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

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[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

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. Cells were grown for various times in the presence or aliquoted to a separate tube containing 200 ␮l of charcoal suspension and absence of 1 ␮g/ml doxycycline, collected, and lysed; mRNA was isolated vortexed. The tubes were then centrifuged for 5 min at 15,000 rpm. The using the Invitrogen mRNA isolation kit, and reverse transcriptase reaction supernatant (100 ␮l) was then added to 5 ml of scintillation mixture in 20-ml performed using the Invitrogen cDNA cycle kit. The relative message quantity scintillation vials and counted in a scintillation counter. Samples were counted was analyzed using probe and primers specific for DHFR cDNA as described in the order of 0, 15, 30, and 45 min. A set of four samples plus the previously (21). In brief, PCR was performed by using a TaqMan 1,000 RXN corresponding time points were used to calculate slope. Thymidylate synthesis Gold with buffer A kit and the ABIPrism 7700 thermocycler (Applied Bio- activity is expressed as cpm/min/107 cells and calculated as follows: Activity systems). The temperature profile was 95°C, 10 min (1 cycle); and T1, 95°C, (cpm/min/107 cells) ϭ [slope/cell number] ϫ 107. 15 s; T2, 60°C, 60 s (42 cycles). The primer and probe sequences are listed Analysis of Intracellular Tritium-Labeled Thymidine Metabolites. below. The first primer is the TaqMan probe, the second primer is the forward Cells were plated in 100-mm plates. One batch of cells was treated with 1 PCR primer, and the third primer is the reverse PCR primer. The DHFR primer ␮g/ml doxycycline. After 72 h, the doxycycline-treated and untreated groups and probe sequences are as follows: (a) TTGGTTCGCTAAACTG- were incubated for an extra4hinthepresence of 25 ␮M thymidine containing CATCGTCGC; (b) GTCCTCCCGCTGCTGTCAT; and (c) ATGCCCATGT- 10 ␮Ci [methyl-3H]thymidine. The cells were then trypsinized and collected TCTGGGACAC. by centrifugation. After washing with cold PBS, resuspended cells were Growth Assays and Fluorescence-Activated Cell Sorter Analysis. The transferred to a 1.5-ml tube and centrifuged for 15 s at 12,000 ϫ g at 4°C. short-term growth assay was carried out by plating 50,000 cells in each well of Pellets were immediately resuspended in 0.3 ml of 0.6 M trichloroacetic acid a six-well plate in growth medium, with or without doxycycline. Every 24 h, (4°C) and incubated at 4°C for 10 min followed by centrifugation. The acid starting on the day of plating, cells exposed or not exposed to 1 ␮g/ml supernatant was recovered and added to an equal volume of cold freon doxycycline cells were detached from the wells by treatment with 0.25% containing 0.5 M tri-n-octylamine. The mixture was vortexed for 15 s and trypsin/0.25% EDTA, resuspended in 1 ml of growth medium, and counted centrifuged for 30 s at 12,000 ϫ g. The aqueous upper phase was recovered using the Coulter Counter ZM from Coulter Electronics (Hialeah, FL). The and analyzed by high performance liquid chromatography, as described pre- percentage of viable cells was determined by trypan blue staining. The exper- viously (28). Thymidine metabolite peaks from cell extracts were identified by iment was repeated three times. For the long-term survival assay, 1,000 cells comparison of retention times using thymidine mono-, di-, and triphosphates as were plated in each well of a six-well plate in growth medium with or without standards. 