From the Geraldine Brush Cancer Research Institute, California Pacific Medical Center, San Francisco, California 94115
Received for publication, August 1, 2000, and in revised form, January 8, 2001
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ABSTRACT |
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Id proteins are dominant negative regulators of
basic helix-loop-helix transcription factors. Previous work in our
laboratory has shown that constitutive expression of Id-1 in SCp2 mouse
mammary epithelial cells inhibits their differentiation and induces
proliferation, invasion, and migration. Id-1 expression also correlates
with the invasive and aggressive potential of human breast cancer
cells. However, little is known about Id-1 target genes that are
important for regulating normal and transformed breast epithelial cell
phenotypes. Now we report the cloning of a novel zinc finger protein,
Zfp289, using degenerate primers to specifically amplify cDNAs from
Id-1-transfected SCp2 cells. Zfp289 has homology with a yeast zinc
finger protein, the GTPase-activating protein Gcs-1, which was
initially identified as a gene required for the re-entry of cells into
the cell cycle after stationary phase growth. Zfp289 mRNA
expression pattern correlates with Id-1 expression in SCp2 mammary
epithelial cells under various experimental conditions as well as in
the mouse mammary gland at different stages of development. It is
predominantly present in the cytoplasm of the cells as evident from
green fluorescent protein fusion protein localization. SCp2
mammary epithelial cells with constitutive expression of Zfp289 have a
higher S-phase index, compared with control cells, when cultured in a
serum-free medium. We conclude that the novel zinc finger protein
Zfp289, which may represent the mammalian homologue of Gcs-1, is
potentially an important mediator of the Id-1-induced proliferation
pathway in mammary epithelial cells.
Basic helix-loop-helix
(bHLH)1 factors are
transcription factors that bind DNA as homo- or heterodimers and
regulate transcription of target genes containing E-boxes (CANNTG) or
E-box-like sequences in their promoters. Dimerization occurs through
interactions of the HLH domains, while binding to DNA is mediated by
the basic domain. These factors have been shown to regulate the
expression of tissue-specific genes in a number of mammalian and
nonmammalian organisms (1).
Id proteins (for "inhibitors of differentiation or DNA binding")
are dominant negative regulators of the bHLH transcription factors. Id
proteins contain an HLH domain, allowing them to form dimers with bHLH
proteins, but they lack the basic domain, and therefore such dimers,
Id/bHLH, do not bind DNA (2). Therefore, Id proteins do not regulate
transcription directly, but indirectly, by preventing bHLH proteins
from interacting with the promoter of various target genes. The role of
Id proteins in the tissue-specific regulation of growth and
differentiation has been examined in several systems. For example, Id-1
has been found to inhibit differentiation in myoblast (2), trophoblast
(3), erythroid (4), B-lymphocyte (5, 6), and myeloid cells (7).
Previous studies in our laboratory have shown that constitutive
expression of Id-1 results in the inhibition of differentiation of SCp2
mouse mammary epithelial cells (8). It also induces proliferation,
invasion, and migration of the same cells (9) and increased secretion
of a 120-kDa matrix metalloproteinase, the level of which
correlates well with the invasive ability of these cells. In addition,
Id-1 is highly expressed in aggressive and invasive human breast cancer
cell lines as compared with noninvasive cell lines (9) and in biopsies
from invasive ductal carcinomas as compared with ductal carcinomas
in situ (10).
Investigations have shown that Id-1 is a positive regulator of
G1-S phase transition during cell cycle progression and is also involved in inducing apoptosis (11-16). A recent report
demonstrated that Id-1 and Id-3 might also control angiogenesis by
regulating the growth and invasion of endothelial cells (17). However, little is known about Id target genes, which are important for regulating growth, differentiation, invasion, and apoptosis of normal
and transformed mammary epithelial cells.
