From the Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania 19107
Received for publication, November 20, 2000, and in revised form, January 16, 2001
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ABSTRACT |
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The Id proteins play an important role in
proliferation, differentiation, and tumor development. We report here
that Id gene expression can be regulated by the insulin-like growth
factor I receptor (IGF-IR), a receptor that also participates in the regulation of cellular proliferation and differentiation. Specifically, we found that the IGF-IR activated by its ligand was a strong inducer
of Id2 gene expression in 32D murine hemopoietic cells. This activation
was not simply the result of cellular proliferation, as Id2 gene
expression was higher in 32D cells stimulated by IGF-I than in cells
exponentially growing in interleukin-3. The up-regulation of Id2 gene
expression was largely dependent on the presence of insulin receptor
substrate-1, a major substrate of the IGF-IR and a potent activator of
the phosphatidylinositol 3-kinase (PI3K) pathway. The role of
PI3K activity in the up-regulation of Id2 gene expression by the IGF-IR
was confirmed by different methods and in different cell types. In 32D
cells, the up-regulation of Id2 gene expression by the PI3K pathway
correlated with interleukin-3 independence and inhibition of differentiation.
The Id proteins are helix-loop-helix proteins that form
heterodimers with a large family of other helix-loop-helix proteins, mostly transcriptional activators, that have a basic region in addition
to the helix-loop-helix region (1). These heterodimers cannot bind to
DNA because the Id proteins lack a DNA-binding region; Id proteins
therefore negatively regulate the DNA-binding capacity of other basic
helix-loop-helix proteins (1). There are at least four Id proteins
encoded by individual genes, which, in humans, are located on different
chromosomes (2). Id gene expression is elevated in undifferentiated
cycling cells and tumor cell lines (3, 4), and high levels of Id gene
expression inhibit the differentiation of a variety of cell types (3, 5-7). Id gene expression is also cell cycle-regulated and has been
implicated in playing a major role in the G1-to-S
transition (2, 8, 9). The Id1 protein has been reported to inhibit mammary cell differentiation (6), to promote mammary epithelial cell
invasion (10), and to increase the aggressive phenotype of human breast
cancer cells (11). A connection between N-Myc, the retinoblastoma
protein, and Id2 has been recently established in neuroblastoma cells
(9). Id proteins are also required for angiogenesis and vascularization
of tumor xenografts (12).
Id gene expression is up-regulated by serum (8, 9), by platelet-derived
growth factor (13), and by some cytokines that induce cellular
differentiation (14, 15). The dual role of Id proteins in proliferation
and differentiation has prompted us to examine their regulation by the
insulin-like growth factor I receptor
(IGF-IR),1 which is also
involved in the proliferation and differentiation of cells. We have
chosen for these studies the 32D murine myeloid cell line (16), in
which induction of differentiation requires a short but intense period
of cell proliferation, as it happens for other hemopoietic cells
induced to differentiate (16-18). This dual response has been
interpreted as indicating that differentiating growth factors send two
simultaneous signals, one for proliferation and one for
differentiation, with the latter eventually prevailing.
The IGF-IR offers an ideal paradigm for studying how growth factors
regulate the balance between cell proliferation and cell differentiation. In many cell types (mouse embryo fibroblasts like 3T3
cells, human diploid fibroblasts, some epithelial cells, etc.), the
IGF-IR sends an unambiguous mitogenic signal (19). However, in other
cell types, IGF-I and IGF-II can stimulate either proliferation or
differentiation, or both (20). These contradictory signals of the
IGF-IR have been studied to advantage in 32D cells, a cell line that is
interleukin-3 (IL-3)-dependent for growth (16). In the
absence of IL-3, 32D cells undergo apoptosis (21). The expression of a
human IGF-IR in 32D cells (32D IGF-IR cells) prevents apoptosis caused
by IL-3 withdrawal (22, 23). In fact, 32D IGF-IR cells are stimulated
to grow exponentially by IGF-I for ~48 h (22-24). Then, the cells
stop growing and begin to differentiate along the granulocytic pathway
(25). Thus, 32D IGF-IR cells recapitulate the program induced in
hemopoietic cells by other differentiating growth factors (see above).
A characteristic of 32D cells is that they do not express insulin
receptor substrate-1 (IRS-1) and IRS-2 (25, 26). The IRS proteins are
major substrates of both the IGF-IR and the insulin receptor and play
an important role in the mitogenic signaling of both receptors (27).
Ectopic expression of IRS-1 in 32D IGF-IR cells (32D IGF-IR/IRS-1
cells) causes inhibition of differentiation (25). In fact, 32D
IGF-IR/IRS-1 cells become permanently IL-3-independent and form tumors
in mice (24).
We have taken advantage of this dual signaling from the IGF-IR
(differentiation in the absence of IRS-1 and continuous proliferation in its presence) to investigate the signal transduction pathways leading to Id gene expression, specifically Id1 and Id2. We found that
the IGF-IR can induce the expression of Id2 mRNA and proteins, especially when IRS-1 is present. Since IRS-1 is a strong activator of
PI3K (27, 28), we investigated the role of PI3K in the expression of
Id2 mRNA. Using inhibitors of PI3K and a mutant IRS-1 that fails to
activate PI3K, we show that, in 32D IGF-IR cells, PI3K activation plays
an important role in the up-regulation of Id2 gene expression. We have
confirmed these findings in two different cell types, 293T and LNCaP
cells. With 293T cells (29), we transiently transfected a
constitutively active p110 Plasmids and Retroviral Transduction--
A truncated
mouse IRS-1 was generated that encodes the first 309 amino acid
residues and comprises the pleckstrin homology (PH) domain and the
phosphotyrosine-binding (PTB) domain. For its construction, wild-type
IRS-1 was used as a template for PCR amplification. The sequence of
the sense primer was
5'-ATACCGTTACACaagcttGGCGCAGTTACCTCGTCCTTCGG-3' (positions
301-327 of the mouse IRS-1 genome (GenBankTM/EBI accession
number X69722); the sequence of the HindIII restriction site
is indicated in lowercase letters, and the overhang sequence of the
primer is underlined). The sequence of the antisense primer was
5'-TTCATAGCATTTCGTCATTATaagcttTTTCCCACCCACCATTCAGGCAGG-3' (positions 1418 to 1394 of the mouse IRS-1 genome (accession number X69722); the sequence of the HindIII restriction site is
indicated in lowercase letters, and the overhang sequence of the primer is underlined). The PCR conditions were as follows: a denaturation step
of the template at 94 °C for 1 min, followed by a 1-min interval at
52 °C to allow for the annealing of the primers to the template and
a 3-min incubation at 72 °C for polymerase elongation of the primers. This cycle was repeated 35 times. At the end of these 35 cycles, an additional incubation at 72 °C for 15 min was included to
allow completion of the amplification. The PCR product was electrophoresed on a 1% agarose gel. The correct size product was
excised from the gel and purified with a gel extraction kit (QIAGEN
Inc.) following the manufacturer's recommendations. The end PCR
product was in 50 µl of water and was digested with the HindIII restriction enzyme. The HindIII-digested
PCR product was gel-purified again and ligated into the
HindIII cloning site of a murine stem cell virus
based retroviral vector carrying the puromycin resistance gene. The
HindIII PCR fragment was sequenced to monitor the mutation.
