V600E B-Raf requires the Hsp90 chaperone
for stability and is degraded in response
to Hsp90 inhibitors
O. M. Grbovic, A. D. Basso*, A. Sawai, Q. Ye, P. Friedlander, D. Solit, and N. Rosen†
Program in Molecular Pharmacology and Chemistry and Department of Medicine, Memorial Sloan–Kettering Cancer Center, New York, NY 10021
Communicated by Samuel J. Danishefsky, Memorial Sloan–Kettering Cancer Center, New York, NY, November 16, 2005 (received for review May 25, 2005)
The Raf family includes three members, of which B-Raf is fre-
quently mutated in melanoma and other tumors. We show that
Raf-1 and A-Raf require Hsp90 for stability, whereas B-Raf does
not. In contrast, mutated, activated B-Raf binds to an Hsp90–cdc37
complex, which is required for its stability and function. Exposure
17-demethoxygeldanamycin results in the degradation of mutant
B-Raf, inhibition of mitogen-activated protein kinase activation
and cell proliferation, induction of apoptosis, and antitumor ac-
tivity. These data suggest that activated mutated B-Raf proteins
are incompetent for folding in the absence of Hsp90, thus sug-
gesting that the chaperone is required for the clonal evolution of
melanomas and other tumors that depend on this mutation. Hsp90
inhibition represents a therapeutic strategy for the treatment of
17-allylamino-17-demethoxygeldanamycin ? cdc37 ? melanoma
regulated kinase kinase (MEK)?MAPK signaling pathway is mu-
tationally activated in most melanomas. One member of the Ras
family, N-Ras, is mutated in ?25% of melanomas (1), whereas
mutations in the H-ras and K-ras genes are rare (2). The Raf gene
family (Raf-1, A-Raf, and B-Raf) encodes closely related serine?
threonine protein kinases that are important effectors of Ras
recently, when Davies et al. (3) showed that Raf-1 and A-Raf are
rarely mutated but that mutations in the B-Raf gene are common
in human cancer, especially in melanoma.
Refs. 3 and 4 showed that B-Raf is mutated in ?70% of human
melanomas, 35–70% of papillary thyroid carcinomas, and less
always in the B-Raf kinase domain and, in melanomas, the vast
majority are V600E missense mutations (3). Marais and coworkers
(5) have shown in heterologous systems that the V600E mutation
leads to activation of B-Raf kinase.
The frequency and activating nature of the B-Raf mutations
and perhaps other tumors in which they have been detected.
Moreover, small interfering RNA against mutated B-Raf but not
Raf-1 inhibits the transformed phenotype of melanoma cells har-
be mutually exclusive in melanoma, suggesting that they make
similar contributions to transformation and that activation of this
pathway is a key event in the development of this disease (3, 8).
Together, these data suggest that inhibition of B-Raf?MEK?
MAPK signaling could be a powerful means for treating melano-
mas and other tumors with B-Raf mutation. There is no validated
therapy that potently inhibits mutated B-Raf function in patients.
Selective inhibitors of MEK have been developed and have anti-
tumor activity in xenograft models of melanoma (9). Putative Raf
inhibitor is currently in trial but has low potency against Raf and
inhibits multiple other kinases (10, 11).
n the last several years, it has become clear that the Ras?Raf?
mitogen-activated protein kinase (MAPK)?extracellular signal-
The details of Raf regulation suggest another strategy for its
inhibition. The protein chaperone Hsp90 is required for the con-
formational maturation of several key signaling proteins, including
Raf-1 (12). Inhibition of Hsp90 function with natural products like
geldanamycin that bind to its N terminus causes the ubiquitin-
mycin derivative, 17-allylamino-17-demethoxygeldanamycin (17-
we reasoned that B-Raf is also likely to require Hsp90 function and
that 17-AAG would induce its degradation and cause inhibition of
melanoma growth. Surprisingly, we found that although Raf-1 and
A-Raf are degraded in cells that are exposed to 17-AAG, WT
B-Raf is not found in an Hsp90 complex and is unaffected by the
inhibitor. However, mutationally activated B-Raf apparently ac-
quires a dependence on Hsp90 for its stability; it is associated with
Hsp90 and is selectively degraded in the proteasome in cells
exposed to 17-AAG. Degradation of mutated B-Raf leads to
antitumor activity in murine xenograft models.
