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INTRODUCTION |
Intrinsic radiosensitivity is one of the critical factors that
determines the probability of successful tumor cure or local control
following radiotherapy (1, 2). Activation of oncogenes, including
ras, and mutation of tumor suppressors such as p53 are well
known to induce radioresistance (3-5), and the mechanisms by which
this occurs have been investigated from a number of aspects, including
cell cycle progression and signal transduction (6-8). It has been
reported that phosphatidylinositol 3'-kinase
(PI3-K),1 but not
mitogen-activated protein kinase (MAPK), is important for mutant
Ras-induced radioresistance, although both are known to convey potent
survival signals (8). Some of the growth factors, which activate a
variety of downstream pathways including Ras, also mediate cell
survival functions through their cognate receptors (9). Of these,
epidermal growth factor receptor (EGFR), which is often overexpressed
in various tumor types, has been shown to induce radioresistance;
specific antibodies for EGFR or expression of dominant negative EGFR
significantly radiosensitizes tumor cells both in vitro and
in vivo (10-12). It has been suggested that stimulation of
survival signals such as the PI3-K and MAPK pathways following EGFR
activation contributes to radioresistance (13, 14).
The insulin-like growth factor I receptor (IGF-IR) is a transmembrane
tyrosine kinase, the amino acid sequence of which is highly homologous
to that of the insulin receptor (IR) (15). It is a generally held view
that IGF-IR activation plays a key role in cell growth, establishment,
and maintenance of a transformed phenotype, cell survival, and
differentiation (16-20). Elevated levels of IGF-IR have been observed
in human tumors of breast (21), brain (22), and lung and colon (23)
and, when observed, are associated with a poor prognosis (21). As
IGF-IR was found to possess the ability to induce radioresistance (21,
24-26), directed study of this receptor is likely to shed light on the downstream pathways leading to this phenomenon. Comprehensive study
from such a viewpoint has not been previously conducted, except that
antisense targeting of IGF-IR reduces the activity of ataxia
telangiectasia-mutated (ATM), a necessary factor for proper double
strand break repair, resulting in enhanced radiosensitivity (27).
Direct connection, however, between the IGF-IR pathway and ATM has not
been established to date.
Two major pathways are thought to originate from IGF-IR, one through
insulin receptor substrate-1 (IRS-1), which activates the PI3-K/Akt
pathway, and the other through Shc, which activates the Ras/Raf/MEK/ERK
pathway (28). These two substrates bind to the
NPXY950 motif in the juxtamembrane domain, and
Tyr-950 plays an important role in binding as revealed by a yeast
two-hybrid assay (29, 30). The Raf/MEK/ERK pathway is also activated
through 14-3-3 proteins, which bind to the C terminus of IGF-IR, a site
not available on the IR (31-33). In addition to these main pathways,
activation of c-Raf kinase by 14-3-3 proteins bound to the IGF-IR also
results in its translocation to the mitochondria, where it exerts a
survival effect with Nedd4 (28, 34). How these downstream pathways of
IGF-IR influence radioresistance is not known.
In this study, we sought to determine the contributions of the
different downstream pathways of IGF-IR to IGF-IR-mediated radioresistance. For this purpose, we used a series of mutant IGF-IRs,
potentially relevant to PI3-K and MEK/ERK activation, expressed in R
cells deficient in endogenous IGF-IR (17). Radiosensitivity was further
analyzed in combination with specific inhibitors of PI3-K and MEK.
Here, we show that IGF-IR mediates clonogenic radioresistance through a
number of redundant survival signals of differently weighted relevance,
including PI3-K, MEK/ERK, and signals stemming from the C terminus
domain, presumably through 14-3-3 proteins.
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EXPERIMENTAL PROCEDURES |
Materials--
Wortmannin, LY294002, and PD98059 were purchased
from Sigma. Antibodies against IGF-IR
- and
-subunits, ERK2, goat
IgG conjugated with horseradish peroxidase (HRP), rabbit IgG-HRP, and
Protein A/G PLUS-agarose were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phosphotyrosine antibody (PY20) was purchased from Transduction Laboratory (Lexington, KY) and anti-phosphokinase B/AKT phosphoserine 473 antibody was purchased from
BIOSOURCE (Camarillo, CA). Anti-ACTIVETM
MAPK antibody was purchased from Promega (Madison, WI), and
anti-
-actin antibody from Chemicon International (Temecula, CA).
Anti-hemagglutinin (HA) antibody was purchased from Roche (Mannheim,
Germany). The ECL Western blotting analysis system,
125I-IGF-I, and [
-32P]ATP were purchased
from Amersham Biosciences. Recombinant human IGF-I was purchased
from Invitrogen. Phosphatidylinositol was purchased from Avanti
(Alabaster, AL). pEGFP-C1 vector was purchased from
Clontech (Palo Alto, CA). A plasmid containing
constitutively active MEK was kindly provided by Dr. E. Nishida (Kyoto
University, Kyoto, Japan).
