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  current news   Press   selected story    
     
  19th January 2009  
 

Focused PCR Screen Reveals p53-Dependence of Nitric Oxide-Induced Apoptosis and Up-Regulation of Maspin and Plasminogen Activator Inhibitor-1 in Tumor Cells.

 
 




Authors
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: shlim@imcb.a-star.edu.sg

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 =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.

 
 


 
 


FIGURE 8
. Failure of maspin and PAI-1 up-regulation by NO in p53-/- HCT116 cells. A. Quantitative real-time PCR analysis. HCT116-p53+/+ and HCT116-p53-/- cells were treated with 1mmol/L DETA-NO for the indicated times, and total RNA was extracted and reverse-transcribed. The levels of mRNA transcripts for maspin (left) or PAI-1 (right) were determined by Q-PCR. Columns, mean fold of untreated HCT116-p53+/+ cells (n=3) after normalization to -actin expression; bars, SE. B to C. Western blot analysis. HCT116-p53+/+ and HCT116-p53-/- cells were treated with 1mmol/L DETA-NO for various times. Total cell lysates were separated in a 10% polyacrylamide gel, and the membrane was probed with anti-maspin (B) or anti-PAI-1 (C) antibodies.

Published in Mol Cancer Res 2009;7(1):55-66.


Lim Shuhui - Personal thoughts

In 2007, I joined Prof Alan Porter’s laboratory as a fresh graduate from NUS with little experience in research work. In 2009, we managed to get a paper published in Molecular Cancer Research. The vital factors making this accomplishment possible are a good supervisor, a good mentor and very good funding! Alan in my opinion is a true gentleman who is always very kind to everyone in the laboratory. Instead of relentlessly pushing us to work harder, he would encourage us to take a well-deserved break. This makes the working environment a much more relaxed one and everything done was motivated by pure interest in the topic rather than simply to churn out data. He was also very approachable and discussions with him are just a door-knock away. Alan placed me under an excellent post-doc, Amos, who never once complained even though he had to painstakingly go through every single protocol with me step-by-step, starting from how to cast a gel. Amos taught me the virtue of patience and persistency, and not to be discouraged when things do not work out the way you expected (which by-the-way is quite common in research). This is where I learnt not only to do research, but also to produce reliable and consistent results. And most importantly, the strong financial support provided by A*STAR equipped us with the resources required to generate quality data acceptable for publication. Coupled with the moral support and valuable suggestions of members in the laboratory, we are very fortunate to produce a paper just before Alan retires. As I now commence on my PhD program in a different laboratory, I am very grateful to everyone in the AGP lab who has made my 1-year research attachment such a memorable and fruitful one.

For more information on Alan Porter’s Lab, Please Click here.