Pw1/Peg3 is a potential cell death mediator and cooperates with Siah1a in p53-mediated apoptosis

Abstract

Induction of wild-type p53 in mouse fibroblasts causes cell cycle arrest at the G(1) phase, whereas coexpression of p53 and the protooncogene c-myc induces apoptosis. Although p53 transcriptional activity generally is required for both pathways, the molecular components mediating p53-dependent apoptosis are not well understood. To identify factors that could mediate p53-induced cell death, we used a comparative RNA differential display procedure. We have identified Pw1/Peg3 as a gene product induced during p53/c-myc-mediated apoptosis. Pw1/Peg3 is not induced during p53-mediated G(1) growth arrest nor by c-myc alone. Although it is not clear whether the induction of Pw1/Peg3 depends on p53 activity, we show that Pw1/Peg3 interacts with a p53-inducible gene product Siah1a. We demonstrate that coexpression of Pw1/Peg3 with Siah1a induces apoptosis independently of p53 whereas expression of Pw1/Peg3 or Siah1a separately has no effect on cell death. These data suggest that Siah1a and Pw1/Peg3 cooperate in the p53-mediated cell death pathway. Furthermore, we show that inhibiting Pw1/Peg3 activity blocks p53-induced apoptosis. The observation that Pw1/Peg3 is necessary for the p53 apoptotic response suggests a pivotal role for this gene in determining cell death versus survival.

Figure 1

Identification of ASF-1 (Pw1/Peg3) as a gene specifically induced during p53-mediated apoptosis. (A) Northern blot analysis of Pw1/Peg3 (ASF-1) during p53-mediated cell cycle arrest and apoptosis. Total RNA was prepared from VHD, VM10, and VM7 cells growing at 38°C and cells shifted to 32°C for 24 and 48 hr. Total RNA was extracted and RNA samples of the two time points at 32°C were pooled together. Fifteen micrograms of total RNA from each sample was subjected to electrophoresis on a 1% formaldehyde agarose gel, transferred to a nylon membrane, and hybridized with ³²P-labeled ASF-1 probe and a mouse p21 probe. The positions of 28S and 18S ribosomal RNA are indicated. RNA loading was normalized by hybridization with glyceraldehyde-3-phosphate dehydrogenase (GAPDH). (B) Induction of Pw1/Peg3 during p53/E2F-1 mediated apoptosis. The C18 cell is a murine fibroblast cell line expressing tsp53 and E2F-1 and undergoes apoptosis at 32°C as described (1). Total RNA were extracted and analyzed by Northern blotting using ASF-1 as a probe as described in A. A 5-fold increase in Pw1/Peg3 expression was observed in C18 cells at 32°C. (C) Time course of Pw1/Peg3 induction in VM10 cells at 32°C. The VM10 cell and control VHD cells were shifted to 32°C at the time point indicated. Total RNA were extracted and blotted with ASF-1 probe. The control of RNA loading was measured by probing with GAPDH.

Figure 2

Coexpression of p53 and Pw1/Peg3 induces apoptosis. (A) 10.1 cells were transfected with empty vector, Pw1/Peg3, p53 and Pw1/Peg3, p21 and Pw1/Peg3. A GFP expressing plasmids pEGEP was cotransfected at molar ratio of 3 to 1. The transfected cells were fixed, stained with propidium iodide, and subjected to FACS analyses. The transfected cells represented by GFP-positive population were gated and analyzed for their DNA content. The level of apoptosis was measured by cells in the sub-G₁ population. The level (percent) of apoptotic cells was averaged from three independent experiments. SDs are given. The coexpression of p53 and Pw1/Peg3 results in 15% apoptotic cells, whereas 0.5% of control cells shows cell death. Coexpression of p21^WAF1/CIP1 and Pw1/Peg3 does not induce cell death. (B) GFP-positive cells display apoptotic morphology. The 10.1 cells transfected with GFP, p53, and Pw1/Peg3 were fixed on coverslips, stained with propidium iodide, and viewed under a fluorescent microscope. The GFP-positive cells show condensed chromatin and cell membrane blebbing that are characteristics of apoptosis. (C) Expression of Pw1/Peg3 and p53 proteins in transfected cells. The 10.1 cells were transfected with DNA as in the FACS experiments. The Pw1/Peg3 and p53 proteins indicated by arrows were detected by immunoprecipitation and blotting with either anti-HA antibody or pAB421, respectively.

