Download Polyamines Regulate Growth Factor

Cancer Research and Treatment 2002;34(3):198-204
Polyam ines Regulate Growth Factor-Induced Protein
Phosphorylation in M CF-7 H um an Breast Cancer Cells
Ji Young Lee, Ph.D., Kyeong Hee Lee, Ph.D. and Byeong Gee Kim, Ph.D.
Division of Biological Sciences, College of Natural Sciences, Pusan National University, Busan, Korea
Purpose: Growth factors stim ulate protein phosphorylation resulting in transm ission of m itogenic signals. In
breast cancer, protein kinases and their substrate pro teins are importnat in cell proliferation and phathogenesis.
Polym ine is known as a m ediator of stim uli-induced
proliferation in m any cell system s. In the present study,
we report the im portance of polyam ines in protein phosphorylation in M CF-7 hum an breast cancer cells.
M aterials and Methods: Protein phosphorylation study
was done by incubating cells in the DMEM containing [γ32
P]-ATP. Quantitation of phosphorylation was analysed
by fluorescene image analyzer. Tyrosine phosphorylation
was detected by anti-phosphotyrosine antibody. Shc was
detected by radioim m unoprecipitation and W estern blotting.
Results: E 2 , TGF-α, and EGF enhanced the protein
phosphorylation in very similar pattern. Among those pro teins, 67 kDa protein was m ost strongly phosphorylated.
But the m ost prom inent tyrosine phosphoprotein was 52
kDa protein. DFM O at 5 m M strongly inhibited the phosphorylation of the m ost proteins. Externally added polyam ine could recover the inhibitory effect of DFM O in
protein phosphorylation. Am ong the 5 m ajor tyrosine
phosphoproteins, 52 and 46 kDa proteins appeared to be
Shc proteins.
Conclusion: Polyam ines m odulate signal transduction
in relation with estrogen receptor and EGF receptor
through multiple steps of protein phosphorylations. Tyro sine phosphorylation of Shc proteins were m ost significantly influenced by polyam ines in growth factor-stimulate breast cancer cell proliferation. (Cancer Research
and Treatm ent 2002;34:198-204)
ꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏ
Key W ords: Alpha-difluorom ethylornithine, Epiderm al
growth factor, Polyamines, Transforming
growth factor alpha, MCF-7 cells
Grb2 or Shc via its SH2 domain, to bind to phosphotyrosine
residue. Shc becomes phosphorylated on tyrosine residues in
response to growth factors and src family kinases (3). This
signaling protein is constitutively phosphorylated at tyrosine
residue (s) in a number of breast cancer cell lines but not in
a breast cell line derived from normal tissue (4). Tyrosine
phosphorylated Shc leads to the activation of p21 Ras, and
subsequent formation of the Shc/Grb2 complex plays a major
role in the breast cancer cell lines (5). Shc proteins were also
involved in transmitting the signal of HER2/neu in breast
cancer cell lines (6,7).
Polyamine is a mediator of stimuli-induced proliferation of
the cells in vitro and in vivo. Its contents increases massively
during the onset of proliferation in cells induced by a variety
of growth stimuli. Therefore polyamine biosynthes is a critical
event in cellular growth and tumor formation. Alpha-difluoromethylornithine (DFMO) is a highly specific and irreversible inhibitor of ornithine decarboxylase (ODC), which is
the first and rate limiting enzyme in polyamine biosynthesis (8).
Malignant breast tumors contain higher polyamine levels than
levels measured in normal mammary tissue.
Activation of the polyamine pathway by promoting several
key steps involved in proliferation causes the transition from
a hormone-dependent to a hormone-independent breast cancer
phenotype (9). Tamoxifen, an anti-estrogen, reduced ODC
mRNA and polyamine levels in MCF-7 cells (10). Recently,
INTRODUCTION
The pathogenesis and progression of human breast cancer
appear to involve a complex array of molecular events. One
such mechanism is the phosphorylation of the protein related
to growth signaling. The identification of specific kinase targets
is also important to understand the mechanisms that control cell
growth and oncogenesis.
