From the Cancer Biology Laboratories, Department of Molecular Medicine, Cornell University College of Veterinary Medicine, Ithaca, New York 14853
Received for publication, April 2, 2003 , and in revised form, April 18, 2003.
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ABSTRACT |
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INTRODUCTION |
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The involvement of DPPIV in the colonization of the lungs by blood-borne cancer cells was first discovered when we screened cancer cells with different organ colonization preferences for binding to endothelial cells from various tissue sources. Rather than relying on tissue culture-isolated endothelial cells, screening was performed with outside-out membrane vesicles freshly harvested from the luminal membrane of endothelial cells by in situ vascular perfusion of rat lungs with a formaldehyde/dithiothreitol solution to induce endothelial cell-surface vesiculation (6, 15). Consistently, lung endothelial vesicles adhered in large numbers to lung-metastatic cancer cells only, but not to liver-metastatic or non-metastatic cancer cells, whereas control vesicles prepared from a rarely metastasized organ (e.g. leg musculature) adhered at background levels to lung- and liver-metastatic and non-metastatic tumor cells. Using a passive/active immunization schedule involving emulsified leg endothelial vesicles for passive immunization and lung endothelial vesicles for active immunization, we identified a monoclonal antibody that totally blocked the adhesion of lung endothelial vesicles to lung-metastatic cancer cells. The molecule blocked by the antibody was identified as DPPIV (6). Subsequently, we showed that vascular arrest of lung-metastatic cancer cells was mediated by DPPIV adhesion to cancer cell surface-associated FN (1618). In contrast to the poor ability of DPPIV to bind to soluble plasma FN, the binding to cell surface-associated FN was of high avidity, suggesting that cell surface-associated FN presents itself in a different configuration than soluble plasma FN (2527). Indeed, detailed biochemical analyses of cancer cell surface-associated FN revealed that FN is assembled into large fibrillar polymers (poly-FN) that are dispersed over the cancer cell surface in multiple globular aggregates, apparently exposing multiple DPPIV-binding sites (16, 17). Participation of the DPPIV/poly-FN adhesion in lung metastasis was substantiated by the findings that (a) a soluble DPPIV polypeptide representing the entire extracellular domain totally abolishes adhesion of lung-metastatic breast cancer cells to DPPIV and, accordingly, prevents lung colonization (17); (b) the Fischer 344/CRJ rat substrain, which harbors a G633R substitution in DPPIV that leads to retention and degradation of much of the mutant protein in the endoplasmic reticulum (18, 28), as well as DPPIV/ mice (obtained from Dr. Marquet, Centre d'Immunologie de Marseille Luminy, INSERM-CNRS) (11) allow lung metastasis at dramatically reduced levels relative to wild-type Fischer 344 rats (18) and C57BL/6 mice,2 respectively; (c) lung-metastatic tumor cell lines derived from various human, mouse, and rat cancers invariably carry a strong message for endogenous FN and are able to assemble poly-FN on their surfaces; (d) FN surface expression in clones derived from a rhabdomyosarcoma correlates with lung metastasis (29); and (e) human and mouse melanoma cell lines selected for enhanced lung colonization overexpress FN (30, 31). Together, these data suggest that the DPPIV/poly-FN adhesion might be a quite common mechanism in the colonization of the lungs by blood-borne cancer cells, where it may operate alone or collaborate with other adhesion molecules, including FN ligands other than DPPIV (e.g. integrins, heparan sulfate proteoglycans, and CD44), to mediate vascular arrest (3235).
In this study, we authenticate and refine the role played by the DPPIV/poly-FN adhesion in pulmonary metastasis, identifying DPPIV-binding sites within FN and generating synthetic peptides of these sites that, by their anti-metastatic effect, clearly distinguish DPPIV/poly-FN adhesion from other FN adhesions. The binding sites were found by generating and screening a series of successively shorter FN sequences for DPPIV binding. The sites are located in type III repeats 13, 14, and 15 (FNIII13, -14, and -15, respectively) and represent a common consensus motif. Their DPPIV-binding specificity is confirmed in biochemical, mutational, adhesion, and lung colony assays.
