Cancer Research Center, Boston University School of Medicine, Boston, Massachusetts 02118
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ABSTRACT |
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Hypoxia is known to induce extravasation of lymphocytes and leukocytes during ischemic injury and increase the metastatic potential of malignant lymphoid cells. We have recently identified a new adhesion molecule, hypoxia-activated ligand-1/13 (HAL-1/13), that mediates the hypoxia-induced increases in lymphocyte and neutrophil adhesion to endothelium and hypoxia-mediated invasion of endothelial cell monolayers by tumor cells. In this report, we used expression cloning to identify this molecule as the lupus antigen and DNA-dependent protein kinase-associated nuclear protein, Ku80. The HAL-1/13-Ku80 antigen is present on the surface of leukemic and solid tumor cell lines, including T and B lymphomas, myeloid leukemias, neuroblastoma, rhabdomyosarcoma, and breast carcinoma cells. Transfection and ectopic expression of HAL-1/13-Ku80 on (murine) NIH/3T3 fibroblasts confers the ability of these normally nonadhesive cells to bind to a variety of human lymphoid cell lines. This adhesion can be specifically blocked by HAL-1/13 or Ku80-neutralizing antibodies. Loss of expression variants of these transfectants simultaneously lost their adhesive properties toward human lymphoid cells. Hypoxic exposure of tumor cell lines resulted in upregulation of HAL-1/13-Ku80 expression at the cell surface, mediated by redistribution of the antigen from the nucleus. These studies indicate that the HAL-1/13-Ku80 molecule may mediate, in part, the hypoxia-induced adhesion of lymphocytes, leukocytes, and tumor cells.
lupus antigen; lymphocyte; fibroblast
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INTRODUCTION |
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ENDOTHELIAL CELLS FROM DIFFERENT SOURCES increase their adhesiveness to leukocytes when subjected to hypoxia (3, 20, 46, 51, 58, 69, 73). We have also shown that human muscle rhabdomyosarcoma (RD) cells respond to hypoxia in the same way, becoming more adhesive for leukocytes (19). In a functional screen of antibodies generated against membrane antigens on hypoxic human endothelial and RD cells, we identified and characterized a new cell surface molecule, hypoxia-activated ligand (HAL-1/13), which is expressed on both endothelial and RD cells and mediates the increased adhesion of leukocytes to both RD cells and endothelium under hypoxic conditions (21).
In addition, HAL-1/13 was shown to be expressed on the surface of several leukemia or lymphoma cell lines, including Jurkat T cell leukemia and U-937 histiocytic lymphoma, as well as on the surface of solid tumor cells, including hepatoma (Hep 3B) cells and several neuroblastoma and breast carcinoma cell lines (18). We also recently reported that hypoxic exposure of Kelly neuroblastoma and MCF7 breast carcinoma cells resulted in upregulation of HAL-1/13 surface expression, coincident with an increased ability of these tumor cells to invade endothelial monolayers. This enhanced invasion could be partially attenuated by the anti-HAL-1/13 antibody. Anti-HAL-1/13 antibody also inhibited locomotion of hypoxic tumor cells on laminin (18). These studies indicate that the HAL-1/13 antigen plays a crucial role in cell-cell and perhaps also cell-matrix interactions under hypoxic conditions.
To elucidate the nature of the HAL-1/13 antigen and mechanism of its surface upregulation upon hypoxic treatment, the antigen recognized by the HAL-1/13 monoclonal antibody (MAb) was isolated by expression cloning. We report here that the sequence of the HAL-1/13 antigen is identical to the p80/p86 subunit (Ku80) of the DNA-binding protein, Ku, previously identified as a target for autoantibodies produced by patients with systemic lupus erythematosus and related rheumatic disorders (41, 42, 52, 53). Ku80 has also been identified as a regulatory subunit of the DNA-dependent protein kinase (10, 15, 22, 25). In addition, antibodies raised against Ku80 recognize the HAL-1/13 antigen. Furthermore, we demonstrate that transfection of HAL-1/13-Ku80 into (murine) NIH/3T3 fibroblasts results in its expression on the plasma membrane, with concomitant induction of adhesive properties in the transfectants for human lymphoid cells, in a HAL-1/13-Ku80-dependent fashion. These data conclusively demonstrate the ability of HAL-1/13-Ku80 to function as an adhesion molecule. We also present evidence that hypoxic conditions upregulate cell surface expression of HAL-1/13-Ku80 via intracellular redistribution of HAL-1/13-Ku80 to the plasma membrane.
