P14arf Expression Increases Dihydrofolate Reductase Degradation and Paradoxically Results in Resistance to Folate Antagonists in Cells with Nonfunctional P53

[CANCER RESEARCH 64, 4338–4345, June 15, 2004] p14ARF Expression Increases Dihydrofolate Reductase Degradation and Paradoxically Results in Resistance to Folate Antagonists in Cells with Nonfunctional p53 Pellegrino G. Magro,1,2 Angelo J. Russo,1,2 Wei-Wei Li,2 Debabrata Banerjee,3 and Joseph R. Bertino3 1Joan and Sanford I. Weill Graduate School of Medical Sciences of Cornell University, New York, New York; 2Memorial Sloan Kettering Cancer Center, New York, New York; and 3The Cancer Institute of New Jersey, Robert Wood Johnson Medical School, New Brunswick, New Jersey ABSTRACT Although much is known about the function of ARF in the p53 pathway, less is known about its functions in human tumor growth and ARF The p14 protein, the product of an alternate reading frame of the chemosensitivity independently of its regulation of p53 via binding to INK4A/ARF locus on human chromosome 9p21, disrupts the ability of MDM2. Mouse embryonic fibroblasts expressing E1A and exogenous MDM2 to target p53 for proteosomal degradation and causes an increase ARF are more sensitive to killing by doxorubicin than their normal in steady-state p53 levels, leading to a G1 and G2 arrest of cells in the cell cycle. Although much is known about the function of p14ARF in the p53 counterparts that only express E1A (7). Alterations in the INK4A/ pathway, not as much is known about its function in human tumor growth ARF locus reduce p53 induction and cytotoxicity of cyclophospha- and chemosensitivity independently of up-regulation of p53 protein levels. mide in mouse lymphomas (16). Also, recombinant adenovirus-me- To learn more about its effect on cellular proliferation and chemoresis- diated p14ARF overexpression sensitizes MCF-7 breast cancer cells to tance independent of p53 up-regulation, human HT-1080 fibrosarcoma cisplatin (17). Thus, expression of ARF appears to increase sensitivity cells null for p14ARF and harboring a defective p53 pathway were stably to chemotherapeutic drugs. However, this increase in sensitivity has ARF transfected with p14 cDNA under the tight control of a doxycycline- been associated with up-regulation of p53. The question of whether ARF inducible promoter. Induction of p14 caused a decrease in cell prolif- p14ARF can also increase sensitivity to chemotherapeutics independ- eration rate and colony formation and a marked decrease in the level of ARF ently of p53 up-regulation has remained unanswered. To investigate dihydrofolate reductase (DHFR) protein. The effect of p14 on DHFR ARF protein levels was specific, because thymidylate kinase and thymidylate the effect of p14 on growth and sensitivity to chemotherapeutic synthase protein levels were not decreased nor were p53 or p21WAF1 agents independent of p53 up-regulation, we transfected the HT-1080 ARF protein levels increased. The decrease in DHFR protein was abolished human fibrosarcoma cell line that lacks p14 and harbors a non- when the cells were treated with the proteasome inhibitor MG132, dem- functional p53 pathway (18–20) with the pTET-ON, pTRE2 vector onstrating that p14ARF augments proteasomal degradation of the protein. containing p14ARF cDNA, in which expression is tightly regulated by Surprisingly, induction of p14ARF increased resistance to the folate antag- doxycycline. Herein, we report that restoration of p14ARF causes a onists methotrexate, trimetrexate, and raltitrexed. Depletion of thymidine marked decrease in dihydrofolate reductase (DHFR) protein levels by ARF in the medium reversed this resistance, indicating that p14 induction altering DHFR protein stability; decreases the proliferation rate of increases the reliance of these cells on thymidine salvage. these cells by reducing colony formation; and selectively confers resistance to the antifolates methotrexate, trimetrexate, and raltitrexed INTRODUCTION by increasing the reliance of these cells on thymidine salvage. The INK4A/ARF locus on human chromosome 9p21 codes for the p16INK4A and the p14ARF protein. The p16INK4A protein is the MATERIALS AND METHODS product of alternatively spliced exons 1␣, 2, and 3 and functions as a Cell Lines and Culture Conditions. The HT-1080 human fibrosarcoma cyclin-dependent kinase inhibitor, regulating the G -phase exit by 1 cell line and p14JM p14ARF-inducible clone were grown in RPMI 1640 inhibiting the phosphorylation of the retinoblastoma protein (1–3). ARF ARF supplemented with penicillin/streptomycin and 10% Tet approved fetal bovine The p14 protein (p19 in mouse) is transcribed from distinct serum (FBS) from Clontech (Palo Alto, CA). exon 1␤ and shared exons 2, and 3 and has been shown to bind the Cloning and Establishment of p14ARF-Inducible Expression in HT-1080 MDM2 protein, alleviating MDM2-driven degradation of p53, which Cells. The p14ARF cDNA cloned between BamHI and EcoR1 in the Bluescript