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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 7 (April 1), 1998:
pp. 2467-2474
Enhanced MDR1 Gene Expression in Human T-Cell Leukemia
Virus-I-Infected Patients Offers New Prospects for Therapy
By
Alan Lau,
Simon Nightingale,
Graham P. Taylor,
Timothy W. Gant, and
Alan J. Cann
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.
 |
ABSTRACT |
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.
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INTRODUCTION |
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
 and  have failed to show clinical improvement. Limited clinical
improvement in some patients has been reported using deoxycoformycin
(DCF), an adenosine deaminase inhibitor,5 MST-16, a
topoisomerase II inhibitor,6 and anti-Tac
antibodies.7 Improved remission rates have recently been
reported in small studies combining  -interferon and
azidothymidine (AZT).8,9 Generally, ATL is
highly resistant to traditional chemotherapeutic strategies, of which
none confer significant clinical improvement. This observation suggests
that ATL cells have innate drug resistance, a phenotype that could be
one result of HTLV-I-mediated gene deregulation.
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.
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MATERIALS AND METHODS |
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 -TTAAGCTTGCGAGCTGCATGCCCAAGAC-3; antisense, 5-AACTCGAGTCAGACTTCTGTTTCTCGGAAATG-3). The amplified
tax/rex fragment was inserted into pBC12/CMV/IL-230 under
the control of the huCMV immediate early promoter, replacing the
interleukin-2 (IL-2) sequences. pBC-rex only contains the rex orf and
was constructed by deletion of a 534-bp Ava I-Ava I
fragment from pBC-tax/rex (Fig 1). This deletion removes the
3-end 470 bp of the tax orf, which is not present in rex.
pRK7-tax expresses tax, but not rex from the huCMV
immediate early promoter.31
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| Fig 1.
Construction and structure of recombinant plasmids
pBC-tax/rex, pBC-rex, and pMDR-CAT. (A) RT-PCR amplification from HTLV tax/rex mRNA was used to generate a 1,098-bp fragment, which encodes the complete tax, rex, and p21 proteins. (B) The tax/rex protein expression construct, pBC-tax/rex, was derived by insertion of the
1,098-bp tax/rex cDNA into pBC12/CMV/IL-2 under the control of the CMV
immediate early (CMV IE) promoter. The rex protein expression
construct, pBC-rex, was derived by deletion of an Ava I-Ava I fragment in pBC-tax/rex, which removes the C-terminal half of tax. (C) MDR1 promoter-CAT reporter gene constructs.
pMDR(+)CAT contains the MDR1 promoter in tandem with the CAT
gene. pMDR(-)CAT contains the MDR1 promoter in a
reverse orientation to the CAT gene.
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pMDR-CAT contains the 1kb Pst I-Pst I promoter region
( 426 to +536) surrounding the transcription start site of the
human MDR1 gene derived from plasmid pMDR-P3,32
kindly provided by M. Cornwell (Fred Hutchinson Cancer Research Centre,
WA). The SV40 promoter and origin of replication were
excised from pSV2CAT (ATCC #37155, positions 1-328) and replaced with
the MDR1 fragment. pMDR(+)CAT contains the MDR1
promoter in tandem with the CAT gene; pMDR(-)CAT contains the
promoter opposed to the CAT gene and was used as a negative control.
pMDR-CAT (M.M. Gottesman, Laboratory of Cell Biology, NCI,
NIH) contains a longer 1.8 kb promoter region of the
human MDR1 gene inserted upstream of the CAT reporter
gene33 and was kind gift of M.M. Gottesman. pLTR-I-CAT
contains the HTLV-I LTR promoter inserted upstream of the CAT reporter
gene and is described elsewhere.34
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.
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RESULTS |
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).
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| Fig 2.
Analysis of P-gp-mediated drug efflux activity in
freshly isolated PBMCs from HTLV and non-HTLV-infected subjects.