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EXPERIMENTAL PROCEDURES |
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Plasmid Constructs and PROTOMAT SearchRat FNIII16, FNIII815, and FNIII1215 were obtained as MBP fusion proteins from Dr. J. E. Schwarzbauer (Princeton University, Princeton, NJ) (36). To generate MBP-FN fusion proteins, FNIII811 (amino acids 14141720), FNIII1214 (amino acids 18112081), FNIII1213 (amino acids 18111991), FNIII1314 (amino acids 19032081), FNIII1415 (amino acids 19922081 and 22022286), FNIII12 (amino acids 18111902), FNIII13 (amino acids 19031991), FNIII14 (amino acids 19922081), and FNIII15 (amino acids 22022286) were PCR-amplified from FNIII815 and inserted into the EcoRI and XbaI sites of the pMAL-c2 vector (New England Biolabs Inc.). All plasmids were verified by double-stranded sequencing. Overlap extension PCR was used to swap the consensus motif of FNIII13 (amino acids 19591968), FNIII14 (amino acids 20492058), or FNIII15 (amino acids 22472257) with the corresponding region of FNIII12 (amino acids 18681877) (37). The same methods were used to replace amino acids 18681877 in FNIII12 with amino acids 20492058 in FNIII14. GST-PEP14-HA and GST-PEPcon-HA fusion proteins were generated by PCR amplification of FNIII14-(20452062) and FNIIIEDA-(17741791). The amplified sequence included the HA tag (YPYDVPDYA) and was inserted into the EcoRI/HindIII sites of the pGEX-KG vector (38).
PROTOMAT was used to search for conserved motifs in FNIII13, -14, and -15. The MOTIF program (H. O. Smith, Institute for Genomic Research, Rockville, MD) was first run to provide candidate blocks in the FNIII repeats. It was followed by an automated implementation of the GIBBS sampler program (C. E. Lawrence, Department of Statistics, Harvard University, Cambridge, MA) for reality checking (39). A data base of blocks was constructed by successive application of the automated PROTOMAT system to individual entries in the PROSITE catalog (40) of FNIII repeats keyed to the Swiss Protein Sequence Database (41).3 If sequences truly have motifs in common, then both runs typically yield similar or, more significantly, identical sets of blocks (39).
Purification of Fusion Proteins and Rat DPPIVMBP-FN fusion proteins (New England Biolabs Inc.) were purified according to the manufacturer's instructions. Briefly, 2 liters of Escherichia coli culture were spun after a 2-h isopropyl--D-thiogalactopyranoside (0.3 mM) induction. Cell pellets were sonicated in 100 ml of column buffer (20 mM Tris-HCl (pH 7.4), 200 mM NaCl, and 1 mM EDTA) and then spun at 19,000 rpm for 20 min at 4 °C. Supernatants were diluted 1:3 with column buffer and passed through an amylose resin column. Columns were washed with 10 volumes of column buffer and eluted with 10 mM maltose in column buffer. The purity of the elutes were evaluated by Coomassie Blue staining of SDS-polyacrylamide gels and Western blotting with anti-MBP pAb. Protein concentrations were measured by the Bradford method (Bio-Rad). DPPIV was immunopurified from rat lungs using mAb 6A3 (1618). HA-tagged GST fusion proteins were purified with anti-HA mAb-conjugated agarose beads.
Gel Overlay Assay (Far-Western)Equal amounts of N-terminal FN proteolytic fragments (29, 45, and 70 kDa) and MBP-FNIII fusion proteins were subjected to SDS-PAGE under nonreducing conditions (36, 42). Gel-separated proteins were transferred to nitrocellulose membranes and denatured with 4 M guanidine HCl in Tris-buffered saline containing 0.1% Tween 20 (TTBS) for 2 h. Proteins were then slowly renatured in TTBS. After blocking with 5% skim milk in TTBS for 2 h at room temperature, membranes were incubated overnight with 2 µg/ml DPPIV in 5% skim milk at 4 °C, followed by extensive washing with TTBS and incubation with anti-DPPIV pAb CU31. Bound anti-DPPIV antibodies were detected with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody and visualized by ECLTM (Amersham Biosciences).
