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Diabetes Branch (M.P., D.L.), National Institute of Diabetes, Digestive and Kidney Diseases, NIH, Bethesda, Maryland 20892; Department of Biological Chemistry (A.G., A.L.), Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem, Jerusalem 91904, Israel; and Department of Pathology (E.W.), Sackler School of Medicine, Tel Aviv University, Ramat Aviv, Tel Aviv 69978 Israel
Address all correspondence and requests for reprints to: Derek LeRoith, Diabetes Branch, NIDDK, NIH, Building 10, Room 8S235A, 10 Center Drive, MSC-1770, Bethesda, Maryland 20892-1770. E-mail: Derek{at}helix.nih.gov
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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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-subunits
with binding capacity and two transmembrane ß-subunits with tyrosine
kinase activity that are activated in response to ligand binding (1, 2). Thus, binding of the hormone to its respective receptor triggers a
cascade of events that includes receptor autophosphorylation,
phosphorylation of intracellular substrates, and activation of
signaling pathways involved in metabolic processes and growth
regulation. Both peptide hormones induce pleiotropic responses in many cell types and share a host of common functions. However, in spite of their high degree of homology, the principal role of insulin under normal physiological conditions is to maintain metabolic homeostasis, whereas IGF-1 stimulates growth and differentiation (3). Accordingly, with its role in cell growth, overexpression of IGF-1 receptors confers tumorigenic potential to cells (4, 5) as well as protection from apoptosis (5, 6, 7).
The first event in the signal transduction cascade of both insulin and IGF-1 is the autophosphorylation of their respective receptors. The importance of receptor autophosphorylation on the subsequent cellular actions of the hormones remains controversial. Several lines of evidence have suggested that tyrosine phosphorylation of the insulin receptor may not be essential for all of its functions (8, 9). Similarly, high levels of autophosphorylation are not required for mediation of all the biological activities of the IGF-1 receptor (10, 11). However, mutant IGF-1 receptors with decreased autophosphorylation present severely impaired mitogenic and tumorigenic activities (4, 11). Thus, autophosphorylation and tyrosine kinase activity of those receptors play an important role in their signaling functions. Accordingly, enhanced activity of tyrosine kinases has been implicated in many cancers and other proliferative diseases (12). Tyrosine kinases and the signaling pathways in which they participate have therefore been identified as potential targets for drug design.
The tyrphostins are a family of synthetic protein tyrosine kinase
inhibitors that selectively inhibit receptor autophosphorylation (13)
and represent an excellent tool to examine receptor function. The
tyrphostins are derived from a benzylidene malononitrile nucleus, which
resembles the phenolic group of tyrosine, with additional
substitutions. Many of these substitutions have resulted in different
compounds that inhibit specific tyrosine kinases (Table 1
). In this report, we have evaluated several tyrphostins
for inhibition of the tyrosine kinase activity of the IGF-1 and insulin
receptors, as well as for their effect on cell proliferation in
response to their respective ligands.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell culture
NIH-3T3 mouse fibroblast cells overexpressing wild-type IGF-1
(clone NWTc43) or insulin receptors (clone WT2) were previously
described (10, 17). Cells were cultured in DMEM supplemented with 10%
FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin, in the presence
of 500 µg/ml G418 (Geneticin, Life Technologies, Gaithersburg, MD),
in a humidified atmosphere of 95% air and 5% CO2 at 37
C.
MTT cellular proliferation assays
MTT assays were performed as described (4). NIH-3T3 cells
overexpressing IGF-1 or insulin receptors were plated on 96-well plates
(2,000–5,000 cells/well) and maintained overnight in complete medium.
Cells were then changed to DMEM containing 1% FBS in the absence or
presence of 10-8 M IGF-1 or insulin and the
different tyrphostins in triplicate wells (four different
concentrations ranging from 0.1–5 µM), for 6 days (120
h). Medium was replaced every 48 h. At the indicated periods of
time, the medium was aspirated from the wells and 100 µl MTT reagent
(3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide,
Sigma Chemical Co., St. Louis, MO) were added to each well. The cells
were then incubated for 4 h at 37 C and lysed by addition of 100
µl isoamylic alcohol and shaking for 20 min. Plates were then read in
a ELISA reader at 570 and 690 nm. The IC50 values provided
were calculated at the 120-h time point.
