The expression of retinoblastoma and Sp1 is increased by low
concentrations of cyclin-dependent kinase inhibitors
Silvia Pen
˜
uelas*, Cristina Alemany*, Ve
´
ronique Noe
´
and Carlos J. Ciudad
Department of Biochemistry and Molecular Biology, School of Pharmacy, University of Barcelona, Spain
We examined the effect of suboptimal concentrations of
cyclin-dependent kinase inhibitors, which do not interfere
with cell proliferation, on retinoblastoma expression in
hamster (Chinese hamster ovary K1) and human (K562 and
HeLa) cells. To achieve this, we used the chemical inhibitors
roscovitine and olomoucine (which inhibit CDK2 prefer-
entially), UCN-01 (which also inhibits CDK4/6) and p21 (as
an intrinsic inhibitor). All chemical inhibitors and over-
expression of p21 strongly induced retinoblastoma protein
expression. UCN-01-mediated retinoblastoma expression
was caused by an increase in both the levels of retino-
blastoma mRNA and the stability of the protein. The
expression of the transcription factor Sp1, a retinoblastoma-
interacting protein, was also enhanced by all the
cyclin-dependent kinase inhibitors tested. However, Sp1
expression was caused by an increase in the levels of Sp1
mRNA without modification in the stability of the pro-
tein. By using luciferase experiments, the transcriptional
activation of both retinoblastoma and Sp1 promoters by
UCN-01 was confirmed. Bisindolylmaleimide I, at concen-
trations causing a similar or higher inhibition of protein

nonphosphorylated when newly synthesized; hypophos-
phorylated in early G1; and hyperphosphorylated in late
G1, S, and G2/M phases. In mitosis, a protein phosphatase
1-like protein removes all phosphates from phosphorylated
Rb to reset the phosphorylation status of Rb in early G1.
The hypophosphorylated form is involved in the growth
inhibitory potential of Rb [12,13], which has been related to
its capacity to bind and block the activity of the family of
transcription factors, E2F [14,15], thus inhibiting the
expression of genes that contain the E2F response element
in their promoters, e.g. dihydrofolate reductase (DHFR)
[16–18], DNA polymerase alpha [19], thymidine kinase
[20,21], histone H2A [22], proliferating cell nuclear antigen
[23], B-myb [24,25], cyclin D [26,27], cyclin E [28], cyclin A
[29,30] and cdc2 [31,32]. Rb is phosphorylated by the action
of various combinations of cyclin/CDK complexes, such as
cyclin D/CDK4-CDK6 in early G1 and cyclin E/CDK2 in
late G1 and G1/S phases. After mitosis, Rb returns to its
nonphosphorylated state [10,11]. Cyclin/CDK complexes
can be regulated by small inhibitory proteins, known as
intrinsic CDK inhibitors, which suppress cell growth. The
INK4 CDK inhibitors (p15, p16, p18 and p19) inhibit
CDK4 and CDK6, whereas the family of p21, p27 and p57
inhibit or sequester the different known cyclin/CDK
complexes [33–35]. Numerous human cancers present
abnormalities in some components of the Rb pathway as
a result of CDK hyperactivation, decrease in endogenous
Correspondence to C. J. Ciudad, Departamento de Bioquı
´
mica y

[43–45] and inhibition of angiogenesis [46]. CDK inhibitors
also modify the transcript levels of 2–3% of the genes in
Saccharomyces cerevisiae, as measured by array methods
[47].
Given that the modulation of CDK activity is an
attractive target for cancer chemotherapy, we studied the
changes produced by low concentrations of different CDK
inhibitors at the molecular level, with a special focus on their
natural substrate, retinoblastoma.
In addition to the primary function of Rb as a transcrip-
tional co-repressor in cell cycle regulation, this tumor
suppressor protein can function as a transcription
co-activator through its physical interaction with selective
transcriptional factors such as hBRm, C/EBP, AP2 and Sp1
[48–51]. Rb activates a set of gene promoters, e.g. c-fos,
c-myc, transforming growth factor-b1 [52,53], transforming
growth factor-b2 [54], c-jun [55], Cyclin D1 [26], thymidine
kinase and dihydrofolate reductase (DHFR) [53] that
control the cell cycle through stimulation of Sp1 mediated
transcription. Furthermore, the Rb promoter contains
potential binding sites for transcription factors such as
ATF-2, Sp1 and RBF-1, through which these proteins may
regulate Rb expression.
Sp1 and Rb are especially inter-related at the transcrip-
tional level and in their degradation fate. Sp1 is a ubiquitous
transcription factor involved in the activation of a large
number of genes. Its activity can be modulated during
differentiation [56,57], cell growth [58,59], and development
[60]. Sp1 and Rb interact physically, forming a complex that
enhances the transcriptional activation of Sp1 [51]. Rb has

