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[Cancer Research 62, 3322-3326, June 1, 2002]
© 2002 American Association for Cancer Research

Tumor Biology

Overexpression of Icat Induces G2 Arrest and Cell Death in Tumor Cell Mutants for Adenomatous Polyposis Coli, ß-catenin, or Axin1

Takashi Sekiya, Tsutomu Nakamura, Yasuhiro Kazuki, Mitsuo Oshimura, Kazuyoshi Kohu, Ken-ichi Tago, Susumu Ohwada and Tetsu Akiyama2

Laboratory of Molecular and Genetic Information, Institute of Molecular and Cellular Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo 113 [T. S., T. N., K. T., T. A.]; Department of Molecular and Cell Genetics, School of Life Sciences, Faculty of Medicine, Tottori University, Yonago, Tottori 683-8503 [Y. K., M. O.]; and Second Department of Surgery, Gunma University School of Medicine, Maebashi, Gunma 371 [K. T., S. O.], Japan


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Aberrant activation of Wnt signaling caused by mutations in adenomatous polyposis coli (APC) orß-catenin is a critical event in the development of human colorectal tumors. Wnt signaling stabilizes ß-catenin, which in turn associates with TCF/LEF family transcription factors, ultimately altering the expression of Wnt target genes. We have recently identified ICAT, a ß-catenin-interacting protein that interferes with the interaction between ß-catenin and TCF-4, thereby negatively regulating Wnt signaling. In the present study, we generated a recombinant adenovirus encoding ICAT and examined its effect on the growth of tumor cells. We found that Icat inhibits proliferation of colorectal tumor cells mutated in APC or ß-catenin and hepatocellular carcinoma cells mutated in Axin. By contrast, Icat did not inhibit growth of either normal or tumor cells containing the wild-type APC, ß-catenin, and Axin genes. Icat also inhibited the anchorage-independent growth of colorectal tumor cells and tumorigenic growth of colorectal tumor xenografts. Furthermore, we found that Icat inhibits both dephosphorylation of Cdc2 and nuclear translocation of cyclin B1 and induces G2 arrest followed by cell death in colorectal tumor cells. These results suggest that Wnt signaling is critical for the growth of colorectal tumors and some hepatocellular carcinomas and that expression of ICAT or drugs which mimic its effects may be useful in the treatment of these tumors.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The Wnt signaling transduction pathway is involved in a number of developmental processes and tumorigenesis (1, 2, 3) . Activation of the Wnt signaling pathway leads to the intracellular accumulation of ß-catenin, which interacts with TCF/LEF family transcription factors to activate the expression of target genes, such as c-myc, cyclin D1, and peroxisome proliferator-activated receptor-{delta}. In the absence of the Wnt signal, ß-catenin is subjected to degradation because of its association with Axin, GSK-3ß,3 and the tumor suppressor APC (1, 2, 3) . ß-catenin in this complex is phosphorylated by GSK-3ß and degraded by the ubiquitin-proteasome pathway. On activation by the Wnt signal, GSK-3ß activity is inhibited, and ß-catenin accumulates.

APC is mutated in the majority of sporadic colorectal tumors, as well as familial adenomatous polyposis (1, 2, 3, 4, 5) . Both somatic and germ-line mutations are almost exclusively nonsense or frameshift mutations that generate truncated APC gene products unable to induce the degradation of ß-catenin. Furthermore, in colorectal tumors that retain the wild-type APC gene, mutations in ß-catenin have been observed (6 , 7) . Common sites for ß-catenin mutations are the four consensus GSK-3ß phosphorylation motifs found in its NH2-terminal domain. ß-catenin that is mutated at these sites is stabilized and forms constitutively active complexes with TCF. In addition, Axin, as well as ß-catenin, is mutated in a certain subset of hepatocellular carcinomas (8, 9, 10) . Genetic alterations in the components of the Wnt signaling pathway, APC, ß-catenin, and Axin appear to occur in a mutually exclusive fashion but have a similar outcome: the deregulated accumulation of ß-catenin (1, 2, 3, 4, 5) . The accumulation of ß-catenin is also observed in many other types of neoplasms, such as melanoma, ovarian cancer, endometrial cancer, medulloblastoma, pilomatricoma, and prostate cancer (5 , 11) . Thus, constitutive activation of ß-catenin-TCF-mediated transcription may be a critical step in tumorigenesis among divergent types of cancers.