Plasmid Construction--
Wild-type, Y950F, Y1316F, and
1245
receptor mutants were derived from human IGF-IR cDNA (15) as
described previously (35, 36). For Y950F/1316F, the
HindIII-BamHI fragment of pBluescript SK Y950F
(35) was replaced by the HindIII-BamHI fragment
of pBluescript SK Y1316F (36) and was designated pBluescript SK Y950F/Y1316F. The XhoI-NotI fragment of pBPV
IGF-IR (17), an expression plasmid for the wild-type (WT) receptor, was
then replaced with the XhoI-NotI fragment of
pBluescript SK Y950F/Y1316F, including the double mutation. For the
Y950F/
1245 mutant, the HindIII-BamHI fragment
of pBluescript SK Y950F was replaced by the corresponding fragment from
pBluescript SK
1245 (36). Then, the XhoI-NotI fragment of pBPV IGF-IR was replaced by the
XhoI-NotI fragment of the Y950F/
1245 cDNA
in pBluescript SK. For the construction of IGF-IR truncated at residue
950, the XhoI-BamHI fragment of pBPV IGF-IR was
transferred into the vector pEGFP-C1. The
ScaI-BamHI fragment of the IGF-IR cDNA in
this vector was replaced by the following double-stranded
oligodeoxynucleotides: 5'-ACTGAGAATTCG and 3'-TGACTCTTAAGCCTAG. The
oligodeoxynucleotides were designed to terminate translation at residue
950 followed by the stop codon TGA, and they contained a
BamHI restriction overhang for ligation to the
BamHI site of the IGF-IR cDNA in pEGFP-C1 and an
EcoRI restriction site for confirmation. The
XhoI-BamHI fragment of pBluescript SK IGF-IR was
replaced by the corresponding fragment of truncated IGF-IR cDNA in
vector pEGFP-C1. The XhoI-NotI fragment of pBPV
IGF-IR was then replaced with the corresponding fragment containing
950 in pBluescript SK.
Cell Lines, Culture Conditions, and Transfections--
R
cells
were obtained from mouse embryo fibroblasts possessing a null mutation
of the IGF-IR gene (17). Plasmids containing WT or mutant IGF-IR
cDNAs were stably transfected into R
cells with a pPDV6+ plasmid
carrying the puromycin resistance gene (37) by calcium phosphate
precipitation. Cells were selected in 4 µg/ml puromycin, and the
resultant clones were mixed and sorted as described previously (38).
Mixed populations or clones were used in the present study. For
transient expression of constitutively active MEK in R
cells, a
plasmid containing LA-SDSE MAPK kinase cDNA (39) was transfected
into R
cells, and the cells were prepared for Western blotting and a
colony-forming assay 48 h after transfection. Mock-transfected R
cells were treated similarly as a control. All cell lines were
maintained at 37 °C in a humidified atmosphere containing 5%
CO2 in Eagle's minimal essential medium containing 1 mM sodium pyruvate, 100 units/ml penicillin, and 100 µg/ml streptomycin supplemented with 10% (v/v) fetal bovine serum.
Exponentially growing cells were used for all experiments.
Colony-forming Assay--
Radiosensitivity was determined by
colony-forming assay as described previously (25). To assess the effect
of exogenously added IGF-I or inhibitors of PI3-K or MEK, cells in
plastic flasks grown for roughly 10 h were treated with inhibitors
for 1 h and then
-irradiated. Cells were transferred to a
37 °C incubator and rendered to form colonies. Surviving fraction
was calculated based on the plating efficiency determined from the
IGF-I- or inhibitor-treated cells. Cell survival was corrected using
the equation S = 1
(1
f)1/N, where S is the
single cell survival rate, f is the measured surviving
fraction, and N is the multiplicity determined by the average number of cells per microcolony at the time of irradiation. Multiplicity ranged from 1.1 to 1.2 for all cell lines under the described conditions.
Western Blotting--
Cells were digested in a lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5% Triton
X-100, 0.1% SDS, 1 mM EDTA, 100 mM NaF, 1 mM sodium orthovanadate, 1 mM
phenylmethylsulfonyl fluoride, and 1 µg/ml aprotinin). Equal amounts
of cell lysates were separated in SDS-polyacrylamide gel (PAGE), and
proteins were transferred to a nitrocellulose membrane in a
Tris-glycine buffer containing 20% methanol. The membrane was blocked
in 5% nonfat milk in TBST (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.1% Tween 20). Filters were probed with
primary antibodies against target proteins for 1 h at room
temperature or overnight at 4 °C. Filters were washed three times in
TBST, incubated with secondary antibodies conjugated with HRP in TBST
for 1 h at room temperature, and then washed three times in TBST.
