[Cancer Research 60, 4222-4230, August 1, 2000]
© 2000 American Association for Cancer Research
Molecular Biology and Genetics |
Stabilization of the Ras Oncoprotein by the Insulin-like Growth Factor 1 Receptor during Anchorage-independent Growth1
Martina Gatzka,
Marco Prisco and
Renato Baserga2
Kimmel Cancer Center, Thomas Jefferson University Hospital, Philadelphia, Pennsylvania 19107
 |
ABSTRACT
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R- cells are 3T3 cells derived from mouse embryos with a
targeted disruption of the type 1 insulin-like growth factor receptor
(IGF-IR) genes. R- cells are refractory to
transformation by a variety of viral and cellular oncogenes, including
an activated Ras. R- cells stably transfected with an
activated Ha-Ras (R-Ras cells) fail to form colonies in
soft agar. An IGF-IR truncated at residue 1245 cannot transform
R- cells, even when strongly overexpressed. However, the
combination of the truncated IGF-IR and an activated Ras induces
transformation of R- cells. We show here that the Ras
oncoprotein is rapidly degraded when R-Ras cells are grown
under anchorage-independent conditions and that signaling from the
truncated IGF-IR stabilizes Ras. In monolayer cultures, Ras levels
remain constant regardless of the presence or absence of IGF-IR
signaling. These results directly explain why Ras cannot transform
mouse embryo fibroblasts devoid of IGF-IR. They also suggest a more
generalized, alternative mechanism for transformation by Ras and,
implicitly, another possible way for targeting Ras in tumor cells.
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INTRODUCTION
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The
IGF-IR3
plays a crucial role in the establishment and maintenance of the
transformed phenotype (1
, 2)
. 3T3-like fibroblasts from
mouse embryos with a targeted disruption of the IGF-IR genes (3
, 4)
, designated as R- cells (5
, 6)
, are refractory to transformation (colony formation in soft
agar) by a variety of viral and cellular oncogenes that readily
transform cells with endogenous IGF-IR. The list of agents that fail to
transform R- cells include the SV40 T antigen
and/or an activated Ha-ras oncogene (5
, 6)
, the
bovine papilloma virus E5 protein (7)
, the human papilloma
virus E7 protein (8)
, the Ewing sarcoma fusion protein
(9)
, an activated c-src (10)
, an
overexpressed IRS-1 (11)
and overexpressed growth factor
receptors, such as the epidermal growth factor (12)
,
platelet-derived growth factor (13)
, and insulin
(14)
receptors. The only oncogene, thus far, known to
transform R- cells is v-src (10)
.
All of these agents readily transform mouse embryo fibroblasts with
endogenous IGF-IR (1
, 2)
.
R- cells were generated by a 3T3 protocol and
should be considered as 3T3 cells; indeed, they have growth
characteristics indistinguishable from BALB/c 3T3 cells, except for
their inability to respond to IGF-I (5
, 6)
. It is well
known that 3T3 cells and mouse embryo fibroblasts in general are prone
to spontaneous transformation. The finding that
R- cells are refractory to transformation by a
variety of viral and cellular oncogenes is therefore remarkable in
itself. The resistance of R- cells to
transformation is abrogated if the IGF-IR is re-introduced into these
cells. In fact, in R- cells, as well as in other
cell types, overexpression of the wild-type IGF-IR can cause, by
itself, cell transformation (15
, 16)
, again defined as
ability to form colonies in soft agar.
Among the cellular oncogenes that failed to transform
R- cells and are relevant to this investigation
are an activated Ras (6)
and an overexpressed IRS-1
(11)
, which is one of the major substrates of the IGF-IR
(reviewed in Ref. 17
). Both Ras and IRS-1 can transform
cells with endogenous IGF-IR (11
, 18)
.
R-Ras cells grow in monolayers in serum-free
medium, although the parental R- cells cannot
(6)
. R-Ras cells form foci in
monolayer cultures supplemented with serum but fail to form colonies in
soft agar or form very few (Ref. 6
and this paper).
Several reports have indicated that both Ras proteins
(19, 20, 21, 22)
and the IGF-IR (23)
are crucial for
anchorage-independent growth (usually colony formation in soft agar or
growth in experimental animals).
The purpose of this investigation was to explore the mechanism(s) by
which the absence of an IGF-IR in R- cells
interferes with the intermediate transformation step (colony formation
in soft agar) by an activated Ha-ras. For these studies, we could not
simply re-introduce the wild-type IGF-IR into R-
cells, because, as mentioned above, the receptor, when overexpressed,
transforms R- cells by itself. We took
advantage, though, of the fact that an IGF-IR truncated at the COOH
terminus no longer transforms R- cells, even
when strongly overexpressed (24
, 25)
. This IGF-IR lacking
the COOH terminus is still mitogenic in monolayer cultures and protects
cells from apoptosis (24, 25, 26, 27)
. It is therefore a
functional receptor that has simply lost its ability to transform
R- cells. We have inquired whether a combination
of two nontransforming agents, Ras and a truncated IGF-IR, could
transform R- cells, and if so, how they do it.
The results show that a truncated IGF-IR causes
R-Ras cells to form colonies in soft agar and
that transformation is accompanied by the stabilization of the Ras
protein levels when the cells are grown under anchorage-independent
conditions.
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MATERIALS AND METHODS
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Plasmids
pBPVIGF-IR
1245.