Rhodamine 123 (R123) dye efflux assays were used to measure the extent
of drug resistance. This figure shows the results of a single
representative sample; data from other subjects examined is summarized
in Table 1. (Left panel) FACscan analysis of cells from a typical
HTLV-negative control subject. No decrease in fluorescence is evident
after a 90-minute incubation, indicating no P-gp activity. (Center
panel) FACscan analysis of cells from a typical HTLV-I-infected
subject. After a 90-minute incubation, two populations of cells are
present, a high efflux, low fluorescence population and a low efflux,
high fluorescence population. (Right panel) Inhibition of R123 efflux by P-gp inhibitors. In the presence of 5 µmol/L reserpine or 5 µmol/L verapamil (shown here), the dye efflux is blocked, indicating the specificity of this assay for P-gp.
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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.
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| Fig 3.
Immunofluorescent detection of P-gp on the surface of
freshly isolated PBMCs from an HTLV-infected subject. Staining of cells with MRK-16 shows the presence of both high and low P-gp-expressing cell populations. A matched isotype IgG2a antibody was used instead of
MRK-16 as a control.
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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.
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| Fig 4.
Quantitative RT-PCR analysis of MDR1 mRNA. Reactions were
performed as described in Materials and Methods. A representative experiment is shown. All reactions contained 100 ng total RNA. Internal
standard (upper band) 212 bp, MDR1 mRNA (lower band) 161 bp. SM: Size
marker (HaeIII digest  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.
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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.
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| Fig 5.
Trans-activation of the MDR1 gene promoter by the
HTLV-I tax protein. (A) Representative experiment showing
chloramphenicol acetyl transferase (CAT) assays of COS cell extracts.
(B) Quantification of CAT activity in COS cell extracts from three
independent experiments. The percentage of chloramphenicol converted
into acetylated forms is shown. COS cells were electroporated, as
described in the text, with the following plasmids: 1: No DNA; 2:
pBC-tax/rex; 3: pBC-rex; 4: pRK7-tax; 5: pMDR(+)CAT; 6: pMDR(-)CAT;
7: pMDR-CAT (M.M. Gottesman); 8: pLTR-I-CAT; 9: pMDR(+)CAT + pBC-tax/rex; 10: pMDR(-)CAT + pBC-tax/rex; 11: pMDR-CAT (M.M.
Gottesman) + pBC-tax/rex; 12: pLTR-I-CAT + pBC-tax/rex; 13:
pMDR(+)CAT + pBC-rex; 14: pMDR(-)CAT + pBC-rex; 15: pMDR-CAT
(M.M. Gottesman) + pBC-rex; 16: pLTR-CAT + pBC-rex; 17:
pMDR(+)CAT + pRK7-tax; 18 pMDR(-)CAT + pRK7-tax; 19: pMDR-CAT (M.M. Gottesman) + pRK7-tax; 20: pLTR-I-CAT + pRK7-tax. Therefore, lanes 1 to 8, 10, 14, and 18 are negative controls; lanes 12 and 20 are
positive controls. Trans-activation of the MDR1 promoter-CAT constructs by tax can be seen in lanes 9, 11, 17, and 19. Lanes 13 to
16 show negligible activation of the MDR1 promoter-CAT
constructs by rex.
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DISCUSSION |
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.
Our results show that the MDR1 gene is expressed at relatively
high levels in all categories of HTLV-I-infected individuals (asymptomatic, HAM/TSP, ATL, and lymphoma). The molecular mechanisms involved in the normal cellular control of MDR1 gene expression remain unclear. Upstream promoter sequences from the MDR1 gene have been isolated and sequenced.40 The MDR1
promoter does not contain a TATA box, but does contain several distinct
regulatory motifs including a CAAT box (113 to 118), two
GC-rich regions (-51 to 61, 103 to 110), which are
putative Sp-1 binding sites, a heat shock consensus element, and an
AP-1-like binding site. Sequences immediately downstream of the
transcription start site are also involved in MDR1
expression.41,42 The region of DNA present in pMDR-CAT and
pMDR-CAT (M.M. Gottesman) contains all of these putative regulatory
elements.
