CG&D
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cell Growth & Differentiation

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kim, P. N.
Right arrow Articles by Janssen, R. A. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kim, P. N.
Right arrow Articles by Janssen, R. A. J.
Cell Growth & Differentiation Vol. 12, 543-550, November 2001
© 2001 American Association for Cancer Research

Radicicol Suppresses Transformation and Restores Tropomyosin-2 Expression in Both ras- and MEK- transformed Cells without Inhibiting the Raf/MEK/ERK Signaling Cascade1

Phillia N. Kim2, Eric Jonasch2,,3, Barbara C. Mosterman, James W. Mier and Richard A. J. Janssen4

Laboratory of Immunobiology, Division of Monoclonal Antibodies, Center for Biologics Evaluation & Research, Bethesda, Maryland 20892-4555 [P. N. K., R. A. J. J.], Department of Medicine, Beth Israel Deaconess Medical Center [E. J., B. C. M., J. W. M.], and Harvard Medical School, Boston, Massachusetts 02215 [E. J., J. W. M.]


    Abstract
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
The antibiotic radicicol suppresses transformation in a variety of transformed cells. The antineoplastic effects of the drug have been attributed to the degradation of Raf and the inactivation of the Ras/Raf/ mitogen-activated protein kinase kinase (MEK)/ extracellular signal-regulated kinase (ERK) signaling cascade. Here we demonstrate that radicicol induces cell spreading, suppresses anchorage-independent cell growth, and increases the expression of the high-molecular weight tropomyosin isoform TM-2 in cells stably expressing a constitutively active form of MEK-1 as well as in ras-transformed cells. Furthermore, the reverting effects of the drug are achieved at concentrations below those required to deplete Raf from the cell or to inhibit the phosphorylation of ERK or its substrates Elk and pp90RSK. In contrast, low concentrations of radicicol significantly inhibited activator protein (AP-1) and serum response factor (SRF)-mediated transcription. The lack of correlation between the effects of radicicol on cell phenotype and on the signaling activities of the Raf/MEK/ERK pathway indicate that Raf depletion or disruption of proximal signaling events in the mitogen-activated protein kinase pathway are not the predominant mechanisms by which the drug suppresses the transformed phenotype. Our observation that low concentrations of radicicol block transcriptional activities mediated by AP-1 and SRF suggests that interference with signaling upstream of these transcription factors may contribute to the reverting effects of the drug.


    Introduction
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Transformation of fibroblasts by v-ki-Ras induces rounding of cells, the disappearance of stress fibers, loss of serum- and anchorage-dependence, as well as the formation of tumors in vivo and the down-modulation of actin-binding proteins such as vinculin, {alpha}-actinin, and tropomyosin (1) . The transforming effects of oncogenic Ras result from the combined activities of the numerous downstream Ras effectors such as Raf, Rac, Rho, and PI3K,5 each of which may contribute to the malignant phenotype (2 , 3) . Of the numerous signaling pathways activated by oncogenic Ras, the Raf/MEK/ERK kinase cascade plays the most important role in the transformation of fibroblasts. Constitutively active forms of Raf, MEK-1, and ERK are sufficient to transform immortalized fibroblasts (4, 5, 6) . The kinase ERK phosphorylates multiple targets including ETS TCFs such as Elk-1 (7 , 8) . This transcription factor binds cooperatively with dimers of SRF to form a ternary complex on the cis-acting SRE (9) . This enhancer element is essential for mitogen-induced expression of many early response genes, including c-fos and c-jun, which drive proliferation and play an important role in transformation (10 , 11 , 12) .

In addition to the TCF-dependent activity described above, SRF is also able to mediate TCF-independent gene transcription. This is achieved through interaction of SRF with enhancer elements lacking TCF binding sites. SRF is activated in an ERK-independent manner by pathways downstream of PI3K and the Rho-family members Rac, Rho, and Cdc42 (13 , 14) . It is, however, unclear which of the multiple pathways downstream of each of these effectors are responsible for SRF activation. SRF plays an important role in cellular proliferation because its activity is required for PI3K-mediated cell cycle progression, and overexpression of a constitutively active SRF is sufficient for cell cycle entry (15) .

Transformation is almost always accompanied by the down-modulation of various actin-binding proteins including {alpha}-actinin, vinculin, and the high-molecular weight isoforms of the nonmuscle cytoskeletal protein TM (1) . The down-regulation of TM is an essential event during transformation, because restoration of TM is sufficient to suppress the transformed phenotype of ras- and src-transformed cells (16, 17, 18, 19) . In addition, the forced deletion of TM-1 induces a transformed phenotype in immortalized cells (20) , suggesting that the expression of high-molecular weight TM is essential to the maintenance of a nontransformed phenotype.

The macrocyclic antifungal antibiotic radicicol, has been shown to suppress transformation by src, ras, and mos oncogenes (21, 22, 23) , to restore stress fiber formation, and to induce gelsolin expression (23 , 24) . It is unclear how radicicol mediates these tumor suppressive effects. Radicicol associates with members of the Hsp90 family of proteins (Hsp90{alpha} and ß, Grp94, and Trap-1; 25, 26, 27 ). Hsp90 is a folding and scaffolding protein that binds the src family kinases and the serine/threonine kinase Raf-1 among other proteins (see also review by Csermely et al., Ref. 28 ). Association of these signaling proteins with Hsp90 is essential for their stability, activity, and localization. Treatment of cells with radicicol dissociates Hsp90 from Raf, leading to destabilization and degradation of Raf-1 (26 , 29) . The depletion of Raf results in the inhibition of the MEK/ERK pathway (29) . Radicicol further suppresses Raf-mediated activation of the MEK/ERK pathway by disrupting the interaction between activated Ras and Raf (30) . The effects of radicicol are considered to be specific for the Raf/MEK/ERK pathway because treatment of cells with radicicol has been reported to have no effect on the activities of JNK and p38 (31) . The depletion and inactivation of Raf is currently thought to account for the detransforming effect of radicicol on tumor cell lines (29) .

