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Preparation of Cell-lines for Conditional Knockdown of Gene Expression and Measurement of the Knockdown Effects on E4orf4-Induced Cell Death
Published on: October 21, 2012
Biological and molecular characterization of an ECV-304-derived cell line resistant to p53-mediated apoptosis
1Department 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
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.
Insights
A new cell line, DECV, resists p53-induced apoptosis, unlike parental ECV-304 cells. This apoptosis-resistant variant exhibits distinct gene expression and differentiation markers, aiding p53 research.
Area of Science:
- Cell Biology
- Molecular Biology
- Cancer Research
Background:
- The tumor suppressor protein p53 induces apoptosis in ECV-304 cells upon infection with recombinant p53 adenovirus.
- Understanding the mechanisms of p53-mediated apoptosis is crucial for cancer research.
Purpose of the Study:
- To establish a system for elucidating molecular mechanisms of p53-mediated apoptosis.
- To create a variant of ECV-304 cells resistant to p53-induced apoptosis.
Main Methods:
- Developed a resistant cell line (DECV) through repeated recombinant p53 adenovirus infections.
- Compared gene expression using cDNA microarray technology.
- Assessed cell behaviors including soft agar growth, contact inhibition, and 3D culture differentiation.
Main Results:
- DECV cells showed resistance to p53-induced apoptosis, with >95% survival.
- DECV cells exhibited reduced soft agar growth, contact inhibition, and formed differentiated structures.
- Differential gene expression was observed, with Protocadherin-1 in DECV and Id-3 in ECV-304; EPHB2 and ephrin-B1 were upregulated by p53 in ECV-304.
Conclusions:
- The DECV cell line is resistant to p53-mediated apoptosis and displays distinct cellular behaviors and gene expression profiles.
- DECV cells serve as a valuable tool for investigating p53-mediated apoptosis and differentiation mechanisms.
