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Experimental Therapeutics |
Advanced Bioscience Laboratories-Basic Research Program [C. P. W., S. K., M. O.] and Science Applications International Corporation [C. D. H., A. M.], National Cancer Institute-Frederick Cancer Research Development Center, Frederick, Maryland 21702; Curagen Corporation, Branford, Connecticut 06405 [M. J.]; and Division of Cancer Treatment and Diagnosis [E. S.] and Division of Basic Sciences [G. F. V. W.], National Cancer Institute, Bethesda, Maryland 20892
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell invasion is a major component of the complex multistep process of tumor metastasis. Invasion requires both cell motility and degradation of the surrounding ECM, the latter of which is mediated by a number of proteolytic enzymes (reviewed in Ref. 14 ). We and others have shown that HGF/SF stimulation of a variety of cells expressing Met induces the expression of the serine protease urokinase (uPA) and its receptor (uPAR), resulting in an increase of uPA at the cell surface (4 , 7 , 15) through a pathway involving MAPK1/2 signaling (16) . Although uPA is directly involved in the degradation of some components of the ECM, such as fibronectin, most of its ECM/BM-degrading properties are believed to arise through its ability to cleave plasminogen into the broader specificity protease plasmin (17 , 18) . Like uPA, the active plasmin protease is predominantly associated with the cell surface, but its broader substrate specificity allows for plasmin to play a more direct role in ECM/BM degradation. In addition, plasmin can activate metalloproteinases, proteases with potent ECM/BM-degrading capabilities (reviewed in Ref. 19 ). Because uPA plays a central role in catalyzing ECM/BM degradation, it is not surprising that a strong association between uPA expression and induction of the invasive/metastatic phenotype has been demonstrated (reviewed in Ref. 20 ). Thus, activation of the uPA-plasmin proteolytic network is likely of great importance for HGF/SF-mediated cell invasion and metastasis.
Using the induction of uPA/uPAR and subsequent conversion of plasminogen to plasmin as a biological end point, we have developed a cell-based assay to screen for inhibitors of the HGF/SF-Met-mediated signaling pathways that lead to activation of plasmin protease activity. Among a number of interesting inhibitors of this pathway, we show that the geldanamycin family of anisamycin antibiotics inhibit HGF/SF-Met-mediated plasmin activation. The geldanamycins inhibit HGF/SF-mediated cell motility and invasion associated with a reduction in Met expression, and may have potential as a therapeutic in preventing invasion and metastasis associated with aberrant signaling through HGF/SF-Met.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Reagents.
Human plasminogen and a specific plasmin chromophore substrate
(Chromozyme PL) were purchased from Boehringer Mannheim. Human
uncleaved HGF/SF was purified from the supernatant of transformed
NIH3T3 cells engineered to overexpress the factor as previously
described (23)
. Polyclonal antiserum (NCI-53) was
raised against HGF/SF by immunization of rabbits with full-length
purified human HGF/SF. The uPA inhibitor B428 (24)
was a
kind gift from Dr. B. Littlefield (Eisai Research Institute, Andover,
Ma). The MKK inhibitor PD 98059 was purchased from New England Biolabs.
Anthrax LF and PA were a kind gift from Dr. S. Leppla (Institute of
Dental Research, NIH, Bethesda, MD). Drugs were obtained from the Drug
Synthesis and Chemistry Branch of the National Cancer Institute
(Rockville, MD).
The HGF/SF-Met-uPA-Plasmin Cellular Assay.
The following procedure was used to determine the effect of test
reagents on HGF/SF-mediated plasmin activation and cell growth. MDCK-2
cells were seeded at 1500 cells/well of a 96-well microtiter plate and
grown overnight in DMEM/10% FBS growth medium. Duplicate plates were
made for the determination of plasmin activation and cell growth. Drugs
were serially diluted from stock concentrations in DMEM/10% FBS media
and added to the relative wells. A 1:100 dilution of the NCI-53
neutralizing antiserum to human HGF/SF was added to the relevant wells
as a standard control on each microtiter plate. Immediately after drug
or reagent addition, HGF/SF (10 units/ml) was added to all wells (with
the exception of wells used to calculate basal growth and plasmin
activation). Twenty-four h after drug/HGF/SF addition, one of two
duplicate plates was processed for the determination of plasmin
activity as follows. Wells were washed twice with DMEM (without phenol
red; Life Technologies, Inc.), and 200 µl of reaction buffer
[50% (v/v) 0.05 units/ml plasminogen in DMEM (without phenol
red), 40% (v/v) 50 mM Tris buffer (pH 8.2), and 10% (v/v)
3 mM Chromozyme PL in 100 mM glycine solution]
were added to each well. The plates were then incubated at 37°C, 5%
CO2 for 4 h, at which time the absorbances
generated were read on an automated spectrophotometric plate reader at
a single wavelength of 405 nm. The determination of cell growth on a
duplicate plate was performed by measuring SRB staining of cellular
proteins as described previously (25)
. In brief, cells
were fixed in situ with 50% trichloroacetic acid,
and the plates were washed five times with deionized water and dried.