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MATERIALS AND METHODS |
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Molecular cloning and expression plasmids.
The Escherichia coli strain XL1 Blue (Stratagene, La Jolla,
CA) was used for all -phage work, and DH5
(Life Sciences,
Gaithersburg, MD) was used in the transfection and preparation of
plasmid DNAs. A
-ZAP (Stratagene) cDNA library of MCF7 cells
[complexity of 1 × 107; generous gift of Dr. Mark
Sobel, National Cancer Institute (9)] was screened with
expression cloning after isopropyl
-D-thiogalactopyranoside (IPTG) induction
(40) with the MAb HAL-1/13 (21). Recombinant phage were plated at a density of 50,000 plaque-forming units per
150-mm petri dish. Plaque lifts were prepared on 135-mm nitrocellulose discs (Schleider and Scheull, Keene, NH) as described
(40). The membrane filters were subjected to Western
blots, and positive plaques were isolated and purified to homogeneity
(40). Plasmids were prepared from candidate bacteriophage
clones by cotransfection of XL1 Blue with the helper M13 phage R407K
and the
-phage. The resulting phagemids were transfected into
DH5
, positive clones were selected and screened, and plasmid
preparations were prepared. The insert DNA was sequenced in the Boston
University DNA Core Facility with an Applied Biosystems International
3000 DNA sequencer.
Cell lines and culture conditions. HeLa, a human cervical carcinoma cell line, U-937, a line derived from a histiocytic lymphoma with myelomonocytoid characteristics (62), and MCF7 (HTB 22), a human breast carcinoma cell line, were purchased from American Type Culture Collection (ATCC; Rockville, MD) and grown in Dulbecco's modified Eagle's medium (DMEM). DMEM was supplemented with 10% fetal bovine serum (FBS; Sigma Chemical, St. Louis, MO). NIH/3T3, a murine fibroblast cell line, was grown in DMEM supplemented with 10% donor calf serum (DCS). Jurkat, a T cell leukemia line (68), was grown in DMEM with 10% newborn calf serum (NCS). JY, a human Epstein-Barr virus (EBV)-transformed B cell line (39), was grown in RPMI 1640 supplemented with 10% FBS. All DMEM and RPMI 1640 were further supplemented with L-glutamine (2 mM), penicillin (100 U/ml), and streptomycin (100 µg/ml; Life Technologies, Grand Island, NY) and buffered to pH 7.4 with sodium bicarbonate. Cells were grown at 37°C in a humidified 5% CO2 atmosphere and were in a log phase of cell growth when used in adhesion assays. All nonadherent cell lines were resuspended at 2 × 106 cells/ml, labeled with 2 µM 2',7'- bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF; Molecular Probes, Eugene, OR) for 20 min at 37°C, washed, and resuspended in normoxic DMEM/NCS. RD cells, a human muscle cell line (38), were obtained from ATCC and grown using the same medium. MCF, 3T3, and RD cell lines were grown to confluence on 10-cm cell culture dishes, trypsinized, washed, and plated onto 96-well flat-bottom microtiter plates (Nunclon, Nunc, Denmark) at ~1-2 × 105 cells/well and used 24-48 h later (when confluent) in adhesion experiments.
Flow cytometry and antibodies. The HAL-1/13 murine MAb (IgG2a) has been previously described (17). W6/32 is an IgG2a directed against a human leukocyte antigen class I framework antigen. Anti-Ku80 (MAbs 111, N9C1, and S10B1), anti-Ku70 (MAb H3H10), and anti-Ku70/80 (MAb 162) murine MAbs were manufactured by Neomarkers (Fremont, CA) and purchased from Lab Vision. Secondary antibody for fluorescence-activated cell sorter (FACS) analysis cell surface staining was a fluorescein-conjugated sheep (Fab')2 fragment to mouse IgG (whole molecule; Cappel).
