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• W2023204266 abstract "Synucleins are a family of highly conserved small proteins predominantly expressed in neurons. Recently we and others have found that γ-synuclein is dramatically up-regulated in the vast majority of late-stage breast and ovarian cancers and that γ-synuclein over-expression can enhance tumorigenicity. In the current study, we have found that γ-synuclein is associated with two major mitogen-activated kinases (MAPKs),i.e. extracellular signal-regulated protein kinases (ERK1/2) and c-JunN-terminal kinase 1 (JNK1), and have shown that over-expression of γ-synuclein leads to constitutive activation of ERK1/2 and down-regulation of JNK1 in response to a host of environmental stress signals, including UV, arsenate, and heat shock. We also tested the effects of γ-synuclein on apoptosis and activation of JNK and ERK in response to several chemotherapy drugs. We have found that γ-synuclein-expressing cells are significantly more resistant to the chemotherapeutic drugs paclitaxel and vinblastine as compared with the parental cells. The resistance to paclitaxel can be partially obliterated when ERK activity is inhibited using a MEK1/2 inhibitor. Activation of JNK and its downstream caspase-3 by paclitaxel or vinblastine is significantly down-regulated in γ-synuclein-expressing cells, indicating that the paclitaxel- or vinblastine-activated apoptosis pathway is blocked by γ-synuclein. In contrast to paclitaxel and vinblastine, etoposide does not activate JNK, and γ-synuclein over-expression has no apparent effect on this drug-induced apoptosis. Taken together, our data indicate that oncogenic activation of γ-synuclein contributes to the development of breast and ovarian cancer by promoting tumor cell survival under adverse conditions and by providing resistance to certain chemotherapeutic drugs. Synucleins are a family of highly conserved small proteins predominantly expressed in neurons. Recently we and others have found that γ-synuclein is dramatically up-regulated in the vast majority of late-stage breast and ovarian cancers and that γ-synuclein over-expression can enhance tumorigenicity. In the current study, we have found that γ-synuclein is associated with two major mitogen-activated kinases (MAPKs),i.e. extracellular signal-regulated protein kinases (ERK1/2) and c-JunN-terminal kinase 1 (JNK1), and have shown that over-expression of γ-synuclein leads to constitutive activation of ERK1/2 and down-regulation of JNK1 in response to a host of environmental stress signals, including UV, arsenate, and heat shock. We also tested the effects of γ-synuclein on apoptosis and activation of JNK and ERK in response to several chemotherapy drugs. We have found that γ-synuclein-expressing cells are significantly more resistant to the chemotherapeutic drugs paclitaxel and vinblastine as compared with the parental cells. The resistance to paclitaxel can be partially obliterated when ERK activity is inhibited using a MEK1/2 inhibitor. Activation of JNK and its downstream caspase-3 by paclitaxel or vinblastine is significantly down-regulated in γ-synuclein-expressing cells, indicating that the paclitaxel- or vinblastine-activated apoptosis pathway is blocked by γ-synuclein. In contrast to paclitaxel and vinblastine, etoposide does not activate JNK, and γ-synuclein over-expression has no apparent effect on this drug-induced apoptosis. Taken together, our data indicate that oncogenic activation of γ-synuclein contributes to the development of breast and ovarian cancer by promoting tumor cell survival under adverse conditions and by providing resistance to certain chemotherapeutic drugs. Breast carcinoma is the second leading cause of cancer-related deaths in women of the Western world. In the United States alone over 180,000 new cases are diagnosed annually and more than 40,000 women die from this disease each year (1Landis S.H. Murray T. Bolden S. Wingo P.A. CA Cancer J. Clin. 1999; 49: 8-31Crossref PubMed Scopus (3135) Google Scholar). Epithelial ovarian cancer continues to be the leading cause of death from gynecologic malignancies in the United States (1Landis S.H. Murray T. Bolden S. Wingo P.A. CA Cancer J. Clin. 1999; 49: 