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INTRODUCTION |
The insulin-like growth factors
(IGF1-I, IGF-II) belong to a
pleiotropic family of soluble peptide factors that circulate bound to
one of six IGF-binding proteins. The IGFs, their receptors, and
IGF-binding proteins constitute a family of cellular modulators that
play essential roles in the regulation of growth and development. The
actions of IGFs are believed to result primarily from the activation of
the IGF-I receptor (IGF-IR) (1). The IGF-IR resembles the insulin
receptor in structural as well as functional aspects (2). This
heterotetrameric transmembrane glycoprotein consists of 2
- and 2
-subunits. The tyrosine kinase catalytic site and the ATP-binding
site are located in the cytoplasmic portion of the
-subunit. The
-subunit of the IGF-IR has intrinsic tyrosine kinase activity that
is stimulated when IGF-I binds to the
-subunit. The receptor
tyrosine kinase phosphorylates intracellular substrates such as the
insulin receptor substrates IRS-1, IRS-2, and Shc. Two different major
pathways then emerge; one pathway involves ERK1/2 through Ras/Raf/MEK
and the other pathway proceeds through PI3K (see for review Ref. 1).
Thus, the IGF-IR activated by its ligands plays an important role in
the growth of cells inducing mitogenesis, transformation, and
protection of cells from a variety of apoptotic injuries (3). The
IGF-IR was also shown to be involved in mitogenesis through the IRS
molecules (4), and IGF-I can also protect fibroblasts from apoptosis
induced by UV-B light through the activation of the
phosphatidylinositol 3'-kinase (PI3K)/Akt pathway (5). Moreover, the
MAPK pathway may play a role in cell survival independent of PI3K/Akt
as was demonstrated in fibroblasts overexpressing the IGF-IR (6, 7).
IGF-I can also stimulate the proliferation of hematopoietic cells in
combination with other hematopoietic growth factors (8), and IGF-I is
known to support growth and to prevent apoptosis in neuronal cells (9, 10). The viability of most cells is dependent on the presence of growth
factors; recently, IGF-IR-mediated neuroprotection was shown to be
mediated via induction of NF-
B that is dependent upon activation of
the PI3K pathway (11). Keratinocytes are more susceptible to
UVB-induced apoptosis if the growth medium is depleted of exogenous
growth factors; the activation of the IGF-IR was shown to promote the
survival of human keratinocytes following UVB irradiation (12).
To investigate further the mechanisms involved in IGF-IR activation on
rescuing cells against UV-like DNA damage, different cell lines were
damaged with 4-NQO, an UV-like mimetic agent. We used NWTb3 cells, a
NIH-3T3-derived cell line overexpressing the normal human IGF-IR (13).
To compare cell rescue of NWTb3 cells, parental NIH-3T3 and
NIH-3T3-derived cell lines expressing dominant negative IGF-IRs (NKA8
and NKR1) were used (14). We investigated the effect of IGF-IR
stimulation on the cellular proliferation of cells after DNA damage,
and we determined which signaling pathways are involved in this cell
rescue. With the use of an UV-damaged adenoviral vector, we also show
the direct effect of IGF-IR activation on DNA repair.
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EXPERIMENTAL PROCEDURES |
Materials--
4-Nitroquinoline 1-oxide (4-NQO) and
PI3-kinase-specific inhibitor LY 294002 were purchased from Sigma, and
recombinant human IGF-I (rhIGF-I) was a gift of Genentech (San
Francisco, CA). Both MEK1/2-specific inhibitor U 0126 and p38 MAP
kinase-specific inhibitor SB 202190 were from Calbiochem. The stock
solutions of pharmacological inhibitors such as LY 294002, U 0126, and
SB 202190 were all prepared in Me2SO at a concentration of
1000-fold so that when they were added to the culture medium, the
concentration of Me2SO was below 0.1%.
The antibodies used for Western blotting were purchased from New
England Biolabs Inc. (Beverly, MA); they include phospho-Akt (Ser-473),
phospho-MAPK (ERK1/2) (Thr-202/Tyr-204) and phospho-p38 MAPK
(Thr-180/Tyr-182) polyclonal antibodies as well as the Akt, MAPK, and
p38 protein polyclonal antibodies and the anti-rabbit IgG secondary
antibody (horseradish peroxidase-conjugated). Cell culture media and
reagents were purchased from Life Technologies Inc. The p38 dominant
negative construct (pCMV5-p38AGF) was kindly provided by Dr. R. J. Davis (Howard Hughes Medical Institutes, Worcester, MA); the
T*GY* motif of the p38 MAPK (phosphorylation site) was replaced
by AGF, thereby preventing p38 MAPK phosphorylation.
AdCA35lacZ, a replication-deficient recombinant type 5 adenovirus expressing the lacZ gene under the control of the
mouse cytomegalovirus (MCMV) immediate early promoter (15) was
kindly provided by Dr. F. Graham from the McMaster University,
Hamilton, Ontario, Canada.