Flow cytometry of fluorescently labeled cells was performed on a Becton Dickinson FACScan (San Jose, CA), as previously described (17, 19, 20). Briefly, cells (1 × 106) were washed twice with PBS, collected, resuspended in Earle's balanced salts (EBSS)/0.5% serum, and pelleted gently, and the pellet was resuspended in 50-100 µl of primary antibody diluted as required [no dilution for hybridoma supernatant (HAL-1/13 and W6/32); 1:100 dilution for ascites fluid (anti-Ku80, anti-Ku70, and anti-Ku70/80)]. The suspension was incubated with gentle mixing for 45 min at 4°C and then washed three times with 100 µl of ice-cold EBSS/0.5% serum to remove excess primary antibody. The pellet was resuspended in 50-100 µl of diluted secondary antibody, fluorescein isothiocyanate-conjugated sheep (Fab')2 fragment to mouse IgG (whole molecule) at a 1:25 dilution, incubated with gentle mixing for 45 min at 4°C, washed three times with wash buffer, and resuspended in 200 µl of wash buffer to which 200 µl of 2% paraformaldehyde was added to fix the cells. Samples were stored covered at 4°C before FACS analysis. A W6/32-stained sample was included in each experiment as a positive control for staining.Hypoxic treatment of cells. Subconfluent cell cultures were covered with fresh culture medium (DMEM/10% FBS) that contained 20 mM HEPES to prevent a pH drop during hypoxic exposure. They were incubated in chambers that contained gas mixtures of 5% CO2-95% N2-0% O2 (BOC Gases, Boston, MA) for various intervals at 37°C (attaining a measured PO2 = 10-30 Torr in the medium). Chambers were regassed for 1 h after 24 h, when cells were required for 48-h experiments. Cell viability was maintained under these hypoxic conditions. Incubation of tumor cells under these hypoxic conditions had no significant effect on their viability: 90% of cells were alive at the end of incubation, as measured by trypan blue exclusion. Cells continued to proliferate after reoxygenation.
Transfections. To generate cell lines stably expressing HAL-1/13-Ku80, 3T3 cells were cotransfected with 30 µg of the pCINeo-PRNZ vector (or the empty pCINeo vector as a control). Twenty-four hours before transfection, 3T3 cells were plated in 60-mm tissue culture plates at a density of 2.5 × 105 cells/plate. Cells were transfected with PRNZ DNA using Lipofectamine Plus (GIBCO Life Technologies). DNA (5 µg), PLUS reagent (10 µl), and serum-free medium (235 µl) were mixed together and incubated for 15 min at room temperature. Lipofectamine (15 µl) and serum-free medium (235 µl) were mixed and added to the DNA-containing mixture. The Lipofectamine/DNA mixture was vortexed briefly and incubated for 15 min at room temperature. Growth medium was removed from the 60-mm tissue culture plates and replaced by 2 ml of serum-free medium. The Lipofectamine/DNA mixture was added to the cells and incubated for 3 h at 37°C. Serum-free medium was replaced with complete growth medium, and cells were selected 24-48 h later using DMEM/10% DCS supplemented with geneticin (G418; GIBCO Life Technologies) at 0.5 mg/ml. Medium was changed regularly to remove dead cells. Colonies were selected for cloning 10-14 days after transfection. G418-resistant colonies were screened for cell surface expression of HAL-1/13-Ku80 by FACS analysis. Transfected cells were maintained in medium containing G418 at 0.5 mg/ml. Experiments were performed using pooled colonies of G418-resistant cells (to avoid artifacts due to clonal selection of aberrant cells) or clones derived from G418-resistant colonies.
Adhesion assay. Cell adhesion assay was a modified method of an adhesion centrifugation assay previously described (19, 20). Monolayer cells (1-2 × 105 cells/ml) were plated in flat-bottomed 96-well tissue culture plates (Nunclon) and exposed to either normoxic or hypoxic conditions for 24 or 48 h before cell adhesion assay. Normoxic and hypoxic tumor cells were washed twice with ice-cold EBSS/0.5% BSA and centrifuged at 1,000 rpm for 5 min at 4°C. The cell pellet was resuspended at 5 × 106 cells/ml in PBS/0.5% BSA, and calcein (BCECF-AM) was added to 2 µM. Calcein-labeled cells were incubated for 25 min at 37°C, washed with PBS/0.5% BSA three times, and added to cell monolayers in 96-well plates (100 µl/well). Plates were incubated for 30 min at 37°C. Adhesion of calcein-labeled cells to monolayer cells was visualized by both light and fluorescence microscopy before discarding the residual nonadherent cells and washing the plate twice with PBS/0.5% BSA. Quantitation of adherent cells was performed by measuring the fluorescence in each well with a CytoFluor 2300 plate scanner (Millipore, Burlington, MA) at excitation and emission wavelengths of 485 and 530 nm, respectively. Background fluorescence was measured for each plate and subtracted from all readings. Because the intrinsic labeling efficiency by calcein differs among cell types, adhesion data are expressed in "relative adhesion units" rather than absolute values to facilitate comparison of adhesion experiments among cell types. Adhesion (baseline fluorescence) of each cell line to the parental NIH/3T3 cells was arbitrarily given a value of one adhesion unit. Changes in adhesion (fluorescence) values under different experimental conditions are expressed relative to this baseline value of one. In blocking experiments using MAbs, anti-HAL-1/13 (diluted 1:1) or anti-Ku80 (diluted 1:25) MAbs were incubated with cell monolayers for 30 min at 37°C. The monolayers were washed twice, calcein-labeled cells were added, and cell adhesion was assayed. Each experiment was performed in triplicate and the SD calculated.
