From the Southern Alberta Cancer Research Centre,
Department of Biochemistry and Molecular Biology, Cancer Biology
Research Group, University of Calgary, Calgary, Alberta T2N 4N1, Canada
and ¶ Incyte Genomics, Beverly, Massachusetts 01915
Received for publication, October 23, 2002, and in revised form, December 2, 2002
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ABSTRACT |
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Senescent human diploid fibroblasts are unable to
initiate DNA synthesis following mitogenic stimulation and adopt a
unique gene expression profile distinct from young or quiescent cells. In this study, a novel transcriptional regulatory element was identified in the 5'-untranslated region of the cyclin D1 gene. We show
that this element differentially suppresses cyclin D1 expression
in young versus senescent fibroblasts. Electrophoretic mobility shift assays revealed abundant complexes forming with young
cell nuclear extracts compared with senescent cell nuclear extracts.
Binding was maintained in young quiescent cells, showing that loss of
this activity was specific to senescent cells and not an effect of cell
cycle arrest. Site-directed mutagenesis within this cyclin D1
inhibitory element (DIE) abolished binding activity and selectively
increased cyclin D1 promoter activity in young but not in senescent
cells. Sequences with homology to the DIE were found in the
5'-untranslated regions of other genes known to be up-regulated during
cellular aging, suggesting that protein(s) that bind the DIE might be
responsible for the coordinate increase in transcription of many genes
during cellular aging. This study provides evidence that loss of
transcriptional repressor activity contributes to the up-regulation of
cyclin D1, and possibly additional age-regulated genes, during cellular senescence.
Normal human diploid fibroblasts
(HDFs)1 are widely used as a
model system to study the process of replicative or cellular senescence
(1, 2). These cells have a finite proliferative life span, at the end
of which they are unable to enter the S phase in response to mitogenic
stimuli, but they remain metabolically active for long periods of time
(3). They have prominent and active Golgi apparati, a large, flat
morphology, invaginated and lobed nuclei, large lysosomal bodies, and
an increase in cytoplasmic microfibrils compared with young cells
(reviewed in Ref. 4). They also show a senescence-associated
Cyclin D1 is a member of the D-type family of cyclins that
associates with cyclin-dependent kinases 4 and 6 (14, 15). Cyclin D1- cyclin-dependent kinase 4 promotes the
G1 to S phase transition of the cell cycle by cooperating
with cyclin E-cyclin-dependent kinase 2 to sequentially
phosphorylate the retinoblastoma tumor suppressor protein (16).
However, cyclin D1 knockouts are rescued by cyclin E expression,
whereas the reverse does not hold, suggesting that cyclin D1 does not
play a central role in regulating the cell cycle, which is further
suggested by the phenotype of cyclin D1 knockout mice (17-19). Normal
human diploid fibroblasts that have reached the end of their in
vitro life span (senescent cells) express 3-fold higher levels of
cyclin D1 protein than low passage cells. Individual cells in mass
culture that fail to initiate DNA synthesis in response to serum
addition have severalfold higher levels of this cyclin than
proliferation competent cells (7). It has been shown that cyclin D1
overexpression may inhibit this entry into S phase through binding to
proliferating cell nuclear antigen and cyclin-dependent
kinase 2 (7, 20). Cyclin D1 dysregulation and gene amplification have
been implicated in a variety of cancers (21), suggesting that
deregulated expression of cyclin D1 contributes to abnormal cell proliferation.
Ectopic overexpression of cyclin D1 in normal HDFs, the mammary
epithelial cell line MCF-7, Dami megakaryotic cells, and rat embryo
fibroblasts inhibits DNA synthesis and cell growth (7, 20, 22-25).
Cyclin D1 has also been shown to be up-regulated in numerous
nonproliferative differentiated cell types (26-29) and during
apoptosis (30), lending further mass to the idea that it may also
provide growth suppressive functions. In senescent fibroblasts, the
cyclin D1 mRNA and protein levels are constitutively up-regulated
by ~3-5-fold compared with serum-stimulated young cells (6-8,
31).
The 5'-regulatory region of the cyclin D1 gene has been well
characterized, and the regions responsible for serum-inducible transcription have been identified (32, 33). However, the regulation of
cyclin D1 gene expression is not well understood under conditions of
senescence-associated growth arrest where induction is at least as
great as seen in response to serum. The increased expression of cyclin
D1 is specific for senescence-associated growth inhibition in HDFs and
is not apparent in contact-inhibited or serum-deprived cells arrested
at a similar place in the cell cycle (24). This is in contrast to the
closely related cyclin D2 where expression is increased in senescent,
quiescent and contact-inhibited cells (24). Thus, unique
transcriptional mechanism(s) may strongly contribute to cyclin D1
expression during senescence. In this report, we examine potential
5'-regulatory regions and mechanisms that may be responsible for the
up-regulation of cyclin D1 seen in aging HDFs. Analysis of the cyclin
D1 promoter via transient transfections of nested promoter deletions
into young and old fibroblasts has identified a 15-bp cyclin D1
inhibitory element (DIE). This element is located in the 5'-UTR of
cyclin D1 and has been shown to bind a low molecular mass protein more
avidly in young cells compared with senescent cells, suggesting that loss of binding to this element in senescent cells contributes to the
increased expression of cyclin D1 seen during cellular senescence.