Here, we demonstrate that radicicol is able to restore TM-2 expression and to inhibit the transformed phenotype of cells either transformed with the oncogene v-Ki-ras or infected with a constitutively activated form of the Raf target MEK-1. Furthermore, we show that the reverting effects of the drug are apparent at concentrations (i.e., 0.5–2 µM) below those required to down-modulate Raf or interfere with ERK activation. Interestingly, however, radicicol significantly suppresses the activities of the transcription factors SRF and AP-1. Collectively, these data suggest that radicicol exerts multiple effects on transformed cells and that the down-modulation of Raf is not the primary mechanism by which radicicol suppresses transformation.


    Results
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Radicicol Blocks Anchorage Independence in Both ras-transformed and MEK-1-S218D,S222D-infected NIH3T3 Fibroblasts.
The ability of radicicol to induce phenotypic reversion in tumor cells has been attributed to its association with Hsp90 and the ensuing destabilization of Raf. To the extent that this is the predominant mechanism underlying the action of radicicol, one might expect that the drug would only affect cells transformed by oncogenes that act upstream of Raf (e.g., ras) and not cells transformed by activated forms of its downstream effectors such as MEK and ERK. To determine whether radicicol does indeed possess this sort of selectivity, we evaluated the effects of the drug on NIH3T3 fibroblasts transformed with v-Ki-ras or infected with MEK-1-S218D,S222D, a constitutively activated form of MEK-1.

In the absence of radicicol, both the v-Ki-ras-transformed and MEK-1-transformed cells form large colonies in soft agar. Both cells lines are highly sensitive to the growth-suppressive effects of the drug (Table 1)Citation , inasmuch as even low concentrations (i.e., 0.5 µM) completely block colony formation. These results indicate that radicicol inhibits the growth of cells transformed by a signaling molecule acting downstream of Raf, the putative target of the drug. These data suggest that the destabilization of Raf cannot be the sole or even predominant mechanism of action of radicicol.


View this table:
[in this window]
[in a new window]
 
Table 1 Radicicol completely blocks anchorage independent growth

 
Radicicol Induces Cell Spreading in Both ras-transformed and MEK-1-S218D,S222D-infected NIH3T3 Fibroblasts.
MEK-1-S218D,S222D-infected cells are similar to ras-transformed NIH3T3 cells with respect to their spindle-shaped morphology (Fig. 1)Citation . In this study, both cell lines were treated with 0.5, 1, 2, and 5 µM radicicol in DMEM containing 5% serum for 40 h. As shown in Fig. 1Citation , both the ras-transformed and MEK-1-S218D,S222D-infected cells spread dramatically when exposed to concentrations of radicicol as low as 0.5 µM. The fact that radicicol has comparable effects on the morphology of the MEK-1- and v-Ki-ras-transformed cells corroborates the notion that its mechanism of action cannot critically depend on Raf destabilization.



View larger version (105K):
[in this window]
[in a new window]
 
Fig. 1. Radicicol induces spreading of transformed cells. Cells were incubated for 40 h with the indicated concentrations of radicicol in DMEM with 5% DCS. A, ras-transformed NIH3T3 cells. B, MEK-1-S218D, S222D-infected NIH3T3 cells.

 
Radicicol Restores TM Levels in v-Ki-ras- and MEK1-transformed cells.
Ras-induced transformation is associated with rounding of the cells, the disassembly of the cytoskeleton, and reduced expression of numerous actinbinding proteins such as vinculin, {alpha}-actinin, and the high-molecular weight TMs (1) . To determine whether the reverting effect of radicicol on cell shape is accompanied by changes in cytoskeletal protein levels, we evaluated the effects of the drug on the expression of high-molecular weight TMs in both ras-transformed and MEK-1-S218D,S222Dinfected NIH3T3 cells by Western blotting. As shown in Fig. 2Citation , the expression levels of all three high-molecular weight isoforms of TM are significantly decreased in both ras-transformed and MEK-1-S218D,S222D-infected cells compared with normal NIH3T3 cells. Treatment of these cells with as little as 0.5 µM radicicol restores TM-2 expression in ras-transformed cells nearly to the levels observed in nontransformed NIH3T3 cells (Fig. 2)Citation . Radicicol restored TM-2 levels partially in MEK-1-S218D,S222D-infected cells. Thus, the effects of radicicol on the morphology and anchorage-independence of both v-Ki-ras- and MEK-1-transformed cells correlate with an increased expression of the actin-binding protein TM. These data further support our hypothesis that Raf destabilization may not be the sole basis for the phenotypic reversion induced by the drug.



View larger version (49K):
[in this window]
[in a new window]
 
Fig. 2. Radicicol restores TM-2 expression. Whole cell extracts with equal amounts of total protein from cells treated as described in Fig. 1Citation were analyzed by Western blotting for TM expression (top panel) and tubulin expression (bottom panel). A, ras-transformed NIH3T3 cells. B, MEK-1-S218D,S222D-infected NIH3T3 cells. TM isoforms TM-1, TM-2, and TM-3 are indicated. Blots were stripped and reprobed for tubulin.

