p53 expression in nitric oxide‐induced apoptosis

Abstract

Nitric oxide (NO) is a diffusible messenger involved in several patho-physiological processes including immune-mediated cytotoxicity and neural cell killing. NO or the products of its redox chemistry can cause DNA damage and activate subsequent lethal reactions including energy depletion and cell necrosis. However, regardless of whether it is endogenously produced in response to cytokines, or generated by chemical breakdown of donor molecules, NO can also induce apoptosis in different systems. Here, we report that NO generation in response to a cytokine induced NO-synthase or by NO donors stimulates the expression of the tumor suppressor gene, ~53, in RAW 264.7 macrophages or pancreatic RINm5F cells prior to apoptosis. NO-synthase inhibitors such as No-monomethyl+arginine prevent the inducible NO generation as well as p53 expression and apoptosis. Since ~53 expression is linked to apoptosis in some cells exposed to DNA damaging agents, we suggest that NO-induced apoptosis in these cell systems is the consequence of DNA damage and subsequent expression of this tumor suppressor gene.Key words: Nitric oxide; ~53; Apoptosis; DNA fragmentation IntraductiollNitric oxide (NO) is a messenger molecule involved in several processes including relaxation of smooth muscle, neurotransmission, and tumor cell as well as bacteria killing [l-3]. However, induction of a high output system for NO in response to cytokines or a massive production of NO following accumulation of the excitatory neurotransmitter glutamate [4,5] can result in cell killing. Neurons [6], pancreatic B-cells [7] or macrophages [8,9] seem to be particularly sensitive to NO toxicity. While in some systems NO can react with other radicals and effectively cause cell death by necrosis, in others the progressive intra-or extracellular generation of NO has been suggested to cause apoptosis [8,10,11]. Mechanisms proposed for NO toxicity include its interaction with protein thiol groups [3,12] or iron-sulfur proteins [13], or by direct DNA damage [14]. The latter, regardless of whether it is induced by radiation or by drugs such as etoposide, can result in apoptosis [ 15,161. Expression of wild-type ~53, a tumor suppressor gene, seems to be closely linked to apoptosis caused by most of the DNA-damaging agents [15,16]. The wild-type nuclear phosphoprotein ~53, originally characterized as a tumor suppressor protein [17], acts as a checkpoint control in the cell cycle, permitting the repair of damaged DNA. The block in GJS transition which results from ~53 activation has been suggested to cause apoptosis in the case of severe DNA damage [18,19]. More recently, it has become apparent that the ~53 gene product can take part directly in the apoptotic process [20]. Materials and methods MaterialsThe mouse macrophage-like cell line RAW 264.7 was provided by Prof. A. Wendel, Faculty of Biology, University of Konstanz, Germany. LPS (Escherichia coli serotype 0127:B8), NMMA, protein A-Sepharose, and SNP were purchased from Sigma, Deisenhofen, *Corresponding author. Fa...

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