig. 4.
Up-regulation of MDR1
mRNA in drug-induced K562 cells does not lead to expression
of P-glycoprotein. A, flow cytometric analysis of surface
P-glycoprotein expression using the UIC2-phycoerythrin-conjugated
antibody (filled peak) and the corresponding IgG isotype
control (clear peak). For naïve and drug-induced
cells, the IgG and UIC2 peaks overlap, showing that active
P-glycoprotein is not present at the cell surface. A representative
drug-induction experiment with colchicine after 3 days is shown.
Qualitatively similar data were obtained with other drugs (vinblastine,
colcemid, doxorubicin, or cytarabine) or if measured 1, 2, or 4 days
after drug induction (data not shown). For the drug-resistant cell
lines KC40 and KD30, UIC2 fluorescence was higher than the IgG control,
indicating expression of P-glycoprotein on the surface of the cells.
Approximately 40% of cytarabine-resistant (KA25) cells showed limited
P-glycoprotein expression. B, Western blot analysis of
P-glycoprotein (P-gp) in plasma membrane-enriched and
-depleted fractions (200 µg protein) from K562 naïve
(K), drug-induced (D, doxorubicin; A,
cytarabine; CD, colcemid), or drug-resistant cells
(KD, KD30; KA, KA25; KC, KC40). An
anti-Na+/K+-ATPase antibody was used as loading
control. Drug inductions were carried out for 3 days.
View larger version (23K):
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Fig. 5.
Lack of P-glycoprotein expression by
drug-induced K562 cells is not due to cell cycle arrest.
A, K562 cells were treated with colcemid for 4 days and then
grown drug-free for a further 14 days (filled circles). The
growth curves of naïve (untreated) K562 cells (open
circles) are comparable. B, MDR1 and
GAPDH transcripts from the cells were amplified by RT-PCR
and detected by Southern blot hybridization using oligonucleotide
probes. C, flow cytometric analysis of surface
P-glycoprotein expression in the cells using the
UIC2-phycoerythrin-conjugated antibody (filled peak) and the
corresponding IgG isotype control (clear peak). IgG and UIC2
fluorescence signals overlapped, indicating no P-glycoprotein in the
surface of the cells after colcemid withdrawal, despite the presence of
MDR1 mRNA.
View larger version (46K):
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Fig. 6.
MDR1 mRNA is not associated
with translating polyribosomes in cells failing to express
P-glycoprotein. Detection of MDR1 mRNA in fractions
from a 15-45% sucrose gradient of cell extracts by semi-quantitative
RT-PCR is shown. Fractions (0.5 ml) were collected from the top of the
gradient, and RNA was isolated. Where indicated, ribosomes were
dissociated by the inclusion of EDTA in both the extraction and
gradient buffers. A representative induction with colcemid for 3 days
is shown. Similar results were obtained after doxorubicin induction. In
drug-resistant cells expressing P-glycoprotein MDR1 mRNA
migrates with the heavy (containing polyribosomes) fractions. In both
drug-induced K562 and MANN (EBV-transformed human B lymphocytes) cells,
which do not express P-glycoprotein, MDR1 mRNA migrates
with the light fractions lacking polyribosomes, indicating that it is
not being translated.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
---|
We thank Kathleen Scotto and Mercedes Rincon for the gift of pMDR1 and pAP1-luc, respectively, Katie Smith for assistance with flow cytometry, and Nic Jones and Anne Willis for helpful discussions.
![]() |
FOOTNOTES |
---|
* This work was supported by the Medical Research Council UK and Cancer Research UK.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.
¶ Present address: Cardiovascular Molecular Medicine Unit, Stopford Bldg., Manchester University, Manchester M13 9PT, United Kingdom.
To whom correspondence should be addressed. Tel.:
44-20-8383-8269; Fax: 44-20-8383-8337; E-mail:
s.raguz@csc.mrc.ac.uk.
Published, JBC Papers in Press, January 13, 2003, DOI 10.1074/jbc.M211093200
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ABBREVIATIONS |
---|
The abbreviations used are: MDR, multidrug resistance; RT, reverse transcription; TPA, 12-O-tetradecanoylphorbol-13-acetate; CT, threshold cycle; luc, luciferase; EBV, Epstein-Barr virus.
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