SRB [100 µl/well, 0.4 (w/v) in 1% acetic acid] was added to each
well and incubated for 30 min at room temperature. Unbound SRB was
removed by washing three times with 1% acetic acid. Plates were then
air-dried, and bound stain was solubilized with 10
mM Tris. Absorbances were read on an automated
spectrophotometric plate reader at a single wavelength of 570 nm.
Plasmin activation (A405 nm) was first normalized for the amount of protein in each well. After subtracting the background plasmin activity of unstimulated control cells, percent inhibition of chromozyme production [% chromozyme inhibition (CI)] for all test agents was calculated relative to HGF/SF-stimulated cells in the absence of the drug. IC50 values (concentration of drug inhibiting HGF/SF response by 50%) and GI50 values (concentration of drug inhibiting growth by 50%) were then calculated for each drug or reagent from plotted graphs. For the calculation of GI50, one replica plate per experiment was fixed before 24-h HGF/SF stimulation to determine the growth at t = 0 (= 0% growth).
Cell Scattering, Branching Morphogenesis/Invasion, and Motility
Assays.
MDCK-2 cells were used in cell scattering assays as previously
described (26)
. Drugs were diluted in growth media
containing serially diluted human HGF/SF to determine the ability of
the drugs to inhibit cell scattering over 24 h.
Branching morphogenesis/invasion in a three-dimensional Matrigel matrix was analyzed as described previously (6) . In brief, cells at a density of 50,000 cells/ml in DMEM + 10% FBS media were mixed with an equal volume of Matrigel (Becton Dickinson), plated at 0.1 ml/well of a 96-well culture plate, and incubated for 20 min at 37°C/5% CO2 to allow gel formation. Growth media containing 200 units/ml of human HGF/SF with or without drugs at various concentrations were then added to each well. After 48 h, representative fields of view were photographed.
Cell motility assays were performed using 24-well transwell units with 8-µm polycarbonate filters (Costar) as previously described (27) . In brief, 1 x 104 cells (in 100 µl) were plated onto the upper surface of the filter in DMEM media + 1% BSA in the absence or presence of drug. The filter was then lowered into the lower compartment containing DMEM + 1% FBS media ± 200 units/ml human HGF/SF in the absence or presence of drug. After 16 h of incubation at 37°C/5% CO2, cells were fixed in methanol and stained with Diff-Quick (Dade, Aguada, Puerto Rico). Nonmigratory cells on the upper filter surface were removed using a cotton swab, and the total number of cells on each filter were counted at x200 magnification using a phase-contrast microscope accommodated with an ocular grid.
Western Blotting.
Western analysis was performed essentially as described previously
(6
, 28) with the following modifications. In brief, cells
were grown to 
50% confluence in DMEM/10% FBS growth medium before
treatment with HGF/SF (100 units/ml) ± inhibitors at
the indicated concentrations for 24 h. At the end of the
incubation period, cells were lysed by washing twice with ice cold PBS
and resuspending in lysis buffer [20 mM PIPES (pH 7.4),
150 mM NaCl, 1 mM EGTA, 1.5 mM
MgCl2, 1% Triton-X-100, 10 µg/ml aprotinin, 10
µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, 100
µM pervanadate, and 0.1% SDS]. Cell lysates were
clarified by centrifugation at 15,000 g for 15 min at 4°C,
and protein determination was performed on the soluble protein
supernatants using a standard assay (Pierce). Ten µg of cell lysates
were resuspended 3:1 into 4x reducing or nonreducing Laemmli buffer
[± DTT, respectively; 0.4 M Tris-HCl (pH 6.8),
8% SDS, 39% glycerol, 0.04% bromphenol blue ± 0.4
M DTT], boiled for 5 min, and resolved by
SDS-PAGE. Proteins were then transferred to nitrocellulose, and blots
were stained with Ponseau S stain to visualize protein bands and ensure
equal protein loading. Blots were then washed and blocked for 1 h
with a 5% solution of BSA in TBS buffer [20 mM
Tris-HCl (pH 7.4), 150 mM NaCl, 0.1% Tween 20].