 
Low Concentrations of Radicicol Do Not Affect the Raf/MEK/ERK Signaling Cascade.
To elucidate further the mechanism of radicicol-induced inhibition of the transformed phenotype, we attempted to correlate the effects of the drug on the activity of the constituents of the Raf/MEK/ERK signaling pathway with its effects on anchorage independence, morphology, and cytoskeletal protein expression. Similar to the studies described above, ras-transformed and MEK-1-S218D,S222D-infected NIH3T3 cells were exposed to 0, 0.5, 1, 2, or 5 µM radicicol, 50 µM PD98059, or 10 µM U0126 in DMEM containing 5% serum for 40 h. Raf and ERK levels and the extent of ERK phosphorylation were then determined by Western blot analysis. As shown in Figs. 3Citation and 4Citation , radicicol had no discernible effect on any of these parameters in either cell line at concentrations of <5 µM. Raf levels declined slightly at 5 µM, and Raf was completely depleted at 10 µM (not shown), well above the concentration required to suppress colony formation, induce cell flattening, and restore TM expression. ERK phosphorylation was completely unaffected by radicicol, whereas the MEK inhibitors PD98059 and U0126 induced significant reduction in the levels of phosphorylated ERK (Fig. 4)Citation . These data indicate that the reverting effects of radicicol on cell shape and cytoskeletal protein (TM) expression can be achieved at concentrations (0.5–2 µM) that have no effect on Raf levels or on the state of activation of ERK.



View larger version (36K):
[in this window]
[in a new window]
 
Fig. 3. Low concentrations of radicicol do not affect Raf expression. The same extracts as used for Fig. 2Citation were analyzed by Western blotting for expression levels of Raf. Top, ras-transformed NIH3T3 cells. Bottom, MEK-1-S218D,S222D-infected NIH3T3 cells.

 


View larger version (74K):
[in this window]
[in a new window]
 
Fig. 4. Radicicol does not inhibit ERK phosphorylation. Cells were treated for 36–40 h with various concentrations of radicicol or MEK inhibitors in DMEM with 5% DCS. Whole cell extracts with equal amounts of total protein from these cells were analyzed by Western blotting for phosphorylation status of ERK. Blots were first probed with anti-phospho-ERK (top panels) and subsequently stripped and reprobed with antiERK (bottom panels). A, ras-transformed NIH3T3 cells B, MEK-1-S218D,S222D-infected NIH3T3 cells.

 
Radicicol Does Not Affect Elk or pp90RSK Phosphorylation.
Radicicol induces cell flattening, inhibits anchorage independence, and restores the expression of TM in both ras-transformed and MEK-1-S218D,S222D-infected NIH3T3 cells at concentrations insufficient to affect Raf levels or the degree of ERK phosphorylation. This observation suggests that the drug may affect oncogenic signaling events unrelated to or downstream of the Raf/MEK/ERK signaling cascade. To determine whether radicicol affects signaling downstream of ERK, we evaluated the effects of radicicol on the state of phosphorylation of two known ERK substrates, Elk-1 and pp90RSK.

To test the effects of radicicol on the phosphorylation of Elk, cells were transiently transfected with Elk-1. One day after transfection, cells were treated with radicicol or MEK inhibitors. As shown in Fig. 5Citation , radicicol had no effect on the phosphorylation of Elk. In contrast, almost no phosphorylated Elk-1 could be observed in cells treated with the MEK inhibitors PD98059 and U0126. As shown in Fig. 6Citation , radicicol had also no effect on the phosphorylation of pp90RSK in ras-transformed cells and MEK-1-S218D,S222D-infected cells. The inability of radicicol to suppress the phosphorylation of Elk-1 and pp90RSK is consistent with its lack of effect on ERK phosphorylation. These results demonstrate that radicicol achieves its detransforming effects without interfering with proximal events in the Raf/MEK/ERK pathway.



View larger version (74K):
[in this window]
[in a new window]
 
Fig. 5. Radicicol does not inhibit Elk phosphorylation. Cells were transiently transfected with Elk and subsequently treated for 36–40 h with radicicol or MEK inhibitors. Cell extracts were analyzed by Western blotting for phosphorylation status of Elk. Blots were first probed with anti-phospho-Elk (top panels) and subsequently stripped and reprobed with anti-Elk (bottom panels). A, ras-transformed NIH3T3 cells. B, MEK-1-S218D,S222D-infected NIH3T3 cells.

 


View larger version (73K):
[in this window]
[in a new window]
 
Fig. 6. Radicicol does not inhibit p90RSK phosphorylation. The same extracts as those used for Fig. 4Citation were analyzed by Western blotting for phosphorylation status of p90RSK. Blots were first probed with anti-phospho-p90RSK (top panels) and subsequently stripped and reprobed with anti-p90RSK (bottom panels). A, ras-transformed NIH3T3 cells. B, MEK-1-S218D,S222D-infected NIH3T3 cells.

 
Radicicol Inhibits SRF and AP-1 Activity.
Ras-induced transformation is partially dependent on pathways downstream of PI3K and the small GTPases Rac, Rho, and Cdc42 (13 , 14) . It is thus possible that radicicol may inhibit ras-induced transformation by interfering with one of these collaborative pathways. Rho activates multiple targets including the serine kinase ROCK and the transcription factor SRF (32 , 33) . We have observed, however, that treatment with the ROCK-specific inhibitor Y-27632 does not restore TM expression in ras-transformed NIH3T3 cells,6 suggesting that the activation of this kinase is not a critical factor in the down-modulation of TM in tumor cells and therefore is an unlikely target for radicicol. PI3K, Rho, Rac, and Cdc42 all contribute to the activation of the transcription factor SRF (9 , 34) . SRF is crucial for cell cycle entry and progression and also plays an important role in the expression of immediate early genes. To test whether radicicol affects TCF-independent SRF activity, ras-transformed cells were transiently transfected with an SRF-luciferase reporter construct containing five copies of the SRF-binding domain but lacking the binding sites for TCFs. Treatment of these cells with 0.5 µM radicicol resulted in a 5-fold decrease in SRF activity compared with untreated cells. In comparison, the drug had only a minor effect on the control reporter vector pMCS-Luc, containing no enhancer elements (Fig. 7)Citation . These results demonstrate that radicicol suppresses TCF-independent transcriptional activity of SRF in ras-transformed cells.



