Integrin mediated growth and apoptosis in human gastric adenocarcinoma

Integrins are important as the primary cell adhesion molecule providing information about the extracellular microenvironment to the interior of the cell to influence cellular behavior such as differentiation, proliferation and apoptosis. Apoptotic death due to loss of adhesion is termed anoikis. In this study we have obtained a parental human gastric adenocarcinoma cell line that yielded two variant lines that had differing responses to lack of adhesion. The STAD.APO cell line undergoes apoptosis when denied adherence and the STAD.ARR cell line enters into cell cycle arrest under the identical suspended conditions. We have shown that cyclin A and cyclin D mRNA and protein are down regulated when cells are denied adherence for 24 hours in tissue culture wells previously coated with poly-HEMA. To test whether cyclin A was able to rescue cells from cell cycle arrest and/or anoikis by overriding the cell cycle machinery we transfected the full length cDNA in to each cell type. Surprisingly we found that anoikis and cell cycle arrest due to suspended conditions was not affected by overexpression of cyclin A protein, but that growth under adhered conditions was reduced compared to vector alone control transfectants. Further, we transfected other cell lines; ST7, gastric cancer, MDA-MB-4.35, breast cancer, and HPB T-cell leukemic and in no case were suspended culturing conditions overcome by cyclin A. This result indicates an additional level of regulation for the cell cycle machinery. Additionally, soluble collagen was shown to be able to save from anoikis and also from cell cycle arrest while the β1 specific mAb 33B6 was only able to save from anoikis. Immunofluorescent studies show that soluble collagen creates clusters of β1 with FAK and also β1 with actin in the STAD.ARR cells but does not in the STAD.APO cells. This result indicates that the phenotypes under suspended conditions between these cell lines may diverge at their requirements for integrin ligation. Additionally we characterized the nature of anoikis by showing cytochrome c release, caspase 3, p21 and p53 activation in STAD.APO cells. Thus, our results have implications in the understanding of integrin biology and neoplastic progression. ^

Alteration of CEACAM1 gene transcription in prostate cancer cells: The role of AR, Sp2, and HDAC

Cell to cell adhesion molecule (CEACAM1), a type II tumor suppressor, has been found to be down-regulated in prostate cancer cells. The mechanism that causes CEACAM1's down-regulation in tumorigenesis is unknown. Here we show that the transcriptional activity of CEACAM1 is decreased in prostate cancer cells. This decrease is not due to methylation of the CEACAM1's promoter, but rather to the alteration of transcription factors regulating CEACAM1 expression. ^ Since androgen/androgen receptors (AR) are potent regulators of prostate growth and differentiation, their role on CEACAM1 gene transcription was examined. The androgen receptor could directly increase CEACAM1 transcriptional activity in a ligand dependent manner by interacting with an AR consensus element that resides in the CEACAM1 promoter. However, AR binding to the CEACAM1 promoter is not related to the loss of CEACAM1 during prostate cancer progression. ^ Further analysis enabled us to determine the particular region in the CEACAM1 promoter that mediates a decrease in CEACAM1 transcriptional activity in prostate cancer cells. Upon further examination, we found that this CEACAM1 promoter region interacts with the Sp1, Sp2, and Sp3 transcription factors. However, only Sp2 expression was found to increase in prostate cancer cells. Inhibiting Sp2 from binding to the CEACAM1 promoter caused an increase in CEACAM1 transcriptional activity in prostate cancer cells. In addition, over-expressing Sp2 in normal prostate cells resulted in a decrease in CEACAM1 transcriptional activity and endogenous protein expression. These observations suggest that Sp2 is a transcription repressor of CEACAM1. Furthermore, prostate cancer cells treated with trichostatin A (TSA), a specific histone deacetylase (HDAC) inhibitor, activated CEACAM1 transcriptional activity. This implies that HDACs are involved in CEACAM1 transcriptional activity. Mutation of the Sp2 DNA binding region on the CEACAM1 promoter inhibited TSA activation of CEACAM1 transcriptional activity. This indicates that HDACs inhibit CEACAM1 transcriptional activity through Sp2. Base on these results, we propose that Sp2 is critical for down-regulating CEACAM1 expression, and one mechanism by which Sp2 represses CEACAM1 expression is by recruiting HDAC to the CEACAM1 promoter in prostate cancer cells. Collectively, these findings provide novel insights into mechanisms that cause the down-regulation of CEACAM1 expression in prostate cancer cells. ^

