Dimethyl fumarate suppresses metastasis and growth of melanoma cells by inhibiting the nuclear translocation of NF-κB.

BACKGROUND: Malignant melanoma is among the deadliest forms of skin cancers, and its incidence has been increasing over the past decades. In malignant melanoma, activation of the nuclear factor kappa B (NF-κB) promotes survival, migration, and invasion of cancer cells. Anti-NF-κB agents for treating metastatic melanoma would be beneficial, but no such drug is approved as either monotherapy or adjuvant therapy. Dimethyl fumarate (DMF) is an approved anti-inflammatory drug already in clinical use for psoriasis and multiple sclerosis. OBJECTIVE: We investigated the anti-tumour effect of DMF treatment in metastatic melanoma in vitro and in vivo. METHODS: The cell viability was assessed via trypan blue exclusion assay. The migration and invasion was analyzed in a Boyden chamber assay. The anti-metastatic effects and anti-tumour activity of DMF was determined in an in-vivo model. The expressions of NF-κB pathway and NF-κB regulatory proteins were detected via western blotting. RESULTS: DMF decreased the cell viability, migration and invasion in vitro. In addition, DMF inhibited spontaneous metastasis and tumour growth. Mechanistically, DMF prevented the nuclear translocation of NF-κB, whereas no changes were observed in the phosphorylation levels of inhibitor of kappa B (IκB). In addition, DMF inhibited the expression of matrix metalloproteinases (MMPs) and very late antigens (VLAs). Furthermore, DMF treatment decreased the expression of Survivin and Bcl-extra large (Bcl-XL) proteins. CONCLUSION: Our results suggest that DMF as a novel inhibitor of NF-κB may be a potential therapeutic agent for metastatic melanoma.

Use of MR cell tracking to evaluate targeting of glial precursor cells to inflammatory tissue by exploiting the very late antigen-4 docking receptor.

PURPOSE: To determine if glial precursor cells can be targeted to inflamed brain through overexpression of very late antigen-4 (VLA-4) and whether this docking process can be monitored with magnetic resonance (MR) cell tracking after intraarterial injection. MATERIALS AND METHODS: All experimental procedures were performed between August 2010 and February 2012 and were approved by the institutional animal care and use committee. Human glial precursor cells (hGPs) were transfected with VLA-4 and labeled with superparamagnetic iron oxide that contained rhodamine. A microfluidic adhesion assay was used for assessing VLA-4 receptor-mediated cell docking in vitro. A rat model of global lipopolysaccharide (LPS)-mediated brain inflammation was used to induce global vascular cell adhesion molecule-1 (VCAM-1) expression. hGPs were infused into the carotid artery in four animal cohorts (consisting of three rats each): rats that received VLA-4-naive hGPs but did not receive LPS, rats that received VLA-4-expressing hGPs but not LPS, rats that received VLA-4-naive hGPs and LPS, and rats that received VLA-4-expressing hGPs and LPS. MR imaging was performed at 9.4 T before and 1, 10, 20, and 30 minutes after injection. Brain tissue was processed for histologic examination. Quantification of low-signal-intensity pixels was performed with pixel-by-pixel analysis for MR images obtained before and after cell injection. RESULTS: With use of the microfluidic adhesion assay, cell binding to activated brain endothelium significantly increased compared with VLA-4-naive control cells (71.5 cells per field of view±11.7 vs 36.4 cells per field of view±3.3, respectively; P<.05). Real-time quantitative in vivo MR cell tracking revealed that VLA-4-expressing cells docked exclusively within the vascular bed of the ipsilateral carotid artery and that VLA-4-expressing cells exhibited significantly enhanced homing as compared with VLA-4-naive cells (1448 significant pixels±366.5 vs 113.3 significant pixels±19.88, respectively; P<.05). Furthermore, MR cell tracking was crucial for correct cell delivery and proper ligation of specific arteries. CONCLUSION: Targeted intraarterial delivery and homing of VLA-4-expressing hGPs to inflamed endothelium is feasible and can be monitored in real time by using MR imaging in a quantitative, dynamic manner.

Echoviruses 1 and 8 are closely related genetically, and bind to similar determinants within the VLA-2 I domain.