View larger version (10K):
[in this window]
[in a new window]
 
Fig. 7. Radicicol inhibits SRF and AP-1 activity. ras-transformed cells were treated with 0.5 µM radicicol for 36–40 h and reporter activities were analyzed by a dual-luciferase reporter assay as described in "Materials and Methods." Reporter activity in radicicol-treated cells is expressed as the percentage of that in untreated cells. Shown is the average ± SD of quadruplicates from a representative experiment of three performed. pMCS-luc, a reporter construct without enhancer elements.

 
SRF and TCF (Elk-1, Sap-1a/b, Sap-2) cooperatively form a ternary complex on the SRE. DNA binding and transcriptional activity of the ternary complex is fully dependent on the binding of SRF to the SRE (9) . It was therefore expected that radicicol would also inhibit transcription from the SRE. To test whether radicicol inhibits TCF-dependent SRF activity, ras-transformed NIH3T3 cells were transfected with an SRE-luciferase reporter construct containing binding sites for both SRF and Elk-1. Surprisingly, radicicol inhibited SRE-mediated transcriptional activity only up to 2-fold. These results suggest that radicicol affects TCF-dependent and TCFindependent transcriptional activities of SRF to a different degree.

AP-1 is a dimeric transcription factor composed of Jun, Fos or ATF subunits, and its activity plays an important role in transformation (11 , 12) . Takehana et al. (31) reported that radicicol inhibits phorbol 12-myristate 13-acetate-induced AP-1 activity in HEK293 cells without suppressing the activities of JNK or p38, two kinases involved in the activation of AP-1 complexes. To test whether radicicol is also able to inhibit AP-1 activity in transformed cells, we transiently transfected ras-transformed NIH3T3 cells with an AP-1-luciferase reporter construct. Treatment of these cells with 0.5 µM radicicol resulted in a 3–4-fold decrease in AP-1 activity compared with untreated cells (Fig. 7)Citation . In addition, we found that radicicol does not inhibit the phosphorylation of c-Jun in ras-transformed cells.7 These results together with the results of Takahana et al. (31) demonstrate that radicicol is able to suppress AP-1 activity without affecting the phosphorylation of one of its potential subunits.


    Discussion
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
The macrocyclic antifungal antibiotic radicicol has been shown to suppress the transformed phenotype of mos-, ras- and src-transformed fibroblasts and the human urinary bladder carcinoma cell line T24 (21, 22, 23) . Radicicol-induced phenotypic reversion is associated with the reappearance of stress fibers, enhanced gelsolin expression, cell cycle arrest, and inhibition of growth on poly(2-hydroxyethyl methacrylate)-coated surfaces (23 , 24 , 35 , 36) . The ability of radicicol to induce phenotypic reversion in tumor cells has been attributed to its association with Hsp90 and the resultant destabilization of Raf (25, 26, 27 , 29) . To the extent that this is the predominant mechanism underlying the action of radicicol, one might expect that the drug would only affect cells transformed by oncogenes upstream of Raf and not cells that have been transformed by downstream kinases, such as activated forms of MEK-1. However, this prediction is clearly not supported by our data, showing that radicicol also suppresses the transformed phenotype of MEK-transformed cells. Radicicol induces significant cell flattening, complete inhibition of the ability to grow in soft agar, and an increase in the expression of the high-molecular weight TM isoform TM-2, one of the actin-binding proteins down-modulated in transformed cells in both ras-transformed and MEK-1-S218D,S222D-infected cells. Moreover, if the mechanism by which radicicol suppresses transformation is caused by destabilization of Raf and its subsequent degradation, one would also expect that its effects on morphology and TM-2 expression would correlate with inhibition of the MEK/ERK pathway. However, we find that radicicol is able to induce cell flattening and to restore TM-2 levels at a concentrations (0.5–2 µM) at which Raf levels remain stable and ERK phosphorylation and activity are not affected. Radicicol does indeed induce some loss of Raf expression and a decrease in ERK-1 phosphorylation, but this effect is observed only at concentrations (5–10 µM) higher than those required to induce cell flattening and TM-2 expression. These observations suggest that the destabilization of Raf and/or the inhibition of the Raf/MEK/ERK signaling cascade is not the dominant mechanism by which radicicol suppresses transformation.

The hypothesis that the biological effects of radicicol are not limited to the disruption of the mitogen-activated protein kinase pathway is supported by the dissimilarity between the effects of radicicol and those of the MEK inhibitor PD98059. Levels of the high-molecular weight isoforms of TM are markedly reduced in transformed cells and only modestly increased by exposure to PD98059 (37 , 38) . In contrast, radicicol restores TM-2 expression in both ras- and MEK-1-transformed cells, in some instances to levels observed in nontransformed cells. This suggests that radicicol mediates the up-regulation of TM-2 and possibly also its tumor suppressive effects by interfering with signals different from the Raf/MEK/ERK pathway that contribute to ras-induced transformation.

PI3K and the Rho family of small GTPases, which includes Rac, Rho, and Cdc42, play an essential role in ras-induced transformation (9) . Rho GTPases activate many downstream targets including JNK, p38, ROCK, and SRF. Thus far it is unclear which of the many pathways downstream of these Rho GTPases contribute to ras-mediated transformation, although the activation of AP-1 through Rac and JNK/p38 and the Rho-mediated activation of ROCK seem to play essential roles (10 , 32) . In addition, PI3K and the Rho GTPases activate independently from each other the transcription factor SRF, which plays an essential role in cell cycle entry and progression.

