 |
Introduction |
It is now widely accepted that malignant tumors contain
heterogeneous populations of cells with regard to metastatic potential (1). The process of cancer metastasis consists
of linked sequential steps, including invasion, detachment,
intravasation, circulation, adhesion, extravasation, and growth
in distant organs (2). Thus, high- and low-metastatic cells in a
tumor should be different each other with respect to several
biological properties, such as invasiveness, adhesiveness, motility, and proliferation potential. There is much evidence
to support the concept that each discrete step of metastasis
is regulated by transient or permanent changes at the DNA,
messenger RNA (mRNA),1 and/or protein levels in different genes (3). By using the mRNA differential display
method, we previously identified a partial 3
cDNA fragment of a novel gene, Elm1 (for expressed in low-metastatic type 1 cells), that is expressed in low- but not in high-metastatic K-1735 murine melanoma cells (6). Elm1 was
also differentially expressed between high- and low-metastatic B16 murine melanoma cells. A partial Elm1 cDNA
fragment of 211 nucleotides showed no significant homology to any recorded sequence in the DDBJ/GenBank/ EMBL DNA databases.
Here we determined the full-length cDNA structure of
the Elm1 gene. The Elm1 gene encoded a novel type of
CCN (connective tissue growth factor [CTGF], Cyr61/
Cef10, and neuroblastoma overexpressed gene [Nov]) family proteins that consisted of secreted cysteine-rich molecules, CTGF, Cyr61/Cef10, and Nov (7). CTGF and
Cyr61 were induced within minutes of stimulation by serum or growth factors (10, 12) and have a growth regulatory function in fibroblasts or endothelial cells (13, 14).
Nov is a protooncogene isolated from myeloblastomatosis-associated virus-induced nephroblastoma (9). Because of the
high level of sequence similarities between Elm1 and the
other members of the CCN family, it is likely that Elm1
has a function to regulate the growth of normal and/or
cancerous cells. Since Elm1 gene expression was high in
low-metastatic tumor cells and declined with increasing
metastatic potential in two rodent experimental systems,
we investigated the biological effects of the Elm1 gene on
in vitro growth, in vivo growth, and metastatic potential of
K-1735 murine melanoma cells. By introduction of an
Elm1 expression vector into high-metastatic K-1735 M-2
cells that did not express endogenous Elm1, it was revealed
that expression of Elm1 can inhibit tumor growth in vivo
as well as metastasis of K-1735 melanoma cells.
 |
Materials and Methods |
Cell Lines.
K-1735-derived mouse melanoma cell lines were
obtained from I.J. Fidler (University of Texas, Houston, TX; reference 15). Clone 23 (designated C-23) was classified as low metastatic, whereas clone M-2 was high metastatic in syngeneic recipients (16, 17). Serum stimulation was performed using BALB/c
3T3 cells.
Cell Culture and mRNA Isolation.
K-1735 cells and BALB/c
3T3 cells were maintained in tissue culture in the DME-10
(DME supplemented with 10% fetal calf serum, sodium bicarbonate solution, L-glutamine, and penicillin-streptomycin). Cells
were maintained on plastic and were incubated in 5% CO2/95% air at 37°C. Cells at 70% confluency were harvested and subjected to mRNA extraction. mRNA was isolated using Fast Track mRNA
Isolation Kit (Invitrogen Corp., Carlsbad, CA) according to the
manufacturer's recommendations.
Screening of cDNA Library.
To isolate full-length cDNA clones,
cDNA libraries from K-1735 C-23 (low-metastatic clone) constructed in lambda ZAP II (Stratagene Corp., La Jolla, CA) using cDNA Synthesis System Plus (Amersham Corp., Arlington
Heights, IL) was screened with a cloned cDNA fragment of Elm1
that was previously isolated by an mRNA differential display (6).
Isolated clones were sequenced by the A.L.F. DNA Sequencer II
with the AutoRead Sequencing Kit (Pharmacia Biotech, Piscataway, NJ). DNA sequences were aligned, examined for open reading frames, and compared with DNA sequences in the DDBJ/
GenBank/EMBL and amino acid sequence in the SWISS-PROT
and PIR protein databases using the FASTA and BLAST programs.
Northern and Southern Blot Analyses.
