[CANCER RESEARCH 60, 2825–2830, June 1, 2000]
Advances in Brief
Interaction between Sp1 and Cell Cycle Regulatory Proteins Is Important in
Transactivation of a Differentiation-related Gene1
Oliver G. Opitz and Anil K. Rustgi2
Gastroenterology Division [O. G. O., A. K. R.], Cancer Center [A. K. R.], Department of Genetics [A. K. R.], University of Pennsylvania, Philadelphia, Pennsylvania 19104
Abstract
The stratified squamous epithelium is a model system in which to define
molecular mechanisms underlying the switch from proliferation to differentiation. This can be achieved through the functional dissection of keratin gene promoters. Having previously established the importance of
keratin 4 in maintaining the differentiated phenotype in corneal epithelial
cells, we investigated the role of Sp1-mediated transactivation of the
keratin 4 promoter given the role of Sp1 in differentiation and cell cycle
progression. Sp1 transactivation of the keratin 4 promoter was diminished
in cyclin D1-overexpressing cells, which may be mediated through a newly
described direct interaction between Sp1 and cyclin D1 and opposed by a
complex between Sp1 and pRB.
Introduction
The stratified squamous epithelium comprises proliferating basal
cells, differentiating suprabasal and intermediate cells, as well as
terminally differentiated superficial squamous cells that line the surface. Ultimately, these superficial cells desquamate, and the process
then is continuously renewed. The stratified squamous epithelium is
found in many sites in human tissues, including the cornea, esophagus, oropharynx, larynx, skin, and anogenital tract.
Basal cells are subjected to a tightly regulated program of differentiation as they migrate toward the surface accompanied by a series
of morphological, biochemical, and genetic changes. Insights into the
biochemical and molecular genetic mechanisms that orchestrate the
switch from proliferation to early differentiation in this cell type can
be achieved through an understanding of the K3 genes that modulate
this process and their transcriptional regulation. In this context, K5
and K14 heterodimerize in proliferating basal cells. The relatively
ubiquitous K1 and K10 are linked to early differentiation genes and
heterodimerize in suprabasal cells, whereas loricrin, profillagrin, and
transglutaminase are among the late differentiation genes in superficial squamous cells (1, 2). Of significance, the suprabasal K4 and K13
are relatively tissue restricted, with highest expression in esophageal
and corneal squamous epithelial cells (3).
We previously generated a model in which K4 is disrupted through
homologous recombination in murine embryonic stem cells, resulting
in impaired differentiation and basal cell hyperplasia, specifically in
esophageal and corneal epithelia of homozygous null mice (4). This
formed the basis for studying transcriptional regulatory mechanisms
of the human K4 promoter in esophageal and corneal squamous
Received 12/1/99; accepted 4/10/00.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by NIH Grants R01-DK53377 (to A. R.), P01-DE12467 (to
A. R. and O. O.), American Cancer Society Jr. Faculty Research Award JFRA-649 (to
A. R.), The Leonard and Madlyn Abramson Family Cancer Research Institute (to A. R.),
and Deutsche Krebshilfe Grant D/96/17197 (to O. O.).
2
To whom requests for reprints should be addressed, at Gastroenterology Division,
600A CRB, University of Pennsylvania, 415 Curie Boulevard, Philadelphia, PA 19104.
Phone: (215) 898-0154; Fax: (215) 573-5412; E-mail: [email protected].
