Previous Article | Next Article 
J Virol, March 1998, p. 2532-2537, Vol. 72, No. 3
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Suppression of v-src Transformation by
the drs Gene
Hirokazu
Inoue,*
Jing
Pan, and
Akira
Hakura
Department of Tumor Virology, Research
Institute for Microbial Diseases, Osaka University, Osaka 565, Japan
Received 2 September 1997/Accepted 6 December 1997
 |
ABSTRACT |
Previously, we isolated a novel gene, drs, which was
downregulated by retroviral oncogenes such as v-src and
v-K-ras, from a cDNA library of primary rat embryo
fibroblasts. Experiments using a temperature-sensitive mutant of the
v-src gene indicated that downregulation of drs
mRNA was dependent on functional expression of v-Src. In addition,
expression of drs mRNA was also reduced by serum
stimulation of G0-arrested normal rat fibroblast cells. To
clarify the function of the drs gene in cell transformation and proliferation, we introduced drs linked to a potent
promoter into a normal rat cell line, F2408, and examined the effect of ectopic expression of exogenous drs on the transformation
by the v-src gene and growth properties. Cells expressing
exogenous drs gene showed significantly decreased
efficiency of transformation by v-src irrespective of
functional expression of v-Src kinase, while the growth rate and
G1/S progression of the cells were not suppressed by
expression of exogenous drs gene, indicating that drs has the ability to suppress v-src
transformation without disturbing cell proliferation.
| |
TEXT |
Transformation by viral oncogenes
induces a variety of cellular changes such as cell rounding, loss of
contact inhibition, decrease of serum requirement for cell
proliferation, and anchorage-independent growth. The v-src
oncogene of Rous sarcoma virus (RSV) has been most intensively
investigated (29). The product of v-src is a
membrane-associated phosphoprotein, pp60v-src,
which has tyrosine-specific protein kinase activity (9, 20, 32). The v-src gene also positively or negatively
alters the expression of many cellular genes (1, 6, 12, 14, 16, 28, 33, 40, 44, 45, 49). A few of the genes whose expression is
negatively regulated by v-src have been shown to function as
tumor suppressor genes (11, 33, 41). We have reported that a
suppressive factor(s) for v-src transformation is expressed
in primary rat embryo fibroblasts (REF) (25, 51, 52). On
searching for such transformation suppressor genes, we recently
isolated a novel gene, drs (downregulated by
v-src), which was expressed in normal rat fibroblast cells
but completely suppressed in the cells transformed by the
v-src gene, from a cDNA library of REF (42). The
drs gene was also demonstrated to be downregulated by other
retroviral oncogenes such as v-fps, v-ras,
v-mos, v-sis, and v-abl. The
molecularly cloned cDNA of drs was about 1.8 kb in size,
containing an open reading frame composed of 464 amino acid residues.
This protein had one transmembrane domain in the C terminus and three
consensus repeats conserved in various numbers in the extracellular
domain among the selectin family of adhesion molecules and complement
binding proteins (4, 30). Marked downregulation of
drs mRNA in oncogene-transformed cells suggests that the
drs gene plays a role in suppression of transformation.
To clarify the function of drs for cell transformation,
we initially investigated the correlation between
downregulation of drs mRNA and functional expression of the
v-src gene. To perform this experiment, we used a rat
F2408 cell clone (OS7-2) containing a temperature-sensitive
mutant (OS122) of RSV (13, 38, 39, 54). As shown in
Fig. 1, OS7-2 displays round
refractile morphology typical of transformed cells when incubated at
35°C. When the temperature was shifted to 39°C from 35°C, the
morphology of OS7-2 was gradually converted to a flat phenotype within
24 h. Tyrosine kinase activity of v-Src by in vitro kinase assay
with anti-Src serum was also reduced by temperature shift from 35 to
39°C (Fig. 2A). The change of tyrosine
kinase activity roughly paralleled the morphological change in OS7-2
cells. The level of drs mRNA gradually increased after the
temperature increase to 39°C in parallel with the decrease of the
kinase activity in OS7-2 cells (Fig. 2B). In the F2408 cell clone S7-1
(23), containing wild-type RSV, the transformed morphology, tyrosine
kinase activity, and level of drs mRNA were not changed by
temperature shift (Fig. 1 and 2). The expression of drs mRNA
in untransformed F2408 cells was also not affected by temperature shift
(Fig. 2B). These results indicate that downregulation of drs
mRNA depends on functional expression of v-Src tyrosine kinase and
parallels the expression of transformed morphology.
View larger version (48K):
[in this window]
[in a new window]
|
FIG. 1.
Morphological change of OS7-2 cells by temperature
shifting. F2408 is a rat fibroblast cell line established from REF of
Fisher rat (13). S7-1 is an F2408 cell line transformed by
the SR-D strain of RSV (23). OS7-2 is an F2408 cell line
containing a temperature-sensitive mutant (OS122) of RSV (38, 39,
54). The cells were cultured in Dulbecco's modified Eagle's
medium supplemented with 5% FCS (growth medium). OS7-2, S7-1, and
F2408 cells were inoculated into plastic dishes containing growth
medium and incubated at 35°C (permissive temperature). After
incubation for 24 h, the culture temperature was increased to
39°C. At 0, 4, 8, 12, 16, 20, and 24 h after temperature
shifting (0 and 24 h for S7-1 and F2408), cell morphologies were
observed with a phase-contrast microscope and photographed.
|
|
View larger version (62K):
[in this window]
[in a new window]
|
FIG. 2.