UCN-01 was kindly provided by H. Nakano (Kyowa
Hakko Co., Tokyo, Japan). Roscovitine and olomoucine
were purchased from Calbiochem. Bisindolylmaleimide I
(BSM-I) was obtained from Sigma-Aldrich. Stock solutions
were prepared in dimethylsulfoxide and maintained at
)20 °C.
Cell culture
Conditions for the monolayer culture of CHO cells were as
described previously [67]. CHO K1 and CHO DG44 cells
[68] were grown in Ham’s F12 medium supplemented with
7% (w/v) fetal bovine serum (both from Gibco) and
maintained at 37 °C in a humidified 5% (v/v) CO
2
-
containing atmosphere. Human K562 and HeLa cells were
grown under the same culture conditions. When determin-
ing the activity of DHFR by the deoxyuridine method, cells
were incubated in F12 selective DHFR medium (–GHT)
lacking glycine, hypoxanthine and thymidine, the final
products of DHFR activity.
Flow cytometry analysis
Cell cycle phase distribution upon incubation with CDK
inhibitors was monitored by flow cytometry. Nuclei were
stainedwith25lgÆmL
)1
propidium iodide (Sigma-Aldrich)
and analyzed on a Becton-Dickinson flow cytometer.
mRNA levels
mRNA levels were determined by quantitative RT-PCR
using total cell lysates as the starting material for the RT

M
MgCl
2
,0.2m
M
dNTPs, 2.5 lCi of [
32
P]dATP[aP]
4810 S. Pen
˜
uelas et al. (Eur. J. Biochem. 270) Ó FEBS 2003
(3000 CiÆmmol
)1
; Amersham Ibe
´
rica), 1.5 U of Taq poly-
merase (Ecogen) and 500 ng of each of the four primers.
For the determination of mRNA levels, the primers were:
5¢-CGCCAAACTTGGGGGAAGCA-3¢ and 5¢-GAACC
AGGTTTTCCGGCCCA-3¢ for DHFR; 5¢-GTGCCAAT
GGCTGGCAGATCA-3¢ and 5¢-ACCATCCTGCTGCA
CTTGGGC-3¢ for Sp1; 5¢-CTCCACACACTCCAGTT
AGGA-3¢ and 5¢-CTGATTTAAGCATGGATTCCA-3¢
for Rb; and 5¢-CGCAGTTTCCCCGACTTCCC-3¢ and
5¢-GGCAGCGCACATGGTTCCTC-3¢ for adenine phos-
phoribosyltransferase (APRT), which was used as an
internal control.
The reaction mixture was separated into two phases by
a solid paraffin wax layer (melting temperature 58–60 °C;
Fluka), which prevents complete mixing of PCR reactants

by the Bio-Rad protein assay based on the Bradford method
[71], using bovine serum albumin as a standard (Sigma),
and extracts were frozen in liquid N
2
andstoredat)80 °C.
Total extracts
Whole extracts were obtained from K1 or K562 cells
according to the method of Kraus et al.[72].Cellswere
collected in ice-cold F-12 medium and centrifuged at 1000 g
for 5 min. The cell pellet was gently resuspended in 5 mL of
hypotonic buffer (15 m
M
NaCl, 60 m
M
KCl, 0.5 m
M
EDTA, 1 m
M
phenylmethanesulfonyl fluoride, 1 m
M
2-mercaptoethanol, 15 m
M
Tris/HCl, pH 8). After centri-
fugation (1000 g, 5 min), the cell pellet was resuspended in
100 lL of a buffer containing deoxycholate (100 m
M
NaCl,
10 m
M
NaH

