Insulin-Like
Growth Factor-l Induces
Cyclin-D 1 Expression
in MG63
Human Osteosarcoma
Cells in Vitro
Richard
W. Furlanetto,
Shari E. Harwell,
and Kevin K. Frick
Strong Children’s Research Center
Department of Pediatrics
University of Rochester School of Medicine and Dentistry
Rochest&,
New York 14642
each. Although the IGFs differ in their potencies, sites
of synthesis, affinities for various IGF-binding species,
and metabolism, they have a similar spectrum of biological activities and their major metabolic effects appear
to be mediated by the same cell surface receptor, the
type I IGF receptor (3,4). In viva, the IGFs are produced
by a number of cell types and appear to have autocrine
and paracrine as well as classic endocrine activities.
Most growth factors, including the IGFs, regulate cell
division by modulating events occurring during the prereplicative (Gl) phase of the cell cycle (5, 6) but identification of the critical events has proved difficult. However, recent observations made in widely diverse areas
of biology have identified a likely candidate.
These
studies indicate that progression through the eukaryotic
cell cycle is dependent on the sequential activation of a
group
of serine-threonine-specific
protein
kinases
whose activities are regulated by accessory proteins,
termed cyclins because their concentrations
vary during
the cell cycle (reviewed in Refs. 7 and 8). Both the
kinases [termed cyclin-dependent
kinases (cdks)] and
the cyclins exist as families, with 10 kinases (9) and
nine cyclins (10-14) identified thus far in mammalian
cells. A specific cyclin subtype appears to interact preferentially with a limited number of cdk subtypes, and
the different cyclin-cdk complexes appear to function
at different portions of the cell cycle (8, 9, 13-l 6). The
recent identification of cyclin species that are expressed
during Gl has led to the hypothesis that modulation of
Gl cyclin expression may be the critical event that is
regulated by growth factors (8, 1 O-l 3, 16, 17).
Of the nine cyclins thus far identified, six (cyclin-C,
-Dl, -D2, -D3, -E, and -F) are expressed during Gl.
Although the multiplicity of Gl cyclins suggests a functional redundancy, some differences have been noted.
Thus, a specific temporal pattern of Gl cyclin expression has been observed, with cyclin-C and -Dl appearing early in Gl , and cyclin-D2 and -E appearing later (8,
10, 12, 13, 17). The Gl cyclins are also expressed in
cell type-specific patterns; thus, in macrophages, cyclinDl and -D2 are expressed, but cyclin-D3 is not (lo),
whereas in fibroblasts cyclin-Dl and -D3 are expressed,
but D2 is not (17). In contrast, cyclin-A, -Bl, and -82
are expressed universally by replicating cells. Cyclin-A
The insulin-like
growth factors (IGFs) stimulate cell
division by modulating
events occurring
during the
prereplicative
(Gl) phase of the cell cycle, but identification of the critical events has proved difficult.
Recent
observations
suggest
that progression
through the cell cycle is dependent
on the activation
of a group
of serine-threonine-specific
protein
kinases whose activities are regulated by accessory
proteins, termed cyclins. The identification
of cyclin
species expressed during Gl has led to the hypothesis that modulation
of cyclin expression
may be the
critical event regulated by growth factors. The present studies were undertaken
to determine
whether
the IGFs regulate
the expression
of specific
Gl
cyclins in MG63, a human cell line that is unusually
responsive
to IGF, and to characterize
this effect.
We found that in these cells IGF-I stimulates
the
cyclin-dependent
kinases, and that stimulation
is
associated
with an increase in cyclin-Dl
mRNA and
protein expression.
The increase in cyclin-Dl
occurs
early in Gl and corresponds
to the portion of the
cell cycle in which IGF acts on these cells. The
increase in cyclin-Dl mRNA is due at least in part to
an increase in the rate of transcription
initiation of
the gene. The mRNA levels of cyclin-Bl
(a G2 cyclin)
and two cyclin-dependent
kinases, cdc2 and cdk2,
also increased in response to IGF, but at later times.
These results are consistent
with the hypothesis
that IGF modulation
of D-type cyclin expression
plays a role in the regulation
of cell replication.
(Molecular
Endocrinology
8: 510-517,
1994)
INTRODUCTION
The insulin-like growth factors (IGFs) are polypeptide
hormones that stimulate the replication of a wide variety
of normal and transformed cell types (reviewed in Refs.
