Plasminogen activator inhibitor (PAI)-1 is predictive of poor outcome in several types of cancer. The present study investigated the biological role for PAI-1 in ovarian cancer and potential of targeted pharmacotherapeutics. In patients with ovarian cancer, PAI-1 mRNA expression in tumor tissues was positively correlated with poor prognosis. To determine the role of PAI-1 in cell proliferation in ovarian cancer, the effects of PAI-1 inhibition were examined in PAI-1-expressing ovarian cancer cells. PAI-1 knockdown by small interfering RNA resulted in significant suppression of cell growth accompanied with G2/M cell cycle arrest and intrinsic apoptosis. Similarly, treatment with the small molecule PAI-1 inhibitor TM5275 effectively blocked cell proliferation of ovarian cancer cells that highly express PAI-1. Together these results suggest that PAI-1 promotes cell growth in ovarian cancer. Interestingly, expression of PAI-1 was increased in ovarian clear cell carcinoma compared with that in serous tumors. Our results suggest that PAI-1 inhibition promotes cell cycle arrest and apoptosis in ovarian cancer and that PAI-1 inhibitors potentially represent a novel class of anti-tumor agents.
Second-site suppressor (SSS) mutations in p53 found by random mutagenesis have shown to restore the inactivated function of some tumor-derived p53. To screen novel SSS mutations against common mutant p53s, intragenic second-site (SS) mutations were introduced into mutant p53 cDNA in a comprehensive manner by using a p53 missense mutation library. The resulting mutant p53s with background and SS mutations were assayed for their ability to restore the p53 transactivation function in both yeast and human cell systems. We identified 12 novel SSS mutations including H178Y against a common mutation G245S. Surprisingly, the G245S phenotype is rescued when coexpressed with p53 bearing the H178Y mutation. This result indicated that there is a possibility that intragenic suppressor mutations might restore the protein function in an intermolecular manner. The intermolecular mechanism may lead to novel strategies for restoring inactivated p53 function and tumor suppression in cancer treatment. ' 2007 Wiley-Liss, Inc.Key words: p53; second-site suppressor mutation; intragenic suppressor; transactivation Tumor-suppressor p53 protein is a 393 amino-acid nuclear protein that acts as a tetramer. It is divided into 3 domains, an NH 2 -terminal domain containing a transactivation domain, a central core DNA-binding domain and a COOH-terminal domain containing a tetramerization domain. 1-6 Among these, the core DNAbinding domain is well conserved and retains high homology between a variety of lower species and humans 7 as well as human p53 homologues. 8,9 The structure of the core DNA-binding domain has been resolved by X-ray crystallographic analysis. 2 The p53 forms a homotetramer through the tetramerization domain and the p53 tetramer forms a sequence-specific DNA-binding interface. The p53 protein is activated by a variety of cellular stresses including DNA damage and hypoxia, and is phosphorylated and acetylated after translation. The activated p53 binds to the specific DNA sequence in the regulatory region of downstream genes, resulting in cellular events including cell-cycle arrest and apoptosis. So far, a number of downstream genes involved in cellcycle, apoptosis, DNA-repair, angiogenesis and p53 stability have been identified. The loss of p53 function therefore fails to activate these genes after cellular stresses and is thought to be a critical cause of carcinogenesis and/or tumor progression.Although the frequency of TP53 mutations differs among tumor types, 50% of tumors contained the TP53 mutation. [10][11][12] The published mutations have been summarized in the 2 major TP53 mutation databases that contain more than 20,000 mutations. 13,14 According to the databases, 74% of mutations are missense mutations. So far, 1,200 distinct missense mutations have been reported and there are mutation hot spots at residues R175, G245, R248, R273 and R282. These residues reside in the L2 or L3 loop, or the LSH motif and may be particularly critical because the stability of the structures is thought to depend on interactions among ...
Abstract. After DNA damage, p53 is accumulated in the nucleus and transactivates downstream genes and induces apoptosis. There are two pathways in p53-dependent apoptosis, the transactivation-dependent and -independent pathway. In this study, we constructed p53-inducible glioblastoma cell lines and analyzed them for the induction of apoptosis and transactivation of p53-downstream genes after the nuclear or cytoplasmic expression of p53. To sequester p53 in the cytoplasm, we used p53 mutant with arginine to glycine substitution at residue 306 (R306G). Wild-type p53 retained the ability to arrest the cell cycle, and a p53 mutant with serine to phenylalanine substitution at residue 121 (S121F), which has a strong ability to induce apoptosis, retained this ability even when both the wild-type and p53 and S121F mutant were exclusively sequestered from the nucleus into the cytoplasm. Notably, cytoplasmically sequestered wild-type p53 and S121F mutant transactivated the downstream genes with distinct expression profiles, and the strong apoptotic ability of S121F was not associated with its transactivation activity. These results underscore the existence of transactivation-independent apoptosis and cytoplasmic function of p53.
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