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Biomedical subjects

I Stamenkovic

Publications and source records attributed to I Stamenkovic.

At least 37 records · Page 2Linked to original sources
PubMed1997

Induction of apoptosis of metastatic mammary carcinoma cells in vivo by disruption of tumor cell surface CD44 function.

To understand how the hyaluronan receptor CD44 regulates tumor metastasis, the murine mammary carcinoma TA3/St, which constitutively expresses cell surface CD44, was transfected with cDNAs encoding soluble isoforms of CD44 and the transfectants (TA3sCD44) were compared with parental cells (transfected with expression vector only) for growth in vivo and in vitro. Local release of soluble CD44 by the transfectants inhibited the ability of endogenous cell surface CD44 to bind and internalize hyaluronan and to mediate TA3 cell invasion of hyaluronan-producing cell monolayers. Mice intravenously injected with parental TA3/St cells developed massive pulmonary metastases within 21-28 d, whereas animals injected with TA3sCD44 cells developed few or no tumors. Tracing of labeled parental and transfectant tumor cells revealed that both cell types initially adhered to pulmonary endothelium and penetrated the interstitial stroma. However, although parental cells were dividing and forming clusters within lung tissue 48 h following injection, >80% of TA3sCD44 cells underwent apoptosis. Although sCD44 transfectants displayed a marked reduction in their ability to internalize and degrade hyaluronan, they elicited abundant local hyaluronan production within invaded lung tissue, comparable to that induced by parental cells. These observations provide direct evidence that cell surface CD44 function promotes tumor cell survival in invaded tissue and that its suppression can induce apoptosis of the invading tumor cells, possibly as a result of impairing their ability to penetrate the host tissue hyaluronan barrier.

Animals↗
PubMed1997

Selective suppression of CD44 in keratinocytes of mice bearing an antisense CD44 transgene driven by a tissue-specific promoter disrupts hyaluronate metabolism in the skin and impairs keratinocyte proliferation.

CD44 is a broadly distributed polymorphic glycoprotein that serves as the principal cell-surface receptor for hyaluronate. Although CD44-mediated cell interaction with hyaluronate has been implicated in a variety of physiologic events, including cell-cell and cell-substrate adhesion, cell migration, proliferation, and activation, as well as hyaluronate uptake and degradation, the biologic role of CD44 in vivo in various tissues remains to be determined. In the present work we have developed transgenic mice that express an antisense CD44 cDNA driven by the keratin-5 promoter. These mice lack detectable CD44 expression in skin keratinocytes and corneal epithelium and display abnormal hyaluronate accumulation in the superficial dermis and corneal stroma, distinct morphologic alterations of basal keratinocytes and cornea, and defective keratinocyte proliferation in response to mitogen and growth factors. These alterations are reflected by a decrease in skin elasticity, impaired local inflammatory response and tissue repair, delayed hair regrowth, and failure of the epidermis to undergo hyperplasia in response to carcinogen. Our observations indicate that two major functions of CD44 in skin are the regulation of keratinocyte proliferation in response to extracellular stimuli and the maintenance of local hyaluronate homeostasis.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1997

The role of E-selectin for neutrophil activation and tumor metastasis in vivo.

The selectin class of adhesion molecules plays a critical role in facilitating leukocyte adhesion to and subsequent transmigration of endothelium. On this basis, selectins have been suggested to promote metastasis of certain types of tumors, although direct evidence is lacking. To explore this hypothesis two sets of transgenic mice were developed, one TgNES, which constitutively expresses cell surface E-selectin in all tissues, the other TgNEsol, which expresses truncated, soluble E-selectin in the liver. Both transgenic mice showed apparently normal phenotype. However in TgNES mice, but not in TgNEsol mice, the number of neutrophil decreased to 50% compared with that in their negative littermate. And also these neutrophils were markedly activated. On the other hand, B16 F10 melanoma cells were stably transfected with alpha 1-3/4 fucosyltransferase-specific cDNA (B16F10 ft), allowing them to express E-selectin ligands. Normal mice injected with B16F10 ft developed lung tumors exclusively. In contrast, TgNES mice developed massive, infiltrating liver tumors. TgNEsol mice also developed liver tumors that grow more slowly. These observations suggest the important role of E-selectin for neutrophil activation and tumor metastasis in vivo.