The HTLV-I trans-activator protein, tax, is a 40-kD
nonstructural nuclear protein encoded by a doubly spiced subgenomic
viral mRNA. The tax protein is a positive trans-activator of both viral and cellular transcription and is thought to be the main cause of
cellular transformation, due to aberrant expression of a large range of
heterologous cellular genes.2 The mechanism of tax trans-activation is indirect, mediating enhanced transcription through
interactions with cellular transcription factors rather than by direct
binding to DNA. Such transcription factors include the cAMP-responsive
element binding protein/activating transcription factor (CREB/ATF),
NF- B, and serum response factor (SRF) families.43
Evidence for trans-acting transcriptional activation of MDR1
comes from observations of differential expression of MDR1 in normal tissues and tumors in the absence of gene amplification, which
suggests transcription factors are involved.11 Enhanced transcription has also been shown in cells that additionally contain MDR1 gene amplifications.44 MDR1 gene
expression can be activated when cells are subjected to chemical or
physical stress45,46 and increased MDR1 promoter
activity is seen in transfected cells treated with anticancer
agents.47 NF-IL-6 (or C/EBP-), a bZIP motif-containing member of the C/EBP (CCAAT enhancer binding protein) family of transcription factors involved in regulating cytokine IL-6
transcription, has been associated with trans-activation of
MDR1 promoter.48 This suggests a possible mechanism
for trans-activation of MDR1 by tax
(Fig 6), as the tax protein is capable of
modulating the transcription factor CREB through interactions with the
basic domain of bZIP proteins, increasing dimerization, and DNA binding affinities to increase transcription from CRE-containing
promoters.2 A similar mechanism could be postulated with
NF-IL-6, whereby associations with tax via the bZIP domain may lead to
increased active NF-IL-6 dimers and upregulation of MDR1
transcription, overexpression of P-gp, and development of the MDR
phenotype. Although no direct protein associations between tax and the
CEBP family of proteins have been reported, tax has been shown to be able to modulate granulocyte colony-stimulating factor (G-CSF) expression via a NF-IL-6 consensus sequence.49
The presence of GC-rich elements (putative SP1 binding sites) and an
AP-1 like element in the MDR1 promoter are also other potential
targets for tax trans-activation (Fig 6). Both of these elements have
been implicated in mediating trans-activation by tax the protein.
HTLV-I tax upregulates c-fos50 and HTLV-infected
cells show increased AP-1 binding activity.51 AP-1
induction may be critically involved in the trans-activation of other
genes, such as transforming growth factor-1
(TGF-1).52 Moreover, development of the MDR phenotype in
mouse carcinoma cells has been associated with drug inducible increased
c-fos expression,53 suggesting a possible mechanism
whereby the induction of AP-1 complexes stimulate MDR1 gene
expression. Hence, possible trans-activation mechanisms known to be
active for tax can also be implied for the MDR1 gene,
suggesting a molecular basis for the involvement of HTLV-I infection
and MDR development in the resulting tumor. Studies of tax-mediated
activation of MDR1 may aid in the elucidation of the complex
molecular mechanisms involved in normal MDR1 gene regulation.
The possibility of drug resistance phenotypes manifesting themselves in
HTLV-induced tumors seems likely based on the evidence outlined above.
The obvious clinical benefits could include alternative treatments
based on reversion of the MDR phenotype, such as using chemosensitizing
drugs such as cyclosporin A, reserpine, tamoxifen, or
verapamil11,54 or newer specific MDR inhibitors currently in clinical trials. Such compounds act as competitive inhibitors for
drug binding or transport by P-gp and, if used in conjunction with
cytotoxic drugs, may allow full chemotherapeutic potentials to be
achieved. Ultimately, this could lead to a greatly improved prognosis
for patients with HTLV-induced tumors. Moreover, an improved
understanding of the mechanisms underlying control of expression of the
MDR1 gene would be a very important contribution to the
management and therapy of many different types of leukemia and other
tumors in which this gene is believed to play a role.
| |
FOOTNOTES |
Submitted April 22, 1997;
accepted November 16, 1997.
