臺灣博碩士論文加值系統

MBNL3屬於Muscleblind-like (MBNL) 基因家族，與成人中常見的神經肌肉疾病-強直型肌肉萎縮症(DM)-之病理機制有密切關係。已知MBNL3主要表現在高度增生的組織且會抑制肌肉分化的進行，但其在細胞週期調節所扮演的角色尚不清楚。本論文以病毒干擾性核醣核酸技術抑制MBNL3表現，研究MBNL3功能降低對增生中肌母細胞(C2C12)的影響。我們首先建立持續表現MBNL3 shRNA之細胞並獲得三株不同抑制效率之穩定細胞株。由細胞計數和MTT assay實驗結果顯示抑制MBNL3表現會降低C2C12細胞增生速率，將細胞染propidium iodide (PI)並以流式細胞儀分析發現抑制MBNL3表現會造成G1族群比例上升，S族群比例下降。進一步利用real-time PCR及西方點墨法調查調節細胞週期相關基因表現發現其cyclin D1表現量明顯下降，並影響到下游 pRB的磷酸化。另外我們同時也觀察到此類細胞呈擴大且扁平等異常型態，測量證實其直徑變大，而進行老化指標beta-galactosidase染色也發現老化比例明顯增加。若將MBNL3及cyclin D1 cDNA表現於MBNL3 shRNA之細胞，則會顯著回復其正常的增生速率及減少老化細胞比例。這些結果表示MBNL3功能喪失(loss-of-function)會透過cyclin D1相關路徑減緩肌母細胞生長並誘導其老化。

Muscleblind-like protein 3 (MBNL3) belongs to MBNL family implicated in the pathogenesis of myotonic dystrophy, the most common neuromuscular disease in adults. MBNL3 expression is abundant in proliferating myoblasts and greatly down-regulated during differentiation. In this study, we have investigated the effect of MBNL3 loss-of-function on C2C12 myoblasts by using lentiviral knockdown system. The results indicated that MBNL3 knockdown cells had lower proliferation rate than scramble control as revealed by cell counting and MTT assay. Propidium iodide staining revealed that myoblasts with MBNL3 knockdown were enriched in G1 population. Consistent with these findings, real-time PCR and western blotting analyses revealed a decrease in cyclin D1 and S780 phosphorylated pRb protein level considered critical for G1 phase progression. In addition, microscopic observation and measurement of the cell size revealed a flattened and enlarge beta morphology, characteristic of senescent cells. Further senescence-associated beta-galactosidase (SA-beta-gal) assay confirmed that a significant portion of MBNL3 knockdown cells entered senescent state prematurely. Overexpression of MBNL3 and cyclin D1 significantly restored the proliferation rate and reduced senescence of these cells. Our findings suggest that MBNL3 may regulate G1 progression and senescence through cyclin D1-mediated pathway in C2C12 myoblasts.

中文摘要---------------------------------------------------------------1
英文摘要----------------------------------------------------------------2
序論--------------------------------------------------------------------4
材料與方法--------------------------------------------------------------14
一、細胞培養----------------------------------------------------------14
二、shRNA質體細菌株---------------------------------------------14
三、慢病毒製備-------------------------------------------------------15
四、慢病毒感染-------------------------------------------------------16
五、MBNL3-pLKO AS3W. neo及cyclin D1-pLKO AS3W. neo質體製備---------------16
六、細菌轉型----------------------------------------------------------18
七、及時定量之反轉錄聚合酵素連鎖反應(Real-time PCR)--18
八、蛋白質萃取與西方點墨法-------------------------------------20
九、細胞計數及細胞分裂時間-------------------------------------22
十、MTT Assay--------------------------------------------------------22
十一、細胞大小測量----------------------------------------------------23
十二、細胞週期分析----------------------------------------------------23
十三、老化指標beta-Galactosidase染色--------------------------------24
十四、免疫螢光染色----------------------------------------------------24
結果------------------------------------------------------------------26
一、以慢病毒做為載體將MBNL3 shRNA送入C2C12並確定抑制MBNL3基因表現效率-------26
二、抑制MBNL3表現效率導致C2C12細胞增殖速率變慢--27
三、抑制MBNL3表現效率誘導C2C12細胞老化--------------29
四、抑制MBNL3表現延緩G1細胞週期的進行----------------30
五、抑制MBNL3表現對細胞週期調節及肌肉增生分化相關基因表現的影響---------------30
六、MBNL3是C2C12細胞正常增生所必須--------------------33
七、MBNL3調控細胞週期是透過cyclin D1-mediated路徑--36
討論-------------------------------------------------------------------------------38
圖表-------------------------------------------------------------------------------46
附錄-------------------------------------------------------------------------------65
參考文獻------------------------------------------------------------------------74

1.Adereth, Y., Dammai, V., Kose, N., Li, R., and Hsu, T. (2005). RNA-dependent integrin alpha3 protein localization regulated by the Muscleblind-like protein MLP1. Nat Cell Biol 7, 1240-1247.