The truncated receptor is a pBPV (Pharmacia)-based expression vector
described previously by Hongo et al. (25)
,
containing a SalI-NotI fragment of the mutant
IGF-IR cDNA. This mutant was engineered to terminate after the arginine
codon at residue 1245 by replacing a NruI-BamHI
fragment of the wild-type IGF-IR cDNA with a double-stranded
oligodeoxynucleotide containing the stop codon TAG. Plasmid pGR96 was
generated from mutant IGR-IR1245 cDNA (pGR36) with an additional
point mutation at residue tyrosine 950 (to phenylalanine).
pREPIRS-1.
The complete mouse IRS-1 cDNA (11)
was subcloned in the
HindIII site of expression vector pREP4 (Invitrogen)
carrying the Rous sarcoma virus promoter and the hygromycin
phosphotransferase gene.
|
Retroviral Transduction
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The fetal human kidney carcinoma 293T cell line used for
retroviral packaging was purchased from American Type Culture
Collection and cultured as described elsewhere (28)
.
pHIT60 containing the murine leukemia virus gag-pol cassette under
control of the human cytomegalovirus immediate early (hCMVi.e.)
promoter and pHIT123 with the hCMVi.e.-driven murine leukemia virus
ecotropic envelope (both carrying the SV40 origin of replication in
their backbone) were kind gifts from Dr. A. Kingsman. The retroviral
vector MSCV.neoEB was kindly provided by Dr. R. G. Hawley.
These vectors are described in detail in Romano et al.
(28)
. Retroviral transduction was carried out as described
by Prisco et al. (29)
.
|
Cell Lines
|
|---|
R-ras (act. 8).
The R--derived cell line expressing an activated
Ras was generated by stable transfection of pBSPac containing a
BamHI fragment of the human Ha-ras gene derived
from T24 bladder carcinoma cells with a codon 12 mutation. The
Ha-ras gene is under control of its own promoter and the
plasmid carries the puromycin resistance gene, as described by Sell
et al. (6)
.
R-ras1245.
This line was obtained from R-ras cells by
cotransfection of pBPVIGF-IR1245 and pHygro, carrying the gene for
the hygromycin phosphotransferase, conferring resistance to hygromycin
B. Clones were selected using DMEM supplemented with 200 µg/ml
hygromycin B.
R-rasIRS-1.
This cell line was developed from R-ras cells by
stable transfection with the expression vector pREPIRS-1 and subsequent
selection in DMEM containing 200 µg/ml hygromycin B.
R-ras1245(RV).
Ras cells were retrovirally transduced with pGR36. Selection of the
mixed population was carried out in DMEM using 1 mg/ml G418.
R-ras1245/Y950 (RV).
To generate this mixed population, R-ras cells
were retrovirally transduced with pGR96, and selection was carried out
in DMEM containing 1 mg/ml G418.
|
Growth in Soft Agar
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The ability of the various Ras-derived cell lines to grow in
anchorage independence was assessed by colony formation in soft agar
according to the procedure given in Sell et al.
(5)
. Briefly, 103
cells were
suspended in 1 ml of DMEM supplemented with 10% FBS containing 0.2%
agarose and plated in 35-mm tissue culture dishes with 1 ml of a 0.4%
agarose underlay containing DMEM. After 7 days, cultures were fed with
0.2 ml DMEM (10% FBS) to prevent drying. Anchorage-independent growth
was allowed for 2 weeks before scoring the number of colonies >125
µm in diameter.
|
Growth in Poly(HEMA)
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To study signal transduction in anchorage independence, cells
were seeded on poly{HEMA}-coated plates according to a protocol
originally proposed by Folkman and Moscona (30)
. Under
these conditions, cells are denied attachment to a substratum (see
below). Either 35- or 100-mm Petri dishes were coated with
poly{HEMA} as described in detail by Valentinis et al.
(31)
. Cells were made quiescent on regular culture plates
in SFM (DMEM supplemented with 0.1% BSA and 50 µg/ml transferrin)
for 24 h and then washed once with HBSS, twice with
Versene, and subsequently detached from the plate in Versene solution
for 10 min to achieve a single-cell suspension. For growth experiments,
105 cells were seeded in either 2 ml of DMEM or
DMEM supplemented with 10% FBS on 35-mm poly{HEMA}-coated dishes.
After incubation for 24 h at 37°C, cells were harvested by
centrifugation and resuspended in trypsin/EDTA, and the number of
viable cells was determined using a hemocytometer and trypan blue
exclusion. For protein detection (Western blots), the usual number of
plated cells was 106 in 10 ml of DMEM plus the
indicated growth factors.
|
DNA Synthesis (BrdUrd Labeling)
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Cell growth in monolayer was assessed as fraction of cells
entering S-phase by BrdUrd incorporation using the BrdU Labeling kit
(Boehringer Mannheim, Indianapolis, IN).
Cells were seeded at a density of 1–2 x 104 on 35-mm cell culture dishes, serum-starved
for 48 h, and stimulated with 10% FBS or insulin (50
µM) or IGF-I (20 ng/ml) or left in SFM for an additional
24 h. BrdUrd was added at a concentration of 10 µM
after 6 h of stimulation. Cells were subsequently fixed in a
solution containing 95% ethanol, 0.1% acetic acid, and 0.1% Triton
X-100 (Sigma) and washed with PBS, before incubating successively with
the primary anti-BrdUrd (mouse monoclonal BMC 9318) and secondary
(sheep antimouse IgG FITC-labeled) antibodies, each at a dilution of
1:10 in PBS. To visualize all nuclei, total DNA was additionally
stained with 500 µg/ml disbenzimide H33258 (Sigma).