In this paper, we report the cloning of a novel Id-1-induced
zinc finger protein, Zfp289, which is predominantly localized in the
perinuclear compartment of the cells and which appears to function as a
GTPase-activating protein (GAP). Zfp289 expression is correlated with
the proliferative stages of mammary epithelial cells in culture and
during mammary gland development, and this novel zinc finger protein is
able to induce higher S-phase entrance when constitutively expressed in
epithelial cells.
cDNA Cloning of Zfp289--
We used PCR amplification to
isolate genes specifically regulated by Id-1, as indicated by their
up-regulation in SCp2 cells transfected with Id-1. Our rational for
selecting the degenerate primers was that we previously demonstrated a
novel matrix metalloproteinase family member to be up-regulated in SCp2
cells transfected with Id-1 (9). Since we were particularly interested
in cloning this novel metalloproteinase, and since most of the known
metalloproteinases have a "Cys" motif and a "zinc"
binding motif in their sequence, we designed degenerate primers against
two regions of interest, one containing a cysteine residue Cys
(PRCGXPD), the other a catalytic domain binding zinc ions
(VAAHEFGHALGLH). Cys and zinc sequences were as followed:
5'-S(c/g)GR(g/a) TGT GGY(c/t) S(c/g)W (a/t)R(g/a)
CCN(a/c/g/t) GA-3' and 5'-GCR(g/a) TGS(g/c) CCV(a/c/g) AAY(c/t)
TCR(g/a) TGS(c/g) GC-3'.
Total RNA was isolated from SCp2-Id-1-transfected cells and SCp2
control cells, and cDNA was prepared. Specific AP-1 adaptors (Marathon cDNA amplification kit) were ligated at both ends of the
cDNAs, and a first round of PCR amplification was performed using
adaptor-specific primers on one side and zinc primers on the other. A
second round of amplification was performed using the PCR product from
the first round and using Cys and zinc primers. Only two amplified
products of 0.8 and 2.6 kb were visible. We used annealing temperatures
of 45 and 50 °C to obtain sharp bands at 0.8 and 2.6 kb,
respectively. Both of these bands were extracted and cloned into a TOPO
vector for sequencing.
Cell Culture--
SCp2 mouse mammary epithelial cells were grown
in a 1:1 mixture of Dulbecco's modified Eagle's medium and F-12
(Dulbecco's modified Eagle's medium-F-12) containing 5%
heat-inactivated fetal bovine serum and insulin (5 µg/ml), at
37 °C in a humidified 5% CO2 atmosphere, as described
previously (18). For experiments in serum-starved conditions, fetal
bovine serum was omitted. Pool populations of SCp2 cells transfected
with an empty vector or with the murine Id-1 sense cDNA driven by
the mouse mammary tumor virus promoter were as described previously
(8). Single cell-derived clones from SCp2 cells transfected with an
Id-1 antisense cDNA (8) were derived by plating cells at limiting
dilutions in 24-well plates.
Plasmids Construction and Transfection--
The Zfp289 encoding
region including the Kozak sequence was amplified from the
BamHI site at the 5'-end, to either BamHI (for LXSN vector) or SalI site (for pBabe vector) at the 3'-end.
The restriction digested fragments were then cloned into appropriate sites of LXSN and pBabe vectors. These viral vectors were then packaged
in TSA-54 cells (Cell Genesis, Foster City, CA). After infecting the
SCp2 cells with control or Zfp289 vectors, stably transfected cells
were selected with neomycin and puromycin (for LXSN and pBabe vectors, respectively).
For intracellular localization studies, the full-length coding sequence
of Zfp289 was cloned into a pEGFP vector (CLONTECH) between the SalI and BamHI sites. The pEGFP
vector was transfected using Superfect transfection reagent (Qiagen).
Neomycin-resistant cells were subcultured, and localization of the GFP
fusion protein was determined under inverted fluorescent microscopy.
RNA Isolation and Northern Analysis--
Total cellular RNA was
isolated and purified as described by Chomczynski and Sacchi (19). RNA
(15 µg) was size-fractionated by electrophoresis through denaturing
formaldehyde-agarose gels and transferred to Nylon membrane (Hybond-N
from Amersham Pharmacia Biotech). The blots were hybridized with
32P-labeled probes prepared by random oligonucleotide
priming, washed, and exposed to Kodak XAR-5 film for autoradiography
(20). The multiple tissue Northern blot was purchased from
CLONTECH and probed as above. The Zfp289 probe was
the PCR-amplified 2.6-kb fragment described under "cDNA Cloning
of Zfp289," whereas Preparation of Mammary Gland RNA--
BALB/c wild type
virgin female mice were purchase from Simonsen Laboratories, Inc.
