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J Biol Chem, Vol. 275, Issue 6, 4499-4506, February 11, 2000
From the Division of Gastroenterology, Department of Medicine and
Genetics, University of Pennsylvania,
Philadelphia, Pennsylvania 19104
Cdx1 is a homeodomain transcription factor that
regulates intestine-specific gene expression. Experimental evidence
suggests that Cdx1 may be involved in cell cycle regulation, but its
role is ill defined and the mechanisms have not been explored. We used stable transfection of inducible constructs and transient expression with a replication-deficient adenovirus to induce Cdx1 expression in
rat IEC6 cells, a non-transformed intestinal epithelial cell line that
does not express Cdx1 protein. Expression of Cdx1 markedly reduced
proliferation of IEC6 cells with accumulation of cells in the
G0/G1 phase of the cell cycle. Cell cycle
arrest was accompanied by an increase in the hypophosphorylated forms
of the retinoblastoma protein (pRb) and the pRb-related p130 protein.
Protein levels of multiple cyclin-dependent kinase
inhibitors were either unchanged (p16, p18, p21, p27, and p57) or were
not detected (p15 and p19). Most significantly, levels of cyclins D1
and D2 were markedly diminished with Cdx1 expression, but not cyclins
D3, E, or the G1 kinases. Additionally,
cyclin-dependent kinase-4 activity was decreased in
association with decreased cyclin D protein. We conclude that Cdx1
regulates intestinal epithelial cell proliferation by inhibiting
progression through G0/G1, most likely via
modulation of cyclin D1 and D2 protein levels.
Homeobox genes encode for transcription factors that have been
implicated as critical regulatory proteins for a variety of developmental and differentiation processes in organisms throughout the
phylogenetic tree (reviewed in Refs. 1-4). Moreover, there is evidence
that members of this family of transcriptional proteins are involved in
the control of cellular proliferation and oncogenesis (5). The
Cdx1 gene family
includes mammalian homeobox genes that are related to the
Drosophila melanogaster gene caudal
(6). Three mouse Cdx genes have been identified,
Cdx1 (7), Cdx2 (8-10), and Cdx4 (11);
two genes have been identified thus far in humans, CDX1 (12)
and CDX2 (13). Cdx1 and Cdx2 are
expressed in many tissues early in development, but are restricted to
the intestinal and colonic epithelium later in fetal life and in the
adult (7, 11, 14-17).
Several lines of evidence suggest that Cdx1 and Cdx2 are important
nuclear proteins involved in the regulation of the phenotype of
intestinal epithelial cells (18-22). Cdx1 (23) and Cdx2 (10, 23-28)
interact with DNA elements that have an AT-rich consensus sequence
(TTTAT), similar to other homeodomain proteins. Binding of Cdx1 (23)
and Cdx2 (10 ,23) to intestinal gene promoters leads to transcriptional
activation that is mediated through an activation domain localized to
the amino-terminal region of the protein (29, 30). Most studies have
shown that Cdx2 activates transcription of various genes (10, 23, 27,
28, 31), whereas fewer studies have examined Cdx1 as an activator of
gene transcription (17, 23). The role of these homeobox genes in regulation of proliferation is of particular interest, given the tightly controlled patterns of proliferation in the intestinal epithelium. Cdx2 is able to inhibit of cellular growth as shown by
studies in transfected cell lines (18), but the effect of Cdx1 on
cellular proliferation is not as well defined. The genes that are known
to be regulated by either Cdx1 or Cdx2 are generally ones that define
functional phenotype (for example, sucrase-isomaltase (Ref. 10),
glucagon (Refs. 27 and 28), intestinal phospholipaseA/lysophospholipase (Ref. 23), and carbonic anhydrase (Ref. 32)) and are not known regulators of cellular proliferation.
Several studies have examined the role of Cdx1 on proliferation in
intestinal cell lines. Cdx1 was shown to transform NIH-3T3 cells in a
focus-forming assay in a study that tested many homeobox genes for
transforming properties (5). In another study, a colon cancer cell line
that expresses small amounts of endogenous CDX1 and CDX2 was
transfected with CDX1 sense and antisense expression constructs (19).
These investigators found that overexpression of CDX1 with the sense
construct had no effect on growth, but that the CDX1
antisense-expressing cells had a significant reduction in their growth
rate. Based on these findings, the authors hypothesized that CDX1 acted
as a growth promoter, although CDX protein levels were not examined to
determine whether there were indeed changes in CDX1 levels. Recently,
another human colon cancer cell line, HT29, was transfected with CDX1,
CDX2, or both CDX1 and CDX2 expression constructs (21). HT29 clones
expressing CDX2 or both CDX1 and CDX2 had significantly reduced growth
rates compared with controls. CDX1 expression alone had no effect on
the growth of these cells, but neither CDX1 nor CDX2 protein levels
were measured in this study. Significantly, only CDX1 and CDX2
expressed together could reduce the tumor growth rate of the HT29
clones in nude mice. Two important weaknesses of these studies include
the use of cells that constitutively express CDX genes and that levels
of the expressed proteins were not measured. This experimental design
might lead to a selection of cells that were able to grow despite CDX
gene expression, or for cells that expressed very low levels of CDX1.
The expression of Cdx genes has been investigated in human
colorectal tumors. Expression of CDX2 mRNA (33) and protein (13) is
decreased in a large percentage of colorectal cancers. Expression of
CDX1 mRNA (33) and protein (34) is also decreased in the majority
of colorectal cancers. In studies using immunohistochemistry to detect
CDX1, there appears to be a graded decrease in immunoreactivity between
normal, adenomatous, and cancer cells (34). From these data, there
appears to be a marked decrease in the expression of both CDX1 and CDX2
in colorectal cancers.
Therefore, it is currently unclear how Cdx1 is involved in the control
of cellular proliferation. The purpose of our study was to examine the
effects of Cdx1 on growth regulation of a non-transformed intestinal
cell line. We found that Cdx1 inhibits cellular proliferation by
blocking progression through the cell cycle in G1. This
G1 arrest was accompanied by decreased expression of the
D-cyclins, inhibition of cyclin-dependent kinase activity,
and accumulation of the hypophosphorylated forms of the retinoblastoma
protein (pRb)2 and the
pRb-related p130. These findings have important implications for
understanding the functional role of Cdx1 in the regulation of
intestinal epithelial cell proliferation.
Construction of Expression Vectors--
An inducible Cdx1
expression vector, pMT-Cdx1, was constructed by subcloning the coding
sequence of Cdx1, released by digestion of pEVRF1Cdx1 (16) with
KpnI and XbaI, into pMTCB6+, which
contains the promoter of the sheep metallothionein I gene (35).
