Chromosomal integration of transduced recombinant baculovirus DNA in mammalian cells.

Our group and others have recently demonstrated the ability of recombinant baculoviruses to transduce mammalian cells at high frequency. To further characterize the use of baculovirus as a mammalian gene delivery system, we examined the status of transduced DNA stably maintained in Chinese hamster ovary (CHO) cells. Four independent clones carrying two introduced markers, the genes for neomycin resistance (Neo) and green fluorescent protein (GFP), were selected. PCR analysis, Southern blotting, and DNA sequencing showed that discrete portions of the 148-kb baculovirus DNA were present as single-copy fragments ranging in size from 5 to 18 kb. Integration into the CHO cell genome was confirmed by fluorescent in situ hybridization (FISH) analysis. For one clone, the left and right viral/chromosomal junctions were determined by DNA sequencing of inverse PCR products. Similarly, for a different clone, the left viral/chromosomal junction was determined; however, the right junction sequence revealed the joining to another viral fragment by a short homology (microhomology), a hallmark of illegitimate recombination. The random viral breakpoints and the lack of homology between the virus and flanking chromosomal sequences are also suggestive of an illegitimate integration mechanism. To examine the long-term stability of reporter gene expression, all four clones were grown continuously for 36 passages in either the presence or absence of selection for Neo. Periodic assays over a 5-month period showed no loss of GFP expression for at least two of the clones. This report represents the first detailed analysis of baculovirus integrants within mammalian cells. The potential advantages of the baculovirus system for the stable integration of genetic material into mammalian genomes are discussed.

Transient and stable gene expression in mammalian cells transduced with a recombinant baculovirus vector.

Recombinant baculoviruses can serve as gene-transfer vehicles for transient expression of recombinant proteins in a wide range of mammalian cell types. Furthermore, by inclusion of a dominant selectable marker in the viral vector, cell lines can be derived that stably express recombinant genes. A virus was constructed containing two expression cassettes controlled by constitutive mammalian promoters: the cytomegalovirus immediate early promoter/enhancer directing expression of green fluorescent protein and the simian virus 40 (SV40) early promoter controlling neomycin phosphotransferase II. Using this virus, efficient gene delivery and expression was observed and measured in numerous cell types of human, primate, and rodent origin. In addition to commonly used transformed cell lines such as HeLa, CHO, Cos-7, and 293, this list includes primary human keratinocytes and bone marrow fibroblasts. In all cases, addition of butyrate or trichostatin A (a selective histone deacetylase inhibitor) to transduced cells markedly enhanced the levels of reporter protein expression observed. When transduced cells are put under selection with the antibiotic G418, cell lines can be obtained at high frequency that stably maintain the expression cassettes of the vector DNA and exhibit stable, high-level expression of the reporter gene. Stably transduced derivatives have been selected from a substantial number of different cell types, suggesting that stable lines can be derived from any cell type that exhibits transient expression.

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.

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 microg 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.

Gene gun delivered DNA-based immunizations mediate rapid production of murine monoclonal antibodies to the Flt-3 receptor.

Class-switched, affinity-matured murine monoclonal antibody (MAb)-producing cell lines were generated against the Flt-3 receptor in less than 4 weeks following polynucleotide immunizations, used in conjunction with repetitive immunizations, multiple sites (RIMMS). Plasmid DNA encoding Flt-3/Fc was coated onto gold particles, which were subsequently propelled into the epidermis of mice using biolistic particle bombardment using the Accell gene gun. Pools of immune peripheral lymph node cells were somatically fused 13 days after the onset of delivery of DNA encoding the target antigen. To determine if early responses could be augmented, DNA-encoding murine GM-CSF was delivered 3 days prior to the Flt-3/Fc DNA immunizations. The data presented demonstrates the successful identification and characterization of class-switched, affinity-matured MAbs that bind to the Flt-3 receptor. When compared to conventional methodologies or intramuscular targeted DNA-based immunization for the generation of MAbs, use of the gene gun in conjunction with RIMMS allows for a more rapid production of affinity-matured MAb-producing cell lines.

Expression and function of pancreatic beta-cell delayed rectifier K+ channels. Role in stimulus-secretion coupling.