8-31Crossref PubMed Scopus (3135) Google Scholar, 2Greenlee R.T. Murray T. Bolden S. Wingo P.A. CA Cancer J. Clin. 2000; 50: 7-33Crossref PubMed Scopus (3980) Google Scholar). One woman in 70 in the U.S. will develop ovarian cancer in her lifetime, and one woman in 100 will die of this disease. Breast and ovarian cancer etiology are multifactorial, involving environmental factors, hormones, genetic susceptibility, and genetic changes during progression. Both cancers are a heterogeneous group of tumors with no unifying molecular alteration yet identified. A certain number of breast and ovarian cancer cases (∼5–10%) are attributed to inherited mutations in highly penetrant breast cancer susceptibility genes, such as BRCA1 and BRCA2(reviewed in Ref. 3Bove B. Dunbrack R. Godwin A.K. Pasqualini J. Breast Cancer: Prognosis, Treatment and Prevention. Marcel Dekker Inc., New York2002Google Scholar). However, the majority of the tumors occur in women with little or no family history, and the molecular basis of these sporadic cancers is still poorly defined. In an effort to identify other genes involved in the development and/or progression of breast and ovarian cancer, we and others used differential gene expression approaches and have found that γ-synuclein, initially termed breast cancer-specific gene 1 (BCSG1), 1The abbreviations used are: BCSG1, breast cancer-specific gene 1; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated protein kinase; JNK, c-JUN N-terminal kinase; MEK, MAPK kinase; LBs, Lewy bodies; AD, Alzheimer's disease; pNA, p-nitroaniline; PD, Parkinson's disease; PKC, protein kinase C; PARP, poly(ADP-ribose) polymerase; CMV, cytomegalovirus; FBS, fetal bovine serum; PBS, phosphate-buffered saline; SAPK, stress-activated protein kinase; GST, glutathioneS-transferase. is up-regulated in the majority of late-stage breast (4Bruening W. Giasson B.I. Klein-Szanto A.J. Lee V.M. Trojanowski J.Q. Godwin A.K. Cancer. 2000; 88: 2154-2163Crossref PubMed Scopus (159) Google Scholar, 5Ji H. Liu Y.E. Jia T. Wang M. Liu J. Xiao G. Joseph B.K. Rosen C. Shi Y.E. Cancer Res. 1997; 57: 759-764PubMed Google Scholar) and ovarian cancer (4Bruening W. Giasson B.I. Klein-Szanto A.J. Lee V.M. Trojanowski J.Q. Godwin A.K. Cancer. 2000; 88: 2154-2163Crossref PubMed Scopus (159) Google Scholar). 2Z.-Z. Pan and A. K. Godwin, unpublished data. In addition, we showed that there was a correlation between γ-synuclein expression in breast ductal carcinomas and the staging of the cancer suggesting that γ-synuclein may be a potential marker for both late stage breast and ovarian cancer. Additional studies have revealed that γ-synuclein over-expression leads to increased invasiveness of breast tumor cells (6Jia T. Liu Y.E. Liu J. Shi Y.E. Cancer Res. 1999; 59: 742-747PubMed Google Scholar) and stimulated cell proliferation (7Liu J. Spence M.J. Zhang Y.L. Jiang Y. Liu Y.E. Shi Y.E. Breast Cancer Res. Treat. 2000; 62: 99-107Crossref PubMed Scopus (43) Google Scholar). Synucleins are a family of small, highly soluble proteins that are predominantly expressed in neurons. The functions of the synucleins are not entirely understood. There are four known members: α-synuclein (also referred to as synelfin or non-Aβcomponent of Alzheimer's disease (AD) amyloid precursorprotein) and β-synuclein (also referred to as phosphoneuroprotein 14) are neuronal proteins primarily expressed in brain and are predominantly found at axonal terminals. γ-Synuclein (also known as persyn) is predominantly expressed in certain regions of the peripheral nervous system such as dorsal root ganglia and trigeminal ganglia. Synoretin, the newest member of the synuclein family, is expressed at high levels in the retina and at lower levels in the brain (8Lavedan C. Genome Res. 1998; 8: 871-880Crossref PubMed Scopus (271) Google Scholar, 9Surguchov A. Surgucheva I. Solessio E. Baehr W. Mol. Cell Neurosci. 1999; 13: 95-103Crossref PubMed Scopus (81) Google Scholar). The synuclein proteins contain several repeated domains that display variations of a KTKEGV consensus sequence. The β-synuclein protein contains five of these domains, whereas the α- and γ-synucleins have six. Interestingly, the third domain of each protein is completely conserved, and this same type of domain is present in proteins of the Rho family (10Maroteaux L. Campanelli J.T. Scheller R.H. J. Neurosci. 