Anti-apoptotic action by hypoxia inducible factor 1-alpha in human pituitary adenoma cell line, HP-75 in hypoxic condition.
Hypoxia-inducible factor-1 (HIF-1) alpha is the major transcription factor involved in the adaptive response to hypoxia. The purpose of this study was to investigate whether HIF 1-alpha protects HP75 cells, pituitary adenoma cell line from hypoxia induced apoptosis. HP75 was transfected with siRNA targeting HIF 1-alpha mRNA sequences or scrambled RNA duplexes, followed by subjected to hypoxia (1% oxygen) for 24 h, compared with normoxia (21%). The efficacy of RNAi was assessed via real-time RT-PCR and immunohistochemistry. Apoptosis was determined by Tdt-mediated dUTP nick end-labeling (TUNEL) assay and agarose gel electrophoresis. Membrane cDNA microarray was examined to detect gene profiling among the cell in normoxia, hypoxia, or hypoxia following the RNAi. A significantly greater proportion of HP75 cells transfected with specific siRNA duplexes and subsequently exposed to hypoxia demonstrated apoptosis to a large extent when compared with non-transfected cells. Transfection with specific siRNA duplexes knocked down HIF 1-alpha mRNA and protein expression in hypoxia-exposed cells by approximately 80%, whereas transfection with scrambled siRNA duplexes had no noticeable effect on HIF 1-alpha expression. Microarray analysis indicated that HIF1-alpha down-regulated caspase-10. These findings strongly suggest that HIF 1-alpha exerts an antiapoptotic role in HP75 in hypoxia.
Yoshida
Daizo
D
Department of Neurosurgery, Nippon Medical School, Tokyo, Japan.
Kim
Kyonsong
K
Noha
Masahiro
M
Teramoto
Akira
A
D000222:Adaptation, Physiological; D000236:Adenoma / Q000235:genetics / Q000378:metabolism*; D017209:Apoptosis / Q000235:genetics / Q000502:physiology*; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000235:genetics / Q000502:physiology; D016172:DNA Fingerprinting; D015972:Gene Expression Regulation, Neoplastic; D020868:Gene Silencing; D006801:Humans; D000860:Hypoxia / Q000378:metabolism*; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics / Q000378:metabolism*; D010911:Pituitary Neoplasms / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000032:analysis; D034741:RNA, Small Interfering; D014162:Transfection
D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D012333:RNA, Messenger; D034741:RNA, Small Interfering