Actins↗
PubMed1996

Identification of a novel inducible cell-surface ligand of CD5 on activated lymphocytes.

CD5 is a 67-kD glycoprotein that is expressed on most T lymphocytes and on a subset of mature B cells. Although its physiologic function is unknown, several lines of evidence suggest that CD5 may play a role in the regulation of T cell activation and in T cell-antigen presenting cell interactions. Using a CD5-immunoglobulin fusion protein (CD5Rg, for receptorglobulin) we have uncovered a new CD5 ligand (CD5L) expressed on the surface of activated splenocytes. Stimulation of murine splenocytes with anti-CD3 and anti-CD28 antibodies induce transient expression of CD5L on B lymphocytes that lasts for approximately 72 h. Binding of CD5Rg to activated splenocytes is trypsin resistant and independent of divalent cations. However, it is pronase sensitive and dependent on N-linked glycosylation of CD5, since treatment of CD5Rg with PNGaseF on N-glycanase completely abrogates its ability to bind activated splenocytes. It addition to splenocytes, CD5L is expressed on activated murine T cell clones. Immunoprecipitation, antibody, and recombinant protein blocking studies indicate that CD5L is distinct from CD72, which has been proposed to be a CD5 ligand. To determine whether CD5-CD5L interaction might play a role in vivo, we tested the effect of CD5Rg in a murine model of antibody-mediated membranous glomerulonephritis. Injection of CD5Rg was found to abrogate development of the disease. Taken together, our results help identify a novel ligand of CD5 and propose a role for CD5 in the regulation of immune responses.

Animals↗
PubMed1996

A single N-linked glycosylation site is implicated in the regulation of ligand recognition by the I-type lectins CD22 and CD33.

CD22 is an immunoglobulin superfamily B lymphocyte-specific adhesion receptor and a member of the recently identified I-type class of lectins. Recent work has shown that CD22 specifically recognizes sialic acid linked alpha2,6 to terminal N-linked oligosaccharides on selected cell surface glycoproteins. CD22-ligand interaction is regulated by the activity of a beta-galactoside alpha2, 6-sialyltransferase that can inactivate CD22-mediated binding by sialylating the CD22 receptor itself. These observations suggest that N-linked glycosylation sites on the CD22 molecule may play a role in the regulation of CD22-mediated adhesion. In this work we have performed site-specific mutagenesis of potential N-linked glycosylation sites on CD22 in an effort to determine whether they might be involved in ligand recognition. We show that mutation of a single potential N-linked glycosylation site in the first immunoglobulin domain of CD22 completely abrogates ligand recognition. Interestingly, this site is characterized by the sequence NCT, where the cysteine is thought to be involved in an intrachain disulfide bond. Site-directed mutagenesis of similar NC(T/S) motifs in the first or second Ig domains of the I-type lectins myelin-associated glycoprotein, and sialoadhesin did not disrupt their ability to mediate sialic acid binding. In contrast, mutation of a NCS motif in the first Ig domain of the I-type lectin CD33 unmasked its sialic acid binding activity. These observations suggest that a single N-linked glycosylation site located at a similar position in the CD22 and CD33 glycoproteins is critical for regulating ligand recognition by both receptors.

Amino Acid Sequence↗
PubMed1996

Keratan sulfate modification of CD44 modulates adhesion to hyaluronate.

