Oncogene (1997) 15, 2625 ± 2631 1997 Stockton Press All rights reserved 0950 ± 9232/97 $12.00 Suppression of morphological transformation by radicicol is accompanied by enhanced gelsolin expression Ho Jeong Kwon1,3, Minoru Yoshida1, Rie Nagaoka2, Takashi Obinata2, Teruhiko Beppu1,4 and Sueharu Horinouchi1 1Department of Biotechnology, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Bunkyo-ku Tokyo 113; 2Department of Biology, Faculty of Science, Chiba University, Chiba-shi, Chiba 263, Japan Radicicol, an inhibitor of Src-family protein-tyrosine ras and mos oncogenes, both of which act downstream kinases, causes morphological reversion of v-src- and v- of src, although its molecular mechanism is unclear Ha-ras-transformed ®broblasts and arrest of the cell (Kwon et al., 1995; Zhao et al., 1995). In this study, we cycle at both the G1 and the G2 phases. Radicicol was investigated the eects of radicicol on several found to inhibit the growth of several other oncogene- oncogene-transformed cell lines and human carcinoma transformed cell lines and human carcinoma cell lines cell lines and found that radicicol induced a ¯atter and to revert their cell morphology to be ¯at. In the morphology, which was accompanied by restoration of radicicol-treated ¯at cells, actin stress ®ber bundles were actin stress ®bers in all of the cell lines tested. Because reorganized. Since this eect of radicicol on these cell radicicol did not induce the morphological reversion of lines was inhibited by cycloheximide, de novo protein transformed cells in the presence of cycloheximide, we synthesis is required for the morphological reversion. searched for proteins that were inducibly produced by Screening of cellular proteins enhanced in response to radicicol by two-dimensional gel electrophoresis and radicicol by two-dimensional gel electrophoresis sug- Western blot analysis. The present study demonstrated gested that the amount of gelsolin, an actin regulatory that gelsolin was overproduced in response to radicicol. protein, was distinctly increased upon radicicol treat- Gelsolin, a protein which is highly conserved in ment. Western blot and Northern blot analyses showed vertebrates, controls the length of actin ®laments in that radicicol enhanced transcription of the gelsolin gene vitro and the cell shape and motility in vivo by a variety in human carcinoma cell lines, as a result of which the of mechanisms (Cunningham et al., 1991). Gelsolin amount of gelsolin was increased several folds. Injection expression also appears to be involved in cell with an anti-gelsolin antibody into cells and successive dierentiation and neoplasia; gelsolin is produced in treatment with radicicol resulted in approximately 80% an undetectable amount in mouse embryonic and reduction of the number of ¯at cells with stress ®bers in immature myeloid cells but increasingly produced comparison with controls treated with an irrelevant during cell dierentiation (Dieenbach et al., 1989; antibody. These results show that elevated expression Kwiatkowski, 1988). The down-regulation of gelsolin of gelsolin is associated, at least in part, with the expression is observed in a variety of transformed cells suppression of transformation and the restoration of and human invasive tumor cells (Chaponnier and actin stress ®bers in human carcinoma cells by radicicol. Gabbiani, 1989; Vandekerckhove et al., 1990). Furthermore, it was recently reported that overexpres- Keywords: radicicol; transformation; oncogene; actin sion of gelsolin causes tumor cells to appear ¯atter, stress ®ber; gelsolin and the chromosomal alteration of 9q found in human bladder carcinoma correlates with diminished expres- sion of the gelsolin gene that is mapped onto chromosome 9q33 (Tanaka et al., 1995). These Introduction observations strongly suggest that gelsolin has a tumor suppressive function. In this paper, the relation- Radicicol, which had been originally isolated from a ship between the gelsolin expression and the morpho- fungus Monosporium bonorden as a microbial cell logical changes induced by radicicol was evaluated by dierentiation modulator, was found by our group as inactivating the gelsolin function via microinjection of a potent inhibitor of p60v-src protein kinase. It caused an anti-gelsolin antibody that neutralizes gelsolin morphological reversion of v-src-transformed fibro- activity. blasts and reorganized actin stress ®bers (Kwon et al., 1992a,b). Other protein-tyrosine kinases in the Src- family, such as p60c-src and p53/56lyn, were also inhibited Results by radicicol (Chanmugam et al., 1995). Furthermore, radicicol was found to suppress transformation by the Eects of radicicol on micro®lament reorganization in oncogene-transformed ®broblasts and human carcinoma cells Correspondence: Minoru Yoshida Previous studies have shown that radicicol (Figure 1), Present addresses: 3 Department of Chemistry & Chemical Biology, an inhibitor of Src-family protein-tyrosine kinases, Harvard University, Cambridge, MA 02138, USA, 4 Department of Applied Biological Sciences, College of Bioresource