Rapid adaptation to CDK2 inhibition exposes intrinsic cell-cycle plasticity.

CDK2 is a core cell-cycle kinase that phosphorylates many substrates to drive progression through the cell cycle. CDK2 is hyperactivated in multiple cancers and is therefore an attractive therapeutic target. Here, we use several CDK2 inhibitors in clinical development to interrogate CDK2 substrate phosphorylation, cell-cycle progression, and drug adaptation in preclinical models.

Risk reduction in biotherapeutic products.

Ensuring the safety of therapeutic modalities produced and purified from biological systems is of high concern. Regulatory authorities and the biopharmaceutical industry are continuously seeking to improve methods for the detection, identification, inactivation and removal of potentially contaminating pathogens in biotherapeutic products. Current methods for pathogen detection and identification are designed to discover adventitious as well as known microbial species in product samples.

Propofol hemisuccinate suppression of experimental autoimmune encephalomyelitis.

Propofol hemisuccinate is a prodrug water soluble form of the lipophilic, phenolic compound propofol (2,6-di-isopropylphenol), that is the active ingredient in the widely used anesthetic agent Diprovan. Propofol binds to GABA(A) receptors but also has a phenolic structure that confers antioxidant properties to the molecule. The effects of propofol hemisuccinate in rat experimental autoimmune encephalomyelitis (EAE) were studied using different doses and time regimes.

Gene therapy progress and prospects: recent progress in transgene and RNAi expression cassettes.

Plasmid expression cassette design must include a thoughtful analysis of potentially every nucleotide comprising a covalently closed circular or end-protected linear DNA. This review will discuss recent studies in unraveling the mechanisms of postdelivery gene silencing, codon optimization and promoter identification. The recent discovery of potent RNA interference (RNAi) mechanisms for sequence-specific gene silencing has also invoked a great deal of interest in development of expression cassettes that can produce double-stranded RNA molecules for RNAi.

Identification of reciprocally regulated gene modules in regenerating dorsal root ganglion neurons and activated peripheral or central nervous system glia.

Differential gene expression in the rat after injury of dorsal root ganglion neurons in vivo, and simulation injury of Schwann cells and oligodendrocytes in vitro was analyzed using high-density cDNA microarrays. The analyses were carried out to study the genetic basis of peripheral nerve regeneration, and to compare gene regulation in glia of the central (oligodendrocyte) and peripheral (Schwann cell) nervous systems. The genes showing significant differential regulation in the three study groups represented all aspects of cellular metabolism.

Gene therapy strategies for the treatment of chronic viral hepatitis.

Chronic viral hepatitis is a major clinical problem, with over half a billion persons infected worldwide. Current therapies, principally treatment with recombinant IFN-alpha protein, have limited benefit. Recent studies suggest that gene-based expression of IFN-alpha is a possible therapeutic alternative that may improve the effectiveness of treatment.

Gene expression monitoring for gene discovery in models of peripheral and central nervous system differentiation, regeneration, and trauma.

Gene expression monitoring using gene expression microarrays represents an extremely powerful technology for gene discovery in a variety of systems. We describe the results of seven experiments using Incyte GEM technology to compile a proprietary portfolio of data concerning differential gene expression in six different models of neuronal differentiation and regeneration, and recovery from injury or disease. Our first two experiments cataloged genes significantly up- or down-regulated during two phases of the retinoic acid-induced differentiation of the embryonal carcinoma line Ntera-2.

Sustained correction of bleeding disorder in hemophilia B mice by gene therapy.

Mice generated by disrupting the clotting factor IX gene exhibit severe bleeding disorder and closely resemble the phenotype seen in hemophilia B patients. Here we demonstrate that a single intraportal injection of a recombinant adeno-associated virus (AAV) vector encoding canine factor IX cDNA under the control of a liver-specific enhancer/promoter leads to a long-term and complete correction of the bleeding disorder. High level expression of up to 15-20 microgram/ml of canine factor IX was detected in the plasma of mice injected with 5.

Optimization of the human factor VIII complementary DNA expression plasmid for gene therapy of hemophilia A.

While the gene delivery vehicle is critical for the efficacy of human factor VIII gene therapy, optimization of the potency and duration of the factor VIII gene that is delivered is equally important in light of the poor transcription and translation characteristics of this gene. We discuss here a systematic approach to optimization of factor VIII complementary DNA expression by analysis of specific elements engineered into the transcription unit and other positions in the expression plasmid. Within the transcription unit we have engineered different 5' and 3' sequence modifications and tested them for factor VIII expression in human liver cells.

Design and construction of a hybrid immunoglobulin domain with properties of both heavy and light chain variable regions.

The complementarity-determining regions (CDRs) of a human kappa light chain were replaced with CDRs from a murine gamma-1 heavy chain and, by use of molecular modeling, key heavy chain framework residues were identified and thus included to preserve the native conformation of the heavy chain CDRs. Co-expression of this hybrid human kappa chain (V[HB]C[L]) with a human kappa chain counterpart (V[L]C[L], engineered to contain murine light chain CDRs) resulted in the secretion of high levels of a heterodimeric protein (V[HB]C[L]::V[L]C[L]) termed 'kappabody'. This protein also had equivalent affinity for antigen as the Fab' of the parent murine IgG1.