Half-life of retinoblastoma and Sp1
The stability of Rb and Sp1 proteins was assessed by
calculating their half-life from the concentration of protein
remaining at different time-points after addition of cyclo-
heximide to the cell culture. CHO K1 control cells, or those
treatedwithUCN-01,5· 10
)8
M
for 48 h, were incubated
with 50 lgÆmL
)1
cycloheximide for various periods of time.
Total protein extracts were prepared and analyzed by
Western blot using Rb and Sp1 specific antibodies. The
results are expressed as the number of cells collected.
Cloning of the retinoblastoma promoter
Human genomic DNA from HeLa cells was used to isolate,
by PCR, a clone containing a 630 bp fragment correspond-
ing to the Rb promoter region. The PCR fragment was
generated using two Rb-specific primers whose sequences
were taken from GenBank accession number L11910. For
the forward primer, the specific sequence (shown below in
upper case text) was preceded by an arbitrary sequence
(shown below in lower case text) that included an NheI
restriction site (underlined). The reverse primer followed
a similar structure but contained a XhoI restriction site
(underlined) in the arbitrary sequence. The numbers indi-
cated after the primer sequences correspond to the distance,
in nucleotides (nt), from the translational start site: Rbprm-
for, 5¢-tcaagtcag

supernatants were transferred to a fresh tube. A 10 lL
aliquot of the cell extract was added to 25 lLofthe
luciferase assay substrate (Promega) and the luminescence
of the samples was read immediately on a TD-20/20
Luminometer, in which light production (relative light
units) was measured for 10 s. Luciferase activity was
normalized to cellular protein concentration, determined
using the Bio-Rad protein assay reagent in accordance with
the manufacturer’s protocol.
Transfections, cotransfections and the DHFR transient
activity assay
CHO K1 cells were cotransfected with increasing amounts
(1, 3 and 5 lg) of a eukaryotic expression vector for p21
(pCMV-Cip1), together with 0.4 lgofBPV-Neo,byusing
the calcium phosphate method [73]. After 24 h of expres-
sion, selection with GeneticinÒ (800 lgÆmL
)1
) was applied.
Three weeks later, the surviving colonies were pooled.
Transient expression experiments were carried out in
dhfr-deficient cells (CHO-DG44) by transfecting a dhfr
minigene in the presence and absence of CDK inhibitors.
When using p21, the expression vector corresponding to this
protein was co-transfected together with the dhfr minigene.
All transient transfections were also performed by the
calcium phosphate method. The plasmid providing basal
DHFR activity was p410, corresponding to a dhfr minigene
driven by its minimal promoter [74]. After 24 h of expres-
sion, the medium was replaced with –GHT medium
(DHFR selective medium) and the resulting DHFR activity

mediate mobility, which appears when cells are in G1; and
a low-mobility hyperphosphorylated form, which is pre-
sent mainly in S and G2/M phases (Fig. 1C). Only the
nonphosphorylated form of Rb was detected upon
incubation with high concentrations of UCN-01
(10
)6
M
) (Fig. 1C), revealing that this compound inhibits
the CDK–cyclin complexes that phosphorylate Rb in
these cells. At this concentration, K1 cells were arrested.
However, 50 n
M
UCN-01, at which the maximum
increase of Rb was observed, did not affect cell prolifer-
ation (data not shown). As UCN-01 is also able to inhibit
protein kinase C (PKC) activity (IC
50
¼ 7 · 10
)9
M
)we
investigated the different effects of UCN by using the
PKC inhibitor, BSM-I (IC
50
 10
)8
M
). In CHO cells,
10