Vectashield (Vector Laboratories, Burlingame, CA) mounting medium was
subsequently applied, and the BrdUrd labeling index was determined
using a Zeiss microscope working in epifluorescence mode (x500). In
randomly selected fields, at least 500 cells/dish were counted.
|
IGF-IR and IRS-1 Expression Levels
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Exponentially growing cells were lysed in lysis buffer [50
mM HEPES (pH 7.5), 150 mM NaCl, 1.5
mM MgCl2, 1 mM EDTA, 10%
glycerol, 1% Triton X-100, 100 mM NaF, 10 mM
sodium PPi, 0.2 mM sodium
orthovanadate, 1 mM PMSF, and 10 µg/ml aprotinin].
Protein concentration was determined with a Bio-Rad protein assay.
After separation of 20 or 10 µg of total proteins on a 4–15%
gradient SDS-PAGE (Bio-Rad) and transfer to a nitrocellulose membrane,
the blots were successively immunostained with an antibody to the 
subunit of the IGF-IR (Santa Cruz) or an anti-IRS-1 antibody (Upstate
Biotechnology, Inc., Lake Placid, NY) and a secondary antirabbit IgG
conjugated to horseradish peroxidase (Oncogene Science, Manhasset, NY),
which was visualized by ECL detection reagents (Amersham Life Science,
Arlington Heights, IL).
|
Ras Detection (Western Blotting)
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After 24 h of serum starvation, 106
cells were either seeded on 100-mm poly{HEMA}-coated dishes as
described above and harvested by centrifugation after either 8, 16, or
24 h of culture or allowed to grow for an additional 24 h in
monolayer in SFM or DMEM with 10% FBS. PBS-washed pellets were
resuspended in 200 µl of ice-cold lysis buffer containing 50
mM Tris (pH 8.0), 400 mM NaCl, 5 mM
EDTA, 1% NP40, 0.2 mM sodium orthovanadate, 1
mM PMSF, and 10 µg/ml aprotinin. The lysates were
incubated for 15 min at 4°C, and the supernatant was recovered by
centrifugation at 10,000 rpm for 10 min. Twenty µg of whole-cell
lysate were resolved by SDS-PAGE on a 15% gel (Bio-Rad, Richmond, CA)
and electroblotted to a nitrocellulose filter. The filters were
successively immunoprobed with a polyclonal anti-Ha-Ras antibody
(sc-520; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and a
horseradish peroxidase-conjugated antirabbit IgG (Oncogene Life
Science). Detection of the immune complexes was carried out using the
ECL Detection system (Amersham Life Science).
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Shc Phosphorylation
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After 48 h of serum starvation, cells were either
stimulated with IGF-I (20 ng/ml) or insulin (50 µM) for
15 min or left untreated in SFM. Cytoplasmic lysates were prepared
using a lysis buffer containing 50 mM HEPES (pH 7.5), 150
mM NaCl, 1.5 mM MgCl2, 1
mM EDTA, 10% glycerol, 1% Triton X-100, 100
mM NaF, 10 mM sodium PPi,
0.2 mM sodium orthovanadate, 1 mM PMSF, and 10
mg/ml aprotinin. Subsequently, 400 µg of proteins were
immunoprecipitated with an anti-Shc polyclonal antibody (Transduction
Laboratories) and Protein G plus A agarose (Oncogene Life Science) in
HNTG buffer [20 mM HEPES (pH 7.5), 150 mM
NaCl, 0.1% Triton X-100, 10% glycerol, 0.2 mM sodium
orthovanadate, 0.2 mM PMSF, and 2 µg/ml aprotinin].
After resolution on a 4–15% gradient SDS polyacrylamide gel and
transfer to a nitrocellulose membrane, blots were hybridized with an
anti-phosphotyrosine monoclonal antibody conjugated with horseradish
peroxidase (Transduction Laboratories) and developed with ECL detection
reagents (Amersham).
|
Northern Blots
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|---|
Cells were seeded on poly{HEMA} dishes under similar
conditions as for protein detection and collected by centrifugation.
After extraction using the RNeasy Kit (Qiagen), 15 µg of total RNA
were separated on a 1% denaturing agarose gel and subsequently blotted
to a nitrocellulose membrane. A human H-Ras cDNA fragment generated by
PCR was used as a probe to detect Ras mRNA after labeling with the
Random Primed DNA labeling kit (Boehringer Mannheim) and 3000 Ci/mmol
[-32P]dCTP.
|
Protein Synthesis
|
|---|
Cells plated in monolayer were starved for 24 h in SFM
without methionine, then treated with Versene, and detached from the
plate. Cells were seeded on poly{HEMA}-coated plates in the same
medium supplemented with 100 µCi/ml of
[35S]methionine (NEN Life Science Products,
Inc.) plus or minus 50 ng/ml of IGF-I (Life Technologies, Inc.) for
4 h. Cell lysates were immunoprecipitated with anti-Ras antibody
(Transduction Laboratories), and the Ras bands, after autoradiography,
were cut out from the membrane and counted in a liquid scintillation
counter.
|
Protein Stability
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Cells in monolayer cultures were starved for 24 h in
methionine-free SFM. Cells were labeled with the same medium
supplemented with 100 µCi/ml of
[35S]methionine (NEN Life Science Products,
Inc.) for 6 h. After washing, cells were detached with Versene and
plated in poly{HEMA}-coated plates for 0, 6, 12, 18, and 24 h,
in complete medium. After lysis at the times indicated, cell lysates
were immunoprecipitated with an anti-Ras antibody (Transduction
Laboratories), and, after autoradiography, the Ras bands were cut out
from the membrane and counted in a liquid scintillation counter.
|
RESULTS
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Our aim in these experiments was to investigate the mechanism(s)
by which the absence of an IGF-IR prevents the Ras oncoprotein from
transforming R- cells. For this purpose, we
combined in R- cells the expression of Ras with
the expression of IGF-IR mutants that, by themselves, are defective in
transformation.
