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1 Tumor Progression and Metastasis Program, Karmanos Cancer Institute, Wayne State University, Detroit, Michigan;
2 Department of Urology, The University of Tokushima School of Medicine, Tokushima, Japan;
3 Department of Otolaryngology and Sensory Organ Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, Japan; and
4 Department of Pathology, Wayne State University, School of Medicine, Detroit, Michigan
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Plasmid Constructions.
Plasmid pGEX-Gal-3 was constructed by insertion of an EcoRI-EcoRI fragment of pBK-CMV-Gal-3 (26)
, coding the full-length wild-type human Gal-3 sequence into the EcoRI site in pGEX-6p-3 (Amersham Biosciences, Arlington Heights, IL). Control plasmid (pGEX-reverse-Gal-3) was constructed by a reverse insertion of the wild-type human Gal-3 sequence into pGEX-6p-3.
Preparation of Recombinant Human Gal-3 and Control.
Gal-3 recombinant proteins and control sample were prepared by using the GST Gene Fusion System (Amersham Biosciences) in Escherichia coli strain BL21(DE3) transformed with expression vector pGEX-Gal-3 or pGEX-reverse-Gal-3. YT medium containing 50 µg/ml ampicillin was incubated overnight with a culture of BL21 transformed with plasmids containing the human sense or antisense Gal-3 cDNA insertion. When the bacterial cells were grown to A600 nm = 0.6, isopropyl-thio-ß-D-galactoside was added to a final concentration of 0.1 mM. Bacterial cells were incubated for an additional 4 h and harvested by centrifugation at 1,250 x g at 4°C. The pellet was suspended in PBS containing 1% Triton X-100 and lysed by sonication. Lysate was centrifuged at 12,000 x g for 10 min, and supernatants were incubated with 50% slurry of glutathione-Sepharose 4B (Amersham Biosciences) equilibrated with 1x PBS for 30 min. Glutathione-Sepharose was washed with 10 beds volume of 1x PBS and once with cleavage buffer [50 mM Tris-HCL (pH 7.0), 150 mM NaCl, 1 mM EDTA, and 1 mM DTT] and then incubated with Prescission Protease (Amersham Biosciences) for 5 h in a rotator at 4°C. Eluted samples were quantitated using BCA protein assay reagent (Pierce, Rockford, IL) and analyzed by SDS-PAGE and immunoblot analysis. All samples were sterilized by Acrodisc Syringe Filter (Pall Corporation, Port Washington, NY).
Generation of Stable Transfectants.
CEM cells were electroporated with 10 µg of linearized vector containing human Gal-3 cDNA or vector only. Cells were pulsed at 200 V/960 µF in a Gene Pulsar (Bio-Rad Laboratories Inc., Hercules, CA). Cells were cultured for 48 h in complete media and transferred to complete media containing 300 µg/ml zeocin (Invitrogen, Carlsbad, CA). Limiting dilution isolated individual clones.
Preparation of PBMCs.
PBMCs were isolated from 6 ml of whole blood using Ficoll-paque (Sigma-Aldrich, St. Louis, MO) density gradient separation solution. Whole blood was collected into sodium heparin tubes, diluted with an equal volume of PBS, and then layered over Ficoll-paque. After centrifugation at 300 x g for 20 min at room temperature, PBMCs were collected from the interphase layer and washed three times with RPMI 1640. PBMCs were resuspended in RPMI 1640 supplemented with 10% FBS and 2 mM glutamine at a concentration of 1 x 106 cells/ml.
Apoptosis Assay.
A total of 2 x 105 cells were treated with several concentrations of Gal-3 or reverse-inserted control in the presence or absence of 50 mM lactose in a total volume of 200 µl for 6 h at 37°C. Apoptotic cells were measured by annexin V binding and PI permeability (Oncogene, San Diego, CA) and analyzed using a Becton Dickinson FACScan and CellQuest software (BD Biosciences, San Jose, CA) (25)
. The fold increase of annexin V-positive rate was determined as follows: annexin V-positive percentage (Gal-3 in the presence or absence of 50 mM lactose)/annexin V-positive percentage (controls).
To detect fragmentation of chromosomal DNA, the percentage of hypoploid DNA was measured by flow cytometric analysis as described previously (27) . Briefly, a total of 1 x 106 cells were treated with 10 µM Gal-3 or reverse-inserted control in the presence or absence of 50 mM lactose for 8 h at 37°C. After washing, cells were fixed with 80% ethanol for 30 min at 4°C, washed with PBS, and treated with RNase A (1 mg/ml in PBS) for 15 min at 37°C, followed by staining with PI (50 µg/ml) for 15 min at room temperature.
Cell Surface Binding Assay.
A total of 2 x 105 cells were incubated with 10 µM Gal-3 in the presence or absence of 50 mM lactose in a total volume of 200 µl for 2 h at 37°C. The cells were washed three times with PBS and incubated with anti-Gal-3 mAb (TIB166; ATCC) for 45 min at 4°C, followed by appropriate antirat FITC-conjugated secondary Ab for 45 min at 4°C. After washing three times with PBS, the stained cells were fixed with 1% formaldehyde in PBS and analyzed for fluorescence intensity using flow cytometry as described above (25)
.
Cell Morphology.