1 ␮g/ml doxycycline; and 14 days later, the medium was removed, and the Proteasome Inhibitor MG-132 Assay. Cells treated or not treated with 1 cells were washed with PBS and then stained with 2% crystal violet solution. ␮g/ml doxycycline for 48 h were treated or not treated with 100 nM MG-132 For thymidine deplete media studies, FBS was incubated with thymidine (Calbiochem, La Jolla, CA) or 0.1% DMSO for 12 h. Cells were lysed, and phosphorylase (TP) at 37°C for 1 h and then incubated at 65°C for 30 min to proteins analyzed by Western blotting. inactivate the enzyme, and the serum was finally added to RPMI 1640 at a Protein Synthesis Inhibition Assay. Cells treated or not treated with 1 concentration of 10%. For cell cycle analysis, asynchronous cells were grown ␮g/ml doxycycline for 24 h were treated for various times with 10 ␮g/ml for 48 h in the presence or absence of 1 ␮g/ml doxycycline. Cells were cycloheximide. Cells were then harvested, whole-cell lysate was recovered, collected and then fixed with ice-cold 70% methanol. DNA was stained with and proteins were analyzed by Western blotting. The approximate half-life of propidium iodide (Sigma) as described previously (22). The stained cells were DHFR was determined by averaging band intensity of at least three separate then analyzed on a Becton Dickinson fluorescence-activated cell sorter. experiments for the various time points by use of Image-Pro Plus software. Cytotoxicity Assay. For cytotoxicity studies, 1,000 cells were plated in each well of 96-well microtiter plates (plating density approximately the same as the one used in short-term growth assay) in replicates of six, with or without RESULTS 1 ␮g/ml doxycycline in regular growth medium or in medium in which Selection of a p14ARF-pTRE2-Inducible Clone. After screening thymidine was depleted using TP. The following drugs were added at the indicated concentrations 24 h later and left in the medium for 96 h. Metho- several clones, one clone designated p14JM was isolated that ex- ARF ␮ trexate, trimetrexate, raltitrexed, and cytarabine were added at concentrations pressed p14 in the presence of 1 g/ml doxycycline. Expression of ARF ranging from 256 pM to 100 ␮M. Doxorubicin was added at concentrations of p14 was detected by the appearance of a band that comigrated 51 pM to 10 ␮M, and hydroxyurea was added at concentrations of 26 nM to with another band detected in BT-549 cells that are known to express ARF ARF 2mM. After 96 h, a solution containing 1 mg/ml 2,3-bis[2-methoxy-4nitro-5- p14 (Fig. 1A). Induction of p14 could be detected 8 h after sulfophenyl]-2H-tetrazolium-5-carboxyanilide sodium salt (XTT) and 25 ␮M exposure to 1 ␮g/ml doxycycline, with maximal expression occurring phenazine methosulfate was added to achieve a final concentration of 0.2 at 24 h (data not shown). Induction of p14ARF was detected with ␮ mg/ml XTT and 5 M phenazine methosulfate and incubated for 2–4hat concentrations as low as 100 ng/ml doxycycline, with maximal in- 37°C, and absorbance was measured at 450 nm with a microplate reader, as duction occurring at 1 ␮g/ml doxycycline (Fig. 1B). described previously (23). The above experiments were repeated at least three Induction of p14ARF in HT-1080 Cells Inhibits Cellular Prolif- times. TK Activity Assay. This is a modification to a previously shown method eration and Colony Formation. ARF has been shown to induce cell (24, 25). In brief, cells treated or not treated with 1 ␮g/ml doxycycline were cycle arrest in a p53-dependent and -independent manner (4, 5, 10). ARF incubated at 37°C for 72 h, then lysed in 50 mM Tris-HCl (pH 7.9), containing To examine whether p14 restoration had any effect on growth rate ARF 1mM ␤-mercaptoethanol, by three repeated freeze-thaw cycles, centrifuged, in the HT-1080 cells, the effect of expressing p14 on p14JM cell and the supernatant recovered. Four ␮g of whole-cell protein lysate were added survival was examined using a clonogenic assay. Colony formation in 4339

