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M Bissonnette,
S Khare,
F C von Lintig,
R K Wali,
L Nguyen,
Y Zhang,
J Hart,
S Skarosi, N Varki,
G R Boss,
T A Brasitus
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ABSTRACT: Azoxymethane (AOM)-induced colonic carcinogenesis involves a number of mutations, including those in the K-ras gene and CTNNB1, that codes for beta-catenin. Prior in vitro studies have also demonstrated that wild type p21(K-ras) can be activated by epigenetic events. We identified 15 K-ras mutations in 14 of 84 AOM-induced colonic tumors by three independent methods. By single strand conformational polymorphism, we also observed mutations in 22 of 68 tumors in exon 3 of CTNNB1. A highly sensitive method was then used to measure p21ras activation levels. All tumors assayed possessing K-ras mutations had significantly higher p21ras activation levels (8.8 +/- 1.5%; n = 13) compared with that of control colon (3.7 +/- 0.4; n = 6; P < 0.05) or tumors without such mutations (4.2 +/- 0.4%; n = 70; P < 0.05). Among tumors with wild-type K-ras, there was a subset of tumors (18 of 70) that had significantly higher p21ras activation levels (8.0 +/- 0.9%; n = 18) compared with control colons. In three of four tumors examined with activated wild-type p21ras, we observed increased c-erbB-2 receptor expression and decreased Ras-GAP expression. In contrast, only one of eight tumors examined with wild-type ras and nonactivated p21ras demonstrated these alterations. Mitogen-activated protein kinase (MAPK) activation and cyclooxygenase-2 (COX-2) expression were increased in tumors with mutated or activated wild-type p21ras, compared with their nonactivated counterparts. Although beta-catenin mutations did not alter COX-2 expression or MAPK activity, mutations in either K-ras or beta-catenin significantly increased cyclin D1 expression. In contrast, in tumors with wild-type but activated p21-ras, cyclin D1 expression was not enhanced. Thus, the spectrum of changes in MAPK, COX-2, and cyclin D1 is distinct among tumors with ras or beta-catenin mutations or nonmutational activation of p21ras.
Cancer Research 08/2000; 60(16):4602-9. · 7.86 Impact Factor
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ABSTRACT: The common sialic acids of mammalian cells are N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc). Humans are an exception, because of a mutation in CMP-sialic acid hydroxylase, which occurred after our common ancestor with great apes. We asked if the resulting loss of Neu5Gc and increase in Neu5Ac in humans alters the biology of the siglecs, which are Ig superfamily members that recognize sialic acids. Human siglec-1 (sialoadhesin) strongly prefers Neu5Ac over Neu5Gc. Thus, humans have a higher density of siglec-1 ligands than great apes. Siglec-1-positive macrophages in humans are found primarily in the perifollicular zone, whereas in chimpanzees they also occur in the marginal zone and surrounding the periarteriolar lymphocyte sheaths. Although only a subset of chimpanzee macrophages express siglec-1, most human macrophages are positive. A known evolutionary difference is the strong preference of mouse siglec-2 (CD22) for Neu5Gc, contrasting with human siglec-2, which binds Neu5Ac equally well. To ask when the preference for Neu5Gc was adjusted in the human lineage, we cloned the first three extracellular domains of siglec-2 from all of the great apes and examined their preference. In fact, siglec-2 had evolved a higher degree of recognition flexibility before Neu5Gc was lost in humans. Human siglec-3 (CD33) and siglec-6 (obesity-binding protein 1) also recognize both Neu5Ac and Neu5Gc, and siglec-5 may have some preference for Neu5Gc. Others showed that siglec-4a (myelin-associated glycoprotein) prefers Neu5Ac over Neu5Gc. Thus, the human loss of Neu5Gc may alter biological processes involving siglec-1, and possibly, siglec-4a or -5.
Journal of Biological Chemistry 04/2000; 275(12):8633-40. · 4.77 Impact Factor
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ABSTRACT: The intestinal epithelium produces and responds to cytokines and lipid mediators that play a key role in the induction and regulation of mucosal inflammation. The lipid mediator platelet-activating factor (PAF) can be produced and degraded by the human intestinal epithelium and is known to mediate a range of proinflammatory and other biological effects in the intestinal mucosa. In the studies herein, we assessed whether or not human intestinal epithelial cells express cell surface or intracellular PAF receptors (PAF-R), whether expression of these receptors can be regulated, and whether human intestinal epithelial cells respond to PAF. Several human colon epithelial cell lines (HT-29, Caco-2, T84, HCT-8, HCA-7, I407, and LS-174T) were shown by RT-PCR to constitutively express mRNA for PAF-R. In addition, PAF-R expression was demonstrated by immunoblot analysis and PAF-R was shown to be constitutively expressed on the cell surface of several of these cell lines, as assessed by flow cytometry. PAF-R expression by human colon epithelial cells was upregulated by stimulation with retinoic acid but not by stimulation with PAF, proinflammatory agonists (tumor necrosis factor-alpha, interleukin-1, interferon-gamma), or transforming growth factor-alpha. PAF-R on intestinal epithelial cells were functional, as PAF stimulation of the cells increased tyrosine phosphorylation of several cellular proteins, including proteins of 75 and 125 kDa, and this response was blocked by a PAF-R antagonist. Consistent with the findings using cell lines, PAF-R were also constitutively expressed by normal human colon and small intestinal epithelium in vivo, as shown by immunohistology. The constitutive and regulated expression of functional PAF-R by human intestinal epithelium suggests PAF produced by the intestinal epithelial cells or cells underlying the epithelium has autocrine or paracrine effects on intestinal epithelial cells.