In parallel, direct counting of the cells attached to the plate was performed. After treatment, medium was removed and plates were washed twice with 1 x PBS. Cells remaining attached to the plate were trypsinized and counted in a Neuebauer chamber.
Intact cell tyrosine phosphorylation
Tyrosine phosphorylation of the ß-subunits of the IGF-1 and
insulin receptors was analyzed in total cell lysates by immunoblotting
with an antiphosphotyrosine antibody as described (11). Briefly,
confluent cells in 60-mm plates were incubated overnight in serum-free
(SF) medium (DMEM with 1% BSA and 20 mM HEPES, pH 7.5).
Tyrphostins at different concentrations were added in fresh SF-medium
for 1 h. Cells were then stimulated with 10-8
M IGF-1 or insulin for 1 min. After treatment, cells were
washed twice with ice-cold 1 x PBS and lysed in the presence of
50 mM HEPES, pH 7.9, 100 mM NaCl, 10
mM EDTA, 1% Triton X-100, 4 mM sodium
pyrophosphate, 2 mM sodium orthovanadate, 1 mM
phenylmethylsulphonyl fluoride and 10 mM sodium fluoride.
Lysates were cleared by centrifugation and equal amounts of protein
were separated by a 7.5% SDS-PAGE and transferred to a nitrocellulose
membrane (Protran, Schleicher & Schuell, Keene, NH). Tyrosine
phosphorylated proteins were immunoblotted with monoclonal
antiphosphotyrosine antibody (clone 4G10, Upstate Biotechnology Inc.,
Lake Placid, NY) and detected with horseradish peroxidase-conjugated
secondary antibody using an ECL system (Amersham Life Sciences,
Arlington Heights, IL). The autoradiograph films were then scanned and
analyzed by densitometry using the NIH Image program (version
1.57).
Receptor semipurification
Semipurification of solubilized IGF-1 and insulin receptors was
performed as described (11) with some modifications. Briefly, confluent
NIH-3T3 cells overexpressing IGF-1 or insulin receptors were washed
twice with ice-cold 1 x PBS and lysed in the presence of 50
mM HEPES, pH 7.6, 150 mM NaCl, 1% Triton
X-100, 1 mM phenylmethylsulfonyl fluoride and 100 U/ml
bacitracin. Lysates were cleared by centrifugation, and the
supernatants were recycled three times through a disposable
polypreparative column packed with 2 ml of wheat germ agglutinin (WGA)
bound to agarose (Vector Laboratories, Burlingame, CA). The column was
washed with 100 ml of a buffer containing 50 mM HEPES, pH
7.6, 150 mM NaCl and 0.1% Triton X-100. Glycoproteins were
eluted from the column with the same buffer supplemented with 0.3
M N-acetyl-D-glucosamine.
Phosphorylation of exogenous substrates
Tyrosine kinase activity of the semipurified receptors was
assayed as described (11) with minor modifications. Briefly, aliquots
of the WGA eluate were incubated overnight at 4 C in the presence or
absence of 10-8 M IGF-1 or insulin and the
different tyrphostins, in a final volume of 40 µl, with 50
mM HEPES buffer (pH 7.6) supplemented with 100
mM NaCl, 0.04% Triton X-100, and 0.01% BSA.
Phosphorylation was allowed to begin by addition of 60 µl kinase
buffer (1 mg/3 ml exogenous substrate poly(Glu:Tyr)4:1, 1.5
mM CTP, 75 mM MgCl2, 75
µM ATP, and 5 µCi/sample 
32P-ATP). The
reaction was stopped by spotting 70 µl of the reaction mixture onto
3 x 3 cm Whatman 3MM filter paper squares and washing them in
10% trichloroacetic acid and 10 mm sodium pyrophosphate. Papers were
then air-dried and counted in a ß-counter with scintillation
liquid.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) were tested for their ability to
inhibit IGF-1-stimulated cell proliferation of NIH-3T3 fibroblasts
overexpressing IGF-1 receptors (NWTc43 clone). The MTT assay was used
to that end. Several of the tyrphostins displayed a dramatic effect,
almost completely blocking IGF-1-stimulated cell growth at doses as low
as 5 µM (Fig. 1). Table 2
shows the IC50 values for several of the more potent
tyrphostins used in the study. The MTT assay measures mitochondrial
activity, which under normal circumstances correlates with cell number.