1988; 8: 2804-2815Crossref PubMed Google Scholar). Another type of organization of the synuclein proteins that has been noted is an 11-residue repeat. This motif, repeated six to seven times in the N-terminal portion of the protein, is reminiscent of the amphipathic α-helical domains of the apolipoproteins and suggests possible lipid binding properties (11Clayton D.F. George J.M. Trends Neurosci. 1998; 21: 249-254Abstract Full Text Full Text PDF PubMed Scopus (664) Google Scholar). The γ-synuclein gene maps to 10q23, is composed of five coding exons, and is transcribed into an mRNA of ∼1 kb (8Lavedan C. Genome Res. 1998; 8: 871-880Crossref PubMed Scopus (271) Google Scholar). The human γ-synuclein is 127 amino acids long and is 87.7% and 83.3% identical to the mouse and rat proteins, respectively. In addition, comparison of the amino acid sequences indicates that γ-synuclein is highly homologous to α-synuclein and β-synuclein except for the last 27 amino acids of γ-synuclein. Overall, γ-synuclein shares 54%, 56%, and 84% amino acid sequence identity with α-synuclein, β-synuclein, and synoretin, respectively (8Lavedan C. Genome Res. 1998; 8: 871-880Crossref PubMed Scopus (271) Google Scholar, 9Surguchov A. Surgucheva I. Solessio E. Baehr W. Mol. Cell Neurosci. 1999; 13: 95-103Crossref PubMed Scopus (81) Google Scholar). Among the synucleins, α-synuclein is the best characterized because of its significant role implicated in neurodegenerative diseases (12Mukaetova-Ladinska E.B. Hurt J. Jakes R. Xuereb J. Honer W.G. Wischik C.M. J. Neuropathol. Exp. Neurol. 2000; 59: 408-417Crossref PubMed Scopus (38) Google Scholar). Mutations in the α-synuclein gene have been identified in rare kindreds with Parkinson's disease (PD) (12Mukaetova-Ladinska E.B. Hurt J. Jakes R. Xuereb J. Honer W.G. Wischik C.M. J. Neuropathol. Exp. Neurol. 2000; 59: 408-417Crossref PubMed Scopus (38) Google Scholar, 13Arima K. Hirai S. Sunohara N. Aoto K. Izumiyama Y. Ueda K. Ikeda K. Kawai M. Brain Res. 1999; 843: 53-61Crossref PubMed Scopus (152) Google Scholar, 14Baba M. Nakajo S., Tu, P.H. Tomita T. Nakaya K. Lee V.M. Trojanowski J.Q. Iwatsubo T. Am. J. Pathol. 1998; 152: 879-884PubMed Google Scholar), and intracytoplasmic aggregates comprised of α-synuclein fibrils are characteristic of several neurodegenerative diseases as exemplified by the intraneuronal Lewy bodies (LBs), neuroaxonal spheroids, and dystrophic neurites (i.e. Lewy neurites) that are prominent in PD, LB variant of Alzheimer's disease (AD), and dementia with LBs (14Baba M. Nakajo S., Tu, P.H. Tomita T. Nakaya K. Lee V.M. Trojanowski J.Q. Iwatsubo T. Am. J. Pathol. 1998; 152: 879-884PubMed Google Scholar, 15Tu P.H. Galvin J.E. Baba M. Giasson B.I. Tomita T. Leight S. Nakajo S. Iwatssubo T. Trojanowski J.Q. Lee V.M.-Y. Ann. Neurol. 1998; 44: 415-422Crossref PubMed Scopus (596) Google Scholar, 16Arawaka S. Saito Y. Murayama S. Mori H. Neurology. 1998; 51: 887-889Crossref PubMed Scopus (159) Google Scholar, 17Arima K. Ueda K. Sunohara N. Hirai S. Izumiyama Y. Tonozuka-Uehara H. Kawai M. Brain Res. 1998; 808: 93-100Crossref PubMed Scopus (218) Google Scholar, 18Irizarry M.C. Growdon W. Gomez-Isla T. Newell K. George J.M. Clayton D.F. Hyman B.T. J. Neuropathol. Exp. Neurol. 1998; 57: 334-337Crossref PubMed Scopus (361) Google Scholar, 19Spillantini M.G. Crowther R.A. Jakes R. Hasegawa M. G. M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6469-6473Crossref PubMed Scopus (2443) Google Scholar). The normal physiological functions of synucleins are not well characterized. The N-terminal portion of α-synuclein (residues 1–61) shares 40% amino acid homology with members of the 14-3-3 protein family (20Ostrerova N. Petrucelli L. Farrer M. Mehta N. Choi P. Hardy J. Wolozin B. J. Neurosci. 1999; 19: 5782-5791Crossref PubMed Google Scholar). The 14-3-3 family of proteins helps regulate many different signal transduction pathways and is thought to act by directly binding to various protein kinases and bringing them into close proximity with substrate and regulatory proteins. 