Estrogen exerts its effects on cell growth by autocrine and
paracrine regulation of growth factors such as EGF, and IGF-I,
TGF-α, and PDGF. These growth factors and estrogen stimulate phosphorylation resulting in transmission of mitogenic
signals. Since several tyrosine kinase genes have been found
to be amplified in human breast cancer, the family of protein
kinase and intracellular substrates are important in the proliferation and pathogenesis in breast cancer (1,2).
Growth factor receptor activation enables the adaptor protein,
Correspondence: Byeong Gee Kim, Division of Biological Science,
College of Natural Science, Pusan National University, Busan
609-735, Korea. (Tel) 051-510-2286, (Fax) 051-581-2962,
(E-mail) [email protected]
Received December 18, 2001, Accepted April 29, 2002
198
Ji Young Lee, et al：Polyamines Regulate Protein Phosphorylation 199
it was suggested that polyamine may regulate the cell cycle
through protein phosphorylation.
In breast cancer cell lines it was suggested that signal
transduction by the growth factor and E2 may be modulated by
polyamine, but the exact mechanisms for this cross-talk are
poorly understood (11). Protein phosphorylation and dephosphorylation represent fundamental regulatory mechanisms of
cellular activity. Growth signaling of breast cancer was also
mediated in part by a signal cascade involving EGFR, c-src,
Shc, and MAP kinase.
Taken together, the intracellular level of an activated second
messenger protein is a useful prognostic indicator and therapeutic target in human breast cancer. Therefore, the purpose of
the present study is to identify polyamine-sensitive phosphoproteins in MCF-7 human breast cancer cells.
MATERIALS AND METHODS
1) Chemicals
TGF-α, EGF, 17β-estradiol, putrescine (tetramethylenediamine), spermidine (N-[3-aminopropyl]-1,4-butanediamine), and
spermine (N, N' bis-[3-aminopropyl]-1, 4-butanediamine) were
purchased from Sigma Chemical Co. [γ-32P]ATP (＞5000
mCi/mmol) was obtained from Amersham Korea Ltd. DFMO
was obtained from Dr. Levenson ILEX Oncology Inc. (San
Antonio, Tx). Anti-phosphotyrosine antibody 4G10 (mouse
monoclonal IgG) and anti-Shc (rabbit polyclonal IgG) were
purchased from Upstate Biotechnology. All other reagents were
of the highest grade commercially available.
2) Cell line and culture condition
MCF-7 cells obtained from the Korean Cell Line Bank were
grown in Dulbecco's modified Eagle's medium (DMEM) with
L-glutamine and 1,000 mg/L glucose containing 10,000 units/
ml penicillin G, 10 mg/ml streptomycin, and 10% heat-inactivated fetal bovine serum (FBS). The cell number was determined by Coulter Counter Z-1 (Coulter Corporation, Miami,
FL). For all experiments, cells were grown for 72 hr in medium
without phenol red prior to any treatment in order to avoid the
estrogenic effect of phenol red (12).
3) Phosphorylation assay
Cells were seeded at the density of 2×105 cells/ml in phenol
red free DMEM supplemented with 2% FBS, which was pretreated with dextran-coated charcoal (DCC) to remove the
endogenous hormone. After 72hr incubation, cultured cells were
treated with 10 nM 17β-estradiol (E2), 5 ng/ml TGF-α, 5
ng/ml EGF, or 5 mM DFMO.
For protein phosphorylation studies, the cultures were gently
washed twice in 50 mM tris-HCl (pH 7.4), and scraped with
a rubbed policeman in lysis buffer containing 50 mM tris-HCl
(pH 7.4), 5 mM EDTA, 5 mM dithiothreitol, 2 mM phenylmethylsulfonylfluoride (PMSF), and 125μM leupeptin. Cells were
then disrupted by sonication at 4oC in lysis buffer. Aliquots of
50μl (about 50μg protein) were pre-incubated in a final
volume of 0.1 ml of kinase buffer (10 mM MgCl2 and 20μM
ATP) containing the experimental treatments at 37oC for 2 min.