Pharmacologic Inhibition of Hsp90 Function Leads to a Decrease in the
Expression of Raf-1 and A-Raf But Not B-Raf.Raf-1(c-Raf)isaknown
Hsp90 client protein that binds and depends on Hsp90 chaperone
function for its proper folding and stability (14). Hsp90 inhibitors
such as 17-AAG disrupt the Raf1?Hsp90 association, resulting in
degradation of Raf-1 via the proteasome (13). To determine
examined the effects of 17-AAG on expression of each of the Raf
family members in a panel of 16 human tumor cell lines, primarily
As reported previously, we found that 100 nM 17-AAG causes
?90% decline in Raf-1 expression levels in all tested cell lines after
24 h of treatment (Figs. 1A and 2A, and data not shown). A-Raf
expression was lost with similar kinetics and sensitivity, suggesting
that it is likely to be an Hsp90 client as well (Figs. 1A and 2A, and
data not shown). In contrast, exposure of cells to up to 2.5 ?M
17-AAG for 24 h had no significant effect on the expression of WT
B-Raf in SK-Mel-31 melanoma cells or in other tumor cell lines
expressing WT B-Raf (Fig. 1). Similarly, 17-AAG had no effect on
expression of WT B-Raf in normal human epithelial melanocytes
that B-Raf differs from A-Raf and Raf-1 in its requirement for
Hsp90 for folding and stability and that WT B-Raf is unaffected by
Hsp90 inhibition. In support of this idea, Hsp90 was not detected
Conflict of interest statement: No conflicts declared.
Abbreviations: 17-AAG, 17-allylamino-17-demethoxygeldanamycin; MAPK, mitogen-acti-
vated protein kinase; MEK, MAPK?extracellular signal-regulated kinase kinase; PI3-kinase,
phosphatidylinositol 3-kinase; IP, immunoprecipitation.
*Present address: Schering–Plough, 2015 Galloping Hill Road, Kenilworth, NJ 07033.
†To whom correspondence should be addressed. E-mail: email@example.com.
© 2005 by The National Academy of Sciences of the USA
January 3, 2006 ?
vol. 103 ?
no. 1 ?
in immunoprecipitates of epitope-tagged WT B-Raf (see below
and Fig. 5).
Activating Mutants of B-Raf Acquire Sensitivity to Hsp90 Inhibitors.
Although most proteins do not require Hsp90 for stability, gain-
of-function mutants, such as v-src, bcr-abl, mutant c-kit, and p53
oncogenes, may acquire dependence on Hsp90 for correct folding
(15–19). In each case, the mutated oncoprotein binds to Hsp90 and
is degraded in cells exposed to Hsp90 inhibitors, whereas the
corresponding normal protooncogene product is much less sensi-
tive to degradation by these drugs. This selective, mutation-
for mutationally activated B-Raf as well.
A V600E substitution in the activation segment of B-Raf kinase
cancers (3). SK-Mel-28 and seven other cancer cell lines with this
a dose-dependent loss of V600E B-Raf expression and a loss of
A-Raf and Raf-1 expression in those cells (Fig. 2 A and B). Also,
to determine whether the effect of 17-AAG on V600E B-Raf
expression was limited to this particular B-Raf mutation, we
cells, which express the V600D B-Raf mutated protein and in two
non-small-cell lung cancer cell lines (H1666 and H1755) with
mutations within the G-loop of B-Raf kinase, 17-AAG causes loss
of B-Raf expression, with kinetics and sensitivity that are indistin-
guishable from that of V600E B-Raf (Fig. 2C).
