Insulin effects and receptor function in bovine aorta endothelial cells

The interaction of insulin with bovine aorta endothelial (BAE) cells has been studied to determine the effect of insulin on endothelial cells, and investigate the function of the insulin receptor in this cell type. BAE cell insulin receptor is similiar to insulin receptor in other cell types in the time to attain equilibrium binding, its physical properties in a solubilized assay system and affinity for insulin in the low nanomolar range. However, BAE cell insulin receptor has unusual properties in its interaction with insulin at 4$\sp\circ$C that include: (1) the inability to completely dissociate prebound $\sp{125}$I-insulin by dilution with excess insulin or acid rinse treatment, indicating that binding is not completely reversible (2) the inability to remove prebound insulin with trypsin and other proteases (3) the implication of disulfide complex formation during binding (4) the inability of pretreatment with trypsin to lower cell surface binding capacity and (5) the suppression of insulin binding by bacitracin. Interactions of insulin with the receptor at 37$\sp\circ$C showed that (1) BAE cells degrade insulin, but not as extensively as other cell types, and (2) an unusual biphasic interaction of insulin with the BAE cells is observed which is indicative of some regulatory mechanism which modulates binding affinity. Functional characterization of the BAE cell insulin receptor revealed that insulin-induced downregulation and phosphorylation of the receptor was observed, and the extent of these processes were comparable to that demonstrated in non-endothelial cell types. However, in contrast to other cell types, insulin did not stimulate deoxyglucose uptake in BAE cells. We were unable to confirm the receptor-mediated transport of insulin by the receptor across the endothelial cell monolayer as reported by a previous investigator. We could not demonstrate a role for the receptor to promote acute intracellular accumulation of insulin as postulated by several investigators. Thus, while BAE cell insulin receptor has many properties that are similiar to those in other cell types, it is distinctly different in its nondissociable binding at 4$\sp\circ$C, its interaction with insulin at 37$\sp\circ$C, and its functional role in the BAE cell. ^

Gene transfer by viral vectors

To initiate our clinical trial for chemotherapy protection, I established the retroviral vector system for human MDR1 cDNA gene transfer. The human MDR1 cDNA continued to be expressed in the transduced bone marrow cells after four cohorts of serial transplants, 17 months after the initial transduction and transplant. In addition, we used this retroviral vector pVMDR1 to transduce human bone marrow and peripheral blood CD34$\sp+$ cells on stromal monolayer in the presence of hematopoietic growth factors. These data suggest that the retroviral vector pVMDR1 could modify hematopoietic precursor cells with a capacity for long-term self renewal. Thus, it may be possible to use the MDR1 retroviruses to confer chemotherapeutic protection on human normal hematopoietic precursor cells of ovarian and breast cancer patients in whom high doses of MDR drugs may be required to control the diseases.^ Another promising vector system is recombinant adeno-associated virus (rAAV) vector. An impediment to use rAAV vectors is that production of rAAV vectors for clinical use is extremely cumbersome and labor intensive. First I set up the rAAV vector system in our laboratory and then, I focused on studies related to the production of rAAV vectors for clinical use. By using a self-inactivating retroviral vector carrying a selection marker under the control of the CMV immediate early promoter and an AAV genome with the deletion of both ITRs, I have developed either a transient or a stable method to produce rAAV vectors. These methods involve infection only and can generate high-titer rAAV vectors (up to 2 x 10$\sp5$ cfu/ml of CVL) with much less work.^ Although recombinant adenoviral vectors hardly infect early hematopoietic precursor cells lacking $\alpha\sb v\beta\sb5$ or $\alpha\sb v\beta\sb3$ integrin on their surface, but efficiently infect other cells, we can use these properties of adenoviral vectors for bone marrow purging as well as for development of new viral vectors such as pseudotyped retroviral vectors and rAAV vectors. Replacement of self-inactivating retroviral vectors by recombinant adenoviral vectors will facilitate the above strategies for production of new viral vectors. In order to accomplish these goals, I developed a new method which is much more efficient than the current methods to construct adenoviral vectors. This method involves a cosmid vector system which is utilized to construct the full-length recombinant adenoviral vectors in vitro.^ First, I developed an efficient and flexible method for in vitro construction of the full-length recombinant adenoviral vectors in the cosmid vector system by use of a three-DNA fragment ligation. Then, this system was improved by use of a two-DNA fragment ligation. The cloning capacity of recombinant adenoviral vectors constructed by this method to develop recombinant adenoviral vectors depends on the efficiency of transfection only. No homologous recombination is required for development of infectious adenoviral vectors. Thus, the efficiency of generating the recombinant adenoviral vectors by the cosmid method reported here was much higher than that by the in vitro direct ligation method or the in vivo homologous recombination method reported before. This method of the in vitro construction of recombinant adenoviral vectors in the cosmid vector system may facilitate the development of adenoviral vector for human gene therapy. (Abstract shortened by UMI.) ^