1 and 2). There are two prototypical IGFs, IGF-I and
IGF-II, but multiple isoforms have been identified for
oeee-8809/94/0510-0517$03.00/0
Molecular Endocrinology
Copyright 0 1994 by The Endocrine
Socety
510
IGF-I Induction
of Cyclin-Dl
Expression
is first detected early in the S phase, reaches peak
levels in G2, and is degraded as cells enter M. The
B-type cyclins appear in the S phase slightly after cyclinA, reach peak levels at G2/M, and are degraded in midM phase. The complex of cyclin-B with cdkl (traditionally referred to as cdc2) has been identified as M-phasepromoting factor (18).
In addition to their appropriate temporal occurrence,
three observations support a critical role for Gl cyclins,
and the D-type cyclins in particular, in regulating cell
replication. The first is the observation
that specific
growth factors regulate D-type cyclin expression. Thus,
colony-stimulating
growth factor-l (CSF-1) (10) and
platelet-derived
growth factor (PDGF) (17) rapidly induce cyclin-Dl expression in responsive macrophages
and fibroblasts, respectively. The second observation
supporting a role for the D-type cyclins in growth control
is that cyclin-D-cdk complexes can interact with other
growth regulator molecules. Specifically, it has been
shown that cyclin-D-cdk
complexes
are capable of
phosphorylating
the retinoblastoma
gene product (pRb)
and its relative (~107) in vitro and in vivo (8, 15, 19, 20).
This suggests that tumor suppressor proteins may be
the proximal targets of the D-type cyclin-cdk complexes. The third observation supporting a critical role
for the D-type cyclins in growth control is that alterations in cyclin-D expression occur in certain tumors.
This was first noted in a subset of benign parathyroid
tumors, in which it was found that a chromosomal
inversion juxtaposes the PTH gene promoter with the
complete coding sequence for cyclin-Dl (21). Similarly,
overexpression
of truncated cyclin-Dl transcripts has
been reported in certain B-cell lymphomas (the W-1
oncogene) (22) and cyclin-Dl overexpression
has been
reported in 15-20% of breast carcinomas and squamous cell tumors of the head and neck (23). These
findings suggest that in some cell types activation of
cyclin-D expression alone may be sufficient to initiate
the mitogenic cascade.
The observations
noted above have led to the hypothesis that growth factor modulation of D-type cyclin
expression plays a critical role in the regulation of cell
replication. The present studies were undertaken
to
determine whether the IGFs regulate the expression of
specific D-type cyclins in MG63, a human osteosarcoma
cell line that is unusually responsive to IGF-stimulated
mitogenesis, and to determine the time course of this
induction.
511
MG63 cells, but increased 6- to lo-fold in cells treated
with IGF-I for 36 h (Fig. 1). Time-course studies showed
that cdk activity was low during the Gl and S phases,
increased as the cells progressed through the G2 and
M phases, and then declined as the cells reentered Gl
(not shown). Thus, IGF-I stimulates the cdk pathway
activity in MG63 cells.
Time Course
in MG63
of IGF-I Stimulation
of DNA Synthesis
Under the serum-free conditions used in these experiments, MG63 cells have a doubling time of 34-40 h
and a Gl phase of 16-18 h (Fig. 2, upper panel). To
determine the portion of Gl during which IGF action is
required, quiescent MG63 cells were stimulated with
IGF-I, and at various times after addition, the IGF-I was
neutralized by the addition of an antibody to IGF-I @GFI) (25) or to the type I IGF receptor (cull+3) (26). The
two antibodies gave similar results, and the data for
culR-3 are shown in the lower panel of Fig. 2. In this
experiment, nlR-3 inhibited IGF-l-stimulated
DNA synthesis by 88% when added 30 min before IGF-I. alR-3
remained effective in inhibiting DNA synthesis when
added within the first 12 h after IGF-I addition, but at
longer times it lost its inhibitory effect. Also shown in
the lower panel of Fig. 2 is the effect of 5,6-dichlorobenzimidazole
riboside (DRB), an inhibitor of RNA synthesis, on IGF-l-stimulated
DNA synthesis in these cells.
DRB inhibited IGF-l-stimulated
DNA synthesis by 72%
when added 30 min before IGF-I; it remained effective
if added any time during the first 16 h after IGF-I
addition, but lost its inhibitory effect thereafter. These
findings indicate that the signals necessary for IGFstimulated mitogenesis are promulgated within the first
12 h of the Gl phase, but that events occurring later in
Fig. 1. Effect of IGF-I on Cyclin-Dependent
MG63
Quiescent
RESULTS
MG63
cells
were
treated
Kinase
with
either
Activity
in
medium
alone or medium containing 8.7 nM IGF-I. After 24 h, both sets
IGF-I Stimulates
cdk Activity
To determine whether the IGFs stimulate cdk activity
in MG63 cells, a 21-amino acid peptide modeled after
a portion of the simian virus-40 large T-antigen and
which has been shown to be a substrate for G2/M
cyclin-cdk complexes, was employed (24). Using this
specific substrate, cdk activity was low in untreated
of cultures were treated with 33 PM nocodazole
(to arrest the
cells in G2). After an additional
12 h, the cells were harvested
by scraping
and lysed, and extracts
were prepared.
