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Modulation of Cell Cycle and Gene 1338 Modulation of cell cycle and gene expression in pancreatic tumor cell lines by methionine deprivation (methionine stress): implications to the therapy of pancreatic adenocarcinoma Demetrius M. Kokkinakis, XiaoYan Liu, regulation and LMNB1, AREG, RhoB, CCNG, TYMS, F3, and Russell D. Neuner and MGMT down-regulation suggest that methionine stress sensitizes the tumor cells to DNA-alkylating drugs, Department of Pathology and the Cancer Institute, University of 5-fluorouracil, and radiation. Increased sensitivity of Pittsburgh, Pittsburgh, Pennsylvania pancreatic tumor cell lines to temozolomide is shown under methionine stress conditions and is attributed in part 6 Abstract to diminished O -methylguanine-DNA methyltransferase and possibly to inhibition of the cell cycle progression. [Mol The effect of methionine deprivation (methionine stress) on Cancer Ther 2005;4(9):1338–48] the proliferation, survival, resistance to chemotherapy, and regulation of gene and protein expression in pancreatic tumor lines is examined. Methionine stress prevents Introduction successful mitosis and promotes cell cycle arrest and Pancreatic adenocarcinoma is the fourth leading cause of accumulation of cells with multiple micronuclei with death in the United States, with an estimated incident of decondensed chromatin. Inhibition of mitosis correlates 30,000 victims annually. Most patients who are diagnosed with CDK1 down-regulation and/or inhibition of its function with this tumor die within a year and the 5-year survival is 15 161 by Tyr phosphorylation or Thr dephosphorylation. <5%. Only a very few tumors are as aggressive as pancreatic Inhibition of cell cycle progression correlates with loss of cancer, and unlike many other cancers, early discovery does hyperphosphorylated Rb and up-regulation of p21 via p53 not always lead to improved survival (1). Frequent B B and/or transforming growth factor- (TGF- ) activation mutations in p53 (75%), p16/RB1 (90%), BRCA2 (50%), depending on p53 status. Although methionine stress– and K-ras (90%; ref. 2) are likely to yield a tumor that is induced toxicity is not solely dependent on p53, the gain in resistant to a variety of conventional chemotherapies and to p21 and loss in CDK1 transcription are more enhanced in radiation (3, 4). Interestingly, pancreatic tumors are sen- B wild-type p53 tumors. Up-regulation of SMAD7, a TGF- sitive to the restoration of p53 (5) but not to restoration of signaling inhibitor, suggests that SMAD7 does not restrict p16/RB1 or silencing of K-ras (6, 7), which only result in B the TGF- -mediated induction of p21, although it may suppression of growth without significant effect on apopto- prevent up-regulation of p27. cDNA oligoarray analysis sis. Clinically, the coexpression of RB1 and p53 pathway- indicated a pleiotropic response to methionine stress. Cell related proteins, such as p21WAF1/CIP1 and BAX, render a cycle and mitotic arrest is in agreement with up-regulation significantly higher survival rate in patients with ductal A B ; of NF2, ETS2, CLU, GADD45 , GADD45 , and GADD45 carcinoma of the pancreas (8). Research in this laboratory (9) and down-regulation of AURKB, TOP2A, CCNA, CCNB, has identified that the two prominent factors in the resis- PRC1, BUB1, NuSAP, IFI16, and BRCA1. Down-regulation tance of pancreatic neoplasms to alkylating chemothera- of AREG, AGTR1, M-CSF, and EGF, IGF, and VEGF peutic agents are (a) the high level of O6-methylguanine- receptors and up-regulation of GNA11 and IGFBP4 signify DNA methyltransferase (MGMT) in the tumor and (b) the loss of growth factor support. PIN1, FEN1, and cABL up- widespread mutation of p53 that prevents p53-mediated cell cycle arrest in response to DNA damage (10). In wild- type (wt) p53 tumors, prolonged suppression of MGMT with O6-benzylguanine and concomitant activation of p53/ Received 5/4/05; revised 6/17/05; accepted 7/15/05. p21 pathway to induce a G1-S arrest results not only in The costs of publication of this article were defrayed in part by the elevated killing due to an increase of BAX to BCL2 ratio but payment of page charges. This article must therefore be hereby marked also in an enhancement of the efficacy of mismatch repair advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. via the up-regulation of mismatch repair components. The Note: R.D. Neuner is currently at the Department of Molecular and Cellular above observations set the stage for an effective treatment of Biology, University of Massachusetts, Amherst, MA 01003. pancreatic malignancies having high levels of MGMT and Requests for reprints: Demetrius M. Kokkinakis, Division of Basic a functional p53/p21 system. However, most pancreatic Research, Hillman Cancer Center, University of Pittsburgh