The American journal of physiology 11/1999; 277(4 Pt 1):G810-8.
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ABSTRACT: Human ATPase (hASNA-I) is a novel human gene recently cloned on the basis of homology to the arsA gene of bacteria. Its protein product is an ATPase that is free in the cytoplasm and bound in the perinuclear area and nucleolus in human cells. We prepared the hASNA-I-specific 5G8 monoclonal antibody and used it to investigate the expression of hASNA-I in normal human tissues and breast cancers. hASNA-I was detected immunohistochemically only in the epithelial cells of the liver, kidney, and stomach wall, in the adrenal medulla, in the islet cells of the pancreas, in the red pulp of the spleen, and in cardiac and skeletal muscle. No staining was observed in the uterus, testis, lung, thyroid, cerebellum, and large intestine. Although no staining was also observed in normal breast tissue, all four cases of breast fibroadenomas and all 15 cases of either primary or metastatic breast carcinoma demonstrated increased staining. No embryological or functional common denominator is readily apparent. However, the increased expression in malignant breast cells is of particular interest with respect to the use of this antibody for screening of cytological specimens.
Journal of Histochemistry and Cytochemistry 12/1998; 46(11):1243-8. · 2.72 Impact Factor
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B J Hicke,
S R Watson,
A Koenig,
C K Lynott,
R F Bargatze,
Y F Chang,
S Ringquist,
L Moon-McDermott,
S Jennings,
T Fitzwater,
H L Han, N Varki,
I Albinana,
M C Willis,
A Varki,
D Parma
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ABSTRACT: Selectins participate in the initial events leading to leukocyte extravasation from the blood into tissues. Thus the selectins have generated much interest as targets for antiinflammatory agents. Therapeutic molecules based on the monomeric carbohydrate ligand sialyl Lewis X (SLe(X)) have low affinities and are not specific for a given selectin. Using SELEX (Systematic Evolution of Ligands by EXponential Enrichment) technology, we have generated aptamers specific for L-selectin that require divalent cations for binding and have low nanomolar affinity. In vitro, the deoxyoligonucleotides inhibit L-selectin binding to immobilized SLe(X) in static assays and inhibit L-selectin-mediated rolling of human lymphocytes and neutrophils on cytokine-activated endothelial cells in flow-based assays. These aptamers also block L-selectin-dependent lymphocyte trafficking in vivo, indicating their potential utility as therapeutics.
Journal of Clinical Investigation 01/1997; 98(12):2688-92. · 15.39 Impact Factor
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ABSTRACT: The selectins are calcium-dependent C-type lectins that recognize complex anionic carbohydrate ligands, initiating many cell-cell interactions in the vascular system. Selectin blockade shows therapeutic promise in a variety of inflammatory and postischemic pathologies. However, the available oligosaccharide ligand mimetics have low affinities and show cross-reaction among the three selectins, precluding efficient and specific blockade. The SELEX (systematic evolution of ligands by exponential enrichment) process uses combinatorial chemistry and in vitro selection to yield high affinity oligonucleotides with unexpected binding specificities. Nuclease-stabilized randomized oligonucleotides subjected to SELEX against recombinant L-selectin yielded calcium-dependent antagonists with approximately 10(5) higher affinity than the conventional oligosaccharide ligand sialyl LewisX. Most of the isolated ligands shared a common consensus sequence. Unlike sialyl LewisX, these antagonists show little binding to E- or P-selectin. Moreover, they show calcium-dependent binding to native L-selectin on peripheral blood lymphocytes and block L-selectin-dependent interactions with the natural ligands on high endothelial venules.
Proceedings of the National Academy of Sciences 07/1996; 93(12):5883-7. · 9.68 Impact Factor
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ABSTRACT: Patients with cancer experience a much higher than expected incidence of thromboembolic disorders, commonly referred as Trousseau syndrome. Although this association has been well documented, the etiology of the hypercoagulable state is not known. The expression on tumor cells of tissue factor (TF), a membrane-bound lipoprotein that functions as a cofactor to factor VIIa in the initiation of the extrinsic pathway of blood coagulation, has been postulated as a possible mechanism. Whereas the distribution of TF in normal tissues is known, no large survey of TF expression in malignant tissues has been reported. In this study a polyclonal, monospecific rabbit anti-human TF IgG was used for immunohistochemical localization of TF antigen in 85 different tumor specimens. In general, cell types which normally express TF continued to do so after malignant transformation (41 of 60 epithelial tumor specimens were positive for TF). Tumors of nonepithelial origin frequently lacked TF, with only 3 of 19 specimens containing evidence of TF antigen. In addition five of six benign tumors did not express TF. Many tumor types commonly associated with Trousseau syndrome, for example lung, pancreatic, breast, colon and gastric carcinomas, stained positively for TF. Based on this survey, it appears that TF expression by tumors may be an important factor in the pathogenesis of a hypercoagulable state in some patients with cancer.