To ascertain that tyrphostins were not affecting cellular respiration
and slowing metabolism only, thereby resulting in an underestimation of
the cell number, we manually performed cell counts in a parallel study.
Cell counts gave similar results to the MTT assays (data not shown),
thus indicating that tyrphostins were really inhibiting cell
proliferation. The tyrphostins were also assayed for their ability to
block serum-stimulated cellular proliferation (in the presence of 10%
FBS). Under these conditions, only AG1557 showed a significant effect
decreasing the rate of serum-stimulated cell proliferation with an
IC50 of 5.3 ± 0.2 µM, whereas all the
other tyrphostins tested did not affect the rates of cellular
proliferation (data not shown).
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).
As was expected, insulin receptor overexpressing cells, showed a much
slower proliferation rate than IGF-1 receptor overexpressing cells. In
a typical experiment, IGF-1 receptor overexpressing cells would
increase their number 20-fold in 6 days, whereas insulin receptor
overexpressing cells multiply only by a factor of 5 in the same time
period (Figs. 1 and 2). The effect of the tyrphostins on
cellular proliferation in cells overexpressing insulin receptors was
similar to that described on cells overexpressing IGF-1 receptor (Fig. 2). Tyrphostins AG1024, AG1034, and AG1557 all presented
IC50 doses on the 10-7 M range.
When tested in their ability to block serum-stimulated cell
proliferation, only AG1557 significantly decreased it, whereas all the
other tyrphostins tested did not alter the rates of cell growth (data
not shown).
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) determined that
1 h incubation was the optimal time point of tyrphostin effect.
The inhibition of the receptor autophosphorylation by the different
tyrphostins tested was transient, with a complete recovery of normal
autophosphorylation levels seen after 12 h of incubation with the
inhibitors. This is possibly due to the metabolism of the compounds, as
indicated by the loss of the orange coloration they give to the culture
medium. Nevertheless, the effect of the tyrphostins on cellular
proliferation was not affected by these changes and persisted at least
until the subsequent doses were added at 48-h intervals. The different
tyrphostins were then assayed in a dose-response manner after 1-h
incubation. A typical experiment for NWTc43 cells is shown in Fig. 4a. The IC50 of the tyrphostins were
significantly higher for inhibiting IGF-1 receptor autophosphorylation
than for the inhibition of cellular proliferation (Table 2).
Tyrphostins AG825 and AG1500 were also tested, showing no significant
effect on the IGF-1 receptor autophosphorylation.
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). As with
IGF-1 receptors, the IC50 dose was higher than the one
needed to inhibit insulin-stimulated cellular proliferation. Some of
the tyrphostins assayed, particularly AG1024 and AG1034, showed a
degree of preference of IGF-1 vs. insulin receptors
with regard to inhibition of ligand-stimulated receptor
autophosphorylation (Table 2). Tyrphostins AG825 and AG1500 did not
affect insulin receptor autophosphorylation.
A number of the tyrphostins were then tested for their ability to
inhibit tyrosine kinase activity of the receptors toward exogenous
substrates in vitro. The IC50 values for
tyrosine kinase inhibition were also on the micromolar range (Table 2).
Again, tyrphostins AG1024 and AG1034 showed the highest degree of
discernment between insulin and IGF-1 receptors with regard to
inhibition of ligand-stimulated tyrosine kinase activity of the
receptor (Fig. 5).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The IGF-1 receptor belongs to the family of receptor tyrosine kinases. IGF-1 effects multiple functions in different cell types, but is primarily involved in cell growth and differentiation (3). Accordingly, IGF-1 receptor levels have been correlated with tumorigenic potential of the cells (4, 5), and increased IGF-1 receptor mRNA has also been detected in malignant states, such as Wilms tumor (20). Moreover, IGF-1 is also able to act as survival factor, preventing apoptosis in a number of cell types (6, 7, 21, 22).
Tyrosine kinases and the signaling pathways in which they participate
have therefore been identified as potential targets for drug design
with therapeutic purposes. Along this line of interest, tyrphostins are
a series of protein tyrosine kinase inhibitors that were originally
modeled after the microbial inhibitor erbstatin (23). These compounds
were derived from the benzylidene malononitrile nucleus, resembling the
phenolic group of tyrosine, with additional substitutions directed to
increase their biological activity. Some of these substitutions
resulted in significant discrimination among tyrosine kinases (13).