3 µg of poly(A)+ RNA
were size fractionated on a denaturing formaldehyde agarose gel
(1.0%) and transferred onto Hibond-N+ membrane (Amersham
Corp.). Mouse multiple tissue Northern (MTN) blot, containing
2 µg of poly(A)+ RNA from a variety of tissues, and ZOO BLOT,
containing 8 µg of EcoRI-digested genomic DNA from various
species, were obtained from Clontech (Palo Alto, CA). Northern
blot hybridization was performed at 42°C for 24 h with hybridization buffer containing 50% formamide, 5× NET (750 mM
NaCl, 5 mM EDTA, and 75 mM Tris-HCl), 0.5% dry milk, 0.4%
SDS, 10% dextran sulfate, and 0.2 mg/ml salmon sperm DNA.
Then the membrane was washed with 0.1× SSC and 0.1% SDS
at 65°C for 60 min. Southern blot hybridization was performed at
37°C with hybridization buffer described above, and the membrane was washed with 0.1× SSC and 0.1% SDS at 55°C. To confirm
the amounts of mRNA loaded in each lane, the blots were hybridized afterwards with a human
-actin probe (Clontech). A
DNA probe for the Elm1 gene corresponded to nucleotides 33-
2399 of the Elm1 cDNA fragment. A DNA probe corresponding
to nucleotides 337-1119 of the Cyr61 mRNA (DDBJ/GenBank/EMBL accession number: M32490) was synthesized by reverse transcription PCR.
Chromosome Mapping of the Elm1 Gene.
An interspecific backcross
panel formed from (DBA/2J × MSM)F1 × DBA/2J (MSM, Mus
musculus molossinus) was used for mapping of the Elm1 gene (18).
Genotypes of Elm1 for 138 individuals in this panel were determined by RFLP observed in PCR-amplified products. A genomic
DNA fragment of 1,193 bp in the Elm1 locus was amplified by
primers 5
-CGATATCTTTGCTGACTTGG-3
(sense strand) and 5
-CTGAGGCTGTAAAGTAGGTC-3
(antisense strand),
corresponding to nucleotides 1223-1242 and 2396-2415, respectively. The restriction enzyme Sau3AI yielded an easily distinguishable polymorphism between two parental strains, MSM and
DBA/2J. The data generated in this study was analyzed (Map
Manager v 2.5.6; Roswell Park Cancer Institute, NY; reference 19).
Serum Stimulation of BALB/c 3T3 Cells.
Quiescent BALB/c 3T3
cells were prepared by growth in DME-10 to confluence followed
by incubation in DME-0.5 (0.5% serum) for 2 d. For stimulation
of quiescent cells, the medium was changed to DME-20 (20%
serum).
Construction of Expression Vector and DNA Transfection.
The
pcDNA3 expression vector was purchased from Invitrogen. A
cDNA fragment of Elm1 (146-1,309 nucleotides [nt]) consisting of 28 nts of the 5
-untranslated region, 1,101 nts of the coding region, and 35 nts of the 3
-untranslated region was amplified using the primer set Elm1-B (146-165 nts; GTAGCTCCTGTGACGCTGAC) and Elm1-C (complemented 1,290-1,309 nts;
GCATGGAACTTTACCCTGAG), and ligated to the pcDNA3
vector at the BamHI site (pcDNA3-Elm1) in the direction of plus
strand. K-1735 M-2 cells were transfected with pcDNA3-Elm1
using LipofectAMINE reagent (GIBCO BRL, Gaithersburg, MD).
After 16 h, the medium was changed to DME-10. At 38 h of transfection, G418 selection was imposed (800 µg/ml; Geneticin; GIBCO BRL). G418-resistant cells were cloned by using the
penicillin cap method and maintained in the medium containing
G418. Cell clones resistant to G418 were assayed for the expression of Elm1 mRNA by Northern blotting.
Cell Growth and Tumorigenicity.
K-1735 M-2, C-23, and transfectants were seeded at the density of 5 × 103 cells/ml in a 24-well
plate. The cells were counted every day from days 1 to 6. 5-wk-old
female C3H/HeN mice were obtained from the Animal Production of Japan Kurea Corporation (Tokyo, Japan). Tumorigenicity
was examined by injecting 106 cells/0.2 ml into the subcutis of
the mice (n = 5). The length and width of each tumor were recorded three times per week. The tumor volume (V) was calculated by the formula V = 1/2 × length × (width)2.
Experimental Metastasis Assay.
