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• W2095484461 abstract "Selectin-mediated binding of tumor cells to platelets, leukocytes, and vascular endothelium may regulate their hematogenous spread in the microvasculature. We recently reported that CD44 variant isoforms (CD44v) on LS174T colon carcinoma cells possess selectin binding activity. Here we extended those findings by showing that T84 and Colo205 colon carcinoma cells bind selectins via sialidase-sensitive O-linked glycans presented on CD44v, independent of heparan and chondroitin sulfate. To assess the functional role of CD44v in selectin-mediated binding, we quantified the adhesion to selectins of T84 cell subpopulations sorted based on their CD44 expression levels and stable LS174T cell lines generated using CD44 short hairpin RNA. High versus low CD44-expressing T84 cells tethered more efficiently to P- and L-selectin, but not E-selectin, and rolled more slowly on P- and E-selectin. Knocking down CD44 expression on LS174T cells inhibited binding to P-selectin and increased rolling velocities over P- and L-selectin relative to control-transfected cells, without affecting tethering and rolling on E-selectin, however. Blot rolling analysis revealed the presence of alternative sialylated glycoproteins with molecular masses of ∼170 and ∼130 kDa, which can mediate selectin binding in CD44-knockdown cells. Heparin diminishes the avidity of colon carcinoma cells for P- and L-selectin, which may compromise integrin-mediated firm adhesion to host cells and mitigate metastasis. Our finding that CD44v is a functional P-selectin ligand on colon carcinoma provides a novel perspective on the enhanced metastatic potential associated with tumor CD44v overexpression and the role of selectins in metastasis. Selectin-mediated binding of tumor cells to platelets, leukocytes, and vascular endothelium may regulate their hematogenous spread in the microvasculature. We recently reported that CD44 variant isoforms (CD44v) on LS174T colon carcinoma cells possess selectin binding activity. Here we extended those findings by showing that T84 and Colo205 colon carcinoma cells bind selectins via sialidase-sensitive O-linked glycans presented on CD44v, independent of heparan and chondroitin sulfate. To assess the functional role of CD44v in selectin-mediated binding, we quantified the adhesion to selectins of T84 cell subpopulations sorted based on their CD44 expression levels and stable LS174T cell lines generated using CD44 short hairpin RNA. High versus low CD44-expressing T84 cells tethered more efficiently to P- and L-selectin, but not E-selectin, and rolled more slowly on P- and E-selectin. Knocking down CD44 expression on LS174T cells inhibited binding to P-selectin and increased rolling velocities over P- and L-selectin relative to control-transfected cells, without affecting tethering and rolling on E-selectin, however. Blot rolling analysis revealed the presence of alternative sialylated glycoproteins with molecular masses of ∼170 and ∼130 kDa, which can mediate selectin binding in CD44-knockdown cells. Heparin diminishes the avidity of colon carcinoma cells for P- and L-selectin, which may compromise integrin-mediated firm adhesion to host cells and mitigate metastasis. Our finding that CD44v is a functional P-selectin ligand on colon carcinoma provides a novel perspective on the enhanced metastatic potential associated with tumor CD44v overexpression and the role of selectins in metastasis. Polymorphonuclear (PMN) 2The abbreviations used are: PMN, polymorphonuclear; CD44s, CD44 standard isoform; CD44v, CD44 variant isoforms; CHO, Chinese hamster ovary; D-PBS, Dulbecco's-phosphate-buffered saline with Ca2+/Mg2+; BSA, bovine serum albumin; mAb, monoclonal