Consistent with a role in cell aging, it was noted that several other
genes that are up-regulated during cell aging were found to have
DIE-like elements in their 5'-UTRs, strongly supporting the idea that
this element may bind a common regulatory protein that contributes to
senescence-specific gene expression.
Cells and Cell Culture--
HDFs (Hs68: ATCC CRL 1635 from
newborn foreskin, reaches 85 mean population doublings (MPDs) in
culture; WI-38: CCL 75, from embryonic lung, reaches 56 MPDs under our
culture conditions) were maintained in Dulbecco's modified Eagle's
medium (Invitrogen) supplemented with 10% (v/v) fetal bovine serum and
1% penicillin/streptomycin. In all experiments, low passage (young)
cells were between 28-32 MPDs and high passage (senescent) cells were
used at 82-86 MPDs (Hs68) or 53-56 MPDs (WI-38). Senescence was
defined as a state in which less than 10% (typically 3-5%) of cells
enter DNA synthesis in response to mitogens in a 36-h period. Senescent
cells used in this study did not reach confluence within 2 weeks after
subculturing at a 1:2 split ratio, demonstrated a characteristic large,
flattened senescent cell morphology, and were positive for
senescence-associated Senescence-associated Nested Deletion Constructs--
The 1.3-kb human cyclin D1
promoter in pUC118 vector was a generous gift from Yue Xiong
(University of North Carolina, Chapel Hill). The vector was digested
with SacI/PvuII to release the 1291-bp promoter
containing 138 bp of the 5'-UTR. The ends of the fragment were filled
in with Klenow polymerase (Invitrogen), and the fragment was
subcloned into the SmaI site of pBluescript II KS. Nested
deletions were performed with a double-stranded nested deletion kit
(Pharmacia Corp.). For 5' nested deletions, the cyclin D1 promoter in
pBluescript was digested with HindIII, filled in with
thionucleotides, and redigested with EcoRV at the 5' end.
The deletions were made by exonuclease III digestion for various time
points followed by S1 nuclease treatment. The deleted constructs were
analyzed by agarose gel electrophoresis, ligated, and transformed to
obtain clones, which were screened and sequenced for the desired
deletions. Deleted clones in pBluescript were digested with
SalI/BamHI and subcloned into the
SalI/BamHI site of the pBLCAT3 vector. The clones
were designated as
For 3' deletions of the 5'-UTR, the Transient Transfections
Electroporation--
5 × 105 log phase young
and senescent Hs68 fibroblasts were seeded in 150-mm plates and
harvested when ~80% confluent. The cells were trypsinized, suspended
in 400 µl of serum-free Dulbecco's modified Eagle's medium, and
transferred to 4-mm gap cuvettes (BTX Inc., San Diego, CA). Cyclin D1
promoter-chloramphenicol acetyltransferase (CAT) reporter plasmid (30 µg) was added to 5 µg of a cytomegalovirus-driven LipofectAMINE 2000--
5 × 103 log phase
(unsynchronized) young and senescent (arrested) Hs68 fibroblasts were
seeded in 6-well plates and treated when ~95% confluent. The cells
were washed in PBS (pH 7.2), and the medium was replaced with OPTI-MEM
(Invitrogen). LipofectAMINE 2000/OPTI-MEM (Invitrogen) solution (250 µl) was incubated for 20 min with DNA/OPTI-MEM solution (250 µl).
Immediately after incubation 500 µl was added to each well of the
6-well plate. Approximately 6 h later, the medium was replaced
with L-Dulbecco's modified Eagle's medium supplemented
with 10% (v/v) fetal bovine serum. The cells were harvested 48 h
later using passive lysis buffer (Invitrogen) and assayed for
luciferase expression using an EG & G Berthold luminometer.
Electrophoretic Mobility Shift Assays--
Nuclear extracts from
young and old Hs68 HDFs were prepared as described previously (36, 37).
A double-stranded 64-bp oligonucleotide spanning nucleotides +75 to + 138 and the overlapping oligonucleotides (both wild type and mutant)
were synthesized with 5' overhangs, end-labeled with Klenow enzyme (0.5 unit; Invitrogen) and 50 µCi of [
Oligonucleotides corresponding to the cyclic AMP response element (CRE)
and binding conditions for this element have been described previously
(36). Briefly, 20,000 cpm (0.1-0.5 ng) of gel-purified
[ Site-directed Mutagenesis--
A total of 8 bp between +117 and
+131 of the 5'-UTR were mutated using a QuikChange site-directed
mutagenesis kit (Stratagene). A mutagenic primer (46-mer) was
synthesized and annealed to the double-stranded pBLCAT3 construct
containing the full-length cyclin D1 promoter. Pfu DNA
polymerase was used to synthesize the mutagenic promoters followed by
digestion of the parental plasmid by DpnI as per the
manufacturer's instructions. The mutated plasmid was transformed into
XL1-Blue competent cells, and the resulting plasmid was isolated and
sequenced to confirm the mutations.