Preparation of membrane, nuclear, cytoplasmic, and whole cell
extract fractions.
The membrane, cytoplasmic, nuclear, and whole cell extract fractions
were prepared separately for immunoprecipitation studies. After
collection of 3 × 107- 4.5 × 107 cells, cells were washed and the cell pellet was
resuspended in 10 ml of ice-cold PBS, vortexed briefly, and spun down.
A minimal volume (0.5 ml/1 × 107 cells) of ice-cold
lysis buffer [10 mM Tris (pH 7.5), 1% Triton X-100, 5 mM EDTA, 50 mM
NaCl, 10 µg aprotinin/ml, 10 µg leupeptin/ml, and 100 µM
phenylmethylsulfonyl fluoride (PMSF)] was added to the cell pellet,
which was then vortexed for 1 h at 4°C and centrifuged at 2,500 rpm for 10 min to pellet nuclei and debris. The supernatant (cytoplasmic fraction) was removed to a fresh microcentrifuge tube and
centrifuged at 11,000 rpm for 30 min to remove debris, and the
supernatant was stored at 80°C. The pellet (nuclear fraction) was
resuspended in 0.5-1 ml of nuclear lysis buffer [10 mM Tris (pH
7.5), 1% Triton X-100, 5 mM EDTA, 500 mM NaCl, 10 µg aprotinin/ml, 10 µg leupeptin/ml, and 100 µM PMSF] and vortexed for 15 min at 4°C. The supernatant was removed and stored at
80°C. The
whole cell extract fractions were prepared by combining separately
prepared nuclear and cytoplasmic fractions in a ratio proportional to
their extraction volumes (1:3). Protein concentration was determined using a Bio-Rad assay standardized against a BSA curve. For preparation of membrane fractions, subconfluent cells were washed and pelleted, and
the cell pellet was resuspended in 1.0 ml of TMSDE [50 mM Tris (pH
7.6), 75 mM sucrose, 6 mM MgCl2, 1 mM dithiothreitol, 1 mM
EDTA, 10 µg aprotinin/ml, and 10 µg leupeptin/ml] and incubated at
0°C for 10 min. Samples were frozen at
70°C, freeze-thawed at
least three times, and then sheared using a 27-gauge needle and
tuberculin syringe (8-10 times on ice). Nuclei and debris were
removed by centrifugation at 13,000 rpm for 20-30 min at 4°C.
The supernatant (membrane fraction) was spun at 45,000 g (22,000 rpm) for 1 h at 4°C. The pellet was resuspended in
0.1-0.2 ml TMSDE. Protein concentration was quantitated using a
Bio-Rad assay standardized against a BSA curve. Membrane fractions were stored at
70°C in 10- to 20-µl aliquots.
Immunoprecipitation and immunoblotting.
Protein G-Sepharose beads (Sigma) were resuspended in lysis
buffer (62.5 mg/ml) and washed three times before use. The lysates (and
mock lysates: PBS/10% serum) were precleared by adding 50 µl of
protein G-Sepharose/ml of lysate in polypropylene tubes. The mixture
was shaken with an orbital shaker either for at least 1 h or
overnight at 4°C and then centrifuged at 1,565 rpm (200 g)
for 5 min at 4°C to remove the beads. The supernatant (precleared fraction) was removed to a fresh microcentrifuge tube to which primary
antibody was added. The primary antibody was added to the precleared
lysate and mixed for 2 h at 4°C. Anti-HAL-1/13 mouse monoclonal
hybridoma culture supernatant was used in a 1:1 ratio of MAb to lysate.