Takehana et al. (31) recently reported that radicicol does not inhibit the phosphorylation of c-jun and ATF-2 in anisomycin-treated HeLa cells, and we have demonstrated its failure to affect the phosphorylation of c-jun in ras-transformed cells.8 However, we and Takehana et al. (31) have both observed that radicicol significantly inhibits AP-1 activity in ras-transformed cells (Fig. 7)Citation and phorbol 12-myristate 13-acetate-stimulated HEK293 cells. AP-1 is a heterodimeric transcription factor composed of Jun, Fos, or ATF subunits, and its activity is partially regulated by the phosphorylation of these subcomponents (11 , 12) . In addition, AP-1 activity is regulated through interactions with a variety of transcriptional coactivators (39) . Because radicicol suppresses AP-1 activity without suppressing the phosphorylation of c-Jun or ATF-2, it is most likely that the drug mediates its effects through the inhibition of transcriptional coactivators or the inhibition of phosphorylation of subunits other than c-Jun or ATF-2.

SRF plays an essential role in cell cycle entry and progression. Interestingly, radicicol blocks cell cycle at G1 and G2 in HL60 cells and inhibits exit from mitosis in Src-transformed fibroblasts (35 , 40) . Here we demonstrate that radicicol inhibits transcription from an SRF reporter construct that binds SRF independently of TCF, one of its potential cofactors. This result suggests that radicicol interferes with signaling downstream of PI3K and/or the Rho GTPases. Moreover, this inhibition of TCF-independent SRF activity may be one of the mechanisms through which radicicol suppresses cell cycle entry and progression.

The enhancer element SRE binds SRF in concert with one of the TCFs, and transcription from this element is dependent on this interaction (9) . Surprisingly, the radicicol-induced decrease in TCF-independent SRF activity is not accompanied by a comparable decrease in TCF-dependent SRF activity, which is only decreased up to 2-fold in the presence of radicicol compared with the 5-fold decrease in TCF-independent activity. This effect has also been observed for the Rho inhibitor C3 toxin, which significantly inhibits growth factor-induced TCF-independent but not TCF-dependent SRF activities (15 , 34 , 41) . The activities of both TCFdependent and TCF-independent SRF activity are influenced by many other transcriptional coactivators. It is possible that radicicol affects the activity of cofactors that are important for the isolated activity of SRF but not for that of TCF-dependent SRF activity.

Taken together these results show that radicicol can interfere with both ras- and MEK-induced transformation without affecting the Raf/MEK/ERK signaling cascade, but by interfering with signals leading to the activation of AP-1 and/or SRF-dependent transcription.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Reagents.
TM311, a monoclonal antibody which recognizes a common epitope in the first exon of TM-1, TM-2, and TM-3 (42) , was purchased from Sigma Chemical Co. (St. Louis, MO). Anti-phospho-Erk, anti-Elk, anti-phospho-Elk, and anti-phospho-p90RSK antibodies and the MEK inhibitor PD98059 were obtained from New England Biolabs (Beverly, MA). The MEK inhibitor U0126 was obtained from Promega (Madison, WI). Anti-Raf (clone C-12) and anti-Erk-1 (clone K-23) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and anti-p90RSK was purchased from Transduction Laboratories (Lexington, KY). Anti-tubulin was obtained from Sigma Chemical Co., and Y-26732 was obtained from Biomol (Plymouth Meeting, PA).

Cells and Culture Conditions.
v-Ki-ras-transformed NIH3T3 fibroblasts were obtained from Larry Feig (Tufts University, Boston, MA), and NIH3T3 fibroblasts infected with a retrovirus carrying MEK-1-S218D,S222D were obtained from Raymond Erikson (Harvard University, Cambridge, MA). The latter cells correspond to the DD1 cells described previously (43) . These cells were cultured at 37°C and 5% C02 in DMEM (Mediatech, Herndon, VA) supplemented with 10% DCS (BioWhittaker, Walkersville, MD) and 1 µg/ml gentamicin (Life Technologies, Inc., Gaithersburg, MD).

Treatment with Radicicol or MEK Inhibitors.
Cells (2.5 x 105 cells/well) were seeded in six-well plates in DMEM containing 10% DCS. After 24 h, the medium was replaced with DMEM containing 5% DCS plus the indicated concentrations of radicicol or the MEK inhibitors PD98059 or U0126. The cells were incubated for an additional 36–40 h. As a negative control, cells were incubated with DMEM containing 5% DCS plus 0.1% ethanol (solvent for radicicol) or 0.2% DMSO (solvent for the MEK inhibitors). Morphology of the cells was determined by phase-contrast microscopy. After the treatment, whole cell extracts were prepared by washing the cells twice in cold PBS. Cells were scraped into SDS cell-lysis buffer [62.5 mM Tris (pH 6.8) and 1% SDS] containing protease inhibitors (10 µg/ml leupeptin, 0.1 unit/ml aprotinin, and 2 mM phenylmethylsulfonyl fluoride) and phosphatase inhibitors (1 mM sodium orthovanadate, 10 mM sodium PPi, and 10 mM sodium fluoride). The samples were passed three to five times through a 25-gauge needle to reduce viscosity. Samples were blotted onto nitrocellulose as described below.

Anchorage Independence Assay.
Cells (103) were mixed into 0.33% semisolid agar medium (DMEM containing 10% DCS) containing the indicated amounts of radicicol (or equivalent volume of ethanol for the negative control). This cell suspension was subsequently layered on top of a 0.6% agar bottom layer. New 0.33% semisolid agar medium containing radicicol or ethanol was added every 2 or 3 days. After 3 weeks, the number of colonies >25 µm were counted. Three independent assays in triplicate were performed.

Elk Phosphorylation Assay.
Western blotting is not sensitive enough to detect endogenous levels of Elk in whole cell extracts. To determine the levels of Elk phosphorylation in the various experiments, cells were transiently transfected with the mammalian expression vector pAUCT containing the Elk-1 gene. This vector was created by subcloning Elk-1 from pGT14b (kindly provided by Dr. R. Davis, University of Massachusetts, Worcester, MA) into pAUCT. Before transfection, 2.5 x 105 cells/well were seeded in six-well plates. After 24 h, cells were transfected by adding 1 ml of DMEM containing 10 µl lipofectamin (Life Technologies, Inc.) and 1 µg of pAUCT/Elk-1. Four h after transfection, the entire medium was exchanged for DMEM with 10% DCS. Eighteen h after transfection, indicated concentrations of radicicol or MEK inhibitors were added, and the cells were allowed to incubate for an additional 36 h. Whole cell extracts were made as described above and analyzed by Western blotting as described below.