UV Cross-linking of DNA-Protein Complexes--
To estimate the
relative molecular mass of the DNA-binding proteins, binding reactions
using various oligonucleotides as described for the band shift assays
were performed. Twice the amount of labeled probe (40,000 cpm) and
nuclear extract (10 µg) were used. After 30 min of incubation at room
temperature, the binding reaction was subjected to UV light, and
unprotected DNA was digested. The samples were irradiated by a 305-nm
inverted UV transilluminator at 7 mW/cm2 for 5 min. The
cross-linked reactions were electrophoresed through 15%
SDS-polyacrylamide gels, dried, and visualized by autoradiography.
Sequence Analysis--
All of the sequence analyses were
performed using the Wisconsin PackageTM (version 9.1) from the Genetics
Computer Group available through the Canadian Bioinformatics Resources
website. Promoter sequences in the GenBankTM DNA sequence
data base were obtained from the NCBI. The accession numbers
identifying particular sequences are listed under "Results." The
sequences were searched for the 15-bp DIE using the FINDPATTERNS program, allowing for 33.3% (5 of 15) random mismatch. Further DIE-like element comparison was done using the PRETTY program to find
those elements with highest sequence similarity to the DIE.
Dual Luciferase Reporter Assays--
Constructs containing wild
type and mutant DIE elements were made using pGL3 Control reporter
plasmids (Promega). The 15-bp high pressure liquid
chromatography-purified oligonucleotides were cloned into the
HindIII site found between the SV40 promoter and the
luc+ reporter gene. This cloning site is found outside of
the pGL3 control multi-cloning site and was chosen because of its
proximity to the functional gene (i.e. the in
vivo DIE is located in the 5'-UTR of the cyclin D1 gene).
Therefore, the DIE was cloned upstream of the luc+ start codon but
downstream of the SV40 promoter (see Fig. 8A). These
constructs were then co-transfected into young and old HDFs with a
Renilla luciferase construct (pRL-TK) as a transfection
efficiency control. Firefly luciferase expression was measured using an
EG & G Berthold luminometer and normalized based on pRL-TK expression.
Removal of a 64-bp Region in the 5'-UTR of Cyclin D1 Abolishes
Increased Promoter Activity in Old Cells--
To determine the basal
activity of the cyclin D1 promoter in young and old cells, a 1.3-kb
cyclin D1 promoter fused to a CAT reporter gene (Fig.
1A) was used in transient
transfection studies. Various 5' deleted cyclin D1 promoter-CAT
reporter constructs were also generated (Fig. 1B) and
transiently co-transfected with a cytomegalovirus-driven
We then generated further 5' and 3' nested deletions of the Decreased DNA Binding Activity in Extracts of Old Cells to the +75
to +138 Region of the Cyclin D1 Promoter--
To determine whether any
proteins could be detected binding to this 64-bp region,
electrophoretic mobility shift assays with young and old cell nuclear
extracts were performed. Specific complexes were formed with young cell
extracts, whereas the levels were dramatically reduced with old cell
extracts (Fig. 3A, lanes
2 and 3). Specificity of the complexes was confirmed by
incubation with a 100-fold excess of unlabeled oligonucleotide, which
effectively competed with the labeled probe (Fig. 3A,
lanes 4 and 5), whereas a 100-fold excess of
unlabeled, unrelated CRE oligonucleotide did not compete efficiently
(Fig. 3A, lanes 6 and 7).
To rule out the possibility that the nuclear extracts from old Hs68
fibroblasts were deficient in their ability to bind DNA through some
nonspecific mechanism, we performed gel shift studies using the same
extracts with an oligonucleotide containing the CRE present in the
c-fos promoter (36). As shown in Fig. 3B, CRE
binding activity was actually about 50% higher in old cell extracts
than in young (lanes 2 and 3). As expected,
100-fold competition with unlabeled oligonucleotide bearing the same
CRE sequence competed with the complexes formed on the labeled CRE (Fig. 3B, lanes 4 and 5), whereas
mutant sequences did not (Fig. 3B, lanes 6 and
7). Given previous reports that CRE binding activity is
about equal in young and old cells, specific binding activity of the
complex in Fig. 3A may actually be even more dramatically reduced in extracts from old compared with young cells (36).
Although extracts from young cells showed more binding activity than
extracts from old cells, it was possible that reduced binding in old
cells was due to cells exiting the cell cycle when senescent rather
than to a senescence-specific event per se. For example, a
clear passage-related up-regulation of cyclin D2 is seen in all
strains of primary HDFs examined, but increased expression is also seen
upon serum withdrawal or contact inhibition-induced cell cycle arrest
(24). To test whether the loss of binding was senescence-specific, we
incubated the 64-bp element with extracts from young proliferating
cells, young quiescent cells, and subconfluent old cells. As shown in
Fig. 3C, reduced binding was not seen in young quiescent
cells (lanes Q) with either the 64-bp element or with
subdomains (see below) of this element, indicating that the loss of
binding is senescence-dependent rather than
growth-dependent.
Identification of a 15-bp DIE in the 5'-UTR with Overlapping
Oligonucleotides--
DNase I footprinting assays were attempted but
did not prove useful in identifying sequences within the 64-bp region
that were differentially protected using young and old cell nuclear extracts (data not shown). An alternative approach using overlapping oligonucleotides was undertaken in an attempt to better define the
region involved in protein binding. As shown in Fig.
