Setor de Vetores Virais, Laboratório de Genética e Cardiologia Molecular/LIM 13, InCor, FM-USP, São Paulo, Brasil.
BMC Cancer. 2010 Jun 22;10:316. doi: 10.1186/1471-2407-10-316.
Reactivation of p53 by either gene transfer or pharmacologic approaches may compensate for loss of p19Arf or excess mdm2 expression, common events in melanoma and glioma. In our previous work, we constructed the pCLPG retroviral vector where transgene expression is controlled by p53 through a p53-responsive promoter. The use of this vector to introduce p19Arf into tumor cells that harbor p53wt should yield viral expression of p19Arf which, in turn, would activate the endogenous p53 and result in enhanced vector expression and tumor suppression. Since nutlin-3 can activate p53 by blocking its interaction with mdm2, we explored the possibility that the combination of p19Arf gene transfer and nutlin-3 drug treatment may provide an additive benefit in stimulating p53 function.
B16 (mouse melanoma) and C6 (rat glioma) cell lines, which harbor p53wt, were transduced with pCLPGp19 and these were additionally treated with nutlin-3 or the DNA damaging agent, doxorubicin. Viral expression was confirmed by Western, Northern and immunofluorescence assays. p53 function was assessed by reporter gene activity provided by a p53-responsive construct. Alterations in proliferation and viability were measured by colony formation, growth curve, cell cycle and MTT assays. In an animal model, B16 cells were treated with the pCLPGp19 virus and/or drugs before subcutaneous injection in C57BL/6 mice, observation of tumor progression and histopathologic analyses.
Here we show that the functional activation of endogenous p53wt in B16 was particularly challenging, but accomplished when combined gene transfer and drug treatments were applied, resulting in increased transactivation by p53, marked cell cycle alteration and reduced viability in culture. In an animal model, B16 cells treated with both p19Arf and nutlin-3 yielded increased necrosis and decreased BrdU marking. In comparison, C6 cells were quite susceptible to either treatment, yet p53 was further activated by the combination of p19Arf and nutlin-3.
To the best of our knowledge, this is the first study to apply both p19Arf and nutlin-3 for the stimulation of p53 activity. These results support the notion that a p53 responsive vector may prove to be an interesting gene transfer tool, especially when combined with p53-activating agents, for the treatment of tumors that retain wild-type p53.
通过基因转移或药物手段使 p53 重新激活，可能有助于补偿黑色素瘤和神经胶质瘤中常见的 p19Arf 缺失或 mdm2 表达过多。在我们之前的工作中，我们构建了 pCLPG 逆转录病毒载体，其中转基因的表达受 p53 通过 p53 反应性启动子控制。利用该载体将 p19Arf 导入含有 p53wt 的肿瘤细胞，将导致病毒表达 p19Arf，继而激活内源性 p53，并增强载体表达和肿瘤抑制。由于 nutlin-3 通过阻断其与 mdm2 的相互作用来激活 p53，我们探讨了 p19Arf 基因转移与 nutlin-3 药物治疗联合应用是否可能在刺激 p53 功能方面具有相加作用。
用 pCLPGp19 转导携带有 p53wt 的 B16（小鼠黑色素瘤）和 C6（大鼠神经胶质瘤）细胞系，并对其进行 nutlin-3 或 DNA 损伤剂阿霉素处理。通过 Western、Northern 和免疫荧光检测证实病毒表达。通过 p53 反应性构建体提供的报告基因活性评估 p53 功能。通过集落形成、生长曲线、细胞周期和 MTT 检测评估增殖和活力的变化。在动物模型中，B16 细胞在皮下注射到 C57BL/6 小鼠前用 pCLPGp19 病毒和/或药物处理，观察肿瘤进展并进行组织病理学分析。
我们发现，在 B16 中激活内源性 p53wt 特别具有挑战性，但当联合应用基因转移和药物治疗时，p53 的转录激活、显著的细胞周期改变和培养中的活力降低得以实现。在动物模型中，用 p19Arf 和 nutlin-3 处理的 B16 细胞产生了更多的坏死和减少的 BrdU 标记。相比之下，C6 细胞对任一治疗均非常敏感，但 p19Arf 和 nutlin-3 的联合使用进一步激活了 p53。
据我们所知，这是首次应用 p19Arf 和 nutlin-3 来刺激 p53 活性的研究。这些结果支持这样的观点，即 p53 反应性载体可能被证明是一种有趣的基因转移工具，特别是当与 p53 激活剂联合应用时，可用于治疗保留野生型 p53 的肿瘤。