Growth Characteristics of the Cell Lines
The cell lines used are described in "Materials and Methods."
They were first tested for their ability to form colonies in soft agar.
The results from several experiments are summarized in Fig. 1
. As expected, neither R- cells nor
R-/1245 cells formed colonies in soft agar
(5
, 25)
. p6 cells are a control cell line derived from
BALB/c 3T3 cells (16)
that overexpress the IGF-IR and
reliably produce a large number of colonies in soft agar. The
R-ras cells or the R-ras
cells transduced with the appropriate empty vectors formed only a few,
small colonies. However, R-ras/1245 cells
readily formed numerous colonies (Fig. 1
shows three clones and one
mixed population). When R-ras cells were
transduced with another mutant IGF-IR (truncated at residue 1245 and
with a mutation at tyrosine 950), they still formed colonies in soft
agar. These experiments have been repeatedly confirmed (see also
below).
The levels of IGF-IR expression in the indicated cell lines are shown
in Fig. 2
. The receptor levels are compared with those of established cell lines
with known numbers of IGF-IR (16
, 32)
. From these data,
one can calculate that the mixed population of
R-ras cells transduced with the mutant IGF-IR
express 3 x 104 or more
receptors/cell. Other cell lines and clones express higher levels, with
clone #3 expressing roughly 5 x 105 receptors/cell. Mouse embryo fibroblasts with
3 x 104 wild-type receptors/cell
are already transformed (32)
. The arrows in Fig. 2
indicate both the proreceptor and the subunit of the receptor. The
proreceptor of cells expressing the 1245 mutant is, as expected,
shorter than the wild-type proreceptor. No IGF-I receptor is detectable
in R-ras cells.
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Fig. 2. IGF-IR levels in various cell lines. Western blots from
lysates of various cell lines, using an antibody to the subunit of
the IGF-I receptor, are shown. The cell lines are indicated
below each lane. The arrows indicate both
the proreceptor and the subunit of the IGF-IR (see "Materials and
Methods"). On the left side, for comparison, are four
cell lines, the IGF-IR levels of which have been established by
Scatchard plots. p6, R600, R503, and R508 cells are all mouse
fibroblasts with, respectively, 500, 30, 22, and 15 x 103
receptors/cell. Notice that the proreceptors of cells expressing the
1245 mutant are, as expected, shorter than the wild-type
proreceptors.
|
|
IRS-1 Cannot Transform R-ras Cells
An overexpressed IRS-1 can transform cells with endogenous IGF-IR
(11
, 18) and can combine with SV40 T antigen to induce
transformation of R- cells (33)
. We
therefore asked whether IRS-1 could combine with Ras to transform
R- cells. For this purpose, we transfected a
plasmid expressing IRS-1 into R-ras cells. Fig. 3A
shows that IRS-1 expression was increased in the
transfected cells, 2–5-fold (by densitometry; not shown). It should be
remembered that IRS-1 is already strongly expressed in
R- cells. We have calculated that the expression
of IRS-1 in parental R- cells is 
5-fold the
expression in BALB/c 3T3 cells. Fig. 3B
shows, in tabular
form, that overexpression of IRS-1 fails to transform
R-ras cells. In this figure, we also tested
again the cell lines of Fig. 1
, for comparison and confirmation. These
results therefore show that Ras can cooperate with a nontransforming,
mutant IGF-IR in transforming R- cells.
Interestingly, an additional mutation in the truncated receptor at
Y950, a binding site for Shc proteins (34)
, still resulted
in a receptor, which is transforming when combined with Ras. However,
IRS-1, in the absence of an endogenous IGF-IR, cannot transform
R-ras cells.
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Fig. 3. Effect of an overexpressed IRS-1 on colony formation in
soft agar. A, levels of expression of IRS-1 (Western
blots) in R- cells and R-Ras cells stably
transfected with a plasmid expressing IRS-1 (see "Materials and
Methods"). The cell lines are indicated below each
lane. R-IRS-1 cells are R- cells
overexpressing IRS-1 (no Ras). The R-raspCep were cells
transfected with an empty vector. Three different clones of
R-Ras/IRS-1 cells are shown. B, colony
formation in soft agar of various cell lines, including some of the
cell lines already tested in Fig. 1
. The last column (n)
gives the number of determinations.
|
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Growth in Monolayer Cultures
We have shown previously that both R-ras
cells and R-/1245 cells grow in SFM (6
, 25)
, although the parental R- cells do
not (6
, 32)
. We have confirmed that
R-ras and R-ras-derived
cells grow in SFM by labeling with BrdUrd (see "Materials and
Methods"). Serum-starved cells were supplemented with fresh medium
with or without the indicated growth factors for 6 h, before
BrdUrd was added. The percentage of cells labeled with BrdUrd was
determined after an additional 24 h and is shown in Fig. 4
. Clearly, the cell lines tested can all synthesize DNA in SFM. Addition
of 10% serum can significantly increase BrdUrd incorporation in these
cells over SFM. Insulin and IGF-I are less effective. An exception is
the modest stimulation by insulin of R-ras/IRS-1
cells, not surprising in view of the higher levels of IRS-1.
