Cancer Research The Future of Cancer Research: Science and Patient Impact Cancer Health Disparities Conference 2009
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Yoshida, J.
Right arrow Articles by Nishio, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yoshida, J.
Right arrow Articles by Nishio, M.
[Cancer Research 61, 6151-6157, August 15, 2001]
© 2001 American Association for Cancer Research

Experimental Therapeutics

Growth-inhibitory Effect of a Streptococcal Antitumor Glycoprotein on Human Epidermoid Carcinoma A431 Cells

Involvement of Dephosphorylation of Epidermal Growth Factor Receptor

Junko Yoshida1, Takaharu Ishibashi and Matomo Nishio

Department of Pharmacology, Kanazawa Medical University, Ishikawa 920-0293, Japan


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
An antitumor glycoprotein [streptococcal acidic glycoprotein (SAGP)] purified from an extract of Streptococcus pyogenes inhibited the growth of human epidermoid carcinoma A431 cells overexpressing epidermal growth factor receptor (EGFR) in a time- and a concentration-dependent manner. The antiproliferative effect of SAGP was diminished by preincubating the cells with pertussis toxin and by coadministration of sodium orthovanadate, an inhibitor of protein tyrosine phosphatases (PTPases). Western blot analysis showed that the immunoreactivity of a Mr 170,000 band of cell lysate to antiphosphotyrosine antibody was reduced by SAGP, and the effect was abolished by sodium orthovanadate. The phosphotyrosine level of the precipitant with anti-EGFR antibody was reduced by SAGP, which was abolished by preincubation with pertussis toxin or by a coadministration with sodium orthovanadate. The PTPase activity transiently increased in the lysate of cells incubated with SAGP and was inhibitable by sodium orthovanadate. Additionally, preincubation of serum-starved A431 cells with SAGP decreased the epidermal growth factor-induced tyrosine phosphorylation of EGFR, and the effect of SAGP was sodium orthovanadate sensitive. These findings indicate that dephosphorylation of the Mr 170,000 EGFR by activation of PTPase(s) may be responsible in part for the antiproliferative effect of SAGP on A431 cells.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The Streptococcus pyogenes Su strain, from which immunomodulating agent OK-432 (1) was prepared, provides a novel antitumor SAGP2 as reported previously (2 , 3) . SAGP is a glycoprotein with an apparent molecular weight of 140,000–150,000 that consists of identical subunits with a molecular weight of 48,000. This protein has been shown to prolong the life span of mice inoculated i.p. with Ehrlich ascite carcinoma cells (2) or murine Meth A cells (4) . The life-prolonging effect of SAGP on the mice inoculated with Ehrlich ascite carcinoma cells or Meth A cells is known to be reduced by immunosuppression with X-ray irradiation or antimacrophage agent carrageenan injection, indicating that the antitumor effect of SAGP depends in part on the activation of the host immune system. Indeed, an in vitro assay revealed considerable cytostatic activity of spleen cells (carrageenan sensitive and/or asialo GM1 positive) from Meth A-inoculated and SAGP-injected mice against Meth A cells (3 , 4) .

On the other hand, the direct inhibitory effect of SAGP on cell proliferation has been reported on transformed hamster embryonic lung cells (2) , murine leukemia L1210 cells (5) , and Meth A cells (6 , 7) in culture. Sulfhydryl groups on SAGP appear to be essential for the expression of SAGP activity because the cell growth-inhibitory effect on Meth A cells was reduced by sulfhydryl-oxidizing agents such as cystamine and 5,5'-dithio-bis(2-nitrobenzoic acid) (6) . The activation of IAP-sensitive GTP-binding protein (G protein) is known to be required for expression of the antiproliferative effect of SAGP, and the inhibition of nucleic acid synthesis may contribute to the direct effect of SAGP (7) . Although the cAMP level in Meth A cells exposed to SAGP was reduced slightly (7) , it was unlikely that the reduced cAMP levels contributed to the SAGP-induced cell growth inhibition because dibutyryl-cAMP or a cAMP phosphodiesterase inhibitor could not reverse the SAGP activity.3

Our recent study with Meth A cells revealed that the antiproliferative effect of SAGP was diminished by sodium orthovanadate, an inhibitor of PTPases, but not by a serine/threonine phosphatase inhibitor (8) . Western blot analysis revealed that immunoreactivity of a Mr 170,000 cellular protein to antiphosphotyrosine Ab was reduced in the cells incubated with SAGP, and the effect was abolished by sodium orthovanadate (8) . These findings led us to a working hypothesis that SAGP binds to an IAP-sensitive G protein-coupled unknown receptor and activates PTPases, resulting in a decrease of tyrosine phosphorylation of the Mr 170,000 cellular protein. It was suggested that the dephosphorylated Mr 170,000 cellular protein may be a growth factor receptor because some studies have demonstrated that stimulation of G protein-coupled receptor activates intracellular PTPases (9 , 10) and that PTPases directed to growth factor receptors inhibit the autophosphorylation of growth factor receptors (11, 12, 13) . Therefore, we intended to identify the Mr 170,000 protein, to confirm the above-mentioned hypothesis, and to further elucidate the mechanism of the antiproliferative action of SAGP. Although most previous studies have been performed with Meth A cells, we used human epidermoid carcinoma A431 cells here because this cell line is known to overexpress EGFR, and a variety of immunological tools such as Abs to the cell growth signaling components are commercially available.

Similar to the results obtained from Meth A cells, the growth of A431 cells was inhibited by SAGP in a concentration- and a time-dependent manner. The antiproliferative effect of SAGP was reduced by preincubating the cells with IAP or by coadministration of sodium orthovanadate. In this study, we identified the Mr 170,000 protein of A431 cells as an EGFR and showed that stimulation of the IAP-sensitive G protein-coupled receptor by SAGP caused dephosphorylation of the EGFR via an activation of PTPases, leading to the inhibition of cell growth.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Preparation of SAGP.
SAGP was prepared as described previously (2 , 7) .