iodide and analyzed by fluorescence-activated cell sorting, there was

a modest increase in the amount of cells in G1 versus S phase in the induced cells (Fig. 2D), with no change in the amount of cells in

sub-G1 (data not shown). Therefore, the increase in the amount of ARF cells in G1 observed indicated that p14 is affecting the progression

of cells from G1 to S, rather than due to an increase in cell death. In addition, no difference was observed in the percentage of viable cells between the induced and uninduced cells, as measured by trypan blue staining after 72 h of doxycycline induction. p14ARF Expression Decreases DHFR Stability in a Proteosome- Dependent Manner. DHFR is a key enzyme in the de novo pathway for thymidylate synthesis, allowing for the recycling of dihydrofolate, the product of this reaction, to tetrahydrofolate. One of the expected consequences of inhibiting this enzyme would be the decreased pro- duction of thymidylate and therefore a reduction in DNA replication, leading to a decrease in growth rate. Induced expression of p14ARF resulted in a greater than 9-fold decrease in DHFR protein levels beginning at 48 h, with no significant effect on the levels of TK or TS (Fig. 3A, 1 and 3). There was no significant decrease of DHFR protein levels seen in the first 24 h of p14ARF induction (data not shown). Fig. 1. Isolation of a p14ARF-inducible clone and detection by Western blot. A, Western Addition of the same concentration of doxycycline for 72 h did not blot using NB-200-111 polyclonal p14ARF antibody of 50 ␮g of whole-cell lysate from affect DHFR levels in HT-1080 parental cells (Fig. 3A, 2). There was HT-1080 parental cells, BT-549, or p14JM clone uninduced (ϪDOX) or induced (ϩDOX) for 24 h with 1 ␮g/ml doxycycline (DOX). B, Western blot using NB-200-111 polyclonal a small difference in DHFR mRNA levels between the induced and p14ARF antibody of p14JM cell lysate from cells that were incubated with various amounts uninduced cells, as shown by quantitative PCR (Fig. 3B), indicating of doxycycline for 24 h. Ponceau S staining revealed equal transfer. Equal intensities of that this decrease was primarily posttranscriptional. This decrease, higher M nonspecific bands served as loading controls. r which may be related to the known effect of p14ARF on E2F1 transcription, would not explain the large difference in DHFR protein the cells induced to express p14ARF was reduced by more than 50% levels. Addition of cycloheximide to inhibit protein translation re- compared with cells that were not induced (Fig. 2A). This was not due vealed a decreased stability of DHFR in the presence of p14ARF to a doxycycline-induced increase in p53 or p21 levels (Fig. 2B). The expression; the t1/2 of DHFR changed from over 20 to 8 h (Fig. 3C). effect of p14ARF on the short term growth of p14JM cells was also When p14JM cells were exposed to 100 nM proteasome inhibitor examined by seeding cells with or without doxycycline (1 ␮g/ml) for MG-132, upon induction with p14ARF, there was complete restoration 5 days. The doxycycline-treated cells expressing p14ARF had a slower of DHFR protein levels (Fig. 3D). These results indicate that p14ARF growth rate than that of the untreated cells not expressing p14ARF affects DHFR protein stability via increased proteasomal degradation. (Fig. 2C). One reason for the reduced colony formation and growth The decreased stability of DHFR upon p14ARF induction appears to be rate of the p14ARF-expressing cells might be the ability of p14ARF to indirect, because efforts to detect p14ARF-DHFR binding were unsuc- arrest cells in G1 (4, 6). When cells were stained with propidium cessful (data not shown).

Fig. 2. Effect of p14ARF on growth and cell cycle state of cells. A, observed number of p14JM or HT-1080 colonies (14 days in ϩ/Ϫ doxycycline- containing medium) after staining with crystal violet solution. The results represent averages of three separate experiments, and the error bars represent the respective SD. B, Western blot using respective antibodies of whole-cell lysate from p14JM cells induced to express p14ARF for various times. C, trypan blue viability assay of p14JM cells or HT-1080 parental cells in the presence or absence of 1 ␮g/ml doxycycline. The average of three separate experiments and their respective SD are shown. D, fluorescence-activated cell sorter analysis after propidium iodide staining of cell cycle stage of p14JM and HT-1080 cells at 48 h post- induction. The change in percentage in the amount of induced cells compared with the uninduced ones for each state of the cell cycle is shown in the bar graph (Change in % ϭ [(% induced Ϫ % uninduced)/% uninduced] ϫ 100). The results are the average of three experiments. Error bars represent the SD.

