Mitotic Arrest, Apoptosis, and Sensitization to Chemotherapy of Melanomas by Methionine Deprivation Stress
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Mitotic Arrest, Apoptosis, and Sensitization to Chemotherapy of Melanomas by Methionine Deprivation Stress Demetrius M. Kokkinakis,1,4 Anthony G. Brickner,2,3,4 John M. Kirkwood,2,4 XiaoYan Liu,4 Jason E. Goldwasser,4 Anastasiya Kastrama,4 Cindy Sander,2 Dora Bocangel,5 and Sunil Chada5 Departments of 1Pathology, 2Medicine, and 3Immunology and 4Cancer Institute, University of Pittsburgh, Pittsburgh, Pennsylvania and 5Introgen Therapeutics, Inc., Houston, Texas Abstract it is likely that such control is exerted via the Methionine deprivation stress (MDS) eliminates mitotic induction/up-regulation of tumor suppressors/growth activity in melanoma cells regardless of stage, grade, inhibitor genes, such as TGFB, PTEN, GAS1, EGR3, or TP53 status, whereas it has a negligible effect on BTG3, MDA7, and the proteoglycans (LUM, BGN, and normal skin fibroblasts. In most cases, apoptosis DCN), as well as the down-regulation/loss of function of accounts for the elimination of up to 90% of tumor prosurvival genes, such as NFkB, MYC, and ERBB2. cells from the culture within 72 hours after MDS, leaving Although MDS targets several common genes in a scattered population of multinucleated resistant tumors, mutational variability among melanomas cells. Loss of mitosis in tumor cells is associated may decide which metabolic and signal transduction with marked reduction of cyclin-dependent kinase pathways will be activated or shutdown. (Mol Cancer Res (CDK) 1 transcription and/or loss of its active form 2006;4(8):575–89) (CDK1-P-Thr161), which is coincident with up-regulation of CDKN1A, CDKN1B, and CDKN1C (p21, p27, and p57). Expression of the proapoptotic LITAF, IFNGR, EREG, Introduction TNFSF/TNFRSF10 and TNFRSF12, FAS, and RNASEL Melanoma is a prevalent disease among fair-skinned is primarily up-regulated/induced in cells destined to Caucasians and has been linked to sunlight exposure (1). High undergo apoptosis. Loss of Aurora kinase B and BIRC5, constitutive expression of extracellular signal-regulated kinase which are required for histone H3 phosphorylation, 1 and 2 and the amplification of cyclin D1 (CCND1) in most is associated with the accumulation of surviving melanomas has been linked with the activation of the RAS- multinucleated cells. Nevertheless, noncycling survivors RAF mitogen-activated protein kinase signaling pathway (2-4). of MDS are sensitized to temozolomide, carmustin, Mutations in RAS and BRAF, which occur in 15% and 66% of and cisplatin to a much greater extent than normal skin melanomas, respectively, could account for enhanced prolifer- fibroblasts possibly because of the suppression of ative activity (5-8). In addition, loss of PTEN’s regulatory MGMT/TOP1/POLB, MGMT/RAD52/RAD54, and control of AKT and epigenetic silencing of APAF1 confer cMET/RADD52, respectively. Sensitivity to these and a high rate of proliferation (9-11). In metastatic melanoma, additional genotoxic agents and radiation may also be resistance to treatment could be linked to defective apoptosis acquired due to loss of cMET/OGG1, reduced due to high expression of the antiapoptotic BCL-2 (12-14). glutathione reductase levels, and a G2-phase block that The majority of melanomas (>80%) are curatively treated when is a crucial step in the damage response associated with surgery is applied early before metastatic disease. However, enhancement of drug toxicity. Although the genes advanced metastatic melanoma is incurable (15, 16). The controlling mitotic arrest and/or apoptosis in response methylating agents dacarbazine given i.v. and its oral analogue to low extracellular methionine levels are unknown, temozolomide are the most frequently used drugs with a response of only 15% (17-21). Nitrosoureas (22, 23), platinum analogues (24, 25), Vinca alkaloids (26), and taxanes (27) have shown limited promise. Attempts to combine chemotherapy Received 11/15/05; revised 5/9/06; accepted 5/15/06. Grant support: NIH grant RO1-CA78561 (D.M. Kokkinakis), Grant Channel with surgery have been fruitless in significantly extending Memorial Fund (J.M. Kirkwood), and Elsa U. Pardee Foundation (A.G. survival (28). The poor response to chemotherapy and the Brickner). steady increase of the incidence of melanoma worldwide show The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in the need for the development of new therapeutic approaches. accordance with 18 U.S.C. Section 1734 solely to indicate this fact. One of the best understood reasons for the failure of Note: Supplementary data for this article are available at Molecular Cancer methylating agents and nitrosoureas to kill tumor cells is the Research Online (http://mcr.aacrjournals.org/). 