ANTICANCER RESEARCH 25: 895-902 (2005)

Dextran Sulfate Suppresses Cell Adhesion and

Cell Cycle Progression of Melanoma Cells
TSUYOSHI TAKAGI1, CHOUHEI SAKAKURA1, SHUICHI KIN1, YUEN NAKASE1,
KENICHIRO FUKUDA1, KATSUMI SHIMOMURA1, TADAO ITO1, JUNSHIN FUJIYAMA1,
JUNYA YAMASAKI1, HIROYUKI TSUJIMOTO1, YASUSHI OKAZAKI2,
YOSHIHIDE HAYASHIZAKI2, HISAKAZU YAMAGISHI1 and AKEO HAGIWARA1

1Department of Digestive Surgery, Kyoto Prefecture University of Medicine, Kamigyo-ku, Kawaramachi-dori, Kyoto 602-8566;
2RIKEN Genomic Sciences Center, Yokohama 230-0045, Japan

Abstracts. We have reported that dextran sulfate is a possible
candidate for an antimetastatic drug because it inhibits cell
adhesion. It has been demonstrated that dextran sulfate can
detach cancer cells adhering to the bottom of plastic flasks, and
that the detached cells do not readhere or proliferate. In this
study, we investigated the effects of dextran sulfate on cancer
cells, focusing on cell cycle regulators as well as cell adhesion
molecules. The effects of dextran sulfate on the cell cycle were
examined by flow cytometry, and changes in gene expression
caused by dextran sulfate were analyzed by cDNA microarray to
identify changes in adhesion and cell cycle genes. By flow
cytometry, treatment with dextran sulfate increased the
percentage of cells in the G1/G0-phase, and decreased those in
the S- and G2/M-phases. Analysis by cDNA microarray revealed
decreased expression of several genes essential for progression of
the G1- and S-phases. The expression of the adhesion factors
involved in metastases was also suppressed. Furthermore, we
confirmed these changes in the gene expression by Northern and
Western blotting. Our results indicate that dextran sulfate
suppresses cell adhesion and cell cycle progression, both of
which are essential for metastasis, suggesting that dextran sulfate
could be used as an antimetastatic agent.
Peritoneal recurrences are a critical problem that occur after
surgery for gastrointestinal cancers. Although many
Abbreviations: Dextran sulfate, DS; intercellular adhesion molecule
2, ICAM-2; proliferating cell nuclear antigen, PCNA.
Correspondence to: Tsuyoshi Takagi, M.D., Department of Digestive
Surgery, Kyoto Prefecture University of Medicine, Kamigyo-ku,
Kawaramachi-dori, Kyoto 602-8566, Japan. Tel: (+81) 75 251-5527,
Fax: (+81) 75 251-5522, e-mail: nao3@d2.dion.ne.jp
Key Words: Anti-adherence therapy, anticancer cell adherence
agent, peritoneal metastasis, dextran sulfate, peritoneal metastasis.
investigators have tried to develop effective methods for
preventing recurrences, no curative treatment has been
established for peritoneal recurrences. Cytotoxic
chemotherapeutic drugs have been developed and are now
widely used for metastatic foci. In many cases, however, the
intensive administration of such chemotherapeutic drugs to
suppress cancers results in significant side-effects. For the
formation of peritoneal metastases, free cancer cells must
disperse into the abdominal cavity, adhere onto the
peritoneum and, subsequently, proliferate. Therefore, we
believe that the most effective treatment for peritoneal
metastases is to prevent the cancer cells from adhering to the
peritoneum. We focused on dextran sulfate (DS), an anticell-
adherence agent, for the prevention of cancer cell adhesion to
the peritoneum. Previous studies showed that DS significantly
decreased cancer cells adhering to plastic flasks in vitro (1). In
addition, in vivo experiments demonstrated that the adhesion
of malignant tumor cells to the abdominal wall was
significantly suppressed in mice after DS treatment and that,
when cancer cells were injected into the abdominal cavity of
mice, the survival curve for the mice was improved by DS
administration (2, 3). The housing and all treatments of mice
were in accordance with national and institutional guidelines.
Our above-mentioned studies indicated that DS might
suppress the formation of micrometastatic foci by inhibiting
one or more processes of adhesion and proliferation.
In this study, to clarify the mechanism of action of DS on
cancer cells, we examined: i) the cell cycle by flow
cytometry, ii) changes in the expression of the genes relating
to cell adhesion and cell cycle by cDNA microarray, iii)
gene expression by Northern blotting, and iv) protein
expression by Western blotting.
Materials and Methods
Cell lines and preparation of dextran sulfate. The B-16 melanoma cell
line was used as the experimental tumor, because it exhibits high
adhesion. The cells were cultured in RPMI1640 (Nacalai Tesque Inc,