Science, Nihon induces rapid reversal of transformed morphology of University, Fujisawa-shi, Kanagawa 252, Japan. ®broblasts transformed with the v-src,v-ras, and v-mos Received 14 April 1997; accepted 22 July 1997 oncogenes (Kwon et al., 1992b, 1995; Zhao et al., Flat reversion and gelsolin induction by radicicol HJ Kwon et al 2626 1995). To examine whether this eect of radicicol on upon treatment with radicicol. These cells apparently morphology is observed in other transformed cells, we possess reorganized thick bundles of actin stress ®bers, examined the ®broblasts transformed with oncogenic one of the most important markers of normal agents, such as v-Ha-ras,v-raf,v-fos and simian virus ®broblasts. We further examined two dierent human 40 (SV40) by visualizing the intracellular distribution carcinoma cell lines, T24 and HeLa. T24 is a human of actin micro®laments after radicicol treatment. urinary bladder carcinoma cell line that contains an Radicicol (0.5 mg/ml) caused morphological changes activated ras mutation (Goldfarb et al., 1982), and of raf, fos and SV40-transformed cells within 24 h after HeLa is an epithelium-like cervix carcinoma cell line the radicicol challenge, in addition to v-Ha-ras- that expresses the human papillomavirus gene pro- transformed cells (Figure 2). These transformed ducts, E6 and E7 (Schwarz et al., 1985). As shown in ®broblasts normally show a spindle-like shape in the Figure 3, these cells became ¯at upon radicicol absence of radicicol, but they became larger and ¯at treatment, in which actin stress ®bers were restored. The ability of radicicol to revert the morphology of a variety of transformed cells, irrespective of their origins and oncogenes, suggests that radicicol does not inhibit directly the oncogene functions that are activated or ampli®ed in these cells. Eect of cycloheximide on radicicol-induced morphological changes We have previously shown that reversal of the transformed morphology of ras-transformed fibro- blasts by radicicol was almost completely inhibited by cycloheximide (CHX), a protein synthesis inhibitor (Kwon et al., 1995). We investigated the eect of cycloheximide on the radicicol-induced morphological changes of the above-described cells (Figures 2 and 3). Simultaneous addition of 1 mg/ml of CHX with Figure 1 Chemical structure of radicicol radicicol to ras, raf, fos and SV40-transformed cells Control Radicicol Radicicol/CHX ras v-H- raf v- fos SV40 v- Figure 2 Morphological changes of oncogene-transformed ®broblasts. v-Ha-ras- and v-raf-transformed NIH3T3 cells and v-fos- and SV40-transformed 3Y1 cells were treated for 24 h with 0.5 mg/ml radicicol in the absence or presence of 1 mg/ml cycloheximide (CHX). Actin stress ®bers were stained with FITC-phalloidin Flat reversion and gelsolin induction by radicicol HJ Kwon et al 2627 Control Radicicol Radicicol/CHX T24 HeLa Figure 3 Morphological changes of human carcinoma cells. T24 and HeLa were treated for 24 h with 0.5 mg/ml radicicol in the absence or presence of 1 mg/ml cycloheximide (CHX). Actin stress ®bers were stained with FITC-phalloidin abolished the formation of actin stress ®bers and the kDa cells remained still a spindle-like shape, almost 106 identical to those of untreated cells. A similar eect of CHX was also observed in T24 and HeLa cells; restoration of stress ®bers did not occur upon radicicol 80 treatment. These results suggest strongly that de novo protein synthesis is required for the morphological reversion of transformed cells induced by radicicol. 49.5 Identi®cation of gelsolin as a protein overproduced in 32.5 radicicol-treated cells 27.5 To identify the proteins responsible for the radicicol- induced morphological changes, we searched for the cellular proteins enhanced in radicicol-treated cells versus untreated cells. We prepared total cell extracts 106 from v-Ha-ras-transformed NIH3T3 cells both treated and untreated with radicicol, and compared their 80 cellular protein patterns analysed by 2D gel electro- phoresis. As shown in Figure 4, the amount of a 90 kDa protein (p90) whose pI value is about 6 (p90) 49.5 was distinctly larger in the radicicol-treated cells. Several other proteins were also enhanced by the radicicol treatment, but the most prominent one was 32.5 p90. The increase in the amount of p90 was in a dose- 27.5 and a time-dependent manner (data not shown). The migration of the radicicol-sensitive spot matches Figure 4 Two-dimensional electrophoresis of total proteins that of gelsolin, which is 84 kDa and is thought to be a isolated from radicicol-treated or untreated cells. v-Ha-ras- candidate for tumor suppressors of human bladder transformed NIH3T3 cells were treated with or without 1 mg/ml cancer (Fujita et al., 1990; Kuzumaki et al., 1989; radicicol. After cultivation for 24 h, the cells were lyzed and Mullauer et al., 1993; Tanaka et al., 1995). A cellular proteins were analyzed as described in Materials and È methods. (a) control without radicicol treatment. (b) radicicol signi®cant reduction of gelsolin protein is associated (1 mg/ml) treatment.