title: The expression of human papillomavirus type 16 (HPV16 E7) induces cell cycle arrest and apoptosis in radiation and hypoxia resistant glioblastoma cells.
abstract: p53 is a widely known tumor-suppressor gene product that plays a key role in apoptotic cell death induced by DNA-damaging chemotherapeutic agents. Human glioma cells with functional p53 are known to be more resistant to γ-radiation. The aim of this study was to investigate whether the mutant glioblastoma cells (U87MG) transfected with human papilloma virus-type 16 E7 (HPV16 E7) genes were capable of increasing sensitivity towards irradiation and hypoxia-induced cell death. The pLXSN retroviral vector expressed HPV-16E7 genes and was infected into the p53 mutated U87MG cell line. A specific amplification band of HPV16 E7 genes was detected in E7 genes and transfected in the U87MG cell line using a reverse transcriptase polymerase chain reaction. The experimental groups included the mutant glioblastoma cell line (U87MG), empty vector (pLXSN) transfected to mutant glioblastoma cell line (U87MG-LXSN), and retrovirus carrying HPV16 E7 genes transfected to the mutant glioblastoma cell line (U87MG-E7). Hypoxic conditions were optimized using LDH assay and the subjects were exposed to hypoxia (16 and 20 h) and irradiation (9 h). Hoechst-propidium iodide (PI) staining results showed that hypoxia and irradiation increased the number of dead cells in the U87MG-E7 cells compared to U87MG and U87MG-LXSN cells. Results of the FACS analysis showed a similar pattern and recorded 80.44 and 58.94% of apoptotic cells in U87MG-E7 and U87MG cells, respectively. Cell cycle analysis by FACS revealed that, following irradiation and hypoxia, cells showed G2-M arrest. Additionally, the Western blot analysis results showed altered expression of E2F-1, Rb and p53 in the irradiation- and hypoxia-induced U87MG-E7 cells compared to U87MG and U87MG LXSN cells. In conclusion, the over-expression of HPV16 E7 genes in U87MG cell lines increasd cell apoptosis and E2F1 expression compared to the HPV non-infected U87MG cells following irradiation and hypoxia. These results indicate that tumor-specific therapies that increase sensitivity towards radiation and hypoxic conditions may be beneficial for suppression of cancers.
authors.affiliation: Department of Anatomy, Yonsei University College of Medicine, Seoul 120-752, Republic of Korea.
authors.forename: Sung-Ung
authors.initials: SU
authors.lastname: Moon
authors.forename: Soo Kyoung
authors.initials: SK
authors.lastname: Choi
authors.forename: Han Jo
authors.initials: HJ
authors.lastname: Kim
authors.forename: Kiran
authors.initials: K
authors.lastname: Kumar Bokara
authors.forename: Kyung Ah
authors.initials: KA
authors.lastname: Park
authors.forename: Won Taek
authors.initials: WT
authors.lastname: Lee
authors.forename: Jong-Eun
authors.initials: JE
authors.lastname: Lee
mesh_terms: D017209:Apoptosis / Q000528:radiation effects*; D059447:Cell Cycle Checkpoints / Q000528:radiation effects*; D015687:Cell Hypoxia; D045744:Cell Line, Tumor; D050687:E2F1 Transcription Factor / Q000378:metabolism; D005720:Gamma Rays; D005822:Genetic Vectors / Q000737:chemistry / Q000378:metabolism; D005909:Glioblastoma / Q000473:pathology / Q000532:radiotherapy*; D052162:Human papillomavirus 16 / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D050725:Papillomavirus E7 Proteins / Q000378:metabolism; D016160:Retinoblastoma Protein / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism
chemical_list: D050687:E2F1 Transcription Factor; D050725:Papillomavirus E7 Proteins; D016160:Retinoblastoma Protein; D016159:Tumor Suppressor Protein p53