ISSN 0388‑0192

CANCERRESEARCH
INSTITUTEREPORT

2003 ‑ 2005

KANAZAWA UNIVERSITY

Cancer ResearchInstitute Report
2003－2005

SCIENTIFIC REPORTS

Unpublished data should not cited without
permission of the authors.

Divisionof
Molecular Biology

General Summary of Division of Molecular Biology
The division has been dedicated to basic researches on Hepatitis B Virus (HBV) and Hepatitis
C Virus (HCV), the two predominant causes of chronic hepatitis and eventual hepatocellular
carcinoma (HCC) among the world. We focused two molecular targets of the viruses; HBV X
protein (HBx) that has been long-suspected but not approved to be oncogenic, and HCV NS5B
that is RNA-dependent RNA polymerase (RdRP) responsible for HCV replication. Also wehave opened the researches on telomere and telomerase in yeast and human, that are tightly
regulated in normal cells but deregulated in cancer cells. The outcomes of these projects in thelast three years have been much contributed to understanding new aspects of the regulatory
processes and the putative molecular partners of these target molecules. Unfortunately, thedivision will be closed by the end of March 2006 since the chief has to retire.
A) HBx and related targets.
To elucidate the molecular role of HBx in transcriptional modulation, we further analyzed thenuclear target of HBx, RNA polymerase II subunit 5 (RPB5) by a two or three-step alanine
scanning strategy. Among the identified residues critical for the bindings to HBx and RAP30,
T111 and S113 are close to DNA and critical for both TFIIF- and HBx-binding. We found the
double-strand DNA-binding ability of RPB5 and identified the critical and important residues
including T111 and S113 for the DNA-binding. These results may imply that HBx interfereswith DNA-binding of RPB5 to modulate transcription initiation or initiation to elongation.
We tried to evaluate biological roles of HBx in two aspects. Firstly the roles of HBx in HBVtranscription and replication using a HBV replicon system were examined. The results
demonstrate that HBx augments HBV transcription and replication though the coactivation
ability since the HBx sequences critical for the coactivation and for the augmentation of HBVreplication are the same, and HBV 3.5 kb RNA and DNA are affected at the similar extent by theectopic expressed HBx. Secondly we evaluated whether HBx retains ability to immortalize
human primary cells and/or to transform human immortalized primary cells in vitro since theoncogenic property of HBx has been all addressed with rodent systems, and it remains strongly
controversial during last decades. We found that HBx overcomes active RAS-induced
senescence and affords ability to the immortalized cells expressing RAS and HBx to makecolonies in soft agar assay and tumors in nude mice. Such overcoming ability of HBx requires
full-length HBx, indicating that the coactivation domain is not enough and that the N-terminal
domain has some critical role(s) for the ability.
B) HCV NS5B, replicase, and HCV replication.
We previously identified the novel 5 residues of NS5B critical for RdRP activity and the― 1 ―interaction between NS5B and NS5A, the HCV non-structural regulatory protein, using bacterial
recombinant proteins in vitro. On the way to elucidate subcellular localization of NS5B, wefound the tight interaction of NS5B and nucleolin, a RNA chaperon. In last several years, weconcentrated to evaluate whether the findings obtained in vitro have biological relevance in
HCV replication in vivo by introducing HCV RNA subreplicon system (provided by Dr.
Shimotohno, Kyoto Univ.) and by establishing an improved system. We confirmed that the 5
residues of NS5B defined in vitro are also critical for HCV replication in vivo. By scanning withclustered alanine substitution libraries of NS5A in HCV replicon, the critical nature of theinteraction of NS5A and NS5B was found to be crucial in HCV replication. We are now going tocharacterize the important role of nucleolin in HCV replication through the direct binding to
NS5B.
C) Telomere and telomerase in yeast and human.
We characterize regulatory steps of telomere and telomerase in yeast and human.
In order to investigate relationships between life span and telomere function and to identify
signal-transmitting pathway, which responds to ageing stress, we screened telomere length andsilencing in the knock-out series of protein phosphatase (PPase) genes in budding yeast. Only
the sit4 mutant encoding the type 2A of PPase (PP2A) was selected. The mutant cells
accumulated highly phosphorylated Sir3 and decreased amount of Sir3 on subtelomere region.
Moreover, shortened life span comparable to sgs1 cells, which lost RecQ type helicase, wasobserved in sit4 cells. These suggest that PP2A regulates phosphorylation state of Sir3
responding to ageing stress.
In human system, we have tried to purify insect-expressed recombinant hTERT. During
purification, we found the highly purified protein failed to exhibit reconstituted telomerase
activity in vitro. Then, we established an insect coexpression system of the two-components, andare trying to purify the recombinant telomerase. We collaborated with Drs. Hahn and Weinberg
to raise anti-hTERT mAbs that enabled to characterize telomerase in human primary fibroblasts.
The results together with knock-down experiments of hTERT indicate that telomerase activity isimportant for cell proliferation and cell lifespan. We also found the interaction between hTERT
and nucleolin that is important in regulating subcellular localization of telomerase.

― 2 ―The transcriptional transactivation function of HBx protein is
important for its augmentation role in hepatitis B virus replication1.
Tang H*, Delgermaa L, Huang F*, Oishi N, Liu L*, He L*, Zhao L*, Murakami S.
The roles of HBx in regulating HBV transcription and replication were examined with atransient transfection system in HepG2 using wild-type or HBx-minus HBV genome constructs,
and a series of mutation HBx expression plasmids. HBx has augmentation effects on HBVtranscription and replication as HBV mutant genome with defective X gene led to decreased
levels of 3.5-Kb HBV RNA and HBV replication intermediates, and these decreases can becomplemented in trans by ectopic expression of HBx. The truncation mutant HBx-D1 (aa.
51-154) containing the coactivation domain is enough for this function. By alanine scanmutagenesis strategy, the regions between aa 52 - 65 and aa 88 -154 are important for theaugmentation function of HBx in HBV replication. With reporter gene analysis, thetransactivation and coactivation activities of HBx are well coincide with its augmentation
function in HBV transcription and replication. Therefore HBx has important role in stimulating
HBV transcription and replication, and the coactivation function of HBx may be critical for theaugmentation effect on HBV replication. (*The project is based on the international
collaboration between Sichuan Univ. and Kanazawa Univ,)
Reference 1: Tang H, et al., (2005) J. Virol., 79: 5548-5556.
Figureillustrates

quantitative

analysis of the 3.5-kb HBV RNA andHBV DNA replication intermediatesof 1.2 genome-unit of wild anddefective in X-ORF in the absence orin the presence of varying amount of
the HBx-expression plasmid.

― 3 ―HBx contributes to the oncogenic conversion of human immortalized
cells with active RAS by overcoming oncogene-induced senescence1.
Oishi T, Masutomi K, Khurts S, Nakamoto T, Kaneko S, Murakami S.
HBx has been long suspected to be oncogenic although it remains still controversial.
Pathological roles of HBx in the carcinogenic process have been previously examined only withrodent systems. Therefore we addressed effect of HBx on the immortalization andtransformation abilities in human primary cells.
As HBx alone failed to immortalize human primary cells, BJ and TIG3 cells, nor to transformthe hTERT-introduced immortalized BJ cells, we next examined HBx overcomes active
RAS-induced cellular as oncogene-induced senescence (OIS) has been recently regarded as oneof the antitumor processes of cells. The immortalized BJ cells expressing active RAS and HBxproliferate more than 80 population doublings and could form colonies in soft agar and tumors
in nude mice (Figure) although RAS alone actually induced senescence of the immortalized BJ
cells. A truncated mutant, HBx-D1 (aa 51-154), had the ability to overcome OIS in a population
doubling analysis, but failed to exhibit colonigenic and tumorigenic abilities. These observations
indicate that HBx-D1, which harbors the coactivation domain is not equivalent to full-length
HBx in the ability to overcome active RAS-induced senescence, strongly suggesting that HBx
can contribute to carcinogenesis by overcoming OIS in human cells.
Reference 1: Oishi N, et al., (2005) International Meeting on the Molecular Biology of Hepatitis
B Viruses, Heidelberg, Germany, Sep. 18-21, 2005
Figure illustrates tumor formation in nude
mice. Each point on the graph represents
the average volume of tumors. SV40
LT+ST+RAS is a positive control.

― 4 ―Mutational analysis of the central part of human RNA polymerase II
subunit 5 (RPB5) by two-step alanine scanning: the residues criticalfor interactions with TFIIF subunit RAP30 and Hepatitis B Virus X
protein and those for DNA-binding ability.
Le TTT, Zhang S, Hayashi N, Yasukawa M, Delgermaa L, Murakami S.
RPB5 is close to DNA downstream of initiation site and interacts with several regulators.
HBx binds the central part of RPB5 to modulate activated transcription, and TFIIF subunit
RAP30 interacts with the same part of RPB5 that is critical for the association between TFIIF
and RNAPII. By introducing systematic mutagenesis of the central part of RPB5 using two-stepalanine scanning libraries to pinpoint critical residues for its binding to RAP30 in the TFIIF
complex and/or to HBx, and identified these residues in both mammalian cells and in an in vitro
binding assay. Four residues, F76, I104, T111 and S113, are critical for both TFIIF- andHBx-binding, indicating the overlapping nature of the sites of interaction. In addition, V74 andN98 are required for HBx-binding, and T56 and L58 are needed for RAP30-binding.
Interestingly the residues exposed to solvent, T111 and S113, are very close to the DNA,
implying that two factors may modulate the interaction between DNA and RPB51.
Reference 1: Le TTT, Zhang S, Hayashi N, Yasukawa M, Delgermaa L, Murakami S. (2005) J.
Biochem (Tokyo), 138(3): 215-224.

― 5 ―DNA-binding ability of RNA polymerase II subunit 5 (RPB5)
Zhang S, Le TTT, Tang H, Wei W, Hayashi N, Murakami S.
We found that RPB5 retains DNA-binding ability trapped by double-stranded DNA cellulose.
The 6 residues critical for binding DNA were identified within the middle part of RPB5, by athree-step alanine scanning with clustered and point substitution libraries. Among them, T111 issolvent-exposed and the nearest neighbor of P112 that is the residue predicted to be closest to
DNA. Three residues are important for the structural integrity of the mixed ß-sheet that may
indirectly affect DNA-binding ability. We evaluated these residues conserved among human andyeast by introducing a point mutant in yeast in place of its wild-type counterpart. T117A, T117G(hT111) and the glycine substitution of the three residues in the ß-sheet affected cell growth atsuboptimal temperatures. Interestingly, most of these residues are also indispensable for RPB5
to bind HBx and/or RAP30. Actually these factors inhibited DNA-binding of RPB5, strongly
supporting the notion that these regulators may modulate transcription by inhibiting RPB5 frominteracting with DNA.
Zhang S, Le TTT, Tang H, Wei W, Hayashi N, Murakami S. 2005 International Meeting on
Molecular Biology of HBV viruses. Sept. 20 at Heidelberg Univ.

― 6 ―Subcellular Localization of RPB5-Mediating Protein, RMP, and its
putative functional partner1.
Delgermaa L, Hayashi N, Dorjsuren D, Nomura T, Thuy LT, Murakami S.
We previously identified a novel cellular protein RMP that retains corepressor activity andfunctionally antagonizes transcriptional modulation by Hepatitis B virus X protein (Dorjsuren D.
et al., Mol. Cell. Biol., 18: 7546-7555, 1998). Here, subcellular localization of RMP was
examined with GFP-fused forms. We found that a nuclear localization signal (NLS) and acoiled-coil (CC) domain functioning as a cytoplasmic localization signal (CLS), are both
important for the subcellular localization of RMP. The CLS apparently acts dominantly since
RMP is mostly localized in cytoplasm with weak and diffuse signals in nuclei, and the NLS isindispensable for the nuclear localization of RMP only in the absence of the CLS. Using a yeast
two-hybrid method, we isolated a putative corepressor, DNA methyltransferase 1 (DNMT1)
associating protein, DMAP1, which was demonstrated to bind the CC domain of RMP. DMAP1
facilitates nuclear localization of RMP and the corepressor activity of RMP in a dose-dependent
manner through the interacting with the CC domain of RMP. The results were discussed in lightof the recent paper showing a novel evolutionary conserved role of RMP/URI in TOR signaling(Gstaiger, M. et al., Science, 302: 1208-1212, 2003).
Reference 1: Delgermaa L, Hayashi N, Dorjsuren D, Nomura T, Thuy LT, Murakami S. (2004)
Mol. Cell. Biol., 24(19):8556-8566.
Figure illustrates the summary of the DMAP1-binding
region of RMP that is within the coiled-coil domain by
GST pull-down in vitro using the partially purified
proteins. The result is consistent with that using extractsof cells transiently expressing two differentially tagged
proteins1.

― 7 ―Direct Interaction between nucleolin and HCV NS5B1.
M. Hirano, S. Kaneko, T. Yamashita, H. Luo, W. Qin, Y. Shirota, T. Nomura, K.
Kobayashi, S. Murakami.
HCV NS5B is an RNA-dependent RNA polymerase (RdRP), a central catalytic enzyme in
HCV replication. While studying the subcellular localization of a NS5B mutant lacking the
C-terminal membrane-anchoring domain, NS5Bt, we found that expression of the GFP-fused
form was exclusively nucleolar. Interestingly, the distribution of endogenous nucleolin changed
greatly in the cells expressing GFP-NS5B, with nucleolin colocalized with GFP-NS5B inperinuclear regions in addition to the nucleolus. The interaction between nucleolin and NS5B
was demonstrated by GST pull-down assay. The results indicated that C-terminal region of
nucleolin was important for its binding to NS5B. Scanning a clustered- alanine substitution
mutant library of NS5B revealed that two sequences of NS5B, aa 208-214 and aa 500-506, wereboth found to be indispensable for the nucleolin binding. We reported that the latter sequence isessential for oligomerization of NS5B which is a prerequisite for the RdRP activity. C-terminal
nucleolin inhibited the NS5B RdRP activity in a dose dependent manner. Taken together, thebinding ability of nucleolin may be involved in NS5B functions.
Reference 1: M. Hirano, S. Kaneko, T. Yamashita, H. Luo, W. Qin, Y. Shirota, T. Nomura, K.
Kobayashi, S. Murakami. (2003) J. Biol. Chem., 278: 5109-5115.

The critical sequences of NS5B shown at the
top

of

NS5Bt

which

were

clusteredalanine-substituted as shown at the bottom
constructs1.

― 8 ―Effect of Interaction between Hepatitis C Virus NS5A and NS5B on the
Hepatitis C Virus Replicon1.
Shimakami T, Hijikata M*. Luo H, Ma Y, Kaneko S, Shimotohno K*, Murakami S.
*Virus Research Inst. Kyoto Univ.
HCV NS5A is important for the establishment of replication by adaptive mutations or
localization, although its role in viral replication remains unclear. It was previously reported that
NS5A interacts with NS5B via two regions of NS5A in the isolate JK-1 and modulates theactivity of NS5B RdRp (Y. Shirota et al., J. Biol. Chem., 277:11149–11155, 2002), but thebiological significance of this interaction has not been determined. We examined the effect of
this interaction on HCV RNA replication with an HCV replicon system derived from the isolate
M1LE (H. Kishine et al., Biochem. Biophys. Res. Commun., 293:993–999, 2002). We
constructed three internal deletion mutants, M1LE/5Adel-1 and M1LE/5Adel-2, each encoding
NS5A which cannot bind NS5B, and M1LE/5Adel-3, encoding NS5A that can bind NS5B. Aftertransfection into Huh-7 cells, M1LE/5Adel-3 was replication competent, but both
M1LE/5Adel-1 and M1LE/5Adel-2 were not. By scanning with 20 alanine-substituted clusteredmutants within both NS5B-binding regions, only 5 of the 20 mutants were replication competent.
Subsequently, we established more efficient replicon system by introducing a point mutation,
S232I, into NS5A using cured Huh-7 cells as recipient cells. In this system, only the same fivemutants were replication competent. These results strongly suggest that the interaction between
NS5A and NS5B is critical for HCV RNA replication in the HCV replicon system. This
improved system was applied to examine whether the 5 residues of NS5B indispensable for
RdRP activity in vitro are critical in HCV replication. An alanine-substitution mutant of one of
the 5 residues was replication incompetent in the HCV replicon system2.
Reference 1: Shimakami, T., Hijikata, M. Luo, H., Ma, Y., Kaneko, S., Shimotohno, K.,
Murakami, S. (2004) J. Virol., 78: 2738-2748.
Reference 2: Ma Y, Shimakami T, Luo H, Hayashi N, Murakami S.
279(24):25474-25482.

― 9 ―(2004) J Biol Chem,Genetic studies of signal transduction pathway, responding to ageing
stress and regulating telomere function.
N. Hayashi, S. Murakami
Cellular senescence is irreversible arrest of cell cycle.

The probability to be into thesenescence increases with the passage of cell division. Accumulation of many kinds of stress,
such as ROS (reactive oxygen species), DNA lesion, shortened telomere, loss ofheterochromatin structure and so on, would cause senescence.
work for the maintenance of its cellular life span.

So, many signal pathways may

Budding yeast, Saccharomyces cerevisiae,

has 32 protein phosphatases (PPase), and 30 in them are nonessential.

We screened yeast

knock-out series of PPases to investigate the regulatory pathway of cellular senescence.

We

examined telomere length of them by Southern blot analysis, and isolated the sit4 cell that had
slightly short telomere. SIT4 gene encodes one of type 2A PPases (PP2A), and involved in cell
cycle control.

Silencing ability at telomere region in the sit4 cells was also deficient, andphosphorylated Sir3 protein was accumulated. Life span in the sit4 cells was as short as in thesgs1 cells. Furthermore, we found altered structure of nucleoli and frequent recombination at
rDNA region in them.

These suggest that PP2A regulates phosphorylation of the Sir3 protein

to maintain heterochromatin structure at telomere, and also these may suggest that formation ofheterochromatin structure affects cellular life span.
Reference: Hayashi et al. The SIT4 gene, which encodes protein phosphatase 2A, is required fortelomere function in Saccharomyces cerevisiae. Curr Genet 47:359-367. 2005.

Figure.

Modelofregulatory

systemofheterochromatin status via Sir3 phosphorylation
by PPase and MAP kinase.

― 10 ―

Telomerase maintains telomere structure in normal human cells.
Masutomi K, Yu EY, Khurts S, Ben-Porath I, Currier JL, Metz GB, Brooks MW, Kaneko S,
Murakami S, DeCaprio JA, Weinberg RA, Stewart SA, Hahn WC.
In normal human cells, telomeres shorten with successive rounds of cell division, andimmortalization correlates with stabilization of telomere length. These observations suggest thathuman cancer cells achieve immortalization in large part through the illegitimate activation of
telomerase expression.
We found that the rate-limiting telomerase catalytic subunit hTERT is expressed in cycling
primary presenescent human fibroblasts, previously believed to lack hTERT expression andtelomerase activity. Disruption of telomerase activity in normal human cells by knock-down
hTERT slows cell proliferation, restricts cell lifespan, and alters the maintenance of the 3'
single-stranded telomeric overhang without changing the rate of overall telomere shortening.
Together, these observations support the view that telomerase and telomere structure are
dynamically regulated in normal human cells and that telomere length alone is unlikely totrigger entry into replicative senescence.
(This project was carried out by an international collaboration with Dr. Hahn WC, Dana-Farber
Cancer Institute, Harvard Medical School, and Dr. Weinberg RA, Whitehead Inst. MIT.)
Reference 1: Masutomi K, Yu EY, Khurts S, Ben-Porath I, Currier JL, Metz GB, Brooks MW,
Kaneko S, Murakami S, DeCaprio JA, Weinberg RA, Stewart SA, Hahn WC. (2003)
114(2):241-253.

― 11 ―

Cell,

Nucleolin interacts with telomerase1.
Khurts S, Masutomi K, Delgermaa L, Arai K, Oishi N, Mizuno H, Hayashi N, Hahn W,
Murakami S.
Telomerase is a specialized reverse transcriptase composed of core RNA and protein subunits
that plays essential roles in maintaining telomeres in actively dividing cells. Recent work
indicates that telomerase shuttles between subcellular compartments during assembly and in
response to specific stimuli. In particular, telomerase co-localizes with nucleoli in normal humanfibroblasts. Here, we show that nucleolin, a major nucleolar phosphoprotein, interacts withtelomerase and alters its subcellular localization. Nucleolin binds hTERT through interactions
with its RNA-binding domain 4 and carboxyterminal RGG domain, and this binding also
involves the telomerase RNA subunit hTERC. The protein-protein interaction between nucleolin
and hTERT is critical for the nucleolar localization of hTERT. These findings indicate thatinteraction of hTERT and nucleolin participates in the dynamic intracellular localization of
telomerase complex.
Reference 1: Khurts S, Masutomi K, Delgermaa L, Arai K, Oishi N, Mizuno H, Hayashi N,
Hahn W,Murakami S. (2004) J Biol Chem, 279: 51508-51515.
Figureillustrates

hTERT-bindingtheregiondelineated by in vivo and invitro with GST pull-down
experiments1.

― 12 ―

Publications
1. Hirano M, Kaneko S, Yamashita T, Luo H, Qin W, Shirota Y, Nomura T, Kobayashi K,
Murakami S. (2003) Direct interaction between nucleolin and Hepatitis C Virus NS5B. J Biol
Chem. 278: 5109-5115.
2. Wei W, Gu JX. Zhu CQ, Sun FY, Dorjsuren D, Lin Y, Murakami S. (2003) Interaction withgeneral transcription fator IIF (TFIIF) is required for the suppression on activatedtranscription by RPB5-mediating protein RMP. Cell Research. 13:111-120.
3. Masutomi K, Yu EY, Khurts S, Ben-Porath I, Currier JL, Metz GB, Brooks MW, Kaneko S,
Murakami S, DeCaprio JA, Weinberg RA, Stewart SA, Hahn WC. (2003) Telomerase
maintains telomere structure in normal human cells. Cell. 114(2): 241-253.
4. Kyo S, Masutomi K, Maida Y, Kanaya T, Yatabe N, Nakamura M, Tanaka M, Takarada M,
Sugawara I, Murakami S, Taira T, Inoue M. (2003) Significance of immunological detectionof human telomerase reverse transcriptase: re-evaluation of expression and localization ofhuman telomerase reverse transcriptase. Am J Pathol. 163(3): 859-867.
5. Shimakami T, Hijikata M, Luo H. Ma Y, Kaneko S, Shimotohno K, Murakami S. (2004)
Effect of interaction between Hepatitis C Virus NS5A and NS5B on the Hepatitis C Virusreplicon. J Virol. 78: 2738-2748.
6. Moon EJ, Jeong CH, Jeong JW, Kim KR, Yu DY, Murakami S, Kim CW, Kim KW. (2004)
Hepatitis B virus X protein induces angiogenesis by stabilizing hypoxia -inducible
factor-1alpha. FASEB J. 18(2): 382-384.
7. Luo H, Qin W, Ma Y, Shimakami T, Murakami S, He F, Zhao L.(2004) Purification andpartial characterization of hepatitis C virus (HCV) non-structural protein 5A (NS5A)
expressed in Escherichia coli. Sheng Wu Yi Xue Gong Cheng Xue Za Zhi. Oct;21(5):795-799.
[Article in Chinese]
8. Ma Y, Shimakami T, Luo H, Hayashi N, Murakami S. (2004) Mutational analysis of hepatitis
C virus NS5B in the subgenomic replicon cell culture. J Biol Chem. 279 (24) : 25474-25482.
9. Delgermaa L, Hayashi N, Dorjsuren D, Nomura T, Le TTT, Murakami S. (2004) Subcellular
localization of RPB5-mediating protein, RMP, and its putative functional partner. Mol Cell
Biol. 24(19): 8556-8566.
10. Khurts S, Masutomi K, Delgermaa L, Arai K, Oishi N, Mizuno H, Hayashi N, Hahn WC,
Murakami S. (2004) Nucleolin interacts with telomerase. J Biol Chem. 279: 51508-51515.
11. Tang H, Delgermaa L, Huang F, Oishi N, Liu L, He L, Zhao L, Murakami S. (2005)
Transcriptional transactivation function of HBx protein is important for the augmentation role

― 13 ―

in Hepatitis B Virus replication. J Virol. 79(9): 5548-5556.
12. Hayashi N, Nomura T, Sakumoto N, Mukai Y, Kaneko Y, Harashima S, Murakami S. (2005)
The SIT4 gene, which encodes protein phosphatase 2, is reqired for telomere function in
Sccharomyces cerevisiae. Current genetics. 47: 359-367
13. Le TTT, Zhang S, Hayashi N, Yasukawa M, Delgermaa L, Murakami S.(2005) Mutational
analysis of human RNA polymerase II subunit 5 (RPB5): the residues critical for interactions
with TFIIF subunit RAP30 and Hepatitis B Virus X protein. J Biochem (Tokyo). 138(3):215-224.
14. Shimakami T, Honda M, Kusakawa T, Murata T, Shimotohno K, Kaneko S, Murakami S.
(2006) Effect of HCV NS5B-nucleolin interaction on HCV replication with HCV subgenomic
replicon. J Virol., in press.

Other achievements
Murakami S. Hokkoku Bunka Prize by Hokkoku Shinbun. Nov. 3, 2004
Murakami S. Invited professor of Soochaw University, Souzhou, China
From April, 2005~
Murakami S. Head of Collaboration Research Center of Kanazawa University.
From April, 2004~March 2006.

― 14 ―

Divisionof
Molecular
Virology and Oncology

General Summary of Division of Molecular Virology and Oncology
Degradation of extracellular matrix (ECM) is one of the first steps for tumor invasion andmetastasis. Matrix metalloproteinases (MMPs) have been strongly implicated in this step.
Membrane-type MMP-1 (MT1-MMP) was identified in this laboratory as an activator of
MMP-2 expressed on the surface of tumor cells (Nature, 370, 61-65, 1994), and later not only
ECM macromolecules but also various biologically important molecules were shown to serve as
substrates for MT1-MMP. Accumulating evidences have demonstrated that MT1-MMP
expression level is closely associated with invasiveness and malignancy of tumors, suggestingthat MT1-MMP is one of the most critical factors for tumor invasion and metastasis. Despite
enthusiasm for MMP inhibitors, phase III trials have not yet demonstrated significance inoverall survival and side effects remain an issue. We believe that understanding of the functionsof MT1-MMP could supply clues to develop novel therapeutic strategies targeting MT1-MMP.
The goal of study in Molecular Virology and Oncology Division is to elucidate the molecularmechanism of tumor invasion and metastasis, and identify the molecular targets for diagnosis
and therapy of malignant tumors.
A) Novel Functions of MT1-MMP:
MT1-MMP was first identified as an activator of pro-MMP-2, and later MT1-MMP wasshown to degrade a various ECM components including collagens. While degradation of ECM
is an important aspect of MMP biology, growing evidence has demonstrated specificprocessing/activation or degradation of cell surface receptors and ligands. To identify molecules
which interact with MT1-MMP including its substrates, we have developed an expression
cloning strategy and been screening cDNA libraries of human placenta, fetal brain and fetal
kidney in which ECM turnover is active. So far we have identified claudin-5 and N-Tes(Testican-3), which regulates MT1-MMP activity. We also identified Kiss-1/metastin,
syndecan-1 and lumican as substrates for MT1-MMP, and demonstrated that degradation ofthese substrates contributes to invasive growth of tumor cells.
B) Stimulation of Cell Migration by MT1-MMP:
Cell migration is a complex process that can be regulated by multiple mechanisms, including
MAPK/ERK, phosphatidylinositol 3-kinase and ECM degrading proteinases such as MMPs.
Adhesion-mediated ERK activation is dependent on integrin engagement. ERK activity has been
implicated in ECM-dependent cell spreading and migration, concomitant with a role for ERK in
the regulation of integrin-dependent adhesion/cytoskeletal organization. Although MMPs were
known to be involved in cell migration, the molecular mechanism had remained elucidated. We
examined the effect of MT1-MMP expression on ERK activation, and found that MT1-MMP
positively regulates cell-collagen interactions which generate a sustained signal through the
MEK/ERK pathway, and this sustained ERK activation in turn up-regulates MT1-MMP
expression. Thus, MT1-MMP functions in a positive feedback loop to induce sustained ERK
activation, which consequently stimulates cell migration on type I collagen.
C) JSAP1 Cooperates with FAK to Regulate JNK and Cell Migration
Focal adhesion kinase (FAK) plays a central role in adhesion, spreading and migration of cells.
Previously we found that c-Jun N-terminal kinase (JNK)/stress activated protein
kinase-associated protein 1 (JSAP1), a scaffold factor in the MAPK cascades forms a complex
with FAK. Our recent finding showed that the JSAP1/FAK complex functions cooperatively asa scaffold for the JNK signaling pathway and regulator of cell migration on fibronectin, and that
JSAP1 is also associated with malignancy in brain tumors.
― 15 ―

Testican 2 Abrogates Inhibition of Membrane-Type
Metalloproteinases by Other Testican Family Proteins.

MatrixM. Nakada*, H. Miyamori, J. Yamashita* and H. Sato. (*Dept. Neurosurgery, Med. Sch.,
Kanazawa Univ.)
Testican family protein are putative extracellular heparan/chondroitin sulfate proteoglycans of
unknown function. Recently we identified N-Tes, which is a product of testican 3 splicing
variant gene, as an inhibitor of membrane-type matrix metalloproteinases (MT-MMPs). The
inhibitory function is common among testican family members except for testican 2, which wasshown to uniquely abolish inhibition of MT1-MMP- or MT3-MMP-mediated pro-matrix
metalloproteinase-2 activation by other testican family members. Testican 2 inactivates N-Tes
by binding to the C-terminal extracellular calcium-binding (EC) domain of N-Tes through its
N-terminal unique domain as demonstrated by co-immunoprecipitation analysis, and thus
testican 2 was unable to inactivate an N-Tes deletion mutant lacking the EC domain(N-Tes-∆122). Migration of U251 cells on collagen which was dependent on MT1-MMP
activity under serum-free condition, was inhibited by N-Tes or N-Tes-∆122 deposited oncollagen. Testican 2 was not incorporated into collagen by itself, and was deposited only in thepresence of N-Tes, suggesting that testican 2 bounds to N-Tes deposited on collagen. Binding of
testican 2 to N-Tes deposited on collagen allowed migration of cells expressing MT1-MMP.
Unlike wild-type N-Tes, N-Tes-∆122 did not bind to testican 2, and thus expression of testican 2
did not recover cell migration blocked by N-Tes-∆122. In situ hybridization showed thatneurons are major source of all testican family members in the normal brain. The quantitative
reverse transcription-polymerase chain reaction analysis demonstrated that all testican family
members are expressed prominently in normal brain, and their expression levels decrease as
tumor grade increases. The expression level of testican 2 was the highest among testican family
members regardless of histological grade of astrocytic tumors. These results suggest thatabundant distribution of testican 2 may contribute to glioma invasion by inactivating othertestican family members including N-Tes, which all inhibit MT-MMPs. We propose that
N-Tes-∆122, which is resistant to testican 2, may have therapeutic potential as a barrier against
glioma invasion.
Fig. 1 Effects of testican on migration of
U251

gliomacells

expressing

MT1-MMP. U251 cells transfected with
MT1-MMP and indicated plasmids were
subjected to wound-induced migration
assay on plates coated with collagen.
Refference: Nakada, M., Miyamori, H., Yamashita, J. and Sato, H. (2003) Testican 2
abrogates inhibition of membrane-type matrix metalloproteinases by other testican family
proteins. Cancer Res., 63, 3364-3369.

― 16 ―

CleavageofSyndecan-1
byMembrane-Type
Metalloproteinase-1 Stimulates Cell Migration.

MatrixK. Endo*, T. Takino, H. Miyamori, H. Kinsen*, T. Yoshizaki*, M. Furukawa* and H. Sato(*Dept. Otolaryngology, Med. Sch., Kanazawa Univ.)
The transmembrane heparan sulfate proteoglycan syndecan-1 was identified from a humanplacenta cDNA library by the expression cloning method as a gene product which interacts withmembrane-type matrix metalloproteinase-1 (MT1-MMP). Co-expression of MT1-MMP withsyndecan-1 in HEK293T cells promoted syndecan-1 shedding, and concentration ofcell-associated syndecan-1 was reduced. Treatment of cells with MMP inhibitor BB-94 or tissue
inhibitor of MMP (TIMP)-2 but not TIMP-1 interfered with the syndecan-1 shedding promotedby

MT1-MMP

expression.

Incontrast,syndecan-1

shedding

inducedby

12-O-tetradecanoylphorbol-13-acetate (TPA) treatment was inhibited by BB-94 but not by either
TIMP-1 or TIMP-2. Shedding of syndecan-1 was also induced by MT3-MMP, but not by other
MT-MMPs. Recombinant syndecan-1 core protein was shown to be cleaved by recombinant
MT1-MMP or MT3-MMP at G82-L83 and G245-L246 peptide bonds. HT1080 fibrosarcoma cells
stably transfected with the syndecan-1 cDNA (HT1080/SDC), which express endogenous
MT1-MMP

spontaneously,shedsyndecan-1.

MigrationofHT1080/SDC

cells

on

collagen-coated dishes was significantly slow compared with that of control HT1080 cells.
Treatment of HT1080/SDC cells with BB-94 or TIMP-2 induced accumulation of syndecan-1 oncell surface, concomitant with further retardation of cell migration. These results suggest that theshedding of syndecan-1 promoted by MT1-MMP stimulates cell migration.
Fig.

1

Mock-transfected

HT1080(Mock

lanes)or

HT1080/syndecan-1 (SDC lanes) cells were subjected towound-induced migration assay on collagen-coated dishes inmedium containing 0.1 µM BB-94 or 2 µg/ml TIMP-2
protein. Note that migration of syndecan-1-expressing cells
was severely inhibited by MMP inhibitors. *, P< 0.01.

Reference: Endo, K., Takino, T., Miyamori, H., Kinsen, H., Yoshizaki, T., Furukawa, M. andSato, H. (2003) Cleavage of Syndecan-1 by Membrane-Type Matrix Metalloproteinase-1
Stimulates Cell Migration. J. Biol. Chem. 278, 40764-40770.

― 17 ―

Membrane-Type
1
Matrix
Metalloproteinase
Regulates
Collagen-dependent
Mitogen-ActivatedProtein/Extracellular
Signal-Related Kinase Activation and Cell Migration
T. Takino, H. Miyamori, Y. Watanabe, K.Yoshioka1, M. Seiki2 and Hiroshi Sato (1Dept.
Cell Cycle Regulation, Cancer Res. Inst. Kanazawa Univ., 2Dept. Cancer Cell Research,
Inst. of Medical Science, University of Tokyo)
Mitogen-activated protein kinase (MAPK)-extracellular signal-related kinase (ERK) kinase 1(MEK1)/ERK signaling has been implicated in regulation of tumor cell invasion and metastasis.
Migration of HT1080 cells on type I collagen was suppressed by matrix metalloproteinase(MMP) inhibitors BB94 and tissue inhibitor of metalloproteinase (TIMP)-2 but not by TIMP-1.
TIMP-2-specific inhibition suggests that membrane type-1 MMP (MT1-MMP) is involved in it.
Activation of ERK was induced in HT1080 cells adhered on dishes coated with type I collagen,which was inhibited by BB94. MMP-2 processing in HT1080 cells, which was also stimulated
by cultivation on type I collagen was inhibited by a MEK inhibitor PD98059. Expression of
constitutively active form of MEK1 promoted MMP-2 processing concomitant with the increaseof MT1-MMP level, suggesting that MT1-MMP is regulated by MEK/ERK signaling. In
addition, expression of hemopexin-like domain of MT1-MMP in HT1080 cells interfered with
MMP-2 processing, ERK activation and cell migration, implying that the enzymatic activity of
MT1-MMP is involved in collagen-induced ERK activation which results in enhanced cell
migration. Thus, adhesion of HT1080 cells to type I collagen induces MT1-MMP-dependent
ERK activation, which in turn causes increase of MT1-MMP level and subsequent cell
migration.
Fig. 1.

MT1-MMP is involved in cell migration.

HT1080cells cultured in serum-free DMEM containing DMSO (Control),BB94 (1 µM), PD98059 (25 µM), recombinant TIMP-1 or
TIMP-2 (10 µg/ml) and HT1080 cells transfected with
MT1-MMP or CA-MEK were examined for migration on type I
collagen. Error bars indicate S.D. for at least 30 cells percondition. Asterisk, P < 0.01 versus control.