Production and Characterization of Adenovirus Vectors--
Mouse
Cdx1 cDNA was subcloned into the adenoviral transfer vector,
pAdCMVlink, and used to produce the expression vector Adeno-Cdx1. pAdCMVlink without a cDNA insert was used to generate the control Adeno-NULL virus. The linearized adenoviral transfer plasmid was cotransfected with adenoviral DNA into 293 cells to allow for homologous recombination. Viral plaques were then picked and expanded on 293 cells. Recombinent virus was expanded, purified by CsCl centrifugation, and the infectious titers determined by an adenoviral plaque assay.
Multiplicity of infection (m.o.i.) required for expression of Cdx1 in
>90% of cell was determined for IEC6 cells subcultured at a density
of 1 × 104 cells/well in 24-well culture dishes.
Cells were infected in 0.2 ml of infection medium (culture medium with
only 2% fetal bovine serum) containing various dilutions of Adeno-Cdx1
to yield m.o.i. between 25 and 1000. Twenty-four hours after infection, the medium was changed to growth medium, and 24 h later cells were
fixed and assayed for Cdx1 expression by immunohistochemistry using
anti-Cdx1 antibody (1:200) (34) and a rhodamine-conjugated anti-rabbit
secondary antibody. The optimal m.o.i. for expression of Cdx1 in >90%
of cells was 50 for IEC6 cells.
Cell Culture, Transfection, and Infection--
IEC6 cells were
maintained as described previously (18) and transfected with pMT-Cdx1
by electroporation at 250 V and 960 microfarads with a Gene Pulser
(Bio-Rad). NIH3T3 cells were grown in Dulbecco's modified Eagle's
medium with 4.5 g/liter glucose and transfected by CaPO4
precipitation, as described previously (10). Briefly, plasmids were
incubated for 20 min in a solution containing 2× BBS (50 mM BES, pH 6.95, 280 mM NaCl, and 1.5 M Na2HPO4) and 0.25 M
CaCl2 and then added to NIH3T3 cells dropwise. The plates
were incubated in 3% CO2 for 24 h. The cells were
washed with PBS, split 10:1, and incubated in 5% CO2
incubator. Clones were selected in the presence of 0.6 mg of G418/ml,
isolated, and screened for Cdx1 or Cdx2 expression by electrophoretic
mobility shift assay (EMSA) and Northern blot analysis.
Adenovirus infections were performed in 24-well culture plates (cell
count and BrdUrd assay) or 100-mm culture dishes (Western and Northern
analysis). Twelve to 18 h before infection, exponentially growing
IEC6 cells were subcultured at a density of 1 × 104
cells/well or 1.4 × 105 cells/100-mm culture dish.
Cells were infected the following morning in 0.2 or 5.7 ml of infection
medium (culture medium with only 2% fetal bovine serum) containing no
virus (control) or enough Adeno-Cdx1 or Adeno-Null virus to yield an
m.o.i. = 50. Twenty-four hours after infection, the medium was changed
to growth medium. At 48 h, the cells were processed for the
various end points. For cell counts, the cells were trypsinized at
several time points after infection, pelleted by centrifugation, and
resuspended in PBS. Duplicate counts were performed for each well on a
hemocytometer (Hausser Scientific).
EMSA--
Nuclear proteins were isolated from cell cultures and
EMSA was performed exactly as described previously (10).
Soft Agar and Focus Assay--
104 or 5 × 104 cells were suspended in 0.3% agar (Difco) solution in
Dulbecco's modified Eagle's medium containing 10% fetal calf serum
and overlaid onto 0.5% agar solution in Dulbecco's modified Eagle's
medium containing 10% fetal calf serum in 60-mm plates. Regular medium
were added after top agar was solidified. For induction,
ZnSO4 were added to bottom, top agar, and top medium to
final concentration of 50 µM. Colonies were stained 2-4
weeks after plating with 50 µg/ml p-iodanitrotetrazolium
violet (Sigma) and scored 24 h later.
For the focus assay, 7 × 103 cells from each stable
lines (3T3MT1, 3T3Cdx1MT1, and 3T3Cdx2MT17) were mixed with 9.3 × 104 NIH3T3 cells and plated in six-well plates. The culture
medium was changed twice weekly for 4 weeks. At 2 and 4 weeks, cells were fixed with Bouin's solution for 1 h, rinsed with PBS, and stained with 4% Giemsa reagents for 24 h. Dishes were rinsed
briefly with distilled water and then scored for the number of foci.
Cell Proliferation and Flow Cytometry Analysis--
BrdUrd
incorporation was measured using the 5-bromo-2'-deoxyuridine labeling
and detection kit I (Roche Molecular Biochemicals). BrdUrd labeling of
IEC6 cells was performed over 1 h, and then the cells were
processed according to the instructions provided with the kit, except
that in the final washing step, Hoechst dye (bisbenzimide; Sigma) was
added to the wash reagent (final concentration = 2 µg/ml) to
stain all nuclei. At least 200 cells from each well were counted using
a fluorescent microscope, and those incorporating the BrdUrd label were
expressed as a percentage of all cells counted. These percentages were
transformed using the arcsine transformation prior to statistical
analysis with analysis of variance and Tukey tests.
For flow cytometry, IEC6 cells were infected and cultured as described.
DNA content was measured using a rapid propidium iodide staining
method. Approximately 250,000 cells (one 100-mm dish) were trypsinized,
washed once in PBS, and then resuspended in 0.35 ml of STAIN solution
(3% polyethyleneglycol 8000, 50 µg/ml propidium iodide, 360 units/ml
RNase A, 0.1% Triton X-100, 3.6 mM citric acid buffer, pH
7.2). The cells were incubated for 30 min at 37 °C, then 0.35 ml of
SALT solution (3% polyethyleneglycol 8000, 50 µg/ml propidium
iodide, 0.1% Triton X-100, 0.4 M NaCl, pH 7.2) was added,
and the solution incubated in the dark at 4 °C for at least 1 h
prior to flow cytometry and DNA quantitation using a FACScan (Becton Dickson).
Immunoblot Analysis--
Protein extracts of IEC6 cells were
made from trypsinized cells resuspended in 80 µl of buffer A (1× PBS
with 2 µg/ml aprotinin, 2 µg/ml leupeptin, 0.2 mM PMSF,
1 mM NaF, and 1 mM NaVO4). Two volumes of 2× lysis buffer was added (250 mM Tris-Cl, pH
7.4, 10% SDS, 20% glycerol, and 2% Northern Blot and Ribonuclease Protection Analysis--
Total
RNA was isolated by the guanidinium thiocyanate/CsCl method from
several 100-mm plates infected and cultured as described (18). 50 µg
of RNA was run on a 0.8% agarose and 6% formaldehyde gel with 1×
MOPS buffer. The RNA was transferred to a nitrocellulose membrane, the
filter was air-dried, and the RNA was UV cross-linked to the membrane
using a UV Stratalinker 1800 (Stratagene). The RNA was stained with
methylene blue after blotting to evaluate for equal transfer.