(Gilroy, CA) and some were mated at the age of 12 weeks. The animals
were sacrificed, and biopsies of the fourth inguinal mammary gland were
performed at 3, 7, and 12 weeks of age for the virgin stage; at days 2 and 12 of pregnancy; and at days 2, 7, 20, and 21 of lactation. Mammary
glands were immediately frozen at DNA Synthesis and Autoradiography--
Cells (104 or
5 × 104) plated on coverslips were labeled with
[3H]methylthymidine (10 µCi/ml; 60-70 Ci/mmol) for at
least 7 h, washed twice with phosphate-buffered saline, then fixed
for 5 min with 1:1 (v/v) mixture of acetone and methanol kept at
Purification of Zfp289 and ARF-1--
The coding region of
Zfp289 was amplified by PCR and then cloned into a bacterial expression
vector pTrcHis A (Invitrogen). The 6×His-tagged protein was expressed
in the Escherichia coli strain DH5 ARF-GAP Assay--
ARF-GAP activity was assayed by measuring the
effects of the putative GTPase-activating protein (Zfp289) on the
hydrolysis of ARF-bound GTP to GDP, with some modifications of the
assay previously described by Goldberg (22). Briefly, ARF-1 was
incubated with [ Isolation of Two Id-1-inducible Genes in SCp2 Cells--
Using
degenerate primers as described under "Materials and Methods," we
isolated two cDNA clones (0.8 and 2.6 kb) preferentially up-regulated in SCp2-Id-1-transfected cells. A partial sequencing of
the 0.8-kb band revealed that it corresponded to a known gene encoding
histone H3.3. Since we only performed a partial sequencing of this
clone and did not analyze it further, we could not ascertain the
presence of the zinc and Cys motifs. Using this cDNA as a probe,
two mRNAs of 1.2 and 1.8 kb were detected in SCp2 cells cultured in
5% serum (Fig. 1A, lane
1) and in SCp2-Id-1 serum-starved for 2 days (Fig. 1A,
lane 3).
The other 2.6-kb band corresponded to a gene encoding a mRNA of
about 2.9 kb. We found a higher expression of this 2.9-kb transcript in
serum-starved Id-1-transfected cells (Fig. 1A, lane 3) as compared with control cells, which were also serum-starved for 2 days and which showed a reduced amount of Id-1 protein (Fig. 1A, lane 2).
We detected a high level of expression of this 2.9-kb transcript in
control SCp2 cells cultured in the presence of 5% serum (Fig.
1A, lane 1). This level of expression was not
further increased in Id-1-transfected SCp2 cells also cultured in 5%
serum (data not shown). This may be due to the large amount of
endogenous Id-1 proteins, up-regulated by serum in control cells, which
may be sufficient to interact with all bHLH proteins present. The level
of expression of target genes, such as the one encoding the 2.9-kb
transcript, may then correspond to a maximum.
As a control of the loading, we used ethidium bromide staining of
ribosomic RNA as well as
To establish further that Id-1 up-regulates expression of Zfp289, we
analyzed mRNA levels of both genes in nine different clones from
SCp2 cells transfected with Id-1 antisense vectors (8) and treated with
growth factors, lactogenic hormones, and extracellular matrix. As shown
in Fig. 1B, these clones expressed variable amounts of Id-1.
In each of these clones, the levels of Zfp289 strongly correlated with
that of Id-1. Four clones (lanes 1, 4,
6, and 8) expressed high levels of Id-1 and
Zfp289, whereas in five clones (lanes 2, 3,
5, 7, and 9) Id-1 expression was
considerably reduced, and therefore Zfp289 mRNA levels were
significantly down-regulated. As described previously (8, 9), the
expression of Sequence Analysis--
The nucleotide sequence analysis of the
2.6-kb fragment (plus the sequence of overlapping expressed sequence
tags at the 5'- and 3'-ends) revealed an open reading frame of
1560 base pairs, encoding a 519-amino acid polypeptide with a
predicted molecular mass of about 57 kDa (Fig.