14-3-3 proteins bind to phospho-serine residues critical for the functions of many kinases and phosphatases that are involved in diverse cell functions (21Fu H. Subramanian R.R. Masters S.C. Annu. Rev. Pharmacol. Toxicol. 2000; 40: 617-647Crossref PubMed Scopus (1334) Google Scholar, 22Muslin A.J. Tanner J.W. Allen P.M. Shaw A.S. Cell. 1996; 84: 889-897Abstract Full Text Full Text PDF PubMed Scopus (1195) Google Scholar, 23Yaffe M.B. Elia A.E. Curr. Opin. Cell Biol. 2001; 13: 131-138Crossref PubMed Scopus (290) Google Scholar, 24Yaffe M.B. Rittinger K. Volinia S. Caron P.R. Aitken A. Leffers H. Gamblin S.J. Smerdon S.J. Cantley L.C. Cell. 1997; 91: 961-971Abstract Full Text Full Text PDF PubMed Scopus (1353) Google Scholar). Like the synucleins, 14-3-3 proteins are ubiquitously expressed in the brain and have been shown to associate in a chaperone-like manner with PKC, BAD, ERK, and RAF-1 (16Arawaka S. Saito Y. Murayama S. Mori H. Neurology. 1998; 51: 887-889Crossref PubMed Scopus (159) Google Scholar, 21Fu H. Subramanian R.R. Masters S.C. Annu. Rev. Pharmacol. Toxicol. 2000; 40: 617-647Crossref PubMed Scopus (1334) Google Scholar). α-Synuclein binds 14-3-3 as well as to PKC, BAD, ERK, and the microtubule-associated protein tau (25Tzivion G. Luo Z. Avruch J. Nature. 1998; 394: 88-92Crossref PubMed Scopus (390) Google Scholar). In addition to shared regions of homology to 14-3-3, α-synuclein as well as β- and γ-synuclein also appear to act as a protein chaperone, at least in vitro, by disrupting protein aggregation (26Souza J.M. Giasson B.I. Lee V.M. Ischiropoulos H. FEBS Lett. 2000; 474: 116-119Crossref PubMed Scopus (190) Google Scholar). To help further unravel the function of γ-synuclein and establish its role in the oncogenesis of breast and ovarian cancer, we searched for proteins that could interact with γ-synuclein and identified the MAPKs ERK1/2 and JNK1. In this study we provide evidence that γ-synuclein contributes to tumor development by protecting cancer cells under adverse conditions through modulating the ERK and JNK pathways. In addition, we observed that paclitaxel- or vinblastine-induced cell death is protected by γ-synuclein, indicating that chemotherapeutic drugs that take effect through activating the JNK apoptosis pathway may not be effective for cancer with high γ-synuclein expression. Paclitaxel, vinblastine, and etoposide were purchased from Sigma (St. Louis, MO). The MEK1/2 inhibitor U0126 was purchased from Promega (Madison, WI). Anti-ERK1, anti-ERK2, and anti-JNK1 antibodies and normal IgG were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phospho-ERK1/2 and anti-PARP antibodies were obtained from Cell Signaling (Beverly, MA). The mouse antibody Syn303 was raised to recombinant human α-synuclein but recognizes α-, β-, and γ-synuclein. γ-2 is a rabbit polyclonal antibody raised to recombinant human γ-synuclein that specifically recognizes human γ-synuclein (27Giasson B.I. Duda J.E. Forman M.S. Lee V.M.-Y. Trojanowski J.Q. Exp. Neurol. 2001; 172: 354-362Crossref PubMed Scopus (49) Google Scholar). Mouse monoclonal antibodies Syn204 and Syn207 were raised against human α- and β-synucleins, respectively. Ovarian cancer cell lines A2780 and OVCAR5 were maintained in 10% FBS Dulbecco's modified Eagle's medium and 10% FBS RPMI 1640, respectively. HEK293, human embryonic kidney cells, were maintained in 10% FBS Dulbecco's modified Eagle's medium supplemented with sodium pyruvate and non-essential amino acids. To create the CMV plasmid for establishing stable cell lines over-expressing human α-, β-, or γ-synuclein, human cDNAs were amplified by PCR and subcloned into pcDNA3 (Invitrogen). GenePorter Transfection Reagent (GTS Inc., San Diego, CA) was used for transfection, and stable cell lines were selected by G418 (Invitrogen). Expression of human α-, β-, or γ-synuclein was confirmed by immunoblotting with Syn204, Syn207, or γ-2 antibody, respectively. For UV treatment, cells at 70–80% confluence were washed once with PBS before UV irradiation (254 nm, 20 J/m2). Complete medium was added to the plates after treatment. The intensity of the UV light source was measured with a BLAK-RAY meter (UVP, Inc., San Gabriel, CA) prior to each experiment. Heat-shock was carried at 42 °C for 15 min. 50 μmsodium arsenite was used to