― 18 ―

Cleavage of Lumican by Membrane-Type Matrix Metalloproteinase-1
Abrogates This Proteoglycan-Mediated Suppression of Tumor Cell
Colony Formation in Soft Agar
Y. Li, T. Aoki, Y. Mori, M. Ahmad, H.Miyamori, T. Takino and H. Sato
The small leucine-rich proteoglycan lumican was identified from a human placenta cDNA
library by the expression cloning method as a gene product which interacts with membrane-typematrix metalloproteinase-1 (MT1-MMP). Co-expression of MT1-MMP with lumican in
HEK293T cells reduced the concentration of lumican secreted into culture medium, and thisreduction was abolished by addition of the MMP inhibitor BB94. Lumican protein from bovine
cornea and recombinant lumican core protein fused to glutathione S-transferase were shown to
be cleaved at multiple sites by recombinant MT1-MMP. Transient expression of lumican in
HEK293 cells induced expression of tumor suppressor gene product p21/Waf-1, which was
abrogated by the co-expression of MT1-MMP concomitant with a reduction in lumican
concentration in culture medium. Stable expression of lumican in HeLa cells induced expressionof p21 and reduction of colony formation in soft agar, which were both abolished by theexpression of MT1-MMP. HT1080 fibrosarcoma cells stably transfected with the lumican cDNA
(HT1080/Lum), which express endogenous MT1-MMP, secreted moderate levels of lumican,
however, treatment of HT1080/Lum cells with BB94 resulted in accumulation of lumican inculture medium. The expression levels of p21 in HT1080/Lum were proportional to theconcentration of secreted lumican, and showed reverse co-relation with colony formation in soft
agar. These results suggest that MT1-MMP abrogates lumican-mediated suppression of tumor
cell colony formation in soft agar by degrading this proteoglycan which down-regulates it
through the induction of p21.
Fig. 1. Degradation of lumican by HT1080
cells enhances colony formation in soft agar.
Control

HT1080(-)

orHT1080cells

transfected with lumican plasmid (Lum) wereplated into 0.3% agarose with or without 0.1µM BB94, and colonies were observed under
microscopy 2-weeks after incubation.

― 19 ―

Cleavage of Apolipoprotein E by Membrane-Type Matrix
Metalloproteinase-1 Abrogates Suppression of Cell Proliferation
T. Aoki, D. Sato, Y. Li, T. Takino, H. Miyamori and H. Sato
Apolipoprotein E (apoE) in a human fetal brain cDNA library was identified, using theexpression cloning method, as a gene product that formed a complex with latent matrix
metalloproteinase (MMP)-2. Co-expression of membrane-type MMP-1 (MT1-MMP) with apoE
in HEK293T cells reduced the amount of apoE secreted into the culture medium, whereas
cell-associated apoE core protein was not affected. Incubation of native apoE protein withrecombinant MT1-MMP resulted in the cleavage of apoE. Recombinant apoE protein fused to
glutathione S-transferase (apoE-GST) was cleaved by MT1-MMP at the following peptide
bonds; T85-M86, K93-S94, R246-L247, A255-E256 and G296-L297. HT1080 cells transfected with theapoE gene, which express endogenous MT1-MMP, secreted a low level of apoE protein and its
cleaved fragments, and treatment with MMP inhibitor BB94 induced accumulation of apoE andretardation of cell proliferation. Addition of apoE-GST protein to the culture of HEK293T cells
suppressed cell proliferation, and stable transfection of the MT1-MMP gene partly abrogated the
suppression. These results suggest that cleavage of apoE protein by MT1-MMP abrogatesapoE-mediated suppression of cell proliferation.

Fig. 1. Cleavage of apoE by MT1-MMP. ApoE-GST fusion
protein (5 µg) was incubated with recombinant MT1-MMP
catalytic

domain(0.5µg),

separatedon

15%

SDS-polyacryamide gel, and then blotted onto PDF
membrane. The N-terminal amino acid sequence of eachfragment was determined with a Beckman Coulter LF300
amino acid sequencer.

― 20 ―

JSAP1/JIP3 Cooperates with FAK to Regulate c-Jun N-terminal
Kinase and Cell Migration
T. Takino, M. Nakada, K. Yoshioka1, H. Miyamori, Y. Watanabe, K. M. Yamada2 and H.
Sato (1Dept. Cell Cycle Regulation, Cancer Res. Inst. Kanazawa Univ., 2CDBRB,NIDCR, NIH, USA)
c-Jun N-terminal kinase (JNK)/stress-activated protein kinase-associated protein 1(JSAP1) (also termed JNK interacting protein 3; JIP3) is a member of a family of scaffold
factors for the mitogen-activated protein kinase (MAPK) cascades, and it also forms a complex
with focal adhesion kinase (FAK).

Here we demonstrate that JSAP1 serves as a cooperative

scaffold for activation of JNK and regulation of cell migration in response to fibronectin (FN)
stimulation.

JSAP1 mediated an association between FAK and JNK, which was induced byeither co-expression of Src or attachment of cells to FN. Complex formation of FAK with
JSAP1 and p130 Crk-associated substrate (p130Cas) resulted in augmentation of FAK activity
and phosphorylation of both JSAP1 and p130Cas, which required p130Cas hyperphosphorylation
and was abolished by inhibition of Src.

JNK activation by FN was enhanced by JSAP1, which

was suppressed by disrupting the FAK/p130Cas pathway by expression of a dominant-negative
form of p130Cas or by inhibiting Src.

We also documented the co-localization of JSAP1 withJNK and phosphorylated FAK at the leading edge and stimulation of cell migration by JSAP1
expression, which depended on its JNK binding domain and was suppressed by inhibition of
JNK. The level of JSAP1 mRNA correlated with advanced malignancy in brain tumors, unlike
other JIPs.

We propose that the JSAP1/FAK complex functions cooperatively as a scaffold for
the JNK signaling pathway and regulator of cell
migration on FN, and we suggest that JSAP1 isalso associated with malignancy in brain tumors.
Fig. 1. Model depicting a FAK-JSAP1 scaffold for JNK
activation and cell migration. JSAP1 seems to be amodulator of cell migration by associating with MAPK
signaling pathways and possibly microtubules.

― 21 ―

Publications
1. Takino, T., Miyamori, H., Kawaguchi, N., Uekita, T., Seiki, M., Sato, H. (2003)
Tetraspanin CD63 promotes targeting and lysosomal proteolysis of membrane-type 1
matrix metalloproteinase. Biochem Biophys Res Commun., 304, 160-166.
2. Mori, N., Sato, H., Hayashibara, T., Senba, M., Geleziunas, R., Wada, A., Hirayama, T.,
Yamamoto, N. (2003) Helicobacter pylori induces matrix metalloproteinase-9 throughactivation of nuclear factor kappaB. Gastroenterology., 124, 983-992.
3. Takino, T., Koshikawa, N., Miyamori, H., Tanaka, M., Sasaki, T., Okada, Y., Seiki, M.,
and Sato. H. (2003) Cleavage of Metastasis Suppressor Gene Product KiSS-1
Protein/Metastin by Matrix Metalloproteinases. Oncogene, 22, 4617-4626, 2003.
4. Takino, T., Nakada, M., Miyamori, H., Yamashita, J., Yamada, K.M., Sato, H. (2003) CrkI
adapter protein modulates cell migration and invasion in glioblastoma. Cancer Res., 63,
2335-2337.
5. Nakada, M., Miyamori, H., Yamashita, J. and Sato, H. (2003) Testican 2 abrogatesinhibition of membrane-type matrix metalloproteinases by other testican family proteins.
Cancer Res., 63, 3364-3369.
6. Endo, K., Takino, T., Miyamori, H., Kinsen, H., Yoshizaki, T., Furukawa, M., and Sato, H.
(2003) Cleavage of Syndecan-1 by Membrane-Type Matrix Metalloproteinase-1
Stimulates Cell Migration. J. Biol. Chem. 278, 40764-40770.
7. Takino T, Tamura M, Miyamori H, Araki M, Matsumoto K, Sato H, Yamada KM. (2003)
Tyrosine phosphorylation of the CrkII adaptor protein modulates cell migration. J Cell
Sci., 116, 3145-3155.
8. Takahara, T., Zhang, L.P., Yata, Y., Xue, F., Minemura, M., Sato, H., Watanabe, A. (2003)
Modulation of matrix metalloproteinase-9 in hepatic stellate cells by three-dimensional
type I collagen: its activation and signaling pathway. Hepatol Res., 26, 318-326.
9. Takino, T., Miyamori, H., Watanabe, Y., Yoshioka, K., Sato, H. (2004) Membrane-Type 1
Matrix

Metalloproteinase

Regulates

Collagen-dependent

Mitogen-ActivatedProtein/Extracellular Signal-Related Kinase Activation and Cell Migration. Cancer Res.,64, 1044-1049.
10. Inaki, N., Tsunezuka, Y., Kawakami, K., Sato, H., Takino, T., Oda, M. and Watanabe, G.
(2004)

Increasedmatrix

metalloproteinase-2

andmembrane

type1

matrix

metalloproteinase activity and expression in heterotopically transplanted murine tracheas.
J. Heart and Lung Transplantation, 23, 218-227.
11. Li, Y., Aoki, T., Mori, Y., Ahmad, M., Miyamori, H., Takino T. and Sato, H. (2004)― 22 ―

Cleavage of lumican by membrane-type matrix metalloproteinase-1 abrogates thisproteoglycan-mediated suppression of tumor cell colony formation in soft agar.

CancerRes., 64, 7058-7064.
12. Gum, R., Lengyel, E., Juarez, J., Chen, J.H., Sato, H., Seiki, M., Boyd, D. (2004)
Stimulation of 92-kDa gelatinase B promoter activity by ras is mitogen-activated protein
kinase -1-independent and requires multiple transcription factor binding sites including
closely spaced PEA3/ets and AP-1 sequences. J. Biol. Chem., 271, 10672-10680.
13. Uekita, T., Gotoh, I., Kinoshita, T., Itoh, Y., Sato, H., Shiomi, T., Okada, Y., Seiki, M.
(2004) Membrane-type 1 matrix metalloproteinase cytoplasmic tail-binding protein-1 is a
new member of the cupin superfamily: A possible multifunctional protein acting as aninvasion suppressor down-regulated in tumors. J. Biol. Chem., 279, 12734-12743.
14. Kondo, S., Horikawa, T., Takeshita, H., Kanegane, C., Kasahara, Y., Sheen, T.-S., Sato, H.,
Furukawa, M., Yoshizaki, T. (2004) Diagnostic value of serum EBV-DNA quantification
and antibody to viral capsid antigen in nasopharyngeal carcinoma patients. Cancer
Science, 95, 508-513.
15. Ren, Q., Sato, H., Murono, S., Furukawa, M., Yoshizaki, T. (2004) Epstein-Barr Virus(EBV) Latent Membrane Protein 1 Induces Interleukin-8 through the Nuclear Factor-κB
Signaling Pathway in EBV-Infected Nasopharyngeal Carcinoma Cell Line. Laryngoscope,
114, 855-859.
16. Uekita T, Yamanouchi K, Sato H, Tojo H, Seiki, M, Tachi C. (2004) Expression andlocalization of matrix metalloproteinases (MT1-MMP, MMP-2) and tissue inhibitor of
metalloproteinase-2 (TIMP-2) during synepitheliochorial placentation of goats (Capra
hircus). Placenta, 10, 810-819.
17. Aoki, T., Sato, D., Li, Y., Takino, T., Miyamori, H. and Sato, H. (2005) Cleavage of
Apolipoprotein E by Membrane-Type Matrix Metalloproteinase-1Abrogates Suppressionof Cell Proliferation. J. Biochemistry, 137, 95-99.
18. Kondo S, Wakisaka N, Schell MJ, Horikawa T, Sheen TS, Sato H, Furukawa M, Pagano
JS, Yoshizaki T. (2005) Epstein-Barr virus latent membrane protein 1 induces the matrix
metalloproteinase-1 promoter via an Ets binding site formed by a single nucleotide
polymorphism: enhanced susceptibility to nasopharyngeal carcinoma. Int J Cancer., 115,
368-376.
19. Takino, T., Nakada, M., Miyamori, H., Watanabe, Y., Sato, T., Gantulga, D., Yoshioka, K.,
Yamada, K.M., Sato, H. (in press) JSAP1/JIP3 cooperates with FAK to regulate c-Jun
N-terminal kinase and cell migration. J. Biol. Chem.

― 23 ―

20. Nakada, M., Miyamori, H., Kita, D., Takahashi, T., Yamashita, J., Sato, H., Miura, R.,
Yamaguchi, Y., Okada, Y. (in press) Human glioblastomas overexpress ADAMTS-5 that
degrades brevican. Acta. Neuropathol. (Berl).

― 24 ―

Divisionof
Molecular Bioregulation

General Summary of Division of Molecular Bioregulation
Tumor cells and resident cells can produce various chemokines and pro-inflammatory
cytokines, during carcinogenesis and metastasis processes. The produced chemokines andpro-inflammatory cytokines can modulate the microenvironment of tumor tissues, thereby
affecting carcinogenic and/or metastatic processes. A major research object in our division is toelucidate the roles of these endogenously produced chemokines and pro-inflammatory cytokines
in these processes, in order to use these molecules and/or their antagonists for the treatment ofcancer.
We discovered recently that Pim-3, a proto-oncogene with serine/threonine kinase
activity, was enhanced in chronic inflammation-mediated hepatoma tissues in mice and areinvestigating the roles of Pim-3 in the carcinogenesis processes of various types of cancers.
A) Chemokines and pro-inflammatory cytokines in carcinogenesis and metastasis
processes
Tumor cells as well as resident normal cells can produce a wide variety of chemokinesand pro-inflammatory cytokines, thereby affecting the course of carcinogenesis and metastasis
processes. Until present, we have provided definitive evidence to indicate that tumor necrosis
factor receptor p55 has crucial roles in inflammation-mediated colon carcinogenesis, liver
metastasis, and lung metastasis. Moreover, we demonstrated that tumor cells and resident
normal cells can produce various chemokines and that the produced chemokines have effects on
tumor cells as well as inflammatory cells, thereby contributing to carcinogenesis and metastasis
processes.
B) Pim-3, a proto-oncogene with serine/threonine kinase activity, in carcinogenesis
Pim-3 is aberrantly expressed in various types of tumor tissues and tumor cell lines,
including hepatocellular carcinoma and pancreatic cancer. Cell proliferation was attenuated andapoptosis was enhanced in human hepatocellular carcinoma and pancreatic cancer cell lines by
the ablation of Pim-3 gene with RNA interference. Therefore, Pim-3 may be a good moleculartarget for treating tumors, which exhibit the enhanced expression of Pim-3. We are now
investigating the detailed molecular and pathological consequences of Pim-3-mediatedphosphorylation of various substrates.

― 25 ―

Essential contribution of tumor necrosis factor receptor (TNF-R)
p55-mediated signals in metastasis and carcinogenesis
Y. Tomita, X. Yang, H. Kitakata, B.K. Popivanova, Y. Nemoto-Sasaki and N. Mukaida
We have previously demonstrated that TNF-Rp55-mediated signals could up-regulate theexpression of an adhesion molecule, vascular adhesion molecule (VCAM)-1 in sinusoidal
endothelial cells and eventually facilitate liver metastasis by intrasplenic injection of a colon
carcinoma cell line into mice (1).
Intravenous injection of a mouse renal carcinoma cell line, Renca, caused a large number oflung metastasis foci in wild-type mice, with TNF-α protein at tumor sites. Metastasis fociexpanded at similar rates in both wild-type and TNF-Rp55-deficient mice until 21 days after the
injection. On the contrary, later than 21 days, metastasis foci regressed spontaneously in
TNF-Rp55-deficient mice, but not wild-type mice. Concomitantly, the number of apoptotic
tumor cells were greater in TNF-Rp55-deficient mice, whereas neovascularization was lessevident in TNF-Rp55-deficient mice than wild-type mice, with depressed expression of a potent
angiogenic factor, hepatocyte growth factor gene in TNF-Rp55-deficient mice (2) (see below
Figure). These observations implied that TNF-Rp55-mediated signals have distinct but
important roles in metastasis to liver and lung.

Mice developed multiple adenomatous lesions in colon after repeated oral intake of dextran
sulfate sodium solution, following an intraperitoneal injection of a potent chemical carcinogen,azoxymethane. The same treatment caused few adenomatous lesions in TNF-Rp55-deficientmice. Because this model may recapitulate adenocarcinoma developed in patients with chronic
ulcerative colitis, TNF-Rp55 may be a good molecular target for preventing this severe
complication in chronic colitis.
References
1. Kitakata H et al. Cancer Res.62: 6682, 2002.
2. Tomita Y et al. Intl. J. Cancer 112: 927, 2004.

― 26 ―

Essential contribution of a chemokine, CCL3, and its receptor, CCR1,
to hepatocarcinogenesis
P. Lu, X. Yang, C. Fujii, and N. Mukaida.
We previously observed that a chemokine, macrophage inflammatory protein-1 α/CCL3, and its
receptor, CCR1, were aberrantly expressed in human hepatocellular carcinoma (HCC) tissues(1). We further demonstrated that CCL3 and CCR1 are also expressed in two different modelsof this cancer; N-nitrosodiethylamine (DEN)-induced HCC and HCC induced by hepatitis B
virus surface (HBs) antigen-primed splenocyte transfer to myelo-ablated syngeneic HBs antigentransgenic mice (2). At 10 months after DEN treatment, tumor incidence was marginally but
significantly higher in CCR1- and CCL3-deficient mice than WT mice, in line with the in vitro
observation that CCL3 can inhibit the proliferation of hepatoma cell lines. On the contrary, focinumbers and sizes were remarkably reduced in CCR1- and CCL3-deficient mice, compared withwild-type mice. Of note is that tumor angiogenesis was also markedly diminished in CCL3- andCCR1-deficient mice, with a concomitant reduction in the number of intratumoral Kupffer cells,
a rich source of growth factors and matrix metalloproteinases (MMPs). Among growth factors
and MMPs that we examined, only MMP9 and MMP13 gene expression was augmented
progressively in liver of WT mice after DEN treatment. Moreover, MMP9 but not MMP13
gene expression was attenuated in CCR1- and CCL3-deficient mice, compared with wild-type
mice. Furthermore, MMP9 was expressed mainly by mononuclear cells, and MMP9-expressing
cell numbers were decreased in CCR1- or CCL3-deficient mice, compared with wild-type mice(2). These observations suggest that the CCR1-CCL3 axis has different roles in HCC
progression, depending on its phases (see below Figure).

References
1) Lu P, et al. Am. J. Pathol. 162: 1249, 2003.
2) Yang X, et al., Intl. J. Cancer [Epub ahead of print]― 27 ―

PMID: 16284949

Essential involvement of a chemokine receptor, CCR2, to liver
metastasis
X. Yang, C. Fujii, and N. Mukaida.
The liver parenchyma is populated by hepatocytes and several non-parenchymal cell types
including Kupffer cells (KC) and hepatic stellate cells (HSC). Both KC and HSC are responsive
to the chemokine CCL2, but the precise roles of CCL2 and these cells in liver tumor formation
remain undefined. Hence, we investigated the effects of the lack of the major CCL2 receptor,
CCR2, on liver tumor formation induced by intraportal injection of the murine colon
adenocarcinoma cell line, colon 26. Wild-type mice showed macroscopic tumor foci in the liver
10 days after injection of colon 26 cells. After 10 days, CCL2 proteins were detected
predominantly in tumor cells, coincident with increased intratumoral KC and HSC numbers.
Although tumor formation occurred at similar rates in wild-type and CCR2-deficient mice up to
10 days after tumor cell injection, the number and size of tumor foci were significantly
attenuated in CCR2-deficient mice relative to wild-type mice thereafter.
Moreover,neovascularization and matrix metalloproteinase (MMP) 2 expression were diminished in
CCR2-deficient mice with a concomitant reduction in the accumulation of KC and HSC. MMP2
was detected predominantly in HSC but not in KC. Thus, CCR2-mediated signals can regulate
the trafficking of HSC, a main source of MMP2, and consequently can promote
neovascularization during liver tumor formation.

Reference
Yang X, et al. Intl. J. Cancer 118: 335, 2006.

― 28 ―

Enhanced expression of a proto-oncogene, Pim-3,
serine/threonine kinase activity, in various types of tumors

with

C. Fujii, Y. Li, B. Popivanova, Y. Nakamoto, and N. Mukaida.
Most cases of human hepatocellular carcinoma develop after persistent chronic infection withhuman hepatitis B virus or hepatitis C virus, and host responses are presumed to have majorroles in this process. To recapitulate this process, we have developed the mouse model ofhepatocellular carcinoma using hepatitis B virus surface antigen transgenic mice. In order to
identify the genes associated with hepatocarcinogenesis in this model, we compared the gene
expression patterns between pre-malignant lesions surrounded by hepatocellular carcinoma
tissues and control liver tissues by using a fluorescent differential display analysis. Among thegenes which were expressed differentially in the pre-malignant lesions, we focused on Pim-3, amember of a proto-oncogene Pim family. Due to the unavailability of the nucleotide sequence offull-length human Pim-3 cDNA, we cloned full-length Pim-3 cDNA, consisting of 2,392 bp,
which encodes a predicted open reading frame consisting of 326 amino acids. Pim-3 mRNAwas selectively expressed in human hepatoma cell lines, but not in normal liver tissues.
Moreover, Pim-3 protein was detected in human hepatocellular carcinoma tissues and cell lines
but not in normal hepatocytes. Furthermore, cell proliferation was attenuated and apoptosis
was enhanced in human hepatoma cell lines by the ablation of Pim-3 gene with RNAinterference (reference). These observations suggest that aberrantly expressed Pim-3 can cause
autonomous cell proliferation and/or prevent apoptosis in hepatoma cell lines.

We recently observed that Pim-3 expression was enhanced selectively also in human pancreas
cancer tissues and pancreatic cancer cell lines, but not normal pancreas tissue. Moreover,similarly to human hepatoma cell lines, cell proliferation was attenuated and apoptosis wasenhanced also in human pancreatic cancer cell lines by the ablation of Pim-3 gene with RNAinterference. Thus, Pim-3 is aberrantly expressed in various types of tumors and can counteract
apoptosis process, thereby contributing to carcinogenesis. Therefore, Pim-3 may be a goodmolecular target for treating tumors, which exhibit the enhanced expression of Pim-3.
Reference
Fujii C, et al. Intl. J. Cancer 114: 209, 2005.
― 29 ―

Publications
1. Wang H, Nemoto-Sasaki Y, Kondo T, Akiyama M, and Mukaida N. (2003) Potential
involvement of monocyte chemoattractant protein (MCP)-1/CCL2 in IL-4-mediated tumor
immunity through inducing dendritic cell migration into the draining lymph nodes. Int.
Immunopharmacol. 3: 627-642.
2. Kim H, Seo JY, Lim JW, Mukaida N, and Kim KH.

(2003) Helicobacter pylori-induced

IL-8 expression in AGS cells: mediation by NF-κB and AP-1. Dig. Dis. Sci. 48: 257-265.
3. Tsuchiyama T, Kaneko S, Nakamoto Y, Sakai Y, Honda M, Mukaida N, and Kobayashi K.
(2003)

Enhanced anti-tumor effects of a bicistronic adenovirus vector expressing both

herpes simplex virus thymidine kinase and monocyte chemoattractant protein-1 against
hepatocellular carcinoma. Cancer Gene Ther. 10: 260-269.
4. Furuichi K, Wada T, Iwata Y, Kitagawa K, Kobayashi K, Hashimoto H, Ishiwata Y,
Tomosugi N, Mukaida N, Matsushima K, Egashira K, and Yokoyama H. (2003) Genetherapy expressing amino-terminal truncated monocyte chemoattractant protein-1 prevents
renal ischemia-reperfusion injury. J. Am. Soc. Nephrol. 14: 1066-1071.
5. Lu P, Nakamoto Y, Nemoto-Sasakai Y, Fujii C, Wang H, Hashii M, Ohmoto Y, Kaneko S,
Kobayashi K, and Mukaida N. (2003) Potential interaction between CCR1 and its ligand,
CCL3, induced by endogenously produced interleukin-1, in human hepatomas. Am. J. Pathol.
162: 1249-1258.
6. Popivanova B, Koike K, Tonchev AB, Ishida Y, Kondo T, Ogawa S, Mukaida N, Inoue M,
and Yamashima T.

(2003) Accumulation of microglial cells expressing ELR motif-positive

CXC

and

chemokines

their

receptor

CXCR2

in

monkeyhippocampus

after

ischemia-reperfusion. Brain Res. 970: 195-204.
7. Lin Z-Q, Kondo T, Ishida Y, Takayasu T, and Mukaida N.

(2003) Essential involvementof IL-6 in skin wound healing process as evidenced by delayed wound healing in
IL-6-deficient mice. J. Leukoc. Biol. 73: 713-721.
8. Inaoki M, Sato F, Shirasaki F, Mukaida N, and Takehara K. (2003) The frequency oftype 2 CD8+ T cells is increased in peripheral blood from patients with psoriasis vulgaris. J.
Clin. Immunol. 23: 269-278.
9. Kuwada Y, Sasaki T, Morinaka K, Kitadai Y, Mukaida N, and Chayama K.

(2003)

Potential involvement of IL-8 and its receptors in the invasiveness of pancreatic cancer cells.
Int. J. Oncol. 22: 765-771.

― 30 ―

10. Ohta M, Kitadai Y, Tanaka S, Yoshihara M, Yasui W, Mukaida N, Haruma K, and Chayama
K.

(2003)

Monocyte chemoattractant protein-1 expression correlates with macrophage

infiltration and tumor vascularity in human gastric carcinomas. Int. J. Oncol. 22: 773-778.
11. Furuichi K, Wada T, Iwata Y, Kitagawa K, Kobayashi K-i, Hashimoto H, Ishiwata Y, Asano
M, Wang H, Matsushima K, Takeya M, Kuziel WA, Mukaida N, and Yokoyama H. (2003)
CCR2 signaling contributes to ischemia-reperfusion injury in kidney. J. Am. Soc. Nephrol.
14: 2503-2515.
12. Mukaida N. (2003) The pathophyiological roles of interleukin (IL)-8/CXCL8 in pulmonary
diseases. Am. J. Physiol.Lung Cell. Molec. Physiol. 284: L566-L577, 2003.
13. Mukaida N, Ketlinsky SA, and Matsushima K. (2003)
chemokines.

Interleukin-8 and other CXC

In The Cytokine Handbook (Thomson A and Lotze MT eds.), Elsevier

Science Ltd, London, UK, pp. 1049-1081.
14. Nakamura M, Kyo S, Kanaya T, Yatabe N, Maida y, Tanaka M, Ishida Y, Fujii C, Kondo T,
Inoue M, and Mukaida N.

(2004)hTERT-promoter-based tumor-specific expression of

MCP-1 effectively sensitizes cervical cancer cells to a low dose of cisplatin. Cancer Gene
Ther. 11: 1-7.
15. Ishida Y, Kondo T, Tsuneyama K, Lu P, Takayasu T, and Mukaida, N.

(2004) The

pathogenic roles of tumor necrosis factor receptor p55 in acetaminophen-induced liver injury
in mice.

J. Leukocyte Biol. 75: 59-67.

16. Amano H, Morimoto K, Senba M, Wang H, Ishida Y, Kumatori A, Yoshimine H, Oishi K,
Mukaida N, and Nagatake T.

(2004) Essential contribution of monocyte chemoattractant

protein (MCP)-1/C-C chemokine ligand-2 to resolution and repair process in acute bacterial
pneumonia.

J. Immunol. 172: 398-409.

17. Ishida Y, Kondo T, Takayasu T, Iwakura Y, and Mukaida N.

(2004)The essential

involvement of cross-talk between IFN-γ and TGF-β in the skin wound-healing process. J.
Immunol. 172: 1848-1855.
18. Ishida Y, Maegawa T, Kondo T, Kimura A, Iwakura Y, Nakamura S, and Mukaida N. (2004)
Essential involvement of IFN-γ in Clostridium difficile toxin A-induced enteritis. J.
Immunol. 172: 3018-3025.
19. Wada T, Furuichi K, Sakai N, Iwata Y, Kitagawa K, Ishida Y, Kondo T, Hashimoto H,
Ishikawa Y, Mukaida N, Tomosugi N, Matsushima K, Egashira K, and Yokoyama H.
(2004) Gene therapy via blockade of monocyte chemoattractant protein-1 for renal fibrosis.
J. Am. Soc. Nephrol. 15: 940-948.

― 31 ―

20. Ryoo S, Won M, Kim DU, Kim L, Han G, Park SK, Mukaida N, Maeng P, Yoo HS, and Hoe
KL.

(2004)

PPARα

activation abolishes LDL-stimulated IL-8 production via AP-1

deactivation in human aortic smooth muscle cells. Biochem. Biophys. Res. Commun. 318:
329-334.
21. Hwang YS, Jeong M, Park JS, Kim MH, Lee DB, Shin BA, Mukaida N, Ellis LM, Kim HR,
Ahn BW, and Jung YD.

(2004) Interleukin-1β stimulates IL-8 expression through MAP

kinase and ROS signaling in human gastric carcinoma cells. Oncogene 23: 6603-6611.
22. Kitagawa K, Wada T, Iwata Y, Kitagawa K, Kobayashi K, Tomosugi N, Matsushima K,
Mukaida N, and Yokoyama H.
fibrosis.

(2004)

Blockade of CCR2 ameliorates progressive

Am. J. Pathol. 165: 237-246.

23. Qiu Z, Fujimura M, Kurashima K, Nakao S, and Mukaida N.

(2004) Enhanced airway

inflammation and decreased subepithelial fibrosis in interleukin 6-deficient mice following
chronic exposure to aerosolized antigen.

Clin. Exp. Allergy 34: 1321-1328.

24. Imamura R, Konaka K, Matsumoto N, Hasegawa M, Fukui M, Mukaida N, Kinoshita T, andSuda T.

(2004)

Fas ligand induces cell-autonomous NF-κ B activation and IL-8

production by a mechanism distinct from that of TNF-α.

J. Biol. Chem. 279:

46415-46423.
25. Tomita Y, Yang X, Ishida Y, Nemoto-Sasaki Y, Kondo T, Oda M, Watanabe G, Chladakov
GN, Fujii C, and Mukaida N. (2004) Spontaneous regression of lung metastasis in theabsence of tumor necrosis factor receptor p55. Intl. J. Cancer 112: 927-933.
26. Kitamura K, Nakamoto Y, Kaneko S, and Mukaida, N.

(2004) Pivotal roles ofinterleukin-6 in transmural inflammation in murine T cell transfer colitis.

J. Leukocyte

Biol. 76: 1111-1117.
27. Yanaba K, Mukaida, N, Matsushima K, Murphy PM, Takehara K, and Sato S. (2004) A roleof C-C chemokine receptors 1 and 5 and macrophage inflammatory protein-1α on the
cutaneous Arthus reaction: Possible attenuation of their inhibitory effects by compensatory
production. Eur. J. Immunol. 34: 3553-3561.
28. Mukaida N. (2004) Chemokines.

In The Encylopedia of Endocrine Diseases, Four-Volume

Set (Martini L Editor-in-Chief), Elsevier Science Ltd, London, UK, vol. 1, pp.490-494.
29. Mukaida N. (2004) Cancer gene therapy using cytokine and chemokine genes.

Ann.

Cancer Res. Ther. 12: 33-51.
30. Kimura A, Ishida Y, Wada T, Yokoyama H, Mukaida N, and Kondo T.

(2005) MRP-1

expression levels determine strain-specific susceptibility to arsenic-induced renal injury
between C57BL/6 and BALB/c mice. Toxicol.Appl. Pharmacol. 203: 53-61.

― 32 ―

31. Fujii C, Nakamoto Y, Lu P, Tsuneyama K, Popivanova B, Kaneko S, and Mukaida N. (2005)
Aberrant expression of serine/threonine kinase Pim-3 in hepatocellular carcinoma
development and its role in the proliferation of human hepatoma cell lines.

Intl. J. Cancer

114: 209-218.
32. Iizasa H, Yoneyama H, Mukaida N, Kataoka Y, Naito M, Yoshida N, Nakashima E, andMatsushima K. (2005) Exacerbation of granuloma formation in IL-1 receptor
antagonist-deficient mice with impaired dendritic cell maturation associated with Th2
cytokine production.

J. Immunol. 174: 3273-3280.

33. Edwards MR, Mukaida N, JohnsonM,and Johnston SL. (2005) IL-1β induces IL-8 inbronchial cells via NF-κB and NF-IL6 transcription factors and can be suppressed by
glucocorticoids. Pulmon. Ther. Pharmacol. 18: 337-345.
34. Sato K, Imai Y, Higashi N, Kumamoto Y, Mukaida N, and Irimura T.

(2005)

Redistributions of macrophages expressing the macrophage galactose-type C-type lectin(MGL) during antigen-induced chronic granulation tissue formation.

Intl. Immunol. 17:

559-568.
35. Kurashiama K, Tamura J, Fujimura M, Qiu Z, Nakako S, and Mukaida N. (2005)

Reducedserum antibody production and acute airway inflammation in interleukin-6-dericient micechallenged with ovalbumin. Allergology Intl. 54: 331-338.
36. Kuroda T, Kitadai Y, Tanaka S, Yang X, Mukaida N, Yoshihara M, and Chayama K. (2005)
Monocyte chemoattractant protein-1 transfection induces angiogenesis and tumorigenesis ofgastric carcinoma in nude mice via macrophage recruitment.

Clin. Cancer Res. 11:

7629-7636.
37. Yang X, Lu P, Ishida Y, Kuziel WA, Fujii C, and Mukaida, N. (2006) Attenuated liver tumor
formation in the absence of CCR2 with a concomitant reduction in the accumulation ofhepatic stellate cells, macrophages, and neovascularization. Intl. J. Cancer 118: 335-345.
38. Kagaya T, Nakamoto Y, Sakai Y, Tsuchiyama T, Yagita H, Mukaida N, and Kaneko S. (2006)
Monocyte chemoattractant protein-1 gene delivery enhances antitumor effects of herpes
simplex virus thymidine kinase/ganciclovir system in a model of colon cancer. Cancer Gene
Ther. [Epub ahead of print]

PMID: 16224495.

39. Yang X, Lu P, Fujii C, Nakamoto Y, Gao J-L, Kaneko S, Murphy PM, and Mukaida N.
(2006) Essential contribution of a chemokine, CCL3, and its receptor, CCR1, to
hepatocellular carcinoma progression.

Intl. J. Cancer [Epub ahead of print]

16284949.

― 33 ―PMID:

Other achievements
Patent No. 2003-291060
Hepatoma-specific polypeptide, the polynucleotide encoding the polypeptide, and the inhibitory
RNA for the polypeptide. (Pending on August 11, 2003)― 34 ―

Divisionof
Molecular Genetics

General Summary of Division of Molecular Genetics
In 2005, Dr. Hirao was appointed to the professor and chairman of this division. Since then, thedivision has been dedicated mainly to basic researches on the mechanisms of self-renewal ofstem cells.

Stem cells are defined as cells that have the ability to perpetuate throughself-renewal, and develop into mature cells of a particular tissue through differentiation.
Appropriate controls of stem cell functions are critical for maintaining tissue homeostasis. It has
been suggested that dysregulation of stem cell could lead to cell senescence or cancer. Most
cancers comprise a heterogeneous population of cells with marked differences in their
proliferative potential as well as the ability to reconstitute the tumor on transplantation. Cancer
stem cells are a minor population of tumor cells that possess the stem cell property ofself-renewal. To understand molecular mechanisms by which normal tissue stem cells andcancer stem cells are regulated, several approaches have been done as follows. In October 2005,
the Japan Science and Technology Agency (JST) started to support the group as a part of BasicTechnologies for Controlling Cell Functions Based on Metabolic Regulation Mechanism
Analysis.
A) Identification of molecules essential for self-renewal of tissue stem cells.
Normal tissue stem cells are defined by three common properties: 1. Presence of an extensive
capacity for self-renewal that allows maintenance of the undifferentiated stem cell pool over thelife time of the host; 2. Strict regulation of stem-cell number; 3. Ability to undergo a broad rangeof differentiation events to clonally reconstitute all of the functional elements within the tissues.
Since hematopoiesis provides the most well-characterized stem cell system, roles of several
molecules on self-renewal of stem cells have been analyzed using hematopoietic system in thisdivision. Further, for some molecules, other type of stem cells including germ or neural stem
cells have been analyzed to address a question whether self-renewal of several tissue stem cells
is regulated by common molecules or signaling pathways.
B) Development of identification system for cancer stem cells.
Recent evidence has demonstrated that in leukemia and solid tumors only a minority ofcancer cells has the capacity to proliferate extensively and form new tumors. These tumorigenic
or tumor-initiating cells, which are called cancer stem cells, have been identified and enrichedon the basis of their expression of cell-surface markers. To identify the cancer stem cells,
molecules that commonly express in several tissue stem cells were identified. Promoter DNA― 35 ―

fragments of the candidate molecules were used for a tool by which tissue stem cells, but notdifferentiated cells, can be labeled with EGFP in mice. Cancer stem cell has been analyzed byusing the stem-cell marking system.