Targeting of {\it HER\/}-{\it 2\/}/{\it neu\/} with E1A gene therapy

Amplification or overexpression of HER-2/neu has been demonstrated in human cancers of the ovary, breast, lung and correlated with chemoresistance and poor clinic prognosis. We have previously found that the adenovirus type 5 early region 1A (E1A) gene product can repress the overexpression and suppress the tumorigenic potential of HER-2/neu-overexpressing cancer cells. In addition, E1A has been reported to induce apoptosis and inhibit the metastatic potential of tumor cells. Therefore, E1A could be considered as a tumor suppressor gene in HER-2/neu-overexpressing cancer cells. To develop an efficient HER-2/neu-targeting gene therapy with E1A, adenoviral vector or cationic liposome was used to introduce E1A into human ovarian, breast and lung cancer cells. Successful therapeutic effects were achieved.^ A replication-deficient adenovirus containing the E1A gene, Ad.E1A(+), was used to infect HER-2/neu-overexpressing human ovarian cancer cell line. Ovarian cancer growth in vitro and colony formation in soft agarose were greatly inhibited.^ To examine tumor suppressor function of E1A in breast cancer, we introduced E1A in vitro by adenovirus into both HER-2/neu-overexpressing and low-expressing human breast cancer cell lines. In HER-2/neu-overexpressing cells, E1A greatly inhibited tumor cell growth in vitro and colony formation in soft agarose. However, in low HER-2/neu expressing cancer cell lines, E1A could only reduce colony formation in soft agarose but had no significant effect on cell growth in monolayer, indicating different effects of E1A in these two types of cancer cells. To test the local therapeutic efficacy of E1A, we used either adenovirus- or liposome-mediated E1A gene delivery systems in an orthotopic breast cancer animal model.^ To test the therapeutic efficacy of systemically-delivered E1A in vivo lung cancer, we treated mice bearing intratracheal lung cancer by i.v. tail injections of Ad.E1A(+). As a result, Ad.E1A(+) suppressed HER-2/neu overexpression and inhibited intratracheal lung cancer growth. However, no significant tumor suppression effect of Ad.E1A(+) was observed in mice bearing HER-2/neu low expressing cell line when the same therapeutic procedure was followed. (Abstract shortened by UMI.) ^

Analysis of pp60$\sp{{\rm c}{-}src}$ and pp62$\sp{{\rm c}{-}yes}$ intracellular protein tyrosine kinase function in colon tumor cell growth regulation using herbimycin A, a tyrosine kinase specific inhibitor

Two approaches were utilized to investigate the role of pp60c-src activation in growth control of model colon tumor cell lines. The first approach involved analysis of pp60c-src activity in response to growth factor treatment to determine if transient activation of the protein was associated with ligand induced mitogenic signal transduction as occurs in non-colonic cell types. Activation of pp60c-src was detected using colon tumor cell lysates after treatment with platelet derived growth factor (PDGF). Activation of pp60c-src was also detected in response to epidermal growth factor (EGF) treatment using cellular lysates and intact cells. In contrast, down-regulation of purified pp60c-src occurred after incubation with EGF-treated EGFr immune complexes in vitro suggesting additional cellular events were potentially required for the stimulatory response observed in intact cells. The results demonstrated activation of pp60c-src in colon tumor cells in response to PDGF and EGF which is consistent with the role of the protein in mitogenic signal transduction in non-colonic cell types.^ The second approach used to study the role of pp60c-src activation in colonic cell growth control focused on analysis of the role of constitutive activation of the protein, which occurs in approximately 80% of colon tumors and cell lines, in growth control. These studies involved analysis of the effects of the tyrosine kinase specific inhibitor Herbimycin A (HA) on monolayer growth and pp60c-src enzymatic activity using model colon tumor cell lines. HA induced dose-dependent growth inhibition of all colon tumor cell lines examined possessing elevated pp60c-src activity. In HT29 cells the dose-dependent growth inhibition induced by HA correlated with dose-dependent pp60c-src inactivation. Inactivation of pp60c-src was shown to be an early event in response to treatment with HA which preceded induction of HT29 colon tumor cell growth inhibition. The growth effects of HA towards the colon tumor cells examined did not appear to be associated with induction of differentiation or a cytotoxic mechanism of action as changes in morphology were not detected in treated cells and growth inhibition (and pp60c-src inactivation) were reversible upon release from treatment with the compound. The results suggested the constitutive activation of pp60c-src functioned as a proliferative signal in colon tumor cells. Correlation between pp60c-src inactivation and growth inhibition was also observed using HA chemical derivatives confirming the role of tyrosine kinase inactivation by these compounds in inhibition of mitogenic signalling. In contrast, in AS15 cells possessing specific antisense mRNA mediated inactivation of pp60c-src, HA-induced inactivation of the related pp62c-yes tyrosine kinase, which is also activated during colon tumor progression, was not associated with induction of monolayer growth inhibition. These results suggested a function for the constitutively activated pp62c-yes protein in colon tumor cell proliferation which was different from that of activated pp60c-src. (Abstract shortened by UMI.) ^