Proteins were visualized using the ECL system. For the detection of
activated proteins, cells were incubated in serum-free medium
containing 1 µg/ml bovine serum albumin overnight. Serum-starved
cells either treated or untreated with indicated concentrations of
IGF-I for 10 min were processed as described above.
IGF-I Binding Assay--
The number of IGF-I binding sites was
determined in each cell line as described previously (38). Cells grown
on six-well cell plates were washed with Hanks' balanced salt solution
and incubated for 4 h at 4 °C in binding buffer (Eagle's
minimal essential medium plus 25 mM Hepes, pH 7.4, and 1 mg/ml bovine serum albumin) containing 0.5 ng/ml 125I-IGF-I
and/or increasing concentrations of unlabeled IGF-I. After washing with
cold Hanks' balanced salt solution, cells were lysed with 0.03% SDS,
and cell-associated radioactivities were measured by an autowell
-counter. Specific binding was expressed by subtracting nonspecific
binding as determined in the presence of excess unlabeled IGF-I (200 ng/ml). Relative number of specific binding sites in cells incubated in
buffer containing 0.5 ng/ml 125I-IGF-I alone was determined
in each cell line with values of WT 11 cells normalized to 1.0. Receptor number per cell and dissociation constants
(Kd) were also estimated in some cell lines by
Scatchard analysis as described previously (38).
Phosphatidylinositol 3-Kinase Activity--
Activity of PI3-K
was measured as described previously (40). Briefly, IGF-I-treated or
untreated cells were lysed in a buffer containing 20 mM
Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, and 1% Nonidet P-40, and phosphotyrosine-containing proteins were immunoprecipitated with anti-phosphotyrosine antibody (PY20) bound to
Protein A/G-agarose. PI3-K activity in the immunoprecipitates was
measured in a reaction mixture containing phosphatidylinositol and
[
-32P]ATP. After 10-40 min, the reaction was stopped
by the addition of a solution (chloroform:methanol:HCl = 2:1:0.1)
and analyzed by thin layer chromatography.
-Irradiation--
-Irradiation was performed using a
60Co
-ray therapeutic machine, RCR-120 (Toshiba, Tokyo,
Japan), at a dose rate of 1.4-1.6 Gy/min.
Statistical Analysis--
Statistical comparison of mean values
was performed using the Student's t test or one-way
analysis of variance followed by Fisher's protected least significant
difference. Differences with a p value of <0.05 were
considered statistically significant.
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RESULTS |
Expression of Wild-type IGF-IR Confers Clonogenic Radioresistance
in R
Cells--
We previously reported that introduction of IGF-IR
into R
cells, which are deficient in endogenous IGF-IR, confers
clonogenic radioresistance (25). To confirm this, we newly established several clones and their radiosensitivities were determined by colony-forming assay. Of these, mixed populations (WTmix), clones 9 and
11, which expressed similar levels of IGF-IR, exhibited a significant
radioresistance (to a similar extent) compared with R
or R
(puro)
cells expressing a marker gene alone (Fig.
1, A and B).
Because the extent of radioresistance was relatively modest, we
attempted to examine whether radioresistance was increased when cells
were stimulated with exogenously added IGF-I, although growth medium
already contained IGF-I in serum. Addition of 10, 20, and 50 ng/ml
IGF-I in growth medium 1 h before irradiation, however, did not
confer any further significant increase in survival fractions at 6 Gy
in WTmix and WT 11 cells (Fig. 1C). One may argue that the
structure of IGF-IR per se, irrespective of its signaling
function, could somehow affect the radiosensitivity of R
cells. We
therefore tested R
cells expressing
950 IGF-IR, which lack most of
the
-subunit including the tyrosine kinase domain. The expression
levels of
950 clones 5 and 7 are shown in Fig. 1D. An
antibody specific for the
-subunit of IGF-IR detected levels of
receptor similar as or even higher than clone WT 9. The proreceptor
could rarely be distinguished because of overlapping with the
-subunit, and the observed size of
-subunit was small because of
the large deletion. When an antibody specific for the C terminus of the
-subunit was used, only WT receptors were detected. Both
950
clones displayed the same radiosensitivity as R
or R
(puro) cells
(Fig. 1E), demonstrating that the IGF-IR-mediated clonogenic
radioresistance is attributable to signal transduction via the tyrosine
kinase of the receptor. Furthermore, considering that IGF-IR kinase
activation was not clearly detectable in the growth medium following a
6-Gy irradiation (data not shown), a very low level of receptor
activation should be sufficient to saturate clonogenic radioresistance
in WT cells, because no further radioresistance was obtained by
exogenously added IGF-I (Fig. 1C).

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Fig. 1.