Different groups of tyrphostins that are highly discriminating among
particular growth factors have been described so far. Specific
tyrphostins against NGF have been shown to block NGF-induced
phospholipase C- (PLC-) phosphorylation, phosphatidylinositol 3'
kinase activation, and neurite outgrowth in PC12 cells (15).
Tyrphostins against PDGF inhibited PDGF receptor kinase and
PDGF-dependent DNA synthesis in Swiss 3T3 cells and porcine aorta
endothelial cells (24). Effects of tyrphostins on EGF-mediated
functions have been widely studied. Tyrphostins against EGF inhibited
EGF-dependent phosphorylation of exogenous substrates and cellular
proliferation (14, 16, 23) as well as EGF-dependent activation of the
src-family kinases (25), with IC50s in the submicromolar or
micromolar range. Recently, a tyrphostin blocking Jak-2 activity and
cell growth in leukaemic cells both in vitro and in
vivo has been described (26), with important implications for
cancer control and treatment. In this report, we tested the effect of
several tyrphostins on the IGF-1- and insulin-stimulated cellular
proliferation, as well as autophosphorylation in intact cells and
tyrosine kinase activity toward exogenous substrates in
vitro.
Several of the tyrphostins tested dramatically blocked hormone-stimulated cellular proliferation. Low doses of the inhibitor (in the 10-6 M range) were enough to completely block the effect of the ligand, with the IC50 for most of the inhibitors being in the 10-7 M range. On the other hand, the IC50 concentrations needed to block the autophosphorylation and tyrosine kinase activity of the receptors were about 10-fold higher. Despite these differences, the effect of the tyrphostins on cellular proliferation is specific for IGF-1 and insulin receptors because the inhibitory effect on serum-induced proliferation is negligible for all tyrphostins tested except AG1557 (data not shown). Interestingly, a similar result was found with tyrphostins inhibiting EGF receptor-dependent tyrosine kinase activity and [3H]thymidine incorporation (14) Moreover, other well known tyrosine kinase inhibitors such as genistein and erbstatin inhibit EGF-induced [3H]thymidine uptake at far lower concentrations than those needed to inhibit tyrosine phosphorylation in intact cells (27, 28). One possible explanation is that, in addition to inhibiting the IGF-1 and insulin receptor tyrosine kinase activities, the tyrphostins inhibit some downstream tyrosine kinase governing cellular proliferation, the activation of which is IGF-1- and insulin-dependent.
Most of the tyrphostins tested showed no distinct specificity distinguishing between insulin and IGF-1 receptors, except for AG1024 and AG 1034, which showed a tendency to lower IC50s for the IGF-I than the insulin receptors.This difficulty in finding specific inhibitors is not unusual in view of the strict evolutionary conservation of the tyrosine kinase domain and the high degree of homology of the ATP-binding region of both receptors. It has been proposed by the use of computer simulations that tyrphostins bind to the active center of the receptors, distorting it in such a way that in most cases neither the substrate nor ATP can bind to the receptor (29). In that case, the presence of the different substituents in the benzylidene malononitrile nucleus can make possible the design of inhibitors capable of fine discrimination among receptors.
We have identified a family of tyrphostins that inhibit IGF-1 and insulin receptor autophosphorylation and function. Interestingly, the two tyrphostins that showed the highest activities in our system (AG1024 and AG1034) have been previously assayed for their effects in blocking NGF function (15), showing no distinct effect. Conversely, during this study we also tested tyrphostin AG825, which has been shown to act against the EGF receptor (14), finding it to be inactive with IGF-1 and insulin receptors.
In addition to the potential therapeutic uses of receptor-specific tyrphostins, the ultimate design of specific insulin and IGF-1 receptor kinase inhibitors may also help to establish the precise signaling mechanisms that discriminate between the different effects of these two hormones, which at the present time have not been clearly elucidated.
Received November 20, 1996.
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 </ |
, medium supplemented with 10-8 M
IGF-1; 
: medium supplemented with 10-8 M
IGF-1 and 1 µM tyrphostin; 
: medium supplemented with
10-8 M IGF-1 and 5 µM
tyrphostin; •, medium in the absence of IGF-1 and tyrphostins; 
,
addition of fresh media. Values are means ± SEM of
three separate wells.
