Diminished G1 Checkpoint after
-Irradiation and
Altered Cell Cycle Regulation by Insulin-like Growth Factor II
Overexpression*
Lijuan
Zhang
,
Min
Kim§,
Yung Hyun
Choi¶,
Bianca
Goemans
,
Choh
Yeung
,
Zongyi
Hu
,
Shili
Zhan
,
Prem
Seth§, and
Lee J.
Helman
**
From the
Molecular Oncology Section,
§ Medical Breast Cancer Section, ¶ Cell Signaling and
Oncogenesis Section, NCI,
Liver Diseases Section, NIDDK,
National Institutes of Health, Bethesda, Maryland 20892-1928
 |
ABSTRACT |
High levels of insulin-like growth factor II
(IGFII) mRNA expression are detected in many human tumors of
different origins including rhabdomyosarcoma, a tumor of skeletal
muscle origin. To investigate the role of IGFII in tumorigenesis, we
have compared the mouse myoblast cell line C2C12-2.7, which was stably
transfected with human IGFII cDNA and expressed high and constant
amounts of IGFII, to a control cell line C2C12-1.1. A rhabdomyosarcoma cell line, RH30, which expresses high levels of IGFII and contains mutated p53, was also used in these studies. IGFII overexpression in
mouse myoblast C2C12 cells causes a reduced cycling time and higher
growth rate. After
-irradiation treatment, C2C12-1.1 cells were
arrested mainly in G0/G1 phase. However,
C2C12-2.7 and RH30 cells went through a very short G1 phase
and then were arrested in an extended G2/M phase. To verify
further the effect of IGFII on the cell cycle, we developed a Chinese
hamster ovary (CHO) cell line with tetracycline-controlled IGFII
expression. We found that CHO cells with high expression of IGFII have
a shortened cycling time and a diminished G1 checkpoint
after treatment with methylmethane sulfonate (MMS), a DNA base-damaging
agent, when compared with CHO cells with very low IGFII expression. It
was also found that IGFII overexpression in C2C12 cells was associated with increases in cyclin D1, p21, and p53 protein levels, as well as
mitogen-activated protein kinase activity. These studies suggest that
IGFII overexpression shortens cell cycling time and diminishes the
G1 checkpoint after DNA damage despite an intact p53/p21
induction. In addition, IGFII overexpression is also associated with
multiple changes in the levels and activities of cell cycle regulatory components following
-irradiation. Taken together, these changes may
contribute to the high growth rate and genetic alterations that occur
during tumorigenesis.
 |
INTRODUCTION |
Insulin-like growth factor II
(IGFII)1 has been shown to
play an important role in the development, growth, and survival of normal cells. IGFII is encoded by the imprinted Igf2
gene expressed only from the paternal allele in most tissues (1). The
signaling of IGFII is mediated by the type I IGF receptor. We have
previously shown that forced overexpression of IGFII in C2C12 cells
leads to transformed characteristics (2). IGFII expressing cells exhibit a proliferative advantage. Moreover, elevated levels of IGFII
have been detected in human tumors of various origins and may act
through autocrine or paracrine signaling loops, which are often
associated with increased levels of type I IGF receptor (3-7). In a
transgenic carcinogenesis model involving simian virus 40 T antigen
targeted to pancreatic
-islet cells, the initial proliferation
switch is correlated with focal activation of IGFII, and reduced IGFII
expression impairs tumor cell growth in vitro and in
vivo (8). In addition, IGFII is further up-regulated in all
pancreatic islet-cell tumors in the T antigen transgenic mice, and
Igf2 gene disrupted mice developed fewer tumors of reduced size, a lower grade malignancy, and higher number of apoptotic cell
bodies (8). IGFII may also act as a survival factor and inhibits
apoptosis induced by cytokine deprivation, DNA damage, and a variety of
chemotherapeutic agents, although this anti-apoptotic activity may not
be a primary consequence of IGFII signaling (9-11). The anti-apoptotic
activity of IGFII could promote the accumulation of additional
genetic abnormalities that lead to cell proliferation.
Cell cycle progression of eukaryotic cells is finely regulated by an
intrinsic molecular clock comprised of cyclins and cyclin-associated kinases (12). The final decision of mammalian cells to replicate their
DNA or to withdraw from the cell cycle with an unduplicated genome
takes place in mid- to late G1 phase, referred to as the restriction point (13). The commitment process at the G1
checkpoint reflects a complicated integration of positive and negative
extracellular and intracellular signals transduced by multiple cascades
into the cell nucleus (14-17). The existence of internal checkpoints at different stages of the cell cycle is an important feature to
prevent the cell from prematurely entering the next phase before all
the necessary macromolecular events have been completed. A key
regulatory component of cell cycle progression is the tumor suppressor
p53 which is normally expressed at very low levels in many different
tissues due to the short half-life of the protein (18). Following DNA
damage, p53 protein levels rise dramatically and promote the
transcription of WAF1/CIP1 gene, the product of which,
p21WAF1CIP1, causes growth arrest and delayed entry into the S
phase until the genomic lesions are fully repaired (19, 20).
