Anti-apoptotic role of cenexin1

국문 초록 (Abstract)

Apoptosis는 불필요한 세포나 노화된 세포, 각종 상해를 받은 세포에서 enzyme의 순차적인 활성에 의한 자살프로그램에 의해 조절되는 세포사멸의 경로이다. Apoptosis에 의한 죽음은 세포 숫자와 조직의 크기를 유지하며 세포내의 항상성을 위협하는 자극으로부터 보호하도록 하는 세포의 정상적인 현상이다. p53은 일종의 전사인자로 세포분열의 제어나 apoptosis등을 포함한 세포의 다양한 반응을 유도한다. p53은 암세포에서 과발현 시켰을 경우 apoptosis를 유발한다. p53을 과발현 시키는 아데노바이러스로부터 살아남은 clone으로부터 Cenexin1 의 C-terminal fragment를 얻었다. 얻어진 C-terminal fragment와 cenexin1 의 full length는 p53에 의한 extrinsic death signal 과 intrinsic death signal을 통한 apopotosis에서 저항성을 갖는다. siRNA에 의한 Cenexin1의 knock down은 TNF-a에 의해서 유도되는 caspase-8와 PARP의 활성을 늦췄다. extrinsic pathway에서 ligands(FasL, TNFα, etc.)나 receptor recognizing antibodies에 의한 death receptors (Fas, TNF receptor etc.)의 binding은 adaptor molecules (FADD, TNF receptor 1-associated death domain protein (TRADD), etc.)의 보충을 유도한다. TNF에 의한 apoptosis는 TNF Receptor Associated Death Domain Protein (TRADD) protein, Fas-associated death domain protein (FADD)과 pro-caspase-8으로 구성된 DISC를 형성하여 apoptosis의 활성에 관여한다. Cenexin1은 FADD과 co-localization됨을 확인했고 TNF receptor complex와의 localization의 가능성을 제시했다.
이 논문에서 우리는 cenexin1의 anti-apoptotic으로서의 기능과 TNF receptor complex와의 localize의 가능성, caspase-8 cleavage의 억제를 연구했다. 게다가 cenexin1은 새로운 anti-apoptotic protein family 의 구성원일 수도 있음을 나타냈다.

다국어 초록 (Multilingual Abstract)

Apoptosis is a pathway of cell death that is controlled by tightly regulated suicide program in which cells eventually enter to death by sequential activations of enzymes. Death by apoptosis is a normal phenomenon that serves to get rid of cells that are no longer needed and to maintain a steady number of cell populations in tissue. Apoptosis eliminates cells that are injured beyond repair without inducing a host inflammatory response, thus protecting surrounding tissue from damage. From functional cloning of gene that provides viabilities under the adenovirus mediated overexpression of p53, C-terminal fragment of Cenexin1 could be obtained among the survived cell clones. Both the rescued C-terminal fragment and full length of Cenexin1 had given resistance against apoptosis under the extrinsic death signal activation and intrinsic death signal activation by p53. The siRNA mediated knock down accelerated TNF-α mediated cell death and over expression of this gene showed delayed activation of caspase-8 and cleavage of PARP. In the extrinsic pathway, binding of the death receptors (Fas, TNF receptor etc.) by the ligands (FasL, TNFα, etc.) or by the receptor recognizing antibodies leads to the recruitment of adaptor molecules (FADD(Fas-associated death domain protein), TRADD(TNF receptor 1-associated death domain protein) etc.) and an initiator caspase, caspase-8, to the receptor complex forming DISC(death inducing signalling complex). Under the condition of induction of apoptosis by TNF, the DISC is comprised of TRADD, FADD and pro-caspase-8. The Cenexin1 colocalized with FADD, suggesting that it might be one of the factors in TNF receptor complex.
In this study we evaluated the antiapoptotic function of Cenexin1 and its possible action mechanism through localization of TNF receptor complex and inhibition of caspase-8 cleavage. In addition to its centrosomal function as centrosomal scaffold protein, Cenexin1 might be a novel member of antiapoptotic protein family.

목차 (Table of Contents)

• Ⅰ.Introduction 1
• Ⅱ.Materials and Methods 4
• 1.Cell Culture and transfection of plasmids and siRNA 4
• 2.Preparations of recombinant GST-Cenexin1 protein and generation of antisera 4
• 3.Construction of stable cell lines expressing rescued ODF2/Cenexin1 clones 5
• 4.Cell death counting - cell viability assay 5
• 5.Western blot analysis 6
• 6.Construction of stable cell lines expressing full length of ODF2 and Cenexin1 6
• 7. Immunofluorescence staining and confocal microscopy 7
• Ⅲ.Results 8
• 1.Purification of recombinant GST-Cenexin1 protein and raise of polyclonal antisera 8
• 2.Generation of stable cell lines expressing rescued gene 8
• 3.Stable overexpression of C-terminal Cenexin protects SaOs-2 cell from cell death stress induced by p53, TNF-α 9
• 4.Stable overexpression of C-terminal Cenexin1 inhibits the activation of Caspase-3, -9 and cleavage of PARP 9
• 5.SiRNA mediated knock down of ODF2/Cenexin1 can accelerates apoptosis 10
• 6.Investigation of activation of caspase-8 10
• 7.Colocalization of EGFP-Cenexin and FADD 11
• 8.Overexpression of full length of ODF2/Cenexin1 shows delayed activation of caspase-8 11
• Ⅳ.Discussion 23
• References 26
• Abstract in Korean 30