BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 225, 427–435 (1996) ARTICLE NO. 1190 Decreased Expression of a Single Tropomyosin Isoform, TM5/TM30nm, Results in Reduction in Motility of Highly Metastatic B16-F10 Mouse Melanoma Cells Kenji Miyado,* , ² ,1 Minoru Kimura,* and Shun’ichiro Taniguchi² , ‡ *Molecular Medicine Research Center, The Institute of Medical Sciences, Tokai University, Isehara 259-11, Japan; ² Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, Fukuoka 812, Japan; and ‡Department of Molecular Oncology and Angiology, Research Center on Aging and Adaptation, Shinsyu University School of Medicine, Matsumoto 390, Japan Received July 10, 1996 Tropomyosin is an actin-associated cytoskeletal protein expressed in muscle and non-muscle cells. There are several tropomyosin isoforms, and their cellular expression is known to be associated with transformation events caused by retroviral infection and chemical mutagens. We found that expression of a low-molecular weight tropomyosin isoform, TM5/TM30nm, was higher in a high-metastatic B16 mouse melanoma cell line, B16-F10, than in B16-F1, a low-metastatic mouse melanoma cell line. In order to determine whether this elevated level of TM5/TM30nm plays a role in malignant phenotype, B16-F10 cells were transfected with recombinant DNA containing antisense rat TM5/TM30nm cDNA linked to the human metallothionein- IIa promoter, which is inducible by heavy metals such as zinc and cadmium. When the stably transfected clones were treated with ZnSO 4 , decreased expression of TM5/TM30nm and reduction in cell motility, which is thought to be an indicator of cellular malignancy, were observed. These findings suggest that TM5/TM30nm plays a fundamental role in regulating cell motility, which is essential for metastasis and invasion of tumor cells.  1996 Academic Press, Inc. Tropomyosins (TMs) are actin-associated cytoskeletal proteins of muscle and non-muscle cells and integral components of the actin-based contractile apparatus. Multiple isoforms of TMs are found in vertebrate non-muscle cells (1-3). These non-muscle TM isoforms differ in size, from the 284 residues typical of muscle TMs, the so-called high-molecular weight TMs including TM1, TM2 and TM3, to 248 residues (1), the so-called low-molecular weight TMs including TM4, TM5a, TM5b and TM30nm (also termed TM5 for mouse; hereafter referred to as TM5/TM30nm), which is ubiquitously expressed in non-muscle cells. These different isoforms are encoded by a multigene family, and in mammals four TM genes have been characterized. TM5/TM30nm is encoded by a single gene located on a chromosomal locus different from those of other TM genes, and thus is probably regulated in a different manner from the other TM genes (4). Although it can be expected that each isoform has unique functional characteristics, e.g., for actin-binding and polymerization, this has yet to be demonstrated. In yeast, disruption of a TM gene results in reduced growth rate, heterogeneity in cell size, and disruption of actin cables (5). These findings suggest that TM plays a structural and/or regulatory role in the organization of microfilaments. In mammals, numerous studies have suggested that decrease 1 All correspondence should be addressed to Kenji Miyado, Department of Molecular and Developmental Science, Molecular Medicine Research Center, The Institute of Medical Sciences, Tokai University, Bohseidai, Isehara, Kana- gawa 259-11, Japan. Fax: /81/463-94-8524. Abbreviations: TMs, tropomyosins; UTR, untranslated region; EBNA, EB virus-associated nuclear antigen I; Hygr R , hygromycin-resistant gene; MI, motility index. 0006-291X/96 $18.00 Copyright  1996 by Academic Press, Inc. All rights of reproduction in any form reserved. 427