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Fig. 4. Growth of R--derived cells in monolayer
cultures. Serum-starved cells were treated as indicated (right
ordinate). Six h later, BrdUrd (10 nM) was added,
and the percentage of BrdUrd-labeled cells was determined after an
additional 24 h. The cell lines are indicated on the
abscissa. Bars, SD.
|
|
These experiments confirm the dissociation between growth factor
requirements and colony formation in soft agar. Both
R-ras and R-ras/IRS-1
cells are nontransformed, but they grow in SFM as well as the
transformed R-ras/1245 cells. We have
observed repeatedly that the IGF-IR is not an absolute requirement for
monolayer growth but is crucial for anchorage-independent growth
(reviewed in Ref. 23
).
Growth on poly{HEMA} Plates
The standard test for intermediate transformation is colony
formation in soft agar (35)
, which is a very good method,
highly reproducible, that has stood the test of time and gives an
objective evaluation of the capacity of cells to grow under
anchorage-independent conditions. As an alternative method, one can use
poly{HEMA}-coated plates, a method originally proposed by Folkman
and Moscona (30)
, in which the cells are denied attachment
to the substratum. This last test is no more artificial than colony
formation in soft agar, is a good measure of anchorage independence
(20
, 36) , and allows the recovery of intact cells for
molecular or biochemical analyses, as from monolayer cultures.
It is known that, at variance with epithelial cells, fibroblasts
survive on poly{HEMA} plates in 10% serum (37)
. We
have confirmed these findings and provided the fluorescence-activated
cell sorter analysis documentation that, under these conditions, there
is no appreciable cell death of R--derived cells
(36)
. We have reported the same observation in
R-/Ras cells, which grow on poly{HEMA}
plates, provided again that they are supplemented with serum
(31)
. We have consistently monitored cell death also in
the present experiments, and although an occasional dead cell could be
observed, there was no appreciable differences among the three cell
lines examined.
Because overexpression of IRS-1 does not alter the ability of
R-ras cells to form colonies in soft agar,
subsequent experiments were largely confined to the three cell lines
R-ras, R-ras/1245, and
R-ras/1245/Y950. The selected cell lines were
tested for survival and/or growth on poly{HEMA} plates (see
"Materials and Methods"). The results are shown in Fig. 5
. None of the cell lines tested did well in SFM on poly{HEMA}-coated
plates. This is not surprising, because most cells require
supplementation with growth factors and/or serum for survival in
poly{HEMA}-coated plates. In the absence of growth factors, most
cells on poly{HEMA} plates undergo a form of apoptosis that has
been called anoikis (20
, 38
, 39)
. When supplemented with
10% serum, R-ras and
R-/1245 cells survive and even show a modest
growth (increase in number, 8–10% over plated number).
R-ras/1245 and
R-ras/1245/Y950 cells grew well, confirming
that these last two cell lines are transformed. There is indeed a
correlation between colony formation in soft agar and growth in
poly{HEMA} plates in 10% serum. It should be remembered that
colony formation in soft agar is also done in 10% serum.
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Fig. 5. Growth of R--derived cells on poly{HEMA}
plates. Cells were plated on poly{HEMA}-coated plates. The cell
lines are indicated on the abscissa, and the treatment
(SFM; FBS, fetal bovine serum) are indicated on the
right ordinate. The number of viable cells was
determined after 24 h (see "Materials and Methods").
|
|
Ras Protein Levels in R--derived Cell Lines
In previous reports, we have shown that, in cells expressing the
IGF-IR, growth in poly{HEMA} plates does not interfere with IGF-I
(or serum)-mediated signaling. The IGF-IR is autophosphorylated
(36)
, and phosphatidylinositol 3-kinase and Akt are
activated (31)
. In fact, IGF-I can even induce an increase
in Ras GTP:GDP ratio, provided the cells have a sufficient number of
IGF-IR (31)
. In exploring signal transduction in our cell
lines, seeded in poly{HEMA}-coated plates, we examined several
aspects of IGF-IR signaling. Some of them will be mentioned in the
"Discussion," but we would like to focus in this report on the most
striking observation, which was a reduction in Ras levels in
R-ras cells seeded on poly{HEMA}-coated
plates. This is already evident when one compares Ras levels in
R-ras in poly{HEMA} plates versus
monolayer cultures (Fig. 6
). Whereas Ras levels remain high in R-ras cells
cultured in monolayers, they decrease in cells in poly{HEMA}-coated
plates, regardless of the addition of growth factors. Ras levels also
decrease in R-ras/1245 cells in SFM in
poly{HEMA}-coated plates (when these cells do not survive well) but
not when the cells are incubated in 10% serum (when these cells grow
well). Addition of IGF-I or insulin also influences Ras levels
favorably in R-ras/1245 cells in suspension
but not as clearly as with 10% serum.
