Project description:The 60 cell lines of the NCI Anti-Cancer Drug Screen (NCI60) constitute the most extensively characterized in vitro cancer cell model, and have been tested for sensitivity to over 100,000 potential chemotherapy agents. We have used the NCI60 cell lines and three additional lymphoblast lines to develop a database of responses of cancer cells to ionizing radiation. We compared clonogenic survival, apoptosis induction, and gene expression response by microarray analysis. While several studies have measured relative basal gene expression in the NCI60, this is the first comparison of large-scale gene expression changes in response to genotoxic stress. We found genes differentially regulated in cells with low survival after 2 or 8 Gy γ-rays. In contrast to reported basal gene expression patterns, little tissue-of-origin effect was detected in the radiation response pattern of gene expression, with the exception of lymphoblastoid cell lines. The most striking patterns in the radiation data were a set of genes upregulated preferentially in the p53 wild-type lines, and a set of cell-cycle regulatory genes strongly down-regulated across the entire NCI60 panel. The response of these genes to γ-rays appears to be unaffected by the myriad of genetic differences across this very diverse cell set, and represents the most universal gene expression response to ionizing radiation yet observed. Keywords: radiation response
Project description:The 60 cell lines of the NCI Anti-Cancer Drug Screen (NCI60) constitute the most extensively characterized in vitro cancer cell model, and have been tested for sensitivity to over 100,000 potential chemotherapy agents. We have used the NCI60 cell lines and three additional lymphoblast lines to develop a database of responses of cancer cells to ionizing radiation. We compared clonogenic survival, apoptosis induction, and gene expression response by microarray analysis. While several studies have measured relative basal gene expression in the NCI60, this is the first comparison of large-scale gene expression changes in response to genotoxic stress. We found genes differentially regulated in cells with low survival after 2 or 8 Gy γ-rays. In contrast to reported basal gene expression patterns, little tissue-of-origin effect was detected in the radiation response pattern of gene expression, with the exception of lymphoblastoid cell lines. The most striking patterns in the radiation data were a set of genes upregulated preferentially in the p53 wild-type lines, and a set of cell-cycle regulatory genes strongly down-regulated across the entire NCI60 panel. The response of these genes to γ-rays appears to be unaffected by the myriad of genetic differences across this very diverse cell set, and represents the most universal gene expression response to ionizing radiation yet observed. Cells were exposed to 8Gy and gene expression ratios between untreated (Cy5) cells and exposed cells (Cy3) were measured four hours later
Project description:Kynureninase is a member of a large family of catalytically diverse but structurally homologous pyridoxal 5'-phosphate (PLP) dependent enzymes known as the aspartate aminotransferase superfamily or alpha-family. The Homo sapiens and other eukaryotic constitutive kynureninases preferentially catalyze the hydrolytic cleavage of 3-hydroxy-l-kynurenine to produce 3-hydroxyanthranilate and l-alanine, while l-kynurenine is the substrate of many prokaryotic inducible kynureninases. The human enzyme was cloned with an N-terminal hexahistidine tag, expressed, and purified from a bacterial expression system using Ni metal ion affinity chromatography. Kinetic characterization of the recombinant enzyme reveals classic Michaelis-Menten behavior, with a Km of 28.3 +/- 1.9 microM and a specific activity of 1.75 micromol min-1 mg-1 for 3-hydroxy-dl-kynurenine. Crystals of recombinant kynureninase that diffracted to 2.0 A were obtained, and the atomic structure of the PLP-bound holoenzyme was determined by molecular replacement using the Pseudomonas fluorescens kynureninase structure (PDB entry 1qz9) as the phasing model. A structural superposition with the P. fluorescens kynureninase revealed that these two structures resemble the "open" and "closed" conformations of aspartate aminotransferase. The comparison illustrates the dynamic nature of these proteins' small domains and reveals a role for Arg-434 similar to its role in other AAT alpha-family members. Docking of 3-hydroxy-l-kynurenine into the human kynureninase active site suggests that Asn-333 and His-102 are involved in substrate binding and molecular discrimination between inducible and constitutive kynureninase substrates.