Supported by Grant No. 92/13 from the Leukaemia Research Fund (to
A.J.C.) and MRC Grant No. G9622810. A.L. was the recipient of an MRC
PhD studentship.
Address reprint requests to Alan J. Cann, PhD, Department
of Microbiology and Immunology, University of Leicester, Leicester LE1
9HN, United Kingdom.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
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.
| |
REFERENCES |
1.
De The G,
Bomford R:
An HTLV-I vaccine: Why, how, for whom?
AIDS Res Hum Retroviruses
9:381,
1993[Medline]
[Order article via Infotrieve]
2. Cann A, Chen I: Human T-cell leukemia virus types I and II, in
Fields BN, Knipe DM, Howley PM (eds): Fields Virology. New York, NY,
Raven, 1996, p 1501
3.
Minato K,
Araki K,
Hanada S:
An interim report of LSG4 treatment for advanced peripheral T-cell lymphoma.
Int J Hematol
54:179,
1991
4.
Shimoyama M:
Treatment of patients with adult T-cell leukemia-lymphoma: An overview.
Gann Monogr Cancer Res
39:43,
1992
5.
Dearden C,
Matutes E,
Catovsky D:
Deoxycoformycin in the treatment of mature T-cell leukaemias.
Br J Cancer
64:903,
1991[Medline]
[Order article via Infotrieve]
6.
Ohno R,
Masaoka T,
Shirakawa S,
Sakamoto S,
Hirano M,
Hanada S,
Yasunaga K,
Yokomaku S,
Mitomo Y,
Nagai K,
Yamada K,
Furue H:
Treatment of adult T-cell leukemia/lymphoma with MST-16, a new oral antitumor drug and a derivative of bis(2,6-dioxopiperazine).
Cancer
71:2217,
1993[Medline]
[Order article via Infotrieve]
7.
Waldmann TA,
White JD,
Goldman CK,
Top L,
Grant A,
Bamford R,
Roessler E,
Horak ID,
Zaknoen S,
Kasten-Sportes C,
England R,
Horak E,
Mishra B,
Dipre M,
Hale P,
Fleisher TA,
Junghans RP,
Jaffe ES,
Nelson DL:
The interleukin-2 receptor: A target for monoclonal antibody treatment of human T-cell lymphotrophic virus I-induced adult T-cell leukemia.
Blood
82:1701,
1993[Abstract/Free Full Text]
8.
Gill PS,
Harrington WJ,
Kaplan MH,
Ribeiro RC,
Bennett JM,
Liebman HA,
Bernstein-Singer M,
Espina BM,
Cabral L,
Allen S,
Kornblau S,
Pike MC,
Levine AM:
Treatment of adult T-cell leukemia-lymphoma with a combination of interferon alfa and zidovudine.
N Engl J Med
332:1744,
1995[Abstract/Free Full Text]
9.
Hermine O,
Bouscary D,
Gessain A,
Turlure P,
Leblond V,
Franck N,
Buzyn Veil A,
Rio B,
Macintyre E,
Dreyfus F,
Bazarbachi A:
Brief report: Treatment of adult T-cell leukemia-lymphoma with zidovudine and interferon alfa.
N Engl J Med
332:1749,
1995[Free Full Text]
10.
Endicott JA,
Ling V:
The biochemistry of P-glycoprotein-mediated multidrug resistance.
Annu Rev Biochem
58:137,
1989[Medline]
[Order article via Infotrieve]
11.
Gottesman MM,
Pastan I:
Biochemistry of multidrug resistance mediated by the multidrug transporter.
Ann Rev Biochem
62:385,
1993[Medline]
[Order article via Infotrieve]
12.
Gros P,
Ben Neriah Y,
Croop JM,
Housman DE:
Isolation and expression of a complementary DNA that confers multidrug resistance.
Nature
323:728,
1986[Medline]
[Order article via Infotrieve]
13.
Sugimoto Y,
Tsuruo T:
DNA-mediated transfer and cloning of a human multidrug-resistant gene of Adriamycin-resistant myelogenous leukemia K562.