We explored further the Ras decrease in the following experiments, in
which we grew three cell lines in 10% serum on poly{HEMA}-coated
plates. The results of such an experiment is shown in Fig. 7
. Ras levels were first determined in monolayer cultures, either in
serum (Fig. 7
, Lane 1) or in SFM (Fig. 7
, Lane
2). Then the cells were plated on poly{HEMA}-coated plates,
and Ras levels were determined while the cells were kept in 10% serum.
Ras levels decreased in R-ras cells, almost
disappearing by 24 h in poly{HEMA} plates (see for instance
the experiment in the second row of Fig. 7
). Ras levels remained
constant under the same conditions in
R-ras/1245 cells (Fig. 7
A, upper
row). They also remained high in
R-ras/1245/Y950 cells (Fig. 7
A, second
row), although they did decrease a little. Note however that
growth of these cells in poly{HEMA} plates is somewhat less than
growth of R-ras/1245 cells. Fig. 7B
shows a densitometric analysis of the Ras levels
displayed in Fig. 7A
. Ras oncoprotein levels decreased a
little in R-ras/1245 cells (15% after
24 h) but not as dramatically as in R-ras
cells, where Ras levels decreased in 24 h to <10% of the
original amount. The Ras levels in
R-ras/1245/Y950F cells decreased 50% by
24 h, still significantly higher than the Ras levels in
R-ras cells. We also looked at Ras levels in
R-/1245 cells, but in these cells Ras was not
detectable under the conditions we used (not shown).
Mechanism of Ras Down-Regulation in R-/Ras Cells
The experiments described above show that Ras levels decrease in
the nontransformed R-/Ras cell line in anchorage
independence, whereas they remained higher in the two transformed cell
lines. The obvious next question is why Ras levels decreases in
R-ras cells on poly{HEMA} plates. To study
the mechanism of Ras down-regulation in R-ras
cells on poly{HEMA} plates, we first addressed the possibility that
Ras mRNA may be decreased in these conditions.
R-ras cells, as mentioned in "Materials and
Methods," carry a Ras gene under the control of its own
promoter, and it would therefore be possible that Ras protein
expression in this cell line may be regulated at the transcriptional or
pretranslational levels. We therefore determined Ras mRNA levels in
both R-ras and
R-ras/1245 cells. Ras mRNA levels decreased
slightly in both cell lines when they are transferred from monolayer
cultures to poly{HEMA} plates, roughly by 20%. However, there
were no significant differences between the two cell lines (data not
shown).
The next possibilities for explaining the down-regulation of Ras
oncoprotein levels are either protein synthesis or protein stability.
For this purpose, we carried out two separate experiments. In the first
experiment, R-ras and
R-ras/1245 cells were labeled for 4 h
with [35S]methionine while suspended in
poly{HEMA}-coated plates. The cells were then collected, lysates
were made, the Ras protein was immunoprecipitated, and both immunoblots
and autoradiography were carried out (see "Materials and Methods").
The autoradiography is shown in the inset of Fig. 8
. There is no appreciable difference in Ras protein synthesis between
the two cell lines. This was confirmed by counting the radioactivity
from the cut-out bands (not shown). In the second experiment, the cells
were labeled with [5S]methionine in monolayer
cultures, then the cells were collected, and an aliquot was taken to
determine the amount of radioactivity in the labeled Ras protein. The
rest of the cells were suspended in poly{HEMA}-coated plates, and
aliquots were taken at various times afterward. Radioactivity in the
Ras protein was determined as described in "Materials and Methods."
Fig. 8
gives, for clarity, the initial amount of radioactivity and
subsequent measurements at 12 and 24 h. The initial amount of
radioactivity is given as 100%. Although there is a slight decrease in
the amount of radioactivity in Ras even in
R-ras/1245 cells, it is evident that the Ras
oncoprotein decays more rapidly in R-ras cells
than in R-ras/1245 cells in suspension.
|
Shc Phosphorylation
|
|---|
It is well established that the Shc proteins play an important
role in Ras activation by the insulin and IGF-I receptors, although a
contribution by IRS-1 has been also confirmed (17)
.
Binding sites for the Shc proteins in the IGF-IR have been localized at
Y950 (34)
and in the COOH terminus (40)
. We
examined Shc phosphorylation after IGF-I stimulation in the three cell
lines of Fig. , 7R
-ras cells,
R-ras/1245, and
R-ras/1245/Y950F cells. Fig. 9
shows that IGF-I induces Shc phosphorylation in
R-ras/1245 cells but not in
R-ras or in
R-ras/1245/Y950F cells. As a control, we used
p6 cells (16)
, which overexpress the IGF-IR and induce
marked tyrosyl phosphorylation of Shc. With IGF-I, only the Mr
52,000 isoform of Shc is phosphorylated (29)
.
Although this result was somewhat expected, its significance lies in
the fact that Shc phosphorylation does not seem to be a requirement for
maintaining high levels of Ras protein, as
R-ras/1245/Y950 cells do maintain high levels
of Ras protein, even on poly{HEMA} plates. Other studies on IGF-IR
signaling in these cells were less informative and will be only
mentioned in the "Discussion."
|
DISCUSSION
|
|---|
The primary conclusions of these investigations are:
(a) the failure of Ras to transform
R- cells (colony formation in soft agar) can be
explained by the fact that, under anchorage-independent conditions, the
Ras oncoprotein in these cells is unstable and is rapidly degraded;
(b) Ras protein levels in anchorage-independent conditions
are stabilized by the presence of an IGF-IR. The COOH terminus of the
IGF-IR is dispensable for this function. An additional mutation at Y950
causes only a slight decrease both in survival in poly{HEMA} plates
and in Ras levels; (c) the down-regulation of Ras in
R-ras cells is not attributable to inhibition of
transcription or RNA stability but to a decrease in the stability of
the Ras protein in these cells. Secondary conclusions include:
(d) maintenance of Ras protein levels on poly{HEMA}
plates does not require the phosphorylation of Shc proteins;
(e) the stability of Ras levels in monolayer cultures
explains why R-ras cells have a first step
transformed phenotype (see below) in monolayers (decreased growth
factor requirements and foci formation), but not in suspension; and
(f) overexpression of IRS-1 is not sufficient for
transformation of R-/Ras cells. These
conclusions will be discussed separately.
