CDK2 Transcriptional Repression Is an Essential Effector in P53
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Published OnlineFirst August 22, 2014; DOI: 10.1158/1541-7786.MCR-14-0163 Cell Cycle and Senescence Molecular Cancer Research CDK2 Transcriptional Repression Is an Essential Effector in p53-Dependent Cellular Senescence— Implications for Therapeutic Intervention Hasan Zalzali1, Bilal Nasr1, Mohamad Harajly1, Hussein Basma1, Farah Ghamloush1, Sandra Ghayad1,Noel€ Ghanem2, Gerard I Evan3, and Raya Saab1 Abstract Cellular senescence, a form of cell-cycle arrest, is a tumor- cence. Pharmacologic inhibition of CDK2 in an in vivo model of suppressor mechanism triggered by multiple tumor-promoting pineal tumor decreased proliferation and promoted early senes- insults, including oncogenic stress and DNA damage. The role of cence, and it also decreased tumor penetrance and prolonged time cyclin-dependent kinase 2 (CDK2) regulation has been evaluated to tumor formation in animals lacking p53. In conclusion, for in models of replicative senescence, but little is known regarding both oncogene- and DNA damage–induced cellular senescence, its role in other senescence settings. Using in vitro and in vivo CDK2 transcript and protein are decreased in a p53- and RB- models of DNA damage–and oncogene-induced cellular senes- dependent manner, and this repression is necessary for cell-cycle cence, it was determined that activation of the tumor-suppressor exit during senescence. protein p53 (TP53) resulted in repression of the CDK2 transcript that was dependent on intact RB. Ectopic CDK2 expression was Implications: These data show that CDK2 inhibition may be sufficient to bypass p53-dependent senescence, and CDK2-spe- useful for cancer prevention in premalignant hyperproliferative cific inhibition, either pharmacologically (CVT313) or by use of a lesions, as well as established tumors. Mol Cancer Res; 13(1); 29–40. dominant-negative CDK2, was sufficient to induce early senes- Ó2014 AACR. Ink4b settings, other CDK inhibitors, such as CDKN2B (p15 ) and Introduction Ink4c CDKN2C (p18 ), have also been shown to play roles in this Cellular senescence is a well-documented tumor-suppressor process (11, 12). response, which in most instances is dependent on functional In contrast with the well-documented roles of the CDK inhi- TP53 (p53), and results in irreversible cell-cycle exit, so preventing Ink4a Cip1 Ink4b Ink4c bitors (p16 , p21 , p15 , and p18 ) in cellular senes- further tumor progression under the influence of oncogenic or cence, the roles of the respective CDKs are not well defined. genotoxic insults (reviewed in refs. 1, 2). Replicative senescence The role of CDK2 has been primarily studied in the setting of occurs due to telomere shortening, but senescence can also be replicative senescence, in which several investigators have shown induced by oncogene activation, loss of tumor suppressors, or that activity of CDK2 decreases in senescent fibroblasts, likely direct DNA damage (reviewed in ref. 3). Cell-cycle regulators are Cip1 through inhibition of Cyclin A–CDK2 complexes by p21 , critically involved in the execution of the senescence response. In whereas CDK2 protein levels remain unchanged (13–15); where- human fibroblasts, the cyclin-dependent kinase (CDK) inhibi- Ink4a Cip1 as in replicative senescence of cultured human umbilical vein tors, CDKN2a (p16 ) and CDKN1A (p21 ), are activated in endothelial cells (HUVEC), reduction of both CDK2 activity and response to oncogenic signals and are essential for cell-cycle exit protein levels were noted (16). Transduction of replicatively during cellular senescence (4–8). In senescence caused by geno- senescent cells with Cyclin E–CDK2 complexes results in cell- toxic insults, such as irradiation or topoisomerase inhibition, p53 Cip1 cycle reentry in a subset of cells, indicating that reduction of CDK2 is activated and this results in p21 induction, which is essential activity is indeed essential for replicative senescence (17). for cell-cycle exit and senescence (5, 9, 10). In yet other in vivo In oncogene-induced senescence, limited data are available, but suggest a physiologic role for CDK2 in opposing senescence. V12 For instance, in RAS -induced senescence, endogenous CDK2 1Department of Pediatric and Adolescent Medicine, American Univer- activity is reported to be decreased (6). MYC-induced senescence sity of Beirut, Beirut, Lebanon. 