The change of cell morphology with Gal-3 treatment was assessed by microscopy. A total of 2 x 105 cells were incubated with 5 µM Gal-3 in the presence or absence of 50 mM lactose in a total volume of 200 µl at 37°C. Cells were photographed by microscopy 2 h after incubation.
Caspase Activation Assay.
The active form of caspase-3 was determined by direct staining of cells with a FITC-conjugated rabbit anti-active caspase-3 mAb (BD Biosciences) followed by fluorescence-activated cell-sorting analysis (28)
. In brief, Jurkat cells treated with control buffer or 3 µM Gal-3 in the presence or absence of lactose for 7 h were washed with PBS and fixed with 1% formaldehyde (in PBS) for 15 min at 4°C. Cells were permeabilized using fluorescence-activated cell-sorting permeabilizing solution for 15 min at 4°C. The cells were then incubated for 15 min at 4°C with mouse IgG (Zymed Laboratories, Inc., San Francisco, CA) to block nonspecific binding, followed by a 45-min incubation at 4°C with a FITC-conjugated rabbit anti-active caspase-3 mAb (BD Biosciences). The stained cells were fixed with 1% formaldehyde in PBS and analyzed for fluorescence intensity using FACScan as described above. For studies of the effect of the caspase inhibitor, 50 µM Z-VAD-fmk (Sigma-Aldrich) was added to the cells 16 h before analysis. As controls, cells were treated with anti-Fas Ab (200 ng/ml; CH-11; Kamiya Biomedical Company, Seattle, WA) for 6 h in the presence or absence of Z-VAD-fmk.
To further investigate caspase activity, we measured DEVDase and IETDase activity in Jurkat cells treated with 5 µM Gal-3 in the presence or absence of 50 mM lactose as described previously (29) . Cells were lysed with cell extract buffer [20 mM HEPES (pH 7.5), 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, and 1 mM DTT] containing 0.03% NP40. Lysates were centrifuged at 15,000 x g for 10 min, and 50 µl of the cytosolic fraction were incubated for 60 min at 37°C in a total volume of 200 µl of caspase buffer [10 mM HEPES (pH 7.5), 50 mM NaCl, and 2.5 mM DTT] containing 25 µM Ac-DEVD-AMC or Ac-IETD-AMC (Bachem). AMC fluorescence, released by caspase activity, was measured at 460 nm using 360 nm excitation wavelength with a Spectra Maxi Gemini fluorescence plate reader (Molecular Devices). Caspase activity was normalized per microgram of protein determined by the BCA protein assay kit (Pierce Chemical Co.).
Cytochrome c Release.
A total of 2 x 108 cells were harvested for 10 h after treatment with control sample or 10 µM Gal-3 in the presence or absence of 50 mM lactose, washed twice with ice-cold PBS, resuspended in ice-cold cell extract buffer [20 mM HEPES-KOH (pH 7.5), 10 mM KCl, 1.5 mM MgCl2, 1 mM sodium EDTA, 1 mM sodium EGTA, and 4 mM DTT] containing 250 mM sucrose and protease inhibitor mixture (Sigma-Aldrich), and incubated for 1 h at 4°C as described previously (29)
. The lysates were then passed through a 27.5-gauge syringe 10 times and centrifuged at 15,000 x g for 30 min at 4°C. The resulting supernatant was analyzed by immunoblot analysis using anti-cytochrome c Ab (Zymed Laboratories, Inc.).
Ab Inhibition Assay.
Each mAb was incubated with a total of 4 x 105 MOLT-4 cells in PBS and 1% BSA for 30 min at 4°C in a 96-well plate. After the wash with PBS and resuspension in RPMI 1640 including 10% FBS, Gal-3 was added to a final concentration of 10 µM, and the plate was incubated for 20 h at 37°C. After washing, apoptotic cells were detected by PI staining as described above. Abs were obtained from the following sources. UCHT1 (CD3) and M-T701 (CD7) were from BD Biosciences, TDM29 (CD29) was from Southern Biotechnology Associates (Birmingham, AL), LCA (CD45) was from Dako, and Q-20 (CD51) and N-20 (CD61) were from Santa Cruz Biotechnology (Santa Cruz, CA).
Confocal Immunofluorescence Microscopy.
A total of 1 x 106 cells were fixed with paraformaldehyde (2%) for 30 min on ice and stained by incubation for 1 h at 4°C with R-phycoerythrin-conjugated mouse antihuman CD7 mAb (BD Biosciences) and FITC-conjugated mouse antihuman CD29 mAb (Southern Biotechnology Associates). The cells were washed, dropped on glass microscope slides, and mounted using Prolong Anti-fade mounting media (Molecular Probes). The stained cells were analyzed using Zeiss Laser Scanning Microscope 310 (Zeiss, Chester, VA). The cells were scanned by dual excitation of fluorescein (green) and R-phycoerythrin (red) fluorescence. Areas of green and red overlapping fluorescence are represented by a yellow signal.
Immunoprecipitation.
Cells were lysed with ice-cold lysis buffer as described previously (27)
. The cell lysates were precleared by overnight incubation at 4°C with 30 µl of 1:2 slurry of protein A-Sepharose 6MB (Amersham Biosciences) in ice-cold lysis buffer. Immunoprecipitation was initiated by adding 2 µg of polyclonal rabbit anti-Gal-3 Ab to the precleared supernatant (300 µg) followed by 30 µl of 1:2 slurry of protein A-Sepharose 6MB. The reaction mixture was incubated at 4°C for 2 h, followed by five washes with ice-cold lysis buffer. The washed precipitates were boiled for 5 min in SDS-PAGE sample buffer containing 5% ß-mercaptoethanol and separated by 12.5% SDS-PAGE.
