[TAp63gamma-induced apoptosis mediated by apoptosis inducing factor in human esophageal squamous carcinoma EC9706 cells].
OBJECTIVE
To study the molecular mechanism of TAp63gamma-induced cell apoptosis.


METHODS
Transcription and protein expression of apoptosis inducing factor and p63 were investigated by immunohistochemistry and RT-PCR in human esophageal squamous carcinoma cell line EC9706 respectively. Twenty-four hours after transfection with pcDNA3.1-TAp63gamma, the apoptosis and translocation of apoptosis inducing factor in EC9706 cells were studied by flow cytometry, laser confocal microscopy and mitochondrial/cytosol/nuclear extraction analysis respectively. Down-regulation of apoptosis inducing factor protein was achieved by RNAi and pretreatment with caspase inhibitor zVAD.fmk of EC9706 cells.


RESULTS
Presence of protein expressions of apoptosis inducing factor and absence of TAp63gamma was observed in the cytoplasm of untransfected cells. RT-PCR verified the subtype of p63 in EC9706 cells was DeltaNp63. After 24 hours of transfection, both nuclear and cytoplasmic expression of apoptosis inducing factor protein were observed in cells transfected with TAp63gamma and p53 expression vectors, but not in cells transfected with control vector. Cell apoptosis rates were 1.37%, 13.64%, 4.52%, 4.03% and 1.91% in the pcDNA3.1 transfection group, pcDNA3.1-TAp63gamma transfection group, apoptosis inducing factor siRNA and pcDNA3.1-TAp63gamma transfection group, zVAD.fmk treatment group, and the group receiving apoptosis inducing factor siRNA, plus zVAD.fmk treatment and pcDNA3.1-TAp63gamma transfection, respectively.


CONCLUSIONS
Apoptosis inducing factor of EC9706 cells is released from mitochondria into both the cytoplasm and nucleus during TAp63gamma induced apoptosis. Down-regulation of apoptosis inducing factor inhibits TAp63gamma-induced apoptosis. Overall, TAp63gamma-induced apoptosis is dependent on the expression of apoptosis inducing factor and caspase.
Fan
Tian-li
TL
Department of Pathology, Henan Institute of Medical Science, Zhengzhou University, Zhengzhou 450052, China.
Hao
Yi-bin
YB
Xu
Pei-rong
PR
Hou
Gui-qin
GQ
Jiang
Guo-zhong
GZ
Yang
Guan-rui
GR
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D017209:Apoptosis; D051033:Apoptosis Inducing Factor / Q000235:genetics / Q000378:metabolism*; D002294:Carcinoma, Squamous Cell / Q000378:metabolism / Q000473:pathology*; D061945:Caspase Inhibitors; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D015536:Down-Regulation; D004938:Esophageal Neoplasms / Q000378:metabolism / Q000473:pathology*; D006801:Humans; D008928:Mitochondria / Q000378:metabolism; D010957:Plasmids; D021381:Protein Transport; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors; D014162:Transfection; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism*