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Blood, Vol. 91 No. 7 (April 1), 1998:
pp. 2467-2474
By
From the Department of Microbiology and Immunology, and MRC
Toxicology Unit, University of Leicester, Leicester; the Queen
Elizabeth Neuroscience Centre, Queen Elizabeth Hospital, Birmingham;
and St Mary's Hospital Medical School, London, UK.
Overexpression of P-glycoprotein (P-gp), the protein product of the
multidrug resistance gene (MDR1), confers a drug resistant phenotype on cells. This phenotype is reminiscent of human T-cell leukemia virus (HTLV)-transformed leukemic cells, for which no consistently effective chemotherapeutic regime has been found. The
presence of an active multiple drug resistance (MDR) phenotype in
freshly isolated peripheral blood mononuclear cells (PBMC) from
HTLV-I-infected subjects was investigated. Significant P-gp-mediated efflux activity and enhanced MDR1 mRNA expression was observed in nine of 10 HTLV-infected subjects. The development of MDR phenotypes was found to be independent of disease type or status with significant MDR activities being observed in adult T-cell leukemia (ATL), HTLV-associated myelopathy (HAM)/tropical spastic paraparesis (TSP),
and asymptomatic HTLV-infected individuals. P-gp-mediated drug efflux
was also found to be restricted to CD3+ T-cell
populations. Furthermore, we show the novel finding that the
MDR1 gene promoter is transcriptionally activated by the HTLV-I tax protein, suggesting a molecular basis for the development of drug
resistance in HTLV-I infections. These observations open up the
possibility of new chemotherapeutic approaches to HTLV-associated diseases through the use of P-gp inhibitors.
ATOTAL OF 10 to 20 million people
worldwide are estimated to be infected with human T-cell leukemia virus
(HTLV).1 Infection is associated with at least two distinct
disease syndromes.2 Adult T-cell leukemia (ATL) is an
aggressive T-cell malignancy, which occurs worldwide in populations
where HTLV-I infection is endemic. HTLV-I infection has also been
linked with a syndrome known as HTLV-associated myelopathy (HAM) or
tropical spastic paraparesis (TSP), and more recently, a variety of
other pathologies. The pathogenesis of these conditions remains
unclear.
Treatment of ATL patients has traditionally consisted of combination
chemotherapy, but this approach has limited clinical benefit. Four
generations of combination chemotherapy have shown an increase in
remission rates from 11% to 42%, but a corresponding improvement in
overall or disease-free survival time has not occurred.3,4 Similarly other novel treatments, including bone marrow
transplantation, total body irradiation, and treatment with interferons
The multiple drug resistance phenotype (MDR) results in a broad
spectrum of resistance to chemotherapy. There are several known
mechanisms by which an MDR phenotype may arise in cells, but one of the
best characterized is that caused by the overexpression of
P-glycoprotein (P-gp).10,11 P-gp, the protein product of the MDR gene family, is able to confer a MDR phenotype by pumping a
wide spectrum of chemotherapeutic agents (including anthracyclines, vinca alkaloids, epipodophylline, and dactinomycins) from the cell. The
result is lower intracellular drug concentrations and reduced drug
efficacy. There are two MDR genes in humans, 1 and 2. Only MDR1
is able to confer a MDR phenotype in transfection analysis.12-14 Increased MDR1 gene expression has
been associated with poor prognosis and drug resistance in acute
myeloid leukemia (AML), adult lymphoblastoid leukemia
(ALL),15-17 and various other leukemias, lymphomas,
myelomas, and sarcomas.18-22 The expression of membrane
bound P-gp and MDR1 RNA has been shown in cells from ATL
patients.23-25 The incidence of P-gp expression was also
suggested to be higher in ATL than in other non-HTLV-induced
leukemias.25 However, these preliminary experiments did not
establish an association between P-gp expression and drug resistance
activity, only that membrane P-gp and MDR1 RNA could be
detected in ATL cells. Similarly, no evidence for a mechanism or the
extent of enhancement of P-gp expression in HTLV infections was given.
In the present study, we show significant P-gp-mediated drug efflux
associated with enhanced MDR1 gene transcription in T cells of
individuals infected with HTLV regardless of disease status. Using
specific assays to measure MDR1 gene expression and
P-gp-mediated drug efflux activity, we have confirmed and extended
preliminary studies to include asymptomatics, HAM/TSP, and ATL
subjects. The molecular mechanism of P-gp upregulation in HTLV
infections was also investigated, and we show here for the first time
activation of the MDR1 gene promoter by the HTLV tax protein.
