Comparative Analysis of Protein Expression Concomitant with DNA Methyltransferase 3A Depletion in a Melanoma Cell Line


DNA methyltransferase 3A (Dnmt3a), a de novo methyltransferase, has attracted a great deal of attention for its important role played in tumorigenesis. We have previously demonstrated that melanoma is unable to grow in-vivo in conditions of Dnmt3a depletion in a mouse model. In this study, we cultured the Dnmt3a depletion B16 melanoma (Dnmt3a-D) cell line to conduct a comparative analysis of protein expression con-comitant with Dnmt3a depletion in a melanoma cell line. After two-dimensional separation, by gel electro-phoresis and liquid chromatography, combined with mass spectrometry analysis (1DE-LC-MS/MS), the re-sults demonstrated that 467 proteins were up-regulated and 535 proteins were down-regulated in the Dnmt3a-D cell line compared to the negative control (NC) cell line. The Genome Ontology (GO) and KEGG pathway were used to further analyze the altered proteins. KEGG pathway analysis indicated that the MAPK signaling pathway exhibited a greater alteration in proteins, an interesting finding due to the close relation-ship with tumorigenesis. The results strongly suggested that Dnmt3a potentially controls the process of tu-morigenesis through the regulation of the proteins (JNK1, p38α, ERK1, ERK2, and BRAF) involved in tu-mor-related pathways, such as the MAPK signaling pathway and melanoma pathway.

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X. Liu, S. Tang, T. Li, H. Wang, J. Sun, Q. Qiao, J. Yao and J. Fei, "Comparative Analysis of Protein Expression Concomitant with DNA Methyltransferase 3A Depletion in a Melanoma Cell Line," American Journal of Analytical Chemistry, Vol. 2 No. 5, 2011, pp. 539-572. doi: 10.4236/ajac.2011.25064.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] A. J. Miller and M. C. Jr Mihm, “Melanoma,” The New England Journal of Medicine, Vol. 355, 2006, pp. 51-65.
[2] A. D. Sasse, E. C. Sasse, L. G. Clark, L. Ulloa and O. A. Clark, “Chemoimmunotherapy Versus Chemotherapy for Metastatic Malignant Melanoma,” Cochrane Database System Reviews, 2007, p. D5413.
[3] M. F. Paz, M. F. Fraga, S. Avila, M. Guo, M. Pollan, J. G. Herman and M. Esteller, “A Systematic Profile of Dna Methylation in Human Cancer Cell Lines,” Cancer Re-search, Vol. 63, 2003, pp. 1114-1121.
[4] W. M. Gallagher, O. E. Bergin, M. Rafferty, Z. D. Kelly, I. M. Nolan, E. J. Fox, A. C. Culhane, L. McArdle, M. F. Fraga, L. Hughes, C. A. Currid, F. O’Mahony, A. Byrne, A. A. Murphy, C. Moss, S. McDonnell, R. L. Stallings, J. A. Plumb, M. Esteller, R. Brown, P. A. Dervan and D. J. Easty, “Multiple Markers for Melanoma Progression Re-gulated by Dna Methylation: Insights From Transcriptomic Studies,” Carcinogenesis, Vol. 26, No. 11, 2005, pp. 1856-1867.
[5] P. A. Van der Velden, W. Zuidervaart, M. H. Hurks, S. Pavey, B. R. Ksander, E. Krijgsman, R. R. Frants, C. P. Tensen, R. Willemze, M. J. Jager and N. A. Gruis, “Ex-pression Profiling Reveals that Methylation of Timp3 is Involved in Uveal Melanoma Development,” International Journal of Cancer, Vol. 106, No. 4, 2003, pp. 472-479.
[6] E. Li, T. H. Bestor, and R. Jaenisch, “Targeted Mutation of the Dna Methyltransferase Gene Results in Embryonic Lethality,” Cell, Vol. 69, No. 6, 1992, pp. 915-926.
[7] H. Lei, S. P. Oh, M. Okano, R. Juttermann, K. A. Goss, R. Jaenisch and E. Li, “De Novo Dna Cytosine Methyl-transferase Activities in Mouse Embryonic Stem Cells,” Development, Vol. 122, 1996, pp. 3195-3205.
[8] M. Okano, D. W. Bell, D. A. Haber and E. Li, “Dna Me-thyltransferases Dnmt3a and Dnmt3B are Essential for De Novo Methylation and Mammalian Development,” Cell, Vol. 99, No, 3, 1999, pp. 247-257.