Blots were then probed for 1 h using either 2 µg/ml of a rabbit
antihuman uPA antiserum (American Diagnostica), 1 µg/ml of a rabbit
antihuman uPAR antiserum (American Diagnostica), 1 µg/ml of a rabbit
antimouse Met antiserum (clone SP-260, Santa Cruz, CA), or 1 µg/ml of
a rabbit antihuman Met antiserum (clone C-28, Santa Cruz, CA). Blots
were then probed with a 1:15,000 dilution of a goat antirabbit
antiserum coupled to peroxidase (Sigma), followed by detection using
the enhanced chemiluminescence system (Amersham) and X-ray film
development. Protein bands were quantified using a Linocolor scanner
and Quantiscan analysis software.
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RESULTS |
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shows the relative responses of different cell lines to 10
units/ml HGF/SF above control untreated cells. Of the various cell
lines tested, a clone of the MDCK canine kidney cell line (MDCK-2) was
found to display the greatest increase in plasmin activity following
24-h stimulation with HGF/SF. Doses ranging from 1 to 10000 units/ml
HGF/SF were tested in MDCK-2 cells. Ten units/ml HGF/SF were found to
be submaximal and were used in subsequent experiments. The maximum
response (4.5-fold increase) was observed using 32 units/ml HGF/SF
(Fig. 1B
). Higher doses (>1000 units/ml) induced plasmin
activation to a lesser extent, and the dose-response curve displayed
typical bell-shaped characteristics (data not shown). An additional
variant MDCK cell clone (MDCK-1) displayed no uPA-plasmin response to
HGF/SF concentrations up to 10,000 units/ml consistent with its
previously reported insensitivity to HGF/SF (21)
. SKLMS-1,
EMT6, HT-29, A431, A549, and C127 cells displayed between
1.1–1.7-fold increases in plasmin activity following 10 units/ml
HGF/SF treatment (Fig. 1A
). MDCK-2 cells were, therefore,
used in further studies because they gave the best response to HGF/SF.
In addition, these cells scatter in response to HGF/SF
(26)
and, therefore, provide an additional means to study
inhibition of HGF/SF-Met signaling.
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90% (Fig. 2A
). HGF/SF had no effect on MDCK-2 cell proliferation as
previously observed (3
, 21)
. The NCI-53 antiserum
displayed no cytotoxic/cytostatic properties and had no effect on the
basal level of plasmin activity displayed by MDCK-2 cells (data not
shown), demonstrating that both cell growth and basal uPA/plasmin
activity are HGF/SF-independent. The NCI-53 antiserum was included as
an inhibitory control in all assays in the search for inhibitors.
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; 24
). Furthermore, B428 displayed no
toxicity and was a more potent inhibitor if added only during the
second stage, where cells are incubated for 4 h with exogenous
plasminogen after the 24-h period of HGF/SF stimulation (see
"Materials and Methods"). Because B428 is a competitive inhibitor
of uPA activity (24)
, this is likely due to B428 directly
inhibiting uPA activity. Thus, the reduced efficacy of B428 when added
during the first stage is likely due to the washing steps before
plasminogen addition. Interestingly, B428 does not prevent the
scattering of MDCK cells in response to HGF/SF (data not shown),
indicating that scattering occurs independently of uPA/plasmin
activation.
Inhibitors of the MAP Kinase Pathway Inhibit HGF/SF-mediated
Plasmin Generation.
It has previously been suggested that activation of the MAP kinase
1/2 pathway plays an important role in the induction of uPA
expression (16
, 29, 30, 31)
. We tested the activity of two
known inhibitors of the MAP kinase pathway in this assay, anthrax LF
and PD 98059. LF proteolytically inactivates MKK by cleaving within the
amino terminus (32)
. Thus, by the addition of LF together
with PA, which allows LF to enter cells through cell surface
PA-receptors (33
, 34)
, the MKK-MAP kinase signaling
pathway is inactivated. Treatment of MDCK-2 cells with LF and PA
resulted in a dose-dependent inhibition of HGF/SF-mediated plasmin
activation with minimal effects on cell proliferation (Fig. 2C
). Similarly, the MKK inhibitor PD 98059 (35)
inhibited plasmin activation in response to HGF/SF with little effect
on cell proliferation during the 24-h period (Fig. 2D
).
These results show that HGF/SF-mediated uPA/plasmin activation requires
activation of the MKK-MAP kinase pathway and furthermore demonstrates
the ability of this assay to detect inhibitors of multiple targets
within the HGF/SF-Met-uPA-plasmin signaling pathway.