― 36 ―

The identification of hematopoietic stem cell niche.
A. Hirao, F. Arai, T. Suda
Hematopietic stem cells (HSC) in adult mouse bone marrow are quiescent, while mobilized
HSCs are cycling rapidly following depletion of mature and progenitor cells by bleeding,infection, cytotoxic reagents or irradiation. Although interaction between microenvironments
and HSCs contributes to maintenance of quiescence of HSCs resulting in the long-term
self-renewal, mechanisms are not understood. In this study, we demonstrate that side population(SP), based on FACS analysis with Hoechst33342, in c-kit+ Sca-1+ Lin-(KSL) cells represents a
population of quiescent HSC. Cell cycle analysis with BrdU labeling showed that SP were
slowly cycling in G0 phase. HSCs mobilized in peripheral blood from a bone marrow niche by
G-CSF or 5-FU treatment were cycling and those cycling HSCs were not in SP. HSCs indeveloping mice, which were cycling and expanding, were also in main population (MP). SP
cells in KSL fraction were resistant to myelosuppressive stress including X-ray and 5-FU
treatment which depletes cycling hematopoietic cells. Histological examination revealed that the
5-FU resistant HSCs were surrounded by bone-lining osteoblast-like cells on the surface of thebone. These data indicate that the osteoblastic zone is a niche for quiescent HSCs in bonemarrow.

FACS,

RT-PCR and histological examination showed that the expression of Tie2, a

receptor tyrosine kinase, was limited to the HSCs in a niche. Angiopoietin-1 (Ang-1), a ligand
for Tie2, increased the number of LTC-IC and mixed CFC, accompanied with enhanced
adhesion of hematopoietic cells to stroma cells in vitro.

Furthermore, Ang-1 induced SP

phenotype in HSC in vivo. These data suggest that Ang-1/Tie2 plays a key role for recruitmentof stem cells to a niche, and remains cells in quiescent state, resulting in stem cell maintenance.

― 37 ―

Role of ATM on self-renewal capacity of hematopoietic stem cells.
A. Hirao, K. Ito, T. Suda
The hematopoietic stem cells (HSC) are maintained in an undifferentiated quiescent state in aniche. Quiescent stem cells show resistance to various stresses, suggesting that mechanisms forprotection of HSC from stress contribute to maintenance of self-renew through a whole life inanimals. In this study, we demonstrate that cell-cycle checkpoint molecule, ATM (ataxia
telangiectasia mutated), is essential for maintenance of capacity of regeneration in HSC.
Ataxia telangiectasia (A-T) is an autosomal recessive disorder caused by mutational
inactivation of the ATM. ATM and its homologue have a central role in maintenance of genomic
stability via regulating cell cycle checkpoint in response to DNA damage, stability of telomere
and oxidative stress. A-T patients display variety of symptoms including premature aging. We
hypothesized that a signaling pathway for regulating aging and longevity may be involved instem cell functions. Hence we analyzed a role of ATM in self-renewal and repopulating capacityof HSCs.
In this study, we demonstrate that ATM has an essential role in the reconstitutive
capacity of the haematopoietic stem cells (HSC), but is less required for the differentiation or
proliferation into progenitors. ATM-/- mice over the age of 24 weeks showed progressive bonemarrow failure due to a defect in HSC function that was associated with up-regulation of
p16INK4A and p19ARF in response to elevated reactive oxygen species (ROS). Treatment ofanti-oxidative agents restored the reconstitutive capacity of ATM-/- HSCs. These data
demonstrate that the self-renewal capacity of HSC depends on ATM-mediated inhibition ofoxidative stress.

― 38 ―

Role of Chk2 on tumorigenesis
A. Hirao, K. Naka, N. Motoyama, R. Hakem, JP. McPherson, T. Mak,
In response to ionizing radiation (IR), the tumor suppressor p53 is stabilized and promotes either
cell cycle arrest or apoptosis. Chk2 activated by IR contributes to this stabilization, possibly bydirect phosphorylation. Like p53, Chk2 is mutated in patients with Li-Fraumeni syndrome.
Since the ATM gene is required for IR-induced activation of Chk2, it has been assumed that
ATM and Chk2 act in a linear pathway leading to p53 activation. To clarify the role of Chk2 intumorigenesis, we generated gene-targeted Chk2-deficient mice. Unlike ATM-/- and p53-/- mice,Chk2-/- mice do not spontaneously develop tumors, although Chk2 does suppress
DMBA-induced skin tumors. Tissues from Chk2-/- mice, including thymus, CNS, fibroblasts,
epidermis and hair follicles, show significant defects in IR-induced apoptosis or impaired G1/S
arrest. Quantitative comparison of the G1/S checkpoint, apoptosis, and expression of p53
proteins in Chk2-/- versus ATM-/- thymocytes suggested that Chk2 regulates p53-dependent
apoptosis in an ATM-independent manner. These data indicate that distinct pathways regulate
the activation of p53 leading to cell cycle arrest or apoptosis.
Disruption of Brca1 results in cellular demise or tumorigenesis depending on cellular
context. Inactivation of p53 contributes to Brca1-associated tumor susceptibility. We show that
Chk2 inactivation is partially equivalent to p53 inactivation, in that Chk2 deficiency facilitates
the development, survival, and proliferation of Brca1-deficient T cells at the expense of genomic
integrity. Brca1 deficiency was found to result in Chk2 phosphorylation and the Chk2-dependentaccumulation and activation of p53. Furthermore, inactivation of Chk2 and Brca1 wascooperative in breast cancer. Our findings identify a critical role for Chk2 as a component of the
DNA damage-signaling pathway activated in response to Brca1 deficiency.

― 39 ―

Publications
1. Keramaris E, Hirao A, Slack RS, Mak TW, Park DS. (2003) ATM can regulate p53 andneuronal death independent of Chk2 in response to DNA damage.

J Biol Chem.

278:37782-9
2. Kato N., Sugiyama K., Nanba K., Dansako H., Nakamura T., Takami M., Naka K., Nozaki A.
and Shimotohno K. (2003) Establishment of a hepatitis C virus subgenomic replicon derived
from human hepatocytes infected in vitro. Biochem. Biophys. Res. Commun. 306: 756-766
3. McPherson JP, Lemmers B, Hirao A, Hakem A, Abraham J, Migon E, Matysiak-Zablocki E,
Tamblyn L, Sanchez-Sweatman O, Khokha R, Squire J, Hande MP, Mak TW, Hakem R.
(2004) Collaboration of Brca1 and Chk2 in tumorigenesis. Genes Dev. 18:1144-53.
4. Arai F, Hirao A, Ohmura M, Sato H, Matsuoka S, Takubo K, Ito K, Koh GY and Suda T.
(2004) Tie2/Angiopoietin-1 Signaling Regulates Hematopoietic Stem Cell Quiescence in theBone Marrow Niche. Cell, 118:149-61
5. Ito K, Hirao A, Arai F, Matsuoka S, Takubo K, Hamaguchi I, Nomiyama K, Hosokawa K,
Sakurada K, Nakagata N, Ikeda Y, Mak TW, Suda T. (2004) Regulation of oxidative stress by
ATM is required for the self-renewal of haematopoietic stem cells. Nature, 431:997-1002
6. Naka K., Tachibana A., Ikeda K. and Motoyama N. (2004) Stress-induced prematuresenescence in hTERT-expressing Ataxia Telangiectasia fibroblasts. J. Biol. Chem. 279 (3),2030-2037
7. Kondo T., Oue N., Yoshida K., Mitani Y., Naka K., Nakayama H. and Yasui W. (2004)
Expression of POT1 is associated with tumor stage and telomere length in gastric carcinoma.
Cancer Res. 64 (2): 523-529
8. Namba K., Naka K., Dansako H., Nozaki A., Ikeda M., Shiratori Y., Shimotohno K. and KatoN.

(2004)

Establishmentof

hepatitis

C

virus

replicon

cell

lines

possessing

interferon-resistant phenotype. Biochem. Biophys. Res. Commun. 323(1): 299-309
9. Azuma M, Hirao A, Takubo K, Hamaguchi I, Kitamura T, Suda T. (2005) A quantitative
matrigel assay for assessing repopulating capacity of prostate stem cells. Biochem Biophys
Res Commun, 338(2):1164-70
10. Abe K., Ikeda M., Dansako H., Naka K., Shimotohno K. and Kato N. (2005) cDNA
microarray analysis to compare HCVsubgenomic replicon cells with their cured cells, Virus
Res. 107 (1): 73-81
11. Kato N., Nakamura T., Dansako H., Namba K., Abe K., Nozaki A., Naka K., Ikeda M. andShimotohno K. (2005) Genetic variation and dynamics of hepatitis C virus replicons in― 40 ―

long-term cell culture. J. Gen. Virol. 86(3): 645-656.
12. Ikeda M., Abe K., Dansako H., Nakamura T., Naka K. and Kato N. (2005) Efficient
replication of a full-length hepatitis C virus genome, strain O in cell culture, anddevelopment of a luciferase reporter system. Biochem. Biophys. Res. Commun 329(4),1350-1359.
13. Tamura T., Nozaki A., Abe K., Dansako H., Naka K., Ikeda M., Tanaka K. and Kato N.
(2005) cDNA microarray analysis of lactoferrin expression in non-neoplastic humanhepatocyte PH5CH8 cells. Biochim. Biophys. Acta. 1721: 73-80
14. Tahara E., Tahara H., Kanno M., Naka K., Takeda Y., Ishihara H., Yasui W., Barrett J.C., Ide
T. and Tahara E. (2005) G1P3, an interferon inducible gene 6-16, is expressed in gastriccancers and inhibits mitochondorial mediated apoptosis in gastric cancer cell line TMK-1cell. Cancer Immunol. Immunother. 54 (8): 729-740.
15. Dansako H., Naka K., Ikeda M. and Kato N. (2005) Hepatitis C virus proteins exhibit
conflicting effects on the interferon system in human hepatocyte cells. Biochem. Biophys.
Res. Commun. 336(2): 458-468
16. Naka K., Ikeda M., Abe K., Dansako H. and Kato N. (2005) Mizoribine inhibits hepatitis C
virus RNA replication: effect of combination with interferon-alpha. Biochem. Biophys. Res.
Commun. 330(3): 871-879
17. Naka K., Takemoto K., Abe K., Dansako H., Ikeda M., Shimotohno K. and Kato N. (2005)
Interferon resistance of hepatitis C virus replicon-harbouring cells is caused by functional
disruption of type I interferon receptors. J. Gen. Virol., 86(10): 2787-2792.
18. Naka K., Dansako H., Kobayashi N., Ikeda M. and Kato N. (2006) Hepatitis C virus NS5B
delays cell cycle progression by inducing interferon-beta via Toll-like receptor 3 signaling
pathway without replicating viral genomes. Virology in press (VIRO-05-455R1).

Other achievements
The 41st Erwin von Bälz Prize: A. Hirao(2004)― 41 ―

DivisionofCell Biology

General Summary of Division of Cell Biology
In the past three years the division has been dedicated to basic researches on new RNA
functions, novel RNA polymerase II associating proteins and infection mechanism ofhepadnaviruses.
A) Functional analysis of low molecular weight RNAs
Various low molecular weight RNAs function at many stages in gene expression and concern
with maintenance and proliferation of the cells. Although functions of many kinds of low
molecular weight RNAs were determined in the last decade, functions of several low molecularweight RNAs are still unknown. We are currently interested in the structure, function andbiogenesis of low molecular weight RNAs. In this period we focused on the functional
characterization of U13 small nucleolar RNA (snoRNA) and micro RNA (miRNA).
B) Novel proteins associated with phosphorylated RNA polymerase II
The carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II consists ofmultiple repeats of a highly conserved seven amino acid sequence YSPTSPS. The CTD can beextensively phosphorylated, especially at the serine 2 and 5 positions in the consensus repeat.
CTD phosphorylation has been reported to act as an important signal not only for transcription
but also for the recruitment of pre-mRNA processing factors to transcription sites. To approachto the molecular mechanism for coupling transcription with pre-mRNA processing, we haveidentified and characterized novel factor PCIF1 that can directly interact with thephosphorylated CTD.
C) Infection mechanism of hepatitis B viruses
Although much is known about genomic replication and gene expression of hepatitis B
viruses (hepadnaviruses), little is known about the molecular mechanisms of the entry and therelease pathways for this virus. To understand the nature of these pathways, we tried to search
for the host proteins that interact to capsid protein of the duck hepatitis B virus (DHBV). We
have obtained a 33-kDa protein (p33) as a DHBV-core binding protein. The human hepatitis B
virus (HBV) core protein also binds to human p33. p33 was identified as complement
component 1q receptor (gC1qR) by mass spectrometry. The binding mechanism between gC1qR
and viral core protein was examined.

― 43 ―

Functional analysis of microRNAs by the using of gene disruption
F. Harada, S. Ishii and Y. Kido
MicroRNAs (miRNAs) are small (approximately 22 nucleotides) RNAs that negatively
regulate gene expression through interactions with 3’-untranslated regions of their target
mRNAs, which leads to mRNA cleavage or translational repression. To date, 326 miRNA genes
have been identified from human genome, the precise functions of these non-coding RNAs
remains largely obscure. Interestingly, many miRNAs are found in close proximity to other
miRNAs, and these clustered miRNAs are transcribed from a single polycistronic transcription
unit by RNA polymerase II. Therefore clustered miRNAs might have very important roles inmaintenance of the specific cell lineage. Human chromosome 13 has a one of the clustered
miRNA gene, which encodes 6 miRNAs (miR-17-5P, 18a, 19a, 20a, 19b-1 and 92-1) in 800bp.
To clarify the function of these miRNAs, we tried to obtain homologue of this clustered genefrom chicken B cell line DT40 and disrupt it by targeted integration.
First of all, we obtained a chicken DNA fragment containing miRNA gene cluster
homologous to human chromosome 13 by PCR amplification using human miR-17-5P and miR
92-1 sequences as primers. Screening of DT40 genomic library with this DNA fragment as aprobe, we obtained several chicken genomic clones. These clones contained 6 miRNA genes inthe same order as the human gene and the nucleotide sequence of each miRNA was completely
identical with that of human. Therefore these clustered miRNAs are conserved between humanand chicken cells and might have essential roles in these animals. To disrupt the clustered
miRNA gene in DT40 cells, the targeting vectors containing blasticidin S and puromycin
resistance genes were constructed and then introduced into DT40 cells by sequential
homologous recombination. Three knockout cell lines on this clustered miRNA gene wereobtained. The growth rate of these mutant cells was slightly slower than that of wild-type DT40
cells. The expression of each miRNA was analyzed by Northern hybridization of total RNA
isolated from mutant and wild-type cells. In the mutant cells, miR-20a was undetectable whileother five miRNAs still expressed about 50% of wild-type cells. In human genome, paralogous
cluster in X chromosome, which encodes miR-106a, 18b, 20b, 19b-2 and 92-2 has been reported.
Diminished expression of 5 miRNAs in mutant cells must be attributable to this miRNA cluster.
We prepared DT40 genomic clone containing new miRNA cluster and construction of double
knockout cells on miRNA clusters is now in progress.

― 44 ―

Involvement of U13 small nucleolar RNA in cytidine modification of
18S rRNA.
Y. Kido and F. Harada
Since 1990’s, many small nucleolar RNAs (snoRNAs) are shown to be involved in
2’-O-methylation or pseudouridylation of nucleosides on rRNAs. U13 snoRNA was first
identified in 1986 as a trimethylguanosine-capped, box C/D class snoRNA isolated from HeLa
cell. According to the complementarity to 3’-end of 18S rRNA, U13 RNA has been
hypothesized to be involved in maturation of 3’-end region of 18S rRNA. Cavaille et al. (1996)
discussed the involvement of U13 snoRNA in cleavage of pre-rRNA at the 18S rRNA / ITS1
junction. There is, however, no direct evidence supporting the suggested function of U13
snoRNA so far.
In order to elucidate the function of U13 snoRNA we have established U13-knockout DT40
cell lines and have compared all the nucleoside (and its modification) of the 3’-end region of
18S rRNA between wild-type and knockout DT40 cells by 2-dimentional TLC. In all
independent U13-knockout cell lines examined so far a modified cytidine (N4-acetylcytidine)on position 1796, which detected in wild-type cell, was not detected. By transfection of U13
RNA gene into a knockout strain, we have detected expression of U13 snoRNA at the samelevel as that of wild-type. In all independent transfectants examined so far, cytidine acetylation
at the position 1796 was restored. These results strongly suggest that U13 snoRNA is involvedin the modification of C1796 of 18S rRNA.
U13 snoRNA has a complementarity to the 3’-end of 18S rRNA (see Figure). To test theimportance of the complementarity in acetylation activity, we are introducing several nucleotide
substitutions on the complementary region of U13 snoRNA gene and are analyzing themodification status of 18S rRNA isolated from the individual transfectants of the mutant U13
snoRNA genes.
To study acetylation of the 3’-end of
18S rRNA biochemically, in vitro
system of cytidine acetylation in a cell
extract is required. So far, however, wehave not been able to detect rRNA
acetylation in vitro.

― 45 ―

Functional characterization of the vertebrate phosphorylated RNA
polymerase II interacting factor PCIF1
Y. Hirose, M. Yuda, I. Yunokuchi, K. Sakuraba, H. Fan and F. Harada
The carboxy-terminal domain of the largest subunit of RNA polymerase II (CTD) consists ofmultiple repeats of an evolutionally conserved seven amino acid sequence YSPTSPS. The CTD
is subject to reversible phosphorylation at Ser2 and Ser5 of the heptapeptide during thetranscription cycle. Ser5 phosphorylation occurs in the early stage of transcription cycle andpromotes 5’ capping of pre-mRNA. Transcription elongation is accompanied by Ser2
phosphorylation, which is also required for efficient 3’ end formation of pre-mRNA. Thus CTD
phosphorylation plays important role in coordinating transcription and pre-mRNA processing.
To better understand the molecular mechanism by which transcription couples with pre-mRNA
processing, we have identified and characterized novel human factors that can directly interact
with the phosphorylated CTD (pCTD).
We recently identified a novel human nuclear WW domain protein, PCIF1 as a pCTD
interacting factor. The WW domain of PCIF1 is responsible for specific binding to pCTD andexhibits the considerable homology to the WW domain of human peptidyl-prolyl isomerase Pin1
that has been shown to bind to a phophorylated Ser/Thr-Pro motif. An over expression of PCIF1
in human cells resulted in repressing trans-activation of the co-transfected reporter gene
expression driven by various transcription activators. PCIF1 WW preferentially binds to a CTD
peptide phosphorylated at only Ser5 position against a CTD peptide phosphorylated at only Ser2
position. We further examined the effect of PCIF1 on dephosphorylation of pCTD by a human
CTD phosphatases SCP1 which have been shown to preferentially dephosphorylate pSer5
within the CTD repeat. We found that PCIF1 WW domains strongly inhibited CTD phosphataseactivity of SCP1 in vitro. We speculate that PCIF1 play a role in early stage of transcription
cycle and/or in regulating phosphorylation dynamics of the CTD.
To further investigate cellular functions of PCIF1, we have disrupted PCIF1 gene in thechicken B-cell line DT40. The growth curve for the PCIF1 deficient mutant DT40 cells wasindistinguishable from that for wild-type DT40 cells. Thus chicken PCIF1 is not essential forcell growth. PCIF1 depletion in DT40 cells significantly affected neither global phosphorylation
status of the CTD nor global methylation status of the histone H3. Interestingly, however, weobserved that PCIF1 depletion resulted in augmentation of chicken Pin1 expression both atmRNA and protein level.

― 46 ―

Hepatitis B virus core proteins associate with host cell protein gC1qR
K. Kuroki, Y. Yamano, T. Kawakami1, Y. Fukushima2, T. Nishimura1, T. Ishikawa2 and
F. Harada (1Clinical Proteome Center, Tokyo Medical University, 2Dept. of Internal
Medicine, University of Tokyo)
The human hepatitis B virus (HBV) is a small and enveloped DNA virus of the prototype of afamily of Hepadnaviridae that causes acute and chronic liver disease and increases the risk ofdeveloping hepatocellular carcinoma. Despite of considerable understanding of the details of
hepadnaviral replication and gene expression, little is known about the nature of the entry andthe release pathways for this virus.
To understand the nature of the uptake and the maturation pathways for the hepadnaviruses,
we have begun the search for the host proteins that interacts to capsid proteins of HBV and the
duck hepatitis B virus (DHBV) as a model of these viruses.
We have identified a 33-kDa protein as a DHBV-core binding protein in duck liver extracts
using glutathione-S-transferase (GST)-DHBV core-CBD (chitin binding domain) fusion
proteins. The same molecular sized protein was also identified with GST-HBV core-CBD fusion
protein in HepG2 extracts, so human p33 was subjected to reversed-phase liquid
chromatography (LC) coupled with electrospray-tandem mass spectrometry (MS/MS). p33 hasturned out to be gC1qR.
To elucidate the function of gC1qR in hepadnavirus life cycle, we examined the binding
domain on DHBV and HBV core proteins by constructing the DHBV and HBV core deletionmutants. Our data show 1) the cellular gC1qR protein binds to arginine-rich domain ofcarboxyl-terminal of core proteins 2) DHBV and HBV has a common binding motif witharginine repeated region, 3) gC1qR binds at least two domains in both DHBV and HBV coreproteins (DHBV a.a.213-229, a.a.225-235, HBV a.a.157-169, a.a.164-177), 4) DHBV coreproteins substituted two serine residues (a.a.230 and 232) with aspartic acid to mimic thephosphorylated form could not bind to gC1qR. These arginine-rich domains of the hepatitis B
virus core protein have been shown to be required for pregenome encapsidation, productive viral
DNA synthesis and sorting viral genome to the nuclei. gC1qR may regulate these processes by
the interaction with nonphosphorylated core proteins.

― 47 ―

Publications
1.

Fan, H., Sakuraba, K., Komuro, A., Kato, S., Harada, F. and Hirose, Y. (2003) PCIF1, anovel human WW domain-containing protein, interacts with the phosphorylated RNA
polymerase II. Biochem. Biophys. Res. Commun., 301:378-385.

2.

Xu Y-X., Hirose Y., Zhou X.Z., Lu K.P. and Manley. J.L. (2003) Pin1 modulates the
structure and function of human RNA polymerase II.

3.

Genes ＆ Dev., 17:2765-2776

Kawakami, T., Tateishi, K., Yamano, Y., Ishikawa, T., Kuroki, K., Nishimura, T. (2005)
Protein identification from product ion spectra of peptides validated by correlation between
measured and predicted elution times in liquid chromatography/mass spectrometry.
PROTEOMICS, 5:856-864.

4.

Enomoto, M., Sawano, Y., Kosuge, S., Yamano, Y., Kuroki, K., Ohtsuki, K. Highphosphorylation of HBV core protein by two a-type CK2-activated cAMP-dependent
protein kinases in vitro. FEBS Lett. in press

Other achievements
Patent No. 3593482 (September 3, 2004)
Human nuclear proteins containing WW domain and cDNA encoding them.
(M. Kato, A. Komuro and Y. Hirose)― 48 ―

DivisionofStem Cell Biology

General Summary of Division of Stem Cell Biology
In the past three years, we have been addressing the mechanism of developmental, physiologic
and pathologic blood vessel formation and self-renewal of hematopoietic stem cells (HSCs). In theformer, we analyzed interaction among endothelial cells (ECs) and hematopoietic cells (HCs)and mural cells (MCs) especially focusing on receptors, Flk-1, Tie2, EphB4, and nueropilin-1. In thelater, we have been analyzing the function of Tie2 receptor tyrosine kinase. In both system,
angiogenesis and self-renew of HSCs, Tie2 plays very important role, therefore we have cloned
various genes that might be affected by Tie2 activation. The function of those genes have beenstudied by molecular biological technique and determined.
A) Role of hematopoietic cells in vascular development
Angiogenesis is an important event for embryonic organogenesis as well as for tissue repair in theadult. Among various kinds of HCs, we reported that HSCs play important roles for angiogenesis
during embryogenesis. HSCs, which express angiopoietin-1 (Ang1), directly promoted migrationof ECs and capillary formation. Moreover, we found that NP-1 expressed on HCs such as B
lymphocyte, erythroblast, and monocyte binds VEGF and stimulates VEGF receptor on ECs
strongly by an exogenous manner resulted in enhancement of angiogenesis.
1) In the past three years, we found that HSCs differentiate into MCs through CD11b positive
monocyte lineage. This transdifferentiation is observed in physiological conditions as well as
pathological conditions. Interestingly, we found that those CD11b positive cells produce
Angiopoietin-1 and induce non-leaky vessel by the stabilization of blood vessel structure.
2) Based on the findings that HCs regulate angiogenesis by many ways, we evaluated the function of
HCs for tumor angiogenesis. Bone marrow suppression by injection anti-c-Kit antibody
suppressed the tumor angiogenesis resulted in delay of tumor growth. Therefore, we concluded
that suppression of HC migration into tumor is one of promising approach for managing cancer.
B) Role of Tie2 for Self-renewal of HSCs
Tie2 is expressed on HSCs and plays a critical role for development of HSCs. In bone marrow,
HSCs localize in trabecular bone and adhere to osteoblast. In such niche, it is suggested that
osteoblasts produce angiopoietin-1 and stimulate Tie2 on HSCs, resulted in quiescent state of
HSCs. In order to confirm this action of Tie2 on HSCs genetically, we constructed constitutive
active form of Tie2 and generated conditional transgenic mice expressing constitutive active Tie2
in HSCs by cre-LoxP system. We previously reported the first site where HSCs proliferate isomphalomesenteric artery at E9.5 in mice. In this tg mice, we found that HSCs could not
proliferate in this artery and died by severe anemia, suggesting that differentiation of HSCs is also
impaired. Therefore, we confirm that Tie2 should be inactivated transiently for self-renewal of
HSCs. Additonally, in this tg mice, severe defective angiogenesis was observed. Upon activation ofTie2 expressed on ECs, cell adhesion of MCs and ECs are induced. Therefore, we concluded thatfor the dissociation between MCs and ECs, inactivation of Tie2 is necessary for initiation of― 49 ―

sprouting angiogenesis.
C) Isolation of genes affected by Tie2 activation relating to stemness.
We and the other group have reported that Tie2 regulates cell adhesion of HSCs to osteoblasts andECs to MCs, resulted in maintaining of dormant state and long term survival in both ECs and
HSCs. Then, we have tried to isolate genes associating with such dormancy and anti-apoptotic
effects regulated by Tie2.
1) Psf1: We isolated cell cycle related gene, psf1. Anaysis of this PSF1 has been well performed inyeast and it has been reported that PSF1 is critical for cell division by regulating the formation of
DNA replication fork. We found that PSF1 expression is restricted in a certain stem /immature cell
population. In knock out study of PSF1 gene in mice showed that lack of PSF1 lead deficiency ofcells composing with inner cell mass (ES cells) proliferation.
2) Galectin-3: A member of galactose binding lectin, galectin-3 (Gal-3) is upregulated under Tie2
activation. We found that Gal-3 might induce anti-apoptotic effect under hypoxic condition. Nicheis the place locating distant from vascular area, so HSCs in niche are exposed with hypoxia. Then,
HSCs produce Gal-3 under Tie2 activation and might protect cell apoptosis under hypoxia.
D) Isolation of genes affected by Tie2 activation relating to blood vessel formation.
It is well known that Tie2 activation induces non-leaky and enlarged blood vessel. However, whichmolecules actually carry out such effects are not clearly known. Therefore, we isolated genes
which regulate permeability or caliber change of blood vessel and analyzed the function of thosemolecules.
1) ACE (angiotensin converting enzye): It was previously reported that VEGF mediatedhyperpermeability was suppressed by angiopoietin-1. We found that ACE is downregulated by
Tie2 activation. ACE is well known molecule that converts angiotensin I to angitensin II and it isreported that angiotensin II induces vascular permeability in in vitro system. Therefore, wehypothesized that ACE is negatively regulated by Tie2 activation and inhibits leakiness. Using
Miles assay, we confirmed that VEGF mediated hyperpermeability was inhibited by angiotensin
type 1 receptor blocker, ARB. It has been reported that VEGF mediated leakiness was caused by
the production of NO from ECs. We found that VEGF mediated NO production was inhibited by
ARB.
2) Apelin: Apelin is a ligand for APJ, GPCR with seven transmembrane domain expressed on ECs
and MCs. We found that Tie2 activation on ECs induces apelin production from ECs. To know thefunction of apelin on blood vessel formation, we induced apelin into tumor cells, inoculated theminto mice and observed tumor angiogenesis. We found that apelin induced enlarged blood vessels
compared with control. A variety of experiments suggested that apelin regulates cell adhesion in
ECs and enhances assembly of ECs resulted in formation of enlarged vessels.

― 50 ―

PSF1 Is Essential for Immature Cells Proliferation
M. Ueno and N. Takakura
Eukaryotic chromosomal replication is tightly regulated to maintain the integrity of genomic
information. In yeast, Orc (origin recognition complex) is bound to replication origins throughout
the cell cycle. From late M to G1 phase, MCM (minichromosome maintenance) protein is loaded
onto the origin, marked by Orc, by Cdc6 and Cdt1, and forms the pre-replication complex (pre-RC).
On activation and recruitment of additional factors, such as CDC45, the pre-RC is converted to thepre-initiation complex (pre-IC), which is the complex essential for the transition to DNA replication.
In yeast, CDC45 is essential for the initiation and elongation of DNA replication.
Recently, a novel multi-protein complex “GINS” was identified. This GINS complex contains,Psf1 (partner of sld five 1), Psf2, Psf3, and Sld5 and forms a ring-like structure. During the S-phase,
the GINS complex is loaded onto chromatin after the formation of pre-RCs, and then tightly
associates with the replication origin. This binding is suppressed by p21 and geminin by inhibiting
the loading of CDC45 onto chromatin and the pre-RC formation by binding to Cdt1. Moreover, thechromatin binding of GINS complex and of CDC45 are mutually dependent processes, but they do
not associate with each other. The association of PSF1 and Dpb11/Cut5 with the origins is alsomutually dependent. All genes encoding GINS components are evolutionarily conserved and areessential for cell growth. However, the functions of GINS complex in mammalian cells have notbeen reported.
We originally cloned the mouse ortholog of PSF1 from a hematopoietic stem cell cDNA
library and found that PSF1 is expressed in blastocysts, adult bone marrow, and testis, in which thestem cell system is active. We used the gene-targeting technique to determine the physiological
function of PSF1 in vivo. Mice homozygous for a non-functional mutant of PSF1 died in utero
around the time of implantation. PSF1-/- blastocysts failed to outgrowth in culture and exhibited acell proliferation defect. Our data clearly indicate that PSF1 is required for early embryogenesis.
Ref:
Ueno, M., et al. (2005) Mol Cell Biol. 25:10528-10532.
Kong, L., et al. (2006) Biochem. Biophys. Res. Commun. 339:1204-1207.

― 51 ―

The identification of cardio-vascular stem cells and clarification its
differentiation mechanism
Yoshihiro Yamada, Nobuyuki Takakura
Purpose: Ischemic disease is the main cause of death as well as cancer. To overcome it, we need toreveal the mechanism how cardio-vascular stem cells in tissue differentiate into cardiomyocytes or
vascular cells. Furthermore, we would like to approach cancer stem cells, which have supposed as
the cause of drug resistant cancer.
Results
1-1. In embryonic stage, vascular stem cells (VPCs) are originated from hematopoietic stem cells(HSCs) fraction and they differentiate into both ECs and SMCs (vascular cells) in vitro and in vivo.
1-2. HSCs(lin-ckit+CD45+), which differentiate into vascular cells, have tissue specific mechanism.
HSCs in head region differentiate into vascular cells, but HSCs in fetal liver can not differentiate
into vascular cells.
1-3. Tissue specificity in HSCs is due to the induction of Flk-1 expression by brain ECs.
1-4. In adult stage, especially in ischemic condition, vascular stem cells are exsited inmonocyte/M-phage fraction, such as CD45+CD11b+ cells. Moreover, very interestingly, there are
two populations, such as CD45+CD11blow+ and CD45+CD11bhigh+cells, and CD45+CD11blow+ cells
mainly contribute to angiogenesis as vascular cells in both short term and long term.
2-1. We identified cardiac stem cells in brown adipose tissue. We named them BATCM (brown
adipose tissue derived cardiomyocyte)
2-2. BATCMs expressed CD29, CD105, and CD133, such as mesenchymal stem cells marker, but
not expressed linage marker, c-kit, and CD31, such as hematopoietic and endothelial cells marker.
2-3. In vitro, BATCMs differentiated into sarcomeric-actin, troponinT, MEF2C, and GATA-4
positive CMs, furthermore, they differentiated into CD31, VE-cadherin, Flk-1 positive ECs and
SMA, PDGF-β and desmin-positive SMCs.
2-4. In vivo infarction model, BATCMs contributed as CM, ECs and SMCs in ischemic border zone,
and improved the function of heart and mortality.
3-1. Cancer cells interacted with hematopoietic cells and performed as cancer stem like cells in vivo.
Future plan
1. To detect vascular stem cells in other condition, such as in tumorangiogenesis and brain ischemia.
To detect the inducible factor for Flk-1 expression in HSCs.
2. To detect the regulating factor from stem cells into CMs in adipose tissue.
3. To analyze the features of cancer stem cell like cells precisely in vitro and in vivo.
Publication
1. Yamada. Y et al (2003) Blood 101:1801-1809.
2. Yamada. Y et al (inpress) Biochem. Biophys. Res. Commun.

― 52 ―

Development of blood cells in the ascidian Halocynthia roretzi
S. Amano, N. Takakura and I. Hori* (*Dept. of Biol., Kanazawa Medical Univ., Uchinada,
Ishikawa)
In recent years our understanding of the ontogenesis of mammalian hematopoietic cells has been
improved greatly. However, we have only a limited knowledge about the ontogenesis of ascidian
hemocytes. The trunk lateral cells (TLCs) of the tail bud embryo are the origin of hemocytes andbody wall muscle in the ascidian Halocynthia roretzi. The TLCs migrate posteriorly and ventrally,and differentiate to hemocytes and muscle cells in the juveniles. Each ascidian species has nearly ten
types of hemocytes in adulthood. It has been shown repeatedly that the hemocyte types of adults arevery variable among ascidian species, and this fact made difficult to reveal the relationships of thehemocyte types between species. All ascidians ever studied, however, have a common type ofhemocytes, the hemoblast. They are undifferentiated cells having typical morphology with aconspicuous nucleolated nucleus and scanty cytoplasm. The hemoblasts are thought to behematopoietic stem cells (HSCs). Although the hemoblasts are found in the hematopoietic tissue andin circulation, it is not clear that both of them are truly HSCs.
In this study, the development of hematopoiesis was studied in detail with electron
microscopy in the ascidian H. roretzi. We found only four types of hemocytes in the juveniles, andthe scarcity of the hemocyte types allowed us to follow the process of their differentiation distinctly.
In the swimming larvae and juveniles, nascent mesoderm cells infiltrate between the epidermis andendoderm. The mesoderm cells have yolk granules and compact masses of glycogen granules, andthose nutritive granules are gradually consumed after metamorphosis. Consequently, in 5d juveniles,
the mesoderm cells transform to hemoblasts with a scanty cytoplasm and a conspicuous nucleolated
nucleus. Before the appearance of the hemoblasts, hematopoiesis initiates soon after metamorphosis.
This fact means that the mesoderm cells directly differentiate to giant cells, vacuolated cells, andgranular cells. About 5 days after metamorphosis the hemoblasts begin to differentiate to the three
types of hemocytes instead of the mesoderm cells. The proportion of hemocyte types in thejuveniles is very different from that in adults, for example the hemoblasts and giant cells are rare in
the adults but they are major hemocyte types in the juveniles. The hemoblasts seem to be rapidly
dividing cells because we found their mitotic figures in the hemocoel of the juveniles. We concluded
Tail bud
Swimming larva
Juvenile 1~5dthat the circulating hemoblasts are
Juvenile 5d~
HSCs that self-renew and
TLC
Mesoderm cell
Mesoderm cell
Hemoblast
replenish multiple types ofhemocytes in the ascidian H.
×××
roretzi.
Giant
Vacuolated
Granular
Giant
Vacuolated
Granular
cell

cell

cell

cell

cell

cell

Figure shows the ontogenesis of hemocytes revealed in this study in the ascidian H. roretzi. Nascent mesoderm cells never differentiate to hemocytes inthe swimming larvae. They begin to differentiate to three types of hemocytes in the juveniles after metamorphosis. Prior to the 5th day after
metamorphosis, the nascent mesoderm cells transform to hemoblasts. Thereafter the hemoblasts differentiate to the three types of hemocytes, that is, giant
cells, vacuolated cells, and granular cells. All these results distinctly show that the nascent mesoderm cells in the juveniles are multipotential cells. In
addition, it is evident that the hemoblasts, derived from the nascent mesoderm cells, are HSCs.