Western Blot Analysis.
Western blot analyses were performed using the enhanced chemiluminescence detection system (Amersham Biosciences) as described previously (27)
. The other Abs used were as follows: anti-Gal-3 mAb (TIB166; ATCC) and anti-actin polyclonal Ab (Santa Cruz Biotechnology).
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. To assess activity of extracellular Gal-3 on T cells, MOLT-4 cells were treated with 5 µM recombinant Gal-3 in the presence or absence of 50 mM lactose, and we investigated its effects on cell death by flow cytometry after staining with PI as described in "Materials and Methods." It should be noted that unlike Gal-1, Gal-3 induction of apoptosis does not require the presence of a reducing agent. Control samples were prepared from control vector-transformed bacteria. Recombinant human Gal-3 induced apoptosis of the human T-cell line MOLT-4 after an 8-h (Fig. 1B, I)
or a 16-h (Fig. 1B, II)
incubation at 37°C. After treatment with Gal-3, 12.19% and 23.07% of MOLT-4 cells underwent apoptotic cell death at 8 and 16 h, respectively, whereas in the control-treated cells, only 3–5% of the cells were apoptotic during the same treatment periods (Fig. 1B, I)
. As a control, we have examined the effect of a recombinant deletion mutant Gal-3 at the matrix metalloproteinase cleavage site, in which the first 62 amino acids were deleted. At the same concentration of intact Gal-3, the deletion mutant Gal-3 did not induce apoptosis (data not shown). Gal-3-induced apoptosis was completely inhibited by its sugar inhibitor, lactose (50 mM), reducing apoptosis in the sub-G1 population to the control level [3.54% for 8-h incubation and 4.72% for 16-h incubation (Fig. 1B, I and II)
].
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and other human T-cell lines including H9, Jurkat, and CEM (data not shown) in a carbohydrate-dependent manner (Fig. 2A)
; noncompetitive control sugars such as sucrose had no effect (data not shown). We observed that MOLT-4 cells underwent homotypic aggregation with 5 µM Gal-3 (Fig. 2B, b)
as compared with control cells (Fig. 2B, a)
, which was completely abrogated by lactose (Fig. 2B, c)
. These results imply that Gal-3 binds to the cell surface of T cells, leading to cell-cell adhesion.
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3 µM Gal-3 was required for minimal induction of apoptosis (Fig. 3A)
, as compared with 10 µM Gal-1 (13)
. To investigate the generality of apoptotic activity of Gal-3 on T cells, we have tested the apoptotic effects of Gal-3 on human B-cell line SKW6.4; human T-cell lines H9, CEM, Jurkat, and MOLT-4; and normal human PBMCs (Fig. 3B)
. Apoptosis sensitivity to Gal-3 varied among cell lines. Interestingly, in SKW6.4 and H9 cells, which expressed intracellular Gal-3 (Fig. 3C)
, only 2.87- and 2.47-fold induction of annexin V-positive cells was detected after exposure to Gal-3. In contrast, Jurkat and CEM cells, which did not express Gal-3 (Fig. 3C)
, were significantly more sensitive to Gal-3-mediated apoptosis, as evidenced by the 9.34- and 9.18-fold increase of annexin V-positive cells (Fig. 3B)
. MOLT-4 cells, which are resistant to anti-Fas Ab (30)
, were also very sensitive to Gal-3-induced apoptosis, as detected by the 11.1-fold increase in annexin V-positive cells. Furthermore, Gal-3 induced apoptosis of human PBMCs cells by 4.03-fold (Fig. 3B)
. Similar results were obtained when apoptosis was assessed with PI staining of DNA (data not shown). Gal-3-induced apoptosis of each cell line was completely inhibited by lactose, suggesting that apoptosis signaling of Gal-3 was carbohydrate dependent. Lactose treatment alone had no effect on either cell growth or death (data not shown). We did not detect a significant apoptotic effect of Gal-3 on anchorage-dependent cancer cells such as human breast cancer BT549 cells and human cervical cancer HeLa cells (data not shown). Taken together, these results indicate that soluble Gal-3 protein could play a significant role in the induction of apoptosis of circulating immune cells.
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, Gal-3 bound to and induced apoptosis of a significant fraction of BALB/c mouse T cells. In control-treated samples, 18.8% and 16.2% of the cells were apoptotic, whereas in Gal-3-treated sample, 38.8% and 33.7% of the cells were apoptotic (Fig. 4A)
. The difference was statistically significant (P < 0.05). These binding and apoptosis results were also completely inhibited by lactose.