Metastatic potential of the cells
was measured by the quantitative lung colony assay as described
by Fidler et al. (20). In brief, the cells were injected into the tail
vein of 5-wk-old female C3H/HeN mice at the density of 105
cells/0.2 ml (n = 5). Mice were killed when three of five mice with K-1735 M-2 injection were dead, that is, 22 d (Table 2, experiment 1) and 23 d (Table 2, experiment 2) after cell inoculation. The number of lung metastatic colonies >1 mm in diameter
was counted with the aid of magnifying glass.
Statistical Analysis.
The in vivo data were analyzed by the
Mann-Whitney U test.
DDBJ/GenBank/EMBL Accession Numbers.
Cyr61: M32490;
mouse Nov (NovM): X96585; fibroblast-inducible secreted protein (Fisp)12: M70642.
 |
Results |
Isolation and Characterization of the Elm1 Gene.
We screened
cDNA libraries from low-metastatic K-1735 C-23 cells
constructed with oligo dT and random primers using an
Elm1 cDNA fragment of 211 bp as a probe. Several clones
were obtained, sequenced, and aligned. We have assembled a composite 5,020-bp transcript using the sequences of
seven independent clones (Fig. 1). We verified that the assembled sequence was derived from a single gene by reverse transcription PCR. Searches of the DDBJ/GenBank/ EMBL nucleotide databases indicated that this sequence has
not been reported.

View larger version (114K):
[in this window]
[in a new window]
|
Fig. 1.
Nucleotide and predicted amino acid sequences of the Elm1
gene. Amino acids are shown in their one letter form under the corresponding nucleotide sequence. An in frame 5 stop codon and the predicted termination stop codon are in bold. A potential polyadenylation signal is indicated by bold italic letters. A signal sequence is lined under
the corresponding amino acid sequence, and underlined regions with Roman numerals represent the approximate locations of the IBP-like domain, VWC domain, TSP1 domain, and a CT domain. The nucleotide
and amino acid numbering are indicated on the right side of the sequence. The nucleotide sequence data reported in this paper are available
from EMBL/GenBank/DDBJ under accession number AB004873.
|
|
The composite cDNA contained an open reading frame
of 1,101 bp with a potential start codon of ATG, which was
located 10 bp downstream of an in-frame stop codon. Using this methionine as a translation start site, a peptide of
367 amino acids (40.7 kD) was predicted (Fig. 1). Sequence
homology was detected between the predicted amino acid
sequence of the Elm1 protein and those of the CCN family
proteins. The Elm1 protein showed 38.1, 44.0, and 42.6%
identity with Cyr61, Fisp12 (mouse orthologue of CTGF; reference 10), and NovM, respectively. Elm1 has a signal
peptide that is conserved in all of the recorded CCN family
proteins. The deduced Elm1 amino acid sequence contains
a hydrophobic amino terminus with a predicted signal cleavage site between Ala (position 24) and Leu (position 25;
reference 21; Fig. 1). CCN family proteins are cysteine rich
(10% of all residues) and conserve the four domains: insulin-like growth factor binding protein (IBP)-like domain,
von Willebrand factor type C repeat (VWC), thrombospondin type 1 repeat (TSP1) domain, and COOH-terminal (CT) domain (7) (Figs. 1 and 2). The deduced Elm1
amino acid sequence contains 38 of the cysteine residue
(10.4% of all residues) and showed 40-60% identity with
Cyr61, Fisp12, NovM in each of the four domains (Fig. 2).
Similarities of the amino acid sequence outside the four domains were insignificant for any of the three subclasses. A
dendrogram indicates that Elm1 is not an orthologue of
other CCN family members (Fig. 2 B).

View larger version (45K):
[in this window]
[in a new window]
|
Fig. 2.
(A) Alignment and homology of amino acid sequences corresponding to the IBP-like, VWC, TSP1, and CT domains of the Elm1
gene and the genes of CCN family. The predicted amino acid sequence of the Elm1 protein is compared with those of the mouse Cyr61, NovM,
and mouse Fisp12 proteins. Positions of the identical amino acids are indicated by asterisks. Consensus features in the four domains are indicated in
the consensus line (C, cysteines; t, turn-like or polar [E, D, Q, N, K, R,
T, S, P, G, A]; h, hydrophobic [I, L, V, W, Y, F, M, A, G]; a, aromatic
[Y, F, W]; reference 7). The percentage values on the right side of the
four domains of Cyr61, NovM, and Fisp12 refer to the percentage identity with the corresponding domains of Elm1. (B) Dendrogram of the
members of CCN family. The dendrogram was generated using the UPGMA program (GENETYX; Shibuya-ku, Tokyo, Japan; reference 31).