Other Biochemical MethodsA modified enzyme-linked immunosorbent assay (ELISA) was used to measure the binding of DPPIV to FN fusion proteins. FN fusion proteins were coated at the indicated concentrations onto 96-well microtitration plates overnight at 4 °C. The plates were washed three times with phosphate-buffered saline (PBS) and then incubated overnight with 5 µg/ml immunopurified DPPIV at 4 °C. DPPIV binding was detected with anti-DPPIV pAb CU31, horse-radish peroxidase-conjugated goat anti-rabbit antibody, and ECL. DPPIV affinity precipitation (pull-down assays) was carried out with DPPIV-conjugated Affi-Gel 10 beads (16). FN fusion proteins (5 µg/ml in 300 µl of PBS containing 0.1% -octyl glucoside) were incubated overnight with DPPIV beads at 4 °C. The beads were washed with 0.1%
-octyl glucoside-containing PBS and then boiled for 10 min in SDS sample buffer. Bound SDS-PAGE-separated proteins were Western-probed with anti-MBP pAb. For the co-immunoprecipitation of DPPIV and MBP-FNIII repeats, DPPIV (5 µg/ml in 300 µl of 0.5%
-octyl glucoside-containing PBS) was incubated with the same concentrations of wild-type or mutant MBP-FNIII single repeats for 6 h at 4 °C. Complexes were then incubated overnight with anti-MBP pAb-conjugated protein A-agarose beads at 4 °C. After washing with 0.5%
-octyl glucoside-containing PBS, immunoprecipitates were subjected to SDS-PAGE and immunoblotting with anti-DPPIV pAb CU31 or anti-MBP pAb. To examine the heparin-binding abilities of wild-type and mutant MBP-FNIII fragments, fusion proteins were incubated overnight with heparin-agarose beads at 4 °C. After washing with 0.5%
-octyl glucoside-containing PBS, the beads were boiled for 10 min in SDS sample buffer and subjected to SDS-PAGE, and the resolved proteins were immunoblotted with anti-MBP pAb.
Peptide Binding to Purified DPPIV and DPPIV-expressing Cells Immunopurified DPPIV (10 µg/ml) was coated onto 96-well microtitration plates overnight at 4 °C. After blocking with 1% bovine serum albumin (BSA), the coated plates were incubated with biotinylated PEP14 or PEPcon (or GST-PEP14 or GST-PEPcon) for 2 h at room temperature. Bound peptide was detected by ELISA using horseradish peroxidase-streptavidin or anti-GST pAb. HEK293 cells were transfected with either rat DPPIV cDNA or vector (pRc-CMV) alone (18). Surface expression of DPPIV was evaluated by fluorescence-activated cell sorting using anti-DPPIV mAb 6A3. To determine the ability of biotinylated PEP14 and PEPcon to bind to cell surface-expressed DPPIV, transfected cells were incubated with 0.1 mg/ml biotinylated peptide for 1 h at room temperature. The cells were then washed with PBS, and bound peptide was detected by staining with Texas Red-conjugated streptavidin for 30 min at 4 °C. Staining patterns were observed by fluorescence microscopy.
Cell Adhesion and Lung Colony Inhibition AssaysCell adhesion and lung colony assays were described previously (16, 42). For adhesion inhibition experiments, DPPIV-binding MBP-FNIII fusion proteins (160 nM) were coated onto the wells of microtitration plates overnight at 4 °C (control, 30 µg/ml poly-L-lysine) and then blocked with 1% BSA in PBS and conjugated with DPPIV at a concentration selected to equate the amount of DPPIV bound to plastic as determined by ELISA with anti-DPPIV pAb CU31. The wells were then seeded with tumor cells (35 x 104 cells/well) and incubated for 30 min at 37 °C. The percent tumor cell adhesion was determined as described (16). Alternatively, DPPIV (10 µg/ml) was coated onto the microtitration plate directly overnight at 4 °C, blocked with 1% BSA in PBS, and incubated with GST-PEP14 (control, GSP-PEPcon) for 2 h at room temperature. After washing with PBS, tumor cells were added, and adhesion was determined as described above. For lung colony inhibition assays, GST-PEP14 or GST-PEPcon (1 mg/mouse) was injected intravenously 15 min prior to tumor cells (1 x 105 cells/0.2 ml of Dulbecco's modified Eagle's medium/mouse). To exclude adverse reactions of GST-PEP14 with DPPIV-expressing T-cells, lung colony assays were performed in T-cell-deficient female 4-week-old Scid/beige mice (Charles River Laboratories, Wilmington, MA).