used to normalize the results.
To test whether the effect of UCN-01 was also caused by
an increase in the stability of Rb and Sp1 proteins, we
determined the decay of these proteins after inhibiting
protein synthesis by cycloheximide. CHO cells were
incubated with 50 n
M
UCN-01 for 48 h, which yields
maximal expression of Rb and Sp1, and control and UCN-
01-treated cells were then incubated with 50 lgÆmL
)1
cycloheximide for different periods of time. Whole protein
extracts were prepared and used to determine the levels of
Rb and Sp1 by Western blot, as described in the Materials
and methods. The half-life of the Rb protein increased
from 8.7 h to 14.2 h upon treatment with UCN-01
(Fig. 3A,B), which corresponds to a 62% increase in the
stability of the protein, whereas the difference in Sp1
stability between control and UCN-01 treated cells was not
significant (data not shown).
4812 S. Pen
˜
uelas et al. (Eur. J. Biochem. 270) Ó FEBS 2003
Therefore, the effect of UCN-01 on the levels of Rb
protein may be caused by an increase in the synthesis of Rb
and by a decrease in its degradation. However, the effect on
Sp1 could be accounted for by the increase observed in Sp1
mRNA.
Effects of roscovitine, olomoucine and p21 in CHO cells
To examine whether the effects caused by UCN-01 were

product (Rb) and Sp1 proteins, respsectively. K1 cells (10
6
cells per 100 mm diameter dish) were incubated with the indicated concentrations of
UCN-01 for 48 h. Nuclear extracts were prepared and resolved by SDS/PAGE. Rb protein was detected by Western blotting using a 1 : 100
dilution of the C-15 antibody against Rb and enhanced chemiluminescence. Sp1 protein was detected using PEP 2 antibody. The same blots were
reprobed with a 1 : 100 dilution of the C-21 antibody against Oct-1 to normalize the results. Quantification of the signal is shown in the bottom
panel. Rb and Sp1 protein levels were determined from the ratio of the intensities between the Rb or the Sp1 and Oct-1 signals, respectively. Results
represent the mean ± SEM of three experiments. (C) Phosphorylated forms of Rb in K1 cells. Nuclear extracts (20 lg) from cells in each phase of
the cell cycle (G0, G1, S and G2/M), exponentially growing cells (Exp) or cells treated with 10
)6
M
UCN-01 for 24 h, were resolved by SDS/PAGE.
A Western blot was performed, as described above for (A). The phosphorylated forms of Rb are indicated by arrows. Rb (nonphosphorylated
form), Rb-P (hypophosphorylated form), Rb-PP (hyperphosphorylated form). (D). Determination of Rb protein levels with 50 n
M
UCN-01 or
1000 n
M
bisindolylmaleimide I by Western blot analysis using C-15 antibody in CHO K1 cells.
Ó FEBS 2003 Rb and Sp1 regulation by CDK inhibitors (Eur. J. Biochem. 270) 4813
Effects of CDK and PKC inhibition on Rb and Sp1
expression in human cells
Next, we determined whether the increased expression of
Rb and Sp1 protein upon incubation with UCN-01,
roscovitine and olomoucine were also produced in
human cells. It was observed that low concentrations
of UCN-01 (50 n
M
), roscovitine (100 n
M

48 h, followed by the addition of 50 lgÆmL
)1
cycloheximide to the
culture medium. At different time-points, cells were collected and used
to prepare total protein extracts. The total amount of the extract was
resolved by SDS/PAGE and the Rb protein levels were determined by
Western blot, as described in the legend to Fig. 1A. (A) Semi-log plot
of the levels of Rb protein as a function of chase time. A representative
experiment of the five performed is shown. The remaining Rb protein
levels are expressed as the percentage of the Rb protein present at 0 h
of CHX, for either the control or the UCN-01-treated cells. (B) Effect
of UCN-01 on Rb stability. The half-life was calculated using the
exponential curve fit method depicted in the legend to Fig. 3A. Results
represent the mean ± SEM from five experiments.
4814 S. Pen
˜
uelas et al. (Eur. J. Biochem. 270) Ó FEBS 2003
upon transfection of reporter constructs Rb-luc and Sp1-
luc in K562 (Fig. 6A,B,C,D). Transient transfection with
a p21 expression vector also caused an activation of
Rb- and Sp1 promoters (data not shown). In addition,
UCN01 also increased Rb and Sp1 transcription in
HeLa cells (Fig. 6E,F). Cell cycle distribution was
determined in K562 to study whether the effect of the
three compounds on Rb expression was related to CDK
inhibition. UCN-01, at 50 n
M
, caused a change in the
distribution of the cell cycle towards the G1 phase, at the
24 h time-point, without affecting cell proliferation. At