antibody; PE, phycoerythrin; AP, alkaline phosphatase; HRP, horseradish peroxidase; GAG, glycosaminoglycan; sLex, sialyl Lewis x; MFI, mean fluorescence intensity; shRNA, short hairpin RNA; siRNA, short interfering RNA; benzyl-GalNAc, benzyl-2-acetamido-2-deoxy-α-d-galactopyranoside. leukocyte recruitment to sites of inflammation/infection proceeds in a cascade-like fashion that requires the sequential involvement of the following distinct types of receptors: the selectins (P-, E-, and L-selectin), integrins, and immunoglobulin superfamily members. According to this model, free-flowing PMNs first loosely attach (tether) and roll on the layer of activated endothelial cells via selectin-ligand interactions, then stop, flatten, and squeeze between endothelial cells into the afflicted tissues in an integrin/immunoglobulin superfamily member-dependent manner (1Konstantopoulos K. Kukreti S. McIntire L.V. Adv. Drug Delivery Rev. 1998; 33: 141-164Crossref PubMed Scopus (106) Google Scholar, 2Simon S.I. Green C.E. Annu. Rev. Biomed. Eng. 2005; 7: 151-185Crossref PubMed Scopus (224) Google Scholar). The mechanisms used for PMN trafficking may be appropriated for the dissemination of tumor cells via the bloodstream and lymphatics. In particular, the paradigm of the coordinated action of a selectin-dependent tethering/rolling pathway followed by an integrin-mediated firm adhesion process has been extended to account for maximal binding of colon carcinoma cells to activated endothelium (3Burdick M.M. Konstantopoulos K. Am. J. Physiol. 2004; 287: C539-C547Crossref PubMed Scopus (55) Google Scholar, 4Burdick M.M. McCaffery J.M. Kim Y.S. Bochner B.S. Konstantopoulos K. Am. J. Physiol. 2003; 284: C977-C987Crossref PubMed Scopus (97) Google Scholar, 5Tozeren A. Kleinman H.K. Grant D.S. Morales D. Mercurio A.M. Byers S.W. Int. J. Cancer. 1995; 60: 426-431Crossref PubMed Scopus (118) Google Scholar), platelets (6McCarty O.J. Jadhav S. Burdick M.M. Bell W.R. Konstantopoulos K. Biophys. J. 2002; 83: 836-848Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 7McCarty O.J. Mousa S.A. Bray P.F. Konstantopoulos K. Blood. 2000; 96: 1789-1797Crossref PubMed Google Scholar), and PMNs (8Jadhav S. Bochner B.S. Konstantopoulos K. J. Immunol. 2001; 167: 5986-5993Crossref PubMed Scopus (72) Google Scholar, 9Jadhav S. Konstantopoulos K. Am. J. Physiol. 2002; 283: C1133-C1143Crossref PubMed Scopus (47) Google Scholar) under physiological shear. Several lines of evidence suggest that selectins facilitate cancer metastasis and tumor cell arrest in the microvasculature by mediating specific interactions between selectin-expressing host cells and ligands on tumor cells. The most convincing evidence for the direct role of P- and L-selectin in the metastatic process is the pronounced inhibition of metastasis in P- and/or L-selectin-deficient mice (P-sel-null, L-sel-null, and PL-sel-double null) compared with wild-type controls in a colon carcinoma cell model (10Borsig L. Wong R. Feramisco J. Nadeau D.R. Varki N.M. Varki A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3352-3357Crossref PubMed Scopus (602) Google Scholar, 11Borsig L. Wong R. Hynes R.O. Varki N.M. Varki A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 2193-2198Crossref PubMed Scopus (356) Google Scholar). Along these lines, enzymatic removal of O-linked mucin-like glycoproteins from colon carcinoma cells resulted in marked reduction of experimental metastasis in a wild-type mouse model (10Borsig L. Wong R. Feramisco J. Nadeau D.R. Varki N.M. Varki A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3352-3357Crossref PubMed Scopus (602) Google Scholar). Similarly, endothelial E-selectin has been shown to support metastatic spread in vivo (12Izumi Y. Taniuchi Y. Tsuji T. Smith C.W. Nakamori S. Fidler I.J. Irimura T. Exp. Cell Res. 1995; 216: 215-221Crossref PubMed Scopus (123) Google Scholar, 13Brodt P. Fallavollita L. Bresalier R.S. Meterissian S. Norton C.R. Wolitzky B.A. Int. J. Cancer. 