The most important conclusion of this investigation is that, at least
in this system, the IGF-IR stabilizes Ras expression when the cells are
placed in anchorage-independent condition. This conclusion is supported
directly by comparing the levels of Ras proteins in
R-ras cells and in
R-ras/1245 cells seeded on poly{HEMA}
plates. If the cells lose Ras when in anchorage-independent conditions,
it is not surprising that R-ras cells cannot
form colonies in soft agar. Three important points are relevant to a
discussion of this finding:
(a) We had to use the 1245 mutant of the IGF-IR because
it is nontransforming (25)
. An overexpressed wild-type
IGF-IR transforms mouse embryo fibroblasts by itself (15
, 16)
. Its use would have obscured the effect of the IGF-IR on the
ability of R-ras cells to form colonies in soft
agar. Thus, R-ras cells are not transformed
(colony formation in soft agar), R-/1245
cells are also nontransformed, but the combination of both results in
transformation. The establishment of a transformed phenotype is
accompanied by Ras stabilization, confirmed also with another
nontransforming mutant of the IGF-IR, the 1245/Y950F mutant (see
below).
(b) A second important point is that
R-Ras cells grow in monolayer cultures in
serum-free medium (31)
and form foci in 10% serum
(6)
. Thus, Ras can give R- cells
the phenotype associated with the first step in transformation but not
the intermediate steps of transformation (2
, 41)
. Although
we have not yet investigated this aspect in detail, it is tempting to
speculate that, in monolayer cultures, the integrity and the activation
of Ras may be mediated by the attachment of cells to a substratum. The
attachment to a substratum is known to activate integrin function,
which, in turn, can activate Ras (42
, 43)
. Whatever the
mechanism, Ras levels remain high in monolayer cultures, contributing
to the partially transformed phenotype, which disappears in suspension
cultures.
(c) The Ras-transfected cells in poly{HEMA} plates are
fully viable (31
, 36)
, provided they are supplemented with
10% serum. Cell death, under these conditions, is negligible, the
percentage of viable cells constantly remaining close to 100%, at
least for the first 3–4 days of culture (we never extended our
experiments beyond this time, because cells form huge aggregates, which
are very difficult to disperse).
A requirement for Ras in anchorage-independent growth has also been
suggested by previous reports. Thus, Rak et al.
(19)
transfected an activated Ras into a cell line that
could grow very well in monolayer cultures but underwent apoptosis when
cultured as multicellular spheroids on a nonadhesive surface. The
transfection of Ras allowed these cells to grow in suspension.
Similarly, Lebowitz et al. (44)
found that a
FTase inhibitor lacked significant cell toxicity in monolayer cultures
but became a potent activator of apoptosis when cell attachment to
substratum was prevented. Also compatible with our results is the
observation by Du et al. (45)
that activation
of the IGF-IR protects cells from the proapoptotic effect of FTase
inhibitors. The same authors concluded that the survival of
Ras-transformed cells in monolayers depends on the activation of the
phosphatidylinositol 3-kinase/Akt pathway. A specific protective effect
of Ras in anchorage-independent growth was also reported by Khwaja
et al. (20)
and by Valentinis et al.
(31)
. In the latter case, it was shown that an activated
Ras protected from anoikis cells with endogenous IGF-IR seeded on
poly{HEMA} plates, but not R- cells, that do
not have endogenous receptors (as in this report). Finally, an
essential role of oncogenic Ras in tumor growth was confirmed in
experimental animals by the elegant experiments of Chin et
al. (46)
. Our data provide a mechanism to explain the
role of Ras in anchorage independence, i.e., the ability of
the IGF-IR to stabilize Ras when the cells are not attached to a
substratum. In all of the reports not using R-
cells, it can be assumed that the cells had IGF-IR, because this
receptor is ubiquitous, hepatocytes and B lymphocytes being the only
exceptions (1)
.
It is generally accepted that when cells are stimulated with growth
factors (including IGF-I), Ras is translocated to the membrane
(47
, 48)
; this translocation is actually necessary for Ras
activation (49)
. Membrane anchorage of Ras oncoproteins
depends on their COOH-terminal farnesylcysteine (48)
.
Prenylation of the COOH terminus of Ras is in turn dependent on the
activation of FTase. It is generally agreed that inhibition of Ras
farnesylation brings about degradation of Ras and reversal of the
transformed phenotype. When the function of farnesylcysteine is
interfered with, Ras is dislodged from the membrane and is degraded
(50)
. Incidentally, in our experiments we have used
Ha-ras, which can be farnesylated but does not undergo
geranylgeranylation, as other Ras proteins can do. This mechanism of
Ras stabilization has been connected to insulin and the IGF system.