For details of the retroviral vector, see Romano et al.
(32).
The PI3K constructs were hemagglutinin-tagged and included
wild-type p110 Cell Lines--
32D IGF-IR cells were derived from the 32D
murine hematopoietic cell line clone 3 (16), stably transfected with a
plasmid expressing the human IGF-IR cDNA (25). 32D IGF-IR/IRS-1
cells are 32D IGF-IR cells expressing wild-type IRS-1 (25), and 32D IGF-IR/PH/PTB cells are 32D IGF-IR cells expressing a truncated form of
IRS-1 under the control of the cytomegalovirus promoter. 293T cells
were originally obtained from the laboratory of David Baltimore
(29). LNCaP, LNCaP/PTEN, and LNCaP/PTEN/IRS-1 cells have been
described by Reiss et al. (31). LNCaP/PTEN/IGF-IR cells were
generated by transducing LNCaP/PTEN cells with a retrovirus expressing
the human IGF-IR cDNA. Selection was carried out with 2 µg/ml
puromycin and 500 µg/ml G418 (both from Life Technologies, Inc.). All
these cell lines are mixed populations.
32D cells were grown in RPMI 1640 medium supplemented with 10%
heat-inactivated fetal bovine serum (Life Technologies, Inc.), 10%
WEHI cell-conditioned medium (as a source of IL-3), 2 mM
L-glutamine (Life Technologies, Inc.), and the required
antibiotic to maintain the selective pressure (250 µg/ml G418 for 32D
IGF-IR cells, 250 µg/ml G418 plus 250 µg/ml hygromycin (Calbiochem)
for 32D IGF-IR/IRS-1 cells, or 250 µg/ml of G418 plus 1 µg/ml
puromycin for 32D IGF-IR/PH/PTB cells). For brevity, the WEHI
cell-conditioned medium will be referred to as IL-3. In the experiments
for Id1 or Id2 detection, exponentially growing cells (in
complete medium with 10% fetal bovine serum and IL-3) were
washed in Hank's balanced solution to remove IL-3 and incubated
for the indicated times in medium with 10% fetal bovine serum plus 50 µg/ml IGF-I.
LNCaP cells were cultured in RPMI 1640 medium supplemented with 10%
fetal bovine serum. In the experiment for Id2 detection, cells
were serum-starved in RPMI 1640 medium and 0.1% bovine serum albumin
(Sigma) for 48 h and then 50 ng/ml IGF-I was added for the
indicated times.
293T cells were grown in Dulbecco's modified Eagle's medium and 10%
fetal bovine serum. For the transient expression experiments, 293T
cells (29) were transfected with the PI3K constructs (see above) using
the FuGENE transfection reagent (Roche Molecular Biochemicals)
according to the manufacturer's instructions. After 48 h, the
cells were harvested, and lysates were prepared as previously described
(25).
Northern Blots--
For the detection of the Id1 and Id2
mRNAs, exponentially growing cells were washed three times and
seeded in IL-3-free medium (RPMI 1640 medium containing only 10%
heat-inactivated fetal bovine serum and L-glutamine), and
then IGF-I (50 ng/ml) was added. Cells incubated with IL-3 served as
controls. In some experiments, an inhibitor was added (30 µM LY294002; BIOMOL Research Labs Inc.) for 15 min prior
to the stimulation with IGF-I. At the indicated time points, cells were
collected, and total RNA was extracted using the RNeasy kit (QIAGEN
Inc.) following the manufacturer's instructions. Northern blotting was
carried out by standard techniques. The labeled probes used were a
250-base pair PCR product for Id1 (spanning bases 259-530) and the
full-length cDNA for Id2.
Western Blots--
For the detection of the truncated form of
IRS-1 (the PH/PTB mutant), an anti-N-terminal IRS-1 antibody (A-19,
sc560, Santa Cruz Biotechnology) was used at 1:500 dilution in
Tris-buffered saline containing 0.1% Tween and 5% milk
(TBS-T/5% milk). The antibody for the Id2 protein (C-20, sc489,
Santa Cruz Biotechnology) was diluted 1:500 in TBS-T/5% milk. The
anti-hemagglutinin antibody was a mouse monoclonal antibody from
BABCO (MMS-101P) used at a dilution of 1:500 in TBS-T/5% milk.
The anti-PTEN antibody (sc7974, Santa Cruz Biotechnology) was also used
at 1:500 in TBS-T/5% milk. Western blotting was carried out by
standard techniques as previously described (22, 25). The anti-Grb2
antibody was a mouse monoclonal antibody from Transduction Laboratories
and was used at 1:1000 in TBS-T/5% milk.
PI3K Activity Assay--
Exponentially growing cells were
collected, washed three times, and starved for 5 h in serum-free
medium (RPMI 1640 medium with 0.1% bovine serum albumin and
L-glutamine). After starvation, cells were stimulated with
50 ng/ml IGF-I for 5 min and lysed. One milligram of proteins was then
immunoprecipitated with an anti-Tyr(P) monoclonal antibody (PY20,
Transduction Laboratories) overnight at 4 °C, and the associated
PI3K activity was assessed by incubating the immunoprecipitate with
phosphatidylinositol and [ Growth and Differentiation Analyses--
These analyses were
carried out by routine methods, as already described in detail for 32D
cells in previous works from this laboratory (23-25). Cell growth was
also assessed as the fraction of cells in S phase by
5-bromo-2'-deoxyuridine (BrdUrd) incorporation using the BrdUrd
labeling kit (Roche Molecular Biochemicals). Exponentially growing
cells were collected, washed three times, and resuspended in IL-3-free
medium or supplemented with 50 ng/ml IGF-I or 10%
IL-3-conditioned medium. Cells were seeded at a density of
5 × 104 cells/ml on 6-well multiwell plates. At the
indicated time points, 10 µM BrdUrd was added to the
medium for 1 h before collecting the cells. Cytospins were
performed, and slides were fixed for 20 min in 3% paraformaldehyde,
permeabilized for 5 min in 0.2% Triton, treated for 5 min with 1.5 N HCl, and subsequently incubated for 30 min at 37 °C
with the primary anti-BrdUrd monoclonal antibody and for 30 min at room
temperature with the secondary Texas Red-conjugated anti-mouse
antibody. Hoechst 33258 (Sigma) staining of the nucleus was performed
for 5 min at room temperature before applying Vectashield mounting
medium (Vector Laboratories, Inc., Burlingame, CA). The BrdUrd
incorporation index was determined using a Zeiss microscope working in
epifluorescence mode (magnification × 500). In randomly selected
fields, at least 300 cells/slide were counted.