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, Gal-3 is abundantly expressed in SKW6.4 and H9 cells, whereas no Gal-3 was detected in Jurkat, CEM, and MOLT-4 cells. Thus, among these five cell lines, there was an inverse correlation between cellular Gal-3 expression and Gal-3-induced apoptosis, suggesting that intracellular Gal-3 may protect these cells similarly to other cells (19, 20, 21, 22
, 27)
. To further determine whether intracellular Gal-3 protects T cells for extracellular Gal-induced apoptosis, we transfected human Gal-3 cDNA into CEM cells (Fig. 5A)
. As shown in Fig. 5B
, 56.1% and 50.5% of the CEM control cells underwent apoptotic cell death by 10 µM Gal-3, whereas only 34.3% (clone 1) and 30.4% (clone 2) of CEM-Gal-3 cells were apoptotic. These differences were statistically significant (P < 0.05), suggesting that apoptotic sensitivity to Gal-induced apoptosis may result, in part, from cytoplasmic Gal-3 protection.
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demonstrate that CEM-Gal-3 cells were resistant to C2-ceramide-induced apoptosis, whereas CEM control cells were sensitive to the drug treatment, suggesting that endogenous Gal-3 blocked C2-ceramide apoptotic signaling on the mitochondria.
Gal-3-Induced Apoptosis Involves Activation of the Caspase Pathway.
Caspase-3 is a critical downstream protease in the apoptotic cascade (35)
, which is involved in cell death in response to numerous apoptotic stimuli including Fas ligand or tumor necrosis factor 
ligation with its receptor (36
, 37)
. To investigate whether extracellular Gal-3-induced apoptosis is the result of the activation of the caspase pathway, we simultaneously evaluated the levels of active caspase-3 and apoptotic cells treated with Gal-3 in the presence or absence of lactose, respectively (Fig. 6, A and B)
. In addition, we determined the effect of a broad-range caspase inhibitor, Z-VAD-fmk, on Gal-3-induced apoptosis. The results show that Gal-3 induced activation of caspase-3 (Fig. 6A)
and that the percentage of cells expressing the active form of caspase-3 was proportional to the percentage of annexin V-positive cells (Fig. 6, A and B)
. Lactose treatment, which reduced the proportion of annexin V-positive cells (Fig. 6B)
, simultaneously reduced the activation level of caspase-3 (Fig. 6A)
. Moreover, Z-VAD-fmk treatment reduced the level of annexin V-positive and active caspase-3-positive cells to the background levels despite the treatment of Gal-3 (Fig. 6, A and B)
.
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. Lactose treatment, which reduced the proportion of annexin V-positive cells, simultaneously reduced the cytochrome c release to 1.1-fold above basal levels. These results showed that Gal-3 induced cytochrome c release followed by activation of the caspase pathway.
We further measured DEVDase and IETDase activity in H9 and Jurkat cells treated with Gal-3 in the presence or absence of lactose 6 h after the induction of apoptosis. DEVDase activity in Gal-3-insensitive H9 cells treated with Gal-3 increased by only 2-fold above basal levels (Fig. 7A)
. whereas activity in Gal-3-sensitive Jurkat cells increased by more than 7-fold above basal levels in the Gal-3-treated cells (Fig. 7B)
. In contrast, Gal-3 did not induce IETDase activity in both H9 and Jurkat cells (Fig. 7, A and B)
. These results imply that Gal-3 induces activation of caspase-3 but not caspase-8, suggesting that the signaling pathway of Gal-3-induced apoptosis directly affects mitochondria integrity, leading to the activation of the downstream effector caspase-3.
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, Ab to CD7 and CD29 antigens significantly inhibited Gal-3-induced apoptosis in MOLT-4 cells, whereas CD7 was not expressed on SKW6.4 and H9 cells, which are not sensitive to Gal-3 (Fig. 8B, I)
. On the other hand, CD29 was expressed in all cell lines (SKW6.4, H9, Jurkat, CEM, and MOLT-4). To further understand the mode of action of Gal-3, we questioned whether both antigens are colocalized at the cell surface. Thus, single and double immunofluorescence staining was done to localize them. CD7 (red) and CD29 (green) colocalized (yellow) on the cell surface of MOLT-4 cells (Fig. 8C)
, and blocking both CD7 and CD29 antigen did not inhibit Gal-3-induced apoptosis synergistically, suggesting that CD7 and CD29 may act as a complex during the delivery of the Gal-3-induced death signal. The above result thus allows us to propose that in Gal-3-sensitive cells, CD7 and CD29 cooperate, triggering the apoptosis signal through mitochondria and resulting in the activation of caspase-3, whereas in Gal-3-insensitive cells, which express cytoplasmic Gal-3, the cells do not express CD7, and signaling is through CD29 dimerization (39)
.
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DISCUSSION |
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In this report, we evaluated apoptosis using annexin V staining of phosphatidylserine and PI staining of DNA. Although a recent report showed that dimeric Gal-1 induces phosphatidylserine exposure without inducing apoptosis (40) , Gal-3-induced annexin V staining of T cells was associated with apoptosis. After treatment of Gal-3-resistant cells (SKW6.4 and H9) with Gal-3, only a small percentage of cells were at sub-G1 and stained with annexin V, giving credence to the results reported here. On the contrary, after treatment of Gal-3-sensitive cells (Jurkat, CEM, and MOLT4) with Gal-3, a dose-dependent increase of annexin V-positive cells and cells at sub-G1 was observed. Therefore, the mechanism leading to phosphatidylserine exposure seems to differ between Gal-1 and Gal-3.
