Katherine E. Kilpatrick

TNFα-Mediated Inhibition and Reversal of Adipocyte Differentiation Is Accompanied by Suppressed Expression of PPARγ without Effects on Pref-1 Expression1

Tumor necrosis factor α (TNFα) is a polypeptide hormone with pleiotropic effects on cellular proliferation and differentiation. To investigate how TNFα inhibits and reverses adipocyte differentiation, we studied the expression of two factors involved in the adipocyte differentiation process. Peroxisome proliferator-activated receptor γ (PPARγ) is a positive regulator of adipogenesis, whereas preadipocyte factor 1 (Pref-1) inhibits adipocyte differentiation. The expression patterns of both PPARγ and Pref-1 change during early stages of adipocyte differentiation. Decreased expression of Pref-1 and increased expression of PPARγ occur 1 day and 2 days, respectively, after 3T3-L1 cells reach confluence. During TNFα-mediated inhibition of adipocyte differentiation, PPARγ messenger RNA (mRNA) expression stays at low levels. In contrast, TNFα treatment has no effect on the normal decrease in Pref-1 gene expression that occurs during adipogenesis. We observed that certain cytokine and growth factors[ such as TNFα,...

GRID: a novel Grb-2-related adapter protein that interacts with the activated T cell costimulatory receptor CD28.

Adapter proteins such as Grb2 play a central role in the formation of signaling complexes through their association with multiple protein binding partners. These interactions are mediated by specialized domains such as the well-characterized Src homology SH2 and SH3 motifs. Using yeast three-hybrid technology, we have identified a novel adapter protein, expressed predominantly in T lymphocytes, that associates with the activated form of the costimulatory receptor, CD28. The protein is a member of the Grb2 family of adapter proteins and contains an SH3-SH2-SH3 domain structure. A unique glutamine/proline-rich domain (insert domain) of unknown function is situated between the SH2 and N-terminal SH3 domains. We term this protein GRID for Grb2-related protein with insert domain. GRID coimmunoprecipitates with CD28 from Jurkat cell lysates following activation of CD28. Using mutants of CD28 and GRID, we demonstrate that interaction between the proteins is dependent on phosphorylation of CD28 at tyrosine 173 and integrity of the GRID SH2 domain, although there are also subsidiary stabilizing contacts between the PXXP motifs of CD28 and the GRID C-terminal SH3 domain. In addition to CD28, GRID interacts with a number of other T cell signaling proteins, including SLP-76 (SH2 domain-containing leukocyte protein of 76 kDa), p62dok, and RACK-1 (receptor for activated protein kinase C-1). These findings suggest that GRID functions as an adapter protein in the CD28-mediated costimulatory pathway in T cells.

Production of a urokinase plasminogen activator-IgG fusion protein (uPA-IgG) in the baculovirus expression system ☆

Numerous studies have demonstrated the importance of urokinase plasminogen activator (uPA) and its receptor, uPAR, in the processes of tumor progression and metastasis. Thus, the uPA/uPAR interaction may represent an important target for inhibiting metastatic disease. The baculovirus expression system was used to produce high levels of a secreted uPA-Immunoglobulin G fusion protein (uPA-IgG) which could then be used for displacing uPA from the surface of tumor cells. The recombinant uPA-IgG fusion protein was placed under the control of either the viral polyhedrin promoter or a copy of the viral basic protein promoter. Recombinant viruses were then used to infect Sf9 and BTI-Tn-5B1-4 cells. Infection of both cell types resulted in the production of secreted uPA-IgG. The molecular mass of the secreted protein as determined by SDS-PAGE was approximately 40 kDa. The highest level of secreted uPA-IgG, 444 microg/ml, was found in the culture medium of BTI-Tn-5B1-4 cells 72 h post-infection with the basic protein promoter-uPA-IgG virus. In the case of Sf9 cells, the highest level of secreted protein was 195 microg/ml. The amount of cell-associated uPA-IgG in infected BTI-Tn-5B1-4 cells was significantly less than that of infected Sf9 cells, reflecting the superior secretory capability of the BTI-Tn-5B1-4 cells. The uPA-IgG was readily purified using a combination of zinc chelate and sephacryl S-100 column chromatography. Routinely, greater than 100 mg of greater than 95% pure protein could be obtained per liter of culture medium collected at 72 h post-infection of BTI-Tn-5B1-4 cells with the basic protein promoter virus. BIAcore analysis and competition binding assays using LOX human malignant melanoma cells expressing uPAR indicated that the purified recombinant protein possessed similar ligand binding characteristics to that of human uPA.

