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Blood, Vol. 94 No. 5 (September 1), 1999:
pp. 1747-1754
By
From Laboratory of the Biology and Treatment of Metastasis and
Laboratory of Cellular and Molecular Biology of the Immune Response and
Auto Immunity, Mario Negri Institute for Pharmacological Research,
Bergamo, Italy; International Centre for Genetic Engineering and
Biotechnology (ICGEB), Trieste, Italy; and Institute for Cancer
Research and Treatment (I.R.C.C.), School of Medicine, University of
Turin, Candiolo, Italy.
Human immunodeficiency virus-1 (HIV-1)-Tat, the transactivating gene
product of HIV-1, has been shown to interact with different cell types,
inducing gene expression, altering their growth and migratory behavior.
In this study we examined whether Tat might affect functions of
acquired immunodeficiency syndrome (AIDS)-related non-Hodgkin's
lymphoma (NHL), relevant to the in vivo dissemination. Our results show
that Tat significantly augmented the motility of the two AIDS-related
Burkitt's lymphoma cell lines (AS283 and PA682PB) and AIDS-primary
effusion lymphoma cell line (HBL-6-AIDS-PEL). Mutations in RGD or basic
domain of Tat (KGE-MBP and LxI-MBP, respectively) sharply reduced
migration compared with wild type, suggesting that both domains are
required for migration. In contrast, a Tat protein
mutation outside the active domains (NH2-TAT-GST) did not
reduce lymphoma cell migration. The treatment of lymphoma cells with
Tat did not influence their adhesion to matrix proteins or to human
vascular endothelial cells, but endothelial cells treated with Tat
became more adhesive to lymphoma cells. Flow cytometric analysis showed
that treatment of endothelial cells with Tat induced the cell surface
expression of the adhesion molecules vascular cell adhesion molecule-1
(VCAM-1) and E-selectin and increased the expression of intercellular
adhesion molecule-1 (ICAM-1). Only antibodies against
VCAM-1 on endothelial cells or against the VLA-4 integrin expressed on
AS283 cells inhibited the increment of adhesion, indicating the
relevance of this pathway in the adhesion of lymphoma cells to vascular
endothelium. In our work, we show for the first time that Tat can
enhance the migration of lymphoma cells and their adhesion to
endothelial cells, two processes that may contribute to the malignant
behavior of NHL in patients with AIDS.
NON-HODGKIN'S LYMPHOMA (NHL) represents
one of the most frequent complications in acquired immunodeficiency
syndrome (AIDS).1,2 The majority of
AIDS-related NHLs are diffused aggressive B-cell lymphomas with
extranodal involvement of the central nervous system in
particular.1,3 Their histology includes four categories,
namely small noncleaved-cell (including Burkitt's lymphoma),
large-cell immunoblastic plasmocytoid, anaplastic-large cell, and
AIDS-related body-cavity-based lymphoma.1,4 Small noncleaved-cell lymphomas are characterized by activating mutations of
c-myc, frequent alterations of p53, and occasional
Epstein-Barr virus infection.5,6 Large cell subtype
lymphomas are strictly associated with Epstein-Barr virus infection and
with rearrangements of c-myc and BCL-6.5,7
Finally, AIDS-related body-cavity-based lymphoma is consistently
associated with the coinfection of human herpesvirus type-8 and
Epstein-Barr virus,8 whereas the anaplastic subtype has not
been studied extensively. A feature common to the four AIDS-NHL
subtypes is the presence of mutations in the noncoding region of
BCL-6 in the proximity of the gene promoter.9
The mechanism by which AIDS-NHL cells home into the central nervous
system is largely unknown and could involve signals from endothelial
cells of blood-brain barrier infected by human immunodeficiency virus-1
(HIV-1)10,11 or specific chemoattractants for lymphoma cells. Microvascular endothelial cells originated from the brain or the
bone marrow and infected by HIV-1 support the growth and the adhesion
of neoplastic B cells through the surface expression of CD40 that binds
CD40 ligand present on lymphoma cells.12 Tat is a potent
inducer of the expression of endothelial cell adhesion molecules,
including vascular cell adhesion molecule-1 (VCAM-1)13
that is involved in the migration of B cells to and within the germinal
center of lymphonodes.14
Tat is a viral protein that transactivates viral genes and increases
the replication rate of virion.15 Tat is also able to
regulate the expression of host genes, including tumor necrosis factors
Furthermore, it has been recently shown that Tat activates the
migration and the tissue invasion of monocytes.27,29
Finally, Huang et al have reported that Tat regulates the expression of Fas in B cells.32
In light of the fact that the infection is a key event in the
progression of AIDS-NHLs12 and the viral product Tat can
influence the functions of B cells32 and vascular
endothelial cells,13,31,34-36 we have investigated whether
Tat regulates the homing mechanisms of AIDS-NHL cells.
Cells.