We have recently identified a ß-catenin-interacting protein, ICAT, which inhibits the interaction of ß-catenin with TCF, preventing formation of the transactivator complex and thereby negatively regulating ß-catenin-TCF-mediated transcription (12) . Therefore, we examined whether ICAT exhibits any effect on the growth of tumor cells. To address this issue, we generated a replication-deficient adenovirus that expresses ICAT (Ad-Icat) with high efficiency. Here, we show that Ad-Icat inhibits the growth of colorectal tumor cells carrying an APC or ß-catenin mutation, as well as hepatocellular carcinoma cells carrying an Axin mutation. We further show that this inhibition of colorectal tumor cell proliferation involves the induction of G2 arrest and cell death.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Generation of Recombinant Adenovirus Expressing Murine ICAT.
The Ad-Icat vector was constructed using the COS-TPC method (13) .

Luciferase Assay.
Cells (2 x 106 cells/60-mm dish) were infected with Ad-Icat or Ad-control at m.o.i. 50, incubated for 1 h, and then transfected by LipofectAMINE-PLUS (Life Technologies, Inc.) with 2 µg of reporter plasmid (TOPtkLuciferase or FOPtkLuciferase) and 0.2 µg of the internal control pRL-TK (Promega). Luciferase activities were measured 24 h after transfection using the Dual-Luciferase Reporter Assay System (Promega).

Cellular Proliferation Assay.
Cells (1 x 104 cells/well) were in six-well culture plants, cultured for 24 h, and infected with Ad-Icat or Ad-control at m.o.i. 50. Cells from each treated sample were counted in triplicate by using trypan blue exclusion by hemocytometer on days 0, 2, 4, and 6 after infection.

Cell Cycle Analysis.
Cells (5 x 105 cells/10-cm dish) were cultured for 24 h and infected with Ad-Icat or Ad-control at m.o.i. 50. After 48, 96, and 144 h of infection, cells were harvested and fixed in 70% ethanol. Cells were then pelleted and resuspended in 1 ml of 50 µg/ml propidium iodide in PBS containing 20 µg/ml RNase for 30 min. DNA content of 1 x 104 cells was analyzed by flow cytometry.

Soft Agar Colony Formation Assay.
Cells (5 x 106 cells/10-cm dish) were infected with Ad-Icat or Ad-control at m.o.i. 50 and cultured for 24 h. Cells (5 x 103 cells) were then resuspended in 0.35% agar in DMEM supplemented with 10% fetal bovine serum and plated on 3.5-cm plates containing a solidified bottom layer (0.5% agar in growth medium). The numbers of colonies were counted 2 weeks after plating.

Antibodies.
Rabbit pAb to ICAT was prepared as described (12) . pAb to phospho-cdc2 (Tyr15) was purchased from New England Biolabs. Monoclonal antibody to the Myc tag, pAb to Cdc25C (sc-327), monoclonal antibody to Cdc2 (sc-54), and cyclin B1 (sc-245) were from Santa Cruz Biotechnology.

Immunoprecipitation and Immunoblotting.
Cells (5 x 106 cells) were lysed in 500 µl of buffer A [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 2 mM Na3VO4, and 10 mM NaF] containing 1% Triton X-100. The lysates were incubated with 2 µg of antibodies for 1 h at 4°C, and then the immunocomplexes were adsorbed to protein G-Sepharose 6B for 2 h at 4°C. After washing extensively with buffer A containing 0.1% Triton X-100, samples were resolved by SDS-PAGE and transferred to a polyvinylidene difluoride membrane filter (Immobilon P; Millipore). The blot was subjected to immunoblotting analysis using alkaline phosphatase-conjugated mouse antirabbit or goat antimouse IgG (Promega) as a second antibody.