treat the cells for 6 h. Paclitaxel, vinblastine, etoposide, and U0126 were dissolved in Me2SO, and cells were treated with various concentrations of the drugs as indicated in each experiment. Cells at 70–80% confluence were washed twice with ice-cold d-PBS before scraping on ice with lysis buffer (20 mm Tris-HCl, pH 7.5; 150 mm NaCl; 2.5 mm sodium pyrophosphate; 1 mm sodium β-glycerophosphate; 5 mm NaF, 1 mm Na3VO4, 1 mmphenylmethylsulfonyl fluoride, 1% Triton X-100, and 1 tablet of protease inhibitor mixture (Roche Molecular Biochemicals, Indianapolis, IN) per 40 ml of lysis buffer). Cellular debris was removed by centrifugation (14,000 × g for 15 min at 4 °C) and precleared with protein G-agarose (Invitrogen, Rockville, MD). Protein concentrations were determined with Bio-Rad DC protein assay reagents. Syn303 (3 μl of ascites) or control IgG (3 μg) were preincubated in 500 μl of PBS with 50 μl of protein-G-agarose overnight at 4 °C, and washed twice with PBS before incubation with 300 μg of total cellular lysate for 4 h at 4 °C. The beads were washed four times with the lysis buffer, resuspended in 50 μl of 2× SDS sample buffer before boiling for 5 min. 10 μl of immunoprecipitates was separated by SDS-PAGE electrophoresis on 4–20% linear gradient Tris-HCl ready gels (Bio-Rad). Proteins separated on SDS-PAGE gels were transferred onto an Immobilon-P polyvinylidene difluoride membrane (Millipore, Bedford, MA). The primary antibodies were diluted 1:1000, and the horseradish peroxidase-conjugated second antibodies were diluted 1:10,000 (Amersham Biosciences, Piscataway, NJ). PerkinElmer Life Sciences Renaissance Enhanced Luminol Reagents (Boston, MA) were used as substrates for detection. For re-use of the same membrane with another primary antibody, Restore Western blot Stripping buffer (Pierce, Rockford, IL) was used to strip the membrane. The results of immunoblotting were quantitated using the program IMAGE (National Institutes of Health) for the integrated density of each band. The kinase activity of JNK was measured using the SAPK/JNK assay kit (Cell Signaling Technologies). Briefly, 250 μl of cell lysate (1 μg/μl protein) was incubated with 2 μg of c-JUN fusion protein beads (in 20 μl) overnight at 4 °C. After washing, the proteins on the beads were incubated in the kinase reaction buffer supplemented with 100 μm ATP for 30 min at 30 °C. To measure the JNK activity, the phosphorylated c-JUN was detected by SDS-PAGE and immunoblotting with the specific antibody (Cell Signaling Technologies). Cell viability was determined by Trypan blue exclusion assay and/or WST-1 assay. For Trypan blue assay, cells were stained with 0.2% Trypan blue for 2–5 min. The number of viable cells (non-stained) and dead cells (stained) were counted under a microscope using a cell hemacytometer. For the WST-1 assay, cells under different culture conditions were incubated with WST-1 (Roche Molecular Biochemicals) for 4 h. Cleavage of WST-1 to formazan was monitored at 450 nm using a microplate reader. Colorimetric CaspACE assay (Promega) was used to detect the caspase-3 activity. Briefly, pNA released from the substrate Ac-DEVD-pNA by caspase-3 in the cell lysate was monitored at 405 nm using a microplate reader. Where indicated, a two-tailed Studentt test was used to test for significance. We have previously reported that γ-synuclein is highly expressed in the vast majority of late-stage breast and ovarian tumors (4Bruening W. Giasson B.I. Klein-Szanto A.J. Lee V.M. Trojanowski J.Q. Godwin A.K. Cancer. 2000; 88: 2154-2163Crossref PubMed Scopus (159) Google Scholar), suggesting a potentially important role for γ-synuclein in the development of these diseases. To help unravel the function of γ-synuclein, we established several in vitro models. The ovarian tumor cell lines A2780 and OVCAR5, which express low levels of γ-synuclein, as well as kidney HEK293 cells, which do not express detectable levels of γ-synuclein, were transfected with CMV-γ-synuclein or with vector alone and were selected with G418. Resistant colonies were screened by Western blotting for stable expression of γ-synuclein protein, and positive colonies were pooled into A2780gam, OVCAR5gam, and 293gam cell lines (Fig. 1). Cell lines stably expressing α-synuclein and β-synuclein were also derived from A2780 cells as described for the γ-synuclein expressing lines (Fig. 1). Like A2780gam, OVCAR5gam, and 293gam cells, there were no obvious alterations in cell doubling time of the A2780alpha or A2780beta cell lines (data not shown). α-Synuclein has recently been reported to bind directly to the ERK2 kinase (28Iwata A. Miura S. Kanazawa I. Sawada M. Nukina N. J. Neurochem. 