DNA vaccination against the novel tumor antigen, MUC18, for the therapy of metastatic melanoma

Melanoma patients with metastases have a very low survival rate and limited treatment options. Therefore, the targeting of melanoma cells when they begin to invade and metastasize would be beneficial. A specific adhesion molecule that is upregulated at the vertical growth phase is the melanoma cell adhesion molecule (MCAM/MUC18). MUC18 is expressed in late primary and metastatic melanoma with little or no expression on normal melanocytes. MUC18 has been demonstrated to have a role in the progression and metastasis of human melanoma. We utilized the alphavirus-based DNA plasmid, SINCp, encoding full length human MUC18 for vaccination against B16F10 murine melanoma cells expressing human MUC18. The alphavirus-based DNA plasmid leads to the expression of large quantities of heterologous protein as well as danger signals due to dsRNA intermediates produced during viral replication. In a preventative primary tumor model and an experimental tumor model, mice vaccinated against human MUC18 had decreased tumor incidence and reduced lung metastases when challenged with B16F10 murine melanoma cells expressing human MUC18. In a therapeutic tumor model, vaccination against human MUC18 reduced the tumor burden in mice with pre-existing lung metastases but did not have a significant effect on therapeutic vaccination in a primary tumor model. We next cloned murine MUC18 into SINCp for use in determining the efficacy of vaccination against murine MUC18 in a syngeneic animal model. Mice were vaccinated and challenged in a primary tumor and experimental metastasis model. In both models, vaccination significantly reduced tumor incidence and lung metastases. Humoral and cell-mediated responses were then determined. Flow cytometry and immunohistochemistry showed that specific antibodies were developed from vaccination against both human and murine MUC18. IgG2a antibody isotype was also developed indicating a Th1 type response. ELISPOT results showed that mice vaccinated against human MUC18 created a specific T cell response to targets expressing human MUC18. Mice vaccinated against murine MUC18 raised specific effector cells against target cells expressing murine MUC18 in a cell killing assay. These results indicate that vaccination against MUC18 developed specific immune responses against MUC18 and were effective in controlling tumor growth in melanoma expressing MUC18. ^

Use of Echogenic Immunoliposomes for Delivery of both Drug and Stem Cells for Inhibition of Atheroma Progression

Use of Echogenic Immunoliposomes for Delivery of both Drug and Stem Cells for Inhibition of Atheroma Progression By Ali K. Naji B.S. Advisor: Dr. Melvin E. Klegerman PhD Background and significance: Echogenic liposomes can be used as drug and cell delivery vehicles that reduce atheroma progression. Vascular endothelial growth factor (VEGF) is a signal protein that induces vasculogenesis and angiogenesis. VEGF functionally induces migration and proliferation of endothelial cells and increases intracellular vascular permeability. VEGF activates angiogenic transduction factors through VEGF tyrosine kinase domains in high-affinity receptors of endothelial cells. Bevacizumab is a humanized monoclonal antibody specific for VEGF-A which was developed as an anti-tumor agent. Often, anti-VEGF agents result in regression of existing microvessels, inhibiting tumor growth and possibly causing tumor shrinkage with time. During atheroma progression neovasculation in the arterial adventitia is mediated by VEGF. Therefore, bevacizumab may be effective in inhibiting atheroma progression. Stem cells show an ability to inhibit atheroma progression. We have previously demonstrated that monocyte derived CD-34+ stem cells that can be delivered to atheroma by bifunctional-ELIP ( BF-ELIP) targeted to Intercellular Adhesion Molecule-1 (ICAM-1) and CD-34. Adhesion molecules such as ICAM-1 and vascular cell adhesion molecule-1 (VCAM-1) are expressed by endothelial cells under inflammatory conditions. Ultrasound enhanced liposomal targeting provides a method for stem cell delivery into atheroma and encapsulated drug release. This project is designed to examine the ability of echogenic liposomes to deliver bevacizumab and stem cells to inhibit atheroma progression and neovasculation with and without ultrasound in vitro and optimize the ultrasound parameters for delivery of bevacizumab and stem cells to atheroma. V Hypotheses: Previous studies showed that endothelial cell VEGF expression may relate to atherosclerosis progression and atheroma formation in the cardiovascular system. Bevacizumab-loaded ELIP will inhibit endothelial cell VEGF expression in vitro. Bevacizumab activity can be enhanced by pulsed Doppler ultrasound treatment of BEV-ELIP. I will also test the hypothesis that the transwell culture system can serve as an in vitro model for study of US-enhanced targeted delivery of stem cells to atheroma. Monocyte preparations will serve as a source of CD34+ stem cells. Specific Aims: Induce VEGF expression using PKA and PKC activation factors to endothelial cell cultures and use western blot and ELISA techniques to detect the expressed VEGF.  Characterize the relationship between endothelial cell proliferation and VEGF expression to develop a specific EC culture based system to demonstrate BEV-ELIP activity as an anti-VEGF agent. Design a cell-based assay for in vitro assessment of ultrasound-enhanced bevacizumab release from echogenic liposomes.  Demonstrate ultrasound delivery enhancement of stem cells by applying different types of liposomes on transwell EC culture using fluorescently labeled monocytes and detect the effect on migration and attachment rate of these echogenic liposomes with and without ultrasound in vitro.