Reporter Assays.
To determine the effects of radicicol or MEK inhibitors on SRE, SRF, or AP-1 activity, dual-luciferase reporter assays were performed using the pSRE-Luc, pSRF-Luc, and pAP-1-Luc reporter constructs from Stratagene (La Jolla, CA) in combination with pRL-null from Promega (Madison, WI). The SRE reporter contains five copies of the sequence AGGATGT CCATATTAGG ACATCT upstream of a basic TATA element and the firefly luciferase reporter gene. The SRF reporter contains five copies of the enhancer sequence GT CCATATTAGG AC, and the AP-1 reporter contains seven copies of the enhancer sequence TGACTAA. pRL-null contains the renilla luciferase gene without enhancer elements and serves as an internal control for transfection efficiency and protein levels (44) . As a negative control, the SRE, SRF, or AP-1 reporter constructs were substituted by pMCS-Luc (Stratagene) containing the luciferase gene without any enhancer elements. Briefly, 3 x 104 cells/well were seeded in 24-well plates. After 24 h, cells were transfected by adding 200 µl of Optimem (Life Technologies, Inc.) containing 1 µl lipofectamin (Life Technologies, Inc.), 4 µl PLUS reagent (Life Technologies, Inc.), 0.4 µg of pSRE-Luc, pSRF-Luc, pAP-1-Luc, or pMCS-Luc reporter plasmid, and 2 ng of pRL-null. Four h after transfection, the entire medium was exchanged for DMEM with 10% DCS. Eighteen h after transfection, indicated concentrations of radicicol were added, and the cells were allowed to incubate for an additional 36 h. At that time, cells were washed once with cold PBS and lysed in 100 µl passive lysis buffer (Promega) containing protease inhibitors (10 µg/ml leupeptin, 0.1 units/ml aprotinin, and 2 mM phenylmethylsulfonyl fluoride) and phosphatase inhibitors (1 mM sodium orthovanadate, 10 mM sodium PPi, and 10 mM sodium fluoride). Dual luciferase-reporter assays were performed on a LB96V Microlumat luminometer (Perkin-Elmer, Gaithersburg, MD) using 20 µl of lysate/assay. Reporter activities were normalized for pRL-null activity as described before (44) .

Western Blotting.
Protein concentrations of the whole cell lysates were determined using the bicinchoninic acid assay (Pierce, Rockford, IL). Equal amounts of protein were run on SDS-polyacrylamide gels and subsequently blotted onto nitrocellulose membranes. Equal loading of the samples was assessed by staining the blots with a 0.1% Ponceau S and 5% acetic acid solution (Sigma Chemical Co.). The membranes were probed with antibodies directed against the relevant proteins and then goat antimouse or goat antirabbit antibodies conjugated to horseradish peroxidase (Pierce). Proteins were visualized by chemiluminescence using SuperSignal as substrate (Pierce). Blots were exposed to Kodak X-Omat autoradiography film.


    Acknowledgments
 
We thank Dr. Raymond Erikson (Harvard University, Boston, MA) for the kind gift of DD1 fibroblasts and Wendy C. Weinberg and Ezio Bonvini (Center for Biologics Evaluation & Research, Bethesda, MD) for critical comments.


    Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This research was supported by a grant from the Alexander and Margaret Stewart Trust, Washington, D C (to R. A. J. J.), and NIH Grant CA74401 (to J. W. M.). Back

2 These authors contributed equally to this work. Back

3 Present address: Division of Oncology, Massachusetts General Hospital and Harvard Medical School, 55 Fruit Street, Boston, MA 02114. Back

4 To whom requests for reprints should be addressed, at FDA/CBER/OTRR/DMA, Building 29B, Room 3NN22, 29 Lincoln Drive, HFM 564, Bethesda, MD 20892-4555. Phone: (301) 827-0713; Fax: (301) 827-0852; E-mail: janssen{at}cber.fda.gov Back

5 The abbreviations used are: PI3K, phosphatidylinositol 3-kinase; MEK, mitogen-activated protein (MAP) kinase kinase; ERK, extracellular signal-regulated kinase; TCF, ternary complex factor; SRF, serum response factor; SRE, serum response element; TM, tropomyosin; Hsp90, heat shock protein 90; JNK, c-Jun NH2-terminal kinase; AP-1, activating protein; ATF, activating transcription factor; DCS, donor calf serum; pp90RSK, 90-kDa ribosomal S6 kinase; ROCK, Rho-associated kinase. Back

6 P. N. Kim and R. A. J. Janssen, unpublished observations. Back

7 P. N. Kim, E. Jonasch, J. W. Mier, and R. A. J. Janssen, unpublished observations. Back

8 Unpublished observation. Back

Received for publication 2/28/00. Revision received 7/ 6/01. Accepted for publication 9/17/01.