Echoviruses (EV) 1 and 8 were originally considered to be distinct serotypes, but more recently have been considered strains of the same virus. In experiments with chimeric recombinant fusion proteins, both viruses bound to the I domain of the integrin VLA-2, and both required the same receptor residues for attachment. A full-length, infectious cDNA clone encoding EV1 was obtained; its nucleotide sequence was determined, as were the sequences encoding the EV8 capsid. EV1 and 8 show 94% amino acid identity within the capsid region and are more similar to each other than to any other human picornavirus.

High affinity very late antigen-4 subsets expressed on T cells are mandatory for spontaneous adhesion strengthening but not for rolling on VCAM-1 in shear flow.

The very late Ag-4 (VLA-4) integrin supports both rolling and firm adhesion of leukocytes on VCAM-1 under shear flow. The molecular basis for the unique ability of a single adhesion molecule to mediate these versatile adhesive processes was investigated. VLA-4 occurs in multiple activation states, with different affinities to ligand. In this study we tested how these states regulate VLA-4 adhesiveness under shear flow in Jurkat T cells and PBL. VLA-4 on nonstimulated Jurkat cells supported rolling and spontaneous arrest on VCAM-1, whereas a Jurkat activation mutant with reduced VLA-4 affinity failed to spontaneously arrest after tethering to or during rolling on VCAM-1. The contribution of VLA-4 affinity for ligand to rolling and spontaneous arrests on immobilized VCAM-1 was dissected using soluble VLA-4 ligands, which selectively block high affinity states. VLA-4 saturation with ligand completely blocked spontaneous adhesion strengthening post-tethering to VCAM-1, but did not impair rolling on the endothelial ligand. High affinity VLA-4 was found to comprise a small subset of VLA-4 on resting Jurkat cells and PBL. This subset is essential for firm adhesion but not for tethering or rolling adhesions on VCAM-1. Interestingly, low and high affinity VLA-4 states were found to mediate similar initial tethering to ligand. High affinity VLA-4, constitutively expressed on circulating T cells, may control their early adhesion strengthening on VCAM-1-expressing endothelium before exposure to vascular chemokines and activation of additional integrins.

Distinct effect of retroviral-mediated IFN-alpha gene transfer on human erythroleukemic and CD34+ cell growth and differentiation.

Human interferon-alpha (IFN-alpha) has been used in the management of leukemia, but its diverse adverse effects may influence the ability of IFN-alpha to treat this disease. We constructed two retroviral vectors, LSN-IFN-alpha and LNC-IFN-alpha, in which IFN-alpha cDNA was driven by viral LTR and CMV promoters, respectively. After transduction into the PA317 and PG13 retroviral packaging cells, high titers of retrovirus were produced and were used to infect K562 and human BM CD34+ hematopoietic cells. The IFN-alpha gene expression in transduced K562 cells was confirmed by Northern blot, RT-PCR, RIA, and biologic assay. Cell proliferation and cell viability in IFN-alpha-transduced K562 cells were significantly suppressed as compared with control K562 cells. Although the IFN-alpha expression in K562 cells did not affect BCR/ABL expression, it apparently upregulated the production of adhesion molecules (VLA-4 and Mac-1). We evaluated the effect of IFN-alpha gene transfer on human CD34+ cells infected with LSN-IFN-alpha retrovirus with the aid of fibronectin (FN) fragment CH-296 and growth factors. RIA showed that IFN-alpha-transduced CD34+ cells produced 72.2+/-15 U/ml of IFN-alpha compared with 4.3+/-1.2 U/ml in control CD34+ cells. Methylcellulose clonogenic assay indicated that IFN-alpha-transduced CD34+ cells produced similar numbers of burst-forming units-erythrocytes (BFU-E)/colony-forming units-GM (CFU-GM) colonies as compared with control CD34+ cells. Selected colonies expressed IFN-alpha and neo(r) mRNA, as measured by RT-PCR. These studies indicate that retrovirus-mediated IFN-alpha gene transfer may provide a useful tool for studying the effect of IFN-alpha gene transfer on leukemic cells and long-lived CD34+ cells.