― 53 ―

Publications
1. Yamada, Y., Oike, Y., Ogawa, H., Ito, Y., Fujisawa, H., Suda, T., Takakura, N. (2003)
Neuropilin-1 on hematopoietic cells as a source of vascular development. Blood 101:1801-1809.
2. Nakajima, M., Yuasa, S., Ueno, M., Takakura, N., Koseki, H., Shirasawa, T. (2003) Abnormal
blood vessel development in mice lacking presenilin-1. Mech Dev. 120: 657-667.
3. Li, Z., Takakura, N., Oike, Y., Imanaka, T., Araki, K., Suda, T., Kaname, T., Abe, K. and Yamamura,
K. (2003) Defective smooth muscle development in qkI deficient mice. Development Growth.
Differ. 45:449-461.
4. Takakura, N. (2003) Hematopoietic stem cells and angiogenesis. Cardiomyopathies and Heart
Failure. Kluwer Academic Publishers. 469-480.
5. Contribution of hematopoietic stem cells in blood vessel formation. Okamoto R, Yamada Y, Sano
H, Ueno M, Takakura N. Biomed Rev. 14: 1-8, 2003(accepted &published in 2004).
6. Ueno, M., Itoh, M., Kong, L., Sugihara, K., Asano, M., and Takakura, N. PSF1 is essential for
Early Embryogenesis in mice. Mol. Cell. Biol. In press
7. Fukuhara, S., Sakurai, A., Sano, H., Yamagishi, A., Somekawa, S., Takakura, N., Saito, Y.,
Kangawa, K., and Mochizuki, N. (2005) Cyclic AMP potentiates VE-cadherin-mediated cell-cell
contact to enhance endothelial barrier function through an Epac-Rap1 signaling pathway. Mol.
Cell. Biol. 25:136-146.
8. Okamoto R, Ueno M, Yamada Y, Takahashi N, Sano H, and Takakura N: Hematopoietic cells
regulate the angiogenic switch during tumorigenesis. Blood 105: 2757-2763, 2005
9. Lingyu Kong, Masaya Ueno, Machiko Itoh, Katsuji Yoshioka, and Nobuyuki Takakura:
Identification and Characterization of Mouse PSF1 Binding Protein, SLD5. BBRC
339:1204-1207.
10. Yamada Y. et al Cardiac progenitor cells in brown adipose tissue repaired damaged myocardium.
BBRC in press

Other achievements
Patent
1. Title:
Myocardialcell
inductionutilizing
adiposetissueinmammal.
No.2003-429088,PCT/JP2004/19666
2. Title: Methods and machine for expansion of organ specific stem cells. No.
2004-119179,PCT/JP2005/006424
3. Title: Methods for usage of PSF1deficient mice. No. 2004-242022
4. Title: anti-ephrinB2 antibody. No. 2004-244433
5. Title: drug for blood vessel enlargement. No. 2004-300653
6. Title: Method for generation of cancer stem cells. No. 2005-126266― 54 ―

Divisionof
Molecular Pathology

General Summary of Division of Molecular Pathology
Our main research interest is to clarify how cells recognize DNA damage and transduce signals
to cell cycle control, DNA repair and apoptotic machineries. To achieve this goal, we are
currently using a gene knockout approach in chicken DT40 cell lines.
A) Direct ATM Activation by Sulfhydryl-Reactive Inflammatory Cyclopentenone
Prostaglandins
It is not clear whether or not ATM responds to oxidative DNA damage or rather responds to achange in intracellular redox state independent of DNA damage. We found that ATM is
directly activated by protein sulfhydril-group-modifying agents,
N-methyl-N'-nitro-nitrosoguanidine and 15-deoxy-∆12,14-prostaglandin J2, in chicken DT40
cells deficient for DNA damage sensors, such as NBS1 or MSH6.
B) c-Abl tyrosine kinase family is involved in the formation of stable chromatin-associatedRad51 complex
It is unclear how these Abl tyrosine kinase members mechanistically regulate Rad51 functions
during homologous recombination repair (HRR).

We found that the c-Abl tyrosine kinase

family enhances the formation of stable chromatin-associated Rad51 complexes throughphosphorylation of Rad51 mainly on Tyr-315 during HRR.
C) Involvement of the ATR- and ATM-dependent checkpoint responses in cell cycle arrest
evoked by pierisin-1
Piersin-1 identified from the cabbage butterfly, Pieris rapae, is a novel mono
ADP-ribosylating toxin that transfers the ADP-ribose moiety of NAD at N2 of dG in DNA.
We found that mono ADP-ribosylation of DNA causes a specific type of fork blockage which
induces checkpoint activation and signaling.
D) Msh6 and Nbs1 are essential for ATR-mediated Chk1 activation induced by DNA
replication stalling in higher vertebrate cells
We found that ATR-mediated Chk1 and Chk2 phosphorylation linked to methylated DNA
damage requires DNA damage sensors, Msh6 and Nbs1, in addition to early acting checkpoint
proteins, such as Rad17 and Rad9, and ATRIP, the DNA binding subunit of ATR.

― 55 ―

We are very grateful to investigators outside of this institute for their contributions to our
projects; Kenshi Komatsu, Radiation Biology Center, Kyoto University; Koji Uchida, Graduate
School of Bioagricultural Science, Nagoya University; Hiroshi Tauchi, Department ofEnvironmental Science, Faculty of Science, Ibaraki University; Masayuki Takahashi, UMR
6204 Centre National de la Recherche Scientifique & Universite de Nantes; Hitoshi
Kurumisaka, Graduate School of Science and Engineering, Waseda University; Shunichi
Takeda, Graduate School of Medicine, Kyoto University; Minoru Takata, Kawasaki Medical
School; Keiji Wakabayashi, National Cancer Center Research Institute; Ashok R.
Venkitaraman, Cancer Research UK Department of Oncology, University of Cambridge; Yosef
Shiloh, Sackler School of Medicine, Tel Aviv University; Steve Jackson, The Wellcom Trustand Cancer Research UK Gurdon Institute, Cambridge University.

― 56 ―

Direct ATM Activation by Sulfhydryl-Reactive Inflammatory
Cyclopentenone Prostaglandins
Masahiko Kobayashi, Hiroto Ono, Keiko Mihara, Hiroshi Tauchi, Kenshi Komatsu,
Hiroko Shimizu, Koji Uchida, and Ken-ichi Yamamoto
ATM (ataxia-telangiectasia mutated) is essential for cellular response to double strand breaks invertebrate cells. However, ATM is activated by a variety of noxious agents, including oxidative
stress, and ATM deficiency results in anomalous cellular response to oxidative stress. While thisdefective response to oxidative stress may underlie the pathogenesis of cerebellar ataxia,
premature aging and cancer predisposition in ataxia-telangiectasia, mechanisms for ATM
activation by oxidative stress remain to be established. Furthermore, it is not clear whether or
not ATM responds to oxidative DNA damage or rather responds to a change in intracellular
redox state independent of DNA damage. To address these questions, we studied ATM activationby protein sulfhydril (SH)-group-modifying agents, N-methyl-N'-nitro-nitrosoguanidine(MNNG) and 15-deoxy-∆12,14-prostaglandin J2 (15d-PGJ2), in chicken DT40 cells deficient for
NBS1 or MSH6 generated by targeted disruption. We found that ATM is effectively activatedwith MNNG and 15d-PGJ2 in NBS1-/-/- and MSH6-/- cells. We further found that ATM is directly
activated by treatment of chromatin-free ATM immunoprecipitates with MNNG or 15d-PGJ2
through modification of SH groups and that 15d-PGJ2 covalently binds to ATM. Interestingly,
15d-PGJ2-induced ATM activation leads to p53 phosphorylation and apoptosis but not Chk2
phosphorylation in human tumor cells. These results indicate that ATM is activated throughdirect modification by free SH-group-modifying reagents independently of DNA damage,resulting in apoptotic downstream response. However, it remains to be established how ATM
regulates intracellular oxidative stress and how ATM abnormality leads to various defective
manifestations in oxidative stress response. More extensive proteomics analysis is, thus,
required for establishing ATM functions in oxidative stress response. For such analysis,
15d-PGJ2 might be a useful agent, since ATM activation by 15d-PGJ2 does not result in theactivation of DNA damage-linked Chk1 and Chk2 phosphorylation.

― 57 ―

c-Abl tyrosine kinase family is involved in the formation of stable
chromatin-associated Rad51 complex

Hiroko Shimizu, Satoko Kitayama, Masahiko Kobayashi, Hitoshi Kurumisaka, Masayuki
Takahashi, and Ken-ichi Yamamoto
c-Abl tyrosine kinase is activated by DNA damage, such as ionizing radiation (IR), in an
ATM-dependent manner, and plays important roles in growth arrest and cell death. Several
recent studies also indicate that c-Abl, Arg (the only other known member of the c-Abl family)
and Bcr-Abl (the oncogenic form of c-Abl fusion kinase) are involved in DNA repair throughthe phosphorylation of Rad51, a key molecule in homologous recombination repair (HRR).
However, it is unclear how these Abl tyrosine kinase members mechanistically regulate Rad51
functions. We found that Rad51 with the mutation of Arg-167 to Gly (Rad51-R167G) is
defective in IR-induced nuclear focus formation, chromatin association and in vivo
self-association, though this Rad51 mutant retains the capacity to bind BRCA2 in vivo.
Interestingly, we also found that this Rad51 mutant is not effectively phosphorylated by c-Abl
and Arg. However, co-transfection of wild-type but not kinase-dead c-Abl or Arg with
Rad51-R167G enhanced the chromatin association of Rad51-R167G, suggesting that
phosphorylation of tyrosine residues, probably Tyr-315, enhances the chromatin association of
Rad51-R167G. We subsequently confirmed the phosphorylation of Tyr-315 by antibodies
specific to tyrosine phosphorylated Tyr-315. To study further roles for c-Abl or Arg in thechromatin association of Rad51, we studied effects of Glivec, a relatively specific inhibitor forthe c-Abl tyrosine kinase, on the chromatin association of transiently expressed Rad51. We firstexamined

effects

ofGlivecon

Rad51

tyrosine

phosphorylation

and

on

phosphorylation-mediated interactions between Rad51 and Arg, and confirmed that Glivec
inhibits Rad51 tyrosine phosphorylation mediated by c-Abl or Arg and the interaction between
Rad51 and Arg when wild-type Rad51 is transiently co-expressed with Arg or c-Abl. We
subsequently found that Glivec effectively inhibits the enhancement of chromatin association of
Rad51-R167G mediated by c-Abl or Arg. These results therefore indicate that the c-Abl tyrosine
kinase family enhances the formation of stable chromatin-associated Rad51 complexes throughphosphorylation of Rad51 mainly on Tyr-54 or Tyr-315 during HRR.

― 58 ―

Involvement of the ATR- and ATM-dependent checkpoint responses incell cycle arrest evoked by pierisin-1

Bunsyo Shiotani, Masahiko Kobayashi, Masahiko Watanabe, Ken-ichi Yamamoto,Takashi Sugimura and Keiji Wakabayashi
Piersin-1 identified from the cabbage butterfly, Pieris rapae, is a novel mono ADP-ribosylating
toxin that transfers the ADP-ribose moiety of NAD at N2 of dG in DNA.

Resulting mono

ADP-ribosylated DNA adducts cause mutations and the induction of apoptosis.

However, little

is known about checkpoint responses elicited in mammalian cells by the formation of suchbulky DNA adducts.

In the present study, it was demonstrated that DNA polymerases were

blocked at the specific site of mono ADP-ribosylated dG which might lead to the replicationstress.

Pierisin-1-treatment of Bcl-2-overexpressing HeLa cells was found to induce theS-phase arrest and the G2/M-phase delay. In the colony survival assays, Rad17-/- DT40 cells
showed greater sensitivity to pierisin-1-induced cytotoxicity than wild type and ATM-/- DT40
cells, possibly due to defects of checkpoint responses, such as the Chk1 activation. Further
investigation of HeLa cells confirmed that Chk1 was activated ATR- and Rad17-dependently
and that mitotic delay was inhibited in ATR- and Rad17-knockdown HeLa cells, but not in
ATM-knockdown cells. The results thus suggest ATR-Rad17-Chk1 pathway mainly
contributes to the S-phase arrest and G2/M-phase delay induced by pierisin-1.

Simultaneously,

pierisin-1-treatment activated Chk2 pathway ATM-dependently and ATM-independently.
Furthermore characteristic 50 kbp DNA fragmentation, known to be blocked by Bcl-2
overexpression was observed and activation of ATM and Chk2 was only partially inhibited.
Thus, the roles of ATM-Chk2 or Chk2 pathways appear minor in cell cycle arrest, but may be
involved in the induction of apoptosis.

From these findings, it is suggested that mono

ADP-ribosylation of DNA causes a specific type of fork blockage which induces checkpoint
activation and signaling.

― 59 ―

Msh6 and Nbs1 are required for ATR-mediated Chk1 activationinduced by DNA replication stalling in higher vertebrate cells
Masahiko Kobayashi, Hiroto Ono, Keiko Mihara, Aki Takahashi, Makiko Ikei, Hiroshi
Tauchi, Kenshi Komatsu, Hiroko Shimizu, and Ken-ichi Yamamoto
MutSα, which consists of Msh2 and Msh6, is known to recognize O6-MeG generated by
MNNG and to bind to this DNA lesion. This is consistent with the recent findings that Msh2 andMsh6 interact with ATR and is involved in ATR-mediated Chk1 phosphorylation induced by
MNNG. We confirmed the requirement of Msh6 for MNNG-induced and ATR-mediated Chk1
phosphorylation. However, MNNG-induced Chk2 phosphorylation did not require Msh6, andwas rather enhanced in MSH6-/- DT40 cells. Nbs1, in addition to its role in DSB-induced ATM
activation, is involved in ATR-mediated Chk1 phosphorylation induced by HU and UV. Thus,
consistent with these findings, we found that MNNG-induced Chk1 phosphorylation on Ser-345
was severely diminished in NBS1-/-/- cells. We also observed MNNG-induced Chk2
phosphorylation, which was previously shown to be independent of human ATM and Mlh1
mismatch repair proteins, particularly with higher MNNG concentrations such as those used in
the present study. However, we observed greatly reduced MNNG-induced Chk2
phosphorylation in NBS1-/-/- cells, indicating that ATM and mismatch repair proteins are
dispensable but Nbs1 is required for Chk2 phosphorylation induced by high concentrations of
MNNG. Many recent studies have provided evidence for a role for Nbs1 as a damage sensor or
activator acting upstream of ATM in cellular response to DSB. There is also evidence for a
direct physical interaction between ATM and Nbs1. In addition, both of Nbs1 and ATM are
involved in DSB repair by the homologous recombinational DNA repair system. These findings
explain shared clinical features of A-T and NBS (Nijmegen breakage syndrome caused by ahypomorphism in NBS1), such as immunodeficiency, radiosensitivity, chromosome instability
and cancer predisposition. However, the results presented in the present study as well as thosereported recently by other investigators indicate that Nbs1 is also involved in ATR-mediatedChk1 phosphorylation, which is induced by MNNG, HU, UV or cisplatin, which cause
replication stalling. It is also likely that a kinase involved in Msh6/ATM- independent but
Nbs1-dependent Chk2 phosphorylation induced by high concentrations of MNNG is ATR. Thisfunctional relationship between Nbs1 and ATR may explain the embryonic lethality of NBS1
knockout in mice, which is distinct from the non-essential feature of ATM in mice and human.

― 60 ―

Publications
1.

M. Hashimoto, S. Rao, K. Yamamoto, M. Takata, S. Takeda, and H. Utsumi. DNA-PK: themajor target for wortmannin-mediated radiosensitization by the inhibition of DSB repair via
NHEJ pathway. J. Radiat. Res., 44, 155-163, 2003.

2.

Wang, W., Seki, M., Otsuki, M., Tada, S., Takao, N., Yamamoto, K., Hayashi, M., Honma, M.,
and Enomoto, T. The absence of a functional relationship between ATM and BLM, thecomponents of BASC, in DT40 cells. Biochem. Biophys. Acta, 1688, 137-144, 2004.

3.

Kobayashi, M., Hirano, A., Kumano, T., Shuang-Lin Xiang, S-L., Mihara, K., Haseda, Y.,
Matsui, O., Shimizu, H., and Yamamoto, K. Critical role for vertebrate Rad17 and Rad9 in thecellular response to DNA damage and stalled DNA replication. Genes to Cells, 9, 291-303,
2004.

4.

Barzilai, A., and Yamamoto, K. DNA damage responses to oxidative stress. DNA Repair, 3,1109-1115, 2004.

5.

Fukao, T., Chen, P., Ren, J., Kaneko, H., Yamamoto, K., Takeda, S., Kondo, N., and Lavin, M.
F. Disruption of the BLM gene in ATM – null DT40 cells does not exacerbate either
phenotype. Oncogene, 23, 1498-1506, 2004.

6.

He, J., Tohyama, Y., Yamamoto, K., Kobayashi, M., Shi, Y., Takano, T., Noda, C., Tohyama,K., and Yamamura, H. Lysosome is a primary organelle in B cell receptor-mediated apoptosis:
an indispensable role of Syk in lysosomal function. Genes to Cells, 79, 2597-2603, 2005

7.

Yamamoto, K., Kobayashi, M., and Shimizu, H. ATM, a paradigm for a stress-responsive
signal transducer in higher vertebrate cells. In DT40 Handbook, in press, 2006.

8.

Shibata, T., Iio, K., Kawai, Y., Shibata, N., Kawaguchi, M., Toi, S., Kobayashi, M., Kobayashi,M., Yamamoto, K., and Uchida, K. Identification of a lipid peroxidation product as a potential
trigger of the p53 pathway. J. Biol. Chem., in press, 2006

9.

Shiotani, B., Kobayashi, M., Watanabe, M., Yamamoto, K., Sugimura, T., and Wakabayashi, K.
Involvement of the ATR- and ATM-dependent checkpoint responses in cell cycle arrest
evoked by pierisin-1. Mol. Cancer Res., in press, 2006

― 61 ―

DivisionofCell Cycle Regulation

General Summary of Division of Cell Cycle Regulation
Since 1998 the division has been dedicated to basic researches on MAP kinase (MAPK)
signaling pathways, which play key roles in a wide array of cellular processes, including
proliferation, differentiation, and apoptosis.
The MAPK pathway is an intracellular cascade consisting of MAPK, MAPK kinase(MAPKK) and MAPKK kinase (MAP3K). Recent rapid progress in identifying members of
MAPK cascades suggests that there are a number of these distinct signaling pathways in cells.
However, how the specificity and efficiency of the MAPK cascades is maintained is poorly
understood. Furthermore, the function of each MAPK cascade in vivo is largely unknown.
We have identified two novel JNK MAPK-binding proteins, termed JSAP1 and JNKBP1,
respectively. The subsequent biochemical analyses revealed that the proteins function as scaffold
proteins in the JNK MAPK cascades. The goal of our projects is to demonstrate the functions of
the scaffold proteins in vivo, and furthermore, to clarify the molecular mechanisms of how thesescaffolds ensure the specific and efficient activation of the MAPK cascades in vivo.

― 63 ―

Scaffold protein JSAP1 is transported to growth cones of neurites
independently from JNK signaling pathways in PC12h cells
S. Sato, M. Ito, T. Ito and K. Yoshioka
The c-Jun NH2-terminal kinase (JNK)/stress-activated protein kinase-associated protein 1(JSAP1; also known as JNK-interacting protein 3) has been identified as a scaffold protein for
JNK mitogen-activated protein kinase signal transduction pathways and as a cargo adapter inconventional kinesin-mediated transport system. Furthermore, the functional relationshipbetween UNC-16, C. elegans ortholog of JSAP1, and JNK signaling has been geneticallyestablished. In this study, we first investigated expression properties of endogenous JSAP1 indifferentiating PC12h cells, and demonstrated the requirement of kinesin light chain for thetargeting and localization of JSAP1 to the tips of the neurites. Furthermore, to understandwhether JNK signaling is involved in the kinesin-mediated JSAP1 trafficking, we established
PC12h rat pheochromocytoma stable cell lines that express wild type and mutant JSAP1 lacking
JNK-binding domain, respectively. Immunocytochemical studies of the cell lines indicated thatthe mutant JSAP1 is localized to growth cones of differentiating PC12h cells in a similar mannerto the wild type JSAP1. Taken together, these results suggest that the proper subcellular
localization of JSAP1 along microtublues does not require JNK signaling.

Figure Expression and subcellular distribution of JSAP1b in established PC12h cell lines. Cellsof established PC12h lines (wt#13 and ∆JBD#12) that differentiated in response to treatment
with NGF were fixed and processed for double-label indirect immunofluorescence microscopy.
Arrows (in a and d) indicate signals over the tips of neurites.
Reference:
S. Sato, M. Ito, T. Ito and K. Yoshioka (2004) Gene 329, 51-60.

― 64 ―

Selective expression of the scaffold protein JSAP1 in spermatogonia
and spermatocytes
M. Bayarsaikhan, A. Shiratsuchi, D. Gantulga, Y. Nakanishi and K. Yoshioka
Scaffold proteins of mitogen-activated protein kinase (MAPK) intracellular signaltransduction pathways mediate the efficient and specific activation of the relevant MAPK
signaling modules. Our group and others previously identified c-Jun NH2-terminal kinase(JNK)/stress-activatedproteinkinase-associatedprotein1(JSAP1,

also

known

as

JNK-interacting protein 3) as a scaffold protein for JNK MAPK pathways. Although JSAP1 isexpressed in the testis in adults, its expression during development has not been investigated. In
addition, it is unknown which types of cells in the testis express the scaffold protein. Here, we
examined the expression of JSAP1 in the testis of mice aged 14 days, 20 days, 6 weeks, and 12
weeks by immunohistochemistry and Western blotting. The specificity of the anti-JSAP1
antibody was evaluated from its reactivity to exogenously expressed JSAP1 and a structurally
related protein, and by antigen-absorption experiments. The immunohistochemical analyses withthe specific antibody showed that the JSAP1 protein was selectively expressed in the
spermatogonia and spermatocytes, but not in other cell types, including spermatids and somatic
cells, during development. However, not all spermatogonia and spermatocytes were
immunopositive either, especially in the 12-week-old mouse testis. Furthermore, we found byWestern blotting that the expression levels of JSAP1 protein vary during development; there ishigh expression until 6 weeks after birth, which approximately corresponds to the end of thefirst wave of spermatogenesis. Collectively, these results suggest that JSAP1 function may be
important in spermatogenic cells during early postnatal development.

Figure JSAP1 immunoreactivity in the testis of a 12-week-old mouse. The positive signals wereseen in the spermatogonia (openarrowhead) and spermatocytes (arrow), but not in Sertoli cells(arrowhead) or spermatids (*).
Reference: M. Bayarsaikhan, A. Shiratsuchi, D. Gantulga, Y. Nakanishi and K. Yoshioka (2005)
Reproduction (in press)― 65 ―

Impairment of cardiomyogenesis in embryonic stem cells lacking
scaffold protein JSAP
T. Sato, K. Hidaka, A. Iwanaga, M. Ito, M. Asano, Y. Nakabeppu, T. Morisaki and K.
Yoshioka
We previously reported that c-Jun NH2-terminal kinase (JNK)/stress-activated protein
kinase-associated protein 1 (JSAP1), a scaffold protein for JNK signaling pathways, is importantin embryonic stem (ES) cells during neurogenesis. In that study, we also observed alteredexpression of mesodermal marker genes, implying that JSAP1 is involved in differentiation
during mesodermal lineages. In the present study, we investigated the function of JSAP1
focusing on cardiomyocyte development using JSAP1-null ES cells, and found thatcardiomyogenesis is impaired in the JSAP1-null mutant. We also found that JSAP1-deficiency
results in lower expression of the cardiac transcription factor Nkx2.5 and contractile proteins,and contrarily, in significantly higher expression of mesoderm-related markers other than the
cardiomyocyte lineage. Taken together, these results suggest that JSAP1 may play an importantrole in differentiation during mesodermal lineages, where JSAP1 functions as a positive factor
for cardiomyocyte differentiation, and as an inhibitory factor for differentiation into the otherlineages.

Figure Impairment of cardiomyogenesis in Jsap1-/- Nkx2.5GFP ES cells. (A) EBs derived fromwild-type and Jsap1-/- ES cells were almost identical morphologically at d5. Phase-contrast
photomicrographs of the EBs (upper panels) and the corresponding fluorescent micrographs(lower panels) are shown. Scale bar represents 250 µm. (B) Cardiomyogenesis of Jsap1-/- ES
cells was largely impaired at d5+3. Micrographs are as in (A). Scale bar represents 250 µm. (C)Quantification of contracting EBs derived from wild-type and Jsap1-/- ES cells.
Reference: T. Sato, K. Hidaka, A. Iwanaga, M. Ito, M. Asano, Y. Nakabeppu, T. Morisaki and K.
Yoshioka (2005) BBRC, 338, 1152-1157.

― 66 ―

Publications
1. Ito, M., and Yoshioka, K. (2003) Scaffold proteins of the c-Jun NH2-terminal kinase (JNK)
signaling pathways in neuronal cells. In Pandalai, S.G. ed., Recent Res. Devel. Biochem.
4:271-282
2. Enomoto, A., Suzuki, N., Morita, A., Ito, M., Liu, C.Q., Matsumoto, Y., Yoshioka, K., andShiba, T., Hosoi, Y. (2003) Caspase-mediated cleavage of JNK during stress-induced
apoptosis. Biochem. Biophys. Res. Commun. 306(4):837-842
3. Xu, P., Yoshioka, K., Yoshimura, D., Tominaga, Y., Nishioka, T., Ito, M., and Nakabeppu, Y.
(2003) In vitro development of mouse embryonic stem cells lacking JNK/stress-activatedprotein kinase-associated protein (JSAP1) scaffold protein revealed its requirement during
early embryonic neurogenesis. J. Biol. Chem. 278(48):48422-48433
4. Takino, T., Miyamori, H., Watanabe, Y., Yoshioka, K., Seiki, M., and Sato, H. (2004)
Membrane-type 1 matrix metalloproteinase regulates collage-dependent mitogen-activatedprotein/extracellular signal-regulated kinase activation and cell migration. Cancer Res.
64(3):1044-1049
5. Ikeda, F., Nishimura, R., Matsubara, T., Tanaka, S., Inoue, J., Reddy, S.V., Hata, K.,
Yamashita, K., Hiraga, T., Watanabe, T., Kukita, T., Yoshioka, K., Rao, A., and Yoneda, T.
(2004) Critical roles of c-Jun signaling in regulation of NFAT family and RANKL-regulated
osteoclast differentiation. J. Clin. Invest. 114(4):475-484
6. Akiyama, S., Yonezawa, T., Kudo, T., Li, M.G., Wang, H., Ito, M., Yoshioka, K.,
Ninomiya-Tsuji, J., Matsumoto, K., Kanamaru, R., Tamura, S., and Kobayashi, T. (2004)
Activation mechanism of c-Jun amino-terminal kinase in the course of neural differentiationof P19 embryonic carcinoma cells. J. Biol. Chem. 279(35):36616-36620
7. Yoshioka, K. (2004) Scaffold proteins in mammalian MAP kinase cascades. J. Biochem.
13(6)5:657-661
8. Tsuruta, F., Sunayama, J., Mori, Y., Hattori, S., Shimizu, S., Tsujimoto, Y., Yoshioka, K.,
Masuyama, N., and Gotoh, Y. (2004) JNK promotes Bax translocation to mitochondria
through phosphorylation of 14-3-3 proteins. EMBO J. 23(8):1889-1899
9. Sato, S., Ito, M., Ito, T., and Yoshioka, K. (2004) Scaffold protein JSAP1 is transported to
growth cones of neurites independent of JNK signaling pathways in PC12h cells. Gene
329:51-60
10. Yoshimura, S., Yoshioka, K., Barr, F.A., Lowe, M., Nakayama, K., Ohkuma, S., andNakamura, N. (2005) Convergence of cell cycle regulation and growth factor signals on
GRASP65. J. Biol. Chem. 280(24):23048-23056― 67 ―

11. Takino, T., Nakada, M., Miyamori, H., Watanabe, Y., Sato, T., Gantulga D., Yoshioka, K.,
Yamada, K.M., and Sato, H. (2005) JSAP1/JIP3 cooperates with FAK to regulate c-Jun
N-terminal kinase and cell migration. J. Biol. Chem. 280(45): 377772-377781
12. Sato, T., Hidaka, K., Iwanaga, A., Ito, M., Asano, M., Nakabeppu, Y., Morisaki, T. andYoshioka, K. (2005) Impairment of cardiomyogenesis in embryonic stem cells lacking
scaffold protein JSAP1. Biochem. Biophys. Res. Commun. 338(2): 1152-1157
13. Bayarsaikhan, M., Shiratsuchi, A., Gantulga, D., Nakanishi, Y. and K. Yoshioka
Reproduction (in press)
14. Kong, L., Ueno, M., Itoh, M., Yoshioka, K. and Takakura, N. Identification andcharacterization of mouse PSF1-binding protein, SLD5. Biochem. Biophys. Res. Commun.
(in press)
15. Yoshioka, K. Control of granule cell precursor proliferation in the developing cerebellum
and medulloblastoma Biomed. Rev. (in press)― 68 ―

DivisionofGenetics

General Summary of Division of Genetics
Division of Genetics has been started at June 2005 as a laboratory of “Cancer Stem Cell
Research Center” that will be launched at April 2006. In the past decade, we have been
studying molecular pathogenesis and prevention of gastrointestinal tumors by constructing
genetically engineered mouse models (i.e., knockout mice for Apc, Tgfbr2 or COX-2; andtransgenic mice for COX-2, mPGES-1 or Wnt1). Using these mouse models, we demonstrated
that host reactions through COX-2 pathway in addition to oncogenic mutations are required forgastrointestinal tumor formation. It has been suggested that tissue injury stimulates replicationof tissue stem cells to repair the damaged tissues. Importantly, COX-2 pathway is also inducedin the injured tissues, resulting in elevated prostaglandin production. Accordingly, it ispossible that induction of COX-2 is required for maintenance and replication of the cancer stem
cells as well as those of normal tissue stem cells. For the future cancer stem cell research, weneed to use genetically engineered mouse models because host responses through COX-2
pathway must be critical for stem-cell maintenance in the tumor tissues. In the past 6 months,
we have been concentrating to the following two projects to establish the rationale and mousemodels for future approach.
A) Host inflammatory responses and gastrointestinal tumorigenesis
We have demonstrated using mouse models that COX-2 and downstream PGE2 play a keyrole in gastrointestinal tumorigenesis. To investigate the molecular mechanism, we constructedtransgenic mice (K19-C2mE) expressing two enzymes for PGE2 synthesis, COX-2 andmPGES-1. K19-C2mE mice developed hyperplastic tumors, suggesting that PGE2 signalingenhances proliferation of normal progenitor cells of the gastric gland. Moreover, we haveshown that TNF-α-dependent inflammatory responses are responsible for suppression of gastricepithelial differentiation. These results suggest that COX-2 and PGE2 play a pivotal role intumor development through keeping progenitor cells undifferentiated.
B) Construction of transgenic mouse model for gastric cancer
It has been shown that activation of Wnt pathway is important for tumor formation.

We

have investigated 80 samples of human gastric cancer and found that Wnt signaling is activatedin more than 50% of cases.

To investigate whether Wnt activation induces gastric tumor

development, we constructed transgenic mice (K19-Wnt1) expressing Wnt1 gene in the gastricmucosa. K19-Wnt1 mice showed limited preneoplastic lesions, indicating that Wnt signaling
triggers gastric tumorigenesis.

― 69 ―

Host inflammatory responses and gastrointestinal tumorigenesis
H. Oshima, A. Matsunaga*, M.M. Taketo* and M. Oshima(*Department of Pharmacology, Kyoto University Graduate School of Medicine)
Cyclooxygenase-2 (COX-2), a rate-limiting enzyme for prostanoid biosynthesis, plays a keyrole in gastrointestinal carcinogenesis. Among various prostanoids, prostaglandin E2 (PGE2)
appears to be most responsible for cancer development.

It has been shown that Helicobacter

pylori infection induces expression of COX-2 and microsomal prostaglandin E synthase(mPGES)-1 in the gastric mucosa, resulting in increased level of PGE2 in the stomach.

To

investigate the role of PGE2 in gastric tumorigenesis, we constructed transgenic mice
(K19-C2mE) expressing COX-2 and mPGES-1 in the gastric mucosa.

The transgenic micedeveloped hyperplastic gastric tumors associated with inflammatory responses.

To further

investigate the roles of PGE2-dependent host inflammatory and immune responses in gastrictumorigenesis, we introduced knockout mutations for tumor necrosis factor (TNF)-α, interleukin(IL)-1 receptor α chain and Rag 2 genes, respectively, into K19-C2mE mice.

Among thecompound mutants, only TNF-α (−/−) K19-C2mE mice showed significant suppression of
hyperplastic tumors with reduced epithelial cell proliferation (Figure).

Importantly,

spasmolytic polypeptide-expressing metaplasia (SPEM) in the K19-C2mE stomach was also
suppressed in the TNF-α (−/−) K19-C2mE mice.

Gastric metaplasia to the SPEM lineage has

been considered as a preneoplastic lesion of gastric cancer.

These results indicate that

TNF-α-dependent inflammation caused by increased PGE2 is responsible for development ofhyperplastic tumors and SPEM (Figure).

Therefore, it is possible that inhibition of

TNF-α-dependent inflammation together with eradication of Helicobacter is an effective
prevention strategy for gastric cancer.
H. pylori infection

gastric stem cell

COX-2/mPGES-1

progenitor cells
TNF-α-associatedinflammation

Terminal differentiation

SPEM cells― 70 ―

Construction of transgenic mouse model for gastric cancer
H. Oshima, T. Fujimura*, M.M. Taketo+ and M. Oshima(*Gastroenterologic

Surgery,

Kanazawa

University

Hospital,+

Department

ofPharmacology, Kyoto University Graduate School of Medicine)
It has been demonstrated that nuclear translocation of β-catenin, a hallmark of Wnt
activation, is found in human gastric cancer, suggesting the causal role of Wnt pathway ingastric carcinogenesis. We examined 80 cases of gastric cancer by immunohistochemistry, andfound nuclear β-catenin in 54% (51% and 58% for intestinal- and diffuse types, respectively).
Accordingly, it is conceivable that Wnt pathway activation is one of the major causes of gastriccancer development regardless of the histological type.

To investigate the genetic mechanism

further, we constructed transgenic mice (K19-Wnt1) expressing Wnt1, one of the ligands that
activate the canonical Wnt signaling, in the gastric epithelial cells. Importantly, trefoil factor 2(TFF2)-expressing cell population was expanded in the gastric gland of the K19-Wnt1 mice.
TFF2 is a marker for undifferentiated gastric epithelium which expresses in the small isthmal
cells where β-catenin accumulated.

Moreover, we found dysplastic preneoplastic lesions

consisted of β-catenin-accumulated epithelial cells in the K19-Wnt1 mice (Figure). These
results, taken together, suggest that Wnt signaling keeps gastric stem cells and progenitors
undifferentiated and that activation of Wnt pathway leads to development of preneoplastic
lesions in the stomach.

For gastric cancer development, activation of an additional pathway(e.g., COX-2) appears to be required.

It is of important to investigate compound mutants of

K19-Wnt1 and K19-C2mE.

