Báo cáo khoa học: The expression of retinoblastoma and Sp1 is increased by low concentrations of cyclin-dependent kinase inhibitors ppt

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

kinase C than UCN-01, provoked a lower activation of
retinoblastoma and Sp1 expression. Finally, the effects of
cyclin-dependent kinase inhibitors on dihydrofolate reduc-
tase gene expression were evaluated. Treatment with UCN-
01 increased cellular dihydrofolate reductase mRNA levels,
and dihydrofolate reductase enzymatic activity was
enhanced by UCN-01, roscovitine, olomoucine and p21, in
transient transfection experiments. These results support a
mechanism for the self-regulation of retinoblastoma
expression, and point to the need to establish the appropriate
dose of cyclin-dependent kinase inhibitors as antiprolifera-
tive agents in anticancer treatments.
Keywords: retinoblastoma gene product; Sp1; UCN-01;
roscovitine; dihydrofolate reductase.
Cyclin-dependent kinases (CDKs) are key regulators of cell
cycle progression. They constitute the catalytic subunits of
holoenzymes formed in combination with regulatory sub-
units named cyclins. Thirteen CDKs [1,2] and at least 25
cyclins [2] have been reported to date. Cyclin expression
varies during the cell cycle and the cyclin/CDK holoenzyme
is activated by phosphorylation of specific residues in the
CDK catalytic subunit by the cdk-activating kinase [3,4].
CDKs are involved in transcriptional control [5], mitotic
progression [6], DNA repair (CDK7) [7], differentiation of
brain neurons (CDK5) [8] and play a crucial role in the
progression of cells from G1 to S phase by regulating the
phosphorylation state of the retinoblastoma gene product
(Rb). The tumor suppressor Rb is a nuclear protein of 928
amino acids [9] that is present in distinct phosphorylation
states depending on the phase of the cell cycle [10,11]: it is

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

Biologı
´
a Molecular, Division IV, Facultad de Farmacia, Universidad
de Barcelona, Avenue Diagonal 643, E-08028 Barcelona, Spain.
Fax: + 34 93 402 4520, Tel.: + 34 93 403 4455,
E-mail:
Abbreviations: APRT, adenine phosphoribosyltransferase; BSM-I,
bisindolylmaleimide I; CDK, cyclin-dependent kinase; CHO, Chinese
hamster ovary; DHFR, dihydrofolate reductase; IC
50
,50%inhibitory
concentration; PKC, protein kinase C; Rb, retinoblastoma gene
product.
*Note: The two first authors contributed equally to this work.
(Received 17 June 2003, revised 10 September 2003,
accepted 7 October 2003)
Eur. J. Biochem. 270, 4809–4822 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03874.x
CDK inhibitors or Rb gene mutations. Therefore, the use of
pharmacologic CDK inhibitors offers great potential for the
treatment of many neoplasms [36]. In this regard, chemical
CDK inhibitors, such as UCN-01, roscovitine and olomou-
cine, have been developed. Roscovitine and olomoucine are
more specific towards CDK2, whereas UCN-01 shows a
similar 50% inhibitory concentration (IC
50
)forCDK2and
CDK4/6. Flavopiridol (another CDK inhibitor), UCN-01
and roscovitine are currently used in clinical studies on
cancer therapy [2,37]. CDK inhibition leads to cell cycle
arrest [38–41], apoptotic cell death [39,41,42], differentiation

[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

also been described as a transcriptional activator of the p21
gene in epithelial cells through Sp1 and Sp3 transcription
factors [61,62]. The transcriptional interaction of Rb, Sp1
and p21 implies an auto-loop of regulation between Rb and
CDK activities.
Sp1 can be phosphorylated by a cyclinA/CDK complex
that probably includes CDK2, as phosphorylation is
inhibited by olomoucine. Dephosphorylated Sp1 shows
decreased DNA binding and transcriptional activity
[63,64]. Moreover, Rb and Sp1 proteins are degraded by
the same proteolytic enzyme, SPase, a nuclear and cytosolic
protease with cathepsin B- and L-like proteolytic activity
[65]. The levels of SPase vary along the cell cycle,
correlating with Rb degradation, suggesting that SPase
regulates Rb [66].
Taken together, these data prompted us to analyze the
changes in the expression (transcription, mRNA and
protein levels), of both Rb and Sp1 after CDK inhibition
by the chemical inhibitors UCN-01, roscovitine and
olomoucine, and by the intrinsic inhibitor p21. As DHFR
activity is enhanced by the association between Rb and Sp1,
we used DHFR as a target model to study the final effects of
CDK inhibitors.
We report that the expression of retinoblastoma and Sp1
is increased by low concentrations of CDK inhibitors in
Chinese hamster ovary (CHO) K1 and human cells by a
mechanism involving transcriptional activation and, also in
the case of Rb, by an increase in its stability.
Materials and methods
Materials

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

reaction, as described in Noe
´
et al. [69]. K1 cells were plated
in 35 mm-diameter dishes and, after several periods of
incubation with 5 · 10
)8
M
UCN-01, they were collected in
500 lL of F-12 medium. The cells were then centrifuged and
washed with NaCl/P
i
, and the final pellet was resuspended
in 11.25 lL of diethylpyrocarbonate-treated water. The cells
were lysed at 80 °C for 5 min. cDNA was synthesized in
20 lL of reaction mixture containing 125 ng of random
hexamers (Roche), 10 m
M
dithiothreitol, 20 U of RNasin
(Promega), 0.5 m
M
dNTPs (AppliChem), 4 lLof5· RT
buffer, 200 U of M-MLV reverse transcriptase (BRL-
Gibco) and the cell lysate. The reaction mixture was
incubated at 37 °C for 60 min. Five microlitres of the
cDNA mixture was used for PCR amplification.
PCR reactions were typically carried out as follows. A
standard 50 lL mixture contained 5 lL of the cDNA
mixture, 4 lLof10· PCR buffer (Mg
2+
-free), 1.5 m