Expression of IGF-IR and dose-survival curves
in cell lines expressing WT or mutant receptors lacking the tyrosine
kinase domain. Figure shows Western blots for IGF-IR expression in
R , R (puro clones), WT 9, WT 11, and WTmix cells (A), and
in 950 clone 5 and 950 clone 7 cells (D, #5
and #7). Equal amounts of cell lysates were subjected to
SDS-PAGE, and IGF-IR - or -subunits were detected as described
under "Experimental Procedures." The unprocessed form of the
proreceptor was also detected with both antibodies. The antibody used
to detect the IGF-IR -subunit recognizes the C terminus of the
subunit. -Actin was used as a loading control. C, effect
of exogenously added IGF-I on surviving fractions. WTmix and WT 11 (#11) cells were irradiated at a dose of 6 Gy in the
presence or absence of exogenously added IGF-I in growth medium, and
surviving fractions were determined. Lane 1, 0 ng/ml; lane 2, 10 ng/ml; lane
3, 20 ng/ml; lane 4, 50 ng/ml. Data
represent the means ± S.D. of triplicate determinants. No
significant differences were obtained versus cells
irradiated in the absence of exogenously added IGF-I. Dose-survival
curves of the cell lines in panels A and
D are shown in panels B and
E, respectively. Data shown are the means of at least two
independent experiments.
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Characteristics of Mutant Receptors--
We next attempted to
determine which domains of the IGF-IR
-subunit are necessary for
clonogenic radioresistance. For this purpose, we made various mutant
receptors with specific mutations potentially relevant to activation of
downstream pathways. For clarity, the mutant receptors used in this
study are shown in Fig. 2. The tyrosine
residue at position 950 is part of the NPXY950
motif, a major binding site for IRS-1 and Shc, which is conserved in IR
(41) and interleukin 4 receptor (42). IRS-1 activates PI3-K, which
phosphorylates phosphatidylinositol (PtdIns) phosphates, converting
PtdIns 4,5-P2 to PtdIns 3,4,5-P3. This lipid
activates phosphoinositide-dependent kinases 1 and 2, which
in turn activate Akt (43). Shc strongly activates the Ras/Raf/MEK/ERK
pathway (44, 45). Tyrosine 1316 is a constituent of the
Y1316XXM motif, a binding site for the
regulatory subunit p85 of PI3-K, and is able to stimulate its activity
(46). This is also conserved in IR (47). Because each tyrosine, Tyr-950
and Tyr-1316, is reported to play a critical role in each binding
function (29, 30, 46), these residues were mutated to
phenylalanines to attenuate the relevant pathways. The C
terminus of IGF-IR includes a quartet of serine residues 1280-1283,
which is a binding site for 14-3-3 proteins (33) that in turn lead to
activation of c-Raf and the MAPK pathway. c-Raf undergoes mitochondrial
translocation and exerts a survival effect in cooperation with Nedd4
(34). We will refer to this pathway as 14-3-3/c-Raf hereafter to
differentiate this c-Raf signaling event as separate from its
activation of the MAPK pathway. To eliminate the binding site of the
14-3-3 proteins, the receptor was truncated at residue 1245. Double
mutation at Tyr-950 and the C terminus was also introduced to exclude
both signals.

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Fig. 2.
Schematic presentation of the WT and mutant
IGF-IR -subunits used in this study. For
simplicity, only a single -subunit is presented. TK,
tyrosine kinase domain; Y, tyrosine; F,
phenylalanine; S, serine. The number denotes
amino acid number according to Ullrich et al. (15).
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Mutant receptors were expressed in R
cells, and their expression
levels were assayed by Western blotting using antibodies specific for
the IGF-IR
- or
-subunit (Fig.
3A). Clones expressing levels
of receptor almost similar to those of WT 9 or 11 were selected. To
assess the number of mature cell-surface receptors, 125I-IGF-I-binding assay was also done (Fig.
3B). Although there were some variations in the number of
IGF-I binding sites among cell lines, all the mutants possessed levels
of binding sites at least more than WT 11 cells. Specific binding was
undetectable in R
cells. As an example, 125I-IGF-I
binding competition in WT 11 and Y950F/
1245 clone 3 cells is shown
in Fig. 3C, exhibiting similar displacement properties (IC50 = ~1 nM). Scatchard analysis revealed
that receptor number per cell and dissociation constant
(Kd) in WT 11 cells were 9 × 105
receptors and 0.6 nM, respectively. We could thus confirm
that all cell lines express almost similar levels of high affinity surface receptors.

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Fig. 3.
