Induction of apoptosis in imatinib sensitive and resistant chronic myeloid leukemia cells by efficient disruption of bcr-abl oncogene with zinc finger nucleases.
Induction of apoptosis in imatinib sensitive and resistant chronic myeloid leukemia cells by efficient disruption of bcr-abl oncogene with zinc finger nucleases.
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通过锌指核酸酶有效破坏 bcr-abl 癌基因诱导伊马替尼敏感和耐药的慢性粒细胞白血病细胞凋亡
DOI：
10.1186/s13046-018-0732-4
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发表时间：
2018-03-20
期刊：
影响因子：
--
通讯作者：
Feng W
中科院分区：
文献类型：
--
作者：
Huang N;Huang Z;Gao M;Luo Z;Zhou F;Liu L;Xiao Q;Wang X;Feng W
背景bcr-abl融合基因是慢性粒细胞白血病(CML)的病理起源，在伊马替尼耐药中起重要作用。因此，基于bcr-abl阻断的新的治疗策略可能值得探索。在我们的研究中，我们惊讶地发现BCR-ABL序列的特征符合锌指核酸酶(ZFN)的设计要求。Western blotting检测bcr-abl在CML细胞中的表达及其下游信号转导通路5、ERK和CRKL的磷酸化。采用CCK8比色法、集落形成实验和流式细胞仪(FCM)检测ZFN对CML细胞和CML CD34+细胞存活率和凋亡率的影响。结果ZFNS通过同源定向修复(Hdr)技术，巧妙地在伊马替尼敏感和耐药CML细胞的bcr-abl基因上增加了8base NotI酶切位点，导致终止密码子，终止了bcr-abl蛋白的翻译。正如预期的那样，bcr-abl基因的破坏诱导了细胞的凋亡，抑制了细胞的增殖。值得注意的是，我们在CML患者的CD34+细胞中也得到了类似的结果。结论以ZFN为基础的bcr-abl基因突变可能为治疗伊马替尼耐药或不耐受的CML患者提供了一种选择。
BackgroundThe bcr-abl fusion gene is the pathological origin of chronic myeloid leukemia (CML) and plays a critical role in the resistance of imatinib. Thus, bcr-abl disruption-based novel therapeutic strategy may warrant exploration. In our study, we were surprised to find that the characteristics of bcr-abl sequences met the design requirements of zinc finger nucleases (ZFNs).MethodsWe constructed the ZFNs targeting bcr-abl with high specificity through simple modular assembly approach. Western blotting was conducted to detect the expression of BCR-ABL and phosphorylation of its downstream STAT5, ERK and CRKL in CML cells. CCK8 assay, colony-forming assay and flow cytometry (FCM) were used to evaluate the effect of the ZFNs on the viablity and apoptosis of CML cells and CML CD34+cells. Moreover, mice model was used to determine the ability of ZFNs in disrupting the leukemogenesis of bcr-abl in vivo.ResultsThe ZFNs skillfully mediated 8-baseNotI enzyme cutting site addition in bcr-abl gene of imatinib sensitive and resistant CML cells by homology-directed repair (HDR), which led to a stop codon and terminated the translation of BCR-ABL protein. As expected, the disruption of bcr-abl gene induced cell apoptosis and inhibited cell proliferation. Notably, we obtained similar result in CD34+cells from CML patients. Moreover, the ZFNs significantly reduced the oncogenicity of CML cells in mice.ConclusionThese results reveal that the bcr-abl gene disruption based on ZFNs may provide a treatment choice for imatinib resistant or intolerant CML patients.
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影响因子：
64.8
作者：
Genovese, Pietro;Schiroli, Giulia;Escobar, Giulia;Di Tomaso, Tiziano;Firrito, Claudia;Calabria, Andrea;Moi, Davide;Mazzieri, Roberta;Bonini, Chiara;Holmes, Michael C.;Gregory, Philip D.;van der Burg, Mirjam;Gentner, Bernhard;Montini, Eugenio;Lombardo, Angelo;Naldini, Luigi
通讯作者：
Naldini, Luigi
影响因子：
158.5
作者：
Druker, Brian J.;Guilhot, Francois;Larson, Richard A.
通讯作者：
Larson, Richard A.
影响因子：
56.9
作者：
Boch, Jens;Scholze, Heidi;Bonas, Ulla
通讯作者：
Bonas, Ulla
影响因子：
48
作者：
Gaj, Thomas;Guo, Jing;Kato, Yoshio;Sirk, Shannon J.;Barbas, Carlos F., III
通讯作者：
Barbas, Carlos F., III
影响因子：
2.9
作者：
Eide, Christopher A.;O'Hare, Thomas
通讯作者：
O'Hare, Thomas