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Molecular and Cellular Biology, May 1999, p. 3696-3703, Vol. 19, No. 5
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Reversible Tumorigenesis Induced by Deficiency of
Vasodilator-Stimulated Phosphoprotein
Keyi
Liu,1
Limin
Li,1
Paul E.
Nisson,2
Chris
Gruber,2
Joel
Jessee,2 and
Stanley
N.
Cohen1,3,*
Departments of
Genetics1 and
Medicine,3 Stanford University School of
Medicine, Stanford, California 94305-5120, and Life
Technologies, Inc., Rockville, Maryland 208502
Received 28 October 1998/Returned for modification 3 December
1998/Accepted 15 February 1999
 |
ABSTRACT |
Random homozygous knockout (RHKO) is an antisense RNA
strategy capable of identifying genes whose homozygous functional
inactivation yields a selectable phenotype in cells growing in culture.
Using this approach, we isolated NIH 3T3 fibroblast clones that showed the ability to form colonies on 0.5% agar and tumors in nude
mice. The gene inactivated in one of these clones was found to encode VASP (vasodilator-stimulated phosphoprotein), a previously identified protein that binds to components of the cadherin-catenin junctional complex and has been implicated in cell-cell interactions, the formation of actin filaments, and the transmission of signals at the
cytoskeleton-membrane interface. Fibroblasts made deficient in VASP by
RHKO showed loss of contact inhibition, and consequently, continued
cell division past confluence. Restoration of VASP function by
reversal of RHKO yielded cells that had lost the neoplastic capabilities acquired during RHKO. Overproduction of VASP mRNA in the
sense or antisense orientation from expression constructs introduced by transfection into naive NIH 3T3 fibroblasts also resulted
in neoplastic transformation, implying that normal cell growth may
require the maintenance of VASP expression within a narrow range. Our
results implicate VASP in tumorigenesis and/or cancer progression.
| |
INTRODUCTION |
Vasodilator-stimulated
phosphoprotein (VASP), a proline-rich founding member of the
Ena-VASP protein family, was discovered both as a substrate of cyclic
AMP- and cyclic GMP-dependent protein kinases and a component of the
actin-based cytoskeleton (10, 25-27, 65). VASP is
associated with focal adhesions, actin filaments, and highly dynamic
membrane regions (50) and has been shown to be a multiligand
protein that binds to actin, profilin, ActA, zyxin, and vinculin
(9, 12, 51, 52). Vinculin in turn associates with tensin,
-actinin, -catenin, and talin (4, 31, 63, 66, 68), and
zyxin and profilin are involved in actin filament assembly and
organization (3, 14, 43, 56, 60). In vivo evidence indicates
that VASP is a crucial factor in the formation of actin filaments and
the integration of signals transmitted between the cytoskeleton, the
cytoskeleton-membrane interface, and two cyclic nucleotide signal
transduction pathways (2, 25, 65). Additionally, recent data
indicate that VASP is functionally homologous to the Drosophila
melanogaster Enabled (Ena) locus, which was identified initially
as a dominant genetic suppressor of mutations in the Abelson tyrosine
kinase (1). However, the biological role of VASP has
remained largely unknown.
Recently, a novel gene discovery strategy termed random homozygous
knockout (RHKO) was shown to be capable of identifying genes whose
functional inactivation in murine fibroblasts leads to reversible
cellular transformation (40). This approach uses a promoter
within a randomly inserted, chromosomally integrated gene search vector
(GSV) to produce antisense transcripts complementary to those
originating in the chromosomal gene containing the GSV, and
consequently, also complementary to transcripts from other copies of
that gene. Use of a 
-geo reporter gene within the
GSV allows selection of integration events in transcriptionally active genes and also enables the monitoring of antisense effects. Reversal of
antisense inhibition is accomplished by Cre- and lox-mediated deletion
of a gene encoding an activator of the antisense promoter (40,
69).
Using the RHKO strategy, we generated multiple NIH 3T3 cell clones able
to form colonies on 0.5% agar and metastatic tumors in nude mice. We
report here that the gene affected by RHKO in one of these clones is
the previously identified cell adhesion protein and signal
transducer, VASP. We further show that the tumorigenic
capabilities associated with VASP deficiency are reversible and that
overexpression of VASP, as well as underproduction, can lead to
neoplastic transformation. Our results suggest that VASP has a
previously unsuspected role in tumorigenesis and/or cancer progression.
| |
MATERIALS AND METHODS |
Cell culture and transformation.