K19-Wnt1
stomachH&E

β-catenin

preneoplastic
lesion― 71 ―

Publications
1. Thumkeo, D., Keel, J., Ishizaki, T., Hirose, M., Nonomura, K., Oshima, H., Oshima, M.,
Taketo, M.M. and Narumiya, S. (2003) Targeted disruption of the mouse rho-associatedkinase 2 gene results in intrauterine growth retardation and fetal death. Mol. Cell. Biol.
23:5043-5055.
2. Takeda, H., Sonoshita, M., Oshima, H., Sugihara, K., Chulada, P.C., Langenbach, R., Oshima,
M. and Taketo, M.M. (2003) Cooperation of cyclooxygenase 1 and cyclooxygenase 2 inintestinal polyposis. Cancer Res. 63:4872-4877.
3. Aoki, K., Tamai, Y., Horiike, S., Oshima, M. and Takto, M.M. (2003) Colonic polyposis
caused by mTOR-mediated chromosomal instability in Apc∆716 Cdx2+/− compound mutant
mice. Nat. Genet. 35:323-330.
4. Harada, N., Oshima, H., Katoh, M., Tamai, Y., Oshima, M. and Taketo, M.M. (2004)
Hepatocarcinogenesis in mice with β-catenin and Ha-ras gene mutations. Cancer Res.
64:48-54.
5. Takeda, H., Miyoshi, H., Tamai, Y., Oshima, M. and Taketo, M.M. (2004) Simultaneous
expression of COX-2 and mPGES-1 in mouse gastrointestinal hamartomas. Br. J. Cancer
90:701-704.
6. Oshima, H., Oshima, M., Inaba, K., and Taketo, M.M. (2004) Hyperplastic gastric tumors
induced by activated macrophages in COX-2/mPGES-1 transgenic mice. EMBO J.
23:1669-1678.
7. Kawada, K., Sonoshita, M., Sakashita, H., Takabayashi, A., Yamaoka, Y., Manabe, T., Inaba,K., Minato, N., Oshima, M. and Taketo, M.M. (2004) Pivotal role of CXCR3 in melanoma
cell metastasis to lymph nodes. Cancer Res. 64:4010-4017.
8. Fujishita, T., Doi, M., Sonoshita, M., Hiai, H., Oshima, M., Huebner, K., Croce, C.M. and
Taketo, M.M. (2004) Development of spontaneous tumors and intestinal lesions in Fhit
knockout mice. Br. J. Cancer 91:1571-1574.
9. Li, Q., Ishikawa, T., Miyoshi, H., Oshima, M. and Taketo, M.M. (2005) A targeted mutationof Nkd1 impairs mouse spermatogenesis. J. Biol. Chem. 280:2831-2839.
10. Shimizu, Y., Thumkeo, D., Keel, J., Ishizaki, T., Oshima, H., Oshima, M., Noda, Y.,
Matsumura, F., Taketo, M.M. and Narumiya, S. (2005) ROCK-I regulates closure of theeyelids and ventral body wall by inducing assembly of actomyosin bundles. J. Cell Biol.
168:941-953.
11. Oshima, M., Oshima, H. and Taketo, M.M. (2005) Hypergravity induces expression of― 72 ―

cyclooxygenase-2 in the heart vessels. Biochem. Biophys. Res. Commun. 330:928-933.
12. Li, Q., Ishikawa, T., Oshima, M. and Taketo, M.M. (2005) The threshold level of APC
protein for mouse intestinal tumorigenesis. Cancer Res. 65:8622-8627.
13. Oshima, M., Oshima, H., Matsunaga, A. and Taketo, M.M. (2005) Hyperplastic gastrictumors with spasmolytic polypeptide-expressing metaplasia (SPEM) caused by
TNF-α-dependent inflammation in COX-2/mPGES-1transgenic mice. Cancer Res.
65:9147-9151.
14. Takeda, H., Miyoshi, H., Kojima, Y., Oshima, M. and Taketo, M.M. (2005) Accelerated
onsets of gastric hamartomas and hepatic adenomas/carcinomas in Lkb1+/-p53+/- compoundmutant mice. Oncogene [advance online 07 Nov, doi:10.1038/sj.onc.1209207]― 73 ―

DivisionofMedical Oncology

General Summary of Division of Medical Oncology
We have have been basically and clinically analyzing the conditions of digestive cancers from theprecancerous state to onset to determine the groups at high risk for each kind of cancer, and have also been
evaluating the biological characters and clinical usefulness of tumor-related genes such as oncogenes,
oncosuppressive genes, apoptosis-related genes, and so on in various digestive cancers. Through theseresearches, we aim to develop new tumor markers that are useful for early diagnosis, selection oftreatment methods, and prediction and evaluation of outcomes. In addition, pancreatic disease such as
pancreatic cancer (PCa) and pancreatitis has been our major target of our research. We have beenworking on research projects focused on cancer-related substances, and biological factors associated withpancreatitis.
We already reported that K-ras mutations were detectable in more than 90% of endoscopicallyaspirated pancreatic juice (PJ) from patients with PCa,using a highly sensiteve method such as MASA.
However, the frequent presence of K-ras mutation was shown in PJ from noncancerous patients, rasing
quenstions as to the cancer specificity of this marker. On the other hand, the prevalence of p53 mutations
in PJ was about 50%, but not as high as that of K-ras mutations. However, p53 mutations were specicallyfound only in PCa, suggesting that it has high specificity for PCa. Recent studies have demonstrated that
not only genetic changes but also epigenetic changes in various factors have an important role incancinogenesis. It is increasingly apparent that many important genes can be inactivated in association
with promoter hypermethylation in carcinogenesis of each kind of cancer. Using methylation-specific
PCR or real-time quantitative MSP, we have been analyzing the methylation status of 6 CpG islands of
ppENK, SARP2, TFPI-2, NPTX-2, CLDN5 and RASSFIA in PJ samples from pancreatic diseases.
Aberrant methylation of ppENK in the PJ was found in 50% of PCa patients and only in 5% of CP
patients, and that of TFPI-2 was detectable in 62% of PCa patients and in 14% of CP patients, suggestingthat aberrant methylation of ppENK or TFPI-2 has high specificity for cancer, although its incidence is not
so high. Furthermore, we have detected aberrant methylation of SARP2 in 85% of PCa, in 70% of
intraductal papillary mucinous neoplasm of the pancreas and in 26% of CP. Although cancer specificity ofaberrant methylation of SARP2 in PJ is not sufficient, its prevalence in PJ from PCa is strikingly high,indicating usefulness for the detection of pancreatic neoplasm. In addition, we have investigated someoverexpressed genes such as mesothelin and midkine. Mesothelim mRNA expression in PJ has been
discovered using RT-PCR in 52% of PCa patients, but also in 14% of CP patients. Serum levels of
midkine measured by ELISA elevated in a varity of digestive cancer, especially in gastric and coloniccancer patients in whom the positive rate of serum midkine was relatively high and has no relation to thestage of cancer.
On the assumption that in pancreatic diseases, substances derived from the pancreas reflect
abnormalities more sensitively in the PJ than in the blood, lactoferrin, reg-protein identical to pancreaticstone protein and tumor carbohydrate andtigens in the PJ were measured, and interesting results were

― 75 ―

already reported. We have been also analyzing cancer-related genes in the PJ as above mentioned, andstuding the pathophysiological and clinical significance of pancreatitis-associated protein (PAP) belonging
to reg-gene family,which is almost absent in mormal pancreas and is markdly induced in pancreatitis.
Furthermore, the monitoring of serum PAP levels in acute pancreatitis has been shown the most useful for
estimating the severity and clinical healing of the disease. In an attempt to elucidate the role of PAP in thepathogenesis of pancreatitis, we have analyzed expression kinetics of PAP, various cytokines/chemokines,
and apoptosis-related factors in spontaneous CP of the WBN/Kob rat in vivo, and in arginine-induced rat
pancreatic acinar AR4-2J cell injury in vitro. TNF-alpha peaked at 8 weeks prior to the onset of CP, PAP,
IL-6, IL-8, TGF-beta, and vacuole membrane protein 1 peaked at 12 weeks, and IFN-gamma peaked at
16 weeks in the pancreas of WBN/Kob rats. Thus, PAP is strongly induced in pancreatic acinar cells at theonset of CP in the cascade of cytokines/chemokines. Acinar cell apoptosis as well as the expressions of
pro-apoptotic factors such as Fas/FasL, c-myc and stress-induced protein (TP53INP1) showed two peaks
at 12 and 20 weeks, suggesting different roles of apoptosis in the onset and progression of CP. The firstpeak at 12 weeks is both for pro- and anti-apoptotic factors (PAP, bcl-2, clusterin), whereas the second
peak at 20 weeks is only for pro-apoptosis. The first peak of apoptosis indicates the acute-phase reactionof the pancreas in response to various stimuli such as oxidative stress, and the second peak suggests theremodeling and pancreatic regeneration in the course of CP. Therapeutic drugs such as camostat mesilate,
TJ-10 and IS-741, suppressed the expressions of these factors, suggesting molecular action mechanismsof the drugs.
The use of endoscopy has been extended from diagnosis to treatment. We have been enthusiastically
performing endoscopy to treat cancer such as EMR (endoscopic mucosal resection) or ESD (endoscopic
submucosal dissection), although the use of this method requires accurate evaluation of the depth ofcancerous invasion. In this respect, endoscopic ultrasonography (EUS) that allows three-dimensional
evaluation is of diagnostic value, and we have been conducting fundamental and clinical studies on the
diagnostic usefulness of this metbod in gastrointestinal cancers. Furthermore, EUS has been expected todetect mild lesions or small tumor in pancreas where other tests including endoscopic retrograde
pancreatocholangiography (ERCP) cannot show diagnostic findings, since EUS is capable of providing
not only ductal but also parenchymal features in pancreas. We have intended to elucidate the diagnostic
ability of EUS in detecting small pancreatic cancer, applying EUS examination before ERCP. Additionally,
we are currently attempting to develop the ability of early diagnosis for PCa in which biopsy specimens
cannot be easily obtained in combination with imaging test by EUS and molcular biological analysis oftumor-related substances in endoscopically aspirated PJ as above mentioned. Autoimmune pancreatitis(AIP) is a newly proposed clinical entity in which autoimmune mechanisms are involved in thepathogensis. However, it is sometimes difficult to differentiate AIP from PCa. We have reviewed morethan 40 patients with AIP in cooperative study with the affiliated medical facilities, and reported theclinical and imaging features of AIP, differential diagnosis of AIP from PCa, its long-term prognosis,
outcome of steroid therapy in AIP, and so on.

― 76 ―

Aberrant methylation of secreted apoptosis-related protein 2 (SARP2)
in pure pancreatic juice in diagnosis of pancreatic neoplasms
H. Watanabe, G. Okada, K. Ohtsubo, F. Yao, PH. Jiang and N. Sawabu.
Secreted apoptosis-related protein (SARP) families are considered to counteract the oncogenic
Wnt signaling pathway and inactivation of this gene may aid cancer development andprogression. Recently, the aberrant methylation of SARP2 was detected frequently in pancreatic
carcinoma (PCa) tissues, but not in normal pancreatic tissues.
We evaluated the hypermethylation of SARP2 in pure pancreatic juices (PPJ) aspirated
endoscopically from patients with PCa, intraductal papillary mucinous neoplasm of the pancreas(IPMN), chronic pancreatitis (CP), and a control group (C) who was consequently free ofpancreatic disease by methylation-specific PCR (MSP) and real-time MSP.
The incidence of the aberrant methylation of SARP2 using MSP was 79% (26/33) in the PPJ
with PCa, and 85% (17/20) with IPMN. However, it was only 5% (1/19) in the PPJ with CP and
0% (0/10) in the PPJ of C, respectively. The incidences of aberrant methylation of SARP2 in the
PPJ with PCa and IPMN were significantly higher than that in the PPJ with CP (p< 0.001, p<
0.001). Melting curve analysis by real-time MSP as shown in Figure revealed that the incidenceof aberrant methylation of SARP2 in PPJ was 85% (28/33) with PCa, 82% (9/11) with themalignant group of IPMN, 56% (5/9) with the benign group of IPMN and 26% (5/19) with CP.
In this analysis, there were significant differences between PCa and CP (p<0.001), and between
the malignant group of IPMN and CP (p<0.005). In the quantitative analysis by real-time MSP
with a suitable cut-off value, the incidences of aberrant methylation of SARP2 in the PPJ with
PCa, the malignant group of IPMN, the benign group of IPMN and CP were 58 % (19/33), 55%(6/11), 33% (3/9) and 11% (2/19), respectively. The incidence of the aberrant methylation of
SARP2 in the PPJ was significantly different between PCa and CP, and between the malignant
group of IPMN and CP (p<0.005, p<0.05).
These results suggest that promoter methylation of SARP2 in the PPJ may be a highly
sensitive and useful marker for the detection of pancreatic neoplasms, including PCa and themalignant group of IPMN.
Control(Mia PaCa-2)
Figure Melting curve analysis of the quantitative MSP
products by real-time PCR
Representative case of aberrant methylation of
SARP2 in the PPJ sample from patients with
PCa revealed a similar melting curve (red curve)
and the same melting temperature (83℃),
compared with methylation profiles of thehuman PCa celll line Mia PaCa-2 as a methylated
control of SARP2.
Melting temperature (83℃)― 77 ―

Usefulness of aberrant methylation of tissue factor pathway inhibitor
2 (TFPI-2) in pure pancreatic juice in diagnosis for pancreatic
carcinoma
H. Watanabe, PH. Jiang, G. Okada, F. Yao, K. Ohtsubo and N. Sawabu.
Tissue factor pathway inhibitor 2 (TFPI-2) is a Kunitz-type serine proteinase inhibitor and isthought to protect the matrix from degradation therefore counteracting tumor invasion andmetastasis. Recently, aberrant methylation of TFPI-2 was detected frequently in pancreatic
carcinoma (PCa) tissues and not in normal pancreatic tissues. We analyzed the aberrant
methyaltion of TFPI-2 in the pure pancreatic juice (PPJ) from patients with various pancreaticdiseases and evaluated its clinical usefulness in the diagnosis of PCa.
We evaluated the hypermethylation of TFPI-2 in PCa cell lines and PPJ aspirated
endoscopically from patients with PCa, benign and malignant intraductal papillary mucinous
neoplasm of the pancreas (IPMN), chronic pancreatitis (CP) by methylation-specific PCR (MSP)and real-time quantitative MSP (Q-MSP).
The incidence of the aberrant methylation of TFPI-2 using MSP was 7 (77.8%) of 9 PCa celllines by Q-MSP and the expression of TFPI-2 mRNA by quantitative RT-PCR showed inverse
correlation to the aberrant methylation of TFPI-2. Moreover, the incidence of the aberrant
methylation of TFPI-2 in the PPJ was 21 (58.3%) of 36 PCa patients, 2 (20%) of 10 malignant
IPMN and 1 (14.3%) of 7 benign IPMN and 1 (4.8%) of 21 CP by MSP assay. The incidence of
the aberrant methylation of TFPI-2 in the PPJ with PCa was significantly higher than that with
IPMN (p< 0.05) or CP (p< 0.001) as shown in Table. Using Q-MSP analysis, the ratio of
hypermethylation TFPI-2/MyoD1 in the PPJ from patients with PCa, IPMN and CP was 6.62±
7.20, 0.58±1.02 and 1.28±1.73, respectively. The ratio of hypermethylationTFPI-2/MyoD1 in
the PPJ with PCa was significantly higher than that with IPMN (p< 0.01) or CP (p< 0.05). Using
the suitable cut-off value set as 2.5 according to the ROC curve, the incidence of the aberrant
methylation of TFPI-2 in the PPJ by real-time MSP was 18 (62.1%) of 29 PCa patients, 1 (5.9%)of 17 IPMN and 3 (14.3%) of 21 CP, respectively. The incidence of quantitative TFPI-2
hypermethylation in the PPJ with PCa was significantly higher than that with IPMN (p< 0.001)
or CP (p< 0.001).
These results suggest that promoter methylation of TFPI-2 in the PPJ may be specific to PCa
and a useful marker in the diagnosis of PCa.
Table

The incidence of aberrant methylation of TFPI-2 in the pure pancreatic juice frompatients with various pancreatic diseases by methylation-specific PCR
Pancreatic disease
Incidence of aberrant methylation of TFPI-2
Pancreatic carcinoma
58.3% (21/36)
Malignant IPMN
20.0% (2/10)****
Benign
IPMN
14.2% (1/7)
Chronic pancreatitis
4.8% (1/21)
IPMN: intraductal papillary mucinous neoplasm, * : p< 0.05, ** : p< 0.001.
― 78 ―

Detection of preproenkephalin (ppENK) hypermethylation in thepancreatic juice from patients with pancreatic carcinomas:comparedwith p53 mutations
K. Ohtsubo, H. Watanabe, F. Yao, G. Okada, H. Mouri, Y. Yamaguchi, and N. Sawabu
Aberrant methylation of CpG islands is a common mechanism for tumor suppressor genes
in a variety of human malignancies. The preproenkephalin (ppENK) gene encodes a native
opioid peptide, met-enkephalin, which is known to be a potent regulator of development, cellproliferation and angiogenesis. ppENK hypermethylation is recognized in 90% in pancreatic
carcinoma (PCa), but not normal pancreas tissues. We analyzed the ppENK hypermethylation inpure pancreatic juice (PPJ) in patients with PCa and chronic pancreatitis (CP), and elucidated
the usefulness for diagnosis of PCa. Furthermore, p53 mutations were also studied in the samePCa patients.
PPJ was collected endoscopically from 28 patients with PCa and 20 patients with CP. DNA
was extracted from PPJ. Methylation specific PCR was performed for hypermethylation
analyses with the DNA extracted from these samples. In addition, single-strand conformation
polymorphism and sequencing were performed simultaneously for the analyses of p53
mutations.
The incidence of ppENK hypermethylation in PPJ was 50% (14 of 28) in patients with PCa.
In contrast, only one of 20 cases presented hypermethylation in patients with CP (p＜0.002).
p53 mutations were observed in 12 of 28 PCa cases (42.9%), but not in 20 CP cases. In
combination analyses of ppENK hypermethylation and p53 mutations both were positive in 7
and both were negative in 9 of 28 patients. ppENK hypermethylation alone was positive in 7 andp53 mutations alone in 5 patients. ppENK hypermethylation or p53 mutations were observed in
19 of 28 shown in Table, so that positivity improved to 67.9% upon their combination.
These results suggest that ppENK hypermethylation in PPJ would be specific for cancer,
and the combination assay with p53 could enhance the genetic diagnosis of PCa.

Table. Comparison of ppENK hypermethylation and p53
mutations from patients with pancreatic cancer

ppENK
hypermethylation
Total

+
-

p53 mutations
+
-

Total

7
5

7
9

14
14

12

16

28

― 79 ―

Quantitative measurement of hTERT mRNA in sera of the patients withpancreatic carcinoma by real-time RT-PCR
G. Okada, H. Watanabe, H. Mouri, K. Ohtsubo and N. Sawabu
Human telomerase reverse transcriptase (hTERT) is regarded as a catalytic component oftelomerase and a rate-limiting determinant of the enzymatic activity of human telomerase. Its
expression reflects the telomerase activity. Usually, telomerase is inactivated or its activity
repressed in the majority of normal somatic cells but is activated in germ cells, embryonal cells,
proliferating cells of renewable tissues, and activated lymphocytes as well as in most malignant
tumor cells. We examined whether the detection of hTERT mRNA in sera by real-time RT-PCR
is useful or not for the diagnosis of pancreatic carcinoma (PCa).
The serum samples were obtained from 16 patients with PCa, 4 with chronic pancreatitis(CP), and 23 healthy subjects and the RNA was extracted from the serum immediately andstored at –80℃ until the assay. The RNA samples were reverse-transcribed after DNase I
treatment, and measured by the real-time RT-PCR.
The hTERT mRNA in sera was detected in 13 (57%) of the 23 control subjects and 16(100%) of 16 PCa patients by the real-time RT-PCR and the agarose gel electrophoresis. Basedupon the standard curve using hTERT cDNA as a positive control, a linear relationship between
PCR products and cycle number of hTERT cDNA was confirmed in the range from 0.03pg to
30ng. A linear relationship about 18S rRNA was also confirmed by real-time RT-PCR. The mean
±SD of the ratio of hTERT mRNA X 10-2/18S in sera from the control subjects, patients with
PCa, and patients with CP was 0.07±0.18, 0.13±0.25, and 0.002±0.05, respectively. With astandard value of 0.07 as the mean value of normal subjects, 3 (13%) of 23 normal subjects, 6(38%) of 16 patients with PCa, and none (0%) of 4 with CP showed elevated values. The PCapatients with the high values of hTERT mRNA/18S in sera showed advanced clinical stages
such as liver metastasis and peritoneal dissemination, whereas some of the PCa patients with its
low value less than 0.07 in sera underwent surgical resection and obtained longer survival.
However, the values of hTERT mRNA/18S in sera were elevated in a part of normal subjects aswell as PCa patients. Moreover, the values of hTERT mRNA/18S with PCa patients
considerably overlapped with those with normal subjects even in the high range. It is probably
because the hTERT mRNA derived from proliferating cells of renewal tissues or activatedlymphocytes may be included in the serum. Therefore, assay of hTERT mRNA in the serum may
be troublesome and not useful for the clinical application from the results of our study although
hTERT mRNA has been reportedly supposed to be a useful tumor marker.

― 80 ―

Elevated levels of serum midkine as a tumor marker in patients withdigestive cancer.
H. Mouri, K. Ohtsubo, Y. Yamaguchi, H, Watanabe, and N. Sawabu
Midkine (MK) is one of a family of heparin-binding growth factors whose gene was identifiedin embrypnal carcinoma. MK messenger RNA (mRNA) is highly expressed in various tissues
during the midgestation period of mouse embryogenesis, and it is considered to be involved inregulation of organogenesis. Increased MK mRNA and protein expression are exhibited in many
human carcinomas such as gastric, pancreas, bile duct, colorectal, hepatocellular cancers as wellas in Wilms' tumor and neuroblastoma. Serum level of MK is reportedly elevated in patientswith esophageal cancer or neuroblastoma However, little is unknown about usefulness ofdetermination of serum MK as a tumor marker in patients with digestive cancers. In the present
study, we measured serum levels of MK in various digestive cancers and benign diseases, andevaluate its clinical significance as a tumor marker.
Serum levels of MK were measured by enzyme-linked immunosorbent assay (ELISA) using
rabbit anti-human MK antibody as a first antibody, and biotinylated mouse anti-MK antibody asa second one in the sera from digestive malignant diseases (n=139) and benign diseases (n=87).
The cut-off value of serum MK was set at 450pg/ml chiefly based on the ROC curve. The
positive rate of MK in each cancer was as follows: 40% (14/35) in pancreatic cancer, 64%(23/36) in gastric cancer, 67% (16/24) in colorectal cancer, 38% (17/45) in hepatocellular cancer,
and 71% (5/7) in bile duct cancer. Among patients with gastric or colorectal cancer, the positive
rate of serum MK was relatively high and had no relation to the stage of cancer shown in Fig.
High positivity of MK of gastric or colorectal cancer in stageⅠis worthy of notice. On the other
hand, positivity of serum MK in benign digestive disease was as follows: 31% (5/16) in chronic
hepatitis, 33% (4/12) in chronic pancreatitis, 16%(2/12) in acute pancreatitis, and 67% (6/9) inMK(pg/ml)

cholangitis., indicating that false positivity ishigh in inflammatory diseases.
In conclusion , serum MK level increased ina variety of digestive cancers, especially in

1000
450
100

gastric and colorectal cancer. These results

68%suggest that determination of serum MK

67% 33%50%

ⅡⅣ

may be useful as an aid in initial screening

Stage Ⅰof gastrointestinal cancers, although false

Fig. Relationships between serum MK

positivity is high in inflammatory diseases.

Ⅲlevels and tumor stage in gastric cancer― 81 ―

Prolonged, NK cell-mediated antitumor effects of suicide genetherapy combined with monocyte chemoattractant protein-1 against
hepatocellular carcinoma.
T. Tsuchiyama, Y. Nakamoto, Y. Sakai, N. Mukaida, N Sawabu and S. Kaneko
Tumor recurrence rates remain high after curative treatments for hepatocellular carcinoma(HCC). Immunomodulatory agents, including chemokines, are believed to enhance theantitumor effects of tumor cell apoptosis induced by suicide gene therapy. Recently, theantitumor effects of the herpes simplex virus thymidine kinase / ganciclovir (HSV-tk / GCV)
system were observed to be enhanced by codelivery of monocyte chemoattractant protein-1(MCP-1). The current study was designed to evaluate the immunomodulatory effects of abicistronic adenovirus vector expressing both HSV-tk and MCP-1 on HCC cells.
Using an athymic nude mouse model (BALB/c-nu/nu), subcutaneous tumors were completely
eradicated by rAd followed by treatment with ganciclovir, subsequently these mice werere-challenged with HCC cells and tumor development was monitored. In addition, after theadministration of re-challenged tumors, the serum levels of interleukin (IL)-12 and IL-18 were
measured, and the recruitment or activation of natural killer (NK) cells was analyzed
immunohistochemically or by measuring interferon (IFN)-γ mRNA expression. Finally, weevaluated the antitumor response in immunocompetent wild type mice (BALB/c-jcl) using thesame experimental protocol.
Tumor growth was markedly suppressed compared with mice treated with rAd expressing
HSV-tk gene alone (P<0.001). Suppression of tumor growth was associated with elevation ofserum IL-12 and IL-18. During suppression, NK cells were recruited exclusively, and T helper 1
cytokine gene expression was enhanced in tumor tissues. The antitumor activity, however, wasabolished when the NK cells were inactivated with anti-asialo GM1 antibody. Moreover, theeffects of the anti-tumor response in wild type mice were comparable to them in nude mice.
In conclusion, these results indicate that suicide gene therapy, together with delivery of MCP-1,
eradicates HCC cells and exerts prolonged NK cell-mediated antitumor effects in a model of
HCC, suggesting a plausible strategy to prevent tumor recurrence.

― 82 ―

Tumor protein p53-induced nuclear protein 1 (TP53INP1) in
spontaneous chronic pancreatitis in the WBN/Kob rat: drug effects on
its expression in the pancreas
Pei-Hong Jiang, Yoshiharu Motoo, Gensaku Okada, and Norio Sawabu
TP53INP1 is a nuclear factor that is rapidly and strongly induced by various stresses.
TP53INP1 is overexpressed in acinar cells during acute pancreatitis in the mouse. TP53INP1
overexpression promotes cellular apoptosis. It is known acinar cell apoptosis is involved inacinar destruction/degeneration in the course of chronic pancreatitis. Therefore, examining
TP53INP1 gene expression in CP and related to acinar cell apoptosis will contribute to a better
understanding of the molecular mechanisms of the onset and progression of chronic pancreatitis.
Four-week-old male WBN/Kob rats were fed a special breeding diet MB-3 to induce
chronic pancreatitis. Anti-inflammatory drug such as camostat mesilate (0.01%) and
Saiko-keishi-to (0.08%) were mixed in the diet MB-3 fed to the rats from 4 weeks. Sections
were stained with hematoxylin and eosin for histological evaluation. TP53INP1 mRNA
expression was determined by RT-PCR with a semi-quantitative analysis and in situ
hybridization. TUNEL was used to detect cell apoptosis. In vitro, 5 mg /ml L-arginine wasdissolved in the medium of AR4-2J cell.
TP53INP1 mRNA expression began at 4 weeks, reached its first peak at 12 weeks, after
decreased at 16 weeks, TP53INP1 mRNA reached its second peak at 20 weeks, finallydecreased at 24 weeks. By in situ hybridization analysis, TP53INP1 mRNA was strong in theacinar cells but absent in duct or islet cells. The apoptotic index was significantly increased at
12 and 20 weeks showing two peaks, so that, the kinetics of TP53INP1 mRNA expression wasparalleled to apoptotic index. TP53INP1 mRNA expression was significantly suppressed in the
camostat mesilate group at 8, 12, and 20 weeks, and it was also significantly suppressed in the
Saiko-keishi-to treated group at 8 and 12 weeks. TP53INP1 mRNA was expressed in AR4-2J
cells from 2 to 24 hours. The expression level was significantly higher at 6 hr than those at othertime points.
In the present study, TP53INP1 expression showed two peaks in the course of WBN/Kob
rat. The first peak of TP53INP1 would reflect the concomitant induction of pro-, andanti-apoptotic factors at the onset of chronic pancreatitis. Around this time point, thehistopathology of the pancreas resembles acute edematous pancreatitis, although there is alreadypancreatic fibrosis. Acinar cell apoptosis would attenuate the severity of pancreatitis,
preventing the excessive release of harmful pancreatic enzymes. The second peak of acinar cell
apoptosis and pro-apoptotic factors including TP53INP1 could reflect acinar cell loss and acinar
remodeling or regeneration during the progression of chronic pancreatitis. Suppression of
TP53INP1 mRNA expression by therapeutic drugs may confirm that TP53INP1 expression is
deeply involved in the onset and progression of chronic pancreatitis in this model.
In conclusion, we have shown that TP53INP1 mRNA expression is induced in the onset
and progression of chronic pancreatitis in
WBN/Kob rats. TP53INP1 may be involvedin the pathogenesis of chronic pancreatitis
as a pro-apoptotic factor.
Figure: TP53INP1 mRNA was expressed from 4 to
24 weeks. It showed the first peak at 12 weeks andthe second peak at 20 weeks.

― 83 ―

Expression of vacuole membrane protein 1 (VMP1) in spontaneous
chronic pancreatitis in the WBN/Kob rat
Pei-Hong Jiang, Yoshiharu Motoo, Gensaku Okada, and Norio Sawabu
VMP1 is a transmembrane protein located in the Golgi and endoplasmic reticulum. It isstrongly and rapidly induced in pancreas during acute pancreatitis whereas it is hardly detectablein normal rats pancreas. Its overexpression promotes formation of intracellular vacuoles and cell
death through apoptosis. Intracellular vacuolation is a cellular response to injury in the pancreas
during pancreatitis to maintain function under stress. The vacuoles have been shown to containboth digestive and lysosomal enzymes suggesting an abnormal intracellular processing of newly
synthesized enzymes in acute pancreatitis. Physiologically, digestive and lysosomal enzymes aresynthesized in ribosomes attached to the rough endoplasmic reticulum. Vacuoles represent earlymorphological changes during apoptosis. The aim of this work was to examine the kinetics of
VMP1 gene expression in spontaneous chronic pancreatitis in WBN/Kob rat.
Four-week-old male WBN/Kob rats were fed a special breeding diet MB-3 to induce
chronic pancreatitis. Sections were stained with hematoxylin and eosin for histologicalevaluation and vacuoles examination. VMP1 mRNA expression was determined by RT-PCR
with a semi-quantitative analysis and in situ hybridization. TUNEL was used to detect cell
apoptosis. Cytoplasmic vacuoles with different sizes were observed in the pancreas of the
WBN/Kob rat from 4 to 24 weeks. The vacuoles increased in size and number at the onset of CP
at 12 weeks. VMP1 mRNA expression began at 8 weeks, reached a peak at 12 weeks, decreased
at 16 and 20 weeks, then disappeared at 24 weeks. It was showed VMP1 mRNA was located inacinar cells and absent in the islets of Langerhans, duct cells, inflammatory infiltrates, and otherstromal cells by in situ hybridization analysis. The vacuoles formation was paralleled to VMP1mRNA expression. The kinetics of VMP1 mRNA expression also corresponded to the apoptosisof acinar cells.
Vacuolation is an early phenomenon proceeding cell death, and is related to apoptosis. We
have shown, for the first time, that cytoplasmic vacuoles in acinar cells are increased during thecourse of CP in the WBN/Kob rat and that the number of vacuoles was most increased at theonset of the CP. Acinar cell vacuolation is possibly a general feature of the development of acutepancreatitis and therefore, may also have important pathophysiological implications for CP.
VMP1 gene activation in chronic pancreatitis is part of the acinar cell response to aggression. In
conclusion, we have revealed cytoplasmic vacuolization in CP of the WBN/Kob rat, anddemonstrated that VMP1 mRNA expression is strongly induced at the onset of CP. VMP1mRNA expression, relating to acinar cell apoptosis, may reflect important pathophysislogicalchanges in CP.
Figure: A: VMP1 mRNA of 604 bp was
expressed from 8 to 20 weeks. B: VMP1mRNA expression began at 8 weeks reached
its peak at 12 weeks decreased at 16 and 20
weeks.

― 84 ―

Duct-narrowing chronic pancreatitis without immunoserologicalabnormality: comparison with duct-narrowing chronic pancreatitis withpositive serological evidence and its clinical management.
N. Sawabu, T. Wakabayashi, Y. Satomura, H. Watanabe, Y. Motoo
We reviewed the clinical features and clinical course of patients with duct-narrowing chronic pancreatitis
who were negative for immunoserological test results (n=16) in comparison with the findings forserological test-positive patients (n=20) in order to determine an adequate treatment for those who had
typical morphology of autoimmune pancreatitis in the absence of immunoserological abnormality. No
significant differences were found between the two groups of patients in clinical profiles including
associated autoimmune-related diseases, pancreatic histology, and response to steroid therapy. Of theseronegative patients, eight patients who showed an improvement in narrowing of the main pancreatic
duct with steroid therapy and three who did no show an improvement or who relapsed after surgicalresection without this therapy had stenosis of the common bile duct with increased levels of serum
hepatobiliary enzymes, except for two patients with affected sites limited to the body or tail of the gland.
For the remaining five patients, who showed an improvement in pancreatic duct changes or long-term
remission after surgery without steroid administration, normal biochemistry test results for liver functions
were obtained, with no abnormal cholangiographic findings in the 3 patients examined.
Duct-narrowing chronic pancreatitis without immunoserological abnormality overlaps in clinical featureswith that fulfilling the immunoserological criteria for a diagnosis of autoimmune pancreatitis. In particular,
the disease with bile duct involvement should be treated clinically as autoimmune pancreatitis, for whichsteroid therapy is recommended, even if an autoimmune mechanism is not demonstrated serologically.
Table. Comparison of clinicopathologic findings for seropositive and seronegative DNCP patients
Clinicopathologic finding

seropositive(n=20)

seronegative(n=16)

P valueSex (male/female)Age (mean±SD; yr)
Location (diffuse/focal)
DM
Extrabilopancreatic autoimmunerelated disease
Jaundice
Upper abdominal pain
Increased cholestatic enzymes
Increased pancreatic enzymes
CBD stenosis
Dense inflammatory cell infiltration
Effective steroid therapy

17/3
62.9±10.0
17/3
12 (60%)6 (30%)

10/6
57.6±13.9
12/4
6 (38%)
3 (19%)

p＞0.10p=0.20p＞0.90p＞0.25p＞0.5011 (55%)4 (20%)
15 (75%)10 (50%)18/19 (95%)
7/8 (88%)
14/15 (93%)

5 (31%)
7 (44%)
9 (56%)
13 (81%)
9/12 (75%)
4/6 (67%)
8/8 (100%)p＞0.25p＞0.10p＞0.25p＞0.10p＞0.25p＞0.75p＞0.50

DNCP, duct-narrowing chronic pancreatitis; DM, diabetes mellitus; CBD, common bile duct

― 85 ―

Long-term prognosis of duct-narrowing chronic pancreatitis: strategy forsteroid treatment
N. Sawabu, T. Wakabayashi, Y. Satomura, H. Watanabe and Y. Motoo.
The purpose of this study was to assess the long-term prognosis in patients with duct-narrowing
pancreatitis (DNCP) and determine the indications for steroid therapy. We evaluated clinical and imaging
outcomes in patients with DNCP classified into three groups (A, B, and C) according to the treatment given.
Group A included 6 patients who underwent surgical resection. Groups B and C included 21 and 10
patients treated medically with and without steroid therapy, respectively.
In group A, 2 patients relapsed in the remnant pancreas. In group B, pancreatic swelling was resolved in
all the patients, and moreover, both the irregular narrowing of the main pancreatic duct (MPD) and thestrictures of the common bile duct (CBD) improved after initiation of corticosteroid therapy in all but 1 ofthe patients, including 5 without immunoserological abnormality. However, clinical recurrences occurred in
4 patients (19%) during or after the maintenance therapy. In group C, all the patients showed an
improvement in swelling of the gland, while only 5 showed improvement of pancreatic duct involvement.
Four of these 5 patients did not show any serological data suggesting autoimmune abnormality or CBD
involvement. In all of the other 5 patients in whom MPD irregularities did not improve, bile duct stricturesor positive immunoserological test results were noted.
Steroid therapy is effective for improving pancreatic duct and bile duct lesions in patients with DNCP,
and is indicated particularly in those who show CBD strictures or immunoserological abnormality,
although some patients have recurrences.
0

6

12

18

24

30

follow-up period (months)
36 after starting of steroid

Patient No.
B-1
B-2
B-3
B-4
B-5
B-6
B-7
B-8
B-9
B-10
B-11
B-12
B-13
B-14
B-15
B-16
B-17
B-18
B-19
B-20
B-21

Figure. Prognosis after clinical remission in 21 patients of group B.
●, relapsed during maintenancetherapy;● , relapsed after the cessation of maintenance therapy;, continued to be inclinical remission with maintenance therapy;, continued to be in clinical remission after the
cessation of maintenance therapy.