For mouse ascites MAbs anti-Ku80, anti-Ku70, or anti-Ku70/80, 3-5
µl of MAb were added per milligram of protein lysate. Protein
G-Sepharose (62.5 mg/ml) was added to the lysate-antibody mixture (50 µl/ml) and mixed for 1 h at 4°C. The mixture was centrifuged at 14,000 rpm for 5 min to precipitate the antigen. The
immunoprecipitated (IP) fraction was washed two to three times with
immunoprecipitation buffer [50 mM Tris-Cl (pH 7.5), 150 mM NaCl,
0.02% (wt/vol) sodium azide, 100 µg PMSF/ml, 1 µg aprotinin/ml
(0.5%), 0.1% Tween 20, and 1 mM EDTA (pH 8.0)]; 1 ml buffer was
added and centrifuged at 14,000 rpm for 2 min. The supernatant
(immunodepleted fraction) and IP fractions were stored at 80°C.
Reproducibility and statistical analysis. All immunoblots and FACS histograms shown are representative of results from experiments that were performed at least three times. Similar results were obtained on each occasion. All data points shown are means of duplicates or triplicates, which differed by <5% unless stated otherwise.
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RESULTS |
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Cloning of gene product recognized by HAL-1/13 antibody and
identification of the antigen as human Ku80.
Expression cloning was used to isolate the cDNA encoding the HAL-1/13
antigen. A -ZAP cDNA library of MCF7 cells (complexity of 1 × 107 genes) was screened using expression cloning after IPTG
induction. Plaque lifts on membranes were probed with the MAb HAL-1/13,
and positive plaques were isolated and purified to homogeneity.
Candidate phagemids were rescued, and plasmid preparations were
prepared and sequenced. One clone, pPRNZ-19, had a 3.05-kb
EcoRI/XhoI insert that, by BLAST searches, was
identical to human Ku80 (72). Expression of this clone in
E. coli yielded a protein of ~80 kDa, which was immunoreactive with the HAL-1/13 MAb. This clone was used for all
subsequent manipulations. Other shorter clones were also obtained, which were immunoreactive with HAL-1/13 MAb, had nucleotide sequence identical to human Ku80 cDNA, and which may represent a truncated Ku80
transcript previously identified (6, 30).
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Ectopic expression of the cDNA encoding the HAL- 1/13-Ku80
antigen confers adhesion to lymphoid and myeloid cells.
The HAL-1/13 antigen was initially described as an adhesion molecule
and the Ku antigen as a multifunctional nuclear effector. The
unexpected identity of these two molecules suggested a new role for Ku
antigen as a surface adhesion receptor. To verify and elucidate the
adhesion properties of HAL-1/13-Ku80, murine NIH/3T3 fibroblasts were
transfected with the HAL-1/13-Ku80 cDNA. If Ku antigen can indeed
function as an adhesion molecule, then transfection of fibroblasts
should result in surface expression of HAL-1/13-Ku80, which would
confer to fibroblasts the ability to adhere to human lymphoid cells.
Murine fibroblast NIH/3T3 cells were chosen as the background for
ectopic expression of HAL-1/13-Ku80 because the Ku antigens are
expressed only at very low levels in nonprimate cells, and the
antibodies against primate Ku antigens are not cross-reactive with
murine Ku proteins (67). We have also demonstrated that
neither the anti-HAL-1/13 nor the anti-Ku antibodies cross-reacted with
any NIH/3T3 cell surface or intracellular antigens (Fig.
2).
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Effect of hypoxia on cell surface expression of
HAL- 1/13-Ku80 and its association with Ku70.
The HAL-1/13 antibody was initially selected for its ability to
block adhesion of leukocytes to hypoxic endothelium and RD cells
(21). More recently, we have shown that this antibody can
inhibit invasion of hypoxic neuroblastoma and breast carcinoma cells in
vitro and that hypoxia increases expression of HAL-1/13 on the surface
of these cells (18). Having ascertained that HAL-1/13 is
identical to the Ku80 antigen and that the Ku antigen is predominantly
expressed in the nucleus, we investigated whether hypoxic
treatment affects the distribution of HAL-1/13 between the nucleus and
plasma membrane. U-937 and Jurkat cells, both of which express
significant levels of HAL-1/13 on their surface under normoxic
conditions, were chosen for these experiments. Forty-eight hours of
hypoxic (PO2 = 20 Torr) exposure of U-937 cells resulted in a greater than fivefold increase in cell surface expression of the HAL-1/13-Ku80 antigen, as determined using a MAb
specific for each for staining (Fig.
6A). Shorter periods of
hypoxic exposure (24 h) did not result in consistent induction in U-937
cells (not shown). Hypoxic conditions also induced HAL-1/13-Ku80 antigen expression on Jurkat cells, although to a lesser
extent (~2.5-fold), and induction of expression on these cells was
apparent at 24 h (Fig. 6B) and did not increase further
at 48 h (not shown).