NIH 3T3 cells
(American Type Culture Collection) were grown in Dulbecco modified
Eagle medium supplemented with 100 U of penicillin per ml, 100 µg of
streptomycin per ml, and 10% calf serum (Life Technologies, Inc.,
Rockville, Md.). RHKO was done as previously described (40).
Briefly, pLLGSV, a Moloney murine leukemia virus-derived retroviral GSV
containing the -geo reporter gene, was introduced into
NIH 3T3 cells, where it integrated at multiple chromosomal sites.
Infected cells were selected by using 800 µg of G418 per ml for 2 to
3 weeks. Suspensions of G418-resistant NIH 3T3 cells were transfected
with pLLTX DNA by electroporation. Following selection in hygromycin
(500 µg/ml) for 2 to 3 weeks, hygromycin-resistant clones were plated
onto 0.5% agar (11, 13, 39) and the colonies that formed
after 4 to 5 weeks were isolated and expanded to cell lines. Cells
transfected with pRSV-Cre for removal of the transactivator were
selected by using 1 µM gancyclovir for 2 to 3 weeks. Resistant clones
were isolated and expanded individually, and the status of cellular
transformation was confirmed by soft agar assays as described above.
Tumorigenicity assays.
Tumorigenicity was assayed by
injection of 105 cells into athymic nude mice (NIH
nu/nu, female, and 6 weeks of age) subcutaneously over the
lateral thorax. Mice were examined twice weekly and sacrificed 32 days
later (40).
In vitro cell proliferation.
Cells were seeded at a density
of 5 × 105 in 10-cm-diameter tissue culture dishes.
The live cells were counted 24, 48, 72, and 96 h later with a hemacytometer.
Identification of fusion transcripts.
Polyadenylated mRNA
was isolated from cells from which the transactivator gene had been
removed and a cDNA library was constructed with the GIBCO BRL
SuperScript plasmid system (Life Technologies). Oligo(dT) was used to
prime the mRNA for synthesis of the first cDNA strand, and cDNAs were
cloned into pCMVSPORT 3.0. Fusion transcripts containing the
chromosomal vasp gene and the gene search vector were
identified by using the GeneTrapper cDNA positive selection system
(Life Technologies). To confirm that fusion transcripts contained both
vasp and GSV sequences, reverse transcription-PCR (RT-PCR)
was done by using a specific forward primer for a mouse vasp
sequence (5'-ACA GTA GTA AGA GTA ACC GCG-3'), and a specific reverse
primer for the gene search vector sequence (5'-GAT CCG CCA TGT CAC AGA
TC-3').
Isolation of cDNA clones.
cDNAs corresponding to fusion
transcripts were prepared by RT-PCR with poly(A) RNA as an initial
template. The vasp-specific reverse primer was 5'-ACT CCA
GAG ACC TTT GCA TT-3', and the forward primer was 5'-TGG AAC GAA ATG
TAG CAA AGA GAG-3'. PCR was performed as follows: 48°C for 45 min;
94°C for 2 min; 40 cycles, with 1 cycle consisting of 94°C for
30 s, 60°C for 1 min, and 68°C for 2 min; and 68°C for 7 min. PCR products were resolved in 1.5% agarose gels stained with
ethidium bromide, and DNA fragments were cut from gels and purified
with a Qiagen gel extraction kit. The sequences of the purified DNA
were determined with an Applied Biosystems model 310 genetic analyzer.
Full-length vasp cDNA was cloned into the pCR3.1 vector by
using a eukaryotic TA cloning kit (Invitrogen). The mouse
vasp cDNA was inserted between the cytomegalovirus (CMV)
promoter and polyadenylation site in either the sense or antisense
direction. A mutation was introduced into the construct containing
vasp cDNA in the sense orientation by a 4-bp insertion at
codon 38 (BglII digestion and Klenow fill-in), which
produces a frameshift and a translation stop signal 7 codons later.
Western blot analysis.