― 86 ―

Publications
1. Sabet EA, Okai T, Minamoto T, and Sawabu N: Visualizing the gastric wall with a30-MHz ultrasonic miniprobe: ex vivo imaging of normal gastric sites and sites of earlygastric cancer. Abdom Imaging 28：252-256, 2003.
2. Okai T, Minamoto T, Ohtsubo K, Minato H, Kurumaya H, Oda Y, Mai M, and Sawabu N:
Endosonographic evaluation of c-kit-positiv gastrointestinal stromal tumor. Abdom
Imaging 28: 301-307, 2003.
3. Xie M-J, Motoo Y, Iovanna JL, Su S-B, Ohtsubo K, Matsubara F, and Sawabu N:
Overexpression of pancreatitis-associated protein (PAP) in human pancreatic ductal
adenocarcinoma. Dig Dis Sci 48(3): 459-464, 2003.
4. Ohtsubo K, Watanabe H, Yamaguchi Y, Hu Y-X, Motoo Y, Okai T, and Sawabu N:
Abnormalities of tumor suppressor gene p16 in pancreatic carcinoma:
immunohistochemical and genetic findings compared with clinicopathological parameters.
J Gastroenterol 38: 663-671, 2003.
5. Okai T, Shirasaki F, and Sawabu N: Erythematous maculopapular eruption due to ribavirin
administration in a patient with chronic hepatitis C. J Clin Gastroenterol 36(3): 283-284,
2003.
6. Motoo Y, Xie M-J, Su S-B, and Sawabu N: Molecular mechanisms of therapeutic effects of
Saiko-keishi-to on spontaneous chronic pancreatitis in the WBN/Kob rat. J Trad Med
20(4): 132-138, 2003.
7. Ito Y, Yoshida H, Motoo Y, Miyoshi E, Iovanna JL., Tomoda C, Uruno T, Takamura Y,
Miya A, Kobayashi K, Matsuzuka F, Matsuura N, Kuma K, and Miyauchi A: Expression
and cellular localization of p8 protein in thyroid neoplasms. Cancer Letters 201: 237-244,
2003.
8. Wakabayashi T, Kawaura Y, Satomura Y, Watanabe H, Motoo Y, Okai T, and Sawabu N:
Clinical and imaging features of autoimmune pancreatitis with focal pancreatic swelling or
mass formation: comparison with so-called tumor-forming pancreatitis and pancreatic
carcinoma. Am J Gastroenterol 98(12): 2679-2687, 2003.
9. Motoo Y, and Uchide K: Regulations of ephedra in the United States. Am J Chin Med 31:
1003-1004, 2003.
10. Wang Y, Yamaguchi Y, Watanabe H, Ohtsubo K, Motoo Y, and Sawabu N: Detection of
p53 gene mutations in the supernatant of pancreatic juice and plasma from patients withpancreatic carcinomas. Pancreas 28(1): 13-19, 2004.
11. Feng S-Y, Ohta K, Yamada Y, Sawabu N and Ito T: A yeast one-hybrid system to detect
methylation-dependent DNA-protein interactions. Biochem Biophy Res Commun 313:
919-922, 2004.
12. Sawabu N, Watanabe H, Yamaguchi Y, Ohtsubo K, and Motoo Y: Serum tumor markers
and molecular biological diagnosis in pancreatic cancer. Pancreas 28(3): 263-267,
2004.
13. Motoo Y, Xie MJ, Mouri H, and Sawabu N. Expression of interleukin-8 in humanobstructive pancreatitis. Journal of the Pancreas (JOP), 5(3): 138-144, 2004.
14. Jiang PH, Motoo Y, Iovanna JL, Pebusque MJ, Xie MJ, Okada G, and Sawabu N. Tumor― 87 ―

protein p53-induced nuclear protein 1(TP53INP1) in spontaneous chronic pancreatitis in
the WBN/Kob rat : drug effects on its expression in the pancreas. Journal of the Pancreas(JOP), 5(4): 205-216, 2004.
15. Jiang PH, Motoo Y, Vaccaro MI, Iovanna JL, Okada G, and Sawabu N. Expression of
vacuole-membrane protein-1 (VMP1) in spontaneous chronic pancreatitis in the WBN/Kob
rat. Pancreas, 29(3): 225-230, 2004.
16. Watanabe H, Yamaguchi Y, Ohtsubo K, Mouri H, Motoo Y, Yamashita K, Minamoto T,
Gabata T, and Sawabu N. Epidermoid cyst of the intrapancreatic accessory spleen
producing CA 19-9. Dig Endosc, 16(3): 244-248, 2004.
17. Wakabayashi T, Kawaura Y, Satomura Y, Watanabe H, Motoo Y, and Sawabu N. Long-term
prognosis of duct-narrowing chronic pancreatitis: strategy for steroid treatment. Pancreas,30(1): 31-39, 2005.
18. Motoo Y, Mouri H, Ohtsubo K, Yamaguchi Y, Watanabe H, and Sawabu N. The herbal
medicine Ninjinyoeito ameliorates ribavirin-induced anemia in chronic hepatitis C: arandomized controlled trial. World J Gastroenterol, 11(26): 4013-4017, 2005.
19. Takahashi Y, Mai M, Sawabu N, and Nishioka K. A pilot study of individualized maximum
repeatable dose (iMRD), a new dose finding system, of weekly gemcitabine for patientswith metastatic pancreas cancer. Pancreas, 30(3): 206-210, 2005.
20. Wakabayashi T, Kawaura Y, Satomura Y, Urabe T, Watanabe H, Motoo Y, and Sawabu N.
Duct-narrowing chronic pancreatitis without immunoserological abnormality: comparison
with duct-narrowing chronic pancreatitis with positive serological evidence and its clinicalmanagement. Dig Dis Sci, 50(8): 1414-1421, 2005.
21. Sawabu N, and Watanabe H. Altered expression of p27 protein in pancreatic carcinomas(Molecular pathology). Elsevier Science/ Academic Press, Hagat M.A. (eds); 331-333,
2005.
22. Ito Y, Yoshida H, Motoo Y, Iovanna JL, Nakamura Y, Nakudo K, Uruno T, Takamura Y,
Miya A, Noguchi S, Kuma K, and Miyauchi A. Expression of P8 protein in breast
carcinoma; an inverse relationship with apotosis. Anticancer Res 25: 833-838, 2005.
23. Watanabe H, Okada G, Ohtsubo K, Yamaguchi Y, Mouri H, Motoo Y, Wakabayashi T, andSawabu N. Expression of mesothelin mRNA in pure pancreatic juice from patients withpancreatic carcinoma, intraductal papillary mucinous neoplasm of the pancreas, andchronic pancreatitis. Pancreas 30(4):349-354,2005.
24. Jiang PH, Motoo Y, Iovanna JL, Garcia S, Pebusque MJ, and Sawabu N. The
down-expreesion of tumor protein 53-induced nuclear protein 1 (TP53INP1) in humangastric cancer. World J Gastroenterol, (in press).
25. Watanabe H, Ohtsubo K, Yamaguchi Y, Mouri H, Motoo Y, Noto M, Kitagawa H, Kayahara
M, Ohta T, Gabata T, Sakamoto S, and Sawabu N. Successful cyotic fenestration for a
macrocyotic serous cyotadenoma of the pancreas causing obstructive jaundice: report of acase. Surg Today 36: 89-93, 2006.
26. Jiang PH, Motoo Y, Minamoto T, and Sawabu N. Gemcitabine changes the expression of
apoptosis-related genes in human pancreatic cancer cells. World J, Gastroenterol (in press).
27. Kamisawa T, Wakabayashi T, and Sawabu N. Autoimnune pancreatitis in young patients. J.
Gastroenterol, (in press).
― 88 ―

28. Watanabe H, Okada G, Ohtsubo K, Yao F, Jiang PH, Mouri H, Wakabayashi T, and Sawabu
N. Aberrant methylation of secreted apoptosis-related protin 2(SARP2) in pure pancreatic
juice in diagnosis of pancreatic neoplasms. Pancreas, (in press).

― 89 ―

DivisionofSurgical Oncology

General Summary of Division of Surgical Oncology
Since Cancer Institute Hospital had unified into University Hospital at October 2001, we havevery busily occupied with hospital work and they had no time to spare for research work.
Under this circumstance we continued several research work on cancer metastasis, molecularpathology, and tumor immunology. Recent projects may be summarized as follows.
Project on cancer metastasis
To clarify molecular mechanism of cancer metastasis, especially matrix metalloproteinase andangiogenesis using clinical materials, and to inhibit metastasis. After we have firstly reported
that vessel density and expression of VEGF and its receptor, KDR, correlates with metastasis,and proliferation of human colon cancer, many researchers have reported VEGF is one ofimportant angiogenic factor in patients with colon cancer. Moreover, many clinical trials using
these VEGF targeting drugs were done in several cancers such as breast, lung and colon cancer.
Among them, phase II and III of recombinant human monocolonal antibody to VEGF (Avastin,
Genentech) showed positive data, and accepted a new molecular targeting drug for metastaticcolon cancer by FDA at February 2004. Therefore we continued this project for some new
indication of this drug.
We have shown that mRNA upregulation of metastasis-related genes such as angiogenic
factors, metalloproteinases, growth factors, and adhesion molecules, occur just prior tometastasis in human colon cancer in an orthotopic nude mice model of colon cancer liver
metastasis. We have also shown that upregulation of angiogenesis, i.e. angiogenic switch may
occur between Tis and T1, simultaneous to initiation of invasion, in the early development of
human colon cancer. Finally, we showed that VEGF inhibit angiogenic switch and liver
metastasis of the orthotopic xenograft model in site-dependent tumor of VEGF. These results
suggest that VEGF antibody have another possibility as a drug for chemoprevention of coloncancer, especially premalignant lesions such as polyposis and long-termed ulcerative colitis.
We have shown that VEGF expression was higher in patients with liver metastasis of gastriccancer. Recently, we showed the correlation between alpha-fetroprotein (AFP) and VEGF
expression in AFP producing gastric cancer, which is well known to have a high incedence ofliver metastasis. From these results, we are planning to indicate the Avastin in patients withliver metastasis of gastric cancer.
Project on chemotherapy including tumor dormancy therapy
The goal of chemotherapy in patients with far advanced cancer should be aimed to prolong
the survival of cancer patients, tumor dormancy in other words, rather than tumor shrinkage(killing cancer cells), with fairly good QOL. In order to induce tumor dormancy two approaches
are considered: one is a molecular targeting therapy to prolong the dormant period itself and― 91 ―

other is obtain prolonged NC with chemotherapy. We developed and established a new
dose-finding system, the individualized maximum repeatable dose (iMRD), suitable to induce
prolonged TTP rather than tumor shrinkage. And we started a randomized Phase II clinical
trial of tailored CPT-11 + S-1 vs S-1 in patients with advanced or recurrent gastric carcinoma as
the first line chemotherapy to study the significance of iMRD. Moreover we started andplanned some similar clinical studies for colon, breast, ovary, pancreas cancers.
Project on minimally invasive surgery (MIS)
Laparoscopic surgery in patients with early GI cancer allows minimally invasive surgery to be
carried out to maintain the patient’s quality of life in future. Dr. Omote has conducted thisproject and extended the indication of MIS in advanced gastric cancer and colorectal cancer.
He showed that the laparoscopic surgery was associated with less bleeding, rapid recovery ofbowel movement, faster initiation of oral intake and shorter hospital stay. He also showed thatthe recovery after laparoscopy assisted partial gastrectomy is as successful as after laparoscopic
local resection.
Project on cytokines
Dr. Yasumoto studied the significance of CSCL12 in peritoneal caricinomatosis of gastriccancer. Finally, he showed that three out of seven human gastric carcinoma cell lines
selectively expressed CXCR4 mRNA and protein at high levels, and which were all highly
efficient in generating malignant ascites in nude mice upon intraperitoneal inoculation. NUGC4
cells expressed CXCR4 mRNA at high levels and showed vigorous migratory responses to its
ligand CXCL12 (also called stromal-derived factor-1a, SDF-1a). Furthermore, CXCL12-treated
NUGC4 cells showed enhanced proliferation and rapid increases in phosphorylation of protein
kinase B/Akt and extracellular signal-regulated kinase (ERK). He also CXCR4-positivity ofprimary gastric carcinomas significantly correlated with the development of peritoneal
carcinomatosis, and concluded that CXCR4/CXC12 axis plays an important role in thedevelopment of peritoneal carcinomatosis from gastric carcinoma.
Project on gene expression in human gastric cancer
Dr. Yamashita analyzed gene expression in human gastric cancer by serial analysis of gene
expression (SAGE). SAGE is a method for comprehensive analysis of gene expression patterns.
A short sequence tag (10-14bp) contains sufficient information to uniquely identify a transcript.
He showed that total of 60621 transcripts were identified, and higher expression of SIAT6,
APOC1, galectin-1 and COL1A1 in lymph node metastasis versus primaly tumor among them.

― 92 ―

A pilot study of individualized maximum repeatable dose (iMRD), a
new dose finding system, of weekly gemcitabine for patients withmetastatic pancreas cancer
Y. Takahashi and K. Yamashita
We developed and established a new dose-finding system, the individualized maximum
repeatable dose (iMRD), suitable to induce prolonged TTP rather than tumor shrinkage.
We applied this system in weekly gemcitabine therapy for 18 metastatic pancreas cancer
patients. We determined the iMRD at the 5th week, after weekly dose adjustments.

We

2

started at 500 mg/m (1/2 MTD (maximum tolerated dose)) of gemcitabine and repeated thetreatment with an increase or a decrease of 100 mg/m2 each week, if toxicity was 0 or more thangrade 1, respectively (Figure 1).
The iMRD of weekly gemcitabine was 300 mg/m2 in 2 patients, 400 mg/m2 in 3 patients, 500
mg/m2 in 5 patients, 600 mg/m2 in 6 patients, and 700 mg/m2 in 2 patients, demonstrating
significant differences among individual patients. Grade 3 marrow depression occurred in only
1 patient (5.6%).

Of these 18 patients, 3 (16.7%), 13 (72.2%) and 2 (11.1%) patients showed

PR, SD and PD, respectively.

The median of TTP and survival was 4.5 months and 9.5 months,

respectively. There were no significant differences in 1 year survival time and more than 50 %reduction rate of serum CA19-9, a tumor marker for pancreatic cancer, between patients withlower (500 mg/m2 or less) and higher (600 mg/m2 or more) iMRD.
These results suggest that iMRD is a simple method to determine an individual’s tailored dosefor chemotherapy and could be the optimal dose for patients with non-curable cancers such as
metastatic pancreas cancer.

Toxicity

500 mg/m2

grade 0600 mg/m2

grade 1500 mg/m2

grade 2400 mg/m2

Figure 1. The method of determination of iMRD

― 93 ―

repeat until 5th weekChemotherapy under cachectic conditions and the possibility ofcachexia-controlled chemotherapy
Y. Takahashi and K. Yasumoto
We studied the role of chemotherapy in cancer cachexia, which is well known to lower QOL
as well as responsiveness to chemotherapy.

In BALB/c mice we used two clones derived fromthe murine colon 26 adenocarcinoma cell line, clone 5, a non-cachexigenic tumor and clone 20,a cachexigenic tumor, in which IL-6 mRNA was selectively detected.

While maximum

tolerated dose (MTD) of CPM and CPT-11 showed significantly smaller tumor weight andhigher survival than 1/2 MTD in both drug groups with clone 5, the results were reversed withclone 20 (Fig 1). The tumor weights with MTD of CPM or CPT-11 in combination with
anti-IL-6 antibody treatment, which decreases serum IL-6 level and improves cachexia status,
were significantly smaller than those in the MTD treatment-alone group with clone 20, but notwith clone 5 (Fig 2).
From these results, we suggest that lower dose chemotherapy or cachexia-controlled
chemotherapy such as some chemotherapeutic agents with neutralizing cachexia-related
cytokine effects, elicits superior antitumor effects in cachectic individuals than conventional
MTD chemotherapy.
A

100%

A

CPT-11 100 mg *100%

survival rate

survival rateanti-IL-6 + CPM 200 mg *CPT-11 50 mg
50%

50%

Control
CPM 200 mg
0

0

5

5

10

10

days from treatmentdays from treatment

B

100%

survival rate

survival rate

B

100%

CPT-11 50 mg * *50%anti-IL-6 + CPT-11 100 mg *50%

Control
CPT-11 100 mg

CPT-11 100 mg
0

0
5

10

days from treatment

Fig 1. Survival curves of CPT-11 therapy

5

10

days from treatment

Fig. 2. The effect of anti-IL-6 antibody― 94 ―

Laparoscopic Surgery for Early Gastric Cancer
K. Omote, H. Kitakata, K. Yamashita, K. Yasumoto and Y. Takahashi
Resent advancement of endoscopy and its technique lead to early detection of gastriccancer, and enable us to perform an entirely new surgical approach to gastric cancer.
Laparoscopic surgery for early gastric cancer is minimally invasive and offers patients theadvantages of reduced pain, shorter hospital stay and quicker convalescence, compared with
open surgery. In Japan, laparoscopic local (wedge) resection of the stomach (lesion lifting
method) and laparoscopic mucosal resection (intra-gastric surgery) were first performed in 1992
and in 1993 respectively, and laparoscopy assisted partial resection of the stomach with lymph
node dissection in 1994. Laparoscopic surgery for early gastric cancer has been introduced inour department since 1995. Since then, we have experienced 20 cases with laparoscopic local
resection of the stomach and 24 cases with laparoscopy assisted partial gastrectomy.

Recently,

the opportunity to select laparoscopy assisted partial gastrectomy has been increased. Comparedwith open surgery, the laparoscopic surgery is associated with less bleeding, rapid recovery ofbowel movement, faster initiation of oral intake and shorter hospital stay. Laparoscopic surgery
for early gastric cancer is feasible, with favorable short-term outcome. Although procedure oflaparoscopy assisted partial gastrectomy is much different in that of laparoscopic local resection,
the recovery after laparoscopy assisted partial gastrectomy is as successful as after laparoscopic
local resection.

― 95 ―

Role of the CXCL12/CXCR4 axis in Peritoneal Carcinomatosis of
Gastric Cancer
K. Yasumoto1, K. Koizumi2, I. Saiki2, and Y. Takahashi1
1Division of Surgical Oncology, Cancer Research Institute, Kanazawa University

2

Division of Pathogenic Biochemistry, Institute of Natural Medicine, Toyama University
Peritoneal carcinomatosis is a frequent cause of death in patients with advanced

gastric carcinoma. Since chemokines are now considered to play an important role in themetastasis of various malignancies, we hypothesized that they may also be involved in thedevelopment of peritoneal carcinomatosis by gastric carcinoma. We found that three out ofseven human gastric carcinoma cell lines selectively expressed CXCR4 mRNA and protein athigh levels. These three cell lines were those that were all highly efficient in generating
malignant ascites in nude mice upon intraperitoneal inoculation. In particular, NUGC4 cells
expressed CXCR4 mRNA at high levels and showed vigorous migratory responses to its ligand
CXCL12 (also called stromal-derived factor-1a, SDF-1a). Furthermore, CXCL12-treated
NUGC4 cells showed enhanced proliferation and rapid increases in phosphorylation of protein
kinase B/Akt and extracellular signal-regulated kinase (ERK). We also demonstrated that
AMD3100 (a specific CXCR4 antagonist) effectively reduced tumor growth and ascitic fluid
formation in nude mice inoculated with NUGC4 cells. We next examined human clinicalsamples. We found that malignant ascites fluids from patients with peritoneal carcinomatosis
contained CXCL12 (average, 4.67 ng/mL) enough to exhibit biological effects on NUGC4 cells.
Moreover, immunohistochemical analysis revealed that 22 out of 33 primary gastric tumors withperitoneal metastasis were scored positive for CXCR4 expression (67%), and only 4 out of 16
with other distant metastasis were positive (25%). Notably, 22 out of 26 CXCR4-expressing
primary tumors developed peritoneal metastases (85%). CXCR4-positivity of primary gastriccarcinomas significantly correlated with the development of peritoneal carcinomatosis.
Collectively, our results strongly suggest that the CXCR4/CXC12 axis plays an important role inthe development of peritoneal carcinomatosis from gastric carcinoma. Thus, CXCR4 may be apotential therapeutic target for peritoneal carcinomatosis of gastric carcinoma.

― 96 ―

Serial Analysis of Gene Expression in Human Gastric Cancer
Kaname Yamashita1), Hidekazu Kitakata1), Kazuo Yasumoto1), Kazuhiko Omote1),
Yutaka Takahashi1) Toshinari Minamoto2)(Div. of Surg. Oncol1). and Diag. Mol. Oncol2)., Cancer Res. Inst., Kanazawa Univ.)
It is important to identify genes responsible for metastasis of gastric cancer, as thisdisease is one of the leading causes of cancer death in the world. Here we analyzed gene
expression in human gastric cancer by serial analysis of gene expression (SAGE). SAGE is amethod for comprehensive analysis of gene expression patterns. A short sequence tag (10-14bp)
contains sufficient information to uniquely identify a transcript. The tag is obtained from aunique position within each transcript. Sequence tags can be linked together to from long serial
molecules that can be cloned and sequenced and quantitation of the number of times a particular
tag is observed provides the expression level of the corresponding transcript. We obtained
primaly tissue, normal mucosa, and lymph node metastasis from a advanced gastric cancer
patient. Total RNA was isolated and SAGE was performed. Tag sequences were analyzed using
SAGE software. A total of 60621 transcripts were identified. We show tags which highly
expressed, in primaly tumor, normal mucosa, and lymph node metastasis. The lymph nodemetastasis library was compared with primaly tumor library, we could identify certain set ofgenes up-regulated in lymph node metastasis in tag ratio of two fold or more. Validation by
RT-PCR on RNA from another patients shows higher expression of SIAT6, APOC1, galectin-1
and COL1A1 in lymph node metastasis versus primaly tumor. These results together withcurrently accumulating data would provide basic knowledge toward understanding molecularmechanism underlying progression and metastasis of gastric cancer.
Summary of SAGE tag libraries
Library

Sequences

Total Tags

Unique Tags

Normal

731

14843

5243

Tumor

933

22681

8337

LN meta

1030

23097

7976

― 97 ―

Up-regulation of VEGF in Alpha-fetoprotein-producing gastric cancer
H.Kitakata, Y.Takahashi
Alpha-fetoprotein (AFP)-producing gastric carcinoma (APGC) is well known to have a poor
prognosis and high incidence of lymph node and liver metastases compared to non-producing
carcinoma.
known.

Although the reason why APGC frequently metastasizes to the liver is not well

We hypothesizes that angiogenesis is one of the important reasons for frequent liver

metastases from APGC.

In order to clarify this hypothesis, we examined the vessel density andvascular endothelial growth factor (VEGF) expression in APGC.

Furthermore, we studied theeffects of anti-AFP antibody for angiogenesis using APGC xenotransplanted mouse model.
Archival specimens of APGC (n=25) and non-APGC (n=68) were studied.

Expressions ofvessel density and VEGF were significantly higher in APGC than those in non-APGC (p<0.001).
Immunohistochemical analysis of patients with gastric cancer shows that expression of VEGF
and factor VIII are significantly higher in APGC than non-APGC.

There is correlation among

the AFP expression, the vessel density and the VEGF expression in APGC (p<0.001).
Next, we studied the effects of anti-AFP antibody on APGC xenotransplanted in nude mice.
There is significant inhibition of tumor growth in the treatment groups compared to the control
groups in two APGC lines (p<0.01). But, there is no difference in non-APGC cell line.
Moreover, vessel densities of the treatment groups were significantly lower than those of thecontrol groups in these two lines. These findings thus suggest that the biological behavior of
APGC is angiogenesis-dependent. Down regulation of angiogenesis by anti-AFP antibody
suggest that AFP itself may upregulate the angiogenesis, and the treatment by antibody could
have anti-angiogenic effects, inhibiting metastasis, especially liver metastasis in APGC.

― 98 ―

Publications
1.

Takahashi, Y., Ellis, LM., Mai, M. (2003) The angiogenic switch of human colon cancer
occurs simultaneous to initiation of invasion. Oncology Report. 10: 9-13.

2.

Takahashi, Y., Takeuchi, T., Sakamoto, J., Touge, T., Mai, M., Ohkura, H., Kodaira, S.,
Okajima, K., Nakazato, H. (Tumor Marker Committee, Japanese Foundation for
Multidisciplinary Treatment of Cancer) (2003) The Significance of CEA and/or CA19-9 in
Monitoring for Recurrence in Gastric Cancer Cases. A Prospective Clinical Study. Gastric
Cancer. 6: 142-145.

3.

Ougolkov, A., Yamashita, K., Bilim, V., Takahashi, Y., Mai, M., Minamoto, T. (2003)
Abnormal expression of E-cadherin, β-catenin and c-erbB-2 in gastric cancer with liver
metastasis. Int J Colorect Dis. (Clin Mol Gastroenterol Surg) 18: 160-166.

4.

Zhang, B., Ougolkov, A., Yamashita, K., Takahashi, Y., Mai, M., Minamoto, T. (2003)
β-catenin and ras oncogenes detect most human colorectal cancer. Clin. Cancer Res. 9:3073-3079.

5.

Zhang, B., Fujimoto, T., Kaneda, A., Mai, M. (2003) Irinotecan Enhances IL-12 Production
by OK-432-activated Murine Macrophages. Anticancer Res. 23(3B): 2499-2504.

6.

Sakamoto, J., Hamashima, H., Suzuki, H., Ito, K., Mai, M., Saji, S., Fukushima, M.,
Matsushima, Y., Nakazato, H., For the CIP Genetic Marker Committee. (2003)
Thymidylate synthase expression as a predictor of the prognosis of curatively resectedcolon carcinoma in patients registered in an adjuvant immunochemotherapy clinical trial.
Oncology Report. 10: 1081-1090.

7.

Sabet, EA., Okai, T., Minamoto, T., Mai, M., Sawabu, N. (2003) Visualizing the gastricwall with a 30-MHz ultrasonic miniprobe: ex vivo imaging of normal gastric sites and sitesof early gastric cancer. Abdom Imaging. 28: 252-256.

8.

Okai, T., Minamoto, T., Ohtsubo, K., Minato, H., Kurumaya, H., Oda, Y., Mai, M.,
Sawabu, N. (2003) Endosonographic evaluation of c-kit-positive gastrointestinal stromal
tumor. Abdom Imaging. 28: 301-307.

9.

Takahashi, Y., Kitakata, H., Hirano, K., Yamashita, K., Yasumoto, K., Omote, K.,
Minamoto, T., Mai, M. (2004) Pilot study of low dose, divided MTD of CPT-11 on 21
consecutive patients with metastatic colorectal or gastric cancer. Surgery Today.
34 :246-250.

10. Takahashi, Y., Ohta, T., Mai, M. (2004) Angiogenesis of AFP producing gastric carcinoma:
Correlation with frequent liver metastasis and its inhibition by anti-AFP antibody.
Oncology Reports. 11: 809-813.

― 99 ―

11. Takahashi, Y., Sakamoto, J., Takeuchi, T., Mai, M., Kubota, T., Kitajima, M., Tanigawara ,Y.,
Komatsu, Y., Toge,T., Saji, S. (2004) A randomized phase II clinical trial of tailored CPT-11+ S-1 vs S-1 in patients with advanced or recurrent gastric carcinoma as the first line
chemotherapy. Japanese Journal of Clinical Oncology. 34: 342-345.
12. Takahashi, Y., Yamashita, K., Endo, Y., Sasaki, T., Mai, M. (2004) Oral administration of
uracil-tegarfur (UFT) inhibits liver micrometastasis of human colon cancer in an orthotopic
model of nude mice and its early detection system. Surgery Today. 34: 855-859.
13. Takahashi, Y., Mai, M., Sawabu, N., Nishioka, K. (2005) A pilot study of individualized
maximum repeatable dose (iMRD), a new dose finding system, of weekly gemcitabine forpatients with metastatic pancreas cancer. Pancreas. 30: 206-210.
14. Takahashi, Y., Mai, M., Nakazato, H. (2005) Preoperative CEA and PPD values as
prognostic factors for immunochemotherapy using PSK and 5-FU. Anticancer Res. 25:
1377-1384.
15. Takahashi, Y., Yasumoto, K., Mai, M. (2005) Chemotherapy under cachectic conditions andthe possibility of cachexia-controlled chemotherapy. Oncology Reports. 14: 135-140.
16. Takahashi, Y., Mai, M. (2005) Antibody against Vascular Endothelial Growth Factor(VEGF) Inhibits Angiogenic Switch and Liver Metastasis in Orthotopic Xenograft Modelwith Site-dependent Expression of VEGF. Journal of Experiment and Clinical Research.
24: 237-243.
17. Yasumoto, K., Takahashi, Y., Mai, M. and Kawashima, A. (2005) Meningeal
carcinomatosis preceded by a rapid increase in serum CA19-9 levels in a patient withbreast cancer. Int J Clin Oncol. 10:276-280.
18. Yasumoto, K., Koizumi, K., Kawashima, A., Saitoh, Y., Arita, Y., Shinohara, K., Minami, T.,
Nakayama, T., Sakurai, H., Takahashi, Y., Yoshie, O., and Saiki, I. Role of
CXCL12/CXCR4 axis in Peritoneal Carcinomatosis of Gastric Cancer. Cancer Res. inpress.
19. Shakoori, A., Ougolkov, A., Yu, XW., Zhang, B., Modarresi, MH., Biladeau, DD., Mai, M.,
Takahashi, Y., Minamoto, T. (2005) Deregulated GSK3beta activity in colorectal cancer: Itsassociation with tumor cell survival and proliferation. Biochem Bioph Res Co. 334:
1365-1373.
20. Kaibara, N., Otani, Y., Takahashi, Y., et al. (2004) Meeting report of the 76th Japanese
gastric cancer congress. Gastric Cancer. 7: 185-195.

― 100 ―

DivisionofDiagnostic
Molecular Oncology

General Summary of the Division of Diagnostic Molecular Oncology
To strengthen its capabilities for responding to the rapid pace of advance in basic andclinical cancer research, and to better meet its evolving responsibilities, the Cancer ResearchInstitute of Kanazawa University was restructured in April of 1997. At the time the Division of
Diagnostic Molecular Oncology was established as a clinical oncology division. Following asemester of four years for building up research activity and organizing laboratory, the division
assumed an active role in July of 2001, when Dr. T. Minamoto was elected Professor and Chiefof the division. Through twice movements, the division has been enlarged having more researchmembers and two exclusive laboratories in the Institute.
The mission of the division, broadly, centers on basic and clinical research to develop thenovel strategies and methods for diagnosis and treatment of cancer on the basis of molecular andcellular assessment of tumor aggressiveness and biological characteristics of individual tumor
types relevant to metastatic potential, recurrence and outcome.
Table. Research directions and activities in the Division of Diagnostic Molecular Oncology
I. Activities toward molecular diagnosis of hereditary nonpolyposis colorectal cancer (HNPCC)
1. Clinical and genetic analysis of the patients and their families
2. Genetic testing for DNA mismatch repair enzymes’ genes
3. Surveillance and (surgical) intervention
II. Molecular diagnosis of pathology and malignant potential of cancers
1. Oncogenic signaling in colorectal cancers, targeting Wnt pathway and its regulators
2. Molecular pathology of clinical gastrointestinal cancers
3. Development of new modalities for diagnosis and treatment of cancer on the basis ofmolecular and cellular characteristics of cancer
III. Genome-wide analysis of gene expression in gastrointestinal cancers
1. Genome level: restriction landmark genomic scanning (RLGS)
2. Transcriptome level: serial analysis of gene expression (SAGE)
3. Epigenetic level: development of a new strategy covering all epigenetic alterations
spanning through the genome
Current efforts in this division are focused on 1) clinical and basic activities towardmolecular diagnosis of HNPCC; 2) the molecular characterization of gastrointestinal cancer by
an interdisciplinary approach bringing together clinical oncology, molecular and cellularpathology and biology; and 3) comprehensive (genome-wide) analysis of gene expression andepigenetic alterations in stomach and colorectal cancers by the highly sophisticated methods
including RLGS and SAGE, to cover the post-genome research fields. The most importantachievements in this semester have been (1) discovery of previously unrecognized tumor
supportive properties of GSK3β; and (2) identification of the genes responsible for lymph nodemetastasis in stomach cancer. We will intend to translate as much the results of these studies as
possible to those responsible for diagnosis and treatment of cancer patients in clinical setting.

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β-catenin and ras oncogenes detect most human colorectal cancer *B. Zhang, A. Ougolkov, K. Yamashita, Y. Takahashi, M. Mai, T. Minamoto
PURPOSE AND STUDY DESIGN: Recent studies have shown that β-catenin translocatedinto the cell nucleus functions like an oncogene. Accumulating evidence suggests that activationof the β-catenin oncogenic signaling cascade along with its twin, the K-ras cascade, may exert
syngeneic or synergistic effects on tumor development and progression. In the study reported
here, we analyzed oncogenic β-catenin activation on the basis of its nuclear accumulation (NA)and compared the results with those of mutational activation of K-ras in 74 patients withcolorectal cancer to determine whether the two oncogene-mediated signaling cascades interact.
RESULTS: We found two distinct patterns of β-catenin activation, i.e., diffuse NA in 20
cases (27%) and selective NA at the tumor invasion front (NAinv) in 19 cases (26%). The
presence of the NAinv pattern was significantly correlated with advanced Dukes' stage tumor (P= 0.0005) and the presence of distant metastases (P = 0.0064). K-ras proto-oncogene wasmutated in the tumors of 31 cases (42%). Activated β-catenin or K-ras was detected in most
(78%) colorectal cancers analyzed, although a weak inverse correlation was found between theactivities of the two oncogenes in the tumors. Importantly, most (7 of 8) patients with tumor
showing both K-ras activation and the NAinv pattern of β-catenin activation were in Dukes'
stage C at surgery, and half of them developed distant metastases to the liver and lungs.
CONCLUSION: The results suggest that although oncogenic activation of β-catenin andK-ras is independent in the process of clinical cancer development, combined analysis of thetwo major oncogenes can detect most colorectal cancers and identify a subset of patients withpoorer outcomes. Consequently, activation of either or both of these oncogenes may serve as a
genetic marker for molecular diagnosis.
*Reference:
Zhang B, Ougolkov A, Yamashita K, Takahashi Y, Mai M, Minamoto T. β-catenin and ras
oncogenes detect most human colorectal cancers. Clin Cancer Res 9: 3073-3079, 2003.

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Associations among β-TrCP, an E3 ubiquitin ligase receptor, β-catenin,
and NF-κB in colorectal cancer *A. Ougolkov, B. Zhang, K. Yamashita, V. Bilim, M. Mai, S.Y. Fuchs, T. Minamoto
BACKGROUND: The ubiquitin-proteasome pathway is important in regulating protein
signaling pathways that are involved in tumorigenesis. β-transducin repeat-containing proteins(β-TrCP) are components of the ubiquitin ligase complex targeting β-catenin and IκBα forproteasomal degradation and are thus a negative regulator of Wnt/β-catenin signaling and apositive regulator of NF-κB signaling. We analyzed expression of β-TrCP in colorectal cancers
and its association with types of β-catenin subcellular localization, an indirect measure ofactivation. METHODS: Levels of β-TrCP1 mRNA and protein were measured by quantitative
reverse transcription-polymerase chain reaction and immunoblotting, respectively, in samples oftumor and normal tissues from 45 patients with colorectal cancer. Types of β-catenin activation(diffuse or invasion edge) and NF-κB activation were examined by immunohistochemistry.
Apoptosis was determined by the terminal deoxynucleotidyl transferase-mediated deoxyuridine
triphosphate-biotin nick-end labeling (TUNEL) assay. All statistical tests were two-sided.
RESULTS: Compared with the β-TrCP1 levels in normal tissues, 25 (56%) of 45 tumors had
increased β-TrCP1 mRNA and protein levels. Of the 22 (49%) tumors with β-catenin activation,
12 had the diffuse type (i.e., nuclear accumulation throughout the tumor) and 10 had theinvasion edge type (i.e., nuclear accumulation predominantly in the tumor cells that formed theinvasion edge). Increased β-TrCP1 levels were statistically significantly associated with
β-catenin activation (P =.023) and decreased apoptosis (P =.035). β-TrCP accumulated in the
nuclei of tumor cells that contained increased levels of β-TrCP1 mRNA and the active form of
NF-κB. Higher levels of β-TrCP1 mRNA were detected in primary tumors of patients who had
metastases (0.960 arbitrary units, 95% confidence interval = 0.878 to 1.042) than in the tumorsof patients who did not (0.722 arbitrary units, 95% confidence interval = 0.600 to 0.844; P=.016). CONCLUSION: In colorectal cancer, increased expression of β-TrCP1 is associatedwith activation of both β-catenin and NF-κB, suggesting that the integration of these signaling
pathways by increased β-TrCP expression may contribute to an inhibition of apoptosis andtumor metastasis.
*References: Ougolkov A, Zhang B, Yamashita K, Bilim V, Mai M, Fuchs SY, Minamoto T.
Associations among β-TrCP, an E3 ubiquitin ligase receptor, β-catenin and NF-κB incolorectal cancer. J Natl Cancer Inst 96:1161-1170, 2004.