3
The abbreviations used are: K, keratin; EMSA, electrophoretic mobility shift assay;
IP, immunoprecipitation; cdk, cyclin-dependent kinase.
epithelial cells. Previously, we have demonstrated that, in esophageal
squamous epithelial cells, the human K4 promoter is transcriptionally
regulated by esophageal-specific transcription factors (5). To further
elucidate how the regulation of K4 is linked to differentiation, we
undertook analysis of its 5⬘ untranslated regulatory region and promoter in corneal epithelial cells. Given that one of the roles of Sp1 is
to modulate cellular differentiation and influence cell cycle progression in cooperation with the retinoblastoma protein (pRB), we focused
on putative Sp1 DNA binding motifs in the K4 promoter and the role
of Sp1 in regulating the K4 promoter. This was further investigated by
comparing Sp1-mediated transactivation of the K4 promoter in parental normal corneal epithelial cells and stably transfected corneal
epithelial cells with the cell cycle regulator, cyclin D1, the latter
achieved through retroviral transduction. We describe herein that Sp1
transactivation of the K4 promoter in corneal epithelial cells is suppressed by cyclin D1, and that this can be rescued by concurrent
ectopic expression of pRB. In particular, we demonstrate that cyclin
D1 interacts with Sp1 in vivo, suggesting perhaps a model in which
Sp1 may influence differentiation through interactions with either
pRB or cyclin D1.
Materials and Methods
Cell Culture and Retroviral Infection. A rabbit normal corneal cell line
SIRC (American Type Culture Collection, Rockville, MD) and the cyclin
D1-overexpressing SIRC cell line, designated C3D1, were grown in Eagle’s
minimal essential medium (Sigma) supplemented with 10% fetal bovine serum
(Sigma) and antibiotics. Additionally, the human esophageal squamous cancer
cell line TE-12, the human osteosarcoma cell line U2OS, the human bladder
cancer cell line J82, and the lymphoma B-cell line DT40 were grown under
standard conditions.
The amphotrophic packaging cell line Phoenix A was grown in supplemented DMEM (Sigma) and transiently transfected with the respective retroviral vector to generate amphotrophic retroviruses. Fresh retroviral supernatant
was used for infection of exponentially growing SIRC cells. Several clones
were harvested for further processing after puromycin selection. The retroviral
expression vectors pBPSTR-D1 and pBabe-lacZ were obtained from S. Reeves
(Massachusetts General Hospital, Charlestown, MA). The retroviral vector
pBPSTR-D1 is described previously (6) and contains both elements of the
tetracycline-regulated system. The retroviral expression vector pBPSTR is
based on the vector pBabe, which accounts for comparable infectivity. The
puromycin resistance gene under the control of the promoter within the 5⬘ long
terminal repeat is present in both retroviral vectors.
Transient Transfection. Transient transfection of different K4 promoter
deletion-luciferase reporter constructs (K4-940, K4-540, K4-163, K4-140, and
K4-76; Ref. 5) and cotransfections of expression plasmids pRC/RSV-empty,
pRC/CMV-Sp1, pSG5-RB (wild-type pRB), pJ3 RB-592 (mutant pRB), and
pJ3 RB-209 (mutant pRB) were carried out using the calcium phosphate
precipitation technique (5⬘33⬘, Inc.), as described previously (5, 7, 8). Incubations were performed in triplicate, and results were calculated as the
mean ⫾ SE values for luciferase activity. Values were then expressed as fold
increase or decrease compared with the control for each set of experiments.
Activities were expressed as the mean of at least three independent transfection
experiments. Transfection efficiency was controlled by cotransfection with
pGreen Lantern-1 (Life Technologies, Inc.) and found not to vary within a
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SP1 TRANSACTIVATION OF KERATIN 4
given transfection experiment, indicating that transfection efficiency was uniform.
Flow Cytometry. Exponentially growing cells were collected, and the cell
pellet fixed overnight in 70% ethanol and then resuspended in a 1-ml solution
containing 3.8 mM sodium citrate and 10 ␮g/ml propidium iodide. After 10
mg/ml RNase treatment at 37°C for 20 min, the samples were analyzed by a
fluorescence-activated cell sorter (FACScan; Becton Dickinson).