Filters were prehybridized for several hours at 42 °C in buffer
containing 50% formamide, 5× SSPE, 5× Denhardt's solution, 100 µg/ml sonicated salmon sperm DNA, and 0.1% SDS. A human cyclin D1
cDNA (kindly provided by Anil Rustgi, University of Pennsylvania) was restriction enzyme-digested and gel-purified. A random-primed, 32P-labeled probe was prepared from 30 ng of the purified
cDNA using the Random Primed DNA labeling kit (Roche Molecular
Biochemicals) according to manufacturer's instructions. The probe was
purified from unincorporated nucleotides using a G-50 Quick Spin Column (Roche Molecular Biochemicals). The membrane was hybridized with 1 × 106 cpm of purified probe/ml of hybridization solution
at 42 °C for 24 h. The blot was the washed twice (10 min each,
room temperature) in 250 ml of 2× SSC and 0.1% SDS, with gentle
agitation. The filter was then washed for 1 h at 55 °C in 250 ml of 0.2× SSC and 0.1% SDS, with the buffer changed after 30 min.
The membrane was briefly air-dried and exposed to Kodak XAR film with
an intensifying screen at
To reprobe blots for 36B4, the membrane was first stripped of the
previous probe by washing in 1× Blot Wash Buffer at 60 °C for
1 h. Blot was and exposed to Kodak XAR film with an intensifying screen at
The same RNA was subjected to ribonuclease protection analysis using
rat-specific probes to cyclins D1, D2, and D3, as well as an Cyclin-dependent Kinase Activity
Analysis--
Several 100-mm plates of IEC6 were infected and cultured
as described. At 48 h, they were briefly trypsinized, washed twice in PBS, then resuspended in IP buffer (50 mM Hepes (pH
7.5), 150 mM NaCl, 1 mM EDTA, 2.5 mM EGTA, 10% glycerol, 0.1% Tween 20, 1 mM
NaF, 0.1 mM Na3VO4, 1 mM PMSF, 1 mM dithiothreitol, 25 µg/ml leupeptin, 25 µg/ml aprotinin, 150 µg/ml benzamidine, and 10 µg/ml trypsin inhibitor). Cells were briefly sonicated, then
incubated in IP buffer at 4 °C with rotation for 30 min. Cellular
debris was pelleted by centrifugation at 14,000 rpm for 10 min at
4 °C. The supernatant was saved and was further cleared by repeat
centrifugation as before. The protein concentration of the supernatant
was determined by the BCA protein assay (Pierce). Duplicate 100 µg
samples of protein extract was used in each kinase reaction. The
lysates were first precleared with prewashed Protein G beads (Life
Technologies, Inc.), and then with prewashed Protein A beads (Life
Technologies, Inc.). These cleared lysates were then subject to
immunoprecipitation with 1 µg of either anti-Cdk4 (sc-260, Santa
Cruz) or 1 µg of rabbit pre-immune IgG (Santa Cruz) for 2 h at
4 °C with rotation. Prewashed Protein A beads were added, and the
incubation continued for an additional 1 h. The IP products were
pelleted by centrifugation at 4000 rpm for 5 min at 4 °C, and washed
twice with IP buffer, then three times with kinase buffer (50 mM Hepes (pH 7.5), 10 mM MgCl2, 5 mM MnCl2, 1 mM NaF, 0.1 mM Na3VO4, 1 mM PMSF, 1 mM dithiothreitol, 1 µg/ml leupeptin, 0.2 µg/ml
aprotinin). To initiate the reaction, 30 µl of kinase buffer with 2.5 mM EGTA, 50 µM cold ATP, 0.2 mg/ml BSA, 10 µCi of [ Inhibition of Intestinal Cell Proliferation by Cdx1 in Stably
Transfected IEC6 Cells--
IEC6 cells are non-transformed intestinal
epithelial cells that were originally isolated from neonatal rat small
intestine (37). They have some characteristics of intestinal crypt
cells, but are morphologically undifferentiated and do not express
genes that are associated with a differentiated phenotype (37). Since they lack expression of both Cdx1 and Cdx2, we have used IEC6 cells to
evaluate the functional role of Cdx genes in intestinal epithelial differentiation (18). The pattern of IEC6 cell accumulation in tissue culture was markedly affected by the expression of Cdx2 (18).
For 2-3 days following induced expression of Cdx2 in stably transfected IEC6 cells, there was a marked decrease in the accumulation of cells in culture (18). This period was followed by exponential growth of cells, reaching confluence at a higher density than cells
that were not induced to express high levels of Cdx2 (18).
To determine the effect of Cdx1 on IEC6 cell proliferation, stable cell
lines were made using an inducible expression construct for Cdx1.
Immunoblots for Cdx1 proteins showed that stable cell lines containing
the Cdx1 expression vector expressed low constitutive amounts of
protein, which was induced following addition of zinc sulfate to the
culture medium (Fig. 1A). The
expressed Cdx1 protein was competent to bind to a known DNA binding
element, the SIF1 element in the sucrase-isomaltase gene promoter (10)
(Fig. 1B). Since the SIF1 element has been shown to have two
adjacent Cdx binding sites (23), the gel shift has two specific
complexes labeled A and B, which represent the monomer and dimer
complexes, respectively. The cell lines transfected with the Cdx1
construct grew more slowly than cell lines transfected with the vector
alone (Fig. 2). Induction of Cdx1
resulted in slower accumulation of cells in culture when compared with
uninduced cells. However, the effect of induction was not great since
the rate of growth of the uninduced cells was already diminished. It is
possible that the decreased growth of uninduced cells is due to the
small amount of expressed Cdx1 in the absence of inducing media (Fig. 1A).
Cdx1 Inhibits Proliferation of Non-intestinal Cells--
To
determine whether the effect of Cdx1 on proliferation is specific for
intestinal IEC cells, we examined the effect on growth of the NIH3T3
fibroblast cell line. A stable cell line was isolated that expressed
Cdx1 under the control of an inducible promoter (3T3 Cdx1 MT1), as
demonstrated by immunoblot (Fig. 1A). The cell lines
transfected with Cdx1 expression vector grew more slowly than those
transfected with empty vector, growing to 2 × 105
cells/100-mm plate at confluence versus 3-4 × 105 cells for vector control cells (data not shown).
Induction of Cdx1 resulted in an additional decrease in the number of
cells at confluence to 1 × 105 cells/100-mm plate
(data not shown). Thus, the inhibitory effect on cellular growth by
Cdx1 is not specific for intestinal cell lines.