The Geldanamycins Are Potent Inhibitors of the
HGF/SF-Met-uPA-Plasmin Proteolytic Network.
Approximately 1000 compounds were tested for inhibitory properties
using the cell-based screen in MDCK-2 cells. Our major interest was to
identify compounds that prevented plasmin activation but had
insignificant cytotoxic or cytostatic activities. Whereas three
fluorinated steroids and two staurosporine analogues as well as others
were identified as displaying these
properties4
, geldanamycin (National Service Center No. 122750) and a
geldanamycin analogue (National Service Center No. 255109) were
discovered as highly potent inhibitors in this assay (Fig. 3, A and B
, respectively).
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. For example, 320877 was the most potent inhibitor of those tested,
with an IC50 value of 3.04 x 10-15 M, whereas 255110 displayed
inhibitory properties only at cytotoxic concentrations
(IC50 = 7.84 x 10-6 M; Fig. 3, C and D
, respectively and Table 1
). This suggests that certain
structural-functional relationships exist within these geldanamycin
analogues that govern their relative abilities to inhibit
HGF/SF-mediated uPA/plasmin activation.
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, top and middle panel, Lanes
1 and 2), both 122750 and 255109 inhibited the
induction of uPA and uPAR (Lanes 3 and 4,
respectively). HGF/SF induced uPA expression by 1.4-fold above control
cells, but this was inhibited by 122750 and 255109 (1.0-fold and
0.6-fold relative to control unstimulated cells, respectively). Both
compounds also inhibited the formation of the uPA-plasminogen-activator
inhibitor complex induced by HGF/SF (6)
. uPAR expression
was induced 2.5-fold in the presence of HGF/SF, and this was inhibited
by both 122750 and 255109 (1.6-fold and 0.8-fold relative to control
unstimulated cells, respectively). It should be noted, however, that
these effects were only observed at nanomolar concentrations (data not
shown), suggesting that the inhibition of HGF/SF-mediated uPA/uPAR
induction may not be the sole mechanism by which the geldanamycins
inhibit HGF/SF-mediated plasmin activation at lower concentrations
(Table 1)
. Identical results were obtained when using the MDCK-2 cells
originally screened (data not shown).
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, bottom panel).
SKLMS-1 cells express high levels of both the
p170Met precursor and the mature
p140Met ß-chain (Lane 1). HGF/SF
stimulation results in a reduction in p140Met
expression due to increased receptor turnover (Lane 2; Ref.
37
). However, HGF/SF treatment in the presence of either
122750 or 255109 resulted in an even greater reduction in
p140Met expression (Lanes 3 and 4,
respectively). At 100 nM, treatment with 255109
induced a near complete loss of p140Met
expression, as well as a reduction in the expression of the
p170Met precursor. Treatment with 100
nM 122750 down-modulated the expression of
p140Met alone. In addition, both compounds
reduced Met expression in SKLMS-1 and MDCK-2 cells when added in the
absence of HGF/SF (data not shown), demonstrating that ligand
stimulation is not necessary for this effect and that
geldanamycin-mediated Met down-regulation occurs in multiple cell
types. However, loss of Met expression was not observed at subnanomolar
concentrations (data not shown), suggesting that at lower
concentrations, the geldanamycins function to inhibit plasmin
activation independently of Met down-regulation.
The Geldanamycins Inhibit HGF/SF-mediated Cell Motility and
Branching Morphogenesis/Invasion.
We tested the ability of the geldanamycins to inhibit HGF/SF-mediated
motility and invasion in responsive cell lines in vitro.
Both 122750 and 255109 were potent inhibitors of HGF/SF-mediated MDCK-2
cell scattering (Fig. 5A
). Concentrations of 122750 and 255109 as low as 1
nM inhibited scattering (data not shown). At 100
nM, both 122750 and 255109 displayed noticeable
cytotoxicity consistent with that observed during the cell-based
plasmin activation assay (compare Fig. 5A
and Fig. 3, A and B
). To ensure that the inhibitory
properties were not selective to MDCK-2 cells, we performed branching
morphogenesis/invasion assays using human SKLMS-1 cells and a
three-dimensional Matrigel ECM. At 100 nM, both
122750 and 255109 inhibited HGF/SF-mediated branching/invasion of
SKLMS-1 cells (Fig. 5B
). In addition, in human glioblastoma
cells (U118) and human renal cell carcinoma cells (ARZ-2), which
efficiently branch/invade in response to HGF/SF (27
, 38)
, this response was inhibited by the geldanamycins
(data not shown). We also tested the ability of the geldanamycins to
inhibit HGF/SF-mediated motility (chemotaxis) across 8-µm filters in
SK-LMS-1 cells (Fig. 5C
). Both compounds inhibited
HGF/SF-mediated SKLMS-1 cell motility at 100 nM,
with 255109 displaying the greatest degree of inhibition. However, the
effects on cell motility and branching morphogenesis/invasion were only
observed at concentrations >1 nM (data not
shown). These data demonstrate that the geldanamycins are potent
inhibitors of HGF/SF-mediated cell motility and branching
morphogenesis/invasion.