Cells were grown to 90% confluence,
and whole-cell lysates were prepared in RIPA buffer (1×
phosphate-buffered saline, 1% Nonidet P-40 or lgepa CA-630, 0.5%
sodium deoxycholate, 0.1% sodium dodecyl sulfate). Fifty-microgram
amounts of these lysates were separated by electrophoresis on sodium
dodecyl sulfate-10% polyacrylamide gels, and electroblotted onto
nitrocellulose membranes. The blots were probed with anti-VASP
monoclonal antibody (a generous gift of Michael Zimmer and Ulrich
Walter) and detected by an enhanced chemiluminescence procedure
(Amersham). The same blots were stripped and reprobed with monoclonal
antibody to -tubulin (clone DM 1A; Sigma) as a loading control.
Protein levels were determined by scanning autoradiograms of Western
blots with Scanmaster 3 (Howtek), which were analyzed by the Quality
One program (pdi, Huntington Station, N.Y.).
Southern blot analysis.
Genomic DNA was isolated from VK
cells by standard procedures, and 25-µg samples were digested
with restriction enzymes, subjected to electrophoresis on a 1%
agarose gel, blotted onto Hybond N nylon membrane (Amersham), and fixed
to the membrane by UV cross-linking. The blots were probed with a
1.3-kb -geo fragment labelled by the random priming
method (18).
| |
RESULTS |
Isolation of cells in which homozygous gene inactivation
produces colony formation on 0.5% agar and tumor formation in nude
mice.
We employed the RHKO procedure as previously described
(40) to isolate clones of G418-resistant NIH 3T3 cells able
to form colonies on 0.5% agar, which has commonly been used to select neoplastically transformed cells that have high metastatic potential (13, 21, 39). One of these clones (Fig.
1A) was expanded into the cell line
designated VK; Southern blot analysis showed that VK cells contain the
provirus form of the pLLGSV retroviral gene search vector integrated as
a head-to-tail tandem repeat at a single chromosomal site (Fig.
2).
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FIG. 1.
Transactivation of antisense promoter leads to cell
transformation and tumorigenesis. (A) Effect of transactivation of
antisense promoter on cell growth. Cell line VK contained the
transactivator vector, pLLTX, introduced as previously described
(40). The LAP 348 transactivator gene has been deleted in
cell line VK T2 by introducing pRSV-Cre and selecting cells resistant
to ganciclovir. All cells were incubated for 3 weeks in 0.5% agar.
Bar, 100 µm. (B) Assay for tumorigenicity of the two cell lines shown
in panel A. A total of 105 cells of each cell line were
injected subcutaneously over the lateral thorax into four animals as
described in Materials and Methods; a representative mouse receiving
each cell line is shown 32 days after injection.
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FIG. 2.
Structure of integrated provirus and results of Southern
blot analysis. (A) Map of integrated provirus. RP, regulated antisense
promoter (arrow indicates direction of transcription). 3'dLTR or
5'dLTR, defective 3' or 5' retroviral long terminal repeat lacking
sequences required for production of virions; SA, splice acceptor site;
-geo, reporter gene fusion of Escherichia coli
lacZ and neo (aph) genes (arrow indicates
sense direction of transcription into the fusion gene). Relevant
restriction enzyme sites are shown, along with the regions used as
probes for Southern blots. (B) Southern blot. Genomic DNA (25 µg)
used for each restriction enzyme digestion was separated by
electrophoresis and analyzed as described in Materials and Methods.
Blots were probed with a 1.3-kb DNA probe complementary to the region
of the provirus. The positions of size markers are indicated to the
right of the blot.
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To establish that the transformed phenotype observed for VK cells is
dependent on expression of antisense RNA from the pLLGSV-derived
chromosomally integrated provirus, the antisense promoter in the
provirus was turned off. This was accomplished by transfecting
VK cells
with pRSV-Cre, which deletes the LAP 348 (
42) transactivator
gene and an adjacent fusion of hygromycin resistance and thymidine
kinase genes by site-specific recombination at lox sites bracketing
a
segment containing these genes (
40). Cells losing thymidine
kinase activity were identified by their consequent resistance
to
ganciclovir (
40). Examination of 19 cell clones from a
population
of ganciclovir-resistant, hygromycin-sensitive cells
indicated
that 7 had lost the ability to form colonies on agar. The
reversion
of phenotype of VK
T2, one of these clones that was chosen
for
further study, is shown in Fig.
1A. None of 20 individual subclones
of VK cells, which retain the transactivator, lost their ability
to
produce colonies on 0.5%
agar.
Properties of VK and VKT2 cell lines.
