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Regulation of Microfilament Organization and Anchorage- Independent Growth by Tropomyosin 1 JEFF BOYD*, JOHN I

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Regulation of Microfilament Organization and Anchorage- Independent Growth by Tropomyosin 1 JEFF BOYD*, JOHN I Proc. Natl. Acad. Sci. USA Vol. 92, pp. 11534-11538, December 1995 Cell Biology Regulation of microfilament organization and anchorage- independent growth by tropomyosin 1 JEFF BOYD*, JOHN I. RISINGER, ROGER W. WISEMAN, B. ALEX MERRICK, JAMES K. SELKIRK, AND J. CARL BARRETTt Laboratory of Molecular Carcinogenesis, Environmental Carcinogenesis Program, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC Communicated by Martin Rodbell, National Institute of Environmental Health Sciences, Research Triangle Park NC, July 12 1995 (received for review March 6, 1995) ABSTRACT Variants of chemically immortalized Syrian To explore the molecular basis of anchorage-independent hamster embryo cells that had either retained (supB+) or lost growth and the potential relationship of the cytoskeleton to (supB-) the ability to suppress tumorigenicity when hybrid- this phenomenon, we utilized a series of Syrian hamster ized with a fibrosarcoma cell line were subcloned. Both supB embryo (SHE) cell lines in which loss of a tumor suppressor cell types are nontumorigenic; however, the supB- but not function correlates with the acquisition of conditional anchor- supB+ cells exhibit conditional anchorage-independent age-independent growth potential (21-25). We describe the growth. Alterations of actin microfilament organization were use of these closely related clonal variants to examine the observed in supB- but not supB+ cells that corresponded to potential relationships among microfilament organization, tro- a significant reduction of the actin-binding protein tropomy- pomyosin expression, and anchorage-independent growth.t osin 1 (TM-1) in supB- cells. To examine the possibility of a direct relationship between TM-1 expression and the supB- MATERIALS AND METHODS phenotype, supB+ cells were transfected with an expression vector containing the TM-1 cDNA in an antisense orientation. Cell Lines and Culture. The establishment, characterization, The antisense-induced reduction of TM-1 levels in supB+ and culture of the SHE cell lines used in this study have been clones caused a microfilament reorganization and conferred described in detail (23, 24). anchorage-independent growth potential that were indistin- Photomicroscopy. To visualize actin microfilaments, sub- guishable from those characteristic of supB- cells. These data confluent cells were fixed, permeabilized, and stained with the provide direct evidence that TM-1 regulates both microfila- F-actin-specific rhodamine-phalloidin conjugate as described ment organization and anchorage-independent growth and by the supplier (Molecular Probes). Photomicrographs were suggest that microfilament alterations are sufficient for an- taken with a Leitz Orthomat microscope equipped with an epifluorescence light source, a Leitz N2 filter unit, and an oil chorage-independent growth. immersion objective at 630X and with Kodak Tri-X film (ASA 400). Cytoskeletal alterations are frequently observed in tumor cells Two-Dimensional PAGE. For comparison of total proteins (1), but the significance and molecular basis of these changes among the various supB clones, metabolic labeling of total are unclear. The actin microfilament component of the cy- cellular proteins and quantitative two-dimensional PAGE toskeleton appears to regulate cell shape in many contexts were performed as described (26, 27). [14C]Methylated pro- (2-4), and growth control is one of several biological processes teins (14.3-200 kDa, Amersham) were used as molecular mass that appears tightly linked to cell shape in normal cells (5-8). markers on slab gels. The proliferating cell nuclear antigen One common change in neoplastically transformed cells is the protein was utilized as a quantitative control because of its loss of anchorage- or shape-dependent controls on growth (9). electrophoretic migration in the TM region and its apparently It has been hypothesized that the cytoskeletal perturbations invariant expression in these cells under the conditions de- observed in tumor cells, particularly of the microfilament scribed. The identity of TMs on two-dimensional gels was component, may mediate the uncoupling of cell shape and confirmed by two independent methods. High molecular growth control. weight TM isoforms were immunoprecipitated from radiola- The regulation of microfilament organization is complex, beled SHE cell extracts by using rabbit polyclonal antiserum and numerous actin-binding proteins are involved in this against smooth muscle TM from chicken gizzard (T651, Sigma) process (10). Synthesis of several members of the tropomyosin as described (14). The