Cell Culture--
The parental mouse embryonic fibroblast
NIH-3T3 cell line expresses ~16 × 103 IGF-IR/cell
(13). The NWTb3 cell line established in this laboratory expresses the
normal human IGF-IR at a level of ~4 × 105
receptors/cell (13). The NKA8 and NKR1 cell lines, where the Lys-1003
residue at the ATP-binding site was substituted by Ala (NKA8 mutant) or
Arg (NKR1 mutant), respectively (14), express the dominant negative
human IGF-IR (~3-7 × 105 receptors/cell). The
parental NIH-3T3 cell line was routinely cultured in Dulbecco's
modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated
fetal bovine serum (FBS), 100 units/ml penicillin, 100 µg/ml
streptomycin, and 300 µg/ml L-glutamine in a humidified
atmosphere of 95% air and 5% CO2 (37 °C). NWTb3, NKA8,
and NKR1 cells were cultured in the same medium with the addition of
0.5 g/liter G418 (Geneticin).
Immunoblotting--
NWTb3 cells were cultured in 100-mm dishes
(1 × 106 cell/dish) in the medium described under the
cell culture method. The day after seeding, cells were starved
overnight in FBS-free medium. After incubation (1 h at 37 °C) with
inhibitors (appropriate dilutions) and stimulation with 50 nM IGF-I (10 min at 37 °C), the cells were washed with
ice-cold PBS, and total cell lysates were prepared on ice with lysis
buffer (20 mM Tris, pH 7.5, 100 mM NaCl, 2.5 mM EDTA, 1% Triton X-100, 0.5% deoxycholate, 0.1% SDS,
5% glycerol, 10 mM NaF, 0.3 mM NaMo, 1 mM Na3VO4, and 0.5 mM
4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride). The cell
lysates were centrifuged for 30 min at 4 °C, and the supernatant was
separated. The total protein concentration was measured using the BCA
protein assay kit (Pierce). Phosphorylated Akt, ERK1/2, and p38 as well
as Akt, ERK1/2, and p38 protein were detected in 15 µg of protein
cell lysate by Western blotting using antibodies against phospho-Akt,
phospho-ERK1/2, and phospho-p38 as well as against total Akt, ERK1/2,
and p38 protein, respectively. All antibodies used were diluted 1:1000 in Tris-buffered saline/Tween 0.1% plus 5% bovine serum albumin.
4-NQO Treatment--
The UV-mimetic drug 4-NQO was dissolved in
absolute ethanol at a concentration of 2 mM, whereupon a
100 µM stock solution was prepared by diluting the
concentrated solution in serum-free medium and stored at
20 °C for
use throughout the study. All 4-NQO treatments were performed with a
final concentration of 2.5 µM 4-NQO in serum-free medium
for 30 min at 37 °C in a CO2 incubator. Following 4-NQO
treatment, cultures were rinsed with pre-warmed (37 °C) 5% FBS/DMEM
and then incubated in the same medium containing different compounds
(IGF-I and specific pathway inhibitors) for various times as indicated
in the cell proliferation method.
Cell Proliferation--
The cell proliferation assay was
performed using the CyQUANT Cell Proliferation kit (Molecular Probes,
Eugene, OR) and an HTS 7000 BioAssay reader (PerkinElmer Life
Sciences). The NWTb3 overexpressing IGF-IR and the other cell lines
were cultured in 75-cm2 flasks. When flasks were 90%
confluent, cells were counted using a hemocytometer. Cells were seeded
on 96-well Costar Cluster plates (Corning Glass) in 200 µl of DMEM
with 10% FBS. For the standard curve, the cell number at base line
ranged from 1,000 to 40,000 per well. After 15 h of incubation,
the medium was changed to 1% FBS/DMEM for an overnight starvation. 2.5 µM 4-NQO was added to the medium during 30 min at
37 °C, and cells were then washed and cultured in 5% FBS/DMEM with
or without 50 nM IGF-I, in the absence or in the presence
of different inhibitors (see figure for the concentrations used). Cells
were then incubated for 5 days. The same experiment was performed on
NWTb3 cells transiently transfected with a dominant negative p38
construct (pCMV5-p38AGF). The transfection was performed the day after
seeding the cells, and cell proliferation assay was performed as
described, with 4-NQO and IGF-I treatments. At the
indicated times, the medium from plates was aspirated, and
the plate was stored at
70 °C until all plates had been processed.
The cells were then lysed using the lysis buffer from the CyQUANT kit.
The fluorescence intensity of the DNA binding dye was measured using an
HTS 7000 microplate reader with excitation at 485 nm and emission at
535 nm.
Transient Transfection of p38 Dominant Negative Construct
(pCMV5-p38AGF)--
NWTb3 cells were seeded the day before the
transfection. Cells were then transiently transfected using the FuGENE6
transfection reagent (Roche Molecular Biochemicals) according to the
manufacturer's instructions with 3.6 µg of a p38 dominant negative
construct (16) or vector alone (p38 dn(+) or vector alone -(
)). In
order to determine the transfection efficiency under the conditions used in this experiment, 3.6 µg of DNA from pSV-
-galactosidase control vector (Promega, Madison, WI) was used to transiently transfect
NWTb3 cells. 24 h after transfection, in situ
-galactosidase staining for enzyme expression was performed, and
transfected cells (blue staining) were counted over the total cell
number. To check p38 phosphorylation in NWTb3 cells transfected with
the p38 dn construct and compare with cells transfected with vector alone, cells were starved overnight 24 h after transfection and stimulated with 50 nM IGF-I (10 min at 37 °C). Protein
extracts were prepared as described previously, and Western blot
analyses were performed with anti-phospho-p38 antibody to detect p38
phosphorylation induced by IGF-I stimulation as well as with a p38
antibody to detect both endogenous and transfected p38 proteins
(transfection efficiency).