0250-7005/2005 $2.00+.40 895

 ANTICANCER RESEARCH 25: 895-902 (2005)

Figure 1. Flow cytometry analysis. Control cells, untreated with DS, are shown in panel A. Cells treated with DS for 12, 24 and 36 h are shown in panels
B, C and D, respectively. The G0-G1 population in the DS group (panels B, C, and D) were increased compared with the control (panel A) (68.4, 72.4,
74.7% versus 58.5%).

Kyoto, Japan) media supplemented with 10% fetal bovine serum and
incubated under standard conditions (5% CO2 atmosphere at 37ÆC).
DS (dextran sulfate sodium salt: mean molecular weight 5x105;
Sigma, St. Louis, MO, USA) was dissolved in the medium for cell
culture at a final concentration of 0.2 mg/ml, which caused no toxic
symptoms in the mice but did have a significant therapeutic effect
on the peritoneal metastasis of inoculated B-16 melanoma cells. This
concentration also caused no cytotoxicity as assessed by the MTT
assay (data not shown). The DS solution was filtered through a
200 nm filter to sterilize it before use.
Flow cytometry analysis. B-16 melanoma cells were suspended at a
density of 106 cell/ml in culture flasks and allowed to grow. After
incubation for 24 h, each medium was exchanged with normal
medium (control group) or the same media containing 0.2 mg/ml of
DS (DS group). Between 12 and 36 h later, at the time of harvest, the
cultures were 60 to 80% confluent. The adherent cells, which were
harvested by mild trypsinization, were pooled together with the
detached cells. All the cells were fixed in 70% ethanol for 12 h and
then pelleted by centrifugation. The ethanol solution was
subsequently removed after centrifugation, and the cells were treated
with RNase (Sigma) and stained with PI. The DNA content of the
cells was determined using a FACScan cytometer (Becton Dickinson,
Cockeysville, MD, USA). Each population of cells in the G1/G0-, S-
and G2/M-phases was calculated using the SFIT method (Becton
Dickinson Immunocytometry Systems, Cockeysville, MD, USA).
cDNA microarray analysis. B-16 melanoma cells were grown in the
same medium for 24 h, as described previously, and then the
medium was exchanged with normal medium (control group) or 0.2
mg/ml DS-containing medium (DS group). Aliquots of cells treated
with DS were harvested at 1, 3, 6, 12 and 24 h (DS subgroup).
We used the RIKEN set of 18,816 full-length enriched mouse
cDNA arrays to monitor the expression profiles of the B-16
melanoma cells that were treated with DS (4). One microgram of
mRNA extracted from each group was prepared using the Fast Track
mRNA Isolation kit (Invitrogen). For each microarray hybridization,
two separate probes were made: one labelled with Cy3 (control
group) and one with Cy5 (DS-treated group). The labelling was
carried out at 42ÆC for 1 h in a total volume of 30 Ìl containing 400

