Project description:To find candidate circRNAs and to unravel their molecular functions during colorectal cancer progression and during LDM topotecan chemotherapy, we utilized a xenograft mouse model based on the HT29.hCG.Luc colorectal cancer cell line (referred to as HT29) implanted into SCID mice. HT29 cells were injected into the spleen and primary tumors developed. Three mice served as control group while two mice served as LDM topotecan treated group. After another 4-6 weeks, liver metastasis can be detected and resected for further investigation. For a global view on miRNA changes, we performed miRNA-Seq from HT29 cells, primary tumors and liver metastases from control mice (C-PT and C-LM) and liver metastases from treated mice (T-LM).
Project description:We report the single cell RNA sequencing on HT29 colorectal cancer line with 100 nM dasatinib treatment after double thymidine block.
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:In the present study we have isolated and characterized cancer stem cells and non-cancer stem cells (bulk tumor cells) from high grade human colorectal cancer cell line HCT116 and low grade human colorectal cancer cell line HT29. For this study, cancer stem cells and non-cancer stem cells (bulk tumor cells) were isolated from HCT116 and HT29 human colorectal cancer cell line. For isolating cancer stem cells by FACS, CD44 and CD166 tagged with V450 and PE respectively were used. CD44+CD166+ was the cancer stem cell population and CD44-CD166- was designated as the non-cancer stem cell (bulk tumor cells) population for this study. RNA was isolated from the isolated cell populations and microarray was done to study the whole genome transcriptomic changes.
Project description:Affymetrix HG-U133 Plus2 Array was applied to identify differentially expressed genes between FACS sorted CD26+ and CD26- subpopulations of HT29 colorectal cancer cell-line.
Project description:To find candidate circRNAs and to unravel their molecular functions during colorectal cancer progression and during LDM topotecan chemotherapy, we utilized a xenograft mouse model based on the HT29.hCG.Luc colorectal cancer cell line (referred to as HT29) implanted into SCID mice. HT29 cells were injected into the spleen and primary tumors developed. Three mice served as control group while two mice served as LDM topotecan treated group. After another 4-6 weeks, liver metastasis can be detected and resected for further investigation. For a global view on gene expression changes, we performed rRNA-depletion RNA-Seq from HT29 cells, primary tumors and liver metastases.