Tumor Cell Lines.
The human epidermoid carcinoma A431 cells were kindly supplied by Prof. Katsuzo Nishikawa (Second Department of Biochemistry, Kanazawa Medical University) and cultured in DMEM containing 10% heat-inactivated FBS, 2 mM L-glutamine, 12.7 mM HEPES, 0.12% sodium bicarbonate, 100 units/ml penicillin G, and 100 µg/ml streptomycin at 37°C in humidified air containing 5% CO2. Cells were seeded at 3 x 105 cells/plate in 10-cm diameter plastic culture dishes and passaged every 3–4 days. The doubling time of A431 cells at densities of 3–30 x 104 cells/ml was 22–24 h.

Cell Growth Assay.
A431 cells were seeded at 3 x 104 cells/ml in 35-mm-diameter culture dishes (2.5 ml/plate). After an overnight attachment phase, the DMEM containing 10% FBS was refreshed, and SAGP (0.03–1.0 µg protein/ml) or the same volume of PBS alone was added to the wells. Following the specified times of incubation, the number of cells was determined by trypan blue dye exclusion after trypsinization. The cell growth rate (percentage of control) was expressed as the percentage of the number of the cells in the wells with SAGP: the number of cells in the control wells.

Stimulation and Extraction of Cells.
A431 cells were plated at 3 x 104 cells/ml in 100-mm-diameter culture plates (10 ml/plate). After an overnight attachment phase, the DMEM containing 10% FBS was refreshed, and the cells were incubated with or without SAGP (0.1 and 0.3 µg protein/ml) for 48 h. The stimulated cells were washed twice with PBS and lysed in 1 ml of radioimmunoprecipitation assay buffer (PBS containing 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM phenylmethylsulfonyl fluoride, 1 mM Na3VO4, and 57 µg/ml aprotinin) by repeated passage through a 23-gauge needle. The lysates were centrifuged at 12,500 x g for 20 min at 4°C to remove insoluble material. After protein concentrations were determined with Bio-Rad protein assay reagent (Bio-Rad Laboratories), the cell lysate was used for Western blot analysis or immunoprecipitation with anti-EGFR Ab as described below. For Western blot analysis on total cell lysate, an equal amount of cell lysate (10–25 µg of protein) was denatured in the same volume of 2-fold concentrated Laemmli electrophoresis buffer [250 mM Tris-HCl, 4% SDS, 10% glycerol, 0.006% bromphenol blue, and 2% 2-mercaptoethanol (pH 6.8)]. The samples were used immediately or stored at -30°C until Western blot analysis.

Immunoprecipitation with Anti-EGFR Ab.
An equal amount of cell lysate (300–500 µg of protein) was immunoprecipitated with a monoclonal anti-EGFR Ab (1 µg Ab/100 µg protein; Upstate Biotechnology) by overnight incubation at 4°C, and 20 µl of protein G PLUS-agarose (Santa Cruz Biotechnology) were added and incubated for 2 h at 4°C. The gels were washed three times with 1 ml of radioimmunoprecipitation assay buffer, denatured in 30 µl of 2-fold concentrated Laemmli electrophoresis buffer, and used immediately or stored at -30°C until Western blot analysis.

Western Blot Analysis.
The A431 cell lysate or immunoprecipitated EGFR was separated by 7.5% SDS-PAGE and transferred to a nitrocellulose membrane (Hybond ECL; Amersham Pharmacia Biotech) using transfer buffer [25 mM Tris, 192 mM glycine, and 20% (v/v) methanol] at 2 mA/cm2 for 40 min. The membrane was blocked with 5% BSA in PBS-T and subsequently probed for 1 h at room temperature with an antiphosphotyrosine monoclonal Ab (clone 4G10; Upstate Biotechnology) at 0.3 µg/ml in 5% BSA/PBS-T. Following a wash with PBS-T, the membrane was incubated with antimouse IgG conjugated to horseradish peroxidase (Santa Cruz Biotechnology) and diluted in 5% nonfat milk in PBS-T at room temperature for 1 h. Bound horseradish peroxidase was visualized with ECL Western blotting detection reagents (Amersham Pharmacia Biotech) by exposure to X-ray films (XAR-5; Eastman Kodak Company). To examine the content of EGFR in some experiments, EGFR precipitates from half of each cell lysate were analyzed by Western blot with anti-EGFR Ab (Upstate Biotechnology). Quantification of bands was performed by densitometric scanning of the films and analysis using the NIH Image 1.61 program.

Assay of PTPase Activity.
PTPase activity was measured using a nonradioactive kit from TaKaRa Biochemicals (Shiga, Japan). A431 cells were plated at 3 x 104 cells/ml in 45-mm-diameter culture plates (5 ml of medium). After 24 h of incubation, the DMEM containing 10% FBS was refreshed, and the cells were incubated with SAGP or PBS alone for various times. Preparation of cell lysate and assay of PTPase activity were carried out according to the manufacturer’s procedure. Briefly, the cells were lysed in 0.3 ml of cell lysis buffer and centrifuged at 10,000 x g for 10 min at 4°C. The cell lysate was diluted at 1:100 with PTPase assay buffer. The lysate dilution (50 µl) was transferred to a microplate well, where the phosphorylated substrate [(Glu, Tyr) polymer] was immobilized, and incubated for 40 min at 37°C. After washing the wells with PBS containing 0.05% Tween 20, the reaction was blocked for 30 min at 37°C with 100 µl of blocking buffer. The fraction of dephosphorylated substrate was determined immunochemically with antiphosphotyrosine Ab (PY-20) conjugated to peroxidase. The absorbance was measured at 450 nm using a microplate reader. The results from duplicate wells for each sample were compared with a standard curve with CD45 (Calbiochem-Novabiochem) and expressed as phosphate released/min/µg protein.