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).
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). Collectively, these data demonstrate that the geldanamycins revert
the Met-transformed phenotype and down-regulate Met expression
independently of endogenous promoter activity and autocrine HGF/SF-Met
signaling.
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DISCUSSION |
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To validate the assay, we demonstrated that a polyclonal neutralizing
antiserum against HGF/SF (NCI-53) and a proven uPA inhibitor (B428;
Ref. 24
) inhibited HGF/SF-dependent plasmin activation
(Fig. 2, A and B
). B428 competitively binds and
selectively inhibits uPA catalytic activity and has virtually no effect
on the tissue-type plasminogen activator (24)
. Coupled
with our results, this suggests that the activation of plasmin after
HGF/SF stimulation in MDCK cells occurs predominantly through uPA. We
also show that the MAP kinase 1/2 pathway is important for
HGF/SF-mediated plasmin activation because both the MKK inhibitor (PD
98059) and anthrax LF inhibited HGF/SF-mediated plasmin activation.
This supports previous findings reporting the role of the MKK/MAP
kinase 1/2 pathway in the regulation of uPA expression (16
, 29, 30, 31)
. Recently, we have shown the importance of the
Raf-MKK-MAPK kinase 1/2 pathway in the acquisition of the metastatic
phenotype in NIH3T3 cells (28)
. Thus, agents that inhibit
this important signaling pathway could be useful as anti-invasive drugs
and ultimately useful for the treatment of tumor progression and
metastasis. In this regard, B428 has already been shown to inhibit
tumor growth and metastasis in a variety of experimental systems
in vivo (44
, 45)
.
Some of the compounds identified also served to validate the assay. For example, a series of fluorinated steroids and some staurosporine analogues were identified as weak to moderate inhibitors of HGF/SF-mediated plasmin activation.4 The fluorinated steroids appear to block uPA/uPAR expression, but do not effect the expression or activation of the Met receptor.4 Staurosporine and its analogues are known inhibitors of protein kinase C isoforms and have shown potential as anticancer drugs (46) , although their precise mechanism of action in relation to their inhibitory properties in the HGF/SF-Met-uPA-plasmin assay remains unclear.
The geldanamycins were identified as potent inhibitors in this assay
with some inhibitory activity at femtomolar concentrations. Complete
inhibition of HGF/SF-mediated plasmin activity was observed at >1
nM, a concentration that was sufficient to inhibit
HGF/SF-induced motility and invasion in various cell types (Fig. 3, A and B
and Fig. 5
). The geldanamycin family of
anisamycin antibiotics were first identified as inhibitors of the Src
family of tyrosine kinases (47)
. More recently, however,
they have been shown to strongly bind the heat shock protein, Hsp90
(48
, 49)
. Hsp90 is a molecular chaperone that, in
association with other proteins, serves to ensure the correct folding
of several regulatory and signal transduction proteins
(50)
. Geldanamycin has been shown to interfere with the
chaperone function of Hsp90, leading to the destabilization and
degradation of several key cellular proteins, including
pp60v-src (48)
, ErbB2
(51)
, Raf-1 (52)
, and mutated p53
(53)
. Although further work is required to detail the
mechanisms by which geldanamycin inhibits the HGF/SF-Met-uPA-plasmin
response, we have shown that Met is down-regulated after exposure to
nanomolar concentrations of geldanamycin. This suggests that Met
degradation is controlled, in part, by Hsp90 or related proteins and
provides a partial explanation for its inhibitory properties in this
cellular assay. However, because some inhibition of HGF/SF-mediated
plasmin activity is also observed at concentrations where there is no
apparent effect on Met expression (<1 nM), it is
likely that additional targets lying within the HGF/SF-Met-uPA-plasmin
pathway are inhibited by geldanamycin. For example, Raf-1 function is
inhibited by geldanamycin (52)
, and based upon the role of
the Raf-MKK-MAPK1/2 pathway in HGF/SF-mediated uPA/uPAR induction (Fig. 2, C and D
; Ref. 16
), this pathway
is likely to be influenced by geldanamycin.