4A, five double-stranded 22-bp
oligonucleotides designated 5'-UTR-1 to 5'-UTR-5 were synthesized and
used in mobility shift assays using young and old cell nuclear
extracts. No detectable binding activity was observed when the 5'-UTR-1
and 5'-UTR-3 oligonucleotides were used (Fig. 4B,
lanes 8, 9, 12, and 13),
whereas a very weak complex was detected in young cell extracts when
the 5'-UTR-2 probe was used (lanes 10 and 11).
Complexes were readily detected when using 5'-UTR-4 and 5'-UTR-5 as
probes with young cell but not with old cell extracts (lanes
14-17). The migration of the complexes with these
oligonucleotides was slightly slower than the migration of the
complexes with the 64-bp oligonucleotide (lanes 18 and 19), probably reflecting the smaller charge-to-mass ratio
with the shorter oligonucleotides. Complexes with the shorter
oligonucleotides were also less readily detectable than those with the
64-bp oligonucleotide, perhaps reflecting stabilization of the
complexes by peripheral sequences present in the 64-bp oligonucleotide.
Because both 5'-UTR-4 and 5'-UTR-5 share a 15-bp overlap as shown in
Fig. 4A, the region involved in protein binding was mapped
to this site in the 5'-UTR that we have termed the cyclin D1 inhibitory
element (DIE).
To determine whether this interaction was specific, the labeled
5'-UTR-4 and 5'-UTR-5 probes were incubated with a 100-fold excess of
unlabeled 5'-UTR-4, 5'-UTR-5, the 64-bp oligonucleotide, or 5'-UTR-3 as
a negative control. As shown in Fig. 4C, incubation with
unlabeled oligonucleotides containing the DIE (lanes 3-8 and 13-18), but not the 5'-UTR-3 oligonucleotide
(lanes 9, 10, 19, and 20)
competed with the labeled probe for binding proteins, and the degree of
competition varied when using different unlabeled probes.
Mutations within the DIE Abolish Binding Activity--
To identify
the bases within the DIE that were responsible for protein binding,
oligonucleotides of 5'-UTR-4 containing various mutations (5'-UTR-4a to
5'-UTR-4d) were generated as shown in Fig.
5A. In each case, purines and
pyrimidines were exchanged for noncomplementary pyrimidines and
purines, respectively. Electrophoretic mobility shift assays of young
and old cell nuclear extracts with these oligonucleotides showed that
the mutations in 5'-UTR-4a, -4b, and -4c reduced binding in young cell
extracts significantly compared with 5'-UTR-4 (Fig. 5B,
compare lanes 8-13 with lanes 6 and
7), and that 5'-UTR-4d, which contains all of the individual mutations, nearly abolished binding in both young and old extracts (lanes 14 and 15). Densitometric scanning of
different exposures of the lanes in Fig. 4B indicated that
the binding to these oligonucleotides was reduced by greater than 90%
in the case of the most mutated (5'-UTR-4d). Relative binding values
for the oligonucleotides were 5'-UTR-4 (100%), 5'-UTR-4a (53%),
5'-UTR-4b (42%), 5'-UTR-4c (22%), and 5'-UTR-4d (9%). Therefore, the
base changes in the 5'-UTR-4d oligonucleotide were used for
site-directed mutagenesis of the DIE in the context of the full-length
cyclin D1 promoter.
Mutation of the DIE Increases Cyclin D1 Promoter Activity in Young
Cells--
An 8-bp change in the full-length cyclin D1 promoter-CAT
reporter construct from +117 to +131, corresponding to the mutations in
5'-UTR-4d, was introduced by site-directed mutagenesis. The 1.3-kb
full-length cyclin D1 promoter-CAT reporter construct and the mutated
construct were transfected into young and old cells, and the CAT
activity resulting from each construct was measured after normalizing
for Low Molecular Mass Proteins Bind the DIE--
To estimate the
molecular mass of any protein(s) that bound specifically to the DIE,
nuclear extracts from young and old cells were incubated with 5'-UTR-4,
5'-UTR-5, or the 64-bp oligonucleotide and were subjected to UV
cross-linking. Following resolution by SDS-PAGE, complexes were
apparent in young cell extracts that were decreased in old cell
extracts (Fig. 7). Three different complexes were formed with the 5'-UTR-4, suggesting that this sequence
might contain a more complete binding site(s) than 5'-UTR-5, with which
only two complexes were seen (compare lane 4 with lane 9). Three complexes were also apparent with the 64-bp
oligonucleotide (lanes 14 and 15). After
subtraction of the molecular mass of the labeled probe (i.e.
649 Da/bp), an estimated molecular mass range of 20-45 kDa was
calculated for proteins bound to both the 22- and 64-bp
oligonucleotides, depending upon the amount of DNA protected by UV
cross-linking.
The DIE Preferentially Inhibits Gene Expression in Young
Fibroblasts--
To test the activity of the 15-bp wild type and
mutant, DIE elements in luciferase reporters containing these elements
(Fig. 8A) were transfected
into young and senescent Hs68 fibroblasts. Transfection efficiencies of
55 and 40% in young and senescent fibroblasts, respectively, were
obtained using LipofectAMINE 2000. Transfection efficiencies were
normalized based on Renilla luciferase (pRL-TK)
expression.
As shown in Fig. 8B, young HDFs showed more than 80%
reporter inhibition by the wild type DIE compared with the mutant
element, whereas senescent HDFs were inhibited ~30%. Therefore,
although the DIE element had an inhibitory effect upon transcription in both young and senescent cells, the inhibitory effect was markedly greater in senescent cells, even though the assay is done with a single
DIE element (compared with several seen in most genes) outside the
context of the native cyclin D1 promoter.