2001; 77: 239-252Crossref PubMed Google Scholar). Therefore, we evaluated whether γ-synuclein could also interact with the ERK kinases as well as other MAPKs. By co-immunoprecipitation approaches, we were able to demonstrate a novel association of γ-synucleins with ERK1/2 and JNK1 kinase but not with the p38 kinase (Fig. 2). We also confirmed that α-synuclein is associated with ERK1/2 as well with JNK1 (Fig. 2), which is consistent with the recent studies using neuro2a, a neuronal cell line (29Iwata A. Maruyama M. Kanazawa I. Nukina N. J. Biol. Chem. 2001; 276: 45320-453229Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). These data indicate that γ- and α-synuclein can interact with ERK1/2 and JNK1 in cancer cells that over-express these proteins. We next evaluated whether these protein interactions would affect the activity of ERK1/2 and/or JNK1 (see below). In A2780 and OVCAR5 cancer cells over-expressing γ-synuclein, the activated ERK1/2 was increased 2- to 3-fold as evidenced by immunoblotting with an anti-phospho-ERK specific antibody (Fig.3). In contrast, α- and β-synucleins appeared to have little or no effect on the activity of ERK1/2 (Fig.3 A), although α-synuclein was also found to be associated with ERK (as described above and shown in Fig. 2) in A2780 cells. In HEK293 cells, the basal level of ERK activation is undetectable and γ-synuclein over-expression does not increase its activation level (Fig. 3 B). Structural analysis indicated that γ-synuclein does not contain any kinase domain, suggesting that the activation of ERK is mediated by other kinase. Because MEK1/2 is required for the activation of ERK1/2 in response to many mitogens, we determined whether MEK1/2 is still required for γ-synuclein-mediated activation of ERK1/2. When cells over-expressing γ-synuclein were treated with the MEK1/2 inhibitor U0126, the activation of ERK1/2 was suppressed (Fig. 4 A). We further studied the relation of γ-synuclein-ERK interaction and the activation status of ERK1/2. In cells treated with U0126 or serum-starved, the association of γ-synuclein and ERK1/2 was still present (Fig.4 B). These data indicate that γ-synuclein may be constitutively associated with ERK1/2, which could facilitate the activation of ERK by MEK1/2 and lead to the constitutive activation of ERK1/2 in cells over-expressing γ-synuclein.Figure 4Requirement of MEK1/2 for γ-synuclein enhanced ERK1/2 activation. A, A2780 and A2780/gam cells untreated or treated with the MEK1/2 inhibitor, U0126 (10 μm), were lysed, and 30 μg of proteins was loaded into each lane. As in Fig. 3, anti-phospho-ERK1/2-specific antibody was used to detect activated ERK1/2; the antibody against ERK1/2 was used to detect the protein level of total ERK1/2. B, the interaction between ERK and γ-synuclein is independent of the activation status of ERK1/2. A2780/gam cells in normal 10% FBS medium, 10% FBS medium with U0126 (10 μm), or serum-free medium were lysed and immunoprecipitated with Syn303 or control IgG. The proteins in the immunoprecipitates were detected with the antibodies against ERK1/2 or against human γ-synuclein (γ-2). The autoradiogram shown is representative of three independent experiments with comparable results.View Large Image Figure ViewerDownload (PPT) JNK is activated by stress signals, including UV, which leads to mitochondria-mediated apoptosis (30Davis R.J. Cell. 2000; 103: 239-252Abstract Full Text Full Text PDF PubMed Scopus (3666) Google Scholar). The basal level of JNK activity in A2780 and OVCAR5 ovarian cancer cells is very low in untreated cells whether γ-synuclein was over-expressed or not (Fig. 5). JNK was highly activated in the parental cells when treated with UV (Fig. 5). In cells over-expressing γ-synuclein, the activation of JNK was almost completely blocked in A2780/gam