Coimmunoprecipitation and Western Blot Analysis. Lysates from exponentially growing cells were harvested in a buffer [50 mM HEPES (pH 7.4),
0.1% NP40, and 250 mM NaCl] with 1 mM protease and 10 mM phosphatase
inhibitors. Total protein (150 ␮g) was incubated with 1 ␮g of primary antibody
at 4°C overnight, followed by incubation with Protein A/Protein G Plus
Agarose (Santa Cruz Biotechnology) for 2 h. Proteins were separated on
6⫺12% SDS-polyacrylamide gels and transferred to Immobilon membranes
(Millipore). Incubation with primary antibodies was performed as indicated
(1:3000). The secondary antibody was either peroxidase-conjugated antimouse
or antirabbit immunoglobulin (Amersham Corp.; 1:2500). Detection was by
chemiluminescence (ECL; Amersham Corp.).
EMSAs. Nuclear extracts from SIRC and C3D1 cell lines were prepared,
and EMSAs were performed as described previously (5, 7, 8) using probes
Sp1-A (5⬘-AGCTT AACGG GTGCG GGAAG GATGG CTTGC-3⬘), Sp1-C
(5⬘-AGCTT AGGCT AAGGC TGGAC-3⬘), and Sp1-consensus (5⬘-AGCTT
ATTCG ATCGG GGCGG GGCGA GCC-3⬘). For competition experiments,
the nuclear extract was preincubated with 50-fold excess of unlabeled doublestranded Sp1 consensus oligonucleotide prior to the addition of the ␣32Plabeled oligonucleotide probe. AP1 consensus oligonucleotide was used as a
control. Immune supershift assays were performed using a polyclonal anti-Sp1
(PEP2) or anti-AP1 antibody (D; Santa Cruz Biotechnology). The antibody
was preincubated with the nuclear extract at room temperature for 30 min prior
to the addition of the ␣32P-labeled oligonucleotide DNA probe.
IP-EMSA. IPs were done as described. Protein-agarose complexes were
then washed three times in gelshift buffer (7, 8) treated with 32 ␮l of 0.8%
deoxycholate to dissociate protein complexes on ice and neutralized with 1%
NP40. Ten ␮l of the supernatant were then used for EMSAs as described
above.
Results
Ectopic Cyclin D1 Overexpression in Normal Corneal Epithelial Cells Causes Cell Cycle Abnormalities. We used a tetracyclineregulated retroviral vector system, pBPSTR-D1 (6), containing the
cyclin D1 cDNA, to infect the normal corneal squamous epithelial cell
line termed SIRC. Parallel experiments used a lacZ gene containing
retrovirus (pBabe-lacZ) to determine infection efficiency. Infection
efficiencies of retroviral supernatants of the two vectors are comparable because pBPSTR is a derivative of pBabe. Infected SIRC cells
were puromycin selected, and the clone used for further studies was
designated C3D1, although several clones were expanded (e.g.,
B4D1) without any differences observed in cyclin D1 overexpression
and cell cycle kinetics. The levels of endogenous and ectopically
expressed cyclin D1 in the parental SIRC and cyclin D1-transduced
cell lines were determined using Western blot analysis (Fig. 1A).
Ectopic cyclin D1 was induced in asynchronously growing cells in the
absence of tetracycline. Of note, the levels of cyclin D1 in the
presence of tetracycline were essentially the same observed in the
parental cell line (data not shown). Flow cytometry analysis revealed
pronounced cell cycle redistribution in C3D1 cells with a 20% increase in cells cycling in S-phase compared with parental SIRC cells
(Fig. 1B). Furthermore, C3D1 cells demonstrated an altered morphology and grew in clusters with smaller size and rounded appearance
compared with the pleomorphic parental SIRC cells (data not shown).
Sp1 Transactivation in Corneal Epithelial Cells Is Cell Cycle
Dependent. We next assessed transcriptional regulation of the differentiation-linked human K4 promoter in corneal epithelial cells.