A COOH-terminal peptide confers regiospecific orientation and facilitates atomic force microscopy of an IgG1.

An antibody (IgG1) was designed for oriented adherence to a metal-containing surface. This was achieved by adding a metal-chelating peptide, (CP = His-Trp-His-His-His-Pro), to the COOH-terminus of the heavy chain through genetic engineering. Electroporation of the engineered heavy chain gene into cells expressing the complimentary light chain yielded colonies secreting an intact antibody containing the metal-chelating peptide (IgG1-CP) which had high affinity for a nickel-loaded iminodiacetate column.

Species specificity of iron delivery in hybridomas.

Studies with Human X Human (H X H), Human X Mouse (H X M), and Mouse X Mouse (M X M) hybridomas have enabled us to define specific factors that affect hybridoma growth in a species-specific manner. Three transferrins and three lipophilic iron chelates have been tested for their ability to support hybridoma proliferation and antibody production. The results of these studies demonstrate that H X H hybridomas do not respond to bovine transferrin a+ concentrations up to 100 micrograms/ml and are approximately 100-fold less responsive to mouse transferrin than to human transferrin.

Association of thrombin-antithrombin III complex with vitronectin in serum.

Purification of vitronectin by identical procedures from serum instead of plasma results in the coisolation of an additional protein component with mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of 82 kDa. We show that this component is the thrombin-antithrombin III complex based on the following evidence. Similar to a complex constructed using purified thrombin and antithrombin III, the 82-kDa component has a reduced molecular size of 69 kDa if it is not boiled prior to SDS-PAGE.

Adhesion of platelets to laminin in the absence of activation.

The binding of platelets to components in the subendothelial matrix is an initial event in hemostasis and thrombosis. The glycoprotein components of the matrix are considered important in this interaction. Of these, collagen binds and activates platelets and induces their aggregation.

Permissive effect of insulin on the adenosine 3',5'-monophosphate-dependent up-regulation of low density lipoprotein receptors and the stimulation of steroid release in bovine adrenal cortical cells.

Bovine adrenal cortical cells growth on extracellular matrix-coated dishes in the presence of F-12 medium supplemented with high density lipoprotein (30 micrograms protein/ml), insulin (50 ng/ml), transferrin (1 microgram/ml), and fibroblast growth factor (100 ng/ml) expressed high affinity low density lipoprotein (LDL) receptors (Kd = 2.0 X 10(-8) M) present at a density of 3 X 10(4) receptors/cell. The density of LDL receptors per cell could be increased 3-fold (9 X 10(4) receptors/cell) by incubating the cells for 24 h with cholera toxin (CT; 10 ng/ml), but not with insulin (100 ng/ml).

Factors involved in supporting the growth and steroidogenic functions of bovine adrenal cortical cells maintained on extracellular matrix and exposed to a serum-free medium.

Bovine adrenal cortex cells maintained on extracellular matrix (ECM)-coated dishes will proliferate actively when serum is replaced by HDL (25 micrograms protein/ml), insulin (10 ng/ml), and FGF (100 ng/ml). The cells have an absolute requirement for HDL in order to survive and grow. The omission of insulin, FGF, or both results in a slower growth rate and lower final cell density of the cultures.

Control of proliferation of human fetal adrenal cells in vitro.

The influence of fibroblast growth factor (FGF) and epidermal growth factor (EGF) on the proliferation of cultured human fetal adrenal cells has been examined. Separated human definitive zone and fetal zone adrenal cells plated at low density in the presence of 10% serum and maintained on plastic culture dishes proliferated slowly. If the cultures were exposed to either FGF or EGF, the growth rate of the cells from each zone increased significantly.

Kinetics of cAMP inhibition of DNA synthesis in bovine adrenocortical cells.

cAMP-treated bovine adrenocortical cells are arrested in the G1 phase of the cell cycle. Removal of serum also arrests bovine adrenocortical cells in G1. In the presence of cAMP, serum and fibroblast growth factor stimulate increases in medium cell volume, but DNA synthesis is not initiated.

Extracellular matrix and control of proliferation of vascular endothelial cells.

Bovine vascular endothelial cells plated at low cell density in the presence of high (10%) concentrations of serum and maintained on plastic tissue culture dishes proliferate slowly. If the cultures were exposed to fibroblast growth factors (FGF), the cells proliferated actively and, after a week, a monolayer composed of closely apposed and highly contact-inhibited mononucleated cells formed. In contrast to cultures maintained on plastic, cultures maintained on dishes coated with an extracellular matrix produced by corneal endothelial cells proliferated rapidly and no longer required FGF to reach confluence.

Do plasma and serum have different abilities to promote cell growth?

The abilities of plasma and serum to support the growth of vascular smooth muscle cells maintained on uncoated tissue culture dishes or dishes coated with an extracellular matrix (ECM) have been compared. Vascular smooth muscle cells maintained on plastic dishes and exposed to plasma proliferate poorly; when exposed to serum they proliferate actively. Addition of fibroblast growth factor (FGF) incrases the growth rate of the cultures in both cases.