Arafat W O, Gómez-Navarro J, Xiang J, Barnes M N, Mahasreshti P, Alvarez R D, Siegal G P, Badib A O, Buchsbaum D, Curiel D T, Stackhouse M A
Department of Medicine, and Gene Therapy Center, University of Alabama at Birmingham, 35233, USA.
Mol Ther. 2000 Jun;1(6):545-54. doi: 10.1006/mthe.2000.0071.
Overexpression of proapoptotic Bax favors death in cells resistant to ionizing radiation. We hypothesized that expression of Bax via adenoviral-mediated gene delivery could sensitize radiation-refractory cells to radiotherapy. An inducible Bax recombinant adenovirus (Ad/Bax) had been generated using the Cre/loxp system. Human ovarian cancer cell lines and primary, patient-derived cancer cells from ascites were irradiated and infected with the Ad/Bax and an expression-inducing vector, Ad/Cre. Cell death was evaluated by crystal violet staining, fluorescence-activated cell sorter analysis of Annexin V, and colony formation assay (cell lines only). To further characterize the mechanism of death, cell morphology was examined by nuclear staining with Hoechst 33258. Lastly, to evaluate the capacity of the combined treatment to inhibit tumor growth, mice were injected subcutaneously with ovarian cancer cells exposed to Bax, radiation therapy (RT), or both, and tumor size was measured periodically. Infection of the cancer cell lines and primary cells with both Ad/Bax and Ad/Cre significantly enhanced sensitivity to ionizing radiation, achieving high levels of cell killing in short-term assays. In addition, the combination of Bax and radiotherapy reduced the survival fraction of cell lines 2 logs in standard colony-forming assays. Investigation into the involved mechanism suggests that Bax-mediated radiosensitization occurs through both apoptosis and necrosis pathways. Further, mice subcutaneously injected with ovarian tumor cells previously treated with radiation, or with radiation and irrelevant viruses, consistently developed tumor nodules. In addition, approximately 80% of injections were followed by tumor formation after treatment with Ad/Bax and Ad/Cre alone. In contrast, tumor formation was completely inhibited after combined treatment with Ad/Bax and Ad/Cre and radiation. Augmentation of the effect of radiotherapy on human ovarian cancer cells and primary cancer cells from patients via a recombinant adenovirus encoding Bax is feasible.
促凋亡蛋白Bax的过表达有利于对电离辐射有抗性的细胞发生死亡。我们推测，通过腺病毒介导的基因传递来表达Bax可以使对放疗有抗性的细胞对放射治疗敏感。利用Cre/loxp系统构建了一种可诱导的Bax重组腺病毒（Ad/Bax）。对人卵巢癌细胞系以及来自腹水的原发性患者来源癌细胞进行照射，并分别用Ad/Bax和一种表达诱导载体Ad/Cre进行感染。通过结晶紫染色、膜联蛋白V的荧光激活细胞分选分析以及集落形成试验（仅用于细胞系）来评估细胞死亡情况。为了进一步明确死亡机制，用Hoechst 33258进行核染色来检查细胞形态。最后，为了评估联合治疗抑制肿瘤生长的能力，将暴露于Bax、放射治疗（RT）或两者的卵巢癌细胞皮下注射到小鼠体内，并定期测量肿瘤大小。用Ad/Bax和Ad/Cre同时感染癌细胞系和原代细胞显著增强了对电离辐射的敏感性，在短期试验中实现了高水平的细胞杀伤。此外，在标准集落形成试验中，Bax与放射治疗的联合使用使细胞系的存活分数降低了2个对数。对相关机制的研究表明，Bax介导的放射增敏作用是通过凋亡和坏死途径发生 的。此外，皮下注射先前经过放射治疗或经过放射治疗及无关病毒处理的卵巢肿瘤细胞的小鼠持续长出肿瘤结节。另外，单独用Ad/Bax和Ad/Cre处理后，约80%的注射部位出现肿瘤形成。相比之下，用Ad/Bax和Ad/Cre与放射治疗联合处理后肿瘤形成被完全抑制。通过编码Bax的重组腺病毒增强放射治疗对人卵巢癌细胞和患者原发性癌细胞的作用是可行的。
