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YPEL3, a P53-Regulated Gene That Induces Cellular Senescence

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YPEL3, a P53-Regulated Gene That Induces Cellular Senescence Published OnlineFirst April 13, 2010; DOI: 10.1158/0008-5472.CAN-09-3219 Molecular and Cellular Pathobiology Cancer Research YPEL3, a p53-Regulated Gene that Induces Cellular Senescence Kevin D. Kelley1, Kelly R. Miller1, Amber Todd1, Amy R. Kelley1, Rebecca Tuttle2, and Steven J. Berberich1 Abstract Cellular senescence, the limited ability of cultured normal cells to divide, can result from cellular damage triggered through oncogene activation (premature senescence) or the loss of telomeres following successive rounds of DNA replication (replicative senescence). Although both processes require a functional p53 signaling pathway, relevant downstream p53 targets have been difficult to identify. Discovery of senescence activators is important because induction of tumor cell senescence may represent a therapeutic approach for the treat- ment of cancer. In microarray studies in which p53 was reactivated in MCF7 cells, we discovered that Yippee- like-3 (YPEL3), a member of a recently discovered family of putative zinc finger motif coding genes consisting of YPEL1-5, is a p53-regulated gene. YPEL3 expression induced by DNA damage leads to p53 recruitment to a cis-acting DNA response element located near the human YPEL3 promoter. Physiologic induction of YPEL3 results in a substantial decrease in cell viability associated with an increase in cellular senescence. Through the use of RNAi and H-ras induction of cellular senescence, we show that YPEL3 activates cellular senescence downstream of p53. Consistent with its growth suppressive activity, YPEL3 gene expression is repressed in ovarian tumor samples. One mechanism of YPEL3 downregulation in ovarian tumor cell lines seems to be hypermethylation of a CpG island upstream of the YPEL3 promoter. We believe these findings point to YPEL3 being a novel tumor suppressor, which upon induction triggers a permanent growth arrest in human tumor and normal cells. Cancer Res; 70(9); 3566–75. ©2010 AACR. Introduction senescence; ref. 6). However, unlike apoptosis and transient cell cycle arrest, less is known about the p53 targets critical Biochemical and genetic data show that in response to a to cellular senescence. Although it is accepted that cyclin- variety of cellular stresses, the p53 tumor suppressor protein dependent kinase inhibitor and p53 target gene p21 is in- plays a critical role in the inhibition of cell proliferation. Al- duced as cells undergo senescence, in some cell systems, though p53 can elicit transcription-independent apoptosis the loss of p21 leads to senescence bypass (7); yet in other cell (1), it also functions as a transcription factor inducing genes systems, p21-null cells can still undergo a ras-mediated and miRNAs to mediate its complex biological functions (2). senescence (8). In contrast to p21, plasminogen activator Although p53 transcriptional and nontranscriptional me- inhibitor 1 represents a critical p53-regulated gene in cellular chanisms to induce apoptosis have been linked to tumor senescence of both murine and human cells (9). inhibition (3), recent in vivo studies have shown that p53- In studies in which we triggered a p53-dependent cell cycle dependent cellular senescence represents an important arrest in MCF7 cells through the RNAi targeting of HdmX or mechanism to block tumor development (4, 5). These find- Hdm2, both p53-negative regulators, we uncovered several ings suggest that studies focused on understanding signaling novel p53-regulated genes, one of which was YPEL3 (10). pathways in senescence may lead to new targets. Located on the short arm of chromosome 16, YPEL3 is part It is known that p53 and Rb signaling pathways are at the of a five-member family of closely related paralogues— nexus of senescence resulting from oncogene activation YPEL1-5—that are named in reference to their Drosophila (premature senescence) or telomere shorting (replicative orthologue (11). The Drosophila Yippee protein was identified as a putative zinc finger motif containing protein with a high Authors' Affiliations: 1Wright State University, Biochemistry & Molecular degree of conservation among the cysteines and histidines Biology Department and 2Wright State University, Department of Surgery, Dayton, Ohio that form the motif (12). Note: Supplementary data for this article are available at Cancer Murine YPEL3 has been shown to have an association with Research Online (http://cancerres.aacrjournals.org/). the induction of cell growth inhibition through apoptosis. K.D. Kelley and K.R. Miller contributed equally to this work. Originally named SUAP for small unstable apoptotic protein, Corresponding Author: Steven J. Berberich, Wright State University, murine