When growth factors interact with their receptors at the cell surface,
a cascade of phosphorylation events is triggered which transduce
mitogenic signals to the nucleus, leading to DNA synthesis and
subsequently to mitosis. Since all these extracellular stimuli must
ultimately pass their signals to the cell cycle machinery itself, it is
important to investigate the effects of growth factors on the cell
cycle and to understand the molecular basis of the link between the
upstream signaling pathways and the cell cycle clock. In our studies,
we examined the mouse myoblast cell line C2C12, stably transfected with
human IGFII cDNA, CHO cells with tetracycline-regulated IGFII
expression, and a RMS cell line, RH30, which expresses high levels of
IGFII and contains mutated p53. After DNA damage, cells with IGFII
overexpression had a diminished G1 checkpoint arrest and an
amplified G2/M phase despite intact p53/p21 induction. We
also found that IGFII overexpression was associated with increases in
basal levels of cyclin D1, p21, and p53 proteins, as well as
mitogen-activated protein kinase (MAPK) activity. These effects of
IGFII on the cell cycle may contribute to the high proliferation rate
and accumulation of genetic changes during tumorigenesis.
 |
MATERIALS AND METHODS |
Stable Transfection of CHO-AA8 Tet-off Cells with
pTRE-IGFII--
CHO cells were transfected with two plasmids, Tet-off
and pTRE-IGFII separately. CHO cells stably transfected with the
Tet-off plasmid were purchased from CLONTECH
(CHO-AA8 Tet-off, catalog number C3004-1). The tetracycline-regulatable
IGFII plasmid pTRE-IGFII was generated by inserting a full-length human
IGFII cDNA into the tetracycline-responsive vector pTRE
(CLONTECH) between the EcoRI and
XbaI site. The pTRE-IGFII was cotransfected with pTK-Hyg into the CHO-AA8 Tet-off cells as follows. Cells were trypsinized and
resuspended in complete
-MEM medium at 10 million cells per ml. 0.4 ml of the resuspended cells was mixed with 40 µg of pTRE-IGFII and 2 µg of pTK-Hyg in a 0.4-cm cuvette. The cells were electroporated in a
Bio-Rad Gene Pulsar at 960 microfarads, 0.22 kV/cm (t = 20-30 ms) and then allowed to stand at room temperature for 10 min. The cells were plated into 20 10-cm tissue culture plates containing 10 ml of complete
-MEM medium. The cells were allowed to grow for
48 h, and then hygromycin was added at 0.5 mg/ml. Cells were selected for 2 weeks or until single colonies were seen. About 200 clones were picked and subcultured in 6-well plates. Confluent clones
were trypsinized and resuspended in 3 ml of complete media. One ml of
clone was plated in duplicate into another 6-well plate. Complete
-MEM with 10 µg/ml tetracycline was added to one plate and
complete media to the other. The remaining 1 ml of clone was maintained
in culture as a stock. After 48 h of treatment, the duplicate
clone or set of clones with and without tetracycline were trypsinized
with 50-100 µl of trypsin for 3 min, and then 1 ml of complete media
was added to neutralize the trypsin. For dot blot, 50 µl of the
suspension was pipetted on to a dot blotter and filtered through a
Nytran membrane presoaked in 10× SSC. IGFII expression was checked by
Northern blot as described below.
Cell Cultures and Treatment--
The mouse myoblast cell lines,
C2C12-1.1 and C2C12-2.7, were generated by C. P. Minniti and have
previously been described (2). C2C12 cells were grown in Dulbecco's
modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 50 units/ml penicillin, 50 mg/ml
streptomycin, and 1 mg/ml G418 (geneticin) at 37 °C, 5%
CO2 in a humidified incubator. CHO cells were grown in
-MEM supplemented with 10% tetracycline-free fetal bovine serum,
0.5 mg/ml G418, 1 mg/ml hygromycin B, and alternative 10 µg/ml
tetracycline. Cells were irradiated with a 137Cs source at
5.0 Gray/min. In some experiments, cells were exposed to 1 µg/ml
nocodazole (Sigma) alone or plus MMS (Aldrich) at 40 µg/ml for
varying duration.
Cell Cycle Analysis--
Exponentially growing cells were washed
in PBS, fixed in ice-cold 70% ethanol for at least 1 h at
4 °C. After washing in PBS containing 0.1% glucose, cells were
treated with PBS staining buffer containing RNase A at 1 mg/ml,
propidium iodide at 50 µg/ml, 0.1% glucose in the dark at 4 °C
for 30 min, and filtered through a 35 µM strainer cap
(catalog number 2235, Becton Dickinson). A total of 10,000 stained
cells were analyzed in a fluorescence-activated cell sorter (FACScan;
Becton Dickinson). The CELLQUEST software (Becton Dickinson) was used
to determine the distribution of cells in the various cell cycle
compartments as G0/G1, S, and
G2/M.