ϩ results in elevated p53 levels that lead to cell cycle arrest in the G1-S KS vector was a kind gift from Dr. G. Peters (Imperial Cancer Research and G2-M boundaries of the cell cycle (4, 5). Induction of ARF has Fund Laboratories, London, United Kingdom). The plasmid was digested with been shown to occur in response to proliferative signals such as myc, BamHI and HindIII, and the p14ARF cDNA was isolated and cloned into the E1A, Ras, and E2F-1 (6–9). Thus, induction of ARF provides a link pTRE-2 vector that was also digested with BamH1 and HindIII. The pTRE-2 ARF between retinoblastoma protein and p53 tumor-suppressive pathways. vector containing the p14 cDNA was then cotransfected with the pTet-On vector in a ratio of 2:1 in cells that were approximately 30% confluent in a However, p19ARF also causes a G -phase cell cycle arrest independent 1 150-mm tissue culture dish. The pTRE-2 and pTet-On vectors for establish- of p53 (10). This finding is not surprising, because tumors exist in ARF ARF ment of the inducible system were purchased from Clontech. Positive colonies which p53 and p14 are inactivated, indicating that p14 may were selected in medium supplemented with 500 ␮g/ml G-418 for 2 weeks; ARF have other functions (10–12). It has been shown recently that p14 and cell lines were characterized for p14ARF expression by plating the ex- ARF and p19 are able to bind to E2Fs and alter their transcriptional panded clones in six-well plates with or without 1 ␮g/ml doxycycline for 24 h, ability and stability, possibly explaining why ARF is able to inhibit lysed, and then analyzed by Western blot analysis. cell cycle progression independent of p53 (13–15). Western Blotting. Actively growing cells were harvested from culture, and protein lysates were prepared by lysing cells in the presence of lysis buffer [50 mM Tris-HCl (pH 7.4), 100 mM NaCl, 0.5% sodium deoxycholate, and 0.5% Received 4/15/03; revised 4/5/04; accepted 4/21/04. Grant support: United States Public Health Service Grant CA08010. NP40] containing Protease Inhibitor mixture from PharMingen (San Diego, The costs of publication of this article were defrayed in part by the payment of page CA; 10 ␮g/ml phenanthrolene, 10 ␮g/ml aprotinin, 10 ␮g/ml leupeptin, 10 charges. This article must therefore be hereby marked advertisement in accordance with ␮g/ml pepstatin A, and 1 mM phenylmethylsulfonyl fluoride). 18 U.S.C. Section 1734 solely to indicate this fact. The lysates were kept on ice for 15 min, vortexed twice, and centrifuged at Requests for reprints: Joseph R. Bertino, Cancer Institute of New Jersey, 195 Little ϫ Albany Street, New Brunswick, NJ 08903. Phone: (732) 235-8510; Fax: (732) 235-8181; 4°C for 10 min at 10,000 g in an Eppendorf microcentrifuge; and clear E-mail: [email protected]. supernatant recovered. The protein concentration was determined by the Bio- 4338 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2004 American Association for Cancer Research. p14ARF EXPRESSION AND DIHYDROFOLATE REDUCTASE Rad assay. Fifty ␮g of protein were analyzed on SDS-PAGE (varying between to a reaction mix consisting of 10 ␮M thymidine, 50 mM Tris-HCl, 5 mM ␮ 12% and 14% depending on protein analyzed). The proteins were electro- MgCl2,5mM ATP, 4 mM DTT, 10 mM NaF, and 1 Ci tritiated thymidine blotted onto nitrocellulose membrane and probed with the appropriate primary (25-␮l reaction). The mixture was incubated at 37°C for 20, 40, and 60 min. and secondary antibodies. The protein bands were visualized on X-ray film At each time point, 20 ␮l were withdrawn and spotted on DE-81 filter paper. using the enhanced chemiluminescence reagent from Amersham Biosciences The filter paper was allowed to dry, washed three times in 5 mM ammonium (Arlington Heights, IL). The p21 (F-5) and p53 (Bp53-12) antibodies were formate, and placed in a scintillation vial containing 10 ml of Scintiverse BD purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The polyclonal scintillation fluid; and radioactivity was determined. thymidine kinase (TK) antibody was a gift from Dr. T. Kelly (Memorial Sloan In Situ (Whole-Cell) Thymidylate Synthesis Activity Assay. This Kettering Cancer Center, New York). The polyclonal thymidilate synthase method has been described previously (26, 27). In brief, p14JM cells were (TS) antibody was a gift from Dr. F. Maley (Wadsworth Center, New York resuspended in growth medium at concentration of 2 ϫ 106 cells/ml and placed State Department of Health, Albany, NY). in 1.5-ml Eppendorf tubes. The cells were incubated for3hat37°C. After the The ␣-tubulin antibody (B-5-1-2) was purchased from Sigma (St. Louis, 3 h incubation, [5-3H]deoxyuridine solution was added in 40-␮l volume to MO), and the p14ARF antibody (NB 200-111) was purchased from Novus cells to achieve a final concentration of 2 ␮Ci/ml. At time points of 0, 15, 30, Biologicals (Littleton, CO). and 45 min, a 100-␮l cell suspension from the induced or uninduced cells was mRNA Analysis.

Load more