Apoptotic cell death may be triggered by a variety of stimuli, including death ligands such as tumor necrosis factor , Fas ligand, and tumor necrosis factor-related apoptosis-inducing ligand; DNA-damaging agents; and loss of matrix attachment in adherence-dependent cells (35
, 36)
. Many of these stimuli activate the apoptotic cascade through the caspases. Here we investigated human lymphoma and leukemia cell lines, human PBMCs, and mouse T cells for susceptibility to Gal-3-induced apoptosis and its cellular mechanism. Soluble Gal-3 binds to each of the tested human lymphoid cell lines comparably (data not shown) and to mouse T cells (Fig. 4B)
. However, the cells varied in their sensitivity to Gal-3-induced apoptosis. CD95 type I apoptotic cells (SKW6.4 and H9), which require the activation of caspase-8 by the death-inducing signaling complex (DISC), closely followed by activation of caspase-3 in CD95 (APO-1/Fas)-induced apoptosis, were less sensitive to Gal-3 than CD95 type II apoptotic cell lines (Jurkat and CEM cells), which are characterized by a weak caspase-8 activation at the DISC (31
, 41)
. We show here that Gal-3 induced cytochrome c release as well as caspase-3 activation, but not caspase-8 activation, on T cells, suggesting that soluble Gal-3 is involved in the mitochondria-mediated apoptotic pathway in type II cells. Recent studies have revealed that only type II apoptotic cells, such as Jurkat or CEM cells, are sensitive to C2-ceramide or sphingosin, whereas type I cells, such as SKW6.4 or H9 cells, are refractory to it (31
, 42)
. Our results show that soluble Gal-3 directly acts at the level of the mitochondria, similar to C2-ceramide or sphingosin (Fig. 9)
, implying that type I apoptotic cells lack a component downstream of the mitochondria that is necessary for the processing of caspase-3.
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Here, we report that sensitivity to Gal-3-induced apoptosis correlated with the presence of cytoplasmic Gal-3. Gal-3-sensitive cells were found to be Gal-3 null. Moreover, transfection of Gal-3 cDNA into CEM cells endowed cells with a significantly reduced sensitivity to apoptosis induction compared with CEM control cells. Recently, we have shown that cytoplasmic Gal-3 is translocated to the mitochondria after apoptotic stimuli and prevents mitochondrial damage and cytochrome c release (29) . Similarly, it has been suggested that apoptotic sensitivity to Gal-1 is associated with repression of the Gal-1 gene because nonsensitive cells express high levels of Gal-1 (45) . Furthermore, we found that Gal-3-expressing cells were resistant to C2-ceramide, which causes the formation of reactive oxygen intermediates, and directly affected mitochondrial components (31 , 42) . These results suggest that there is an inverse correlation between cytoplasmic Gal-3 expression and soluble Gal-3-induced apoptosis and that intracellular Gal-3 protected cells from mitochondrial damage as described previously (29) .
To understand the novel mechanism of apoptosis triggered by Gal-3, identification of Gal-3 receptors is important. Previously, it has been reported that CD3, CD7, and CD45 are the candidate receptors for Gal-1-induced apoptosis (13
, 24)
. In this study, we have found that CD7-negative SKW6.4 and H9 cells were more resistant to Gal-3-induced apoptosis than the CD7-positive Jurkat, CEM, and MOLT-4 cells. Abs to CD7 antigen blocked Gal-3-induced apoptosis in MOLT-4 cells, suggesting that CD7 is the receptor of the Gal-3 death pathway in human T cells. Abs directed against CD3 and CD45 did not inhibit Gal-3-induced apoptosis, suggesting that the regulation of Gal-3-induced apoptosis is molecularly distinct from that of Gal-1 (24)
. CD7 is a Mr 40,000 member of the immunoglobulin gene superfamily expressed on the majority of human thymocytes and a large subset (85%) of peripheral blood T cells and natural killer cells (46, 47, 48, 49)
. In addition, the surface density of CD7 is increased after T-cell activation, and CD7 has been proposed to participate in the activation and surface adhesion of mature T cells and natural killer cells (48
, 49)
. The experiments using CD7-deficient mice revealed that CD7 regulates thymocyte maturation and CTL immune response (50)
. CD7 possesses two N-linked glycans that are clustered on either side of IgG fold and numerous O-linked glycans arranged closely in a mucin-like domain (46)
. The arrangement of glycans in CD7 makes it a suitable ligand for Gals. Several reports have shown the involvement of CD7 in Gal-1-induced apoptosis (13
, 24
, 25)
. An Ab against CD7 inhibits Gal-1 binding resulting in apoptosis induction (13)
, and CD7 rendered Gal-1-resistant cells susceptible to Gal-1-induced apo-ptosis (25)
. Immunohistochemical analysis of cutaneous T-cell lymphoma specimens has revealed that the T-cell lymphoma cells have lost CD7 expression, and this loss of CD7 expression in lymphoma cells contributes to resistance to Gal-1 and tumor progression (51
, 52)
. Although both Gal-1- and Gal-3-induced apoptosis require CD7, the other cell surface molecules required are different, suggesting that Gal-1 and Gal-3 induce apoptosis of different subsets of T cells and regulate immune response.