1997; 71: 612-619Crossref PubMed Scopus (195) Google Scholar). Since the selectins are known to recognize HECA-452-reactive sialofucosylated oligosaccharides, such as sialyl Lewis x (sLex), and overexpression of these moieties on tumor cells correlates with poor prognosis and tumor progression (14Kannagi R. Glycoconj. J. 1997; 14: 577-584Crossref PubMed Scopus (301) Google Scholar, 15Kim Y.J. Varki A. Glycoconj. J. 1997; 14: 569-576Crossref PubMed Scopus (494) Google Scholar), it appears likely that selectin-mediated adhesion to sialofucosylated target molecules on tumor cells is an important determinant for metastatic spread. To date, the selectin ligands on colon carcinoma cells have yet to be identified and characterized other than by general classifications (i.e. sialofucosylated mucin-like glycoproteins). The binding affinity of selectins for isolated monovalent sLex and its isomer sLea is very low. Consequently, neither expression of the sLex nor the sLea groups per se correlates with the properties of endogenous selectin ligands on cellular targets. As has been argued appropriately in the literature (16Varki A. J. Clin. Investig. 1997; 100: S31-35PubMed Google Scholar), distinctions must be drawn between structures than can bind to selectins under certain conditions in vitro and structures that actually do interact with selectins in vivo. To this end, a “functional” selectin ligand should fulfill certain criteria (16Varki A. J. Clin. Investig. 1997; 100: S31-35PubMed Google Scholar) as follows: it should be expressed in the right place at the right time; the ligand should bind with some selectivity and relatively high affinity; and removal or absence of the ligand should prevent cell adhesive interactions. We recently reported that O-linked sialofucosylated glycans on CD44 variant isoforms (CD44v) on LS174T colon carcinoma cells possess selectin binding activity (17Hanley W.D. Burdick M.M. Konstantopoulos K. Sackstein R. Cancer Res. 2005; 65: 5812-5817Crossref PubMed Scopus (103) Google Scholar, 18Hanley W.D. Napier S.L. Burdick M.M. Schnaar R.L. Sackstein R. Konstantopoulos K. FASEB J. 2006; 20: 337-339Crossref PubMed Scopus (105) Google Scholar). However, these studies did not reveal a functional role for LS174T CD44v in selectin-mediated adhesion. Moreover, it is not clear whether the selectin binding activity of CD44v is shared by other metastatic colon carcinoma cells. Here we employ two distinct metastatic colon carcinoma cell lines, LS174T and T84, expressing similar surface levels of CD44, and two complementary strategies to modulate CD44 expression to assess whether CD44v is a functional selectin ligand on colon carcinoma cells under physiological shear conditions. By perfusing subsets of T84 cells, sorted based on their CD44 expression levels, over purified selectin substrates, we show that high versus low CD44-expressing cells (mean fluorescence intensity (MFI), 1731 versus 364) bind more efficiently to P- and L- but not E-selectin and roll more slowly on P- and E-selectin. These data highlight the role of avidity in CD44v binding to P- or L-selectin and implicate CD44v as an auxiliary E-selectin ligand on colon carcinoma, which is engaged in the stabilization of tumor cell-endothelial cell adhesive interactions against fluid shear. Furthermore, stable LS174T cell lines generated using CD44 short hairpin (sh)-RNA display markedly reduced (>95%) CD44 surface expression and tether to P- but not L- or E-selectin with an ∼55% efficiency relative to untreated or control-transfected cells. Interestingly, this intervention did