These observations raise the possibility of new and alternative
chemotherapeutic approaches to HTLV-associated diseases.
Cells.
Preservative-free heparinized fresh whole blood was obtained by
venipuncture of 10 HTLV-I-seropositive subjects and six healthy uninfected controls. Peripheral blood mononuclear cells (PBMC) were
isolated by density gradient centrifugation over Ficoll-Paque (Amersham
Pharmacia Biotech, UK). Isolated PBMCs were assayed immediately for MDR activity or RNA extracted. COS cells26
were maintained in Dulbecco's modified Eagle's medium
(DMEM) medium supplemented with 10% calf serum at
37°C, 5% C02 in a humidified atmosphere.
Rhodamine 123 (R123) dye efflux MDR assay.
R123 efflux assays were performed as described by Davies et
al,27 except that the assays presented here used 2 × 106 PBMC in 2 mL RPMI 1640 medium supplemented with 2.5%
fetal calf serum (FCS) and 0.4 µmol/L R123. After incubation and
propidium iodide staining as described, cells were fixed using 1%
paraformaldehyde before FACscan analysis (Becton Dickinson, Franklin
Lakes, NJ). The effects of P-gp inhibitors were assessed
by performing R123 assays on cells in the presence 5 µmol/L reserpine
or verapamil (Sigma, St Louis, MO). This assay is highly
specific for MDR1 because R123 accumulation correlates with monoclonal
antibody (MoAb) MRK16 binding (specific for MDR1) in cells that express MDR1 and MDR2.27
Determination of cell surface phenotypes of MDR+
cells.
PBMC subset analysis of R123 efflux cells was performed using
phycoerythrin (PE)-conjugated lymphocyte cell typing MoAbs (VERItype) from Harlan Sera-lab (Sussex, UK). R123 efflux
assays were performed on cells as described above except that the cells were not labeled with propidium iodide. The cells after R123 dye efflux
were washed in once in phosphate-buffered saline (PBS) and
resuspended at 1 × 106 cells/mL in PBSH (PBS + 2%
human AB serum). Cells (2 × 105) were incubated with
2 µL (1:100 dilution) of PE-conjugated mouse anti-CD3, CD14, or CD22
VERItype antibodies. Cells were directly stained at 4°C for 30 minutes, then washed in PBS and fixed in 1% paraformaldehyde before
flow cytometric analysis. High and low R123 dye efflux cell populations
were analyzed for PE fluorescence with either anti-CD3, anti-CD14, and
anti-CD22 antibodies on a Becton Dickinson FACscan.
Immunofluorescent detection of P-gp.
Indirect immunofluorescent detection of cell surface-associated P-gp
was performed using the anti-P-gp MoAb MRK-16 (Kamiya Biomedical Co,
Thousand Oaks, CA). Freshly isolated PBMCs were washed in PBS and
resuspended at 1 × 106 cells/mL in PBSG (PBS + 2%
goat serum). Cells (2 × 105) were incubated with 2 µg (10 µg/mL) of either anti-P-gp MRK-16 or IgG2a control primary
antibody at 4°C for 1 hour. Cells were washed once with PBST (PBS + 10% Tween-20), then resuspended in 200 µL cold PBSG containing 10 µL (1:10 dilution) of fluorescein isothiocyanate (FITC)-conjugated
goat antimouse IgG secondary detection antibody (Dako, Glostrup,
Denmark). Cells were incubated at 4°C for 1 hour,
then washed twice with PBST and fixed in 1% paraformaldehyde before
FACscan analysis.
Quantitative reverse transcriptase-polymerase chain reaction
(RT-PCR) of MDR1 mRNA.
RNA for RT-PCR analysis was isolated by guanidium isothiocyanate/phenol
extraction (RNAzol B, Biogenesis, Poole, UK) according to
the supplier's recommend protocol. A total of 100 to 500 ng of total
cellular RNA were reverse transcribed by incubation with 2 U AMV-RT (Promega, Madison,
WI) at 42°C for 1 hour. After inactivation at
90°C for 5 minutes, the entire mix was subjected to PCR
amplification using 1.25 U Taq polymerase (Promega) using the
MDR1-specific primers and internal standard
RNA,27,28 giving rise to an amplified product of 161 bp and
not 425 bp, as originally published. Negative controls consisted of
mock reactions lacking RT, positive controls provided by internal
standard RNA. PCR products were visualized under ultraviolet (UV) light
on 3% GTG Nusieve agarose gels (Flowgen, Staffordshire,
UK). Relative band intensities were calculated by
densitometry.