Assay for EGF-induced EGFR Signaling.
To ascertain the effect of SAGP on the EGF-induced EGFR phosphorylation and downstream elements involved in EGF-induced mitogenic signaling, A431 cells were incubated in DMEM containing 10% FBS for 2 days and then incubated for 24 h in DMEM without FBS. The serum-starved A431 cells were preincubated with or without SAGP for the indicated time at 37°C and then stimulated with 5 ng/ml EGF (Sigma Chemical Co.) for 5 min at 37°C and lysed. Tyrosine phosphorylation of EGFR was assayed by Western blotting of immunoprecipitated EGFR with antiphosphotyrosine Ab (clone 4G10) as described above. To look at the effect of SAGP on p42/44 MAPK, the total cell lysate (25 µg of protein) was resolved by SDS-PAGE (10% gels). A Western blot was probed with an Ab that recognizes phospho-p42/44 MAPK (New England Biolabs). To control the amount of p42/44 MAPK, the blot was stripped and reprobed with a goat polyclonal anti-p42 MAPK Ab (Santa Cruz Biotechnology) that reacts with p42 MAPK and, to a lesser extent, with p44 MAPK. Antimouse or antigoat IgG conjugated to horseradish peroxidase (Santa Cruz Biotechnology) was used as the secondary Ab, and proteins were visualized with ECL-PLUS Western blotting detection reagents (Amersham Pharmacia Biotech).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Growth-inhibitory Action of SAGP on A431 Cells.
SAGP inhibited the growth of A431 cells in a concentration- and time-dependent manner (Fig. 1, a and b)Citation . The median inhibitory concentration (IC50) of SAGP on the growth of A431 cells after 3 days of incubation was 0.08 µg protein/ml. The growth inhibition rate increased gradually up to day 3.



View larger version (13K):
[in this window]
[in a new window]
  		Fig. 1. a, growth-inhibitory effect of SAGP on A431 cells in culture. The cells (3 x 104 cells/ml) were incubated in the absence or presence of SAGP for 3 days. The values are the mean ± SE of six cultures from three separate experiments. b, the time course of the effect of SAGP on A431 cell growth. A431 cells (3 x 104 cells/ml) were incubated in the absence or presence of SAGP for 3 days. SAGP (0.1 or 0.3 µg protein/ml) was added to the cultures on day 0. The values are the mean ± SE of four cultures from two separate experiments.

 
Effects of IAP, Sodium Orthovanadate, and Catalase on the Growth-inhibitory Effect of SAGP.
Similar to the result obtained with the Meth A cell line (7) , pretreatment of A431 cells with 100 ng/ml IAP (Seikagaku, Tokyo, Japan) for 6–16 h decreased the antiproliferative effect of SAGP (Fig. 2)Citation . Sodium orthovanadate, an inhibitor of PTPases, significantly diminished the growth-inhibitory activity of SAGP on A431 cells, although sodium orthovanadate by itself had a toxic effect on the cells in a concentration-dependent manner (Fig. 3)Citation . Sodium fluoride (1 mM), a serine/threonine phosphatase inhibitor, did not affect the growth-inhibitory action of SAGP (data not shown). However, the growth-inhibitory effect of SAGP was augmented by coadministration of catalase (Fig. 4)Citation . The augmentation by catalase has also been observed in Meth A cells.3



View larger version (15K):
[in this window]
[in a new window]
  		Fig. 2. Effect of IAP on the growth-inhibitory effect of SAGP on A431 cells. The cells were preincubated with 100 ng/ml IAP for 6–16 h before incubation with SAGP (0.1 and 0.3 µg protein/ml). Percentage growth rates were determined on day 3. Values are expressed as the mean ± SE of six cultures from three separate experiments. Statistical analyses were performed by unpaired Student’s t test (*, P < 0.05 versus 0.1 µg protein/ml SAGP alone; #, P < 0.05 versus 0.3 µg protein/ml SAGP alone).

 


View larger version (20K):
[in this window]
[in a new window]
  		Fig. 3. Effect of sodium orthovanadate on the growth-inhibitory effect of SAGP on A431 cells. The cells were incubated with SAGP (0.1 and 0.3 µg protein/ml) in the presence of sodium orthovanadate (10 and 30 µM). Percentage growth rates were determined on day 3. Values are expressed as the mean ± SE of six cultures from three separate experiments. Growth rates in parentheses are expressed as a percentage of each control (without SAGP). Statistical analyses of the variables in the groups incubated with 0.1 and 0.3 µg protein/ml SAGP were performed by one-way ANOVA followed by Fisher’s PLSD, respectively (*, P < 0.05 versus 0.1 µg protein/ml SAGP alone; #, P < 0.05 versus 0.3 µg protein/ml SAGP alone).

 


View larger version (16K):
[in this window]
[in a new window]
  		Fig. 4. Effect of catalase on the growth-inhibitory effect of SAGP on A431 cells. The cells were incubated with SAGP (0.06 and 0.1 µg protein/ml) in the presence of 100 units/ml catalase. Percentage growth rates were determined on day 3. Values are expressed as the mean ± SE of six cultures from three separate experiments. Statistical analyses were performed by unpaired Student’s t test (*, P < 0.05 versus 0.06 µg protein/ml SAGP alone; #, P < 0.05 versus 0.1 µg protein/ml SAGP alone).

 
Tyrosine Phosphorylation of Cellular Proteins in A431 Cells.
Western blot analysis on the cell lysate using antiphosphotyrosine Ab revealed that a 48-h exposure of cells to SAGP (0.1 and 0.3 µg protein/ml) caused a decrease in phosphotyrosine immunoreactivity at Mr 170,000 cellular protein. No differences in the phosphotyrosine level between samples from SAGP-treated or PBS-treated cells were seen in the presence of 30 µM sodium orthovanadate (Fig. 5)Citation . This suggests that the reduction in the phosphotyrosine level of Mr 170,000 protein may be due to the activation of PTPases by SAGP.



View larger version (49K):
[in this window]
[in a new window]
  		Fig. 5. The effect of SAGP on tyrosine phosphorylation of cellular protein in A431 cells. A431 cells were incubated with SAGP (0.1 and 0.3 µg protein/ml) in the presence or absence of sodium orthovanadate (30 µM) for 48 h, and the cell lysate was subjected to Western blot analysis by antiphosphotyrosine Ab. EGF-stimulated A431 cell lysate (10 µg; Upstate Biotechnology) was used as a positive control to antiphosphotyrosine Ab. The Mr 170,000 band in EGF-stimulated A431 cell lysate represents the tyrosine-phosphorylated EGFR in response to a stimulation with EGF. Positions of the Mr 170,000 band and the size markers are shown. Similar results were obtained from three independent experiments.