Comet Assay--
A single cell gel electrophoresis assay (17)
was performed using the Trevigen Comet assay kit (Trevigen,
Gaithersburg, MD) according to the manufacturer's conditions. This
comet assay provides an effective method for evaluating DNA damage into
cells. The principle of this assay is based upon the ability of
denatured and cleaved DNA fragments to migrate out of the cell under
the influence of an electric potential, whereas undamaged and
supercoiled DNA stays in the cell when a current is applied.
NWTb3 cells (190,000cell/well) were plated in 6-well plates in 10%
FBS/DMEM. After seeding (15 h), the medium was replaced by 1% FBS/DMEM
for overnight starvation. Cells were treated or not with 2.5 µM 4-NQO during 30 min at 37 °C. Cells were washed and
incubated with or without 50 nM IGF-I in DMEM, 5% FBS for the indicated times. Untreated cells (negative controls) and cells treated with 100 µM H2O2
(positive controls) were included in the assay.
At every 24 h time point, cells were scraped and counted. After
washing once with ice-cold PBS, a dilution of 1 × 105
cell/ml was used to perform the assay. Cells were mixed with molten
agarose (ratio 1:10), and 50 µl were pipetted onto Comet Slides.
Slides were immersed in lysis solution and incubated on ice for 30 min,
followed by an immersion in an alkali buffer for 1 h at room
temperature in the dark. Slides were rinsed in TBE 1× before
electrophoresis (1 V/cm for 10 min). Samples were stained with the
supplied SYBR Green dye, and slides were viewed using an inverted
fluorescence microscope equipped with a FITC filter (AXIOVERT 100S,
Zeiss). The presence of a tail, so called "comet," reflects DNA
damage (single- and double-strand breaks, alkali-labile sites). For
each condition, "comet cells" were counted in a total of 200 cells.
Flow Cytometry--
Every 48 h following treatment with 2.5 µM 4-NQO (in control experiments 4-NQO was omitted) and
incubation with or without 50 nM IGF-I in the presence of
5% FBS/DMEM, cells were collected and washed twice in PBS. Cell
pellets were then resuspended in 100 µl of PBS, fixed in 1 ml of 70%
ethanol, 30% saline buffer, and stored at
20 °C until analysis.
Cells were washed twice with PBS followed by an incubation for 40 min
in 0.5 ml of PBS containing 0.1% Triton and 50 µg of RNase at room
temperature. 20 µg of propidium iodide (Sigma) was added, and the
suspension was incubated in the dark at room temperature for 15 min.
DNA content staining was finally determined by flow cytometry. All
analyses were carried out on a FACSCalibur using CellQuest Software
(Becton Dickinson, Mountain View, CA).
Adenovirus Amplification, Purification, and Titration--
The
recombinant adenovirus AdCA35lacZ used in this study was
constructed as described previously (15). In this vector, the Escherichia coli lacZ reporter gene is under the control of
a 1.4-kilobase pair MCMV immediate early promoter. The
virus was propagated in human 293 cells grown in modified Eagle's
medium enriched with L-glutamine (2 mM),
penicillin (100 units/ml), streptomycin (0.1 mg/ml), and 10%
heat-inactivated FBS.
AdCA35lacZ was amplified and purified as described
previously (18). Briefly, purified adenovirus was obtained from a crude viral lysate by purification on a CsCl gradient resulting in a titer of
4 × 109 plaque-forming units (pfu)/ml. 100-µl
aliquots of the adenovirus were frozen at
70 °C and kept until use.
UV Irradiation of the Virus--
UV irradiation of the virus has
been described previously (19). Briefly, 40 pfu/cell of viral
suspension, diluted in 1% FBS/DMEM, was irradiated in a 35-mm dish on
ice with continuous stirring using an EL UV lamp (model UVLS-26, 254 nm) and a UVX radiometer (both from UVP, Inc., Upland, CA) at an
incident fluence rate of 2 watts/m2. For the host cell
reactivation curve (HCR), the UV dose rates used were 0, 100, 200, 300, 500, 800, and 1000 J/m2. Aliquots of 200 µl were removed
for each exposure to the virus and diluted appropriately with 1%
FBS/DMEM.
IGF-I Treatment of the UV-irradiated Reporter Gene--
NWTb3
cells were seeded in 6-well plates at a density of 190,000 cells/well.