The horizontal distances to the subclusters correspond to the relative degrees of sequence identities among members. Schemata for Elm1 and
three types of mammalian CCN family proteins represented by mouse
Cyr61/chicken Cef10, chicken Nov/NovM, and human CTGF/mouse
Fisp12 were shown.
|
|
The Elm1 gene is conserved among different species
(Fig. 3 A), and highly expressed in the kidney and lung,
and at lower levels in the heart, brain, spleen, liver, skeletal
muscle, and testis (Fig. 3 B). The Elm1 locus was mapped
to chromosome 15, between the D15Mit17 and D15Mit3
loci, which is not a site of known mouse CCN family genes
(22, 23; Fig. 4).

View larger version (70K):
[in this window]
[in a new window]
|
Fig. 3.
(A) Southern blot
analysis of DNA from various
species using the Elm1 cDNA
probe. Lane 1, human; lane 2,
monkey; lane 3, rat; lane 4,
mouse; lane 5, dog; lane 6, cow;
lane 7, rabbit; lane 8, chicken;
lane 9, yeast. (B) Northern blot
analysis of the Elm1 gene in various mouse tissues. Mouse multiple tissue Northern blot (Clontech) was hybridized with the
Elm1 cDNA probe (top) or a human -actin probe (bottom). Lane
1, heart; lane 2, brain; lane 3,
spleen; lane 4, lung; lane 5, liver;
lane 6, skeletal muscle; lane 7,
kidney; lane 8, testis.
|
|

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 4.
Linkage analysis of the Elm1 locus on mouse chromosome
15. (A) Haplotype data of 138 progenies of (DBA/2J × MSM)F1 × DBA/2J for the loci flanking the Elm1 locus. The microsatellite and Elm1 loci are listed at the left. Each column represents a chromosomal haplotype identified in the progenies. Filled box, MSM allele; open box, DBA/2J
allele. The number of progenies for each haplotype is listed at the bottom
of each column. (B) A genetic map around the Elm1 locus constructed
from the haplotype data. Recombination frequencies expressed as genetic
distance in centiMorgan are shown on the left.
|
|
Quiescent BALB/c 3T3 cells were stimulated by serum,
and the expression of Elm1 and Cyr61 were examined by
Northern blot analysis (Fig. 5). Elm1 was not induced
within 30 min, but was induced after 3 h of serum stimulation. Although the amount of
-actin mRNA in lane 1 quantified by densitometric analysis was about half of that
in lane 3, the relative level of Elm1 expression in lane 3, that is, the ratio of Elm1 mRNA over
-actin mRNA, was
seven times higher than that in lane 1. On the other hand, Cyr61 expression was markedly induced within 30 min
(Fig. 5).

View larger version (59K):
[in this window]
[in a new window]
|
Fig. 5.
Expression of Elm1
and Cyr61 after serum stimulation
of BALB/c 3T3 cells. 2 µg of
poly(A)+ RNA resolved electrophoretically were hybridized with
the Elm1 (A) or Cyr61 (B) cDNA
probe. The membrane was rehybridized with a human -actin
probe (C). poly(A)+ RNA from
quiescent cells (lane 1), cells
stimulated with serum for 30 min (lane 2), and 3 h (lane 3)
were loaded.
|
|
Effects of Elm1 Expression on the Growth and Metastasis of
K-1735 Cells.
A full-length Elm1 cDNA was transfected
into K-1735 M-2 cells using the pcDNA3 vector, and several G-418 resistant clones were tested for continued expression of Elm1 mRNA by Northern blot analysis (Fig. 6
and Table 1). M-2.Elm1.23-1 cells showed the highest
level of Elm1 mRNA expression among clones examined. M-2.Elm1.20-3 and M-2.Elm1.6-2 cells showed a similar
level of Elm1 expression to endogenous Elm1 expression in
low-metastatic K-1735 C-23 cells. M-2.Elm1.0-1 and
M-2.Elm1.20-2 cells showed a lower level of Elm1 expression
than endogenous Elm1 expression in K-1735 C-23 cells.