Expression levels of IGF-IR in cell lines
expressing WT or various mutant IGF-IRs. A, Western
blots for IGF-IR expression. Equal amounts of cell lysates were
subjected to SDS-PAGE, and IGF-IR - or -subunits were detected as
described under "Experimental Procedures." B, IGF-I
binding assay. Panel shows relative number of IGF-I binding sites per
cell in each cell line. Cells were incubated in binding buffer
containing 0.5 ng/ml 125I-IGF-I, and relative numbers of
specific binding sites were determined. Results were presented with
values of WT 11 (#11) cells normalized to 1.0. Data
represent the means ± S.D. of triplicate determinants.
C, 125I-IGF-I binding competition in WT 11 and
Y950F/ 1245 clone 3 (#3) cells. Specific binding in cells
incubated in binding buffer containing 0.5 ng/ml 125I-IGF-I
and/or increasing concentrations of unlabeled IGF-I was determined as
described under "Experimental Procedures." Percentages of maximum
bound/free (B/F) ratios were plotted against unlabeled IGF-I
concentrations.
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To examine the functional properties of the Tyr-950 and the C terminus
receptor mutants, we measured the activation of the PI3-K and MAPK
pathways upon IGF-I stimulation (Fig. 4).
Phosphorylation of IRS-1 and Akt was used as a marker of activation of
the PI3-K pathway. The activation of both IRS-1 and Akt seemed to be
somewhat mitigated in the Y950F, Y950F/Y1316F, and Y950F/
1245
mutants compared with WT after testing the dose dependence of the
response (Fig. 4, A and B). The PI3-K assay was
in agreement with the results regarding those of activation for IRS-1
and Akt (Fig. 4C). Taken together, we concluded that the
PI3-K pathway is not abrogated, but inhibited in Y950F and
Y950F/
1245 mutants. It is unlikely that Tyr-1316 contributes in a
significant manner to the activation of the PI3-K pathway (Fig.
4A), which is consistent with both the results that the
1245 mutant had no significant effect on the activation of the PI3-K
pathway (Fig. 4, B and C), and those from the
studies of the IR C-terminal deletion mutants (48). The ability of the
Y950F and
1245 mutants to activate the ERK pathway was intact, as
determined by measurement of ERK-1 and -2 phosphorylation (Fig. 4,
A and B). Double mutation of Tyr-950 and the C
terminus was required to inhibit the ERK pathway (Fig. 4B),
confirming previous reports (49).

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Fig. 4.
Activity of the PI3-K and MAPK pathways in
cells expressing WT or various mutant receptors upon IGF-I
stimulation. A, IGF-I-induced phosphorylation of IRS-1,
Akt, and ERK on extracts from cell lines expressing WT or mutant
receptors potentially relevant to PI3-K activation (Y950F, Y1316F, and
Y950F/Y1316F). Serum-deprived cells were stimulated with increasing
concentrations of IGF-I (1-125 ng/ml) for 10 min, and whole cell
lysates were subjected to SDS-PAGE, whereupon each phosphorylated form
of protein was analyzed as described under "Experimental
Procedures." Lane 1, 0 ng/ml; lane
2, 1 ng/ml; lane 3, 5 ng/ml;
lane 4, 25 ng/ml; lane 5, 125 ng/ml.
B, Western blots for IGF-I-induced phosphorylation of IRS-1,
Akt, and ERK in WT, 1245, and Y950F/ 1245 cells. Cell lysates
collected 10 min after stimulation with 50 ng/ml IGF-I were analyzed as
described in panel B. C, autoradiogram
of PI3-K activity in cell lines expressing WT or mutant receptors upon
IGF-I stimulation. Serum-deprived cells were either stimulated with 50 ng/ml IGF-I for 10 min or unstimulated, and cell lysates were
immunoprecipitated with anti-PY20 antibody. PI3-K activity in the
immunoprecipitates was measured using phosphatidylinositol and
[ -32P]ATP as substrates. Phosphatidylinositol
phosphate (PIP) was detected by autoradiography after
isolation by thin layer chromatography as described under
"Experimental Procedures."
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Double Mutation at Tyr-950 and the C Terminus Is Required to
Inactivate IGF-IR-mediated Radioresistance--
The radiosensitivities
of the various mutants were determined by colony-forming assays, and
dose-survival curves were obtained as shown in Fig.
5A. Survival fractions after
exposure to 6 Gy are also summarized in Fig. 5B. All the
specific mutants except for Y950F/
1245 exhibited very similar
radioresistance to WT cells, demonstrating that any single mutation
does not influence the phenotype. Double mutation of Tyr-950 and the
C-terminal domain was required to inactivate the phenotype. This
suggests that signals from either of the two different sites, Tyr-950
or the C terminus, may be sufficient to induce clonogenic
radioresistance in R
cells. Because Tyr-950 has the ability to
stimulate both PI3-K and MAPK pathways and the C terminus has both MAPK
and 14-3-3/c-Raf pathways, mutational analysis was thus still
insufficient to clearly define the relative contributions of the three
pathways.

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Fig. 5.
