D. TIS21 inhibited Chk2 phosphorylation through regulation of Mre11
Ⅵ. CONCLUSION
In the present study, downregulation of p-Chk2T68 and p-p53S20 in Ad-TIS21-HA infected Huh7 cells after treatment of etoposide were observed during DNA damage signal.
It was also confirmed in several different cell lines. Inhibition of p-Chk2T68 caused downregulation of p53-dependent-apoptotic protein such as BAX, p21Waf1/Cip1, and NOXA in the same condition, leading to anti-apoptotic responses. We confirmed the apoptosis by using apoptotic marker such as cleaved form of caspase3, PARP, caspase3/7 activity, FACS, and chromatin condensation. Furthermore, TIS21 induced Mre11 protein methylation and we thought TIS21 increased Mre11 DNA repair activity.
Therefore, TIS21 involved DNA damage pathway. However, it is still more identified of which TIS21 inhibits Chk2 phosphorylation.
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- 국문요약 -
TIS21 이 Etoposide 에 의해 유도되는 DNA 손상 후 수복기작에 미치는 영향 연구
아주대학교 대학원 의생명과학과 최규성
(지도교수: 박태준)
TIS21/BTG2/PC3 (12-O-tetradecanoyl phorbol-13-acetate-inducible
sequence 21) 은 p53 의 타깃 유전자로써 갑상선, 전립선, 신장 및 간에서의
암 발생과정 중에서 암을 억제하는 기능을 하는 것으로 알려져 있다. 또한 암을
치료하기 위한 화학요법에 의해 발생하는 세포사멸 중 TIS21 의 발현됨을
관찰함으로써, 세포사멸 과정 중의 TIS21 이 중요시되었다.
이 연구는 암 억제유전자인 TIS21 이 etoposide 에 의해 일어나는 세포
사멸조절 기작의 관여에 대해 연구하기 위하여 실시되었다. DNA 손상의 마커인
gH2AX 의 foci 형성을 wild type MEF 와 TIS21-/-mouse embryo
fibroblasts(MEFs)에서 관찰 해 본 결과 TIS21-/- MEFs 에서 gH2AX foci
형성되는 세포들이 wild type MEFs 보다 더 많이 관찰 되었으며, 이는
TIS21 이 DNA 수복 기작에 관여를 한다는 것을 의미하였다. 다음으로 DNA
손상 마커인 single cell electrophoresis 라고 불리는 comet assay 를 시행
하였다. Etoposide 를 사용하여 adenovirus LacZ 와 TIS21 이 과발현 된
간암세포주인 Huh7 세포에 DNA 손상을 주었으며, 이때 TIS21 이 과발현 된
세포에서의 comet tail 이 더 짧은 것으로 보아, TIS21 이 DNA 수선 역할을 할
수 있다는 증거를 관찰하였다. 다음으로 DNA 손상 시그널에 TIS21 이
관여하는지를 연구하기 위해, ATM-mediated DNA 수복과 p53-mediated
apoptosis 에 관여되는 단백질들의 발현과 인산화 정도를 관찰하였다. Chk2 의
Thr68 잔기의 인산화는 DNA 손상 후 시그널을 하위로 내려 보내는 중요한
단백질로 Ad-TIS21-HA 가 과발현된 세포에서 Thr68 잔기의 인산화와
Chk2 의 직접적인 하위 시그널인 p53 의 Ser20 잔기의 인산화가 Ad-LacZ 가
과 발현 된 세포보다 적은 것을 관찰 하였다. p53 의 인산화는 단백질의 안정화
및 전사조절에 중요한 역할을 하며, 나아가 세포주기 억제와 세포사멸에 영향을
미치게 된다. Wild type p53 을 발현하는 MCF7, HeLa, H9C2 와 같은 다양한
세포주에서도 동일한 현상을 관찰하여 mutant p53 을 가지고 있는 Huh7 에서
관찰되는 Chk2, p53 인산화의 저해가 p53 비 의존적으로 관찰됨을 알게 되었다.
p53 의 Ser20 잔기 인산화의 감소가 세포사멸 기작에 미치는 영향을 연구하기
위해 BAX, p21Waf1/Cip1, NOXA 와 같은 세포사멸에 관계된 단백질 발현을
관찰해본 결과, TIS21 이 과발현 세포에서 세포사멸에 관계된 단백질의 발현이
감소 되어있는 것을 관찰하였다. 또한 caspase 3/7 활성도 및 annexin V 염색이
TIS21 과 발현 세포에서 감소함을 관찰하였다. TIS21 은 protein arginine
methyltransferase 1(PRMT1) 과 결합을 통하여 PRMT1 의 활성도를
조절한다는 보고를 바탕으로 TIS21 이 과 발현되어있을 때 PRMT1 의 기질 중
DNA 수선에 관여하는 Mre11 의 메칠화가 증가하는 것을 관찰 하였다. Mre11
의 과 발현이 또한 Chk2 의 인산화를 억제하는 것으로 관찰되어 TIS21 이
Mre11 의 메칠화를 통해 Chk2 의 인산화에 영향을 미친다는 것을 관찰 하였다.
TIS21 은 Mre11 과의 결합 및 메칠화를 통해 Chk2 의 인산화를 억제하는
것으로 보아 Chk2-p53 를 통한 세포 사멸을 억제하며 DNA 수복에 관여
한다는 결론을 낼 수 있다.
핵심어: ATM, Chk2, BTG2, TIS21, Mre11, DNA damage, apoptosis