Differential gene expression in p53-mediated apoptosis-resistant vs. apoptosis-sensitive tumor cell lines - PubMed (original) (raw)

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

S A Maxwell et al. Proc Natl Acad Sci U S A. 2000.

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.

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Figures

Figure 1

Figure 1

Differential regulation of gene expression by p53 in ECV-304 and DECV cells. ECV-304 and DECV cells were infected with control recombinant β-galactosidase adenovirus (ECV/gal; DECV/gal) or recombinant p53 adenovirus (ECV/p53; DECV/p53) for 12 h. Total RNA was extracted and subjected to RT-PCR to analyze the expression of_XRCC9, Bcl-2, PISSLRE, BTG2, Apaf-1, Bax, Pig-3, Pig-11, Bcl_ XL , E2F, Bak, protocadherin-42 (PRCAD-42), cyclin A1 (CYCLIN A), and HLH 1R21 (HLHP). Glyceraldehyde 3-phosphate dehydrogenase and Bad, which were not influenced by p53, were used as expression controls to ensure that equivalent amounts of cDNA were included in each assay.

Figure 2

Figure 2

Induction of proline oxidase and arginase II in ECV-304 cells up-regulated for p53. (A–D) RT-PCR was performed to analyze p53 (A), arginase II (B), proline oxidase (C), and cyclin A1 (D) in mock-infected (lane 1), GFP recombinant adenovirus-infected (lane 2), p53 recombinant adenovirus-infected (lane 3), DMSO-treated (lane 4), and P5C-treated (lane 5) ECV-304 cells. (E) Northern blot analysis was performed on total RNA isolated from mock-infected (lane 1), GFP-infected (lane 2), and p53-infected (lane 3) ECV-304 cells using a radiolabeled proline oxidase cDNA probe and shows induction of proline oxidase mRNA only in p53-induced cells. (F) After capillary transfer from the agarose gel to a nylon membrane, total RNA was stained with methylene blue before hybridization with the proline oxidase probe and shows equivalent amounts of quality total RNA in each gel lane.

Figure 3

Figure 3

P5C inhibits growth and induces apoptosis in p53-sensitive and p53-resistant cells. (A and B) FACS was performed on ECV-304 cells treated with DMSO (A) and on cells treated with P5C (B) for 16 h. (C) P5C inhibited the proliferation and reduced the survival of DECV and ECV-304 cells. (D) Internucleosomal DNA fragmentation analysis supported the FACS analysis in showing DNA fragmentation ladders characteristic of apoptosis. DMSO-treated ECV-304 and DECV are shown in lanes 1 and 2, and ECV-304 and DECV cells treated with 600 μM P5C for 36 h are shown in lanes 3 and 4, respectively.

Figure 4

Figure 4

A recombinant GFP-proline oxidase induces apoptosis in H1299 non-small cell lung carcinoma cells. (A) H1299 cells were transfected with GFP (lane 1) or GFP-proline oxidase (lane 2) expression vector for 24 h and immunoblotted with a polyclonal GFP antibody. (B) H1299 cells at 40–50% confluence were transfected with GFP or GFP-proline oxidase and subjected to FACS analyses 72 h later. (C) Quantitation of apoptosis occurring in cells transfected with GFP, GFP-proline oxidase fusion protein, or a combination of GFP + p53. The data values shown were compiled from four independent FACS scans.

Figure 5

Figure 5

Recombinant GFP-proline oxidase localizes to mitochondria. Cells were subfractionated into nuclei (N), cytoplasmic/membrane (C), and mitochondrial (M) fractions. (A and B) A portion of each subcellular fraction (5 μg protein) was solubilized in SDS gel loading buffer and immunoblotted with a polyclonal GFP antibody (A) or a monoclonal cytochrome c antibody (B). (C) Confocal microscopy was used to visualize the localization of GFP-proline oxidase in transfected H1299 cells (GFP-POX; green fluorescence). These same cells were counterstained with MitoTracker Red (red fluorescence) to visualize the localized concentration of mitochondria in the cytoplasm. The right panel shows the simultaneous excitation of GFP-proline oxidase and MitoTracker Red in the same field as that shown in the left and middle panels, which yielded a yellowish-red fluorescence at areas of colocalization of GFP-POX and MitoTracker Red. Filtered control analyses indicated that there was no contamination of the red fluorescence window by the green fluorescence signal or of the green fluorescence window by the red fluorescence signal.

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