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Intracellular redistribution of HAL-1/13-Ku80 by hypoxia.
Potential mechanisms for hypoxic induction of cell surface expression
of HAL-1/13-Ku80 include increases in the total cellular levels of
HAL-1/13-Ku80 or redistribution of intracellular HAL-1/13-Ku80 to the
membrane. Isolated or pooled subcellular fractions (nuclear fractions
and plasma membrane fractions) of U-937 cells were assayed for relative
levels of Ku80, with or without exposure to hypoxic conditions. Because
cytoplasmic fractions of U-937 cells contained no detectable Ku80 or
Ku70 (data not shown), the nuclear and membrane fractions were pooled
to provide an estimate of total cellular levels of the protein.
Immunoblotting with a Ku80 MAb (Fig.
7A) showed no significant
changes in total cellular HAL-1/13-Ku80 after hypoxic exposure. Levels
of HAL-1/13-Ku80 in the nucleus, relative to the plasma membrane, were
increased in membranes relative to the nucleus under hypoxic
conditions, with a nuclear-to-membrane (N:M) ratio of 2.3:1 under
normoxic conditions, changing to 1:2.9 under hypoxic conditions.
Parallel experiments using the HAL-1/13 MAb yielded identical results
(not shown). This same hypoxia-induced shift in subcellular
distribution was observed when an anti-Ku80 MAb was used to first
immunoprecipitate proteins from the whole cell lysates or subcellular
fractions, followed by separation and immunoblotting with the
anti-HAL-1/13 MAb (N:M ratio of 1.6:1 for normoxia shifting to 1:3.6
for hypoxia; Fig. 7B). The reciprocal of this latter
experiment (immunoprecipitating with the HAL-1/13 MAb and
immunoblotting with an anti-Ku80 MAb) gave identical results (not
shown).
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DISCUSSION |
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The nuclear localization and nuclear functions of Ku, and more specifically the Ku80 component, have been well characterized by both cell fixation and immunocytochemical techniques (26, 53, 54, 67, 70, 71). Ku, predominantly as the Ku70/Ku80 heterodimer, is dispersed in particulate fashion throughout the nucleus. During mitosis, Ku is concentrated at the nuclear periphery in interphase cells and associated with metaphase chromosomes (26). Ku nuclear staining displays a reticular pattern with sparing of nucleoli (41), although Ku is associated with nucleoli at certain phases of cell cycle (71). Ku80 has been isolated from nucleoli and active (DNAse sensitive) chromatin (70). A wide variety of functions has been reported for the nuclear component of Ku, including DNA double-stranded break repair and V(D)J recombination (4, 5, 24, 36, 37, 59), DNA replication (see Ref. 2 for review), DNA transcription (see Ref. 33 for review), ATP-dependent helicase activity (65), DNA-dependent ATPase activity (7), stimulation of elongation property of RNA polymerase II (14), binding to human immunodeficiency virus-1 transactivating region RNA (31), maintenance of normal telomere length in yeast (23, 34, 45, 48), and chromatin condensation in G2/M (44), among others.
Although the nuclear-localized fraction of the Ku80 protein has been predominantly studied in connection with DNA repair and replication, independent observations demonstrate that Ku80 can also be a cell surface protein (13, 28, 49) and a possible participant in signal transduction (35) and cell-cell interactions (63). Analysis of the predicted amino acid sequence of the Ku80 cDNA identified a large hydrophobic region near the NH2 terminus of the molecule (72). Yet, even if this region represents a membrane-spanning domain, it remains unclear why all primate cells that express Ku80 in the nucleus do not also express it on the surface. Indeed, in limited surveys of the normal tissue distribution of Ku80, we have found cell surface expression of Ku80 only on endothelial cells. In contrast, the HAL-1/13-Ku80 antigen is found on the surface of many leukemic and solid tumor cells and cell lines, including T and B lymphomas, myeloid leukemias, neuroblastoma, RD, and breast carcinoma cells (this report and Refs. 18 and 21). Others have reported that resting (G0) but not proliferating lymphocytes are positive for cell surface expression of Ku antigen (1). It is interesting to note that, in our studies, every transfected NIH/3T3 clone that expressed HAL-1/13-Ku80 in the nucleus also expressed the antigen on the cell surface, suggesting that the lack of expression of Ku80 on the surface of most normal human cells, and its frequent expression on tumor cells, may be an active restriction against transport or expression rather than the result of uncharacterized, aberrant membrane transport processes in tumor cells.