2.Alwazzan, M., Newman, E., Hamshere, M.G., and Brook, J.D. (1999). Myotonic dystrophy is associated with a reduced level of RNA from the DMWD allele adjacent to the expanded repeat. Hum Mol Genet 8, 1491-1497.
3.Ben-Porath, I., and Weinberg, R.A. (2005). The signals and pathways activating cellular senescence. Int J Biochem Cell Biol 37, 961-976.
4.Brook, J.D., McCurrach, M.E., Harley, H.G., Buckler, A.J., Church, D., Aburatani, H., Hunter, K., Stanton, V.P., Thirion, J.P., Hudson, T., et al. (1992). Molecular basis of myotonic dystrophy: expansion of a trinucleotide (CTG) repeat at the 3' end of a transcript encoding a protein kinase family member. Cell 69, 385.
5.Buj-Bello, A., Furling, D., Tronchere, H., Laporte, J., Lerouge, T., Butler-Browne, G.S., and Mandel, J.L. (2002). Muscle-specific alternative splicing of myotubularin-related 1 gene is impaired in DM1 muscle cells. Hum Mol Genet 11, 2297-2307.
6.Bigot, A., Klein, A.F., Gasnier, E., Jacquemin, V., Ravassard, P., Butler-Browne, G., Mouly, V., and Furling, D. (2009). Large CTG repeats trigger p16-dependent premature senescence in myotonic dystrophy type 1 muscle precursor cells. Am J Pathol 174, 1435-1442.
7.Campisi, J. (1996). Replicative senescence: an old lives' tale? Cell 84, 497-500.
8.Charlet, B.N., Savkur, R.S., Singh, G., Philips, A.V., Grice, E.A., and Cooper, T.A. (2002). Loss of the muscle-specific chloride channel in type 1 myotonic dystrophy due to misregulated alternative splicing. Mol Cell 10, 45-53.
9.Conejo, R., and Lorenzo, M. (2001). Insulin signaling leading to proliferation, survival, and membrane ruffling in C2C12 myoblasts. J Cell Physiol 187, 96-108.
10.Dimri, G.P., Lee, X., Basile, G., Acosta, M., Scott, G., Roskelley, C., Medrano, E.E., Linskens, M., Rubelj, I., Pereira-Smith, O., et al. (1995). A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc Natl Acad Sci U S A 92, 9363-9367.
11.Fardaei, M., Larkin, K., Brook, J.D., and Hamshere, M.G. (2001). In vivo co-localisation of MBNL protein with DMPK expanded-repeat transcripts. Nucleic Acids Res 29, 2766-2771.
12.Fardaei, M., Rogers, M.T., Thorpe, H.M., Larkin, K., Hamshere, M.G., Harper, P.S., and Brook, J.D. (2002). Three proteins, MBNL, MBLL and MBXL, co-localize in vivo with nuclear foci of expanded-repeat transcripts in DM1 and DM2 cells. Hum Mol Genet 11, 805-814.
13.Ferri, P., Barbieri, E., Burattini, S., Guescini, M., D'Emilio, A., Biagiotti, L., Del Grande, P., De Luca, A., Stocchi, V., and Falcieri, E. (2009). Expression and subcellular localization of myogenic regulatory factors during the differentiation of skeletal muscle C2C12 myoblasts. J Cell Biochem 108, 1302-1317.
14.Furling, D., Coiffier, L., Mouly, V., Barbet, J.P., St Guily, J.L., Taneja, K., Gourdon, G., Junien, C., and Butler-Browne, G.S. (2001). Defective satellite cells in congenital myotonic dystrophy. Hum Mol Genet 10, 2079-2087.
