Reactive oxygen species promote caspase-12 expression and tubular apoptosis in diabetic nephropathy.

Apoptosis of tubular epithelial cells contributes to the tubular atrophy that accompanies diabetic nephropathy. Reactive oxygen species (ROS) promote tubular apoptosis, but the mechanisms by which this occurs are incompletely understood. Here, we sought proapoptotic genes that ROS differentially upregulate in renal proximal tubular cells of diabetic (db/db) mice. We performed microarray analysis using total RNA from freshly isolated renal proximal tubules of nondiabetic, diabetic, and diabetic transgenic mice overexpressing catalase in the proximal tubule (thereby attenuating ROS). We observed greater expression of caspase-12 in the proximal tubules of the diabetic mice compared with the nondiabetic and diabetic transgenic mice. Quantitative PCR and immunohistochemistry confirmed the enhanced expression of caspase-12, as well as members of the endoplasmic reticulum stress-induced apoptotic pathway. Ex vivo, albumin induced caspase-12 activity and expression (protein and mRNA) and mRNA expression of the CCAT/enhancer-binding protein homologous protein in freshly isolated wild-type proximal tubules but not in catalase-overexpressing proximal tubules. In vitro, albumin stimulated activity of both caspase-12 and caspase-3 as well as expression of caspase-12 and CCAT/enhancer-binding protein homologous protein in a human proximal tubule cell line (HK-2). The free radical scavenger tiron inhibited these effects. Furthermore, knockdown of caspase-12 with small interfering RNA reduced albumin-induced apoptosis in HK-2 cells. Taken together, these studies demonstrate that albuminuria may induce tubular apoptosis through generation of ROS and the subsequent expression and activation of endoplasmic reticulum stress genes in the diabetic kidney.

Scalable serum-free production of recombinant adeno-associated virus type 2 by transfection of 293 suspension cells.

Recombinant adeno-associated virus (rAAV) has emerged in recent years as a promising gene therapy vector that may be used in the treatment of diverse human diseases. The major obstacle to broadening the usage of rAAV vectors remains the limited capacity of available production systems to provide sufficient rAAV quantities for preclinical and clinical trials. The impracticality of expanding commonly used adherent cell lines represents a limitation to large-scale production. This paper describes successful productions of rAAV type 2 using suspension-growing human embryonic kidney (HEK293) cells in serum-free medium. The developed process, based on triple transfection employing polyethylenimine (PEI) as DNA transporter, allowed for a serum-free production of AAV, yielding viral vector titer up to 4.5x10(11) infectious viral particles (IVP) in a 3.5-L bioreactor. A maximum ratio of VG:IVP in the order of 200:1 was obtained, indicating the efficient encapsidation of viral vectors in HEK293 cells. The effect of varying the ratio of three plasmids and the influence of cell density at transfection were studied. The conditioned medium did not limit or inhibit the rAAV production; therefore, the elimination of the medium exchange step before or after transfection greatly simplified the scale-up of rAAV production. The cell-specific viral titers obtained in bioreactor suspension cultures were similar or higher than those obtained with control adherent cell cultures which further supported the scalability of the process. From multiple aspects including process simplicity, scalability, and low operating costs, this transfection method appears to be the most promising technology for large-scale production of rAAV.

Differential response of glioma cells to FOXO1-directed therapy.

Gliomas are the most common adult primary brain tumors, and the most malignant form, glioblastoma multiforme, is invariably fatal. The phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway is altered in most glioblastoma multiforme. PTEN, an important negative regulator of the PI3K-Akt pathway, is also commonly mutated in glioma, leading to constitutive activation of Akt. One ultimate consequence is phosphorylation and inactivation of FOXO forkhead transcription factors that regulate genes involved in apoptosis, cell cycle arrest, nutrient availability, DNA repair, stress, and angiogenesis. We tested the ability of a mutant FOXO1 factor that is not subject to Akt phosphorylation to overcome dysregulated PI3K-Akt signaling in two PTEN-null glioma cell lines, U87 and U251. Adenovirus-mediated gene transfer of the mutant FOXO1 successfully restored cell cycle arrest and induced cell death in vitro and prolonged survival in vivo in xenograft models of human glioma (33% survival at 1 year of animals bearing U251 tumors). However, U87 were much more resistant than U251 to mutant FOXO1-induced death, showing evidence of increased nuclear export and Akt-independent phosphorylation of FOXO1 at S249. A cyclin-dependent kinase 2 inhibitor decreased phosphorylation of S249 and rendered U87 cells significantly more susceptible to mutant FOXO1-induced death. Our results indicate that targeting FOXO1, which is at the convergence point of several growth factor receptor tyrosine kinase pathways, can effectively induce glioma cell death and inhibit tumor growth. They also highlight the importance of Akt-independent phosphorylation events in the nuclear export of FOXO1.