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. 2002 May 14;99(10):6649-54.
doi: 10.1073/pnas.102523299.

The c-Myc target gene PRDX3 is required for mitochondrial homeostasis and neoplastic transformation

Diane R Wonsey  1 , Karen I Zeller, Chi V Dang
Affiliations

The c-Myc target gene PRDX3 is required for mitochondrial homeostasis and neoplastic transformation

Diane R Wonsey et al. Proc Natl Acad Sci U S A. .

Abstract

Deregulated expression of the c-Myc transcription factor is found in a wide variety of human tumors. Because of this significant role in oncogenesis, considerable effort has been devoted to elucidating the molecular program initiated by deregulated c-myc expression. The primary transforming activity of Myc is thought to arise through transcriptional regulation of numerous target genes. Thus far, Myc target genes involved in mitochondrial function have not been characterized in depth. Here, we describe a nuclear c-Myc target gene, PRDX3, which encodes a mitochondrial protein of the peroxiredoxin gene family. Expression of PRDX3 is induced by the mycER system and is reduced in c-myc(-/-) cells. Chromatin immunoprecipitation analysis spanning the entire PRDX3 genomic sequence reveals that Myc binds preferentially to a 930-bp region surrounding exon 1. We show that PRDX3 is required for Myc-mediated proliferation, transformation, and apoptosis after glucose withdrawal. Results using mitochondria-specific fluorescent probes demonstrate that PRDX3 is essential for maintaining mitochondrial mass and membrane potential in transformed rat and human cells. These data provide evidence that PRDX3 is a c-Myc target gene that is required to maintain normal mitochondrial function.

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Figures

Figure 1
Figure 1
PRDX3 is regulated by c-myc expression. (A) RNA from Rat1a (R1a) fibroblasts or Rat1a fibroblasts expressing ectopic c-Myc (R1a-myc). rpL32 is shown as a loading control. RNA was isolated from adherent cells (A) or nonadherent cells grown over a layer of agar (N). (B) PRDX3 expression in logarithmically growing c-myc+/+, +/−, or −/− Rat1 fibroblasts. PRDX3 expression was calculated relative to vimentin. (C) Hepatic RNA from mice injected with either adenoviral LacZ or c-myc. Numbers represent days after injection with adenovirus. 18S RNA is shown as a loading control. (D) Schematic representation of the PRDX3 genomic locus. Exons are indicated by black boxes. Fragments analyzed for Myc binding are indicated by lettered black bars. The sole canonical E box is indicated in bold, and noncanonical E boxes (38, 39) in fragments C and D are also shown. (E) Ethidium bromide-stained gels of PCR products. (F) Sybr green analysis of PCR fragments evaluated for Myc binding. The absolute amount of DNA in each sample was calculated, and the average was plotted ± SD. (G) Relative mRNA levels for c-myc and PRDX3 during serum stimulation. Signals were normalized to the level of 18S RNA and plotted relative to the 0 h time point for each series.
Figure 2
Figure 2
Effect of PRDX3 expression on doubling time, transformation, and apoptosis in R1a-myc cells. (A) Immunoblot analysis of cell lysates from R1a-myc cells transfected with pSG5 empty vector, pSG5-PRDX3, or pSG5-PRDX3AS. (B) Growth curves of R1a-myc transfectants: pSG5 (□), PRDX3 (▵), and PRDX3AS (○). Doubling times were 10.4, 10.9, and 19.0 h, respectively. (C) Photomicrographs of methylcellulose colonies. (Bar = 500 μM.) The bar graph represents the average colony number per 35-mm dish ± SD. (E) Tumor formation in nude mice. The average estimated tumor mass was plotted at 2, 3, and 4 weeks after injection ± SD (n = 8). (D) Percentage of apoptotic cells 24 h after serum deprivation (light bars) or glucose deprivation (dark bars). The average ± SD of three experiments is shown.
Figure 3
Figure 3
Effect of PRDX3 expression on doubling time and apoptosis in MCF7/ADR cells. (A) Immunoblot analysis of cells lysates from MCF7/ADR cells transfected with pSG5, pSG5-PRDX3, or pSG5-PRDX3AS. (B) Growth curves of MCF7/ADR transfectants: pSG5 (□), PRDX3 (▵), and PRDX3AS (○). Doubling times were 43.0, 37.6, and 60.2 h, respectively. (C) Percentage of apoptotic cells 24 h after glucose withdrawal. The average ± SD of three separate experiments is shown.
Figure 4
Figure 4
PRDX3 affects mitochondrial membrane integrity and morphology. (A) Histograms generated by FACS analysis of cells incubated with dye specific for cellular reactive oxygen species (DCF), mitochondrial mass (NAO), or mitochondrial membrane potential (DiOC6): pSG5 (solid black line), PRDX3 (solid gray line), PRDX3AS (dotted line). (B) Transmission electron microscopy of R1a-myc-pSG5 and R1a-myc-PRDX3AS cells. (Bar = 1 μM.) (C) Analysis of ROS after glucose deprivation. Cells were exposed to glucose-free media for 1.5 h before incubation with DCFH-DA.
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