1. Art and Diseño

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Constitutive Overexpression of the L-Selectin Gene in Fresh Leukemic Cells
of Adult T-cell Leukemia That Can Be Transactivated by Human T-cell
Lymphotropic Virus Type 1 Tax
By Makoto Tatewaki, Kazunari Yamaguchi, Masao Matsuoka, Toshinori Ishii, Masayuki Miyasaka, Shigeo Mori,
Kiyoshi Takatsuki, and Toshiki Watanabe
L-selectin is an adhesion molecule of the selectin family that
mediates the initial step of leukocyte adhesion t o vascular
endothelium. Upon cellular activation, expression of the Lselectin gene is downregulated at both the protein
and
mRNA levels. To understand the mechanism of leukemic
cell infiltration into organs, we studied the expression and
regulation of L-selectin mRNA in fresh leukemic cells of adult
T-cell leukemia (ATL) patients and investigatedthe response
of theL-selectin promoter t o human T-cell lymphotropic virus type1(HTLV-1)Tax, which isa viral transcriptional transactivator. Flow cytometry showed that L-selectin was expressed onfresh ATL cells along with otheractivation
antigens. Northern blotanalysis showed that ATL cells overexpressed the L-selectin mRNA and that the
level was aberrantly upregulated after PMA stimulation. Studies using in
situ hybridizationshowed
expression of the L-selectin
mRNA in the infiltrating leukemic cells in the liver of two
ATL patients. Intravenous injection of a rat T-cell line that
overexpresses L-selectin showed increased organ infiltration. The induction ofTax expression in JPX9 cells resulted
in about a twofold increase in the mRNA expression levels
compared with the basal level. Chloramphenicol acetyltransferase(CAT)assay after transient cotransfection showed
about a fivefold transactivation of the L-selectin promoter
by Tax. The serum level of the shed form of L-selectin was
significantly increased in ATL patients (mean f SD, 4,215.4 f
4,111 ng/mL) compared with those of asymptomatic carriers
and healthy blood donors (mean f SD, 1.148.0 & 269.0 ng/
mL and 991.9 f 224 ng/mL, respectively). These results indicated that ATL cells constitutively overexpress the L-selectin
gene that can be transactivated by HTLV-1 Tax. The overexpression of L-selectin, aswell as of inflammatory cytokines,
by ATL cells may provide a basis for ATL cells t o attach the
vascular endothelium, leading t o transmigration and organ
infitration.
0 1995 by The American Societyof Hematology.
A
from the cell surface by shedding16 and transcription of the
mRNA is also downregulated by18 to 24 hours.L7318
This
downregulation is thought to prepare the extravasated cells
for transmigration into tissues. The extracellular domain is
proteolytically shed from the cell surface” and the shed
form of L-selectin (sL-selectin) is present at a relatively high
concentration in the normalserum, in which it is functionally
active, inhibiting leukocyte attachment to the endothelium
at high concentrations.20Thus, the regulation of L-selectin
expression at the transcriptional level as well as in protein
processing appears to be an important determinant of physiologic and pathologic manifestations of the function of this
adhesion molecule.
HTLV- 1 has a transcriptional activator, Tax, that regulates
viral replication at the transcriptional level.21Transactivation
of the viral genes by Tax is mediated through three repeats
of a 21-bp enhancer in the long terminal repeat (LTR).22
Because Tax cannot directly bind to the enhancer sequence,
the cellular transcription factors are thought to mediate the
DULT T-CELL LEUKEMIA (ATL) is an aggressive,
usually fatal T-cell malignancy that is caused by human T-cell lymphotropic virus type 1 (HTLV-1)
ATL is also characterized by unique clinical features such as
a very high frequency of hypercalcemia anda remarkable tendencyoforgan
infiltration by leukemiccellsintotheliver,
spleen,lungs,and
~ k i n . 4 ,HTLV-1
~
isalsoassociated
with
chronic inflammatory disorders such as tropical spastic paraparesis /HTLV-1 -associated myelopathy (TSPMAM), HTLV-l
uveitis (W),alveolitis, and others!-” Infiltration of mononuclear cells and HTLV-1 -infected
T cells is alsoa characteristic
commonly found in these
HTLV-l-associated diseases. Spinal
cord lesions of patients with TSP/HAM are characterized by
demyelinationaccompanied by mononuclearcellinfiltration
and perivascularcuffing.12 Inthe aqueous humor
of the patients
with HU, the infiltrating cells consisted mostly
of lymphocytes
with a predominance of T cells and theHTLV-1-infected
cells
were
aimost
universally
present
among
Thus,
the
infiltration of lymphocytes in various organs could be one of
thecommonpathologicfindingsamongdiseasesassociated
with HTLV-1 infection.