Inhibition of establishment of primary and micrometastatic tumors by a urokinase plasminogen activator receptor antagonist

Tumor establishment and metastasis are dependent on extracellular matrix proteolysis, tumor cell migration, and angiogenesis. Urokinase plasminogen activator (uPA) and its receptor are essential mediators of these processes. The purpose of this study was to investigate the effect of a recombinant human uPAR antagonist on growth, establishment, and metastasis of tumors derived from human cancer cell lines. A noncatalytic recombinant protein, consisting of amino acids 1-137 of human uPA and the CH2 and CH3 regions of mouse IgG1 (uPA-IgG), was expressed, purified, and shown to bind specifically to human uPAR and to saturate the surface of human tumor cells which express uPAR. Daily i.p. administration of uPA-IgG to nude mice extended latencies of unstaged tumors derived from Lox melanoma and SW48 colon carcinoma cells by 7.7 and 5.5 days, respectively. uPA-IgG treatment did not affect the growth of Lox or KB tumors staged to 200 mg before antagonist treatment commenced. The effect of uPA-IgG on the establishment of micrometastases was assessed in SCID mice. KB head/neck tumor cells were injected in the tail vein and allowed to seed for 48 h before initiation of daily i.p. injections of uPA-IgG for 24 days. The number of lung colonies ranged between 5 and 30% of vehicle-treated mice in two separate experiments. Furthermore, a single 800 μg dose of uPA-IgG administered 1 h prior to tail vein injection of KB cells reduced lung colony formation to just 3.5% of vehicle-treated SCID mice. These data demonstrate that antagonism of uPAR arrested metastasis and inhibited the establishment of primary tumors and micrometastases. Thus, small molecule uPAR antagonists may serve as useful adjuvant agents in combination with existing cancer chemotherapy.

Shorter development of immunoassay for drugs: application of the novel RIMMS technique enables rapid production of monoclonal antibodies to ranitidine

High affinity, specific murine monoclonal antibodies have been produced for ranitidine using the novel RIMMS (repetitive immunizations, multiple sites) technique. We demonstrate that this technique can be employed to produce high affinity monoclonal antibodies to drug haptens in approximately 1 month; whereas, conventional techniques typically require 3-9 months. Polyclonal antiserum development typically requires at least 6 months. Consequently, RIMMS has a clear impact allowing reagent antibodies to be available earlier in the drug development process. Isotyping studies demonstrated that the developed antibodies are either IgG1 or IgG2b immunoglobulins which confirms that the technique produces class-switched, affinity matured reagent antibodies. The most promising monoclonal antibody for quantitative applications afforded similar sensitivity, by competitive ELISA, to the established sheep polyclonal anti-ranitidine sera. The calibration range, estimated as the limits between the asymptotic regions of calibration graphs, is 0.5-41.2 ng ranitidine per well. Specificity studies indicated that the monoclonal antibody afforded superior selectivity, yielding only 4.1% cross-reactivity with the ranitidine sulphoxide metabolite; the corresponding value for the antiserum was 8.6%. Both reagents had similar cross-reactivities with the N-oxide metabolite.

High-Affinity Monoclonal Antibodies to PED/PEA-15 Generated Using 5 μg of DNA

Class-switched, affinity-matured murine monoclonal antibody (MAb) producing cell lines reactive with PED/PEA-15 were generated and isolated in less than 4 weeks following polynucleotide immunizations using only 5 μg of DNA in conjunction with the Powderject gene gun. Somatic fusions of peripheral lymph node cells were performed 13 days after initiating delivery of DNA encoding the target antigen. The data presented demonstrates the rapid production, identification, and characterization of class-switched, affinity-matured MAbs that bind PED/PEA-15. The reported strategy enabled the rapid development of MAbs that are useful in enzyme-linked immunoadsorbent assay (ELISA), Western blotting, and immunoprecipitations.

In Vivo Expression of a GST-Fusion Protein Mediates the Rapid Generation of Affinity Matured Monoclonal Antibodies Using DNA-Based Immunizations

Previously we demonstrated the rapid generation of affinity matured monoclonal antibody (MAb) producing cell lines following gene gun delivery of DNA using a mammalian expression vector (pAlpha/hFc), which enables the expression of human Fc-chimera proteins in vivo. Here we compare the pAlpha/hFc vector to modified vectors that replace human IgG(1) with either a Glutathione-S-Transferase (GST) fusion protein or a mouse IgG(2c) (mFc) fusion protein. We report that in vivo expression of a GST-chimera results in the rapid generation of affinity matured MAbs, comparable with antibodies raised using the pAlpha/hFc vector, that were reactive with annexin V. The mFc vector failed to induce early antigen-specific B-cell responses suitable for MAb development.

Monoclonal Antibodies Generated Against Recombinant ATM Support Kinase Activity

We report on the rapid generation of two monoclonal antibodies, ATM A16.35 and ATM D16.11, that bind to the kinase domain of mutated ataxia telangiectasia (ATM). These antibodies were generated against E. coli-expressed recombinant protein using the RIMMS strategy. We show that ATM A16.35 binds ATM by Western blot analysis, and ATM D16.11 forms immune complexes with native ATM in immunoprecipitations without neutralizing kinase activity.