Cancer Res
47:2620,
1987[Abstract/Free Full Text]
14.
Ueda K,
Cardarelli C,
Gottesman MM,
Pastan T:
Expression of a full-length cDNA for the human `MDR1' gene confers resistance to colchicine, doxorubicin, and vinblastine.
Proc Natl Acad Sci USA
84:3004,
1987[Abstract/Free Full Text]
15.
Campos L,
Guyotat D,
Archimbaud E,
Calmard-Oriol P,
Tsuruo T,
Troncy J,
Treille D,
Fiere D:
Clinical significance of multidrug resistance P-glycoprotein expression on acute nonlymphoblastic leukemia cells at diagnosis.
Blood
79:473,
1992[Abstract/Free Full Text]
16.
Goasguen JE,
Dossot JM,
Fardel O,
Le Mee F,
Le Gall E,
Leblay R,
LePrise PY,
Chaperon J,
Fauchet R:
Expression of the multidrug resistance-associated P-glycoprotein (P-170) in 59 cases of de novo acute lymphoblastic leukemia: Prognostic implications.
Blood
81:2394,
1993[Abstract/Free Full Text]
17.
Marie JP,
Zittoun R,
Sikic BI:
Multidrug resistance (mdr1) gene expression in adult acute leukemias: Correlations with treatment outcome and in vitro drug sensitivity.
Blood
78:586,
1991[Abstract/Free Full Text]
18.
Bell DR,
Gerlach JH,
Kartner N,
Buick RN,
Ling V:
Detection of P-glycoprotein in ovarian cancer: A molecular marker associated with multidrug resistance.
J Clin Oncol
3:311,
1985[Abstract]
19.
Bourhis J,
Goldstein LJ,
Riou G,
Pastan I,
Gottesman MM,
Benard J:
Expression of a human multidrug resistance gene in ovarian carcinomas.
Cancer Res
49:5062,
1989[Abstract/Free Full Text]
20.
Chan HSL,
Thorner PS,
Haddad G,
Ling V:
Immunohistochemical detection of P-glycoprotein: Prognostic correlation in soft tissue sarcoma of childhood.
J Clin Oncol
8:689,
1990[Abstract]
21.
Goldstein LJ,
Galski H,
Fojo A,
Willingham M,
Lai SL,
Gazdar A,
Pirker R,
Green A,
Crist W,
Brodeur GM,
Lieber M,
Cossman J,
Gottesman MM,
Pastan I:
Expression of a multidrug resistance gene in human cancers.
J Natl Cancer Inst
81:116,
1989[Abstract/Free Full Text]
22.
Goldstein LJ,
Fojo AT,
Ueda K,
Crist W,
Green A,
Brodeur G,
Pastan I,
Gottesman MM:
Expression of the multidrug resistance, MDR1, gene in neuroblastomas.
J Clin Oncol
8:128,
1990[Abstract/Free Full Text]
23.
Kato S,
Nishimura J,
Muta K,
Yufu Y,
Nawata H,
Ideguchi H:
Overexpression of P-glycoprotein in adult T-cell leukaemia.
Lancet
336:573,
1990[Medline]
[Order article via Infotrieve]
24.
Kuwazuru Y,
Hanada S,
Furukawa T,
Yoshimura A,
Sumizawa T,
Utsunomiya A,
Ishibashi K,
Saito T,
Uozumi K,
Maruyama M,
Ishizawa M,
Arima T,
Akiyama S:
Expression of P-glycoprotein in adult T-cell leukemia cells.
Blood
76:2065,
1990[Abstract/Free Full Text]
25.
Su IJ,
Chang IC,
Cheng AL:
Expression of growth-related genes and drug-resistance genes in HTLV-I-positive and HTLV-I-negative post-thymic T-cell malignancies.
Ann Oncol
2:151,
1991[Abstract/Free Full Text]
26.
Gluzman Y:
SV40-transformed simian cells support the replication of early SV40 mutants.