Novel inhibitors of alpha 4 beta 1 integrin receptor interactions through library synthesis and screening.

A library of 2302 small molecule beta-turn mimetics was screened for inhibition of the alpha 4 beta 1 integrin-CS1 splice variant binding interaction. Preliminary data revealed several active ligands, and validation with purified material culminated in the identification of some of the first small molecule ligands (1, IC50 = 5 microM, and 2, IC50 = 8 microM) to be reported for this class of integrins.

Histopathology of primary biliary cirrhosis with emphasis on expression of adhesion molecules.

In the initiation and progression of immune-mediated destruction of interlobular bile ducts and hepatocytes in primary biliary cirrhosis, T-cell-mediated responses to target antigen(s) expressed on the bile ducts and hepatocytes, as well as cellular adhesions via various adhesion molecules are critical. Intercellular adhesion molecule 1 and, to a lesser degree, vascular adhesion molecule 1 are increasingly expressed on the damaged bile ducts in primary biliary cirrhosis. In addition, lymphocyte function-associated antigens, very late antigens, endothelial-leukocyte adhesion molecule 1, and other adhesion molecules on the vascular endothelial cells and/or inflammatory cells, particularly activated lymphocytes, are also expressed in the portal tracts and hepatic parenchyma. These adhesion molecules are involved in the extravasation as well as epitheliotropic processes of inflammatory cells. Dendritic cells, particularly interdigitating ones in the periductal tissue, are positive for these immune molecules and also for the B-7 family. They may also be important in antigen presentation to CD4+ helper T cells and their activation. However, there is still controversy about whether the B-7 family is expressed on the bile ducts and, then, whether biliary epithelial cells work as an antigen presenting cell. Expression of a very late antigen family on the basolateral surface of bile ducts may be involved in the cell-cell and cell-matrix interactions. Soluble adhesion molecules may be involved in the regulation of immune-mediated bile duct lesions.

The expression of adhesion molecules in cigarette smoke-induced airways obstruction.

Cigarette smoking produces peripheral airway inflammation in all smokers, and chronic airways obstruction in approximately 20% of heavy smokers. The present study was designed to test the hypothesis that airways obstruction is related to changes in the expression of adhesion molecules involved in the recruitment of cells to sites of inflammation in the lung. Freshly resected lungs from heavy smokers with airways obstruction (n = 10) and from heavy smokers with normal lung function (n = 10) were collected in the operating room, inflated with optimal cutting temperature (OCT) medium and frozen over liquid nitrogen. Six micrometres thick cryostat sections cut from random samples of this tissue were stained, using immunohistochemistry, with monoclonal antibodies to the adhesion molecules on leucocytes: L-selectin, very late activation antigen-4 (VLA-4), CD11a/CD18, CD11b/CD18, CD11c/CD18; and on endothelial and epithelial surfaces: E-selectin, P-selectin, vascular cell adhesion molecule (VCAM-1), intercellular adhesion molecule (ICAM)-1 and ICAM-2 using the alkaline phosphatase anti-alkaline phosphatase (APAAP) technique. The slides were coded and the expression of each molecule scored by three observers using a semiquantitative grading system. Two inducible adhesion molecules, E-selectin on endothelium and CD11b on leucocytes, were also evaluated using quantitative morphometric analysis. The results showed a distribution of adhesion molecules that was consistent with the inflammatory response in the airways and parenchyma of all subjects but failed to show any differences between those with or without airways obstruction. We conclude that development of airways obstruction in heavy smokers cannot be explained by differences in the expression of adhesion molecules known to be involved in the control of cell traffic in the lung.

Selective immunomodulation: utilization of CD29/VLA molecules.