Aliquots
of these extracts were assayed for kinase activity by measuring the incorporation
of 32P0., into a 21-amino
acid peptide
substrate,
as described
in Materials and Methods. The counts
per min incorporated
into acid-soluble
material
by 1.2 ~1 cell
extract in 30 min at 30 C are shown. The values shown are
the means of duplicate samples.
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TIME AFTER ADDITION (hours)
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No. 4
transferase-cyclin-Dl
fusion protein, identifying this as
the specific cyclin-Dl band (not shown). The results of
one of four experiments
examining the time course of
IGF-I stimulation of cyclin-D protein expression in MG63
are shown in Fig. 3. The cyclin-Dl protein level was
low in quiescent MG63 cells, but increased 3- to 5-fold
with IGF-I addition. In multiple experiments
maximum
levels were reached between 6-12 h; the levels then
decreased, but in most experiments,
remained above
baseline throughout
the period of observation.
Also
shown in Fig. 3 is the time course of appearance of
three other components
of the cyclin-cdk pathway,
cyclin A, cdc2, and cdk2, as determined
by Western
blot analysis. The cyclin-A [mol wt (Mr), 58 kDa] protein
level increased about 5fold in response to IGF-I, but in
contrast to cyclin-Dl , it was low during Gl and reached
maximal levels at 24 h and beyond. The cdc2 (Mr, 34
kDa) and cdk2 (Mr, 33 kDa) protein levels increased
more rapidly than cyclin-A levels, but also were maximal
at 24 h and later. In some experiments,
the levels of
these three species had declined by 36 h.
TIME OF ADDITION (hours)
Fig. 2. Time Course of IGF-l-Stimulated
DNA Synthesis
and
Inhibition by UIR-3 and DRB in MG63 Cells
At time zero, IGF-I (4.3 nM) was added to quiescent
MG63
cells in the presence
of 1 pCi [methy/-3H]thymidine.
Upper
panel, At the indicated times, the monolayers
were harvested,
&din
Cyclin
cdc
cdk
and [methyL3H]thymidine (3H-THY) incorporation into DNA
was quantitated by scintillation counting. The data are ex-
A
0
2
2
.
pressed as a percentage
of the maximum
incorporation
(i.e.
42 h). Lower pane/, At the indicated times, alI+3 (W) and DRB
(A) were added to give final concentrations
of 100 nM and 47
PM, respectively,
and the ceils were returned
to the incubator.
Forty-two
hours after IGF-I addition, the monolayers were
0
6 12 18 2430
36
TIME (hrs)
harvested, and [methyL3H]thymidine incorporation into DNA
was quantitated by scintillation counting. The data are expressed as a percentage of maximal inhibition, which was that
observed when the agents were added 30 min before IGF-I;
these values were 88% and 72% for 100 nM otlR-3 and 47 PM
DRB, respectively. All values are the mean + 1 SD for triplicate
IA
L’
/I
samples.
Gl , including the synthesis of certain mRNAs essential
for DNA synthesis, are IGF independent. Thus, we have
concentrated
on events occurring early in Gl as those
most likely to be critical for IGF action.
0.0
0
To determine whether IGF-I stimulates the expression
of D-type cyclins in MG63, extracts of quiescent and
IGF-l-treated
cells were examined by Western blot
analysis using an antiserum that detects all three human
D-type cyclins. With this antiserum a major band of 36
kilodaltons (kDa) and minor bands of 34 and 67 kDa
were observed in IGF-l-stimulated
cells; however, only
binding to the 36-kDa band was abolished by preincubating the antibody with an excess of a glutathione S-
'0
6
12
cyclin
Dl
O-O
24
18
TIME AFTER
0-O
IGF-I Stimulates D-Type Cyclin Synthesis in MG63
\
/
---0
cyclin
ADDITION
A
A-
30
36
(hours)
-A
cdc2
A-
-Acdk2
Fig. 3. Effects
of IGF-I on Cyclin and cdk Protein Synthesis
in
MG63
Quiescent
MG63 cells were stimulated
with 8.6 nM IGF-I,
and the cells were harvested
at the times indicated.
Equal
volumes of cell extracts
(10 ~1) were resolved
by electrophoresis on 10% SDS-polyacrylamide
gels and transferred
to
Immobilon-P.