Alterations of protein kinase activity of v-Src (A) and
expression of drs mRNA (B) by temperature shifting in OS7-2
cells. (A) In vitro protein kinase assay. Temperature shifting was
performed as described in the legend to Fig. 1. For examination of
protein kinase activity of v-Src, cells were lysed in
radioimmunoprecipitation assay (RIPA) buffer (1% Triton X-100, 1%
sodium deoxycholate, 0.1% sodium dodecyl sulfate [SDS], 20 mM
Tris-HCl [pH 7.4], 150 mM sodium chloride, 5 mM EDTA, 1 mM
Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 20 µg of aprotinin/ml) and centrifuged at 13,000 × g
for 30 min at 4°C. The resulting supernatant was used for kinase
assay of v-Src as described by Inoue et al. (22). Cell
extracts containing 100 µg of protein were immunoprecipitated with 5 µl of anti-Src serum for 1 h at 4°C. The immunocomplexes were
bound to protein A-Sepharose, washed three times with RIPA buffer, and
suspended in 20 µl of kinase buffer (20 mM Tris-HCl [pH 7.4], 10 mM
MnCl2) containing 5 µCi of [ -32P]ATP
(3,000 Ci/mmol; Amersham). After incubation for 10 min at 20°C, the
reaction was stopped by addition of 15 µl of 4× Laemmli-SDS buffer
(0.25 M Tris-Cl [pH 6.8], 40% glycerol, 8% SDS, 20%
2-mercaptoethanol, 0.01% bromophenol blue) and boiled for 2 min. The
reaction mixtures were centrifuged at 10,000 × g for 2 min, and samples of the supernatant were analyzed by SDS-polyacrylamide
gel electrophoresis. The position of the phosphorylated immunoglobulin
G (IgG) heavy chain is indicated. (B) Northern blot analysis. Cellular
RNA was isolated from cells by the guanidium isothiocyanate-cesium
chloride method (8). Samples (20 µg) of total RNA from the
cells were subjected to electrophoresis on a 1% agarose gel containing
2.2 M formaldehyde and transferred to a nylon filter. The filter was
hybridized at 42°C overnight in a solution containing 50% formamide,
0.6 M sodium chloride, 60 mM sodium citrate, 0.2% SDS, 0.1% bovine
serum albumin, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, and 50 µg of
herring sperm DNA/ml with a labeled DNA probe. The hybridized filter
was washed with 15 mM sodium chloride-1.5 mM sodium citrate-0.1% SDS
at 50°C and autoradiographed. Probes used in this experiment were a
drs DNA fragment and a human  -actin DNA fragment
(37).
|
|
To examine whether drs mRNA expression varies with cell
cycle progression, F2408 cells arrested in G0 phase by
serum starvation were stimulated by serum (10% fetal calf serum
[FCS]) and the change in the amount of drs mRNA was
examined (Fig. 3). Under these
conditions, G0-arrested F2408 cells synchronously
progressed to S phase 12 h after serum stimulation
(26). Expression of drs mRNA was gradually
reduced until 5 h after serum stimulation (Fig. 3). The level of
drs mRNA was unchanged from 5 to 18 h (the peak of S
phase) (Fig. 3). These results suggest that expression of the
drs gene is also regulated during the cell cycle.
View larger version (38K):
[in this window]
[in a new window]
|
FIG. 3.
Expression of drs mRNA during cell cycle
progression of F2408 cells. Confluent cultures of F2408 cells were
serum starved for 24 h and then supplemented with 10% FCS. At 0, 1, 2, 3, 4, 5, 12, and 18 h after serum stimulation, cellular RNA
was isolated. Isolation of cellular RNA and Northern blot hybridization
were carried out as described in the legend to Fig. 2B.
|
|
To assess the function of the drs gene in v-src
transformation and cell proliferation, we constructed a recombinant
plasmid, pSR
Neo/drs, that expresses the drs gene under
the potent promoter-enhancer SR (50), and transfected
this plasmid into an F2408 cell line. After G418 selection, six
independent G418-resistant clones (F-drs-2, -3, -4, -5, -7, and -10)
were isolated, and expression of exogenous drs gene in these
clones was examined by Northern blot hybridization. As shown in Fig.
4A, three clones (F-drs-2, -4, and -7)
expressed high levels of exogenous drs mRNA (upper band) in
addition to expressing endogenous drs mRNA (lower band),
while another three clones (F-drs-3, -5, and -10) expressed only
endogenous drs mRNA. The cell morphologies of F2408 and the
six G418-resistant clones were similar (data not shown). To clarify
whether the drs gene has the activity to suppress
v-src transformation, F2408 and these clones were infected
with a high titer of a recombinant murine retrovirus (MRSV) containing
the v-src gene (2), and the colony-forming abilities in soft agar and the focus-forming abilities in liquid culture of these cells were investigated. As shown in Fig.
