腫瘤抑制蛋白p53 (Tumor protein 53)為最早被發現的腫瘤抑制基因，目前已知當DNA受到外界影響而受損時，細胞便會誘導p53表現，透過影響細胞週期或細胞凋亡，避免使不正常的細胞繼續生長；然而p53在肌肉細胞中所扮演的角色目前並不清楚。因此本研究透過肌肉細胞C2C12，探討p53在肌肉發育過程中所造成的影響。本實驗使用p53或M3-p53 (p53基因與MyoD的轉錄激活區域），觀察如何影響肌肉細胞的分化。結果顯示， 大量表現p53使細胞週期停滯於G1 / G0、降低細胞生長速度、提高細胞的氧化壓力及誘導p21和Rb1的表現量。雖然p53造成細胞週期停滯，但並沒有降低細胞週期蛋白Cyclin D的表現量，並且在M3-p53大量表現時也呈現相同的結果；但M3-p53會稍微的減少Cyclin D表現。此外，我們還探討了p53及M3-p53對肌肉細胞分化的影響，結果顯示兩者皆會減少肌肉細胞分化，並且抑制分化相關基因Mef2c、Myogenin及MRF4的表現；提高Myf5的表現量。實驗結果表示，p53和M3-p53會延遲肌肉細胞分化過程，但並不會抑制肌肉細胞的分化程度。本研究接下來可探討，大量表現被p53和M3-p53抑制的Mef2c或Myogenin，是否可回復肌肉細胞的分化比例。將來可透過此方式提高肌肉細胞的分化，用於治療肌肉相關疾病。

The overexpression of p53 has been known widely to be stimulated by cellular stress; however, its aberrant towards muscle development that leads to muscle disease is not apparent. Therefore, we are investigating how p53 or M3-p53, a chimeric gene of p53 and the activation domain of MyoD, affects cell cycle, metabolism, and muscle differentiation in myoblast. Up-regulation of both genes exhibited interchangeable results. The over-activation of p53 caused cell cycle progression to arrest at G1/G0, increased oxidative stress, decreased cell viability, and up-regulated p21 and Rb1. Intriguingly, Cyclin D was not repressed. Meanwhile, M3-p53 induction also exhibited similar results. It arrested cell cycle at G0/G1, reduced cell number, and up-regulated oxidative stress, p21, and Rb1 but slightly induced cyclin D. We also explored the effects at myotube stage in which the activation of p53 showed lesser fusion index value than uninduced cells as well as M3-p53. Reduction of late differentiation marker, Myosin, was confirmed at the protein level. At this stage, p53 and M3-p53 activation shared a similar result in repressing Mef2c, Myogenin, MRF4, and inducing Myf5. These results suggest that p53 or M3-p53 activation leads to delay but not to terminate the differentiation program. Further research would be performing the restoration of Mef2c or Myogenin in excessive activation of p53 or M3-p53 at the myotube formation stage. Expectedly, this strategy could be developed to enhance the differentiation that may have the prospect to cure muscle-related disease.

Agarwal, M.L., Agarwal, A., Taylor, W.R., and Stark, G.R. (1995). p53 controls both the G2/M and the G1 cell cycle checkpoints and mediates reversible growth arrest in human fibroblasts. Proc Natl Acad Sci U S A 92, 8493-8497.
Barnum, K.J., and O'Connell, M.J. (2014). Cell cycle regulation by checkpoints. Methods Mol Biol 1170, 29-40.
Beaudry, M., Hidalgo, M., Launay, T., Bello, V., and Darribere, T. (2016). Regulation of myogenesis by environmental hypoxia. J Cell Sci 129, 2887-2896.
Benson, E.K., Mungamuri, S.K., Attie, O., Kracikova, M., Sachidanandam, R., Manfredi, J.J., and Aaronson, S.A. (2014). p53-dependent gene repression through p21 is mediated by recruitment of E2F4 repression complexes. Oncogene 33, 3959-3969.
Bentzinger, C.F., Wang, Y.X., and Rudnicki, M.A. (2012). Building muscle: molecular regulation of myogenesis. Cold Spring Harb Perspect Biol 4.
Cam, H., Griesmann, H., Beitzinger, M., Hofmann, L., Beinoraviciute-Kellner, R., Sauer, M., Huttinger-Kirchhof, N., Oswald, C., Friedl, P., Gattenlohner, S., et al. (2006). p53 family members in myogenic differentiation and rhabdomyosarcoma development. Cancer Cell 10, 281-293.
Carvajal, L.A., Hamard, P.J., Tonnessen, C., and Manfredi, J.J. (2012). E2F7, a novel target, is up-regulated by p53 and mediates DNA damage-dependent transcriptional repression. Genes Dev 26, 1533-1545.