The membranes
were blocked and probed with
the antisera described
in Materials
and Methods,
and detected
using an appropriate
peroxidase-linked
second antibody
and
the ECL detection
system.
The autograms
were quantitated
by densitometric
scanning,
and the data were plotted as a
percentage of the maximal signal intensity for each species.
IGF-I Induction
of Cyclin-Dl
IGF-I Stimulates
MG63
Expression
Cyclin-Dl
513
mRNA Expression
in
Both translational and posttranslational
regulation have
been described for various components
of the cyclincdk pathway (10, 15, 27-29). To determine whether
the IGF-l-modulated
increase in cyclin-D protein expression observed in MG63 cells was associated with an
increase in steady state mRNA levels and to better
define the cyclins involved in this response, Northern
and dot blot analyses were performed. By Northern
analysis, all RNAs were detected predominantly
as
single bands (Fig. 4). Of the three D-type cyclins, the
steady state mRNA levels of cyclin-Dl showed the most
dramatic change in response to IGF-I. In MG63 cells,
cyclin-Dl
mRNA was detected as a single band of
approximately
6 kilobases with both IGF-I and serum
stimulation (Fig. 4). The cyclin-Dl mRNA level was low
in quiescent MG63 cells, increased within 1 h of IGF-I
addition, and reached peak values 2- to 5-fold above
baseline by 6 h (Fig. 5). In some experiments the cyclinDl mRNA level then remained relatively constant,
whereas in others it decreased to about 50% of the
maximum as the cells exited Gl (Fig. 5, compare upper
and lower panels). Cyclin-D3 mRNA was inconsistently
detected in MG63 and at much lower levels, which did
not appear to change significantly during the cell cycle,
and cyclin-D2 mRNA was not detectable in these cells
(not shown). As expected, the cyclin-Bl
mRNA level
CYC Dl
cdk2
kb
7.54
4.4-j
2.4,
I .4+
IGF-1 CS
IGF-1 CS
Fig. 4. Northern
Blot Analysis of Cyclin-Dl
and cdk2 mRNAs
in MG63
Quiescent
MG63 cells were treated
with IGF-I (4.3 nM) or
10% calf serum (CS). After 24 h, the monolayers
were washed,
the cells were lysed with 6 M guanidinium
isothiocyanate,
and
the RNA was purified by centrifugation
in CsC12. Thirty micrograms of total RNA were loaded onto each lane of a 1%
agarose
gel. After electrophoresis
and transfer
to nitrocellulose, the RNA was cross-linked
by UV irradiation,
and the
membranes
were hybridized
with 32P-labeled human cyclin-Dl
(left panel) or cdk2 (right panel) cDNA probes. The membranes
were then washed
in 2 x SSC containing
0.1% SDS and
autoradiographed.
kb, Kilobases.
increased in response to IGF-I, but it peaked at 24 h,
i.e. during the S phase (Fig. 5, lower panel).
The effects of IGF-I on the steady state mRNA levels
of cdc2 and cdk2 were also examined (Fig. 5, lower
panel). The steady state levels of both cdks showed
definite cell cycle periodicity, reaching maximum values
2- to 4-fold above baseline 12 h after IGF-I addition (i.e.
late Gl) and then declining.
To determine whether IGF-I increased the rate of
transcription
initiation of the cyclin-Dl
gene, nuclear
run-on assays were performed. In two experiments,
IGF-I treatment increased the level of cyclinD1 transcripts an average of 2.6-fold above baseline 3 h after
addition (Fig. 6). This suggests that the increase in the
level of cyclin-Dl mRNA induced by IGF-I is due at least
in part to an increase in the rate of transcription initiation
of the gene.
DISCUSSION
The present studies were undertaken
to determine
whether the IGFs regulate the expression of specific Dtype cyclins in MG63, an IGF-responsive
human cell
line, and to characterize this effect. We found that IGFI stimulates the cyclin-dependent
kinase pathway in
these cells and that this is associated with an increase
in cyclin-Dl
mRNA and protein expression.
The increase in cyclin-Dl occurs early in Gl and corresponds
to the portion of the cell cycle in which IGF acts on
these cells. This increase in the cyclin-Dl mRNA level
is due at least in part to an increase in the rate of
transcription
initiation of the gene. Both cyclin-D2 and
-D3 mRNAs were present only at levels near or below
the limit of detection of these studies. Cyclin-Bl , cdc2,
and cdk2 mRNA levels also increased in response to
IGF-I in these cells, but at later times.
Three previous reports have examined D-type cyclin
expression in response to specific growth factors. The
first report was that of Matsushime
et al. (lo), who
showed
that CSF-1 stimulates
cyclin-Dl
and -D3
expression
in mouse macrophages.