2). We could clearly detect a sequence on
this cDNA that was homologous to the zinc degenerate primer. We
attempted to determine any sequence homology to the Cys motif, but due
to the level of degeneracy of this Cys primer, we were not able to
locate any region of clear homology. A search of the protein data base
revealed that this predicted 57-kDa protein was a unique sequence,
having, however, a 48% homology and 32% identity with the yeast zinc
finger protein Gcs-1 (23). This putative mouse protein, which we
called Zfp289 after submission to the nomenclature committee, has one
zinc finger domain at the N terminus, with a
CxxCx(16)CxxC motif
encompassing 26-49 amino acids. This zinc finger domain has 66%
homology with the zinc finger domain in the yeast protein Gcs-1.
Tissue Distribution--
Northern blot analysis of Zfp289 revealed
a predominantly 2.9-kb transcript in all the major murine tissues (Fig.
3), although the level of expression
varied widely. Zfp289 mRNA was expressed at very high levels in
liver, followed by heart and kidney. Skeletal muscle and spleen had the
lowest levels of mRNA expression.
During mammary gland development in mice, Zfp289 mRNA expression
pattern was closely correlated with Id-1 expression (Fig. 4). We detected high levels of expression
of Zfp289 as well as Id-1 in the mammary gland from virgin
(V) mice and during pregnancy (P) when there is
extensive ductal cell proliferation and lobulo-alveolar development,
respectively. The expression of both genes declined at the beginning of
lactation (L) when the glands fully differentiate and
express the milk product Cellular Localization--
Localization studies using EGFP
vector-transfected SCp2 cells showed that GFP-Zfp289 fusion protein was
predominantly present in the cytoplasm with a high proportion of cells
showing perinuclear staining (Fig. 5,
B, C, and D). These data suggest that
Zfp289 is not a transcription factor, which is corroborated by the fact that it contains only one zinc finger domain and not several like typical transcription factors belonging to the zinc finger protein family. This also suggests a function in the regulation of exocytic and/or endocytic vesicular transport pathways at the periphery of the
nuclei. The control EGFP plasmid-transfected cells showed homogenous
nonspecific staining all over the cell cytoplasm as well as nucleus
(Fig. 5A).
Functional Analysis of Zfp289--
To investigate the functional
role of Zfp289, we stably transfected the SCp2 mouse mammary epithelial
cells with two different mammalian expression vectors (LXSN and pBabe)
containing the full-length coding region of Zfp289. Northern blot
analysis of total RNA from these cells confirmed the constitutive
transcription of transfected Zfp289, which displayed a larger size than
the endogenous Zfp289 mRNA (Fig.
6A). In low serum conditions,
constitutive expression of Zfp289 resulted in higher S-phase rate of
mammary epithelial-transfected SCp2 cells as compared with control
plasmid-transfected cells (Fig. 6B). The difference was more
significant in the case of cells transfected with LXSN (170% of the
control) than with pBabe (150% of the control). This may be due to a
higher level of the transgene in the LXSN vector than in the pBabe
vector.
Zfp289 does not appear to play a role in the invasive behavior of the
cells. Even after 2 weeks on extracellular matrix, both control as well
as Zfp289-transfected cells remained associated within compact spheres
(data not shown). In contrast, constitutive expression of Id-1 was able
to induce invasion in SCp2 cells (9). We conclude that Zfp289 may be a
downstream gene under the control of the transcriptional regulators of
the helix-loop-helix family. This novel zinc finger protein is
potentially an important mediator of the Id-1-induced proliferation
pathway, but not of Id-1-induced invasiveness and/or migration, in
mammary epithelial cells.
Zfp289 Appears to Be a GAP Protein--
We noted above the
homology of sequence between Zfp289 and the yeast GAP Gcs-1. Since
Zfp289 also contains the zinc finger motif shared by most of the GAP
proteins, we examined whether Zfp289 was functionally a
GTPase-activating protein. We analyzed the GTPase activity of ARF-1 in
the presence of two different amounts of recombinant Zfp289 protein.
ARF (ADP-ribosylation factor) proteins are 20-kDa guanine
nucleotide-binding proteins that are active when GTP is bound. However,
hydrolysis of bound GTP requires interaction with a GAP protein, as ARF
itself has no detectable GTPase activity. Zfp289 displays a strong GAP
activity as demonstrated by the decrease of label associated with ARF-1
(Fig. 7). This is particularly
significant at 200 ng of Zfp289 and already detectable at 50 ng. Zfp289
may therefore correspond to the mammalian counterpart of the yeast GAP
protein, Gcs-1.
