Shuhui Lim2, Amos C. Hung1,3 and Alan G. Porter1.
Cell Death and Human Disease Group, Division of Cancer and Developmental Cell Biology, Institute of Molecular and Cell Biology, A*STAR (Agency for Science, Technology and Research), 61 Biopolis Drive, Singapore 138673, Republic of Singapore
1 These authors contributed equally
3 Present address: Menzies Research Institute, University of Tasmania, Australia.
Key words: nitric oxide; p53; apoptosis; maspin; PAI-1
Running Title: NO-Induced p53-Dependent Apoptosis
2 Requests for reprints: Lim Shuhui, Cell Division and Cancer Laboratory, Institute of Molecular and Cell Biology, 61 Biopolis Drive, Proteos, Singapore 138673, Republic of Singapore, Tel. (+65) 6586 9850; fax: (+65) 6779 1117. E-mail: email@example.com
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 =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.