Having shown before that multiple positive and negative cis-regulatory elements reside in the K4 promoter (5), here we focused our
attention on the role of Sp1 in transcriptional regulation of the K4
Fig. 1. Effects of cyclin D1 overexpression in corneal epithelial cells. A, equal amounts
of protein for parental SIRC and the infected C3D1 and B4D1 cell lines, cultured in either
tetracycline-free or tetracycline containing (⫹tet) medium were separated on a 10%
SDS-PAGE. Subsequent immunoblot was done with cyclin D1 monoclonal antibody
(HD11). B, cell cycle analysis of SIRC and C3D1 cells was done under standard
conditions using a fluorescence-activated cell sorter (FACScan; Becton Dickinson). Cell
cycle analysis is either displayed as a histogram or a table, where the left panel represents
SIRC cells and the right panel is C3D1 cells.
promoter. Sp1 is known to regulate differentiation in several cell types
(9, 10). Different K4 promoter deletion constructs (K4-940, K4-540,
K4-163, K4-140, and K4-76; Ref. 5) were transfected in SIRC cells;
an Sp1 expression vector or an empty vector was cotransfected, and
luciferase activity was assayed as described (5, 7, 8). Several potential
Sp1 binding sites are located throughout the K4 promoter. We found
that Sp1 transactivates the K4-940 promoter ⬃10-fold, which is
reduced by 50% in the K4-540, K4-163, and K4-140 deletion constructs and almost abolished in the K4-76 deletion construct. These
two reductions in Sp1-mediated transactivation are consistent with
two distinct functional Sp1 cis-regulatory elements within the K4
promoter, one within the K4-940 construct and the other one within
the K4-140 construct, respectively (Fig. 2A). One putative Sp1 binding site resides at ⫺649 bp and the other one at ⫺125 bp.
To investigate whether cyclin D1 overexpression influences the
transcriptional regulation of the differentiation-linked K4 promoter in
corneal epithelial cells, the K4-940 construct was transfected in C3D1
cells. Overall, the activity of the K4 promoter was 10-fold lower in
C3D1 cells compared with parental SIRC cells. Furthermore, when
Sp1 was cotransfected, Sp1-mediated transactivation of the K4 promoter was reduced to 5-fold in C3D1 cells compared with 10-fold in
SIRC cells (Fig. 2B).
Because pRB is the major substrate of cyclin D1 and is known to
act as a transcriptional activator through Sp1 (11, 12), the role of pRB
role in influencing the transcriptional regulation of K4 was assessed.
Interestingly, cotransfection with wild-type pRB, but not mutated
pRB (pRB-592 and pRB-209), resulted in a 10-fold transactivation of
the K4 promoter in C3D1 cells (Fig. 2B). Thus, pRB rescues the
reduction in Sp1-mediated transactivation of the K4 promoter in
cyclin D1-overexpressing cells. It has been described previously that
2826
SP1 TRANSACTIVATION OF KERATIN 4
Fig. 2. Sp1 transcriptionally regulates the K4 promoter in SIRC and C3D1 cells. A, SIRC corneal cells were transfected with 2 ␮g of K4 promoter deletion-luciferase reporter
constructs (K4-940, K4-540, K4-163, K4-140, and K4-76) and cotransfected with 1 ␮g of pRC/RSV-empty or pRC/CMV-Sp1 vectors. B, effects of Sp1 and/or pRB on K4 promoter
activity in SIRC and C3D1 cells. SIRC and C3D1 cells were transfected with the K4-940 promoter-luciferase construct and cotransfected with 1 ␮g of pRC/RSV-empty,
pRC/CMV-Sp1, pSG5-RB (wild-type pRB), pJ3 RB-592 (mutant pRB), or pJ3 RB-209 (mutant pRB). In both panels, luciferase activity is expressed as fold increase relative to the
empty vector-cotransfection (means; bars, SE) and is calculated from three independent transfections. C, EMSAs were performed with SIRC nuclear extracts using probes Sp1-A,
Sp1-C, and Sp1-consensus. D, equal amounts of SIRC and C3D1 nuclear extracts were compared using probe Sp1-C (Lanes 1 and 2). The Sp1-DNA complex was competed specifically
by the Sp1-consensus oligonucleotide but not by a nonspecific (AP1-consensus) oligonucleotide (Lanes 3 and 5). The addition of Sp1 polyclonal antibody (PEP2) diminished the
Sp1-DNA complex. AP1 monoclonal antibody (D) served as negative control (Lanes 4 and 6).