[9] K. D. Robertson, E. Uzvolgyi, G. Liang, C. Talmadge, J. Sumegi, F. A. Gonzales and P. A. Jones, “The Human Dna Methyltransferases (Dnmts) 1, 3a and 3B: Coordinate Mrna Expression in Normal Tissues and Overexpression in Tumors,” Nucleic Acids Research, Vol. 27, No. 11, 1999, pp. 2291-2298. doi:org/10.1093/nar/27.11.2291
[10] D. L. Zheng, L. Zhang, N. Cheng, X. Xu, Q. Deng, X. M. Teng, K. S. Wang, X. Zhang, J. Huang and Z. G. Han, “Epigenetic Modification Induced by Hepatitis B Virus X Protein Via Interaction with De Novo Dna Methyltrans-ferase Dnmt3a,” Journal of Hepatology, Vol. 50, No. 2, 2009, pp. 377-387. doi:org/10.1016/j.jhep.2008.10.019
[11] D. V. Maltseva, A. A. Baykov, A. Jeltsch, and E. S. Gro- mova, “Impact of 7,8-Dihydro-8-Oxoguanine On Methy-lation of the Cpg Site by Dnmt3a,” Biochemistry, Vol. 48, No. 6, 2009, pp. 1361-1368.
[12] J. Feng, Y. Zhou, S. L. Campbell, Le T, E. Li, J. D. Sweatt, A. J. Silva and G. Fan, “Dnmt1 and Dnmt3a Maintain Dna Methylation and Regulate Synaptic Function in Adult Forebrain Neurons,” Nature Neuroscience, Vol. 13, 2010, pp. 423-430. doi:org/10.1038/nn.2514
[13] H. Wu, V. Coskun, J. Tao, W. Xie, W. Ge, K. Yoshikawa, E. Li, Y. Zhang and Y. E. Sun, “Dnmt3a-Dependent Nonpromoter Dna Methylation Facilitates Transcription of Neurogenic Genes,” Science, Vol. 329, No. 5990, 2010, pp. 444- 448.
[14] J. Nawarak, R. Huang-Liu, S. H. Kao, H. H. Liao, S. Sinchaikul, S. T. Chen and S. L. Cheng, “Proteomics Analysis of a375 Human Malignant Melanoma Cells in Response to Arbutin Treatment,” Biochim Biophys Acta, Vol. 1794, No. 2, 2009, pp. 159-167.
[15] Y. Zhang, Y. Gao, G. Zhang, S. Huang, Z. Dong, C. Kong, D. Su, Du J, S. Zhu, Q. Liang, J. Zhang, J. Lu, and B. Huang, “Dnmt3a Plays a Role in Switches Between Doxorubicin-Induced Senescence and Apoptosis of Co-lorectal Cancer Cells,” International Journal of Cancer, Vol. 128, No. 3, 2010, pp. 551-561.
[16] Z. Zhao, Q. Wu, J. Cheng, X. Qiu, J. Zhang and H. Fan, “Depletion of Dnmt3a Suppressed Cell Proliferation and Restored Pten in Hepatocellular Carcinoma Cell,” Journal of Biomedicine and Biotechnology, Vol. 2010, Vol. 2010, pp. 737535-737545. doi:org/10.1155/2010/737535
[17] T. Deng, Y. Kuang, L. Wang, J. Li, Z. Wang, and J. Fei, “An Essential Role for Dna Methyltransferase 3a in Me-lanoma Tumorigenesis,” Biochemical and Biophysical Reseasch Communications, Vol. 387, No. 3, 2009, pp. 611-616. doi:org/10.1016/j.bbrc.2009.07.093
[18] E. F. Wagner and A. R. Nebreda, “Signal Integration by Jnk and P38 Mapk Pathways in Cancer Development,” Nature Reviews Cancer, Vol. 9, 2009, pp. 537-549. doi:org/10.1038/nrc2694
[19] A. Zougman, B. Pilch, A. Podtelejnikov, M. Kiehntopf, C. Schnabel, C. Kumar and M. Mann, “Integrated Analysis of the Cerebrospinal Fluid Peptidome and Proteome,” Journal of Proteome Research, Vol. 7, 2008, pp. 386-399. doi:org/10.1021/pr070501k
[20] B. Macek, L. F. Waanders, J. V. Olsen and M. Mann, “Top-Down Protein Sequencing and Ms3 On a Hybrid Linear Quadrupole Ion Trap-Orbitrap Mass Spectrometer,” Mol Cell Proteomics, Vol. 5, 2006, pp. 949-958.