Identification of DIE-like Elements in Other Genes Up-regulated
during Cellular Aging--
We next examined whether sequences similar
to the DIE were present in promoters of other genes up-regulated during
cellular senescence. Well characterized genes whose expression is
increased in senescent cells include 1) plasminogen activator inhibitor type-1 (38, 39), 2) insulin-like growth factor binding protein-3 (12),
3) p14ARF (40), 4) ING1 (41), 5) NF- In this study, a region has been identified in the cyclin D1
5'-UTR that strongly contributes to selectively repressing cyclin D1
gene expression in young cells. Although deletion of most cyclin D1
promoter sequences affected reporter gene expression to a similar extent in young and old cells, deletion of a 64-bp sequence from +75 to
+138 of the 5'-UTR selectively reduced expression in old cells. By
using overlapping oligonucleotides corresponding to fragments of the
64-bp region, the protein-binding region was further narrowed to a
15-bp sequence that we have termed the DIE. Mutation of particular
bases within the DIE reduced or abolished complex formation and
selectively up-regulated cyclin D1 promoter activity in young cells.
Luciferase reporter constructs effectively demonstrate the ability of
this element to differentially regulate transcription in young
versus senescent HDFs and gel shift assays suggest that
protein(s) ranging from 20 to 45 kDa specifically bind the DIE.
Finally, sequence similarity comparisons revealed that sequences with
homology to the DIE were also found clustered in the promoters of a
subset of genes whose expression is also up-regulated in aging cells at
a frequency higher than seen in control genes whose expression is
unaffected or reduced by replicative senescence.
The age-related increase in cyclin D1 transcripts observed in old cells
could be the result of specific transcriptional and/or post-transcriptional regulation. Our observations suggested that D-type cyclin transcripts are similarly stable over
extended time courses (4- to 16-h) in both young and old cells (data
not shown), suggesting that age-related up-regulation of these
transcripts occurs at the level of transcription.
Mutation of the DIE between +117 to +131 increased cyclin D1 promoter
activity in young cells but not to the levels seen in old cells. Thus,
other regions of the cyclin D1 promoter are also likely to contribute
to age-related expression. In fact, differential DNA binding activity
in young and old cells has been reported for various regions of the
cyclin D1 promoter (8), although their contribution to gene expression
is unknown. Interestingly, the sequence similarity comparison revealed
two additional DIE-like sequences within the cyclin D1 5'-UTR (Fig. 9).
This included a sequence with limited homology within the 5'-UTR-2
oligonucleotide, with which weak complex formation was observed in
mobility shift assays (Fig. 4). Based on our results, it appears that
specific interactions of the repressor are stabilized by multiple
copies of the DIE such that mutation of one can only partly reverse
cyclin D1 gene repression in young cells. This would be consistent with the stronger binding seen using the 64-bp versus the shorter
22-bp oligonucleotides.
Although regulation of gene expression via the 5'-UTR has not been
widely reported, such mechanisms might act to repress transcription more commonly than previously thought. For example, p53 suppresses the
expression of bcl-2 at least partly through a p53 response element located in the 5'-UTR of the bcl-2 gene (46).
Similarly, a suppressor element has been identified in the 5'-UTR of
the androgen receptor gene (47). Although the exact mechanisms
operating to repress transcription are unknown, the results presented
here raise the possibility that other genes that are up-regulated
during cellular senescence could be regulated by a mechanism similar to
if not identical with the DIE identified here. Indeed, comparison of
the DIE with a selected number of genes up-regulated during cellular
aging revealed the presence of DIE-like sequences in their 5'-UTRs and
the absence of these elements in control genes. This raises the
possibility that the expression of a group of age-related genes is
coordinately regulated by one principal mechanism and that it may be
possible to influence the expression of many genes by targeting a
single repressor element or protein.
Taken together, these data indicate that a specific transcriptional
mechanism contributes to the increased expression of cyclin D1 during
cellular senescence. Searches of transcription factor data bases using
the DIE revealed no homologies to known human transcription factor
binding sites, suggesting that the protein(s) binding the DIE are
novel. Experiments to purify the putative repressor protein(s) by DNA
affinity chromatography are underway and should reveal important
insights regarding the regulation and identification of additional
genes containing DIE elements and their role(s) in cellular aging.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase (
-gal) activity, staining blue under acidic
conditions (pH 6.0), whereas young cells do not (5). Some of the
biochemical hallmarks that accompany cellular senescence include
up-regulation of cyclin D1 (6-8), p21Waf1/Cip1/Sdi1 (9),
p16INK4a (10, 11), and insulin-like growth factor binding
protein-3 (12). Furthermore, senescent cells arrest with a DNA content characteristic of the G0 and G1 phases of the
cell cycle; yet the expression and activity of many cell cycle
regulatory proteins during G0, G1, and
senescence are distinct, implying that senescent cells exist in a
unique, nonproliferative state that we have termed GS (13).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-gal activity.