5A, the colony-forming efficiencies of
the clones expressing exogenous drs (F-drs-2, -4, and -7)
were significantly lower than those of F2408 and the clones expressing
only endogenous drs. Focus-forming efficiencies of F-drs-2,
F-drs-4, and F-drs-7 by MRSV were also markedly decreased compared with
those of F2408 and F-drs-10 (Fig. 5B). These results suggest that the
drs gene acts to suppress v-src transformation.
To exclude the possibility that expression of functional v-Src protein
was inhibited in the clones expressing exogenous drs gene,
the tyrosine kinase activity of v-Src protein in MRSV-infected cells
was examined by in vitro protein kinase assay with anti-Src serum.
Figure 6 shows the results. v-Src kinase activities of F-drs-2, -4, and -7 infected with MRSV were not reduced
compared with those of MRSV-infected F2408, F-drs-3, -5, and -10, indicating that suppression of v-src transformation in the
clones expressing exogenous drs gene is not due to the
reduced expression of v-Src tyrosine kinase. We also examined the
expression of exogenous and endogenous drs mRNA in mock- and
MRSV-infected F-drs-7 cells. As shown in Fig. 4B, the level of
endogenous drs mRNA was reduced by MRSV infection, whereas
the expression of exogenous drs mRNA driven from the SR
promoter was not affected by MRSV, confirming that v-src
certainly functions to downregulate endogenous drs in
MRSV-infected F2408 cells expressing exogenous drs. However,
the drs gene driven from the exogenous promoter was not
downregulated by v-src. This result further supports our conclusion that an ectopically expressed exogenous drs acts
to suppress transformation by v-src.
View larger version (47K):
[in this window]
[in a new window]
|
FIG. 4.
Expression of endogenous and exogenous drs
mRNA in F2408 and F-drs cells (A and C) and the effect of MRSV
infection on drs mRNA expression (B). (A) The expression
vector in this experiment was pSRNeo, which contains the SR
promoter (50) for efficient expression of inserted cDNA and
the neomycin-resistant gene as a selective marker. A 1.8-kb cDNA
fragment (BamHI) containing the open reading frame of the
drs gene was inserted into the BamHI-cleaved
pSRNeo vector. The recombinant plasmid, pSRNeo/drs, in which a
drs gene was inserted in the sense orientation, was used for
DNA transfection experiments. pSRNeo/drs plasmid DNA was introduced
into F2408 by the calcium-phosphate transfection method
(17), and G418-resistant clones were isolated. Isolation of
cellular RNA and Northern blot hybridization were carried out as
described in the legend to Fig. 2B. (B) Cellular RNA was isolated 3 days after MRSV infection. The upper and lower bands indicate exogenous
and endogenous drs transcripts, respectively. (C) A 1.8-kb
drs cDNA fragment was inserted into BamHI-cleaved
pBabePuro retrovirus vector (36) in the sense orientation.
The recombinant plasmid, pBabePuro-drs, was introduced into a packaging
cell line,  2 (34), by DNA transfection, and
puromycin-resistant clones containing the drs gene were
isolated. F2408 cells were infected with culture medium of cells
containing pBabePuro-drs or vector (pBabePuro) virus, and
puromycin-resistant cells were pooled (F/pBP-drs and F/pBP).
|
|
View larger version (31K):
[in this window]
[in a new window]
|
FIG. 5.
Transformation of F2408 and F-drs cells by MRSV and
Ki-MSV. F2408 and F-drs clones transfected with pSRNeo/drs plasmid
were infected with MRSV (A and B) or Ki-MSV (D and E). F/pBP and
F/pBP-drs cells were infected with MRSV (C). For virus infection,
inocula of 2 × 105 cells were plated in
60-mm-diameter plastic dishes with growth medium and incubated
overnight at 37°C. After treatment of the cultures with Polybrene (2 µg/ml) for 30 min at 37°C, the medium was removed and 0.3 ml of
virus preparation was added to each culture. After adsorption for
1 h at 37°C, the cultures were covered with growth medium and
incubated at 37°C. For soft agar assays (A, C, and D), 3 days after
virus infection, the cells were trypsinized and portions of
104 cells were inoculated into 0.4% soft agar. Colonies
were scored after incubation for 2 weeks. For focus assays (B and E),
transformed foci were scored 10 days after virus infection.
|
|
View larger version (49K):
[in this window]
[in a new window]
|
FIG. 6.
v-Src kinase activities in MRSV-infected F2408 and F-drs
cells. Three days after MRSV infection, the mock- and MRSV-infected
cells were lysed in RIPA buffer. The cell lysates containing 100 µg
of protein were used for protein kinase assay of v-Src as described in
the legend to Fig. 2A.
|
|
To further confirm the correlation between expression of
exogenous drs and suppression of transformation by
v-src, we constructed a recombinant retrovirus
containing the drs gene and the puromycin-resistant gene as
a selective marker (pBabePuro-drs) and infected F2408 cells with
the virus. After incubation in selection medium containing 1 µg of
puromycin/ml, puromycin-resistant cells were pooled, infected with
MRSV, and inoculated into soft agar. F2408 cells infected with
pBabePuro-drs virus expressed a considerable amount of viral mRNA
hybridized with drs cDNA (Fig. 4C). As shown in Fig. 5C, the
colony-forming efficiency of F2408 cells containing pBabePuro-drs virus
(F/pBP-drs) by MRSV was markedly decreased compared with that of F2408
cells containing vector virus (F/pBP). v-Src kinase activities in
MRSV-infected F/pBP and F/pBP-drs cells were also almost similar (Fig.
