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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.

Proline Oxidase Induces Apoptosis in Tumor Cells, and Its Expression Is Frequently Absent or Reduced in Renal Carcinomas

“…To normalize for transfection efficiency, pAdtrack, which expresses green fluorescence protein, was included in the transfection assay where appropriate. Western blotting was performed by using the SuperSignal immunodetection system (Pierce Chemical), as previously described (18). Proline oxidase activity was detected by the * The costs of publication of this article were defrayed in part by the payment of page charges.…”

Differential gene expression in p53-mediated apoptosis-resistant vs. apoptosis-sensitive tumor cell lines

“…Recent investigation by the American Type Culture Collection using DNA profiling has revealed that the ECV-304 cell line is genetically similar to the T24 bladder carcinoma cell line (see http:͞͞www.atcc.org). The DECV cell line was generated from ECV-304 by selection for resistance to p53-mediated apoptosis (22). ECV-304 and DECV cells were maintained and propagated in DMEM containing 10% FCS.…”

“…ECV-304 cells resistant to p53-mediated apoptosis were generated by repeated infections with Ad5CMV-p53 as described (22). The details of the cell behavior differences between DECVs and the parental ECV-304 cells are reported elsewhere (22).…”

“…The details of the cell behavior differences between DECVs and the parental ECV-304 cells are reported elsewhere (22).…”

“…The green fluorescent apoptotic cells expressing GFP or GFPproline oxidase were quantified as the proportion of cells that contained a DNA content of less than 2 M (subG1 DNA content), as described for analysis of p53-mediated apoptosis (22,25). H1299 cell monolayers (3-5 ϫ 10 6 total cells) were trypsinized, washed twice in PBS at 4°C, and fixed in 70% ethanol as described (22).…”