    References
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

  1. Pawlak G., Helfman D. M. Cytoskeletal changes in cell transformation and tumorigenesis. Curr. Opin. Genet. Dev., 11: 41-47, 2001.[Medline]
  2. Campbell S. L., Khosravi-Far R., Rossman K. L., Clark G. J., Der C. J. Increasing complexity of Ras signaling. Oncogene, 17: 1395-1413, 1998.[Medline]
  3. Rodriguez-Viciana P., Warne P. H., Khwaja A., Marte B. M., Pappin D., Das P., Waterfield M. D., Ridley A., Downward J. Role of phosphoinositide 3-OH kinase in cell transformation and control of the actin cytoskeleton by Ras. Cell, 89: 457-467, 1997.[Medline]
  4. Jansen H. W., Lurz R., Bister K., Bonner T. I., Mark G. E., Rapp U. R. Homologous cell-derived oncogenes in avian carcinoma virus MH2 and murine sarcoma virus 3611. Nature, 307: 281-284, 1984.[Medline]
  5. Cowley S., Paterson H., Kemp P., Marshall C. J. Activation of MAP kinase kinase is necessary and sufficient for PC12 differentiation and for transformation of NIH 3T3 cells. Cell, 77: 841-852, 1994.[Medline]
  6. Robinson M. J., Stippec S. A., Goldsmith E., White M. A., Cobb M. H. A constitutively active and nuclear form of the MAP kinase ERK2 is sufficient for neurite outgrowth and cell transformation. Curr. Biol., 8: 1141-1150, 1998.[Medline]
  7. Gille H., Kortenjann M., Thomae O., Moomaw C., Slaughter C., Cobb M. H., Shaw P. E. ERK phosphorylation potentiates Elk-1-mediated ternary complex formation and transactivation. EMBO J., 14: 951-962, 1995.[Medline]
  8. Cruzalegui F. H., Cano E., Treisman R. ERK activation induces phosphorylation of elk-1 at multiple S/T-P motifs to high stoichiometry. Oncogene, 18: 7948-7957, 1999.[Medline]
  9. Cahill M. A., Janknecht R., Nordheim A. Signalling pathways: jack of all cascades. Curr. Biol., 6: 16-19, 1996.[Medline]
  10. Olive M., Krylov D., Echlin D. R., Gardner K., Taparowsky E., Vinson C. A dominant negative to activation protein-1 (AP1) that abolishes DNA binding and inhibits oncogenesis. J. Biol. Chem., 272: 18586-18594, 1997.[Abstract/Free Full Text]
  11. Angel P., Karin M. The role of Jun, Fos and the AP-1 complex in cell-proliferation and transformation. Biochim. Biophys. Acta, 1072: 129-157, 1991.[Medline]
  12. Karin M., Liu Z., Zandi E. AP-1 function and regulation. Curr. Opin. Cell Biol., 9: 240-246, 1997.[Medline]
  13. Zohn I. M., Campbell S. L., Khosravi-Far R., Rossman K. L., Der C. J. Rho family proteins and Ras transformation: the RHOad less traveled gets congested. Oncogene, 17: 1415-1438, 1998.[Medline]
  14. Shields J. M., Pruitt K., McFall A., Shaub A., Der C. J. Understanding Ras: "it ain’t over ’til it’s over". Trends Cell Biol., 10: 147-154, 2000.[Medline]
  15. Poser S., Impey S., Trinh K., Xia Z., Storm D. R. SRF-dependent gene expression is required for PI3-kinase-regulated cell proliferation. EMBO J., 19: 4955-4966, 2000.[Abstract]
  16. Prasad G. L., Fuldner R. A., Cooper H. L. Expression of transduced tropomyosin 1 cDNA suppresses neoplastic growth of cells transformed by the ras oncogene. Proc. Natl. Acad. Sci. USA, 90: 7039-7043, 1993.[Abstract/Free Full Text]
  17. Takenaga K., Masuda A. Restoration of microfilament bundle organization in v-raf-transformed NRK cells after transduction with tropomyosin 2 cDNA. Cancer Lett., 87: 47-53, 1994.[Medline]
  18. Gimona M., Kazzaz J. A., Helfman D. M. Forced expression of tropomyosin 2 or 3 in v-Ki-ras-transformed fibroblasts results in distinct phenotypic effects. Proc. Natl. Acad. Sci. USA, 93: 9618-9623, 1996.[Abstract/Free Full Text]
  19. Janssen R. A., Mier J. W. Tropomyosin-2 cDNA lacking the 3' untranslated region riboregulator induces growth inhibition of v-Ki-ras-transformed fibroblasts. Mol. Biol. Cell, 8: 897-908, 1997.[Abstract/Free Full Text]
  20. Boyd J., Risinger J. I., Wiseman R. W., Merrick B. A., Selkirk J. K., Barrett J. C. Regulation of microfilament organization and anchorage-independent growth by tropomyosin 1. Proc. Natl. Acad. Sci. USA, 92: 11534-11538, 1996.[Abstract/Free Full Text]
  21. Kwon H. J., Yoshida M., Fukui Y., Horinouchi S., Beppu T. Potent and specific inhibition of p60v-src protein kinase both in vivo and in vitro by radicicol. Cancer Res., 52: 6926-6930, 1992.[Abstract/Free Full Text]
  22. Zhao J. F., Nakano H., Sharma S. Suppression of RAS and MOS transformation by radicicol. Oncogene, 11: 161-173, 1995.[Medline]
  23. Kwon H. J., Yoshida M., Muroya K., Hattori S., Nishida E., Fukui Y., Beppu T., Horinouchi S. Morphology of ras-transformed cells becomes apparently normal again with tyrosine kinase inhibitors without a decrease in the ras-GTP complex. J. Biochem., 118: 221-228, 1995.[Abstract/Free Full Text]
  24. Kwon H. J., Yoshida M., Nagaoka R., Obinata T., Beppu T., Horinouchi S. Suppression of morphological transformation by radicicol is accompanied by enhanced gelsolin expression. Oncogene, 15: 2625-2631, 1997.[Medline]
  25. Sharma S. V., Agatsuma T., Nakano H. Targeting of the protein chaperone, HSP90, by the transformation suppressing agent, radicicol. Oncogene, 16: 2639-2645, 1998.[Medline]