cells (p < 0.05) and was down-regulated by ∼50% in OVCAR5/gam cells when treated with UV (Fig. 5) or heat-shock (data not shown). The blockage of JNK activation by UV appears to be γ-synuclein-specific, because over-expression of α- and β-synucleins appeared to have little or no effect on the activation of JNK by UV (Fig. 5 and data not shown). The inhibition of JNK activation in OVCAR5/gam cells is much less than that in A2780/gam cells. The cause of this difference could either be cell-type specific or it could be the high endogenous γ-synuclein expression in OVCAR5 cells. Similarly, the activation of JNK by sodium arsenate was blocked to different extents by γ-synuclein in 293/gam, OVCAR5/gam, and A2780/gam cells (Fig.6). Collectively, these data indicate that stress-induced activation of JNK can be blocked by γ-synuclein over-expression in a variety of cell lines.Figure 6JNK activation is down-regulated by over-expression of γ-synuclein in response to sodium arsenate. HEK293, OVCAR5, and A2780 cells and their γ-synuclein over-expressing counterparts were treated with sodium arsenite (50 μm) for 6 h. Cells were lysed, and the extracts were assayed for JNK activity as described in the legends for Fig. 5. The number beneath each band represents the arbitrary densitometry units of the corresponding band. The blots shown here are representative of three independent experiments with comparable results.View Large Image Figure ViewerDownload (PPT) Based on the data presented above, we hypothesized that γ-synuclein may contribute to cancer cell survival by up-regulating the ERK cell survival pathway and by suppressing the JNK apoptosis pathway under adverse conditions. However, when we treated γ-synuclein over-expressing cells with UV, significant differences in cell survival between A2780 and A2780/gam cells were not observed (data not shown). The reason for this lack of difference is not readily apparent. However, cell survival and cell death are regulated by the counterbalance between the survival factors and the apoptotic signaling pathways. Because UV treatment also activates the cell survival pathways ERK (Ref. 31Rosette C. Karin M. Science. 1996; 274: 1194-1197Crossref PubMed Scopus (945) Google Scholar and data not shown) and PI3K-AKT (32Krasilnikov M. Adler V. Fuchs S.Y. Dong Z. Haimovitz-Friedman A. Herlyn M. Ronai Z. Mol. Carcinog. 1999; 24: 64-69Crossref PubMed Scopus (69) Google Scholar, 33Nomura M. Kaji A., Ma, W.Y. Zhong S. Liu G. Bowden G.T. Miyamoto K.I. Dong Z. J. Biol. Chem. 2001; 276: 25558-25567Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar), the initiation of the mitochondria-associated caspase pathway may be blocked by the activation of ERK or AKT in A2780 cells. In support of these findings we did not observe the cleavage of the caspase-3 substrate PARP in A2780 cells when treated with UV (data not shown). We next evaluated the survival of γ-synuclein over-expressing cells in response to Taxol, a commonly used chemotherapeutic drug. In addition to its role in affecting microtubule assembly, Taxol is known to lead to apoptosis via the mitochondria by activating the JNK signaling pathway and Taxol-induced apoptosis can be enhanced by MEK inhibition (34Lee L.F., Li, G. Templeton D.J. Ting J.P. J. Biol. Chem. 1998; 273: 28253-28260Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar, 35Mandlekar S., Yu, R. Tan T.H. Kong A.N. Cancer Res. 2000; 60: 5995-6000PubMed Google Scholar, 36Wang T.H. Popp D.M. Wang H.S. Saitoh M. Mural J.G. Henley D.C. Ichijo H. Wimalasena J. J. Biol. Chem. 1999; 274: 8208-8216Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). In A2780 cells, Taxol did not affect the basal level activity of ERK or the activation of ERK by γ-synuclein (Fig.7 A). To test the effect of γ-synuclein on cell survival, cells were treated with Taxol for varying lengths of time. At 48 h after treatment, 45–60% of A2780 cells had died, whereas only about 7–15% of A2780/gam cells were dead indicating that Taxol-induced cell death can be rescued by γ-synuclein over-expression (Fig. 7 B). When ERK activation was inhibited using the MEK1/2 inhibitor U0126, the cell death was reduced by ∼25% in A2780 cells but was nearly doubled in A2780/gam cells" @default.