Phosphorylation was initiated with the addition of 10μl of 10
32
μCi [γ- P]ATP and terminated 1 min later by the addition
of Laemmli's stop solution. Proteins were separated on 10%
SDS-PAGE. The results were analyzed and the signal intensities were quantified by Fluorescent Image Analyzer
(FLA-2000, Fuji Photo Film, Japan) (see below).
4) Radioimmunoprecipitation of tyrosine phosphorylated
protein
Radioimmunoprecipitation assay (RIPA) was performed by
the method of Hartley et al. (13) with some modifications. For
each reaction, 32P-labeled lysates were diluted in RIPA buffer
with 1 mM PMSF and 10μg/ml aprotinin to obtain a final
volume of 200μl. The above sample was reacted with 20μl
of monoclonal anti-phosphotyrosine antibody (1：500) overo
night at 4 C with gentle rotation. Immune complexes were
precipitated with 200μl of pre-swelled Protein A Sepharose
(0.05 g protein A/ml RIPA buffer with 1 mg BSA) for an
o
additional 1.5 hr at 4 C. Bound immune complexes were
washed 4 times with 1.0 ml RIPA lysing buffer. The immune
complexes were dissociated from the beads by boiling in 50
μl SDS-PAGE sample buffer for 3 min. Supernatant was
separated by micro centrifugation for 2 min.
5) SDS-Polyacrylamide gel electrophoresis and fluorescence image analyzer system
Phosphorylated proteins were subjected to SDS-PAGE on
10% acrylamide gel using the method of Laemmli (14). Dried
gels were exposed to the Image Plate (Fuji Photo Film, Japan),
and scanned using the Image Reader program of FLA-2000.
The signal intensities were quantified with the Image Gauge of
FLA-2000.
6) Western blot analysis
Cells were homogenized in lysis buffer at 4oC, then the
homogenates were separated in 10% SDS-PAGE. Separated
proteins were transferred to a polyvinylidene difluoride filter
(PVDF) at 50 V for 2 hr, and blocked with 5% BSA in basic
blot buffer overnight ; 10 mM Tris, pH 7.5, 150 mM NaCl,
1.5 M NaN3, 1μM CaCl2. The membrane was incubated for
2 hr with a anti-rabbit Shc antibody (1：2,000) at 4oC. Antirabbit IgG (1：1,000) horseradish peroxidase-conjugated was
used as the second antibody. Following each incubation, the
membrane was washed thoroughly to remove unspecifically
bound antibodies for 5 min each, two times with only basic
blot buffer, and once with basic blot buffer with 0.25% NP-40.
Proteins were detected by an enhanced chemiluminescence
reagent using a commercial ECL kit (Amersham, Arlington
Heights, IL).
7) Statistical analysis
All experiments were carried out at least three times. The
data are presented as mean±standard deviation (SD). Statistical
significance of the difference between untreated control and
treated groups in protein phosphorylation was determined using
one-way analysis of variance (ANOVA), followed by the
Duncan's multiple range test. In all cases, a p value less than
0.05 was considered significant.
200
Cancer Research and Treatment 2002;34(3)
increments were statistically significant compared to the
untreated control (p＜0.05). Maximum stimulation of 116 kDa
protein phosphorylation was obtained at 5 ng/ml EGF treatment, which increased phosphorylation to 162% of the control.
While maximum stimulation of 52 kDa protein was obtained
at 5 nM E2-treatment to 164% of the control. DFMO at 5 mM
concentration strongly inhibited the phosphorylation of the most
proteins to less than the untreated background phosphorylation
at 5 min incubation. Especially, DFMO severely blocked the
phosphorylation of ER induced by E2, TGF-α, or EGF treatment. The administration of 5 mM DFMO decreased E2stimulated phosphorylation of ER to 25% of the control.