6), confirming the suppression function of drs for
v-src transformation. In addition, we also examined colony formation in soft agar and focus formation of F2408, F-drs-2, F-drs-4,
F-drs-7, and F-drs-10 cells by a murine retrovirus containing v-K-ras (Ki-MSV). As shown in Fig. 5D (soft agar assay) and
E (focus assay), transformation efficiencies of F-drs-2, F-drs-4 and
F-drs-7 were also significantly decreased compared with those of F2408
and F-drs-10. These results, together with those of v-src transformation, indicate that ectopic expression of the drs
gene suppresses transformation by viral oncogenes such as
v-src and v-K-ras.
To investigate the effect of ectopic expression of the drs
gene on growth properties of F2408 cells, we examined the growth rates
of F2408, F-drs-2, F-drs-7, and F-drs-10 cells. As shown in Fig.
7A, expression of exogenous
drs gene did not affect the growth rate of the cells. The
growth rates of F/pBP and F/pBP-drs cells were also similar (data not
shown). To examine whether overexpression of exogenous drs
gene suppresses G1/S progression of the cell cycle, F2408,
F-drs-2, F-drs-7, and F-drs-10 cells were arrested in G0
phase by serum starvation and stimulated with serum. Progression of the
cell cycle of these cells was examined by flow cytometry-activated cell
sorting (Fig. 7B). The entry into S phase at 18 h after serum stimulation was not reduced by expression of exogenous drs
gene in F-drs-2 and F-drs-7 cells compared with that of F2408 and
F-drs-10 cells, indicating that the exogenous drs gene does
not suppress G1/S progression of the cell cycle. From these
results, we concluded that the drs gene acts to suppress
transformation induced by v-src and v-ras without
affecting cell proliferation in F2408 cells.
View larger version (20K):
[in this window]
[in a new window]
|
FIG. 7.
Growth curves (A) and cell cycle analyses (B) of F2408
and F-drs clones. Cell cycle analysis was carried out by measuring the
cellular DNA content by flow cytometry-activated cell sorting as
previously described (26). To isolate and stain the cell
nuclei, the Cycle TEST PLUS DNA Reagent Kit (Becton Dickinson) was
used. Cells were washed with phosphate-buffered saline, suspended in
sodium citrate buffer, quickly frozen in a bath of dry ice-methanol,
and stored at  80°C until use. The cells were thawed, and their DNA
was stained with Cycle TEST reagent by the procedures recommended by
the manufacturer. The fluorescence of the cells was measured with a
FACScan system (Becton Dickinson), and the percentages of the cells in
G1, S, and G2/M phases were determined by the
CellFit program (Becton Dickinson).
|
|
Previously, we showed that drs mRNA was markedly reduced in
rat cell lines transformed by v-src, v-fps,
v-ras, v-mos, v-sis, v-abl,
or middle T antigen of polyomavirus but not in cell lines transformed
by large T antigen of simian virus 40 or the E6 and E7 genes of human
papillomavirus type 16 (24, 42). As the oncogenes that
reduced drs mRNA are considered to act upstream of the
p42/p44 mitogen-activated protein (MAP) kinase pathway (19,
43), we speculated that expression of the drs gene is negatively regulated by mitogenic signals from growth factors downstream of the MAP kinase pathway but upstream of the cyclin/CDK-Rb pathway. In fact, as shown in Fig. 3, the level of drs mRNA
was considerably reduced by serum stimulation of
G0-arrested cells although the expression was not
completely suppressed. This result suggested that downregulation of
drs mRNA by mitogenic signals plays a role in the
progression of the cell cycle. However, overexpression of the exogenous
drs gene by the SR promoter did not affect cell proliferation (Fig. 7). Although mitogenic factors in the serum act to
modestly regulate the expression of drs gene, the
downregulation might not be critical for G1/S progression
of the cell cycle. However, we cannot completely rule out the
possibility that the level of exogenous drs mRNA is not
sufficient to affect cell proliferation in this cell line.
Figure 4B shows that introduction of the v-src gene reduced
the level of endogenous drs mRNA but did not affect that of
exogenous drs mRNA driven by the SR promoter. This result
also implies that downregulation of the drs gene by
v-src is caused by transcriptional repression in the 5'
regulatory region of the drs gene. Activated MAP kinase
moves into the nucleus and acts to regulate gene transcription (19, 43). Viral oncogenes such as v-src and
mitogenic factors in serum may repress transcription of the
drs gene through the MAP kinase pathway. The mechanism of
downregulation of the drs gene by oncogenes and mitogens
still remains to be worked out.
Introduction of the v-src gene into normal cells results in
multiple cellular events including morphological change, activation of
the mitogenic pathway, and anchorage-independent growth. Overexpression of the drs gene with the SR promoter significantly
suppressed anchorage-independent growth and focus formation by
v-src without disturbing usual cell proliferation.