Animal Studies.
SW48 (1 x 107) cells were injected s.c. into 8-week-old athymic BALB/c-nu/nu (nude) mice (Sankyo Lab Service). Animals were inspected for tumor formation for 4 weeks after implantation.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Characterization of Ad-Icat.
To examine the effects of Icat on cell proliferation, we generated an adenovirus that encodes myc-tagged ICAT (Ad-Icat). Immunoblotting analysis showed that infection of SW48, SW480, and A431 cells with Ad-Icat but not control virus (Ad-control) produces similar amounts of a protein of the expected size (Fig. 1A)Citation . Consistent with our previous results (12) , overexpression of Icat in SW48, DLD-1, SW480, and SNU423 cells (see below) by infection with Ad-Icat repressed the activity of a reporter plasmid that contains optimal TCF-4-binding sites upstream of a luciferase reporter gene (Fig. 1B)Citation . These results indicate that Ad-Icat generates Myc-tagged ICAT that is active as an inhibitor of ß-catenin-TCF-regulated transcription.



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  		Fig. 1. Characterization of Ad-Icat. In A, infection of SW48, SW480, and A431 cells with Ad-Icat generates Myc-tagged ICAT. Lysates prepared from mock, Ad-control, or Ad-Icat-infected cells were subjected to immunoblot analysis with anti-Myc tag antibody. B, effect of Ad-Icat on ß-catenin-TCF-mediated transcription in tumor cells. SW48, DLD-1, SW480, and SNU423 cells that had been infected with Ad-Icat or Ad-control, or control uninfected cells, were transfected with a luciferase reporter plasmid containing optimal (TOPtkLuciferase) or mutated (FOPtkLuciferase) TCF-binding sites. Values are the average of three experiments.

 
Icat Inhibits Proliferation of Specific Cancer Cell Lines.
We examined the effect of Icat on the growth of several human cancer cell lines in which ß-catenin-TCF4-mediated transcription is aberrantly activated because of a mutation in the ß-catenin (colorectal tumor SW48 and HCT116 cells), APC (colorectal tumor DLD-1 and SW480 cells), or Axin gene (hepatocarcinoma SNU423 and SNU475 cells). Infection with Ad-Icat, but not Ad-control, resulted in growth inhibition of these cells (Fig. 2A)Citation . On the other hand, Ad-Icat infection did not inhibit proliferation of cells containing the normal ß-catenin, APC, and Axin genes; normal diploid TIG103 cells; normal keratinocyte HaCaT cells; and epithelial tumor KB, HeLa, and A431 cells (Fig. 2A)Citation . These results suggest that Icat specifically inhibits the growth of tumor cells in which ß-catenin-TCF4-mediated transcription is aberrantly activated. Furthermore, trypan blue exclusion experiments revealed that overexpression of Icat leads to the induction of cell death in colorectal tumor cells in a time-dependent manner (Fig. 2B)Citation .



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  		Fig. 2. Icat inhibits proliferation of specific tumor cells. A, the 12 indicated cell lines were mock, Ad-control, or Ad-Icat infected and cultured for 144 h. Cell number was determined at the indicated times. Error bars, SD of the average of three experiments. In B, SW48 cells were mock, Ad-Icat, or Ad-LacZ infected, and dead cell number was determined by trypan blue exclusion at the indicated times. Error bars, SD of the average of three experiments.

 
Icat Inhibits Anchorage-independent Growth of Specific Cancer Cell Lines.
One of the hallmarks of the tumorigenic state is anchorage-independent growth. Therefore, we next studied the ability of Ad-Icat to suppress the growth of cancer cells normally able to proliferate in soft agar. We observed that Ad-Icat, but not Ad-control, significantly suppressed colony formation in soft agar of those cell lines that had previously shown susceptibility to Icat-mediated growth suppression, i.e., Ad-Icat inhibited the anchorage-independent growth of SW48, DLD-1, and SW480 cells but not A431 cells (Fig. 3)Citation .



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  		Fig. 3. Icat inhibits anchorage-independent growth of tumor cells. Four cell lines indicated were mock, Ad-control, or Ad-Icat infected and cultured in soft agar for 2 weeks. Overall mean number of colonies ±SE from triplicate experiments is shown in the bar graph.