2Department of Biology, American occurs in a Cdk2-null background (and upon pharmacologic 3 University of Beirut, Beirut, Lebanon. Department of Biochemistry, CDK2 inhibition in fibroblasts), but not in Cdk4-null or Cdk6- University of Cambridge, Cambridge, United Kingdom. null backgrounds (18). We previously showed that in a mouse B. Nasr and M. Harajly contributed equally to this article. model of CCND1 (Cyclin D1)-driven senescence, CDK2 protein Corresponding Author: Hasan Zalzali or Raya Saab, American University of levels are also markedly decreased (19). All the above data Beirut, Riad El Solh Street, Beirut 1107 2020, Lebanon. Phone: 961-1-350000, ext. together prompted us to investigate the role of CDK2 in onco- 4780. Fax: 961-1-377384; E-mail: [email protected]; [email protected] gene-induced and DNA damage–induced senescence. In this doi: 10.1158/1541-7786.MCR-14-0163 report, we uncover a central physiologic role for p53-mediated Ó2014 American Association for Cancer Research. repression of CDK2 during oncogene-induced senescence, and www.aacrjournals.org 29 Downloaded from mcr.aacrjournals.org on September 26, 2021. © 2015 American Association for Cancer Research. Published OnlineFirst August 22, 2014; DOI: 10.1158/1541-7786.MCR-14-0163 Zalzali et al. highlight CDK2 as a possible target to limit premalignant lesions, staining, cells were fixed and stained overnight at 37C, and as well as inhibit progression of established cancer, via induction counterstained with eosin, as in our previous studies (12, 19). of senescence. For BrdUrd incorporation assays, cells were treated with BrdUrd at a concentration of 33 mmol/L for 2 hours, fixed with 50% Materials and Methods methanol/50% acetone solution for 2 minutes, then treated with 2N HCL for 10 minutes, and neutralized by Borate buffer for 12 Mouse studies À À minutes. After blocking, slides were probed with anti-BrdUrd Irbp-Cyclin D1 transgenic mice (20) were bred with p53 / mice antibody (Santa Cruz Biotechnology), Cy3-conjugated secondary (The Jackson Laboratory) or p53ERTAM ki/ki mice (21) and antibody (Jackson Immunoresearch) or Alexa 488–conjugated maintained in a mixed C57BL/6  129/Sv genetic background. antibody (Invitrogen) was used for detection. Stained cells were PCR for targeted alleles was used to verify mouse genotypes as covered with aqueous mounting medium containing DAPI (Vec- described previously (20–22). Animals were euthanized at defined tor Laboratories) and visualized by immunofluorescence micros- time points or when obviously ill in accordance with the American copy. The number of BrdUrd-positive cells was manually counted University of Beirut Institutional Animal Care and Use Committee from at least five representative fields, at Â20 magnification, and guidelines; all animal studies were approved by this committee. normalized to total cell number (counted as DAPI-positive For all in vivo studies, CVT313 was administered by once daily i. nuclei). Digital photomicrographs were obtained using a Zeiss p. injection of 1.25 mg/kg. For bromodeoxyuridine (BrdUrd) 510 NLO multiphoton/confocal laser scanning microscope. incorporation assays, mice were given an i.p. injection of 50 Composite images were constructed using Photoshop CS6 soft- mg/kg of BrdUrd (Sigma-Aldrich), every 2 hours  5 and sacri- ware (Adobe Systems). ficed 2 hours later. Cell accumulation assay Plasmid constructs, virus production, and transduction Cells were plated in 6-well plates at a density of 200,000 cells CMV–CDK2–HA and CMV–CDK2DN–HA plasmid constructs per well. For senescence induction, cells were exposed to 20 Gy were obtained from Addgene [Addgene plasmids 1884 and 1885 irradiation at 1 Gy/min, treated with 10 mmol/L etoposide or (ref. 23); www.addgene.org], and subcloning of the constructs transduced with RasV12-GFP. The cells were collected at 7 days was performed into a lentivirus backbone expressing GFP sepa- V12 (after Ras transduction) or 14 days (after DNA damage), fixed rated by an IRES site. The RasV12-GFP plasmid was purchased with methanol: acetic acid 3:1 for 5 minutes, then stained in 0.5% from Clonetech. HEK 293T cells were used for lentivirus produc- Crystal Violet in methanol for 15 minutes. A dissecting micro- tion (CDK2 constructs) and retrovirus production (RasV12 con- scope was used to view the cells under a magnified field. structs), using calcium phosphate transfection with the appropri- ate respective packaging plasmids. Cell explantation and ex vivo culture Cell culture Mice were euthanized according to the IACUC-approved pro- Human BJ foreskin fibroblasts (ATCC) were cultured at 37C tocol. Pineal cells were explanted at postnatal day 10 (P10), mechanically dispersed, and cells were plated onto 8-well perma- and 5% CO2 in a cell incubator, in DMEM containing 10% FBS, 1% glutamine, and 1% pen/strep. Mouse embryonic fibroblasts nox chamber slides (Nunc), and cultured in DMEM with 10% (MEF) were isolated from E13.5 embryos from wild-type (WT), FBS, 1% glutamine, and 1% pen/strep. Explanted cells were À À ki/ki fl/fl p53 / , p53ERTAM ,andRb mice (in which exon 19 of the treated with CVT313 (Santa Cruz Biotechnology) or DMSO fi Rb gene is flanked by two LoxP sites, allowing induction of vehicle; media were renewed every 3 days, and cells were xed Cre-mediated recombination; ref. 24). MEFs were cultured in after 7 days. BrdUrd and SABG staining and cell counting were DMEM containing 10% FBS, 1% nonessential amino acids, 1% performed as detailed above. Digital photomicrographs were sodium pyruvate, 1% glutamine, and 1% pen/strep. For MEFs analyzed using Adobe Photoshop CS4 software. ki/ki derived from p53ERTAM mice, p53 was reversibly switched on and off by addition or withdrawal of 100 nmol/L 4-OH Histologic studies and immunostaining fl/fl tamoxifen, as specified.