With respect to
cyclin-Dl,
the response of these cells was strikingly
similar to that reported here for MG63 cells; in both cell
types the specific growth factor stimulated cyclin-Dl
expression early in Gl , and in both cell types cyclin-Dl
mRNA and protein levels remained elevated through
the remainder of the cell cycle. These findings contrast
with those of two studies (17, 30) that examined the
induction of D-type cyclin expression in quiescent fibroblasts exposed to specific growth factors. In both of
those studies it was found that PDGF was a potent
stimulator of cyclin-Dl expression in early Gl , whereas
IGF-I had little effect on cyclin-Dl levels. It is noteworthy, however, that in fibroblasts IGF-I alone is a relatively poor mitogen and requires cotreatment
with
PDGF for full activity (5), whereas in MG63 cells and
macrophages
only IGF-I or CSF-1, respectively, is necessary for cell replication. Thus, the different responses
MOL
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B
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,
-I
0
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30
36
TIME AFTER ADDITION (hours)
0-O
cyclin
Dl
O--O
cyclin
B A-
-A
cdc2
A-
-A
cdk2
Fig. 5. Effect of IGF-I on Steady State Levels of Cyclin and cdk mRNAs in MG63
Quiescent
MG63 cells were treated with 4.3 nM IGF-I. At the times indicated,
the monolayers
were washed,
and the RNA was
isolated
and purified as described
in Fig. 4. The different
panels show the results of two separate
experiments.
Upper panel,
Northern
blot analysis. Thirty micrograms
of total RNA were loaded onto each lane of these gels. In this experiment
a murine
cyclin-Dl
probe was used. Similar results were obtained
with a human probe. Lower pane/, Dot blot analysis. Ten micrograms
of
32P-labeled
probes.
Autoradiograms
were
purified RNA were absorbed
onto nitrocellulose
and hybridized
with the appropriate
quantitated
by densitometry.
All points in this panel are from a single experiment.
observed in MG63 cells and macrophages,
on the one
hand, and fibroblasts, on the other, may reflect differences in the growth factor requirements
of these different cell types and are consistent with the hypothesis
that the D-type cyclins function to regulate events occurring early in Gl that are critical for entry into the
proliferative cell cycle.
In the present study we have documented
an effect
of IGF-I on cyclin-Dl gene expression in early Gl and
shown that this is due at least in part to an increase in
the rate of transcription initiation of the gene. However,
other mechanisms, such as increased mRNA stability
or translational efficiency, may also contribute to the
increase in cyclinD1 expression. Moreover, in addition
to the induction of cyclin-Dl expression, other modes
of regulation
of cyclinD1
activity are ‘possible.
For
example, cyclin-Dl
has been shown to undergo cell
cycle-dependent
phosphorylation
(lo), which could affect its activity. Regulation of the synthesis or activity
of the specific catalytic subunit(s) with which the D-type
cyclins interact is another potential control mechanism
(15, 27, 28). In this regard, cyclin-Dl has been shown
to interact with cdk4 (15), a cdk isotype that lacks the
PSTAIRE motif characteristic
of most other members
of the cdk family, and that these complexes can phosphorylate pRb and ~107 in vitro. In preliminary studies
we have found that cdk4 steady state mRNA levels are
not cell cycle regulated, but this finding does not rule
IGF-I
Induction
CYCLIN
ACTIN
of Cyclin-Dl
Expression
DI
515
-
w
3
0
TIME
6
(hrs)
Cell Lines
Fig. 6. Effect of IGF-I on the Rate of Cyclin-Dl
Transcription
Initiation in MG63
Quiescent
MG63 cells were treated with IGF-I (8.6 nM), and
the nuclei were isolated
at the times shown.
Nascent
transcripts were labeled with [(u-~‘P]UTP
at 30 C for 30 min, and
the labeled RNA purified as described
in Materials
and Mefhads. The appropriate
cDNAs were denatured,
applied to nitrocellulose (10 pg/slot),
cross-linked
by UV irradiation,
and hybridized
to the labeled RNA at 68 C for 40 h in 5 x SSC
containing
0.5% nonfat dry milk. After washing
in 2 x SSC0.1% SDS at 50 C, the radiolabeled
bands were detected
by
autoradiography.
The blot was exposed
for 11 days to detect
cyclin-Dl
and for 19 h to detect actin.
out regulation
of
lational
levels.
The observation
cdk4 at the translational
ties of all cDNAs were confirmed
other reagents
and supplies were
sources.
or posttrans-
that IGF-I rapidly stimulates cyclinDl expression in MG63 cells is the first report linking
the IGFs to this putative growth regulatory pathway.