The loss of Met expression that occurs after treatment with
nanomolar concentrations of geldanamycin results in the concomitant
inhibition in the uPA/uPAR response after HGF/SF stimulation. Analogues
of geldanamycin are presently under consideration for trials in human
cancer patients based upon their limited cytotoxic properties
(54)
. We have shown that there is a vast difference
between the concentrations of certain (but not all) geldanamycins
required for cytotoxic effects (GI50 typically
10-6 M) and those necessary for
inhibition of HGF/SF-mediated plasmin activation
(IC50 typically 10-14
M). Moreover, our results suggest that tumors in which
aberrant HGF/SF-Met signaling has been implicated should be considered
for geldanamycin-based therapy. For example, germ-line and sporadic
activating mutations in Met have been identified in patients with
papillary renal carcinoma (10)
. The same mutations in
murine Met transform rodent cell lines and mediate tumorigenesis and
metastasis in mice (11
, 12)
. We have shown that the
geldanamycins revert the transformed phenotype associated with these
activating Met mutations, with a concomitant reduction in the
expression of the mutant Met proteins. These findings suggest that
geldanamycin and its analogues may be effective as
anti-invasive/metastatic agents.
ACKNOWLEDGMENTS
This manuscript is dedicated to Dr. Richard Warn, a mentor,
friend, and scholar. We thank Tracy Webb for critical review of the
manuscript, Richard Frederickson for artwork, Linda Miller for
technical support, and Ave Cline for manuscript preparation.
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FOOTNOTES |
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1 Supported by the National Cancer Institute,
Department of Health and Human Services under contract with Advanced
Bioscience Laboratories. 
2 To whom requests for reprints should be
addressed, at Van Andel Research Institute, Suite 400, 201 Monroe
Avenue NW, Grand Rapids, MI 49503; Phone: (616) 391-3441; Fax: (616)
391-3445; E-mail: craig.webb{at}vai.org
3 The abbreviations used are: HGS/SF,
hepatocyte growth factor/scatter factor; ECM, extracellular matrix;
uPA, urokinase plasminogen activator; uPAR, uPA receptor; BM, basement
membrane; FBS, fetal bovine serum; MDCK, Madine Darby canine kidney;
MAP, mitogen-activated protein; MKK, MAP kinase kinase; LF,
lethal factor; PA, protective antigen; SRB, sulforhodamine B; GI,
growth inhibitory.
4 C. P. Webb, A. Monks, C. D. Hose, and G.
F. Vande Woude, unpublished observations.
Received 7/27/99. Accepted 11/15/99.
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REFERENCES |
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U. Banerji, M. Walton, F. Raynaud, R. Grimshaw, L. Kelland, M. Valenti, I. Judson, and P. Workman Pharmacokinetic-Pharmacodynamic Relationships for the Heat Shock Protein 90 Molecular Chaperone Inhibitor 17-Allylamino, 17-Demethoxygeldanamycin in Human Ovarian Cancer Xenograft Models Clin. Cancer Res., October 1, 2005; 11(19): 7023 - 7032. [Abstract] [Full Text] [PDF]
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R. V. Hay, B. Cao, R. S. Skinner, Y. Su, P. Zhao, M. F. Gustafson, C.-N. Qian, B. T. Teh, B. S. Knudsen, J. H. Resau, et al. Nuclear Imaging of Met-Expressing Human and Canine Cancer Xenografts with Radiolabeled Monoclonal Antibodies (MetSeekTM) Clin. Cancer Res., October 1, 2005; 11(19): 7064s - 7069s. [Abstract] [Full Text] [PDF]
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Z. Jia, L. Barbier, H. Stuart, M. Amraei, S. Pelech, J. W. Dennis, P. Metalnikov, P. O'Donnell, and I. R. Nabi Tumor Cell Pseudopodial Protrusions: LOCALIZED SIGNALING DOMAINS COORDINATING CYTOSKELETON REMODELING, CELL ADHESION, GLYCOLYSIS, RNA TRANSLOCATION, AND PROTEIN TRANSLATION J. Biol. Chem., August 26, 2005; 280(34): 30564 - 30573. [Abstract] [Full Text] [PDF]