Northern Blot--
Total RNA was isolated by using the RNeasy
Mini Kit (Qiagen) and loaded on a Nytran membrane (Schleicher & Schuell). The membrane was washed with 100 ml of 10× SSC and UV
cross-linked with Stratagene UV cross-linker. Dot blot or RNA blot was
prehybridized in QuikHyb Hybridization Solution (Stratagene) at
65 °C with 50 µg/ml sheared salmon sperm DNA for 30 min and then
hybridized with a random primer labeled probe (Amersham Pharmacia
Biotech, rediprime DNA labeling system) in the QuikHyb Hybridization
Solution (Stratagene) at 65 °C for 2 h. The membrane was washed
by using the QuikHyb washing procedures (Stratagene) and exposed to
x-ray film. Quantitative results were obtained by using the NIH Image
program. In some experiments, RNA transcript levels were normalized to
glyceraldehyde-3-phosphate dehydrogenase expression.
Western Blot and Immunoprecipitation Assays--
Exponentially
growing cells were washed twice in cold PBS and lysed in lysis buffer
(Tris-HCl 50 mM, pH 7.5; NaCl 120 mM; Nonidet
P-40 0.5%; NaF 10 mM; Na3VO4 1 mM; Na4P2O7 1 mM; dithiothreitol 1 mM; 4-(2-amino
ethyl)-benzenesulfonyl fluoride hydrochloride 1 mM;
leupeptin 20 µg/ml; aprotinin 20 µg/ml). Lysates were incubated for
30 min in ice (vortexed vigorously every 10 min) and clarified by
centrifugation at 14,000 rpm for 2 min. Protein concentration was
determined by the Bio-Rad Protein Assay (catalog number 500-0006, Bio-Rad). For Western blot analysis, proteins were separated by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (Novex) and blotted
onto a Protran nitrocellulose membrane (catalog number 00830N,
Schleicher & Schuell). Membranes were blocked for 1 h in TBST
(Tris-HCl 20 mM, pH 7.5; NaCl 138 mM; Tween 20 0.1%) containing 5% non-fat milk at room temperature, probed with
primary antibody for 1 h, washed three times with TBST, probed
again with horseradish peroxidase-conjugated secondary antibody for 45 min, and washed again three times in TBST. Antigen-antibody reaction
was revealed by using enhanced chemiluminescence (ECL) procedures
according to the manufacturers' recommendation (Pierce). Mouse
monoclonal anti-p53 and anti-p21 antibodies were purchased from
Calbiochem. All other primary and secondary antibodies were purchased
from Santa Cruz Biotechnology. For immunoprecipitation, 1 mg of
cellular extracts was precleared by incubating extracts with protein A or protein G-agarose (Santa Cruz Biotechnology) for 1 h at
4 °C. Extracts were then incubated with primary antibody overnight
at 4 °C, and immune complexes were collected by incubation (1 h, 4 °C with rocking) with protein A- or protein G-agarose.
Immunoprecipitates were washed four times with ice-cold lysis buffer,
eluted in equal volume of 2× SDS sample buffer (Tris-HCl 125 mM, pH 6.8; SDS 4%, glycerol 20%, and 0.005% bromphenol
blue; Novex), and resolved on SDS-polyacrylamide gel electrophoresis.
Antigen-antibody complexes were visualized with the enhanced
chemiluminescence (ECL) detection system (Pierce).
Kinase Assay--
Immunoprecipitation was performed as described
above. Immunoprecipitates were washed three times with lysis buffer
(content as described above) followed by washing three times with
kinase buffer (Tris-HCl 20 mM, pH 7.5; MgCl2 4 mM; dithiothreitol 1 mM). Protein A- or protein
G-agarose beads were resuspended in 26 µl of kinase buffer containing
3 µCi of [
-32P]ATP (NEN Life Science Products) and 3 µg of histone H1 (Life Technologies, Inc.) for Cdk2 and Cdc2 kinases,
3 µg of GST-Rb (Santa Cruz) for Cdk4 kinase, or 3 µg of myelin
basic protein (Sigma) for MAPK kinase. Kinase reactions were carried
out for 30 min at 37 °C and stopped by addition of 26 µl of 2×
SDS sample buffer. The samples were heated (100 °C, 5 min) and
subjected to SDS-polyacrylamide gel electrophoresis. Gels were fixed
and dried, and kinase activities were visualized and quantitated as described above. To verify the specificity of the Cdk4 kinase activity,
we included a Cdk4 antibody-specific blocking peptide (Calbiochem) in
the reaction and used the same amount of normal rabbit IgG (Santa Cruz)
to replace Cdk4 antibody.