Although this observation supports the hypothesis that
modulation of Gl cyclin expression is the critical event
that is regulated by growth factors, it is possible that
this is an epiphenomenon,
and further studies will be
required to determine whether cyclin-Dl expression is
necessary for IGF-stimulated mitogenesis in MG63 cells
and if it alone is sufficient to stimulate the mitogenic
cascade in these cells. In any case, identification of the
cyclin-Dl
gene as a target of IGF action offers an
opportunity to define the mechanism by which these
hormones regulate the expression of genes involved in
the mitogenic pathway.
AND METHODS
Materials
IGF-I (receptor
grade) was obtained
from GroPep
Pty. (Adelaide, Australia).
Cell culture medium [MCDB-104
and Dulbecco’s Modified Eagle’s Medium (DMEM)]
and the cdk substrate
peptide (24) were purchased
from Gibco (Grand
Island, NY).
Bovine calf serum was obtained
from HyClone
Laboratories,
Inc. (Logan, UT). The ECL detection
reagents
were purchased
from Amersham
(Arlington
Heights, IL). Antisera to IGF-I ((YIGFI) (25) and to the human type I IGF receptor
(ollR3) (26) were
prepared
as previously
described
(3). Rabbit polyclonal
antisera to human cyclin-D,
cdc2, and cdk2 were obtained
from
UBI (Lake Placid, NY). A mouse monoclonal
antibody
to human
cyclin-A
was provided
by Dr. E. Harlow
(Cold Spring Harbor, NY). Peroxidase-conjugated
goat antirabbit
and sheep
antimouse
antisera
were obtained
from Jackson
ImmunoResearch
Laboratories,
Inc. (West Grove, PA). Plasmids
containing cDNAs for the various cyclins and cdks were obtained
from the following
investigators:
mouse cyclin-Dl,
-D2, and
-D3, C. Scherr, St. Judes Children’s
Research
Hospital (Memphis, TN); human cvclin-Dl
, -D2, and -D3, S. I. Reed, Scripps
Research
Institute (La Jolla, CA); human cyclin-Bl
, T. Hunter,
The Salk Institute
(San Dieao. CA): human
cdkllcdc2,
P.
Nurse, University
of Oxford
‘iOxford,
United Kingdom);
and
human cdk2, E. Harlow (Cold Spring Harbor, NY). The identi-
Conditions
MG63
cells were obtained
from the American
Type Culture
Collection
(Rockville,
MD) and were routinely
grown at 37 C in
DMEM
supplemented
with 10% calf serum in a humidified
atmosphere
containing
5% COa. Stock cultures
were split
weekly
at a 1:40 dilution,
and new cultures
were initiated at
approximately
6-week
intervals.
The effects of IGF-I on the various components
of the cyclincdk pathway
were assessed
using the serum-free
culture
system previously
described
(31). Briefly, cells from exponentially growing
cultures
were plated at a density
of 5,00010.000 cells/cm2 in DMEM suoolemented
with alutamine
and
10% calf serum. After 16-24‘ h, this medium
was removed,
the monolayers
were washed
twice with PBS, and fresh
serum-free
MCDB104
was added. After 48-72
h, the cells
were quiescent,
and an appropriate
volume of fresh MCDB104
containing
0.1% BSA, penicillin (100 U/ml), streptomycin
(100
pg/ml), transferrin
(1 pg/ml), dexamethasone
(0.1 FM), and the
desired concentration
of IGF-I, antibody,
or other agents was
added. The cells were then incubated
at 37 C and harvested
at various times as appropriate
for the specific experimental
design.
Quantitation
of DNA
Synthesis
and Cell
Replication
To quantitate
DNA synthesis,
cells were plated in 2-cm2 wells,
and 1 &i
[mefhy/-3H]thymidine
was added simultaneously
with the IGF-I. At appropriate
times, the cultures
were harvested and prepared
for autoradiography
or scintillation
counting, as previously
described
(31). Alternatively,
cell cycle progression
was quantitated
by flow cytometry
using a Coulter
Profile II (Coulter
Electronics,
Hialeah, FL). At appropriate
times, cells were harvested
by trypsinization,
fixed in 75%
ethanol, and stored at 4 C. Immediately
before analysis, the
fixed cells were treated with RNase (1 mg/ml) and stained with
propridium
iodide (10 pg/ml). A minimum of 10,000 cells were
examined.
Measurement
MATERIALS
and Culture
by restriction
analysis.