Plasmids.
Plasmids pBC-tax/rex, pBC-rex, and pMDR-CAT were
constructed as shown in Fig 1. pBC-tax/rex
contains a 1,098-bp fragment of cDNA corresponding to the tax/rex mRNA
of HTLV-I, which encodes both the tax and rex open reading frames
(orf). Total cellular RNA was extracted from virus-infected C91/PL
cells29 by guanidium isothiocyanate/phenol extraction
(RNAzol B, Biogenesis). A total of 400 ng of total cellular RNA was
reverse transcribed with 200 U M-MLV RT (GIBCO-BRL,
Gaithersburg, MD) and 200 ng oligo (dT)12-18 primer at 37°C for 1 hour. One quarter of the cDNA products were then PCR amplified for 30 cycles using 1 U pfu DNA polymerase (Stratagene, La Jolla, CA) and 100 ng each of sense and
antisense tax/rex primer (sense,
5
Trans-activation of the MDR1 promoter by HTLV-I tax.
Electroporation of COS cells and chloramphenicol acetyl transferase
(CAT) assays were performed as previously described35 except that 3 × 106 COS cells were electroporated at
250 V, 960 µF with 25 µg of total plasmid DNA (12.5 µg of each
plasmid to be coelectroporated). Forty-eight hours after
electroporation, cells were harvested and cells lysed by
freeze-thawing. Total protein concentrations were determined using the
Bio-Rad (Hertfordshire, UK) protein assay kit. A
total of 50 µg of cell lysates were incubated with 4 mmol/L
Acetyl-CoA (Sigma) and 0.05 µCi [14C]-chloramphenicol
(ICN, Costa Mesa, CA) at 37°C for 3 hours. Reactions
were extracted with ethyl acetate, then acetylated products resolved by
thin layer chromatography and quantified by scintillation counting.
R123 dye efflux analysis of PBMC from HTLV and non-HTLV-infected
subjects.
PBMC from 10 HTLV-I-seropositive subjects (6 asymptomatic, 2 HAM/TSP,
1 lymphoma, 1 ATL) and 6 healthy uninfected controls were examined
using a rhodamine 123 (R123) dye efflux assay
(Fig 2, Table 1). None of
the non-HTLV-infected control subjects tested showed any significant
level of P-gp efflux activity in PBMC, whereas all but one (no. 1) of
the HTLV-infected subjects showed at least some activity, indicated by
the presence of a high efflux cell population. The role of P-gp in the
observed R123 efflux was confirmed by abolition of the high efflux
population of cells by P-gp inhibitors, reserpine and verapamil (Fig 2,
Table 1).
Cell surface phenotypes of MDR+ PBMC subpopulations from
HTLV-infected subjects.
The identity of cells in the high and low R123 efflux populations was
tested by labeling with cell surface MoAb markers and measuring the
relative distribution of the markers in each of the populations
(Table 2). The majority of stained cells in
the high efflux population were CD3 positive (pan T-cell marker), while
the low efflux population contained a mixture of T cells (CD3+), B cells (CD22+), and
monocyte-macrophages (CD14+). This result indicates that
the high efflux population contains mostly T cells with very few B
cells or monocyte-macrophages.
Immunofluorescent detection of P-gp.
The presence of surface expressed P-gp on PBMC from HTLV-infected
subjects was confirmed by indirect immunofluorescence using anti-P-gp
MoAb MRK-16 (Fig 3). Staining of cells with
MRK-16 antibody showed the presence of a high P-gp cell surface
expressing subpopulation in PBMC. The proportion of high P-gp
expressing cells was found to be around 50% of the total number of
PBMCs. This is consistent with the proportion of MDR+ cells
found by the R123 dye efflux assay method, suggesting that the high
P-gp expressing and high R123 dye efflux populations are the same
cells.
Quantitative RT-PCR analysis of MDR1 mRNA.