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 M. Salerno, D. Palmieri, A. Bouadis, D. Halverson, and P. S. Steeg Nm23-H1 Metastasis Suppressor Expression Level Influences the Binding Properties, Stability, and Function of the Kinase Suppressor of Ras1 (KSR1) Erk Scaffold in Breast Carcinoma Cells Mol. Cell. Biol., February 15, 2005; 25(4): 1379 - 1388. [Abstract] [Full Text] [PDF]
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 J. Sun and J. K. Liao Induction of Angiogenesis by Heat Shock Protein 90 Mediated by Protein Kinase Akt and Endothelial Nitric Oxide Synthase Arterioscler Thromb Vasc Biol, December 1, 2004; 24(12): 2238 - 2244. [Abstract] [Full Text] [PDF]
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S. R. Pavithra, G. Banumathy, O. Joy, V. Singh, and U. Tatu Recurrent Fever Promotes Plasmodium falciparum Development in Human Erythrocytes J. Biol. Chem., November 5, 2004; 279(45): 46692 - 46699. [Abstract] [Full Text] [PDF]
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G. Kaur, D. Belotti, A. M. Burger, K. Fisher-Nielson, P. Borsotti, E. Riccardi, J. Thillainathan, M. Hollingshead, E. A. Sausville, and R. Giavazzi Antiangiogenic Properties of 17-(Dimethylaminoethylamino)-17-Demethoxygeldanamycin: An Orally Bioavailable Heat Shock Protein 90 Modulator Clin. Cancer Res., July 15, 2004; 10(14): 4813 - 4821. [Abstract] [Full Text] [PDF]
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 T. O. Nielsen, H. N. Andrews, M. Cheang, J. E. Kucab, F. D. Hsu, J. Ragaz, C. B. Gilks, N. Makretsov, C. D. Bajdik, C. Brookes, et al. Expression of the Insulin-Like Growth Factor I Receptor and Urokinase Plasminogen Activator in Breast Cancer Is Associated with Poor Survival: Potential for Intervention with 17-Allylamino Geldanamycin Cancer Res., January 1, 2004; 64(1): 286 - 291. [Abstract] [Full Text] [PDF]
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K. Jankowski, M. Kucia, M. Wysoczynski, R. Reca, D. Zhao, E. Trzyna, J. Trent, S. Peiper, M. Zembala, J. Ratajczak, et al. Both Hepatocyte Growth Factor (HGF) and Stromal-Derived Factor-1 Regulate the Metastatic Behavior of Human Rhabdomyosarcoma Cells, But Only HGF Enhances Their Resistance to Radiochemotherapy Cancer Res., November 15, 2003; 63(22): 7926 - 7935. [Abstract] [Full Text] [PDF]
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N. Shinomiya and G. F. Vande Woude Suppression of Met Expression: A Possible Cancer Treatment: Commentary re: S. J. Kim et al., Reduced c-Met Expression by an Adenovirus Expressing a c-Met Ribozyme Inhibits Tumorigenic Growth and Lymph Node Metastases of PC3-LN4 Prostate Tumor Cells in an Orthotopic Nude Mouse Model. Clin. Cancer Res., 14: 5161-5170, 2003. Clin. Cancer Res., November 1, 2003; 9(14): 5085 - 5090. [Full Text] [PDF]
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X. Wang, P. Le, C. Liang, J. Chan, D. Kiewlich, T. Miller, D. Harris, L. Sun, A. Rice, S. Vasile, et al. Potent and selective inhibitors of the Met [hepatocyte growth factor/scatter factor (HGF/SF) receptor] tyrosine kinase block HGF/SF-induced tumor cell growth and invasion Mol. Cancer Ther., November 1, 2003; 2(11): 1085 - 1092. [Abstract] [Full Text] [PDF]
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M. Sattler, Y. B. Pride, P. Ma, J. L. Gramlich, S. C. Chu, L. A. Quinnan, S. Shirazian, C. Liang, K. Podar, J. G. Christensen, et al. A Novel Small Molecule Met Inhibitor Induces Apoptosis in Cells Transformed by the Oncogenic TPR-MET Tyrosine Kinase Cancer Res., September 1, 2003; 63(17): 5462 - 5469. [Abstract] [Full Text] [PDF]
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R. V. Hay, B. Cao, R. S. Skinner, L.-M. Wang, Y. Su, J. H. Resau, B. S. Knudsen, M. F. Gustafson, H.-M. Koo, G. F. V. Woude, et al. Radioimmunoscintigraphy of Human Met-Expressing Tumor Xenografts Using Met3, a New Monoclonal Antibody Clin. Cancer Res., September 1, 2003; 9(10): 3839s - 3844s. [Abstract] [Full Text]
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O. Tikhomirov and G. Carpenter Identification of ErbB-2 Kinase Domain Motifs Required for Geldanamycin-induced Degradation Cancer Res., January 1, 2003; 63(1): 39 - 43. [Abstract] [Full Text] [PDF]