In the current study, we also found that CD29 (ß1 integrin) is an additional receptor required for Gal-3-mediated apoptosis. Integrins mediate T-cell adhesion via interactions with their ligands such as intercellular adhesion molecule-1, intercellular adhesion molecule-2, vascular cell adhesion molecule (VCAM-1), and extracellular matrix components including fibronectin and laminin (53
, 54)
. It has been reported previously that Gal-1, -3, and -9 bind to 7ß1, 1ß1, and M integrin, respectively (55, 56, 57, 58)
. CD29 is a Mr 130,000 ß1 integrin subunit with a broad tissue distribution (53)
. CD29-mediated signaling promotes or inhibits apoptosis, depending on the ligands and apoptotic insults (59)
. CD29 mediates apoptosis of smooth muscle cells, rheumatoid synovila cells, endothelial cells, and invasion-induced cell death of T lymphocytes (60, 61, 62)
. CD29-mediated apoptosis requires recruitment of caspase-8 to the membrane, similar to Fas-induced apoptosis (63)
. In addition to anoikis, CD29 is associated with T-cell activation and induction of T-cell death (64, 65, 66)
. Because both CD29 and Gal-3 were found to modulate TCR signaling and subsequent T-cell activation, it was tempting to speculate that Gal-3-induced apoptosis is mediated in part by CD29 and may be associated with differentiation and selection of thymocytes (10
, 64, 65, 66)
.
It has been shown that adhesion of peripheral T cells to ß1 integrin ligands is induced by ligation of a number of T-cell surface molecules, including CD7 (67 , 68) . We found that both CD7 and CD29 were colocalized on the surface of MOLT-4 cells, suggesting that CD7 and CD29 may work as a complex during the delivery of the Gal-3-induced death signal. Thus, we hypothesize that Gal-3 secreted from tumor cells binds to CD7- and CD29-positive T-lymphoid cells and induces apoptosis, resulting in tumor cell evasion of host immune responses.
Based on the data submitted here, we propose the following model for Gal-3-induced apoptosis (Fig. 9)
. Secreted soluble Gal-3 binds to the cell surface CD7 and CD29 (Fig. 9, 1)
. Oligomerization is important for Gal-3-induced apoptosis because truncated Gal-3 without NH2 terminus, which regulates dimer or oligomer formation, reduced apoptotic function compared with full-length Gal-3 (69)
. After binding, the caspase pathway is activated through loss of mitochondria integrity (Fig. 9, 2)
. Cytoplasmic Gal-3 blocks apoptotic signaling on a mitochondria site (Fig. 9, 3)
.
In summary, induction of T-cell apoptosis by secreted Gal-3, which is dependent in part on the presence or absence of cytoplasmic Gal-3, represents a new mechanism for modulating the immune escape mechanism of cancer cells, a finding that could assist in determining cancer diagnosis and therapy.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Requests for reprints: Avraham Raz, Tumor Progression and Metastasis Program, Karmanos Cancer Institute, 110 East Warren Avenue, Detroit, Michigan 48201. Phone: (313) 833-0960; Fax: (313) 831-7518; E-mail: raza{at}karmanos.org
5 The abbreviations used are: Gal, galectin; Ac, acetyl; AMC, 7-amino-4-methylcoumarin; FBS, fetal bovine serum; GST, glutathione S-transferase; mAb, monoclonal antibody; PBMC, peripheral blood mononuclear cell; Z-VAD-fmk, N-CBZ-Val-Ala-Asp (O-Me) fluoromethyl ketone; Ab, antibody; PI, propidium iodide; ATCC, American Type Culture Collection. 
Received 8/13/03. Revised 9/11/03. Accepted 9/15/03.
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 M. V. Tribulatti, V. Cattaneo, U. Hellman, J. Mucci, and O. Campetella Galectin-8 provides costimulatory and proliferative signals to T lymphocytes J. Leukoc. Biol., August 1, 2009; 86(2): 371 - 380. [Abstract] [Full Text] [PDF]
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 C. Iacobini, S. Menini, C. Ricci, A. Scipioni, V. Sansoni, S. Cordone, M. Taurino, M. Serino, G. Marano, M. Federici, et al. Accelerated Lipid-Induced Atherogenesis in Galectin-3-Deficient Mice: Role of Lipoxidation via Receptor-Mediated Mechanisms Arterioscler Thromb Vasc Biol, June 1, 2009; 29(6): 831 - 836. [Abstract] [Full Text] [PDF]
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 L. V Norling, M. Perretti, and D. Cooper Endogenous galectins and the control of the host inflammatory response J. Endocrinol., May 1, 2009; 201(2): 169 - 184. [Abstract] [Full Text] [PDF]
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 S. D. Liu, T. Tomassian, K. W. Bruhn, J. F. Miller, F. Poirier, and M. C. Miceli Galectin-1 Tunes TCR Binding and Signal Transduction to Regulate CD8 Burst Size J. Immunol., May 1, 2009; 182(9): 5283 - 5295. [Abstract] [Full Text] [PDF]
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K. Loser, A. Sturm, M. Voskort, V. Kupas, S. Balkow, M. Auriemma, C. Sternemann, A. U. Dignass, T. A. Luger, and S. Beissert Galectin-2 Suppresses Contact Allergy by Inducing Apoptosis in Activated CD8+ T Cells J. Immunol., May 1, 2009; 182(9): 5419 - 5429. [Abstract] [Full Text] [PDF]