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RESULTS |
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The functional significance of the DPPIV/FNIII1215 adhesion was substantiated in adhesion inhibition assays with four cancer cell lines. Three of these cell lines, MTF7, B16-F10, and MDA-MB-231, consistently colonized the lungs of rats and mice upon tail vein injection, expressed strong endogenous messages for FN, and accumulated significant amounts of aggregated FN on their surfaces (Fig. 3A). In contrast, MCF7 tumor cells did not colonize the lungs and were negative for endogenous message and surface accumulation of FN. Accordingly, MTF7, B16-F10, and MDA-MB-231 cells strongly adhered to DPPIV-coated dishes as well as to DPPIV bound to dishes coated with poly-L-lysine, but failed to adhere to DPPIV immobilized on dishes coated with FNIII1215 (Fig. 3B). MCF7 cells did not adhere to DPPIV (Fig. 3B). In these experiments, the DPPIV coating concentration was selected such that equal binding to the three substrates (plastic, MBP-FNIII1215, and poly-L-lysine) was achieved as determined by ELISA.
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DPPIV Binds to FNIII13, -14, and -15Further dissection of the DPPIV-binding polypeptide MBP-FNIII1215 was done to yield dual (MBP-FNIII1213, MBP-FNIII1314, and MBP-FNIII1415) and single (FNIII12, -13, -14, and -15) type III repeats to narrow the DPPIV-binding site to a single repeat (Fig. 1). Among these constructs, only the single repeat FNIII12 lost DPPIV-binding ability; all other fragments strongly bound DPPIV as observed by Far-Western and pull-down analyses (Fig. 4A, single repeats shown only). These binding interactions were specific because DPPIV bound neither to FNIII12 nor to MBP, and control beads failed to pull down any of the FN repeats (Fig. 4A). Parallel ELISA analyses also demonstrated that DPPIV bound to FNIII13, -14, and -15 (Fig. 4B). However, these data did not reveal whether DPPIV binding to FNIII13, -14, and -15 was mediated by three distinct binding domains or a common binding site present in each of the three repeats. To address this issue, we performed MTF7/DPPIV adhesion inhibition experiments in which DPPIV was bound to dishes coated with any one of the three repeats. The data show that all three fragments totally abolished the binding interaction between MTF7 cells and DPPIV (Fig. 4C). Similarly, FN fragments containing one or more of the three DPPIV-binding repeats also inhibited MTF7 cell adhesion to DPPIV (data not shown), suggesting that FNIII13, -14, and -15 might harbor a common DPPIV-binding motif (provided DPPIV contains a single FN-binding site).
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FNIII13, -14, and -15 Contain a Consensus DPPIV-binding MotifTo examine whether a common DPPIV-binding motif is indeed present in FNIII13, -14, and -15, we employed the computer algorithm PROTOMAT (39). The sequence T(I/L)-TGLX(P/R)G(T/V)XYXIX(L/V)X(T/A)LX(D/N)(Q/N)X(R/K) was identified. Comparison of this sequence motif in FNIII13, -14, and -15 with the corresponding region in FNIII12 revealed that the N-terminal (underlined) part of this motif (T(I/L)TGLX(P/R)G(T/V)X preceding the core Tyr residue present in all FNIII repeats) (43, 44) is the most conserved among the three DPPIV-binding repeats FNIII13, -14, and -15, but is most divergent in FNIII12, which does not bind DPPIV (Fig. 5A). The underlined motif includes the last four amino acids of the E -strand and extends over the entire length of the solution-accessible EF loop region (44).
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To test this putative DPPIV-binding motif, we swapped the T(I/L)TGLX(P/R)G(T/V)X motif sequence in FNIII13, -14, and -15 with the corresponding loop in FNIII12 rather than deleting the entire loop and causing structural alterations in the repeat that might interfere with function (Fig. 5B). In DPPIV pull-down assays, we showed that wild-type FNIII13, -14, and -15 repeats were successfully pulled down by DPPIV, whereas the three FNIII13(12), FNIII14(12), and FNIII15(12) mutants were not, although equal amounts of wild-type and mutant proteins were subjected to pull down (Fig. 5C, upper and middle panels). To ensure that our swapping strategy did not perturb the inherent repeat structure and inadvertently affect DPPIV binding, we examined the FNIII13(12) and FNIII14(12) mutants, which also contain the second heparin-binding domain (45) located remotely from the swapped sequence, for their abilities to mediate heparin binding. Both wild-type and mutant FNIII13 and FNIII14 were equally competent to mediate heparin binding (Fig. 5C, lower panels). The same DPPIV-binding behavior for wild-type and mutant FNIII13, -14, and -15 was also observed in co-immunoprecipitation experiments. DPPIV co-immunoprecipitated with wild-type (but not mutant) FNIII13, -14, and -15 using anti-MBP antibodies (Fig. 5D). Although the data presented so far clearly show that the identified consensus motif is essential for DPPIV binding to FNIII13, -14, and -15, the ultimate test for its serving as the DPPIV-binding domain was obtained when we introduced the consensus motif of FNIII14 into FNIII12 in lieu of the corresponding sequence. This domain exchange resulted in gain of DPPIV binding in the FNIII12(14) mutant, as observed by DPPIV pull-down assays (Fig. 5E) and co-immunoprecipitation (Fig. 5F).