Cell
23:175,
1981[Medline]
[Order article via Infotrieve]
27.
Davies R,
Budworth J,
Riley J,
Snowden R,
Gescher A,
Gant TW:
Regulation of P-glycoprotein 1 and 2 gene expression and protein activity in two MCF-7/Dox cell line subclones.
Br J Cancer
73:307,
1996[Medline]
[Order article via Infotrieve]
28.
Zhang F,
Riley J,
Gant TW:
Use of internally controlled reverse transcriptase-polymerase chain reaction for absolute quantitation of individual multidrug resistant gene transcripts in tissue samples.
Electrophoresis
17:255,
1996[Medline]
[Order article via Infotrieve]
29.
Popovic M,
Sarin PS,
Robert Gurroff M,
Kalyanaraman VS,
Mann D,
Mirowada J,
Gallo RC:
Isolation and transmission of human retrovirus (human T-cell leukemia virus).
Science
219:856,
1983[Abstract/Free Full Text]
30.
Cullen BR:
Trans-activation of human immunodeficiency virus occurs via a bimodal mechanism.
Cell
46:973,
1986[Medline]
[Order article via Infotrieve]
31.
Chlichlia K,
Moldenhauer G,
Daniel PT,
Busslinger M,
Gazzolo L,
Schirrmacher V,
Khazaie K:
Immediate effects of reversible HTLV-1 tax function  T-cell activation and apoptosis.
Oncogene
10:269,
1995[Medline]
[Order article via Infotrieve]
32.
Ueda K,
Yamano Y,
Kioka N,
Kakehi Y,
Yoshida O,
Gottesman MM,
Pastan I,
Komano T:
Detection of multidrug resistance (MDR1) gene RNA expression in human tumors by a sensitive ribonuclease protection assay.
Jpn J Cancer Res
80:1127,
1989[Medline]
[Order article via Infotrieve]
33.
Chin KV,
Ueda K,
Pastan I,
Gottesman MM:
Modulation of activity of the promoter of the human MDR1 gene by Ras and p53.
Science
255:459,
1992[Abstract/Free Full Text]
34.
Chen ISY,
Slamon DJ,
Rosenblatt JD,
Shah MP,
Quan SG,
Wachsman W:
The x gene is essential for HTLV replication.
Science
229:54,
1985[Abstract/Free Full Text]
35.
Cann AJ,
Koyanagi Y,
Chen ISY:
High efficiency transfection of primary human lymphocytes and studies of gene expression.
Oncogene
3:123,
1988
36.
Koralnik IJ,
Gessain A,
Klotman ME,
Lo Monico A,
Berneman ZN,
Franchini G:
Protein isoforms encoded by the pX region of human T-cell leukemia/lymphotropic virus type I.
Proc Natl Acad Sci USA
89:8813,
1992[Abstract/Free Full Text]
37.
Gupta S,
Kim CH,
Tsuruo T,
Gollapudi S:
Preferential expression and activity of multidrug resistance gene 1 product (P-glycoprotein), a functionally active efflux pump, in human CD8+ T cells: A role in cytotoxic effector function.
J Clin Immunol
12:451,
1992[Medline]
[Order article via Infotrieve]
38.
Gupta S,
Tsuruo T,
Gollapudi S:
Multidrug resistant gene 1 product in human T cell subsets: Role of protein kinase C isoforms and regulation by cyclosporin A.
Adv Exp Med Biol
323:39,
1992[Medline]
[Order article via Infotrieve]
39.
Damiani D,
Michieli M,
Michelutti A,
Geromin A,
Raspadori D,
Fanin R,
Savignano C,
Giacca M,
Pileri S,
Mallardi F,
Baccarani M:
Expression of multidrug resistance gene (MDR-1) in human normal leukocytes.
Haematologica
78:12,
1993[Medline]
[Order article via Infotrieve]
40.
Ueda K,
Pastan I,
Gottesman M:
Isolation and sequence of the promoter region of the human multidrug-resistance (P-glycoprotein) gene.