There is a report in the literature asserting that expression of Cdx1
in NIH3T3 cells, as well as many other homeobox genes, leads to
transformation as determined by in vitro assays (5). Our
finding that Cdx1 inhibited growth was contrary to these published data. Thus, the growth characteristics of stable lines of NIH3T3 cells
expressing either high or low levels of Cdx1 was examined using a soft
agar assay. Comparison of colony formation in cells cultured in either
control or induction media demonstrated no increase in the number of
colonies as a result of high level expression of Cdx1 (data not shown).
Therefore, our results suggest that Cdx1 is unable to transform NIH3T3
cells, consistent with the effects on proliferation.
Inhibition of Intestinal Cell Proliferation by Adenovirus
Expressing Cdx1--
The cell lines stably transfected with Cdx1
vector consistently grew to a lower density than the cells transfected
with the vector alone. This finding suggested that even low level
expression of Cdx1 in uninduced transfected cells may inhibit IEC6 cell
growth. Therefore, we used an adenoviral vector to express Cdx1 in IEC6 cells. Adenovirus expressing Cdx1 was made as described under "Materials and Methods" and shown to express authentic Cdx1 protein by both Western blot (Fig. 1A) and EMSA (Fig.
1C). Comparison of Cdx1 protein levels on the immunoblot
(Fig. 1A) shows that Cdx1 levels were much greater in the
adenovirus-infected cells than in the stable lines.
The effects of Adeno-Cdx1 was compared with the same adenovirus without
a cDNA insert, Adeno-NULL. The number of infectious particles for
both adenovirus were determined by dilutional infections in 293 cells.
The multiplicity of infection (m.o.i.) was varied in a series of IEC6
cell infections to determine the effect on cell viability and efficacy
of infection and protein expression. It was determined by
immunofluorescence for Cdx1 protein that an m.o.i. of 50 resulted in
expression of Cdx1 in greater than 90% of cells in the culture dish
(data not shown). Thus, an m.o.i. of 50 was used for all experiments.
The accumulation of IEC6 cells infected with Adeno-Cdx1 was markedly
inhibited when compared with uninfected control cells or those infected
with Adeno-Null (Fig. 3A).
Additionally, there was a marked decrease in the percentage of cells
incorporating BrdUrd into DNA (Fig. 3B). Thus, expression of
Cdx1 inhibits proliferation of IEC6 cells, as suggested by the
experiments with stably transfected cells.
Cdx1 Gene Expression Blocks Progression of the Cell Cycle in
G1--
Flow cytometry was used to evaluate cell cycle
progression of IEC6 cells. Cells infected with Adeno-Cdx1 showed a
marked increase in the percentage of cells in G1 with a
decrease in cells in S phase and G2 in comparison to
uninfected cells and cell infected with Adeno-Null (Table
I). Of note, the flow cytometry profiles did not show a subdiploid peak of DNA, suggesting that apoptosis did
not result from expression of Cdx1 (data not shown). Therefore, Cdx1
expression in IEC6 cells inhibits proliferation by blocking cell cycle
progression in G1.
Cell Cycle Protein Changes Associated with Cdx1 Expression--
To
explore the mechanism of the G1 block, we first examined
the expression of the retinoblastoma family of proteins, which are key
regulators of the G1/S transition. In cells expressing Cdx1, there was a shift of pRb to the hypophosphorylated form, there
was a marked increase in hypophosphorylated p130, and a decrease in
p107 (Fig. 4). These changes in Rb
proteins are known to alter transcriptional events that inhibit the
transition from G1 to S phase.
We next examined cell cycle proteins that directly or indirectly affect
the phosphorylation of Rb proteins. Cyclins D1 and D2 are both markedly
decreased in cells expressing Cdx1 with only a slight decrease in
cyclin D3 (Fig. 5). Cdks 4 and 6, which
both associate with D cyclins, are only slightly decreased in
comparison to cells infected with null virus. Cyclin A and B1 were both
decreased, as occurs in any event that blocks the G1/S
transition. There was no change in cyclin E. Since an important
mechanism for inhibition of cyclin-dependent kinase
activity is induction of inhibitor proteins, we examined the expression
of multiple known cyclin-dependent kinase inhibitors. There
was no change in expression of p16, p18, p21, p27, or p57 in cells
expressing Cdx1 (Fig. 5). Immunoblots were also performed for p15 and
p19, but there was no detectable protein (data not shown).
Cyclin-dependent Kinase Activity Is Decreased by
Expression of Cdx1--
The decrease in cyclin D1 and D2 protein
levels represents a potential mechanism by which phosphorylation of Rb
proteins is decreased, resulting in inhibition of the cell cycle.
However, the maintained cyclin D3 could potentially support enough
cyclin D-dependent kinase activity to allow
progression through G1 and the G1/S transition.
To test this question, we measured cyclin-dependent kinase
4 (Cdk4) activity in cellular extracts. Cdk4 kinase activity was
decreased 60% of full activity in cells expressing Cdx1 (Fig. 6). Since Cdk4 protein is not
significantly altered in cells expressing Cdx1 (Fig. 6), the decreased
kinase activity is likely due to low levels of D cyclins. These data
establish the link between decreased cyclin D protein levels and
hypophosphorylation of Rb proteins.
Cdx1 Expression Decreases Cyclin D1 mRNA--
The analysis of
the expression of cell cycle proteins indicated that the likely cause
of G1 arrest was a decrease in the levels of D cyclins.
Therefore, we examined the level of expression of cyclin D1 mRNA
following induction of Cdx1. There was a dramatic reduction of cyclin
D1 mRNA upon expression of Cdx1 in IEC6 cells with no change noted
in control-infected cells (Fig. 7A). We next examined the
expression of mRNAs simultaneously for cyclin D1, D2, and D3 using
a ribonuclease protection assay (Fig.
7B). This experiment showed
that the mRNA levels of the three D cyclins decreased, as would be
predicted from the protein levels with cyclin D1 and D2 mRNAs being
markedly reduced, whereas there was little change in mRNA for
cyclin D3 (Fig. 7B). Therefore, expression of Cdx1 in IEC6
cells causes a reduction in cyclin D1 and D2 mRNA and protein, with
little change in cyclin D3.
Cdx transcription factors appear to have multiple functions in the
establishment and maintenance of the intestinal epithelium. One
critical feature of the organization of the intestinal epithelium is
the compartmentation of proliferating cells in crypts and the continual
renewal of differentiated cells on the villus from this proliferating
compartment. The control of the proliferative state of crypt cells is
important for maintaining the cellular equilibrium in the normal
epithelium and for adapting to pathophysiological stimuli. Taken
together, the data presented in this report shows that Cdx1 is able to
inhibit growth of intestinal epithelial cells by blocking progression
of the cell cycle in G1. Moreover, inhibition of cell cycle
progression appears to be due to decreased cyclin D1 and D2 and the
decreased levels of protein correlate with decreased mRNA.
