Zu Bailing, Shi Yi, Xu Min, You Guoling, Huang Zhenglan, Gao Miao, Feng Wenli
Department of Clinical Hematology, Key Laboratory of Laboratory Medical Diagnostics of Ministry of Education, Chongqing Medical University, No.1, Yixueyuan Road, Chongqing, 400016, People's Republic of China.
Department of Clinical Laboratory, Shanghai Pudong Hospital, Fudan University Pudong Medical Center, Shanghai, China.
J Exp Clin Cancer Res. 2015 May 28;34(1):56. doi: 10.1186/s13046-015-0139-4.
Treatment of blast phase chronic myeloid leukemia (BP-CML) remains a challenge, and the median survival is less than 6 months. Because effective treatments are lacking, we studied tight targeting of blast crisis CML cells using adenoviral (Ad) vectors expressing a HSV-TK system under dual control of a specific SUZ12 promoter and an antioxidant response element (ARE).
A potential SUZ12 promoter fragment was designed with bioinformatics databases and identified with a luciferase assay. Next, we cloned the ARE element of the NQO1 gene and developed Ad vectors expressing TK kinase or luciferase under the dual control of a specific SUZ12 promoter and an ARE element. An in vitro transfection assay with Ad-ARE/SUZ12-Luc was used to determine promoter activity of ARE/SUZ12 regulatory element in blast crisis CML cells. After incubating human BP-CML-derived cells with Ad-ARE/SUZ12-TK and ganciclovir, Western blot, CCK8, Immunofluorescent assays and Annexin V assays were conducted to assess the efficacy of an ARE/SUZ12 dual-specific TK/GCV system for BP-CML cell lines.
Here, luciferase data confirmed significantly higher and specificer promoter activity of the ARE/SUZ12 composite component in CML blast crisis-derived cell lines (K562, KCL22, and K562/G01) compared to HepG2 cells, and Ad-AS-TK/GCV system could exhibit enhanced apoptotic effects and decreased cell viability for BP-CML cell lines. Additionally, Ad-AS-TK/GCV system altered expression of cycle-related and apoptosis-related proteins in BP-CML cell lines.
Thus, ARE/SUZ12 dual targeting TK/GCV system was effective in killing BP-CML cells. Moreover, efficacy and specificity of CML cell eradication were enhanced by synergistic effects of ARE/SUZ12 dual-specific regulation. We conclude that suicide gene-targeted therapy might hold promise for BP-CML treatment.
急变期慢性髓性白血病（BP-CML）的治疗仍然是一项挑战，其平均生存期不足6个月。由于缺乏有效的治疗方法，我们研究了使用在特异性SUZ12启动子和抗氧化反应元件（ARE）双重控制下表达单纯疱疹病毒胸苷激酶（HSV-TK）系统的腺病毒（Ad）载体对CML急变期细胞进行精准靶向治疗。
利用生物信息学数据库设计一个潜在的SUZ12启动子片段，并通过荧光素酶测定法进行鉴定。接下来，我们克隆了NQO1基因的ARE元件，并构建了在特异性SUZ12启动子和ARE元件双重控制下表达TK激酶或荧光素酶的Ad载体。采用Ad-ARE/SUZ12-Luc进行体外转染试验，以确定ARE/SUZ12调控元件在CML急变期细胞中的启动子活性。用Ad-ARE/SUZ12-TK和更昔洛韦孵育人BP-CML来源的细胞后，进行蛋白质免疫印迹法、CCK8法、免疫荧光测定法和膜联蛋白V测定法，以评估ARE/SUZ12双特异性TK/GCV系统对BP-CML细胞系的疗效。
在此，荧光素酶数据证实，与HepG2细胞相比，ARE/SUZ12复合元件在CML急变期来源的细胞系（K562、KCL22和K562/G01）中具有显著更高且更特异的启动子活性，并且Ad-AS-TK/GCV系统对BP-CML细胞系可表现出增强的凋亡效应和降低的细胞活力。此外，Ad-AS-TK/GCV系统改变了BP-CML细胞系中周期相关蛋白和凋亡相关蛋白的表达。
因此，ARE/SUZ12双靶向TK/GCV系统在杀伤BP-CML细胞方面是有效的。此外，ARE/SUZ12双特异性调控的协同作用增强了CML细胞根除的疗效和特异性。我们得出结论，自杀基因靶向治疗可能对BP-CML的治疗具有前景。