Subcutaneous
injection of VK cells into nude mice resulted in tumors at the site of
injection in all animals (Fig. 1B and Table
1). The tumor in one of these mice was
observed at autopsy to have metastasized spontaneously to the lungs.
The VKT2 cell line, which had undergone reversal of the ability to
form colonies in agar following excision of the transactivator, showed
no tumorigenic capabilities following injection into nude mice, and no
tumors were observed in any of the four animals receiving these cells (Fig. 1).
Comparison of the morphology and growth properties of VK, VK
T2, and
control cells (Fig.
3) showed that the
tumorigenic capabilities
observed for VK were accompanied by marked
differences in morphology
and growth properties during culture. VK
cells were much smaller,
as determined visually, than control NIH 3T3
cells containing
the transactivator vector only and failed to show the
contact
inhibition at confluence characteristic of NIH 3T3 cells. They
also grew to a cell density at 96 h of culture that was two to
three times the maximum density attained by NIH 3T3 cells (Fig.
3A).
VK
T2 cells resembled the control and showed normal contact
inhibition, consistent with their inability to form colonies on
agar or
tumors in nude mice. VK cells, which contain an active
promoter
producing antisense RNA complementary to VASP transcripts,
assumed a
dramatically round shape for the first 24 h after plating
(Fig.
3B), as has been observed also for cells treated by antisense
oligonucleotides to some cell adhesion proteins (
44,
46,
61),
but became flatter after 2 days in culture. VK
T2 cells
displayed
the characteristically flat shape characteristic of
fibroblasts
in addition to showing contact inhibition at confluence
(Fig.
3B), demonstrating reversibility of abnormal morphology upon
excision
of the transactivator.
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FIG. 3.
Characteristics of VK and VKT2 cell lines compared
with NIH 3T3 cells containing only the pLLTX transactivator vector in
VASP function. (A) Growth properties of cells in vitro. Cells (5 × 105) were plated into 10-cm-diameter culture dishes, and
at the indicated times, cells were removed and counted. Values indicate
averages of three independent experiments, each done in triplicate
(mean ± standard error). (B) Morphology of cells in vitro 24 h following the plating of cultures. All fields were examined at the
same magnification. Bar, 100 µm.
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cDNA cloning and characterization of the fusion transcript.
To
identify the chromosomal gene initiating transcripts fused to the
-geo reporter gene in VK cells, a cDNA library was
constructed by using poly(A) RNA isolated from cells from which the
transactivator gene had been removed. cDNA cloning and sequencing led
to identification of a cDNA clone containing 313 bp of a chromosomally
encoded transcript sequence fused in frame to the splice acceptor site
5' to -geo (Fig. 4). A
database search using the BLAST program showed that the segment 5' to
-geo corresponded to the 5' untranslated region of the
VASP gene plus the first five nucleotides of the VASP protein-coding sequence (70).
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FIG. 4.
Murine vasp cDNA sequence fused to the GSV.
The nucleotides in uppercase indicate the nucleotides in the
vasp sequence which match those of the published
vasp sequence from nucleotides 23 to 335 (70).
The nucleotides in lowercase are those in the sequence at the splice
acceptor site of the GSV. The splice junction 5' to -geo
is underlined. The nucleotides shown in bold type are the first five
nucleotides of the coding sequence of exon 1 of vasp.
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|
Independent evidence that transcripts fusing the VASP sequence to
-
geo are made in VK cells was obtained by RT of poly(A)
RNA from VK
T2 and amplification of the resulting cDNA by PCR,
using
primers corresponding to chromosomal and vector sequences,
as described
in Materials and Methods. Such PCR amplification
yielded cDNA
corresponding to the transcript predicted for the
VASP-
-
geo fusion segment identified by gene trapping, as
demonstrated
by agarose gel electrophoresis and Southern blotting.
Sequencing
of the PCR product confirmed that fusion of VASP and
-
geo sequences
had occurred at the expected splice
acceptor
site.
VASP expression is reduced in VKT2 cells. As seen in Fig.
5 and Table
2, VK
T2 cells, which contain a GSV
insertion
in one chromosomal copy of VASP but lack the transactivator
that
turns on the GSV antisense promoter, maintained a steady-state
level of VASP protein at approximately 58% of the level observed
in
control NIH 3T3 cells. Western blots of protein isolated from
VK cells
showed 28% of the wild-type level of VASP protein, indicating
that
total ablation of VASP production is not required for neoplastic
transformation of NIH 3T3 cells.
