Project description:Background Identification of the molecular determinants of invasion and metastatic potential may guide the development of a rational strategy for devising specific therapies that target the pathways leading to osteosarcoma. Methods In this study, we used pathway-focused GEarrays to screen for candidate genes related to tumor progression. Expression patterns of the selected genes were validated by real time PCR on 61 osteosarcoma patient tumor samples and correlated with clinical and pathological data. Results THBS3, SPARC and SPP1 were identified with pathway-focused GEarrays as genes differentially expressed in osteosarcoma. In particular, THBS3 was expressed at significantly high levels (p=0.0001) in biopsies from patients with metastasis at diagnosis, which is a predictor of worse overall survival, event-free survival and relapse free survival at diagnosis. After chemotherapy, patients with tumors over-expressing THBS3 have worse relapse free survival. High SPARC expression was found in 51 of 55 (96.3%) osteosarcoma samples and correlated with the worst event-free survival (p=0.03) and relapse free survival (p=0.07). Overexpression of SPP1 was found in 47 of 53 (89%) osteosarcomas correlating with better overall survival, event-free survival and relapse free survival at diagnosis. Conclusion With pathway-focused Gearrays, we identified three genes, which interestingly all encode for proteins involved in extracellular remodeling suggesting potential roles in osteosarcoma progression. This is the first report on the THBS3 gene working as a stimulator of tumor progression. Higher levels of THBS3 maintain the capacity of angiogenesis. High levels of SPARC are not required for tumor progression but are necessary for tumor growth and maintenance. SPP1 is not necessary for tumor progression in osteosarcoma and may be associated with inflammatory response and bone remodeling, functioning as a good biomarker. Keywords: disease state analysis using Superarray Pathway focused arrays
Project description:Transcriptional profiling of human mesenchymal stem cells comparing normoxic MSCs cells with hypoxic MSCs cells. Hypoxia may inhibit senescence of MSCs during expansion. Goal was to determine the effects of hypoxia on global MSCs gene expression.
Project description:SaOS2 osteosarcoma cells were cultured with or without atorvastatin (10 µM) for 6, 15 or 24h (2 biological replcates). RNA were isolated and hybridized to RNG microarrays. Background Osteosarcoma is the most common primary tumor of bone. The rapid development of metastatic lesions and resistance to chemotherapy remain major mechanisms responsible for the failure of treatments and poor survival rate for patients. Methods We previously showed that the HMGCoA reductase inhibitors statins exhibit anti-tumoral effects on osteosarcoma cells. Here, using microarray analysis, we identify cyr61/CCN1 as a new target of statins. Modulations of expression of cyr61 were performed in human and murine osteosarcoma cell lines to investigate in vitro cell viability, migratory potential and invasiveness. Cyr61 expression was evaluated in 231 tissue cores from osteosarcoma patients using tissue microarray. Tumor behavior and metastases occurence were analysed by IM injection of modified osteosarcoma cells to BALB/c mice. Results Transcriptome comparisons revealed that statins down-regulate cyr61 expression in SaOS2 cells. Cyr61 silencing in human and murine osteosarcoma cell lines enhanced cell death, but reduced cell migration and cell invasion compared to parental cells whereas cyr61 overexpression had opposite effects. Tissue microarray analysis demonstrated that cyr61 protein expression is higher in human osteosarcoma compared to normal bone tissue and is further increased in metastatic tissues. In vivo, cyr61 overexpression in osteosarcoma cells enhanced lung metastases development whereas cyr61 silencing strongly reduced metastases in mice. Conclusion The results reveal that cyr61 expression increases with tumor grade in human osteosarcoma and demonstrate that cyr61 silencing inhibits in vitro osteosarcoma cell invasion and migration as well as in vivo lung metastases in mice. These data provide a novel molecular target for therapeutic intervention in metastatic osteosarcoma. Dye balance-experiment comparing atorvastatin versus untreated cells at 6, 15 and 24 hours using 2 biological replicates.
Project description:Gene methylation profiling of immortalized human mesenchymal stem cells comparing HPV E6/E7-transfected MSCs cells with human telomerase reverse transcriptase (hTERT)- and HPV E6/E7-transfected MSCs. hTERT may increase gene methylation in MSCs. Goal was to determine the effects of different transfected genes on global gene methylation in MSCs.
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:Transcriptional profiling of human mesenchymal stem cells comparing normoxic MSCs cells with hypoxic MSCs cells. Hypoxia may inhibit senescence of MSCs during expansion. Goal was to determine the effects of hypoxia on global MSCs gene expression. Two-condition experiment, Normoxic MSCs vs. Hypoxic MSCs.