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Figure 2. Chages in gene expression in response to dextran sulfate (DS). The Treeview and Cluster programs were used to group and display sequences
that were either induced (red) or suppressed (green) in expression during the 24-h time course of DS treatment. In panel A, of 18,816 genes in the
microarray, 1,016 were selected for this analysis. Bars to the right identify the location of the inserts displayed in panels B and C. Panel B. Up-regulated
genes in B-16 melanoma cell during the 24-h time course of DS treatment. Panel C. Down-regulated genes in B-16 melanoma cell during the 24-h time
course of DS treatment.

units of SuperScript II (GIBCO/BRL); 0.1 mM Cy3-dUTP (or Cy5-
dUTP); 0.5 mM each dATP, dCTP and dGTP; 0.2 mM dTTP, 10
mM DTT, 6 Ìl of 5x first-strand buffer, and 6 Ìg of random primers.
This labelled probe was mixed with blocking solution containing 3 Ìl
of 10 Ìg/Ìl oligo(dA), 3 Ìl of 20 Ìg/Ìl yeast tRNA, 1 Ìl of 20 Ìg/Ìl
mouse Cot1 DNA, 5.1 Ìl of 20x SSC and 0.9 Ìl 10% SDS.
Before hybridization, aliquots of the probes were heated at 95ÆC
for 1 min and cooled to room temperature. The coverslips were
hybridized overnight at 65ÆC in a Hybricasette. After hybridization,
the slides were washed in 2x SSC/0.1% SDS until the coverslips
dropped off, and the slides were then transferred into 1x SSC,
shaken gently for 2 min, and rinsed with 0.1x SSC for 2 min. After
washing, the slides were spun at 800 rpm in a Sorvall centrifuge
(RC-3B plus; H6000A/HBB6 rotor).
These slides were scanned on a ScanArray 5000 confocal laser
scanner, and the images were analyzed using IMAGENE
(BioDiscovery, Los Angeles, CA, USA). Preceding the clustering,
ratio values from duplicate experiments were averaged, log-
transformed (base 2), and stored in a table. We applied
hierarchical clustering to both axes, using the weighted pair-group
method with a centroid average as implemented by the program
Cluster (5). The distance matrices we used were the Pearson
correlation for clustering the arrays and the inner product of the
vectors were normalized to magnitude 1 for the genes (this is a
slight variation of the Pearson correlation). The results were
analyzed by using Treeview (5).
Northern blot analysis. Northern blot analysis was performed as
described previously (6). In brief, total cellular RNA was prepared by
the guanidine isothiocyanate-phenol-chloroform procedure. The
poly(A)+ RNA was selected by an oligo dT column, then fractionated
on 1% agarose/2.2 M formaldehyde gels. Probes were labelled with
32P by random priming. Each blot was hybridized with the probes. We
analysed the signals with a BAS 2000 image analyzer and calculated
the degree of expression induced by DS compared to the control.
Western blot analysis. The B-16 melanoma cells were grown as
previously described for the cDNA microarray analysis. The control

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 ANTICANCER RESEARCH 25: 895-902 (2005)

Table I. Down-regulated genes in B-16 melanoma cell in response to dextran sulfate. GenBank accession number and one representative function of the
genes are shown.

Gene bank (accession) Gene name Function

2700002H18 Cyclin-dependent kinase 4 Cell cycle

2700091J11 Cyclin-dependent kinase 2 Cell cycle
2810401B17 Cyclin D1 Cell cycle
2410004D05 Cyclin E Cell cycle
3000002M17 Cyclin B1 Cell cycle
2610001P14 Cyclin-dependent kinase regulatory subunit 1 (CKS-1) Cell cycle
1110056L21 Proliferating cell nuclear antigen (PCNA) Cell cycle
3200001H17 Cadherin 11 Adhesion, Cytoskeleton
1810047B10 Intercellular adhesion molecule (ICAM) 2 Adhesion, Cytoskeleton
2210403I21 Integrin alpha 6 Adhesion, Cytoskeleton
2010107I09 Integrin beta 1 Adhesion, Cytoskeleton
1200018A13 Integrin beta 7 Adhesion, Cytoskeleton
2700079H05 Basigin Adhesion, Cytoskeleton
2310044M12 Keratin complex 1, gene 5 Adhesion, Cytoskeleton
2310031G15 Keratin complex 2, basic, gene 4 Adhesion, Cytoskeleton
1020007O09 CD 36 antigen Adhesion, Cytoskeleton
3300002H11 CD 81 antigen Adhesion, Cytoskeleton
2700002C08 Vimentin Adhesion, Cytoskeleton
1190001A18 Calmodulin 2 Signaling
1110033C24 Fyn proto-oncogene Signaling
1810015H17 RAB3D, member RAS oncogene family Signaling
2310010A14 RAB11B, member RAS oncogene family Signaling
1500016L08 RAB1, member RAS oncogene family Signaling

