activation of endogenous p53 by combined p19arf gene transfer and nutlin-3 drug treatment modalities in the murine cell lines b16 and c6激活内源性p53的单独基因转移和nutlin-3联合药物治疗模式在小鼠b16转椅和c6细胞行.pdfVIP

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activation of endogenous p53 by combined p19arf gene transfer and nutlin-3 drug treatment modalities in the murine cell lines b16 and c6激活内源性p53的单独基因转移和nutlin-3联合药物治疗模式在小鼠b16转椅和c6细胞行.pdf

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activation of endogenous p53 by combined p19arf gene transfer and nutlin-3 drug treatment modalities in the murine cell lines b16 and c6激活内源性p53的单独基因转移和nutlin-3联合药物治疗模式在小鼠b16转椅和c6细胞行

Merkel et al. BMC Cancer 2010, 10:316 /1471-2407/10/316 R E S E A R C H A R T I C L E Open Access Research article Activation of endogenous p53 by combined p19Arf gene transfer and nutlin-3 drug treatment modalities in the murine cell lines B16 and C6 Christian A Merkel1,2,3, Rafael B da Silva Soares1,2,3, Anna Carolina V de Carvalho1,2,3, Daniela B Zanatta1,2,3, Marcio C Bajgelman1,2,3, Paula Fratini1,2,3, Eugenia Costanzi-Strauss2,3,4 and Bryan E Strauss*1,2,3 Abstract Background: 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. Methods: 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 s