Expression of v-Src tyrosine kinase by MRSV infection was not affected
by ectopic expression of the drs gene (Fig. 6), indicating
that drs acts to suppress v-src transformation
after function of v-Src kinase. The most prominent biochemical change
induced by v-Src is an extensive tyrosine phosphorylation of cellular
proteins (29). Most of these target proteins of v-Src kinase
are localized in the focal adhesions linked to the plasma membrane.
Deregulated phosphorylation of these focal adhesion proteins, such as
paxillin, focal adhesion kinase, talin, and tensin, is considered to be
the cause of rounding and disordered proliferation of the cells. The
gene structure of drs implies the membrane-associated
localization of Drs protein. It seems possible that the product of the
drs gene interacts with these focal adhesion proteins at the
membrane and interferes with transformation by v-Src. The
drs gene also contains repeated motifs conserved in the
extracellular domain among selectin family adhesion molecules (4,
30). Three selectins (L-selectin, E-selectin, and P-selectin) are
included in this family. They share a similar molecular structure,
consisting of an amino-terminal C-type lectin domain, an epidermal
growth factor-like domain, from two to nine short complement regulatory
repeats, a transmembrane domain, and a short cytoplasmic tail (5,
27, 31, 48, 53). The complement regulatory repeats of the
selectin family had homology with three consensus repeats of the
drs gene (42). The selectin family is considered
to be crucial for the initial step of leukocyte-endothelial interaction
in response to inflamatory stimuli such as injury and infection
(4, 30). Recently, in addition to adhesion function,
E-selectin was shown to associate with actin-associated proteins such
as -actinin, vinculin, filamin, paxillin, and focal adhesion kinase
(57). L-selectin has also been reported to act as a
signaling molecule which activates MAP kinase and Ras pathways through
tyrosine phosphorylation (7, 56). In this regard, selectins
resemble another family of adhesion molecules, integrins. The
integrin-mediated signaling pathway is thought to play an important
role in adhesion-dependent cell cycle progression as well as regulation
of the cytoskeleton (3, 21, 46, 47). Accumulating evidence
indicates that some of the adhesion molecules localized in the plasma
membrane are able to act as tumor suppressor genes (15, 18,
55). The drs gene may also function as a receptor for
adhesion signaling and be involved in the anchorage-dependent pathway.
Further investigation of the drs gene is necessary to clarify the mechanism of suppression of transformation. Recently, we
found that the drs gene is highly homologous (80% in
nucleotide sequence) to a human gene which is deleted in patients with
X-linked retinitis pigmentosa (10, 35), suggesting that
drs has significant physiological functions in a variety of
cell types.
| |
ACKNOWLEDGMENTS |
This work was supported by a Grant-in-Aid for Special Project
Research, Cancer-Bioscience, from the Ministry of Education, Science,
Sports, and Culture of Japan.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Tumor Virology, Research Institute for Microbial Diseases, Osaka
University, 3-1 Yamadaoka, Suita, Osaka 565, Japan. Phone:
81-6-879-8313. Fax: 81-6-879-8315. E-mail:
hiroinou{at}biken.osaka-u.ac.jp.
| |
REFERENCES |
| 1.
|
Adams, S. L.,
J. A. Alwine,
B. de Crombrugghe, and I. Pastan.
1979.
Use of recombinant plasmids to characterize collagen RNAs in normal and transformed chicken embryo fibroblasts.
J. Biol. Chem.
254:4935-4938[Abstract/Free Full Text].
|
| 2.
|
Anderson, S. M., and E. M. Scolnick.
1983.
Construction and isolation of a transforming murine retrovirus containing the src gene of Rous sarcoma virus.
J. Virol.
46:594-605[Abstract/Free Full Text].
|
| 3.
|
Assoian, R. K.
1997.
Anchorage-dependent cell cycle progression.
J. Cell Biol.
136:1-4[Free Full Text].
|
| 4.
|
Bevilacqua, M. P.
1993.
Endothelial-leukocyte adhesion molecules.
Annu. Rev. Immunol.
11:767-804[Medline].
|
| 5.
|
Bevilacqua, M. P.,
S. Stenselin,
M. A. Gimbrone, Jr., and B. Reed.
1989.
Endothelial leukocyte adhesion molecule 1: an inducible receptor for neutrophils related to complement regulatory proteins and lectins.
Science
243:1160-1165[Abstract/Free Full Text].
|
| 6.
|
Birdenall-Roberts, M. C.,
F. W. Ruscetti,
J. Kasper,
H.-D. Lee,
R. Friedman,
A. Geiser,
M. B. Sporn,
A. B. Roberts, and S.-J. Kim.
1990.
Transcriptional regulation of the transforming growth factor 1 promoter by v-src gene products is mediated through the AP-1 complex.
Mol. Cell. Biol.
10:4978-4983[Abstract/Free Full Text].
|
| 7.
|
Brenner, B.,
E. Gulbins,
K. Schlottmann,
U. Koppenhoefer,
G. L. Busch,
B. Walzog,
H. Steinhausen,
K. M. Coggeshall,
O. Linderkamp, and F. Lang.
1996.