Among the major phosphoproteins, 35 kDa protein was not
affected by DFMO in the level of their phosphorylation in the
presence of E2, TGF-α, or EGF. To the contrary, phosphory-
RESULTS
1) Effects of DFMO on E2-, TGF-α-, and EGF-induced
phosphorylation in the whole cell extracts
E2, TGF-α, and EGF enhanced the protein phosphorylation
at 5 min incubation in very similar patterns. The enhancement
of protein phosphorylation by E2 was significantly greater than
by TGF-α and EGF treatment in the most proteins. The results
of quantification of phosphoproteins are shown in Table 1. The
phosphorylation of 67 kDa protein, which is known as ER, was
most prominently increased to 1.8 fold of the untreated control
in 5 min of E2-stimulation. TGF-α and EGF also gave
somewhat similar enhancement in ER phosphorylation. These
Table 1. Inhibition effect of DFMO on protein phosphorylation induced by E2, TGF-α, or EGF in the whole cell protein extracts (%
of the control)
ꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚ
Treatment
M.W. (kDa) ꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏ
E2
E2+DFMO
TGF-α
TGF-α+DFMO
EGF
EGF+DFMO
ꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏ
116
147±6*
32±2*†
135±8*
43±2*‡
162±29*
34±10*§
†
‡
104
131±2*
82±5*
129±17*
93±6
133±3*
97±12*§
†
‡
67
178±11*
25±5*
136±7*
29±4*
130±6*
32±8*§
†
‡
52
164±10*
42±3*
150±9*
48±4*
138±1*
32±6*§
†
‡
48
167±38*
46±10*
143±12*
49±4*
136±5*
35±7*§
†
‡
46
150±10*
32±12*
136±3*
34±5*
127±2*
40±17*§
†
‡
42
136±4*
45±13*
123±2*
52±11*
120±7
45±24*§
35
116±16*
118±13*†
95±9
120±25
97±10
106±6
ꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏ
The data presented here is the mean±SD of at least 3 separate experiments. *p＜0.05 vs. control, †p＜0.05 vs. E2, ‡p＜0.05 vs. TGF-α,
§
p＜0.05 vs. EGF
A
MW (kDa)
B
1
2
3
4
5
6
7
1
200
116
97
116
104
66
67
45
52
48
46
42
2
3
4
5
6
7
116
104
52
48
46
42
35
31
35
Fig. 1. Effect of DFMO on E2, TGF-α, and EGF-induced phosphorylation in the whole cell protein extracts.
(A) autoradiogram of total phosphoprotein. (B) autoradiogram of tyrosine phosphoprotein. Lane 1:
untreated control, 2: 10 nM E2, 3: E2+5 mM DFMO 4: 5 ng/ml TGF-α, 5: TGF-α+DFMO, 6: 5
ng/ml EGF, 7: EGF+DFMO. An equal amount of the membrane proteins were loaded on each lane.
The data presented here is the average of at least 3 separate experiments.
Ji Young Lee, et al：Polyamines Regulate Protein Phosphorylation 201
lation of 35 kDa protein was slightly increased with DFMO
treatment. But this increase was not statistically significant.
phosphorylation of 52 kDa protein was severely decreased to
44% of the untreated control and to 25% of TGF-α-induced
phosphorylation. In 116 kDa protein, E2, TGF-α, and EGF
significantly increased tyrosine phosphorylation but, its tyrosine
phosphorylation was increased by DFMO.