Accumulating evidence suggests that the VLA/CD29 molecule plays an important role in T-cell costimulation, and CD4+CD29/VLA+ memory T cells play a key role in induction of CD8 killer effector T cells which are considered to be a major population involved in graft rejection. To target limited elements of the T-lymphocyte population, we have described the preparation of a bispecific antibody-toxin conjugate designed to target CD4+CD29+ memory T cells. We also showed that the solid-phase crosslinking of VLA-4 by the antibody against this molecule or by its ligand, the CS-1 region of fibronectin, stimulates tyrosine phosphorylation of 140, 120-105, 80-70, 60-55, 50 and 45 kilodalton proteins. In addition, we identified the pp140 protein as PLC gamma, pp120 protein as pp125FAK, pp70 and pp50 proteins as paxillin, and pp60-55 proteins as pp59fyn and pp56lck, and pp45 as MAP kinase, respectively. Moreover, we demonstrated that pp125FAK is directly associated with paxillin. The paxillin binding domain of pp125FAK is homologous to the paxillin binding domain of vinculin. Mutations in the conserved amino acid residues between pp125FAK and vinculin result in the loss of paxillin-binding activity. Because VLA/CD29 is preferentially expressed on CD4 memory T cells, the above described system will be used to develop a novel drug design for providing selective immunosuppression useful for organ transplantation.

Expression of the adhesion molecules ICAM-1, VCAM-1, and E-selectin and their ligands VLA-4 and LFA-1 in chronic venous leg ulcers.

BACKGROUND: Leukocyte binding to endothelial cells (ECs) is thought to contribute to the pathogenesis of leg ulcers caused by chronic venous insufficiency. In other systems, such binding is mediated by the interaction of adhesion molecules such as intracellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule- (VCAM-1) and E-selectin (on ECs), and leukocyte function-associated antigen-1(LFA-1) and very late activated antigen-4 (VLA-4) (on Leukocytes). OBJECTIVE: Our purpose was to determine whether an increased expression of these adhesion molecules contributes to the pathogenesis of chronic venous insufficiency. METHODS: Twenty-seven biopsy specimens of inflamed dermatoliposclerotic skin adjacent to venous leg ulcers were stained immunohistochemically with monoclonal antibodies against ICAM-1, VCAM-1, LFA-1, VLA-4, and E-selectin. Staining intensity was compared with that of normal skin. RESULTS: Specimens of leg ulcers caused by chronic venous insufficiency showed increased expression of ICAM-1 and VCAM-1 but not of E-selectin on The expression of LFA-1 and VLA-4 on perivascular leukocytes was increased dramatically in comparison to healthy skin. CONCLUSION: Upregulation of ICAM-1 and VCAM-1 on ECs may contribute to the increased adherence and extravasation of LFA-1 and VLA-4-positive leukocytes in chronic venous insufficiency.

A region of the integrin VLA alpha 4 subunit involved in homotypic cell aggregation and in fibronectin but not vascular cell adhesion molecule-1 binding.

The VLA-4 (alpha 4 beta 1) integrin is involved in the adhesion of cells to fibronectin and vascular cell adhesion molecule-1 (VCAM-1). In order to study alpha 4 structure-function relationships, we have expressed mutated alpha 4 subunit by transfection into VLA-4-negative K562 cells. Substitutions at alpha 4 residues Arg89-Asp90, which show the highest surface probability indexes inside the N-terminal alpha 4/80 fragment, resulted in a reduction in the reactivity of all anti-alpha 4 epitope A monoclonal antibodies (mAbs) tested, compared with the reactivity with anti-alpha 4 epitopes B1, B2, and C mAb, both by transfectant flow cytometry, and by immunoprecipitation and SDS-polyacrylamide gel electrophoresis analysis of transfectant surface-iodinated proteins. In contrast, substitutions at nearby residues, Gln101, Pro102, and Ile105 did not affect the reactivity of any anti-alpha 4 mAb representing the known alpha 4 epitopes. Homotypic cell aggregation triggered by anti-alpha 4 epitope A mAb was prevented in the transfectants expressing mutated alpha 4 Arg89-Asp90Asp residues, while cell aggregation was fully achieved with either anti-alpha 4 epitope B2 or anti-beta 1 mAb. Mutations at alpha 4 residues Gln101, Pro102, and Ile108 did not affect the homotypic cell aggregation of the transfectants expressing these mutations. In addition, the adhesion of mutant Arg89-Asp90 alpha 4 transfectants to the connecting segment-1-containing fibronectin-40 (FN-40) fragment of fibronectin was diminished compared to wild type alpha 4 transfectants, as well as to other mutant alpha 4 transfectants. This adhesion to FN-40 was restored when the activating anti-beta 1 TS2/16 mAb was present in the adhesion assays. In contrast, adhesion to VCAM-1 was not affected by mutations at Arg89-Asp90, nor at Gln101, Pro102, and Ile108 alpha 4 residues. Altogether, these results indicate that alpha 4 residues Arg89 and Asp90 are included in a region involved in homotypic cell aggregation, as well as in adhesion to FN-40, but not to VCAM-1.