Clonogenic radiosensitivity in WT and various
mutant IGF-IRs. A, dose-survival curves of cell lines
expressing WT or various mutant receptors. Data shown are means of at
least two independent experiments. B, surviving fractions of
each of the cell lines after exposure to 6 Gy of radiation. Data
represent the means ± S.D. of at least four surviving fractions
derived from each clone. *, p < 0.05 versus
WT cells (Student's t test).
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Effects of Specific Inhibitors of PI3-K and MEK on Radioresistance
in WT, Y950F, and C-terminal Mutants--
We reasoned that it would be
possible to analyze the relative contributions of the three pathways
when Tyr-950 or C-terminal single mutants were combined with specific
inhibitors of PI3-K or MEK. The resulting patterns of signal inhibition
in WT 11 cells by each inhibitor are shown in Fig.
6A, and the survival fractions after a radiation exposure to 6 Gy of WT cells or cells expressing Y950F or
1245 receptors are shown in Fig. 6B. Wortmannin
is a well known PI3-K inhibitor and has often been used in studies of
radiosensitization. Previous reports have shown that concentrations of
more than ~5 µM will significantly sensitize cells, and
that significant sensitization is not observed at lesser concentrations (50, 51). This sensitization is a result of inhibition of ATM or
DNA-dependent protein kinase, important factors in DNA double strand break repair, but not to inhibition of PI3-K itself. Specific inhibition for PI3-K is usually obtained at 0.1-1
µM (52). The survival rates of the cell lines used in
this study were unaffected by 1 µM wortmannin (Fig.
6B), at which concentration IGF-I-induced Akt activation is
completely inhibited (Fig. 6A), but they were uniformly
sensitized by 5 and 10 µM concentrations (data not
shown). In addition, we tested another PI3-K inhibitor, LY294002, in a
similar manner. Precise information was not available on the
concentration at which ATM and DNA-dependent protein kinase inhibition occurred using this inhibitor. When 10 µM
LY294002 was applied, a concentration frequently used to inhibit PI3-K (8, 20), IGF-I-induced Akt activation was significantly inhibited, but
somewhat less so than after treatment with 1 µM
wortmannin (Fig. 6A). The use of more than 10 µM LY294002, however, resulted in significant toxicity
and a remarkable decrease in plating efficiency (data not shown). No
effect was observed on the survival of the three cell lines (Fig.
6B), consistent with the results obtained after treatment
with 1 µM wortmannin. The fact that the
1245 mutant
was still resistant after treatment with PI3-K inhibitors was quite
informative; cells displayed radioresistance even under conditions
where the only known surviving pathway of IGF-IR was that of MEK/ERK.
We further used an inhibitor of MEK, PD98059, to inhibit this pathway.
Of interest, cells expressing WT receptors were not influenced, but
both mutants were significantly radiosensitized upon treatment with 25 µM PD98059, a concentration that effected a nearly
complete inhibition of IGF-I-induced ERK activation (Fig. 6,
A and B). These results suggest that the ability
of IGF-IR to activate the PI3-K pathway alone is insufficient to
produce radioresistance, but radioresistance is achievable when PI3-K signals are combined with signals from the C terminus that are independent of the MEK/ERK pathway. The results of these two inhibitor experiments both point toward a strong contribution of the MEK/ERK pathway in the development of radioresistance. The results using inhibitors are summarized in Table I.

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Fig. 6.
Effect of specific inhibitors of PI3-K and
MEK on clonogenic radioresistance in cells expressing WT or mutant
receptors. A, effect of inhibitors on IGF-I-induced
activation of Akt or ERK in WT 11 (#11) cells.
Serum-deprived cells were incubated with inhibitors (Wort, 1 µM wortmannin; LY, 10 µM
LY294002; PD, 25 µM PD98059) for 1 h and
stimulated with 50 ng/ml IGF-I. Cells were lysed after 10 min, and
phosphorylation of Akt or ERK was detected by Western blotting as
described under "Experimental Procedures." -Actin and ERK-2 were
used as loading controls. B, effect of inhibitors on
clonogenic radioresistance. Appropriate numbers of cells grown in
plastic flasks were treated with the same concentrations of inhibitors
as described in panel A for 1 h and
-irradiated at a dose of 6 Gy. Irradiated cells were transferred to
an incubator and rendered to form colonies in the presence of
inhibitors. Surviving fractions were calculated as described under
"Experimental Procedures." Data represent the means ± S.D. of
three separate experiments. *, p < 0.002; **,
p < 0.001 versus cells irradiated in the
absence of inhibitors (analysis of variance).