20 h after seeding, the medium was aspirated, and cells were
infected with either UV-irradiated or non-irradiated AdCA35lacZ in a volume of 300 µl at a multiplicity of
infection (m.o.i) of 40 pfu/cell. Following viral adsorption for 90 min at 37 °C, the cells were washed twice with medium and re-fed with 2 ml of warmed 1% FBS medium. For the IGF-I experiments on the damaged
virus DNA, a 90-min infection of the cells with the adenovirus was
followed by washing the cells to remove any trace of virus, and cells
were then re-fed with 1% FBS/DMEM with or without 50 nM
IGF-I. Infected cells were harvested 48 h after infection, and
cells were resuspended in 400 µl of reporter lysis buffer (Promega,
Madison, WI). Transgene activity was measured using the
-galactosidase assay kit (Promega). Results were normalized, and
-galactosidase activity of the cells infected with non-irradiated virus (J/m2) was set at a level of 1. Similar values
were obtained when cell number was used for normalization.
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RESULTS |
NWTb3 Rescue Induced by IGF-IR Activation after 4-NQO
Treatment--
Given the importance of IGF-I and IGF-IR pathway in
cell proliferation (4, 20), we examined the effect of IGF-IR activation on the cellular proliferation of mouse embryonic fibroblasts upon an
UV-mimetic treatment using 4-NQO. Cell proliferation assays were
performed on NWTb3 cells overexpressing normal human IGF-IR (13). NWTb3
cells were treated with 4-NQO for 30 min at a concentration of 2.5 µM that was determined to induce cell growth arrest.
4-NQO was removed after the treatment, and the cells were washed twice and then stimulated with IGF-I at a physiological concentration (50 nM). As expected, IGF-I induced the stimulation of NWTb3
cell proliferation with an increasing cell number that reached a
plateau after 4 and 5 days when cells became confluent (Fig.
1A). Moreover after 4-NQO
treatment, IGF-IR activation resulted in an enhanced cellular
proliferation of NWTb3, i.e. IGF-I was able to rescue the
cells, whereas no growth was observed in the absence of IGF-I. This
effect of IGF-I could be detected as early as 3 days after the 4-NQO
treatment and was significant at 4 and 5 days (p < 0.001). Not only was IGF-I capable of maintaining the survival of these cells, but in addition an obvious enhancement of the cellular proliferation could be observed 4 and 5 days after 4-NQO treatment. The
same experiment was performed with NIH-3T3 parental cells (Fig.
1B) and two cell lines expressing the dominant negative human IGF-IR, NKA8 and NKR1 cells ADDIN ENRfu (14) as shown in Fig. 1,
C and D, respectively. It has been shown
previously that NIH-3T3 cells express ~16,000 IGF-IR/cell as compared
with the NWTb3 cells (~4 × 105 IGF-IR/cell). The
IGF-I rescue was observed only in NWTb3 cells overexpressing normal
IGF-IRs as compared with parental NIH-3T3, NKA8, and NKR1 cell lines.
Thus, these data demonstrate that IGF-IR activation can overcome the
inhibition of the cellular proliferation induced by 4-NQO, an
UV-mimetic drug.

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Fig. 1.
Effect of the IGF-IR activation on
cellular proliferation following 4-NQO-induced damage. Cell
proliferation assay of NWTb3 (A), NIH-3T3 parental
(B), NKA8 (C), and NKR1 cells (D)
after UV-mimetic 4-NQO treatment. All the figures represent cellular
proliferation assays carried out using the CyQUANT cell proliferation
kit. The four cell lines were seeded on 96-well Costar Cluster plates
in 200 µl of DMEM containing 10% FBS. After starvation the cells
were incubated in the absence or in the presence of 2.5 µM 4-NQO for 30 min at 37 °C. Cells were washed and
incubated in DMEM, 5% FBS for the indicated times either in the
absence or in the presence of 50 nM human IGF-I (rhIGF-I).
Cells were then lysed with the CyQUANT lysis buffer, and the
fluorescence intensity of each sample was measured using an HTS 7000 microplate reader. For each figure, 5 experiments (4 replicates) were
combined and represented as the mean ± S.E. The data have been
normalized and shown as fold increase over the basal cell number.
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4-NQO Cell Cycle Arrest Is Overcome by IGF-I--
In order to
determine the role of 4-NQO in inhibiting cellular proliferation and
the effect of IGF-I in overcoming this inhibition, we carried out
fluorescence-activated cell sorter analysis. We examined the DNA
content of cells treated (or not) with 2.5 µM 4-NQO and
stimulated (or not) immediately after the 4-NQO with 50 nM
IGF-I. 4-NQO induced cell cycle arrest of NWTb3 cells in G0-G1 since the percentage of cells in S phase
was low and most of the cells were present in
G0-G1 (Table I).
No sub-2N (apoptotic) cells were detected in the presence of 4-NQO.
IGF-I treatment was capable of rescuing the cells from growth arrest
induced by 4-NQO as shown by an increase in S phase from 9 to 15% at
day 2 and from 6 to 23% at day 4 after 4-NQO treatment (Table I). As a
control experiment, DNA content analysis was performed on NWTb3 cells
not treated with 4-NQO 2 and 4 days after the addition, or not, of
IGF-I. As shown in the Table I (see numbers in parentheses), IGF-I was
not able to increase significantly the number of cells in S phase
(10-12% on day 4 after IGF-I treatment). These findings support the
results obtained from the cellular proliferation assay of NWTb3 cells
in the presence of 4-NQO and IGF-I.
