(Received for publication, May 4, 1995)
From the
Tumor necrosis factor-
Tumor necrosis factor- Rb protein is known to be cell
cycle-regulated(23, 24, 25, 26, 27, 28) .
This protein has tumor suppressing activity when the wild type Rb gene
is transfected into transformed mammalian cells, which do not express
functional Rb protein. In MCF-7 cells, as well as in osteocarcomas, Rb
protein was shown to be abnormally expressed(29, 30) .
The hypophosphorylated form of Rb protein acts as a functional tumor
suppressor(30) . This hypophosphorylated Rb protein is
associated with oncoproteins such as SV40 T antigen and adenovirus E1A
protein, indicating their involvement in cellular
transformation(31) . Phosphorylation of Rb protein is dependent
on the growth state of cells (23, 26) , and
antimitogenic agents or mitogenic agents exert their effects by
changing the phosphorylation state of Rb protein(32) . As
with Rb protein, cyclins are known to be cell cycle-related (33, 34, 35, 36, 37, 38, 39) .
Levels of cyclin-associated histone H1 kinase activities are also cell
cycle-regulated(35, 37, 39, 40) .
Some of these cyclins are known to have oncogenic activities associated
with Rb activity, and a shortened G Cdc2 and Cdk2 proteins are known to phosphorylate Rb protein both in vitro and in vivo(47, 48) . This
phosphorylation of Rb protein is believed to be achieved by physical
association of these proteins and cyclins. Tumor suppressor cell
cycle inhibitory protein (Cip1) (p21) was found to be a potent
inhibitor of cyclin-dependent kinases(50) . It is known to be
associated with various cyclin-Cdk complexes. The main role of Cip1
(p21) protein is to inhibit cyclin-dependent kinase activities. Cip1
(p21) protein is known to be induced in mammalian cells undergoing
G We investigated the antimitogenic effects of TNF-
For immunoprecipitation, confluent MCF-7 cells
(10
Figure 1:
Dose response of TNF-
To determine whether
antimitogenicity of TNF-
Figure 2:
A, temporal profile of TNF-
Figure 3:
TNF-
TGF-
Figure 4:
Growth-regulated expression of cyclin D1
in MCF-7 cells. Normally proliferating MCF-7 cells
(10
Fig. 5shows that
TNF-
Figure 5:
Specificity of TNF-
To determine which cellular molecules are important for cell cycle
progression, cell synchronization studies were carried out. Normally
proliferating MCF-7 cells (10
Figure 6:
Kinetics of induction of cell
cycle-regulated proteins in MCF-7 cells. Normally proliferating MCF-7
cells (10
Fig. 7A shows that activation
of protein kinase A by Bt
Figure 7:
Growth-regulated expression of Rb protein
in MCF-7 cells. A, confluent MCF-7 cells
(10
Fig. 7B shows that the
serine/threonine kinase inhibitor H7 causes dephosphorylation of Rb
protein in MCF-7 cells. This result suggests that Rb phosphorylation is
achieved by serine/threonine kinases in MCF-7 cells. This is consistent
with the previous results that showed phosphorylation of Rb protein at
serine/threonine residues. To determine whether TNF-
Figure 8:
TNF-
Since Cip1 (p21) protein was
induced in response to TNF-
Figure 9:
Effect of TNF-
Figure 10:
A, induction of p53 correlates
with Cip1 (p21) protein in MCF-7 cells. Confluent MCF-7 cells
(10
Fig. 10B shows the kinetics of induction of p53 and
Cip1 (p21) protein in MCF-7 cells. p53 protein showed earlier induction
than that of Cip1 (p21) protein. This is consistent with the fact that
p53 protein is a transcriptional activator and induction of Cip1 (p21)
protein is closely related to p53 induction. These results suggest that
induction of Cip1 (p21) protein by TNF-
Figure 11:
Molecular interaction between cyclin D1
and cellular molecules in MCF-7 cells. 50 µg of cell lysates
prepared from MCF-7 cells treated with TNF-
Figure 12:
Effect of phosphatase on Rb
phosphorylation. 50 µg of cell lysates were immunoprecipitated with
monoclonal anti-human Rb Ab conjugated with protein A-Sepharose.
