Activation of endogenous p53 by combined p19Arf gene transfer and nutlin-3 drug treatment modalities in the murine cell lines B16 and C6.
Activation of endogenous p53 by combined p19Arf gene transfer and nutlin-3 drug treatment modalities in the murine cell lines B16 and C6.
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DOI：
10.1186/1471-2407-10-316
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发表时间：
2010-06-22
期刊：
影响因子：
3.8
通讯作者：
Strauss BE
中科院分区：
文献类型：
--
作者：
Merkel CA;da Silva Soares RB;de Carvalho AC;Zanatta DB;Bajgelman MC;Fratini P;Costanzi-Strauss E;Strauss BE
通过基因转移或药理学方法重新激活p53可以补偿p19 Arf的丢失或mdm 2过度表达，这是黑色素瘤和胶质瘤中的常见事件。在我们之前的工作中，我们构建了pCLPG逆转录病毒载体，其中转基因表达由p53通过p53应答启动子控制。使用该载体将p19 Arf引入具有p53 wt的肿瘤细胞中应产生p19 Arf的病毒表达，这反过来将激活内源性p53并导致增强的载体表达和肿瘤抑制。由于nutlin-3可以通过阻断其与mdm 2的相互作用来激活p53，因此我们探索了p19 Arf基因转移和nutlin-3药物治疗的组合可能在刺激p53功能方面提供额外益处的可能性。携带p53 wt的B16（小鼠黑色素瘤）和C6（大鼠神经胶质瘤）细胞系用pCLPGp 19转导，并另外用nutlin-3或DNA损伤剂多柔比星处理。通过Western、北方和免疫荧光测定证实病毒表达。通过由p53应答构建体提供的报道基因活性来评估p53功能。通过集落形成、生长曲线、细胞周期和MTT法检测细胞增殖和活力的变化。在动物模型中，在皮下注射C57 BL/6小鼠之前，用pCLPGp 19病毒和/或药物处理B16细胞，观察肿瘤进展和组织病理学分析。在这里，我们表明，内源性p53 wt在B16中的功能激活是特别具有挑战性的，但完成时，基因转移和药物治疗相结合的应用，导致增加的反式激活p53，显着的细胞周期改变和降低的活力在培养。在动物模型中，用p19 Arf和nutlin-3处理的B16细胞产生了增加的坏死和减少的BrdU标记。相比之下，C6细胞对任何一种治疗都非常敏感，但p53被p19 Arf和nutlin-3的组合进一步激活。据我们所知，这是第一项同时应用p19 Arf和nutlin-3刺激p53活性的研究。这些结果支持的概念，p53响应载体可能被证明是一个有趣的基因转移工具，特别是当结合p53激活剂，用于治疗保留野生型p53的肿瘤。
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.
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DOI：
10.1158/1078-0432.ccr-07-5136
发表时间：
2008-09-01
期刊：
Clinical cancer research : an official journal of the American Association for Cancer Research
影响因子：
--
作者：
Shangary S;Wang S
通讯作者：
Wang S
影响因子：
5.8
作者：
Strauss BE;Fontes RB;Lotfi CF;Skorupa A;Bartol I;Cipolla-Neto J;Costanzi-Strauss E
通讯作者：
Costanzi-Strauss E
影响因子：
4.8
作者：
Hu, Baoli;Gilkes, Daniele M.;Chen, Jiandong
通讯作者：
Chen, Jiandong
影响因子：
5.4
作者：
Naviaux, RK;Costanzi, E;Verma, IM
通讯作者：
Verma, IM
影响因子：
5.3
作者：
DUBRIDGE, RB;TANG, P;CALOS, MP
通讯作者：
CALOS, MP