M-2.Elm1.8-3 cells did not express detectable levels of Elm1,
but expressed Neo mRNA. Thus, the level of Elm1 expression in the cloned cells was in the order of M-2.Elm1.23-1 > M-2.Elm1.20-3 > M-2.Elm1.6-2 > K-1735 C-23 > M-2.
Elm1.0-1 > M-2.Elm1.20-2 > M-2.Elm1.8-3 > K-1735
M-2 (Table 1).

View larger version (75K):
[in this window]
[in a new window]
|
Fig. 6.
Northern blot analysis of Elm1 transfectants using the
Elm1 (A), Neo (B), and -actin
(C) probes. The size of exogenous
and endogenous Elm1 transcripts
are 1.5 and 5.0 kb, respectively. Lane 1, K-1735 C-23; lane 2,
K-1735 M-2; lane 3, M-2.Elm1.
23-1; lane 4, M-2.Elm1.20-3; lane
5, M-2.Elm1.0-1; lane 6, M-2.
Elm1.6-2; lane 7, M-2. Elm1.
20-2; lane 8, M-2.Elm1.8-3.
|
|
We examined the in vitro growth properties of K-1735
M-2 and transfectants. Transfectants that express large
amounts of Elm1, M-2.Elm1.20-3, and M-2.Elm1.23-1
cells showed slightly increased population doubling time
and decreased saturation density compared to those of
K-1735 M-2 cells (Table 1). Morphological diversities were not observed among the clones in association with
the level of Elm1 expression. We next injected the clones
subcutaneously into female C3H/HeN mice to evaluate
the effect of Elm1 expression on tumorigenicity. All clones
were tumorigenic, but the incidence of tumor formation
was decreased in the M-2.Elm1.23-1 cells. Moreover, in
vivo growth rates of the transfectants became slower in
proportion to the increase in the level of Elm1 expression
(Fig. 7 and Table 1).

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 7.
In vivo growth of K-1735 M-2 cells transfected with the
Elm1 gene. (A) Experiment 1; (B) experiment 2. 106 cells were inoculated
subcutaneously into syngeneic mice and tumor growth was monitored three
times per week. Average volume in mice with tumors and SD of values
(bars) are shown.
|
|
Two independent experiments were performed to evaluate the metastatic ability of transfectants. The numbers of
metastatic colonies of the transfectants became smaller in
proportion to the increase in the level of Elm1 expression
(Table 2). In particular, the numbers of metastatic colonies
of M-2.Elm1.23-1 cells were significantly lower than those
of K-1735 M-2 cells in both experiments (Table 2). The
numbers of metastatic colonies of M-2.Elm1.20-3 cells
were significantly lower than those of K-1735 M-2 cells in
experiment 1. Low-metastatic C-23 cells did not produce
lung colonies. Metastatic colonies of M-2, M-2.Elm1.8-3,
M-2.Elm1.20-3, and M-2.Elm1.23-1 cells in experiment 1 are shown in Fig. 8.

View larger version (93K):
[in this window]
[in a new window]
|
Fig. 8.
Metastatic potential of K-1735 M-2 cells transfected with the Elm1 gene. Syngeneic mice were injected intravenously with 105 cells and killed
23 d after injection. A, K-1735 M-2; B, M-2.Elm1.8-3; C, M-2.Elm1.20-3; D, M-2.Elm1.23-1.
|
|
 |
Discussion |
We determined the full-length cDNA structure of the
Elm1 gene that was expressed in low-metastatic but not in
high-metastatic murine melanoma cells. Elm1 is a novel
mouse gene and showed a sequence similarity to the CCN
family. CCN family members have been defined as proteins with a mosaic structure because they all contain several motifs shared by various functionally unrelated proteins
(7). The Elm1 gene product highly conserved these motifs,
including the IBP-like, VWC, TSP1, and CT domains
(Fig. 2). Identity of amino acid sequence between the deduced Elm1 and the other CCN family proteins was
~40%, and a dendrogram indicated that no recorded CCN
family members would be an orthologue of Elm1. These
results suggest that the Elm1 gene belongs to a new subclass
of the CCN family.
