Project description:Physical factors can have major influences on the proliferation and differentiation fate of hematopoietic stem cells in culture. Recently, we demonstrated that cord blood CD34+ cells undergo accelerated and increased megakaryocyte differentiation when incubated at 39°C. In this study, we investigated in detail the impacts of mild hyperthermia on the kinetics of megakaryocyte differentiation, maturation, polyploidization and cell viability. The qualitative and quantitative effects on Mk differentiation were found to be rapidly induced, and optimization of the culture length at 39°C led to greater Mk yields. Mild hyperthermia did not promote endomitosis of cord blood-derived Mk, but rather led to a small reduction in the proportion of polyploid Mk. Moreover, it had little impact on viability but induced a significant shift in the proportion of cells undergoing apoptosis at 37°C to necrosis at 39°C. Finally, our results suggest that the effects on megakaryocyte differentiation could be the consequences of aberrant expression of key megakaryocyte transcription factors induced by mild hyperthermia. Keywords: Effect of mild hyperthermia on Mk differentiation
Project description:Physical factors can have major influences on the proliferation and differentiation fate of hematopoietic stem cells in culture. Recently, we demonstrated that cord blood CD34+ cells undergo accelerated and increased megakaryocyte differentiation when incubated at 39°C. In this study, we investigated in detail the impacts of mild hyperthermia on the kinetics of megakaryocyte differentiation, maturation, polyploidization and cell viability. The qualitative and quantitative effects on Mk differentiation were found to be rapidly induced, and optimization of the culture length at 39°C led to greater Mk yields. Mild hyperthermia did not promote endomitosis of cord blood-derived Mk, but rather led to a small reduction in the proportion of polyploid Mk. Moreover, it had little impact on viability but induced a significant shift in the proportion of cells undergoing apoptosis at 37°C to necrosis at 39°C. Finally, our results suggest that the effects on megakaryocyte differentiation could be the consequences of aberrant expression of key megakaryocyte transcription factors induced by mild hyperthermia. Experiment Overall Design: To define potential differences between megakaryocytes (Mk) derived at 37°C or 39°C, we compared their gene expression repertoire by micro-gene chip technology (Affymetrix GeneChip HG133A). Two independent experiments were carried out on Mk at 37 or 39°C. CD41a+ Mk issued from CB CD34+ cells were cultured for 7-days with TPO at 100 ng/ml and purified by positive magnetic selection using a CD41a-FITC monoclonal antibody (Immunotech, Marseille, France) and MACS columns according to manufacturerâs instructions (Miltenyi, Auburn, CA, USA) with a purity of >95%.
Project description:Tumor Treating Fields (TTFields) disturbs mitosis and consequently leads to cell cycle arrest and cell death. Mild hyperthermia induces cancer cell death by apoptosis,leading to DNA damage and disturbing DNA repair. Thus, when mild hyperthermia is combined with TTFields, the anti-tumor effect could be augmented. This prompted the hypothesis for the present thesis: TTFields and mild hyperthermia as synergistic modalities in Pancreatic ductal adenocarcinoma (PDAC) treatment could probably enhance antitumor efficacy and abate individual toxic effects through distinct and overlapping mechanisms target the tumor cell. Our established PDAC cell line (Bx-GEM) was treated with TTFields and TTFields with mild hyperthermia and was examined by microarray. 500 ng mRNA was checked for quality control and the concentration was measured again. Subsequently, gene expression profiling was performed using human HuGene-2_0-st-type array from Affymetrix.
Project description:Hyperthermia is widely used to treat patients with various cancers. Here, the effects of heat stress at 41°C for 30 min (mild hyperthermia) on the gene expression in OUMS-36 human normal fibroblast cells were investigated using an Affymetrix GeneChip system. The cells were treated with mild hyperthermia, followed by incubation for 0, 1, or 3 h at 37°C. No cell death was observed in the mild hyperthermia-treated cells. On the other hand, many genes that were differentially expressed by a factor 1.5 or greater were identified in the cells treated with the mild hyperthermia.
Project description:Hyperthermia is widely used to treat patients with various cancers. Here, the effects of heat stress at 41°C for 30 min (mild hyperthermia) on the gene expression in Hs68 human skin normal fibroblast cells were investigated using an Affymetrix GeneChip system. The cells were treated with mild hyperthermia, followed by incubation for 0, 1, or 3 h at 37°C. No cell death was observed in the mild hyperthermia-treated cells. On the other hand, many genes that were differentially expressed by a factor 1.5 or greater were identified in the cells treated with the mild hyperthermia.
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:The rationale underlying hyperthermia is the fact that temperatures over 42.5˚C are highly cytotoxic to tumor cells. On the other hand, although mild hyperthermia at a range from 39 to 41˚C alone did not induce cytotoxicity in tumor cells, mild hyperthermia is reported to show a synergism with radiotherapy and anti-cancer drugs. Here, the effects of mild hyperthermia (41˚C for 30 min) on the gene expression in human lymphoma U937 cells were investigated using by an Affymetrix GeneChip system. Although the cells treated with the mild hyperthermia did not induce apoptosis, a significant increase in protein levels of heat shock proteins, Hsp40 and Hsp70, was observed following activation of heat shock factor-1. At 3 h post-treatment, 938 probe sets that were differentially expressed by >1.5-fold were identified. Keywords: mild hyperthermia, gene expression, Human lymphoma U937 cell
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.