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에너지 소실에 의한 유사 분열 이탈과 그 기전 연구
- Alternative Title
- Mitotic slippage by ATP depletion and its mechanism
- Author(s)
- Park yunyeon
- Alternative Author(s)
- Yun Yeon Park
- Advisor
- 이재호
- Department
- 일반대학원 의생명과학과
- Publisher
- The Graduate School, Ajou University
- Publication Year
- 2016-08
- Language
- eng
- Keyword
- Mitotic slippage
- Alternative Abstract
- It has been known that tumor cells possess two main energy checkpoints monitoring the amount of available ATP during G1 phase and G2 to M transition. However, it is still uncertain the existence of energy checkpoint during mitotic progression. In our hand, ATP depletion by the co-treatment with 2-deoxy glucose (2-DG) and sodium azide (NaN3) during interphase caused robust cell cycle arrest, supporting the existence of energy checkpoints sensing shortage of ATP supply before mitosis. On the other hand, reduction of ATP during or after prometaphase did not affect mitotic progression, suggesting the absence of energy checkpoint after prometaphase. Interestingly, the ATP depletion during mitotic arrest induced mitotic slippage, which is evidenced by reduction of rounded mitotic cells, degradation of cyclin B, increase of cell attachment, and reassembly of nuclear membrane. The results of EdU incorporation assay and time-lapse imaging revealed that survived cells from mitotic slippage preceded next interphase, but finally died even in normal growth condition. Although degradation of cyclin B during normal mitotic progression is regulated by APCCdc20, decrease of Cdc20 paradoxically occurred prior to cyclin B degradation during mitotic slippage by ATP depletion. The results presenting the changes in Cdc20 level by MG132 or cycloheximide treatment demonstrated that the decrease of Cdc20 is not attributable to enhanced degradation but reduced translation. Moreover, we confirmed that APCCdh1, but not APCCdc20, is responsible for cyclin B degradation following ATP depletion by Cdh1 siRNA experiment and immunoprecipitation analysis. Through the mitotic slippage by ATP depletion, we could not find any changes of several spindle assembly checkpoint proteins including BubR1, Mad2, and MCAK. However, the localization and the kinase activity of Aurora B, which is widely recognized as a component of error correction of microtubule attachment and its role in maintenance of spindle assembly checkpoint, were reduced on kinetochores during the mitotic slippage by co-treatment with 2-DG and NaN3. Decrease of p-CENP-A and p-histone H3 (T3) on kinetochore during the mitotic slippage also supported that kinase activity of Aurora B is inhibited by co-treatment with 2-DG and NaN3. Moreover, phosphorylation of Histone H3 at Thr 3 by Haspin was especially decreased by ATP depletion, suggesting that activity of Haspin to recruit the chromosomal passenger complex (CPC) at centromeres is also attenuated during mitotic slippage. Overall, inactivation of Aurora B and Haspin at centromeres by ATP depletion contributes to the induction of mitotic slippage. We also observed the increase of inhibitory phosphorylation Cdk1 (pY15) by ATP depletion, suggesting the inactivation of Cdk1 kinase during mitotic slippage. Reactivation of Cdk1 during mitotic slippage by treatment with PD166285, a Wee1/Myt1 inhibitor, partially reduced the induction of mitotic slippage by ATP depletion. Induction of mitotic slippage by direct inhibition of Cdk1 kinase activity using RO3306 treatment was also observed the possible involvement of Cdk1 inactivation in mitotic slippage. On the other hand, these results suggest that Cdk1 inactivity was partially involved in the induction of mitotic slippage by ATP depletion. Since AMP-activated protein kinase (AMPK) is a central regulator of cellular energy homeostasis, we assumed the possible involvement of AMPK in the induction of mitotic slippage by ATP depletion. However, inhibition of AMPK using AMPK siRNA did not alter the progression of mitotic slippage by ATP depletion, confirming the irrelevant involvement of AMPK in mitotic slippage. Taken together, ATP depletion by co-treatment with 2-DG and NaN3 during mitotic arrest induces mitotic slippage, through decrease of APCCdc20 and increase of APCCdh1, abrogation of mitotic checkpoint activity via inhibition of Aurora B and Haspin activity at centromere as well as Aurora B centromere-localization, and Cdk1 inactivation by increase of pY15. --------------------------------------------------------- Mitotic slippage, ATP depletion, cyclin B, Cdc20, Cdh1, Aurora B, Haspin, Cdk1
- Fulltext
- Appears in Collections:
- Graduate School of Ajou University > Department of Biomedical Sciences > 4. Theses(Ph.D)
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