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Table I
DNA content analysis by FACS, cell cycle arrest by 4-NQO and rescue
by IGF-I
Numbers in parentheses represent a control experiment with no 4-NQO
treatment (n = 2).
The results are shown at day-2 and day-4 time points after the addition
( /+) of IGF-I. This table shows a representative experiment of DNA
content analysis (n = 3).
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IGF-I Decreases DNA Damage following 4-NQO Treatment--
NWTb3
cells were exposed to the DNA-damaging agent 4-NQO with the aim of
determining whether DNA damage occurring upon 4-NQO treatment could be
restored by IGF-IR activation. Cell DNA damage was detected using a
"comet assay" (also called single cell gel electrophoresis). To
perform such an assay, NWTb3 cells were treated with 2.5 µM 4-NQO as described under "Experimental Procedures" and incubated with medium, with or without 50 nM of IGF-I,
up to 5 days after the 4-NQO treatment. The cells exhibiting DNA damage, named comet cells were counted randomly for a total of 200 cells. As shown in Fig. 2, about 35%
of the cells were shown to be damaged (comet cells) when the experiment
was performed 4 days after the 4-NQO treatment, and 40% of the cells
were comet cells 5 days after the same treatment. At each time point,
IGF-I was able to decrease the percentage of comet cells from 35 (day 4) or 40% (day 5) to 5%. As shown in the figure, the level of DNA-damaged cells following the addition of IGF-I reaches the level of
undamaged cells (negative control). Thus, it appears that these results
correlate with those obtained with the cell proliferation assay; IGF-I
is involved in rescuing NWTb3 cells from DNA damage, leading to normal
cellular proliferation.

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Fig. 2.
Effect of the IGF-IR activation on
4-NQO-induced cell DNA damage. NWTb3 cells were plated in DMEM,
10% FBS in 6-well plates. After 15 h, the medium was changed to
DMEM, 1% FBS for overnight starvation. Cells were untreated or treated
with 2.5 µM 4-NQO for 30 min at 37 °C. The drug was
removed by washing the cells with medium, and cells were incubated for
the indicated time in DMEM, 5% FBS with or without 50 nM
IGF-I. At each day of the experiment, cells were scraped gently and
counted. 1 × 105 cells/ml were used for the comet
assay (single cell gel electrophoresis) that was performed as described
under "Experimental Procedures." Slides containing cells were
viewed by fluorescence microscopy; 200 cells were counted, and
DNA-damaged cells were recognized by their comet (tail DNA). The
percentage of comet cells for 200 total cells is represented on the
figure that shows a representative experiment of comet assay
(n = 2). The figure legend is as follows:
C, negative control (no treatment); +C,
positive control (cell treatment 100 µM
H2O2 for 20 min at 4 °C); +4-NQO,
treatment with 2.5 µM 4-NQO; +IGF-I,
incubation with 50 nM IGF-I. Days 4 and
5 indicate the time after 4-NQO treatment when the assay was
performed.
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p38 MAP Kinase Is Involved in IGF-I-induced Cell Proliferation
following DNA Damage--
In order to study the signaling pathways
involved in the effect of IGF-I to rescue NWTb3 cells damaged following
4-NQO treatment, PI3K-, ERK1/2-, and p38-specific inhibitors were used.
NWTb3 cells were treated with 2.5 µM 4-NQO and incubated
during the indicated periods in the absence or in the presence of 50 nM IGF-I to induce rescue. Fig.
3 indicates that in the presence of the
PI3K pathway inhibitor LY 294002 (Fig. 3A) or in the
presence of the ERK1/2 inhibitor U 0126 (Fig. 3B), no effect
of these inhibitors was observed on the IGF-I rescue. In order to
ensure that the inhibitors were active on protein phosphorylation
induced by IGF-I stimulation, NWTb3 cells were treated for 1 h
with inhibitors prior to the IGF-I stimulation (10 min). Western
blotting analysis (Fig. 4) revealed the
dose-dependent inhibition of LY 294002 (PI3K pathway), U
0126 (ERK1/2 pathway), and SB 202190 (p38 pathway), respectively, on
the IGF-I-induced Akt, ERK1/2, and p38 phosphorylation. As shown in
Fig. 4A, 10 µM LY 294002 was able to inhibit
Akt phosphorylation induced by IGF-I stimulation and in the same
manner; 2 µM U 0126 and 50 µM SB 202190 inhibited ERK1/2 (Fig. 4B) and p38 (Fig. 4C) phosphorylation, respectively. Western blot analyses, performed to
detect all forms of Akt, ERK1/2, and p38 kinases, revealed that protein
levels were similar among the samples.

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Fig. 3.
Determination of the intracellular signaling
pathway involved in the IGF-I rescue of cells after 4-NQO
treatment. The figure represents cellular proliferation assays
carried out using the CyQUANT cell proliferation kit. NWTb3 cells were
seeded in 96-well Costar Cluster plates in DMEM 10% FBS. After
starvation, the cells were treated with 2.5 µM 4-NQO for
30 min at 37 °C and washed with medium. They were then incubated for
the indicated time in DMEM, 5% FBS either with or without 50 nM IGF-I, and each inhibitor was used at the inhibiting
concentration as follows: 10 µM LY 294002 (A),
2 µM U 0126 (B), and 50 µM SB
202190 (C). Four experiments of cellular proliferation (4 replicates per experiment) were combined and represented as the
mean ± S.E.