An intriguing aspect of these experiments is that in the first
48 h after IL-3 withdrawal followed by IGF-I stimulation, 32D IGF-IR cells (eventually undergoing differentiation) and 32D
IGF-IR/IRS-1 cells (transformed) could not be distinguished from each
other in terms of cell proliferation. Both cell lines double in number in each 24-h period (22-24) (see below), although their fates diverge in opposite directions soon after. Furthermore, all 32D-derived cell
lines, even those expressing a disabled IGF-IR grow exponentially in
IL-3 (22, 23, 25). Therefore, any change in Id gene expression in the
first 24 h after shifting from IL-3 to IGF-I would not be simply a
reflection of the proliferative status of the cells. We have
investigated Id mRNA expression in 32D IGF-IR cells and 32D
IGF-IR/IRS-1 cells during this period, limiting ourselves to Id1 and
Id2 gene expression because Id3 and Id4 are not expressed in parental
32D cells (33).
IGF-I Stimulates an Increase in Id2 mRNA Levels--
Fig.
1A shows Id2 mRNA levels
in Northern blots from 32D IGF-IR cells and 32D IGF-IR/IRS-1 cells. Id2
RNA was barely detectable in 32D IGF-IR/IRS-1 cells growing in
IL-3, and its levels increased sharply when the cells were shifted from
IL-3 (Time 0 lane) to IGF-I, with a peak at 6 h.
Significantly, the Id2 RNA levels were lower in IL-3. Even at 24 h, Id2 RNA levels were higher in cells incubated in IGF-I than in cells
incubated in IL-3, although the cells were exponentially growing in
both conditions. The levels of Id2 RNA were much lower in 32D IGF-IR
cells, already indicating that IRS-1 plays a crucial role in
determining Id2 RNA levels after IGF-I stimulation. However, a very
modest but reproducible increase in Id2 RNA could also be observed in
32D IGF-IR cells (see also below). These experiments were repeated
several times by different operators, with similar results.
The results with Id1 mRNA (Fig. 1A, middle
panel) were different. In fact, it was difficult to see a clear
difference in Id1 RNA levels between 32D IGF-IR cells and 32D
IGF-IR/IRS-1 cells. There was perhaps a peak in Id1 RNA levels in the
latter cells at 6 h after IGF-I stimulation, but the effect was
modest. In addition, Id1 RNA levels were already easily detectable in
cells in IL-3, and shifting to IGF-I did not seem to increase them. The
results indicate that regulation of Id1 and Id2 RNAs differs in
32D-derived cells. In the following experiments, we focused mostly
on Id2 gene expression.
To demonstrate that the IGF-IR can indeed up-regulate Id2 gene
expression, we compared Id2 RNA levels in 32 IGF-IR/IRS-1 cells in 10%
serum and in 10% serum supplemented either with IL-3 or IGF-I for
6 h (Fig. 1B). Only the addition of IGF-I caused a
convincing increase in Id2 RNA levels (third lane). The
experiments in Fig. 1 (A and B) are a clear
indication that Id2 gene expression is specifically up-regulated by
IGF-I. In Fig. 1C, we show that Id2 protein levels also
increased in 32D IGF-IR/IRS-1 cells. Id2 protein (~14 kDa) amounts
increased by 6 h after shifting the cells from IL-3 to IGF-I, and
although they eventually decreased, they were still high at 72 h.
Id2 protein levels were much less elevated in 32D IGF-IR cells,
although they were slightly higher in IGF-I than in IL-3. These
experiments conclusively demonstrate the ability of the IGF-IR,
activated by its ligand, to up-regulate the expression of Id2 RNA and
protein, especially in the presence of its substrate IRS-I. In
subsequent experiments with 32D cells, we focused on RNA levels to
emphasize the fact that changes in Id2 gene expression occur at a time
when all cell lines examined are exponentially growing.
PI3K Is Required for Strong Induction of Id2
mRNA--
Wild-type IRS-1 is known to send a powerful
mitogenic stimulus through the PI3K, Akt/protein kinase B, and
p70S6K pathway (28). Since parental 32D and 32D
IGF-IR cells do not express IRS-1 (25, 26), the results of Fig. 1
suggest that PI3K activity may be crucial for the up-regulation of Id2
RNA in 32D-derived cells. As a first approach, we determined the levels of PI3K activity in the two cell lines. As expected, PI3K activity, after IGF-I stimulation, was substantially higher in 32D IGF-IR/IRS-1 cells than in 32D IGF-IR cells (Fig.
2A). There may be a slight increase in the latter cell line after IGF-I stimulation. This slight
increase is compatible with our previous observation that IGF-I causes
a modest but reproducible increase in Akt activation even in 32D IGF-IR
cells (25). We then compared Id2 RNA levels in 32D IGF-IR/IRS-1 cells
untreated or treated with an inhibitor of PI3K, LY294002 (30 µM). The results are shown in Fig. 2B. The addition of LY294002 markedly inhibited the increase in Id2 mRNA levels caused by IGF-I.
Id2 RNA Levels Are Not Increased in 32D IGF-IR Cells Expressing a
Mutant IRS-1--
To confirm a role of PI3K (and IRS-1) in the
regulation of Id2 RNA levels, we generated a third cell line in which
we expressed the PH/PTB mutant of IRS-1 in 32D IGF-IR cells. PH/PTB
mutant IRS-1 has only the PH and PTB domains and lacks the binding
domains for PI3K.This truncated IRS-1 is only 309 amino acids long and can be detected only with an antibody to the amino terminus of IRS-1
(see "Experimental Procedures"). It has been reported that the
PH/PTB mutant does not activate PI3K (26). The effects of this mutant
IRS-1 on Id expression are shown in Fig.
3A. For Id2 RNA, we compared
32D IGF-R/IRS-1 cells with 32D IGF-IR/PH/PTB cells. The mutant IRS-1
failed to elicit a strong induction of Id2 RNA (Fig. 3A,
upper panel). Very little differences were noted when Id1
RNA levels were examined in the same cells, again indicating that Id1
RNA levels, in this model, are regulated by different pathways than Id2
RNA levels. Fig. 3B shows that the PH/PTB mutant was well
expressed in two separate mixed populations derived from 32D IGF-IR
cells. In 32D IGF-IR/PH/PTB cells, both PI3K activity and Akt/protein
kinase B phosphorylation were the same as in 32D IGF-IR cells
(data not shown). The results therefore confirm that PI3K plays a major
role in IGF-I-mediated up-regulation of Id2 gene expression.