Multiple loop structures critical for ligand binding of the integrin α4 subunit in the upper face of the β-propeller mode 1

A non-I-domain integrin, α4β1, recognizes vascular cell adhesion molecule 1 (VCAM-1) and the IIICS portion of fibronectin. To localize regions of α4 critical for ligand binding, we swapped several predicted loops within or near the putative ligand-binding site of α4 (which spans repeats 2–5 of the seven N-terminal repeats) with the corresponding regions of α5. Swapping residues 112–131 in repeat 2, or residues 237–247 in repeat 4, completely blocked adhesion to immobilized VCAM-1 and connecting segment 1 (CS-1) peptide. However, swapping residues 40–52 in repeat 1, residues 151–164 in repeat 3, or residues 282–288 (which contain a putative cation binding motif) in repeat 5 did not affect or only slightly reduced adhesion to these ligands. The binding of several function-blocking antibodies is blocked by swapping residues 112–131, 151–164, and 186–191 (which contain previously identified residues critical for ligand binding, Tyr-187 and Gly-190). These results are consistent with the recently published β-propeller folding model of the integrin α4 subunit [Springer, T. A. (1997) Proc. Natl. Acad. Sci. USA 94, 65–72], in which seven four-stranded β-sheets are arranged in a torus around a pseudosymmetric axis. The regions of α4 critical for ligand binding are adjacent to each other and are located in the upper face, the predicted ligand-binding site, of the β-propeller model, although they are not adjacent in the primary structure.

Nucleotide sequence of the Kaposi sarcoma-associated herpesvirus (HHV8)

The genome of the Kaposi sarcoma-associated herpesvirus (KSHV or HHV8) was mapped with cosmid and phage genomic libraries from the BC-1 cell line. Its nucleotide sequence was determined except for a 3-kb region at the right end of the genome that was refractory to cloning. The BC-1 KSHV genome consists of a 140.5-kb-long unique coding region flanked by multiple G+C-rich 801-bp terminal repeat sequences. A genomic duplication that apparently arose in the parental tumor is present in this cell culture-derived strain. At least 81 ORFs, including 66 with homology to herpesvirus saimiri ORFs, and 5 internal repeat regions are present in the long unique region. The virus encodes homologs to complement-binding proteins, three cytokines (two macrophage inflammatory proteins and interleukin 6), dihydrofolate reductase, bcl-2, interferon regulatory factors, interleukin 8 receptor, neural cell adhesion molecule-like adhesin, and a D-type cyclin, as well as viral structural and metabolic proteins. Terminal repeat analysis of virus DNA from a KS lesion suggests a monoclonal expansion of KSHV in the KS tumor.