 
Icat Inhibits Tumor Growth in an Animal Model.
To study the antitumor effect of Icat in vivo, we implanted SW48 cells infected with Ad-Icat or Ad-control in nude mice and compared their growth for 4 weeks. Consistent with the results obtained with the growth suppression and soft agar assays, Ad-Icat, but not Ad-control, completely inhibited the growth of SW48 cells in nude mice (Table 1)Citation . Theses results provide direct evidence that Icat has an antitumor effect in vivo.


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  		Table 1 Tumorigenicity of mock, Ad-control, or Ad-Icat-infected SW48 cellsa

 
Icat Induces G2 Arrest and Cell Death in Colorectal Tumor Cells.
To elucidate the mechanism by which Icat inhibits proliferation of cancer cell lines, we subjected Ad-Icat-infected cancer cells to flow cytometric analysis. In cells infected with Ad-Icat, but not Ad-control, the relative percentage of SW48 and DLD-1 cells in the G2-M phase increased until 48-h postinfection (Fig. 4)Citation . Thereafter, the population of >4N and sub-G1 cells increased in a time-dependent manner, suggesting that SW48 cells overexpressing ICAT continue DNA synthesis without entering the M phase and eventually undergo cell death.



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  		Fig. 4. Icat induces G2 arrest and cell death in colorectal tumor cells. Three cell lines indicated were mock, Ad-control, or Ad-Icat infected. DNA content of 1 x 104 cells was analyzed by flow cytometry at the indicated times after infection.

 
We next asked whether Icat arrests cells at the G2 or M phase. Because Cdc2 is phosphorylated at Tyr-15 in the G2 phase but is dephosphorylated in the M phase, we examined the phosphorylation state of Cdc2 in nocodazole-treated SW48 cells that had been infected with Ad-Icat. Immunoblot analysis using an antibody that recognizes Cdc2 phosphorylated at Tyr-15 revealed that tyrosine phosphorylation was significantly higher in Ad-Icat-infected cells than Ad-control-infected cells (Fig. 5A)Citation , suggesting that SW48 cells overexpressing Icat are arrested at the G2 phase. Another marker of the G2-M phase is cyclin B1, which is localized in the cytoplasm in the G2 phase but translocates to the nucleus in the M phase. Immunostaining of nocodazole-treated SW48 cells with anticyclin B1 antibody showed that cyclin B1 translocated into the nucleus in Ad-control-infected cells but remained in the cytoplasm in Ad-Icat-infected cells (Fig. 5B)Citation . Chromosomal condensation was also markedly repressed in the Ad-Icat-infected cells (Fig. 5B)Citation . In addition, interphase fluorescence in situ hybridization analysis using a chromosome 1p36.1 probe revealed that infection of SW48 cells with Ad-Icat markedly increases the population of cells having a DNA content of >=4N, whereas infection with an adenovirus encoding ß-galactosidase (Ad-LacZ) does not (Table 2)Citation . Together, these results suggest that Icat induces cell cycle arrest at the G2 phase.



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  		Fig. 5. Analysis of cyclin B1, Cdc2, and Cdc25 in SW48 cells overexpressing Icat. A, expression and phosphorylation of Cdc2. SW48 cells infected with Ad-control or Ad-Icat were cultured for 24 h, then cultured further in the presence of nocodazole (0.2 µg/ml) for 24 h. Cell lysates, prepared at the indicated times after the addition of nocodazole, were subjected to immunoblotting with antiphospho-Cdc2 (Tyr-15) or anti-Cdc2 antibody. B, subcellular localization of cyclin B1. SW48 cells infected with Ad-control or Ad-Icat were cultured for 24 h and then cultured further in the presence of nocodazole (0.2 µg/ml) for 18 h. Cells were stained with anticyclin B1 antibody and 4',6-diamidino-2-phenylindole. C, expression of cyclin B1, Cdc2, and Cdc25 in SW48 cells overexpressing Icat. Mock (M)-, Ad-Icat (I)-, or Ad-LacZ (Z)-infected SW48 cells were subjected to immunoblot analysis with antibodies to indicated proteins. Cell lysates, prepared at the indicated times after infection, were subjected to immunoblotting.