-Gal Staining--
In vitro
senescence-associated
-gal activity was detected as described (5,
34) with minor modifications. The cells were washed with
phosphate-buffered saline (PBS; pH 7.2), fixed with 0.5%
glutaraldehyde in PBS for 3-5 min at room temperature, washed several
times with PBS (pH 6.0), and stained for 16-24 h at 37 °C with 1 mg/ml 5-bromo-4-chloro-3-indolyl-
-galactoside (X-gal) in PBS (pH
6.0) containing 5 mM potassium ferrocyanide, 5 mM potassium ferriccyanide, and 1 mM
MgCl2.
1154 (full length),
858,
580,
459,
425,
198,
85,
23, and +26 relative to the transcriptional start site.
23 clone in pBluescript was cut
with SpeI, filled in with thionucleotides, and redigested with BamHI at the 3' end. After exonuclease III/S1 nuclease
treatment, the deleted clones were ligated, screened, and sequenced.
Clones with 3' deletions were digested with
SalI/XbaI and subcloned into the
SalI/XbaI sites of the pBLCLAT3 vector. Two
clones with the 3' end deleted were designated as
23 to +74 and
23
to +24.
-galactosidase
expression construct and 15 µg of salmon sperm DNA to make a total
DNA content of 50 µg/cuvette. Electroporations were done using a
Bio-Rad gene pulser at 250 V and 960 microfarad, and following
transfection, the samples were transferred to 10-cm plates. The cells
were harvested 48 h post-transfection and assayed for CAT activity.
-Galactosidase and CAT Assays--
Electroporated cells were
washed twice with ice-cold PBS, harvested, and centrifuged. The cell
pellet was washed with PBS, resuspended in 100 µl of lysis buffer
(0.25 M Tris-Cl, pH 7.8), and subjected to three
freeze-thaw cycles in liquid nitrogen with rigorous vortexing between
each step. The cell lysates were centrifuged at 10,000 rpm for 5 min,
and the supernatants containing the cell extract were used for
-galactosidase assays as previously described (5). Briefly, 10 µl
of cell extract was added to a tube containing 90 µl of lysis buffer,
350 µl of LacZ buffer (60 mM
Na2HPO4, 40 mM
NaH2PO4, pH 7, 10 mM KCl, 1 mM MgSO4, 50 mM
-mercaptoethanol) and 50 µl of
chlorophenolred-
-D-galactopyranoside (30 µg/µl) (Roche Molecular Biochemicals). The mixture was incubated for 1 h
at 37 °C, and the absorbance at 574 nm was determined. Activity resulting from the co-transfected cytomegalovirus-
-galactosidase plasmid was determined by subtraction of endogenous basal activity values obtained from mock-transfected young and old fibroblasts. Extracts corresponding to equal units of
-galactosidase activity were used for CAT assays as described previously (35). The proportion of acetylated product was determined by liquid scintillation counting of excised thin layer chromatography spots.
32P]dCTP (3000 Ci/mmol; Amersham Biosciences), and gel-purified. 20,000 cpm of labeled
probe (0.1-0.5 ng) was incubated with 5 µg of nuclear extracts from
young or old cells in binding reactions that contained 20 mM Hepes (pH 8.0), 25 mM KCl, 5 mM
MgCl2, 5% glycerol, 2 mM dithiothreitol, 0.1 mM EDTA, and 2 µg of poly(dI-dC) as described previously
(36). The reactions were incubated for 30 min at room temperature,
electrophoresed through 5% nondenaturing polyacrylamide gels at 150 V
at room temperature, dried, and visualized by autoradiography.
32P]dCTP-labeled probe was incubated with 5 µg of
nuclear protein extract in a buffer that contained 20 mM
Tris (pH 7.6), 4% Ficoll, 50 mM KCl, 1 mM
EDTA, 0.2 mM dithiothreitol, 10 µM
ZnCl2, 1 µg of partially denatured salmon sperm DNA, and
30 µg of bovine serum albumin in a final volume of 20 µl for 30 min
at room temperature. Unlabeled wild type and mutant competitor DNA was
added at a 100-fold excess and incubated for 10 min at room temperature
before the addition of hot probe.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase construct (as internal control) into young and old
cells. CAT activity caused by the full-length cyclin D1 promoter was
4-5-fold greater in old than in young cells (Fig. 1B),
consistent with endogenous levels of cyclin D1 expression in those
cells (7). Deletion of segments from the 5' end from
1154 to
85
progressively reduced the expression difference between young and old
cells to levels observed with the promoterless pBLCAT3 vector (Fig.
1B). However, the smallest deletion construct containing
23 to +138 of the cyclin D1 promoter retained 3-4-fold higher
expression levels in old compared with young cells, indicating that
this region is sufficient to confer increased expression in old
cells.
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Fig. 1.
5'-nested deletion analysis of the cyclin D1
promoter. A, schematic representation of several
potential transcription factor binding sites in the cyclin D1 promoter.
TRE, TPA response element; Egr1, immediate-early
growth response gene; WT1, Wilms' tumor suppressor gene
product; E2F, transcription factor E2F-binding site;
Sp1, promoter-specific transcription factor;
Oct1, octamer-like transcription factor binding site;
E-box, insulin-responsive region. B, a series of
5'-nested deletions of the cyclin D1 promoter in pBluescript vector was
generated by exonuclease III digestion, and the resulting deletions
were subcloned into the pBLCAT3 vector for transient transfections into
young and old fibroblasts (left panel). The bar
graphs in the right panel represent the fold difference
in CAT activity in old versus young cells after normalizing
for transfection efficiencies by measuring activity from co-transfected
-galactosidase constructs. The results represent CAT activity from
three independent transfections with the standard error
indicated.
