Analysis of Molecular Mechanism of ARF-dependent apoptotic cell death

ARF依赖性细胞凋亡的分子机制分析

• 批准号: 13214040
• 负责人: KAMIJO Takehiko
• 金额: $ 21.76万
• 依托单位: Shinshu University Hospital and Shinshu University School of Meclioine
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research on Priority Areas
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-13214040/
• 关键词: ARF; p53; Apoptosis; Mitochondria; MAPK; Caspase; SV4OT; SV40T; Apootosis; apoptosis; ERK; cell cycle; 癌抑制遺伝子; 白血病; アポトーシス

The tumor suppressor p19ARF, p14ARF in humans, is encoded by the Ink4a/ARF locus (Quelle, D. E.et al., (1995) Cell 83, 993-100) and mutated, deleted, or silenced in many forms of cancer. pl9ARF induces growth arrest by antagonizing the activity of the p53-negative regulator, Mdm2, thereby permitting a p53 transcriptional response (Kamijo, T. et al., (1997) Cell 91, 649-659; Kamijo T. et al., (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 8292-829'). Although ARF activation and p53 stabilization alone are not sufficient to induce apoptosis in all cell types, upregulation ofp53 in this way strongly sensitizes cells to die in response to genotoxic stresses or additional apoptotic signals induced by oncogenes themselves. In this project, we first analyzed the mechanism of ARF-dependent apoptosis and demonstrated that ARF induces mitochondria-dependent apoptosis in p53 wild-type, ARF/p16-null cells. We also found that ARF evokes cytocbrome c release from mitochondria, decreases mitochondria mem … More brane potential and activates pro-caspase-9 to induce apoptosis. Our findings suggest that this apoptotic cellular modulation is brought about by up-regulation of the pro-apoptotic Bc1-2 family proteins Bax and Bim, and down-regulation of anti apopt3tic Bcl-2, in mitochondrial fractions. Additionally, ARF seems to down-tegulate Bc1-2 in a p53-dependent manner, while up-regulating Bax/Bim via a p53-independent pathway (Nakazawa Y, Kamuo, Koike K, Noda T. J Biol Chem. 278(30) : 27888-95. 2003)A recent study showed that triple knockout (TKO) mice lacking ARF, p53, and MDM2 develop multiple and more aggressive tumors per animal than mice lacking either gene alone (Weber, J. D et al., (1999) Nat. Cell Biol. 1, 20-26), indicating that ARF may suppress tumor progression by a p53/MDM2-independent manner. Moreover, previous reports presented that murine and human ARFs interact with MDM2 via NH2-terminal domains, but the functional domain of ARF and the molecular mechanism of p53/MDM2-independent tumor suppression has thus far not been identified. Taken together these findings, we next addressed the molecular mechanism of ARF-induced apoptotic cell death in a p53-independent manner using a p53/Rb-inactivated cell line, and, surprisingly, found that the COOH-terminal of ARF has the ability to induce p53-independent cell death. This is the first report that describes the apoptotic cell death-function of the p19ARF COOH-terminal. Furthermore, we address the responsible caspase cascade for p19ARF COOH-terminal induced apoptosis and relationship between the activated caspases and MAPKs controlling p19ARF COOH-terminal-induced apoptosis. For further investigation of ARF-induced cell death, we made p53/Rb-inactivated cells by introducing SV4OT into ARF-null MEFs. Re-introduction of ARF into the p53/Rb-inactivated cells up-regulated mitochondria-dependent apoptotic cell death accompanied cytochrome c release from mitochondria and activation of the intrinsic pathway caspases, i.e. caspase-9,-3 and-7. Interestingly, ARF bound SV4OT in the ARF-induced cells and re-activation of p53 was seemed to be mediated by the SV4OT inactivation by ARF, resulting in the mitochondrial dysfunction (Nakazawa Y and Kamijo T, submitted). Less