 
Tyrosine Phosphorylation of Immunoprecipitated EGFR.
The phosphotyrosine level of immunoprecipitated EGFR was decreased in the cells incubated with SAGP for 48 h in the presence of 10% FBS, whereas the content of EGFR immunoprecipitated from an equal amount of cell lysate was not changed by incubating the cells with SAGP for 48 h (Fig. 6a)Citation . The effect of SAGP was abolished by coadministration of 30 µM sodium orthovanadate, although sodium orthovanadate itself stimulated basal tyrosine phosphorylation (Fig. 6b)Citation . Pretreatment of cells with IAP (100 ng/ml) for 6–16 h diminished the inhibitory effect of SAGP on tyrosine phosphorylation of EGFR (Fig. 6b)Citation . Fig. 6cCitation shows the average (percentage of control) signal intensity on scanning densitometry, demonstrating that the SAGP-induced dephosphorylation of EGFR was abolished by sodium orthovanadate and inhibited in part by IAP, although quantification of the signal by scanning densitometry is nonlinear and therefore generates numbers that cannot be strictly interpreted. Neither IAP nor sodium orthovanadate alone affected EGFR protein levels (data not shown).



View larger version (26K):
[in this window]
[in a new window]
  		Fig. 6. The effect of SAGP on tyrosine phosphorylation of EGFR. a, A431 cells were incubated with SAGP (0.1 and 0.3 µg protein/ml) for 48 h, and the immunoprecipitated EGFRs were analyzed by Western blot with antiphosphotyrosine Ab or anti-EGFR Ab. The relative phosphotyrosine level of EGFR to its protein (percentage of control) is shown. b, effects of a coadministration of 30 µM sodium orthovanadate and a pretreatment of the cells with 100 ng/ml IAP on the dephosphorylation of EGFR by SAGP. c, quantification of the phosphotyrosine level of EGFR. Statistical analyses of the variables (mean ± SE) of each group were performed by one-way ANOVA followed by Fisher’s PLSD (*, P < 0.05 versus control).

 
PTPase Activity.
When A431 cells were exposed to SAGP (0.3 µg protein/ml) for various times in the presence of 10% FBS, significant increases in PTPase activity were detected 1 and 2 min after the addition of SAGP. No differences in the PTPase activity between control and SAGP-stimulated cells were seen at 5, 10, 30, and 60 min of incubation, although control PTPase activity tended to increase gradually and at 60 min reached 150% of that at 1 min (Fig. 7a)Citation . The SAGP-induced stimulation of PTPase activity at 2 min was inhibited by coadministration of 30 µM sodium orthovanadate (Fig. 7b)Citation .



View larger version (17K):
[in this window]
[in a new window]
  		Fig. 7. The effect of SAGP on PTPase activity. A431 cells were incubated with SAGP or PBS for various times. The cell lysates were prepared, and PTPase activity was assayed as described in "Materials and Methods." a, effect of incubation time on PTPase activity. The results (mean ± SE) are expressed as a percentage of basal PTPase activity obtained from PBS-incubated cells at 1 min. *, P < 0.05 versus each control (without SAGP) by unpaired Student’s t test. PTPase activity in PBS-incubated cells at 1 min averaged 0.021 ± 0.002 nmol phosphate/min/µg protein. b, effect of sodium orthovanadate on SAGP-induced PTPase activation at 2 min. The results (mean ± SE) are expressed as a percentage of basal PTPase activity obtained from PBS-incubated cells at 2 min. Statistical analyses of the variables of each group were performed by one-way ANOVA followed by Fisher’s PLSD (*, P < 0.05 versus control cells without SAGP). PTPase activity in PBS-incubated cells at 2 min averaged 0.024 ± 0.005 nmol phosphate/min/µg protein.

 
Effect of SAGP on EGF-stimulated Mitogenic Signaling.
Serum-starved A431 cells were incubated with or without SAGP (0.1 and 0.3 µg protein/ml) for 16 h before stimulation with EGF (5 ng/ml, 5 min), and cells were lysed. The immunoprecipitated EGFR was resolved by SDS-PAGE (7.5% gels), and a Western blot was probed with an antiphosphotyrosine Ab (clone 4G10). As shown in Fig. 8aCitation , EGF-induced tyrosine phosphorylation of EGFR was diminished in the lysate of cells preincubated with SAGP. The effect was not observed in the extract of cells preincubated with SAGP and sodium orthovanadate. A kinetic analysis of the SAGP-induced EGFR dephosphorylation revealed that a decrease in EGFR phosphorylation was observed between 30 and 60 min of cell preincubation with SAGP (Fig. 8b)Citation .



View larger version (36K):
[in this window]
[in a new window]
  		Fig. 8. Effect of SAGP on EGF-stimulated tyrosine phosphorylation of EGFR. a, serum-starved A431 cells were preincubated with SAGP (0.1 and 0.3 µg protein/ml) in the presence or absence of 30 µM sodium orthovanadate for 16 h and then stimulated for 5 min with 5 ng/ml EGF. b, serum-starved A431 cells were preincubated with SAGP (0.3 µg protein/ml) for 16 h, 1 h, 30 min, 15 min, and 5 min and then stimulated for 5 min with 5 ng/ml EGF. After stimulation with EGF, cells were lysed, and cell lysates were immunoprecipitated with an anti-EGFR Ab. Immunoprecipitates were analyzed by SDS-PAGE (7.5% gels) followed by Western blot analysis with an antiphosphotyrosine Ab (clone 4G10). Similar results were obtained from three (a) or two (b) independent experiments.