M
UCN-01 for different periods of time, the levels of DHFR
mRNA transiently increased to a maximum at 36 h
(Fig. 7A). The effect of UCN-01 on DHFR activity was
analyzed in transiently transfected dhfr-deficient cells (CHO
DG44) using a dhfr minigene (p410). DHFR activity
increased to a maximum at 50 n
M
UCN-01; at higher
concentrations of the inhibitor, the activity decreased
(Fig. 7B). Roscovitine and olomoucine also enhanced
DHFR activity, to a maximum at 100 n
M
roscovitine and
Fig. 4. Effect of roscovitine, olomoucine and p21 on the levels of Rb and Sp1 protein. (A), (B) Effect of roscovitine or olomoucine on retinoblastoma
gene product (Rb) and Sp1 protein levels. CHO K1 cells (500 000 per 100 mm diameter dish) were treated with increasing concentrations of the
CDK2 inhibitors, roscovitine (A) or olomoucine (B) for 48 h. Twenty micrograms of nuclear extracts from either control or treated cells was
resolved by SDS/PAGE. Rb and Sp1 proteins were detected by Western blot using C-15 antibody against Rb, PEP 2 antibody against Sp1, or C-21
antibody against Oct-1, which was used as a control to normalize the results. (C) Effect of the overexpression of p21 on Rb and Sp1 protein levels.
Nuclear extracts (20 lg) were obtained from CHO K1 cells stably transfected with different amounts (1, 3 and 5 lg) of a eukaryotic expression
vector for p21. Western blot analysis was performed with C-15 antibody against Rb, PEP 2 antibody against Sp1, or C-21 antibody against Oct-1,
which was used as a control to normalize the results. (D) Determination of the overexpression of p21 in permanent transfectant cells using SX118
antibody against p21 and A2066 antibody against actin to normalize the result.
Ó FEBS 2003 Rb and Sp1 regulation by CDK inhibitors (Eur. J. Biochem. 270) 4815
500 n
M
olomoucine, but the activity decreased thereafter
(Fig. 7C,D). In addition, the effect of overexpression of
p21 on DHFR activity was also analyzed in transient
co-transfection experiments. DHFR activity increased,

M
, respectively. However,
roscovitine and olomoucine have an IC
50
for CDK4/6 that
is  100-fold higher than for CDK2 (0.7 l
M
for CDK2 and
> 100 l
M
for CDK4/6, for roscovitine, and 7 l
M
for
CDK2 and > 1000 l
M
forCDK4/6,forolomoucine,
respectively) [76,77]. Thus, CDK2 can be selectively inhi-
bited by using low concentrations of these inhibitors,
according to their IC
50
values.
A first conclusion of this work is that upon incubation
with submaximal concentrations of CDK inhibitors, the
total amount of Rb protein increases in a dose-dependent
manner. In the case of UCN-01, the maximal effect was
observed at 50 n
M
, a concentration that did not interfere
with cell proliferation. However, this inhibitor was able to
arrest the cells and to prevent Rb phosphorylation

or olomoucine (C), (D). Transfections were
performedinduplicate,andtheresultsrepre-
sent the mean ± SEM of two experiments.
Luciferase activity was normalized to micro-
grams of protein for each sample. (E), (F) As
for (A), (B), but incubation was with increas-
ing concentrations of UCN-01 in HeLa cells.
Ó FEBS 2003 Rb and Sp1 regulation by CDK inhibitors (Eur. J. Biochem. 270) 4817
UCN01 is caused by overlapping effects. However, this
increase in Rb expression is lower than that caused by
UCN-01, when similar inhibition of PKC was achieved. In
addition, 100 n
M
roscovitine and 500 n
M
olomoucine,
which increase Rb expression, do not inhibit PKC (IC
50
values of roscovitine and olomoucine for PKC are
> 100 l
M
and > 1000 l
M
, respectively [76,77]).
Regarding roscovitine and olomoucine, the levels of Rb
protein increase at concentrations that inhibit CDK2 but
not CDK4/6. Thus, inhibition of CDK2 alone, which
prevents hyperphosphorylation of Rb, is sufficient to trigger
the increase in expression of Rb.
The effects on Rb and Sp1 have also been demonstrated