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Fig. 4.
Western blot analysis. Inhibition of
IGF-I-induced Akt (A), ERK1/2 (B), and p38
(C) phosphorylation, respectively, by the PI3K inhibitor LY
294002, the MEK1/2 inhibitor U 0126, and the p38 inhibitor SB 202190. NWTb3 cells were plated in 100-mm dishes (1 × 106
cell/dish). Serum-starved cells were pretreated with LY 294002 (A), U 0126 (B), or SB 202190 (C) for
1 h at the indicated concentrations followed by incubation with or
without 50 nM IGF-I (10 min). Akt, ERK1/2, and p38
phosphorylation was immunodetected with phospho-antibodies as described
under "Experimental Procedures," and the membranes were re-blotted
with antibodies that detected the total proteins as a control.
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Since inhibition of IGF-I-induced rescue of cells could not be
detected with the use of PI3K and ERK1/2 pathway inhibitors, we
employed a specific p38 MAP kinase inhibitor SB 202190, which fully
inhibits p38 kinase activity without affecting JNK or ERK activities
(21). Most interesting, the addition of SB 202190 significantly
inhibited the IGF-I rescue of NWTb3 cell proliferation (Fig.
3C). Four to 5 days after the 4-NQO treatment, IGF-I induced cellular proliferation to a level of 161 ± 8 and 166 ± 4%,
respectively, above cells not receiving IGF-I indicating rescue from
DNA damage. In the presence of SB 202190 cell numbers were respectively
reduced to 118 ± 4 and 122 ± 3% above untreated cells.
When inhibitors of the different signaling pathways were combined,
there was total abrogation of the IGF-I rescue. Thus the rescue of
NWTb3 cells induced by IGF-IR activation seems to be mediated primarily
through the p38 MAPK pathway.
In order to confirm this result, NWTb3 cells were transiently
transfected with a dominant negative p38 construct (p38 dn). Upon IGF-I
stimulation, there was a 60% reduction in p38 phosphorylation after
transfection of the dominant negative construct (data not shown). Cell
proliferation assays were performed on NWTb3 transfected with p38
dominant negative (+) or empty vector (
) as a control (Fig.
5). After induction of 4-NQO-DNA damage
and upon IGF-I stimulation, transfected cells with empty vector could
be rescued from DNA damage and started to grow upon IGF-I treatment
after 4 and 5 days. However, when NWTb3 cells were transfected with a
dominant negative p38 MAPK, the IGF-I rescue was markedly reduced. As
observed in the Fig. 5, the inhibition of the IGF-I rescue was about
75%. This result may be explained by the fact that only 30% of the cells were transfected at the passage used in this experiment (data not
shown). Thus inhibition of the p38 MAP kinase pathway, either by the
specific p38 inhibitor SB 202190 (Fig. 3C) or by the
expression of p38 dn in NWTb3 cells (Fig. 5), results in a marked
reduction of the IGF-I-induced rescue of cells treated with 4-NQO.

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Fig. 5.
Cell proliferation assay on NWTb3 cells
transfected with a dominant negative p38 MAPK. NWTb3 cells were
transiently transfected with pCMV5-p38AGF, a dominant negative p38
construct (p38 dn (+)) or vector alone ( ) as a control experiment.
Cell proliferation assay on transfected NWTb3 was performed after 4-NQO
treatment (30 min at 37 °C). Cells were washed and incubated in
DMEM, 5% FBS for the indicated times either in the absence or in the
presence of 50 nM human IGF-I. Cells were then lysed with
the CyQUANT lysis buffer, and the fluorescence intensity of each sample
was measured as indicated previously. The cell number that was
determined at day 0 was the same as found at days 4 and 5. Two
experiments (3 replicates) were combined and represented as the
mean ± S.E.
|
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HCR of
-Galactosidase Activity for the UV DNA-damaged Adenovirus
Is Enhanced by IGF-I--
In order to determine the role of IGF-I in
DNA repair, we employed an adenoviral vector that readily infects
cells. The recombinant adenoviral vector AdCA35lacZ contains
the
-galactosidase reporter gene under the control of the MCMV
immediate early promoter inserted into the deleted E1 region of the
virus (15). This vector can efficiently infect and express the reporter
gene in cells but is unable to replicate in the absence of exogenous E1
expression. We damaged the adenovirus DNA using UVC irradiation
(wavelength 354 nm), and we determined whether IGF-I was able to induce
repair of this DNA-damaged adenovirus. The experiment consisted of
irradiating the adenovirus DNA with UVC, infecting NWTb3 cells with
damaged or undamaged adenovirus, and incubating these cells for 48 h with or without IGF-I. Thus, transcription of the
-galactosidase
reporter gene present in the virus genome serves as an indicator of DNA repair after UV damage.
Since it has been suggested that increased survival (or enhanced
reactivation) of DNA-damaged viruses results from an induced DNA repair
pathway, we first examined the survival of AdCA35lacZ in
NWTb3 cells. A typical survival curve (host cell reactivation or HCR)
of
-galactosidase activity for the UV-irradiated
AdCA35lacZ in NWTb3 cells is represented in the Fig.