2) Effects of DFMO on E2-, TGF-α-, and EGF-induced
tyrosine phosphorylation
Effects of E2, TGF-α, or EGF on tyrosine phosphorylation
were very similar in the overall protein phosphorylation pattern
(Fig. 1B). The tyrosine phosphorylations of two prominent
proteins (52 and 48 kDa) was extremely enhanced by these
treatments. The tyrosine phosphorylation of 52 kDa protein was
increased to a maximum 204% of control by the treatment of
10 nM E2 (Table 2). The maximum stimulation of TGF-α was
obtained at 48 kDa protein, which increased the tyrosine
phosphorylation to 186% of the untreated control. Even though
the total phosphorylation of 67 kDa protein was enhanced by
E2, TGF-α, or EGF treatment, its tyrosine phosphorylation was
not detected at all. Tyrosine phosphorylations of most proteins
were severely inhibited by the treatment of DFMO in E2, TGFα, or EGF treatments. Particularly, by 5 mM DFMO, tyrosine
3) Effects of polyamines in protein phosphorylation
induced by E2, TGF-α or EGF
Externally added putrescine did not abolish the inhibitory
effects of DFMO on enhanced total phosphorylation induced by
E2. However, putrescine fully recovered the inhibitory effect of
DFMO on enhanced tyrosine phosphorylation induced by E2
(Fig. 2). The same results were also found in spermidine and
spermine treatment. All three polyamines added along with E2
and DFMO could reverse the inhibitory effect of DFMO on
phosphorylation of the most protein in a dose dependent
manner (data not shown). The same recovery effects of polyamines were found in EGF and DFMO co-treated cells. Even
at as low as 0.1 mM, putrescine could reverse the tyrosine
Table 2. Effect of DFMO on tyrosine phosphoprotein induced by E2, TGF-α or EGF in the whole cell protein extracts (% of the control)
ꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚ
Treatment
M.W. (kDa) ꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏ
E2
E2+DFMO
TGF-α
TGF-α+DFMO
EGF
EGF+DFMO
ꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏ
116
137±10*
152±17*
146±4*
159±16*
142±8*
151±17*
52
204±37*
55±6*†
175±21*
44±7*c
157±23*
65±6*§
†
c
48
154±6*
64±11*
186±16*
59±12*
154±23*
65±6*§
†
c
46
152±8*
68±2*
194±28*
72±1*
134±12*
64±13*§
†
c
35
109±9
191±15*
104±4
168±11*
110±11
162±12*§
ꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏ
32
P-labeled tyrosine phosphorylated proteins were analyzed in RIPA using anti-phosphotyrosine antibody. The data presented here is the
mean±SD of at least 3 separate experiments. *p＜0.05 vs. control, †p＜0.05 vs. E2, ‡p＜0.05 vs. TGF-α, §p＜0.05 vs. EGF
A
B
MW (kDa)
1
2
3
4
5
6
1
2
3
4
5
6
200
116
97
116
104
66
67
45
52
48
46
42
116
104
52
48
46
Fig. 2. Reversal of DFMO-altered protein phosphorylation by putresine (% of the untreated control). (A)
autoradiogram of total phosphoprotein (B) autoradiogram of tyrosine phosphoprotein. Bars on the left
of gel represent M.W. standards (see Fig. 1). Lane 1: untreated control, 2: 10 nM E2, 3: E2+5 mM
DFMO 4∼6: E2+DFMO+putrescine (0.1, 0.5, and 1 mM, respectively). The data presented here is
the average of at least 3 separate experiments. +p＜0.05 vs. control, #p＜0.05 vs. treatment (E2, TGF-α,
or EGF), *p＜0.05 vs. DFMO.
202
Cancer Research and Treatment 2002;34(3)
phosphorylations of these proteins up to EGF-stimulated levels.
Tyrosine phosphorylation in both 52 and 48 kDa proteins were
also recovered by all three polyamines in E2, TGF-α, and EGF
co-treated with DFMO (Fig. 3). The inhibitory effect of DFMO
in tyrosine phosphorylations of 52 and 48 kDa proteins could
be completely blocked by 1 mM spermidine cotreated with
TGF-α.
A
kDa
B
1
2
3
4
200
P52 Shc
116
97
P46 Shc
4) Identification and phosphorylation of Shc protein
To characterize the three low molecular weight proteins of
52, 48 and 46 kDa of the whole cell extracts, immunoprecipitation was performed with anti-Shc antibody. As shown in
Fig. 4A, 52 and 46 kDa proteins appeared to be Shc proteins.
It was further examined by Western blotting using anti-Shc
antibodies. Consistently with the data from immunoprecipitation analysis, 52 and 46 kDa proteins were precipitated by
the anti-Shc antibodies (Fig. 4B). Fig. 5 shows the change of
phosphorylation levels of two isoforms in each experimental
treatment. In the case of 52 kDa isoform, tyrosine phosphorylation was increased to 204% of the control by E2 treatment
with maximal level. Also, tyrosine phosphporylation of 46 kDa
protein was enhanced to 194% of control by a maximum level
Fig. 3. Polyamines reverse the inhibitory effects of DFMO (5 mM)
on E2 (10 nM)-,TGF-α (5 ng/ml)-, or EGF (5 ng/ml)induced tyrosine phosphorylation of the 52 and 48 kDa
proteins. Cont: untreated control, 1, 4, 7α E2+DFMO+
1.0 mM put, spd, spm, 2, 5, 8: TGF-α+DFMO+1.0 mM
put, spd, spm, 3, 6, 9: EGF+DFMO+1.0 mM put, spd,
spm. The data presented here is the average of at least 3
separate experiments. *p＜0.05 vs. control, †p＜0.05 vs.
