No abstract

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 wildtype 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/enhancerbinding 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.

No abstract

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 Aktindependent phosphorylation of FOXO1 at S249. A cyclindependent 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. [Cancer Res 2009;69(13):5433-40]

Gene therapy is a promising approach for cancer treatment; however, efficacy of current vectors remains insufficient. To improve the success of suicide gene therapy, we constructed a replication-competent adenoviral vector that has its protease gene deleted and expresses bacterial cytosine deaminase fused with bacterial uracil phosphoribosyltransferase (CU). The prodrug, 5-fluorocytosine, is transformed into the highly toxic and tissue-diffusible 5-fluorouracil by CU in infected cells. This vector is incapable of producing infectious particles but is able to undergo a single round of replication, thereby increasing transgene copy number and expression. In the presence of 5-FC, compared with the first-generation vector (AdCU), the replication-competent vector, Ad(dPS)CU-IRES-E1A, was significantly more efficacious for in vitro tumor cell killing and in bystander assays, whereas 25-fold fewer viral particles were required in a three-dimensional spheroid model. For in vivo experiments, in which virus was injected into preestablished intracranial glioma xenografts, followed by 5-FC treatment, mice receiving Ad(dPS)CU-IRES-E1A had significantly smaller tumors at 35 days postinjection as well as significantly longer median survival than mice treated with the replication-deficient, protease-deleted vector [Ad(dPS)CU]. In an immunocompetent syngeneic model, Ad(dPS)CU + 5-FC-treated mice had a median survival of only 23 days, whereas Ad(dPS)CU-IRES-E1A + 5-FCtreated animals had a survival of 57.1% at 365 days. In conclusion, Ad(dPS)CU-IRES-E1A in the presence of 5-FC produces more potent tumoricidal effects than its replicationdeficient counterparts. [Cancer Res 2007;67(7):3387-95]

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Supplementary Figure 1 from Differential Response of Glioma Cells to FOXO1-Directed Therapy

Supplementary Table 1 from Differential Response of Glioma Cells to FOXO1-Directed Therapy