Ectopic ERK expression induces phenotypic conversion of C10 cells and alters DNA methyltransferase expression
© Sontag and Weber; licensee BioMed Central Ltd. 2012
Received: 2 March 2012
Accepted: 19 April 2012
Published: 4 May 2012
Many lung carcinogens activate mitogen activated protein kinase (MAPK) pathways and DNA methyltransferases (DNMTs) are under investigation as therapeutic targets for lung cancer. Our goal is to determine whether C10 type II alveolar epithelial cells are a sensitive model to investigate ERK-dependent transformation and DNMT expression patterns in experimental lung cancer.
Ectopic expression of an extracellular signal regulated kinase (ERK)-green fluorescent protein (ERK1-GFP) induces acquisition of growth in soft agar that is selectively associated with latent effects on the expression of DNA methyl transferases (DNMT1 and 3b), xeroderma pigmentosum complementation group A (XPA), DNA-dependent protein kinase catalytic subunit (DNA-PKcs), increased phosphatase activity and enhanced sensitivity to 5-azacytidine (5-azaC)-mediated toxicity, relative to controls.
Ectopic expression of ERK alone is sufficient to promote phenotypic conversion of C10 cells associated with altered DNMT expression patterns and sensitivity to DNMT inhibitor. This model may have applications for predicting sensitivity to DNMT inhibitors.
KeywordsEpigenetics ERK DNMT
In previous studies we have used xeroderma pigmentosum complementation group A (XPA) as a loading control for nuclear extracts because the expression of this protein generally showed little change under a variety of experimental conditions. However, we observed a marked increase in the expression of XPA in late passage ERK1-GFP cells, relative to early passage cells and vector controls (Figure 3A). We subsequently defined the expression of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) in late passage ERK1-GFP cells as an additional index for DNA damage signaling which was also increased (Figure 3A). Lamin a/c levels were not increased under these conditions and served as loading control. The combined results of three independent experiments are illustrated in Figure 3B. The reason for increased expression of DNA repair proteins is unclear. One possible interpretation may relate persistent ERK activation to genomic instability, which is a common feature of human cancers . Genomic instability encompasses a broad array of chromosomal rearrangements and DNA damage events  that could generate signals leading to the regulation of repair proteins such as XPA and DNA-PKcs. ERK regulates NADPH oxidase activity , which is associated with a significant generation of oxygen free radicals  and chronic oxidative stress can induce genomic instability . Alternatively, DNA-PKcs is hypothesized to play an important role in maintaining genomic stability  and the increase in DNA-PKcs may reflect effort to maintain stability in an unstable environment.
Ectopic expression of ERK alone is sufficient to induce phenotypic conversion of C10 cells and this model may provide insight into the underlying molecular determinants of this response. The window between early and late passage ERK-transduced variants that encompasses phenotypic conversion (approximately 15 passages) is a reasonable time frame to enable interrogative studies to define molecular determinants. Our expectation is that causal molecular processes will precede the appearance of the transformed phenotype and will be observed in early passage cells. At present, we have characterized changes in DNMT, DNA damage recognition and repair proteins and phosphatase activities that are selectively altered in late, but not early passage cells, suggesting they are secondary to transformation. Additional studies, perhaps with a more global screening approach, may provide insight into those molecular processes perturbed by ERK overexpression in early passage cells. Alternatively, because DNMTs are under investigation as therapeutic targets for lung cancer, the C10 model may provide insight into the molecular processes that confer sensitivity to DNMT inhibitors and regulate their aberrant expression.
chromosome region maintenance protein 1
DNA-dependent protein kinase catalytic subunit
DNA methyl transferase
extracellular signal regulated kinase
ERK1-green fluorescent protein
mitogen activated protein kinase
xeroderma pigmentosum complementation group A.