Peptides Harboring the DPPIV-binding Site of FNIII14 Impede Tumor Cell Adhesion to DPPIV and Pulmonary MetastasisTo examine whether the consensus motif by itself is sufficient for DPPIV binding, we synthesized biotinylated peptides based on the consensus motif of FNIII14 (PEP14). Because the corresponding peptide from FNIII12 was insoluble, we used a sequence from the same loop in FNIIIEDA (the second alternatively spliced repeat) as the control peptide, which displayed similar charge and hydrophobicity characteristics as PEP14, i.e. EDTAELQGLRPGSEYTVS (PEPcon). First, we measured the binding abilities of these biotinylated peptides to immobilized DPPIV by ELISA using streptavidin detection. PEP14 bound to DPPIV in a dose-dependent manner, whereas PEPcon failed to bind DPPIV at all test concentrations (Fig. 6A). Second, we performed peptide affinity precipitation assays in which biotinylated peptides were bound to streptavidin-conjugated beads before being exposed to immunopurified soluble DPPIV (Fig. 6B, upper panel). Only PEP14 was able to precipitate DPPIV (Fig. 6B, middle panel), whereas beads alone or PEPcon-conjugated beads left DPPIV in the flow-through (Fig. 6B, lower panel). To test whether PEP14 also binds to DPPIV protein in its native transmembrane state, we transfected HEK293 cells with rat DPPIV and measured the ability of the transfected cells to bind the biotinylated peptides by Texas Red-conjugated streptavidin staining. Again, PEP14 bound selectively to the DPPIV-expressing HEK293 cells, but not to the mock-transfected HEK293 cells, whereas PEPcon bound to neither cell type (Fig. 6C).
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Finally, we examined PEP14 in tumor cell adhesion and lung colony assays. For economical reasons and to guarantee longer survival in serum, we conducted our experiments with a GST-PEP14 fusion protein rather than with synthetic PEP14. GST-PEP14 blocked adhesion of MTF7 cancer cells to DPPIV in a dose-dependent manner, causing a 50% inhibition of adhesion at a concentration of 3.0 µM (Fig. 7A). The control fusion protein GST-PEPcon had no adhesion inhibitory effect at any of the test concentrations. In lung colony assays, GST-PEP14 consistently reduced the colonization of the lungs by MTF7 cells, allowing the generation of fewer and smaller tumor colonies than GST-PEPcon (Fig. 7B).
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DISCUSSION |
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The three DPPIV-binding domains are located within an FN region (FNIII1315) that is involved in multiple biological functions (34, 48). For example, FNIII1315 is important in FN fibrillogenesis (49); binds human immunodeficiency virus type 1 gp120/160 to reduce virus infectivity (50); and harbors adhesion sites for integrins 4
1 and
IIb
3 (33, 48), the chondroitin sulfate proteoglycan CD44 (35), and heparin (second heparin-binding domain) (34, 44, 48, 5153). The integrin- and heparin-binding domains are located in areas distinct from the DPPIV-binding sites, and each is characterized by a unique sequence (45, 48), suggesting that they may operate in an independent manner. However, studies by Sharma et al. (44) showed that the integrin
4
1- and heparin-binding domains located on opposite faces of FNIII1314 may affect binding of one binding domain by loading or unloading the other. This type of interaction between distinct binding domains has also been observed for the heparin- and DPPIV-binding domains, where occupation of the heparin-binding site with its ligand totally inhibited DPPIV binding to MTF7 cells in vitro and abolished lung colonization of these tumor cells in vivo.4 Although we have no precise explanation for this preliminary result, it is likely that heparin binding acting in an allosteric manner perturbs FNIII13, -14, and -15 binding to DPPIV. Full disclosure of the dynamic interplay between ligand-bound and -unbound cell adhesion domains in FNIII1315 may therefore provide new insights into the conditions that regulate FN binding engagement with integrins, heparan sulfate proteoglycans, and DPPIV and may explain the different binding behavior of FN under different microenvironmental conditions (3335, 44, 4851).