Chao, H.X., Fakhreddin, R.I., Shimerov, H.K., Kedziora, K.M., Kumar, R.J., Perez, J., Limas, J.C., Grant, G.D., Cook, J.G., Gupta, G.P., et al. (2019). Evidence that the human cell cycle is a series of uncoupled, memoryless phases. Mol Syst Biol 15, e8604.
Di Paolo, C., Muller, Y., Thalmann, B., Hollert, H., and Seiler, T.B. (2018). p53 induction and cell viability modulation by genotoxic individual chemicals and mixtures. Environ Sci Pollut Res Int 25, 4012-4022.
Du, Z., Tong, X., and Ye, X. (2013). Cyclin D1 promotes cell cycle progression through enhancing NDR1/2 kinase activity independent of cyclin-dependent kinase 4. J Biol Chem 288, 26678-26687.
Ehrlich, P.F., and Shamberger, R.C. (2012). Wilms't Tumor In Pediatric Surgery A.G. Coran, ed. (Elsevier).
Faralli, H., and Dilworth, F.J. (2012). Turning on Myogenin in Muscle: A paradigm for understanding mechanisms of tissue-specific gene expression. Comp Funct Genomics, 836374.
Francetic, T., and Li, Q. (2011). Skeletal myogenesis and Myf5 activation. Transcription 2, 109-114.
Fricano-Kugler, C.J., Williams, M.R., Salinaro, J.R., Li, M., and Luikart, B. (2016). Designing, Packaging, and Delivery of High Titer CRISPR Retro and Lentiviruses via Stereotaxic Injection. J Vis Exp.
Giacinti, C., and Giordano, A. (2006). RB and cell cycle progression. Oncogene 25, 5220-5227.
Godefroy, N., Lemaire, C., Mignotte, B., and Vayssiere, J.L. (2006). p53 and Retinoblastoma protein (pRb): a complex network of interactions. Apoptosis 11, 659-661.
Gossett, L.A., Kelvin, D.J., Sternberg, E.A., and Olson, E.N. (1989). A new myocyte-specific enhancer-binding factor that recognizes a conserved element associated with multiple muscle-specific genes. Mol Cell Biol 9, 5022-5033.
Haber, D., and Harlow, E. (1997). Tumour-suppressor genes: evolving definitions in the genomic age. Nat Genet 16, 320-322.
Halevy, O. (1993). p53 gene is up-regulated during skeletal muscle cell differentiation. Biochem Biophys Res Commun 192, 714-719.
He, G., Siddik, Z.H., Huang, Z., Wang, R., Koomen, J., Kobayashi, R., Khokhar, A.R., and Kuang, J. (2005). Induction of p21 by p53 following DNA damage inhibits both Cdk4 and Cdk2 activities. Oncogene 24, 2929-2943.
Hirai, H., Tani, T., Katoku-Kikyo, N., Kellner, S., Karian, P., Firpo, M., and Kikyo, N. (2011). Radical acceleration of nuclear reprogramming by chromatin remodeling with the transactivation domain of MyoD. Stem Cells 29, 1349-1361.
Karawajew, L., Rhein, P., Czerwony, G., and Ludwig, W.D. (2005). Stress-induced activation of the p53 tumor suppressor in leukemia cells and normal lymphocytes requires mitochondrial activity and reactive oxygen species. Blood 105, 4767-4775.
Karimian, A., Ahmadi, Y., and Yousefi, B. (2016). Multiple functions of p21 in cell cycle, apoptosis and transcriptional regulation after DNA damage. DNA Repair (Amst) 42, 63-71.
Kastan, M.B., and Kuerbitz, S.J. (1993). Control of G1 arrest after DNA damage. Environ Health Perspect 101 Suppl 5, 55-58.
Kastan, M.B., Onyekwere, O., Sidransky, D., Vogelstein, B., and Craig, R.W. (1991). Participation of p53 protein in the cellular response to DNA damage. Cancer Res 51, 6304-6311.
Kondoh, H., Lleonart, M.E., Gil, J., Wang, J., Degan, P., Peters, G., Martinez, D., Carnero, A., and Beach, D. (2005). Glycolyric enzymes can modulate cellular life span. Cancer Res 65, 177-85.
Ladelva, M.F., Toledo, M.F., Laiseca, J.E., and Monte, M. (2011). Interaction of p53 with tumor suppressive and oncogenic signaling pathways to control cellular reactive oxygen species production. Antioxid Redox Signal 15, 1749-1761.
Levine, A.J. (1997). p53, the cellular gatekeeper for growth and division. Cell 88, 323-331.
Liu, B., Chen, Y., and St Clair, D.K. (2008). ROS and p53: a versatile partnership. Free Radic Biol Med 44, 1529-1535.