15.Fu, Y.H., Pizzuti, A., Fenwick, R.G., Jr., King, J., Rajnarayan, S., Dunne, P.W., Dubel, J., Nasser, G.A., Ashizawa, T., de Jong, P., et al. (1992). An unstable triplet repeat in a gene related to myotonic muscular dystrophy. Science 255, 1256-1258.
16.Gangoiti, P., Bernacchioni, C., Donati, C., Cencetti, F., Ouro, A., Gomez-Munoz, A., and Bruni, P. (2011). Ceramide 1-phosphate stimulates proliferation of C2C12 myoblasts. Biochimie.
17.Gayraud-Morel, B., Chretien, F., Flamant, P., Gomes, D., Zammit, P.S., and Tajbakhsh, S. (2007). A role for the myogenic determination gene Myf5 in adult regenerative myogenesis. Dev Biol 312, 13-28.
18.Goldstein, S. (1990). Replicative senescence: the human fibroblast comes of age. Science 249, 1129-1133.
19.Grammatikakis, I., Goo, Y.H., Echeverria, G.V., and Cooper, T.A. (2011). Identification of MBNL1 and MBNL3 domains required for splicing activation and repression. Nucleic Acids Res 39, 2769-2780.
20.Hao, M., Akrami, K., Wei, K., De Diego, C., Che, N., Ku, J.H., Tidball, J., Graves, M.C., Shieh, P.B., and Chen, F. (2008). Muscleblind-like 2 (Mbnl2) -deficient mice as a model for myotonic dystrophy. Dev Dyn 237, 403-410.
21.Harper, P.S. (1989). Postoperative complications in myotonic dystrophy. Lancet 2, 1269.
22.Hayflick, L., and Moorhead, P.S. (1961). The serial cultivation of human diploid cell strains. Exp Cell Res 25, 585-621.
23.Ho, T.H., Charlet, B.N., Poulos, M.G., Singh, G., Swanson, M.S., and Cooper, T.A. (2004). Muscleblind proteins regulate alternative splicing. EMBO J 23, 3103-3112.
24.Ho, T.H., Savkur, R.S., Poulos, M.G., Mancini, M.A., Swanson, M.S., and Cooper, T.A. (2005). Colocalization of muscleblind with RNA foci is separable from mis-regulation of alternative splicing in myotonic dystrophy. J Cell Sci 118, 2923-2933.
25.Ishibashi, J., Perry, R.L., Asakura, A., and Rudnicki, M.A. (2005). MyoD induces myogenic differentiation through cooperation of its NH2- and COOH-terminal regions. J Cell Biol 171, 471-482.
26.Janga, S.C., and Vallabhaneni, S. (2011). MicroRNAs AS POST-TRANSCRIPTIONAL MACHINES AND THEIR INTERPLAY WITH CELLULAR NETWORKS. Rna Infrastructure and Networks 722, 59-74.
27.Jansen, G., Groenen, P.J., Bachner, D., Jap, P.H., Coerwinkel, M., Oerlemans, F., van den Broek, W., Gohlsch, B., Pette, D., Plomp, J.J., et al. (1996). Abnormal myotonic dystrophy protein kinase levels produce only mild myopathy in mice. Nat Genet 13, 316-324.
28.Jiang, H., Mankodi, A., Swanson, M.S., Moxley, R.T., and Thornton, C.A. (2004). Myotonic dystrophy type 1 is associated with nuclear foci of mutant RNA, sequestration of muscleblind proteins and deregulated alternative splicing in neurons. Hum Mol Genet 13, 3079-3088.
29.Kanadia, R.N., Johnstone, K.A., Mankodi, A., Lungu, C., Thornton, C.A., Esson, D., Timmers, A.M., Hauswirth, W.W., and Swanson, M.S. (2003). A muscleblind knockout model for myotonic dystrophy. Science 302, 1978-1980.
30.Kanadia, R.N., Shin, J., Yuan, Y., Beattie, S.G., Wheeler, T.M., Thornton, C.A., and Swanson, M.S. (2006). Reversal of RNA missplicing and myotonia after muscleblind overexpression in a mouse poly(CUG) model for myotonic dystrophy. Proc Natl Acad Sci U S A 103, 11748-11753.
31.Kino, Y., Washizu, C., Oma, Y., Onishi, H., Nezu, Y., Sasagawa, N., Nukina, N., and Ishiura, S. (2009). MBNL and CELF proteins regulate alternative splicing of the skeletal muscle chloride channel CLCN1. Nucleic Acids Res 37, 6477-6490.