Importance of newly generated neurons in the adult olfactory bulb for odor discrimination

In adult rodents, neurons are continually generated in the subventricular zone of the forebrain, from where they migrate tangentially toward the olfactory bulb, the only known target for these neuronal precursors. Within the main olfactory bulb, they ascend radially into the granule and periglomerular cell layers, where they differentiate mainly into local interneurons. The functional consequences of this permanent generation and integration of new neurons into existing circuits are unknown. To address this question, we used neural cell adhesion molecule-deficient mice that have documented deficits in the migration of olfactory-bulb neuron precursors, leading to about 40% size reduction of this structure. Our anatomical study reveals that this reduction is restricted to the granule cell layer, a structure that contains exclusively γ-aminobutyric acid (GABA)ergic interneurons. Furthermore, mutant mice were subjected to experiments designed to examine the behavioral consequences of such anatomical alteration. We found that the specific reduction in the newly generated interneuron population resulted in an impairment of discrimination between odors. In contrast, both the detection thresholds for odors and short-term olfactory memory were unaltered, demonstrating that a critical number of bulbar granule cells is crucial only for odor discrimination but not for general olfactory functions.

E-cadherin induces mesenchymal-to-epithelial transition in human ovarian surface epithelium

Ovarian carcinomas are thought to arise in the ovarian surface epithelium (OSE). Although this tissue forms a simple epithelial covering on the ovarian surface, OSE cells exhibit some mesenchymal characteristics and contain little or no E-cadherin. However, E-cadherin is present in metaplastic OSE cells that resemble the more complex epithelia of the oviduct, endometrium and endocervix, and in primary epithelial ovarian carcinomas. To determine whether E-cadherin was a cause or consequence of OSE metaplasia, we expressed this cell-adhesion molecule in simian virus 40-immortalized OSE cells. In these cells the exogenous E-cadherin, all three catenins, and F-actin localized at sites of cell–cell contact, indicating the formation of functional adherens junctions. Unlike the parent OSE cell line, which had undergone a typical mesenchymal transformation in culture, E-cadherin-expressing cells contained cytokeratins and the tight-junction protein occludin. They also formed cobblestone monolayers in two-dimensional culture and simple epithelia in three-dimensional culture that produced CA125 and shed it into the culture medium. CA125 is a normal epithelial-differentiation product of the oviduct, endometrium, and endocervix, but not of normal OSE. It is also a tumor antigen that is produced by ovarian neoplasms and by metaplastic OSE. Thus, E-cadherin restored some normal characteristics of OSE, such as keratin, and it also induced epithelial-differentiation markers associated with weakly preneoplastic, metaplastic OSE and OSE-derived primary carcinomas. The results suggest an unexpected role for E-cadherin in ovarian neoplastic progression.

Interaction of voltage-gated sodium channels with the extracellular matrix molecules tenascin-C and tenascin-R

The type IIA rat brain sodium channel is composed of three subunits: a large pore-forming α subunit and two smaller auxiliary subunits, β1 and β2. The β subunits are single membrane-spanning glycoproteins with one Ig-like motif in their extracellular domains. The Ig motif of the β2 subunit has close structural similarity to one of the six Ig motifs in the extracellular domain of the cell adhesion molecule contactin (also called F3 or F11), which binds to the extracellular matrix molecules tenascin-C and tenascin-R. We investigated the binding of the purified sodium channel and the extracellular domain of the β2 subunit to tenascin-C and tenascin-R in vitro. Incubation of purified sodium channels on microtiter plates coated with tenascin-C revealed saturable and specific binding with an apparent Kd of ≈15 nM. Glutathione S-transferase-tagged fusion proteins containing various segments of tenascin-C and tenascin-R were purified, digested with thrombin to remove the epitope tag, immobilized on microtiter dishes, and tested for their ability to bind purified sodium channel or the epitope-tagged extracellular domain of β2 subunits. Both purified sodium channels and the extracellular domain of the β2 subunit bound specifically to fibronectin type III repeats 1–2, A, B, and 6–8 of tenascin-C and fibronectin type III repeats 1–2 and 6–8 of tenascin-R but not to the epidermal growth factor-like domain or the fibrinogen-like domain of these molecules. The binding of neuronal sodium channels to extracellular matrix molecules such as tenascin-C and tenascin-R may play a crucial role in localizing sodium channels in high density at axon initial segments and nodes of Ranvier or in regulating the activity of immobilized sodium channels in these locations.