L-selectin activates the ras pathway via the tyrosine kinase p56lck.
Proc. Natl. Acad. Sci. USA
93:15376-15381[Abstract/Free Full Text].
|
| 8.
|
Chirgwin, J. M.,
A. E. Przybyla,
R. J. MacDonald, and W. J. Rutter.
1979.
Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease.
Biochemistry
18:5294-5299[Medline].
|
| 9.
|
Collett, M.,
A. F. Purchio, and R. L. Erikson.
1980.
Avian sarcoma virus transforming protein, pp60src, shows protein kinase activity specific for tyrosine.
Nature
285:167-169[Medline].
|
| 10.
|
Dry, K. L.,
M. A. Alfred,
A. J. Edgar,
J. Brown,
F. O. Manson,
M.-F. Keuren,
D. M. Kurnit,
A. C. Bird,
M. Jay,
A. P. Monaco, and A. F. Wright.
1995.
Identification of a novel gene, ETX1, for Xp21.1, a candidate gene for X-linked retinitis pigmentosa (RP3).
Hum. Mol. Genet.
4:2347-2355[Abstract/Free Full Text].
|
| 11.
|
Enomoto, H.,
T. Ozaki,
E. Takahashi,
N. Ohnuma,
M. Tanaka,
I. Iwai,
H. Yoshida,
T. Matsunaga, and S. Sakiyama.
1994.
Identification of human DAN gene, mapping to the putative neuroblastoma tumor suppressor locus.
Oncogene
9:2785-2791[Medline].
|
| 12.
|
Fagan, J. B.,
M. E. Sobel,
K. M. Yamada,
B. de Crombrogghe, and I. Pastan.
1981.
Effects of transformation on fibronectin cDNA.
J. Biol. Chem.
256:520-525[Free Full Text].
|
| 13.
|
Freeman, A. E.,
H. J. Igel, and P. J. Price.
1975.
In vitro transformation of rat embryo cells: correlations with the known tumorigenic activities of chemicals in rodents.
Carcinog. In Vitro
11:107-119.
|
| 14.
|
Fujii, M.,
D. Shalloway, and I. M. Verma.
1989.
Gene regulation by tyrosine kinases: src protein activates various promoters, including c-fos.
Mol. Cell. Biol.
9:2493-2499[Abstract/Free Full Text].
|
| 15.
|
Giancotti, F. G., and E. Ruoslahti.
1990.
Elevated levels of the 51 fibronectin receptor suppress the transformed phenotype of chinese hamster ovary cells.
Cell
60:849-859[Medline].
|
| 16.
|
Glen, S.,
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].
|
| 17.
|
Graham, F. L., and A. J. van der Eb.
1973.
A new technique for the assay of infectivity of human adenovirus 5 DNA.
Virology
52:456-467[Medline].
|
| 18.
|
Hedrick, L.,
K. R. Cho, and B. Vogelstein.
1993.
Cell adhesion molecules as tumor suppressor.
Trends Cell Biol.
3:36-39.
|
| 19.
|
Hunter, T.
1997.
Oncoprotein networks.
Cell
88:333-346[Medline].
|
| 20.
|
Hunter, T., and B. M. Sefton.
1980.
Transforming product of Rous sarcoma virus phosphorylates tyrosine.
Proc. Natl. Acad. Sci. USA
77:1311-1315[Abstract/Free Full Text].
|
| 21.
|
Hynes, R. O.
1992.
Integrins: versatility, modulation and signaling in cell adhesion.
Cell
69:11-25[Medline].
|
| 22.
|
Inoue, H.,
M. Isaka,
S. Takeda, and A. Hakura.
1991.
Simple system for isolation of cellular and viral mutants for transformation by retroviruses.
J. Med. Virol.
35:246-249[Medline].
|
| 23.
|
Inoue, H.,
M. K. Owada,
M. Yutsodo, and A. Hakura.
1989.
A rat mutant cell clone showing temperature-dependent transformed phenotypes with functional expression of the src gene product.
Virology
168:57-66[Medline].
|
| 24.
| Inoue, H., J. Pan, and A. Hakura. Unpublished data.
|
| 25.
|
Inoue, H.,
N. Tavoloni, and H. Hanafusa.
1995.
Suppression of v-Src transformation in primary rat embryo fibroblasts.
Oncogene
11:231-238[Medline].
|
| 26.
|
Inoue, H.,
A. Yamashita, and A. Hakura.
1996.
Adhesion-dependency of serum-induced p42/p44 MAP kinase activation is released by retroviral oncogenes.
Virology
225:223-226[Medline].
|
| 27.
|
Johnston, G. I.,
R. G. Cook, and R. P. McEver.
1989.
Cloning of GMP-140, a granule membrane protein of platelets and endothelium: sequence similarity to proteins involved in cell adhesion and inflammation.
Cell
56:1033-1044[Medline].
|
| 28.
|
Joseph, C. K.,
S. A. Qureshi,
D. J. Wallace, and D. A. Foster.
1992.
MARCKS protein is transcriptionally down-regulated in v-src-transformed BALB/c 3T3 cells.