  26. Schulte T. W., Akinaga S., Soga S., Sullivan W., Stensgard B., Toft D., Neckers L. M. Antibiotic radicicol binds to the N-terminal domain of Hsp90 and shares important biologic activities with geldanamycin. Cell Stress Chaperones, 3: 100-108, 1998.[Medline]
  27. Schulte T. W., Akinaga S., Murakata T., Agatsuma T., Sugimoto S., Nakano H., Lee Y. S., Simen B. B., Argon Y., Felts S., Toft D. O., Neckers L. M., Sharma S. V. Interaction of radicicol with members of the heat shock protein 90 family of molecular chaperones. Mol. Endocrinol., 13: 1435-1448, 1999.[Medline]
  28. Csermely P., Schnaider T., Soti C., Prohaszka Z., Nardai G. The 90-kDa molecular chaperone family: structure, function, and clinical applications. A comprehensive review. Pharmacol. Ther., 79: 129-168, 1998.[Medline]
  29. Soga S., Kozawa T., Narumi H., Akinaga S., Irie K., Matsumoto K., Sharma S. V., Nakano H., Mizukami T., Hara M. Radicicol leads to selective depletion of Raf kinase and disrupts K-Ras-activated aberrant signaling pathway. J. Biol. Chem., 273: 822-828, 1998.[Abstract/Free Full Text]
  30. Ki S. W., Kasahara K., Kwon H. J., Eishima J., Takesako K., Cooper J. A., Yoshida M., Horinouchi S. Identification of radicicol as an inhibitor of in vivo Ras/Raf interaction with the yeast two-hybrid screening system. J. Antibiot., 51: 936-944, 1998.[Medline]
  31. Takehana K., Sato S., Kobayasi T., Maeda T. A radicicol-related macrocyclic nonaketide compound, antibiotic LL- Z1640–2, inhibits the JNK/p38 pathways in signal-specific manner. Biochem. Biophys. Res. Commun., 257: 19-23, 1999.[Medline]
  32. Sahai E., Ishizaki T., Narumiya S., Treisman R. Transformation mediated by RhoA requires activity of ROCK kinases. Curr. Biol., 9: 136-145, 1999.[Medline]
  33. Sahai E., Alberts A. S., Treisman R. RhoA effector mutants reveal distinct effector pathways for cytoskeletal reorganization, SRF activation and transformation. EMBO J., 17: 1350-1361, 1998.[Abstract]
  34. Hill C. S., Wynne J., Treisman R. The Rho family GTPases RhoA, Rac1, and CDC42Hs regulate transcriptional activation by SRF. Cell, 81: 1159-1170, 1995.[Medline]
  35. Pillay I., Nakano H., Sharma S. V. Radicicol inhibits tyrosine phosphorylation of the mitotic Src substrate Sam68 and retards subsequent exit from mitosis of Src-transformed cells. Cell Growth Differ., 7: 1487-1499, 1996.[Abstract]
  36. Fukazawa H., Nakano S., Mizuno S., Uehara Y. Inhibitors of anchorage-independent growth affect the growth of transformed cells on poly(2-hydroxyethyl methacrylate)-coated surfaces. Int. J. Cancer, 67: 876-882, 1996.[Medline]
  37. Janssen R. A., Veenstra K. G., Jonasch P., Jonasch E., Mier J. W. Ras-, and Raf-induced down-modulation of non-muscle tropomyosin are MEK- independent. J. Biol. Chem., 273: 32182-32186, 1998.[Abstract/Free Full Text]
  38. Seddighzadeh M., Linder S., Shoshan M. C., Auer G., Alaiya A. A. Inhibition of extracellular signal-regulated kinase 1/2 activity of the breast cancer cell line MDA-MB-231 leads to major alterations in the pattern of protein expression. Electrophoresis, 21: 2737-2743, 2000.[Medline]
  39. Lee J. W., Lee Y. C., Na S. Y., Jung D. J., Lee S. K. Transcriptional coregulators of the nuclear receptor superfamily: coactivators and corepressors. Cell. Mol. Life Sci., 58: 289-297, 2001.[Medline]
  40. Shimada Y., Ogawa T., Sato A., Kaneko I., Tsujita Y. Induction of differentiation of HL-60 cells by the anti-fungal antibiotic, radicicol. J. Antibiot. (Tokyo), 48: 824-830, 1995.[Medline]
  41. Beltman J., Erickson J. R., Martin G. A., Lyons J. F., Cook S. J. C3 toxin activates the stress signaling pathways, JNK and p38, but antagonizes the activation of AP-1 in rat-1 cells. J. Biol. Chem., 274: 3772-3780, 1999.[Abstract/Free Full Text]
  42. Stamm S., Casper D., Lees-Miller J. P., Helfman D. M. Brain-specific tropomyosins TMBr-1 and TMBr-3 have distinct patterns of expression during development and in adult brain. Proc. Natl. Acad. Sci. USA, 90: 9857-9861, 1993.[Abstract/Free Full Text]
  43. Alessandrini A., Greulich H., Huang W., Erikson R. L. Mek1 phosphorylation site mutants activate Raf-1 in NIH 3T3 cells. J. Biol. Chem., 271: 31612-31618, 1996.[Abstract/Free Full Text]
  44. Behre G., Smith L. T., Tenen D. G. Use of a promoterless Renilla luciferase vector as an internal control plasmid for transient co-transfection assays of Ras-mediated transcription activation. Biotechniques, 26: 24-26, 28, 1999.[Medline]



This article has been cited by other articles:


Home page
Mol. Cell. Biol.Home page
R. A. J. Janssen, P. N. Kim, J. W. Mier, and D. K. Morrison
Overexpression of Kinase Suppressor of Ras Upregulates the High-Molecular-Weight Tropomyosin Isoforms in ras-Transformed NIH 3T3 Fibroblasts
Mol. Cell. Biol., March 1, 2003; 23(5): 1786 - 1797.
[Abstract] [Full Text] [PDF]


This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kim, P. N.
Right arrow Articles by Janssen, R. A. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kim, P. N.
Right arrow Articles by Janssen, R. A. J.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cell Growth & Differentiation