CD44 alternative splicing has been implicated in the regulation of CD44 function. CD44 undergoes significant posttranslational modification in all cells, but the functional consequences of these modifications are poorly understood. In the current study, we have demonstrated that keratan sulfate modification of CD44 significantly modulates its ability to bind to hyaluronate. We observed naturally occurring differences in CD44 keratan sulfate substitution between two clonal variants of the KM12 human colon carcinoma cell line. CD44 on the highly metastatic KM12L4 clone is more heavily substituted with keratan sulfate than CD44 on the poorly metastatic KM12C6 clone. Moreover, CD44H on KM12L4 bound to hyaluronate poorly compared to CD44H on KM12C6. Removal of keratan sulfate from CD44 greatly enhanced CD44-mediated cell adhesion to hyaluronate. Removal of keratan sulfate from CD44H-immunoglobulin fusion proteins also enhanced their adhesion to hyaluronate. The influence of glycosaminoglycan substitution on CD44 function was specific to keratan sulfate substitution; treatment to remove chondroitin sulfate, heparan sulfate, or hyaluronate did not affect CD44-mediated cell adhesion to hyaluronate. Use of site-directed CD44H cDNA mutants with arginine changed to alanine at position 41 indicated that keratan sulfate modification of CD44 modulates hyaluronate adhesion through its B loop domain. These findings suggest that keratan sulfate modification of CD44 may play an important regulatory role in the broad spectrum of biological processes attributed to CD44, including normal development, tumor progression, and lymphocyte function.

Alternative Splicing↗
PubMed1996

The CD95 (APO-1/Fas) receptor activates NF-kappaB independently of its cytotoxic function.

Engagement of the CD95 (APO-1/Fas) receptor induces apoptosis in a variety of cell types. However, the nature of the cytotoxic signal and the intermediate messenger molecules remain to be elucidated. In an effort to understand CD95-mediated signaling, we assessed possible changes in the DNA binding activity of NF-kappaB as a result of CD95 engagement in various tumor cells. By performing electrophoresis mobility shift assays, we show that CD95 can stimulate the DNA binding activity of NF-kappaB in a variety of cells, irrespective of their sensitivity or resistance to CD95-mediated cytotoxicity. Moreover, deletion of 37 carboxyl-terminal residues from the cytoplasmic domain of CD95, which abrogates CD95-mediated apoptosis, only marginally affects NF-kappaB activation. Taken together, these observations indicate that CD95 has a function that involves activation of NF-kappaB and that appears to be unrelated to its role as an inducer of apoptotic cell death.

Apoptosis↗
PubMed1996

Distinct regulatory roles of lymphocyte costimulatory pathways on T helper type-2 mediated autoimmune disease.

We assessed the role of CD40-CD40L, cytotoxic T lymphocyte (CTL)A4/CD28-B7s, and CD2-CD48/CD58 lymphocyte costimulatory pathways in the development of mercury chloride (HgCl2)-induced autoimmune disease in mice, which is believed to be mediated by T helper (Th) subset Th2. Inhibition of CD40-CD40-L and CTLA4/CD28-B7s interactions by anti-CD40-L antibody and soluble CTLA4-immunoglobulin (Ig) fusion protein, respectively, abrogated the autoimmune disease without affecting interleukin 4 (IL-4) production, showing the importance of physical contact between T and B lymphocytes in the Th2-mediated process. In contrast, two anti-CD2 antibodies that have been shown to induce immunosuppression of Th1-mediated events exacerbated the autoantibody response and augmented IgG1, IgE, and IL-4 production, transforming a mild mesangial glomerulopathy into a severe systemic immune complex disease. These observations demonstrate that manipulation of lymphocyte accessory counterreceptor interactions may affect the course of Th2-associated autoimmune disease and suggest that signals resulting from CD2 engagement play an essential role in the regulation of the Th1-Th2 effector equilibrium.

Animals↗
PubMed1996

Redirection of tumor metastasis by expression of E-selectin in vivo.