not alter the rolling velocity of CD44-knockdown LS174T cells relative to control cells on E-selectin. The lack of significant inhibition of cell tethering to L-selectin and the absence of modulation of the rolling velocity on E-selectin are attributed to the presence of sialofucosylated glycans on alternative selectin ligands with apparent molecular masses of ∼130 and ∼170 kDa in CD44-knockdown cells. Taken altogether, these data provide functional evidence that CD44v is a major functional P- but not L- or E-selectin ligand on colon carcinoma cells. Our findings offer a unifying perspective on the apparent enhanced metastatic potential associated with tumor cell CD44v overexpression and the critical role of selectins in metastasis. Adhesion Molecules, Antibodies, and Reagents—The chimeric forms of L-selectin-IgG Fc (L-selectin), P-selectin-IgG Fc (P-selectin), and E-selectin-IgG Fc (E-selectin), consisting of the lectin, epidermal growth factor, and consensus repeat domains for human L-, P-, or E-selectin linked to each arm of human IgG1, were generous gifts of Wyeth External Research (Cambridge, MA) (19Somers W.S. Tang J. Shaw G.D. Camphausen R.T. Cell. 2000; 103: 467-479Abstract Full Text Full Text PDF PubMed Scopus (636) Google Scholar). Anti-CD44 (2C5) monoclonal antibody (mAb) was from R&D Systems (Minneapolis, MN). Alkaline phosphatase (AP)- and horseradish peroxidase (HRP)-conjugated anti-mouse IgG and AP-conjugated anti-rat IgM were from Southern Biotech Associates (Birmingham, AL). All other unlabeled and phycoerythrin (PE)- or fluorescein isothiocyanate-conjugated antibodies were from BD Biosciences. All other reagents were from Sigma unless otherwise stated. Cell Culture—The human colorectal carcinoma cell lines T84, Colo205, and LS174T were obtained from the American Type Culture Collection (Manassas, VA) and cultured in the recommended media. Prior to cell lysis, colon carcinoma cells were detached from culture flasks using Enzyme Free Cell Dissociation Media (15 min at 37 °C; Chemicon, Phillipsburg, NJ). For flow cytometric/sorting and flow-based adhesion assays, T84 and LS174T cells were harvested by mild trypsinization (0.25% trypsin/EDTA for 5 min at 37 °C) and subsequently incubated (107 cells/ml) at 37 °C for 2 h to allow regeneration of surface glycoproteins (7McCarty O.J. Mousa S.A. Bray P.F. Konstantopoulos K. Blood. 2000; 96: 1789-1797Crossref PubMed Google Scholar, 8Jadhav S. Bochner B.S. Konstantopoulos K. J. Immunol. 2001; 167: 5986-5993Crossref PubMed Scopus (72) Google Scholar, 20Mannori G. Crottet P. Cecconi O. Hanasaki K. Aruffo A. Nelson R.M. Varki A. Bevilacqua M.P. Cancer Res. 1995; 55: 4425-4431PubMed Google Scholar). Colo205 cells were detached by nonenzymatic means, as described above, and used immediately. CHO cells, stably transfected with cDNA encoding full-length E- (CHO-E) or P-selectin (CHO-P), were kindly donated by Affymax (Palo Alto, CA) and processed as described previously (18Hanley W.D. Napier S.L. Burdick M.M. Schnaar R.L. Sackstein R. Konstantopoulos K. FASEB J. 2006; 20: 337-339Crossref PubMed Scopus (105) Google Scholar). Cell lines were routinely checked and confirmed to be negative for mycoplasma infection (8Jadhav S. Bochner B.S. Konstantopoulos K. J. Immunol. 2001; 167: 5986-5993Crossref PubMed Scopus (72) Google Scholar). Colon Carcinoma Cell Lysis and Immunoprecipitation of CD44—Whole cell lysate was prepared by membrane disruption using 2% Nonidet P-40 followed by differential centrifugation (18Hanley W.D. Napier S.L. Burdick M.M. Schnaar R.L. Sackstein R. Konstantopoulos K. FASEB J. 2006; 20: 337-339Crossref PubMed Scopus (105) Google Scholar, 21Sackstein R. Dimitroff C.J. Blood. 