 

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  		Table 2 Interphase FISHa analysis of SW48 cells overexpressing Icat

 
Icat Suppresses Expression of G2-M Regulatory Proteins.
The above results suggest that inhibition of Cdc2 dephosphorylation and nuclear translocation of cyclin B1 is important for Icat-induced G2 arrest. We next analyzed the effects of Icat on expression of the G2-M regulatory proteins Cdc2, cyclin B1, and Cdc25 in SW48 cells. Immunoblot analysis with anticyclin B1 antibody revealed that the levels of cyclin B1 in Ad-Icat-infected cells decreased to ~50% of those in uninfected and Ad-LacZ-infected cells <=48-h postinfection (Fig. 5C)Citation . Cdc2 and Cdc25C protein levels were also found to decrease modestly after Ad-Icat infection. By contrast, ß-tubulin expression was unaffected by Icat, suggesting that the effects of Icat on cyclin B1, Cdc2, and Cdc25C are specific. We also measured the mRNA levels of these three G2-M regulatory proteins in SW48 cells infected with Ad-control or Ad-Icat. Icat was found to suppress cyclin B1, Cdc2, and Cdc25C mRNA expression at 36 h after infection (data not shown). Consistent with previous reports (14 , 15) , Icat suppressed the expression of cyclin D1, a known target gene of the Wnt signaling pathway, whereas the expression of g3pdh was unaffected. These results suggest that decreases in the G2-M regulatory proteins Cdc2, cyclin B1, and Cdc25 contribute to Icat-mediated G2 arrest.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We have shown here that Icat inhibits the proliferation of colorectal tumor cells bearing mutant APC or ß-catenin and hepatocellular carcinoma cells bearing mutant Axin. This finding is consistent with a previous report showing that down-regulation of ß-catenin mRNA expression by a ß-catenin antisense oligonucleotide specifically suppresses the neoplastic growth of colorectal tumor cells (16) . It has also been reported that expression of the central third of APC, a region that contains ß-catenin- and Axin-binding sites, induces down-regulation of ß-catenin-TCF-4-mediated transcription and inhibits growth of colorectal tumor cell lines that carry a mutated APC (17) . Of particular interest is the fact that both overexpression of Icat and of the ß-catenin antisense oligonucleotide (16) specifically inhibit the growth of tumor cells in which Wnt signaling is aberrantly activated because of mutations in APC, ß-catenin, or Axin. Icat did not show growth inhibitory activity on normal cells or on tumor cells in which Wnt signaling is not constitutively activated. These results suggest that Icat inhibits the growth of tumor cells by down-regulating ß-catenin-TCF-4-mediated transcription and that aberrant activation of Wnt signaling is important not only for the generation of polyps but also the growth of colorectal tumor cells and some hepatocellular carcinoma cells.

Expression of Icat inhibited the anchorage-independent growth of colorectal tumor cells more efficiently than it did anchorage-dependent cell growth. Anchorage-independent growth is well known to be a better in vitro indicator of tumorigenicity. Indeed, the ability of Icat to efficiently inhibit growth of colorectal tumor xenografts in nude mice was greater than would have been predicted from its ability to suppress anchorage-dependent growth.

It has been shown in colorectal tumor cells that ß-catenin activates transcription of cyclin D1, which is a critical regulator of the G1 to S cell cycle progression (14 , 15) . Furthermore, it has been reported that the expression of a dominant-negative form of TCF, a factor which down-regulates ß-catenin-TCF-mediated transactivation, causes cells to arrest in the G1 phase of the cell cycle. Therefore, we expected that Icat would block the G1-S transition of colorectal tumor cells. However, overexpression of Icat was found to induce mainly G2 arrest of the cell cycle in colorectal tumor cells. In addition, overexpression of the central region of APC has been shown to block the cell cycle at the G2 phase (Fig. 6a in Ref. 10 , although not mentioned specifically by the authors). Therefore, it is intriguing to speculate that ß-catenin may regulate not only the G1-S transition but also the G2-M transition. In this regard, it is interesting to note that AF17, a gene that stimulates the G2-M transition, has been reported to be up-regulated by Wnt signaling (18) . Furthermore, in tumor cells that contain a mutation in APC, ß-catenin, or Axin, constitutive activation of ß-catenin-TCF-mediated transactivation may result in an aberrant G2 checkpoint, which may contribute to tumorigenic cell growth.