Growth and Differentiation of 32D IGF-IR/PH/PTB Cells--
The
effect of IGF-I on the growth and differentiation response of 32D
IGF-IR/PH/PTB cells has never been reported. Yenush et al.
(26) have noticed a modest but reproducible proliferative stimulus
originating from the PH/PTB domain of IRS-1 in 32D cells overexpressing
the insulin receptor. We investigated the ability of the PH/PTB mutant
of IRS-1 to send proliferative and/or differentiation signals in the
context of the 32D cells expressing the IGF-IR. Fig.
4 (A and B)
compares the growth and differentiation of the three cell lines: 32D
IGF-IR, 32D IGF-IR/IRS-1, and 32D IGF-IR/PH/PTB. As already reported,
32D IGF-IR cells (black bars) grew for a period of time
(A), but then began to differentiate (B), and the number of cells no longer increased, whereas 32D IGF-IR/IRS-1 cells
(hatched bars) continued to grow. The percentage of
differentiating cells reached 40% by day 6 in 32D IGF-IR cells,
whereas it remained at background levels in 32D IGF-IR/IRS-1 cells,
confirming the results of Valentinis et al. (25). 32D
IGF-IR/PH/PTB cells (stippled bars) fell somewhere in
between in terms of cell proliferation, which was reproducibly better
than that of 32D IGF-IR cells, but not as brisk as that of 32D
IGF-IR/IRS-1 cells. The 32D IGF-IR/PH/PTB cells differentiated as well
as the parental 32D IGF-IR cells. It seems therefore that 32D
IGF-IR/PH/PTB cells have an increased stimulus for cell proliferation,
but that the differentiation program is not extinguished. Therefore, in
this model, PI3K modulates not only the expression of the Id2 genes,
but also the differentiation program. In the absence or with a marked
reduction of PI3K activity, 32D IGF-IR/PH/PTB cells differentiate, even
in the presence of a mild proliferative stimulus.
We have looked at the incorporation of BrdUrd into these three
different cell lines. Table I shows the
results of a 1-h pulse labeling with BrdUrd at various times after IL-3
withdrawal and IGF-I supplementation. 32D IGF-IR/PH/PTB cells
incorporated BrdUrd at a percentage that was substantially higher than
32D IGF-IR cells, although not as high as the percentage obtained with
32D IGF-IR/IRS-1 cells. For completeness, it should be added that all
three cell lines, as usual, grow equally well in IL-3, and all undergo
apoptosis if the IL-3-depleted medium is not supplemented with
IGF-I (22, 24, 25).
Effect of PI3K and PTEN on Id2 Gene Expression--
To confirm the
role of the IGF-IR and the importance of the PI3K pathway in the
regulation of Id2 expression, we investigated the regulation of Id2
gene expression in two other cell lines and by two different methods.
In one series of experiments, we studied Id2 gene expression in 293T
cells (29) transiently transfected with wild-type p110
For a third cell type, we chose LNCaP cells, a human prostatic cancer
cell line with a frameshift mutation of PTEN, a tumor suppressor gene
(also called MMAC1 or TEP1) identified on human chromosome 10q23 (34, 35). PTEN is a phosphatase (36, 37) that
regulates the activity of PI3K (38-41) and blocks cells in the
G1 phase of the cell cycle (42, 43). Accordingly, Akt is
constitutively activated in LNCaP cells (31, 39, 40, 44, 45) because of
the PTEN mutation.
Fig. 5D shows the levels of expression of the Id2 protein in
four different cell lines, three of which have been previously described (31): parental LNCaP, LNCaP/PTEN, and LNCaP/PTEN/IRS-1 cells.
A fourth cell line was generated by introducing a retrovirus expressing
the IGF-IR into LNCaP/PTEN cells (see "Experimental Procedures").
All these cell lines are mixed populations and were tested for
IGF-I-mediated induction of Id2 gene expression. The cells were made
quiescent by serum deprivation for 48 h and then stimulated with
IGF-I (50 ng/ml). Fig. 5D is a Western blot for Id2 protein
at various times after IGF-I stimulation. The Id2 protein was
up-regulated in parental LNCaP cells by IGF-I. The presence of PTEN
abrogated the response. In LNCaP/PTEN/IRS-1 cells, a slight increase
(over LNCaP/PTEN cells) was detectable, as if IRS-1 were restoring the
PI3K modulation of Id2. This increase, albeit extremely modest, was
reproducible. We have no explanation at the present moment for the
constitutive expression of Id2 in these latter cells, even before IGF-I stimulation.
Fig. 5E shows the expression of PTEN in each cell line. PTEN
is expressed strongly in all mixed populations, except the parental LNCaP cells, where PTEN is not detectable (31, 34, 35). Equal loading
was monitored by standard methods (data not shown).
The novel findings of this study are summarized and discussed
below. 1) Id2 gene expression is up-regulated by an activated IGF-IR
both in hemopoietic cells and in other cell types. 2) In 32D cells,
induction of Id2 gene expression is largely dependent on the presence
of IRS-1, a major substrate of the IGF-IR. 3) Activation of the PI3K
pathway (which is strongly promoted by IRS-1) is apparently the main
pathway for the up-regulation of Id2 gene expression. 4) Transient
expression of the wild-type or constitutively active PI3K subunit
induces an increase in Id2 protein expression. 5) In LNCaP prostate
cancer cells, the important role of the PI3K pathway was confirmed by
the inhibition of Id2 gene expression by PTEN, an inhibitor of PI3K. 6)
Up-regulation of Id2 gene expression by IGF-I is not simply due to the
proliferative status of the cells, as Id2 RNA and protein levels
increase when 32D cells are shifted from IL-3 to IGF-I. 7) The
modulation of Id2 gene expression by the IRS-1/PI3K pathway correlates
with the inhibition of IGF-I-mediated differentiation of 32D IGF-IR cells. A secondary finding is the failure of IGF-I to induce
significant changes in Id1 expression.
Although there is a substantial literature on the role of Id proteins
in proliferation and differentiation (see the Introduction), information on the signaling pathways regulating Id gene expression is
limited. Serum, nerve growth factor, platelet-derived growth factor,
and some cytokines have been mentioned as regulators of Id gene
expression, but no information on the signal transducing pathways
leading to Id gene expression has been forthcoming. We show in this
study that activation of the IGF-IR is a signal for up-regulation of
Id2 gene expression. Id2 RNA levels actually increase when 32D
IGF-IR/IRS-1 cells are shifted from IL-3- to IGF-I-supplemented medium.