Figure 3

Interaction of Pw1/Peg3 with Siah proteins and expression of Siah1a during p53-mediated growth arrest and apoptosis. (A) Interaction between Pw1/Peg3 and the Siah family of proteins in yeast. Pw1/Peg3, Siah1a, and Siah2 were fused to the Gal4 DNA binding domain and activation domain separately and were tested in yeast for interaction. The empty vectors were used as negative controls. + indicates growth and LacZ staining, whereas − indicates no growth on selective media. Siah1a and Siah2 showed strong homo- and heterotypic interactions, as indicated by ++. (B) 293 cells plated in 10-cm dishes were transiently transfected with expression vectors encoding Flag-tagged Siah1a and Siah2 with HA-tagged Pw1/Peg3. Transfection of empty vector was used as a control. The cells were harvested and lysed 48 hr later. Half of the lysate was immunoprecipitated with anti-Flag antibody (Sigma), and the other half was precipitated by using antibody against HA (12CA5). The immunocomplex was subjected to SDS/PAGE, and Western blot was carried out by using either α-HA or α-Flag antibodies as indicated. Pw1/Peg3/Siah complex is readily detected. (C) Induction of Siah1a expression by p53. Total RNA was prepared from VHD and VM10 as described in Fig. 1A. Northern analysis of Siah1a mRNA was carried out by using a ³²P-labeled Siah1a probe. The Siah1a mRNA appears as a doublet. The RNA loading is shown below.

Figure 4

Induction of apoptosis by coexpression of Pw1/Peg3 and Siah1a. (A) 10.1 cells transfected with vector, Pw1/Peg3, Siah1a, Siah2, and GFP-expressing plasmids, as well as in combinations were subject to FACS analysis as described in Fig. 2. The percentages of apoptosis were quantified by sub-G₁ cells in three independent experiments. SDs are given. (B) In situ fluorescence staining shows that Pw1/Peg3-expressing cells display condensation and fragmentation of DNA. 10.1 cells grown on coverslips were transfected with Pw1/Peg3 and Siah1a-expressing plasmids. The cells were fixed and stained with antibody against HA epitope tag on Pw1/Peg3 and nuclei by 4′,6-diamidino-2-phenylindole (DAPI). The phase contrast morphology of the cell also is shown. The fragmented nucleus is indicated by an arrow. (C) Expression of Pw1/Peg3 and Siah proteins in transfected cells. The 10.1 cells were transfected with DNA as in the FACS experiments. The Pw1/Peg3 and Siah proteins were detected by immunoprecipitation followed by Western blotting.

Figure 5

Inhibition of p53/c-myc-induced apoptosis by antisense Pw1/Peg3 and a dominant negative mutant. (A) VM10 cells were transfected with vector control, Pw1/Peg3, Pw1/Peg3 antisense mRNA, and a deletion mutant Pw1/Peg3 containing amino acids 1–592 (ΔPw1). The cells were cotransfected with pEGFP as described. The transfected cells were shifted to 32°C for 24 h and analyzed by FACS as in Fig. 2. Percentages of sub-G₁ cells (apoptotic) are the averages of three independent experiments. (B) Antisense Pw1 reduces Pw1/Peg3 protein levels. 293 cells were transfected with either 2 μg of Pw1/Peg3, 2 μg of ΔPW1, or the two together in different ratios (1:1 and 1:3). The HA-tagged Pw1/Peg3 was detected by immunoprecipitation-Western blotting. Transfection efficiency was monitored by cotransfecting with 0.5 μg of pEGFP.