L-selectin is a member of the selectin family of adhesion
molecule^.^^ It is expressed on the surface of most leukocytes, including lymphocytes, neutrophils, monocytes, eosinophils, hematopoietic progenitor cells, and immature thymocytes. L-selectin is a highly glycosylated protein of 95 to
105 kD on neutrophils and of 74 kD on lymphocytes. It is
thought to mediate lymphocyte recirculation to the peripheral
lymph node, specifically recognizing high endothelial venules in lymph nodes. In addition, L-selectin is involved in
the adhesion of neutrophils, monocytes, and lymphocytes to
activated endothelium at sites of inflammation. Extravasation
of circulating lymphocytes consists of three steps? rolling
mediated by selectins, sticking .by integrins, and transendothelial migration. L-selectin is considered to mediate the
initial attachment of lymphocytes to endothelium, which determines the stickiness of the cells.
After cellular activation, this molecule rapidly disappears
B/ood, Vol86, No 8 (October 15). 1995 pp 3109-3117
From the Department of Pathology, The Institute of Medical Science, The University of Tokyo, Tokyo; theSecond Department of
Internal Medicine, Kumamoto University School ofMedicine, Kumamoto: andthe Division of Organ Bioregulation, Biomedical Research Center, Osaka University Medical School, Osaka, Japan.
Submitted December 30, 1994: accepted June S, 1995.
Supported in part by a Grant in Aid for ScientGc Research to
T.W. and a Grant in Aid for Cancer Research to T. W.and K. Y. from
the Ministry of Education, Science and Culture, Japan.
Address reprint requests to Toshiki Watanabe, MD, PhD, Department of Pathology, The Institute of Medical Science, The University
of Tokyo, 4-6-1 Shirokanedai, Minatoku, Tokyo 108, Japan.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. section I734 solely to
indicate this fact.
0 1995 by The American Society of Hematology.
0006-4971/95/8608-0007$3.00/0
3109
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31 10
TATEWAKI ETAL
transa~tivation?~.~~
In addition to the viral genes, Tax induces
the expression of the cellular transcription factor genes25that
are controlled by transmembrane signals and also some
genes that encode cytokines and their rece~tors.*~-*~
Some of
the genes that can be transactivated by Tax are constitutively
expressed in ATL cells that modulate clinical
ATL cells have a membrane phenotype ofactivated CD4+
T cells, expressing interleukin-2 receptor (Y (LL-2Ra), HLADR, and other activated cell antigen^.^',^' The data of Ishikawa et al. suggested that ATL cells are characterized as
CD4+Leu-8’ T cells?2 Because Leu-8 antigen is identical
to L-selectin, ATL cells express typical activation antigens
and L-selectin at the same time, which suggests that the
regulation of L-selectin expression is aberrant.
We studied the expression of L-selectin in ATL cells and
tested the regulation of its gene expression by HTLV-1 Tax,
which would provide a clue to understanding the molecular
mechanism of L-selectin gene expression and leukemic cell
infiltration into various organs in ATL patients.
MATERIALS AND METHODS
Patientsplasma and cell samples. ATL was diagnosed according to the described criteria? Briefly, a T-lymphocyte neoplasm
that resulted from the monoclonal expansion of HTLV-l-infected
T cells was diagnosed as ATL. Seropositivity against HTLV-l was
tested using a PA kit (Fujirebio, Tokyo, Japan) and confirmed using
an enzyme immunoassay (HA) kit (Eizai, Tokyo, Japan). The integration of HTLV-l provirus DNA was studied using Southern blottir~g.~’
Peripheral blood mononuclear cells (PBMCs) from patients with
ATL and normal blood donors were isolated by density gradient
centrifugation with Ficoll Paque (Pharmacia, Uppsala, Sweden).
RNA was immediately extracted from the cells or they were quickly
frozen in liquid nitrogen and stored at -80°C until use. Plasma
samples were collected at the time of PBMC preparation and stored
at -80°C until use.