Immunoprecipitated samples were treated with 100 units of calf
intestine alkaline phosphatase for 30 min at 37 °C. Samples were
boiled and were loaded for SDS-PAGE. Detection of Rb protein was done
by Western immunoblot analysis using monoclonal anti-human Rb Ab (1
µg/ml).
We have found that TNF- Since TNF- G It is
noteworthy that Cip1 (p21) protein was significantly induced in
response to TNF-
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(TNF-
) demonstrated
antimitogenic activity in MCF-7 cells (estrogen receptor-positive human
breast cancer cells) in a dose- and time-dependent manner (EC-50 of 2.5
ng/ml). This antimitogenic effect of TNF-
was accompanied by a
decreased number of cells in S phase in a dose- and time-dependent
manner. Based on growth arrest experiments using aphidicolin, it is
apparent that TNF-
acted in early G
phase. It did not
show antimitogenic effects once cells reentered the S phase based on
[
H]thymidine incorporation into DNA and cell
cycle analysis. Specificity of TNF-
was established by using
monoclonal anti-human TNF-
antibody. On the basis of Western
immunoblot analysis of Rb, p53 and cell cycle inhibitory protein (Cip1)
(p21) proteins, TNF-
decreased Rb protein expression in a dose-
and time-dependent manner whereas it increased the expression level of
tumor suppressor p53 protein. TNF-
also increased the expression
level of Cip1 (p21) protein in a dose-dependent manner. This induction
of Cip1 (p21) protein was preceded by the induction of p53 protein in
MCF-7 cells. Cip1 (p21) protein associated with cyclin D was also
increased. Tumor suppressor Rb protein expression was increased during
G
to S phase progression. Cyclin D protein expression
levels were not changed in response to TNF-
treatment, although
serine/threonine kinase inhibitors such as H7 and the protein kinase C
inhibitor staurosporine decreased cyclin D expression levels in MCF-7
cells. Based on experiments with staurosporine, it appears that
TNF-
does not utilize a protein kinase C pathway in MCF-7 cells.
Other cell cycle-related proteins such as Cdk2, Cdc2, and Cdk4 did not
show any change in response to TNF-
. TNF-
did not affect
complexes between cyclin D and Cdk2, Cdk4, and Rb proteins in MCF-7
cells. Taken together these results suggest that Rb, p53, and Cip1
(p21) proteins mediate TNF-
antimitogenic activity, and TNF-
induces growth arrest in the G
phase in MCF-7 cells.
(TNF-
) (
)is a
macrophage-derived multi-functional cytokine that acts as a cytostatic
or cytotoxic agent in many transformed and normal cells (1, 2, 3, 4, 5) . There
have been reports of functional roles of TNF-
such as
phosphorylation of epidermal growth factor receptor (6, 7) and cellular molecules, notably hsp27 and hsp70
protein(8, 9, 10, 11, 12, 13, 14, 15, 16) .
Overexpression of hsp27 and hsp70 proteins protect cells against the
cytotoxic effects of TNF-
(12, 17, 18) .
It also induces phosphorylation of NF-KB (19) and the
eukaryotic initiation factor EIF 4E(20) . The cellular
responses to TNF-
are necrotic or apoptotic killing, DNA
fragmentation, changes in arachidonic acid metabolism, lipid
peroxidation, inhibition of mitochondrial electron
transfer(21) , and increased production of prostaglandin
E(22) . Although TNF-
is a well known antimitogenic agent,
the mechanisms explaining these pleiotropic effects have not been well
understood.
phase(41) .
These cyclins are associated with other cell cycle-regulated proteins
such as Cdc2 and Cdk2 in many mammalian cells(42) . Complexes
between Rb and these cyclins are assumed to regulate Rb protein
functions in many mammalian
cells(33, 43, 44, 45, 46) .
arrest or apoptosis(51) . G
arrest is
linked to p53 gene induction, and it is believed that induction of p53
in turn induces expression of Cip1 (p21) protein. The fact that
oncogenes such as SV40 T antigen inhibits induction of Cip1 (p21)
protein suggests that Cip1 (p21) protein might act as an anti-oncogene.
in MCF-7
cells as determined by expression levels of cell cycle-related proteins
such as Rb, Cip1 (p21), p53, and cyclins to extend our understanding of
the mechanisms of TNF-
action in MCF-7 cells. We found by cell
cycle analysis that TNF-
acted during the G
phase of
the cell cycle. This was associated with a decrease in the expression
of Rb and increased expressions of Cip1 (p21) and p53. There was no
effect on cyclin D1, Cdk2, or Cdk4 proteins.
