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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. REFERENCES 1. Cance WG, Craven RJ, Weiner TM, Liu ET. Novel protein kinases expressed in human breast cancer. 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