Nerve growth factor control of neuronal expression of angiogenetic and vasoactive factors

In postnatal tissues, angiogenesis occurs in nontumoral conditions on appropriate stimuli. In the nervous tissue, hypoxia, neural graft, increased neural function, and synaptic activity are associated with neoangiogenesis. We have investigated the occurrence of neoangiogenesis in the superior cervical ganglia (scg) of newborn rats treated for 8–21 days with 6-hydroxy-dopamine (6-OHDA), nerve growth factor (NGF), or 6-OHDA + NGF. The two latter treatments induced a significant increase in scg size. However, the increase after combined treatment far exceeded that of NGF alone. Similarly, histological and histochemical analysis revealed neuronal hypertrophy and endothelial cell hyperplasia associated with stromal hypertrophy (as described by laminin immunostaining) and increased vascular bed (as revealed by platelet/endothelial cell adhesion molecule-1 immunostaining) in 6-OHDA + NGF-treated pups. NGF, either alone or associated with 6-OHDA, also induced a significant up-regulation of NADPH diaphorase, neuronal nitric oxide synthase, and vascular endothelial growth factor expression in scg neurons. The present investigation suggests that the increase of scg size induced by NGF and 6-OHDA + NGF is associated with neoangiogenesis, and that the induction of vasoactive and angiogenic factors in neurons represents a further and previously undisclosed effect of NGF.

Differential regulation of beta 1 and beta 2 integrin avidity by chemoattractants in eosinophils.

The CC chemokines regulated on activation normal T expressed and secreted (RANTES) and monocyte chemotactic protein 3 (MCP-3), and the anaphylatoxin C5a, induce activation, degranulation, chemotaxis, and transendothelial migration of eosinophils. Adhesion assays on purified ligands showed differential regulation of beta 1 and beta 2 integrin avidity in eosinophils. Adhesiveness of VLA-4 (alpha 4 beta 1, CD29/CD49d) for vascular cell adhesion molecule 1 or fibronectin was rapidly increased but subsequently reduced by RANTES, MCP-3, or C5a. The deactivation of VLA-4 lead to cell detachment, whereas phorbol 12-myristate 13-acetate induced sustained activation of VLA-4. In contrast, chemoattractants stimulated a prolonged increase in the adhesiveness of Mac-1 (alpha M beta 2, CD11b/CD18) for intercellular adhesion molecule 1. Inhibition by pertussis toxin confirmed signaling via G protein-coupled receptors. Chemoattractants induced transient, while phorbol 12-myristate 13-acetate induced sustained actin polymerization. Disruption of actin filaments by cytochalasins inhibited increases in avidity of VLA-4 but not of Mac-1. Chemoattractants did not upregulate a Mn2+-inducible beta 1 neoepitope defined by the mAb 9EG7, but induced prolonged expression of a Mac-1 activation epitope recognized by the mAb CBRM1/5. This mAb inhibited chemoattractant-stimulated adhesion of eosinophils to intercellular adhesion molecule 1. Thus, regulation of VLA-4 was dependent on the actin cytoskeleton, whereas conformational changes appeared to be crucial for activation of Mac-1. To our knowledge, this is the first demonstration that physiological agonists, such as chemoattractants, can differentially regulate the avidity of a beta 1 and a beta 2 integrin expressed on the same leukocyte.

Poly (alpha 2,8-deaminoneuraminic acid) is expressed in lung on a single 150-kDa glycoprotein and is an oncodevelopmental antigen.