- Grozio A, Catassi A, Cavalieri Z, Paleari L, Cesario A, Russo P: Nicotine, lung and cancer. Anticancer Agents Med Chem. 2007, 7: 461-466.PubMedView ArticleGoogle Scholar
- Zhang J, Lodish HF: Constitutive activation of the MEK/ERK pathway mediates all effects of oncogenic H-ras expression in primary erythroid progenitors. Blood. 2004, 104: 1679-1687. 10.1182/blood-2004-04-1362.PubMedView ArticleGoogle Scholar
- Chung E, Hsu CL, Kondo M: Constitutive MAP kinase activation in hematopoietic stem cells induces a myeloproliferative disorder. PLoS One. 2011, 6: e28350-10.1371/journal.pone.0028350.PubMedPubMed CentralView ArticleGoogle Scholar
- Scholl FA, Dumesic PA, Khavari PA: Effects of active MEK1 expression in vivo. Cancer Lett. 2005, 230: 1-5. 10.1016/j.canlet.2004.12.013.PubMedView ArticleGoogle Scholar
- Boucher MJ, Jean D, Vezina A, Rivard N: Dual role of MEK/ERK signaling in senescence and transformation of intestinal epithelial cells. Am J Physiol Gastrointest Liver Physiol. 2004, 286: G736-G746. 10.1152/ajpgi.00453.2003.PubMedView ArticleGoogle Scholar
- Welch DR, Sakamaki T, Pioquinto R, Leonard TO, Goldberg SF, Hon Q, Erikson RL, Rieber M, Rieber MS, Hicks DJ, et al: Transfection of constitutively active mitogen-activated protein/extracellular signal-regulated kinase kinase confers tumorigenic and metastatic potentials to NIH3T3 cells. Cancer Res. 2000, 60: 1552-1556.PubMedGoogle Scholar
- Alessandrini A, Greulich H, Huang W, Erikson RL: Mek1 phosphorylation site mutants activate Raf-1 in NIH 3 T3 cells. J Biol Chem. 1996, 271: 31612-31618. 10.1074/jbc.271.49.31612.PubMedView ArticleGoogle Scholar
- Shukla A, Timblin C, BeruBe K, Gordon T, McKinney W, Driscoll K, Vacek P, Mossman BT: Inhaled particulate matter causes expression of nuclear factor (NF)-kappaB-related genes and oxidant-dependent NF-kappaB activation in vitro. Am J Respir Cell Mol Biol. 2000, 23: 182-187.PubMedView ArticleGoogle Scholar
- Papaiahgari S, Zhang Q, Kleeberger SR, Cho HY, Reddy SP: Hyperoxia stimulates an Nrf2-ARE transcriptional response via ROS-EGFR-PI3K-Akt/ERK MAP kinase signaling in pulmonary epithelial cells. Antioxid Redox Signal. 2006, 8: 43-52. 10.1089/ars.2006.8.43.PubMedView ArticleGoogle Scholar
- Lounsbury KM, Stern M, Taatjes D, Jaken S, Mossman BT: Increased localization and substrate activation of protein kinase C delta in lung epithelial cells following exposure to asbestos. Am J Pathol. 2002, 160: 1991-2000. 10.1016/S0002-9440(10)61149-2.PubMedPubMed CentralView ArticleGoogle Scholar
- Buder-Hoffmann S, Palmer C, Vacek P, Taatjes D, Mossman B: Different accumulation of activated extracellular signal-regulated kinases (ERK 1/2) and role in cell-cycle alterations by epidermal growth factor, hydrogen peroxide, or asbestos in pulmonary epithelial cells. Am J Respir Cell Mol Biol. 2001, 24: 405-413.PubMedView ArticleGoogle Scholar
- Malkinson AM, Dwyer-Nield LD, Rice PL, Dinsdale D: Mouse lung epithelial cell lines–tools for the study of differentiation and the neoplastic phenotype. Toxicology. 1997, 123: 53-100. 10.1016/S0300-483X(97)00108-X.PubMedView ArticleGoogle Scholar
- Wardlaw SA, Zhang N, Belinsky SA: Transcriptional regulation of basal cyclooxygenase-2 expression in murine lung tumor-derived cell lines by CCAAT/enhancer-binding protein and activating transcription factor/cAMP response element-binding protein. Mol Pharmacol. 2002, 62: 326-333. 10.1124/mol.62.2.326.PubMedView ArticleGoogle Scholar
- Smith GJ, Le Mesurier SM, de Montfort ML, Lykke AW: Development and characterization of type 2 pneumocyte-related cell lines from normal adult mouse lung. Pathology. 1984, 16: 401-405. 10.3109/00313028409084730.PubMedView ArticleGoogle Scholar
- Kitamura T, Onishi M, Kinoshita S, Shibuya A, Miyajima A, Nolan GP: Efficient screening of retroviral cDNA expression libraries. Proc Natl Acad Sci U S A. 1995, 92: 9146-9150. 10.1073/pnas.92.20.9146.PubMedPubMed CentralView ArticleGoogle Scholar
- Lenormand P, Sardet C, Pages G, L’Allemain G, Brunet A, Pouyssegur J: Growth factors induce nuclear translocation of MAP kinases (p42mapk and p44mapk) but not of their activator MAP kinase kinase (p45mapkk) in fibroblasts. J Cell Biol. 1993, 122: 1079-1088. 10.1083/jcb.122.5.1079.PubMedView ArticleGoogle Scholar