2000; 96: 2765-2774Crossref PubMed Google Scholar, 22Dimitroff C.J. Lee J.Y. Fuhlbrigge R.C. Sackstein R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13841-13846Crossref PubMed Scopus (111) Google Scholar). CD44 was immunoprecipitated from colon carcinoma cell lysate with an anti-CD44 mAb, 2C5, using recombinant protein G-agarose beads (Invitrogen) (18Hanley W.D. Napier S.L. Burdick M.M. Schnaar R.L. Sackstein R. Konstantopoulos K. FASEB J. 2006; 20: 337-339Crossref PubMed Scopus (105) Google Scholar). SDS-PAGE and Western Blotting—Whole cell lysate or immunopurified CD44 was diluted with reducing sample buffer and separated using 4–20% SDS-polyacrylamide gels (Bio-Rad) (17Hanley W.D. Burdick M.M. Konstantopoulos K. Sackstein R. Cancer Res. 2005; 65: 5812-5817Crossref PubMed Scopus (103) Google Scholar, 18Hanley W.D. Napier S.L. Burdick M.M. Schnaar R.L. Sackstein R. Konstantopoulos K. FASEB J. 2006; 20: 337-339Crossref PubMed Scopus (105) Google Scholar). Resolved proteins were transferred to Sequi-blot or Immun-blot polyvinylidene difluoride or Sequi-blot nitrocellulose membrane (Bio-Rad) and blocked with StartingBlock (Pierce) for 15 min. Immunoblots were stained with HECA-452 or anti-CD44 (2C5) mAbs and rinsed with Tris-buffered saline, 0.1% Tween 20. In all cases, duplicate immunoblots were stained in parallel with irrelevant isotype control primary antibodies to assess nonspecific binding to protein bands. Subsequently, blots were incubated with appropriate AP- or HRP-conjugated secondary antibodies. Western Blue AP substrate (Promega, Madison, WI) and SuperSignal West Pico chemiluminescent substrate (Pierce) were used to develop the AP- and HRP-conjugated antibody-stained immunoblots, respectively. Blot Rolling Assay—Blots of immunopurified CD44 from T84 or Colo205 whole cell lysate or whole cell lysate from untreated, mammalian scramble, or CD44-knockdown LS174T cells were stained with anti-CD44 (2C5) or HECA-452 mAbs and rendered translucent by immersion in 90% D-PBS, 10% glycerol (23Fuhlbrigge R.C. King S.L. Dimitroff C.J. Kupper T.S. Sackstein R. J. Immunol. 2002; 168: 5645-5651Crossref PubMed Scopus (66) Google Scholar). The blots were placed under a parallel plate flow chamber, and human peripheral blood lymphocytes (18Hanley W.D. Napier S.L. Burdick M.M. Schnaar R.L. Sackstein R. Konstantopoulos K. FASEB J. 2006; 20: 337-339Crossref PubMed Scopus (105) Google Scholar) or CHO transfectants, resuspended at 5 × 106 cells/ml in 90% D-PBS, 10% glycerol, were perfused at the shear stress of 0.5 dyne/cm2. Molecular weight markers were used as guides to aid placement of the flow chamber over stained bands of interest. The number of interacting cells per lane was averaged over ×10 fields of view (0.55 mm2 each). Nonspecific adhesion was assessed by perfusion by adding 10 mm EDTA in the flow medium. Preparation of CD44-coated Microspheres—Immunoprecipitated CD44 from control and metabolically inhibited T84 whole cell lysate was diluted to desired concentrations with binding buffer (0.2 m carbonate/bicarbonate buffer, pH 9.2), and incubated with 10-μm polystyrene microspheres (2.5 × 107 microspheres/ml; Polysciences Inc., Warrington, PA) overnight at 4 °C with constant rotation (18Hanley W.D. Napier S.L. Burdick M.M. Schnaar R.L. Sackstein R. Konstantopoulos K. FASEB J. 2006; 20: 337-339Crossref PubMed Scopus (105) Google Scholar). Microspheres were washed twice with D-PBS and subsequently blocked with D-PBS, 1% BSA for 30 min at room temperature. Microspheres were resuspended (2 × 106 microspheres/ml) in D-PBS, 0.1% BSA for use in flow cytometric and flow chamber assays. Site densities of CD44-coated microspheres were determined by flow cytometry (18Hanley W.D. Napier S.L. Burdick M.M. Schnaar R.L. Sackstein R. Konstantopoulos K. FASEB J. 2006; 20: 337-339Crossref PubMed Scopus (105) Google