CCN family members are considered to function in a
wide variety of biological processes, such as tissue regeneration, tumor formation, and embryonic development, as signal molecules to the extracellular matrix (7, 13, 14). CTGF
is induced by TGF-
and mediates mitogenic effects of
TGF-
on fibroblasts (13). Cyr61 is transcriptionally activated by serum growth factors in fibroblasts, secreted to the
extracellular matrix, and enhances the mitogenic effect of
basic fibroblast growth factor (bFGF) on fibroblasts and endothelial cells (10, 14). Nov was isolated from an integration site of myeloblastomatosis-associated virus (MAV) and
overexpressed in MAV-induced nephroblastoma (9). CTGF
and Cyr61 are classified as immediate early genes involved in the control of cell proliferation because they are induced within 30 min after serum stimulation. (10, 12). In contrast to CTGF and Cyr61, Nov is downregulated in proliferating fibroblasts (24). Elm1 was induced by serum, but not
induced within 30 min. Thus, Elm1 would play a different
role from the other CCN family members in proliferating
fibroblasts. CTGF is a mitogenic growth factor (8). Cyr61
itself does not act as a mitogen, but has the ability to enhance the effect of bFGF on DNA synthesis (14). Nov protein has a growth suppressor activity in fibroblasts (9). To
examine whether Elm1 has such a growth regulatory function, we performed thymidine incorporation assay using
sample mediums of Elm1-transfected K-1735 M-2 cells.
However, we could not detect mitogenic activity of Elm1
by itself or the ability of Elm1 to enhance the effect of
bFGF on NIH3T3 cells (data not shown). Thus, it is unlikely that Elm1 itself has growth regulatory function on
the NIH3T3 fibroblast. However, it is possible that Elm1
might enhance the mitogenic effect of growth factors other
than bFGF.
We found that Elm1 gene transaction resulted in an inhibition of in vivo growth and metastasis formation. Expression of Elm1 had little suppressive effect on in vitro
growth, but a marked suppressive effect on in vivo growth.
Suppressive effect of Elm1 on metastatic potential was associated with suppressive effect of Elm1 on in vivo growth.
In M-2.Elm1.23-1 and M-2.Elm1.20-3 cells that express
high levels of Elm1, a reduction in the incidence of subcutaneous tumor formation as well as metastatic formation was
observed. The reason why the cells failed to form tumors is presently unknown, but may result from alterations in tumor cell-host interactions, such as angiogenesis and/or responses to stimulatory and suppressive cytokines (25). TSP1
inhibits angiogenesis and modulates endothelial cell adhesion, motility, and growth. The antiproliferative activity of
TSP1 is mimicked by a synthetic peptide derived from the
type 1 repeats of TSP1 that antagonizes fibroblast growth factor and induces programmed cell death in bovine aortic endothelial cells (26). Thus, it is possible that an inhibition of
angiogenesis by the TSP1 domain of Elm1 leads to the suppression of in vivo growth. Further studies are now in progress on this subject.
Elm1 was isolated as a gene differentially expressed between high- and low-metastatic K-1735 mouse melanoma
cells. In K-1735 systems, actin organization, cell adhesion,
motility, and a growth rate at a subcutaneous site have been
shown to be different between high- and low-metastastic
cells (27). Elm1 could be a gene that is involved in the
growth of K-1735 cells in vivo. It is noted that the Elm1
gene is one of several genes involved in the regulation of
metastatic potential in K-1735 cells since it was previously shown that expression of the inducible nitric oxide synthase and nm23 genes suppresses tumorigenicity and metastasis of K-1735 cells (25, 30). These sets of genes would
cooperatively affect the metastatic potential of K-1735
cells. A more detailed characterization of the Elm1 gene
should allow a critical analysis of the molecular mechanism
of metastasis in K-1735 cells.
Address correspondence to Jun Yokota, Biology Division, National Cancer Center Research Institute, 1-1, Tsukiji 5-chome, Chuo-ku, Tokyo 104, Japan. Phone: 81-3-3542-2511, ext. 4650; Fax: 81-3-3542-0807;
E-mail: jyokota{at}gan2.ncc.go.jp
We thank Dr. Isaiah J. Fidler of M.D. Anderson Cancer Center (University of Texas, Houston, TX) for
providing K-1735 cells and Dr. Y. Yaoi of National Cancer Center Research Institute (Chuo-ku, Tokyo, Japan) for providing BALB/c 3T3 cells.
This work was supported in part by Grants-in-Aid from the Ministry of Health and Welfare and the Ministry of Education, Science, Sports and Culture of Japan. Y. Nagamachi is a recipient of the research resident
fellowship from the Foundation for Promotion of Cancer Research.