The Cdc2-cyclin B complex is a critical regulator of the G2-M transition (19) . Cdc2 is activated at the end of the G2 phase by dephosphorylation of two inhibitory residues: (a) Tyr-15; and (b) Thr-14. Translocation of Cdc2 and cyclin B from the cytoplasm to the nucleus is further critical for the G2-M transition. We found that overexpression of Icat inhibits both dephosphorylation of Cdc2 at Tyr-15 and nuclear translocation of cyclin B1. Thus, these effects of Icat may be critical to the arrest of colorectal tumor cells in the G2 phase. In addition, overexpression of Icat caused a decrease in the levels of cyclin B1, Cdc2, and Cdc25, suggesting that these alterations may also contribute to cell cycle arrest at the G2 phase. In this regard, it is interesting to note that p53, which also induces G2, as well as G1 arrest, inhibits transcription of cyclin B1 and Cdc2 and interferes with the accumulation of the Cdc2-cyclin B1 complex in the nucleus (20) . Furthermore, a p53-regulated inhibitor of the G2-M transition, 14-3-3{sigma}, has been reported to induce G2 arrest by inhibiting nuclear translocation of the Cdc2-cyclin B1 complex (21 , 22) . Another mediator of p53-induced G2 arrest, Reprimo, has also been reported to induce G2 arrest by inhibiting both Cdc2 activity and nuclear translocation of the Cdc2-cyclin B1 complex (23) . However, although our data are suggestive, the precise mechanism underlying Icat-induced G2 arrest needs to be further confirmed.

Flow cytometric analysis revealed that overexpression of Icat in SW48 cells increases the sub-G1 population, typical of cells undergoing apoptosis. Furthermore, overexpression of Icat was found to increase the population of cells having a DNA content of >=4N. Thus, SW48 cells arrested at G2 phase because of Icat overexpression appear to have entered a DNA synthetic phase illegitimately, i.e., without having undergone prior mitosis. Trypan blue exclusion assay confirmed that these cells are undergoing cell death. Interestingly, although 14-3-3 {sigma} and Reprimo induce G2 arrest, these proteins do not induce cell death (21, 22, 23) . Thus, it will be interesting to examine in more detail how Icat induces cell death.

ß-catenin was identified originally as a cadherin-associated protein and found to play an important role in cell-cell adhesion (24) . In addition to Wnt signaling and cell-cell adhesion, ß-catenin plays a variety of roles in the cell via its interaction with multiple proteins, including the actin-binding protein Fascin and the Presenilins (25 , 26) . Therefore, it is possible that overexpression of Icat affects the function of these proteins.

The results presented in this study suggest that Icat may have potential as a target of gene therapy or drug development. Icat may prove to be a better target than APC or Axin, because Icat is able to inhibit the proliferation of tumor cells harboring a mutant ß-catenin and having aberrantly ß-catenin-TCF-mediated transcription, cells which are resistant to the action of APC and Axin. Thus, drugs that mimic the effects of ICAT may be useful as antitumor reagents. Elucidation of the three-dimensional structure of ICAT may also provide important insights into the development of such drugs.


   ACKNOWLEDGMENTS
 
We thank Mark Lamphier for reading the manuscript and I. Saitoh for providing us the materials for the COS-TPC method.


   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 Supported by Grants-in-Aid for Scientific Research on Priority Areas and the Organization for Pharmaceutical Safety and Research. Back

2 To whom requests for reprints should be addressed, at Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1–1-1 Yayoi, Bunkyo-ku, Tokyo 113, Japan. E-mail: akiyama{at}imcbns.iam.u-tokyo.ac.jp. Back

3 The abbreviations used are: GSK, glycogen synthase kinase; APC, adenomatous polyposis coli; m.o.i., multiplicity of infection; pAb, polyclonal antibody. Back

Received 1/22/02. Accepted 3/26/02.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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