Initial loss of V600E B-Raf expression was observed as early as
12 h after exposure of SK-Mel-28 cells to 1 ?M 17-AAG, whereas
?90% inhibition occurred by 24 h and persisted up to 48 h later
(Fig. 3A). The proteasome inhibitor MG132 prevented loss of
V600E B-Raf expression, resulting in the partition into a Nonidet
P-40-insoluble fraction (Fig. 3B). Lysosome, caspase, or calpain
inhibitors had no effect on 17-AAG-induced loss of V600E B-Raf
expression. Moreover, we found that the combination of protea-
some inhibitor with 17-AAG resulted in the more pronounced
formation of polyubiquitinated, higher-molecular-weight forms of
V600E B-Raf, as compared with treatment of cells with the
proteasome inhibitor alone (Fig. 3C). These findings are consistent
AKT, and Raf-1, in which inhibition of Hsp90 leads to ubiquitina-
tion and trafficking of Hsp90 client protein to an Nonidet P-40-
insoluble fraction of the proteasome where it gets degraded (13,
To determine whether the sensitivity of B-Raf to degradation is
cell-context-dependent, MCF7 breast cancer cells were transiently
transfected with myc-tagged, WT, or V600E B-Raf and subse-
quently exposed to 17-AAG (Fig. 4). Treatment with 1 ?M
17-AAG had no effect on the expression of myc-tagged or endog-
enous WT B-Raf in these cells. However, 17-AAG did cause the
loss of myc-tagged V600E B-Raf expression with similar kinetics
and sensitivity as compared with endogenous mutated B-Raf in
melanoma cells (Fig. 4 A and B). These data confirm differential
sensitivity of B-Raf is a function of its mutational status. Moreover,
17-AAG caused significant inhibition of kinase activity of myc-
tagged mutant B-Raf as revealed by B-Raf kinase cascade assay.
The initial loss of mutant B-Raf kinase activity was observed as
early as 4 h after 17-AAG treatment, whereas ?70% (?8.3%)
inhibition was achieved by 8 h, indicating that the loss of kinase
activity of myc-tagged V600E B-Raf may occur before loss of its
expression. After 24 h of exposure to 1 ?M 17-AAG, a slight (31 ?
7.6%) decrease of myc-tagged WT B-Raf kinase activity was
observed (Fig. 4C).
V600E B-Raf Is Found in a Complex with Cdc37 and Hsp90. The data
presented above suggest that Hsp90 function is required for the
stability of V600E but not WT B-Raf. Serine kinases, such as Raf-1
and cdk4, that depend on Hsp90 are found in cells in a complex
containing Hsp90 and its cochaperone cdc37 (14, 22). We found
V600E but not WT B-Raf (Fig. 5A Middle). Cdc37 was detected in
with 17-AAG caused a dose-dependent decline in A-Raf and C-Raf expression
and also loss of V600E B-Raf expression in SK-Mel-28 cells. (B) (Left) The 24-h
treatment with 17-AAG caused a dose-dependent down-regulation of V600E
p85 PI3-kinase expression was assessed as a loading control. (C) A 24 h
G465V, and G468A B-Raf expression.
17-AAG inhibits expression of mutant B-Raf. (A) A 24-h treatment
sion. (A) SK-Mel-31 cells (WT B-Raf) were treated with the indicated concen-
trations of 17-AAG for 24 h, and levels of A-Raf, B-Raf, C-Raf (Raf-1), and p85
PI3-kinase expression were determined by immunoblotting. 17-AAG caused a
dose-dependent decline in A-Raf and C-Raf expression but had no effect on
expression of WT B-Raf. (B) (Left) 17-AAG had no effect on the expression of
WT B-Raf in a panel of melanoma, colon, and breast cancer cells. (Right) p85
PI3-kinase expression was assessed as a loading control.
17-AAG selectively inhibits A-Raf and C-Raf but not WT B-Raf expres-
www.pnas.org?cgi?doi?10.1073?pnas.0609973103Grbovic et al.
myc immunoprecipitates of both WT and V600E B-Raf; however,
the quantity of cdc37 that is associated with V600E B-Raf was five
After 4 h of exposure to 17-AAG, neither Hsp90 nor cdc37 was
found in association with V600E B-Raf. The association of cdc37
with WT B-Raf was lost as well. The 17-AAG had no effect on
association of V600E B-Raf with Hsp90 or cdc37 preceded the
down-regulation of both its activity and expression, which were not
clearly apparent until 12 h (compare Fig. 5A with Figs. 3A and 4C).