32.Kitagawa, M., Higashi, H., Jung, H.K., Suzuki-Takahashi, I., Ikeda, M., Tamai, K., Kato, J., Segawa, K., Yoshida, E., Nishimura, S., et al. (1996). The consensus motif for phosphorylation by cyclin D1-Cdk4 is different from that for phosphorylation by cyclin A/E-Cdk2. EMBO J 15, 7060-7069.
33.Kitzmann, M., Carnac, G., Vandromme, M., Primig, M., Lamb, N.J., and Fernandez, A. (1998). The muscle regulatory factors MyoD and myf-5 undergo distinct cell cycle-specific expression in muscle cells. J Cell Biol 142, 1447-1459.
34.Klesert, T.R., Cho, D.H., Clark, J.I., Maylie, J., Adelman, J., Snider, L., Yuen, E.C., Soriano, P., and Tapscott, S.J. (2000). Mice deficient in Six5 develop cataracts: implications for myotonic dystrophy. Nat Genet 25, 105-109.
35.Kurz, D.J., Decary, S., Hong, Y., and Erusalimsky, J.D. (2000). Senescence-associated (beta)-galactosidase reflects an increase in lysosomal mass during replicative ageing of human endothelial cells. J Cell Sci 113 ( Pt 20), 3613-3622.
36.Kuyumcu-Martinez, N.M., Wang, G.S., and Cooper, T.A. (2007). Increased steady-state levels of CUGBP1 in myotonic dystrophy 1 are due to PKC-mediated hyperphosphorylation. Mol Cell 28, 68-78.
37.Ladd, A.N., Charlet, N., and Cooper, T.A. (2001). The CELF family of RNA binding proteins is implicated in cell-specific and developmentally regulated alternative splicing. Mol Cell Biol 21, 1285-1296.
38.Lavoie, J.N., L'Allemain, G., Brunet, A., Muller, R., and Pouyssegur, J. (1996). Cyclin D1 expression is regulated positively by the p42/p44MAPK and negatively by the p38/HOGMAPK pathway. J Biol Chem 271, 20608-20616.
39.Le Grand, F., and Rudnicki, M.A. (2007). Skeletal muscle satellite cells and adult myogenesis. Curr Opin Cell Biol 19, 628-633.
40.Lee, K.S., Cao, Y., Witwicka, H.E., Tom, S., Tapscott, S.J., and Wang, E.H. (2010). RNA-binding protein Muscleblind-like 3 (MBNL3) disrupts myocyte enhancer factor 2 (Mef2) {beta}-exon splicing. J Biol Chem 285, 33779-33787.
41.Lee, K.S., Smith, K., Amieux, P.S., and Wang, E.H. (2008). MBNL3/CHCR prevents myogenic differentiation by inhibiting MyoD-dependent gene transcription. Differentiation 76, 299-309.
42.Lee, K.S., Squillace, R.M., and Wang, E.H. (2007). Expression pattern of muscleblind-like proteins differs in differentiating myoblasts. Biochem Biophys Res Commun 361, 151-155.
43.Liquori, C.L., Ricker, K., Moseley, M.L., Jacobsen, J.F., Kress, W., Naylor, S.L., Day, J.W., and Ranum, L.P. (2001). Myotonic dystrophy type 2 caused by a CCTG expansion in intron 1 of ZNF9. Science 293, 864-867.
44.Lu, X., Timchenko, N.A., and Timchenko, L.T. (1999). Cardiac elav-type RNA-binding protein (ETR-3) binds to RNA CUG repeats expanded in myotonic dystrophy. Hum Mol Genet 8, 53-60.
45.Machuca-Tzili, L.E., Buxton, S., Thorpe, A., Timson, C.M., Wigmore, P., Luther, P.K., and Brook, J.D. (2011). Zebrafish deficient for Muscleblind-like 2 exhibit features of myotonic dystrophy. Dis Model Mech 4, 381-392.
46.Mahadevan, M., Tsilfidis, C., Sabourin, L., Shutler, G., Amemiya, C., Jansen, G., Neville, C., Narang, M., Barcelo, J., O'Hoy, K., et al. (1992). Myotonic dystrophy mutation: an unstable CTG repeat in the 3' untranslated region of the gene. Science 255, 1253-1255.
47.Mankodi, A., Lin, X., Blaxall, B.C., Swanson, M.S., and Thornton, C.A. (2005). Nuclear RNA foci in the heart in myotonic dystrophy. Circ Res 97, 1152-1155.