 |
RESULTS |
Cells Stably Expressing IGFII Show a Diminished G1
Checkpoint after DNA Damage--
To study the effects of IGFII on cell
cycle control and the growth properties of mammalian cells, we chose
the mouse myoblast cell line C2C12. Cells stably transfected with human
IGFII cDNA and expressing high and constant levels of IGFII were
termed C2C12-2.7, and a control cell line was called C2C12-1.1 which
contained the vector alone. An RMS cell line, RH30, which expresses
high levels of IGFII and mutated p53, was also used in these
experiments. IGFII expression altered the proliferation rate and the
distribution of the cell cycle profile under normal culture conditions
(Fig. 1A, 0 Gy). As
demonstrated in Fig. 1A, increasing doses of
-irradiation leads to a much more prominent G2/M phase arrest in IGFII
overexpressing C2C12-2.7 cells than in control C2C12-1.1 cells.
Furthermore, the response to
-irradiation in C2C12-2.7 cells appears
quite similar to the response pattern observed in the RMS tumor cell line RH30. To analyze further the effect of IGFII expression on cell
response to DNA damage, we synchronized C2C12 cells by serum starvation
for 48 h. Cells were then stimulated with serum and received
-irradiation at the same time. As shown in Fig. 1B, there
is a prominent G1 arrest in control C2C12 cells by 18 h following
-irradiation (compare C2C12-1.1
IR to +IR). In
contrast, the IGFII overexpressing cells have no detectable
G1 arrest following
-irradiation (compare C2C12-2.7
IR
to +IR). Since the C2C12-1.1 and C2C12-2.7 cells have the same genetic
background except for different levels of IGFII expression, these
results suggest that the diminished G1 checkpoint in
C2C12-2.7 cells after DNA damage is likely due to the overexpression of
IGFII. It is noteworthy that no apoptosis was observed in either
C2C12-1.1 or C2C12-2.7 cells 48 h following both serum depletion
and 3-10 Gray
-irradiation (data not shown). Furthermore, in the
absence of
-irradiation, 14.5% of C2C12-1.1 cells are in
S/G2M compared with 23% of C2C12-2.7 cells 9 h
following serum addition, and by 18 h, 29% of C2C12-2.7 cells
have entered G2M in contrast to only 15% of
C2C12-1.1 cells, strongly suggesting that IGFII overexpression shortens G1 phase (Fig. 1B).

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Fig. 1.
Effect of IGFII overexpression on cell
cycle distribution. A, asynchronously growing cells
were treated with 0-10 Gray (Gy) of -irradiation and harvested
9 h later for C2C12 cells and 24 h later for RH30 cells which
express high levels of IGFII and mutated p53. DNA content was assessed
by flow cytometry as described under "Materials and Methods." The
percentage of cells in each phase of the cell cycle was calculated
using the CELLQUEST (Becton Dickinson) and indicated on the top
right of each cell cycle profile. B, IGFII expression
diminishes G1 checkpoint after DNA damage. Exponentially
growing cells were incubated in serum-free medium for 48 h, then
treated with 7 Gray of -irradiation (IR), and 10% serum
was added to the medium. After the indicated time points, the cells
were harvested for DNA content analysis by using propidium iodide
staining.
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To exclude that clonal variation between C2C12-1.1 and C2C12-2.7 cells
was responsible for the observed changes, we developed another model
using tetracycline-regulated IGFII expression in CHO cells. As shown in
Fig. 2A, the addition of 10 µg/ml tetracycline for 48 h (+Tet) markedly decreased
IGFII expression compared with CHO cells growing without tetracycline
(
Tet). The proliferation rate of CHO
tet cells is
higher than in CHO + tet cells (data not shown). Following 12 h of
treatment with a microtubule polymerization inhibitor, nocodazole, CHO
control cells (+tet) demonstrated a G2/M arrest
as expected (Fig. 2B). When these cells were subjected to a
DNA base-damaging agent, MMS, for 12 h, G1 arrest was
also seen (Fig. 2B, CHO+tet, Noco + MMS). In
contrast, the addition of MMS for 12 h to nocodazole-treated CHO
cells expressing high levels of IGFII (
tet) caused no
detectable G1 block (Fig. 2B, CHO-tet,
Noco + MMS). When
-irradiation was used instead of MMS treatment, the same findings were observed (data not shown). Moreover, with 12 h nocodazole treatment, 91% of CHO
tet cells
accumulate in G2M phase compared with 60% of CHO+tet
cells. Furthermore, 18% of CHO+tet cells stay in G1 phase
in comparison to 2.8% of CHO
tet cells (Fig. 2B). These
results further indicate that IGFII overexpression shortens
G1 phase of the cell cycle and diminishes the
G1 checkpoint after DNA damage.

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Fig. 2.