Voltage-dependent delayed rectifier K+ channels regulate aspects of both stimulus-secretion and excitation-contraction coupling, but assigning specific roles to these channels has proved problematic. Using transgenically derived insulinoma cells (betaTC3-neo) and beta-cells purified from rodent pancreatic islets of Langerhans, we studied the expression and role of delayed rectifiers in glucose-stimulated insulin secretion. Using reverse-transcription polymerase chain reaction methods to amplify all known candidate delayed rectifier transcripts, the expression of the K+ channel gene Kv2.1 in betaTC3-neo insulinoma cells and purified rodent pancreatic beta-cells was detected and confirmed by immunoblotting in the insulinoma cells. betaTC3-neo cells were also found to express a related K+ channel, Kv3.2. Whole-cell patch clamp demonstrated the presence of delayed rectifier K+ currents inhibited by tetraethylammonium (TEA) and 4-aminopyridine, with similar Kd values to that of Kv2.1, correlating delayed rectifier gene expression with the K+ currents. The effect of these blockers on intracellular Ca2+ concentration ([Ca2+]i) was studied with fura-2 microspectrofluorimetry and imaging techniques. In the absence of glucose, exposure to TEA (1-20 mM) had minimal effects on betaTC3-neo or rodent islet [Ca2+]i, but in the presence of glucose, TEA activated large amplitude [Ca2+]i oscillations. In the insulinoma cells the TEA-induced [Ca2+]i oscillations were driven by synchronous oscillations in membrane potential, resulting in a 4-fold potentiation of insulin secretion. Activation of specific delayed rectifier K+ channels can therefore suppress stimulus-secretion coupling by damping oscillations in membrane potential and [Ca2+]i and thereby regulate secretion. These studies implicate previously uncharacterized beta-cell delayed rectifier K+ channels in the regulation of membrane repolarization, [Ca2+]i, and insulin secretion.

Flow cytometric assay of modulation of P-glycoprotein function in whole blood by the multidrug resistance inhibitor GG918.

We sought to develop an assay for measuring the inhibition of P-glycoprotein (Pgp) function in whole blood as an indicator of in vivo drug activity. Since the CD56(+) subset of peripheral blood lymphocytes (PBLs) has been shown to express functional Pgp, the changes in rhodamine 123 (R123) uptake by CD56(+) PBLs from GG918-treated and untreated whole blood were used as the basis for these studies. In an ex vivo study, heparin-treated whole blood was obtained from normal volunteers, and GG918 and R123 were added at various concentrations for time course analyses of dye loading. GG918 concentrations from 2.5 to 800 nm were tested in incubations ranging from 15 min to 3 h prior to R123 addition. R123 loading times ranged from 0 to 80 min. Flow cytometric analyses of the CD56(+) PBLs indicated that the resolution of Pgp inhibition was dependent on inhibitor concentration and time of R123 loading and independent of the R123 concentrations tested. In this ex vivo assay model, a dose-dependent response was seen for GG918 with a 2-fold increase in cellular R123 intensity being produced at a drug concentration of 80 nm. When this assay method was applied to blood samples from volunteers dosed p.o. with GG918, similar shifts in R123 fluorescence of the CD56(+) PBLs were observed with significant increases in R123 intensity occurring at serum concentrations as low as 40 nm. In contrast to assays in which target cell populations are enriched prior to testing, the addition of the substrate (R123) directly to the blood sample combined with the segregation of the target cells by specific immunofluorescence provides the investigator an indication of in situ activity of circulating drug. Thus, CD56(+) PBLs may prove useful as a surrogate target for monitoring multidrug resistance inhibitor activity in situ.

Characterization and myocarditic capabilities of coxsackievirus B3 variants in selected mouse strains.

Two variants of coxsackievirus B3 (CVB3) were compared with the original myocarditic parent variant (CVB3m) for myocarditic properties in several strains of mice. The ts1R variant produced little to no myocarditis in any of the nine mouse strains examined. The ts10R variant and CVB3m could be differentiated on the basis of the extent of myocarditis induced in mice of selected H-2b and H-2k haplotypes and in the female versus the male responses of two other inbred strains. Virus quantities recovered from the hearts of myocarditic mice did not correlate with the extent of disease. The three variants could not be differentiated on the basis of: (i) rate and extent of adsorption to heart tissue homogenates, (ii) kinetic neutralization rates with antiserum directed against CVB3m, (iii) 125I labeling of surface regions of polypeptides on purified particles, or (iv) rates of heat inactivation of infectivity at 50 degrees C. These data suggest that differences in pathogenicity cannot be attributed to major alterations in capsid polypeptides. Oligonucleotide fingerprint maps of T1 RNase digests of the genomes of purified particles of the three CVB3 variants showed distinct differences. Thus, the extent of myocarditis induced by CVB3 variants in a mouse model is affected by some subtle expression of the genome, presumably not involving capsid polypeptides, as well as by the haplotype and sex of a given mouse host species.