48.Mankodi, A., Logigian, E., Callahan, L., McClain, C., White, R., Henderson, D., Krym, M., and Thornton, C.A. (2000). Myotonic dystrophy in transgenic mice expressing an expanded CUG repeat. Science 289, 1769-1773.
49.Mankodi, A., Urbinati, C.R., Yuan, Q.P., Moxley, R.T., Sansone, V., Krym, M., Henderson, D., Schalling, M., Swanson, M.S., and Thornton, C.A. (2001). Muscleblind localizes to nuclear foci of aberrant RNA in myotonic dystrophy types 1 and 2. Hum Mol Genet 10, 2165-2170.
50.Marcotte, R., and Wang, E. (2002). Replicative senescence revisited. J Gerontol A Biol Sci Med Sci 57, B257-269.
51.Megeney, L.A., Kablar, B., Garrett, K., Anderson, J.E., and Rudnicki, M.A. (1996). MyoD is required for myogenic stem cell function in adult skeletal muscle. Genes Dev 10, 1173-1183.
52.Michalowski, S., Miller, J.W., Urbinati, C.R., Paliouras, M., Swanson, M.S., and Griffith, J. (1999). Visualization of double-stranded RNAs from the myotonic dystrophy protein kinase gene and interactions with CUG-binding protein. Nucleic Acids Res 27, 3534-3542.
53.Miller, J.W., Urbinati, C.R., Teng-Umnuay, P., Stenberg, M.G., Byrne, B.J., Thornton, C.A., and Swanson, M.S. (2000). Recruitment of human muscleblind proteins to (CUG)(n) expansions associated with myotonic dystrophy. EMBO J 19, 4439-4448.
54.Molkentin, J.D., and Olson, E.N. (1996). Defining the regulatory networks for muscle development. Curr Opin Genet Dev 6, 445-453.
55.Montarras, D., Lindon, C., Pinset, C., and Domeyne, P. (2000). Cultured myf5 null and myoD null muscle precursor cells display distinct growth defects. Biol Cell 92, 565-572.
56.Morisaki, H., Ando, A., Nagata, Y., Pereira-Smith, O., Smith, J.R., Ikeda, K., and Nakanishi, M. (1999). Complex mechanisms underlying impaired activation of Cdk4 and Cdk2 in replicative senescence: roles of p16, p21, and cyclin D1. Exp Cell Res 253, 503-510.
57.Mukhopadhyay, D., Houchen, C.W., Kennedy, S., Dieckgraefe, B.K., and Anant, S. (2003). Coupled mRNA stabilization and translational silencing of cyclooxygenase-2 by a novel RNA binding protein, CUGBP2. Mol Cell 11, 113-126.
58.Napierala, M., and Krzyzosiak, W.J. (1997). CUG repeats present in myotonin kinase RNA form metastable "slippery" hairpins. J Biol Chem 272, 31079-31085.
59.Pascual, M., Vicente, M., Monferrer, L., and Artero, R. (2006). The Muscleblind family of proteins: an emerging class of regulators of developmentally programmed alternative splicing. Differentiation 74, 65-80.
60.Philips, A.V., Timchenko, L.T., and Cooper, T.A. (1998). Disruption of splicing regulated by a CUG-binding protein in myotonic dystrophy. Science 280, 737-741.
61.Pages, G., Lenormand, P., L'Allemain, G., Chambard, J.C., Meloche, S., and Pouyssegur, J. (1993). Mitogen-activated protein kinases p42mapk and p44mapk are required for fibroblast proliferation. Proc Natl Acad Sci U S A 90, 8319-8323.
62.Peeper, D.S., van der Eb, A.J., and Zantema, A. (1994). The G1/S cell-cycle checkpoint in eukaryotic cells. Biochim Biophys Acta 1198, 215-230.
63.Ranum, L.P., Rasmussen, P.F., Benzow, K.A., Koob, M.D., and Day, J.W. (1998). Genetic mapping of a second myotonic dystrophy locus. Nat Genet 19, 196-198.
64.Reddy, S., Smith, D.B., Rich, M.M., Leferovich, J.M., Reilly, P., Davis, B.M., Tran, K., Rayburn, H., Bronson, R., Cros, D., et al. (1996). Mice lacking the myotonic dystrophy protein kinase develop a late onset progressive myopathy. Nat Genet 13, 325-335.