IGFII expression shortens G1
checkpoint both before and after DNA damage. A, Northern
blot analysis of IGFII mRNA from CHO cells growing without
tetracycline ( Tet) or with 10 µg/ml of tetracycline
(+Tet) for 48 h. After transferring total RNA to
membrane, the blot was hybridized with the random primer-labeled IGFII
probe. rRNA of 28 S and 18 S stained by ethidium bromide was
displayed as a loading control. B, equal number of CHO
cells was plated at low density and incubated for 48 h in medium
with or without tetracycline (10 µg/ml). CHO cells with
tetracycline-controlled IGFII expression were then treated
with 1 µg/ml nocodazole (Noco) alone or plus 40 µg/ml of
base-damaging agent, MMS, for 12 h. Flow cytometric analysis of
cells stained with propidium iodide was then performed.
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Expression Levels of Cell Cycle Regulatory Proteins in C2C12
Cells--
In order to investigate the role of IGFII on the kinetics
of cell cycle regulatory protein expression after DNA damage, we prepared cell extracts from C2C12-1.1 and C2C12-2.7 cells at various time points following 7 Gy
-irradiation and determined the
expression of a variety of cell cycle proteins by Western blotting. As
shown in Fig. 3A, the basal
cyclin D1 levels were consistently higher in C2C12-2.7 cells compared
with control cells. Following exposure to
-irradiation, cyclin D1
levels increased slightly in both control and C2C12-2.7 cells. No
changes were observed in the basal levels of cyclins E, A, B1, and p27
proteins between the two C2C12 cell lines. However, in both cell lines,
cyclin A and cyclin B1 proteins increased at 8 h after irradiation
and then decreased gradually to the level below the non-radiation group
at 36 h. There was an additional lower molecular weight band of
E2F-1 in C2C12-2.7 cells compared with C2C12-1.1 cells. After 7 Gray of
-irradiation, E2F-1 protein levels decreased in both cell lines. The
cyclin-dependent kinases Cdk2, Cdc2, and Cdk4 had
equivalent protein expression in both C2C12 cell lines. Furthermore,
these levels did not change following
-irradiation (Fig.
3B). There were no changes in proliferating cell nuclear
antigen protein levels both with and without
-irradiation (data not
shown).

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Fig. 3.
Effects of ectopic expression of human IGFII
on the levels of cell cycle regulatory proteins. Cell lysates were
prepared at the indicated time points after 7 Gray of -irradiation.
Western blot analysis was performed by using cyclin D1, cyclin E,
cyclin A, cyclin B1, E2F-1, p27 (A); or Cdk2-, Cdc2-, or
Cdk4 (B)-specific antibodies.
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C2C12-2.7 cells with high levels of IGFII expressed elevated levels of
p21 and p53 proteins (Fig. 4,
A and B) compared with C2C12-1.1 control cells.
In both high IGFII and control cell lines, p53 protein levels were
increased after
-irradiation. The increases in p53 levels were
observed at 3 h after irradiation and were dose-dependent in the range of 3-7 Gray. The relative
increase of p53 protein after radiation compared with basal expression in C2C12-1.1 cells is higher than in IGFII overexpressing C2C12-2.7 cells. After 7 Gy
-irradiation, in both C2C12 cell lines, p21 protein levels were also induced compared with the levels observed at
time 0 h. However, the relative increase of p21 levels compared with basal expression in C2C12-1.1 cells was higher than in IGFII overexpressing C2C12-2.7 cells (seen on darker exposure). The induced
p21 protein reached a maximum at 8 h and was sustained at least to
36 h. Constitutive levels of p21 RNA are also higher in IGFII
overexpressing C2C12-2.7 cells compared with control cells. Twenty four
hours following
-irradiation, the relative increase in p21 RNA was
similar in C2C12-1.1 and C2C12-2.7 cells (Fig. 4, C and
D). pRb protein level was similar in high IGFII expressing
C2C12-2.7 cells compared with C2C12-1.1 control cells. After various
doses of
-irradiation, pRb shifted from the hyperphosphorylated inactive form (higher band in Fig. 4E) to the
hypophosphorylated active form (lower band in Fig.
4E) in both cell lines.

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Fig. 4.
Changes in the inhibitors of cell cycle
progression by IGFII expression after DNA damage. Immunoblot
analyses of p53 protein level 24 h after indicated doses of
-irradiation (IR) treatment (A), p21
expression at indicated time points after 7 Gray of -irradiation
(B) were performed using antibodies specific for p53 (mouse
monoclonal antibody) or p21 (rabbit polyclonal antibody). C,
Northern blot analysis of p21 mRNA from C2C12-1.1 and C2C12-2.7
cells 24 h following indicated doses of -irradiation. After
transferring total RNA to a membrane, the blot was hybridized with a
random primer-labeled p21 probe. D, histogram results were
obtained by scanning the autoradiographic film of C and
using 0 Gray of C2C12-1.1 cells treated as 100% control. E,
Western blot analysis of pRb protein levels from C2C12-1.1 and
C2C12-2.7 cells with or without -irradiation.