The selectin class of adhesion molecules plays a critical role in facilitating leukocyte adhesion to and subsequent transmigration of endothelium. On this basis, selectins have been suggested to promote tumor cell attachment to endothelium, thereby facilitating metastasis of certain types of tumors, although direct evidence for such a role is lacking. To explore this hypothesis, two sets of transgenic mice were developed: TgnES, which constitutively expresses cell surface E-selectin in all tissues, under the control of the beta-actin promoter; and TgnEsol, which expresses truncated, soluble E-selectin in the liver, under the control of the alpha 1 antitrypsin promoter. B16F10 melanoma cells were stably transfected with alpha(1,3/1,4) fucosyltransferase-specific cDNA (B16F10ft), allowing them to express E-selectin ligands or with hygromycin resistance selection vector only B16F10hygro). Normal mice injected with B16F10ft and B16F10hygro and transgenic mice injected with B16F10hygro developed lung tumors exclusively. In contrast, TgnES mice injected with B16F10ft cells developed massive infiltrating liver tumors. B16F10ft cells injected into TgnEsol mice also formed liver tumors, but these grew more slowly, with a well-delineated, noninfiltrating distinct histologic pattern. These observations provide direct evidence that expression of E-selectin can redirect metastasis of tumor cells expressing appropriate ligands in vivo.

Animals↗
PubMed1996

Superoxide anion is a natural inhibitor of FAS-mediated cell death.

The cell surface receptor Fas is a major trigger of apoptosis. However, expression of the Fas receptor in many tumor cell types does not correlate with sensitivity to Fas-mediated cell death. Because a prooxidant state is a common feature of tumor cells, we examined the role of intracellular reactive oxygen intermediates in the regulation of Fas-mediated cytotoxicity. Our results show that an oxidative stress induced by increasing the intracellular superoxide anion (O2-) concentration can abrogate Fas-mediated apoptosis in cells which are constitutively sensitive to Fas. Conversely, an O2- concentration decrease is observed to sensitize cells which are naturally resistant to Fas signals. These observations suggest that intracellular O2- may play a key role in regulating cell sensitivity to a potentially lethal signal and provide tumor cells with a natural, inducible mechanism of resistance to Fas-mediated apoptosis.

Antioxidants↗
PubMed1996

Glycosylation of CD44 is implicated in CD44-mediated cell adhesion to hyaluronan.

CD44-mediated cell adhesion to hyaluronate is controlled by mechanisms which are poorly understood. In the present work we examine the role of N-linked glycosylation and Ser-Gly motifs in regulating CD44-hyaluronate interaction. Our results show that treatment of a panel of human cell lines which constitutively express CD44 with the inhibitor of N-linked glycosylation tunicamycin results in the loss of attachment of these cells to hyaluronate-coated substrate. In contrast, treatment of the same cells with deoxymannojirimycin, which inhibits the conversion of high mannose oligosaccharides to complex N-linked carbohydrates, results in either no change or an increase in CD44-mediated adhesion to hyaluronate, suggesting that complex N-linked oligosaccharides may not be required for and may even inhibit CD44-HA interaction. Using human melanoma cells stably transfected with CD44 N-linked glycosylation site-specific mutants, we show that integrity of five potential N-linked glycosylation sites within the hyaluronate recognition domain of CD44 is critical for hyaluronate binding. Mutation of any one of these potential N-linked glycosylation sites abrogates CD44-mediated melanoma cell attachment to hyaluronate-coated surfaces, suggesting that all five sites are necessary to maintain the HA-recognition domain in the appropriate conformation. We also demonstrate that mutation of serine residues which constitute the four Ser-Gly motifs in the membrane proximal domain, and provide potential sites for glycosaminoglycan side chain attachment, impairs hyaluronate binding. Taken together, these observations indicate that changes in glycosylation of CD44 can have profound effects on its interaction with hyaluronic acid and suggest that glycosylation may provide an important regulatory mechanism of CD44 function.

1-Deoxynojirimycin↗
PubMed1995

CD44 hyaluronate binding influences growth kinetics and tumorigenicity of human colon carcinomas.