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Fig. 3. p14ARF inhibition of DHFR protein by proteosomal degradation. A, 1, Western blot with each respective antibody of p14JM lysates of cells that were allowed to reach confluency, detached, replated, and incubated with (ϩDOX) or without (ϪDOX)1␮g/ml doxycycline (DOX) for various amounts of time. 2, as a control, HT-1080 parental cells treated (ϩDOX) or not treated (ϪDOX) with 1 ␮g/ml doxycycline for 72 h were also subjected to Western analysis using DHFR and tubulin antibodies as described above. 3, the p14JM whole-cell lysates from cells incubated with (ϩDOX) or without (ϪDOX)1␮g/ml doxycycline for 72 h for the TS and TK Western blots were electrophoresed on a separate polyacrylamide gel and blotted simultaneously with the TK and TS primary antibody. B, relative DHFR mRNA levels using TaqMan quantitative reverse transcription-PCR analysis (probe and primers specific for DHFR cDNA and normalized by actin message levels) of p14JM cells that were allowed to reach confluency, replated, incubated in the presence (ϩDOX) or in the absence (ϪDOX)of1␮g/ml doxycycline, and allowed to grow for various amount of time. Average values for three experiments of DHFR/ACTIN ratios are shown with their respective SD. C, p14JM cells incubated with (ϩDOX) or without (ϪDOX)1␮g/ml doxycycline for 24 h were treated with 10 ␮g/ml cycloheximide. Right panel, at the indicated times, cells were lysed and Western blot was performed using DHFR and ␣-tubulin antibodies. Intensity of protein bands at different time points after cycloheximide addition relative to band intensity at the 0-h time point was plotted. Left panel, graph represents average and SDs of at least three separate experiments. D, cells were incubated with (ϩ) or without (Ϫ)1␮g/ml doxycycline for 48 h, and 100 nM MG-132 or 0.1% DMSO was added for 12 h. The cells were then lysed, and Western blots were performed using the antibodies as described above.

p14ARF Expression Confers Selective Resistance to the Antifo- methotrexate, trimetrexate, and raltitrexed. Because FBS contains late DHFR Inhibitors Methotrexate, Trimetrexate, and the Anti- high levels of thymidine (29–31), the possibility that increased thy- folate Raltitrexed, a TS Inhibitor. The role of p14ARF in p14JM midine salvage explained the resistance to the antifolates was exam- sensitivity to chemotherapeutics was examined by inducing cells to ined. Cytotoxicity studies were conducted in the presence of 10% FBS express p14ARF and adding various concentrations of methotrexate, depleted of thymidine, by pretreating serum with TP (32). In the trimetrexate, and raltitrexed to separate cells in different plates. After presence of thymidine-depleted FBS, the resistance conferred by continuous doxycycline induction and drug exposure for 4 days, cell p14ARF expression to methotrexate, trimetrexate, and raltitrexed was viability was assessed by the XTT colorimetric assay (23). Expression completely abolished (Fig. 4C). Although depletion of thymidine did of p14ARF conferred resistance to all three drugs (Fig. 4A). In partic- not affect the sensitivity profile of doxorubicin or cytarabine (data not ular, compared with uninduced cells, the expression of p14ARF was shown), to rule out an effect of doxycycline on antifolate sensitivity, associated with marked resistance to methotrexate and raltitrexed, parental HT-1080 cells were separately treated with methotrexate, in even at very high concentrations of these drugs. In contrast, when the presence or absence of 1 ␮g/ml doxycycline; no difference in cytotoxicity experiments were carried out using hydroxyurea, doxo- cytotoxicity was observed (data not shown). Because human serum rubicin, or cytarabine, also S-phase inhibitors with different mecha- contains less thymidine than FBS or mouse serum (29–31), we nisms of action, there were no differences in cytotoxicity observed in repeated the methotrexate cytotoxicity experiment in p14JM cells the absence or presence of p14ARF expression (Fig. 4B). Therefore, uninduced and induced to express p14ARF in 10% FBS thymidine- ARF Ϫ7 p14 expression selectively altered resistance to the antifolates depleted medium, in which physiological levels for humans (10 M) 4341

Fig. 4. Expression of p14ARF is associated with resistance to antifolates. A, p14JM cells uninduced (ϪDOX) or induced (ϩDOX) to express p14ARF were incubated 24 h later with methotrexate, raltitrexed, or trimetrexate added at different concentrations; and 96 h later, cells were assayed for viability using the XTT method (see “Materials and Methods”). Concentration of drug versus percent survival was plotted. Error bars represent SD of three separate experiments. B, the same experiment as above was repeated using cytarabine, hydroxyurea, and doxorubicin. C, cells were plated and treated with methotrexate, trimetrexate, or raltitrexed, as above, in medium containing FBS depleted of thymidine by TP treatment. D, cells were Ϫ7 plated and treated with methotrexate, as above, in medium containing 10 M thymidine.