All
obtained
from commercial
of cdk
Activity
For cdk assays, cell extracts
were prepared
by the method of
Pagan0 et al. (32). At the desired time, the monolayers
were
washed
twice with cold PBS, and the cells were detached
from the plate by scraping
into PBS. The cells were then
pelleted by centrifugation
at 500 x g for 10 min, and the pellet
was resuspended
in buffer containing
20 mrv HEPES, 0.4 M
NaCI, 25% glycerol,
1 mrv EDTA, 2.5 mM dithiothreitol
(DTT),
and 1 mM phenylmethylsulfonylfluoride,
pH 7.6. The cells were
kept on ice for 20 min, frozen to -70 C for 1 h, thawed
on
ice, vot-texed
vigorously,
and centrifuged
at 14,000 x g for 10
min at 4 C. The supernatants
were then removed
and stored
at -70 C.
Cyclin-dependent
kinase
activity
was measured
by the
method of Marshak
et a/. (24). This procedure
uses a synthetic
peptide derived from a portion of the simian virus-40
large Tantigen as substrate;
this peptide
has been shown to be a
substrate
for the G2/M cyclin-cdk
complexes
and for the cyclin
E-cdk 2 complex.
Typically,
a ~-PI aliquot of cell extract
was
added to 10 ~1 assaY buffer, giving a final concentration
of
0.50 mM Tris-HCI
(pH 8.0) 10rn~MgClp,
1 mM DTT, 1 mM
EGTA. 0.10 mM ATP. 1 &if?*Pl-ATP.
and 0.35 mM substrate
peptide. The mixture
was ;ncubated
at 30 C for 30 min, and
the reaction was stopped by the addition of 15 ~1 20% trichloroacetic acid. The mixture
was then incubated
at 4 C for 30
min, and the denatured
proteins were precipitated
by centrifugation at 14,000 x g for 10 min. Duplicate
lo-~1 aliquots of
the supernatants
were spotted
onto separate
phosphocellu-
MOL
516
Vol8
END0.1994
lose discs (Whatman
P81, Clifton, NJ), which were
then
washed three times for 10 min each time in 100 mM phosphoric
acid and twice in deionized
water. The disks were then air
dried, and the bound radioactivity
was measured
by scintillation counting.
Western
lmmunoblot
Analysis
Cell extracts
were prepared
as described
for the kinase assays. Aliquots
of these samples
were mixed with an equal
volume of buffer containing
4% sodium dodecyl sulfate (SDS),
0.2 M Tris-HCI,
0.2 M DTT, 0.2% bromophenol
blue, and 20%
glycerol, pH 6.8, and heated to 95 C for 5-10 min. The proteins
were resolved using a 10% SDS-polyacrylamide
gel with a 4%
stacking
gel according
to the method
of Laemmli
(33) and
were transferred
to Immobilon-P
membrane
(Millipore,
Bedford, MA) over l-2 h at 4 C using a Tris-glycine-methanol
buffer system. The membrane
was then blocked by incubation
in 5% nonfat dry milk in PBS at 4 C overnight.
After washing
twice for 5 min each time in PBS containing
0.05% Tween-20,
the membrane
was incubated
for 2-4 h at room temperature
with an appropriate
dilution of the primary antibody
of interest
diluted in 5% dry milk. The membrane
was then washed three
times in 0.05% Tween-20
and incubated
for l-2 h with the
species-appropriate
horseradish
peroxidase-linked
second antibody in 5% skimmed
milk. The membrane
was again washed
in 0.05%
Tween-20,
and the immunoreactive
bands were
visualized
using the ECL detection
system.
Autograms
were
quantitated
using an LKB Ultrascan
XL Densitometer
(Pharmacia LKB Biotechnology,
Piscataway,
NJ).
RNA Isolation
and Northern
Gel Analysis
Total cellular RNA was isolated from MG63 cells by harvesting
in guanidinium
isothiocyanate
and pelleting
through
5.7 M
CsCI, as described
by Chirgwin
et al. (34). RNA was analyzed
on 1% agarose
gels containing
1.2% formaldehyde
(vol/vol)
and 1 pg/ml ethidium bromide
in buffer containing
200 mM 3[N-morpholinolpropanesulfonic
acid, 8 mM sodium
acetate,
and 1 mM EDTA, pH 7.0. The quality of the RNA preparation
was judged on the basis of the integrity of the ribosomal
RNA
bands, as visualized
by ethidium
bromide
staining.
The RNA
was transferred
to nitrocellulose
(Nitroplus,
MSI,
Westboro, MA)
using
a Posiblot
apparatus
(Stratagene,
La
Jolla, CA) with 20 x SSC (1 x SSC is 0.15 M NaCl and 0.015
M sodium citrate) as buffer and was fixed to the membrane
by
UV cross-linking.