immunoprecipitated proteins were (TM) family of actin-binding proteins (11), particularly the electrophoresed concomitantly with experimental samples to isoforms of apparently higher molecular weight, is frequently confirm TM localization. In addition, second-dimension gels decreased in association with neoplastic transformation by containing unlabeled SHE cell proteins were subjected to various chemical and viral agents (12-16), as well as in human Western blotting procedures (28) with the mouse anti-chicken carcinoma cells (17). In addition, transfection of TM-1 into gizzard TM monoclonal antibody TM311 (T2780, Sigma). viral oncogene-transformed rodent cells suppresses the tumor- cDNA Probes and Cloning. cDNA probes used in the cloning igenic phenotype (18). These observations suggest that down- of homologous Syrian hamster cDNAs and in Northern blot regulation of TM synthesis is related to the microfilament analysis included human ,B-actin clone pHFO3A-3'UT-HF (29), aberrations observed in diverse types of tumor cells (19). human y-actin clone pHF-yA-3'UT-Fnu (29), rat TM-1 clone Consistent with this hypothesis is a report demonstrating that pSM10 (30), and rat TM-4 clone pREF-102 (31). All cDNA disruption of the single TM gene in the yeast Saccharomyces cerevisiae leads to disappearance of cytoplasmic actin cables Abbreviations: SHE, Syrian hamster embryo; TM, tropomyosin. and perturbations in cellular growth rate (20). *Present address: Department of Obstetrics and Gynecology, Univer- sity of Pennsylvania Medical Center, 415 Curie Boulevard, Philadel- phia, PA 19104. The publication costs of this article were defrayed in part by page charge ITo whom reprint requests should be addressed. payment. This article must therefore be hereby marked "advertisement" in *The sequence reported in this paper has been deposited in the accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession no. U29167). 11534 Downloaded by guest on September 28, 2021 Cell Biology: Boyd et al. Proc. Natl. Acad. Sci. USA 92 (1995) 11535 FIG. 1. Fluorescence photomicrographs depicting actin microfilament organization in supB+ and supB- cell lines. (A) 10WsupB+; (B) 1OWsupB-; (C) DES4supB+; (D) DES4supB-. (Photomicrographs taken at a magnification of 630X.) inserts were radiolabeled with [a-32P]dCTP (3000 Ci/mmol; 1 method (35). Following selection in G418 (800 ,ug/ml), resis- Ci = 37 GBq; Amersham) to a specific activity of > 109 cpm/,ug tant cells were further analyzed as either mass cultures or two by using the Prime-It random priming kit (Stratagene). Total subclones each of sense and antisense transfectants. Anchor- cellular RNA was prepared from early passage, normal SHE age-independent growth potential in soft agar was assessed as cells by the guanidinium isothiocyanate/acid/phenol method described (24). All antisense- and sense-expression experi- (32). Poly(A)+ RNA was prepared from total RNA by two ments were carried out in the presence of 1 ,uM dexametha- cycles of selection on an oligo(dT)-cellulose column, and sone to elicit maximal expression of the cloned cDNA se- cDNA was synthesized using oligo(dT) primers, Moloney quence. murine leukemia virus reverse transcriptase, and a Stratagene ZAP-cDNA synthesis kit (Stratagene). The AZAP library was subcloned into pBluescript SK(-), and hamster cDNAs were RESULTS identified by hybridization with the rat TM-1 cDNA probe at low stringency. Candidate cDNA clones were sequenced in Cytoskeletal Organization. Significant differences were ob- both directions with Sequenase 2.0 (United States Biochem- served in actin microfilament organization between supB+ ical). clones and supB- clones (Fig. 1). Well-defined bundles of Northern Blotting. Total RNA was isolated from cultured microfilaments (stress fibers) generally overlapping and ex- cells as above, and Northern blotting was performed as tending entirely across the cells were observed in both described (33). Blots were rehybridized with a f-actin cDNA 10WsupB+ and DES4supB+ cell lines. Conversely, in the to control for RNA integrity and quantity. Quantitation of 1OWsupB- and DES4supB- cells, stress fibers were reduced in TM-1 mRNA levels in transfected clones was performed by number and integrity; many cells lacked them entirely, while in scanning densitometry of autoradiography films with a USB others they were shorter and thinner than in the supB+ cells. SciScan 5000 system (United States Biochemical). The observed differences in microfilament organization were TM-1 Antisense Expression Experiments. A partial hamster not the result of alterations in soluble vs. polymerized actin TM-1 cDNA, beginning 23 bp after the initiation codon and ratios, biosynthesis rates, protein turnover rates, or ratios of extending to the poly(A) terminus, was subcloned into the cytoplasmic ,3- vs. y-actin isoforms (data not shown). Immun- pMAMneo
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