J Biol Chem
262:17432,
1987[Abstract/Free Full Text]
41.
Madden MJ,
Morrow CS,
Nakagawa M,
Goldsmith ME,
Fairchild CR,
Cowan KH:
Identification of 5 and 3 sequences involved in the regulation of transcription of the human mdr1 gene in vivo.
J Biol Chem
268:8290,
1993[Abstract/Free Full Text]
42.
Strauss BE,
Shivakumar C,
Deb SP,
Deb S,
Haas M:
The MDR1 downstream promoter contains sequence-specific binding sites for wild-type p53.
Biochem Biophys Res Commun
217:825,
1995[Medline]
[Order article via Infotrieve]
43.
Wagner S,
Green MR:
HTLV-I Tax protein stimulation of DNA binding of bZIP proteins by enhancing dimerization.
Science
262:395,
1993[Abstract/Free Full Text]
44.
Morrow CS,
Chiu J,
Cowan KH:
Posttranscriptional control of glutathione S-transferase pi gene expression in human breast cancer cells.
J Biol Chem
267:10544,
1992[Abstract/Free Full Text]
45.
Chin KV,
Tanaka S,
Darlington G,
Pastan I,
Gottesman MM:
Heat shock and arsenite increase expression of the multidrug resistance (MDR1) gene in human renal carcinoma cells.
J Biol Chem
265:221,
1990[Abstract/Free Full Text]
46.
Uchiumi T,
Kohno K,
Tanimura H,
Hidaka K,
Asakuno K,
Abe H,
Uchida Y,
Kuwano M:
Involvement of protein kinase in environmental stress-induced activation of human multidrug resistance 1 (MDR1) gene promoter.
FEBS Lett
326:11,
1993[Medline]
[Order article via Infotrieve]
47.
Kohno K,
Sato S,
Takano H,
Matsuo K,
Kuwano M:
The direct activation of human multidrug resistance gene (MDR1) by anticancer agents.
Biochem Biophys Res Commun
165:1415,
1989[Medline]
[Order article via Infotrieve]
48.
Combates NJ,
Rzepka RW,
Chen YNP,
Cohen D:
NF-IL6, a member of the C/EBP family of transcription factors, binds and trans-activates the human MDR1 gene promoter.
J Biol Chem
269:29715,
1994[Abstract/Free Full Text]
49.
Himes SR,
Coles LS,
Katsikeros R,
Lang RK,
Shannon MF:
HTLV-1 tax activation of the GM-CSF and G-CSF promoters requires the interaction of NF-kB with other transcription factor families.
Oncogene
8:3189,
1993[Medline]
[Order article via Infotrieve]
50.
Alexandre C,
Verrier B:
Four regulatory elements in the human c-fos promoter mediate transactivation by HTLV-1 tax protein.
Oncogene
6:543,
1991[Medline]
[Order article via Infotrieve]
51.
Fujii M,
Niki T,
Mori T,
Matsuda T,
Matsui M,
Nomura N,
Seiki M:
HTLV-1 tax induces expression of various immediate early serum responsive genes.
Oncogene
6:1023,
1991[Medline]
[Order article via Infotrieve]
52.
Kim SJ,
Kehrl JH,
Burton J,
Tendler CL,
Jeang KT,
Danielpour D,
Thevenin C,
Kim KY,
Sporn MB,
Roberts AB:
Transactivation of the transforming growth factor beta1 (TGF-beta1) gene by human T lymphotropic virus type 1 Tax: A potential mechanism for the increased production of TGF-beta1 in adult T cell leukemia.
J Exp Med
172:121,
1990[Abstract/Free Full Text]
53.
Bhushan A,
Abramson R,
Chiu JF,
Tritton TR:
Expression of c-fos in human and murine multidrug-resistant cells.
Mol Pharmacol
42:69,
1992[Abstract]
54.
Beck WT,
Qian XD:
Photoaffinity substrates for P-glycoprotein.
Biochem Pharmacol
43:89,
1992[Medline]
[Order article via Infotrieve]
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Abstract
Introduction
Abstract