Scholar). Enzymatic Treatments—To remove terminal sialic acid residues, T84 CD44-coated microspheres were incubated with 0.1 unit/ml Vibrio cholerae sialidase (Roche Applied Science) for 90 min at 37 °C (18Hanley W.D. Napier S.L. Burdick M.M. Schnaar R.L. Sackstein R. Konstantopoulos K. FASEB J. 2006; 20: 337-339Crossref PubMed Scopus (105) Google Scholar). In select experiments, CD44-coated microsphere suspensions were incubated for 6 h at 37°C with 3.6 units/ml Flavobacterium heparinum heparitinase to specifically digest heparan sulfate glycosaminoglycans (GAGs) (24Nader H.B. Dietrich C.P. Buonassisi V. Colburn P. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 3565-3569Crossref PubMed Scopus (146) Google Scholar, 25Wei M. Tai G. Gao Y. Li N. Huang B. Zhou Y. Hao S. Zeng X. J. Biol. Chem. 2004; 279: 29202-29210Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). To cleave chondroitin sulfate GAGs from CD44 isoforms, CD44-coated microsphere suspensions were incubated 2 h at 37 °C with 1 unit/ml Proteus vulgaris chondroitinase ABC (4Burdick M.M. McCaffery J.M. Kim Y.S. Bochner B.S. Konstantopoulos K. Am. J. Physiol. 2003; 284: C977-C987Crossref PubMed Scopus (97) Google Scholar, 26Aruffo A. Stamenkovic I. Melnick M. Underhill C.B. Seed B. Cell. 1990; 61: 1303-1313Abstract Full Text PDF PubMed Scopus (2169) Google Scholar). Site densities of CD44 adsorbed onto microspheres following enzymatic treatments were determined by flow cytometry and confirmed to be equivalent to untreated controls before use in adhesion assays. Inhibitor Treatments—Prior to metabolic inhibitor studies, T84 cell suspensions (107 cells/ml) were pretreated with 0.1 unit/ml V. cholerae sialidase for 60 min at 37 °C to remove terminal sialic acid residues and to ensure de novo synthesis of newly generated HECA-452 reactive carbohydrate structures (17Hanley W.D. Burdick M.M. Konstantopoulos K. Sackstein R. Cancer Res. 2005; 65: 5812-5817Crossref PubMed Scopus (103) Google Scholar). Complete removal of sialic acid was confirmed via flow cytometry using the mAb HECA-452 that recognizes sialic acid-bearing epitopes. Subsequently, T84 cells were cultured for 48 h at 37 °C in medium containing either 2 mm benzyl-2-acetamido-2-deoxy-α-d-galactopyranoside (benzyl-GalNAc) to inhibit O-linked glycosylation (18Hanley W.D. Napier S.L. Burdick M.M. Schnaar R.L. Sackstein R. Konstantopoulos K. FASEB J. 2006; 20: 337-339Crossref PubMed Scopus (105) Google Scholar) or 10 μg/ml Castanospermum australe castanospermine to inhibit N-linked processing of glycoproteins (27Pan Y.T. Hori H. Saul R. Sanford B.A. Molyneux R.J. Elbein A.D. Biochemistry. 1983; 22: 3975-3984Crossref PubMed Scopus (213) Google Scholar); D-PBS diluent was used for control untreated cells. Flow Cytometry—CD29, CD44, and HECA-452 expression levels on LS174T and T84 cells as well as CD44 and HECA-452 site densities on microspheres were quantified by single-color immunofluorescence and flow cytometry (FACSCalibur; BD Biosciences) using PE-conjugated anti-CD29 (MAR4), anti-CD44 (515), or HECA-452 antibodies. Background levels were determined by incubating cell or microsphere suspensions with properly matched PE-conjugated isotype control antibodies (18Hanley W.D. Napier S.L. Burdick M.M. Schnaar R.L. Sackstein R. Konstantopoulos K. FASEB J. 2006; 20: 337-339Crossref PubMed Scopus (105) Google Scholar). Cell Sorting by Flow Cytometry—T84 colon carcinoma cells (107 cells/ml) were incubated with an anti-CD44 mAb (fluorescein isothiocyanate-conjugated G44-26 or PE-conjugated 515) or a fluorescently labeled isotype control antibody for 1 h at room temperature in the dark. Using a flow cytometer/sorter (FACSAria; BD Biosciences), the highest and lowest (above isotype control staining) 10% of CD44-expressing