[21] J. V. Olsen, L. M. de Godoy, G. Li, B. Macek, P. Mor-tensen, R. Pesch, A. Makarov, O. Lange, S. Horning and M. Mann, “Parts Per Million Mass Accuracy On an Orbi-trap Mass Spectrometer Via Lock Mass Injection Into a C-Trap,” Mol Cell Proteomics, Vol. 4, 2005, pp. 2010-2021. doi:org/10.1074/mcp.T500030-MCP200
[22] S. K. Binz, A. M. Sheehan and M. S. Wold, “Replication Protein a Phosphorylation and the Cellular Response to Dna Damage,” DNA Repair (Amst), Vol. 3, No. 8-9, 2004, pp. 1015-1024. doi:org/10.1016/j.dnarep.2004.03.028
[23] M. S. Kobor and J. Greenblatt, “Regulation of Transcrip-tion Elongation by Phosphorylation,” Biochim Biophys Acta, Vol. 1577, No. 2, 2002, pp. 261-275.
[24] H. Ohkura, “Phosphorylation: Polo Kinase Joins an Elite Club,” Current Biology, Vol. 13, 2003, pp. R912-R914. doi:org/10.1016/j.cub.2003.11.012
[25] P. P. Ruvolo, X. Deng and W. S. May, “Phosphorylation of Bcl2 and Regulation of Apoptosis,” Leukemia, Vol. 15, No. 4, 2001, pp. 515-522.
[26] L. Chang and M. Karin, “Mammalian Map Kinase Sig-nalling Cascades,” Nature, Vol. 410, No. 6824, 2001, pp. 37-40.
[27] J. M. Kyriakis and J. Avruch, “Mammalian Mitogen- Activated Protein Kinase Signal Transduction Pathways Activated by Stress and Inflammation,” Physiological Reviews, Vol. 81, No. 2, 2001, pp. 807-869.
[28] A. R. Nebreda and A. Porras, “P38 Map Kinases: Beyond the Stress Response,” Trends in Biochemical Sciences, Vol. 25, No. 6, 2000, pp. 257-260. doi:org/10.1016/S0968-0004(00)01595-4
[29] M. Karin and E. Gallagher, “From Jnk to Pay Dirt: Jun Kinases, their Biochemistry, Physiology and Clinical Importance,” Iubmb Life, Vol. 57, No. 4-5, 2005, pp. 283-295. doi:org/10.1080/15216540500097111
[30] S. Gupta, T. Barrett, A. J. Whitmarsh, J. Cavanagh, H. K. Sluss, B. Derijard and R. J. Davis, “Selective Interaction of Jnk Protein Kinase Isoforms with Transcription Fac-tors,” EMBO Journal, Vol. 15, 1996, pp. 2760-2770.
[31] R. Eferl and E. F. Wagner, “Ap-1: A Double-Edged Sword in Tumorigenesis,” Nature Reviews Cancer, Vol. 3, 2003, pp. 859-868. doi:org/10.1038/nrc1209
[32] K. Sabapathy, K. Hochedlinger, S. Y. Nam, A. Bauer, M. Karin and E. F. Wagner, “Distinct Roles for Jnk1 and Jnk2 in Regulating Jnk Activity and C-Jun-Dependent Cell Proliferation,” Molecular Cell, Vol. 15, No. 5, 2004, pp. 713-725. doi:org/10.1016/j.molcel.2004.08.028
[33] M. Das, F. Jiang, H. K. Sluss, C. Zhang, K. M. Shokat, R. A. Flavell and R. J. Davis, “Suppression of P53-Dependent Senescence by the Jnk Signal Transduc-tion Pathway,” Proceedingof the Natlonal Academy Sciences of the U S A, Vol. 104, 2007, pp. 15759-15764. doi:org/10.1073/pnas.0707782104
[34] D. Morse, L. E. Otterbein, S. Watkins, S. Alber, Z. Zhou, R. A. Flavell, R. J. Davis and A. M. Choi, “Deficiency in the C-Jun Nh2-Terminal Kinase Signaling Pathway Con-fers Susceptibility to Hyperoxic Lung Injury in Mice,” American Journal of Physiology-Lung Cellular Molecular Physiology, Vol. 285, No. 1, 2003, pp. L250-L257.