Table II. Up-regulated genes in B-16 melanoma cell in response to dextran sulfate. GenBank accession number and one representative function of the
genes are shown.

Gene bank (accession) Gene name Function

2210409J05 Cyclin-dependent kinase inhibitor 2B (p15, inhibits CDK4) Cell cycle

2810001M09 Cyclin A2 Cell cycle
2210021M05 Cyclin C Cell cycle
1810059M17 Cyclin H Cell cycle
2310016F12 Cyclin G Cell cycle
1110037C24 Insulin-like growth factor binding protein (IGFBP) 5 Signaling
1110001G24 Myristolated alanine rich protein kinase C substrate Signaling
2310003O03 MAX binding protein Signaling
1600025I01 Cytochrome P-450 Detoxication
1190031H01 Glutathione-S-transferase, a2 Detoxication
1110020I23 Heat shock protein 70 Protein folding
2310033C17 Beta catenin interacting protein Cell adhesion protein
1110039G20 Caspase 14 Apotosis
1500040L20 Caspase 12 Apotosis
2610037G10 Caspase 6 Apotosis
2410015N02 BCL2 interacting protein 1 (Nip1) Apotosis
2610036G18 Programmed cell death 4 Apotosis

cells not exposed to DS and the DS subgroups (DS-1, DS-3, DS-6,
DS-12, DS-24) were harvested as previously described. The cell
pellets containing 5x106 cells were washed twice with PBS and
lysed in ÇP% TritonX-100, 0.15 M NaCl and 10 mM Tris-HCl, pH
7.4, with 50 Ìg/ml of aprotinin at 4ÆC for 1 h. Lysates were
centrifuged at 10,000xg for 10 min, and boiled in sodium
dodecylsulfate (SDS) sample buffer for 5 min. The proteins were
separated by 10 to 20% gradient polyacrylamide gel SDS-
electrophoresis and electrically transferred to a polyvinylydene
difluoride (PVDF) membrane (Immobilon, Nihon Millipore Ltd.,
Tokyo, Japan). After blocking with 5% (w/v) skimmed milk in
PBS containing 0.1% (v/v) Tween 20 (Sigma), the membranes