Project description:As the evolution of miRNA genes has been found to be one of the important factors in formation of the modern type of man, we performed a comparative analysis of the evolution of miRNA genes in two archaic hominines, Homo sapiens neanderthalensis and Homo sapiens denisova, and elucidated the expression of their target mRNAs in bain.A comparative analysis of the genomes of primates, including species in the genus Homo, identified a group of miRNA genes having fixed substitutions with important implications for the evolution of Homo sapiens neanderthalensis and Homo sapiens denisova. The mRNAs targeted by miRNAs with mutations specific for Homo sapiens denisova exhibited enhanced expression during postnatal brain development in modern humans. By contrast, the expression of mRNAs targeted by miRNAs bearing variations specific for Homo sapiens neanderthalensis was shown to be enhanced in prenatal brain development.Our results highlight the importance of changes in miRNA gene sequences in the course of Homo sapiens denisova and Homo sapiens neanderthalensis evolution. The genetic alterations of miRNAs regulating the spatiotemporal expression of multiple genes in the prenatal and postnatal brain may contribute to the progressive evolution of brain function, which is consistent with the observations of fine technical and typological properties of tools and decorative items reported from archaeological Denisovan sites. The data also suggest that differential spatial-temporal regulation of gene products promoted by the subspecies-specific mutations in the miRNA genes might have occurred in the brains of Homo sapiens denisova and Homo sapiens neanderthalensis, potentially contributing to the cultural differences between these two archaic hominines.
Project description:PurposeWe investigated the evidence of recent positive selection in the human phototransduction system at single nucleotide polymorphism (SNP) and gene level.MethodsSNP genotyping data from the International HapMap Project for European, Eastern Asian, and African populations was used to discover differences in haplotype length and allele frequency between these populations. Numeric selection metrics were computed for each SNP and aggregated into gene-level metrics to measure evidence of recent positive selection. The level of recent positive selection in phototransduction genes was evaluated and compared to a set of genes shown previously to be under recent selection, and a set of highly conserved genes as positive and negative controls, respectively.ResultsSix of 20 phototransduction genes evaluated had gene-level selection metrics above the 90th percentile: RGS9, GNB1, RHO, PDE6G, GNAT1, and SLC24A1. The selection signal across these genes was found to be of similar magnitude to the positive control genes and much greater than the negative control genes.ConclusionsThere is evidence for selective pressure in the genes involved in retinal phototransduction, and traces of this selective pressure can be demonstrated using SNP-level and gene-level metrics of allelic variation. We hypothesize that the selective pressure on these genes was related to their role in low light vision and retinal adaptation to ambient light changes. Uncovering the underlying genetics of evolutionary adaptations in phototransduction not only allows greater understanding of vision and visual diseases, but also the development of patient-specific diagnostic and intervention strategies.
Project description:SaOS2 osteosarcoma cells were cultured with or without atorvastatin (10 µM) for 6, 15 or 24h (2 biological replcates). RNA were isolated and hybridized to RNG microarrays. Background Osteosarcoma is the most common primary tumor of bone. The rapid development of metastatic lesions and resistance to chemotherapy remain major mechanisms responsible for the failure of treatments and poor survival rate for patients. Methods We previously showed that the HMGCoA reductase inhibitors statins exhibit anti-tumoral effects on osteosarcoma cells. Here, using microarray analysis, we identify cyr61/CCN1 as a new target of statins. Modulations of expression of cyr61 were performed in human and murine osteosarcoma cell lines to investigate in vitro cell viability, migratory potential and invasiveness. Cyr61 expression was evaluated in 231 tissue cores from osteosarcoma patients using tissue microarray. Tumor behavior and metastases occurence were analysed by IM injection of modified osteosarcoma cells to BALB/c mice. Results Transcriptome comparisons revealed that statins down-regulate cyr61 expression in SaOS2 cells. Cyr61 silencing in human and murine osteosarcoma cell lines enhanced cell death, but reduced cell migration and cell invasion compared to parental cells whereas cyr61 overexpression had opposite effects. Tissue microarray analysis demonstrated that cyr61 protein expression is higher in human osteosarcoma compared to normal bone tissue and is further increased in metastatic tissues. In vivo, cyr61 overexpression in osteosarcoma cells enhanced lung metastases development whereas cyr61 silencing strongly reduced metastases in mice. Conclusion The results reveal that cyr61 expression increases with tumor grade in human osteosarcoma and demonstrate that cyr61 silencing inhibits in vitro osteosarcoma cell invasion and migration as well as in vivo lung metastases in mice. These data provide a novel molecular target for therapeutic intervention in metastatic osteosarcoma.