Project description:To identify patterns of change in gene expression that correlated with drug treatment (saline control, ADH-1 alone, melphalan alone and ADH-1 plus melphalan) in our animal model of regional therapy for extremity melanoma we evaluated gene expression using microarray genechips and a Pearson correlation analysis. Genes were ranked according to the degree to which expression correlated with systemic ADH-1 treatment alone or in combination with regional melphalan. Tissue samples were harvested at 4, 24 and 48 hours after treatment with melphalan. Specific cellular pathways associated with drug treatment were identified using gene set enrichment analysis (GSEA). Keywords: time course; chemotherapy; melanoma; melphalan; N-cadherin; ADH-1
Project description:To identify patterns of change in gene expression that correlated with drug treatment (saline control, ADH-1 alone, melphalan alone and ADH-1 plus melphalan) in our animal model of regional therapy for extremity melanoma we evaluated gene expression using microarray genechips and a Pearson correlation analysis. Genes were ranked according to the degree to which expression correlated with systemic ADH-1 treatment alone or in combination with regional melphalan. Tissue samples were harvested at 4, 24 and 48 hours after treatment with melphalan. Specific cellular pathways associated with drug treatment were identified using gene set enrichment analysis (GSEA). Experiment Overall Design: Sample size was 2 rats for each drug treatment group at both the 4 and 24 hour time points. At the 48 hour time point sample size was 1 rat for each drug treatment group. ADH-1 treatment preceded melphalan by 1 hour. ADH-1 treatment was repeated at 24 and 48 hrs. Tissue was harvested for RNA analysis at 4, 24 and 48 hours after completion of isolated limb infusion with melphalan (and 1 hour after an ADH-1 dose).
Project description:An evaluation of biopsies from patients with in-transit extremity melanoma who have been treated with ADH-1 followed by melphalan in the setting of isolated limb infusion Gene expression profiles were obtained from 28 lesions across 15 patients and evaluated for expression values that correlated with ADH-1 treatment given 4-8hrs prior to melphalan isolated limb infusion Chemotherapy response analysis: complete response - CR; partial response - PR; stable disease - SD; progressive disease - PD; lost to follow-up - LTU/F ADH-1 treatment classification: 'pre' - lesions obtained prior to any treatment with ADH-1; 'post' - lesions obtained just prior to ILI with melphalan after 1 treatment with ADH-1; '2nd' - lesions obtained after a 2nd dose of ADH-1 given 8 days after melphalan ILI
Project description:An evaluation of biopsies from patients with in-transit extremity melanoma who have been treated with ADH-1 followed by melphalan in the setting of isolated limb infusion Gene expression profiles were obtained from 28 lesions across 15 patients and evaluated for expression values that correlated with ADH-1 treatment given 4-8hrs prior to melphalan isolated limb infusion
Project description:Melphalan-induced modulation of miR-221/222 levels in MM cells. Melphalan-resistant U266/LR7 cells showed the highest induction of miR-221/222 after drug exposure. To study the transcriptome perturbation induced in MM cells following the combination of miR-221/222 inhibitors plus melphalan we used the whole gene expression data total RNA was obtained after single or combination treatment of the Melphalan-resistant U266/LR7 cells and the parental cell line U266/s
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.
Project description:To identify patterns of gene expression that correlate with response to treatment with either melphalan or temozolomide we measured both gene expression using microarray genechips and response to drug using a standard in vitro cell proliferation assay. Senstivity to melphalan was measured 48hrs after drug treatment while sensitivity to temozolomide was measured 12 days after drug treatment. Keywords: Patterns of gene expression predictive of response to chemotherapy with melphalan or temozolomide in melanoma
Project description:We have sequenced miRNA libraries from human embryonic, neural and foetal mesenchymal stem cells. We report that the majority of miRNA genes encode mature isomers that vary in size by one or more bases at the 3’ and/or 5’ end of the miRNA. Northern blotting for individual miRNAs showed that the proportions of isomiRs expressed by a single miRNA gene often differ between cell and tissue types. IsomiRs were readily co-immunoprecipitated with Argonaute proteins in vivo and were active in luciferase assays, indicating that they are functional. Bioinformatics analysis predicts substantial differences in targeting between miRNAs with minor 5’ differences and in support of this we report that a 5’ isomiR-9-1 gained the ability to inhibit the expression of DNMT3B and NCAM2 but lost the ability to inhibit CDH1 in vitro. This result was confirmed by the use of isomiR-specific sponges. Our analysis of the miRGator database indicates that a small percentage of human miRNA genes express isomiRs as the dominant transcript in certain cell types and analysis of miRBase shows that 5’ isomiRs have replaced canonical miRNAs many times during evolution. This strongly indicates that isomiRs are of functional importance and have contributed to the evolution of miRNA genes