A recent report came to a different conclusion regarding the effect of
CDX1 on proliferation (21). These investigators constitutively expressed human CDX1 in human adenocarcinoma cell line HT-29 and examined the effect on proliferation and differentiation. They found
that stable expression of CDX1 did not affect the proliferation of
HT-29 cells, whereas co-expression of CDX1 with CDX2 augmented the
growth inhibitory effect of CDX2. There are a number of explanations for the differences in our results in IEC6 cells. First, we used the
mouse Cdx1 cDNA and these investigators used the human cDNA. It
is doubtful that this is the reason for the differences since these two
homologous proteins are highly similar. Second, the cell lines used are
very different. Our studies were performed in a non-transformed rat
intestinal cell line that does not form tumors in nude mice, whereas
HT-29 cells are human adenocarcinoma cells. Cellular growth and
signaling pathways may be very different between these two lines, which
may affect the ability of Cdx1 to regulate proliferation. Third and
most importantly, it is likely that there were very different levels of
expression in the two studies. The expression of Cdx1 in the HT-29
cells was only evaluated using a luciferase reporter construct that is
activated by Cdx proteins, and there was no direct evidence of protein
expression. The luciferase reporter assay is very sensitive and
suggests that the actual levels of Cdx1 protein may have been very low
in the stably transfected lines. Thus, when the different experimental designs are considered, these two apparently contradictory results are
potentially explained by differences in cell lines and levels of Cdx1 expression.
It is unclear whether the cell cycle inhibitory effect of Cdx1 is
important for regulation of intestinal epithelial growth in
vivo. In the adult small intestinal epithelium, Cdx1 protein is
found in greatest amount in the nuclei of crypt cells with decreasing
levels in villus cells as they move toward the villus tip (34).
Similarly, the highest levels of expression in the colonic epithelium
are found in crypt cells with less in the surface epithelial cells
(34). However, it is important to note that Cdx1 is expressed
throughout the villus as well as the crypt, albeit at lower levels. In
Cdx1 null mice, no gross abnormalities were found in the
epithelium, but a careful histological analysis has not yet been
reported (17). Since Cdx1 is also important for expression of
enterocyte genes, there may be other changes in Cdx1 null
mouse epithelium that alters proliferation in addition to loss of Cdx1
alone. Additionally, changes in Cdx2 expression in the Cdx1
null mice may mitigate the effect of loss of Cdx1. Thus, the available
in vivo data are compatible with multiple possibilities
including a pro-proliferative effect, an anti-proliferative effect, or
no effect on proliferation. Our data suggest that Cdx1 may inhibit the
cell cycle and thereby serve as a break on proliferation in crypt
cells. In fact, the cell cycle inhibitory effect is evident over a wide
range of levels of Cdx1 protein (compare IEC6 stable lines to
adenovirus-infected cells; Fig. 1A). However, it is
currently unclear how these protein levels in cell lines compare with
the protein expression in colonocytes in the intact mucosa. Moreover, all conclusions of function in cell lines must be tempered by the fact
that the microenvironment of the intact epithelium in which the
enterocyte resides is very different from cultured cells.
Cdx1 has also been suggested to play a role in neoplasia. A previous
study suggested that Cdx1 was able to transform NIH-3T3 cells (5). We
were unable to confirm these findings since we found no increase in
transformed foci or soft agar colonies in 3T3 cells expressing Cdx1. In
contrast to the previous study, we confirmed the expression of Cdx1 in
the NIH-3T3 clones. Thus, we conclude that Cdx1 does not transform
NIH-3T3 cells. The second association with neoplasia is a change in
expression in human colonic adenocarcinomas. We (34) and others (33)
have shown that the majority of colorectal cancers have decreased
levels of Cdx1 in comparison to normal colonic mucosa. Preliminary data suggest that expression of Cdx1 in some colon cancer cell lines that do
not express Cdx1 results in inhibition of cell growth (38). Since HT-29
cells are not affected by CDX1 expression (21), the role in growth
control of cancer cells will require additional studies.
There are many targets for regulation of the mammalian cell cycle. The
balance between activators and inhibitors of specific kinases
determines progression through the G1 phase and entry into
S phase. The Rb proteins are important gatekeepers of the G1-S transition by regulating the activity of the E2F
family of transcriptional activators. Our data are consistent with Rb
family members as the ultimate targets of Cdx1 protein expression since there was an increase in the inactive hypophosphorylated form of RB and
p130, and loss of p107. Phosphorylation of Rb proteins may be affected
by alterations in many proteins and their multiprotein complexes.
Expression of Cdx1 does not change levels of the
cyclin-dependent kinases or their inhibitors. However,
there is a marked decrease in expression of cyclins D1 and D2, which
serve to activate the kinase activity of CDK4 and CDK6. As a result,
Cdk4-dependent kinase activity is decreased in cells
expressing Cdx1. Thus, it is likely that depletion of these D-type
cyclins results in decreased Rb family phosphorylation by activated
CDKs 4 and 6 and inhibition of progression of the cell cycle. Since
cyclin D1 and D2 mRNAs are decreased in parallel with the proteins,
it appears that Cdx1 affects either transcription of the cyclin D genes
or degradation of the mRNAs, or both. Unraveling this mechanism
will require additional studies.
Taken together, our results suggest a role for Cdx1 in the regulation
of intestinal epithelial cell proliferation as an inhibitor of cell
cycle progression through a decrease in D-type cyclins. We
hypothesize that the potency of the Cdx1 inhibitory effect is dependent
on other cellular signaling pathways and the state of cellular
differentiation. An understanding of the mechanism by which Cdx1
affects the cell cycle will provide insight into maintenance of the
delicate balance of proliferation and differentiation in the intestinal
epithelium and the pathogenesis of colonic neoplasia.
*
This work was supported by National Institutes of Health
NIDDK Grant PO1-DK49210 (to P. G. T.); institutional training
grant support (to J. L.) from the Penn Training Program in
Gastrointestinal Sciences (T32 DK07066); and the Morphology, Vector,
and Molecular Biology Cores of the Center for Molecular Studies in
Digestive Diseases at the University of Pennsylvania (P30-DK50306).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.
1
The nomenclature for caudal-related homeobox
genes is as follows: mouse gene, Cdx1; mouse gene product,
Cdx1; human gene, CDX1; human gene product, CDX1. Because
the functional information derived in this study uses the mouse gene,
this nomenclature will be used as the default. When referring to data
obtained in humans, the human nomenclature will be used.
The abbreviations used are:
pRb, retinoblastoma
protein;
m.o.i., multiplicity of infection;
PBS, phosphate-buffered
saline;
PMSF, phenylmethylsulfonyl fluoride;
GST, glutathione
S-transferase;
EMSA, electrophoretic mobility shift assay;
BrdUrd, 5-bromo-2'-deoxyuridine;
PAGE, polyacrylamide gel
electrophoresis;
RT-PCR, reverse transcription-polymerase chain
reaction;
HRP, horseradish peroxidase;
BES, 2-[bis(2-hydroxyethyl)amino]ethanesulfonic acid;
MOPS, 4-morpholinepropanesulfonic acid;
IP, immunoprecipitation.