Cell lines. Jurkat is a human T-cell line established from T-ALL
cells and JPX-9 is a derivative of Jurkat cells carrying the transfected
Tax gene under the control of metallothionein promoter.34MT-1 is
a human T-cell line derived from leukemic cells of an ATL patient.35
These cells were cultured in RPM11640 supplemented with10%
fetal bovine serum and antibiotics. FL cells were derived from human
amnion cells36and maintained in Dulbecco’s modified Eagle’s medium supplemented with 5% fetal bovine serum and antibiotics. The
expression of Tax in JPX9 cells was induced by adding 20 pmol/L
CdClz into the medium.
Immunophenotyping. Cell surface markers of leukemic cells
from 20 patients with ATL (13 with acute type and 7 with chronic
type) were studied and those of normal T cells from 9 blood donors
served as controls. Heparinized whole blood samples were immunophenotyped without PBMC isolation. Cell surface antigens were
detected by standard immunofluorescence using a panel of directly
fluorescein isothiocyanate (FlTC)- or phycoerythrin-conjugated
monoclonal antibodies. The expression of surface antigens was studied by means of double immunofluorescence using antibodies that
react with CD3, CD4, CD8, CD25, and Leu-8 (Becton Dickinson,
San Jose, CA; Coulter, Hialeath, FL). Double immunofluorescence
was performed using a Spectrum 111 flow cytometer (Ortho, Westwood, MA). A total of more than 5,000 cells were analyzed for each
sample.
Immunohistochemical analysis of the formalin-fixed and paraffinembedded tissues was performed using SAB rneth~d.’~
Analysis of L-selectin gene expression. Total cytoplasmic RNA
was extracted from the fresh PBMCs of ATL patients and cell lines
using the vanadyl ribonucleoside complex.38To study the modulation
of gene expression, PBMC samples were cultured for 18 to 24 hours
with or without phorbol myristate acetate (PMA; 10 ng/mL)and
then cytoplasmic RNA was extracted. Ten micrograms of total RNA
was Northern blotted. After transfer to nylon membranes (BioDyne
B; Pall BioSupport COT, Glen Cove, NY), the RNA was fixed by
UV irradiation. The membrane was hybridized with probes at 42°C
in a solution containing 50% formamide, 4X SSC, lox Denhardt’s
solution, 0.1% sodium dodecyl sulfate (SDS), and 100 pglmL of
denatured salmon sperm DNA. An L-selectin cDNA probe covering
entire protein coding sequence was prepared using reverse transcription-polymerase chain reaction (RT-PCR) and cloning into the pCRIl
plasmid by means of TA cloning (In Vitrogen, San Diego, CA). The
nucleotide sequence of the insert cDNA was confirmed by sequencing using the Sequenase kit (US Biochemicals, Cleaveland, OH).
For detection of L-selectin mRNAin tissue samples, we performed in situ hybridization. Samples of the formalin-fixed and paraffin-embedded liver of two ATL patients and a biopsy specimen of
a patient with chronic active hepatitis were used for the study. Serial
sections of 4 to 6 pm were prepared on the slides coated with 3aminopropyltriethoxysilane. Digoxigenin-labeled antisense and
sense L-selectin cRNA probes were prepared from a plasmid containing 589bp cDNA fragment (nucleotide position 998-1586)17using DIG RNA Labelling Kit (Boehringer Mannheim Biochemica,
Mannheim, Germany). Hybridization was performed at 37°C for 12
hours in a solution containing 50% deionized formamide, 10 mmoll
L Tris HCI, pH 7.6, 10% dextran sulfate, 1 X Denhardt’s solution,
600 mmollL NaCI, 1 mmollL EDTA, 1% SDS, and 200 ng/mL DIGlabeled cRNA probe. After hybridization, the slides wererinsed
twice in a solution containing 2X SSC and 0.1% SDS at room
temperature for 5 minutes and once in a solution containing 0 . 2 ~
SSC and 0.1% SDS at room temperature for 5 minutes. Detection
of the hybridization was performed using DIG Nucleic Acid Detection Kit(Boehringer Mannheim Biochemica) according to the manufacturer’s instructions.