pRB regulates the transcription of target genes through cis-acting
elements referred to as retinoblastoma control elements, which can
bind members of the Sp1 family (11, 13). In contrast, pRB cotransfection showed only a 5-fold transactivation of the K4 promoter in
parental SIRC cells (Fig. 2B). Cotransfection of pRB and Sp1 did not
cause any significant additional increase in K4 promoter activity in
both cell lines (Fig. 2B).
Sp1 Binds to cis Regulatory Elements of the Human K4 Promoter. To examine whether the putative Sp1 cis-regulatory elements
indeed bind Sp1, we performed EMSAs (5, 7, 8) with either SIRC or
C3D1 nuclear extracts. EMSAs were performed with two double-
stranded oligonucleotide probes containing the putative Sp1 elements
within the K4 promoter, residing at positions ⫺649 bp (Sp1-C) and
⫺125 bp (Sp1-A), respectively. As control, a Sp1 consensus oligonucleotide probe was used. One DNA-protein complex was observed
with Sp1-A, Sp1-C, and Sp1-consensus oligonucleotide probes, suggesting that these DNA-protein complexes were formed by Sp1 and
an oligonucleotide harboring an Sp1 DNA binding motif (Fig. 2C).
Adding 50-fold excess of unlabeled Sp1 consensus oligonucleotide
(Fig. 2D) specifically inhibited Sp1-DNA complex formation. Human
Sp1-specific antibody further decreased the Sp1-DNA complex (Fig.
2D). AP1 oligonucleotide and antibody served as controls.
2827
SP1 TRANSACTIVATION OF KERATIN 4
differences in Sp1-mediated transactivation were most apparent. This
IP-EMSA revealed a specific Sp1-DNA complex in both cell lines,
released from either pRB (Fig. 4C, Lanes 5 and 6) or cyclin D1
immunoprecipitates (Fig. 4C, Lanes 9 and 10). However, a more
prominent DNA-protein complex could be observed in immunoprecipitates from C3D1 cells. Addition of Sp1-specific antibody, but not
AP1 antibody, attenuated this immunoprecipitated Sp1 complex (Fig.
4C). Sp1 immunoprecipitates (Fig. 4C, Lanes 1 and 2) and AP1
immunoprecipitates (Fig. 4C, Lanes 15 and 16) served as positive or
negative controls, respectively. In aggregate, our findings are consistent with an interaction between Sp1 and cyclin D1 that leads to
differential regulation of a differentiation-linked K4 promoter.
Fig. 3. Effect of cyclin D1 overexpression on pRB and Sp1 protein levels. Equal
amounts of protein for SIRC and C3D1 cell lines were separated on a 6 or 10%
SDS-PAGE, respectively. Immunoblot of endogenous pRB (A) and Sp1 (B) in SIRC and
C3D1 cell lines was done using pRB monoclonal antibody (G3-245; PharMingen) or Sp1
monoclonal antibody (1C6; Santa Cruz Biotechnology), respectively. The two arrows in
A indicate hyperphosphorylated (upper) and hypophosphorylated (lower) forms of pRB,
respectively.
Comparison of Sp1-DNA complexes in both cell lines indicated a
decrease with C3D1 nuclear extracts compared with SIRC nuclear
extracts, thereby suggesting diminished DNA binding by Sp1 in
cyclin D1-overexpressing cells (Fig. 2D). These results indicate that
cyclin D1 overexpression influences the transcriptional regulation of
the K4 promoter in corneal epithelial cells by interfering with the
function of Sp1 as a transcriptional activator.