The Caudal-related Homeodomain Protein Cdx1 Inhibits
Proliferation of Intestinal Epithelial Cells by Down-regulation of
D-type Cyclins*
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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-mercaptoethanol), and the
cells heated to 100 °C for 5 min. The cells were placed on ice
briefly, then sonicated for 15 s, and stored at 
70 °C.
Protein concentration was determined by the BCA protein Assay (Pierce).
50 µg of whole cell extract was analyzed by a 12% SDS-PAGE
electrophoresis and electroblotted to Immobilon-P membranes. The
membranes were blocked for several hours at room temperature with
PBS/milk, and then incubated overnight at 4 °C with the primary
antibody. The following antibodies were used: for retinoblastoma
protein (pRb), G3-245 (PharMingen); for cyclin D1, 06-137 (Upstate
Biotechnology, Inc.) and SC-246 (Santa Cruz); for p130, SC-317; for
p107, SC-250; for cyclin A, SC-751; for cyclin B1, SC-245; for cyclin
D2, SC-452; for cyclin D3, SC-182; for cyclin E, SC-481; for Cdk2,
SC-163; for Cdk4, SC-601; for Cdk6, SC-177; for p15, SC-613; for p16, SC-1661; for p18, SC-865; for p19, SC-1063; for p21, SC-397; for p27,
SC-1641; for p57, SC-1037 (Santa Cruz); for actin, A-4700 (Sigma); and
anti-Cdx1 antibody (34). After washing membranes three times for 10 min
with PBS/milk, blots were incubated with the secondary antibody
(anti-rabbit/HRP (Amersham Pharmacia Biotech), anti-mouse/HRP, or
anti-goat/HRP (Santa Cruz)) for 45 min at room temperature. Finally,
blots were washed three times and developed with the ECL-Plus Western
blotting kit by Amersham Pharmacia Biotech.
70 °C for 48 h.
70 °C for 48 h to confirm stripping of the previous probe, and then prehybridized and hybridized as before. The 36B4 probe
used as a control is the mouse homologue of the human acidic ribosomal
phosphoprotein P0 (GenBank accession no. M17885). It was generated by
cloning and sequencing the RT-PCR products using the primers
5'-atgtgaagtcactgtgccag-3' and 5'-gtgtaatccgtctccacaga-3'.
-actin
control. The rat-specific probes were generated by cloning and
sequencing RT-PCR products from IEC6 cells using primers to highly
conserved regions of the mouse and human D-cyclins and -actin. The
primers were: D1US, 5'-gatgtgaagttcatttccaa-3'; D1DS,
5'-gttctgctgggcctggcgca-3'; D2US, 5'-tctctgatccgcaagcatgc-3'; D2DS,
5'-ggtgtgggtgatcttggcca-3'; D3US, 5'-cttccctgaggctctccctg-3'; D3DS,
5'-cccaggaccagcacctccca-3'; actin US, 5'-catgtttgagaccttcaaca-3'; and
actin DS, 5'-aggtccagacgcaggatggc-3. RT-PCR products were cloned into
the pCR2.1-TOPO vector (Invitrogen) and sequenced. The clones were
linearized by restriction-enzyme digestion, and antisense
[32P]UTP-labeled riboprobes generated using T7 RNA
polymerase. The riboprobes were then gel-purified. The probe (50,000 cpm) was hybridized with 10 µg of total RNA overnight at 50 °C,
and then subject to RNase T1 digestion using the buffers and conditions recommended by the RPA II kit (Ambion). The products were analyzed on a
7 M urea, 5% acrylamide sequencing gel, dried down, and
exposed to film.
-32P]ATP, and 1 µg of GST-Rb was then
added to the pelleted beads, and the reaction incubated at 30 °C for
30 min with frequent agitation. The reaction was halted with 2×
Laemmli buffer, and the products boiled 5 min and analyzed SDS-PAGE
electrophoresis and autoradiography. The GST-Rb bacterial expression
plasmid was a kind gift of Dr. Greg Enders (University of
Pennsylvania). The fusion protein was expressed and purified as
described (36).
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (69K):
[in a new window]
Fig. 1.
Adeno-CDX1-directed CDX1 protein expression
examined by Western blot and EMSA analysis. A, IEC6 and
3T3 Cell lines stably transfected with an inducible Cdx1 expression
vector, or vector alone, were cultured with (+) or without ( ) 50 µM ZnSO4 for 48 h, and then whole cell
extracts prepared. Additionally, IEC6 cells were sham-infected
(Control), infected with Adeno-Null
(Adeno-Null), and Adeno-CDX1
(Adeno-CDX1) as described under "Materials and
Methods." Whole cell extracts were prepared at 48 h. 20 µg of
each extract was analyzed on a 10% SDS-PAGE for Cdx1 protein using a
polyclonal antibody specific for Cdx1 (CPSP), as described
previously (34). A is a composite figure; the 3T3
lanes were run on a separate gel in parallel. B, stably
transfected IEC6 cells with the pMT-Cdx1 (IEC6Cdx1MT9) were cultured in
the absence or presence of 50 µM ZnSO4 for
24 h. Nuclear proteins were then isolated and analyzed by EMSA to
assess for nuclear binding protein to the SIF1 DNA element of SI
promoter. The A and B DNA-protein complexes represent the interaction
of one and two molecules of Cdx1 on the SIF1 DNA element,
respectively. 10 ng of unlabeled SIF1 oligonucleotide was used for
competition and anti-Cdx1 antibody was added for supershift analysis.
DNA-protein complexes were separated in a native 4% polyacrylamide
gel. The arrowhead marks the position of the complex that
supershifted with addition of anti-Cdx1 antibody. FP, free
probe. C, IEC6 cells were sham-infected (control) or
infected with Adeno-Null and Adeno-CDX1 as before. Nuclear extracts
were prepared 48 h after infection and 5 µg of extract examined
by EMSA using the CDX1 binding SIF1 element from the sucrase-isomaltase
gene as probe. Lanes 1 and 10, probe
only. Specific competition with unlabeled SIF1 (lanes
3, 5, and 7) and supershift using
polyclonal antibodies to CDX1 (CPSP, lane
8) and CDX2 (CANL, lanes 9)
were also performed. A, Cdx1 monomer binding; B,
Cdx1 dimer binding complex. *, SS, supershift complex;
NS, nonspecific complex; FP, free probe.
View larger version (13K):
[in a new window]
Fig. 2.