23 to
+138 region to further define the region involved in the differential
regulation of cyclin D1. As shown in Fig.
2A, removal of the
transcription initiation site resulted in approximately equivalent
levels of inhibition of CAT activity in young and old cells. However,
when 64 bp (+75 to +138) of the 5'-UTR were deleted, it resulted in a
further 44% decrease in old cells, but the CAT activity in young cells
remained relatively unchanged, thus reducing the fold difference in CAT
activity to levels observed with the control vector (Fig.
2B, compare
23 to +138 with
23 to
+74). Further removal of the 5'-UTR did not have any differential
effect on CAT activity because it resulted in an approximately 77%
decrease in young and an 82% decrease in old cells. These results
indicate that together, other regions of the promoter are likely to
contribute as strongly to the differential expression of cyclin D1 in
young and old cells. The 64-bp sequence between +75 and +138 appeared to be a potential binding site for a transcriptional repressor in young
cells and/or a transcriptional activator in old cells.
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Fig. 2.
5' and 3' nested deletion analysis of the
23 to +138 region of the cyclin D1 promoter. A, 5'
and 3' deletions of the cyclin D1 promoter (left panel) and
the resulting CAT activity in young (black bars) and old
(white bars) cells plotted after normalizing for
-galactosidase activity (right panel). B, fold
difference in CAT activity in old versus young cells from
A.
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Fig. 3.
DNA binding activity of the cyclin D1
5'-UTR. A, nuclear extracts from asynchronous young
(Y) and old (O) Hs68 HDFs were incubated with an
oligonucleotide corresponding to the 64-bp sequence of the cyclin D1
5'-UTR (lanes 2 and 3). The arrow
points to the major specific DNA-protein complex. Lane 1 corresponds to the reaction with the labeled oligonucleotide in the
absence of nuclear extracts. Lanes 4 and 5 show
the reaction with the 64-bp oligonucleotide in the presence of a
100-fold excess of unlabeled (Cold) wild type (W)
oligonucleotide or in the presence of a 100-fold excess of cold
unrelated (U) CRE oligonucleotide (lanes 6 and
7). B, control gel shift using a consensus
c-fos CRE binding site with the same young and old nuclear
extracts described in A. Lane 1 shows probe alone
in the absence of nuclear extract. Lanes 2 and 3 show complexes formed on the CRE when young and old nuclear extracts
were added. Lanes 4 and 5 show the reaction in
the presence of a 100-fold cold CRE wild type oligonucleotide
(W) or a 100-fold cold CRE mutant oligonucleotide
(M) (lanes 6 and 7). C,
electrophoretic mobility shift assays using independently isolated
extracts from young growing cells (Y), young quiescent cells
(Q), and old subconfluent cells (O). The
first three lanes show oligonucleotide in the absence of
extract, and lanes 4-12 show the degree of binding activity
of the extracts of the extracts to the 64-bp oligonucleotide, a
subdomain (5'-UTR-4), and a mutant subdomain (5'-UTR-4d) of the
oligonucleotide as identified in Fig. 4.
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Fig. 4.
Identification of the DIE using overlapping
oligonucleotides. A, a portion of the cyclin D1 5'-UTR
showing a 64-bp region divided into five overlapping 22-bp
oligonucleotides (5'-UTR-1 to 5'-UTR-5). B, electrophoretic
mobility shift assay of the overlapping oligonucleotides shown in
A with young (Y) and old (O) cell
nuclear extracts. Lanes 1-7 are control reactions in the
absence of nuclear extract. Detection of complex formation was evident
in young cell nuclear extracts and weaker in old cell extracts when
using 5'-UTR-4 and 5'-UTR-5 as a probe (lanes 14-17), but
signal was not detected when 5'-UTR-1 or 5'-UTR-3 was used as a probe
(lanes 8, 9, 12, and 13). A
very weak complex was detected preferentially in young cell extracts
when 5'-UTR-2 was used as a probe (lanes 10 and
11). Complex formation on the 64-bp and control CRE
oligonucleotide is also shown (lanes 18-21). C,
the 5'-UTR-4 and 5'-UTR-5 oligonucleotides were used as probes for
competition experiments either alone or with the other unlabeled probes
at a 100-fold excess as indicated, to determine the specificity of
binding.
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Fig. 5.
Mutations of oligonucleotides in the 5'-UTR-4
region. A, to determine the effect of various base pair
changes on protein binding activity, four sets of oligonucleotides
(5'-UTR-4a to 5'-UTR-4d) were synthesized and used in mobility shift
assays. B, electrophoretic mobility shift assays with young
and old cell nuclear extracts using the mutant 5'-UTR-4
oligonucleotides shown in A. Lanes 1-5 are
control lanes in the absence of nuclear extracts. Introduction of 2 or
4-bp changes inhibited binding activity (lanes 8-13),
whereas an 8-bp change within the DIE nearly abolished binding activity
(lanes 14 and 15) compared with the wild type
5'-UTR-4 oligonucleotide (lanes 6 and 7).