In a direct comparison, IGF-I induces Id2 RNA, whereas IL-3 fails to do
so. At least in these cells, IGF-I is a more potent activator of Id2
gene expression than IL-3. This is significant, as during the period of
observation, these cell lines (whether or not expressing IRS-1) are
growing exponentially both in IL-3 and in IGF-I. It follows that the
induction of Id2 gene expression by the IGF-IR is not simply due to the
stimulation of cell proliferation and indicates a strong relationship
between the IGF axis and Id2 gene expression. The increase in RNA
levels is mirrored by an increase in the levels of Id2 proteins.
Interestingly, Id2 protein levels (and Id2 RNA levels) are also
slightly increased in 32D IGF-IR cells after shifting from IL-3 to
IGF-I, suggesting that the IGF-IR can activate Id2 gene expression even
in the absence of IRS-1. This effect is, however, in no way comparable
to the effect obtained by the activated IGF-IR in the presence of
IRS-1.
Since the presence of IRS-1 is a determinant factor in the
up-regulation of Id2 RNA by IGF-I, we have naturally focused our attention on the PI3K pathway. IRS-1 is a potent activator of the PI3K
pathway (27, 28) and has binding sites for the p85 subunit of PI3K (26,
46). The literature on IRS-1 and PI3K is abundant both for the IGF-IR
and the insulin receptors (27, 47), and we have confirmed the
importance of IRS-1 in PI3K activation in this study. More important,
we have shown clearly that PI3K is required in 32D cells for strong
up-regulation of Id2 gene expression. Using inhibitors of PI3K and a
mutant IRS-1 defective in the activation of PI3K, we have shown that
up-regulation of Id2 gene expression is markedly inhibited in the
absence of a PI3K signal (or a diminished signal, as in 32D IGF-IR
cells). Interestingly, in 32D cells, PI3K is crucial not only for
up-regulation of Id2 gene expression, but also for inhibition of
IGF-I-mediated differentiation of these cells (24). The PH/PTB mutant
of IRS-1 has a modest proliferative effect in parental 32D IGF-IR
cells, confirming similar results obtained by Yenush et al.
(26) with the insulin receptor. However, the PH/PTB mutant is totally
incapable of extinguishing the differentiation program initiated by the IGF-IR.
The attractive aspect of the 32D model is that, as already emphasized,
all three cell lines under consideration grow exponentially in the
first 48 h after shifting from IL-3 to IGF-I. In IL-3, of course,
all 32D-derived cells grow indefinitely. Thus, the up-regulation of Id2
RNA (already evident 3-6 h after shifting the cells to IGF-I) is
independent of the actual proliferative status of the cells, but it is
indicative of their fate. Our results establish a connection between Id
gene expression and the PI3K pathway. To confirm the importance of the
PI3K pathway in the up-regulation of Id2 gene expression, we have
studied it by different methods in other cell types. We have used a
constitutively active p110 The sequence of events between PI3K activation and up-regulation of Id
gene expression is a very complex question and remains to be fully
elucidated. However, the first events after PI3K activation are well
established, and they include the activation of Akt/protein kinase B
and p70S6K/p85 and the activation of a genetic program for
a number of genes (50). This pathway has also been repeatedly
documented in 32D-derived cells (22-24). Activation of the PI3K
pathway also results in increased expression of c-Myc (51, 52), and
c-Myc expression is induced by IGF-I (53). In fact, the levels of c-Myc
induction by IGF-I are proportional to the number of IGF-IRs/cell (54).
It is therefore of great interest that Lasorella et al. (9)
have recently shown that Id2 is a direct target of N-Myc in
neuroblastoma cells. A connection between N-Myc and the IGF-IR in
neuroblastoma cells has also been reported by Chambery et
al. (55).
The two Id RNAs are somewhat different in their behavior, but this is
not surprising. Differences in expression of Id1 and Id 2 RNAs during
development and differentiation have already been reported (33, 56,
57). Our experiments confirm that there are differences in the
regulation of Id1 and Id2 gene expression. Id2 RNA is clearly regulated
by the IGF-IR through the PI3K pathway, whereas the results with Id1
are inconclusive. As mentioned in the Introduction, Id3 and Id4 are not
expressed in 32D cells (33).
The implications of these findings are worth considering. The Id
proteins have recently been proposed to play a role in malignant transformation and tumor aggressiveness (6, 9-11). In addition, Id2
has been reported to inhibit differentiation and to enhance cellular
proliferation by associating with and inactivating pRb (9, 58). The
activation of the PI3K pathway has also been often implicated in
transformation (59, 60), indeed directly in this model system, where
the expression of IRS-1 makes the difference between terminal
differentiation and transformation (tumor formation in mice) (24).
Considering that parental 32D IGF-IR cells cannot form tumors in mice,
although they grow very well in IL-3, one is tempted to speculate that
Id2 expression may be more important for transformation than for mitogenesis.
There is therefore a reasonable explanation for our results, suggesting
an attractive hypothesis. The IGF-IR, through IRS-1, activates the PI3K
pathway (27), which results in Myc induction (51-54), which induces
Id2 gene expression (9). Preliminary experiments from our laboratory
have indicated that up-regulation of Id2 gene expression in 32D-derived
cell lines is accompanied by the induction of c-Myc
RNA.2 In LNCaP cells,
the PI3K pathway is already constitutively activated by the PTEN
mutation, and IRS-1 is dispensable.
In conclusion, we have shown that IGF-IR signaling can up-regulate Id2
gene expression. The up-regulation is, however, markedly increased by
the presence of one of its major substrates (IRS-1) and, more
specifically, by the activation of the PI3K pathway. The up-regulation
of Id2 gene expression correlates with the extinction of the
IGF-I-mediated differentiation program of 32D IGF-IR cells and their
malignant transformation. Clearly, Id gene expression is activated by
other growth factors (see the Introduction), and we expect that other
signaling pathways will be found leading to up-regulation (or
down-regulation) of Id gene expression. For the moment, we can say that
the IGF-IR is certainly one of the receptors that can modulate Id2 gene
expression and that PI3K is at least one of the pathways leading to it.
These results may further open the way to an investigation of the
growth factors and the signaling pathways that regulate the expression
of Id protein in proliferation, differentiation, and tumor development.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunit of PI3K (30), which caused
an increase in Id2 expression. LNCaP cells are human prostatic cancer
cells (31) with a frameshift mutation of PTEN, an inhibitor of the PI3K
pathway (see below). Re-expression of PTEN inhibits IGF-I-mediated
up-regulation of Id2 proteins. Our results clearly show that the IGF-IR
activated by its ligand is capable of inducing Id2 gene expression
through the IRS-1/PI3K signaling pathway. Although other pathways
probably also lead to Id2 gene expression, this is, to our knowledge,
the first signal transduction pathway to be identified. Significantly, these findings also offer an explanation for the dual signaling of the
IGF-IR in these cells.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(pLHA110) and constitutively active p110
CAAX (pLHACAAX) and were a kind gift of
Christian Sell. The p110
sequences are under the control of the
cytomegalovirus promoter, and the constructs are described by
Didichenko et al. (30).