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The Effects of IGFII Overexpression on MAPK Activity--
Previous
studies have demonstrated that the activation of the MEK-MAPK (also
referred to as extracellular signal-regulated kinase, ERK) pathway is
necessary for mitogen-induced G1/S phase cell cycle
progression (21, 22). Growth factors and oncogenes products can
activate the MAPK pathway through a phosphorylation cascade involving
Ras, Raf, and MAPKK for mitogenesis (14-16, 23). We found that the
MAPK (ERK-1 and ERK-2) protein levels were similar in C2C12-1.1 and
C2C12-2.7 cells with or without irradiation (Fig. 5A). The basal MAPK (ERK-1 and
ERK-2)-associated kinase activity toward the myelin basic protein
substrate in IGFII-overexpressing C2C12-2.7 cells was higher than in
C2C12-1.1 control cells (Fig. 5B, upper panel). After 7 Gray
-irradiation, MAPK activity was decreased in extracts of both C2C12
cells. A Western blot of the immunoprecipitated MAPK used for the above
kinase assay was performed to determine MAPK protein levels. As seen in
Fig. 5B (lower panel), the alteration of kinase
activity is not due to changes in protein levels.

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Fig. 5.
Effects of IGFII overexpression on MAPK
activity. A, 8 h following indicated doses of
-irradiation, cells were harvested and subjected to Western blot by
using rabbit polyclonal anti-p44 (ERK-1) and anti-p42
(ERK-2). B, at different time points following 7 Gray of -irradiation, cells were harvested, and MAPK was
immunoprecipitated by using the same antibody as above and assayed for
activity via phosphorylation of myelin basic protein (MBP,
upper panel). MAPK protein levels in the MAPK
immunoprecipitates were checked by Western blot (lower
panel).
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IGFII Overexpression Partially Releases the Inhibition of Cdk
Kinase Activities by
-Irradiation--
We then examined the
activities of cdks after
-irradiation in IGFII overexpressing and
wild-type C2C12 cells. Histone H1-associated Cdk2 kinase activity
decreased in C2C12-1.1 control cells after
-irradiation (Fig.
6, A and B). Only a
mild decrease, however, in Cdk2 activity in IGFII overexpressing
C2C12-2.7 cells was observed, and no change of Cdk2 activity occurred
in RH30 tumor cells expressing high levels of IGFII. Similarly, histone
H1-associated Cdc2 kinase activity was significantly reduced in
C2C12-1.1 control cells, but this was not seen in high IGFII expressing
C2C12-2.7 cells nor in RH30 tumor cells even following 10 Gy of
-irradiation (Fig. 6, A and C). In addition,
the kinase activity of Cdk4 toward the GST-Rb substrate decreased in
C2C12-1.1 control cells 24 h after 3-7 Gy of
-irradiation,
whereas there were no appreciable changes observed in C2C12-2.7 cells
(upper panel in Fig. 6D). To verify the
specificity of the Cdk4 kinase activity shown above, we included a Cdk4
antibody-specific blocking peptide in the reaction or used normal
rabbit IgG to replace Cdk4 antibody. It was found that Cdk4
antibody-specific blocking peptide could block GST-Rb phosphorylation,
and very weak GST-Rb phosphorylation was observed when Cdk4 antibody
was replaced by normal rabbit IgG (lower panel in Fig.
6D).

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Fig. 6.
Expression of IGFII partially releases the
inhibition of cdks induced by DNA damage. At 24 h after
indicated doses of -irradiation (IR) (A) or at
the indicated time points after 7 Gray of -irradiation
(B) and (C), Cdk2 and Cdc2 kinase assays were
performed by using histone H1 as substrate. D, 24 h
following indicated doses of -irradiation, Cdk4 kinase activities
were measured by using GST-Rb as substrate (upper panel).
The specificity of Cdk4 kinase activity was checked by including Cdk4
antibody (Ab)-specific blocking peptide (Blocker)
in the reaction and using normal rabbit IgG (IgG) to replace
Cdk4 antibody (lower panel).
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Induced p21 Binds to Cyclin-Cdk Complexes--
p21 has previously
been shown to bind and inhibit the activities of cyclin-cdk complexes.
To determine if elevated p21 in high IGFII expressing cells is
associated with the cyclin-cdk complexes, we immunoprecipitated Cdk2,
Cdc2, and Cdk4 from cell extracts that did not receive
-irradiation
or 24 h after
-irradiation. We then examined the p21 protein
levels in the cdk-containing complexes. Our results demonstrate that
more p21 is associated with cdk complexes in IGFII-overexpressing
C2C12-2.7 cells both under basal conditions and following
-irradiation when compared with C2C12-1.1 control cells (Fig.