 
Furthermore, we examined the effect of SAGP on EGF-induced p42/44 MAPK activation by Western blot analysis using an Ab to phosphorylated p42/44 MAPK because trans-inactivation of the EGFR by G protein-coupled receptors, independent of the MAPK pathway, has been demonstrated for bradykinin in A431 cells by Graness et al. (14) . Fig. 9, a and cCitation , shows that the pretreatment of cells with SAGP (0.3 µg protein/ml) for 30 min and 1 h caused a decrease in EGF-induced p42/44 MAPK phosphorylation, whereas the protein levels are equal. The inhibitory effect of SAGP on p42/44 MAPK phosphorylation at 30 min was concentration dependent (Fig. 9b)Citation .



View larger version (28K):
[in this window]
[in a new window]
  		Fig. 9. Effect of SAGP on EGF-stimulated p42/44 MAPK activation. Serum-starved A431 cells were preincubated with SAGP (0.3 µg protein/ml) for the indicated time (a) or preincubated with SAGP (0.1–1.0 µg protein/ml) for 30 min (b), respectively, and then stimulated with EGF (5 ng/ml, 5 min). After stimulation with EGF, cells were lysed, and the cell lysates (25 µg protein/ml) were resolved by SDS-PAGE (10% gels) followed by Western blotting with an Ab to phospho-p42/44 MAPK. The membrane was reprobed with an Ab to p42 MAPK as described in "Materials and Methods." c shows the relative phosphorylation level of p42/44 MAPK to its protein (percentage of control). Statistical analyses of the variables from three independent experiments (mean ± SE) were performed by one-way ANOVA followed by Fisher’s PLSD (*, P < 0.05 versus control).

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In previous studies, we demonstrated that the antiproliferative effect of SAGP on murine Meth A cells was diminished by incubating the cells with IAP and that SAGP actually augmented the activity of IAP-sensitive G protein as assessed by IAP-catalyzed ADP ribosylation (7) . In addition, the Meth A cell growth-inhibitory effect of SAGP was diminished by coadministration of sodium orthovanadate, an inhibitor of PTPases. Western blot analysis in Meth A cell lysate with antiphosphotyrosine Ab demonstrated that phosphotyrosine content of a Mr 170,000 cellular protein was decreased by incubating the cells with SAGP and that the decrease was inhibited by coadministration of sodium orthovanadate, suggesting the involvement of activation of PTPases.

The present study shows that SAGP inhibited proliferation of A431 cells, human epidermoid carcinoma cells overexpressing EGFR, in a concentration- and a time-dependent manner (Fig. 1, a and b)Citation . The growth-inhibitory effect of SAGP on A431 cells was diminished by preincubating the cells with IAP (Fig. 2)Citation . The activity of SAGP was inhibited in part by coadministration of sodium orthovanadate (Fig. 3)Citation . Western blot analysis on A431 cell lysate revealed that the immunoreactivity of a Mr 170,000 cellular protein to antiphosphotyrosine Ab was decreased by incubating the cells with SAGP. The effect was abolished by coadministration of sodium orthovanadate (Fig. 5)Citation . Thus, the involvement of both IAP-sensitive G protein and inhibition of tyrosine phosphorylation of the Mr 170,000 cellular protein by PTPases in a tumor cell growth-inhibitory effect of SAGP was confirmed in the A431 human epidermoid cell line.

Then, to determine the Mr 170,000 cellular protein reactive to antiphosphotyrosine Ab, we investigated the effect of SAGP on the tyrosine phosphorylation of EGFR in A431 cells. The phosphotyrosine content of the precipitant with anti-EGFR Ab was decreased in the extract of cells incubated with SAGP, although the amounts of EGFR were equal (Fig. 6a)Citation . The SAGP-induced inhibitory effect on EGFR phosphorylation was diminished by incubating the cells with SAGP and sodium orthovanadate or by preincubating the cells with IAP (Fig. 6, b and c)Citation . These findings indicate that the dephosphorylated Mr 170,000 protein corresponds to EGFR and also indicate that the dephosphorylation of the EGFR is mediated by sodium orthovanadate-sensitive PTPases and IAP-sensitive G protein.

To clarify whether SAGP could activate PTPases, we next measured the PTPase activity in the cell lysate using a nonradioactive assay kit. When the cells were exposed to SAGP in the presence of 10% FBS, the PTPase activity in cell lysate increased significantly as early as 1–2 min after the addition of SAGP (Fig. 7a)Citation . The activation of PTPase by SAGP at 2 min was abolished by coadministration of sodium orthovanadate (Fig. 7b)Citation . The reason for the discrepancy in the kinetics between the EGFR dephosphorylation and the PTPase activation by SAGP is unclear, but it might be due to the experimental conditions for the PTPase assay. In this assay, we measured the total cellular and membrane-bound PTPases, but we did not measure the specific PTPase being activated by SAGP and associating with the phosphorylated EGFR. Net SAGP-induced PTPase activation might be detected only 1 and 2 min after the cells were stimulated with SAGP in the presence of 10% FBS because growth factors such as insulin and platelet-derived growth factors in FBS would activate cellular PTPases. Some reports demonstrated that PTPase plays a positive role in growth factor-stimulated cell proliferation (15 , 16) .

With regard to the activation of PTPase by SAGP, we observed in the cell growth assay that the antiproliferative effect of SAGP on A431 cells was augmented by a coadministration of catalase as shown in Fig. 4Citation . Bae et al. (17) reported that stimulation of A431 cells with EGF resulted in a transient increase in the intracellular concentration of reactive oxygen species (predominantly H2O2) and that the effect of EGF was abolished by the incorporation of catalase into the cells. In addition, Lee et al. (18) demonstrated that H2O2 inactivated recombinant PTP1B in vitro by oxidizing its catalytic site cysteine, suggesting the concurrent inhibition of PTPases by H2O2 produced in response to EGF. The present finding that the SAGP activity was augmented by catalase may be additional evidence suggesting the involvement of the activation of PTPases in antiproliferative signaling by SAGP.