Liu, G., and Chen, X. (2006). Regulation of the p53 transcriptional activity. J Cell Biochem 97, 448-458.
Lovering, R.M., Porter, N.C., and Bloch, R.J. (2005). The muscular dystrophies: from genes to therapies. Phys Ther 85, 1372-1388.
Lu, T.C., Zhao, G.H., Chen, Y.Y., Chien, C.Y., Huang, C.H., Lin, K.H., and Chen, S.L. (2016). Transduction of Recombinant M3-p53-R12 Protein Enhances Human Leukemia Cell Apoptosis. J Cancer 7, 1360-1373.
Migdal, C., and Serres, M. (2011). [Reactive oxygen species and oxidative stress]. Med Sci (Paris) 27, 405-412.
Molchadsky, A., Rivlin, N., Brosh, R., Rotter, V., and Sarig, R. (2010). p53 is balancing development, differentiation and de-differentiation to assure cancer prevention. Carcinogenesis 31, 1501-1508.
Molkentin, J.D., Black, B.L., Martin, J.F., and Olson, E.N. (1995). Cooperative activation of muscle gene expression by MEF2 and myogenic bHLH proteins. Cell 83, 1125-1136.
Moretti, I., Ciciliot, S., Dyar, K.A., Abraham, R., Murgia, M., Agatea, L., Akimoto, T., Bicciato, S., Forcato, M., Pierre, P., et al. (2016). MRF4 negatively regulates adult skeletal muscle growth by repressing MEF2 activity. Nat Commun 7, 12397.
Moulder, D.E., Hatoum, D., Tay, E., Lin, Y., and McGowan, E.M. (2018). The Roles of p53 in Mitochondrial Dynamics and Cancer Metabolism: The Pendulum between Survival and Death in Breast Cancer? Cancers (Basel) 10.
Ozaki, T., and Nakagawara, A. (2011). Role of p53 in Cell Death and Human Cancers. Cancers (Basel) 3, 994-1013.
Puzio-Kuter, A.M. (2011). The Role of p53 in Metabolic Regulation. Genes Cancer 2, 385-391.
Ridgeway, A.G., Wilton, S., and Skerjanc, I.S. (2000). Myocyte enhancer factor 2C and myogenin up-regulate each other's expression and induce the development of skeletal muscle in P19 cells. J Biol Chem 275, 41-46.
Sambasivan, R., and Tajbakhsh, S. (2007). Skeletal muscle stem cell birth and properties. Semin Cell Dev Biol 18, 870-882.
Schieber, M., and Chandel, N.S. (2014). ROS function in redox signaling and oxidative stress. Curr Biol 24, R453-462.
Singh, B.N., Rao, K.S., and Rao Ch, M. (2010). Ubiquitin-proteasome-mediated degradation and synthesis of MyoD is modulated by alphaB-crystallin, a small heat shock protein, during muscle differentiation. Biochim Biophys Acta 1803, 288-299.
Soddu, S., Blandino, G., Scardigli, R., Coen, S., Marchetti, A., Rizzo, M.G., Bossi, G., Cimino, L., Crescenzi, M., and Sacchi, A. (1996). Interference with p53 protein inhibits hematopoietic and muscle differentiation. J Cell Biol 134, 193-204.
Sun, X., Guo, W., Shen, J.K., Mankin, H.J., Hornicek, F.J., and Duan, Z. (2015). Rhabdomyosarcoma: Advances in Molecular and Cellular Biology. Sarcoma 2015, 232010.
Tamir, Y., and Bengal, E. (1998). p53 protein is activated during muscle differentiation and participates with MyoD in the transcription of muscle creatine kinase gene. Oncogene 17, 347-356.
Waldman, T., Kinzler, K.W., and Vogelstein, B. (1995). p21 is necessary for the p53-mediated G1 arrest in human cancer cells. Cancer Res 55, 5187-5190.
Weintraub, H., Hauschka, S., and Tapscott, S.J. (1991). The MCK enhancer contains a p53 responsive element. Proc Natl Acad Sci U S A 88, 4570-4571.
Wu, M.P. (2013). Enhancing Myoblast Fusion for Therapy of Muscular Dystrophies (Harvard University).
Yang, Z.J., Broz, D.K., Noderer, W.L., Ferreira, J.P., Overton, K.W., Spencer, S.L., Meyer, T., Tapscott, S.J., Attardi, L.D., and Wang, C.L. (2015). p53 suppresses muscle differentiation at the myogenin step in response to genotoxic stress. Cell Death Differ 22, 560-573.
Yee, K.S., and Vousden, K.H. (2005). Complicating the complexity of p53. Carcinogenesis 26, 1317-1322.
Zilfou, J.T., and Lowe, S.W. (2009). Tumor suppressive functions of p53. Cold Spring Harb Perspect Biol 1, a001883.