The p53 pathway : role of telomerase and identification of novel targets : acts of a master regulator of tumor suppression

Abstract

A key role of the p53 protein in tumor suppression is reflected by its frequent mutations in human tumors. p53 is activated by various types stress conditions such as DNA damage, hypoxia and oncogene activation that results in p53-mediated cellular responses including DNA repair, cell cycle arrest, and apoptosis. p53 is a transcription factor that upon stabilization regulates expression of multiple genes involved in the above cellular responses. Thus, depiction of novel p53 targets is important for a better understanding of its tumor suppressive function. In addition, p53 also induces transcription-independent apoptotic activity.

The catalytic subunit of human telomerase, hTERT involved in telomere length maintenance is repressed in normal cells but is highly expressed in most human tumors. We have previously shown that hTERT is downregulated upon activation of wtp53 suggesting that p53-mediated repression of hTERT is important for p53-induced apoptosis. We investigated the effects of constitutive expression of hTERT on p53-dependent apoptosis using two different cell systems, the BL41-tsp53 and HCT116 wtp53+/+ and wtp53-/-. We showed that hTERT expression antagonizes p53-induced apoptosis in both cells. This inhibitory effect was independent of hTERT telomerase activity as expression of catalytically inactive hTERT efficiently blocked apoptosis. To further explore the mechanism of hTERT anti-apoptotic function we studied the mitochondrial pathway and found that hTERT acts upstreams of the mitochondria. hTERT expression specifically inhibits activation of Bax. Thus, hTERT exerts its anti-apoptotic function by inhibition of Bax. Our data suggests that p53-dependent downregulation of hTERT is important for p53-induced Bax activation and apoptosis.

Identification of novel p53 targets is important for elucidation of p53 function. Several studies have addressed p53-dependent gene expression by microarray analysis. However, analysis of p53-dependent expression at the protein level can help to identify targets that are regulated by transcription-independent mechanisms. We examined the effects of p53 activation on the proteome using 2D gel electrophoresis analysis of mitomycin C-treated HCT116 colon carcinoma cells and identified 55 novel proteins, differentially expressed in a p53-dependent manner by mass spectrometry. The proteins identified are involved in different cellular processes. Several of them lack putative p53-binding sites and thus are likely to be regulated independently of p53-mediated transcription. This could be due to posttranslational modifications such as phosphorylation of these targets. To further investigate this possibility, we analyzed p53-dependent phosphorylation of proteins using a fluorescent phosphoprotein dye and 2DE and mass spectrometry. Forty-four proteins showed changes in phosphorylation in a p53-dependent manner. This suggests that active p53 not only regulates gene expression but also triggers posttranslational modification of proteins.

This thesis provides two new aspects of p53 function, namely the significance of p53-mediated downregulation of hTERT for induction of apoptosis and regulation of protein expression by different mechanisms that further demonstrate the multiple dimensions of p53’s role as tumor suppressor.