The identity of activated PTPases is currently unknown. It has been shown that the cellular PTPases play a prominent role in growth factor-mediated signal transduction (19, 20, 21) . The list of PTPases shown to possess the capacity for interaction with growth factor receptor includes, at the least, PTP1B (22 , 23) , PTP1C (24 , 25) , PTP-PEST (26) , and transmembrane PTP LAR (27) . In A431 cells, Tomic et al. (24) demonstrated that PTP1C participates in the dephosphorylation of the EGFR, and Pestana et al. (20) proposed recently that the transmembrane PTPase RPTP{sigma} modulates signaling of the EGFR. Our experiments using some available anti-PTPase Abs such as PTP1B, PTP1C, and PTP1D (Transduction Laboratories) revealed that these PTPases are detectable in A431 cell lysate, but it was unclear which PTPase was associated with the EGFR by the coimmunoprecipitation experiment with anti-EGFR Ab or anti-PTPase Abs.

Furthermore, we assessed the effect of SAGP on EGF-stimulated mitogenic signaling. Preincubation of the serum-starved A431 cells with SAGP caused a decrease in phosphorylation of EGFR (Fig. 8, a and b)Citation and of p42/44 MAPK after EGF stimulation (Fig. 9, a–c)Citation . To examine any immediate downstream signaling effector(s) uncoupled from EGFR as a consequence of SAGP treatment, a Western blot of EGFR immunoprecipitate was probed with an antiphosphotyrosine Ab by prolonged exposure of blotting membrane to X-ray film. We could not observe any other protein besides EGFR in which the phosphotyrosine level was significantly decreased by SAGP. We then examined the effect of SAGP on adaptor Shc proteins, which have been shown to be immediate substrates of receptor tyrosine kinase activity and to relay receptor tyrosine kinase-induced signals to downstream signaling components (28 , 29) . After serum-starved A431 cells were preincubated with SAGP and stimulated with EGF (5 ng/ml, 5 min), Shc proteins (Mr 46,000, Mr 52,000, and Mr 66,000 isoforms) were immunoprecipitated by an anti-Shc rabbit polyclonal Ab (Transduction Laboratories) and probed with antiphosphotyrosine Ab. Our preliminary data showed that tyrosine phosphorylation of Shc isoforms and a Mr 170,000 protein (the latter of which migrated with the same mobility as EGFR) was increased in the extracts of cells stimulated with EGF. The increase in tyrosine phosphorylation of a Mr 170,000 protein by EGF seems to be decreased by preincubating the cells with SAGP, which suggests that the association of adaptor Shc proteins with the tyrosine-phosphorylated EGFR might be decreased in cells preincubated with SAGP.

All of the above-mentioned findings combine to suggest a model for signal transduction of the antiproliferative effects of SAGP, as shown in Fig. 10Citation . Namely, SAGP binds to an IAP-sensitive G protein-coupled receptor that has yet to be identified and activates PTPases followed by dephosphorylation of EGFR, resulting in inhibition of cell proliferation, probably via inhibition of p42/44 MAPK. However, we cannot conclude that the antiproliferative action of SAGP is achieved solely through PTPase activation because SAGP activity is incompletely abolished by sodium orthovanadate or IAP.



View larger version (17K):
[in this window]
[in a new window]
  		Fig. 10. A suggested model for signal transduction of the antiproliferative effect of SAGP. Tyr-P, phosphotyrosine.

 
With regard to the antiproliferative mechanism, interest has focused on the antitumor effect of the neuropeptide somatostatin and its analogues (30, 31, 32, 33) . Stimulation of G protein-coupled receptor by somatostatin (34) has been shown to exhibit an antiproliferative effect on tumor cells via an activation of PTPases (10 , 35, 36, 37, 38) . Recent studies demonstrated that the activated PTPases dephosphorylate the growth factor receptors, resulting in cell growth inhibition (13 , 19 , 39) . It has also been shown that the antiproliferative effect of TNF is associated with alterations in specific PTPases (11 , 40) . Recently, Perez et al. (41) demonstrated that TNF induced a translocation of PTP1B-related proteins in tumor cells that interacts with EGFR, resulting in growth inhibition. Guo et al. (21) also showed that a PTPase activated by TNF inhibits activation of vascular endothelial growth factor receptor KDR and vascular endothelial growth factor-induced endothelial cell proliferation. The present findings may support the concept that activation of growth factor receptor-directed PTPases could contribute to a mechanism for novel antiproliferative agents.

Furthermore, SAGP may also provide a tool to examine a cross-regulation between stimulation of G protein-coupled receptor and growth factor receptor signaling.


   ACKNOWLEDGMENTS
 
We thank Prof. Katsuzo Nishikawa for generously donating the A431 cell line and for valuable discussion. We also thank Drs. Y. Nagao and Dr. I. Takeuchi of Kanazawa Medical University for advice and critical discussions and Yasuko Shinzawa for secretarial assistance.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 To whom requests for reprints should be addressed, at Department of Pharmacology, Kanazawa Medical University, Uchinada, Ishikawa 920-0293, Japan. Phone: 81-76-286-2211; Fax: 81-76-286-8191; E-mail: yayuyo{at}kanazawa-med.ac.jp Back

2 The abbreviations used are: SAGP, streptococcal acidic glycoprotein; EGF, epidermal growth factor; EGFR, EGF receptor; IAP, pertussis toxin; FBS, fetal bovine serum; PTPase, protein tyrosine phosphatase; Ab, antibody; MAPK, mitogen-activated protein kinase; Meth A, methylcholanthrene-induced fibrosarcoma A; cAMP, cyclic AMP; PBS-T, PBS containing 0.1% Tween 20; ECL, enhanced chemiluminescence; TNF, tumor necrosis factor; PLSD, Protected Least Significant Difference. Back