J. Biol. Chem.
267:1327-1330[Abstract/Free Full Text].
|
| 29.
|
Jove, R., and H. Hanafusa.
1987.
Cell transformation by the viral src gene.
Annu. Rev. Cell Biol.
3:31-56.
|
| 30.
|
Kansas, G. S.
1996.
Selectins and their ligands: current concepts and controversies.
Blood
88:3259-3287[Free Full Text].
|
| 31.
|
Larsen, E.,
A. Celi,
G. E. Gilbert,
B. C. Furie,
J. K. Erban,
R. Bonfanti,
D. D. Wagner, and B. Furie.
1989.
PADGEM protein: a receptor that mediates the interaction of activated platelets with neutrophils and monocytes.
Cell
59:305-312[Medline].
|
| 32.
|
Levinson, A. D.,
H. Opperman,
H. E. Varmu, and J. M. Bishop.
1980.
The purified product of the transforming gene of avian sarcoma virus phosphorylate tyrosine.
J. Biol. Chem.
255:11973-11980[Abstract/Free Full Text].
|
| 33.
|
Lin, X.,
P. J. Nelson,
B. Frankfort,
E. Tombler,
R. Johnson, and I. H. Gelman.
1995.
Isolation and characterization of a novel mitogenic regulating gene, 322, which is transcriptionally suppressed in cells transformed by src and ras.
Mol. Cell. Biol.
15:2754-2762[Abstract].
|
| 34.
|
Mann, R.,
R. C. Mulligan, and D. Baltimore.
1983.
Construction of a retrovirus packaging mutant and its use to produce helper-free defective retrovirus.
Cell
33:153-159[Medline].
|
| 35.
|
Meindl, A.,
M. R. S. Carvolho,
K. Herrmann,
B. Lorenz,
H. Achatz,
B. Lorenz,
E. Apfelstedt-Sylla,
B. Wittwer,
M. Ross, and T. Meitinger.
1995.
A gene (SRPX) encoding a sushi-repeat-containing protein deleted in patients with X-linked retinitis pigmentosa.
Hum. Mol. Genet.
4:2339-2346[Abstract/Free Full Text].
|
| 36.
|
Morgenstern, J. P., and H. Land.
1990.
Advanced mammalian gene transfer: high titre retroviral vector with multiple drug selection markers and a complementary helper-free packaging cell line.
Nucleic Acids Res.
18:3587-3596[Abstract/Free Full Text].
|
| 37.
|
Nakajima-Iijima, S.,
H. Hamada,
P. Reddy, and T. Kakunaga.
1985.
Molecular structure of the human cytoplasmic -actin gene: interspecies homology of sequences in the introns.
Proc. Natl. Acad. Sci. USA
82:6133-6137[Abstract/Free Full Text].
|
| 38.
|
Nakayama, T.,
M. Irikura,
Y. Setoguchi,
H. Matsuo,
H. Inoue,
M. Yutsudo, and A. Hakura.
1987.
Purification and properties of a non-histone chromatin protein with a molecular weight of 38,000 daltons specific for transformed rat cells.
Biochem. Biophys. Res. Commun.
149:1156-1164[Medline].
|
| 39.
|
Owada, M., and K. Moelling.
1980.
Temperature-sensitive kinase activity associated with various mutants of avian sarcoma viruses which are temperature sensitive for transformation.
Virology
101:157-168[Medline].
|
| 40.
|
Ozaki, T., and S. Sakiyama.
1993.
Molecular cloning and characterization of a cDNA showing negative regulation in v-src-transformed 3Y1 rat fibroblasts.
Proc. Natl. Acad. Sci. USA
90:2593-2597[Abstract/Free Full Text].
|
| 41.
|
Ozaki, T., and S. Sakiyama.
1994.
Tumor-suppressive activity of N03 gene product in v-src-transformed rat 3Y1 fibroblasts.
Cancer Res.
54:646-648[Abstract/Free Full Text].
|
| 42.
|
Pan, J.,
K. Nakanishi,
M. Yutsudo,
H. Inoue,
Q. Li,
K. Oka,
N. Yoshioka, and A. Hakura.
1996.
Isolation of a novel gene down-regulated by v-src.
FEBS Lett.
383:21-25[Medline].
|
| 43.
|
Pelech, T. G., and J. S. Sarshern.
1992.
Mitogen-activated protein kinases: versatile transducer for cell signaling.
Trends Biochem. Sci.
17:235-238.
|
| 44.
|
Pierani, A.,
C. Pouponnot, and G. Calothy.
1993.
Transcriptional down regulation of the retina-specific QR1 gene by pp60v-src and identification of a novel v-src-responsive unit.
Mol. Cell. Biol.
13:3401-3414[Abstract/Free Full Text].
|
| 45.
|
Qureshi, S. A.,
M. H. Rim,
K. Alexandropoulos,
K. Berg,
V. P. Sukhatme, and D. A. Foster.
1992.
Sustained induction of egr-1 by v-src correlates with a lack of Fos-mediated repression of the egr-1 promoter.
Oncogene
7:121-125[Medline].
|
| 46.
|
Ruoslahti, E., and J. C. Reed.
1994.