CYTOLOGICAL, ULTRASTRUCTURAL, AND BIOCHEMICAL STUDIES OF THE STRUCTURE OF PREMATURELY CONDENSED CHROMOSOMES

The phenomenon of premature chromosome condensation, resulting from fusion between mitotic and interphase cells, includes dissolution of the interphase nuclear framework, thus allowing a direct visualization of interphase chromosomes. Light microscope morphology of prematurely condensed chromosomes (PCC) from synchronized HeLa cells supports the model of an interphase "chromosome condensation cycle". PCC are increasingly attenuated as cells progress through G(,1). A maximum degree of decondensation is observed at active sites of DNA replication during S phase, and a condensed morphology is rapidly resumed following completion of replication of a chromosome segment.^ To permit ultrastructural and biochemical studies of PCC, a procedure was developed to induce premature chromosome condensation at high frequency. This was achieved by polyethylene glycol (PEG)-mediated fusion of a dense monolayer of mitotic and interphase cells induced by centrifugation onto lectin-coated culture dishes. Using this method, PCC induction frequencies of 60-90% are routinely obtained.^ Scanning electron microscope analysis of PCC spreads revealed that the extension of PCC during progression through G(,1) is accompanied by a transition of the basic 30 nm chromatin fiber from tightly packed looping fibers to extended longitudinal fibers. Sites of active DNA replication is S-PCC were indicated to be organized a single longitudinal fibers. Following replication of a chromosome segment, a rapid reorganization from the extended longitudinal fiber to packed looping fibers occurs. The postreplication maturation process appears to include the assembly of a chromosome core consisting of multiple longitudinal fibers.^ The role of histone H1 phosphorylation in PCC formation was investigated by acidurea polyacrylamide gel electrophoresis of total histone extracted from metaphase chromosomes and PCC following high frequency fusion. This investigation failed to demonstrate an extensive phosphorylation of H1 associated with PCC formation. However, significant dephosphorylation of superphosphorylated metaphase chromosome H1 was observed, indicating that interphase H1-phosphatase activity is dominant over metaphase H1 kinase activity. These observations provide evidence against models suggesting a role for H1 superphosphorylation in triggering mitotic condensation of chromosomes. ^

Identification and characterization of antigens and potential virulence factors in Enterococcus

Enterococci are normal flora in the human intestinal tract, and also one of the leading causes of nosocomial infections, with most of the clinical isolates being Enterococcus faecalis and Enterococcus faecium. Despite extensive studies on the antibiotic resistance, the pathogenicity of enterococci is not well understood, especially for E. faecium. To identify potential virulence factors based on their antigenicity during infection, E. faecium genomic libraries were constructed and screened using sera from patients with E. faecium endocarditis. ^ As one of my projects, total polysaccharides were extracted from E. faecalis OG1RF and from two epa mutants constructed previously, TX5179 and TX5180, and western blots with patient sera showed that an immuno-reactive polysaccharide present in wild type OG1RF was not produced by either of the two epa mutants. The epa mutants were more sensitive to ethanol stress, neutrophil killing and neutrophil phagocytosis than the wild type OG1RF. ^ Expression of virulence factors is commonly regulated by two component systems. A BLAST search was performed to identify potential two component systems in the E. faecalis V583 genome database using PhoP/PhoS as query sequences, and 11 gene pairs were identified, seven of which were disrupted in E. faecalis OGIRF. ^ Finally, an in vitro translocation model was established for enterococci. E. faecalis strain OG1RF and E. faecium strain DO were shown to be able to translocate across a T84 monolayer, while E. coli strain DH5α and E. faecalis strain E1 could not. ^ In conclusion, several E. faecium antigens expressed in infection (whose antibodies present in sera from patients with E. faecium endocarditis) were identified, two of which, SagA and GlyA, were characterized and suggested to be involved in cell wall metabolism. E. faecalis epa gene cluster (involving in polysaccharide biosynthesis and known to be involved in virulence of E. faecalis in mice) was shown to be involved in hindering neutrophil killing. Several two-component systems were identified in E. faecalis and two of which, EtaRS and EtbRS, were involved in E. faecalis virulence in a mouse peritonitis model.^