T84 cells were collected into separate growth medium-filled centrifuge tubes. Subsequently, cell suspensions were centrifuged, resuspended to a concentration of 5 × 105/ml in D-PBS, 0.1% BSA, and immediately used in flow-based adhesion assays. Preparation of CD44 siRNA Oligonucleotides—Short interfering (si)RNA oligonucleotides (19 nucleotides) targeting exons 1–5 and 15–20 of CD44 were generated using the WI siRNA design program (Whitehead Institute, Massachusetts Institute of Technology). Identified target sequences were subjected to BLAST search of the human genome and subsequently filtered to remove sequences that were not specific to CD44. The siRNA sequences were used to construct 60-mer short hairpin (sh)RNA oligonucleotides, which were then synthesized (Operon, Inc., Huntsville, AL), and ligated into the pSUPER.neo.gfp expression vector (Oligoengine, Inc, Seattle, WA) under the control of the H1 promoter. The following oligonucleotide was used (underlined, sense and antisense sequences; boldface, restriction enzyme sites; lightface italics, polIII termination signals; boldface italics, loop with linker): (5′-GATCCCCTGTGCTACTGATTGTTTCATTCAAGAGATGAAACAATCAGTAGCACATTTTTC-3′). The ligated product was transformed into competent DH5α Escherichia coli cells, amplified in the presence of ampicillin, and the plasmid was purified using the EndoFree maxi kit (Qiagen, Valencia, CA). Sequence insertion was verified by restriction digestion and confirmed by direct sequencing. A tested mammalian scramble sequence (Oligoengine, Inc) was used as a negative control in all siRNA experiments. Generation of Stable CD44-Knockdown Colon Carcinoma Cell Lines—8 × 106 LS174T cells were plated in 100-mm dishes and grown overnight reaching an ∼50% confluency. The cells were then transfected with 32 μg of pSUPER.neo.gfp.CD44S using Lipofectamine 2000 for 24 h. Upon reaching confluency, transfected LS174T cells were passed and 5 × 106 cells seeded per Petri dish in growth medium in triplicate. After 24 h, the medium was replaced by a fresh aliquot containing 500 μg/ml neomycin. Cells were then grown continually without passaging for 15 days, replenishing the neomycin-containing medium every 2–3 days. Single cell colonies were isolated and cultured using standard techniques. Flow-based Adhesion Assays—To simulate the physiological shear environment of the vasculature, colon carcinoma cells or T84 CD44-coated microspheres suspended in D-PBS, 0.1% BSA were perfused over immobilized E-, P- or L-selectin-coated dishes at prescribed wall shear stresses using a parallel plate flow chamber (250 μm channel depth, 5.0 mm channel width) (4Burdick M.M. McCaffery J.M. Kim Y.S. Bochner B.S. Konstantopoulos K. Am. J. Physiol. 2003; 284: C977-C987Crossref PubMed Scopus (97) Google Scholar, 18Hanley W.D. Napier S.L. Burdick M.M. Schnaar R.L. Sackstein R. Konstantopoulos K. FASEB J. 2006; 20: 337-339Crossref PubMed Scopus (105) Google Scholar). The extent of adhesion was quantified by perfusing cells/microspheres at either 2 × 106/ml and averaging the total number of cells/beads interacting during 15-s intervals over six ×10 fields of view (0.55 mm2 each) or 1 × 106/ml and enumerating the total number of tethering events in a single ×10 field of view during a 5-min period. Average rolling velocities were computed as the distance traveled by the centroid of the translating cell/microsphere divided by the time interval at the given wall shear stress (4Burdick M.M. McCaffery J.M. Kim Y.S. Bochner B.S. Konstantopoulos K. Am. J. Physiol. 2003; 284: C977-C987Crossref PubMed Scopus (97) Google Scholar, 7McCarty O.J. Mousa S.A. Bray P.F. Konstantopoulos K. Blood. 