Cell Culture and Reagents
MCF-7 (estrogen
receptor-positive human breast cancer) cells were cultured in a
humidified air atmosphere (5% CO) and Dulbecco's
modified Eagle's medium (DMEM) supplemented with 10% FCS. Human
recombinant TNF-
was purchased from Sigma. Monoclonal antibodies
to human cyclin A, cyclin B, Rb protein, and TNF-
were purchased
from Upstate Biotechnology, Inc. (UBI, Lake Placid, NY). Polyclonal
antibodies to human Cdc2, Cdk2, Cdk4, and cyclin D were also purchased
from UBI. Polyclonal rabbit anti-human Cip1 (p21) Ab was purchased from
Santa Cruz Biotech (Santa Cruz, CA). Monoclonal anti-human Rb antibody
was purchased from Pharmingen (San Diego, CA). Aphidicolin and
propidium iodide were purchased from Sigma and RNase A (Bovine) was
purchased from Boehringer Mannheim. ECL (enhanced chemiluminescence)
kit was purchased from Amersham Corp.
[
H]Thymidine was purchased from DuPont NEN. H7
and H8 were purchased from Sigma. Staurosporine was purchased from UBI.
[
MCF-7 cells (2 H]Thymidine Incorporation
Assay
10
/well) were incubated
with TNF-
for various time intervals.
[
H]Thymidine (20 µCi/ml) was added to each
well for 1 h, washed with 1
HBSS (Hanks' buffered salt
solution) three times, and cell lysates were prepared with 0.5% (w/v)
SDS solution. To the latter was added 20% trichloroacetic acid (v/v)
solution and incubation continued for 30 min on ice. Trichloroacetic
acid-precipitated samples were washed with 20% trichloroacetic acid
(v/v), 10% trichloroacetic acid (v/v), and 90% ethanol (v/v)
successively, and filters were dried. Radioactivity was determined in a
scintillation counter. [
H]Thymidine incorporation
data were normalized with cell counts and protein concentration of each
sample.
Cell Cycle Analysis
MCF-7 cells treated with
various concentrations of TNF- were trypsinized. Cell pellets were
resuspended in 1 ml of HBSS buffer, and were fixed by HBSS buffer
containing 70% (v/v) ethanol for 1 h at 4 °C. Cells were
centrifuged at 1,000 rpm for 10 min. Cell pellets were resuspended in
HBSS buffer containing 50 µg/ml RNase A and 50 µg/ml propidium
iodide. Incubation continued for 1 h at room temperature. Cells were
filtered through nylon mesh (41 µm) and DNA content was measured in
an Epics II flow cytometer. Propidium iodide staining does not
distinguish G
cells from G
.
Western Blot Analysis
Cell lysates were prepared
from MCF-7 cells treated with TNF- by extraction buffer (20 mM Hepes (pH 7.2), 1% Triton X-100 (v/v), 10% glycerol (v/v), 2
mM sodium fluoride, 1 mM sodium orthovanadate, 50
µg/ml leupeptin, and 0.5 mM phenylmethylsulfonyl
fluoride). Cell lysates were cleared by centrifugation at 15,000 rpm
for 30 min in a microcentrifuge and were loaded for SDS-PAGE. Samples
were transferred to nitrocellulose filters by electroblot transfer for
2 h at 200 mA. The nitrocellulose filter was incubated with blocking
buffer (1
TBS (Tris-buffered saline), Fraction V 3% BSA (w/v),
0.2% (v/v) Tween 20) for 1 h at room temperature and washed with 1
TBS buffer for 20 min. The nitrocellulose filter was incubated
with primary antibody and dissolved in blocking buffer overnight at 4
°C (anti-human Rb Ab was used at a concentration of 1 µg/ml).
On the following day the nitrocellulose filter was washed with 1
TBS for 20 min and incubated with either anti-mouse or
anti-rabbit horseradish peroxidase conjugated with IgG for 1 h at room
temperature. Secondary antibody was at a concentration of 1:1000
dilution. After washing with 1
TBS for 20 min at room
temperature, detection of proteins of interest was carried out by the
ECL method.