UCN-01 (B), roscovitine (C) or olomoucine (D) were added at the indicated concentrations, and maintained in the culture medium throughout the
expression experiments. After 24 h of expression, the medium was replaced with 1 mL of F12 selective DHFR medium (to renew the cyclin-
dependent kinase inhibitors), and 2 lCi of 6[
3
H]deoxyuridine was then added. Cells were collected 24 h later, and the incorporated radioactivity
was measured in a scintillation counter. DHFR activity is expressed as c.p.m. incorporated to DNA. Results represent the mean ± SE of three
experiments. (E) Effect of the overexpression of p21 on DHFR activity upon transient co-transfection with a dhfr minigene. CHO-DG44 cells
(225 000 cells per 35 mm diameter dish) were co-transfected with 1 lgofthedhfr minigene p410 plus 1 or 2 lg of an eukaryotic expression vector
for p21, using the calcium phosphate method. DHFR activity was determined as described in the legend to Fig. 6B. Results represent the mean ±
SEM of three experiments.
4818 S. Pen
˜
uelas et al. (Eur. J. Biochem. 270) Ó FEBS 2003
[80], using transfections of a mutant form of Rb (Dp34)
refractory to phosphorylation, demonstrated that non-
phosphorylated Rb represses Rb transcription. On the
other hand, Park et al. [81] described that the Rb
promoter is positively self-regulated by its own gene
product when Rb is overexpressed in exponentially
growing cells, in which the hypophosphorylated form is
the major species. Sandig et al. [82] reported that over-
expression of the CDK inhibitor, p16, which prevents the
phosphorylation of Rb by CDK4/6, down-regulates Rb.
The hypophosphorylated form of Rb may thus activate
Rb transcription. Our results are in keeping with those
observations, as low concentrations of CDK inhibitors
trigger a response of increased Rb transcription. Of
particular interest are the results obtained upon incuba-
tion with specific CDK2 inhibitors, as their action would
partially decrease the step of phosphorylation towards

in both the levels of mRNA and the stability of the protein,
while the degradation rate of Sp1 does not vary and thus the
increase in the protein levels seems to depend only on
the increase in transcription, and (b) the time dependency of
the increase in mRNAs is shorter for Sp1 than for Rb.
As CDK2 phosphorylates Sp1 and enhances Sp1-
mediated transcription [63], and high concentrations of
olomoucine reduce Sp1 transcriptional activity [64], the
phosphorylated form of Sp1 may be needed for the
transcriptional activation of a variety of Sp1-controlled
genes. A slight decrease in the phosphorylated form of Sp1,
caused by CDK inhibitors, may trigger Sp1 synthesis to
re-establish its normal levels. Given that Sp1 mRNA
increases earlier than Rb mRNA, and that the Rb
promoter contains GC boxes, Sp1 may contribute to the
rise in Rb expression.
Finally, we used the dhfr gene as a target model to study
the effect of CDK inhibitors, on a late response gene, upon
stimulation to proliferate. The dhfr gene was selected
because it is activated mainly by Sp1, especially when the
latter is associated with Rb [51]. DHFR mRNA was
increased by UCN-01 in a time-dependent manner in
CHO K1 cells. In addition, DHFR activity was increased
by UCN-01, roscovitine, olomoucine and p21 in assays of
transient transfections. This increase takes place at concen-
trations of the inhibitors where the expression of Rb and
Sp1 starts to increase. At higher concentrations of these
inhibitors, the effect on DHFR is no longer noticeable.
In summary, we describe that CDK inhibitors, when used
at submaximal concentrations, enhance expression of the

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