We have previously reported that Id-1, a dominant negative
regulator of bHLH transcription factors, is not only involved in the
inhibition of differentiation, but also induces proliferation, migration, and invasion in SCp2 mouse mammary epithelial cells (8, 9).
To learn more about its action, we sought here to identify transcripts
up-regulated in cells transfected with Id-1. Of the two transcripts we
identified, one was the histone H3.3. This is consistent with the
up-regulation of H3.3 during the Go to S-phase transition
in mouse kidney cells (24) and suggests that histone H3.3 is one of the
mediators of Id-1-controlled proliferation.
Of more interest was the second up-regulated transcript, Zfp289,
which encodes for a zinc finger protein with homology to the yeast zinc
finger protein Gcs-1 and its related protein Glo-3. Gcs-1 was first
identified because of its requirement for transition of yeast cells
from stationary to proliferation phase (23). Reports have also shown
that Gcs-1 protein is a GAP for the ARF (25) and is involved in
regulation of vesicular trafficking and actin cytoskeleton network
(26). Yeast cells containing a functionally mutant Gcs-1 gene were
unable to transit from the stationary phase to growth phase (23) and
exhibited vesicle trafficking defects at the nonpermissive 15 °C
temperature (27).
Zfp289 seems to be evolutionarily a well conserved protein, with about
50% homology between mammalian and yeast sequences, suggesting a
conservation of function as well. The GAP activity of Gcs-1 has been
localized in the N-terminal zinc finger domain (28), and the Zfp289
sequence in this finger domain
(CxxCx(16)CxxC) is
66% homologous to that of yeast. We found that Zfp289 appears to have
similar function, as it activates ARF-1-GTPase activity. The
intracellular localization experiments in SCp2 cells confirmed that
Zfp289 functions predominantly in the cytoplasm, and in the majority of
the cells, Zfp289-GFP fusion proteins seemed to be concentrated around
the perinuclear region.
When we compared mRNA expression of Zfp289 with that of Id-1
in the multiple tissue Northern blot, we found a direct correlation in
five out of seven tissues (Fig. 3). The liver exhibited a relatively low level of Id-1 message compared with that of Zfp289, while in lung,
Id-1 expression was quite high as compared with that of Zpf289.
In these tissues, there may be additional or other types of controls
than Id-l for regulating Zfp289 gene expression. However, Zfp289
expression paralleled that of Id-1 during mammary gland
development, with Zfp289 expressed during ductal (virgin) as well as
lobulo-alveolar (pregnant) morphogenesis, when there is extensive
proliferation of mammary epithelial cells. Its down-regulation followed
the decrease in Id-1 expression in differentiated, growth-arrested lactating epithelial cells.
The data presented in Fig. 1, A and B, provide
indirect evidence that Zfp289 mRNA expression may be controlled by
Id-1 levels. To establish this relationship directly, it will be
necessary to sequence the Zfp289 promoter, to determine the presence of E-box motifs and to isolate the Id-1-interacting bHLH proteins, work
now in progress. Nevertheless, this novel zinc finger protein Zfp289 appears to mediate some of the Id-1-dependent
phenotypic effects on mouse mammary epithelial cells. Functional
analysis using retroviral-mediated transfection in SCp2 cells indicates that it may be an important mediator of Id-1-dependent
S-phase entrance (Fig. 6B), a conclusion consistent with the
known function of the yeast protein. The increased expression of Zfp289
may confer an advantage in cell cycle entrance to Zfp289-transfected
cells in comparison with control cells. This may represent an example of the involvement of ARF-GAP proteins in many fundamental cellular processes such as cell growth and survival, as well as vesicular trafficking and cytoskeletal organization.
Although we have no direct evidence that Zfp289 plays a role in
migration or invasion of cells, this question should remain open.
Besides the role of the yeast homologue Gcs-1 in the cytoskeletal organization, it has been found that members of the GTPase family (such
as Rho and Rac) and their activators can regulate cell migration through their control of actin polymerization and cytoskeletal distribution (29). Our negative results obtained from invasion assays
in Zfp289-transfected cells may be due to the fact that constitutive
expression of Zfp289 alone is not sufficient to induce the invasive
phenotype, an event that may also require the induction of some other
genes, such as matrix metalloproteinases.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-casein and Id-1 probes were as described
previously (8). The clusterin probe was obtained by subtractive
hybridization,2 and the
-actin probe was obtained from CLONTECH.