Therefore, these results suggest that 17-AAG inhibits both the
kinase activity of V600E B-Raf and V600E B-Raf??Hsp90 hetero-
Hsp90 Inhibition Causes Inhibition of Melanoma Cell Growth, G1
Cell-Cycle Arrest, and Induction of Apoptosis. In melanoma, the
frequent occurrence of either N-Ras or B-Raf mutation suggests
represents a key element in melanocyte transformation (3, 8). All
melanoma cells, with or without B-Raf and N-Ras mutation, were
6A). Moreover, 17-AAG treatment causes down-regulation of
MAPK activity, loss of cyclin D1 expression, hypophosphorylation
(Fig. 6B). This effect is observed in parallel with A-Raf, Raf-1, and
member of the Raf family in melanomas leads to suppression of
17-AAG causes increase in apoptosis of the SK-Mel-28 (from
5.07% to 15.14% in 48 h) and SK-Mel-31 (from 4.7% to 9.23% in
48 h), as measured by an increase in the sub-G1fraction by FACS.
Growth of SK-Mel-28 Xenograft Tumors. We sought to determine
whether the degradation of V600E B-Raf by 17-AAG could be
elicited in xenograft tumors by 17-AAG. In SK-Mel-28 mouse
dation of V600E B-Raf. (A) After SK-Mel-28 treatment with 1 ?M 17-AAG, loss
of V600E B-Raf expression was first observed as early as 12 h, whereas 90%
inhibition was achieved by 24 h and persisted up to 48 h of treatment. (B)
?M calpeptin, 10 ?M caspase inhibitor (Casp. Inhib.), and 20 mM ammonium
chloride, followed by 24 h of exposure to 1 ?M 17-AAG. Cells were lysed in
Nonidet P-40 buffer, and the Nonidet P-40-insoluble fraction was solubilized
(WB). The proteasome inhibitor abrogated 17-AAG-induced loss of V600E
B-Raf. The protected V600E B-Raf protein accumulated in a Nonidet P-40
and blotted for ubiquitin (UB) and B-Raf.
17-AAG induced ubiquitination and proteasome-mediated degra-
tion of kinase activity and protein expression. Myc-tagged WT or V600E
mutant B-Raf was transiently expressed in MCF-7 cells. At 24 h after transfec-
tion, cells were treated with 1 ?M 17-AAG for 12 or 24 h. (A) Expression of
myc-tagged WT and V600E mutant B-Raf was analyzed by immunoblotting
(Top). The effect of 17-AAG on endogenously expressed WT B-Raf (Middle)
and C-Raf (Bottom) were also assessed. (B) Results of a representative exper-
expression levels of indicated proteins vs. control. (C) Kinase activity of B-Raf
assayed by IP with myc after 17-AGG treatment over 24 h was measured by
using a kinase assay.
V600E B-Raf mutation confers sensitivity to 17-AAG-induced inhibi-
Grbovic et al. PNAS ?
January 3, 2006 ?
vol. 103 ?
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down-regulation of V600E B-Raf, A-Raf, and Raf-1 (Fig. 7A).
Down-regulation of all three Raf isoforms was accompanied by
inhibition of MAPK activity and a loss of cyclin D1 expression. No
change was detected in the expression of several control proteins,
including MAPK and p85 phosphatidylinositol 3-kinase (PI3-
kinase) (Fig. 7A). Treatment of these mice with 17-AAG for 3
consecutive days each week for 4 weeks resulted in a dose-
dependent inhibition of tumor growth, with 100 mg?kg causing
?80% inhibition as compared with vehicle-treated mice at day 25
(Fig. 7B). The 17-AAG was well tolerated at these dose levels, with
no treatment-associated mortality.
The frequent activation by mutation of a specific signaling pathway
in a particular tumor type generally suggests a rational strategy for
mechanism-based therapy. All strategies that effectively inhibit
mutationally activated protooncogene products have proven to be
useful in treating the relevant cancer. Thus, pharmacologic inhibi-
tion of bcr-abl, c-kit, EGF receptor, and HER2, as well as modu-
lation of PML-RXR have proven to be beneficial, sometimes
remarkably so, in tumors in which these targets are mutated.
Recent data suggest that deregulation of Ras?Raf signaling is
characteristic of melanomas and necessary for their continued
proliferation. Mutational activation of N-Ras (25%) and B-Raf
(60–70%) occur in high percentage of cases but not together in the
same tumor (1, 3, 23, 24). The implication is that neither of the two
has a selective advantage if the other is present but that activation
of signaling through Raf is a common event in the development of
the disease. Also, experiments in cell culture show that MAPK
is constitutive in tumor cells (25). These data imply that mutational
activation of N-Ras or B-Raf leads to growth-factor-independent
deregulation of melanoma cell proliferation and that this is a key
step in tumorigenesis. In support of this inference, small interfering
RNA for B-Raf but not Raf-1 inhibits the transformed phenotype
(7). Suppression of oncogenic N-Ras by RNA interference induces
apoptosis in human melanoma cells, suggesting that N-Ras is
important for avoidance of apoptosis in melanomas that harbor the
codon 61 N-Ras mutation (26).
