Biological and molecular characterization of an ECV-304-derived cell line resistant to p53-mediated apoptosis.
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.
Maxwell
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SA
Department of Pathology and Laboratory Medicine, College of Medicine, Texas A&M University System Health Science Center, College Station 77843-1114, USA. s-maxwell@tamu.edu
Davis
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GE
D000256:Adenoviridae / Q000235:genetics / Q000378:metabolism*; D017209:Apoptosis / Q000502:physiology*; D015820:Cadherins / Q000235:genetics / Q000378:metabolism; D002454:Cell Differentiation; D002469:Cell Separation; D048429:Cell Size; D036387:Ephrin-B1; D005434:Flow Cytometry; D020869:Gene Expression Profiling; D017930:Genes, Reporter / Q000235:genetics; D006801:Humans; D051796:Inhibitor of Differentiation Proteins; D008565:Membrane Proteins / Q000378:metabolism; D009363:Neoplasm Proteins; D020411:Oligonucleotide Array Sequence Analysis; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*
D015820:Cadherins; D036387:Ephrin-B1; D051796:Inhibitor of Differentiation Proteins; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D016159:Tumor Suppressor Protein p53; C075640:ID3 protein, human