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 J. Saegusa, D. K. Hsu, H.-Y. Chen, L. Yu, A. Fermin, M. A. Fung, and F.-T. Liu Galectin-3 Is Critical for the Development of the Allergic Inflammatory Response in a Mouse Model of Atopic Dermatitis Am. J. Pathol., March 1, 2009; 174(3): 922 - 931. [Abstract] [Full Text] [PDF]
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 S. R. Stowell, S. Karmakar, C. M. Arthur, T. Ju, L. C. Rodrigues, T. B. Riul, M. Dias-Baruffi, J. Miner, R. P. McEver, and R. D. Cummings Galectin-1 Induces Reversible Phosphatidylserine Exposure at the Plasma Membrane Mol. Biol. Cell, March 1, 2009; 20(5): 1408 - 1418. [Abstract] [Full Text] [PDF]
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 W. Peng, H. Y. Wang, Y. Miyahara, G. Peng, and R.-F. Wang Tumor-Associated Galectin-3 Modulates the Function of Tumor-Reactive T Cells Cancer Res., September 1, 2008; 68(17): 7228 - 7236. [Abstract] [Full Text] [PDF]
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 Y. Zhuo, R. Chammas, and S. L. Bellis Sialylation of {beta}1 Integrins Blocks Cell Adhesion to Galectin-3 and Protects Cells against Galectin-3-induced Apoptosis J. Biol. Chem., August 8, 2008; 283(32): 22177 - 22185. [Abstract] [Full Text] [PDF]
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S. Bi, L. A. Earl, L. Jacobs, and L. G. Baum Structural Features of Galectin-9 and Galectin-1 That Determine Distinct T Cell Death Pathways J. Biol. Chem., May 2, 2008; 283(18): 12248 - 12258. [Abstract] [Full Text] [PDF]
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S. R. Stowell, C. M. Arthur, P. Mehta, K. A. Slanina, O. Blixt, H. Leffler, D. F. Smith, and R. D. Cummings Galectin-1, -2, and -3 Exhibit Differential Recognition of Sialylated Glycans and Blood Group Antigens J. Biol. Chem., April 11, 2008; 283(15): 10109 - 10123. [Abstract] [Full Text] [PDF]
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S. R. Stowell, Y. Qian, S. Karmakar, N. S. Koyama, M. Dias-Baruffi, H. Leffler, R. P. McEver, and R. D. Cummings Differential Roles of Galectin-1 and Galectin-3 in Regulating Leukocyte Viability and Cytokine Secretion J. Immunol., March 1, 2008; 180(5): 3091 - 3102. [Abstract] [Full Text] [PDF]
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S. L. Farnworth, N. C. Henderson, A. C. MacKinnon, K. M. Atkinson, T. Wilkinson, K. Dhaliwal, K. Hayashi, A. J. Simpson, A. G. Rossi, C. Haslett, et al. Galectin-3 Reduces the Severity of Pneumococcal Pneumonia by Augmenting Neutrophil Function Am. J. Pathol., February 1, 2008; 172(2): 395 - 405. [Abstract] [Full Text] [PDF]
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 V. L. J. L. Thijssen, F. Poirier, L. G. Baum, and A. W. Griffioen Galectins in the tumor endothelium: opportunities for combined cancer therapy Blood, October 15, 2007; 110(8): 2819 - 2827. [Abstract] [Full Text] [PDF]
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 F. H.M. de Melo, D. Butera, R. S. Medeiros, L. N. d. S. Andrade, S. Nonogaki, F. A. Soares, R. A. Alvarez, A. M. Moura da Silva, and R. Chammas Biological Applications of a Chimeric Probe for the Assessment of Galectin-3 Ligands J. Histochem. Cytochem., October 1, 2007; 55(10): 1015 - 1026. [Abstract] [Full Text] [PDF]
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J. Kubach, P. Lutter, T. Bopp, S. Stoll, C. Becker, E. Huter, C. Richter, P. Weingarten, T. Warger, J. Knop, et al. Human CD4+CD25+ regulatory T cells: proteome analysis identifies galectin-10 as a novel marker essential for their anergy and suppressive function Blood, September 1, 2007; 110(5): 1550 - 1558. [Abstract] [Full Text] [PDF]
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 S. J. Van Dyken, R. S. Green, and J. D. Marth Structural and Mechanistic Features of Protein O Glycosylation Linked to CD8+ T-Cell Apoptosis Mol. Cell. Biol., February 1, 2007; 27(3): 1096 - 1111. [Abstract] [Full Text] [PDF]
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E. Silva-Monteiro, L. Reis Lorenzato, O. Kenji Nihei, M. Junqueira, G. A. Rabinovich, D. K. Hsu, F.-T. Liu, W. Savino, R. Chammas, and D. M. S. Villa-Verde Altered Expression of Galectin-3 Induces Cortical Thymocyte Depletion and Premature Exit of Immature Thymocytes during Trypanosoma cruzi Infection Am. J. Pathol., February 1, 2007; 170(2): 546 - 556. [Abstract] [Full Text] [PDF]
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 L.-H. Lu, R. Nakagawa, Y. Kashio, A. Ito, H. Shoji, N. Nishi, M. Hirashima, A. Yamauchi, and T. Nakamura Characterization of Galectin-9-Induced Death of Jurkat T Cells J. Biochem., February 1, 2007; 141(2): 157 - 172. [Abstract] [Full Text] [PDF]