The AS283 and PA682PB AIDS-related Burkitt's lymphoma and KD488
Burkitt's lymphoma cell lines were obtained through the courtesy of Dr
I.T. Magrath (National Cancer Institute, Bethesda, MD) and are
described by Kiwanuka et al.37 The Namalwa and
BJAB Burkitt's lymphoma, the diffuse histocytic
lymphoma-4 (DHL-4) follicular lymphoma, and the Capo Epstein-Barr virus
(EBV)-transformed lymphoblastoid cell lines were all obtained from Dr
J. Golay (Mario Negri Institute, Milan, Italy). We also
used the HBL-6-AIDS-primary effusion lymphoma cell line
(HBL-6-AIDS-PEL) obtained from Dr G. Gaidano (University of Piemonte
Orientale "A. Avogadro", Turin, Italy). All these lines were maintained in suspension in RPMI 1640 supplemented with 2 mmol/L glutamine and 10% fetal calf serum (FCS) 3T3-NIH mouse
fibroblasts (American Type Cell Collection, Bethesda, MD) and human
vascular smooth muscle cells were maintained in Dulbecco's modified
Eagle's medium (DMEM) supplemented with 2 mmol/L glutamine and 10% FCS.
Preparation and biological activity of HIV-1-Tat molecules.
The 358-bp Pst I-BamHI fragment of Tat gene
containing exon I and II was kindly donated by Dr A. Caputo (Institute
of Microbiology, University of Ferrara, Ferrara, Italy) and has been
subcloned in the pMAL-c2 vector (New England Biolabs, Beverly, MA) and
then expressed in Escherichia coli according to the
manufacturer's instructions. Tat fused to the maltose binding protein
(Tat-MBP) was purified by affinity chromatography on amylose resin and
used as fusion proteins. The purified protein gave a unique band
detected by silver staining (Gelcode; Pierce Chemical Co, Rockford, IL) after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE); 10%) and was able to induce transcriptional activation of
HIV-1 long terminal repeat (LTR) in HL3T1 cells that contain the
bacterial gene of chloramphenicol acetyltransferase (CAT) directed by
HIV-1 LTR.39 Tat-MBP dose dependently stimulated CAT
expression. Briefly, in prewarmed phosphate-buffered saline (PBS), our Tat-MBP preparation was added to confluent
HL3T1 cells in a 100-mm diameter dish. Cells were immediately scraped
from the plastic surface, then resuspended in fresh medium and
centrifuged. The cells were again plated in CO2 incubator,
and a CAT assay was performed after 6 hours according to Ausubel et
al.40 In our experimental condition, just scraping of the
cells in absence of Tat-MBP had no effect on LTR-directed CAT gene
expression (125 ± 56 [3H]acetylated
chloramphenicol, n = 3). However, CAT gene expression was markedly
elevated in cells that received Tat-MBP (0.5 µg/mL: 423 ± 86
[3H]acetylated chloramphenicol; 1 µg/mL:
2,345 ± 167 [3H]acetylated chloramphenicol; 5 µg/mL: 6,543 ± 567 [3H]acetylated chloramphenicol).
Reagents.
Human recombinant IL-1 Chemotaxis.
Chemotaxis was conducted in the Boyden chamber, as described
previously.43 Different dilutions of Tat-MBP, KGE-MBP,
LxI-MBP, MBP, NH2-Tat-GST, and GST with 0.5 µg/mL heparin
were added to the lower compartment of the chamber. To
block the chemotactic effect of Tat, Tat proteins were mixed with
monoclonal antibodies to Tat (dilution 1:50), or Tat proteins were
boiled for 10 minutes and then added to the lower compartment of the
chamber. Eight-micrometer pore size polyvinylpyrrolidone (PVP)-free
polycarbonate Nucleopore filters (Costar, New York, NY) were coated
with gelatin by immersing them overnight in a solution of 100 µg/mL
gelatin in 0.1% acetic acid and then air
dried. The filter separated the
attractants from the upper part of the chamber in which AS283 cells
were added. After 4 hours of incubation at 37°C, filters were stained
with Diff-Quick (Merz-Dade, Dudingen, Switzerland), and the migrated cells in 20 high-power fields were counted. Checkerboard analysis of
the chemotactic response was performed by varying the concentrations of
Tat-MBP in the upper and lower compartment of the Boyden chamber, as
described previously.43
Adhesion assay.
EC grown to confluent monolayers in 96-well plates were activated for 4 hours with the indicated concentrations of Tat-MBP, KGE-MBP, LxI-MBP,
or MBP diluted in culture medium. IL-1 at 2 ng/mL was used as a
reference control to activate EC.44 After incubation,
monolayers were washed twice, and wells were refilled with 50 µL
medium 199 containing 1% bovine serum albumin (BSA; test medium).
AS283 cells were radiolabeled with
[125I]-Iododeoxyuridine (Amersham), as described
previously.45 Fifty microliters of the
cell suspension (3 × 104 cells) was added to each well
and incubated at 37°C for 30 minutes. After incubation, nonadherent
cells were removed by careful aspiration and three washes with test
medium. Wells were incubated for 10 minutes with 100 µL of 0.1 mol/L
NaOH, and the lysate was counted in a gamma counter.