Analysis of organ injiltrution by an L-selectin-overexressing rut
T-cell line. An expression vector for the rat L-selectin was constructed using BCMGSNeo
and
an
HTLV-lhnfected rat
T-cell line TARL-2,“‘ which does not express L-selectin, was transformed and selected by neomycin. Expression of L-selectin by the
transformed cell line was confirmed by Nothem blotting and flow
cytometry (data not shown). About 1 X 10’ cells of the L-selectinoverexpressing TARL-2 [TARL-2 L(+)] and the original cell line
were labeled by FITP’ and injected into WKAH rat intravenously.
Two hours after injection, an immunohistichemical study was performed using anti-FITC antibody (DAKOPAITS AIS, Glostrup,
Denmark) and SAB method.37
Plasmid, transfections, and chloramphenicol acetyltransferase
(CAT) assays. L-selectin promoter-CAT plasmid, pLS-I CAT, has
a genomic fragment of about 900-bp that flanks the 5’ end of exon
2 of the L-selectin gene.4’ The L-selectin promoter has been identified and characterized (Tatewaki et al, submitted for publication).
Transcriptional transactivation was tested by transient cotransfection
assays using a Tax expression plasmid.26FL and Jurkat cells were
transfected using CaPO, precipitation and diethyl aminoethyl
(DEAE)-dextran, respectively.4 About 1 X IO7 cells of each type
were transfected with 10 pg of CAT reporter plasmids, together with
2 pg of the Tax expression vector. After 48 hours, cells were harvested and extracts were assayed for CAT activity according to the
method of Gorman et
except the radioactivity was measured by
Bio lmage Analyzer, BA100 (Fuji, Tokyo, Japan). The pp-Gal plasmid was cotransfected to monitor the efficiency of transfection and
CAT activity was corrected by the activity of p-Gal in each experiment.
Quantitation of the serum level of sL-selectin and sIL-2Ra. Serum levels of the shed form L-selectin (sL-selectin) were quantified
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OVEREXPRESSION OF L-SELECTIN IN ATL
3111
Table 1. Leu4 Expression in the Peripheral Blood of ATL Patients
and Normal Controls
Case No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
Type
CD4'
Leu8'
Acute
24.8 92.6
Acute
75.9 86.4
Acute
63.8 73.6
Acute
44.9 80.2
Acute
47.1 89.6
Acute
57.6 96.5
Acute
55.9 64.1
Acute
78.5 95.6
Acute
65.6 97.7
Acute
53.3 86.4
Acute
86.9 94.4
Acute
73.8 77.2
Acute
51.7 84.2
Chronic 84.3 91.o
Chronic 90.3
92.3
Chronic 84.1 94.6
Chronic 62.8 66.0
Chronic 87.0 96.4
Chronic 59.2 71.6
Chronic
97.2
Control 43.6
48.3
Control 41.0
46.4
Control 31.1
37.8
Control 43.3
47.1
Control
30.9
Control 39.4
47.1
Control 33.4
32.0
Control 37.6
ND
Control 33.8
ND
17.7'
68.1'
85.0'
57.9'
69.3*
44.9'
68.8'
63.6'
53.4'
1.8'
84.8'
77.6'
54.0'
85.1'
4.8
82.5'
69.9'
79.4*
63.5'
88.7'
70.1
81.2
70.7
79.1
69.5
70.4
65.5
73.8
54.0
*
100%
I .
Leu-8'1CD4'
20
I
n
0
0
I
I
ATL Control
ND
Fig 1. The ratio of Leu&positive cells among CD4+ cells in the
peripheral blood ofATL patients and normal controls. The percentages in ATL patients are significantly higher than those of normal
controls (Mann-Whitney U test). 'P = .0002.
25.1
The percentages of positive cells for each antigen are shown.
Abbreviations: CD4', Leu-8*, percentages of cells positive for respective antigen among lymphocytes; Leu-8'/CD4', percentage of
Leu-8-positive cells among CD4' cells;acute,
acute-type ATL;
chronic, chronic-type ATL; control, control samples collected from
normal volunteers; ND, not determined.