Although Ku70 has also been reported to be expressed on the surface of some (tumor) cells and also possesses several predicted hydrophobic domains, at least one of which is large enough to span the membrane (8, 49), our data suggest that it is not likely that Ku70 targets Ku80 to the membrane, or vice versa. We could demonstrate some association of Ku70 and Ku80 on the cell surface by coimmunoprecipitation studies. In addition, an antibody specific for the Ku70/Ku80 heterodimer showed reactivity with membrane components of certain cells. [This latter antibody is known to recognize a conformational epitope, depending on the quaternary structure of Ku (67). Our unpublished studies indicate that the epitope recognized by this antibody actually resides on the Ku70 molecule.] However, induction of cell surface expression of Ku80 by hypoxia did not produce a commensurate increase in Ku70 antigen levels, or in Ku70/Ku80 complexes, at the cell membrane. Furthermore, cell surface expression of Ku70 and Ku80 is dissociated in certain tumors (unpublished observations), and independent regulation or translocation of the subunits of Ku has been described (16, 32). Posttranslational modifications of Ku80 [serine phosphorylation by DNA-PK (7, 29) and tyrosine phosphorylation, possibly by CD40 (43)] have been reported, but our preliminary studies have found no evidence of differences in the phosphorylation state of Ku80 on the cell surface compared with the nuclear-localized fraction.
In the cell lines examined in this report, exposure to hypoxia was marked by a reversal in the ratios of nuclear-associated to membrane-associated Ku80, without changes in the total cellular amounts of Ku80. Furthermore, HAL-1/13-Ku80 transcript levels in endothelial cells do not change in response to hypoxia (unpublished observations). Thus the induction of cell surface expression of Ku80 by hypoxia may be the result of redistribution among cellular compartments rather than of new synthesis. Translocation of Ku80 from the cytosol to the nucleus after treatment of a cell line with somatostatin has been reported (64). Intracellular redistribution of Ku70 and Ku80 from the nucleus to the cytoplasm as a function of cell density or confluence has also been described (16), and apparent translocation from the cytoplasm to the cell surface has been observed in some myeloma cell lines after stimulation with CD40 ligand (63). Translocation of Ku80 from the cytoplasm to the nucleus has been observed after stimulation through CD40 in B cell lines (43), and Ku80 dissociates from chromosomes during mitosis (71). Although there does not appear to be a transcriptional component to the induction of cell surface expression of Ku80 we observed, Ku gene transcription is thought to be regulated by cell cycle, being activated in late G1 (71), and modulation of the total cellular levels of Ku in response to phorbol 12-myristate 13-acetate, calcium signals, or serum have led to the proposal that Ku may function as a sensor of the cellular environment (50, 61).
Hypoxic exposure resulted in no induction of HAL-1/13 expression on HAL-1/13-Ku80-transfected NIH/3T3 cells (unpublished observations). Although it is possible that this was because the NIH/3T3 transfectants were already expressing "maximal" levels of HAL-1/13-Ku80 on the plasma membrane, the fact that we selected for transfectants expressing a range of levels of expression, with none expressing higher levels than any of the human cell lines we have examined spontaneously express, make this unlikely. Interestingly, there appears to be a selection against expression of Ku80 in these murine cells, with every independently isolated murine clone losing expression within 6 wk, despite continuous selection in G418.
Ectopic expression of HAL-1/13-Ku80 in murine cells was used to assay for a direct role of Ku80 as an adhesion molecule. Murine fibroblasts have often been used as a neutral or null background for the study of ectopic expression of human adhesion molecules because of the very low background binding of human leukocytes to these cells. In general, adhesion molecules appear to be poorly conserved between primate and rodent species. This is also the case with Ku80, where there is little or no antigenic conservation between murine and human (47). HAL-1/13-Ku80 transfectants, which expressed levels of human Ku80 comparable with those expressed on human cells were isolated to avoid the potentially confounding effects of nonphysiological overexpression. Ectopic cell surface expression of HAL-1/13-Ku80 antigen invariably conferred increased adhesiveness of NIH cells, both clones and pools, to three different lymphoid cell lines, providing direct evidence for the ability of Ku80 to function as an adhesion molecule. Furthermore, spontaneous loss of expression of HAL-1/13 correlated with loss of adhesive properties, thereby strengthening the correlation. Finally, MAbs against HAL-1/13-Ku80 attenuated the adhesion, in agreement with other studies that have indirectly suggested a role for HAL-1/13-Ku80 as an adhesion molecule (21, 63).