YPEL3 was linked to apoptosis in a screen for genes 3640 Colonel Glenn Highway, Dayton, OH 45435. Phone: 937-775- involved in the programmed cell death of murine myeloid 4494; Fax: 937-775-3730; E-mail: [email protected]. precursor cells during differentiation (13). doi: 10.1158/0008-5472.CAN-09-3219 In the present study, we set out to examine whether the ©2010 American Association for Cancer Research. human YPEL3 gene was a novel p53 transcriptional gene 3566 Cancer Res; 70(9) May 1, 2010 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2010 American Association for Cancer Research. Published OnlineFirst April 13, 2010; DOI: 10.1158/0008-5472.CAN-09-3219 YPEL3 Induces Cellular Senescence target. Using DNA damage, chromatin immunoprecipitation, gene in pGL3-Basic. The YPEL3(−485:+120) reporter plasmid and luciferase reporter assays, we show that YPEL3 is regu- contains −485 to +120 of the YPEL3 promoter cloned lated by p53. In assessing the biological activity of YPEL3, we into pGL3-Basic. A 50-bp region of the YPEL3 promoter show that it encodes a protein that induces cellular senes- (Fig. 2D), a triple mutant (containing mutations to the criti- cence in human tumor and normal cells. Consistent with this cal nucleotides in each half site), and a randomly generated growth-suppressive activity, YPEL3 is seen downregulated in scramble sequence were cloned into pGL3-Basic. H1299 cells ovarian tumor samples. Using ovarian tumor cell lines, we were plated at 60 k cells per well in 12-well plates 24 h before observed that CpG hypermethylation seems to be one cotransfection with the indicated expression and luciferase mechanism by which YPEL3 gene expression is suppressed. vectors. The following day, the cells were lysed on the plate Overall, these studies identify YPEL3 as a p53-dependent, using 1× passive lysis buffer (Promega) and were analyzed by tumor suppressor gene. the Dual Luciferase Assay kit (Promega) according to the manufacturer's instructions. The experimental reporter's lu- Materials and Methods ciferase activity was normalized to Renilla luciferase activity measured in relative light units. All experiments for each YPEL3 expression plasmids. The tetracycline-inducible treatment were performed in triplicate. Error bars show var- (tet-on) lentiviral YPEL3-V5–tagged expression construct iation as the SD of each set of biological triplicates. Statistical was designed by cloning the YPEL3 open reading frame into significance was determined by using the paired student's pENTR/SD/D-TOPO and then into the pLenti4-T/O-V5 DEST t test (Sigma Plot, P <0.05). vector through the Gateway cloning system (Invitrogen). Immunoblotting. Whole-cell extracts and Western blotting Reverse transcription-PCR. Total RNA was isolated using were performed as previously described with the following the e.Z.N.A. Total RNA kit (Omega Bio-Tek) according to the modifications (14). Cell pellets were lysed in three freeze- manufacturer's instructions. RNA quality and quantity was thaws using single lysis buffer [50 mmol/L Tris (pH 8.0), assessed on a RNA nanochip (Agilent Technologies). One 150 mmol/L NaCl, 1% NP40] containing a protease inhibitor microgram of total RNA was used as a template for cDNA cocktail (Sigma). For endogenous YPEL3 and p53 Westerns, synthesis using the reverse transcriptase core reagent kit 10% to 20% gradient Tricine gels were used to separate 100 μg (Applied Biosystems). Taqman-based PCR was performed of protein extract. Proteins were transferred to polyvinylidene in triplicate using Assay on Demand probe sets (Applied Bio- difluoride using a semidry transfer apparatus (Fisher Biotech). systems) and an Applied Biosystems 7900 Sequence Detec- Antibodies were obtained from Proteintech (YPEL3) and tion System. Glyceraldehyde-3-phosphate dehydrogenase Calbiochem (p53, Ab-4, and Ab-6). (GAPDH) was used as an endogenous control. All Assay on Chromatin immunoprecipitation. Chromatin immuno- Demand probes used in these experiments (YPEL3, p21, Bax, precipitation of the YPEL3 promoter region was performed and GAPDH) were validated gene targets. using the ChIP-It Express Chromatin Immunoprecipitation Cell lines. H1299 cells are derived from non–small cell kit (Active Motif) according to the manufacturer's instruc- lung carcinoma devoid of p53. U2OS osteosarcoma cells, tions. Briefly, six 15-cm culture plates of HCT116 cells ex- HepG2 hepatocarcinoma, and MCF7 breast carcinoma cells pressing wild-type p53 were grown to 70% confluence after all express wild-type p53. HCT116 +/+ and −/−p53 colon which doxorubicin (0.3 μg/mL) was applied to three plates. carcinoma cell lines are isogenic with the exception of Following a 20-h incubation, the cells were fixed in formal- p53 status. IMR90 cells are primary human diploid fibro- dehyde and sonicated to average chromatin fragments of blasts. All
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