Differential gene expression in p53-mediated apoptosis-resistant vs. apoptosis-sensitive tumor cell lines
- Department of Pathology and Laboratory Medicine, Texas A&M University Health Science Center, College Station, TX 77843-1114
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Edited by Bert Vogelstein, Johns Hopkins Oncology Center, Baltimore, MD, and approved August 31, 2000 (received for review October 15, 1999)
Abstract
Induction of wild-type p53 in the ECV-304 bladder carcinoma cell line by infection with a p53 recombinant adenovirus (Ad5CMV-p53) resulted in extensive apoptosis and eventual death of nearly all of the cells. As a strategy to determine the molecular events important to p53-mediated apoptosis in these transformed cells, ECV-304 cells were selected for resistance to p53 by repeated infections with Ad5CMV-p53. We compared the expression of 5,730 genes in p53-resistant (DECV) and p53-sensitive ECV-304 cells by reverse transcription–PCR, Northern blotting, and DNA microarray analysis. The expression of 480 genes differed by 2-fold or more between the two p53-infected cell lines. A number of potential targets for p53 were identified that play roles in cell cycle regulation, DNA repair, redox control, cell adhesion, apoptosis, and differentiation. Proline oxidase, a mitochondrial enzyme involved in the proline/pyrroline-5-carboxylate redox cycle, was up-regulated by p53 in ECV but not in DECV cells. Pyrroline-5-carboxylate (P5C), a proline-derived metabolite generated by proline oxidase, inhibited the proliferation and survival of ECV-304 and DECV cells and induced apoptosis in both cell lines. A recombinant proline oxidase protein tagged with a green fluorescent protein at the amino terminus localized to mitochondria and induced apoptosis in p53-null H1299 non-small cell lung carcinoma cells. The results directly implicate proline oxidase and the proline/P5C pathway in p53-induced growth suppression and apoptosis.
Footnotes
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↵ * To whom reprint requests should be addressed at: Department of Pathology and Laboratory Medicine, Room 252, College of Medicine, Texas A&M University System Health Science Center, College Station, TX 77843-1114. E-mail: s-maxwell{at}tamu.edu.
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This paper was submitted directly (Track II) to the PNAS office.
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Article published online before print: Proc. Natl. Acad. Sci. USA, 10.1073/pnas.230445997.
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Article and publication date are at www.pnas.org/cgi/doi/10.1073/pnas.230445997
- Abbreviations:
- RT-PCR,
- reverse transcription–PCR;
- P5C,
- pyrroline-5-carboxylate;
- DECV,
- differentiated ECV, a derivative line of ECV-304 cells that is resistant to p53-mediated apoptosis;
- GFP,
- green fluorescent protein;
- FACS,
- fluorescence-activated cell sorter;
- DNP,
- 2,4-dinitrophenylhydrazine
- Copyright © 2000, The National Academy of Sciences