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FIG. 5.
Western blot analysis of VASP. Whole-cell lysate (50 µg) in each lane was separated by electrophoresis on a sodium dodecyl
sulfate-10% polyacrylamide gel. Anti-VASP monoclonal antibody was
used to detect the protein levels. Anti--tubulin antibody was used
as a loading control. Blots were prepared as described in Materials and
Methods.
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Abnormal VASP expression transforms naive NIH 3T3 fibroblasts.
Transcription of chromosomally integrated VASP cDNA in an antisense
orientation in stably transfected naive NIH 3T3 cells under control of
the CMV early promoter resulted in the ability of 0.51% of
105 plated cells to form colonies in 0.5% agar (Fig.
6B), providing independent
confirmation of the effects of VASP inactivation in these cell
lines. Additionally, overexpression of chromosomally integrated
VASP cDNA from the CMV promoter in the sense direction surprisingly
also resulted in colony formation by NIH 3T3 cells (frequency, 0.48%
of plated cells) (Fig. 6C). This was abolished by a 4-bp insertion in
codon 38 of the VASP protein-coding sequence that causes premature
termination of the VASP protein (Fig. 6D), indicating specifically that
VASP protein overexpression is responsible for the observed effect.
Cells receiving vectors lacking a VASP insert also showed no evidence
of cellular transformation (Fig. 6A). Examination of transfected
populations of NIH 3T3 cells expressing VASP cDNA in either the sense
or antisense direction indicated that during the first 24 h after
plating, approximately 10% of the cells had an abnormal round
shape similar to that observed for the VK cell line. The lower
frequency of morphologically abnormal cells in these populations
than that in VK cells, which are clonal, correlates with the lower
frequency of cellular transformation by these VASP cDNA
transfectants (Table 3). We speculate
that VASP cDNA may be expressed to different extents at different
chromosomal insertion sites in cells of the population of cDNA
transfectants.
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FIG. 6.
Colony growth in 0.5% agar by NIH 3T3 cells transfected
with vasp cDNA and control constructs. Representative
results are shown for soft agar assays measuring anchorage-independent
growth of NIH 3T3 fibroblasts expressing different forms of
vasp cDNA and control constructs. Transfected cells were
selected for growth in 800 µg of G418 per ml for 18 days, and
105 resistant cells were plated in 0.5% agar and incubated
for 3 weeks. Cells were transfected with different vectors. Vector
control lacking vasp cDNA (A), vectors containing
full-length vasp cDNA in antisense (B) or sense (C)
orientation, and vector containing vasp cDNA in sense
orientation but containing a protein-terminating insertion as described
in Materials and Methods (D) were used. Bar, 100 µm.
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| |
DISCUSSION |
The discovery that the VASP gene, which was
identified previously in another context (29, 65), is
implicated in the pathogenesis and/or progression of cancer is an
unexpected outcome of the use of an RHKO screen to isolate candidate
tumor suppresser genes. However, given the evidence that a variety of
genes having a role in focal adhesions and cytoskeleton interactions
have a role in neoplasia, it is perhaps not surprising to have detected
VASP, which is a component of the actin-based cytoskeleton (10,
25-27, 65) and has been demonstrated to affect actin filaments
and the transmission of signals at the cytoskeleton-membrane interface, in this screen. Consistent with indications that cytoskeletal alterations may be primary events in tumorigenesis (6, 20, 32, 49,
57) are indications of tumor suppressor function of actin
regulatory proteins such as -actinin, tropomyosin 1, gelsolin,
merlin, DCC, tensin, and vinculin (16, 17, 19, 22, 41, 48, 58, 59,
66).
The neoplastic transformation, lack of contact inhibition at
confluence, and morphological alterations that resulted from the
production of antisense RNA complementary to VASP transcripts were
reversed in 7 of 19 transformed NIH 3T3 cell clones upon inactivation
of the GSV antisense promoter, whereas no spontaneous reversion was
observed in any of 20 VK cell subclones retaining the transactivator.
Of 19 clones tested following restoration of VASP activity, 12 retained
abnormal growth properties and the capacity for tumorigenesis,
suggesting that permanent genetic alterations may have occurred in
these cells during VASP inactivation. Analogous observations have been
made following restoration of function of the tsg101 gene
(40, 69), whose role in tumorigenesis was also discovered by
an RHKO screen, for retinoblastoma (Rb) (8, 30) and for
tumor suppressor genes that are believed to function as caretakers
(28, 34).
