J. Cell Cycle Analysis
V. CONCLUSION
In conclusion of part I, our study demonstrates that HD doxorubicin-induced apoptosis and LD doxorubicin-induced mitotic cell death are largely independent, and the activation of distinct signaling pathways may be responsible for the induction of these morphologically distinct cell death modes. Elucidation of the factors that regulate the various aspects of treatment-induced apoptosis and mitotic catastrophe should assist in improving the efficacy of cancer therapy.
In conclusion of part II, p21 play pivotal role in maintaining of G1 or G2
arrest by DNA damage and so preventing mitotic catastrophe by induction of cell cycle arrest. Even though G2 arrest was abrogated by caffeine, mitotic catastrophe was prevented because p21 induced cell cycle arrest at G1 phase.
96
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납작해지면서 senescence associated β-galactosidase (SA-β-gal)의 활성이
증가하는 노화유사 표현형 (senescence-like plenotype; SLP)이 관찰되었다.
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세포의 생존도는 서서히 apoptosis가 아닌 mitotic catastrophe 가 원인이
되는 세포사에 의해 감소하기 시작하여 doxorubicin 처리 9일째에 50%
정도의 세포사가 관찰되었다. Mitotic catastrophe에 의한 cell death 시에는
세포막을 구성하는 phosphatidyl serine의 위치변화 및 mitochondria로부터
cytochrome c 가 세포질로 방출되는 현상이 apoptosis 시와 공통되게
관찰되었으나, stress activated MAPK (p38, JNK) 및 caspases 들의 활성화가
apoptosis와 다르게 거의 나타나지 않았으며, 또한, 핵막의 integrity가
소실됨이 관찰 되었는데 이러한 결과들은 Huh-7 세포에서 관찰되는
mitotic catastrophe에 의한 cell death는 apoptosis와는 전혀 다른 necrosis와
유사한 세포사멸 기전임을 확인하였다. 이러한 LD doxorubicin에 의한 cell
death의 원인인 mitotic catastrophe (혹은, mutinucleation)의 조절 기전을
이해하기 위하여, p21 유전자를 가지고 있거나 (P21+/+) 혹은 가지고 있지
않은 (P21-/-) human colorectal carcinoma cell line인 HCT116 세포에
doxorubicin을 처리한 후 multinucleation 정도를 비교하였다. p21을 발현할
수 있는 세포에서는 (p21+/+ cells) multinucleation 이 유도되지 않는 반면에,
p21이 없는 세포 (p21-/- cells) 에서는 multinucleation이 관찰되었다. 또한, 50
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nM의 doxorubicin이 처리된 p21+/+ 세포는 G1 cell cycle에 세포들이 arrest
되는 반면 p21-/- 세포들에서는 특정 cell cycle에 arrest 되지 않으면서 Cdc2
및 Cdk2 활성이 그대로 유지되었다. Cdc2 와 Cdk2 활성 억제제인
roscovitine 이나 purvalanol A를 p21-/- 세포에 처리하는 경우, multinucleation
생성을 감소시킬 수 있었는데, 이러한 사실은 p21-/- 세포에서 Cdc2 와
Cdk2의 활성 유지가 multinucleation 생성에 매우 중요하게 작용함을
시사한다. 그러나 500 nM의 doxorubicin을 처리하면 p21의 유무에 상관없이
ATM/Chk signaling pathway가 활성화 됨으로서 G2 cell cycle arrest 가
유도되고 multinucleation은 생성되지 않았다. 그러나, ATM/Chk signaling
pathway의 활성을 억제할 수 있는 caffeine을 함께 처리하면 p21+/+ 세포는
G2 phase arrest 가 풀린 후 G1 phase에서 다시 arrest가 되면서
multinucleation은 유도되지 않았다. p21-/- 세포에서는 G1 cell cycle arrest 가
관찰되지 않았고 multinucleation 생성은 caffeine 농도에 의존적으로 크게
증가하였다. 결론적으로 doxorubicin은 abnormal mitosis가 원인이 되어
multinucleation 을 유도하는데, 이러한 multinucleation의 유도를 위해서는
Cdc2 및 Cdk2 활성이 요구되며, 또한, DNA damage 후 특정 cell cycle arrest
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(G1 or G2)가 일어나지 않는 조건을 필요로 한다는 사실을 확인하였다.
따라서 p21은 LD doxorubicin 에 노출된 세포에서 Cdc2 및 Cdk2 의 활성을
억제함으로서 multinucleation 생성을 막는데 반드시 필요하지만, MD
doxorubicin은 p21의 존재유무에 상관 없이 G2 arrest를 유도시킴으로서
multinucleation 생성을 억제하지만 이러한 G2 arrest를 극복하는 세포에서는
p21이 G2 arrest 이외에 부가적으로 작용하여 이 세포들을 다시 G1 phase 에
arrest 를 시킴으로서 multinucleation 생성을 억제할 수 있다.
핵심되는 말: Doxorubicin, Mitotic catastrophe, Senescence-like phenotype, Apoptosis, p21, Multinucleation,
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LIST OF PUBLISHED PAPERS
1: Involvement of c-Src kinase in the regulation of TGF-beta1-induced apoptosis. :Oncogene. 2004 Aug 19;23(37):6272-81.
Park SS, Eom YW, Kim EH, Lee JH, Min do S, Kim S, Kim SJ, Choi KS.
Institute for Medical Sciences, Ajou University School of Medicine, 5 Wonchon-Dong, Paldal-Gu, Suwon 442-749, Korea.
Transforming growth factor-beta1 (TGF-beta1) is a potent inducer of apoptosis in normal hepatocytes, and acquiring resistance to TGF-beta1 may be a critical step in the development of hepatocellular carcinoma (HCC). In this study, we investigated the possible involvement of c-Src in the regulation of TGF-beta1-induced apoptosis.
TGF-beta1 induced transient activation of c-Src and its subsequent caspase-mediated degradation concomitant with cell death in FaO hepatoma cells, which are sensitive to TGF-beta1. In response to TGF-beta1, activated c-Src was translocated into the cytoplasmic membrane, then relocated to the nuclei of apoptotic cells during its cleavage. In TGF-beta1-induced apoptotic cells, c-Src maintained its tight association with p85 FAK fragment cleaved by caspases, possibly contributing to focal adhesion disassembly. TGF-beta1-induced apoptosis was enhanced by either
115
inhibition of c-Src activity using PP1 or PP2, or by overexpression of dominant-negative c-Src. In contrast, overexpression of constitutively active c-Src inhibited apoptosis suppressing TGF-beta1-induced activation of p38, JNK and caspases. In many HCC cell lines resistant to TGF-beta1, enhanced c-Src activity was detected.
We hypothesize that activated c-Src in HCC may contribute to resistance against the apoptotic and/ or antiproliferative properties of TGF-beta1.
PMID: 15208664 [PubMed - indexed for MEDLINE]
2: Leukotriene B(4) stimulates Rac-ERK cascade to generate reactive oxygen species that mediates chemotaxis. :J Biol Chem. 2002 Mar 8;277(10):8572-8.
Epub 2001 Dec 27.
Woo CH, You HJ, Cho SH, Eom YW, Chun JS, Yoo YJ, Kim JH.
Woo CH, You HJ, Cho SH, Eom YW, Chun JS, Yoo YJ, Kim JH.