Dielectrophoresis-based analyte separation and analysis

Dielectrophoresis—the tendency of a material of high dielectric permittivity to migrate in an electrical field gradient to a region of maximum field strength—provides an ideal motive force for manipulating small volumes of biological analytes in microfluidic microsystems. The work described in this thesis was based on the hypothesis that dielectrophoresis could be exploited to provide high-resolution cell separations in microsystems as well as a means for the electrically-controllable manipulation of solid supports for molecular analysis. To this end, a dielectrophoretic/gravitational field-flow-fractionation (DEP/G-FFF) system was developed and the separation performance evaluated using various types and sizes of polystyrene microspheres as model particles. It was shown that separation of the polystyrene beads was based on the differences in their effective dielectrophoretic properties. The ability of an improved DEP/G-FFF system to separate genetically identical, but phenotypically dissimilar cell types was demonstrated using mixtures of 6m2 mutant rat kidney cells grown under transforming and non-transforming culture conditions. Additionally, a panel of engineered dielectric microspheres was designed with specific, predetermined dielectrophoretic properties such that their dielectrophoretic behaviors would be controllable and predictable. The fabrication method involved the use of gold-coated polystyrene microsphere cores coated with a self-assembled monolayer of alkanethiol and, optionally, a self-assembled monolayer of phospholipid to form a thin-insulating-shell-over-conductive-interior structure. The successful development of the DEP/G-FFF separation system and the dielectrically engineered microspheres provides proof-of-principle demonstrations of enabling dielectrophoresis-based microsystem technology that should provide powerful new methods for the manipulation, separation and identification of analytes in many diverse fields. ^

Roles of Alix in regulating cell morphology

Mammalian Alix (ALG2-interacting protein X&barbelow;) is a conserved adaptor protein that is involved in endosomal trafficking, apoptosis and growth factor receptor turnover. Accumulating evidence also indicates that Alix plays roles in promoting/maintaining spread and aligned fibroblast morphology in monolayer culture. Since cell morphology is determined by the structure and dynamics of an integrin-mediated transmembrane protein network that links extracellular matrix to intracellular cytoskeleton, we hypothesized that Alix plays direct or indirect roles in regulating certain components or steps in this transmembrane protein network. To test this hypothesis, we first examined the subcellular localization of Alix and discovered that, as a predominantly cytoplasmic protein, Alix is also present on the substratum/cell surface and in the conditioned medium of fibroblast cultures. Further, precoating of culture surfaces with recombinant Alix promotes spreading and fibronectin assembly to NIH/3T3 cells, and siRNA-mediated Alix knockdown in W138 cells has the opposite effects. These findings indicate the extracellular functions of Alix in regulating cell spreading and extracellular matrix assembly. In a separate study, we analyzed Alix immunocomplexes from normal fibroblast W138 cells by mass spectrometry and identified actin as a major partner protein of Alix. Follow-up studies demonstrated that Alix preferentially binds filamentous actin (F-actin) in vitro and is required for maintaining normal F-actin content and proper actin cytoskeleton assembly in W138 cells. These findings establish direct and essential roles of Alix in regulating actin cytoskeleton. Finally, we investigated the effects of Alix knockdown on the activation and subcellular localization of FAK and Pyk2, the focal adhesion kinases required for cell spreading/migration by promoting turnover of integrin-mediated cell adhesions. We discovered that Alix knockdown inhibits FAK and Pyk2 localizations to focal adhesions or plasma membrane, in association with characteristics of reduced turnover of focal adhesions. These findings reveal a positive role of Alix in focal adhesion turnover. Based on these results, we conclude that Alix targets both intracellularly and extracellularly components to regulate extracellular matrix remodeling, actin cytoskeleton assembly and focal adhesion turnover. A combination of these three functions of Alix explains its crucial role in regulating spread and aligned fibroblast morphology. ^