Cancer 10/1992; 70(5):1194-201. · 4.77 Impact Factor
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ABSTRACT: Chromogranin A (CgA) is a 48 kDa acidic protein in neuroendocrine secretory vesicles whose primary structure is now known. We used synthetic peptides, synthetic peptide antisera, intact molecule antisera, chymotryptic peptide mapping, microsequencing, immunoblotting, and immunoprecipitation to probe the location of immunodominant domains within the CgA molecule. Polyclonal anti mid-molecule, anti N-terminal and anti C-terminal antibodies specifically visualized CgA (both bovine and human) in one and two dimensional immunoblots of adrenal chromaffin vesicles, and the stain CgA fragments further suggested bidirectional (both N- and C-terminal) cleavage or processing of CgA. Anti intact CgA immunoblotting of HPLC-separated peptides from chymotrypsin-digested bovine CgA revealed several strongly immunoreactive internal peptides, two of which were positioned by N-terminal amino acid sequencing: CgA91ff. and CgA197ff.. A single synthetic peptide (CgA79-113) was recognized by three antibodies developed against the intact CgA molecule: two polyclonal rabbit antisera as well as a monoclonal mouse antibody. Not all antigenicity algorithm-predicted domains were immunogenic, suggesting that some of these predicted domains may not be accessible. Polyclonal anti mid-molecule, anti N- and anti C-terminal synthetic peptide antisera specifically immunoprecipitated 125I-labeled bovine CgA from aqueous solution; mid-molecule antisera precipitated substantially greater amounts than terminal antisera. The immunoprecipitation results suggested exposed terminal as well as interior hydrophilic epitopes in the molecule in its intact, native conformation. 125I-human CgA was best precipitated by anti N-terminal antisera, consistent with greatest interspecies sequence conservation at the N-terminus of CgA. The terminal antisera reacted immunohistochemically in a granular pattern with adrenal medullary chromaffin cells (but not adrenal cortical cells) and pancreatic islet cells (but not pancreatic exocrine acini). Thus, synthetic and chymotryptic peptides yielded novel and specific insights into the structure, conformation, vesicular processing and interior immunodominant domains of CgA.
Neuropeptides 03/1992; 21(2):105-18. · 1.55 Impact Factor
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ABSTRACT: Tissue factor, the cofactor for factor VIIa-catalyzed activation of factors IX and X, plays an important role in the initiation of hemostasis. However, the distribution of tissue factor in the body has not been defined until recently. In the present study frozen sections of non-malignant human tissues were immunostained using polyclonal, monospecific rabbit anti-human tissue factor antibodies. Specificity of the anti-tissue factor antibody was established by Western blotting. Sensitivity of the immunostaining technique for tissue factor antigen was confirmed by correlating staining of non-perturbed and perturbed cultured human umbilical vein endothelial cells with their surface membrane tissue factor coagulant activity. Brain, lung and placenta, all known to possess large amounts of tissue factor procoagulant activity, stained strongly for tissue factor, as did peripheral nerves and autonomic ganglia. Epithelium of skin, mucosa, and glomeruli also stained; however, epithelium lining excretory ducts failed to stain. Skeletal muscle did not stain, but cardiac muscle stained faintly. Smooth muscle also did not stain except for the muscularis mucosa of the esophagus, which stained brightly. Fibroblasts varied in stainability; those found in the adventitia of vessels stained strongly. The endothelium, tunica intima and tunica media of blood vessels consistently failed to stain. The distribution of tissue factor antigen as demonstrated by immunostaining supports the hypothesis that maintenance of a physical barrier between tissue factor activity and blood is key to the normal regulation of hemostasis.
Thrombosis Research 08/1990; 59(2):421-37. · 2.44 Impact Factor
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ABSTRACT: Two human tumors, an adenoid cystic carcinoma and a yolk sac tumor, were found by immunocytochemical, ultrastructural, and biochemical studies to contain abundant basement membrane matrix in contrast to the vast majority of human tumors which contained either an absent or scant basement membrane matrix. These tumors were established as xenografts in athymic (nude) mice. Both xenografts maintained characteristic histologic features, immunocytochemical localization of basement membrane components, and reasonable yields of native laminin and Type IV collagen throughout three successive transplant generations. Although only a small fraction of the yield of that of the murine Engelbreth-Holm, Swarm (EHS) sarcoma, the yield of the human basement membrane-producing tumors could be increased by rendering the mice lathyritic. The human basement membrane proteins so extracted were identical on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to their murine counterparts. These human tumors then represent a potential source of human basement membrane proteins.
Cancer 06/1988; 61(9):1798-806. · 4.77 Impact Factor