-galactosidase activity. As shown in Fig. 6A, mutation of the DIE in the
5'-UTR resulted in nearly a doubling of CAT activity in young cells
relative to old cells, compared with the activity of the wild type
construct. In contrast, the CAT activity from the mutant construct was
similar to that from the wild type construct in old cells. Fig.
6B shows the results of the same experiment plotted as fold
difference in CAT activity in old versus young cells.
Mutation of the DIE resulted in a nearly 50% decrease in the fold
difference in CAT activity compared with the unmutated control. These
results suggest that the DIE within the 5'-UTR of cyclin D1 constitutes
a binding site for a potential transcriptional repressor in young
cells, the activity and/or levels of which are dramatically reduced in
old cells.
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Fig. 6.
CAT activity in young
versus old cells using a cyclin D1 promoter containing
a mutant DIE. The 8-bp change corresponding to 5'-UTR-4d was
introduced into the full-length cyclin D1 promoter by site-directed
mutagenesis, and the construct was transfected into young and old
cells. A, the graph represents CAT activity from
young (black bars) and old (overlapping white
bars) cells after normalizing for transfection efficacy using
co-transfected -galactosidase construct. B, results from
A plotted as fold difference in CAT activity in old
versus young cells. An average of three independent
transfections were performed with the standard deviations indicated by
error bars.
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Fig. 7.
Determination of the approximate molecular
mass of protein(s) binding to the DIE. Binding reactions of the
indicated probes with young and old cell nuclear extracts were
subjected to UV cross-linking, digested with DNase I, and subsequently
analyzed by SDS-PAGE. The lanes without UV cross-linking served as
negative controls. The arrows identify various DNA-protein
complexes.
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Fig. 8.
Dual luciferase reporter assays using wild
type and mutant DIE sequences. 26-bp sequences containing wild
type or mutant forms of the 15-bp DIE were cloned into pGL3-control
firefly luciferase reporter plasmids in the sense orientation. The
assays show differential inhibition by the wild type versus
mutant DIE on luc+ expression. Young HDFs show more than 80% reporter
inhibition, whereas senescent HDFs show less than 30% inhibition.
Mutant constructs show little to no inhibition of reporter
expression.
B (42, 43), and 6)
osteonectin (44). As controls, the "housekeeping" genes
glyceraldehyde-phosphate dehydrogenase expressed similarly in young and
senescent fibroblasts, (10) and phenylalanine tRNA synthetase, whose
expression decreases during cellular senescence (45), were used. The
15-bp DIE was searched for in the promoters of these genes using the
FINDPATTERNS program (see "Experimental Procedures"). As shown in
Fig. 9A, sequences with
similarity to the DIE were found to be clustered within 400-bp of the
transcription initiation site (often within the 5'-UTR) of the genes
that are up-regulated in aging cells, whereas the frequency of DIE-like
sequences in the control genes was much lower. Alignment of the DIE
with the sequences detected in the other promoters showed the presence
of six perfectly conserved bases in the group of genes up-regulated
during cellular senescence, whereas only two bases were conserved in
the controls (Fig. 9B). Furthermore, p14ARF had
a second DIE that was very well conserved. Taken together, these
results suggest that the regulatory function of the DIE may not be
restricted to cyclin D1 gene expression but also to a selected number
of genes that are overexpressed in senescent cells.
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Fig. 9.
DIE elements in the promoter regions of
other genes transcriptionally up-regulated during replicative
senescence. A, schematic representation of the promoter
regions of genes well established to be up-regulated with in
vitro age. The white boxes indicate all DIE-like
elements with at least 66.6% (10 of 15) homology to the DIE. The
black boxes indicate the DIE-like elements most similar to
the cyclin D1 DIE that are expanded in B. B,
DIE-like elements from the 5'-UTR of the indicated genes from
A showing the closest sequence homology to the cyclin D1
DIE.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
---|
We thank Yue Xiong for the cyclin D1 promoter, Dylan Edwards for useful vectors, Christoph Sensen for help with bioinformatical analysis, Ivan Chebib and Kevin Schade for help with experiments, and Claude Veillette for technical assistance.
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FOOTNOTES |
---|
* This work was supported by grants from the National Cancer Institute of Canada and the Canadian Institutes of Health Research.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.
§ Recipient of an Alberta Heritage Foundation for Medical Research Studentship award.
Scientist of the Alberta Heritage Foundation for Medical
Research and the Canadian Institutes of Health Research. To whom correspondence should be addressed: Southern Alberta Cancer Research Centre, Dept. of Biochemistry and Molecular Biology, Cancer Biology Research Group, University of Calgary, Heritage Medical Research Bldg.,
3330 Hospital Dr. N.W., Calgary, Alberta T2N 4N1, Canada. Tel.:
403-220-8695; Fax: 403-270-0834; E-mail: karl@ucalgary.ca.
Published, JBC Papers in Press, December 2, 2002, DOI 10.1074/jbc.M210864200
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ABBREVIATIONS |
---|
The abbreviations used are:
HDF, human diploid
fibroblast;
-gal,
-galactosidase;
DIE, cyclin D1 inhibitory
element;
UTR, untranslated region;
MPD, mean population doubling;
PBS, phosphate-buffered saline;
CAT, chloramphenicol acetyltransferase;
CRE, cyclic AMP response element.
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REFERENCES |
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