Anchorage dependence, integrins, and apoptosis.
Cell
77:477-478[Medline].
|
| 47.
|
Schwartz, M. A.,
M. D. Schaller, and M. H. Ginsberg.
1995.
Integrins: emerging paradigms of signal transduction.
Annu. Rev. Cell Biol.
11:549-599[Medline].
|
| 48.
|
Siegelman, M. H.,
M. Van de Rijin, and I. L. Weissman.
1989.
Mouse lymph node homing receptor cDNA clone encodes a glycoprotein revealing tandem interaction domains.
Science
243:1165-1172[Abstract/Free Full Text].
|
| 49.
|
Sugano, S.,
M. Y. Stoeckle, and H. Hanafusa.
1995.
Transcription by Rous sarcoma virus induces a novel gene with homology to a mitogenic platelet protein.
Cell
49:321-328.
|
| 50.
|
Takebe, Y.,
M. Seiki,
J. Fujisawa,
P. Hoy,
K. Yokota,
K. Arai,
M. Yoshida, and N. Arai.
1988.
SR promoter: an efficient and versatile mammalian cDNA expression system composed of the simian virus 40 early promoter and the R-U5 segment of human T-cell leukemia virus type 1 long terminal repeat.
Mol. Cell. Biol.
8:466-472[Abstract/Free Full Text].
|
| 51.
|
Tavoloni, N., and H. Inoue.
1997.
Cellular aging is a critical determinant of primary cell resistance to v-src transformation.
J. Virol.
71:237-247[Abstract].
|
| 52.
|
Tavoloni, N.,
H. Inoue,
H. Sabe, and H. Hanafusa.
1994.
v-src transformation of rat embryo fibroblasts. Inefficient conversion to anchorage-independent growth involves heterogeneity of primary cultures.
J. Cell Biol.
126:475-483[Abstract/Free Full Text].
|
| 53.
|
Tedder, T. E.,
C. M. Isaacs,
T. J. Ernst,
G. D. Dometri,
D. A. Adler, and C. M. Disteche.
1989.
Isolation and chromosomal localization of cDNAs encoding a novel human lymphocyte cell surface molecule, LAM-1.
J. Exp. Med.
170:123-133[Abstract/Free Full Text].
|
| 54.
|
Toyoshima, K.,
M. Owada, and Y. Kozai.
1973.
Tumor producing capacity of temperature sensitive mutants of avian sarcoma viruses in chicks.
Biken J.
16:103-110[Medline].
|
| 55.
|
Tsukita, S.,
M. Itoh,
A. Nagafuchi,
S. Yonemura, and S. Tsukita.
1994.
Submembranous junctional plaque proteins include potential tumor suppressor molecules.
J. Cell Biol.
123:1049-1053[Free Full Text].
|
| 56.
|
Woddell, T. K.,
L. Fialkow,
C. K. Chan,
T. K. Kishimoto, and G. P. Downey.
1995.
Signaling functions of L-selectin. Enhancement of tyrosine phosphorylation and activation of MAP kinase.
J. Biol. Chem.
270:15403-15411[Abstract/Free Full Text].
|
| 57.
|
Yoshida, M.,
W. F. Westlin,
N. Wang,
D. E. Ingber,
A. Rosenzweig,
N. Resnick, and M. A. Gimbrone, Jr.
1996.
Leukocyte adhesion to vascular endothelium induces E-selectin linkage to the actin cytoskeleton.
J. Cell Biol.
133:445-455[Abstract/Free Full Text].
|
J Virol, March 1998, p. 2532-2537, Vol. 72, No. 3
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Tambe, Y., Yoshioka-Yamashita, A., Mukaisho, K.-i., Haraguchi, S., Chano, T., Isono, T., Kawai, T., Suzuki, Y., Kushima, R., Hattori, T., Goto, M., Yamada, S., Kiso, M., Saga, Y., Inoue, H.
(2007). Tumor prone phenotype of mice deficient in a novel apoptosis-inducing gene, drs. Carcinogenesis
28: 777-784
[Abstract]
[Full Text]
-
Bommer, G. T., Jager, C., Durr, E.-M., Baehs, S., Eichhorst, S. T., Brabletz, T., Hu, G., Frohlich, T., Arnold, G., Kress, D. C., Goke, B., Fearon, E. R., Kolligs, F. T.
(2005). DRO1, a Gene Down-regulated by Oncogenes, Mediates Growth Inhibition in Colon and Pancreatic Cancer Cells. J. Biol. Chem.
280: 7962-7975
[Abstract]
[Full Text]
-
Boerner, J., McManus, M., Martin, G., Maihle, N.
(2000). Ras-independent oncogenic transformation by an EGF-receptor mutant. J. Cell Sci.
113: 935-942
[Abstract]
-
Yoshioka, N., Inoue, H., Nakanishi, K., Oka, K., Yutsudo, M., Yamashita, A., Hakura, A., Nojima, H.
(2000). Isolation of Transformation Suppressor Genes by cDNA Subtraction: Lumican Suppresses Transformation Induced by v-src and v-K-ras. J. Virol.
74: 1008-1013
[Abstract]
[Full Text]
Top
Abstract
Text