Flow cytometric analysis.
EC and AS283 cells were treated for 4 hours with Tat-MBP (20 ng/mL),
equimolar concentrations of MBP or IL-1 (2 ng/mL). The expression of
adhesion molecules on EC and AS283 cells was measured by indirect
immunofluorescence using a FACSort (Becton Dickinson). After incubation
with the appropriate primary antibody (1:50 dilution) in PBS with 2.5%
FCS for 30 minutes at 4°C, cells were washed and incubated with an
affinity-purified fluorescein isothiocyanate (FITC)-labeled goat
antimouse Ig antiserum for 30 minutes at 4°C (Tecno Genetics, Italy).
Cells were washed again and fixed with PBS containing 1% formalin.
Results were expressed as percentage positive cells and the mean of the
channel fluorescence intensity.
Statistics.
The Student's t-test was used to determine statistical
differences. Statistical significance was set at P < .05.
Induction of AS283 lymphoma cell migration by Tat-MBP.
Tat-MBP (2 to 200 ng/mL) used as chemoattractant stimulate the motility
of AS283 cells in a concentration-dependent manner (Fig
1). At the optimal concentration of 20 ng/mL Tat-MBP, the number of migrated AS283 cells augmented from 14.5 ± 0.7 in the control wells to 50.5 ± 4.5 in wells with Tat-MBP
(3.5-fold increase). The fusion protein MBP alone did not affect
migration (Fig 1), nor did heparin alone (data not shown). Similar
results were obtained with the AIDS-related lymphoma lines PA682PB and
HBL-6-AIDS-PEL, in which 20 ng/mL Tat-MBP induced a 3.1-fold and a
2-fold increase in migration, respectively (Table
1). In contrast, the migration of the
Burkitt's lymphomas (KD488, Namalwa, and BJAB), the follicular lymphoma (DHL-4), and the EBV-transformed lymphoblastoid cell line
(Capo) was not increased by Tat-MBP (Table 1). Fibroblasts and vascular
smooth muscle cells used as a normal cell control did not respond to
Tat-MBP (Table 1).
Induction of EC adhesiveness by Tat-MBP.
The adhesion of AS283 lymphoma cells was significantly increased on EC
activated with Tat-MBP for 4 hours with maximal stimulation at 20 ng/mL
Tat-MBP (Fig 5). Tat-MBP induced
approximately a 1.6-fold increase of adhesion, reproducible in all the
experiments performed. MBP alone had no effect.
It has been found that biological functions of mesenchymal cells, like
monocytes and endothelial cells, can be modulated by the HIV-1-Tat
protein.20,29,31,34,36 Recently, it has been described that
Tat can upregulate Fas expression in B lymphocytes,32 suggesting that Tat may contribute to B-cell hyperactivation during HIV
infection. Polyclonal hyperactivation and proliferation of B cells has
been associated with the development of NHL in AIDS patients.46 In this study, we show that Tat induces the
migration of two AIDS-related Burkitt's lymphoma cell lines (AS283 and
PA682PB)37 and increases their adhesiveness to human
endothelium, two processes that are required for the spreading of
highly malignant AIDS-NHL cells. We also tested other kinds of lymphoma
cells and found that only the HBL-6-AIDS-PEL lymphoma cell line
migrated against Tat-MBP (Table 1), thus suggesting a selective role
for the Tat protein in the migration of lymphomas derived from
HIV-infected patients. An extensive study on primary lymphoma cells
from patients with AIDS is likely to be necessary to address this hypothesis.
The authors thank Dr L.T. Magrath (National Cancer Institute, Bethesda,
MD), Dr G. Golay (M. Negri Institute for Pharmacological Research,
Milan, Italy), and Dr G. Gaidano (University of Piemonte Orientale
"A. Avogadro", Turin, Italy) for providing some of the lymphoma
cell lines used in this study.
Submitted June 22, 1998; accepted April 26, 1999.
Supported by grants from the Istituto Superiore di Sanità (AIDS
Project and Program on Tumor Therapy), and Associazione Italiana per la
Ricerca sul Cancro (AIRC).
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
1734 solely to indicate this fact.
Address reprint requests to Raffaella Giavazzi, PhD,
Mario Negri Institute for Pharmacological Research, Via Gavazzeni 11, 24125 Bergamo, Italy; e-mail: giavazzi{at}irfmn.mnegri.it.
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TOP
ABSTRACT
INTRODUCTION
and 
, interleukin-2 (IL-2), IL-6, major histocompatibility complex of class I, IL-2 receptor, p53 tumor suppressor gene, c-fos, and superoxide dismutase in T cells.
80°C in the dark, in
buffered-phospate saline containing 0.1 mmol/L ZnCl2, 0.1%
human serum albumin (Farma Biagini, Lucca, Italy), and 1 mmol/L dithiothreitol.
B activation and cytotoxicity by altering the cellular redox state.
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