2000; 96: 1789-1797Crossref PubMed Google Scholar, 18Hanley W.D. Napier S.L. Burdick M.M. Schnaar R.L. Sackstein R. Konstantopoulos K. FASEB J. 2006; 20: 337-339Crossref PubMed Scopus (105) Google Scholar). In select experiments, LS174T cells were preincubated for 10 min at room temperature with either 5 units/ml heparin or diluent (D-PBS, 0.1% BSA) and used in perfusion assays. Statistical Analysis—Data are expressed as the mean ± S.E. for at least three independent experiments. Statistical significance of differences between means was determined by analysis of variance. If means were shown to be significantly different (p < 0.05), multiple comparisons were performed by the Tukey test. The Selectin Binding Determinants of CD44v on Colon Carcinoma Cells Are Displayed on Sialofucosylated O-Linked Glycans and Are Independent of GAGs—We recently reported that the colon carcinoma cell line LS174T expresses CD44 variant isoforms that display selectin binding activity, implicating these molecules as a link between the up-regulation of CD44v expression and increased metastatic potential (17Hanley W.D. Burdick M.M. Konstantopoulos K. Sackstein R. Cancer Res. 2005; 65: 5812-5817Crossref PubMed Scopus (103) Google Scholar, 18Hanley W.D. Napier S.L. Burdick M.M. Schnaar R.L. Sackstein R. Konstantopoulos K. FASEB J. 2006; 20: 337-339Crossref PubMed Scopus (105) Google Scholar). We sought to quantify the expression of these isoforms on other colon carcinoma cell lines, such as T84 and Colo205, to begin to reveal the breadth of its distribution as a selectin ligand as well as to further characterize its selectin binding characteristics. Western blot analysis of CD44 immunopurified from whole cell lysates of 5 × 106 T84 or Colo205 cells, using the anti-CD44 mAb 2C5, revealed the presence of a relatively weak level of CD44s at ∼100 kDa and a more prominent CD44 signal at ∼150 kDa, which corresponds to CD44v (Fig. 1A, T84, lane 1; Colo205, not shown). Fig. 1A, lane 2, stained with HECA-452, identified the presence of sialofucosylated epitopes, such as sLex, solely on the variant isoforms of CD44 (Fig. 1A, T84, lane 2; Colo205, not shown). A blot rolling assay was next employed to assess the ability of immunopurified CD44 to mediate selectin-dependent adhesion under physiologically relevant levels of shear stress. To this end, L-selectin-expressing human peripheral blood lymphocytes or P- or E-selectin-transfected CHO cells, perfused over SDS-PAGE-resolved immunopurified CD44 protein bands, tethered predominantly over the ∼150-kDa region that corresponds to sialofucosylated CD44v, whereas minimal binding was detected at the ∼100-kDa band of CD44s (T84, Fig. 1B; Colo205; data not shown). Taken together, these data suggest that CD44v, but not CD44s, on T84 and Colo205 colon carcinoma cells is capable of interacting efficiently with selectins under physiological flow conditions. We next used a cell-free flow-based adhesion assay to evaluate the adhesion capabilities of microspheres coated with CD44 immunopurified from T84 cells over immobilized E-, P-, or L-selectin. This technique allows quantitative comparisons of CD44-mediated adhesion to purified selectin substrates at identical concentrations under physiological flow conditions. Microspheres, coated with T84 CD44, which displays strong HECA-452 reactivity (supplemental Table 1S), tethered and rolled over all three selectins, albeit with varying efficiencies which correlate with those of intact T84 cells, with E-selectin mediating the slowest rolling and L-selectin the fastest rolling (supplemental Fig. 1S). To characterize the biochemical nature of CD44" @default.
• W2095484461 created "2016-06-24" @default.
• W2095484461 creator A5049898092 @default.
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• W2095484461 date "2007-02-01" @default.
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