7A). However, the relative increase of p21 protein after irradiation in C2C12-1.1 cells is higher
than in IGFII-overexpressing C2C12-2.7 cells. We have performed a
Western blot of immunoprecipitated cdks used in the above experiments. All Cdk2, Cdc2, and Cdk4 protein levels did not change (data not shown)
as had been previously seen in Fig. 3B. However, because this assay reflects both free and p21-associated cdks, we next sought
to compare the levels of cdks in the p21 complex. We immunoprecipitated p21 from cell extracts without or 24 h after
-irradiation. We then examined the cdk protein levels by Western blot. Our results demonstrate that more cdks are associated with p21 in C2C12-2.7 cells
than C2C12-1.1 cells under normal growth conditions (Fig. 7C, 0 Gray). After
-irradiation, the levels of cdks associated with
p21 were also higher in C2C12-2.7 cells especially following 10 Gray
treatment (Fig. 7B).

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|
Fig. 7.
Induced p21 binds to cdk complexes with or
without -irradiation. A,
lysates from cells treated with different doses (0, 7, 10 Gray) of -irradiation for 24 h were immunoprecipitated
(IP) with various cdk antibodies followed by Western blot
for p21 with monoclonal p21 antibody. B, after treatment
with different doses (0, 7, 10 Gray) of -irradiation for
24 h, cell lysates were immunoprecipitated (IP) with
p21 antibody followed by Western blot for cdk proteins with various cdk
antibodies.
|
|
 |
DISCUSSION |
We have shown that the IGFII mitogenic signaling pathway shortens
the G1 interval and appears to inhibit the block of the G1-to-S transition following DNA damage. IGFII
overexpression is associated with relatively high cdk kinase activity
after DNA damage which enables high IGFII expressing cells to quickly
override the G1 checkpoint. This short G1
checkpoint arrest may lead to the lack of appropriate repair of damaged
DNA, and subsequent propagation of genetic alterations may contribute
to genetic instability in tumor cells overexpressing IGFII.
Furthermore, the increased percentage of IGFII overexpressing cells in
S and G2/M phase indicates that the cells have a higher
proliferative rate.
We also found that cyclin D1 levels were increased in IGFII
overexpressing cells which indicates that cyclin D1 may be a downstream target of the IGFII signaling pathway. Since cyclin D1 is required and
is a rate-limiting factor for entry into S phase, the overexpression of
cyclin D1 may accelerate the G1-to-S transition in high
IGFII expressing cells (24-26). Many alterations in
G1-associated processes have been observed in cancer cells,
of which cyclin D1 overexpression is the most common (27-30).
Overexpression of cyclin D1 contributes to the oncogenic transformation
of cells in vitro and in vivo (31-34). Induction
of cyclin D expression is part of the mitogenic response of various
mesenchymal or epithelial cells to serum or growth factors such as
platelet-derived growth factor, epithelial growth factor, and
insulin-like growth factor I (35-38). All of these mitogens operate
via signaling cascades involving tyrosine kinase receptors and G
proteins at least in part through the Ras-Raf-MAPK pathway (14-16).
The role of D-type cyclins as potential sensors and integrators of
diverse mitogenic stimuli with the cell cycle machinery is also
confirmed by induction of cyclin D1 expression and G1 phase
acceleration by activated ras or raf oncogenes
themselves (39, 40).
Another significant finding of this study is that the p21 and p53
proteins are constitutively expressed in the high IGFII-expressing cell
line C2C12-2.7. The increase of p21 expression corresponds to an
increased level of p21 mRNA. Despite the elevated basal levels of
p53 and p21 protein, the proliferation rate of IGFII-overexpressing cells was higher compared with the control C2C12 cells. It has previously been reported that ectopic expression of cyclin D1 in
asynchronously growing cells was accompanied by increased levels of the
p53 tumor suppressor protein as well as the cyclin/cdk inhibitor p21
through an E2F transactivation mechanism (41, 42). The induction of p21
does not lead to growth arrest of cells but rather to stabilization of
cyclin D1/cdk function (41, 42). It is known that p21 does not always
function as an inhibitor of cyclin-cdk complexes. At low
concentrations, p21 promotes the assembly of stable and active Cdk4 and
D-type cyclin kinase complexes which allows the cells to continue to
enter into S phase, resulting in a transformed phenotype. At high
concentrations, such as following the stress of DNA damage, p21
inhibits kinase activity and cell growth (43-46). A proposed
explanation for this paradox is that cyclin D1-cdk complexes containing
a single p21 molecule are still catalytically active, whereas complexes
containing multiple p21 subunits are inactive (46, 47). We found that
following DNA damage, the relative increases in both p53 and p21 were
higher in C2C12-1.1 cells than the increases in IGFII overexpressing C2C12-2.7 cells, which may lead to the longer G1 checkpoint
arrest in C2C12-1.1 cells compared with C2C12-2.7 cells. Following
-irradiation, cdk activities are decreased in both cell lines.