-32P]ATP for 10 min at
37 °C. The products of the kinase reaction were then visualized by
autoradiography and quantified by the radioactivity of the excised
bands in a scintillation counter.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
View larger version (29K):
[in a new window]
Fig. 1.
Up-regulation of Id2 RNA and proteins by the
IGF-IR. The 32D-IGF-IR and 32D IGF-IR/IRS-1 cell lines were
examined in the first few hours after shifting from IL-3 to IGF-I. At
this point, both cell lines were growing exponentially, but 32D IGF-IR
cells were fated to stop proliferating and to begin differentiation
into granulocytes. 32D IGF-IR/IRS-1 cells instead grew indefinitely in
the absence of IL-3 and in the presence of IGF-I. A,
Northern blot of Id1 and Id2 mRNAs at the times indicated after
shifting to IGF-I. B, Id2 RNA levels in 32D IGF-IR/IRS-1
cells at 6 h after incubation in 10% fetal bovine serum
(FBS; first lane), IL-3 (second lane),
or IGF-I (third lane). Levels of ribosomal RNA were used to
monitor the amounts of RNA in each lane for all the experiments.
C, levels of Id2 protein in 32D IGF-IR and 32D IGF-IR/IRS-1
cells at the indicated times after shifting from IL-3 to IGF-I. Levels
of the Grb2 protein were used to monitor the loading in each
lane.
View larger version (61K):
[in a new window]
Fig. 2.
Role of PI3K in the induction of Id2 mRNA
in 32D IGF-IR/IRS-1 cells. A, PI3K activity in
32D-derived cell lines. The cell lines were starved and then stimulated
with IGF-I (50 ng/ml) for 5 min; after which the cells were collected,
and PI3K activity was determined as described under "Experimental
Procedures." B, effect of an inhibitor of PI3K (30 µM LY294002) on Id2 mRNA levels. The inhibitor was
added 15 min before shifting the cells to IGF-I. Treatment and times
are indicated. PIP, phosphatidylinositol phosphate;
Ori, origin; DMSO, dimethyl sulfoxide.
View larger version (45K):
[in a new window]
Fig. 3.
Mutant IRS-1 devoid of PI3K-binding domains
fails to induce Id RNA expression in 32D IGF-IR cells. The
experiments are essentially the same as those described in the legend
to Fig. 1, except that 32D IGF-IR/IRS-1 cells and 32D IGF-IR cells
expressing the PH/PTB mutant of IRS-1 are compared. A,
results with Id1 and Id2 RNAs; B, expression of PH/PTB
mutant IRS-1 (Western blot) in two separate mixed populations of
transduced 32D IGF-IR cells.
View larger version (28K):
[in a new window]
Fig. 4.
Growth and differentiation of 32D-derived
cell lines. The three cell lines examined were 32D IGF-IR, 32D
IGF-IR/IRS-1 (expressing wild-type IRS-1), and 32D IGF-IR/PH/PTB
(expressing a mutant form of IRS-1). A, growth of the three
cell lines as measured by the number of cells at the indicated times
after shifting from IL-3 to IGF-I; B, percentage of
differentiating cells at the same times. The determinations of cell
number and percentage of differentiating cells are given under
"Experimental Procedures."
BrdUrd incorporation
or a
constitutively active mutant of the p110
subunit of PI3K (30).
Levels of Id2 proteins were determined 48 h after transfection,
and the results are shown in Fig.
5A. Both the wild-type and
constitutively active p110
subunits caused an increase in Id2
protein levels (second and third lanes). Fig. 5B shows the levels of expression of p110
(with a
hemagglutinin tag) in the transfected cells. Equal loading was
monitored by anti-Grb2 hybridization (Fig. 5C). The
experiments were also repeated with two other constructs, wild-type
p110
and constitutively active p110
tagged with a Myc sequence.
The results were essentially the same, except that the wild-type
construct, in this case, did not produce an increase in Id2 protein
(data not shown).
View larger version (21K):
[in a new window]
Fig. 5.
IGF-I-mediated regulation of Id2 proteins in
293T and LNCaP cells. 293T cells were transiently transfected with
the wild-type or constitutively active p110 subunit of PI3K (see
"Experimental Procedures"). Levels of Id proteins were determined
48 h after transfection and incubation in serum-supplemented
medium. In A, lysates from 293T cells that were
mock-transfected (first lane), transfected with wild-type
(wt) p110
(second lane), or transfected with
constitutively active p110
(third lane) are shown. The
expression of p110
(using an antibody to the hemagglutinin
(Ha) tag) is shown in B. C shows the
levels of Id2 proteins in LNCaP and LNCaP-derived cell lines (mixed
populations). The cell lines are described under "Experimental
Procedures." Lysates were prepared from cells starved in
serum-free medium for 48 h (time 0) and then stimulated with IGF-I
(50 ng/ml) for the indicated hours. D shows the levels of
PTEN expression in the LNCaP cells stably transfected with a PTEN
plasmid (parental LNCaP cells have a frameshift mutation of
PTEN).
DISCUSSION
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INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunit of PI3K transiently expressed in
293T cells, obtaining a modest but reproducible increase in Id2
proteins. The modest increase also obtained with wild-type p110
is
probably due to the fact that these experiments had to be done in 10%
serum, which contains, among many growth factors, also the IGFs. LNCaP
cells are a prostatic cancer cell line that originated from a
metastatic tumor. They express the IGF-IR, albeit at a low level,
~7-8 × 103 receptors/cell (31). Peptide analogs to
IGF-I inhibit their growth (48), and they respond to IGF-I with an
increase in cell proliferation (49). Their requirement for IGF-I is
more clearly evident under conditions of anchorage-independent growth
(49). As mentioned, they have a frameshift mutation of PTEN (34, 35). Because of the PTEN mutation, the PI3K pathway is constitutively activated (31, 39, 40, 44, 45). By using these cells, we have confirmed
in another cell line that IGF-I up-regulates Id2 gene expression and
that this regulation is inhibited by PTEN, an antagonist of PI3K.
Id2 protein levels are increased in LNCaP cells by IGF-I, but the
increase is abrogated in cell lines (mixed populations), expressing
wild-type PTEN.
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FOOTNOTES |
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* This work was supported by Grants CA 56309, CA 78890, and AG 16291 from the National Institutes of Health. A.M. is a recipient of a NIDDK Career Development Award (K01 DK 02896-01).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: Kimmel Cancer Center,
Thomas Jefferson University, 233 S. 10th St., 624 BLSB, Philadelphia,
PA 19107. Tel.: 215-503-4507; Fax: 215-923-0249; E-mail:
r_baserga@lac.jci.tju.edu.