For preparation
of DNA probes,
plasmids
containrng
the appropriate
cDNA sequences
were purified
using Magic MaxIPreps
(Promega
Corp.,
Madison,
WI), digested with the appropriate
restriction
enzymes,
and electrophoresed
on low melting agarose
gels. The DNA fragments
containing
the sequences
of interest were visualized
by ethidium bromide staining, cut from the gel, diluted with 3 vol water,
and boiled for 3 min to denature
the DNA. The DNA was
labeled by the random primer method (35) using [a32P]deoxyCTP and reagents
obtained
from U.S. Biochemical
Corp.
(Cleveland,
OH). The nitrocellulose
filters were then incubated
in 5 x SSC, 0.5% nonfat dry milk, and 1% diethylpyrocarbonate at 68 C for at least 1 h before addition
of the denatured
probe (36). Hybridization
was allowed to continue for approximately 40 h at 68 C. The filter was then washed three times
with 2 x SSC-0.1%
SDS at 50 C, and the radiolabeled
bands
were detected
by autoradiography.
Nuclear
Run-On
No. 4
7.4) at 1 ml/l 0’ cells, mixed by gentle vortexing,
and allowed
to swell on ice for 5 min. The nuclei were pelleted by centrifugation at 500 x g at 4 C for 5 min, then washed
once in a
double volume of nuclear extraction
buffer and repelleted.
The
washed nuclei were resuspended
in 250 ~1 50 mM Tris-HCI, 5
mM MgCl*. 100 PM EDTA, and 40% glycerol,
pH 8.3; frozen
at -70 C; thawed
on ice; and added to an equal volume of
transcription
buffer (10 mM Tris-HCI,
5 mM MgCI,,
and 300
mM KCI, pH 8.0) containing
0.5 mM each ATP, CTP, and GTP
and 80 PCi [n-32P]UTP
to label nascent transcripts.
This mixture was incubated
at 30 C for 30 min. Nuclear DNA was
digested
by the addition
of RNase-free
DNase to l-5 pg/pl
and CaCI, to 1 mM and incubation
at room temperature
for 5
min.
Labeled RNA was purified by the method of Celano et al.
(38). The nuclei were lysed in 0.5% SDS, 5 mM EDTA, and 10
mM Tris-HCI,
pH 7.4, and digested
with 0.1 pg/pl proteinaseK at 37 C for 30 min. To serve as a carrier in subsequent
precipitation
steps, yeast RNA was then added to give a final
concentration
of 200 ng/gl. Proteins
were extracted
by the
addition of 2.2 vol GITC (4 M guanidinium
isothiocyanate,
25
mM sodium citrate, 0.5% sarcosyl,
and 0.1 M 2-mercaptoethanal. pH 7.0) sufficient
2 M sodium acetate (pH 4.0) to give a
final concentration
of 0.2 M, an equal volume of water-saturated (acidic) phenol,
and 0.1 total volume
of chloroformisoamyl alcohol (24:l).
These components
were thoroughly
mixed and incubated
on ice for 15 min. The phases were
separated
by centrifugation,
the supernatant
was removed,
and the RNA was precipitated
by the addition
of an equal
volume of isopropyl
alcohol and chilling at -70 C. After resuspension and reprecipitation
with isopropyl
alcohol, the RNA
was dissolved
in water, partially hydrolyzed
by the addition of
NaOH to a final concentration
of 0.2 N, and incubated
on ice
for 10 min. The solution was then neutralized
by the addition
of an equal volume of 0.5 M HEPES (free acid) and precipitated
with sodium acetate-ethanol
at -70 C. The labeled RNA was
recovered
by centrifugation,
dissolved
in water, and used as
a probe for hybridization.
Targets for hybridization
were prepared
by adding plasmid
DNA (0.1-10
fig) to 0.1 N NaOH and boiling for 15 min. After
cooling
on ice, the solution
was neutralized
with 1 N HCI,
adjusted
to 10 x SSC-250
mM Tris-HCI (pH 8.0). dotted onto
nitrocellulose,
cross-linked,
and hybridized
as described
above.
Acknowledgments
The authors would like to thank Drs. Peter Keng and Yi Ding
for their technical
assistance
and suggestions,
and Mrs.
Sharon Sibble for her help in preparing
this manuscript.
Received
October
14. 1993. Revision
received
December
14, 1993. Rerevision
received
January
20, 1994. Accepted
January
21, 1994.
Address requests for reprints to: Dr. Richard W. Furlanetto,
Division of Endocrinology,
Department
of Pediatrics,
University
of Rochester
School of Medicine and Dentistry,
Box 777, 601
Elmwood
Avenue,
Rochester,
New York 14642.
This work was supported
by USPHS Grant ROl-CA-38981
from the NCI, DHHS.
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