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 Takagi et al: Dextran Sulfate Effects on Cell Adherence and Growth
Figure 3. Alterations of genes involved in cell adhesion after dextran sulfate(DS) treatment. The time-dependent (1 to 24 h) alterations of gene expression
in B-16 melanoma cells treated with DS.
were incubated at room temperature with the following 1:1000-
diluted primary antibodies: mouse anti-p15, rabbit anti-CDK4,
mouse anti-PCNA, rabbit anti-Integrin ‚1 (Santa Cruz
Biotechnology, Inc, CA, USA) After washing with PBS-T, the
blots were incubated with the 1:1000-diluted anti-mouse IgG,
anti-rabbit IgG (Santa Cruz Biotechnology, Inc.) for 1 h at room
temperature. The membranes were subsequently washed with
PBS-TÅCand the protein bands were detected with ECL Western
blotting detecting reagents (Amersham International Plc,
Buckinghamshire, UK).
Results
Flow cytometry analysis. The results of triplicate experiments
for cell cycle analysis were almost the same (data not
shown). Figure 1 shows the typical results of cell cycle
analysis: Panel A for the untreated control group and Panels
B, C and D for the DS groups at 12, 24 and 36 h after DS
treatment, respectively. The percentages of G1/G0-phase
cells in the DS groups were increased at 68.4%, 72.4% and
74.7% at 12, 24 and 36 h, respectively, compared to 58.5%
in the control group.
cDNA microarray analysis. The Cluster and Treeview
programs were used to analyze the data from all
experiments to determine how DS affects the genes of
cancer cells. The results are shown in Figure 2, with red
representing increased expression and green representing
decreased expression. The group of genes up-regulated after
DS treatment are shown in Table I, while down-regulated
genes are shown in Table II. Among the genes which had
an altered expression pattern after DS treatment, some of
the adhesion-related genes are shown in Figure 3.
Decreased expression of several metastasis-related adhesion
factors, such as integrin beta 1, integrin alpha 6 and
cadherin 11, was observed, beginning 1 h after DS
treatment.
The results for several cell cycle genes are shown in
Figure 4. The expression level of proliferating cell nuclear
antigen (PCNA) did not significantly change 1 h after DS
treatment, but decreased beginning 3 h after DS treatment
in a time-dependent manner, showing about a two-fold
decrease compared to the control group 24 h after DS
treatment. The expression levels of cyclin dependent kinase
4 (CDK4) and CDK2 were decreased by DS treatment, but
not in a time-dependent manner within 24 h after DS
treatment. In contrast, the expression of the CDK4 inhibitor
p15 increased time-dependently after DS treatment. The
expression levels of cyclin D1 and cyclin E decreased from
1 and 6 h after DS treatment, respectively.
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 ANTICANCER RESEARCH 25: 895-902 (2005)
Figure 4. Alterations of genes involved in cell cycle progression after dextran sulfate(DS) treatment. The time-dependent (1 to 24 h) alterations of gene
expression in B-16 melanoma cells treated with DS.
Northern blot analysis. Compared with the control group
(0 h), the DS subgroups (1 to 24 h) showed decreased
expression of the cell cycle genes PCNA, CDK2 and CDK4.
The expression of cyclin D1 did not decrease within 6 h, but
did from 12 h after DS treatment. The Northern blot analysis
also demonstrated time-dependent decreases in expression
levels after DS treatment. Several adhesion genes such as
intercellular adhesion molecule 2 (ICAM-2) also showed
decreased expression levels after DS treatment (Figure 5).
Western blot analysis. Similar to Northern analysis, Western
analysis showed that the protein levels of p15 in the DS
group (1 to 24 h) increased time-dependently compared
with that in the control (0 h). The DS group showed time-
dependent decreases in the expression of PCNA, CDK4 and
integrin beta 1 from 6 h after DS treatment, compared with