were restored, and a difference in sensitivity to methotrexate was still shown). Decreased polyglutamylation and retention as a mechanism observed (Fig. 4D). of resistance was ruled out by the fact that these cells were incubated p14ARF Confers Resistance to Antifolates by Altering the Bal- continuously in the presence of drugs and therefore polyglutamylation ance of Thymidine Supply. The fact that resistance to antifolates would not affect cytotoxicity, due to the continuous availability of the was abolished upon depleting the serum of thymidine indicated that extracellular level of the drug (34) and by the fact that trimetrexate expression of p14ARF may increase thymidine salvage by either in- does not undergo polyglutamylation. Paradoxically, resistance of creasing thymidine uptake and/or increasing TK activity. Cells were p14JM cells to antifolates occurred in a situation in which DHFR collected 72 h post induction with 1 ␮g/ml doxycycline and lysed, and protein levels were not increased but rather were drastically de- TK activity was measured. There was no difference in TK activity creased. An explanation for this finding lies in the understanding of between the cells uninduced and induced to express p14ARF (Fig. 5A). the balance between de novo thymidylate synthesis and the salvage In another experiment, radiolabeled thymidine-treated cells were pathway for thymidylate. DHFR is an important enzyme in the de also collected at 72 h and analyzed for thymidine mono-, di-, and novo pathway in that it provides the necessary reduced folate cofactor, triphosphates by high performance liquid chromatography. There 5,10 methylene tetrahydrofolate, for the conversion of dUMP to were no differences found in the thymidine metabolite peak profiles dTMP via reduction of dihydrofolate to tetrahydrofolate and subse- between doxycycline-treated and untreated cells (Fig. 5B). Thus nei- quent conversion to 5,10-methylene tetrahydrofolate via serine hy- ther TK activity nor thymidine uptake or anabolism explained the droxy methyl transferase. However, dTMP can also be generated via thymidine reversal of antifolate resistance. Other mechanisms of thymidine in the medium. As a consequence of the marked decrease known antifolate resistance include decreased influx of drug, de- in DHFR activity, p14JM cells that are induced to express p14ARF creased polyglutamate formation, and increased DHFR expression would be depleted of 5,10-methylene tetrahydrofolate. This in turn (33). The fact that resistance is seen also with trimetrexate, which would cause a decrease in TS activity and therefore a reduction in the does not use the same active transport mechanism as methotrexate, amount of de novo dTMP being produced. Fig. 5C shows that indeed, renders impaired uptake an unlikely mechanism of resistance. Con- whole-cell thymidylate synthesis activity is decreased. Because the firming this observation, uptake of tritiated methotrexate was similar supply of thymidine is not reduced, as seen by the unaltered TK in the p14ARF-expressing and non-p14ARF-expressing cells (data not activity and thymidine pools (Fig. 5, A and B) in the p14ARF-express- 4342

Fig. 5. p14ARF alters the balance of thymidine supply. A, p14JM cells were incubated with (ϩDOX) or without (ϪDOX)1␮g/ml doxycycline (DOX) for 72 h, lysed, and protein extracted for TK activity assay (see “Materials and Methods”). TK activity, as measured by radioactivity per 4 ␮gof whole-cell extract every 20 min for 1 h, was plotted against time. The results are the average of three experiments, and the error bars represent the respective SD. B, p14JM cells were plated in the presence (ϩDOX) or absence (ϪDOX)of1␮g/ml doxycycline. After 72 h, cells were collected, and extracts were analyzed by high performance liquid chromatography, as described in “Materials and Methods.” The collected samples were then counted for radioactivity and converted to cpm/ 3.0 ϫ 106 cells. The average and SD of three experiments are shown. C, p14JM cells were incubated with (ϩDOX) or without (ϪDOX)1␮g/ml doxycycline for 48 h and then incubated with [5-3H]deoxyuridine for 0, 15, 30, and 45 min, and in situ thymidylate synthesis activity was calculated as described in “Materials and Methods.” In situ thymidylate synthesis activity is expressed as cpm/min/107 cells. The average and SD of four experiments are shown. D, p14JM cells (1,000) were plated in each well of a six-well plate with (ϩDOX) or without (ϪDOX)1␮g/ml doxycycline, with or without TP-treated FBS. After 14 days, the plates were stained with crystal violet solution and colonies counted. The average of three experiments and their respective SDs are shown.