/100-mm dish) were lysed and cell lysates were
immunoprecipitated with polyclonal anti-human cyclin D Ab conjugated
with protein A-Sepharose. Reaction was carried out at 4 °C for 4 h
on a rotary shaker. Immune complexes were washed three times with lysis
buffer, and 2
sample buffer was added to the beads. Boiled
samples were loaded for SDS-PAGE. Western immunoblot analyses were
followed according to standard procedures.
Antimitogenicity of TNF-
To
determine whether TNF- in MCF-7 Cells
has antimitogenic effects on MCF-7 cells,
various concentrations of TNF-
were added to MCF-7 cells for 24 h
at 37 °C and [
H]thymidine incorporation into
DNA was carried out. The antimitogenic effect of TNF-
was
dose-related as shown in Fig. 1. TNF-
did not show any
effect at low concentrations but showed clear antimitogenic effects at
concentrations exceeding 0.5 ng/ml. TNF-
did not show mitogenic
activity at any concentration. The antimitogenic effect was also seen
in MCF-7 cells grown under SFM conditions, suggesting that the
antimitogenic effect is due not to serum factors but to direct
antimitogenic effects of TNF-
on MCF-7 cells. To determine the
specificity of TNF-
, a blocking experiment using monoclonal
anti-human TNF-
Ab was carried out. Briefly, TNF-
(10 ng/ml)
was preincubated with various concentrations of monoclonal anti-human
TNF-
Ab at 37 °C for 1 h before addition to each well. The
next day [
H]thymidine incorporation into DNA was
carried out. As shown in Table 1, the antimitogenic effect of
TNF-
was efficiently blocked by an excess concentration of
monoclonal anti-human TNF-
Ab while antibody itself did not show
any effect on MCF-7 cell growth. Table 2shows that this
inhibition of TNF-
action is consistent with the cell cycle
analysis of MCF-7 cells treated with TNF-
(10 ng/ml) alone or in
combination with anti-TNF-
Ab (500 ng/ml).
in MCF-7
cells. Exponentially growing MCF-7 cells (10
cells/well)
were treated with various concentrations of TNF-
at 37 °C.
[
H]thymidine incorporation into DNA was carried
out at 24 h.
is cell cycle-related, FACS
(fluorescence-activated cell sorter) analysis was carried out. As shown
in Fig. 2A, TNF-
changed the percentage of cells
in S phase. The decrease in the fraction of cells in S phase was
dose-dependent, as evidenced by larger changes in response to higher
concentrations of TNF-
. Changes in cell cycle phases were also
time-dependent (data not shown). These results suggest that TNF-
might prevent cells from entering S phase. Based on these results, it
is likely that TNF-
acts at the G
/S phase boundary. To
prove this point, MCF-7 cells were growth-arrested at the
G
/S boundary by aphidicolin treatment (5 µg/ml) for 24
h, washed with 1
HBSS buffer, and refed with 10% FCS/DMEM. At
each interval after release from aphidicolin growth arrest, TNF-
(10 ng/ml) was added. MCF-7 cells were harvested for cell cycle
analysis, and cell lysates for [
H]thymidine
incorporation into DNA. As shown in Fig. 2B, TNF-
added at 0, 3, 6, and 12 h after release from aphidicolin treatment
showed antimitogenic effects, but not at 20 h. This result suggests
that TNF-
acts in the G
phase but not after cells
reenter the S phase. This suggests that TNF-
does not offset the
mitogenic stimulation of serum. The antimitogenic effect of TNF-
was more apparent in a synchronized population of MCF-7 cells. To
determine whether the blocking effect of TNF-
by antibody is also
accompanied by changes in cell cycle phases, growth arrest experiments
using aphidicolin were carried out as before. As shown in Table 1, monoclonal anti-human TNF-
Ab itself did not affect
cellular growth, suggesting that endogenous TNF-
did not play a
role in MCF-7 cell growth. TNF-
added at 0, 3, 6, and 10 h but not
20 h showed antimitogenic effects. When TNF-
was added along with
monoclonal TNF-
Ab, the latter efficiently inhibited the
antimitogenic effect of TNF-
.
action in
MCF-7 cells. MCF-7 cells (10
cells/100-mm dish) were
treated with various concentrations of TNF-
for 16 h. Cell cycle
analysis was carried out according to standard procedures. Columns
a-f refer to observations made at different concentrations
of TNF-
of 0, 1, 5, 10, 50, and 100 ng/ml. B, temporal
profile of TNF-
action in MCF-7 cells. MCF-7 cells were
growth-arrested by aphidicolin treatment (5 µg/ml) for 24 h, washed
with 1
HBSS buffer extensively, and refed with 10% FCS/DMEM.