3 Unpublished data. Back

Received 7/25/00. Accepted 6/19/01.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Okamoto H., Shoin S., Koshimura S. Streptolysin s-forming and antitumor activities of group A streptococci Jeljaszewicz J. Wadström T. eds. . Bacterial Toxins and Cell Membranes, : 259-289, Academic Press London 1978.
  2. Yoshida J., Yoshimura M., Takamura S., Kobayashi S. Purification and characterization of an antitumor principle from Streptococcus hemolyticus (Su strain). Jpn. J. Cancer Res., 76: 213-223, 1985.[Medline]
  3. Yoshida J., Takamura S., Nishio M. Characterization of a streptococcal antitumor glycoprotein (SAGP). Life Sci., 62: 1043-1053, 1998.[Medline]
  4. Yoshida J., Takamura S., Suzuki S. Antitumor action of an acidic glycoprotein (SAGP) from Streptococcus pyogenes in mice. Biotherapy, 3: 331-336, 1991.[Medline]
  5. Yoshida J., Takamura S., Suzuki S. A simplified method for purification of an antitumor acidic glycoprotein from Streptococcus pyogenes (Su strain) by immunoabsorbent chromatography. J. Antibiot. (Tokyo), 42: 448-453, 1989.[Medline]
  6. Yoshida J., Takamura S., Suzuki S. Evidence for the involvement of sulfhydryl groups in the expression of antitumor activity of streptococcal acid glycoprotein (SAGP) purified from crude extract of Streptococcus pyogenes. Anticancer Res., 14: 1833-1838, 1994.[Medline]
  7. Yoshida J., Takamura S., Suzuki S., Nishio M. Streptococcal glycoprotein-induced tumour cell growth inhibition involves the modulation of a pertussis toxin-sensitive G protein. Br. J. Cancer, 73: 917-923, 1996.[Medline]
  8. Yoshida J., Takamura S., Ishibashi T., Nishio M. Antiproliferative and apoptosis-inducing effects of an antitumor glycoprotein from Streptococcus pyogenes. Anticancer Res., 19: 1865-1872, 1999.[Medline]
  9. Pan M. G., Florio T., Stork P. J. S. G protein activation of a hormone-stimulated phosphatase in human tumor cells. Science (Wash. DC), 256: 1215-1217, 1992.[Abstract/Free Full Text]
  10. Florio T., Rim C., Hershberger R. E., Loda M., Stork P. J. S. The somatostatin receptor SSTR1 is coupled to phosphotyrosine phosphatase activity in CHO-K1 cells. Mol. Endocrinol., 46: 1289-1297, 1994.
  11. Ahmad F., Goldstein B. J. Effect of tumor necrosis factor-{alpha} on the phosphorylation of tyrosine kinase receptors is associated with dynamic alterations in specific protein-tyrosine phosphatases. J. Cell. Biochem., 64: 117-127, 1997.[Medline]
  12. Pestana E. S., Greiser U., Sánchez B., Fernández L. E., Lage A., Perez R., Böhmer F-D. Growth inhibition of human lung adenocarcinoma cells by antibodies against epidermal growth factor receptor and by ganglioside GM3: involvement of receptor-directed protein tyrosine phosphatase(s). Br. J. Cancer, 75: 213-220, 1997.[Medline]
  13. Bousquet C., Delesque N., Lopez F., Saint-Laurent N., Estèeve J-P., Bedecs K., Buscail L., Vaysse N., Susini C. sst2 somatostatin receptor mediates negative regulation of insulin receptor signaling through the tyrosine phosphatase SHP-1. J. Biol. Chem., 273: 7099-7106, 1998.[Abstract/Free Full Text]
  14. Graness A., Hanke S., Boehmer F. D., Presek P., Liebmann C. Protein-tyrosine-phosphatase-mediated epidermal growth factor (EGF) receptor transinactivation and EGF receptor-independent stimulation of mitogen-activated protein kinase by bradykinin in A431 cells. Biochem. J., 347: 441-447, 2000.[Medline]
  15. Vogel W., Lammers R., Huang J., Ullrich A. Activation of a phosphotyrosine phosphatase by tyrosine phosphorylation. Science (Wash. DC), 259: 1611-1614, 1993.[Abstract/Free Full Text]
  16. Xiao S., Rose D. W., Sasaoka T., Maegawa H., Burke T. R., Jr., Roller P. P., Shoelson S. E., Olefsky J. M. Syp (SH-PTP2) is a positive mediator of growth-factor stimulated mitogenic signal transduction. J. Biol. Chem., 269: 21244-21248, 1994.[Abstract/Free Full Text]
  17. Bae Y. S., Kang S. W., Seo M. S., Baines I. C., Tekle E., Chock P. B., Rhee S. G. Epidermal growth factor (EGF)-induced generation of hydrogen peroxide. Role in EGF receptor-mediated tyrosine phosphorylation. J. Biol. Chem., 272: 217-221, 1997.[Abstract/Free Full Text]
  18. Lee S-R., Kwon K-S., Kim S-R., Rhee S. G. Reversible inactivation of protein-tyrosine phosphatase 1B in A431 cells stimulated with epidermal growth factor. J. Biol. Chem., 273: 15366-15372, 1998.[Abstract/Free Full Text]
  19. Srikant C. B., Shen S-H. Octapeptide somatostatin analog SMS 201–995 induces translocation of intracellular PTP1C to membranes in MCF-7 human breast adenocarcinoma cells. Endocrinology, 137: 3461-3468, 1996.[Abstract]
  20. Pestana E. S., Tenev T., Gro B. S., Stoyanov B., Ogata M., Böhmer F-D. The transmembrane protein tyrosine phosphatase RPTP{sigma} modulates signaling of the epidermal growth factor receptor in A431 cells. Oncogene, 18: 4069-4079, 1999.[Medline]
  21. Guo D-Q., Wu L-W., Dunbar J. D., Ozes O. N., Mayo L. D., Kessler K. M., Gustin J. A., Baerwald M. R., Jaffe E. A., Warren R. S., Donner D. B. Tumor necrosis factor employs a protein-tyrosine phosphatase to inhibit activation of KDR and vascular endothelial cell growth factor-induced endothelial cell proliferation. J. Biol. Chem., 275: 11216-11221, 2000.[Abstract/Free Full Text]