Functional analysis with a peptide representing the DPPIV-binding domain of FNIII14 showed competitive blocking of the DPPIV/poly-FN adhesion and a powerful anti-metastatic effect. The extent of these anti-adhesive and anti-metastatic effects was similar to that reported for adhesion-blocking antibodies and the soluble extracellular domain of DPPIV (17). A similar anti-metastatic effect by a 22-mer synthetic peptide harboring the complete DPPIV-binding motif of FNIII14 has also been reported for T lymphoma cells colonizing liver and spleen (52). Although various integrins (for which the 22-mer peptide had no binding affinity) were implicated in the anti-metastatic effect, it is likely that the peptide blocked the binding interaction between hepatic and splenic sinusoidal FN and DPPIV, which has been shown to be prominently expressed on the T lymphoma cells used (53).
Here, we have identified and characterized a novel cell-binding domain on FN and have presented data showing that binding of endothelial DPPIV to this FN domain plays an important role in pulmonary metastasis. However, at this time, it is unclear whether the DPPIV/poly-FN adhesion acts alone in mediating vascular arrest and metastasis or whether it requires the cooperation of a secondary adhesion event, as reported for the adhesion interaction between immune/inflammatory cells and endothelial cells (reviewed in Refs. 54 and 55). Because all lung-metastatic cancer cells tested in our laboratory, including MDA-MB-231, B16-F10, 4T1, MTF7, LLC1, and K7M2, exhibit high message and protein levels for both FN and integrin 4 and adhere to both of the respective pulmonary addressins (DPPIV and CLCA), it is conceivable that pulmonary vascular arrest of blood-borne cancer cells is initiated by the DPPIV/poly-FN adhesion and is stabilized by the CLCA/integrin
4 adhesion discovered in this laboratory (42, 56). Circumstantial support for this notion came from studies of the CLCA/integrin
4 adhesion under flow conditions (57). These experiments showed that integrin
4-expressing tumor cells adhere to CLCA-coated dishes only at low shear stresses. However, once adhesion is established, high shear stress levels are unable to break it, suggesting that, for engagement of the CLCA/integrin
4 adhesion, a "slowdown" of tumor cells may be beneficial. We predict that such a slowdown is mediated by the DPPIV/poly-FN adhesion, which is facilitated by the prominence of tumor cell-surface FN polymers that easily engage in binding interactions with endothelial cells. The necessity for a dual tumor cell/endothelial cell adhesion principle in lung metastasis is also supported by our finding that lung-metastatic, integrin
4-expressing tumor cells colonize the lungs of DPPIV/ mice at reduced levels relative to DPPIV+/+ mice.2 Finally, the expression of DPPIV on cells other than lung endothelial cells could extend the importance of our findings beyond metastasis. A principal DPPIV expresser cell is the T lymphocyte (3, 4, 13, 19), in which DPPIV serves the role of a coactivator (19). Although DPPIV enzymatic activity and the collaboration of DPPIV with other molecules such as CD45 and adenosine deaminase have been implicated in this activating role (2024), future studies will be needed to explore whether the ability of T lymphocytes to bind to fibrillar FN via surface-expressed DPPIV, operating independently or in concert with integrins (e.g.
4
1) (5861), is the missing link in the activation mechanism of T lymphocytes. If this is indeed the case, then the DPPIV/poly-FN adhesion could also be involved in T-cell-mediated cytolysis of tumor cells.
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FOOTNOTES |
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To whom correspondence should be addressed. Tel.: 607-253-3343; Fax: 607-253-3708; E-mail: bup1{at}cornell.edu.
1 The abbreviations used are: DPPIV, dipeptidyl peptidase IV (CD26); FN, fibronectin; poly-FN, polymeric fibronectin; mAb, monoclonal antibody; pAb, polyclonal antibody; MBP, maltose-binding protein; HA, hemagglutinin; GST, glutathione S-transferase; EDA, extra domain A; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; BSA, bovine serum albumin.
2 H.-C. Cheng, M. Abdel-Ghany, and B. U. Pauli, unpublished data.
3 Available at www.blocks.fhcrc.org.
4 H.-C. Cheng, M. Abdel-Ghany, and B. U. Pauli, manuscript in preparation.
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ACKNOWLEDGMENTS |
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REFERENCES |
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