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Deregulated GSK3β activity in colorectal cancer: its association withtumor cell survival and proliferation *A. Shakoori, A. Ougolkov, Z.W. Yu, B. Zhang, M.H. Modaressi, D.D. Billadeau, M. Mai, Y.
Takahashi, T. Minamoto
GSK3β is a multifunctional serine/threonine kinase that regulates various cellular pathways,
depending on its substrates for phosphorylation. It is evident that regulation of Wnt/β-cateninsignaling is only one of its diverse functions. Since oncogenic transcription factors (e.g., c-Jun,c-Myc) and proto-oncoproteins (i.e., β-catenin, Gli proteins) are putative GSK3β substrates forphosphorylation-dependent inactivation, it is hypothesized that GSK3β interferes with cellularneoplastic transformation and tumor development, as exemplified by its activity in
Wnt/β-catenin signaling. However, only a few studies have addressed its role(s) in humancancer, and these studies have reported differing effects of GSK3β on cancer cells. Using
GSK3β deficient mouse embryonic fibroblasts, it was shown that GSK3β plays a crucial role incell survival mediated by the nuclear factor-kappaB (NF-κB) pathway (Nature 2000; 406:86-90).
Interestingly, we have recently shown that the Wnt/β-catenin and NF-κB pathways wereco-activated in colorectal cancer by dysregulation in the ubiquitin system (J Natl Cancer Inst
2004; 96:1161-70). Thus, these observations bring forward apparently opposing notions
regarding the functions of GSK3β in neoplastic cells on the one hand, removing a neoplastic
trigger by phosphorylation-dependent degradation of β-catenin oncoprotein in the ubiquitin
system, and on the other, contributing to a cell proliferation and survival pathways by regulating
NF-κB. The present study was therefore undertaken to clarify the role of GSK3β in cancer by
analyzing expression and activity of this kinase in colon cancer cells and clinical colorectal
cancers and by investigating its effects on cancer cells. In colon cancer cell lines and colorectal
cancer patients, levels of GSK3β expression and its active form were higher in tumor cells thanin their normal counterparts; these findings were independent of nuclear accumulation of
β-catenin oncoprotein in the tumor cells. Inhibition of GSK3β activity by its Ser9phosphorylation was defective in colorectal cancers but preserved in non-neoplastic cells andtissues. Strikingly, inhibition of GSK3β activity by chemical inhibitors and its expression by
RNA interference targeting GSK3β induced apoptosis and attenuated proliferation of coloncancer cells in vitro. Our findings demonstrate an unrecognized role of GSK3β in tumor cell
survival and proliferation and warrant proposing this kinase as a novel and potential therapeutictarget in colorectal cancer.
*Reference: Shakoori A, Ougolkov A, Yu ZW, Zhang B, Modarressi MH, Billadeau DD, Mai
M, Takahashi Y, Minamoto T. Deregulated GSK3β activity in colorectal cancer: its association
with tumor cell survival and proliferation. Biochem Biophys Res Commun 334: 1365-1373,
2005.

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Oncogenic β-catenin signaling networks in colorectal cancer *S.Y. Fuchs, A.V. Ougolkov, V.S. Spiegelman, T. Minamoto
β-catenin has two distinct functions, namely, maintaining cell-to-cell adhesion andmediating the Wnt/β-catenin signal transduction pathway, which plays pivotal roles inembryogenesis and in malignant transformation of cells. The oncogenic properties of
Wnt/β-catenin signaling stem from alteration in phosphorylation-dependent protein degradationand subcellular localization of β-catenin from cell membrane to the nucleus, where it binds toT-cell factor (Tcf) to form a bipartite transcription factor. The β-catenin/Tcf complex facilitatestranscription of target genes that encode effectors for activation of cell proliferation and invasion
and inhibition of apoptosis, leading to colorectal cancer development. In addition, in the tumor
invasion front, stabilized and activated β-catenin interacts with other molecular pathways tofacilitate tumor progression. This review highlighted the β-catenin-dependent oncogenic
signaling network involved in the multi-step process of colorectal tumorigenesis. Wnt signaling
evidently regulates stem cells, leading them to differentiate or self-renew. We are addressingroles of oncogenic β-catenin signaling in the microenvironment of the tumor-host interface that
determine the individual tumor's malignant potential and in regulation of putative cancer stem orprogenitor cells that represent plausible targets for cancer eradication.
*Reference:
Fuchs SY, Ougolkov AV, Spiegelman VS, Minamoto T. Oncogenic β-catenin signaling
networks in colorectal cancer. Cell Cycle 4:1522-1539, 2005 (review).

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Detection and characterization of oncogene
preneoplastic and early neoplastic lesions *mutationsinT. Minamoto
Although it has been more than 20 yr since its discovery, the ras family of genes has not yet
lost its impact on basic and clinical oncology. These genes remain central to the field of
molecular oncology as tools for investigating carcinogenesis and oncogenic signaling, as
powerful biomarkers for the identification of those who have or are at high risk of developing
cancer, and as oncogene targets for the design and development of new chemotherapeutic drugs.
Mutational activation of the K-ras proto-oncogene is an early event in the development andprogression of the colorectal, pancreatic, and lung cancers that are the major causes of cancer
death in the world. The presence of point mutational "hot spots" at sites necessary for theactivation of this proto-oncogene has led to the development of a number of highly sensitive
PCR-based methods that are feasible for the early detection of K-ras oncogene mutations in theclinical setting. In light of these facts, mutation at the K-ras oncogene has the potential to serve
as a useful biomarker in the early diagnosis and risk assessment of cancers with oncogenic Ras
signaling. This chapter described a highly sensitive method for detecting mutant K-ras, enriched
PCR, and its application to early detection of this oncogene in preneoplastic and early neoplastic
lesions of the colon and rectum.
*Reference:
Minamoto T. Detection and characterization of oncogene mutations in preneoplastic and earlyneoplastic lesions. Methods Mol Biol 291:263-78, 2005.

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Publications
1. Ougolkov, A., Yamashita, K., Bilim, V., Takahashi, Y., Mai, M., Minamoto, T. (2003)
Abnormal expression of E-cadherin, β-catenin and c-erbB-2 in gastric cancer with liver
metastasis. Int J Colorect Dis (Clin Mol Gastroenterol Surg) 18:160-166.
2. Sabet, E.A., Okai, T., Minamoto, T., Ma,i M., Sawabu, N. (2003) Visualizing the gastricwall with a 30-MHz ultrasonic miniprobe: ex vivo imaging of normal gastric sites and sitesof early gastric cancer. Abdom Imaging 28:252-256.
3. Morita, H., Hasegawa, T., Minamoto, T., Oda, Y., Inui, K., Tayama, H., Nakao, N., Ideura,T., Yoshimura, A. (2003) Collagenofibrotic glomerulopathy with a widespread expression oftype V collagen. Virchow's Archiv 442:163-168.
4. Okai, T., Minamoto, T., Ohtsubo, K., Minato, H., Kurumaya, H., Oda, Y., Mai, M., Sawabu,
N. (2003) Endosonographic evaluation of c-kit-positive gastrointestinal stromal tumor.
Abdom Imaging 28:301-307.
5. Kawashima, A., Tsugawa, S., Boku, A., Kobayashi, M., Minamoto, T., Nakanishi, I., Oda, Y.
(2003) Expression of αv integrin family in gastric carcinomas: increased αvβ6 is associatedwith lymph node metastasis. Pathol Res Prac 199:57-64.
6. Zhang, B., Ougolkov, A., Yamashita, K., Takahashi, Y., Mai, M., Minamoto, T. (2003)

β-catenin and ras oncogenes detect most human colorectal cancers. Clin Cancer Res 9:3073-3079.
7. Takahashi, Y., Kitakata, H., Yamashita, K., Yasumoto, K., Omote, K., Minamoto, T., Mai,
M. (2004) Pilot study of low dose, divided maximum tolerated dose of CPT-11 in 21
consecutive patients with metastatic colorectal or gastric cancer. Surg Today 34:246-250.
8. Watanabe, H., Yamaguchi, Y., Ohtsubo, K., Mouri, H., Motoo, Y., Yamashita, K., Minamoto,T., Gabata, T., Sawabu, N. (2004) Epidermoid cyst of the intrapancreatic accessory spleen
producing CA19-9. Digest Endosc 16:244-248.
9. Ougolkov, A., Zhang, B., Yamashita, K., Bilim, V., Mai, M., Fuchs, S.Y., Minamoto, T.
(2004) Associations among β-TrCP, an E3 ubiquitin ligase receptor, β-catenin and NF-κB incolorectal cancer. J Natl Cancer Inst 96:1161-1170.
10. Minamoto, T. (2005) Detection and characterization of oncogene mutations in preneoplastic
and early neoplastic lesions. In; Keohavong P and Grant S, eds., Molecular Toxicology
Protocols/Methods of Molecular Biology Vol. 291, Humana Press, Totawa, NJ, pp. 263-278.
11. Fuchs, S.Y., Ougolkov, A.V., Spiegelman, V.S., Minamoto, T. (2005) Oncogenic β-cateninsignaling networks in colorectal cancer. Cell Cycle 4:1522-1539 (review).

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12. Aoki, M., Yamamura, Y., Noshiro, H., Sakai, K., Yokota, J., Kohno, T., Tokino, T., Ishida, S.,
Ohyama, S., Ninomiya, I., Uesaka, K., Kitajima, M., Shimada, S., Matsuno, S., Yano, M.,
Hiratsuka, M., Sugimura, H., Itoh, F., Minamoto, T., Maehara, Y., Takenoshita, S., Aikou, T.,
Katai, H., Yoshimura, K., Takahashi, T., Akagi, K., Sairenji, M., Yamamoto, K., Sasazuki, T.
(2005) A full genome scan for gastric cancer. J Med Genet 42:83-87.
13. Misaki, K., Marukawa, K., Hayashi, Y., Fukusato, T., Minamoto, T., Hasegawa, M.,
Yamashita, J., Fujisawa, H. (2005) Correlation of γ-catenin expression with good prognosis
in medulloblastomas. J Neurosurg (Pediatrics 2) 102:197-206.
14. Kawahara, E., Saito, A., Kobayashi, J., Maenaka, S., Minamoto, T., Akasofu-Imai, M., Oda,
Y. (2005) Adhesiveness of β5 integrin variant lacking FNK767-769 is similar to that of the
prototype containing FNKFNK767-769. Cell Biol Int 29:521-528.
15. Shakoori, A., Ougolkov, A., Yu, Z.W., Zhang, B., Modarressi, M.H., Billadeau, D.D., Mai,
M., Takahashi, Y., Minamoto, T. (2005) Deregulated GSK3β activity in colorectal cancer:
its association with tumor cell survival and proliferation. Biochem Biophys Res Commun
334: 1365-1373.
16. Aoki, M., Yamamoto, K., Ohyama, S., Yamamura, Y., Takenoshita, S., Sugano, K.,
Minamoto, T., Kitajima, M., Sugimura, H., Shimada, S., Noshiro, H., Hiratsuka, M.,
Sairenji, M., Ninomiya, I., Yano, M., Uesaka, K., Matsuno, S., Maehara, Y., Aikou, T.,
Sasazuki, T. (2005) A genetic variant in the gene encoding the stress70 protein chaperone
family member STCH is associated with gastric cancer in the Japanese population. Biochem
Biophys Res Commun, 335:566-574.
17. Kamakura, Y., Hasegawa, M., Kida, S., Tachibana, O., Okamoto, Y., Minamoto, T.,
Yamashita, J., Fujisawa, H. (2006) c-kit gene mutation is common and widely distributed in
intracranial germinomas. J Neurosurg, in press.
18. Maenaka, S., Hikichi, T., Imai-Akasofu, M., Minamoto, T., Kawahara, E. (2006) Loss ofimprinting in IGF2 in colorectal carcinoma assessed by microdissection. Oncol Rep, inpress.
19. Noubissi, F., Elcheva, I., Bhatia, N., Shakoori, A., Ougolkov, A., Liu, J., Minamoto, T., Ross,
J., Fuchs, S.Y., Spiegelman, V.S. CRD-BP mediates stabilization of βTrCP1 and c-myc
mRNA in response to β-catenin signaling, in submission with revision.
20. Yamashita, K., Nakazato, H., Ito, K., Ougolkov, A., Takahashi, Y., Mai, M., Minamoto, T.
Effect of adjuvant immunochemotherapy with PSK for colon cancer patients showing
oncogenic β-catenin activation in primary tumor. J Clin Oncol, in submission.

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21. Yamashita, K., Zhang, B., Tamano, Y., Yu, Z.W., Takahashi, Y., Minamoto, T. Serial
analysis of gene expression identifies genes associated with nodal metastasis in stomach
cancer. Cancer Res, in submission.

Other achievements
1. Minamoto T. A Research Fellow as Scientist Exchange under The U.S.-Japan Cooperative
Cancer Research Program, supported by National Cancer Institute (NCI) and Japan Society
for the Promotion of Science (JSPS).
Sponsor and Host Institution: Dr. Ze’ev Ronai, Ph.D., Derald H. Ruttenberg Cancer Center,
Mount Sinai School of Medicine, New York, NY
Subject of research: Alterations in transcription factors in human cancers: implication inmolecular diagnosis and molecular targeting therapy
Dates of visit: from November 5th, 2003 to December 18th, 2003
2. Patent application
Minamoto T.
Application number: 2005-000133 Country : Japan
Subject: Development of anti-cancer agents (drugs) inhibiting GSK3β (glycogen synthasekinase 3β)Applicant: Kanazawa University
Date of application: January 4th, 2005

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Center for theDevelopment of
Molecular Target Drugs

General Summary Center for the Development of Molecular Target Drugs
The purpose of this center is to perform basic research by the full-time researchers of the center(described below) and to organize collaborative research projects based on creative research
products of our institute or other research groups, aiming the development of clinically truly
effective molecular targeted drugs. We have invited and selected 15 collaborative projects in thelast three years, and have shared 10,000,000 JPY for the selected projects. To spread our
research outcomes promptly, we have held an annual symposium, and have published an annual
report every year. The former director of this center, Prof. Takuma Sasaki retired in March 2005,
and Prof. Takashi Suda succeeded to the post.
Research Projects of Suda’s GroupA) Molecular mechanism of Fas ligand-induced inflammation
Fas ligand (FasL) is a prototype of death factors that induces apoptosis by binding to its receptor,
Fas. We previously demonstrated that anti-Fas ligand antibody administration ameliorates
various inflammatory diseases, and prevent chronic-hepatitis-induced hepatic cancer
development using animal models. We also demonstrated that Fas ligand-induced caspase
activation in neutrophils causes not only apoptosis but also proteolytic maturation and release of
IL-1β, which in turn induces massive neutrophil infiltration in vivo. In the last three years, we
further demonstrated that Fas ligand induces production of various cytokines including IL-1β,
IL-6, IL-17, IL-18, IL-23, KC, and Mip2 in mice. Fas ligand induces IL-23 production in
dendritic cells at the mRNA level in a cell-autonomous manner. The IL-23 then induces IL-17
production in T cells synergistically with IL-1β. We also found that Fas ligand induces IL-8
production in human embryonic kidney (HEK) 293 cells in a manner dependent on the NF-κB
activation. The Fas ligand-induced NF-κB activation is a cell-autonomous response and requires
FADD and caspase-8.
B) Molecular mechanisms of activation and regulation of ASC-mediated inflammation.
ASC is an adaptor molecule that mediates apoptotic and inflammatory signals from several
Apaf-1-like molecules, including CARD12/Ipaf, PYPAF1/cryopyrin, PYPAF5, PYPAF7, and
NALP1. ASC is also implicated in tumor suppression, because the ASC gene expression issuppressed in various cancer cells. We have established an experimental system in whichmuramyl dipeptide, the bacterial component recognized by another Apaf-1-like molecule, Nod2,
induced an interaction between a CARD12-Nod2 chimeric protein and ASC, and elicited
cell-autonomous NF-κB activation leading to IL-8 production. Using this system, we
demonstrated that caspase-8 plays an essential role in ASC-mediated NF-κB activation. On theother hand, we have found that some members of Apaf-1-like proteins such as PYNOD,PYPAF2 and PYPAF3 inhibit ASC-mediated NF-κB activation and or caspase-1-mediated― 111 ―

proteolytic maturation of IL-1β.
Research Projects of Dr. Endo
Antitumor Mechanisms of 3'-Ethynyl Nucleosides: Cellular Metabolic Enzyme and Drug
Resistance
The antitumor 3'-ethynyl nucleosides, ECyd and EUrd, are potent inhibitors of RNApolymerases and show excellent antitumor activity against various human solid tumors in
xenograft models. ECyd and EUrd require the activity of uridine/cytidine kinase (UCK) to
produce the corresponding active metabolite. We established cell lines that are highly resistant to
the 3'-ethynyl nucleosides using human tumor cell lines. All the resistant cell lines showed ahigh cross-resistance to ECyd and EUrd and had mutations in the UCK2 gene. UCK2 protein
was markedly decreased in the resistant cells. In contrast, no mutation in the mRNA or
differences in protein expression levels of UCK1 were shown in the resistant cells. These results
suggest that UCK2 is exclusively responsible for the phosphorylation and activation of theantitumor 3'-ethynyl nucleosides. UCK2 may be a useful marker to predict chemosensitivity toantitumor nucleoside analogs including EUrd and ECyd.
Research Projects of Dr. KunoA) Effects of ADAMTS-1 on tumor growth and metastasis
In 1997, we identified ADAMTS-1, the first member of the ADAMTS family. We have reported
that ADAMTS-1 is an extracellular matrix-anchored metalloproteinase and is able to cleave a
cartilage proteoglycan, aggrecan. As ADAMTS-1 has anti-angiogenic activity, we examined theeffects of ADAMTS-1 on in vivo tumor growth and tumor metastasis. Unexpectedly, full-length
ADAMTS-1 was found to promote tumorigenicity. We also found that ADAMTS-1 significantly
suppresses the experimental metastasis of CHO cells.
B) Roles of ADAMTS-1 in ovarian function
We have reported that ADAMTS-1 null mice display renal anomalies that resemble humanureteropelvic junction (UPJ) obstruction. As ADAMTS-1 null female mice are infertile,
ADAMTS-1 is also important for the function of female reproductive organs. We found thatADAMTS-1 null females showed a reduced ovulation rate of mature follicles in the
superovulatory condition, impairment of follicular growth at the late preantral stage, and theappearance of a number of unusual atretic follicles without the granulosa cell layers. These
results demonstrate that ADAMTS-1 is involved in both follicular development and ovulation.

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Molecular mechanisms of Fas ligand-induced inflammation.
M. Umemura, T. Kawabe, H. Kidoya, K. Shudo, M. Fukui, M. Asano, Y. Iwakura, G.
Matsuzaki,A. Yahagi, R. Imamura, and T. Suda
Fas ligand (FasL) has been well characterized as a death factor. However, recent studies
revealed that ectopic expression of FasL induces inflammation associated with massive
neutrophil infiltration. We previously demonstrated that the neutrophil infiltration-inducing
activity of FasL is partly dependent on but partly independent of IL-1β. Therefore, weinvestigated the cytokine profile of peritoneal lavage fluid obtained from mice that received
intraperitoneal injections of FFL, a FasL-expressing tumor cell line. We found that FFL injection
caused a marked increase of not only IL-1β but also IL-6, IL-17, IL-18, KC/chemokine CXC
ligand 1, and macrophage inflammatory protein (MIP)-2, but not of IL-1α, IFN-γ, TGF-β, or
TNF-α. Among cells transfected to express individually IL-1β, IL-6, IL-17, or IL-18, only thoseexpressing IL-1β and IL-17 induced neutrophil infiltration. Co-administration of the anti-IL-17
antibody with FFL diminished the peritoneal KC levels and neutrophil infiltration in
IL-1-deficient mice. In addition, the expression of IL-17 by the tumor cells inhibited tumor
growth in wild-type and nude mice. These results suggest that IL-17 is involved in FasL-induced
inflammation and tumor rejection in the absence of IL-1β.
Then, we investigated the mechanism of the FasL-induced IL-17 production. We found thatthe culture supernatant of mouse resident peritoneal exudate cells (PEC) cocultured with FFL
cells induced IL-17 production in freshly isolated resident PEC. Anti-IL-1β Ab strongly
inhibited the IL-17-inducing activity. However, recombinant IL-1β by itself induced only weak
IL-17 production. Intriguingly, anti-IL-12 Ab but not an IL-15 neutralizing agent, IL15R-Fc,
strongly inhibited the FasL-induced IL-17-inducing activity. IL-23, which shares the p40 subunitwith IL-12, but not IL-12 itself, induced IL-17 production synergistically with IL-1β in resident
PEC. FasL induced the production of IL-23 in PEC in vivo and in vitro, and IL-17 production
following the i.p. injection of FFL cells was severely impaired in p40-/- mice, indicating that
IL-23 plays an important role in the FasL-induced IL-17 production. FFL also induced the
production of IL-23 in bone marrow- or PEC-derived dendritic cells. Finally, FasL induced only
weak p40 production in a mixture of p40-/- and Fas-/- dendritic cells, indicating that FasL
induces IL-23 production in dendritic cells mainly in a cell-autonomous manner.
1. Umemura, M., et al., Int. Immunol., 16:1099-108, 2004
2. Kidoya, H., et al., J. Immunol., 175:8024-31, 2005

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Molecular Mechanism of Fas ligand-induced IL-8 Production
R. Imamura, N. Matsumoto, M. Hasegawa, K. Konaka, and T. Suda
It has been believed that apoptosis does not induce inflammation. However, there areremarkable similarities between the molecular mechanisms of apoptosis and inflammation. Fas(CD95) is not an exception and recent studies revealed that Fas ligand (FasL)-Fas system
possesses inflammatory activity. We recently found that FasL induces production of theinflammatory chemokine IL-8 in human cell lines and FasL-induced NF-κB and AP-1 activationis required for this IL-8 production. Our further analyses revealed that the death domain of Fas,
FADD, and caspase-8, which are essential for the apoptosis signaling, are required for both
NF-κB and AP-1 activation by FasL. However, rsponses of NF-κB and AP-1 activation are
independent of each other. In the NF-κB signaling pathway, we also showed that TRADD and
RIP, which are essential for the TNF-α-induced NF-κB activation, were not involved in the
FasL-induced NF-κB activation and CLARP/FLIP inhibited the FasL- but not the
TNF-α-induced NF-κB activation. More interestingly, our results revealed that enzymatic
activity of caspase-8 is required for both NF-κB and AP-1 activation induced by FasL. Further
characterization of these pathways will help us to understand and, hopefully, to control the
FasL-induced inflammation. (Imamura, R. et al., J. Biol. Chem., 279:46415-46423, 2004)
FigureIn the case of FasL-Fas system,
contrary to TNF-α-TNFR system,
signaling pathway of apoptosis andtranscription

factoractivation

separates downstream of caspase-8
and enzymatic activity of caspase-8 isrequired for both pathways. Current
our goal is identification of targets(substrates) of caspase-8, which areimportant for FasL-induced NF-κB
and/or AP-1 activation.

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PYNOD, a Novel Apaf-1/CED4-like Protein is an Inhibitor of ASC andCaspase-1
Y. Wang, M. Hasegawa, R. Imamura, T. Kinoshita, C. Kondo, K. Konaka, and T. Suda
Recently, a large subfamily of nucleotide-binding and oligomerization domain-containing
proteins that have an N-terminal pyrin-like domain and C-terminal leucine-rich repeats has been
described. In this study, we identified PYNOD, a novel member of this family that lacks theleucine-rich repeats. We found that human PYNOD mRNA is expressed in various tissues and athigh levels in heart, skeletal muscle, and brain. It is also expressed in various cell lines,
including haematopoietic cell lines. PYNOD oligomerizes and binds to ASC, an adaptor proteinthat plays a role in apoptotic and inflammatory signal transduction, and to caspase-1 and IL-1β.
PYNOD inhibits ASC-mediated NF-κB activation and apoptosis, and caspase-1-mediated IL-1βmaturation, and it does so in the presence and absence of constitutively active mutants of
CARD12 and PYPAF1, which are enhancers of these processes. Thus, PYNOD is a novel
regulator of apoptosis and inflammation. (Wang, Y. et al., Int. Immunol. 16:777-86, 2004)

FigurePYNOD is a member of Apaf-1-like proteins and

CARD12

inhibits ASC-mediated NF-κB activation andapoptosis, and caspase-1-mediated IL-1β activation.

ASC

Caspase-1

PYNOD

NF-κB
apoptosis― 115 ―IL-1β

PYPAF3, a PYRIN-containing Apaf-1-like protein, is a feedback
regulator of caspase-1-dependent interleukin-1β secretion
T. Kinoshita, Y. Wang, M. Hasegawa, R. Imamura, T. Suda
PYPAF3 is a member of the PYRIN-containing apoptotic protease-activating factor-1-likeproteins (PYPAFs) that are thought to function in inflammatory signaling pathways. Among themembers of this family, PYPAF1, PYPAF5, PYPAF7, and NALP1 have been shown to induce
caspase-1-dependent interleukin-1β secretion and NF-κB activation in the presence of theadaptor molecule ASC. On the other hand, we recently identified PYNOD, another member ofthis family, as a suppressor of these responses. In this study, we showed that PYPAF3 is thesecond member that inhibits caspase-1-dependent interleukin-1β secretion (Fig. 1) and that
PYPAF2 does not inhibit this response, but rather inhibits the ASC-mediated NF-κB activation(Fig. 2). Both PYPAF2 and PYPAF3 mRNAs are broadly expressed in a variety of tissues;
however, neither is expressed in skeletal muscle, and only PYPAF2 mRNA is expressed in heartand brain. They are also expressed in many cell lines of both haematopoietic andnon-haematopoietic lineages. Stimulation of monocytic THP-1 cells with lipopolysaccharide orinterleukin-1β induced PYPAF3 mRNA expression (Fig. 3). Furthermore, the stable expressionof PYPAF3 in THP-1 cells abrogated the cells’ ability to produce interleukin-1β in response to
lipopolysaccharide. These results suggest that PYPAF3 is a feedback regulator of interleukin-1β
secretion. Thus, PYPAF2 and PYPAF3, together with PYNOD, may constitute ananti-inflammatory subgroup of PYPAFs. (Kinoshita, T. et al., J. Biol. Chem. 280:21720-5, 2005)― 116 ―

ASC-mediated NF-κB Activation Leading to IL-8 Production Requires
Caspase-8 and Is Inhibited by CLARP
M. Hasegawa, R. Imamura, T. Kinoshita, N. Matsumoto, J. Masumoto, N. Inohara,
and T. Suda
ASC is an adaptor molecule that mediates apoptotic and inflammatory signals from several
Apaf-1-like molecules, including CARD12/Ipaf. ASC is also implicated in tumor suppression,
because the ASC gene expression is suppressed in various cancer cells. To characterize thesignaling pathway mediated by ASC, we established cell lines in which muramyl dipeptide, thebacterial component recognized by another Apaf-1-like molecule, Nod2, induced an interaction
between a CARD12-Nod2 chimeric protein and ASC, and elicited cell-autonomous NF-κB
activation. This response required caspase-8, and was suppressed by CLARP/FLIP, an inhibitorof caspase-8. The catalytic activity of caspase-8 was required for the ASC-mediated NF-κB
activation when caspase-8 was expressed at an endogenous level, although it was not essential
when caspase-8 was overexpressed. In contrast, FADD, the adaptor protein linking Fas andcaspase-8, was not required for this response. Consistently, ASC recruited Caspase-8 and
CLARP but not FADD and Nod2 to its speck-like aggregates in cells. Finally, muramyl
dipeptide induced IL-8 production in MAIL8 cells. These results are the first to indicate that
caspase-8 plays an important role in the ASC-mediated NF-κB activation, and that the
ASC-mediated NF-κB activation actually induces physiologically relevant gene expression.
(Hasegawa M, et al., J. Biol. Chem. 280: 15122-30, 2005.)
FigureDREDD,

Mammalthedrosophila

Drosophila

FasL

Fas

Gram(-)PGRP-LC
caspasehomolog of caspase-8 has been?

shown to play an important

IMD
dFADD

role in NF-κB activation. In

FADD

mammals, caspase-8 also plays

Caspase-8

CARD12ASC

an important role in Fas

Caspase-8

ligand-induced

and

NF-κB

ASC-mediated

NF-κB

IL-8

dTAK1

DREDD

IKKγ
IKKβ

(=Caspase-8)

apoptosis
NF-κB
IL-8

activation.

― 117 ―

apoptosis

Relish(=NF-κB)

DiptericinA CRUCIAL ROLE OF URIDINE/CYTIDINE KINASE 2 IN ANTITUMORACTIVITY OF 3'-ETHYNYL NUCLEOSIDES
Y. Endo, T. Obata, D. Murata, M. Ito, M. Kadohira and T. Sasaki
The antitumor 3'-ethynyl nucleosides, 1-(3-C-ethynyl-β-D-ribopentofuranosyl)cytosine(ECyd) and 1-(3-C-ethynyl-β-D-ribopentofuranosyl)uracil (EUrd), are potent inhibitors of RNApolymerases and show excellent antitumor activity against various human solid tumors in
xenograft models. ECyd is being investigated in phase I clinical trials as a novel anticancer drug
possessing a unique antitumor action. ECyd and EUrd require the activity of uridine/cytidine
kinase (UCK) to produce the corresponding active metabolite (ECTP and EUTP). The UCK
family consists of two members, UCK1 and UCK2, and both UCKs are expressed in manytumor cells. It was unclear, however, whether UCK1 or UCK2 is responsible for thephosphorylation of the 3'-ethynyl nucleosides. We therefore established cell lines that are highly
resistant to the 3'-ethynyl nucleosides from human fibrosarcoma HT-1080 and gastric carcinoma
NUGC-3. All the resistant cell lines showed a high cross-resistance to ECyd and EUrd. As aresult of cDNA sequence analysis, we found that UCK2 mRNA expressed in EUrd-resistant
HT-1080 cells has a 98-base pair deletion of exon 5, whereas EUrd-resistant NUGC-3 cells were
harboring the point mutation at nucleotide position 484 (C to T) within exon 4 of UCK2 mRNA.
This mutation was confirmed by genome sequence analysis of the UCK2 gene. In
ECyd-resistant NUGC-3 cells, the substitution of G to A at 391 nt of cording region of UCK2
cDNA (Val to Met at codon 131) was found. Moreover, the expression of UCK2 protein and the
enzymatic activity were decreased in these resistant cells. In contrast, no mutation in the mRNAor differences in protein expression levels of UCK1 were shown in all the resistant cells. These
results suggest that UCK2 is responsible for the phosphorylation and activation of the antitumor3'-ethynyl nucleosides. Although UCK2 is expressed in many types of tissues and tumor cells,
the expression level of UCK2 appears to be very diverse in cells. UCK2 may be a useful marker
to predict chemosensitivity to antitumor uridine and cytidine analogs including EUrd and ECyd.
In addition, UCK2 may be a novel molecular target for biochemical modulation to potentiate
antitumor effects of such nucleoside analogs.
References
1. Murata D, Endo Y, Obata T, Sakamoto K, Syouji Y, Kadohira M, Matsuda A, Sasaki T. (2004)
A crucial role of uridine/cytidine kinase 2 in antitumor activity of 3'-ethynyl nucleosides.
Drug Metab Dispos. 32(10):1178-82.
2. Tabata S, Tanaka M, Endo Y, Obata T, Matsuda A, Sasaki T. (1997) Anti-tumor mechanismsof 3'-ethynyluridine and 3'-ethynylcytidine as RNA synthesis inhibitors: development andcharacterization of 3'-ethynyluridine-resistant cells. Cancer Lett. 116(2):225-31.
― 118 ―

Enhancement of the tumorigenicity of both Lewis lung carcinoma andChinese hamster ovary cells but suppression of experimental tumor
metastatic potential by ADAMTS-1
K. KunoADAMTS-1 is an extracellular matrix (ECM)-anchored metalloproteinase withproteoglycan-degrading activity. As ADAMTS-1 also has an angiogenesis-inhibiting activity,
we examined the effects of ADAMTS-1 overexpression on in vivo tumor growth and metastasis.
Unexpectedly, we found that overexpression of full-length ADAMTS-1 in both Chinese
hamster ovary (CHO) cells (CHO/ADAMTS-1) and Lewis lung carcinoma (LLC) cells(LLC/ADAMTS1) resulted in enhancement of in vivo tumor growth, suggesting thatADAMTS-1 can promote tumorigenicity. In contrast, overexpression of only the C-terminal
half region of ADAMTS-1 (DPM-1), which lacks the metalloproteinase domain, suppressed
CHO tumor growth in mice, suggesting that that the metalloproteinase activity of ADAMTS-1 isrequired for increased tumorigenicity. We presume that the increased tumorigenicity by
ADAMTS-1 results from its potential action to promote degradation of the ECM barriers
surrounding tumors or to release growth factors from the ECM.
When the CHO/ADAMTS1 was inoculated intravenously into mice, the number of visible
surface lung metastatic nodules markedly decreased in comparison with that of mice implantedwith the control vector clone (CHO/vector). Deletional analyses revealed that the C-terminal
half region of ADAMTS-1 is responsible for its experimental metastasis-inhibitory activity.
Tumor vascular density in the microscopic lung metastases formed by CHO/ADAMTS-1 or
CHO/DPM-1 cells were reduced in comparison with that of the control CHO/vector cells.
Therefore, one possibility is that ADAMTS-1 may suppress experimental metastasis by
inhibiting tumor angiogenesis in the lung metastatic lesion. Our present study suggests that the
C-terminal half region of ADAMTS-1 has therapeutic potential as an inhibitor of tumor growthand metastasis when ADAMTS-1 is applied to anti-cancer therapy.

Number of lung metastases

600
500

S PRO
400

p＜0.001MP

DISlike TSP

Zn

SP

tumor
growth

TSP-sub

ADAMTS-1

300

S

DPM-1

200
100

p＜0.001p＜0.001

DPM-6

0

CHO/
VectorCHO/
CHO/
ADAMTS-1 DPM-1

CHO/
DPM-6

― 119 ―

tumor
metastasis

Involvement of ADAMTS-1 in folliculogenesis, ovulation,organization of the medullary vascular network in the ovary

andM. Shozu, K. Kuno
ADAMTS-1 null mice displayed renal anomalies involving enlarged calices and atrophic
renal papillae, which resemble ureteropelvic junction (UPJ) obstruction in humans. We also
found that ADAMTS-1 null female mice were infertile, indicating that ADAMTS-1 is important
for the function of female reproductive organs. To clarify the role of ADAMTS-1 in ovarianfunction, we examined abnormalities in the ovulatory processes, folliculogenesis, and thevascular system of ADAMTS-1 null ovaries.
First, when immature female mice were treated with pregnant mare serum gonadotropin(PMSG)/human chorionic gonadotropin (hCG), the number of ovulated oocytes was markedly
decreased in ADAMTS-1 null mice when compared with ADAMTS-1 (+/-) controls. The
proportion of anovulated follicles to total mature follicles was significantly higher in
ADAMTS-1 null females when compared with controls. The numbers of growing follicles at
each stage were counted. The number of follicles at type 5b (late preantral) and later stages
was markedly reduced in ADAMTS-1 null mice, irrespective of gonadotropin treatment. These
data demonstrate that impairment of ovarian function to ovulate oocytes in ADAMTS-1 nullmice occurs at two different levels: in the development of growing follicles and in ovulatory
processes. Furthermore, ADAMTS-1 null ovaries was found to include a number of unusual
atretic follicles that showed no sign of oocyte degeneration but that had lost the surrounding
granulosa cell layers and were considered to be derived from type 4 or 5a follicles. These
results suggest that ADAMTS-1 is important for follicular development beyond the type 4
and/or 5a and for maintaining normal granulosa cell layers in follicles. Finally, the number oflarge blood vessels in the medullar zone was significantly decreased in ADAMTS-1 null miceovaries, suggesting that ADAMTS-1 is also involved in the organization of the medullary
vascular network in the ovary.

CL

CL

CL
CL

CL

ADAMTS-1 (-/-)

CL

CL, Corpus luteum

type 3type 4

CL
UF

UF

UF

UF

UF, Unruptured follicles

Unusual follicles
without granulosa cell
layers― 120 ―

Number of unusual
follicles without
granulosa cell layers

ADAMTS-1 (+/-)

200

ADAMTS1 (+/-)
ADAMTS1 (-/-)

150

p<0.01p<0.01

PMSG

PMSG/
hCG

p<0.01

100

50

0

(-)

Publications
1. Kidoya, H., Umemura, U., Kawabe, T., Matsuzaki, G., Yahagi, A., Imamura, R., and Suda, T.
(2005) Fas ligand induces cell-autonomous IL-23 production in dendritic cells, a mechanism
for Fas ligand-induced IL-17 production. J. Immunol. 175:8024-31.
2. Kinoshita, T., Wang, Y., Hasegawa, M., Imamura, R., and Suda, T. (2005) PYPAF3, a
PYRIN-containing APAF-1-like protein, is a feedback regulator of caspase-1-dependentinterleukin-1β secretion. J. Biol. Chem. 280:21720-5
3. Hasegawa M, Imamura R, Kinoshita T, Matsumoto N, Masumoto J, Inohara N, and Suda T.
(2005) ASC-mediated NF-κB activation leading to interleukin-8 production requires
caspase-8 and is inhibited by CLARP. J. Biol. Chem. 280: 15122-30.
4. Suda, T. (2005) Physiological and pathological roles of apoptosis. Nippon Rinsho. Suppl
4:395-400. Review. (Japanese)
5. Moriyama-Kita M, Endo Y, Yonemura Y, Heizmann CW, Miyamori H, Sato H, Yamamoto E,
Sasaki T. (2005) S100A4 regulates E-cadherin expression in oral squamous cell carcinoma.
Cancer Lett. 230:211-218.
6. Nozaki S, Endo Y, Nakahara H, Yoshizawa K, Hashiba Y, Kawashiri S, Tanaka A, Nakagawa
K, Matsuoka Y, Kogo M, Yamamoto E. (2005) Inhibition of invasion and metastasis in oral
cancer by targeting urokinase-type plasminogen activator receptor. Oral Oncol. 41:971-7.
7. Yonemura Y, Endo Y, Tochiori S, Bando E, Kawamura T, Shimada T, Miyamoto K, Tanaka
M, Sasaki T. (2005) Effect of intraperitoneal chemotherapy on experimental peritoneal
dissemination of gastric cancer. Gan To Kagaku Ryoho. 32:1635-9. [in Japanese]
8. Shimada T, Nomura M, Yokogawa K, Endo Y, Sasaki T, Miyamoto K, Yonemura Y.