TNF-
(10 ng/ml) was added at the indicated times. FACS analysis
was carried out according to standard
procedures.
Effects of TNF-
Based on the previous results of the antimitogenic
effects of TNF- on Cell Cycle-related
Proteins
, it was necessary to determine whether TNF-
affects expression levels of cell cycle-regulated proteins. For this
purpose, Western blot analysis using monoclonal anti-human Rb Ab was
carried out. MCF-7 cells were treated with aphidicolin for 16 h, washed
with 1
HBSS buffer, and incubated with 10% FCS/DMEM. TNF-
(10 ng/ml) was added at each time point, and cell lysates were prepared
according to standard procedures. As shown in Fig. 3, TNF-
added at 0, 3, 6, 12, and 20 h after release showed decreased
expression levels of Rb proteins. This result suggests that the
decreased DNA synthesis seen in Fig. 1is consistent with
decreased expression levels of Rb protein in response to TNF-
.
This result also suggests that Rb protein is important for
G
/S transition in the cell cycle. Based on these results,
it is plausible that TNF-
acts, in part, by decreasing Rb tumor
suppressor protein in MCF-7 cells.
acts at G
phase to
decrease Rb expression. Normally growing MCF-7 cells
(10
/100-mm dish) were growth-arrested by incubation with
aphidicolin (5 µg/ml) for 16 h, washed with 1
HBSS buffer
three times, and refed with 10% FCS/DMEM. TNF-
was subsequently
added at the indicated times after aphidicolin release and harvesting
at 24 h. Thus cells were exposed to TNF-
for 24, 21, 18, 12, and 4
h, respectively. Western blot using monoclonal anti-human Rb Ab (1
µg/ml) was carried out according to standard
procedures.
, a well known
antimitogenic agent in many other mammalian cells, did not manifest any
antimitogenic activity or induce decreased Rb protein levels in MCF-7
cells (data not shown). Therefore, it is likely that TNF-
activity
is not mediated by TGF-
, as was the case with interferon-
in
human Burkitt lymphoma Daudi cells(32) . G
cyclins
such as cyclin D did not show any change in expression levels in
response to TNF
in MCF-7 cells. Cdk4 protein did not respond to
TNF-
under these conditions. To determine whether cyclin D protein
reflects the growth state of the MCF-7 cells, normally proliferating
MCF-7 cells were treated with TNF-
(10 and 25 ng/ml),
Bt
cAMP (1 mM), or staurosporine (1 nM)
for 24 h and Western immunoblot analysis using polyclonal anti-human
cyclin D Ab (1 µg/ml) was employed. As seen in Fig. 4, there
was no significant reduction in the expression level of cyclin D in
response to TNF-
. However, in response to H7 as well as
staurosporine, cyclin D expression levels were significantly reduced.
H7 and staurosporine were found to be strong antimitogenic agents in
MCF-7 cells (data not shown). Cdk4 protein levels remained constant
regardless of the treatments.
/100-mm dish) were treated with various concentrations
of TNF-
(10, 25 ng/ml), Bt
cAMP (1 mM), and
staurosporine (1 nM) for 24 h, and cell lysates were prepared
for Western immunoblot analysis. Polyclonal anti-human cyclin D Ab was
used at a concentration of 1 µg/ml.
-mediated regulation of Rb protein expression is specific in
nature, as preincubation of TNF-
with monoclonal anti-human
TNF-
Ab prevented TNF-
antimitogenicity in MCF-7 cells (lanes3, 5, 7, 9, and 11). This result suggests that endogenous TNF-
does not
regulate Rb protein expression. This figure also shows that TNF-
did not have an effect on Rb protein expression once cells enter S
phase (lane13), suggesting an important role of Rb
protein in G
/S transition in the cell cycle of MCF-7 cells.
activity in MCF-7
cells. Normally proliferating MCF-7 (10
/100-mm dish) cells
were treated with aphidicolin (5 µg/ml) for 16 h, washed with 1
HBSS buffer, and refed with 10% FCS/DMEM. TNF-
was added
at the indicated times. For the blocking experiment, TNF-
(10
ng/ml) was preincubated with monoclonal anti-human TNF-
Ab (500
ng/ml) for 1 h at 37 °C before adding to cells. Western immunoblot
analysis using monoclonal anti-human Rb Ab (1 µg/ml) was carried
out according to standard procedures. Detection of Rb protein was done
by the ECL method. Columns1-13 correspond to
the experimental conditions indicated below the
numbers.
