Abstract
We investigated p53-dependent gene expression in nitric oxide (NO)-induced apoptosis of two tumor cell types. Seventy-seven putative p53-regulated genes were screened for NO-mediated expression changes. Twenty-four genes were up-regulated and three genes were down-regulated significantly by NO in human neuroblastoma cells. Genes known to be involved in apoptosis, which were up-regulated by > or = 2-fold, included FAS, CASP-1, BIK, PUMA, DR4 and the serpins maspin (SERPINB5), and plasminogen activator inhibitor-1 (PAI-1). Real-time PCR confirmed maspin and PAI-1 mRNAs exhibited the greatest NO-induced induction, which occurred in a p53-dependent manner. The substantial NO-mediated up-regulation of these serpins mRNAs correlated with large increases in their protein levels, which occurred before or coinciding with apoptosis. p53-deficient neuroblastoma cells were largely resistant to NO killing and showed much reduced maspin and PAI-1 mRNA and protein levels after NO treatment. p53 was activated by NO mainly in the nuclei of neuroblastoma cells. p53(-/-) HCT116 colon carcinoma cells were strongly resistant to NO-induced apoptosis and failed to up-regulate maspin and PAI-1 (in contrast to p53(+/+) HCT116 cells). Our results suggest that both apoptosis and induction of the two serpins by NO require the transcriptional activity of p53. Because maspin is a tumor suppressor and PAI-1 can promote senescence and regulate cell death, it will now be worth investigating whether their p53-mediated expression contributes to the NO-induced p53-dependent death of tumor cells.
Highlights
Nitric oxide (NO) promotes and controls a bewildering range of functions in cells of many tissue origins [1,2,3]
We first verified the kinetics and magnitude of apoptosis induced by the NO donor diethylenetriamine NO adduct (DETA-NO) in SH-Sy5y neuroblastoma cells
Cell fractionation and immunofluorescence studies both showed that p53 protein and Ser-15 phosphorylated protein accumulated in the nuclei of SHSy5y cells (Fig. 1B and C), a minor fraction of Ser15 – phosphorylated p53 was detectable outside the nucleus (Fig. 1B)
Summary
Nitric oxide (NO) promotes and controls a bewildering range of functions in cells of many tissue origins [1,2,3]. Many cells can resist cell death through the ability of moderate amounts of NO to variously up-regulate diverse antiapoptotic/repair proteins or antioxidant pathways including activator protein, neural cell adhesion molecule-140, DNA protein kinase, Bcl-XL, Bcl-2, cyclic AMP-response elementbinding protein, and Nrf2 [7,8,9,10,11]. Excessive NO can elicit apoptosis or necrosis through a huge variety of proposed pathways and mechanisms. These may be transcriptionindependent—for example NO-mediated S-nitrosylation of specific amino acids in certain proteins Cell death may depend on NO-stimulated signaling pathways leading to gene expression, involving the c-Jun-NH2-kinase or p38 mitogen-activated protein kinases, phosphorylated c-Jun, p19ARF, and the tumor suppressor p53 [5, 8, 13,14,15,16,17,18]
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