ing cells, the balance of thymidine supply in the p14ARF-expressing The decrease in growth rate observed upon induction of p14ARF cells shifts to thymidine salvage. These cells will therefore be more demonstrates that p14ARF is able to inhibit growth, independent of its reliant on extracellular thymidine for growth, as seen by their greater ability to increase p53 protein levels. The decrease in cell growth may decrease in colony formation when grown long-term in thymidine be explained by a delay in S-phase entry. It has been shown that ARF ARF deplete medium, compared with p14JM cells that are not expressing p19 , the mouse homologue of p14 , can arrest cells at G1 ARF p14 (7-fold decrease versus 2-fold decrease for cells not express- independently of p53, but the arrest occurs in the absence of MDM2 ARF ing p14 ; Fig. 5D) and therefore are less sensitive to drugs that (10). Because HT-1080 cells express high levels of MDM2 (20), this inhibit the de novo pathway of dTMP synthesis. may explain the lack of a more pronounced G1-S cell cycle arrest after p14ARF induction. However, the decrease in cellular proliferation rate DISCUSSION was delayed until 48 h after induction of p14ARF, at which time a decrease in DHFR protein levels and an increase in the amount of The p53-dependent effects of ARF have been widely studied (35, cells in G were seen, all consistent with a more delayed p53- 36). However, p53-independent effects of ARF have just recently 1 independent response to ARF expression (10). TK and TS levels were been described (10, 13–15). An important question left unanswered not altered after p14ARF expression. Therefore, the decrease in cell has been the effect of p14ARF on chemosensitivity. Although a few growth after restoration of p14ARF expression is not due to a general studies have attempted to answer this question (7, 16, 17, 37), in most decrease in expression of cell cycle genes but rather to a more targeted cases, tumors with an intact p53 response pathway have been used as ARF the model. In this study, we used a human HT-1080 fibrosarcoma cell event. Induction of p14 is associated with a decrease in DHFR line, which is null for p14ARF and harbors a nonfunctional p53 protein levels in p14JM cells, and therefore a slowdown in prolifer- pathway (18–20). Although p53 is expressed in this cell line, it is ation may be due in part to the lack of generation of reduced folate pools, i.e., 5,10-methylene tetrahydrofolate, needed in for conversion nonfunctional, as demonstrated by the lack of a G1-S block after treatment with ␥ radiation or N-(phosphonacetyl)-L-aspartic acid (18). of dUMP to dTMP by TS. A decrease in whole-cell TS activity was The reasons for its lack of function are unclear. MDM2 is also observed correlating with decreased DHFR activity. ARF ARF expressed in HT-1080 cells, and the expression of relatively high p14 and p19 are able to bind to E2F-1 and alter its tran- levels of p53 levels suggests that perhaps MDM2 in this cell line is scriptional activity and stability (13–15). Although neither a decrease mutated or nonfunctional. p14ARF protein expression was induced in E2F-1 protein levels, nor a significant change in DHFR mRNA using a doxycycline-inducible system, thus eliminating clonal varia- levels was found after p14ARF induction in p14JM cells, inhibition of tion. Upon induction of p14ARF, we observed a decreased growth rate, E2F-1 transcription for other E2F-1 targets cannot be ruled out. We decreased colony formation, selective resistance to antifolates, and also cannot rule out the involvement of other downstream targets of increased dependence of the p14JM p14ARF-induced cells on thymi- p14ARF that have not yet been discovered. dine salvage, in the face of a marked decrease in DHFR protein levels. An explanation for the decrease in DHFR levels after p14ARF 4343

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2004 American Association for Cancer Research. p14ARF EXPRESSION AND DIHYDROFOLATE REDUCTASE restoration is a decrease in DHFR stability, reversed in the presence of with the TS polyclonal antibody, and Dr. W. Tong (Memorial Sloan Kettering the proteasome inhibitor MG-132. Therefore, p14ARF expression may Cancer Center, New York, NY) for helping with high performance liquid augment proteasomal degradation of DHFR. Although Martelli et al. chromatography analysis of thymidine metabolites. (14) has found that p19ARF is capable of targeting E2Fs for proteasomal degradation through direct binding of ARF to E2Fs, we were not able to show p14ARF binding to DHFR. Although there are studies REFERENCES that have provided information on DHFR stability in other cell lines 1. Serrano M, Hannon GJ, Beach D. 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Pellegrino G. Magro, Angelo J. Russo, Wei-Wei Li, et al.

Cancer Res 2004;64:4338-4345.

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