  22. Flint A. J., Tiganis T., Barford D., Tonks N. K. Development of "substrate-trapping" mutants to identify physiological substrates of protein tyrosine phosphatases. Proc. Natl. Acad. Sci. USA, 94: 1680-1685, 1997.[Abstract/Free Full Text]
  23. Liu F., Chernoff J. Protein tyrosine phosphatase 1B interacts with and is tyrosine phosphorylated by the epidermal growth factor receptor. Biochem. J., 327: 139-145, 1997.
  24. Tomic S., Greiser U., Lammers R., Kharitonenkov A., Imyanitov E., Ullrich A., Böhmer F-D. Association of SH2 domain protein tyrosine phosphatases with the epidermal growth factor receptor in human tumor cells. Phosphatidic acid activates receptor dephosphorylation by PTP1C. J. Biol. Chem., 270: 21277-21284, 1995.[Abstract/Free Full Text]
  25. Tenev T., Keilhack H., Tomic S., Stoyanov B., Stein-Gerlach M., Lammers R., Krivtsov A. V., Ullrich A., Böhmer F-D. Both SH2 domains are involved in interaction of SHP-1 with the epidermal growth factor receptor but cannot confer receptor-directed activity to SHP-1/SHP-2 chimera. J. Biol. Chem., 272: 5966-5973, 1997.[Abstract/Free Full Text]
  26. Charest A., Wagner J., Kwan M., Tremblay M. L. Coupling of the murine protein tyrosine phosphatase PEST to the epidermal growth factor (EGF) receptor through a Src homology 3 (SH3) domain-mediated association with Grb2. Oncogene, 14: 1643-1651, 1997.[Medline]
  27. Kulas D. T., Freund G. G., Mooney R. A. The transmembrane protein-tyrosine phosphatase CD45 is associated with decreased insulin receptor signaling. J. Biol. Chem., 271: 755-760, 1996.[Abstract/Free Full Text]
  28. Cutler R. L., Liu L., Damen J. E., Krystal G. Multiple cytokines induce the tyrosine phosphorylation of Shc and its association with Grb2 in hemopoietic cells. J. Biol. Chem., 268: 21463-21465, 1993.[Abstract/Free Full Text]
  29. Ravichandran K. S., Burakoff S. J. The adaptor protein Shc interacts with the interleukin-2 (IL-2) receptor upon IL-2 stimulation. J. Biol. Chem., 269: 1599-1602, 1994.[Abstract/Free Full Text]
  30. Weckbecker G., Liu R., Tolcsvai L., Bruns C. Antiproliferative effects of the somatostatin analogue octreotide (SMS 201–995) on ZR-75-1 human breast cancer cells in vivo and in vitro. Cancer Res., 52: 4973-4978, 1992.[Abstract/Free Full Text]
  31. Reubi J-C., Laissue J. A. Multiple actions of somatostatin in neoplastic disease. Trends Pharmacol. Sci., 16: 110-115, 1995.[Medline]
  32. Lamberts S. W., van der Lely A. J., de Herder W. W., Hofland L. J. Somatostatin analogs: future directions. Metabolism, 45: 104-106, 1996.[Medline]
  33. Kéri G. Y., Érchegyi J., Horváth A., Mezõ I., Idei M., Vántus T., Balogh Á., Vadász Z. S., Bökönyi G. Y., Seprõdi J., Teplán I., Csuka O., Tejeda M., Gaál D., Szegedi Z. S., Szende B., Roze C., Kalthoff H., Ullrich A. A tumor-selective somatostatin analog (TT-232) with strong in vitro and in vivo antitumor activity. Proc. Natl. Acad. Sci. USA, 93: 12513-12518, 1996.[Abstract/Free Full Text]
  34. Patel Y. C., Greenwood M., Panetta R., Hukovic N., Grigorakis S., Robertson L-A., Srikant C. B. Molecular biology of somatostatin receptor subtypes. Metabolism, 45: 31-38, 1996.[Medline]
  35. Colas B., Cambillau C., Buscail L., Zeggari M., Esteve J-P., Lautre V., Thomas F., Vaysse N., Susini C. Stimulation of a membrane tyrosine phosphatase activity by somatostatin analogues in rat pancreatic acinar cells. Eur. J. Biochem., 207: 1017-1024, 1992.[Medline]
  36. Lopez F., Estèeve J-P., Buscail L., Delesque N., Saint-Laurent N., Thèeveniau M., Nahmias C., Vaysse N., Susini C. The tyrosine phosphatase SHP-1 associates with the sst2 somatostatin receptor and is an essential component of sst2-mediated inhibitory growth signaling. J. Biol. Chem., 272: 24448-24454, 1997.[Abstract/Free Full Text]
  37. Sharma K., Patel Y. C., Srikant C. B. C-terminal region of human somatostatin receptor 5 is required for induction of Rb and G1 cell cycle arrest. Mol. Endocrinol., 13: 82-90, 1999.[Abstract/Free Full Text]
  38. Douziech N., Calvo E., Coulombe Z., Muradia G., Bastien J., Aubin R. A., Lajas A., Morisset J. Inhibitory and stimulatory effects of somatostatin on two human pancreatic cancer cell lines: a primary role for tyrosine phosphatase SHP-1. Endocrinology, 140: 765-777, 1999.[Abstract/Free Full Text]
  39. Rauly I., Saint-Laurent N., Delesque N., Buscail L., Estèeve J-P., Vaysse N., Susini C. Induction of a negative autocrine loop by expression of sst2 somatostatin receptor in NIH 3T3 cells. J. Clin. Investig., 97: 1874-1883, 1996.[Medline]
  40. Totpal K., Agarwal S., Aggarwal B. B. Phosphatase inhibitors modulate the growth-regulatory effects of human tumor necrosis factor on tumor and normal cells. Cancer Res., 52: 2557-2562, 1992.[Abstract/Free Full Text]
  41. Perez M., Haschke B., Donato N. J. Differential expression and translocation of protein tyrosine phosphatase 1B-related proteins in ME-180 tumor cells expressing apoptotic sensitivity and resistance to tumor necrosis factor: potential interaction with epidermal growth factor receptor. Oncogene, 18: 967-978, 1999.[Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Yoshida, J.
Right arrow Articles by Nishio, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yoshida, J.
Right arrow Articles by Nishio, M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online