Activation of endogenous p53 by combined p19Arf gene transfer and nutlin-3 drug treatment modalities in the murine cell lines B16 and C6

Abstract

BackgroundReactivation 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.MethodsB16 (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.ResultsHere 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.ConclusionsTo 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.

Gene expression of the p16INK4a-Rb and p19Arf-p53-p21Cip/Waf1 signaling pathways in the regulation of hematopoietic stem cell aging by ginsenoside Rg1

“…p19 Arf could directly bind to MDM2 and inhibit the degradation of p53 function, and thus enhance the stability and activity of p53 (Merkel et al, 2010). p21 Cip1/Waf1 , a response gene to p53, could be induced by p53 and then inhibit the CDK4/6-cyclinD1 complex-induced Rb phosphorylation and activity of the CDK2/ cyclinE complex, which could finally result in G 1 phase arrest and cell senescence (van Os et al, 2007;Yu et al, 2010).…”

Reestablishment of p53/Arf and interferon-β pathways mediated by a novel adenoviral vector potentiates antiviral response and immunogenic cell death

“…8 However, the impact of apoptotic cell death may be limited and is not likely to actively promote an antitumor immune response. Therefore, we sought to develop a cancer gene therapy approach that would induce cell death by a mechanism that would have wide-spread anti-cancer effects that reach beyond the treated cell.…”

Autoregulated expression of p53 from an adenoviral vector confers superior tumor inhibition in a model of prostate carcinoma gene therapy

“…For example, we have used retroviruses as reporter constructs of p53 activity. 17,18,30 We have employed the PG-vectors to transfer genes such as p19Arf 30 and interferon-b 20 and have observed striking anti-tumor activity, including an important protective immune response. 21 We have also constructed an adenoassociated virus, AAVPG, 31 to transfer VEGF-A 165 in a model of cardiac hypertrophy, resulting in marked functional improvement using 'on-demand' transgene expression in response to physiologic stress.…”