1.
|
Bishop, J.M..
1987.
The molecular genetics of cancer.
Science.
235:
305-311
[Medline].
|
2.
|
Fidler, I.J..
1990.
Critical factors in the biology of human cancer metastasis: twenty-eighth G.H.A. Clowes memorial award
lecture.
Cancer Res.
50:
6130-6138
[Abstract].
|
3.
|
Fidler, I.J., and
R. Radinsky.
1990.
Genetic control of cancer
metastasis.
J. Natl. Cancer Inst.
82:
166-168
[Medline].
|
4.
|
Kerbel, R.S..
1989.
Towards an understanding of the molecular basis of the metastatic phenotype.
Invasion Metastasis.
9:
329-337
[Medline].
|
5.
|
Liotta, L.A., and
W.G. Stetler-Stevenson.
1991.
Tumor invasion and metastasis: an imbalance of positive and negative regulation.
Cancer Res.
51:
5054s-5059s
[Abstract].
|
6.
|
Hashimoto, Y.,
N. Shindo-Okada,
M. Tani,
K. Takeuchi,
H. Toma, and
J. Yokota.
1996.
Identification of genes differentially expressed in association with metastatic potential of
K-1735 murine melanoma by messenger RNA differential display.
Cancer Res.
56:
5266-5271
[Abstract].
|
7.
|
Bork, P..
1993.
The modular architecture of a new family of
growth regulators related to connective tissue growth factor.
FEBS Lett.
327:
125-130
[Medline].
|
8.
|
Bradham, D.M.,
A. Igarashi,
R.L. Potter, and
G.R. Grotendorst.
1991.
Connective tissue growth factor: a cysteine-rich
mitogen secreted by human vascular endothelial cells is related to the SRC-induced immediate early gene product
CEF-10.
J. Cell Biol.
114:
1285-1294
[Abstract].
|
9.
|
Joliot, V.,
C. Martinerie,
G. Dambrine,
G. Plassiart,
M. Brisac,
J. Crochet, and
B. Perbal.
1992.
Proviral rearrangements and overexpression of a new cellular gene (nov) in myeloblastosis-associated virus type 1-induced nephroblastomas.
Mol. Cell. Biol.
12:
10-21
[Abstract].
|
10.
|
O'Brien, T.P.,
G.P. Yang,
L. Sanders, and
L.F. Lau.
1990.
Expression of cyr61, a growth factor-inducible immediate-early gene.
Mol. Cell. Biol.
10:
3569-3577
[Medline].
|
11.
|
Simmons, D.L.,
D.B. Levy,
Y. Yannoni, and
R.L. Erikson.
1989.
Identification of a phorbol ester-repressible v-src-inducible gene.
Proc. Natl. Acad. Sci. USA.
86:
1178-1182
[Abstract].
|
12.
|
Igarashi, A.,
H. Okochi,
D.M. Bradham, and
G.R. Grotendorst.
1993.
Regulation of connective tissue growth factor
gene expression in human skin fibroblasts and during wound
repair.
Mol. Cell. Biol.
4:
637-645
.
|
13.
|
Kothapalli, D.,
K.S. Frazier,
A. Welply,
P.R. Segarini, and
G.R. Grotendorst.
1997.
Transforming growth factor induced anchorage-independent growth of NRK fibroblasts via
a connective tissue growth factor-dependent signal pathway.
Cell Growth Differ.
8:
61-68
[Abstract].
|
14.
|
Kireeva, M.L.,
F.E. Mo,
G.P. Yang, and
L.F. Lau.
1996.
Cyr61, a product of a growth factor-inducible immediate-early gene, promotes cell proliferation, migration, and adhesion.
Mol. Cell. Biol.
16:
1326-1334
[Abstract].
|
15.
|
Fidler, I.J., and
J.E. Talmadge.
1986.
Evidence that intravenously derived murine pulmonary melanoma metastases can
originate from the expansion of a single tumor cell.
Cancer Res.
46:
5167-5171
[Abstract].
|
16.
|
Aukerman, S.L.,
J.E. Price, and
I.J. Fidler.
1986.
Different deficiencies in the prevention of tumorigenic-low-metastatic
murine K-1735b melanoma cells from producing metastases.
