Focused PCR screen reveals p53 dependence of nitric oxide-induced apoptosis and up-regulation of maspin and plasminogen activator inhibitor-1 in tumor cells.
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.
Lim
Shuhui
S
Cell Division and Cancer Laboratory, Institute of Molecular and Cell Biology, 61 Biopolis Drive, Proteos, Singapore 138673, Republic of Singapore. shlim@imcb.a-star.edu.sg
Hung
Amos C
AC
Porter
Alan G
AG
D017209:Apoptosis / Q000187:drug effects*; D002467:Cell Nucleus / Q000473:pathology; D015536:Down-Regulation; D015972:Gene Expression Regulation, Neoplastic; D045325:HCT116 Cells / Q000187:drug effects / Q000473:pathology; D006801:Humans; D009447:Neuroblastoma / Q000235:genetics* / Q000473:pathology; D009569:Nitric Oxide / Q000494:pharmacology*; D010957:Plasmids; D017395:Plasminogen Activator Inhibitor 1 / Q000235:genetics*; D016133:Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015843:Serpins / Q000235:genetics*; D014162:Transfection; D014226:Triazenes / Q000494:pharmacology; D016159:Tumor Suppressor Protein p53 / Q000235:genetics*; D015854:Up-Regulation
C105351:1-hydroxy-2-oxo-3,3-bis(2-aminoethyl)-1-triazene; D017395:Plasminogen Activator Inhibitor 1; C085344:SERPIN-B5; C051321:SERPINE1 protein, human; D015843:Serpins; C495901:TP53 protein, human; D014226:Triazenes; D016159:Tumor Suppressor Protein p53; D009569:Nitric Oxide