These conclusions lead to the hypothesis that effective inhi-
bition of signaling through the N-Ras?B-Raf pathway could be
very useful in the therapy of this disease, which, when metastatic,
is essentially untreatable. However, potent in vivo inhibition of
neither N-Ras nor B-Raf has been accomplished. Several B-Raf
inhibitors are under development, but the B-Raf inhibitor cur-
V600E mutant B-Raf was transiently expressed in MCF-7 cells. At 24 h after
transfection, cells were treated with 1 ?M 17-AAG for 4 h. Lysates were
of Cdc37 and Hsp90 at this time point. WB, Western blot analysis.
V600E B-Raf associates with Cdc37 and Hsp90. (A) Myc-tagged WT or
cell cycle and an increase in sub-G1fraction. In parallel, 24 h of exposure to indicated concentrations of 17-AAG caused down-regulation in expression of
phospho-MAPK, cyclin D1, and hypophosphorylation Rb in both cell lines and had no effect on MAPK or p85 PI3-kinase expression.
17-AAG inhibits Raf signaling and induces a G1growth arrest. The 24-h treatment with 100 nM 17-AAG caused G1block in SK-Mel-28 and SK-Mel-31
www.pnas.org?cgi?doi?10.1073?pnas.0609973103Grbovic et al.
rently in clinical trial inhibits many protein kinases, is not a
potent Raf inhibitor, and has little single agent activity in
melanoma patients (10, 11). Its clinical antitumor activity has
been attributed to its inhibition of VEGF receptor (10).
Here, we report another mechanism for inhibiting mutated
B-Raf. A chaperone complex containing Hsp90, cdc37, and other
cochaperones is required for the folding, conformational matura-
(14). Raf-1 and other client proteins are degraded in cells exposed
into this class of proteins but that B-Raf does not. Hsp90 is not
detected in B-Raf pull-down experiments and WT B-Raf is not
degraded in melanocytes or tumor cells treated with 17-AAG.
However, V600E B-Raf does associate with Hsp90 and this mutant
is degraded in response to pharmacologic inhibition of Hsp90.
The data suggest that, unlike A-Raf and Raf-1, WT B-Raf does
not require Hsp90 for stability, but mutated V600E B-Raf does.
B-Raf is essential for B-Raf kinase activation. Structural studies by
Wan et al. (5) suggest that this phosphorylation is required to
domain (G-loop), allowing the activation loop to adapt the cata-
lytically active conformation. V600E and most of the other acti-
vating B-Raf mutations found in human cancers are predicted to
disrupt this interaction, obviating the need for phosphorylation of
T598 and accounting for constitutive activation. We show that both
WT and V600E bind to the cdc37 cochaperone, but Hsp90 is
detected in association only with V600E and not WT B-Raf. It is
possible that, whereas WT B-Raf does not require Hsp90 for
efficient folding, V600E does. Alternatively, the activated V600E
conformation may require Hsp90 for stability. Induction of V600E
degradation is preceded by loss of its association with Hsp90, in
support of the latter idea.
It is not clear whether Hsp90 dependence is caused by the
inefficient folding or instability of particular amino acid substitu-
tions or the instability of the active conformation of the protein.
V600E and V600D, which are both predicted to mimic T598
phosphorylation and disrupt activation-loop inhibition, are both
sensitive to 17-AAG. Similarly, two G-loop B-Raf mutants, G465V
and G468A, were also found to be sensitive to 17-AAG exposure.