6A. Cells were infected at an
m.o.i. of 40 pfu/cell with the AdCA35lacZ that was
irradiated with UV doses ranging from 0 to 1000 J/m2. Fig.
6A shows that the decrease in reporter gene activity is related to the degree of UV exposure of the adenovirus used to infect
NWTb3 cells.

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Fig. 6.
A, HCR of the UV-irradiated reporter
gene activity. NWTb3 cells were infected for 90 min at an m.o.i. of 40 pfu/cell with the AdCA35lacZ. The adenovirus was previously
damaged by various doses of UV irradiation as shown on the figure.
Cells were harvested 48 h after infection, and -galactosidase
activity was assayed as described under "Experimental Procedures."
Each point is the average of 3 replicates ± S.E. The
-galactosidase activity determined for cells infected with the
undamaged virus (J/m2) was normalized to 1. B, effect of IGF-I on DNA repair of the UV-damaged
AdCA35lacZ. NWTb3 were grown in 6-well plates in DMEM with
10% FBS. After 20 h of incubation, the medium was removed, and
cells were infected for 90 min at 37 °C at an m.o.i. of 40 pfu/cell
with AdCA35lacZ. The viral DNA was previously damaged (or
not) following exposure to UV irradiation at different doses
(determined based on A). Thereafter, the medium containing
excess virus was removed by washing the cells twice. Cells were then
incubated in the absence (open bars) or in the presence of
50 nM IGF-I in DMEM with 1% FBS. Cells were harvested
48 h after infection, and the -galactosidase activity was
assayed as described under "Experimental Procedures." ***,
p < 0.001 determined by a paired t test at
each UV dose, indicating a significant difference when IGF-I is added
to the infected cells. Four experiments were combined and are
represented as the mean ± S.E. The -galactosidase activity
determined for cells infected with the undamaged adenovirus
(J/m2) was normalized to 1.
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In order to determine whether IGF-I affects DNA repair, we examined its
effect on the HCR of
-galactosidase activity for the UV-damaged
AdCA35lacZ. We infected NWTb3 cells with non-irradiated AdCA35lacZ or virus that had been UV-irradiated over a
dosage range encompassing the IC50 (Fig. 6A,
300, 500, and 800 J/m2). After infection, cells were
incubated for 48 h in the absence or in the presence of 50 nM IGF-I in the medium. Fig. 6B shows the
resulting relative
-galactosidase activity and represents the DNA
repair of the UV-damaged reporter gene. As shown in the figure, IGF-I
is able to enhance the relative
-galactosidase expression from the
damaged adenovirus. This result represents significant DNA repair
induced by the addition of IGF-I.
 |
DISCUSSION |
IGF-I is a major survival factor, protecting cells from apoptosis
induced by a variety of agents such as growth factor withdrawal (22)
and etoposide ADDIN ENRfu (23). In the work reported here, we were
interested in studying the ability of IGF-I, through stimulation of its
receptor, to rescue cells from UV-like DNA damage. Toward this end, we
used 4-NQO as an UV-mimetic agent to damage mouse fibroblast cells.
This agent is one of the few chemical carcinogens whose metabolic
processes are well characterized (24). Treatment with 4-NQO has been
employed in mammalian cells as a paradigm for DNA damage-induced
carcinogenesis. To exert its neoplastic effect, 4-NQO must undergo
metabolic activation to the proximate carcinogen
4-hydroxyaminoquinoline 1-oxide, which, after acylation, reacts with
DNA to form stable quinoline-purine monoadducts, i.e. at the
exocyclic N-2 and N-6 positions of guanine and adenine, respectively
(25). 4-NQO has been characterized as "UV-mimetic" with respect to
its genotoxic properties; it behaves very similar to UVC light (and
ionizing radiation) in several DNA damages systems (26). Both bulky
4-NQO adducts and UV light-induced pyrimidine dimers can be repaired by
nucleotide excision repair pathways (27).
In this study, we used NWTb3 cells, a NIH-3T3-derived cell line
overexpressing the normal human IGF-IR (13). For control cells, we also
used tyrosine kinase-deficient human IGF-I receptors where the lysine
1003 residue at the ATP-binding site was substituted by alanine (KA
mutant) or arginine (KR mutant) (14). The resulting cell lines
overexpressing these constructs were characterized as dominant negative
NIH-3T3-derived cell lines, named NKA8 and NKR1, respectively. Both
were used for comparison with NWTb3. By using a cellular proliferation
assay, we demonstrated that the IGF-IR activation was capable of
rescuing NWTb3 cells from DNA damage induced by 4-NQO. The kinase
activity of IGF-IR is essential for the rescue of cells from DNA damage
since no rescue occurred with NKA8 or NKR1 damaged using 4-NQO. These
mutated IGF-IRs function in a classic dominant negative manner, and it has been shown that addition of IGF-I to cells expressing these mutant
IGF-IRs fails to protect cells from apoptosis (5). In a separate study,
Kuhn et al. (12) demonstrated that the activation of IGF-IR
promotes survival of human keratinocytes following UVB irradiation.