Differential Expression of pRB and Sp1 in Cyclin D1-overexpressing Corneal Epithelial Cells. To clarify differences in Sp1DNA complex formation in C3D1 cells versus SIRC cells, we determined Sp1 and pRB protein levels in these cell lines. Western blot
analysis of pRB levels, using a pRB antibody that equally recognizes
hypo- as well as hyperphosphorylated pRB, revealed a shift toward
the hyperphosphorylated form of pRB in C3D1 cells (Fig. 3A), consistent with cyclin D1 overexpression. Western blot analysis of Sp1
revealed no appreciable differences between C3D1 and SIRC cells
(Fig. 3B).
Sp1 Interacts with pRB and Cyclin D1 in Corneal Squamous
Epithelial Cells. We next asked whether Sp1 physically interacts
with cell cycle regulatory proteins, i.e., pRB and cyclin D1, leading to
this differential transcriptional regulation of K4. Initially, C3D1 and
SIRC cells were immunoprecipitated with either a pRB antibody, a
cyclin D1 antibody, or as a control an AP1 antibody. The coimmunoprecipitates were resolved by SDS-electrophoresis and immunoblotted with an anti-Sp1 antibody (data not shown). Sp1 immunoprecipitates prepared in parallel were immunoblotted with either antipRB or anti-cyclin D1 antibodies, respectively (Fig. 4A and B). We
found that Sp1 physically interacted with either pRB or cyclin D1.
Whereas no differences in levels between the two cell lines could be
determined with the recently described Sp1-pRB interaction (Ref. 12;
Fig. 4A), a more prominent Sp1-cyclin D1 interaction was detected in
C3D1 cells compared with SIRC cells (Fig. 4B). However, in C3D1
cells, Sp1 interacts predominantly with hyperphosphorylated pRB,
whereas in SIRC cells an interaction with both forms of pRB could be
observed (Fig. 4A). As a negative control, J82 cells, where pRB is
truncated, and DT40 cells, where cyclin D1 is deleted, were immunoprecipitated with Sp1 antibody and immunoblotted with either
anti-pRB antibody (Fig. 4A) or anti-cyclin D1 antibody (Fig. 4B),
respectively.
To further confirm these Sp1 protein-protein interactions, IPEMSAs were performed essentially as described (14). C3D1 and
SIRC cells were immunoprecipitated with pRB or cyclin D1 antibodies, complexes were dissociated, and then EMSAs were performed
with radiolabeled Sp1-C probe, the region of the K4 promoter where
Discussion
The equilibrium between proliferation and differentiation is regulated exquisitely by a complex network of signals, both extracellular
and intracellular. This is illustrated by the rapid turnover of cells in the
stratified squamous epithelium at many sites. The switch from proliferation to differentiation is in part governed by the differential
expression of cytokeratins in basal and suprabasal cells (1, 2). K4 is
an excellent model in which to study early differentiation in the
corneal epithelium (4, 5). We now describe that Sp1 transcriptionally
regulates the K4 promoter in corneal epithelial cells, which is mediated through binding of Sp1 to two independent Sp1 cis-acting elements. However, these functional and biochemical effects were partially abrogated in cyclin D1-overexpressing cells, which was restored
by ectopic expression of pRB.
Indeed, a natural question emanating from these findings was
whether cyclin D1 and Sp1 interacted with each other apart from the
recently described Sp1-pRB interaction (12). Our data are consistent
with the notion that cyclin D1 and Sp1 do interact. It is tempting to
speculate that the Sp1-pRB complex may mediate transactivation of
genes, the protein products of which in turn favor growth suppression
and/or commit the cell to early differentiation. The role of Sp1 in
differentiation or growth suppression is further evidenced by its
interaction with Smad proteins to regulate the p21/Cip1/WAF1 promoter (15). Nonetheless, the functional effects of pRb-Sp1 may be
opposed by the Sp1-cyclin D1 complex, a consequence of which is to
retard or partially disrupt the commitment to differentiation. Certainly, such a phenomenon would be evident in an exaggerated form
in some tumor types, for example squamous cell cancers, which
frequently overexpress cyclin D1. It should be emphasized that we
were able to detect the Sp1-cyclin D1 interaction in normal corneal
epithelial cells, indicating that this is not the result of cyclin D1
overexpression in the C3D1 cells. However, the Sp1-cyclin D1 interaction is more dramatic in C3D1 cells with apparent functional
consequences of diminished Sp1-mediated transactivation of the K4
promoter. Mapping of the domains involved in the Sp1-cyclin D1
interaction will be of interest.