70 °C until utilized for total
RNA isolation. Total RNA was isolated using TriPure Isolation reagent
(Roche Molecular Biochemicals).
20 °C. Nuclei were stained with
4,6-diamidino-2-phenylindole-diluted 1:10,000 in
phosphate-buffered saline for 2 min. The coverslips were air-dried,
coated with Kodak NTB2 emulsion (1:2 dilution), and exposed for 16-24
h. The coverslips were developed with D19, fixed with Rapid-fix, and
viewed by phase contrast microscope.
. After inducing
protein expression for 5 h with 1 mM
isopropyl-
-D-thiogalactopyranoside, bacteria were
pelleted and lysed under denaturing conditions in buffer containing 6 M guanidine hydrochloride, 10 mM Tris-HCl, 100 mM Na2PO4, pH 8.0, for 1 h at
room temperature with continuous shaking. Cellular debris was then
pelleted by centrifugation of cell lysate at 10,000 × g for 30 min at room temperature. The supernatant was mixed with nickel-nitrilotriacetic acid beads (Qiagen) and stirred for 1 h at room temperature. The beads were then transferred to a column and sequentially washed with Buffer 1 (6 M urea, 20 mM Tris-HCl, 0.5 M NaCl, pH 8.0) and Buffer 2 (20 mM Tris-HCl, 0.15 M NaCl, pH 8.0) to remove
the nonspecific proteins and to renature the nickel-nitrilotriacetic
acid-bound proteins (21). The 6×His-tagged ZFP-289 protein was
eluted with Buffer 2 containing 50 mM EDTA. The eluted
protein was concentrated by using centricon column, and the purity of
the sample (75-80%) was determined by SDS-polyacrylamide gel
electrophoresis. Full-length coding region of mouse ARF-1 (ADP-ribosylation factor-1) was PCR-amplified and cloned into the
bacterial expression vector pTrcHis A. After induction of recombinant
ARF-1 for 5 h with 1 mM
isopropyl-
-D-thiogalactopyranoside, the bacteria were
lysed and supernatant mixed with nickel-nitrilotriacetic acid
beads as described above. The beads were transferred to a column and
washed with Buffer 3 (8 M urea, 10 mM Tris-HCl,
0.1 M Na2PO4, pH 6.3). The bound
6×His-tagged ARF-1 was eluted with Buffer 4 (8 M urea, 10 mM Tris-HCl, 0.1 M
Na2PO4, pH 5.9) and then again with Buffer 4 containing 250 mM imidazole. The eluted ARF-1 was renatured
by sequential dialysis against 4 M urea, 100 mM
Na2PO4, 10 mM Tris-HCl, pH 8.0, then against 2 M urea 10 mM Tris-HCl, pH 8.0, and finally twice against 10 mM Tris-HCl, pH 8.0. The
renatured ARF-1 was concentrated, and its purity (about 80-85%) was
determined by SDS-polyacrylamide gel electrophoresis analysis.
-32P]GTP for 30 min in binding buffer
(20 mM Tris-HCl, 5 mM MgCl2, 0.1%
Triton X-100, 0.1 M NaCl, 1 mM EDTA, 1 mM dithiothreitol, 1 µg/ml bovine serum albumin,
pH 7.5) containing 0.3 mM [
-32P]GTP. ARF
([
-32P]GTP) was then diluted 10 times with binding
buffer. GTPase assay was done by incubating GTP-bound ARF-1 with or
without Zfp289 at room temperature for 20 min in a 25-µl reaction
mixture. After incubation, the samples were spotted on nitrocellulose
membrane, washed four times with Tris buffer saline, and radioactivity
assayed by scintillation counting. The potential GTPase-activating
protein activity of Zfp289 was determined as the decrease of label
associated with ARF-1.