肿瘤抑制因子p19 ARF，人中的p14 ARF，由Ink 4a/ARF基因座编码（Quelle，D. E.et al.，（1995）Cell 83，993-100），并且在许多形式的癌症中突变、缺失或沉默。p19 ARF通过拮抗p53负调节因子Mdm 2的活性诱导生长停滞，从而允许p53转录应答（Kamijo，T.例如，等人（1997）Cell 91，649-659; Kamijo T.例如，等人（1998）Proc. Acad. Sci.联合S. A. 95，8292- 829 '）。尽管ARF激活和p53稳定化本身不足以在所有细胞类型中诱导凋亡，但p53以这种方式上调强烈地使细胞对基因毒性应激或癌基因本身诱导的额外凋亡信号敏感而死亡。在本研究中，我们首先分析了ARF依赖性细胞凋亡的机制，并证明了ARF诱导p53野生型、ARF/p16缺失细胞中的凋亡依赖性细胞凋亡。我们还发现，ARF引起线粒体释放细胞溴c，减少线粒体钙离子浓度， ...更多信息 膜电位并激活caspase-9前体以诱导细胞凋亡。我们的研究结果表明，这种凋亡细胞的调制是由促凋亡Bcl-1 -2家族蛋白Bax和Bim的上调，以及抗凋亡Bcl-2的下调，在线粒体组分。此外，ARF似乎以p53依赖性方式下调Bcl-2，同时通过p53非依赖性途径上调Bax/Bim（Nakazawa Y，Kamuo，Koike K，Noda T. J Biol Chem.278（30）：27888-95. 2003）最近的一项研究表明，缺乏ARF、p53和MDM 2的三重敲除（TKO）小鼠比单独缺乏任一基因的小鼠在每只动物中发展出多个和更具侵袭性的肿瘤（Weber，J. Det al.，（1999）Nat. Cell Biol.1，20-26），表明ARF可以通过p53/MDM 2非依赖性方式抑制肿瘤进展。此外，以前的报道表明，鼠和人ARF通过NH 2-末端结构域与MDM 2相互作用，但ARF的功能结构域和p53/MDM 2非依赖性肿瘤抑制的分子机制迄今尚未确定。综合这些发现，我们接下来使用p53/Rb失活的细胞系，以p53非依赖性方式探讨了ARF诱导的凋亡性细胞死亡的分子机制，并且令人惊讶地发现，ARF的COOH末端具有诱导p53非依赖性细胞死亡的能力。这是首次报道p19 ARF羧基端的凋亡细胞死亡功能。此外，我们解决了负责caspase级联p19 ARF羧基端诱导的细胞凋亡和激活的caspase和MAPK控制p19 ARF羧基端诱导的细胞凋亡之间的关系。为了进一步研究ARF诱导的细胞死亡，我们通过将SV 4 OT引入ARF无效的MEFs来制备p53/Rb失活的细胞。将ARF重新引入p53/Rb失活的细胞中，上调线粒体依赖性凋亡细胞死亡，伴随着细胞色素c从线粒体释放和内在途径半胱天冬酶（即半胱天冬酶-9、-3和-7）的激活。有趣的是，ARF在ARF诱导的细胞中结合SV 4 OT，并且p53的再活化似乎是由ARF引起的SV 4 OT失活介导的，导致线粒体功能障碍（Nakazawa Y和Kamijo T，提交）。少

项目成果

ST1571 inhibits growth and adhesion of human mast cells in culture.

ST1571 抑制培养物中人类肥大细胞的生长和粘附。

• 发表时间: 2003
• 期刊: J Leukoc Biol. 74
• 作者: Takeuchi K;Koike K;Kamijo T;他10名
• 通讯作者: 他10名

Successful unrelated cord blood transplantation using a reduced-intensity conditioning regimen in a 6・month・old infant with congenital neutropenia complicatod by severe pneumonia.

对一名患有先天性中性粒细胞减少症并发严重肺炎的 6 个月大婴儿，使用降低强度调理方案成功进行无关脐带血移植。

• 发表时间: 2004
• 期刊: Int J Hematol. 80(3)
• 作者: Nakazawa Y;Sakashita K;Kinoshita M;Saida K;Shigemura T;Yanagisawa R;Shikama N;Kamijo T;Koike K.
• 通讯作者: Koike K.

ARF tumor dependent apoptosis by modulation of mitochondrial Bcl

通过调节线粒体 Bcl 来实现 ARF 肿瘤依赖性细胞凋亡

• 发表时间: 2003
• 期刊: J Biol Chem. 278
• 作者: Nakazawa Y;Kamijo T;Koike K.Noda T.
• 通讯作者: Koike K.Noda T.

STI571 inhibits growth and adhesion of human mast cells in culture

STI571 抑制培养物中人类肥大细胞的生长和粘附

• DOI: 10.1189/jlb.0602284
• 发表时间: 2003
• 期刊: Journal of Leukocyte Biology
• 影响因子: 5.5
• 作者: K. Takeuchi;K. Koike;T. Kamijo;S. Ishida;Y. Nakazawa;Y. Kurokawa;K. Sakashita;T. Kinoshita;Shigeyuki Matsuzawa;M. Shiohara;T. Yamashita;M. Nakajima;A. Komiyama
• 通讯作者: A. Komiyama

Hagimoto R: "A possible role for maternal HLA antibodyin a case of alloimmune neonatal neutropenia"Transfusion. 41. 615-620 (2001)

Hagimoto R：“母体 HLA 抗体在同种免疫新生儿中性粒细胞减少症中的可能作用”输血。