MFI of positive cells was lower than that of controlcells.
using an sL-selectin enzyme-linked immunosorbent assay (ELISA)
kit (Bender MedSystems, Vienna, Austria) and those o f the soluble
form of IL-2Ra (sL2Ra)
were quantified with an sIL-2Ra ELISA
kit (Tcell Diagnostics, Cambridge, MA), according to the manufacturer's instructions. The plasma or serum samples numbered 29 from
A T L patients, 10 from asymptomatic carriers, and 10 from seronegative control individuals. Among
the samples f r o m A T L patients,
PBMCs from 5 were included in the analysis o f L-selectin mRNA
expression using Nothem hybridization.
RESULTS
The surface expression of L-selectin on ATL cells. Flow
cytometry showed that the percentage of L-selectin-positive
cells among those that were CD4+ was 65.7% 2 17.6%
(mean 2 SD) in ATL patients, whereas that of the normal
control was 36.5% t 6.15% (mean 2 SD; Table 1). The
results of the flow cytometry are summarized in Fig 1. The
population of Leu-8-positive cells among the CD4+ cells
that almost represents the entire leukemic cell population
was significantly high in ATL patients (P= .0002 by MannWhitney U test).
L-selectin rnRNA expression in ATL cells. Northern blot-
ting showed that normal PBMCs expressed L-selectin transcripts at a relatively high level and that the PBMCs of ATL
patients, which consisted of mainly ATL cells, expressed it
at much higher levels (Fig 2). These results were confirmed
in all 8 patients with ATL that were studied. We then tested
the modulation of gene expression of L-selectin by activating
the cells in vitro. The level of L-selectin expression in the
ATL cells was upregulated after 18 to 24 hours of stimulation
with PMA, whereas the normal PBMCs were downregulated
(Fig 3). All of the three HTLV-l viral transcripts, ie, genomic, env, and pX, were detected after stimulation, whereas
they were not expressed in fresh leukemic cells at a detectable level (Fig 3). Thus, fresh primary ATL cells constitu-
1
L-selectin
2
3
4
4 18s
ribosomal
RNA
Fig 2. Expression of L-selectin mRNA in fresh PBMCs of ATL patients. Ten micrograms of total RNA sample was applied t o each
lane. Lane 1, normal freshPBMC RNA; lanes 2 through 4, PBMC RNA
samples from patients with acute ATL.
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3112
TATEWAKI ET AL
1
Lselectin
2
3
4
induction
The
of L-selectin gene expression byinTax
JPX-9cells. To examine the effect of Tax on the L-selectin
4 18s
HTLV-1
ribosomal
RNA
Fig 3. Aberrant regulation of L-selectin mRNA expression and the
induction of HTLV-1 viral mRNA by PMA stimulation. Lanes 1 and 2,
fresh (1) and PMA-stimulated (2) PBMC samples from a normal control; lanes 3 and 4, fresh (31 and PMA-stimulated (4) PBMC samples
from a patient with acute ATL L-selectin mRNA expression was
upregulated after PMA stimulation in ATL cells. HTLV-1 viral mRNA
was also induced by PMA stimulation of ATL cells.
tively overexpressed L-selectin mRNA in the absence of
viral gene expression, which was further upregulated after
PMA stimulation. The correlation between viral gene expression including pX mRNA and upregulation of L-selectin
gene expression suggested that the viral transactivator, Tax
that is encoded by pX mRNA, is involved in the induction
of L-selectin gene expression, as it is with other cellular
genes.
The expression of L-selectin mRNA in the infiltrating
leukemic cells was studied using in situ hybridization of the
liver samples of two ATL patients that show perivascular
infiltration of ATL cells. A liver biopsy specimen of a
chronic hepatitis patient served as a control. Serial sections
of the liver were hybridized with DIG-labeled antisense (Fig
4A) or sense (Fig 4B) L-selectin cRNA probe. Hybridization
signals for L-selectin mRNA were detected in infiltrating
mononuclear cells in Glisson's sheath of the liver (Fig 4A)
only when the antisense cRNA probe was used. Immunohistochemical staining withUCHL-l
monoclonal antibody
showed that most of the infiltrating cells were T cells (Fig
4C). Almost no hybridization for L-selectin mRNA was detected in the infiltrating lymphocytes in the biopsy specimen
of a chronic active hepatitis patient (data not shown).
Organ infiltration of a rat T-cell line that overexpresses
L-selectin. As shown in Fig 5 , many FITC-labeled cells
were detected immunohistochemically in Glisson's sheath
of the liver when TARL-2 (L+) cells were injected, whereas
only a few were detected when the original cells were used.