J. Natl. Cancer Inst.
77:
915-924
[Medline].
|
17.
|
Talmadge, J.E., and
I.J. Fidler.
1982.
Enhanced metastatic
potential of tumor cells harvested from spontaneous metastases of heterogeneous murine tumors.
J. Natl. Cancer Inst.
69:
975-980
[Medline].
|
18.
| Wakana, S., T. Shiroishi, N. Miyashita, K. Moriwaki, H. Kaneda, M. Okamoto, H. Yonekawa, S. Tsuyoshi, I. Oyanagi, and R. Kominami. 1994. High resoution linkage map
of mouse chromosome 11 consisting of microsatellite markers
based on single intersubspecific backcross. In Genetics in Wild
Mice. K. Moriwaki, T. Shiroishi, and H. Yonekawa, editors.
Japan Sci. Soc. Press, Tokyo. 299-302.
|
19.
|
Manly, K.F., and
R.W. Elliott.
1991.
RI Manager, a microcomputer program for analysis of data from recombinant inbred strains.
Mamm. Genome.
1:
123-126
[Medline].
|
20.
|
Kripke, M.L..
1979.
Speculations on the role of ultraviolet radiation in the development of malignant melanoma.
J. Natl.
Cancer Inst.
63:
541-548
[Medline].
|
21.
|
von Heijne, G..
1983.
Patterns of amino acids near signal-sequence cleavage sites.
Eur. J. Biochem.
133:
17-21
[Abstract].
|
22.
|
Martinerie, C.,
G. Chevalier,
F.J. Rauscher III, and
B. Perbal.
1996.
Regulation of nov by WT1: a potential role for nov
in nephrogenesis.
Oncogene.
12:
1479-1492
[Medline].
|
23.
|
Ryseck, R.P.,
H. Macdonald-Bravo,
M.G. Mattei, and
R. Bravo.
1991.
Structure, mapping, and expression of fisp-12, a
growth factor-inducible gene encoding a secreted cysteine-rich protein.
Cell Growth Differ.
2:
225-233
[Abstract].
|
24.
|
Scholz, G.,
C. Martinerie,
B. Perbal, and
H. Hanafusa.
1996.
Transcriptional down regulation of the nov proto-oncogene
in fibroblasts transformed by p60v-src.
Mol. Cell. Biol.
16:
481-486
[Abstract].
|
25.
|
Leone, A.,
U. Flatow,
C.R. King,
M.A. Sandeen,
I.M. Margulies,
L.A. Liotta, and
P.S. Steeg.
1991.
Reduced tumor incidence, metastatic potential, and cytokine responsiveness of
nm23-transfected melanoma cells.
Cell.
65:
25-35
[Medline].
|
26.
|
Guo, N.,
H.C. Krutzsch,
J.K. Inman, and
D.D. Roberts.
1997.
Thrombospondin 1 and type 1 repeat peptides of Thrombospondin 1 specifically induced apoptosis of endothelial cells.
Cancer Res.
57:
1735-1742
[Abstract].
|
27.
|
Staroselsky, A.H.,
S. Pathak,
Y. Chernajovsky,
S.L. Tucker, and
I.J. Fidler.
1991.
Predominance of the metastatic phenotype in somatic cell hybrids of the K-1735 murine melanoma.
Cancer Res.
51:
6292-6298
[Abstract].
|
28.
|
Raz, A., and
B. Geiger.
1982.
Altered organization of cell-substrate contacts and membrane-associated cytoskeleton in
tumor cell variants exhibiting different metastatic capabilities.
Cancer Res.
42:
5183-5190
[Abstract].
|
29.
|
Hart, I.R.,
J.E. Talmadge, and
I.J. Fidler.
1983.
Comparative studies on the quantitative analysis of experimental metastatic capacity.
Cancer Res.
43:
400-402
[Abstract].
|
30.
|
Xie, K.,
S. Huang,
Z. Dong,
S.H. Juang,
M. Gutman,
Q.W. Xie,
C. Nathan, and
I.J. Fidler.
1995.
Transfection with the
inducible nitric oxide synthase gene suppresses tumorigenicity and abrogates metastasis by K-1735 murine melanoma
cells.
J. Exp. Med.
181:
1333-1343
[Abstract].
|
31.
| Sneath, P.H.A., and R.R. Sokal. 1973. The principles and
practice of numerical classification. In Numerical Taxonomy. W.H. Freeman and Co., San Francisco.
|