Modulation of alpha 4 beta 1 and alpha 5 beta 1 integrin expression: heterogeneous effects of Q-switched ruby, Nd:YAG, and alexandrite lasers on melanoma cells in vitro.

BACKGROUND AND OBJECTIVE: Integrins of the beta 1 family are cellular adhesion molecules that play an important role in cell attachment and migration by interacting with extracellular matrix molecules. Agents such as hormones, cytokines, and ultraviolet radiation have all been shown to have an integrin modulating potential. The present study indicates that radiation of Q-switched lasers is also able to induce transient changes in integrin expression levels on human melanoma cells in vitro. STUDY DESIGN/MATERIALS AND METHODS: Radiation from Q-switched Ruby (694 nm), Alexandrite (755 nm), and Nd:YAG laser (1,064 nm) with fluences comparable to those that are generally used in treating dermatologic lesions were used to irradiate a subconfluent layer of human melanoma cells. After fixed time intervals, the cells were harvested either to analyse the integrin expression by flow cytometry or to investigate changes in cell attachment, spreading, and migration. RESULTS: It was established that all three types of laser were able to cause a significant downregulation of both the alpha 4 and the common beta 1 integrin subunit. The Alexandrite and Ruby lasers also induced a decrease in alpha 5 expression; however, the cells treated with the Nd:YAG laser showed a marked upregulation of the alpha 5 subunit. The expression of the other beta 1 integrin subunits was shown to be unaltered after laser treatment. Downregulation of the alpha 4 upregulation of the alpha 5 integrin subunit expression resulted in, respectively, decreased and increased attachment and spreading on fibronectin, the extracellular matrix ligand for both the alpha 4 beta 1 and alpha 5 beta 1 integrins. Marked upregulation of the alpha 5 subunit also resulted in a higher migration rate. CONCLUSION: Taken together, these results show that nonlethal doses of Q-switched laser radiation are able to induce changes in cellular behavior in vitro by modulating the integrin expression pattern.

A hierarchy for integrin expression and adhesiveness among T cell subsets that is linked to TCR gene usage and emphasizes V delta 1+ gamma delta T cell adherence and tissue retention.

To define the relationship between T cell phenotype and adhesiveness, we examined T cell adhesion to endothelial cell, fibroblast, and epithelial cell monolayers as well as extracellular matrix proteins (collagen and fibronectin) using a three-color flow cytometry-based adherence assay that minimizes basal adhesion levels and facilitates quantitative lymphocyte subtyping. Regardless of monolayer type, monolayer stimulation conditions, or T cell activation status, we found that the gamma delta-TCR-bearing T cells adhered more efficiently than alpha beta T cells. The difference was based predominantly on increased levels of activatable LFA-1 (and to a lesser degree VLA-4) because: 1) it correlated precisely with inhibitability by anti-LFA-1 (and VLA-4) mAbs and the levels of LFA-1 (and VLA-4) on the cell surface, and 2) it persisted after maximal LFA-1 (and VLA-4) activation with phorbol dibutyrate. In contrast to most cases of alpha beta T cell behavior, gamma delta T cell adhesion to cell monolayers was not linked to memory status, i.e., there was no difference between naive V delta 1+ and memory V delta 2+ populations in levels of LFA-1 (or VLA-4) expression or LFA-1- (or VLA-4-) dependent adhesion to cell monolayers. However, V delta 1+ cells exhibited higher levels of VLA-5 that correlated with an increased adhesiveness to fibronectin and to a 120-kDa fibronectin fragment (FN-120) that contains only the VLA-5-binding domain but not to type I collagen or to a fibronectin fragment (FN-40) that binds only VLA-4. Taken together, the results define a hierarchy for integrin (LFA-1, VLA-4, and VLA-5) expression and consequent adhesion among T cell subsets that is linked to TCR gene usage (but not necessarily linked to memory status) and may thereby help to explain the accumulation and retention of V delta 1+ gamma delta T cells in epithelial and connective tissues.