Published, JBC Papers in Press, January 29, 2001, DOI 10.1074/jbc.M010509200
2 M. Prisco, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: IGF-IR, insulin-like growth factor I receptor; IGF, insulin-like growth factor; IL-3, interleukin-3; IRS, insulin receptor substrate; PI3K, phosphatidylinositol 3-kinase; PTEN, phosphatase on chromosome 10; PH, pleckstrin homology; PTB, phosphotyrosine-binding; PCR, polymerase chain reaction; TBS, Tris-buffered saline; BrdUrd, 5-bromo-2'-deoxyuridine.
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---|
1. | Sun, X. H., Copeland, N. G., Jenkins, N. A., and Baltimore, D. (1991) Mol. Cell. Biol. 11, 5603-5611[Medline] [Order article via Infotrieve] |
2. | Norton, J. D., Deed, R. W., Craggs, G., and Sablitzky, F. (1998) Trends Cell Biol. 8, 58-65[CrossRef][Medline] [Order article via Infotrieve] |
3. | Benezra, R., Davis, R. L., Lockshon, D., Turner, D. L., and Weintraub, H. (1990) Cell 61, 49-59[Medline] [Order article via Infotrieve] |
4. | Barone, M. V., Pepperkok, R., Peverali, F. A., and Philipson, L. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 4985-4988[Abstract] |
5. | Kreider, B. L., Benezra, R., Rovera, G., and Kadesch, T. (1992) Science 255, 1700-1702[Medline] [Order article via Infotrieve] |
6. | Desprez, P. Y., Hara, E., Bissell, M. J., and Campisi, J. (1995) Mol. Cell. Biol. 15, 3398-3404[Abstract] |
7. |
Lister, J.,
Forrester, W. C.,
and Baron, M. H.
(1995)
J. Biol. Chem.
270,
17939-17946 |
8. |
Hara, E.,
Yamaguchi, T.,
Nojima, H.,
Ide, T.,
Campisi, J.,
Okayama, H.,
and Oda, K.
(1994)
J. Biol. Chem.
269,
2139-2145 |
9. | Lasorella, A., Noseda, M., Beyna, M., and Iavarone, A. (2000) Nature 407, 592-598[CrossRef][Medline] [Order article via Infotrieve] |
10. |
Desprez, P. Y.,
Lin, C. Q.,
Thomasset, N.,
Simpson, C. J.,
Bissell, M. J.,
and Campisi, J.
(1998)
Mol. Cell. Biol.
18,
4577-4588 |
11. |
Lin, C. Q.,
Singh, J.,
Murata, K.,
Itahana, Y.,
Parrinello, S.,
Liang, S. H.,
Gillett, C. E.,
Campisi, J.,
and Desprez, P. Y.
(2000)
Cancer Res.
60,
1332-1340 |
12. | Lyden, D., Young, A. Z., Zagzag, D., Yan, W., O'Reilly, R., Bader, B. L., Hynes, R. O., Zhuang, Y., Manova, K., and Benezra, R. (1999) Nature 401, 670-677[CrossRef][Medline] [Order article via Infotrieve] |
13. | Christy, B. A., Saunders, L. K., Lau, L. F., Copeland, N. G., Jenkins, N. A., and Mathans, D. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 1815-1819[Abstract] |
14. | Einarson, M. B., and Chao, M. V. (1995) Mol. Cell. Biol. 15, 4175-4183[Abstract] |
15. | Cooper, C. L., and Newburger, P. E. (1998) J. Cell. Biochem. 71, 277-285[CrossRef][Medline] [Order article via Infotrieve] |
16. |
Valtieri, M.,
Tweardy, D. J.,
Caracciolo, D.,
Johnson, K.,
Mavilio, F.,
Altmann, S.,
Santoli, D.,
and Rovera, G.
(1987)
J. Immunol.
138,
3829-3835 |
17. |
Ward, A. C.,
Smith, L.,
de Koning, J. P.,
van Aesch, Y.,
and Touw, I. P.
(1999)
J. Biol. Chem.
274,
14956-14962 |
18. | Brown, G., Choudhry, M. A., Durham, J., Drayson, M. T., and Michell, R. H. (1999) Exp. Cell Res. 253, 511-518[CrossRef][Medline] [Order article via Infotrieve] |
19. | Campisi, J., and Pardee, A. B. (1984) Mol. Cell. Biol. 4, 1807-1814[Medline] [Order article via Infotrieve] |
20. | Baserga, R., and Morrione, A. (1999) J. Cell. Biochem. 32/33, 68-75[CrossRef] |
21. | Askew, D. S., Ashmun, R. A., Simmons, B. C., and Cleveland, J. L. (1991) Oncogene 6, 1915-1922[Medline] [Order article via Infotrieve] |
22. |
Dews, M.,
Prisco, M.,
Peruzzi, F.,
Romano, G.,
Morrione, A.,
and Baserga, R.
(2000)
Endocrinology
141,
1289-1300 |
23. |
Peruzzi, F.,
Prisco, M.,
Dews, M.,
Salomoni, P.,
Grassilli, E.,
Romano, G.,
Calabretta, B.,
and Baserga, R.
(1999)
Mol. Cell. Biol.
19,
7203-7215 |
24. |
Valentinis, B.,
Navarro, M.,
Zanocco-Marani, T.,
Edmonds, P.,
McCormick, J.,
Morrione, A.,
Sacchi, A.,
Romano, G.,
Reiss, K.,
and Baserga, R.
(2000)
J. Biol. Chem.
275,
25451-25459 |
25. |
Valentinis, B.,
Romano, G.,
Peruzzi, F.,
Morrione, A.,
Prisco, M.,
Soddu, S.,
Cristofanelli, B.,
Sacchi, A.,
and Baserga, R.
(1999)
J. Biol. Chem.
274,
12423-12430 |
26. |
Yenush, L.,
Zanella, C.,
Uchida, T.,
Bernal, D.,
and White, M. F.
(1998)
Mol. Cell. Biol.
18,
6784-6794 |
27. | White, M. F. (1998) Mol. Cell. Biochem. 182, 3-11[CrossRef][Medline] [Order article via Infotrieve] |
28. |
Myers, M. G., Jr.,
Grammer, T. C.,
Wang, L. M.,
Sun, X. J.,
Pierce, J. H.,
Blenis, J.,
and White, M. F.
(1994)
J. Biol. Chem.
269,
28783-28789 |
29. |
Pear, W. S.,
Nolan, G. P.,
Scott, M. L.,
and Baltimore, D.
(1993)
Proc. Natl. Acad. Sci. U. S. A.
