journal homepage: www.elsevier.com/locate/yexcr Available online at www.sciencedirect.com Research Article Regulation of nonmuscle myosin II during 3-methylcholanthrene induced dedifferentiation of C2C12 myotubes Sumit K. Dey, Shekhar Saha, Provas Das, Mahua R. Das, Siddhartha S. Jana n Q1 Department of Biological Chemistry, Indian Association for the Cultivation of Science, Jadavpur, Kolkata 32, India articleinformation Article Chronology: Received 17 February 2014 Received in revised form 19 May 2014 Accepted 20 May 2014 Keywords: Myotubes Nonmuscle myosin II Fragmentation Dedifferentiation 3-Methylcholanthrene Myosin regulatory light chain abstract 3-Methylcholanthrene (3MC) induces tumor formation at the site of injection in the hind leg of mice within 110 days. Recent reports reveal that the transformation of normal muscle cells to atypical cells is one of the causes for tumor formation, however the molecular mechanism behind this process is not well understood. Here, we show in an in vitro study that 3MC induces fragmentation of multinucleate myotubes into viable mononucleates. These mononucleates form colonies when they are seeded into soft agar, indicative of cellular transformation. Immunoblot analysis reveals that phosphorylation of myosin regulatory light chain (RLC 20 ) is 5.670.5 fold reduced in 3MC treated myotubes in comparison to vehicle treated myotubes during the fragmentation of myotubes. In contrast, levels of myogenic factors such as MyoD, Myogenin and cell cycle regulators such as Cyclin D, Cyclin E1 remain unchanged as assessed by real-time PCR array and reverse transcriptase PCR analysis, respectively. Interestingly, addition of the myosin light chain kinase inhibitor, ML-7, enhances the fragmentation, whereas phosphatase inhibitor perturbs the 3MC induced fragmentation of myotubes. These results suggest that decrease in RLC 20 phosphorylation may be associated with the fragmentation step of dedifferentiation. & 2014 Published by Elsevier Inc. Introduction The polycyclic aromatic hydrocarbon, 3-Methylcholanthrene (3MC), induces tumor formation in humans and other living organisms [1]. Several studies have revealed that 3MC exerts most of its tumori- genic effect by introducing mutations in genes such as ras and p53 [2,3], which promote cell proliferation, and by modulating structural changes in DNA such as perturbation in vertical nucleotide stacking and the phosphodiester-deoxyribose backbone [4,5]. These muta- tions in ras and p53, and structural changes in DNA are expected to influence gene expression and the fidelity of replication and transcription, having a pivotal effect on the neoplastic transforma- tion of normal tissue [6–9]. A 3MC-induced mouse tumor is a model system for studying the molecular mechanism behind the development of tumors. Koebel et al. [10] showed that cellular transformation of fibroblast-like 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 http://dx.doi.org/10.1016/j.yexcr.2014.05.015 0014-4827/& 2014 Published by Elsevier Inc. Abbreviations: 3MC, 3-Methylcholanthrene; NM II, nonmuscle myosin II; NMHC, nonmuscle myosin heavy chain; GM, growth medium; DM, differentiation medium; RLC 20 , regulatory light chain; DMEM, Dulbecco's Modified Eagle's Medium; BrdU, 5-bromo-2 0 -deoxyuridine n Corresponding author. Fax: þ91 33 2483 6561. E-mail addresses: bcssj@iacs.res.in, siddhartha.jana@gmail.com (S. S. Jana). EXPERIMENTAL CELL RESEARCH ] ( ]]]] ) ]]] – ]]] Please cite this article as: S. K. Dey, et al., Regulation of nonmuscle myosin II during 3-methylcholanthrene induced dedifferentiation of C2C12 myotubes, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.05.015