/100-mm dish) were treated
with aphidicolin (5 µg/ml) for 12 h, washed with 1
HBSS
buffer extensively and refed with 10% FCS/DMEM. At each time after
release from growth arrest by aphidicolin, cells and cell lysates were
harvested for Western blot and FACS analysis. As shown in Fig. 6, tumor suppressor Rb protein showed induction at late
G
phase as expected. This induction of Rb protein remained
through S phase and then declined to normal levels. Cyclin D protein
was induced in late G
phase and remained at high expression
levels through the S phase. Cdk4 protein was similarly induced in the
G
phase and remained elevated during S phase and subsequent
cycles. Well known mitotic cyclin A showed expression patterns similar
to that of Rb protein.
/100-mm dish) were treated with aphidicolin (5
µg/ml) for 10 h, washed with 1
HBSS buffer extensively, and
refed with 10% FCS/DMEM. At each time after release from growth arrest
by aphidicolin, cell lysates and cells were harvested for Western blot
and FACS analysis. Monoclonal anti-human p53 Ab was used at a
concentration of 0.5 µg/ml.
cAMP or inhibition of protein
kinase C by staurosporine decreased Rb protein expression in MCF-7
cells. These results suggest that Rb expression might be regulated by
phosphorylation, and Rb protein expression reflects the growth state of
the MCF-7 cells.
/100-mm dish) were treated with TNF-
, staurosporine
(1 nM) or Bt
cAMP (1 mM) separately for 16
h. Monoclonal anti-human Rb Ab was used at a concentration of 1
µg/ml. B, confluent MCF-7 cells (10
/100-mm
dish) were treated with H7 and Western immunoblot analysis using
monoclonal anti-human Rb Ab was carried
out.
affects
the expression level of another tumor suppressor Cip1 (p21) protein,
MCF-7 cells were treated with TNF-
for 16 h and Western immunoblot
analysis using polyclonal rabbit anti-human Cip1 (p21) Ab was carried
out. As shown in Fig. 8, there was a significant increase in
Cip1 (p21) protein expression level in response to TNF-
treatment.
This is surprising in that Cip1 (p21) protein is known to be induced by
DNA damaging agents such as UV and adriamycin, and requires functional
p53 protein. This result suggests that Cip1 (p21) protein plays a major
role in mediating antimitogenic effects of TNF-
in MCF-7 cells. In
addition to TNF-
, antimitogenic agents such as H7 and
staurosporine also induced Cip1 (p21) protein expression in MCF-7 cells
(data not shown). MCF-7 cells have readily detectable amounts of p53
protein and are also known to have functional p53 protein(42) .
To check whether TNF-
employs any specific kinase pathway,
TNF-
was added to the MCF-7 cells with staurosporine or
Bt
cAMP under SFM conditions and
[
H]thymidine incorporation assays were carried
out according to standard procedures. Neither Bt
cAMP nor
staurosporine was able to block the antimitogenic effect of TNF-
in MCF-7 cells (data not shown).
induces Cip1 (p21) protein in
MCF-7 cells. Confluent MCF-7 cells (10
/100-mm dish) were
treated with TNF-
(10 and 25 ng/ml each) for 16 h under SFM
conditions. Polyclonal rabbit anti-human Cip1 (p21) Ab was used at a
concentration of 0.5 µg/ml in Western immunoblot
analysis.