In the first place, insulin stimulates FTase activity, increasing the
pool of farnesylated Ras from 20–25% to 70% (49
, 51) . Secondly, the activation of FTase by insulin is dependent
on mitogen-activated protein kinase activity (49)
, which
is an important pathway stimulated by both the IGF-I and the insulin
receptors. Another clue is that lovastatin interferes both with the
FTase (reviewed in Ref. 52
) and the levels of IGF-IR
(53)
. Whatever the mechanism(s), we propose that, in
conditions of anchorage-independent growth, the IGF-IR is required for
maintaining the Ras oncoprotein farnesylated and membrane bound and,
therefore, stable. In monolayer cultures, the Ras pathway can be
activated (and Ras stabilized) by growth factor-independent mechanisms
(see above). The attachment to a substratum is known to activate
integrin function, which, in turn, can activate Ras (42
, 43)
. Ras levels remain high in monolayer cultures, contributing
to the partially transformed phenotype, which disappears in suspension
cultures. We would like to emphasize that the novelty of our findings
has little to do with how the Ras oncoprotein is stabilized (which is
known, see above). The question for future studies is how the IGF-IR
activates the machinery that leads to Ras stabilization.
The COOH-terminal truncated IGF-IR is mitogenic (25)
,
protects from apoptosis (26
, 27)
, but is not transforming
(25)
. It seems that, in this system, it is the Ras protein
that is oncogenic, but that it needs the IGF-IR for stabilization in
cells in suspension. The fact that the double mutant receptor
1245/Y950F also induces transformation of
R-ras cells (colony formation in soft agar)
while maintaining Ras levels high shows that tyrosyl phosphorylation of
Shc proteins is not required for these two events. It could be argued
that the double mutant is somewhat less effective than the 1245
truncated mutant, but the difference is modest, and certainly the cells
with the double mutant receptor behave more like the cells with 1245
than the parental R-Ras cells.
An overexpressed IRS-1 is transforming in cells that have IGF-IR
(11
, 18) . However, overexpression of IRS-1 fails to
transform R-ras cells. R-
cells already express substantial levels of IRS-1, which explains why
the overexpression of IRS-1 has no effect on the transformation of
R-Ras cells. The important point, however, is
that the failure of IRS-1 to transform R-ras
cells indicates again that the presence of an IGF-IR (even at modest
levels) is crucial for the establishment and maintenance of the
transformed phenotype (1)
.
Finally, we would like to add that other candidates for Ras-induced
transformation were examined in the course of these experiments and
included p21waf1 (54, 55, 56)
and
p27kip1 (57)
. As expected, both
p21waf1 and p27kip1
decreased sharply when the cells were switched from monolayers to
suspension. However, we could not detect any difference between
R-ras cells and
R-ras/1245 cells in poly{HEMA}-coated
plates (not shown). At least in this model, transformation by Ras is
not dependent on inactivation of these two cyclin-dependent kinase
inhibitors. Ras is oncogenic per se but loses its
oncogenicity in anchorage independence simply because it is degraded.
In conclusion, we have demonstrated a mechanism that can explain the
inability of the Ras oncoprotein to transform R-
cells, i.e., cells devoid of endogenous IGF-IR. Our findings
also explain why Ras is still effective in partially transforming
R- cells in monolayer cultures (growth in
serum-free medium, foci formation). In anchorage-independent growth,
however, the Ras protein requires an IGF-IR for its stabilization,
because, in the absence of the receptor, Ras is no longer membrane
bound. Experiments are in progress to study the mechanism(s) by
which the IGF-IR brings about the stabilization of Ras. Our findings,
besides providing a direct explanation for the failure of Ras to
transform R- cells in anchorage-independent
conditions, suggest a more generalized mechanism for Ras transformation
and the possibility of targeting Ras indirectly by targeting the
IGF-IR.
|
FOOTNOTES
|
|---|
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work is supported by Grants GM 33694 and CA
53484 from the NIH. 
2 To whom requests for reprints should be
addressed, at Kimmel Cancer Center, Thomas Jefferson University, 233
South 10th Street, 624 Bluemle Life Sciences Building,
Philadelphia, PA 19107. Phone: (215) 503-4507; Fax: (215) 923-0249;
E-mail: r_baserga{at}lac.jci.tju.edu
3 The abbreviations used are: IGF-IR, type 1
insulin-like growth factor receptor; BrdUrd, 5-bromo-2'-desoxy-uridine;
PMSF, phenylmethylsulfonyl fluoride; IRS, insulin receptor
substrate; FTase, farnesyltransferase; SFM, serum-free medium.
Received 11/12/99.
Accepted 5/31/00.
|
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Y. S. Chang, L. Wang, D. Liu, L. Mao, W. K. Hong, F. R. Khuri, and H.-Y. Lee
Correlation between Insulin-like Growth Factor-binding Protein-3 Promoter Methylation and Prognosis of Patients with Stage I Non-Small Cell Lung Cancer
Clin. Cancer Res.,
December 1, 2002;
8(12):
3669 - 3675.
[Abstract]
[Full Text]
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A. Chattopadhyay and G. Carpenter
PLC-{gamma}1 is required for IGF-I protection from cell death induced by loss of extracellular matrix adhesion
J. Cell Sci.,
May 15, 2002;
115(10):
2233 - 2239.
[Abstract]
[Full Text]
[PDF]
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ABSTRACT
INTRODUCTION
) are separate clones of R-Ras cells
expressing the 
). The R-ras/