the control group (Figure 6).
Discussion
Our previous results demonstrated that DS has an anticell-
adhesion effect in both in vitro and in vivo experiments using
B-16 melanoma cells, which have strong adhesive properties
and a distinct morphology. In the present study, we also used
900
B-16 melanoma cells to investigate the effects of DS on cancer
cells. We focused on cell adhesion factors and cell cycle
progression essential to metastasis to elucidate the actions of
DS, which had not been clarified in previous studies. To
analyze the expression profiles of a large number of genes
possibly involved in metastasis, cDNA microarray technology
is a powerful tool (7-10). Therefore, we used the RIKEN set
of 18, 816 full-length enriched mouse cDNA arrays.
We first investigated the expression of cell adhesion
molecules. Metastasis is a multistage process consisting of: 1)
loss of cell-cell adhesion, 2) degradation and production of the
extracellular matrix, 3) adhesion to other cells or the matrix,
and 4) changes in the cytoskeleton and subsequent facilitation
of cell motility. It is important to understand the mechanism of
the cell-matrix adhesion in order to ascertain whether DS
prevents the implantation of cancer cells to the basement
membrane. Integrin is a cell-matrix adhesion molecule located
on the cell membrane, which acts as an adherent factor to
various components of the extracellular matrix such as
collagens, laminin and fibronectin. Integrin belongs to a family
consisting of more than 20 species of homologous molecules
(11, 12). There have been other reports on the relationship
between the abnormal expression of integrin beta 1 and
metastasis, using melanoma and colorectal cancer cells (13, 14).
 Takagi et al: Dextran Sulfate Effects on Cell Adherence and Growth
Figure 5. Northern blot analysis. The time course of expression of PCNA,
CDK2, CDK4, cyclinD1 and ICAM2 are shown. Compared with the
control cells (0 h), the expression of PCNA, CDK2, CDK4 and ICAM2
were decreased after DS treatment (1 to 24 h). The expression of cyclinD1
was decreased after 12 h. ‚-actin was used as an internal control.
Our cDNA microarray results demonstrated that the
expression of integrins beta 1 and alpha 6 decreased by DS
treatment compared to the untreated control. The decrease
in the integrin beta 1 expression was further confirmed at the
protein level by Western blot analysis. In addition, the
expression of the cadherin-11 gene was also decreased by DS
in the cDNA microarray analysis. Cadherin-11 is a novel
member of the cadherin supergene family. It has been
suggested that cadherin-11 expression is associated with an
invasive phenotype of cancer cells (15). The present finding
that DS decreased the gene expression of such cell-matrix
adhesion factors suggests that DS prevents cancer cells from
adhering to the extracellular environment, such as the bottom
of culture flasks, and subsequent invasion.
We also investigated the effects of DS on the cell cycle to
determine whether it might be involved in another
mechanism of metastasic foci suppression. The results of flow
cytometry demonstrated that treatment with DS increased
the number of cells in the G1/G0-phase and decreased those
in the S- and G2/M-phases, compared to the untreated
control cells, suggesting that DS inhibits cell cycle progression
by blocking the transition from G1 to S. To further investigate
this result, the expression profiles of various cell cycle genes
were analyzed by cDNA microarray to elucidate which genes
are affected by DS. Differences between the DS-treated and
the control groups were found mainly in the expression level
of genes specific to the G1-S-phase. Among them was cyclin
D1, which is expressed from the G1 metaphase to anaphase,
and binds to the cyclin-dependent kinases CDK4 and CDK6
to activate them (15-18). The cDNA microarray analysis
Figure 6. Western blot analysis. The expression of CDK4, PCNA and
integrin beta 1 were decreased in a time-dependent manner from 6 h after