cDNA cloning of mouse VLA-3 alpha subunit.

cDNA clones for mouse VLA (very late antigen)-3 alpha subunit (alpha 3 integrin) were isolated and sequenced. The encoded mouse alpha 3 integrin subunit was composed of 1,053 amino acid residues. The results of sequence analysis revealed similar structural characteristics of other VLA alpha subunits. For example, the presence of a large extracellular domain including three putative metal binding sequences, a transmembrane domain, and a short cytoplasmic domain. A higher level of its message was detected in thymus than in kidney, stomach, spleen, liver, brain, or lung by Northern blotting analysis.

The VLA-2 (alpha 2 beta 1) I domain functions as a ligand-specific recognition sequence for endothelial cell attachment and spreading: molecular and functional characterization.

The integrin VLA-2 (alpha 2 beta 1), generally considered to represent the specific collagen receptor on human endothelial cells, contains an alpha 2-subunit inserted I domain with structural similarity to the type A domains found within the recently described superfamily of receptor-ligand recognition proteins. This region of the cDNA has now been isolated and used for molecular and functional characterization of this heterodimeric receptor complex. Comparative sequence analysis with the porcine homologue revealed 93% amino acid sequence identity, suggestive of a developmentally conserved function. To complete structure/function studies, this region of the human cDNA was expressed as a chimeric protein in Escherichia coli, and a rabbit polyclonal antibody (anti-I domain) was used to study determinants of endothelial cell attachment and spreading in vitro. Quantifiable and visual disruption of endothelial cell attachment to gelatin, type I collagen, and laminin was evident using the specific anti-I domain antibody, with minimal inhibitory effects demonstrable using fibronectin or fibrinogen matrices. Therefore, these data would suggest that the alpha 2 beta 1 I domain confers ligand-binding specificity for both known alpha 2 beta 1 substrates (laminin and collagen), and that this region subserves a regulatory function in the molecular processes controlling endothelial cell attachment and spreading in vitro.

Cloning and characterization of the promoter region of the murine alpha-4 integrin subunit.

To study the differential expression of the murine VLA-4 (alpha 4 beta 1) integrin, the 5'-flanking region of the gene for the alpha subunit (alpha 4m) was isolated and a cDNA for alpha 4m was obtained with reverse transcriptase polymerase chain reaction (RT-PCR). The cDNA sequence contained a difference in the signal peptide region compared to the previously described cDNA (Neuhaus et al., 1991). As a consequence, another start codon is predicted, resulting in a decrease in size of the signal peptide. This was confirmed by genomic sequencing. The promoter region was delimited by ribonuclease protection assay (RPA) and transfection experiments fusing 5'-upstream fragments to the luciferase gene. A fragment extending from -936 to +221 was capable of controlling the expected cell-type-specific expression. Sequence comparison of the mouse alpha 4m promoter region with the human alpha 4h promoter revealed little homology. Like most integrin subunits, alpha 4m lacks TATA anc CCAAT boxes. Putative recognition sites for DNA-binding nuclear factors (AP1, AP2, Sp1, and PU1) were identified. The characterization of the promoter region and further identification of the transcription regulatory elements should provide insight in the regulation of alpha 4m integrin gene expression.

Expression of early and late activation markers on peripheral blood T lymphocytes does not reliably reflect immune events in transplanted hearts.