(2005)

Pharmacokinetic advantage of intraperitoneal injection of docetaxel in the treatment for
peritoneal dissemination of cancer in mice. J Pharm Pharmacol. 57:177-81.
9. Koshida K, Egawa M, Imao T, Mizokami A, Namiki M, Endo Y, Sasaki T. (2005) In vitro
chemosensitivity test for human genito-urinary tumors using collagen gel matrix. Int J Urol.
12:67-72.
10. Shozu M, Minami N, Yokoyama H, Inoue M, Kurihara H, Matsushima K, Kuno K (2005)
ADAMTS-1 is involved in follicular development, ovulatory process and organization of the
medullary vascular network in the ovary. J. Mol. Endocrinol. 35: 343-355.
11. Imamura, R., Konaka, K., Matsumoto, N., Hasegawa, M., Fukui, M., Mukaida, N.,
Kinoshita, T., and Suda, T. (2004) Fas ligand induces cell-autonomous NF-κB activation andIL-8 production by a mechanism distinct from that of TNF-α. J. Biol. Chem. 279:46415-23.

― 121 ―

12. Umemura, M., Kawabe, T., Shudo, K., Kidoya, H., Fukui, M., Asano, M., Iwakura, Y.,
Matsuzaki, G., Imamura, R., and Suda, T. (2004) Involvement of IL-17 in Fas ligand-induced
inflammation. Int. Immunol. 16:1099-108.
13. Wang, Y., Hasegawa, M., Imamura, R., Kinoshita, T., Kondo, C., Konaka, K., Suda, T.
(2004) PYNOD, a novel Apaf-1/CED4-like protein is an inhibitor of ASC and caspase-1. Int.
Immunol. 16:777-86.
14. Nakamoto Y, Suda T, Momoi T, and Kaneko S. (2004) Different procarcinogenic potentials
of lymphocyte subsets in a transgenic mouse model of chronic hepatitis B. Cancer Res.
64:3326-3333
15. Uekita, T., Gotoh, I., Kinoshita, T., Itoh, Y., Sato, H., Shiomi, T., Okada, Y., and Seiki, M.
(2004). Membrane-type 1 matrix metalloproteinase cytoplasmic tail-binding protein-1 is a
new member of the Cupin superfamily. A possible multifunctional protein acting as aninvasion suppressor down-regulated in tumors. J Biol Chem 279, 12734-43.
16. Takahashi Y, Yamashita K, Endo Y, Sasaki T, Mai M. (2004) Oral administration of
uracil-tegafur (UFT) inhibits liver micrometastasis of human colon cancer in an orthotopic
nude mouse model and its early detection system. Surg Today. 34:855-9.
17. Kaneko M, Kawakami K, Oyama K, Endo Y, Tanaka M, Sasaki T, Watanabe G. (2004) The
association of expression and gene polymorphism of the thymidylate synthase gene with
5-fluoro-5'-deoxyuridine sensitivity in gastric cancer cell lines. Gan To Kagaku Ryoho.
31:1347-50. [in Japanese]
18. Murata D, Endo Y, Obata T, Sakamoto K, Syouji Y, Kadohira M, Matsuda A, Sasaki T.
(2004) A crucial role of uridine/cytidine kinase 2 in antitumor activity of 3'-ethynyl
nucleosides. Drug Metab Dispos. 32:1178-82.
19. Moriyama-Kita M, Endo Y, Yonemura Y, Heizmann CW, Schafer BW, Sasaki T, Yamamoto
E. (2004) Correlation of S100A4 expression with invasion and metastasis in oral squamouscell carcinoma. Oral Oncol. 40:496-500.
20. Yonemura Y, Endo Y, Bando E, Kuno K, Kawamura T, Kimura M, Shimada T, Miyamoto K,
Sasaki T, Sugarbaker PH. (2004) Effect of intraperitoneal administration of docetaxel on
peritoneal dissemination of gastric cancer. Cancer Lett. 210(2): 189-96.
21. Kuno K., Bannai K., Hakozaki M., Matsushima K., Hirose K. (2004) The carboxyl-terminal
half region of ADAMTS-1 suppresses both tumorigenicity and experimental metastatic
potential. Biochem. Biophys. Res. Comm. 319: 1327-1333.
22. Kuno K (2004) ADAMTS-1. in Handbook of Proteolytic Enzymes (Second Edition) (Barrett
A.J., Rawlings N.D., and Woessner J. F., eds.), London, Academic Press 735-737.

― 122 ―

23. Fukui, M., Imamura, R., Umemura, M., Kawabe, T., Suda, T. (2003) Pathogen-associatedmolecular patterns sensitize macrophages to Fas ligand-induced apoptosis and IL-1beta
release. J. Immunol.. 171:1868-74.
24. Tachiiri, A., Imamura, R., Wang, Y., Fukui, M., Umemura, M., and Suda, T. (2003) Genomic
structure and inducible expression of the IL-22 receptor α chain in mice. Gene. Immun.,
4:153-159
25. Endo Y, Tanaka M, Obata T, Sasaki T（2003）Experimental model for studying metastasis
using the developing embryonic chick. Nippon Rinsho. 61(Suppl. 8): 9-54. [in Japanese]
26. Yonemura Y, Sakurai S, Yamamoto H, Endo Y, Kawamura T, Bandou E, Elnemr A,
Sugiyama K, Sasaki T, Akiyama T, Takasawa S, Okamoto H. (2003) REG gene expression isassociated with the infiltrating growth of gastric carcinoma. Cancer. 98:1394-400.
27. Yonemura Y, Bandou E, Kinoshita K, Kawamura T, Takahashi S, Endo Y, Sasaki T. (2003)
Effective therapy for peritoneal dissemination in gastric cancer. Surg Oncol Clin N Am.
12:635-48. Review.
28. Elnemr A, Yonemura Y, Bandou E, Kinoshita K, Kawamura T, Takahashi S, Tochiori S,
Endo Y, Sasaki T. (2003) Expression of collagenase-3 (matrix metalloproteinase-13) inhuman gastric cancer. Gastric Cancer. 6:30-8.

― 123 ―

Meetings
andSeminars

第 24 回 がん研究所セミナープログラム
2003(平成 15).2.26～27
金沢大学医学部記念館 2 階ホール
◆ 腫瘍外科研究分野○ サマリー○ 新しい抗癌剤の濃度設定法，individualized maximum repeatable dose(iMRD)の開発とそれによるテイラード化学療法○ 早期胃癌に対する Minimally Invasive Surgery○ ヒト大腸癌細胞株同所性移植自然転移モデルを用いた転移関連因子群の解析◆ 遺伝子診断研究分野○ サマリー○ β-カテニンがん化シグナルの特異的活性化・制御機構の個別的，網羅的解析◆ 腫瘍内科研究分野○ サマリー○ 超音波内視鏡研究２０年の軌跡○ ヒト膵癌における clusterin の発現とその臨床的意義○ 膵癌患者血漿中 p53 遺伝子変異の検出○ 膵癌組織ならびに膵液における mesothelin mRNA 発現の検討◆ 細胞分子病態研究分野○ サマリー○ DNA 損傷修復経路における c-Abl チロシンキナーゼファミリー分子の機能○ の解析○ DNA 損傷，酸化ストレスに対する ATM 依存的シグナル伝達の解析

磨 伊 正 義高 橋豊
表和 彦
安 本 和 生

源
源

澤
岡
元
山
渡

利 成
利 成

武
井
雄
口
邊

紀 雄
高
良 治
泰 志
弘 之

山 本 健 一清 水 弘 子

小 林 昌 彦◆ 細胞情報調節研究分野○ サマリー○ Ｂ型肝炎ウィルスの構造蛋白質と結合する細胞蛋白質○ リン酸化 RNA ポリメラ－ゼⅡ結合因子の機能検索○ rRNA 生合成における U13 snoRNA の機能

原
黒
廣
木

◆ 細胞周期制御研究分野○ サマリー

善 岡 克 次◆ 細胞分化研究分野○ サマリー○ 世界各地に生息する野生ハツカネズミにこれまで発見された４種類のア
ロタイプの分布と環境との関係○ 線虫で RNAi 法による新規遺伝子 ell の機能解析○ 血管内皮細胞特異的受容体型チロシンキナーゼ TIE2 により活性化される― 125 ―

田 文 夫
木 和 之
瀬
豊
戸 敬 治

髙 倉 伸 幸坂井俊之助
天 野 重 豊上 野 将 也

遺伝子の同定とその機能解析◆ 遺伝子発現研究分野○ サマリー○ テロメア機能と細胞老化○ HCV NS5A Exhibits Distinct Modulation Effects on RNA-dependent RNAPolymerase Activity of NS5B In the Presence of Various Homopolymeric
RNA Templates

村 上 清 史林
直 之羅宏◆ 細胞機能統御研究分野○ サマリー○ がん浸潤における細胞運動と MMP 活性発現の協調的制御機構の解析○ 膜型マトリックスメタロプロテアーゼ活性制御分子の探索佐 藤博
滝 野 隆 久宮 森 久 志◆ 組織分子構築研究分野○ サマリー○ 転移巣形成におけるがん細胞と正常細胞の相互作用の解析

向 田 直 史佐々木洋子

◆ 分子薬理学研究分野○ サマリー○ 腸管 M 細胞の分化機構および細胞輸送機能の解析

大 野 博 司長 谷 耕 二◆ 化学療法研究分野○ サマリー○ エチニルヌクレオシドの感受性規定因子としての CTP synthetase

佐々木琢磨
小 幡
徹

◆ 分子標的薬剤開発センター○ サマリー○ ADAMTS-1 遺伝子欠損マウスのペリスタルシス異常および ADAMTS-1 部分欠損変異体による腫瘍増殖の抑制○ 血管新生阻害性 ANO 化合物の水溶化による bioavailabit の改善○ Fas リガンドによる NF-kB 活性化機構の解析○ アポトーシス促進因子 Bid の制御機構◆ 遺伝子・染色体構築研究分野○ サマリー○ パラレルアフィニティタグ精製法によるユビキチン化蛋白質の網羅的同定○ 質量分析技術を用いた蛋白質複合体の動態解析― 126 ―

須 田 貴 司久 野 耕 嗣遠 藤 良 夫
今 村
龍
木 下
健

伊 藤 隆 司太 田 一 寿紀 藤 圭 治

第 25 回

がん研究所セミナープログラム
2004(平成 16).2.5～6
金沢大学医学部記念館 2 階ホール

◆ 腫瘍外科研究分野○ サマリー○ Tumor dormancy therapy に基づく Tailored dose 化学療法 -根拠，意義，実際○ 視線追従式自動腹腔鏡操作システムの開発 -腹腔鏡手術において術者の負担を軽減する Robotic Assistance○ 胃癌腹膜播種形成における SDF-1a/CXCR4 リガンド・レセプターシステムの関与◆ 遺伝子診断研究分野○ サマリー○ 大腸癌のがん化シグナルの特異的活性化・制御機構の解析◆ 腫瘍内科研究分野○ サマリー○ ラット自然発症慢性膵炎モデルにおけるアポトーシス関連遺伝子の発現
動態○ Real-time PCR 法による膵癌患者の血清中 hTERTmRNA の定量的測定○ 各種消化器癌患者における血清 Midkine 値の測定
○ 膵癌診断を目指した膵液中 ppENK のメチル化異常に関する検討◆ 分子標的薬剤開発センター○ サマリー○ ADAMTS-1 の卵胞成熟，排卵過程への関与と ADAMTS-1 による実験的肺転移抑制○ 血管新生阻害性 ANO 化合物の水溶化による bioavailabit の改善○ Fas リガンドによる NF-kB 活性化機構の解析○ アポトーシス促進因子 Bid の制御機構

磨 伊 正 義
高 橋
豊
表

和 彦

安 本 和 生

源
源

利 成
利 成

澤 武 紀 雄元 雄 良 治渡 邊 弘 之山 口 泰 志大坪公士郎

須 田 貴 司久 野 耕 嗣遠 藤 良 夫
今 村
龍
木 下
健

◆ 化学療法研究分野○ サマリー○ 抗腫瘍性ヌクレオシドの感受性規定因子の解明：CDA の関与と網羅的遺伝
子発現解析法による探索

佐々木琢磨
小 幡
徹

◆ 遺伝子・染色体構築研究分野○ サマリー○ パラレルアフィニティタグ精製法によるユビキチン化蛋白質の網羅的同定

伊 藤 隆 司太 田 一 寿◆ 組織分子構築研究分野○ サマリー○ セリン／スレオニンキナーゼ Pim-3 の肝細胞がんにおける役割― 127 ―

向 田 直 史藤 井 千 文◆ 分子薬理学研究分野○ サマリー○ 腸管 M 細胞の生理作用の解析

大 野 博 司長 谷 耕 二◆ 遺伝子発現研究分野○ サマリー○ 細胞老化における信号伝達系の探索

村 上 清 史林
直 之◆ 細胞機能統御研究分野○ サマリー○ がん浸潤における細胞運動と MMP 活性発現の協調的制御機構の解析○ がん浸潤における膜型マトリックスメタロプロテアーゼ活性制御分子の
検索

佐 藤
博
滝 野 隆 久宮 森 久 志◆ 細胞情報調節研究分野○ サマリー○ Ｂ型肝炎ウイルスのコア蛋白質と結合する細胞蛋白質○ リン酸化 RNA ポリメラ－ゼⅡ結合 WW ドメイン蛋白質の機能解析○ rRNA 生合成における U13 snoRNA の機能

原 田 文 夫
黒 木 和 之廣 瀬豊
木 戸 敬 治◆ 細胞周期制御研究分野○ サマリー○ ミトコンドリア DNA 損傷によるアポトーシス誘導機構の解明

善 岡 克 次西 田
純

◆ 細胞分子病態研究分野○ サマリー○ DNA 損傷修復経路におけるチロシンキナーゼ Arg と Rad51 の相互作用の解析○ DNA 損傷，酸化ストレスに対する ATM 依存的シグナル伝達の解析◆ 細胞分化研究分野○ サマリー○ 開放血管における血管新生関連遺伝子の機能○ 血管内皮細胞特異的受容体型チロシンキナーゼ TIE2 により制御される遺伝子の同定とその機能解析― 128 ―

山 本 健 一清 水 弘 子
小 林 昌 彦髙 倉 伸 幸天 野 重 豊上 野 将 也

第 26 回

がん研究所セミナープログラム
2005(平成 17).3.3～4
金沢大学医学部臨床第一講義室◆ 腫瘍外科研究分野○ サマリー○ VEGF を標的とした Tumor dormancy therapy○ 早期胃大腸癌に対する腹腔鏡手術○ 胃癌腹膜播種形成における SDF-1a / CXCR4 リガンド・レセプターシステ
ムの関与

磨 伊 正 義高 橋豊
表和 彦
安 本 和 生◆ 遺伝子診断研究分野○ サマリー○ 大腸癌における b-TrCP(E3 ユビキチンリガーゼ受容体)の発現とがん化シ
グナル活性化

源
源

利 成
利 成

◆ 腫瘍内科研究分野○ サマリー○ 胃癌における stress-induced protein (SIP)の発現低下について○ 膵液中 SARP2 のメチル化測定による膵癌診断へのアプローチ○ 膵管内乳頭腫瘍(IPMN)における膵液中遺伝子のメチル化異常に関する検討○ 血清 Midkine 値の腫瘍マーカーとしての意義

毛 利 久 継◆ 分子標的薬剤開発センター○ サマリー○ ADAMTS-1 の卵巣機能における役割と実験的肺転移抑制機構の解析○ Fas リガンド刺激による IL-8 産生の分子機構○ PYPAF ファミリーによる炎症抑制機構

須
久
今
木

◆ 化学療法研究分野○ サマリー○ 抗腫瘍性エチニルヌクレオシドの耐性機序と感受性規定因子○ ヌクレオシドトランスポーターと抗腫瘍性ヌクレオシドに対する感受性との関連性

澤 武 紀 雄元 雄 良 治渡 邊 弘 之
大坪公士郎

田 貴 司
野 耕 嗣
村
龍
下
健

佐々木琢磨
遠 藤 良 夫小 幡
徹

◆ 組織分子構築研究分野○ サマリー○ セリン／スレオニンキナーゼ Pim-3 の肝細胞がんにおける役割

向 田 直 史藤 井 千 文◆ 遺伝子発現研究分野○ サマリー○ 細胞老化を制御する信号伝達系の探索

村 上 清 史林
直 之◆ 細胞機能統御研究分野○ サマリー佐 藤― 129 ―

博

○ がん浸潤における細胞運動と MMP 活性発現の協調的制御機構の解析○ がん浸潤における MT1-MMP 活性制御分子の検索◆ 細胞情報調節研究分野○ サマリー○ B 型肝炎ウイルス増殖への gC1qR の関与○ リン酸化 RNA ポリメラーゼ II 結合蛋白質の機能解析○ U13 ノックアウト細胞への変異 U13 RNA 遺伝子の導入とアセチル化活性の検討

滝 野 隆 久宮 森 久 志

原
黒
廣
木

田 文 夫
木 和 之
瀬
豊
戸 敬 治

◆ 細胞周期制御研究分野○ サマリー○ ミトコンドリア DNA 損傷により誘導されるアポトーシス

善 岡 克 次西 田
純

◆ 細胞分子病態研究分野○ サマリー○ Rad51 分子の機能における 301 番目のチロシンの役割について○ アルキル化剤，活性酸素による ATM の活性化機構の解析

山 本 健 一清 水 弘 子
小 林 昌 彦◆ 細胞分化研究分野○ サマリー○ 開放血管における血球分化と Tie homolog○ 幹細胞分裂における新規遺伝子#E11 の機能解析○ 心血管幹細胞の同定及びその分化制御機構の解明

髙
天
上
山

― 130 ―

倉
野
野
田

伸 幸
重 豊
将 也
賢 裕

International Symposium on Tumor Biology in Kanazawa 2003
Program Schedule
March 12,Wed.
Opening Remarks : Ken-ichi Arai (IMS, University of Tokyo)

Session A. Search for Novel Targets for Cancer Treatment
Chairperson: Takashi Ito (Cancer Res. Inst., Kanazawa Univ.)
Masa-aski Muramatsu (Tokyo Medical and Dental University)
Application of single nucleotide polymorphism (SNP) to clinical and epidemiological studies
Satoshi Inoue (University of Tokyo)
Estrogen responsive genes and breast cancer
John N. Weinstein (NIH, USA)
Integromics for cancer drug discovery: integrating genomics, proteomics, andbioinformatics
Chairperson: Tadashi Yamamoto (IMS, University of Tokyo)
Masafumi Shibuya (IMS, University of Tokyo)
A unique signal transduction of VEGF receptors involved in tumor angiogenesis andmetastasis
Kyu-Won Kim (Seoul National University)
Novel HIF-1α interacting proteins and their roles in hypoxia-induced tumor angiogenesis
Chairperson: Hiroshi Sato (Cancer Res. Inst., Kanazawa Univ.)
Motoharu Seiki (IMS, University of Tokyo)
Membrane-type 1 matrix metalloproteinase (MT1-MMP) in cancer invasion andangiogenesis
Toshinari Minamoto (Cancer Res. Inst., Kanazawa Univ.)
Distinct pattern of oncogenic β-catenin activation in colorectal cancer
Makoto Taketo (Kyoto University)
Mouse models for colon cancer: studies on chemoprevention and chemotherapy
March 13,Thu.

Session B. Novel Approaches to Cancer Treatment
Chairperson:

Masafumi Shibuya (IMS, University of Tokyo)

Michael O’Reilly (MD Anderson Cancer Center, USA)
Angiogenesis : from the laboratory to the Clinic― 131 ―

Yutaka Takahashi (Kanazawa University)
Tumor Dormancy Therapy-paradigm shift of cancer therapy
Chairperson: Naofumi Mukaida (Cancer Res. Inst., Kanazawa Univ.)
Mark Smyth (Peter MacCallum Cancer Institute)
Effectors and regulators of cancer immune surveillance
Akira Yamada (Kurume University)
Development of peptide-based vaccine for epithelial cancer
Chairperson: Nobuyuki Takakura (Cancer Res. Inst., Kanazawa Univ.)
Tatsutoshi Nakahata (Kyoto University)
Ex vivo expansion of human hematopoietic stem cells for a variety of clinical applications
Richard Childs (NIH)
Using nonmeyloablative conditioning to expand the role of allogeneic stem celltransplantation
Closing Remarks : Ken-ichi Yamamoto (Cancer Research Institute, Kanazawa University)― 132 ―

International Symposium on Tumor Biology in Kanazawa 2004
Program Schedule
February 12, Thu.
Opening Remarks : Ken-ichi Yamamoto (Cancer Res. Inst. Kanazawa Univ.)
Session A.
Chairperson: Naofumi Mukaida (Cancer Res. Inst., Kanazawa Univ.)
Kenkichi Masutomi (Dana-Farber Cancer Institute)
Functional role of telomerase in normal human cells
Satoru Kyo (Graduate School of Medicine, Kanazawa Univ.)
Recent advances in telomerase-based medicine for targeting cancers
R.Dalla-Favera (Institute for Cancer Genetics, Columbia University)
Molecular pathogenesis of B cell lymphoma
Tadashi Yamamoto (IMS, University of Tokyo)
The tumor suppressor protein Tob functions as a transcriptional coregulator
Chairperson: Tadaomi Takenawa (IMS, University of Tokyo)
Yoshinori Murakami (National Cancer Center Research Institute)
Involvement of a cell adhesion molecule, TSLC1, in human oncogenesis
Yoh Takuwa (Kanazawa Univ. Graduate School of Medicine)
Bimodal regulation by the S1P-Edg signaling system of Rho family GTPase andinvasion/metastasis in cancer cells
Yoshimi Takai (Osaka University Graduate School of Medicine)
Nectins and necls: Roles in cell migration, adhesion, and proliferation
February 13, Fri.
Session B.
Chairperson: Hiroshi Sato (Cancer Research Institute, Kanazawa Univ.)
Seishi Murakami (Cancer Research Institute, Kanazawa University)
Hepatitis C Virus (HCV) NS5B, a central catalytic enzyme for HCV replication
Claus Scheidereit (Max-Delbruck-Center for Molecular Medicine , Germany)
NF-κB/IKK and AP-1 pathways as molecular targets in human lymphomas
Tomohisa Kato (Biosignal Research Center, Kobe University)
NF-κB signaling in the stress response: Cellular response to environmental carcinogens
Jun-ichiro Inoue (IMS, University of Tokyo)
TRAF6-NFκB pathway in cell growth and differentiation― 133 ―

Chairperson: Ken-ichi Yamamoto
Makoto Nakanishi (Nagoya City University)
Chk1, an essential key player that regulates S/M phase transition
Junjie Chen (Mayo Clinic)
Regulation of DNA damage signaling pathways
Closing Remarks : Tadashi Yamamoto (IMS, University of Tokyo)― 134 ―

International Symposium on Tumor Biology in Kanazawa 2005
Program Schedule
January 20, Thu.
Opening Remarks : Ken-ichi Yamamoto (Cancer Res. Inst., Kanazawa Univ.)

Session A. Stem Cell Biology in Cancer and Normal Stem Cell
Chairperson: Yoh Takuwa (Kanazawa Univ. School of Medicine)
Hiroshi Koide (Kanazawa Univ. School of Medicine)
Regulation of proliferation and differentiation in embryonic stem cells
Yumi Matsuzaki (Keio Univ. School of Medicine)
Blood specific differentiation potency of purified HSC
Chairperson: Naofumi Mukaida (Cancer Res. Inst., Kanazawa Univ.)
Atsushi Hirao (Keio University School of Medicine)
Essential role of ATM (ataxia telangiectasia mutated) on stem cell self-renewal and tumor
suppression
In-Kyung Park (University of Michigan, USA)
Regulation of self-renewal in normal and cancer stem cells

Session B.

New Targets for Cancer Treatment

Chairperson: Takashi Yokota (Kanazawa Univ. School of Medicine)
JaeHun Cheong (Pusan National Univ., Korea)
Molecular mechanism of hepatic carcinogenesis by HBx
Shigeki Higashiyama (Ehime University School of Medicine)
Ectodomain shedding of HB-EGF and novel cell signaling
Chairperson: Takashi Suda (Cancer Res. Inst., Kanazawa Univ.)
Margaret Quinlan (MGH Cancer Center, USA)
Tumorigenesis from primary epithelial cells
Osamu Hori (Kanazawa Univ. School of Medicine)
Regulation of ER stress and ER stress-induced cell death in F9 Herp KO cells

Session C.

Molecular Mechanism of Tumor Angiogenesis

Chairperson: Hiroshi Sato (Cancer Res. Inst., Kanazawa Univ.)
Nobuyuki Takakura (Cancer Res. Inst., Kanazawa Univ.)
Molecular regulation of blood vessel maturation
Hajime Kubo (Kyoto University School of Medicine)
Lymphangiogenesis and cancer
Closing Remarks : Ken-ichi Yamamoto (Cancer Res. Inst., Kanazawa Univ.)― 135 ―

Organization
andPersonnel

as of February, 2006

Cancer Research InstituteSuccessive Directors and Acting Directors of the InstituteDr. Hajime Okamoto
Dr. Tachiomaru Ishikawa
Dr. Ryo ItohDr. Saburo Koshimura
Dr. Yoriaki Kurata
Dr. Motoichi Hatano
Dr. Shunsuke Migita
Dr. Tadanori Kameyama
Dr. Morinobu Takahashi
Dr. Masayoshi Mai
Dr. Ken-ichi Yamamoto
Dr. Hiroshi Sato（岡本肇）
（石川太刀雄丸）（伊藤亮）（越村 三郎）（倉田 自章）（波田野基一）（右田 俊介）（亀山 忠典）（高橋 守信）（磨伊 正義）（山本 健一）
（佐藤
博）

June
April
April
April
April
April
April
April
April
April
April
April

1, 1967
1, 1968
1, 1971
2, 1978
2, 1982
2, 1984
1, 1988
1, 1990
1, 1993
1, 1997
1, 2001
1, 2005

～
～
～
～
～
～
～
～
～
～
～
～

March
March
April
April
April
March
March
March
March
March
March
Present

31, 1968
31, 1971
1, 1978
1, 1982
1, 1984
31, 1988
31, 1990
31, 1993
31, 1997
31, 2001
31, 2005

Department of Molecular Oncology
Division of Molecular Biology
Division Chief,Professor
Research Associate
Research Technician
Technician
Graduate Student
Graduate Student
Graduate Student
Research Fellow

Seishi Murakami
Naoyuki Hayashi
Kazuko Kuwabara
Mami Yasukawa
Takashi Kusakawa
Hideki Mizuno
Shijun Zhang
Hong Tang

（村上
（林
（桑原
（安川
（草川
（水野
（張
（唐

Division of Virology
Division Chief,Professor
Associate Professor
Research Associate
Technician
Graduate Student
Graduate Student
Graduate Student
Graduate Student
Graduate Student
Graduate Student

Hiroshi Sato
Takahisa Takino
Hisashi Miyamori
Sayuri Yamagishi
Munirah Ahmad
Abdelaziz Abdalla
Keattikunpairoj Sunisa
Tomoya Kudo
Yumi Saeki
Miyuki Matsui（佐藤博）（滝野 隆久）（宮森 久志）（山岸小百合）（ムニラ アハマド）（アブデエルアジス アブダラ）（ケアティクンパイロ スニサ）
（工藤 知也）（佐伯 裕美）（松井 美雪）― 137 ―

清史）
直之）
和子）
麻美）
貴史）
秀城）
世俊）
紅）

Division of Molecular Bioregulation
Division Chief,Professor
Research Associate
Research Technician
Graduate Student
Graduate Student
Graduate Student
Graduate Student
Research Fellow
Research Fellow

Naofumi Mukaida
Chifumi Fujii
Kuniko Minami
Xiaoqin Yang
Boryana Popivanova
Wu Yu
Jorge Espinoza
Mariko Akiyama
Li Ying Yi（向田 直史）（藤井 千文）
（南
邦子）
（羊
暁勤）
（ボリアナ ポピバノーバ ）（呉俣）（ホルへ エスピノーザ）（秋山萬里子）（李影奕）Division of Molecular Genetics
Division Chief,Professor
Research Associate
Technician
Technician
Research Fellow

Atsushi Hirao
Kazuhito Naka
Miyako Takegami
Chiharu Minami
Masako Omura

（平尾
敦）
（仲
一仁）
（竹上美也子）（南千晴）（大村 昌子）Division of Macromolecular Biochemistry
Visiting Professor
Wei Wenxiang

（魏

文祥）

Department of Molecular and Cellular Biology
Division of Cell Biology
Division Chief,Professor
Associate Professor
Research Associate
Research Associate
Technician
Graduate Student
Graduate Student

Fumio Harada
Kazuyuki Kuroki
Yutaka Hirose
Yukiharu Kido
Toyoko Kikukawa
Shunji Ishii
Masamichi Yuda

（原田
（黒木
（廣瀬
（木戸
（菊川
（石井
（湯田

文夫）
和之）
豊）
敬治）
豊子）
俊二）
昌道）

Division of Stem Cell Biology
Division Chief,Professor
Research Associate
Research Associate
Research Associate
Technician
Graduate Student
Graduate Student
Graduate Student
Graduate Student

Nobuyuki Takakura
Shigetoyo Amano
Masaya Ueno
Yoshihiro Yamada
Yoriko Nakano
Wen-Ling Li
Kong Lingyu
Huang Xiaoyong
Shinya Katoh

（髙倉
（天野
（上野
（山田
（中野
（李
（孔
（黄
（加藤

伸幸）
重豊）
将也）
賢裕)
頼子）
文玲）
令宇）
暁勇）
真也）

― 138 ―

Graduate Student
Graduate Student
Graduate Student
Graduate Student
Graduate Student
Graduate Student
Graduate Student
Research Fellow
JSPS Research Fellow
JST Technical Assistant
JST Technical Assistant
JST Technical Assistant

Hiroyasu Kidoya
Ivan Hristov Vachkov
Han Yonglu
Hisamichi Naitoh
Sanae Akiba
Machiko Itoh
Naoko Takahashi
Rika Okamoto
Hideto Sano
Yuri Shimizu
Kumi Ishida
Miho Sato（木戸屋浩康)（イバン フリストフ ヴァチ）（韓瑛路）
（内藤 尚道）（秋場 早苗）（伊藤真知子）（高橋 直子）（岡本 里香）（佐野 秀人）（清水 友里）（石田 久美）（佐藤 美帆）Division of Molecular Pathology
Division Chief,Professor
Research Associate
Research Associate
Technician
Graduate Student
Graduate Student
Graduate Student
Research Fellow

Ken-ichi Yamamoto
Hiroko Shimizu
Masahiko Kobayashi
Kiyoko Take
Aki Takahashi
Makiko Ikei
Satoko Kitayama
Keiichi Muraoka（山本 健一）（清水 弘子）（小林 昌彦）（武 紀代子）（高橋 亜紀）（池井 牧子）（北山 聡子）（村岡 恵一）Division of Cell Cycle Regulation
Division Chief,Professor
Research Associate
Research Associate
Technician
Graduate Student
Graduate Student
Graduate Student
Graduate Student
Graduate Student
Graduate Student
Research Fellow
Research Fellow

Katsuji Yoshioka
Hiroshi Tanahashi
Naoko Taniura
Tomoe Ohashi
Bayarsaikhan Munkhuu
Davaakhuu Gantulga
Asuka Iwanaga
Tokiharu Sato
Masashi Shimizu
Guangmin Wang
Youichi Yamada
Jun Nishida（善岡 克次）（棚橋浩）（谷浦 直子）（大橋 智江）（バイヤルサハン ムンフォー）（ダバーク ガントウルガ ）（岩永 飛鳥）（佐藤 時春）（清水 雅司）（王光敏）（山田 洋一）（西田純）

Department of Basic and Clinical Oncology
Division of Genetics
Division Chief,Professor
Research Associate
Technician

Masanobu Oshima
Hiroko Oshima
Manami Watanabe― 139 ―

（大島 正伸）（大島 浩子）（渡邊真奈美）

Division of Experimental Therapeutics
Research Associate
Technician
Graduate Student
Graduate Student

Tohru Obata
Michiyo Aikawa
Norimichi Ito
Daisuke Matano（小幡徹）（相川みち代）（伊藤 教道）（俣野 乃輔）Division of Medical Oncology
Division Chief,Professor
Associate Professor
Lecturer
Research Associate
Research Associate
Technician
Technician
Research Technician
Graduate Student
Research Fellow
Research Fellow
Research Fellow
Research Fellow
Research Fellow
Research Fellow

Norio Sawabu
Hiroyuki Watanabe
Koushiro Ohtsubo
Hisatsugu Mouri
Tomoya Tsuchiyama
Tomoyo Irie
Keiko Okamoto
Gensaku Okada
Peihong Jiang
Yoshitake Satomura
Tokio Wakabayashi
Osamu Yamakawa
Hideki Ohta
Yasushi Yamaguchi
Yao Fan（澤武 紀雄）（渡邊 弘之）（大坪公士郎）（毛利 久継）（土山 智也）（入江 朋代）（岡本 恵子）（岡田 源作）（姜培紅）（里村 吉威）（若林 時夫）（山川治）（太田 英樹）（山口 泰志）（姚
凡）

Division of Surgical Oncology
Associate Professor
Lecturer
Research Associate
Resident
Resident
Technician
Technician
Research Fellow
Research Fellow
Research Fellow
Research Fellow
Research Fellow

Yutaka Takahashi
Kazuhiko Omote
Kazuo Yasumoto
Kaname Yamashita
Hidekazu Kitagata
Toshie Tanaki
Hiroko Horita
Tohru Itoh
Takahito Ohta
Michio Watanabe
Yasushi Deguchi
Toshihiro Fujimoto

（高橋
豊）
（表
和彦）
（安本 和生）（山下要）（北方 秀一）（棚木 敏恵）（堀田 博子）（伊藤透）（太田 孝仁）（渡辺美智夫）（出口康）（藤本 敏博）

Division of Diagnostic Molecular Oncology
Division Chief,Professor
Toshinari Minamoto
Postdoctoral Research Fellow
Abbas Shakoori
Research Assistant
Wei MaiTechniclal AssistantRitsuko Kitamura― 140 ―（源利成）
（シャクーリ アッバス）（麦威）（北村 律子）Techniclal AssistantGraduate Student
Graduate Student
Research Fellow
Research Fellow
Research Fellow
Research Fellow
Research Fellow

Masayo Terashima
Katsuyoshi Miyashita
Zhi Wei Yu
Ryo-ichi Asai
Yoshio Oda
Atsuhiro Kawashima
Kazuyuki Kawakami
Hironori Fujisawa

（寺島
（宮下
（干
（旭井
（小田
（川島
（川上
（藤沢

昌代）
勝吉）
志偉）
亮一）
惠夫）
篤弘）
和之）
弘範）

Center for the Development of Molecular Target Drugs
Director of the Center
Dr. Takuma Sasaki （佐々木琢磨）
Dr. Takashi Suda （須田 貴司）
Division Chief,Professor
Associate Professor
Associate Professor
Research Associate
Research Associate
Graduate Student
Graduate Student
Part-time Researcher

April 1, 1997 ～April 1, 2005 ～Takashi Suda
Kouji Kuno
Yoshio Endo
Ryu Imamura
Takeshi Kinoshita
Norihiko Matumoto
Kouji Kawase
Yentao Wang

March 31, 2005
Present（須田 貴司）（久野 耕嗣）（遠藤 良夫）
（今村
龍）
（木下
健）
（松本 則彦）（川瀬 浩司）（王冶陶）Administration Division
Head Official
General Affairs Section
Chief
Clerk

Library:Librarian

Kenji Nakano（中野

Yoshie Matsumoto
Miwako Yamashita
Kumiko Koyama
Reiko Yoneda
Hideki Okumura
Izumi Hashitani
Jyunko Hirose（松本 芳江）（山下美和子）（小山久美子）（米田 玲子）（奥村 英樹）（端谷泉）（広瀬 純子）― 141 ―

賢二）

Cancer Research Institute Report (2003－2005)(Published on March, 2006)

Scientific Report Editor
Fumio Harada

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