Effcient FITC labeling of both cells was confirmed by detection of the injected cells in the lung in which these two cells
did not show a marked difference in the number of trapped
cells (data not shown).
gene expression, we used JPX-9 cells that have an inducible
Tax gene. We studied the time course of the expression of
L-selectin and Tax by means of Northern blotting. CdClz
added to the culture medium induced Tax gene expression
in JPX-9 cells within 1.5 hours.% Although L-selectin gene
is constitutively expressed in JPX-9 cells at a relatively high
level, the expression started to increase along with the induction of Tax expression. It showed a small peak at 2 hours,
a small decrease at 6 hours, and then a continuous increase
until 33 hours, reaching about double the basal level (Fig
6). CdCl2 did not influence the level of L-selectin gene expression in the parental Jurkat cells (data not shown). Thus,
the results suggested that Tax increased the transcription of
the L-selectin gene and that endogenous cellular factors are
involved in the induction.
The L-selectin promoter is transactivated by HTLV-1 Tax.
To examine whether the L-selectin promoter, which we identified (Tatewaki et al, submitted for publication), responds
to Tax, we assayed CAT by means of transient cotransfection
with the Tax expression vector. The reporter plasmid, pLS1 CAT, has the L-selectin promoter fragment up to -885.
The promoter was activated by Tax both in Jurkat and FL
cell lines. Representative results are shown in Fig 7. The
magnitude of transactivation by Tax was about fivefold in
FL cells and a little less in Jurkat cells.
To identify the Tax-responsive element(s), we prepared
CAT reporter plasmids with a serially deleted promoter sequence and tested the response to Tax. However, the levels
of transactivation were gradually decreased with deletion
and no specific region that affected the response was identified (data not shown). These results suggested that multiple
elements in the promoter are involved in the transactivation
by Tax.
Elevated serum levels of sL-selectin in ATL patients. To
examine the overexpression of L-selectin by ATL cells in
vivo, we measured the level of sL-selectin in the sera of 29
ATL patients, 10 asymptomatic carriers, and 10 normal
blood donors. We also measured the serum level of IL-2Ra.
which is a good marker of leukemic cell mass in vivo.*
The mean level of sL-selectin in 29 patients with ATL was
significantly increased (mean t SD, 4,215.4 ? 4,111 ng/
mL) compared with those in asymptomatic carriers and normal blood donors (mean ? SD, 1,148.0 ? 269 ng/mL and
991.9 2 224 ng/mL, respectively; Fig 8). We studied the
correlation between sL-selectin and sIL-2Ra in samples
from 25 patients with ATL. As shown in Fig 9, the level of
sL-selectin correlated well with that of sIL-2Ra ( y = 0.6).
suggesting that the serum level of sL-selectin depends on
the leukemic cell mass. These results provided more evidence that L-selectin is overexpressed by ATL cells in vivo.
DISCUSSION
The adhesion properties of peripheral blood leukemic cells
from 10 patients with ATL have been characterized by Ishikawa et aI?* They showed that E-selectin and vascular cell
adhesion molecule-l (VCAM-1) mediate ATL cell adhesion
to endothelial cells. However, the counter-receptors on ATL
cells for E-selectin and VCA"1 remained to be determined.
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OVEREXPRESSION OF L-SELECTIN IN ATL
3113
Fig 4. Expression of L-selectin mRNA in the infitrating ATL cells
in theliver. (A and B) Hybridizationwith antisense and sense probes
for Laalectin, respectively. (C) Immunohistochemical daection of T
dlr using UCHL-1 monoclonalantibody.
Adhesion molcules expressed on ATL cells have not been
well characterized. Fukudome et a14’ reported that intercellular adhesion molecule-l (ICAM-1) expression was highly
induced in human T cells transformed by HTLV-l and fresh
ATL cells. However, it is not known whether they are involved in organ infiltration of ATL cells. Thus, further characterization of adhesion molecules expressed on ATL cells
should help understand the mechanism underlying organ infiltration.
Fig 5. Infiltration inthe liver by an L-selectin overexpressing T-cell line, TARL-2
L(+), after intravenous
injection. The liver from a rat injected with TARL-2
L(+) is indicated as L(+) and that injected with the
original TARL-2 as L(-) (lower left).A control reaction without anti-MTC antibody using the liver of a
rat injected with TARL-2 L(+) is indicated as PBS
(upper left).