ZnSO4-induced Cdx1 inhibits
proliferation of stably transfected IEC6 cells. Growth curve was
obtained from vector control and cells expressing Cdx1 (IEC6Cdx1),
which were grown in the absence and in the presence of 50 µM ZnSO4 by cell counting at indicated time.
Cell lines tested included IEC6 cells transfected with vector alone
(Vector control), and lines MT1 and MT9 that were
transfected with Cdx1 expression construct. Cell number is expressed as
mean ± standard deviation of three independent determinations.
Square, media with ZnSO4; circle,
media without ZnSO4.
View larger version (19K):
[in a new window]
Fig. 3.
CDX1 expression reduces IEC6 cell
proliferation and DNA synthesis. A, IEC6 cells were
sham-infected or infected with Adeno-Null or Adeno-CDX1. Cell counts
were performed for each treatment on days 1, 2, 3, 4, and 5 after
infection. Cell counts from several separate experiments were averaged
and compiled for this figure. On days 1-4, n = 7, and
day 5, n = 4. Open square,
control uninfected; closed diamond,
Adeno-Null-infected; closed square,
Adeno-CDX1-infected. B, IEC6 cells were infected and
cultured as in A, but on days 1, 2, 3, and 4 after infection
cells were incubated for 1 h with the nucleoside analogue BrdUrd.
Cells were washed and fixed, and then probed with a monoclonal antibody
for nuclear BrdUrd incorporation. Nuclei were counterstained with
bisbenzamide, and BrdUrd (+) nuclei were counted and expressed as a
percentage of all nuclei counted. n = 5 for each
condition, each day. These values were transformed using an arcsin
transformation and evaluated for statistical significance with analysis
of variance and Tukey rank mean tests. *, significant difference from
control sham-infected or Adeno-Null-infected cells, p < 0.01. Representative data from one of three experiments is
presented. Black bars, control uninfected;
dark gray bars, Adeno-Null-infected;
light gray bars,
Adeno-CDX1-infected.
Flow cytometry data for IEC6 cells expressing CDX1
View larger version (79K):
[in a new window]
Fig. 4.
Western blot analysis for pRb and pRB family
proteins in IEC6 cells expressing CDX1. IEC6 cells were cultured
and infected as described previously. At 48 h, cells from each
condition were assayed by flow cytometry to confirm the
G0/G1 block with CDX1 expression. Whole cell
extracts were then prepared. 50 µg of this whole cell extract from
IEC6 cells infected with Adeno-CDX1 (CDX1), Adeno-Null
(Null), or uninfected control (Con) cells were
loaded per lane and subjected to 6% SDS-PAGE gel separation and
Western blotting with antibodies specific for pRb, p130, and p107.
Arrows indicate positions of differentially phosphorylated
species. Control, Adeno-Null, and Adeno-CDX1 extracts were always
prepared simultaneously for each experiment. At least three separate
experiments were performed, and the representative results from one are
presented. Protein loading controls are presented in other
figures.
View larger version (54K):
[in a new window]
Fig. 5.
Western blot analysis for cyclins,
cyclin-dependent kinases, and cyclin-dependent
kinase inhibitors in IEC6 cells expressing CDX1. Western
immunoblotting of whole cell extracts was performed as described
previously using the specific antibodies as noted. All blots were
routinely stripped and reprobed with a monoclonal antibody to -actin
to confirm equal protein loading. A representative blot from one of
three experiments is presented.
View larger version (47K):
[in a new window]
Fig. 6.
Cdx1 expression reduces Cdk4-associated pRb
kinase activity in IEC6 cells. A, IEC6 cells were
sham-infected or infected with Adeno-Null or Adeno-CDX1 as before. At
48 h, whole-cell extracts were obtained, and 100 µg of each
extract was subject to immunoprecipitation with an antibody specific
for Cdk4 or with a control rabbit IgG. The Cdk4-associated kinase
activity present in the IP products was assayed using purified GST-Rb
as substrate. The 32P-labeled products were analyzed on a
12% SDS-PAGE gel. To confirm equivalent levels of Cdk4 in the tested
extracts, an immunoblot for Cdk4 and -actin was performed on 50 µg
of the whole cell extracts. Representative experiments are presented.
B, the dried SDS-PAGE gels were exposed to a PhosphorImager
(Molecular Dynamics) and the labeling present in the GST-Rb kinase
products quantified. Averages and standard deviations were determined
for each condition (n = 4). The 32P
labeling activity present in the rabbit IgG IP products was considered
to be background, and the average values subtracted from the respective
experimental values. To determine the relative Cdk4 kinase activity,
the average 32P labeling of GST-Rb in the control
uninfected IEC6 cells was considered to be 100%, and the labeling
activity in the Adeno-Null- and Adeno-Cdx1-infected cells is expressed
as a percentage of this control value.
View larger version (56K):
[in a new window]
Fig. 7.
Northern blot and RPA analysis analysis for
cyclin D1, D2, and D3 mRNA expression in IEC6 cells expressing
CDX1. IEC6 cells were cultured and infected as described
previously. At 48 h, cells from each condition were assayed by
flow cytometry to confirm the G0/G1 block with
CDX1 expression. Total RNA was then isolated. A, 20 µg of
total RNA from IEC6 cells infected with Adeno-CDX1 (CDX1),
Adeno-Null (Null), or uninfected control (Con)
cells were loaded per lane and subjected to 0.8% agarose, 6%
formaldehyde gel separation and blotting. Blots were probed with
32P-labeled human cyclin D1 cDNA, washed, and exposed
to film. All blots were then stripped of probe and rehybridized for the
36B4 mRNA, an acidic ribosomal phosphoprotein P0 used as a transfer
control. Several separate experiments were performed, and the
representative results from one are presented. B,
32P-labeled riboprobes for rat cyclin D1, D2, D3, and actin
were prepared as described and hybridized to 10 µg of total RNA, and
subject to ribonuclease protection analysis. The products were then
analyzed on a 5% polyacrylamide, 8 M urea gel, dried down,
and exposed to film.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
FOOTNOTES
To whom correspondence should be addressed: 100 Centrex, Hospital
of the University of Pennsylvania, 3400 Spruce St., Philadelphia, PA
19104. Fax: 215-349-5734; E-mail: traberp@mail.
med.upenn.edu.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1.
Duboule, D.
(1994)
Guidebook to the Homeobox Genes
, Oxford University Press, Oxford
2.
Boulikas, T.
(1993)
J. Cell. Biochem.
52,
14-22[CrossRef][Medline]
[Order article via Infotrieve]
3.
Kessel, M.,
and Gruss, P.
(1990)
Science
249,
374-379 4.
Holland, P. W. H.,
and Hogan, B. L. M.
(1988)
Genes Dev.
2,
773-782 5.
Maulbecker, C. C.,
and Gruss, P.
(1993)
Cell Growth Differ.