CD44 is a cell surface receptor of hyaluronate that is implicated in the regulation of tumor growth and metastatic potential. Transformation of colon mucosa to carcinoma is associated with overexpression of several CD44 alternative splice variants. The functional roles of CD44 isoforms in colon carcinoma tumor progression remain unclear. CD44H expression is downregulated in colon carcinomas compared to paired normal mucosa. In the present study we demonstrate that reintroduction of CD44H back into colon carcinoma cells by stable transfection reduces their in vitro growth rate and tumorigenicity. Examination of several colon carcinoma cell lines and use of mutant CD44H reveal that this in vitro and in vivo growth reduction requires the ability of CD44H to bind hyaluronate. These observations indicate that the CD44H downregulation associated with transformation of mucosa to colon carcinoma may provide the carcinoma cells with a growth advantage. Furthermore, the reduction in tumor growth rate mediated by reintroduction of CD44H into colon carcinoma cells is dependent on its ability to bind hyaluronate.

Animals↗
PubMed1995

Regulation of CD45 engagement by the B-cell receptor CD22.

The B-cell receptor CD22 binds sialic acid linked alpha-2-6 to terminal galactose residues on N-linked oligosaccharides associated with several cell-surface glycoproteins. The first of these sialoglycoproteins to be identified was the receptor-linked phosphotyrosine phosphatase CD45, which is required for antigen/CD3-induced T-cell activation. In the present work, we examine the effect of interaction between the extracellular domain of CD45 and CD22 on T-cell activation. Using soluble CD22-immunoglobulin fusion proteins and T cells expressing wild-type and chimeric CD45 forms, we show that engagement of CD45 by soluble CD22 can modulate early T-cell signals in antigen receptor/CD3-mediated stimulation. We also show that addition of sialic acid by beta-galactoside alpha-2,6-sialyltransferase to the CD22 molecule abrogates interactions between CD22 and its ligands. Together, these observations provide direct evidence for a functional role of the interaction between the extracellular domain of CD45 and a natural ligand and suggest another regulatory mechanism for CD22-mediated ligand engagement.

Antigens, CD↗
PubMed1995

Inhibition of the CD40-CD40ligand pathway prevents murine membranous glomerulonephritis.

Several forms of glomerulonephritis are induced by antibodies against self or foreign antigens. Normal B lymphocyte antibody production requires T cell costimulatory signals provided in part by T cell surface expression of gp39/CD40ligand (CD40L) that engages the B cell receptor CD40 and induces B cell differentiation and immunoglobulin class switching. We assessed the effect of disrupting the CD40L-CD40 costimulatory pathway, using a CD40-Ig fusion protein, on the development of membranous glomerulonephritis (MGN) in the mouse. MGN is induced by mouse antibodies that recognize and bind to exogenously administered rabbit anti-mouse renal tubular brush border (RbAMBB) IgG immobilized in the glomerular capillary wall. MGN did not occur in nude mice, showing the need of the T cell function. C57Bl/10 mice immunized with RbAMBB and treated with CD40-Ig fusion protein displayed a delayed autologous response and absence of MGN lesions, while control fusion proteins failed to prevent the development of the disease. These observations provide evidence that disruption of the CD40-CD40L costimulatory pathway can prevent the development of MGN by suppressing T cell-dependent antibody production.

Animals↗
PubMed1995

CD44 isoforms containing exon V3 are responsible for the presentation of heparin-binding growth factor.

Glycosaminoglycan-modified isoforms of CD44 have been implicated in growth factor presentation at sites of inflammation. In the present study we show that COS cell transfectants expressing CD44 isoforms containing the alternatively spliced exon V3 are modified with heparan sulfate (HS). Binding studies with three HS-binding growth factors, basic-fibroblast growth factor (b-FGF), heparin binding-epidermal growth factor (HB-EGF), and amphiregulin, showed that the HS-modified CD44 isoforms are able to bind to b-FGF and HB-EGF, but not AR. b-FGF and HB-EGF binding to HS-modified CD44 was eliminated by pretreating the protein with heparitinase or by blocking with free heparin. HS-modified CD44 immunoprecipitated from keratinocytes, which express a CD44 isoform containing V3, also bound to b-FGF. We examined whether HS-modified CD44 isoforms were expressed by activated endothelial cells where they might present HS-binding growth factors to leukocytes during an inflammatory response. PCR and antibody-binding studies showed that activated cultured endothelial cells only express the CD44H isoform which does not contain any of the variably spliced exons including V3. Immunohistological studies with antibodies directed to CD44 extracellular domains encoded by the variably spliced exons showed that vascular endothelial cells in inflamed skin tissue sections do not express CD44 spliced variants. Keratinocytes, monocytes, and dendritic cells in the same specimens were found to express variably spliced CD44. 35SO4(-2)-labeling experiments demonstrated that activated cultured endothelial cells do not express detectable levels of chondroitin sulfate or HS-modified CD44. Our results suggest that one of the functions of CD44 isoforms expressing V3 is to bind and present a subset of HS-binding proteins. Furthermore, it is probable that HS-modified CD44 is involved in the presentation of HS-binding proteins by keratinocytes in inflamed skin. However, our data suggests that CD44 is not likely to be the proteoglycan principally involved in presenting HS-binding growth factors to leukocytes on the vascular cell wall.