Homopolymers of alpha 2,8-linked N-acetylneuraminic acid [poly(alpha 2,8-Neu5Ac)] of the neural cell adhesion molecule NCAM have been shown to be temporally expressed during lung development and represent a marker for small cell lung carcinoma. We report the presence of a further polysialic acid in lung that consists of oligo/polymers of alpha 2,8-linked deaminoneuraminic acid residues [poly (alpha 2,8-KDN)], as detected with a monoclonal antibody in conjunction with a specific sialidase. Although the various cell types forming the bronchi, alveolar septs, and blood vessels were positive for poly (alpha 2,8-KDN) by immunohistochemistry, this polysialic acid was found on a single 150-kDa glycoprotein by immunoblot analysis. The poly(alpha 2,8-KDN)-bearing glycoprotein was not related to an NCAM protein based on immunochemical criteria. The expression of the poly (alpha 2,8-KDN) was developmentally regulated as evidenced by its gradual disappearance in the rat lung parenchyma commencing 1 week after birth. In adult lung the blood vessel endothelia and the smooth muscle fibers of both blood vessels and bronchi were positive but not the bronchial and alveolar epithelium. The poly (alpha 2,8-KDN)-bearing 150-kDa glycoprotein became reexpressed in various histological types of lung carcinomas and cell lines derived from them and represents a new oncodevelopmental antigen in lung.

Fasciclin II controls proneural gene expression in Drosophila.

Fasciclin II (Fas II), an NCAM-like cell adhesion molecule in Drosophila, is expressed on a subset of embryonic axons and controls selective axon fasiculation. Fas II is also expressed in imaginal discs. Here we use genetic analysis to show that Fas II is required for the control of proneural gene expression. Clusters of cells in the eye-antennal imaginal disc express the achaete proneural gene and give rise to mechanosensory neurons; other clusters of cells express the atonal gene and give rise to ocellar photoreceptor neurons. In fasII loss-of-function mutants, the expression of both proneural genes is absent in certain locations, and, as a result, the corresponding sensory precursors fail to develop. In fasII gain-of-function conditions, extra sensory structures arise from this same region of the imaginal disc. Mutations in the Abelson tyrosine kinase gene show dominant interactions with fasII mutations, suggesting that Abl and Fas II function in a signaling pathway that controls proneural gene expression.

One sequence, two folds: a metastable structure of CD2.

When expressed as part of a glutathione S-transferase fusion protein the NH2-terminal domain of the lymphocyte cell adhesion molecule CD2 is shown to adopt two different folds. The immunoglobulin superfamily structure of the major (85%) monomeric component has previously been determined by both x-ray crystallography and NMR spectroscopy. We now describe the structure of a second, dimeric, form present in about 15% of recombinant CD2 molecules. After denaturation and refolding in the absence of the fusion partner, dimeric CD2 is converted to monomer, illustrating that the dimeric form represents a metastable folded state. The crystal structure of this dimeric form, refined to 2.0-A resolution, reveals two domains with overall similarity to the IgSF fold found in the monomer. However, in the dimer each domain is formed by the intercalation of two polypeptide chains. Hence each domain represents a distinct folding unit that can assemble in two different ways. In the dimer the two domains fold around a hydrophilic interface believed to mimic the cell adhesion interaction at the cell surface, and the formation of dimer can be regulated by mutating single residues at this interface. This unusual misfolded form of the protein, which appears to result from inter- rather than intramolecular interactions being favored by an intermediate structure formed during the folding process, illustrates that evolution of protein oligomers is possible from the sequence for a single protein domain.

Silencing of the E-cadherin invasion-suppressor gene by CpG methylation in human carcinomas.

E-Cadherin, a cell adhesion molecule, which plays a key role in maintaining the epithelial phenotype, is regarded as an invasion-suppressor gene in light of accumulating evidence from in vitro experiments and clinical observations. In an attempt to clarify the mechanism responsible for inactivation of this gene in carcinomas, we investigated the methylation state around the promoter region by digestion of DNA with the methylation-sensitive restriction enzyme Hpa II, as CpG methylation of the promoter has been postulated to be a mechanism of transcriptional inactivation of some genes. We found that E-cadherin expression-negative carcinoma cell lines were accompanied by the hypermethylation state, whereas E-cadherin-positive cell lines were not. Furthermore, treatment of E-cadherin-negative carcinoma cells with the demethylating agent 5-azacytidine resulted in reexpression of the gene and reversion of scattered spindle-shaped cells to cells with epithelial morphology. These results suggest that hypermethylation around the promoter may be a mechanism of E-cadherin inactivation in human carcinomas and that treatment of E-cadherin-inactivated cells with a demethylating agent may cause gene expression reversion leading to epithelial morphogenesis with acquisition of the homophilic cell-cell adhesive property.