Monoclonal antibodies directed against early (receptors for interleukin-2 and transferrin [IL-2R, TfR]) and late (PTA1, alpha 1 integrin VLA-1) activation antigens were used as probes to monitor cardiac transplant patients for episodes of acute graft rejection. Age- and sex-matched patient control groups consisting of 11 patients awaiting cardiac transplantation and 13 kidney transplant recipients with long-term grafts, respectively, were used to define an upper limit for normal activation antigen expression (mean + 3 SD) in patients. Expression of all cell markers was significantly higher in both patient control groups than in healthy control individuals. Therefore, the level of activation marker expression in heart patients awaiting transplantation was used as comparison for the patient population under study. Sequential monitoring of 24 heart transplant recipients failed to demonstrate a significant correlation of increased activation marker expression with clinical events of immune activation. Subsequently 62 consecutive endomyocardial biopsy scores in 36 patients were compared with the expression of IL-2R, TfR and VLA-1 on peripheral blood T cells. Neither increased cellular infiltration of the endocardium, nor of the myocardium, was associated with increasing proportions of IL-2R, TfR, or VLA-1 positive T cells. Elevated T-cell expression of the three markers combined indicated acute graft rejection with a sensitivity, specificity, and overall accuracy of 38%, 52%, and 43%, respectively. Acute graft rejection in biopsies with associated myofiber damage (biopsy rejection scores 2 and 3A,B) was not associated with a change in the proportion of activated T cells in circulation within the first 6 months after transplantation.(ABSTRACT TRUNCATED AT 250 WORDS)

The mouse VLA-2 homologue supports collagen and laminin adhesion but not virus binding.

Human VLA-2 (alpha 2 beta 1) mediates cellular adhesion to collagen and laminin and cell attachment by the human pathogen echovirus 1. We report here the cloning, sequencing and functional expression of the mouse VLA-2 alpha subunit homologue. This integrin subunit is closely related to its human counterpart, with 84% amino acid identity between the human and murine proteins. Conserved structural features include an identical number of amino acids, the presence of an I domain, and identity in the number and position of N-linked glycosylation sites and putative divalent cation binding regions. Murine and human alpha 2 show 30% amino acid divergence within the cytoplasmic tail, a difference that can be detected with antisera directed against the C-terminal peptides. Functionally, mouse alpha 2 was capable of mediating cell attachment to collagen and laminin, and responded to both intra- and extracellular signals with changes in its ligand affinity. In contrast, unlike its human homologue, mouse alpha 2 did not promote binding of echovirus 1. Comparison of the primary structure of the homologues leads us to predict that echovirus 1 may bind in the region of the first two thirds of the human alpha 2 I domain, where the sequences are most divergent, whereas more conserved flanking regions, and the conserved terminal one third of the I domain, may be involved in adhesion to collagen and laminin.

Expression of the integrin alpha 4 beta 1 on melanoma cells can inhibit the invasive stage of metastasis formation.

Among a series of adhesion molecules, expression of integrin alpha 4 beta 1 showed a unique inverse correlation with the invasive potential of B16 melanoma cell lines. When an alpha 4 cDNA was introduced into an alpha 4-beta 1+ highly invasive melanoma line, alpha 4 beta 1 heterodimers were expressed on the surface. Matrigel invasion by the alpha 4+ beta 1+ cells was reduced. Pulmonary metastasis was also suppressed when the transfectants were placed subcutaneously, but not when injected intravenously. Expression of alpha 4 beta 1 promoted homotypic intercellular adhesion. The homotypic adhesion was abrogated, and the alpha 4+ beta 1+ (less invasive cell lines) increased matrigel invasion following the anti-alpha 4 MAb treatment. These results suggest that integrin alpha 4 beta 1 could play a role in controlling melanoma cell metastasis at the invasive stage.

Intercellular adhesion induced by anti-alpha 3 integrin (VLA-3) antibodies.

We generated four monoclonal antibodies (mAbs) specific for human alpha 3 integrin (VLA-3 alpha subunit). All of them were found to induce homotypic cell aggregation of HT1080 fibrosarcoma and SN12C renal carcinoma cells, both of which express high levels of alpha 3 integrin. The antibodies also induced the cell aggregation of K562 erythroleukemic cells transfected with alpha 3 integrin cDNA, but not the parental K562 cells. The aggregation was observed in a temperature-dependent manner and was not inhibited by the addition of EDTA. Immunofluorescence microscopic observation showed that alpha 3 integrin on HT1080 cells was translocated into the contact regions after the mAb treatment. The intercellular adhesion between cells expressing alpha 3 integrin and cells without alpha 3 integrin was also induced by the anti-alpha 3 antibody treatment.