Cancer tumors are p53 dysfunctional and do not arrest in G1-S in Institute, UPCI Research Pavillion, Room G12.e, 5117 Centre Avenue, Pittsburgh, PA 15213-1863. Phone: 412-623-1110; Fax: 412-623-4747. response to DNA damage. Efforts to restore p53 in these E-mail: [email protected] tumors in a clinical setting are cumbersome and not likely to Copyright C 2005 American Association for Cancer Research. become available in the near future. Compounds that doi:10.1158/1535-7163.MCT-05-0141 restore the binding affinity of mutant p53 to DNA, such as Mol Cancer Ther 2005;4(9). September 2005 Downloaded from mct.aacrjournals.org on September 29, 2021. © 2005 American Association for Cancer Research. Molecular Cancer Therapeutics 1339 CP-31398, are promising but not yet suitable for clinical Cultures and Methionine Stress Conditions applications (11, 12). In addition, prolonged inactivation of All cell lines were adapted to DMEM (BioWhittaker, MGMT with currently available MGMT inactivators, such Walkersville, MD) with 10% fetal bovine serum (Media 6 j as O -benzylguanine, is problematic due to nonspecificity Tech, Inc., Herndon, VA) in 6.5% CO2 atmosphere at 37 C resulting in enhanced toxicity and mutagenesis in normal until confluent. Cultures were subsequently adapted to tissue. An alternative approach to induce cell cycle and DMEM-A (Media Tech) supplemented with an additional mitotic arrest and at the same time eliminate MGMT activity 584 mg/L L-glutamine (Sigma, St. Louis, MO) to a final specifically in the tumor can be achieved by depleting concentration of 8 mmol/L, 10 mL/L penicillin/strepto- plasma methionine (13). Application of methionine stress mycin (Sigma), and 100 mL/L dialyzed fetal bovine serum in vivo has shown that it has no deleterious effects on nor- (Life Technologies, Grand Island, NY). Methionine stress mal tissue even under moderate levels of genotoxic stress was induced by transferring cell cultures to methionine- (14). Furthermore, methionine stress has a broad antitumor free DMEM-B (Media Tech) supplemented with 27 mg/L effect that involves the induction/activation of tumor homocysteine thiolactone hydrochloride (Sigma), 62.5 mg/L suppressor genes, such as transforming growth factor-h L-cysteine dihydrochloride (Sigma), and L-glutamine to a (TGF-h), MDA-7, Rb, p21, and p27, to silence prosurvival final concentration of 8 mmol/L, penicillin/streptomycin, pathways, such as phosphatidylinositol 3-kinase (PI3K), and dialyzed fetal bovine serum (full medium B). Medium RAS, nuclear factor-nB, and CDK1, and at the same time was replaced in all cultures every 36 hours. The conditions induce proapoptotic pathways propagated by activation of applied here to induce methionine stress are different than tumor necrosis factor–related apoptosis-inducing ligand those employed previously (13) due to the employment of and death receptor 6 (15). Methionine stress could synergize commercial culture medium and the omission of high with alkylating and perhaps other chemotherapeutic agents concentrations of cobalamin and folic acid, which were in all pancreatic tumors regardless of p53 status by reducing shown to be nonessential for overcoming methionine stress their high MGMT levels and by causing cell cycle arrest via in tumors. the up-regulation of p21 and GADD45, activation of Rb, and MGMT Assay down-regulation of CDK1. In terms of cell cycle kinetics, The biochemical assay for MGMT activity was used to methionine stress can restore cell cycle/mitotic arrest in determine levels of this protein in cells cultured under mutant p53 tumors and prevent immediate transition from control or methionine stress conditions (13). G1 to S and G2, thus enhancing BAX and mismatch repair– Synergy between Methionine Stress and Temo- mediated death. Furthermore, the mutational variability of zolomide pancreatic tumors that is believed to contribute to their Pancreatic tumor cell lines were cultured in medium A as resistance to single therapeutic treatments will be overcome described above and allowed to reach 50% confluence. The by the pleiotropic action of methionine stress. An important following experiments were done to determine the efficacy consideration in choosing methionine stress is that it has of temozolomide. Control: Cells were treated with 0, 50, 100, been tested in primates and the results are supportive for its 200, 500, and 1,000 Amol/L temozolomide for 30 minutes in timely use in clinical trials (16). medium A in the absence of serum and subsequently transferred to full medium A for 96 hours. O6-benzylgua- Materials and Methods nine/temozolomide: Cells in medium A were treated with Cell Lines and Culture O6-benzylguanine (30 Amol/L) for 24 hours and then treated The pancreatic adenocarcinoma cell lines CAPAN1,
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