treatment, it was of interest to
determine whether Cip1 (p21) protein associated with cyclin D was also
increased. To check this, 50 µg of cell lysates from MCF-7 cells
treated with or without TNF-
(10 ng/ml) were immunoprecipitated
with protein A-Sepharose conjugated with polyclonal anti-human cyclin D
Ab and boiled samples were loaded onto 15% SDS-PAGE. As shown in Fig. 9, Cip1 (p21) protein interacts with cyclin D and the
amount of Cip1 (p21) protein associated with cyclin D was increased in
MCF-7 cells treated with TNF-
(10 and 25 ng/ml). Given the fact
that cyclin D remained constant regardless of the treatment, it is
possible that the decreased expression level of Rb protein in response
to TNF-
is due to the induction of Cip1 (p21) protein. Cip1 (p21)
protein is believed to be a part of cyclin D complex and an inhibitor
of cyclin-associated kinase activity. To check this possibility, MCF-7
cells were treated with TNF-
(10 ng/ml) for 16 h and Western
immunoblot analysis was carried out. Fig. 10A shows
that TNF-
induced both p53 and Cip1 (p21) proteins in MCF-7 cells.
on Cip1 protein
associated with cyclin D1 in MCF-7 cells. MCF-7 cells were treated with
TNF-
(10 and 25 ng/ml) for 16 h, and 50 µg of total cell
lysates were immunoprecipitated with protein A-Sepharose conjugated
with polyclonal anti-human cyclin D Ab. Immunoprecipitated samples were
loaded onto 15% SDS-PAGE, and Western immunoblot analysis using
polyclonal anti-human cyclin D (1 µg/ml) or anti-human Cip1 (p21)
Ab (1 µg/ml) was carried out.
/100-mm dish) were treated with TNF-
for 16 h and
Western immunoblot analysis was carried out. Monoclonal anti-human p53
and polyclonal anti-human Cip1 (p21) Ab were used at concentrations of
0.5 and 0.1 µg/ml, respectively. B, induction of p53
precedes that of Cip1 (p21) protein in MCF-7 cells. Confluent MCF-7
cells (10
/100-mm dish) were treated with TNF-
(10
ng/ml) for various times and cell lysates were prepared at each time
point. Western immunoblot analysis was carried out as in panelA.
is mediated through p53.
To determine the cellular molecules that interact with cyclin D, and
the possible effect of TNF-
on these interactions, MCF-7 cells
treated with TNF-
(10 ng/ml) were immunoprecipitated with
polyclonal anti-human cyclin D Ab conjugated with protein A-Sepharose.
These immunoprecipitates were loaded, and Western immunoblot analysis
was carried out. As shown in Fig. 11, TNF-
did not break
complexes formed between cyclin D and Cdk2 or Cdk4. There was no
complex formation between cyclin D and Rb protein in MCF-7 cells. These
results suggest that the decrease in Rb protein in response to
TNF-
might be due to the increase in Cip1 (p21) protein, which in
turn inhibits cyclin D-associated kinase activity. This decreased
kinase activity might play a role in mediating the antimitogenic effect
of TNF-
. No effect on these complexes was noted with TNF-
(Fig. 12).
(10 ng/ml) were
immunoprecipitated with polyclonal anti-human cyclin D Ab conjugated
with protein A-Sepharose. Immunoprecipitates were loaded for SDS-PAGE.
Western immunoblot analysis was carried out. Monoclonal anti-human Rb,
polyclonal anti-human Cdk2, and Cdk4 antibodies were used at
concentrations of 1 µg/ml.
shows antimitogenic effects in a
dose- and time-dependent manner in MCF-7 cells. TNF-
did not show
cytotoxic effects seen in other transformed and normal mammalian
cells(1, 2, 3, 4, 5) .
TNF-
decreased the fraction of cells in the S phase in cell cycle
analysis in a time- and dose-dependent manner. Based on cell
synchronization studies, it seems apparent that TNF-
acted at the
G
phase to prevent cells from entering S phase but did not
show effects once cells entered S phase. This antimitogenic effect of
TNF-
was efficiently blocked by excess concentrations of
monoclonal anti-human TNF-
Ab in MCF-7 cells in a dose-dependent
manner. This blocking effect was also confirmed by cell cycle analysis
and Western immunoblot analysis of Rb protein. This blocking experiment
also proves that TNF-
-mediated regulation of Rb protein expression
is specific in nature because TNF-
preincubated with monoclonal
anti-human TNF-
Ab did not decrease Rb protein expression in MCF-7
cells.
specifically acts at the G
phase
to prevent cells from entering S phase, it was necessary to check
whether TNF-
affects cell cycle-regulated proteins in MCF-7 cells.