Although V600E, V600D, and G468A mutations generate ampli-
fied B-Raf kinase activity, G465A mutation is shown to result in
impaired or lower activity of kinase as compared with WT B-Raf
(5). These findings suggest that activated kinase activity is not
required for sensitization to Hsp90 inhibitors. Also, we found that
inhibition of intracellular V600E B-Raf kinase activity by 17-AAG
begins before detection of loss of B-Raf mutant protein. Several
other mutated oncoproteins and oncogenic fusion proteins, includ-
ing v-src, p53, mutant c-kit, bcr-abl, and NPM-ALK, have an
increased requirement for Hsp90 compared with their normal
sense, Hsp90 may be considered necessary for the development or
clonal evolution of such tumors. The dependence of some tumors
antitumor activity of 17-AAG in various in vivo models (28).
exposed to stress by refolding proteins denatured under these
conditions, it also allows the selection of gain-of-function mutants
that would not fold efficiently in its absence.
However, melanoma cell lines with mutant B-Raf are not more
sensitive to 17-AAG than those with WT B-Raf. The above finding
is perhaps not surprising, considering that Raf-1 is an Hsp90 client
as well and that 17-AAG induces its degradation (13). Several lines
of evidence suggest that, in melanoma, N-Ras or B-Raf mutation
lead to constitutive activation of MAPK by Raf-1- or B-Raf-
dependent pathways and that this activation is required for main-
tenance of the transformed phenotype (25). MAPK is shown to be
constitutively activated in the absence of serum in melanoma cell
lines (25). Marais and coworkers (5) have shown that a class of rare
B-Raf mutants with reduced catalytic activity bind to and activate
Raf-1 kinase and may exert their effects in tumor cells in this way.
mutatant B-Raf are the most sensitive (29). Thus, Hsp90 inhibition
induces the degradation of both mutated B-Raf and Raf-1 with
expression, G1arrest, and an increase in apoptosis in all tested
melanoma cell lines.
Together, these data suggest that induction of B-Raf and Raf-1
degradation by Hsp90 inhibitors could be an important therapeutic
strategy for melanomas and other tumors with B-Raf mutation.
Workman and coworkers (31) have noted significant clinical activ-
ity in several patients with melanoma in the phase-1 clinical trial of
17-AAG. Here, we show that levels of 17-AAG that are achievable
in vivo without significant toxicity are sufficient to cause degrada-
tion of Raf-1, A-Raf, and mutant B-Raf, as well as marked
are associated with significant antitumor activity.
Materials and Methods
Materials. 17-AAG (330507, National Service Center, National
Cancer Institute, Bethesda), MG132, calpeptin, and caspase inhib-
itor I (Calbiochem) were dissolved in 100% DMSO. We used the
following polyclonal Abs: B-Raf, C-Raf, A-Raf, c-myc (Santa Cruz
Biotechnology), p85 PI3-kinase (Upstate Biotechnology, Lake
Placid, NY), cdc37 (Affinity BioReagents, Golden, CO), Hsp90
(Stressgen Biotechnologies, Victoria, Canada), and ubiquitin (Co-
vance, Berkeley, CA). Protein G–sepharose (Amersham Pharma-
cia) was used for immunoprecipitation (IP).
inhibited the growth of SK-Mel-28 xenografts. (A) Mice with
established tumors were treated for 3 consecutive days with
17-AAG (0, 25, 50, or 100 mg?kg) or the egg phospholipid
vehicle alone as a control. At 12 h after the third treatment,
mice were killed and tumor tissue was homogenized in 2%
SDS lysis buffer and analyzed by immunoblotting for various
of SK-Mel-28 xenografts in a dose-dependent manner.
17-AAG causes degradation of mutant B-Raf and
Grbovic et al. PNAS ?
January 3, 2006 ?
vol. 103 ?
no. 1 ?
Cell Culture. The following human melanoma cancer lines were Download full-text
used in this study: SK-Mel-1, SK-Mel-2, SK-Mel-5, SK-Mel-19,
SK-Mel-28, WM-266.4, SK-Mel-31, SK-Mel-103, and SK-Mel-
147; colon cancer lines HT-29 and Colo-205; breast cancer cell
lines MCF-7 and SKBr-3 DU-4475; and non-small-cell lung
cancer lines H1666 and H1755. All cells were maintained in a 1:1
mixture of DMEM?F12, except for Colo-205 (which was main-
tained in RPMI medium 1640) and H1755 and H1666 (which
were maintained in ACL-4 supplemented with 2 mM glutamine,
50 units?ml penicillin, 50 units?ml streptomycin, and 10%
heat-inactivated FBS; Gemini Bioproducts, Calabrasas, CA),
and incubated at 37°C in 5% CO2?95% air. Normal human
melanocytes HEMnL were purchased from Cascade Biologies
(Portland, OR) and maintained at standard tissue culture con-
ditions according to the manufacturer’s instructions.