DS treatment. In contrast, p15 (CDK4 inhibitor) increased in a time-
dependent manner from 1 h after DS treatment.
revealed that the expression levels of cyclin D1 and CDK4 in
the DS-treated cells did not show time-dependent changes
when examined at five time points within 24 h, but was
markedly decreased compared to the control cells. Cyclin D1
is also known to bind to and inactivate PCNA (19). We found
that the expression of PCNA decreased in a time-dependent
manner after DS treatment. On the other hand, the
expression of p15, a specific inhibitor for CDK4 (14),
increased in a time-dependent manner after DS treatment.
Cells normally progress from the G1-phase to the G1-S
checkpoint. This process is regulated by CDK2 associated
with cyclin E (15,20-25). The expression levels of the cyclin E
and CDK2 genes were also decreased by DS treatment.
These cDNA microarray results were confirmed by Northern
and Western blotting.
The results of the cDNA microarray analysis
demonstrated that the treatment of tumor cells with DS
induced changes in the expression of genes associated with
the G1-S-phase, and that there was no significant change in
the expression of genes that function in the other phases of
the cell cycle. These results suggest that DS inhibits the G1-
S-phase transition, and induces G1-S arrest. Analysis by
flow cytometry also showed inhibition of the G1-S transition,
indicating that DS suppresses cell cycle progression by
blocking this process.
These results suggest that DS may function as an
antimetastatic agent that suppresses cancer cell growth and
prevents peritoneal metastasis by inducing the detachment
of implanted cancer cells, inhibiting the re-adhesion of any
free cells, and blocking cell cycle progression.
While the present study clarified the effects of DS on cell
adhesion and cell cycle progression, the intracellular
signaling pass way for regulating path expression of these
affected genes and their relationship with apoptosis should
be studied in the future.
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 ANTICANCER RESEARCH 25: 895-902 (2005)
In contemplating the clinical use of DS, we believe that
it should be administered intraperitoneally immediately
after the resection of a tumor in order to prevent the
implantation of any freely floating cancer cells and the
proliferation of any micrometastases. Chemotherapy can be
administered subsequently to kill any surviving tumor cells.
Acknowledgements
This work was supported by a Grant-in Aid from the Ministry of
Education, Science and Sports and Culture of Japan.
References
1 Hagiwara A, Sawai K, Sakakura C, Shirasu M, Ohgaki M,
Imanishi T, Yamasaki J, Togawa T and Takahashi T: Prevention
of peritoneal metastasis of cancer with dextran sulfate-an
experimental study in mice. Anticancer Drugs 9: 894-897, 1997.
2 Hagiwara A, Sakakura C, Shirasu M, Togawa T, Sonoyama Y,
Fujiyama J, Ebihara Y, Itoh T and Yamagishi H: Intraperitoneal
injection of dextran sulfate as an anti-adherent drug for the
prevention of peritoneal metastasis of cancer show low toxicity
in animals. Anticancer Drugs 5: 393-399, 2000.
3 Hagiwara A, Sakakura C, Yamasaki J, Togawa T, Sonoyama Y,
Fujiyama J and Yamagishi H: Dextran sulfate inhibits injured
abdominal wall-specific tumor implantation in mice. Anticancer
Drugs 10: 873-877, 2000.
4 Miki R, Kadota K, Bono H, Mizuno Y, Tomaru Y, Carninci P,
Itoh M, Shibata K, Kawai J, Konno H, Watanabe S, Sato K,
Tokusumi Y, Kikuchi N, Ishii Y, Hamaguchi Y, Nishizuka I,
Goto H, Nitanda H, Satomi S, Yoshiki A, Kusakabe M, DeRisi
JL, Eisen MB, Iyer VR, Brown PO, Muramatsu M, Shimada H,
Okazaki Y and Hayashizaki Y: Delineating developmental and
metabolic pathways in vivo by expression profiling using the
RIKEN set of 18, 816 full-length enriched mouse cDNA arrays.
Proc Natl Acad Sci USA 98: 2199-2204, 2001.
5 Eisen MB, Spellman PT, Brown PO and Botstein D: Cluster
analysis and display of genome-wide expression patterns. Proc