[35] J. C. Lee, J. T. Laydon, P. C. McDonnell, T. F. Gallagher, S. Kumar, D. Green, D. McNulty, M. J. Blumenthal, J. R. Heys, S. W. Landvatter and Al. Et, “A Protein Kinase Involved in the Regulation of Inflammatory Cytokine Biosynthesis,” Nature, Vol. 372, 1994, pp. 739-746.
[36] K. Ono and J. Han, “The P38 Signal Transduction Path-way: Activation and Function,” Cellular Signalling, Vol. 12, No. 1, 2000, pp. 1-13. doi:org/10.1016/S0898-6568(99)00071-6
[37] D. V. Bulavin and AJ Jr Fornace, “P38 Map Kinase’s Emerging Role as a Tumor Suppressor,” Advances In Cancer Research, Vol. 92, 2004, pp. 95-118. doi:org/10.1016/S0065-230X(04)92005-2
[38] I. Dolado, A. Swat, N. Ajenjo, G. De Vita, A. Cuadrado and A. R. Nebreda, “P38Alpha Map Kinase as a Sensor of Reactive Oxygen Species in Tumorigenesis,” Cancer Cell, Vol. 11, No. 2, 2007, pp. 191-205.
[39] F. B. Engel, M. Schebesta, M. T. Duong, G. Lu, S. Ren, J. B. Madwed, H. Jiang, Y. Wang and M. T. Keating, “P38 Map Kinase Inhibition Enables Proliferation of Adult Mammalian Cardiomyocytes,” Genes Development, Vol. 19, 2005, pp. 1175-1187. doi:org/10.1101/gad.1306705
[40] L. J. Hui, L. Bakiri, A. Mairhorfer, N. Schweifer, C. Has-linger, L. Kenner, V. Komnenovic, H. Scheuch, H. Beug and E. F. Wagner, “P38X3B1; Suppresses Normal and Cancer Cell Proliferation by Antagonizing the JnkX2013;C-Jun Pathway,” Nature Genetics, Vol. 39, 2007.
[41] A. Jones and A. Charlton, “Determination of Metaldehyde in Suspected Cases of Animal Poisoning Using Gas Chromatography-Ion Trap Mass Spectrometry,” Journal Agric Food Chemistry, Vol. 47, No. 11, 1999, pp. 4675-4677. doi:org/10.1021/jf990026d
[42] X. Wang, C. H. Goh and B. Li, “P38 Mitogen-Activated Protein Kinase Regulates Osteoblast Differentiation through Osterix,” Endocrinology, Vol. 148, 2007, pp. 1629-1637.
[43] C. Hikita, S. Vijayakumar, J. Takito, H. Erdjument- Bromage, P. Tempst and Q. Al-Awqati, “Induction of Terminal Differentiation in Epithelial Cells Requires Po-lymerization of Hensin by Galectin 3,” Journal of Cell Biology, Vol. 151, No. 6, 2000, pp. 1235-1246. doi:org/10.1083/jcb.151.6.1235
[44] P. Ordonez-Moran, M. J. Larriba, H. G. Palmer, R. A. Valero, A. Barbachano, M. Dunach, A. G. de Herreros, C. Villalobos, M. T. Berciano, M. Lafarga and A. Munoz, “Rhoa-Rock and P38Mapk-Msk1 Mediate Vitamin D Effects On Gene Expression, Phenotype and Wnt Pathway in Colon Cancer Cells,” Journal of Cell Biology, Vol. 183, No. 4, 2008, pp. 697-710. doi:org/10.1083/jcb.200803020
[45] G. J. Finn, B. S. Creaven and D. A. Egan, “Daphnetin Induced Differentiation of Human Renal Carcinoma Cells and its Mediation by P38 Mitogen-Activated Protein Ki-nase,” Biochemical Pharmacology, Vol. 67, 2004, pp. 1779-1788. doi:org/10.1016/j.bcp.2004.01.014
[46] T. S. Lewis, P. S. Shapiro and N. G. Ahn, “Signal Trans-duction through Map Kinase Cascades,” Advances In Cancer Research, Vol. 74, 1998, pp. 49-139. doi:org/10.1016/S0065-230X(08)60765-4
[47] T. G. Boulton, S. H. Nye, D. J. Robbins, N. Y. Ip, E. Radziejewska, S. D. Morgenbesser, R. A. DePinho, N. Panayotatos, M. H. Cobb and G. D. Yancopoulos, “Erks: A Family of Protein-Serine/Threonine Kinases that are Activated and Tyrosine Phosphorylated in Response to Insulin and Ngf,” Cell, Vol. 65, 1991, pp. 663-675.