65.Sabourin, L.A., and Rudnicki, M.A. (2000). The molecular regulation of myogenesis. Clin Genet 57, 16-25.
66.Sarkar, P.S., Appukuttan, B., Han, J., Ito, Y., Ai, C., Tsai, W., Chai, Y., Stout, J.T., and Reddy, S. (2000). Heterozygous loss of Six5 in mice is sufficient to cause ocular cataracts. Nat Genet 25, 110-114.
67.Savkur, R.S., Philips, A.V., and Cooper, T.A. (2001). Aberrant regulation of insulin receptor alternative splicing is associated with insulin resistance in myotonic dystrophy. Nat Genet 29, 40-47.
68.Sergeant, N., Sablonniere, B., Schraen-Maschke, S., Ghestem, A., Maurage, C.A., Wattez, A., Vermersch, P., and Delacourte, A. (2001). Dysregulation of human brain microtubule-associated tau mRNA maturation in myotonic dystrophy type 1. Hum Mol Genet 10, 2143-2155.
69.Seger, R., and Krebs, E.G. (1995). The MAPK signaling cascade. FASEB J 9, 726-735.
70.Squillace, R.M., Chenault, D.M., and Wang, E.H. (2002). Inhibition of muscle differentiation by the novel muscleblind-related protein CHCR. Dev Biol 250, 218-230.
71.Stein, G.H., Drullinger, L.F., Soulard, A., and Dulic, V. (1999). Differential roles for cyclin-dependent kinase inhibitors p21 and p16 in the mechanisms of senescence and differentiation in human fibroblasts. Mol Cell Biol 19, 2109-2117.
72.Taneja, K.L., McCurrach, M., Schalling, M., Housman, D., and Singer, R.H. (1995). Foci of trinucleotide repeat transcripts in nuclei of myotonic dystrophy cells and tissues. J Cell Biol 128, 995-1002.
73.Tian, B., White, R.J., Xia, T., Welle, S., Turner, D.H., Mathews, M.B., and Thornton, C.A. (2000). Expanded CUG repeat RNAs form hairpins that activate the double-stranded RNA-dependent protein kinase PKR. RNA 6, 79-87.
74.Timchenko, L.T., Miller, J.W., Timchenko, N.A., DeVore, D.R., Datar, K.V., Lin, L., Roberts, R., Caskey, C.T., and Swanson, M.S. (1996). Identification of a (CUG)n triplet repeat RNA-binding protein and its expression in myotonic dystrophy. Nucleic Acids Res 24, 4407-4414.
75.Timchenko, N.A., Welm, A.L., Lu, X., and Timchenko, L.T. (1999). CUG repeat binding protein (CUGBP1) interacts with the 5' region of C/EBPbeta mRNA and regulates translation of C/EBPbeta isoforms. Nucleic Acids Res 27, 4517-4525.
76.Wang, E. (1995). Senescent human fibroblasts resist programmed cell death, and failure to suppress bcl2 is involved. Cancer Res 55, 2284-2292.
77.Wang, G.S., Kearney, D.L., De Biasi, M., Taffet, G., and Cooper, T.A. (2007). Elevation of RNA-binding protein CUGBP1 is an early event in an inducible heart-specific mouse model of myotonic dystrophy. J Clin Invest 117, 2802-2811.
78.Weinberg, R.A. (1995). The retinoblastoma protein and cell cycle control. Cell 81, 323-330.
79.Wei, Q., and Paterson, B.M. (2001). Regulation of MyoD function in the dividing myoblast. FEBS Lett 490, 171-178.
80.Westerlaken, J.H., Van der Zee, C.E., Peters, W., and Wieringa, B. (2003). The DMWD protein from the myotonic dystrophy (DM1) gene region is developmentally regulated and is present most prominently in synapse-dense brain areas. Brain Res 971, 116-127.
81.Zarkowska, T., and Mittnacht, S. (1997). Differential phosphorylation of the retinoblastoma protein by G1/S cyclin-dependent kinases. J Biol Chem 272, 12738-12746.
82.Zhang, J.M., Wei, Q., Zhao, X., and Paterson, B.M. (1999). Coupling of the cell cycle and myogenesis through the cyclin D1-dependent interaction of MyoD with cdk4. EMBO J 18, 926-933.
83.Zhang, H. (2007). Molecular signaling and genetic pathways of senescence: Its role in tumorigenesis and aging. J Cell Physiol 210, 567-574.