formazans and Cell-Transformation--Neoplastic

CD133 (Prominin-1/AC133) is generally treated as a cell surface marker found on multipotent stem cells and tumor stem-like cells, and its biological function remains debated. Genetically modified rat glioma cell lines were generated by lentiviral gene delivery of human CD133 into rat C6 glioma cells (hCD133(+) -C6) or by infection of C6 cells with control lentivirus (mock-C6). Stable hCD133 expression promoted the self-renewal ability of C6-formed spheres with an increase in the expression of the stemness markers, Bmi-1 and SOX2. Akt phosphorylation, Notch-1 activation, and Notch-1 target gene expression (Hes-1, Hey1 and Hey2) were increased in hCD133(+) -C6 when compared to mock-C6. The inhibition of Akt phosphorylation, Notch-1 activation, and Hes-1 in hCD133(+) -C6 cells effectively suppressed their clonogenic ability, indicating that these factors are involved in expanding the growth of hCD133(+) -C6. An elevated expression of GTPase-activating protein 27 (Arhgap27) was detected in hCD133(+) -C6. A decline in the invasion of hCD133(+) -C6 by knockdown of Arhgap27 expression indicated the critical role of Arhgap27 in promoting cell migration of hCD133(+) -C6. In vivo study further showed that hCD133(+) -C6 formed aggressive tumors in vivo compared to mock-C6. Exposure of hCD133(+) -C6 to arsenic trioxide not only reduced Akt phosphorylation, Notch-1 activation and Hes-1 expression in vitro, but also inhibited their tumorigenicity in vivo. The results show that C6 glioma cells with stable hCD133 expression enhanced their stemness properties with increased Notch-1/Hes-1 signaling, Akt activation, and Arhgap27 action, which contribute to increased cell proliferation and migration of hCD133(+) -C6 in vitro, as well as progressive tumor formation in vivo.

Topics: AC133 Antigen; Animals; Antigens, CD; Antineoplastic Agents; Arsenic Trioxide; Arsenicals; Basic Helix-Loop-Helix Transcription Factors; Brain Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Transformation, Neoplastic; Cerebral Cortex; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme Inhibitors; Female; Formazans; Gene Expression Regulation, Neoplastic; Glioma; Glycoproteins; GTPase-Activating Proteins; Homeodomain Proteins; Humans; Intercellular Signaling Peptides and Proteins; Lentivirus; Oncogene Protein v-akt; Oxides; Peptides; Rats; Rats, Sprague-Dawley; Receptor, Notch1; RNA, Messenger; RNA, Small Interfering; Tetrazolium Salts; Time Factors; Transcription Factor HES-1; Transfection; Tumor Stem Cell Assay

This study aimed at examining the effects of hepatitis B viral X protein (HBx) on the biological features and the expression of DNA repair enzymes in non-tumour human hepatic LO2 cells in vitro.. The HBx gene was transfected into LO2 cells to establish stably HBx-expressing LO2/HBx cells. The morphological features, cell growth, cell cycle, apoptosis and colony formation of LO2/HBx cells, vector-transfected LO2/pcDNA3.1 cells and unmanipulated LO2 cells were studied. The expressions of DNA repair enzymes and DNA oxidative stress-related 8-hydroxydeoxyguanosine (8-OHdG) were determined by a real-time quantitative polymerase chain reaction assay and high-performance liquid chromatography coupled with electrochemical detection respectively.. In comparison with controls, significant morphological changes, faster growth, higher frequency of cells at the S phase, but lower at G0/G1 and M/G2 phases, a lower frequency of natural cell apoptosis and a higher percentage of colony formation were observed in the LO2/HBx cells. Furthermore, significantly higher levels of intracellular 8-OHdG and lower levels of human DNA glycosylase alpha (hMYHalpha) mRNA transcripts, but no significant change in human 8-oxoguanine DNA glycosylase 1 (hOGG1), were detected in the LO2/HBx cells.. Our data indicated that HBx promoted growth and malignant transformation of non-tumour hepatic LO2 cells in vitro, which was associated with the downregulation of hMYHalpha expression and accumulation of mutagenic DNA adduct 8-OHdG.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Apoptosis; Cell Cycle; Cell Line; Cell Survival; Cell Transformation, Neoplastic; Colony-Forming Units Assay; Deoxyguanosine; DNA Glycosylases; Down-Regulation; Formazans; Hepatitis B; Hepatitis B virus; Hepatocytes; Humans; Oxidative Stress; Tetrazolium Salts; Trans-Activators; Viral Regulatory and Accessory Proteins