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Table I
Summary of radiosensitivity of WT, Y950F, and 1245 cells after
treatment with specific inhibitors of PI3-K or MEK
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Expression of Constitutively Active MEK Renders R
Cells
Radioresistant--
To confirm the radioresistant effect of MAPK
pathway signaling, constitutively active MEK was transiently expressed
in R
cells, and their radiosensitivity was then assayed and compared with mock-transfected R
cells. At 2 days after transfection, HA-tagged MEK was clearly detected and ERK activity was enhanced in the
growth medium of the transfected cells (Fig.
7A). Comparable radioresistance to WT 11 cells was exhibited by R
cells expressing constitutively active MEK, whereas mock-transfected cells displayed levels similar to those for R
(puro) cells (Fig. 7B).
Cross-activation of IRS-1 or Akt was not detected in R
cells
expressing constitutively active MEK (data not shown).

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Fig. 7.
Effect of constitutively active MEK
expression in R cells on clonogenic radiosensitivity.
A, expression of constitutively active HA-tagged MEK in R
cells. R cells were transfected with a plasmid containing
constitutively active HA-MEK cDNA. Cell lysates were prepared
48 h after transfection and processed for Western blotting as
described under "Experimental Procedures." Proteins were probed
with anti-HA or phosphorylated ERK antibodies. ERK-2 was used as a
loading control. Preparations from mock-transfected cells without
plasmids were also used as controls. B, effect of
constitutively active MEK expression in R cells on clonogenic
radiosensitivity. Cells transfected as described in panel
A were -irradiated at a dose of 6 Gy 48 h after
transfection, and the surviving fractions were determined by
colony-forming assay. Mock-transfected cells were treated similarly as
a control. Data represent the means ± S.D. of three separate
experiments. *, p < 0.05 versus untreated
or mock-transfected cells (Student's t test).
|
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DISCUSSION |
Evidence is rapidly accumulating that IGF-IR is involved in
cellular radioresistance. Mouse embryo fibroblasts exhibit a
radioresistant phenotype when IGF-IR is overexpressed (21, 24, 25), and melanoma cells display an enhanced radiosensitivity after antisense targeting of IGF-IR (27). Furthermore, a specific inhibitor of the
IGF-IR tyrosine kinase, tyrphostin AG 1024, significantly radiosensitized human breast cancer cells (26). The signaling mechanism
whereby IGF-IR leads to radioresistance, however, has never been
comprehensively pursued to date. We therefore wished to determine which
of the downstream pathways of IGF-IR are related to the development of radioresistance.
As a measurement of radiosensitivity, clonogenic cell survival as
determined by colony-forming assay was used during this study. It is a
complex end point influenced by many factors such as cell death and/or
growth, DNA repair, and the ability to escape growth arrest and
re-enter the cell cycle. This in vitro assay has been used
as a representative marker of radiosensitivity thought to reflect the
reproductive integrity of tumor cells (1, 2, 54-56). It may be argued
that clonogenic radioresistance induced by IGF-IR in this study is at
best very modest, leading to no more than a 3-fold increase in survival
fraction at 6 Gy. Although it is well established that activated Ras
(6-8) or overexpression of EGFR (10-14) in tumor cells confers
clonogenic radioresistance, its extent is similar to that obtained by
IGF-IR as shown in our results. This level of difference, however,
critically affects clinical radioresponse or tumor cure (1, 2) because
radiotherapy is usually performed according to fractionated regimen, by
repeating irradiation 20-30 times; the modest difference per
irradiation is enormously enhanced at the end of the therapy (54).
Considering that exogenously introduced WT IGF-IR induces clonogenic
radioresistance in R
cells, which are deficient in the endogenous
IGF-IR, we attempted to use this system to determine which portions of
the receptor structure are necessary for the origin of this signal. A
950 IGF-IR mutant that lacks the tyrosine kinase domain did not
confer radioresistance, confirming that tyrosine
kinase-dependent signal transduction is required for this
activity. We therefore reasoned that the use of mutants with selective
signaling defects could help to pinpoint which downstream pathways are
involved in the development of radioresistance.
How is the IGF-IR activated under the present conditions? Ionizing
radiation by itself activates receptor tyrosine kinases such as EGFR,
which in turn activates its downstream pathways (13). Although such
activation of IGF-IR was not apparent in WT 9 or 11 cells under the
present conditions (data not shown), undetectable levels of activation
could not be ruled out. Another factor that may affect activation of
IGF-IR is relatively high levels of IGF-I (10-20 ng/ml) originally
contained in the serum in growth medium (57). Unlike the case of
stimulation of serum-deprived cells, activation of IGF-IR and its
downstream pathways in cells chronically incubated with growth medium
is very weak (25). Even under these conditions, IGF-IR-overexpressing
cells can proliferate much faster than non-overexpressing cells by the
continuous exposure to 10% serum medium (16, 17, 25). More pronounced
effect is observed under anchorage-independent conditions (16, 17). It
is thus possible that very low levels of IGF-IR activation through
growth medium or irradiation may be enough to induce IGF-IR-mediated clonogenic radioresistance and easily reach to saturation. We inferred
that retained functions of mutant IGF-IRs in growth medium following
irradiation, which are barely detectable, could be qualitatively similar to those activated by IGF-I, which are easily visualized as
shown in Fig. 4.