[48] C. M. Atkins, J. C. Selcher, J. J. Petraitis, J. M. Trzasko-sand J. D. Sweatt, “The Mapk Cascade is Required for Mammalian Associative Learning,” Nature Neuroscience, Vol. 1, 1998, pp. 602-609. doi:org/10.1038/2836
[49] R. Brambilla, N. Gnesutta, L. Minichiello, G. White, A. J. Roylance, C. E. Herron, M. Ramsey, D. P. Wolfer, V. Cestari, C. Rossi-Arnaud, S. G. Grant, P. F. Chapman, H. P. Lipp, E. Sturani and R. Klein, “A Role for the Ras Signalling Pathway in Synaptic Transmission and Long- Term Memory,” Nature, Vol. 390, 1997, PP. 281-286.
[50] G. Pearson, F. Robinson, Gibson T. Beers, B. E. Xu, M. Karandikar, K. Berman and M. H. Cobb, “Mitogen-Ac- tivated Protein (Map) Kinase Pathways: Regulation and Physiological Functions,” Endocrine Reviews, Vol. 22, No. 2, 2001, pp. 153-183. doi:org/10.1210/er.22.2.153
[51] T. Pawson and J. D. Scott, “Signaling through Scaffold, Anchoring, and Adaptor Proteins,” Science, Vol. 278, No. 5346, 1997, pp. 2075-2080.
[52] T. Hunter, “Protein Kinases and Phosphatases: The Yin and Yang of Protein Phosphorylation and Signaling,” Cell, Vol. 80, No. 2, 1995, pp. 225-236.
[53] A. M. Aronov, C. Baker, G. W. Bemis, J. Cao, G. Chen, P. J. Ford, U. A. Germann, J. Green, M. R. Hale, M. Ja-cobs, J. W. Janetka, F. Maltais, G. Martinez-Botella, M. N. Namchuk, J. Straub, Q. Tang and X. Xie, “Flipped Out: Structure-Guided Design of Selective Pyrazolylpyrrole Erk Inhibitors,” Journal of Medicinal Chemistry, Vol. 50, No. 5, 2007, pp. 1280-1287. doi:org/10.1021/jm061381f
[54] K. S. Smalley, “A Pivotal Role for Erk in the Oncogenic Behaviour of Malignant Melanoma?” International Journal of Cancer, Vol. 104, No. 5, 2003, pp. 527-532. doi:org/10.1002/ijc.10978
[55] H. Gear, H. Williams, E. G. Kemp and F. Roberts, “Braf Mutations in Conjunctival Melanoma,” Invest Ophthal-mology Visual Science, Vol. 45, No. 8, 2004, pp. 2484- 2488. doi:org/10.1167/iovs.04-0093
[56] V. K. Goel, A. J. Lazar, C. L. Warneke, M. S. Redston and F. G. Haluska, “Examination of Mutations in Braf, Nras, and Pten in Primary Cutaneous Melanoma,” Journal of Investigative Dermatology, Vol. 126, 2006, pp. 154-160.
[57] H. Davies, G. R. Bignell, C. Cox, P. Stephens, S. Edkins, S. Clegg, J. Teague, H. Woffendin, M. J. Garnett, W. Bottomley, N. Davis, E. Dicks, R. Ewing, Y. Floyd, K. Gray, S. Hall, R. Hawes, J. Hughes, V. Kosmidou, A. Menzies, C. Mould, A. Parker, C. Stevens, S. Watt, S. Hooper, R. Wilson, H. Jayatilake, B. A. Gusterson, C. Cooper, J. Shipley, D. Hargrave, K. Pritchard-Jones, N. Maitland, G. Chenevix-Trench, G. J. Riggins, D. D. Bigner, G. Palmieri, A. Cossu, A. Flanagan, A. Nicholson, J. W. Ho, S. Y. Leun
[58] R. S. Lo and O. N. Witte, “Transforming Growth Fac-tor-Beta Activation Promotes Genetic Context-Dependent Invasion of Immortalized Melanocytes,” Cancer Research, Vol. 68, No. 4248, 2008, pp. 4248-4257. doi:org/10.1158/0008-5472.CAN-07-5671

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