Natl Acad Sci USA 95: 14863-14868, 1998.
6 Sakakura C, Hagiwara A, Yasuoka R, Fujita Y, Nakanishi M,
Masuda K, Shimomura K, Nakamura Y, Inazawa J, Abe T and
Yamagishi H: Tumour-amplified kinase BTAK is amplified and
overexpressed in gastric cancers with possible involvement in
aneuploid formation. Br J Cancer 84: 824-831, 2001.
7 Shalon D, Smith SJ and Brown PO: A DNA microarray system
for analyzing complex DNA samples using two-color fluorescent
probe hybridization. Genome Res 6: 639-645, 1996.
8 Kudoh K, Ramanna M, Ravatn R, Elkahloun AG, Bittner ML,
Meltzer PS, Trent JM, Dalton WS and Chin K-V: Monitoring
the expression profiles of doxorubicin-induced and doxorubicin-
resistant cancer cell by cDNA microarray. Cancer Res 60: 4161-
66, 2000.
9 Zembutsu H, Ohnishi Y, Tsunoda T, Furukawa Y, Katagiri T,
Ueyama Y, Tamaoki N, Nomura T, Kitahara O, Yanagawa R,
Hirata K and Nakamura Y: Genome-wide cDNA microarray
screening to correlate gene expression profiles with sensitivity
of 85 human cancer xenografts to anticancer drugs. Cancer Res
62: 518-527, 2002.
902
10 Kihara C, Tsunoda T, Tanaka T, Yamana H, Furukawa Y, Ono
K, Kitahara O, Zembutsu H, Yanagawa R, Hirata K et al:
Prediction of sensitivity of esophageal tumors to adjuvant
chemotherapy by cDNA microarray analysis of gene-expression
profiles. Cancer Res 61: 6474-6479, 2001.
11 Hynes RO: Integrins. Versatility, modulation, and signaling in
cell adhesion. Cell 69: 11-25, 1992.
12 Friedrichs K, Ruiz P, Franke F, Gille I, Terpe HJ and Imhof
BA: High expression level of alpha 6 integrin in human breast
carcinoma is correlated with reduced survival. Cancer Res 55:
901-906, 1995.
13 Fujita S, Watanabe M, Kubota T, Teramoto T and Kitajima M:
Alteration of expression in integrin ‚1-subunit correlates with
invasion and metastasis in colorectal cancer. Cancer Lett 91:
145-149, 1995.
14 Vink J, Thomas L, Etoh T, Bruijn JA, Mihm MC Jr, Gattoni-
Celli S and Byers HR: Role of beta-1 integrins in organ
specific adhesion of melanoma cells in vitro. Lab Invest 68:
192-203, 1993.
15 Pishvaian MJ, Feltes CM, Thompson P, Bussemakers MJG,
Schalken JA and, Byers SW: Cadherin-11 is expressed in
invasive breast cancer cell lines. Cancer Res 59: 947-952, 1999.
16 Sherr CJ: Cancer cell cycles. Science 274: 1672-1677, 1996.
17 Sherr CJ: D-type cyclins. Trends Biochem Sci 20: 187-190, 1995.
18 Matsushima H, Quelle DE, Shurtleff SA, Shibuya M, Sherr CJ
and Kato J: D-type cyclin-dependent kinase activity in
mammalian cells. Mol Cell Biol 14: 2066-2076, 1994.
19 Keum JS, Kong G, Yang SC, Shin DH, Park SS, Lee JH and
Lee JD: Cyclin D1 overexpression is an indicator of poor
prognosis in resectable non-small cell lung cancer. Br J Cancer
81: 127-132, 1999.
20 Guadagno TM, Otsubo M, Roberts JM and Assoian RK: A link
between cyclin A expression and adhesion-dependent cell cycle
progression. Science 262: 1572-1575, 1993.
21 Botz J, Zerfass-Thome K, Spitkovsky D, Delius H, Vogt B,
Eilers M, Hatzigeorgiou A and Jansen-Durr P: Cell cycle
regulation of the murine cyclin E gene depends on an E2F
binding site in the promoter. Mol Cell Biol 16: 3401-3409, 1996.
22 Geng Y, Whoriskey W, Park MY, Bronson RT, Medema RH,
Li T, Weinberg RA and Sicinski P: Rescue of cyclin D1
deficiency by knock-in cyclin E. Cell 97: 767-777, 1999.
23 Guadagno TM and Newport JW: Cdk2 kinase is required for
entry into mitosis as a positive regulator of Cdc2-cyclin B kinase
activity. Cell 84: 73-82, 1996.
24 Muller-Tidow C, Metzger R, Kugler K, Diederichs S, Idos G,
Thomas M, Dockhorn-Dworniczak B, Schneider PM, Koeffler
HP, Berdel WE and Serve H: Cyclin E is the only cyclin-
dependent kinase 2-associated cyclin that predicts metastasis
and survival in early stage non-small cell lung cancer. Cancer
Res 61: 647-653, 2001.
25 Dulic’ V, Lees E and Reed SI: Association of human cyclin E
with a periodic G1-S phase protein kinase. Science 257: 1958-
1961, 1992.
Received July 26, 2004
Accepted December 29, 2004