However, the inhibition of cdks in high IGFII-expressing C2C12-2.7
cells is diminished. This may be explained by two factors: higher
cyclin D1 levels in IGFII-overexpressing C2C12-2.7 cells and a relative lower increase in p21 protein associated in all the cdk complexes after
radiation when compared with C2C12-1.1 cells. Thus the ratio of p21 to
cyclin D1-cdk complexes in IGFII-overexpressing C2C12-2.7 cells
following DNA damage is less than the ratio in C2C12-1.1 control
cells, which potentially leads to maintenance of stable, active cyclin
D1-cdk-p21 complexes in C2C12-2.7 cells following
-irradiation.
It has been widely reported that expression of p21 is induced in a
p53-dependent manner in response to DNA damage, resulting in G1/S arrest through inhibition of the cyclin-cdk
complexes (48, 49). On the other hand, p21 gene expression is also
induced through a p53-independent mechanism during differentiation of a
number of cell types, as well as in various murine tissues during development and in the adult animal (50-53). Moreover, it was reported that expression of p21 is elevated following stimulation of quiescent cells with serum or purified growth factors, and in proliferating cells
the majority of p21 protein is found in active cyclin-cdk complexes
(46, 52, 54). Induction of p21 in these situations does not appear to
require p53. In addition, ectopic expression of cyclin D1 correlated
with increased levels of p21 protein, which did not lead to growth
arrest (42). It has been reported that higher levels of p21 protein and
RNA are observed in several different human tumors compared with the
corresponding normal tissues and that the p21 is not mutated in these
tumors, although the p21 gene possesses a polymorphism at codon 31 (55). The elevated p21 expression is related to tumor differentiation
and is p53-independent, which is a common feature of in vivo
neoplasms (55). Taken together, our findings support the evidence that the stoichiometry of cyclin D1-cdk-p21 is critical in determining the
activity of this complex.
We also observed that MAPK activity was induced in high
IGFII-expressing C2C12-2.7 cells, which may be a result of the
activation of the IGFII signaling pathway. It has been reported that
the MAPK pathway signals, including Ras, Raf, MAPKK, and MAPK, can all
transcriptionally induce p21 expression in response to growth factors
such as epidermal growth factor, nerve growth factor, serum, and
12-O-tetradecanoylphorbol-13-acetate treatment (54). This
report is consistent with the findings that forced expression of either
v-Src or activated c-Raf in murine hematopoietic Baf-3 cells prevents
down-regulation of p21 expression that normally occurs in these cells
in response to growth factor withdrawal (56). In combination with other
results in our study, it could be suggested that IGFII may directly
activate mitogenic signaling pathways and drive cell cycle progression.
However, it still remains to be investigated whether increased MAPK
activity contributes to the elevated expression of cyclin D1, p21, and
p53 in IGFII-overexpressing C2C12-2.7 cells.
It has been shown that E2F-1 can strongly transactivate the human p21
gene through E2F-binding sites that are located in the p21 gene and
that the transactivation of the p21 gene is correlated with increased
levels of endogenous E2F-1 and p21 proteins at the G1/S
boundary (46). Curiously, we observed a lower molecular weight E2F-1
portion associated with the induced p21 protein levels seen in high
IGFII expression cells compared with control cells. However, the role
of E2F-1 in the regulation of p21 expression is not yet known. We did
not observe obvious differences of cyclin E protein between high
IGFII-expressing cells and control cells with or without DNA damage.
Although cyclin E is also rate-limiting for entry into S phase and may
contribute to phosphorylation of pRb, its peak abundance and its
associated Cdk2 appears to function at the G1/S transition
by phosphorylating some currently unknown substrate(s) other than pRb
(37, 45, 57, 58).
In conclusion, we found that IGFII overexpression in mammalian cells
causes a shortened cycling time and increases in cyclin D1, p21, and
p53 protein levels as well as increases in MAPK activity. However, the
mechanisms underlying the induction of these proteins by IGFII
overexpression and whether the increased levels of cyclin D1, p53, as
well as MAPK activity participate directly or indirectly in the p21
induction are still under investigation. Following DNA damage, IGFII
overexpression is associated with a diminished G1
checkpoint, which may contribute to the high growth rate and genetic
alterations during tumorigenesis.
 |
FOOTNOTES |
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
**
To whom correspondence should be addressed: Molecular Oncology
Section, Pediatric Oncology Branch, Bldg. 10, Rm. 13N240, NCI, National
Institutes of Health, Bethesda, MD 20892-1928. Tel.: 301-496-4257; Fax:
301-402-0575.
 |
ABBREVIATIONS |
The abbreviations used are:
IGFII, insulin-like
growth factor II;
RMS, rhabdomyosarcoma;
CHO, Chinese hamster ovary;
-MEM,
- minimum Eagle's medium;
Gy, gray;
MM, methylmethane
sulfonate;
MAPK, mitogen-activated protein kinase;
MAPKK, MAPK kinase;
PBS, phosphate-buffered saline;
cdk, cyclin-dependent
kinase;
Rb, retinoblastoma;
tet, tetracycline.
 |
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