Alternative Splicing↗
PubMed1995

Regulation of CD44 binding to hyaluronan by glycosylation of variably spliced exons.

The hyaluronan (HA)-binding function (lectin function) of the leukocyte homing receptor, CD44, is tightly regulated. Herein we address possible mechanisms that regulate CD44 isoform-specific HA binding. Binding studies with melanoma transfectants expressing CD44H, CD44E, or with soluble immunoglobulin fusions of CD44H and CD44E (CD44H-Rg, CD44E-Rg) showed that although both CD44 isoforms can bind HA, CD44H binds HA more efficiently than CD44E. Using CD44-Rg fusion proteins we show that the variably spliced exons in CD44E, V8-V10, specifically reduce the lectin function of CD44, while replacement of V8-V10 by an ICAM-1 immunoglobulin domain restores binding to a level comparable to that of CD44H. Conversely, CD44 bound HA very weakly when exons V8-V10 were replaced with a CD34 mucin domain, which is heavily modified by O-linked glycans. Production of CD44E-Rg or incubation of CD44E-expressing transfectants in the presence of an O-linked glycosylation inhibitor restored HA binding to CD44H-Rg and to cell surface CD44H levels, respectively. We conclude that differential splicing provides a regulatory mechanism for CD44 lectin function and that this effect is due in part to O-linked carbohydrate moieties which are added to the Ser/Thr rich regions encoded by the variably spliced CD44 exons. Alternative splicing resulting in changes in protein glycosylation provide a novel mechanism for the regulation of lectin activity.

Alternative Splicing↗
PubMed1995

Restoration of CD44H expression in colon carcinomas reduces tumorigenicity.

OBJECTIVE: The functional consequences of reintroduction of the CD44H cell adhesion molecule into colon carcinomas were investigated. BACKGROUND: CD44 is a cell surface adhesion molecule that is normally present in numerous isoforms as a result of messenger RNA alternative splicing. Individual CD44 isoforms differ in their ability to enhance tumorigenic or metastatic potential when overexpressed on tumor cells. Reverse transcriptase-polymerase chain reaction analysis demonstrates that CD44H is down-regulated during transformation of normal colon mucosa to carcinoma. The functional consequences of CD44H down-regulation in colon carcinomas has not been clarified. METHODS: Tumor cell lines and fresh tissue specimens were examined for CD44 expression by Western blot analysis. CD44H cDNA and site-directed mutants of CD44H cDNA were transfected into colon carcinoma cells. Stable transfectants were examined for adhesion to hyaluronate, in vitro growth, and in vivo growth. RESULTS: CD44H expression was nearly undetectable in primary colon carcinomas and colon carcinoma cell lines. In contrast, normal mucosa expressed high levels of CD44H. When CD44H was reintroduced into colon carcinoma cells, their in vitro and in vivo growth was significantly reduced. This CD44H-mediated growth rate reduction required an intact cytoplasmic domain. CONCLUSIONS: Transformation of normal mucosa to colon carcinoma is associated with a down-regulation of CD44H, which consequently may enhance the growth rate and tumorigenicity.

Blotting, Western↗