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A. D. Basso, D. B. Solit, G. Chiosis, B. Giri, P. Tsichlis, and N. Rosen Akt Forms an Intracellular Complex with Heat Shock Protein 90 (Hsp90) and Cdc37 and Is Destabilized by Inhibitors of Hsp90 Function J. Biol. Chem., October 11, 2002; 277(42): 39858 - 39866. [Abstract] [Full Text] [PDF]
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P. N. Munster, D. C. Marchion, A. D. Basso, and N. Rosen Degradation of HER2 by Ansamycins Induces Growth Arrest and Apoptosis in Cells with HER2 Overexpression via a HER3, Phosphatidylinositol 3'-Kinase-AKT-dependent Pathway Cancer Res., June 1, 2002; 62(11): 3132 - 3137. [Abstract] [Full Text] [PDF]
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 R. J. Motzer, J. Bacik, T. Mariani, P. Russo, M. Mazumdar, and V. Reuter Treatment Outcome and Survival Associated With Metastatic Renal Cell Carcinoma of Non-Clear-Cell Histology J. Clin. Oncol., May 1, 2002; 20(9): 2376 - 2381. [Abstract] [Full Text] [PDF]
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P. Bonvini, T. Gastaldi, B. Falini, and A. Rosolen Nucleophosmin-Anaplastic Lymphoma Kinase (NPM-ALK), a Novel Hsp90-Client Tyrosine Kinase: Down-Regulation of NPM-ALK Expression and Tyrosine Phosphorylation in ALK+ CD30+ Lymphoma Cells by the Hsp90 Antagonist 17-Allylamino,17-demethoxygeldanamycin Cancer Res., March 1, 2002; 62(5): 1559 - 1566. [Abstract] [Full Text] [PDF]
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G. Maulik, T. Kijima, P. C. Ma, S. K. Ghosh, J. Lin, G. I. Shapiro, E. Schaefer, E. Tibaldi, B. E. Johnson, and R. Salgia Modulation of the c-Met/Hepatocyte Growth Factor Pathway in Small Cell Lung Cancer Clin. Cancer Res., February 1, 2002; 8(2): 620 - 627. [Abstract] [Full Text] [PDF]
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 G. Hawcroft, M. D'Amico, C. Albanese, A. F. Markham, R. G. Pestell, and M. A. Hull Indomethacin induces differential expression of {beta}-catenin, {gamma}-catenin and T-cell factor target genes in human colorectal cancer cells Carcinogenesis, January 1, 2002; 23(1): 107 - 114. [Abstract] [Full Text] [PDF]
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 G. A. Gmyrek, M. Walburg, C. P. Webb, H.-M. Yu, X. You, E. D. Vaughan, G. F. Vande Woude, and B. S. Knudsen Normal and Malignant Prostate Epithelial Cells Differ in Their Response to Hepatocyte Growth Factor/Scatter Factor Am. J. Pathol., August 1, 2001; 159(2): 579 - 590. [Abstract] [Full Text]
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P. N. Munster, A. Basso, D. Solit, L. Norton, and N. Rosen Modulation of Hsp90 Function by Ansamycins Sensitizes Breast Cancer Cells to Chemotherapy-induced Apoptosis in an RB- and Schedule-dependent Manner : See The Biology Behind: E. A. Sausville, Combining Cytotoxics and 17-Allylamino, 17-Demethoxygeldanamycin: Sequence and Tumor Biology Matters. Clin. Cancer Res., 7: 2155-2158, 2001. Clin. Cancer Res., August 1, 2001; 7(8): 2228 - 2236. [Abstract] [Full Text] [PDF]
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P. N. Münster, M. Srethapakdi, M. M. Moasser, and N. Rosen Inhibition of Heat Shock Protein 90 Function by Ansamycins Causes the Morphological and Functional Differentiation of Breast Cancer Cells Cancer Res., April 1, 2001; 61(7): 2945 - 2952. [Abstract] [Full Text]
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S. E. Dunn, J. V. Torres, J. S. Oh, D. M. Cykert, and J. C. Barrett Up-Regulation of Urokinase-Type Plasminogen Activator by Insulin-like Growth Factor-I Depends upon Phosphatidylinositol-3 Kinase and Mitogen-activated Protein Kinase Kinase Cancer Res., February 1, 2001; 61(4): 1367 - 1374. [Abstract] [Full Text]
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K. Kuba, K. Matsumoto, K. Date, H. Shimura, M. Tanaka, and T. Nakamura HGF/NK4, a Four-Kringle Antagonist of Hepatocyte Growth Factor, Is an Angiogenesis Inhibitor that Suppresses Tumor Growth and Metastasis in Mice Cancer Res., December 1, 2000; 60(23): 6737 - 6743. [Abstract] [Full Text]
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