We showed that the L-selectin gene is constitutively overexpressed in ATL cells and that the viral transactivator Tax
can induce its expression. We also showed that the serum
levels of sL-selectin in ATL patients were significantly elevated and that they correlated well with those of sIL-2Ra.
These results indicated that the constitutive overexpression
of L-selectin is one of the characteristic phenotypes of ATL
cells.
Our flow cytometric study of surface markers of ATL
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TATEWAKI ET AL
3114
A time (h)
0 2 4 11 18 33 B
n
L-Selectin
HTLV-1 Tax
GAPDH
0
2
4
11 18 33time ( h )
Fig 6. Induction of L-selectin mRNA expression after the expression of HTLV-1 Tax in JPX9 cells. (A) Resutts of Northern blot hybridization.
g
RNA were subjected for formalin-agarose gel electrophoresis. (B) The relative intensities of the hybridization signals.
Samples of l 0 p total
Measured radioactivity was corrected using that of GAPDH.
cells cleary showed that they expressed Leu-8 antigen (Table
1 and Fig 1). The mean fluorescence intensity (MFI) of
positive cells was generally low in ATL cells compared
with that of normal controls. Considering the constitutive
overexpression of mRNA shown in this study, low MFI may
suggest a rapid turnover of the protein. In line with this
notion, other investigatcrs have reported variable levels of
L-selectin expression on ATL cells.3z It has been reported
that L-selectin surface expression is downregulated by various leukocyte isolation procedures?' which may explain
some inconsistency in the data reported by different groups.
Therefore, to characterize the L-selectin expression, mRNA
expression should be studied by such means as Northern
blotting in addition to flow cytometry and immunocytochemistry. The results of our Northern blot analysis showed a
rather constant and high level of mRNA expression in ATL
samples (Fig 2), which is consistent with the results of our
flow cytometric analysis. In addition, demonstration of Lselectin mRNA in the infiltrating cells in the liver of ATL
patients (Fig 4), but not in the liver with inflammatory infiltration of lymphocytes, appears to provide more evidence
for abnormal regulation of gene expression, because it was
reported that L-selectin expression was downregulated after
e~travasation.4~
Subsets of CD4+ T cells that showed enhanced transendothelia1 migration were identified as CD29'nEh', CD45RObngh1,
and CD45RA-.50 These phenotypes are similar to those of
ATL cells." In addition to this, the overexpression of Lselectin may render ATL cells more sticky to vascular endothelial cells than normal T cells. Furthermore, ATL and
HTLV- 1 -infected cells produce various inflammatory cytokine^.^^"^ These phenotypic characteristics of ATL cells
would enable them to activate vascular endothelial cells to
express adhesion molecules and to attach themselves firmly
to activated endothelium. The notion that overexpression of
L-selectin itself can affect the lymphocyte recirculation
seems to be partly supported by our observation that a rat
T-cell line that overexpresses L-selectin showed an increased
A
c
0
.-2
a
2
S
$
4
3
2
1
0
Tax
Tax (+)
(-)
Fig 7. Transactivation of the L-selectin gene promoter by HTLV-1 Tax. Representative results of a
CAT assayafter the cotransfectionof pLS-1 CATand
a Tax expression vector in FL cells (A). The percentage ofconversionofchloramphenicol
that iscorrected by the P-Gal activity is shown in (B). The CAT
activity was transactivatedabout fivefold in the
presTax.of
ence
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3115
OVEREXPRESSiON OF L-SELECTIN IN ATL
Q8
*
I
F
*
I
n
I
01
ATL
AC
Cont.
Fig 8. Elevated serum levels of sL-selectin
in ATL patients. Plasma
levels of sl-selectin in 29 ATL patients, l 0 asymptomatic carriers
(AC), and10 normal blood donors(Cont.)were plotted. Bars indicate
the mean f SD. *P < .05 using the Student's t-test.
level of organ infiltration when injected into a rat intravenously (Fig 5). However, Schleiffenbaum et alzo have shown
that the increased levels of sL-selectin in the medium inhibited the interaction between L-selectin and its ligands. Therefore, it appears more likely that, even though ATL cells
might have enhanced capacity to adhere to and transmigrate
the endothelial cells, organ infiltration is determined by the
dynamic balance in vivo between the increased stickiness of
ATL cells and the inhibitory effect of sL-selectin in the
serum. Thus, to understand the biologic significance of the
L-selectin overexpression, the correlation between the serum
levels of sL-selectin and the clinical manifestation of organ
infiltration in ATL patients of each clinical subtype should
be further studied, and the gene expression of other adhesion
molecules as well as inflammatory cytokines should be characterized.
