Biological and molecular characterization of an ECV-304-derived cell line resistant to p53-mediated apoptosis.

Abstract

Upregulation of the p53 tumor suppressor protein by infection with a recombinant p53 adenovirus resulted in extensive apoptosis in ECV-304 cells and the eventual death of almost all the cells. To establish a system to elucidate the molecular mechanisms involved in p53-mediated apoptosis of these cells, we established a variant of ECV-304 that is resistant to p53-induced apoptosis by repeated infections with a recombinant p53 adenovirus. We have designated this variant as the DECV cell line (Differentiated ECV-304). DECV cells expressed similar amounts of nuclear-localized p53 as ECV-304 cells when infected with recombinant p53 adenovirus, but in contrast to ECV-304 cells, greater than 95% of DECV cells survived and remained viable after 24 hours of infection. In further contrast to ECV-304 cells, DECV cells grew less efficiently in soft agar and exhibited contact inhibition in growth assays. Moreover, DECV cells formed unusual lattice or cyst-like structures in culture and formed lumenal structures indicative of epithelial differentiation in three-dimensional collagen matrices, while parental ECV-304 cells showed minimal evidence of these cellular behaviors. A comparative molecular analysis of gene expression in DECV and ECV-304 cells was conducted by cDNA microarray technology. Protocadherin-1 was found to be expressed in DECV cells but not in ECV-304 cells, while the Id-3 gene was observed expressed in ECV-304 cells but not in DECV cells. Moreover, upregulated expression of p53 in ECV-304 cells induced the EPHB2 (Ephrin) receptor tyrosine kinase and the ephrin-B1 ligand mRNAs compared to DECV cells treated in the same manner. These data demonstrate that a new variant of the ECV-304 cell line, which is resistant to p53-mediated apoptosis, exhibits differential gene expression as well as distinct cell behaviors as compared to the parental ECV-304 cell line. DECV cells should prove to be a useful tool in future studies to elucidate mechanisms of p53-mediated apoptosis and differentiation.