6 Requests for reprints: Demetrius M. Kokkinakis, Hillman Cancer Center presence of high levels of O -methylguanine-DNA methyl- Research Pavillion, Room G12.e, 5117 Centre Avenue, Pittsburgh, PA 15213. transferase (MGMT), a ubiquitously expressed DNA repair Phone: 412-585-0833; Fax: 724-625-3036. E-mail: [email protected] Copyright D 2006 American Association for Cancer Research. protein that is vital for preventing mutations (29-31). MGMT- doi:10.1158/1541-7786.MCR-05-0240 induced resistance can be overcome by MGMT inhibition with Mol Cancer Res 2006;4(8). August 2006 575 Downloaded from mcr.aacrjournals.org on October 2, 2021. © 2006 American Association for Cancer Research. 576 Kokkinakis et al. a pseudosubstrate, such as O6-benzylguanine (32). In the clinic, efficacy of chemotherapy by reducing the expression of genes MGMT is inversely correlated with improved response to involved in the resistance to antitumor agents and radiation. carmustine [1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU)] Most importantly, MDS has a pleiotropic action and kills or and overall survival in patients with gliomas, but the rela- sensitizes tumor cells by using a variety of mechanisms, thus tionship is less clear in patients with melanoma (33, 34). An overriding variable genetic and epigenetic restrictions that are association between the expression of MGMT and the response related to tumor heterogeneity. to dacarbazine has been suggested by one study (33), but this has not been confirmed in patients treated with temozo- lomide (22). The extent of MGMT expression in melanoma Results remains uncertain and the methodologies used to determine Physiologic Changes of Melanoma Cells in Response to such expression have yielded contradictory results. In devel- MDS oping treatments against metastatic melanomas, one must con- MDS retarded the growth of eight melanoma cell lines in sider the resistance of these tumors beyond the involvement of culture. Five of eight cell lines tested (M591, M255, M108, MGMT. M103, and MEWO) showed accumulation of large cells with In this communication, we examine the effect of methionine multiple micronuclei and decondensed chromatin 96 hours after deprivation stress (MDS) on the abrogation of prosurvival MDS (Figs. 1 and 2). These cells survived a burst of cellular pathways and in the induction of cell cycle arrest and apoptosis death that occurred between 48 and 72 hours after replacing in several melanoma cell lines. MDS has been shown to inhibit methionine with homocysteine in the medium (Table 1). AKT, RAS, and nuclear factor-nB(NF-nB) activities in Multinucleated cells persisted for several days in culture; medulloblastomas, glioblastoma multiforme, and pancreatic however, they resumed mitotic activity and proliferated when adenocarcinomas (35, 36) and to greatly enhance the response methionine was replenished (data not shown). In three of these tumors to chemotherapy (37). Resistance of tumors to additional cell lines (M105, WM35, and A375), there was no alkylating chemotherapeutic agents may be eliminated by MDS evidence for progression of the cycle and mitotic activity for at due to the down-regulation of MGMT (38) and the induction of least 10 days following methionine deprivation; however, there tumor suppressor genes, such as MDA7, IFNG, and/or trans- was no accumulation of multinucleated cells. In these tumors, forming growth factor-b (TGFB), which control the expression cellular death was protracted and continued for several days. In of p21 independently of p53 status and may exert tumor growth contrast to tumor cells, the growth of normal human CCD- inhibition in melanomas by the up-regulation and nuclear 187sk and horse fibroblasts E.DERM under MDS was translocation of this cyclin-dependent kinase (CDK) inhibitor moderately affected with no significant evidence of cell death. (35, 36). In addition to p21, MDS can also enhance the To determine the cell cycle distribution during a MDS regimen response of tumors to alkylating agents by a synergistic and derive information regarding possible cell cycle check induction of growth arrest in response to DNA damage points, melanoma and normal skin fibroblast cells cultured in (GADD) genes, which act downstream of p53 and interact either methionine or homocysteine medium for various time with p21 and CDK1, thus regulating the cell cycle and cell intervals were stained with propidium iodide and analyzed by proliferation in a p53-dependent or p53-independent manner flow cytometry. Cells with a multiple to diploid DNA content (35, 36). Furthermore, MDS has the potential to enhance the were exclusively present at 96 hours after MDS in cultures of FIGURE 1. Effect of MDS on mel- anoma cell lines M591 (mutant TP53, infiltrated lymph node), M255 (WT TP53, infiltrated lymph node), M105 (mutant TP53), M103 (mutant TP53, infiltrated lymph node), M108 (WT TP53), A375 (WT TP53), skin mela- nomas WM35(WT TP53)and MEWO (mutant TP53), human normal skin fibroblast CCD-187sk, and horse nor- mal dermis fibroblast E.DERM. Cells were cultured in methionine-efficient medium containing 10% dialyzed se- rum until they were confluent. At that time, methionine was withdrawn and replaced with homocysteine thiolac- tone. At day 4, there was accumula- tion of multinucleated cells in M591, M255, M108, M103, and MEWO. Excessive cell death in the above lines was evident at day 2 but continued to occur in M105, WM35, and A375 for several days.