E2, ‡p＜0.05 vs. DFMO.
65
P52 Shc
45
P46 Shc
Fig. 4. Identification of Shc protein using anti-Shc antibodies in
MCF-7 cells. (A) SDS-acrylamide gel of mmunoprecipitate.
Lane 1: molecular weight standard, 2: untreated whole cell
protein extracts, 3: immunoprecipitate by anti-Shc antibody
(1：1000), 4: immunoprecipitate by anti-Shc antibody (1：
500). (B) Western blot of Shc protein by anti-Shc antibody
(1：2000). The Shc proteins (p52 and p46) are indicated
by arrows.
Fig. 5. Shc protein phosphorylation in E2 (10 nM), TGF-α (5
ng/ml), EGF (5 ng/mL), with or without DFMO (5 mM)
treatments. *p＜ 0.05 vs. control: †p＜0.05 vs. E2, ‡p＜
0.05 vs. TGF-α, §p＜0.05 vs. EGF.
Ji Young Lee, et al：Polyamines Regulate Protein Phosphorylation 203
of stimulation. DFMO administration markedly decreased the
total phosphorylation as well as tyrosine phosphorylation in
both proteins.
DISCUSSION
Protooncogenes are important in normal cell proliferation and
differentiation. Oncogenes originally arose from protooncogenes. Overexpression or certain mutations of protooncogenes
result in transformation. Several tyrosine kinase genes have
been found to be amplified in human breast cancer.
DFMO inhibits ODC by binding to its active site, thereby
preventing polyamine synthesis and cell proliferation. Previous
studies have shown that growth of breast cancer cell lines were
inhibited by DFMO in culture. In the present study, the
phosphorylation of 116, 104, 67, 52, 48, 46 and 42 kDa
proteins were significantly increased by E2 TGF-α or EGF
stimulation. DFMO, a specific inhibitor of ODC, significantly
inhibited E2-, TGF-α-, and EGF-induced proteins phosphorylations, except 35 kDa one of which phosphorylation was rather
slightly enhanced. Exogenous polyamines overcame the inhibitory effect of DFMO in most of proteins. The above results
explain that DFMO inhibits E2-, TGF-α, and EGF-induced cell
proliferations through the reduction of protein phosphorylation.
Several laboratories have investigated the role of polyamines
as mediators of estradiol-stimulated growth of several human
breast cancer cell lines (9,15). Therefore, our data suggest that
polyamine acts as a modulator in growth signaling pathways
of breast cancer through protein phosphorylation.
Among there proteins, 116, 52, 48 and 42 kDa proteins were
found to be the most prominent of the tyrosine phosphoproteins. Tyrosine phosphorylations of 52, 48 and 42 kDa
proteins were significantly decreased by DFMO treatments.
However, 116 kDa protein was mildly increased by DFMO.
Polyamine administration reversed the effect of DFMO on
phosphorylation of 52, 48 and 46, kDa proteins. Most human
breast tumors start as estrogen-dependent, but during the course
of the progression become refractory to hormone therapy. The
transition of breast cancer from estrogen dependent to
independent behavior may be regulated by autocrine and/or
paracrine growth factors that are independent of the ER. The
proteins of 116, 52, 48 and 42 kDa may be related in that
signaling pathway.
There is a lot of evidence that shows polyamines are
involved in protein phosphorylation. Polyamine regulated retinoblastoma protein phosphorylation (16) and regulated protein
tyrosine phosphorylation in the effect of green tea polyphenols
on Ehrlich ascites tumor cells in vitro (17). Polyamine appeared
to increase the protein phosphorylation through the regulation
of polyamine-dependent protein kinase.