To confirm the R123 efflux results, quantitative RT-PCR analysis of
MDR1 mRNA was performed (Fig 4,
Table 3). The results obtained are entirely consistent
with those obtained from the R123 efflux assay. Only very low levels of
MDR1 mRNA were observed in all of the non-HTLV-infected
subjects examined, whereas all but one (no. 1) of the HTLV-infected
subjects showed enhanced MDR1 mRNA levels. These results
suggest that the MDR activity seen in HTLV-infected subjects is the
result of transcriptional activation of the MDR1 gene or
possibly stabilization of MDR1 mRNA.
Trans-activation of the MDR1 promoter by HTLV-I tax.
We examined the transcriptional activation of the human MDR1
gene by constructing tax and rex protein expression vectors and MDR-CAT
reporter constructs (Fig 1). Figure 5 shows
typical CAT assay results when MDR-CAT reporter plasmids were
introduced into COS cells together with HTLV tax or rex protein
expression constructs. When pMDR(+)CAT and pMDR-CAT (M.M. Gottesman)
were coelectroporated with the HTLV-I tax protein expression construct,
pBC-tax/rex, 19-fold and 10-fold activation of the human MDR1
promoter were observed, respectively. The pBC-tax/rex construct
potentially encodes rex2 and p21rex 36 proteins
in addition to tax, and their involvement in MDR1 gene activation was assessed using the rex/p21rex protein
expression construct pBC-rex. Extracts of cells coelectroporated with
pBC-rex and pMDR(+)CAT or pMDR-CAT (M.M. Gottesman) showed negligible
CAT activity and suggests the trans-activation activity is due to
transcriptional activation by the tax protein alone. This was confirmed
using plasmid pRK7-tax, which expresses tax, but not rex31
and still transactivates the pMDR-CAT plasmid. The specificity of tax
trans-activation was further confirmed by coelectroporation with
pMDR(-)CAT in which the MDR1 gene promoter is linked upstream
of the CAT reporter gene in a reverse orientation. This construct was
found to be unable to direct expression of CAT and cell extracts showed
negligible CAT activity. Together these results suggest that the
increase in MDR1 mRNA levels seen may be due to
trans-activation of the MDR1 gene in HTLV-I-infected individuals.
The expression of P-gp has been reported previously on ATL
cells.23-25 These earlier studies detected the presence of
P-gp on membrane fractions derived from ATL cells by immunoblotting using a MoAb. The report by Kuwazuru et al24 also showed
P-gp-like photoaffinity drug labeling and MDR1 RNA in cells
from a single ATL patient after clinical relapse. However, these
reports did not correlate P-gp expression with cellular drug resistance
activity or provide any evidence for a mechanism of P-gp enhancement in HTLV-induced disease. Moreover, these studies did not include appropriate controls from non-HTLV-infected subjects, or did they investigate MDR1 gene expression in HTLV-infected subjects
other than those with full-blown ATL. P-gp has subsequently been found to be expressed at low levels on normal lymphocytes (but not enough to
mediate significant drug resistance) and by the use of the same MoAb
(C219) used in the above studies.37-39 Hence, the actual enhancement of P-gp expression in ATL cells over normal lymphocyte cell
populations was unclear. The data presented here addresses all of these
points and clarifies the involvement of HTLV in the induction of
multiple drug resistance phenotypes.
Submitted April 22, 1997;
accepted November 16, 1997.
We are grateful to the HTLV-infected patients and families who
generously donated materials for this study and to K. Khazaie for
plasmid pRK7-tax.
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|
Abstract
Introduction
Abstract
and 
have failed to show clinical improvement. Limited clinical
improvement in some patients has been reported using deoxycoformycin
(DCF), an adenosine deaminase inhibitor,
-interferon and
azidothymidine (AZT).
-TTAAGCTTGCGAGCTGCATGCCCAAGAC-3
426 to +536) surrounding the transcription start site of the
human MDR1 gene derived from plasmid pMDR-P3,
X174 DNA); 1: negative control (no
RT); 2: 0.05 pg ISC RNA; 3: 0.25 pg ISC RNA; 4: 0.5 pg ISC RNA; 5: 1.0 pg ISC RNA; 6: 2.5 pg ISC RNA.
B, and serum response factor (SRF) families.
T-cell activation and apoptosis.
Oncogene
10:269,
1995