Endostatin, a carboxyl-terminal fragment of collagen XVIII, has been shown to regress tumors in mice. In this study, we have analyzed the mechanism of endostatin action on endothelial cells and nonendothelial cells. Endostatin treatment of cow pulmonary artery endothelial cells caused apoptosis, as demonstrated by three methods, annexin V-fluorescein isothiocyanate staining, caspase 3, and terminal deoxynucleotidyl transferasemediated dUTP nick-end-labeling assay. Moreover, addition of endostatin led to a marked reduction of the Bcl-2 and Bcl-X L anti-apoptotic protein, whereas Bax protein levels were unaffected. These effects were not seen in several nonendothelial cells. Collectively, these findings provide important mechanistic insight into endostatin action.

The p53 tumour-suppressor protein is a sequence-specific DNA-binding transcription factor that induces cell cycle arrest or apoptosis in response to genotoxic stress. Activation of p53 by DNA-damaging agents is critical for eliminating cells with damaged genomic DNA and underlies the apoptotic response of human cancers treated with ionizing radiation (IR) and radiomimetic drugs. The molecular mechanisms by which DNA damage activates p53 have not been elucidated. Both the levels of p53 protein and its affinity for specific DNA sequences increase in response to genotoxic stress. In vitro, the affinity of p53 for DNA is regulated by its carboxy-terminus. We therefore examined whether this region of p53 is targeted by DNA-damage signalling pathways in vivo. In nonirradiated cells, serines 376 and 378 of p53 were phosphorylated. IR led to dephosphorylation of Ser376, creating a consensus binding site for 14-3-3 proteins and leading to association of p53 with 14-3-3. In turn, this increased the affinity of p53 for sequence-specific DNA. Consistent with the lack of p53 activation by IR in ataxia telangiectasia (AT; refs 14,15), neither Ser376 dephosphorylation, nor the interaction of p53 with 14-3-3 proteins occurred in AT cells.

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Endostatin, a type XVIII collagen fragment, is a potent antiangiogenic molecule that inhibits endothelial cell migration, promotes apoptosis, and induces cell cycle arrest in vitro. We have investigated the mechanism by which endostatin causes G 1 arrest in endothelial cells. Endostatin decreased the hyperphosphorylated retinoblastoma gene product and down-regulated cyclin D1 mRNA and protein. Importantly, endostatin was unable to arrest cyclin D1 overexpressing endothelial cells, suggesting that cyclin D1 is a critical target for endostatin action. Next, we analyzed cyclin D1 promoter activity in endothelial cells and found that endostatin down-regulated the cyclin D1 promoter. Using a series of deletion and mutant promoter constructs, we identified the LEF1 site in the cyclin D1 promoter as essential for the inhibitory effect of endostatin. Finally, we showed that endostatin can repress cyclin D1 promoter activity in cells over-expressing ␤-catenin but not in cells over-expressing a transcriptional activator that functions through the LEF1 site and is insensitive to ␤-catenin. Collectively, our data pointed to a role for cyclin D1, and in particular, transcription through the LEF1 site as critical for endostatin action in vitro and suggest that ␤-catenin is a target for endostatin.