BackgroundYes-associated protein (YAP), an essential component of Hippo pathway, was identified as an oncoprotein which participated in the progression of various malignancies. However, its role in chronic myeloid leukemia (CML) remains to be further clarified.MethodsThe expression of YAP in CML cells was determined by western blotting. Next, the effects of YAP knockdown and YAP inhibitor on CML cells were evaluated by MTT assay, flow cytometry (FCM) and Wright’s staining. Moreover, K562 induced mice model was employed to further investigate the role of YAP in vivo.ResultsYAP was overexpressed in CML cells. Knockdown of YAP by si-RNA or inhibition the function of YAP using verteporfin (VP) not only inhibited the proliferation, induced the apoptosis of CML cells but also reduced the expression of YAP target genes c-myc and survivin. Additionally, VP enhanced the efficacy of imatinib (IM) in vitro and suppressed leukemogenesis in vivo.ConclusionOur results indicate that YAP may play an important role in the proliferation and leukemogenesis of CML cells. Genetic or pharmacological inhibition of YAP provides a novel treatment strategy for CML.Electronic supplementary materialThe online version of this article (doi:10.1186/s13046-016-0414-z) contains supplementary material, which is available to authorized users.
BackgroundKaryopherin-β1 (KPNB1) belongs to the karyopherin superfamily, which functions as shuttling proteins from the cytoplasm to nuclear. A high level of KPNB1 has been reported in various cancers which promotes cell proliferation and inhibits apoptosis. However, the role of KPNB1 in chronic myeloid leukemia (CML) remains uncertain.MethodsExpression level of KPNB1 in CML patient samples and cell lines was analyzed by Western blotting. The proliferation assays and colony formation assay were used to study the CML cell proliferation when KPNB1 knockdown in vitro. Next, Western blotting was used to evaluate the effects of KPNB1 on E2F1 and other cell cycle regulators. Then, the location of E2F1 was detected by immunofluorescence. Finally, flow cytometry was used to detect the effect of KPNB1 inhibitor importazole (IPZ) on CML cells.ResultsIn this study, we firstly showed that KPNB1 is over-expressed in CML cells. Targeting KPNB1 with small interfering RNA (siRNA) and IPZ reduced proliferation and induced apoptosis of CML cells. The underlying mechanisms were also investigated that E2F1 nuclear transport was blocked after inhibiting KPNB1 with siRNA, suggesting KPNB1 over-expression mediates the excessive nuclear transport of E2F1 in CML cells. Moreover, the expression of the E2F1 targeted molecule such as c-Myc and KPNA2 was markedly reduced. The IPZ arrested CML cells at G2/M phase and induced cell apoptosis.ConclusionIn summary, our results clearly showed that KPNB1 is over-expressed in CML cells and mediates the translocation of E2F1 into the nucleus of CML cells, thereby inhibition of KPNB1 reduced proliferation and induced apoptosis of CML cells which provides new insights for targeted CML therapies.
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-base NotI 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.Electronic supplementary materialThe online version of this article (10.1186/s13046-018-0732-4) contains supplementary material, which is available to authorized users.
Background The bcr-abl fusion gene encodes BCR-ABL oncoprotein and plays a crucial role in the leukemogenesis of chronic myeloid leukemia (CML). Current therapeutic methods have limited treatment effect on CML patients with drug resistance or disease relapse. Therefore, novel therapeutic strategy for CML is essential to be explored and the CRISPR RNA-guided FokI nucleases (RFNs) meet the merits of variable target sites and specificity of cleavage enabled its suitability for gene editing of CML. The RFNs provide us a new therapeutic direction to obliterate this disease. Methods Guide RNA ( gRNA) expression plasmids were constructed by molecular cloning technique. The modification rate of RFNs on bcr-abl was detected via Not I restriction enzyme digestion and T7 endonuclease 1 (T7E1) assay. The expression of BCR-ABL and its downstream signaling molecules were determined by western blotting. The effects of RFNs on cell proliferation and apoptosis of CML cell lines and CML stem/progenitor cells were evaluated by CCK-8 assay and flow cytometry. In addition, murine xenograft model was adopted to evaluate the capacity of RFNs in attenuating the tumorigenic ability of bcr-abl. Results The RFNs efficiently disrupted bcr-abl and prematurely terminated its translation. The destruction of bcr-abl gene suppressed cell proliferation and induced cell apoptosis in CML lines and in CML stem/progenitor cells. Moreover, the RFNs significantly impaired the leukemogenic capacity of CML cells in xenograft model. Conclusion These results illustrate that the RFNs can target to disrupt bcr-abl gene and may provide a new therapeutic option for CML patients affiliated by drug resistance or disease relapse. Electronic supplementary material The online version of this article (10.1186/s13046-019-1229-5) contains supplementary material, which is available to authorized users.
The chimeric Bcr-Abl oncoprotein, which causes chronic myeloid leukemia, mainly localizes in the cytoplasm, and loses its ability to transform cells after moving into the nucleus. Here we report a new strategy to convert Bcr-Abl to be an apoptotic inducer by altering its subcellular localization. We show that a rapalog nuclear transport system (RNTS) containing six nuclear localization signals directs Bcr-Abl into the nucleus and that nuclear entrapped Bcr-Abl induces apoptosis and inhibits proliferation of CML cells by activating p73 and shutting down cytoplasmic oncogenic signals mediated by Bcr-Abl. Coupling cytoplasmic depletion with nuclear entrapment of Bcr-Abl synergistically enhances the inhibitory effect of nuclear Bcr-Abl on its oncogenicity in mice. These results provide evidence that direction of cytoplasmic Bcr-Abl to the nucleus offers an alternative CML therapy.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.