Our data do not preclude the possibility that the cyclin D1-Sp1
complex may be part of a larger complex that regulates transcription,
and indeed, one such candidate may be TFIID because members of
this family have been demonstrated recently to interact individually
with cyclin D1, Sp1, or pRB (16 –18). It is also conceivable that cyclin
D1 phosphorylates Sp1 because phosphorylation of Sp1 has been
shown to be important in cell cycle regulation of its own transcriptional activity (19). Additionally, the cyclin D1-Sp1 complex may
also contain pRb, a possibility not excluded by our experiments.
Whether cyclin D1-Sp1 complex formation is dependent upon cdk4/
cdk6, as with the phosphorylation of pRB, or independent of cdk
activity as is the case with the cyclin D1-estrogen receptor interaction
(20), requires further investigation. Whereas the ability of Sp1 to
2828
SP1 TRANSACTIVATION OF KERATIN 4
Fig. 4. Interaction of Sp1 with pRB or cyclin D1 in corneal epithelial cells. A, lysates from SIRC, C3D1, and J82 cells were immunoprecipitated with Sp1 polyclonal antibody (PEP2)
and immunoblotted with pRB monoclonal antibody (G3–245). U2OS (osteosarcoma cell line; wild-type pRB) and J82 (bladder cancer cell line; mutated pRB) cell lysates served as
positive or negative control, respectively. The two arrows in A indicate hyperphosphorylated (upper) and hypophosphorylated (lower) forms of pRB, respectively. B, lysates from SIRC,
C3D1, and DT 40 cells were immunoprecipitated with Sp1 polyclonal antibody (PEP2) and immunoblotted with cyclin D1 polyclonal antibody (H-295; Santa Cruz Biotechnology).
IP with AP1 antibody served as negative control and cyclin D1 immunoblot in TE-12 cell-lysate (esophageal squamous cancer cell line) served as positive control. As control for the
primary antibody in immunoblotting, AP1 monoclonal antibody (D) was used (Lane 6). C, lysates of SIRC and C3D1 cells were immunoprecipitated with Sp1 polyclonal antibody
(PEP2; Lanes 1– 4), pRB monoclonal antibody (G3-245; Lanes 5– 8), cyclin D1 monoclonal antibody (HD11; Lanes 9 –14) or AP1 monoclonal antibody (D; Lanes 15–16),
coimmunoprecipitates were dissociated and EMSAs performed using probe Sp1-C. The addition of Sp1 polyclonal antibody (PEP2) specifically diminished Sp1-DNA complex, whereas
AP1 antibody (D) did not. AP1 IP did not lead to Sp1-DNA complex formation.
influence the equilibrium between proliferation and differentiation is
mediated in part through its interactions with cell cycle regulatory
proteins, cyclin D1 in turn may promote the tendency to proliferation
through interaction with proteins such as estrogen receptor in breast
epithelial cells and Sp1 in corneal epithelial cells and possibly, in
different cell types.
Horowitz, and Robert Weinberg for pRb expression vectors, Dr. Jill Lahti for
the DT 40 cells, Dr. Felix Brembeck for providing some K4 promoter constructs, and Drs. Hiroshi Nakagawa, Timothy Jenkins, Felix Brembeck, and
Jun-Ichi Okano for discussions.
References
Acknowledgments
We are grateful to Dr. Steven Reeves for the cyclin D1 retroviral expression
vector; Dr. Ed Harlow for cyclin D1 antibodies, Drs. William Kaelin, Jon
2829
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