To investigate the DFMO-modulated prominent phosphoprotein (52, 48 and 46 kDa), in the whole cell extracts, western
blot analysis and immunoprecipitation assay were performed
using anti-Shc antibodies. The results showed that p52 and p46
proteins are Shc adaptor proteins. Tyrosine phosphorylation of
p52 isoform was increased to 204% of the untreated control
by the treatment of 10 nM E2 and decreased to 44% of the
untreated control by 5 mM DFMO. Tyrosine phosphorylation
of p52 isoform was increased to maximum 193% of control by
the treatment of TGF-α, but DFMO at 5 mM inhibited it to
25% of TGF-α induction. Exogenous polyamines completely
overcame the inhibitory effects of DFMO in p52 and p46
adaptor proteins. Reversal effects of three polyamines were
varied in each proteins and different experimental treatments.
Shc has three isoforms which are all encoded by the same
gene. Because of the differential use of translational initiation
sites and alterative splicing, the three Shc isoforms differ in the
extent of their amino-terminal sequences (18). p66 isoform
tends to be variably expressed, and has recently been reported
to inhibit at least some of the responses normally stimulated
by the p52 and p46 isoforms (19). The carboxy-terminus has
a single src homology 2 domain (SH2) and is one region where
Shc binds to phosphorylated tyrosine kinases (18). The
amino-terminus contains a phoshpotyrosine binding domain
with binding specificity distinct from the SH2 domain.
Shc adaptor signaling protein is constitutively tyrosine
phosphorylated in a number of breast cancer cell lines but not
in a breast cell line derived from normal tissue. In the present
study, polyamines regulate the phosphorylations of p52 and p46
Shc signaling proteins. Our results indicate that Shc is involved
in signaling pathway of E2 and EGFR through elevated
phosphorylation in MCF-7 cells. This result also suggests that
polyamines may be involved in growth signaling pathways of
E2 and TGF-α or EGF through the regulation of p52 and p46
Shc protein phosphorylation in MCF-7 human breast cancer
cells.
MCF-7 cells contained higher amounts of 52 and 46 Shc than
66 kDa Shc. In the present study, 66 isoform was difficult to
detect because the low amount of protein expressed. In
disagreement with our finding, Janes et al. (20) detected
constitutively phosphorylated Shc only in the highest erbB-2
overexpressor, the BT-474 cells, and not in SKBR-3 or MCF-7
cells. In erbB-2 overexpressed breast cancer cell line, Shc may
modulate signal of erbB-2. Although MCF-7 cells have erbB-2
amount not much higher than normal receptor levels (21), Shc
phosphorylation clearly could be regulated by E2, TGF-α, or
EGF in the present study. It has been reported that phosphorylated Shc in erbB-2 high expressed SKBR-3 cells and
erbB-2 low expressed MCF-7 cells (7).
Different Shc isoforms may exert differential physiological
functions. ErbB-2 is highly expressed in SKBR-3, MDAMB-453, and BT-474 cells. Although, p66 isoform is downregulated, both p52 and p46 isoforms were highly expressed in
erbB2-overexpressing breast cancer cell lines. p52 and p46,
without p66, appear to be sufficient to transmit the oncogenic
signal of the activated erbB2 in breast cancer cells. Insulindependent Shc signaling pathways occur through the p52
isoform, whereas the EGF receptor displays similar specificity
for both the p52 and the p46 forms (22). The protein of 42
kDa, in addition to the p52 and p46 isoforms, was also detected
by immunoprecipitation assay with anti-Shc antibody. Shc
binds to activated receptor tyrosine kinase and molecules
containing SH2 domain (5). Thus, 42 kDa protein may be a
subfraction of Shc containing SH2 domain.
204
Cancer Research and Treatment 2002;34(3)
CONCLUSIONS
Our findings suggest that polyamine may modulate signal
transduction in relation with ER and/or EGFR through multiple
phosphoproteins, such as Shc proteins. The results from this
preliminary work can be used further to elucidate the precise
roles of polyamine in the novel cross-talk between estrogen and
growth factor signaling pathway.
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