Although ectopic expression of HAL-1/13-Ku80 in NIH/3T3 cells conferred increased adhesiveness to three different human lymphoid cell lines, the magnitude of the adhesion varied, with JY cells adhering best and Jurkat and U-937 less strongly. The reason for these differences in adhesion level might be related to differing levels of the counterreceptor/ligand for HAL-1/13-Ku80 on these cells. Alternatively, it may be relevant that JY cells do not express HAL-1/13, whereas Jurkat and U-937 cells have high levels of HAL-1/13 on the cell surface, and some type of competitive process may be operative. The counterreceptor/ligand for Ku80 has not yet been determined. We have presented evidence that neutralizing antibodies directed against lymphocyte functions-associated antigens (both the CD11a and the CD18 subunits) can partially block hypoxia-induced, HAL-1/13-dependent adhesion (21). A ligand-dependent association of CD40 and cytoplasmic Ku70/80 has been reported, but this appears to be mediated through an intracytoplasmic domain of CD40 (43). Homotypic Ku-Ku interactions are also a formal possibility. In our previous studies (21), both the adhering cells and the adherent cells expressed HAL-1/13-Ku80 on their surfaces. Pretreatment of either cell adhesion partner with the HAL-1/13 MAb prevented adhesion, but a nonspecific steric effect could not be completely ruled out in those experiments. In the current studies, the presence of HAL-1/13-Ku80 on Jurkat and U-937 cells did not augment their adhesion to Ku80-transfected NIH/3T3 cells, compared with the HAL-1/13-Ku80-negative JY cells, arguing indirectly against homotypic Ku80-Ku80 interactions. Ku70 and Ku80 can heterodimerize (53, 55, 72). The coimmunoprecipitation studies described herein demonstrate a constitutive level of association of Ku70 and Ku80 on the cell surface, but this likely represents association of molecules on the same cell, rather than intercellular associations. Furthermore, anti-Ku70 antibodies do not disrupt Ku80-dependent cell-cell adhesion (although it is also possible that these MAbs may not recognize potentially functional epitopes on Ku70). Studies are underway to determine what other cell surface protein might associate with Ku80 during cell-cell adhesive interactions.
The physiological significance of HAL-1/13-Ku80-mediated cell-cell adhesion is as yet undetermined. Although we have clearly demonstrated that Ku80 mediates the increased adhesion of leukocytes to endothelium under hypoxic conditions (21), whether deficiency in Ku80 might lead to impairment of normal immune function through abrogation of an immune cell-cell adhesion pathway is difficult to assess because mice deficient in Ku80 or Ku70 lack normal B cell or B and T cell development (reviewed in Refs. 10 and 15). There is evidence, however, suggesting that HAL-1/13-Ku80-mediated cell-cell adhesion may play a role in pathological processes such as tumor invasion. Although the HAL-1/13 antigen is not present on most normal lymphoid cells, many leukemic cell lines, including Jurkat, Molt-4, HL-60, and U-937, react strongly with the anti-HAL-1/13 antibody (21). We have also recently shown that hypoxia induces cell surface expression of HAL-1/13-Ku80 on a number of solid tumor cell lines, including neuroblastoma (Kelly, SY-SK), breast carcinoma (MCF7), and RD cells (18, 21). When these cells are exposed to low-oxygen environments, their ability to invade endothelial cell monolayers and to transmigrate through Matrigel-coated filters is increased with, coincident with, and dependent on, increased cell surface expression of HAL-1/13-Ku80. Hypoxia is known to enhance the metastatic potential and invasiveness of tumor cells (11, 12, 27, 56, 57, 60, 74, 75). Together, these findings suggest that HAL-1/13-Ku80 may play a major role in regulating the invasive potential of tumor cells, as well as mediating leukocyte-endothelial cell interactions.
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ACKNOWLEDGEMENTS |
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This work was supported by National Cancer Institute (NCI) Public Health Service Grant CA-50459, by a Department of Defense Breast Cancer Research grant, and by a Leukemia Society of America grant (to D. V. Faller). E. M. Lynch was supported by NCI Oncobiology Training Grant T32-CA-64070.
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FOOTNOTES |
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Address for reprint requests and other correspondence: D. V. Faller, Cancer Research Center, K-701, Boston Univ. School of Medicine, 80 E. Concord St., Boston, MA 02118 (E-mail: dfaller{at}bu.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 10 February 2000; accepted in final form 6 November 2000.
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