As described above, survival signals are transduced by the different
regions of IGF-IR through specific docking proteins, and eventually
converge to cause the activation of the PI3-K, MEK/ERK, and
14-3-3/c-Raf pathways. Mutational analysis revealed that Tyr-950 and
the C terminus are required for conferral of the radioresistant
phenotype, and that both domains must be mutated to inactivate it (Fig.
5). These patterns were reminiscent of those observed for
IGF-IR-mediated differentiation of neuronal cells, where double
mutation of Tyr-950 and the C terminus was similarly required to
inactivate the function (20). This phenotype absolutely depends on
MEK/ERK activity, and indeed cells expressing WT receptor lose
differentiation ability when treated with the MEK inhibitor PD98059
(20). Although this similarity also implicated a contribution of the
MEK/ERK pathway to clonogenic radioresistance, the finding that PD98059
did not radiosensitize cells expressing WT receptors seemed tentatively
puzzling. However, further results helped explain this discrepancy by
revealing the importance of redundancy in this system. Cells expressing
Y950F or
1245 mutant receptors were effectively radiosensitized upon
PD98059 treatment (Fig. 6B), whereas cells expressing either
WT or mutant receptors were unable to be sensitized by PI3-K inhibitor
treatment, i.e. these results suggest that radioresistance
may be obtained as long as the ability of the receptor to stimulate the
MEK/ERK pathway is retained. This was confirmed by the demonstration of
full radioresistance in R
cells transfected with constitutively
active MEK (Fig. 7). Previous reports concluded that the MEK/ERK
pathway is not involved in radioresistance (7, 58), mostly drawn from
the simple finding that PD98059 treatment had no effect on
radiosensitivity. Here, a combination of mutational analysis with the
use of specific inhibitors allowed us to reveal contributions to the
pathway in the absence of redundancy. This study then serves as an
important reminder that careful interpretation is of extreme importance when analyzing the findings from inhibitor studies. In addition, the PI3-K pathway alone is not sufficient to induce radioresistance, but full radioresistance can be achieved by a combination of PI3-K signals with those from the C terminus that are irrelevant to the
MEK/ERK pathway. These non-MEK/ERK C-terminal signals are presumably
mediated by the 14-3-3/c-Raf pathway. Activated c-Raf can migrate to
mitochondria and exerts cell survival effects with Nedd4 through 14-3-3 proteins bound to the C terminus of IGF-IR (28, 34). The PI3-K pathway
finally leads to Bad phosphorylation and induces binding with Bcl-xL in
mitochondria (28). Although it is still unclear exactly how cooperation
of both pathways leads to clonogenic radioresistance, activation of the
PI3-K or the 14-3-3/c-Raf pathway alone is unlikely to be sufficient at
least in these cell lines. Similar combination of pathways is also
required for cell survival of 32D cells expressing IGF-IR induced by
interleukin 3 withdrawal (49). Gupta et al. (8) reported
that Ras-induced clonogenic radioresistance is mediated exclusively by
the PI3-K, and not the MEK/ERK, pathway. Because IGF-IR also activates
Ras pathways, we cannot clearly explain this discrepancy. Considering that IGF-IR signaling and functions vary from one cell type to another,
this variability in signaling may depend on the availability of
substrates and transducing molecules in each cell type, as reported by
Petley et al. (59).
ATM is known to be a sensor of DNA damage, especially as a result of
ionizing radiation, and stimulates double strand break repair via its
kinase activity, whereby it significantly contributes to clonogenic
radioresistance (53). Recently, radiation-induced ATM activation was
reported to be inhibited by antisense against IGF-IR (27), suggesting a
possible connection between IGF-IR and ATM signaling. However, a direct
link between these two molecules has not been established to date.
Discovering a relationship between the MEK/ERK pathway or others and
ATM would be an interesting key to understanding IGF-IR-mediated
clonogenic radioresistance.
The redundancy of the survival signals related to the development of
clonogenic radioresistance revealed by this study may have clinical
implications regarding the use of molecular targeting in radiotherapy.
IGF-IR is overexpressed in several human tumors (21-23) and is
associated with a very poor prognosis following radiotherapeutic
treatment (21). To overcome these problems, the points of downstream
convergence of the IGF-IR pathways known to be involved in the
development of radioresistance, PI3-K and MAPK, may be concomitantly
targeted with radiotherapy to increase the efficacy of treatment for
these cases (8). However, it is possible that the IGF-IR tyrosine
kinase itself, the origin of many features of this robust
radioresistant mechanism, may be a more efficient target.