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
A, correlation between Id-1 expression
and expression of two other genes. Cells were cultured under different
conditions, and RNA extraction and Northern analysis were performed as
described under "Materials and Methods." Lane 1, SCp2
control cells (pool population) growing in 5% fetal bovine serum;
lane 2, SCp2 control cells (pool population) in serum-free
medium for 2 days; lane 3, Id-1 sense-transfected SCp2 cells
(pool population) in serum-free medium for 2 days. B,
Northern blot analysis of Zfp289, Id-1, and -casein comparing nine
different clones expressing variable amounts of Id-1. These clones were
isolated from a pool population of Id-1 antisense transfected SCp2
cells.
-actin, which did not show a difference of
expression at the mRNA level between control and Id-1-transfected cells in serum-starved conditions for 2 days. However, mRNA levels of another gene, clusterin, a glycoprotein involved in cell-cell interaction, were down-regulated in the presence of Id-1.
-casein (a differentiation-related gene) was inversely
correlated with that of Id-1.
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Fig. 2.
Nucleotide sequence and deduced amino acid
sequence of murine Zfp289. The N-terminal zinc finger domain is
underlined, and the putative polyadenylation signal is
shaded.
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Fig. 3.
Tissue distribution of Zfp289 and Id-1 in
multiple tissue Northern blot of mice. -Actin was used as an
internal control.
-casein. Zfp289 and Id-1 were almost undetectable after day 2 of lactation until day 21.
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Fig. 4.
Northern blot analysis of Zfp289, Id-1,
and -casein during mammary gland development
in mice. V3, V7, and V12: glands from
virgin 3-, 7-, and 12-week-old mice, respectively; P2 and
P12, glands from 2- and 12-day pregnant mice, respectively;
L2, L7, L20, and L21:
glands from 2-, 7-, 20-, and 21-day lactating mice, respectively.
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Fig. 5.
Cellular localization of Zfp289.
SCp2 cells were transfected with an empty GFP plasmid (A) or
with a GFP plasmid containing the Zfp289 coding region (B,
C, and D) and analyzed using fluorescent
microscope.
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Fig. 6.
A, Northern blot analysis showing
expression of Zfp289 transgene in SCp2 mammary epithelial cells.
B, thymidine incorporation in SCp2 cells transfected with
either control plasmid (LXSN or pBabe) or plasmid containing
full-length coding region of Zfp289 gene. Data represent the average of
four independent experiments and is presented as percentage of control.
One-way ANOVA comparing Zfp289-transfected cells with control cells was
significantly different at p < 0.0001.
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Fig. 7.
Analysis of the GTPase activity of ARF-1 in
the presence of two different amounts of recombinant Zfp289 protein
after 20-min incubation. The data shown are from one of three
independent experiments which showed similar differences. One-way ANOVA
comparing lane 4 versus lane 1 was not
statistically different (p = 0.074), whereas one-way
ANOVA comparing lane 2 (or lane 3)
versus lane 4 was statistically different at
p < 0.0001.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Dr. Judith Campisi for her critical review of this manuscript and help in preparing the retrovirus, Dr. Andrew P. Smith for editing, Dr. Dan H. Moore for help with the statistical analysis, Dr. Ling-Chun Chen for advice on intracellular localization studies, Dr. Udo Greiser for advice on ARF-GAP assays, and Yoko Iritani for technical assistance.
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FOOTNOTES |
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* This work was supported by Postdoctoral Fellowship 5FB-0112 from the University of California Breast Cancer Research Program (to J. S.), by a fellowship from Fondazione Bonino-Pulejo (Italy) (to S. P.), by Grant 3IB-0123 from the University of California Breast Cancer Research Program, and by the National Institutes of Health-NCI Grant RO1 CA82548 (to P.-Y. D.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF229439.
To whom correspondence should be addressed: Geraldine Brush Cancer
Research Inst., California Pacific Medical Center, Stern Bldg., 2330 Clay St., San Francisco, CA 94115. Tel.: 415-561-1760; Fax:
415-561-1390; E-mail: pdesprez@cooper.cpmc.org.
Published, JBC Papers in Press, January 16, 2001, DOI 10.1074/jbc.M006931200
2 J. Singh and P. Y. Desprez, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: bHLH, basic helix-loop-helix; GAP, GTPase-activating protein; PCR, polymerase chain reaction; kb, kilobase(s); GFP, green fluorescent protein; EGFP, enhanced green fluorescent protein; ARF, ADP-ribosylation factor; ANOVA, analysis of variance.
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