These cells are exquisitely more susceptible to UVB-induced apoptosis
if the growth medium is depleted of exogenous growth factors.
Furthermore, we demonstrate here that IGF-I was also able to act
directly on DNA damage caused by 4-NQO, since we determined that IGF-I
induced a decrease in cell DNA damage as demonstrated by the comet assay.
It has been shown that protection of cells by IGF-I against apoptosis
is commonly mediated by activation of multiple signal transduction
pathways including PI3K and MAPK cascades (7). In the present study, we
have found that the ability of IGF-I to rescue cells from DNA damage is
primarily blocked by SB 202190, a specific inhibitor of p38 MAPK
activity, whereas LY 294002 (PI3K inhibitor) and U 0126 (ERK1/2
inhibitor) did not block the IGF-I-induced rescue. Thus the rescue
pathway used by the IGF-I system apparently acts primarily through the
p38 MAP kinase cascade. Recently, Okamato et al. (28)
described that a dominant negative effect of p38 MAPK enhanced
apoptotic death of differentiating neurons. The p38
/MEF2 pathway
seemed to protect differentiating cells from death during neurogenesis.
Moreover, Scrimgeour et al. (29) showed that neither MAPK
nor PI3K activation is entirely sufficient for the full
mitogenic/tumorigenic effect of the IGF-IR in NIH-3T3 cells. Thus there
must be additional signaling pathways emanating from IGF-IR in addition
to the well described pathways.
Consideration of the results obtained when NWTb3 cells were
transfected with a dominant negative p38 MAP kinase (pCMV5-p38AGF) suggests that the p38 pathway plays a role in the IGF-IR-mediated rescue of cells from DNA damage. Although activation of p38 MAPK was
shown to play a role in apoptosis in the PC12 neuronal cell line (30),
other recent studies have reported the anti-apoptotic effect of p38
MAPK in some cell lines and the role of p38 MAPK in cell proliferation.
For instance, it has been shown that expression of wild type MKK4 and
MKK6 that phosphorylate and activate p38 enhanced cell survival in the
presence of tumor necrosis factor-
(31). Moreover, by using specific
chemical inhibitors of MAP kinases, Rausch and Marshall (32)
demonstrated that both ERK and p38 pathways are critically involved in
the transduction of a proliferative signal and cooperate in granulocyte
colony-stimulating factor (G-CSF)-induced proliferation.
In this study, we found that inhibition of p38 MAPK by SB 202190 blocks
the cell proliferation rescue induced by IGF-I. The finding that
IGF-I-induced activation of p38 can be inhibited by SB 202190 suggests
the activation of p38
or
isoforms by IGF-I, since p38
and
-
are not inhibited by SB 202190 (33). Further experiments will be
necessary to address the activation state of p38
and -
in
IGF-I-stimulated cells since it has been shown that activation of
p38
, but not the other isoforms, is required for
-irradiation-induced G2 arrest (34). Thus in certain cells, p38
and -
may mediate cell cycle arrest, whereas
and
isoforms may mediate rescue from DNA damage and cellular proliferation.
Previous studies performed by McKay et al. (35) have used an
UV-damaged adenovirus as a tool to demonstrate the capacity of
UV-irradiated cells to support viral DNA synthesis involving transcription-coupled repair of UV-damaged DNA. Similarly, a
recombinant adenovirus, Ad5HMCVsp1lacZ, has been used to
study the effect of heat shock on DNA repair of an UV-damaged reporter
gene (36), and an adenoviral vector has been also used to prove the
existence of an UV-inducible error-prone DNA repair mechanism in normal cells (19). Thus, host cell reactivation of UV-damaged nuclear replicating viruses has been used to assess the DNA repair ability of a
variety of cell types. In the present study, we determined rescue of
viral DNA damage induced by IGF-I in normal fibroblasts. Toward this
end, we employed AdCA35lacZ and measured the DNA repair capacity of NWTb3 cells through the host cell reactivation technique. NIH-3T3-derived fibroblasts were infected with AdCA35lacZ in
which the reporter gene expression was under the control of an MCMV immediate early promoter, a promoter known to drive high levels of
expression without any species preferences as has been observed for the
human cytomegalovirus promoter (15). We demonstrate here that IGF-IR
activation is able to facilitate repair of exogenous damaged DNA. The
data show the sensitivity of exogenous DNA repair to IGF-IR activation
and confirm the utility and convenience of using adenoviral vectors to
study this process (35, 37). Indeed, this latter experimental approach
may be of particular value in order to examine repair of DNA damaged by
a variety of different physical and chemical agents.
In summary, our results indicate that IGF-I is able to rescue cells
from DNA damage through activation of its receptor primarily via the
p38 MAP kinase signaling pathway. We additionally demonstrate IGF-I-specific induction of DNA repair on exogenous (adenoviral) UV-damaged DNA. This adenoviral technique may be of importance in
extending DNA repair studies in order to examine the repair of DNA
damaged by a variety of different physical and chemical agents. IGF-I
is a powerful anti-apoptotic agent, and the IGF-IR activation may be
important in inducing DNA repair and allowing the cell cycle to
progress following DNA damage.