Transfections. Myc-tagged B-Raf cDNA was obtained from Walter
Kolch (University of Glasgow, Glasgow, Scotland). B-Raf cDNA
was inserted into the HindIII site of the pcDNA3.1 vector. The
V600E-B-Raf mutant was then constructed by site-directed mu-
tagenesis (Stragene) in which the nucleotides corresponding to
amino acid 600 were changed from GTG to GAG. We transfected
2 million MCF-7 cells with 10 ?g of DNA by using 20 ?l of
Lipofectamine reagent (Invitrogen and Life Technologies, Rock-
ville, MD). Experiments were performed 24 h after transfection.
Protein Analysis. Cells were lysed in Nonidet P-40 buffer (50 mM
EDTA?20 mM NaF?10 mM PMSF?2.5 mM Na3VO4?1 mM
?-glycerol phosphate, with 10 ?M each leupeptin, aprotinin, and
soybean trypsin inhibitor) and cleared by centrifugation. Nonidet
P-40 insoluble fractions were lysed in 2% SDS in 50 mM Tris and
boiled for 15 min. Protein concentration was determined by using
BCA reagent (Pierce). Samples were separated by 7–15% SDS?
PAGE, transferred to nitrocellulose, immunoblotted, and detected
by chemiluminescence by using the ECL detection reagents (Am-
ersham Pharmacia). Results were quantified by using the Gel Doc
IP and in Vitro Kinase Assay. MCF-7 cells were transfected with WT
or V600E myc–B-Raf constructs for 24 h, subsequently incubated
with 1 ?M 17-AAG for various times, and lysed with Nonidet P-40
buffer. We immunoadsorbed 1,000 ?g of total protein with 10 ?g
of 9E10 anti-myc Ab or normal anti-mouse IgG (control), followed
three times with ice-cold wash buffer (0.05% Tween 20?25 mM
Tris, pH.7.5?150 mM NaCl?10 mM MgCl2?1 mM DTT) and
resuspended in 2% SDS sample buffer. B-Raf kinase activity was
assayed by using a B-Raf kinase cascade assay according to the
manufacturer’s protocols (Upstate Biotechnology).
Growth Assays. Cells were seeded in 96-well plates at 1,000 cells per
well. After 24 h, cells were placed in fresh media containing drug
and allowed to grow for 5 days. The cell number in treated vs.
control wells was estimated after staining with Alamar blue (In-
isolated as described (30) and stained with ethidium bromide, and
DNA content was analyzed by using a FACS cytometer (Becton
Animal Studies. Experiments were carried out under protocols
institutional guidelines for the proper, humane use of animals in
1 ? 107SK-Mel-28 cells were mixed with Matrigel (Collaborative
Research) and inoculated s.c. in the right flank of 6-week-old
athymic BALB?c female mice (National Cancer Institute?
Frederick Cancer Research and Development Center). As soon as
tumors reached a minimum of 5 mm in diameter, mice were
randomly assigned to treatment with 17-AAG 50 or 100 mg?kg by
i.p. injection for 3 consecutive days each week for 4 weeks. Control
mice were treated only with the egg phospholipid vehicle. Mice
were weighed, and tumor volumes were calculated with the follow-
ing formula: ??6 ? larger diameter ? (smaller diameter)2. To
or vehicle only as control and then killed 12 h after the third dose.
For immunoblotting, tumor tissue was homogenized in 2% SDS
lysis buffer (pH 7.4).
We thank W. Kolch for providing myc-tagged B-Raf construct; A. Hough-
ton and P. Chapman (both at the Memorial Sloan–Kettering Cancer
Center) for the SK-Mel melanoma cell lines; and I. Osman (New York
University, New York) for analysis of B-Raf and Ras mutational status in
the cell lines used in this study. This work was supported by the Waxman
Foundation and the Breast Cancer Research Foundation.
1. Mercer, K. E. & Pritchard, C. A. (2003) Biochim. Biophys. Acta Rev. Cancer 1653, 25–40.
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