- Zuckerbraun BS, Shapiro RA, Billiar TR, Tzeng E: RhoA influences the nuclear localization of extracellular signal-regulated kinases to modulate p21Waf/Cip1 expression. Circulation. 2003, 108: 876-881. 10.1161/01.CIR.0000081947.00070.07.PubMedView ArticleGoogle Scholar
- Bates RC, Mercurio AM: The epithelial-mesenchymal transition (EMT) and colorectal cancer progression. Cancer Biol Ther. 2005, 4: 365-370. 10.4161/cbt.4.4.1655.PubMedView ArticleGoogle Scholar
- Weber TJ, Markillie LM, Chrisler WB, Vielhauer GA, Regan JW: Modulation of JB6 mouse epidermal cell transformation response by the prostaglandin F2alpha receptor. Mol Carcinog. 2002, 35: 163-172. 10.1002/mc.10079.PubMedView ArticleGoogle Scholar
- Suzukawa K, Weber TJ, Colburn NH: AP-1, NF-kappa-B, and ERK activation thresholds for promotion of neoplastic transformation in the mouse epidermal JB6 model. Environ Health Perspect. 2002, 110: 865-870.PubMedPubMed CentralView ArticleGoogle Scholar
- Deng C, Yang J, Scott J, Hanash S, Richardson BC: Role of the ras-MAPK signaling pathway in the DNA methyltransferase response to DNA hypomethylation. Biol Chem. 1998, 379: 1113-1120.PubMedView ArticleGoogle Scholar
- Scheinbart LS, Johnson MA, Gross LA, Edelstein SR, Richardson BC: Procainamide inhibits DNA methyltransferase in a human T cell line. J Rheumatol. 1991, 18: 530-534.PubMedGoogle Scholar
- Bird A: Perceptions of epigenetics. Nature. 2007, 447: 396-398. 10.1038/nature05913.PubMedView ArticleGoogle Scholar
- Weber TJ, Shankaran H, Wiley HS, Opresko LK, Chrisler WB, Quesenberry RD: Basic fibroblast growth factor regulates persistent ERK oscillations in premalignant but not malignant JB6 cells. J Invest Dermatol. 2010, 130: 1444-1456. 10.1038/jid.2009.383.PubMedView ArticleGoogle Scholar
- Lengauer C, Kinzler KW, Vogelstein B: Genetic instabilities in human cancers. Nature. 1998, 396: 643-649. 10.1038/25292.PubMedView ArticleGoogle Scholar
- Morgan WF: Non-targeted and delayed effects of exposure to ionizing radiation: II. Radiation-induced genomic instability and bystander effects in vivo, clastogenic factors and transgenerational effects. Radiat Res. 2003, 159: 581-596. 10.1667/0033-7587(2003)159[0581:NADEOE]2.0.CO;2.PubMedView ArticleGoogle Scholar
- Pandey D, Fulton DJ: Molecular regulation of NADPH oxidase 5 via the MAPK pathway. Am J Physiol Heart Circ Physiol. 2011, 300: H1336-H1344. 10.1152/ajpheart.01163.2010.PubMedPubMed CentralView ArticleGoogle Scholar
- Abramov AY, Jacobson J, Wientjes F, Hothersall J, Canevari L, Duchen MR: Expression and modulation of an NADPH oxidase in mammalian astrocytes. J Neurosci. 2005, 25: 9176-9184. 10.1523/JNEUROSCI.1632-05.2005.PubMedView ArticleGoogle Scholar
- Limoli CL, Giedzinski E, Morgan WF, Swarts SG, Jones GD, Hyun W: Persistent oxidative stress in chromosomally unstable cells. Cancer Res. 2003, 63: 3107-3111.PubMedGoogle Scholar
- Marampon F, Gravina GL, Di Rocco A, Bonfili P, Di Staso M, Fardella C, Polidoro L, Ciccarelli C, Festuccia C, Popov VM, et al: MEK/ERK inhibitor U0126 increases the radiosensitivity of rhabdomyosarcoma cells in vitro and in vivo by downregulating growth and DNA repair signals. Mol Cancer Ther. 2011, 10: 159-168. 10.1158/1535-7163.MCT-10-0631.PubMedPubMed CentralView ArticleGoogle Scholar
- Tang M, Xu W, Wang Q, Xiao W, Xu R: Potential of DNMT and its Epigenetic Regulation for Lung Cancer Therapy. Curr Genomics. 2009, 10: 336-352. 10.2174/138920209788920994.PubMedPubMed CentralView ArticleGoogle Scholar
- Geiman TM, Sankpal UT, Robertson AK, Zhao Y, Robertson KD: DNMT3B interacts with hSNF2H chromatin remodeling enzyme, HDACs 1 and 2, and components of the histone methylation system. Biochem Biophys Res Commun. 2004, 318: 544-555. 10.1016/j.bbrc.2004.04.058.PubMedView ArticleGoogle Scholar
- Monsey MS, Ota KT, Akingbade IF, Hong ES, Schafe GE: Epigenetic alterations are critical for fear memory consolidation and synaptic plasticity in the lateral amygdala. PLoS One. 2011, 6: e19958-10.1371/journal.pone.0019958.PubMedPubMed CentralView ArticleGoogle Scholar
- Weber TJ, Monks TJ, Lau SS: PGE2-mediated cytoprotection in renal epithelial cells: evidence for a pharmacologically distinct receptor. Am J Physiol. 1997, 273: F507-F515.PubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.