The human VASP gene maps to chromosome 19q13.2-13.3 (70), a
site suspected to harbor tumor suppressor gene(s) involved in a variety
of human cancers, and loss of heterozygosity in this region has been
identified in 35 to 81% of gliomas (5, 53-55) and 53% of
ovarian cancers (7, 47); however, currently there is no
evidence of genomic abnormalities at the VASP locus in human malignancies.
NIH 3T3 cells in which VASP protein was reduced to 58% of the
wild-type level by haploid insufficiency resulting from a GSV insert in
one chromosomal copy of the VASP gene grew normally and showed no
tumorigenic capabilities. However, abnormal cell morphology and the
ability to produce metastatic cancer in nude mice were observed when
VASP protein was reduced by antisense interference to 28% of the
wild-type level, as assayed by Western blotting. Cells in which
the TSG101 protein was diminished but not absent are also
tumorigenic (69), and transforming growth factor 1 and
murine p27kip1 recently have been reported to
show true haploid insufficiency in their ability to protect against
cancer development (45, 62). Moreover, Venkatachalam et al.
(64) have demonstrated that loss of both p53 alleles is not
a prerequisite for neoplasia and that simple reduction in p53 levels
may be sufficient to promote tumorigenesis. These examples of
tumorigenesis associated with reduction in gene expression appear to be
exceptions to the traditional view that neoplasia related to
dysfunction of tumor suppressor genes requires genomic mutations that
ablate gene activity (24, 34-36, 67). Whereas such
loss-of-function mutations classically have provided the framework for
defining, and the basis for the discovery of, tumor suppressor genes,
the RHKO procedure now makes practical the identification of additional
classes of genes whose abnormal regulation may lead to tumorigenesis.
Because RHKO selects for GSV insertions at sites that yield phenotypic
effects and such effects may result from less than total ablation of
protein, RHKO provides a tool for discovering loci implicated in
tumorigenesis by mechanisms such as alterations in transcript splicing
or decay or events that affect proteins at the translational or
posttranslational level (15, 33).
Stable expression of VASP cDNA in naive NIH 3T3 cells in an
antisense orientation resulted in neoplastic transformation, consistent with results obtained by RHKO. Additionally, expression of
chromosomally inserted VASP cDNA in NIH 3T3 cells also resulted in
cellular transformation, and this was reversed by an insertion mutation that leads to premature termination of the VASP protein. Sequencing of
the VASP cDNA derived from VKT2 cells shows two differences from the
sequence reported by Zimmer et al. (70) (i.e., A · T-to-G · C and C · G-to-G · C transitions at 955 and 1192 bp, respectively, and an AGG deletion at 1193 to 1195 bp).
However, the sequences of RT-PCR products generated from total RNA
isolated from VK, VKT2, and parental NIH 3T3 cells were identical,
making it likely that the transformation we observed in cells
overexpressing VASP cDNA in the sense direction is caused by excess
wild-type protein rather than from dominant-negative (38) or
gain-of-function (23) mutations such as those reported for
p53 (37). Together, these results suggest that VASP
expression outside of a narrow range can perturb cell growth, perhaps
by shifting the dynamic equilibrium between pools of assembled and
unassembled cytoskeletal proteins (50). Analogous effects of
over- and underproduction have been observed also for
tsg101, whose excess or deficiency in NIH 3T3 fibroblasts
can lead to tumorigenesis (40).
| |
ACKNOWLEDGMENTS |
We thank Michael Zimmer and Ulrich Walter for anti-VASP antibody
and for helpful discussions.
This study was supported in part by funds from the 1993 Helmut Horten
Foundation Research Award to S.N.C. and by a gift from the Chiron
Corporation. K.L. is a recipient of NIH postdoctoral fellowship awards
from the National Cancer Institute (PHS NRSA CA09302) and Human Genome
Training Program (HG 00044-04).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Genetics, Room M-320, Stanford Medical Center, Stanford, CA 94305-5120. Phone: (650) 723-5315. Fax: (650) 725-1536. E-mail:
sncohen{at}stanford.edu.
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Molecular and Cellular Biology, May 1999, p. 3696-3703, Vol. 19, No. 5
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