As mentioned before, HTLV- 1 Tax induces expression of
cytokines, growth factors, and their receptor^.^^ IL-2Ra and
parathyroid hormone-related protein (FWIrP) are examples
of these genes, and they are constitutively overexpressed in
ATL ~ e l l sThe
. ~overexpression
~ ~ ~ ~
of cellular genes that can
be transactivated by Tax is one of the common features of
ATL cells. The L-selectin gene appears to be the first example of adhesion molecules with this characteristic.
The level of transactivation of the L-selectin promoter
induced by Tax was about fivefold in repeated experiments,
which is lower than that of reported cellular genes. This
difference could be explained by the relatively high level of
background activity of L-selectin promoter both in Jurkat
cells and FL cells (Fig 6 ) .Similar observations were recently
reported by Ohbo et a15' on the IL-2Ry chain gene expression
that was transactivated by HTLV-1 Tax. It may also be due
to the difference in the molecular mechanism underlying
the transactivation. It has been shown that HTLV-1 Tax
transactivates gene expression through interactions with cellular transcription factors such as CREBP, NF-KB, and
No consensus sequence elements for these factors
were found in the L-selectin promoter up to -885 bp. Instead, there are multiple sequence elements for Ets family
oncogenes (Tatewaki et al, submitted for publication), which
reportedly participate in the transactivation of HTLV-1 LTR
and the promoter of the F'THrP
Functional characterization is now under way to answer whether these characteristics of the L-selectin promoter could explain the apparent difference in the response to Tax.
In all the ATL samples that we have so far examined,
viral mRNAs were not detected using Northern blotting.
Furthermore, our RT-PCR studies of fresh primary ATL
cells from 25 patients could not detect pX mRNA in more
than half of them. The amplification of the pX mRNA was
very weak in the remaining samples and it corresponded to
a level of l viral mRNA expressing cell among 1 X lo4
negative cells (Watanabe et al, unpublished observations).
Thus, we could not exclude the possibility that the amplified
pX mRNA was expressed in nontransformed HTLV-1-infected cells that contaminated the samples. It was also shown
that nearly half of the ATL patients have defective provirus
that lacks 5' regions in their tumor
(Matsuoka et al,
unpublished observation). These results indicated that the Lselectin gene is constitutively overexpressed in the absence
of HTLV-1 Tax in vivo. This finding implies that there is
another mechanism independent of HTLV- 1Tax underlying
the overexpression of L-selectin in fresh ATL cells. The
'""1
lo00
Q
1
100
lo00
loo00
1
m
sL-selectln ( ng I m1 1
Fig 9. Correlation between the levels of d-selectin and those of
sIL-2Ra in ATL patients. The results from 25 samples of ATLpatients
are plotted. The correlation coefficientwas .6.
From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
31 16
TATEWAKI ET AL
same notion could be applied to other Tax-responsive genes
such as IL-2Ra, PTHrP, and transforming growth factor p
that are overexpressed in fresh ATL cells.
In conclusion, we showed the constitutive overexpression
of the L-selectin gene in ATL cells in vivo that can be
transactivated by HTLV-1 Tax that provided another clue to
understanding the pathogenesis of organ infiltration of ATL
cells.
ACKNOWLEDGMENT
We thank Drs I. Miyoshi (Kochi Medical School, Kochi, Japan)
and K. Sugamura (Tohoku University School of Medicine, Sendai,
Japan) for the gifts of MT-I and JPX9, respectively.
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1995 86: 3109-3117
Constitutive overexpression of the L-selectin gene in fresh leukemic
cells of adult T-cell leukemia that can be transactivated by human Tcell lymphotropic virus type 1 Tax
M Tatewaki, K Yamaguchi, M Matsuoka, T Ishii, M Miyasaka, S Mori, K Takatsuki and T
Watanabe
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