Tumor suppressor Rb protein has been known to be involved in cellular
growth and differentiation and exists as hypo- and hyperphosphorylated
forms, depending on the growth state of the cells(49) . Its
role in G
to S progression in the cell cycle has been well
documented. Cell cycle-regulated proteins such as Rb protein showed
decreases in response to TNF-
treatment in a time- and
dose-dependent manner, whereas p53 and Cip1 (p21) protein expression
levels increased in response to TNF-
. This result suggests that
TNF-
-mediated antimitogenicity of MCF-7 cells specifically
involves regulation of Rb and p53 protein expression. We did not detect
two Rb protein bands frequently seen in normal mammalian cells. We
assume that the Rb protein band seen in MCF-7 cells is
hyperphosphorylated Rb protein and TNF-
acts by decreasing this
hyperphosphorylated form of Rb protein. Supporting this is the fact
that serine/threonine kinase inhibitor H7 increased mobility of Rb
protein in MCF-7 cells (Fig. 7B).
cyclin, like cyclin D protein, did not show decreased expression
levels in response to TNF-
treatment under normal culture
conditions using asynchronous cells. H7 and Bt
cAMP
decreased cyclin D expression. The expression level of cyclin D is
increased at mid- or late G
phase based on cell
synchronization studies. These results suggest that cyclin D protein
expression is governed by growth states of MCF-7 cells. These results
also suggest that other cell cycle-related proteins mediate the
antimitogenic effect of TNF-
in MCF-7 cells. Mitotic cyclin such
as cyclin A and B did not show apparent changes in their expression
levels in response to TNF-
treatment (data not shown). Other cell
cycle-related proteins such as Cdc2 (data not shown), Cdk2 (data not
shown), and Cdk4 (Fig. 3) did not show any change in their
expression levels in response to TNF-
treatment.
treatment in MCF-7 cells. Cip1 (p21) protein
associated with cyclin D was also increased in response to TNF-
in
MCF-7 cells (Fig. 9). These results provide valuable information
concerning the mechanism of TNF-
antimitogenicity in MCF-7 cells.
The induction of Cip1 (p21) protein was preceded by p53 protein
induction in MCF-7 cells (Fig. 10). This is consistent with the
fact that Cip1 (p21) protein induction needs functional p53 protein and
induction of p53 protein in response to DNA damaging agents. MCF-7
cells are known to have functional p53 protein(42) . The
induction of Cip1 (p21) protein may affect the expression level of Rb
protein in MCF-7 cells treated with TNF-
. Since Rb protein showed
a decrease in response to TNF-
treatment, it was necessary to
determine whether this is due to dissociation of cyclin D-Rb complex or
a decreased cyclin D-associated kinase activity. There was no complex
formation between cyclin D and Rb protein in MCF-7 cells. Therefore, it
is not likely that decreased Rb protein expression in MCF-7 cells is
due to breakdown of a cyclin D-Rb complex in MCF-7 cells. It was of
further interest to determine the effect of TNF-
on molecular
interactions between cyclin D and Cdk proteins since complexes between
cyclins and Cdks have been shown to play a major role in
phosphorylation of Rb protein in other mammalian cells. We detected
complexes of cyclin D with Cdk2 or Cdk4. However, TNF-
did not
have any effect on these complexes. The role of cellular oncogenes is
not clear at present. Studies of these cellular oncogenes will provide
further understanding of the mechanisms of TNF-
-mediated
antimitogenicity in MCF-7 cells. Thus, it would appear that TNF-
acts to increase p53 protein which in turn induces Cip1 (p21) to
decrease Rb protein expression with resultant antimitogenicity.
, tumor necrosis factor-
; Cip1, cell cycle inhibitory
protein; DMEM, Dulbecco's modified Eagle's medium; FACS,
fluorescence-activated cell sorter; FCS, fetal calf serum; H7,
1-(5-isoquinolinylsulfonyl)-2-methylpiperazine; H8, N-(2-methylaminoethyl)-5-isoquinolinesulfoneamide; HBSS,
Hank's balanced salt solution; PAGE, polyacrylamide gel
electrophoresis; TBS, Tris-buffered saline; TGF-
, transforming
growth factor-
; SFM, serum-free medium.
We are grateful to Wayne Douglas for technical
assistance. We thank Sigrid Whaley for help in preparation of the
manuscript.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.