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S. R. Stowell, S. Karmakar, C. J. Stowell, M. Dias-Baruffi, R. P. McEver, and R. D. Cummings Human galectin-1, -2, and -4 induce surface exposure of phosphatidylserine in activated human neutrophils but not in activated T cells Blood, January 1, 2007; 109(1): 219 - 227. [Abstract] [Full Text] [PDF]
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S. Nakahara, V. Hogan, H. Inohara, and A. Raz Importin-mediated Nuclear Translocation of Galectin-3 J. Biol. Chem., December 22, 2006; 281(51): 39649 - 39659. [Abstract] [Full Text] [PDF]
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 F. Zhang, P. Hopwood, C. C. Abrams, A. Downing, F. Murray, R. Talbot, A. Archibald, S. Lowden, and L. K. Dixon Macrophage Transcriptional Responses following In Vitro Infection with a Highly Virulent African Swine Fever Virus Isolate J. Virol., November 1, 2006; 80(21): 10514 - 10521. [Abstract] [Full Text] [PDF]
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S. Nakahara, N. Oka, Y. Wang, V. Hogan, H. Inohara, and A. Raz Characterization of the nuclear import pathways of galectin-3. Cancer Res., October 15, 2006; 66(20): 9995 - 10006. [Abstract] [Full Text] [PDF]
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B. Rossi, M. Espeli, C. Schiff, and L. Gauthier Clustering of Pre-B Cell Integrins Induces Galectin-1-Dependent Pre-B Cell Receptor Relocalization and Activation J. Immunol., July 15, 2006; 177(2): 796 - 803. [Abstract] [Full Text] [PDF]
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E. S. Bernardes, N. M. Silva, L. P. Ruas, J. R. Mineo, A. M. Loyola, D. K. Hsu, F.-T. Liu, R. Chammas, and M. C. Roque-Barreira Toxoplasma gondii Infection Reveals a Novel Regulatory Role for Galectin-3 in the Interface of Innate and Adaptive Immunity Am. J. Pathol., June 1, 2006; 168(6): 1910 - 1920. [Abstract] [Full Text] [PDF]
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M. R. Zubieta, D. Furman, M. Barrio, A. I. Bravo, E. Domenichini, and J. Mordoh Galectin-3 Expression Correlates with Apoptosis of Tumor-Associated Lymphocytes in Human Melanoma Biopsies Am. J. Pathol., May 1, 2006; 168(5): 1666 - 1675. [Abstract] [Full Text] [PDF]
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T. Fukumori, N. Oka, Y. Takenaka, P. Nangia-Makker, E. Elsamman, T. Kasai, M. Shono, H.-o. Kanayama, J. Ellerhorst, R. Lotan, et al. Galectin-3 regulates mitochondrial stability and antiapoptotic function in response to anticancer drug in prostate cancer. Cancer Res., March 15, 2006; 66(6): 3114 - 3119. [Abstract] [Full Text] [PDF]
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B. N. Stillman, D. K. Hsu, M. Pang, C. F. Brewer, P. Johnson, F.-T. Liu, and L. G. Baum Galectin-3 and Galectin-1 Bind Distinct Cell Surface Glycoprotein Receptors to Induce T Cell Death J. Immunol., January 15, 2006; 176(2): 778 - 789. [Abstract] [Full Text] [PDF]
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 J M Ilarregui, G A Bianco, M A Toscano, and G A Rabinovich The coming of age of galectins as immunomodulatory agents: impact of these carbohydrate binding proteins in T cell physiology and chronic inflammatory disorders Ann Rheum Dis, November 1, 2005; 64(suppl_4): iv96 - iv103. [Abstract] [Full Text] [PDF]
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G. C. Fernandez, J. M. Ilarregui, C. J. Rubel, M. A. Toscano, S. A. Gomez, M. Beigier Bompadre, M. A. Isturiz, G. A. Rabinovich, and M. S. Palermo Galectin-3 and soluble fibrinogen act in concert to modulate neutrophil activation and survival: involvement of alternative MAPK pathways Glycobiology, May 1, 2005; 15(5): 519 - 527. [Abstract] [Full Text] [PDF]
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C. Greco, R. Vona, M. Cosimelli, P. Matarrese, E. Straface, P. Scordati, D. Giannarelli, V. Casale, D. Assisi, M. Mottolese, et al. Cell surface overexpression of galectin-3 and the presence of its ligand 90k in the blood plasma as determinants in colon neoplastic lesions Glycobiology, September 1, 2004; 14(9): 783 - 792. [Abstract] [Full Text] [PDF]
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) for 6 h at 37°C. Each experiment was performed in triplicate. Data are the mean ± SD from three independent experiments. B, one human B-cell line (SKW6.4), four human T-cell lines (H9, Jurkat, CEM, and MOLT-4), and human PBMCs were treated with control sample and 15 µM Gal-3 in the presence or absence of 50 mM lactose for 6 h at 37°C. Data are the mean ± SD of duplicates from three independent experiments. C, expression of intracellular Gal-3 in human B-cell and T-cell lines. Western blot analysis of Gal-3 was performed using 50 µg of cell lysates from SKW6.4, H9, Jurkat, CEM, and MOLT-4 cells. Western blot analysis of actin expression was performed using the same membrane used for Gal-3 expression analysis after stripping.