4,
431-441[Abstract]
6.
Burglin, T. R.
(1994)
A Comprehensive Classification of Homeobox Genes
, Sambrook and Tooze Publishing Partnership, New York
7.
Meyer, B. I.,
and Gruss, P.
(1993)
Development
117,
191-203[Abstract]
8.
James, R.,
Erler, T.,
and Kazenwadel, J.
(1994)
J. Biol. Chem.
269,
15229-15237 9.
Gertzenberg, R. H.
(1994)
J. Cell. Biochem.
55,
22-31[CrossRef][Medline]
[Order article via Infotrieve]
10.
Suh, E.-R.,
Chen, L.,
Taylor, J.,
and Traber, P. G.
(1994)
Mol. Cell. Biol.
14,
7340-7351 11.
Gamer, L. W.,
and Wright, C. V. E.
(1993)
Mech. Dev.
43,
71-81[CrossRef][Medline]
[Order article via Infotrieve]
12.
Bonner, C. A.,
Loftus, S. K.,
and Wasmuth, J. J.
(1995)
Genomics
28,
206-211[CrossRef][Medline]
[Order article via Infotrieve]
13.
Ee, H. C.,
Erler, T.,
Bhathal, P. S.,
Young, G. P.,
and James, R. J.
(1995)
Am. J. Pathol.
147,
586-592[Abstract]
14.
Beck, F.,
Erler, T.,
Russell, A.,
and James, R.
(1995)
Dev. Dyn.
204,
219-227[Medline]
[Order article via Infotrieve]
15.
James, R.,
and Kazenwadel, J.
(1991)
J. Biol. Chem.
266,
3246-3251 16.
Duprey, P.,
Chowdhury, K.,
Dressler, G. R.,
Balling, R.,
Simon, D.,
Guenet, J.-L.,
and Gruss, P.
(1988)
Genes Dev.
2,
1647-1654 17.
Subramanian, V.,
Meyer, B. I.,
and Gruss, P.
(1995)
Cell
83,
641-653[CrossRef][Medline]
[Order article via Infotrieve]
18.
Suh, E.-R.,
and Traber, P. G.
(1996)
Mol. Cell. Biol.
16,
619-625[Abstract]
19.
Lorentz, O.,
Duluc, I.,
DeArcangelis, A.,
Simon-Assman, P.,
Kedinger, M.,
and Freund, J. N.
(1997)
J. Cell Biol.
139,
1553-1565 20.
Duluc, I.,
Lorentz, O.,
Fritsch, C.,
Leberquier, C.,
Kedinger, M.,
and Freund, J. N.
(1997)
J. Cell Sci.
110,
1317-1324[Abstract]
21.
Mallo, G. V.,
Soubeyran, P.,
Lissitzky, J.-C.,
Angre, R.,
Farnarier, C.,
Marvaldi, J.,
Dagorn, J.-C.,
and Iovanna, J. L.
(1998)
J. Biol. Chem.
273,
14030-14036 22.
Chawengsaksophak, K.,
James, R.,
Hammond, V. E.,
Kontgen, F.,
and Beck, F.
(1997)
Nature
386,
84-87[CrossRef][Medline]
[Order article via Infotrieve]
23.
Taylor, J. K.,
Boll, W.,
Levy, T.,
Suh, E. R.,
Siang, S.,
Mantei, N.,
and Traber, P. G.
(1997)
DNA Cell Biol.
16,
1419-1428[Medline]
[Order article via Infotrieve]
24.
Traber, P. G.,
and Silberg, D. G.
(1996)
Annu. Rev. Physiol.
58,
275-297[CrossRef][Medline]
[Order article via Infotrieve]
25.
Troelsen, J. T.,
Olsen, J.,
Noren, O.,
and Sjostrom, H.
(1992)
J. Biol. Chem.
267,
20407-20411 26.
Troelsen, J. T.,
Olsen, J.,
Mitchelmore, C.,
Hansen, G. H.,
Sjostrom, H.,
and Noren, O.
(1994)
FEBS Lett.
342,
297-301[CrossRef][Medline]
[Order article via Infotrieve]
27.
Jin, R. R.,
Trinh, D. K. Y.,
Wang, F.,
and Drucker, D. J.
(1997)
Mol. Endocrinol.
11,
203-209 28.
Jin, T.,
and Drucker, D. J.
(1996)
Mol. Cell. Biol.
16,
19-28[Abstract]
29.
Taylor, J. K.,
Levy, T.,
Suh, E. R.,
and Traber, P. G.
(1997)
Nucleic Acids Res.
25,
2293-2300 30.
Trinh, K. T.,
Jin, T.,
and Drucker, D. J.
(1999)
J. Biol. Chem.
274,
6011-6019 31.
Troelsen, J. T.,
Mitchelmore, C.,
Spodsberg, N.,
Jensen, A. M.,
Noren, O.,
and Sjostrom, H.
(1997)
Biochem. J.
322,
833-838
32.
Lambert, M.,
Colnot, S.,
Suh, E. R.,
L'Horset, F.,
Blin, C.,
Calliot, M. E.,
Raymondjean, M.,
Thomasset, M.,
Traber, P. G.,
and Perret, C.
(1996)
Eur. J. Biochem.
236,
778-788[Medline]
[Order article via Infotrieve]
33.
Mallo, G. V.,
Rechreche, H.,
Frigerio, J.-M.,
Rocha, D.,
Zweibaum, A.,
Lacasa, M.,
Jordan, B. R.,
Busetti, N. J.,
Dagorn, J.-C.,
and Iovanna, J. L.
(1997)
Int. J. Cancer
74,
35-44[CrossRef][Medline]
[Order article via Infotrieve]
34.
Silberg, D. G.,
Furth, E. E.,
Taylor, J. K.,
Schuck, T.,
Chiou, T.,
and Traber, P. G.
(1997)
Gastroenterology
113,
478-486[CrossRef][Medline]
[Order article via Infotrieve]
35.
Peterson, M. G.,
Hannan, F.,
and Mercer, J. F. B.
(1988)
Eur. J. Biochem.
174,
417-424[Medline]
[Order article via Infotrieve]
36.
Brent, R.
(1997)
in
Short Protocols in Molecular Biology
(Ausubel, F.
, Brent, R.
, Kingston, R. E.
, Moore, D. D.
, Seidman, J. G.
, Smith, J. A.
, and Struhl, K., eds)
, pp. 16-28-16-31, John Wiley & Sons, Inc., New York
37.
Quaroni, A.,
Wands, J.,
Trelstad, R. L.,
and Isselbacher, K. J.
(1979)
J. Cell Biol.
80,
248-265 38.
Yang, D.,
Lynch, J.,
Silberg, D. G.,
Suh, E. R.,
and Traber, P. G.
(1998)
Gastroenterology
114,
A707
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