90,
8392-8396 |
30. | Didichenko, S. A., Tilton, B., Hemmings, B. A., Ballmer-Hofer, K., and Thelen, M. (1996) Curr. Biol. 6, 1271-1278[Medline] [Order article via Infotrieve] |
31. | Reiss, K., Wang, J. W., Romano, G., Furnari, F. B., Cavenee, W. K., Morrione, A., Tu, X., and Baserga, R. (2000) Oncogene 19, 2687-2694[CrossRef][Medline] [Order article via Infotrieve] |
32. | Romano, G., Prisco, M., Zanocco-Marani, T., Peruzzi, F., Valentinis, B., and Baserga, R. (1999) J. Cell. Biochem. 72, 294-310[CrossRef][Medline] [Order article via Infotrieve] |
33. |
Florio, M.,
Hernandez, M. C.,
Yang, H.,
Shu, H. K.,
Cleveland, J. L.,
and Israel, M. A.
(1998)
Mol. Cell. Biol.
18,
5435-5444 |
34. |
Li, J.,
Yen, C.,
Liaw, D.,
Podsypanina, K.,
Bose, S.,
Wang, S. I.,
Ouc, J.,
Miliaresis, C.,
Rodgers, L.,
McCombie, R.,
Bigner, S. H.,
Giovanella, B. C.,
Ittmann, C.,
Tycko, B.,
Hibshoosh, H.,
Wigler, M. H.,
and Parsons, R.
(1997)
Science
275,
1943-1947 |
35. | Steck, P. A., Pershouse, M. A., Jasser, S. A., Yung, W. K. A., Lin, H., Ligon, A. H., Langford, L. A., Baumgard, M. L., Hattier, T., Davis, T., Frye, C., Hu, R., Swedlund, B., Teng, D. H. F., and Tavtigian, S. V. (1997) Nat. Genet. 15, 356-362[Medline] [Order article via Infotrieve] |
36. |
Furnari, F. B.,
Hong, L.,
Huang, H. J. S.,
and Cavenee, W. K.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
12479-12484 |
37. |
Tamura, M.,
Gu, J.,
Matsumoto, K.,
Aota, S.,
Parsons, R.,
and Yamada, K.
(1998)
Science
280,
1614-1617 |
38. |
Wu, X.,
Senechal, K.,
Neshat, M. S.,
Whang, Y. E.,
and Sawyers, C. L.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
15587-15591 |
39. | Li, J., Simpson, L., Takahashi, M., Miliaresis, C., Myers, M. P., Tonks, N., and Parsons, R. (1998) Cancer Res. 58, 5667-5672[Abstract] |
40. |
Davies, M. A.,
Koul, D.,
Dhesi, H.,
Berman, R.,
McDonnell, T. J.,
McConkey, D.,
Yung, W. K.,
and Steck, P. A.
(1999)
Cancer Res.
59,
2551-2556 |
41. |
Tamura, M.,
Gu, J.,
Danen, E. H. J.,
Takino, T.,
Miyamoto, S.,
and Yamada, K. M.
(1999)
J. Biol. Chem.
274,
20693-20703 |
42. | Furnari, F. B., Huang, H. J. S., and Cavenee, W. K. (1998) Cancer Res. 58, 5002-5008[Abstract] |
43. |
Li, D. M.,
and Sun, H.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
15406-15411 |
44. | Kulik, G., Klippel, A., and Weber, M. J. (1997) Mol. Cell. Biol. 17, 1595-1606[Abstract] |
45. |
Carson, J. P.,
Kulik, G.,
and Weber, M. J.
(1999)
Cancer Res.
59,
1449-1453 |
46. | Sun, X. J., Crimmins, D. L., Myers, M. G., Jr., Miralpeix, M., and White, M. F. (1993) Mol. Cell. Biol. 13, 7418-7428[Abstract] |
47. | Wang, L. M., Myers, M. G., Jr., Sun, X. J., Aaronson, S. A., White, M., and Pierce, J. H. (1993) Science 261, 1591-1594[Medline] [Order article via Infotrieve] |
48. | Pietrzkowski, Z., Mulholl, G., Gomella, L., Jameson, B. A., Wernicke, D., and Baserga, R. (1993) Cancer Res. 53, 1102-1106[Abstract] |
49. | Reiss, K., Yumet, G., Shan, S., Huang, Z., Alnemri, E., Srinivasula, S. M., Wang, J. Y., Morrione, A., and Baserga, R. (1999) J. Cell. Physiol. 181, 124-135[CrossRef][Medline] [Order article via Infotrieve] |
50. | Dufner, A., and Thomas, G. (1999) Exp. Cell Res. 253, 100-109[CrossRef][Medline] [Order article via Infotrieve] |
51. | Mendez, R., Kollmorgen, G., White, M. F., and Rhoads, R. E. (1997) Mol. Cell. Biol. 17, 5184-5192[Abstract] |
52. | Chan, T. O., Rittenhouse, S. E., and Tsichlis, P. N. (1999) Annu. Rev. Biochem. 68, 965-1014[CrossRef][Medline] [Order article via Infotrieve] |
53. | Conover, C. A., and Bale, L. K. (1998) Exp. Cell Res. 238, 122-127[CrossRef][Medline] [Order article via Infotrieve] |
54. | Reiss, K., Valentinis, B., Tu, X., Xu, S. Q., and Baserga, R. (1998) Exp. Cell Res. 242, 361-372[CrossRef][Medline] [Order article via Infotrieve] |
55. |
Chambery, D.,
Mohseni-Zadeh, S.,
de Gallé, B.,
and Babajko, S.
(1999)
Cancer Res.
59,
2898-2902 |
56. |
Ishiguro, A.,
Spirin, K. S.,
Shioara, M.,
Tobler, A.,
Gombart, A. F.,
Israel, M. A.,
Norton, J. D.,
and Koeffler, H. P.
(1996)
Blood
87,
5225-5231 |
57. | Jen, Y., Manova, K., and Benezra, R. (1996) Dev. Dyn. 207, 235-252[CrossRef][Medline] [Order article via Infotrieve] |
58. | Iavarone, A., Garg, P., Lasorella, A., Hsu, J., and Israel, M. A. (1994) Genes Dev. 8, 1270-1284[Abstract] |
59. |
Klippel, A.,
Escobedo, M. A.,
Wachowicz, M. S.,
Apell, G.,
Brown, T. W.,
Giedlin, M. A.,
Kavanaugh, W. M.,
and Williams, L. T.
(1998)
Mol. Cell. Biol.
18,
5699-5711 |
60. |
Aoki, M.,
Schetter, C.,
Himly, M.,
Batista, O.,
Chang, H. W.,
and Vogt, P. K.
(2000)
J. Biol. Chem.
275,
6267-6275 |