Attenuation of p53 expression and Bax down‐regulation during phorbol ester mediated inhibition of apoptosis

Nitric oxide (NO) caused apoptotic cell death in murine RAW 264.7 macrophages. Associated with apoptotic morphology we observed p53 up‐regulation and increased Bax expression. 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA), a protein kinase C (PKC) activator potently blocked NO‐induced apoptosis. To gain insights into the mechanisms involved we investigated the effect of TPA on apoptotic conveying proteins such as p53 and Bax. TPA (100 n M ) attentuated p53 up‐regulation elicited by the NO‐releasing compounds, S‐nitrosoglutathione (1 m M ) and sodium nitroprusside (1 m M ), and suppressed p53 protein accumulation in response to endogenously generated NO. Hence, TPA appeared to lower the steady state p53 level following its up‐regulation by NO. Mezerein, a stage 2 tumour promoter and PKC activating agent was equally active to TPA. Moreover, two potent PKC inhibitors, staurosporine (10 n M ) and Gö 6976 (50 n M ), reversed the inhibitory effect of TPA. However, bisinoylmaleimide I (up to 500 n M ) was ineffective. By extending the studies, we revealed a TPA‐mediated p53 down‐regulation in response to etoposide (50 μ M ), mitomycin C (5 μg ml −1 ) and actinomycin D (2 μg ml −1 ). With the notion that TPA suppressed apoptotic DNA fragmentation in p53 antisense expressing cells as well, we searched for additional inhibitory actions of TPA. As well as affecting p53, TPA elicited a rapid decline of the steady state level of Bax within 30 min. We concluded that down‐regulation of two classical apoptotic promoting proteins contributes to the anti‐apoptotic action of mezerein and TPA.British Journal of Pharmacology (1997) 121 , 625–634; doi: 10.1038/sj.bjp.0701156