• W2023204266 created "2016-06-24" @default.
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• W2023204266 date "2002-09-01" @default.
• W2023204266 modified "2023-10-01" @default.
• W2023204266 title "γ-Synuclein Promotes Cancer Cell Survival and Inhibits Stress- and Chemotherapy Drug-induced Apoptosis by Modulating MAPK Pathways" @default.
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• W2023204266 cites W1530609782 @default.
• W2023204266 cites W1557235501 @default.
• W2023204266 cites W1587753447 @default.
• W2023204266 cites W1587973572 @default.
• W2023204266 cites W1976039245 @default.
• W2023204266 cites W1987564246 @default.
• W2023204266 cites W1989077825 @default.
• W2023204266 cites W1989149919 @default.
• W2023204266 cites W1991130856 @default.
• W2023204266 cites W1996424896 @default.
• W2023204266 cites W1998638154 @default.
• W2023204266 cites W2001237845 @default.
• W2023204266 cites W2003405109 @default.
• W2023204266 cites W2003962192 @default.
• W2023204266 cites W2004315433 @default.
• W2023204266 cites W2008046168 @default.
• W2023204266 cites W2008748100 @default.
• W2023204266 cites W2013085111 @default.
• W2023204266 cites W2024923170 @default.
• W2023204266 cites W2030092320 @default.
• W2023204266 cites W2030570585 @default.
• W2023204266 cites W2030600721 @default.
• W2023204266 cites W2030748454 @default.
• W2023204266 cites W2034164574 @default.
• W2023204266 cites W2034210468 @default.
• W2023204266 cites W2037866233 @default.
• W2023204266 cites W2037915962 @default.
• W2023204266 cites W2037948191 @default.
• W2023204266 cites W2045943650 @default.
• W2023204266 cites W2050786534 @default.
• W2023204266 cites W2060809474 @default.
• W2023204266 cites W2064445049 @default.
• W2023204266 cites W2070760404 @default.
• W2023204266 cites W2071075259 @default.
• W2023204266 cites W2075003503 @default.
• W2023204266 cites W2075927534 @default.
• W2023204266 cites W2076485183 @default.
• W2023204266 cites W2076629995 @default.
• W2023204266 cites W2082107454 @default.
• W2023204266 cites W2091265794 @default.
• W2023204266 cites W2093388612 @default.
• W2023204266 cites W2104912043 @default.
• W2023204266 cites W2116474489 @default.
• W2023204266 cites W2119092360 @default.
• W2023204266 cites W2119807130 @default.
• W2023204266 cites W2122772601 @default.
• W2023204266 cites W2123927174 @default.
• W2023204266 cites W2133068803 @default.
• W2023204266 cites W2138178697 @default.
• W2023204266 cites W2138306686 @default.
• W2023204266 cites W2150656456 @default.
• W2023204266 cites W2161334375 @default.
• W2023204266 cites W2162010804 @default.
• W2023204266 cites W2168155492 @default.
• W2023204266 cites W2330367114 @default.
• W2023204266 cites W2415001185 @default.
• W2023204266 cites W3081445012 @default.
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