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

until the reaction has reached a temperature that minimizes
nonspecific annealing of primers to nontarget DNA. The
lower solution contained the cDNA, the MgCl
2
, the dNTPs,
the [
32
P]dATP[aP] and one half of the buffer, and the upper
solution contained the four primers, the Taq enzyme and
the remaining buffer.
PCR was performed for 30 cycles, in the case of DHFR,
and for 22 cycles, in the case of Sp1 and Rb, after a 1 min
denaturation step at 94 °C. Each cycle consisted of dena-
turation at 92 °C for 30 s; primer annealing at 59 °Cfor
1 min for DHFR and Sp1, and at 55 °C for 1 min for Rb;
and primer extension at 72 °C for 1 min. Five microlitres
of each PCR sample was electrophoresed in a 5% (w/v)
polyacrylamide gel. The gels were dried and the radioactive
bands visualized by autoradiography. Results were quanti-
fied by image analysis using the
BIO
-1
D
, version 99.03,
software from Vilbert-Lourmat. The DHFR, Rb or Sp1
mRNA levels were expressed as the ratio of the intensities of
the DHFR, Rb or Sp1 signals and APRT signals.
Nuclear extracts
Nuclear extracts from K1 cells were prepared as described in
Ciudad et al. [70]. Protein concentrations were determined

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

2
PO
4
, pH 7.4, 0.5% sodium deoxycholate,
0.1% SDS, 1% Triton X-100) and centrifuged at 13 000 g
for 10 min. The resulting supernatant corresponded to the
whole extract. The entire procedure was carried out at 4 °C.
Five microlitres of the extract was used for determining the
protein concentration by using the Bradford assay (Bio-
Rad). The extracts were frozen in liquid N
2
andstoredat
)80 °C.
Western blot analysis
Twenty micrograms of nuclear extract from CHO K1 or
human K562 cells was resolved on SDS/5% polyacrylamide
gels [71] and transferred to poly(vinylidene difluoride)
membranes (Immobilon P; Millipore) using a semidry
electroblotter. The membranes were probed with anti-Rb
[C-15, against amino acids 914–928; IF-8, against amino
acids 300–380 (both from Santa Cruz Biotechnology); and
G3-425, against amino acids 332–344 (BD-Pharmingen)] or
anti-(Sp1 PEP 2) (Santa Cruz Biotechnology). Detection of
p21 was by using anti-SX118 (BD-Pharmingen). Signals
were detected by secondary horseradish peroxidase-conju-
gated antibody and enhanced chemiluminescence, as
recommended by the manufacturer (Amersham). Blots
were reprobed with C-21 antibody against Oct-1, or with
antibody A2066 against actin (Sigma), in the case of
experiments with p21, to normalize the results.

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

gctagcGTTCCGCACCTATCAGCGC
TCC-3¢ (630 nt); Rbprm-rev, 5¢-cagtgctgc
ctcgagGACGC
CTTTCGCGGCGGGAGC-3¢ (1 nt). The PCR product
was sequenced using Big Dye v2.0 (PE Biosystems). After
digestion with NheIandXhoI, the PCR fragment from the
Rb promoter was cloned into the NheI/XhoIsitesof
the reporter luciferase vector pGL3-basic (Promega).
The resulting construct was named prmRB-luc.
Transfections and luciferase assay
HeLa or K562 cells were seeded into six-well plates, the day
before transfection, at a density of 7.5 · 10
4
cells/well in
HAM F-12 containing 5% fetal bovine serum. Eighteen
hours later, the CDK inhibitors were incubated with the
cells and, 18 h later, transfections were performed using
Ó FEBS 2003 Rb and Sp1 regulation by CDK inhibitors (Eur. J. Biochem. 270) 4811
FuGENE6 (Roche Molecular Biochemicals). For each well,
3 lL of FuGENE6 in 100 lL of serum free HAM F-12
medium was incubated at room temperature for 5 min. The
mixture was added to 125 ng of the Rb promoter (prmRB-
luc) or 250 ng of Sp1 promoter (prmSp1-luc) constructs.
The DNA–lipid mixture was incubated at room tempera-
ture for 15 min. The mixture was added to the cells for 30 h.
Luciferase activity was assayed 48 h after treatment with
the CDK inhibitors and 30 h after transfection of the
constructs. Cells were lysed in 500 lL of freshly diluted 1·
reporter lysis buffer (Promega). The lysate was centrifuged
at 10 000 g for 2 min to pellet the cell debris and the

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

was determined by the incorporation of radioactive deoxy-
uridine to cellular DNA, as described by Noe
´
et al.[51].
The chemical CDK inhibitors UCN-01, roscovitine and
olomoucine were added to the medium immediately after
transfection and maintained during the expression and
labeling times of the assay.
Results
Effects of the CDK inhibitor UCN-01 on retinoblastoma
and Sp1 proteins
CHO K1 cells were incubated with increasing concentra-
tions (10 to 100 n
M
) of UCN-01, for 48 h. Nuclear extracts
were then prepared and analyzed by Western blot. UCN-01
resulted in an increase of the total amount of Rb protein in
a dose-dependent manner, with a maximum ( 10-fold) at
5 · 10
)8
M
(Fig. 1A). The results were normalized using the
signal obtained upon reprobing the same blots with an
antibody against Oct-1.
To help define the phosphorylation states of Rb in
these cells, we performed Western blot analysis with
nuclear extracts from CHO cells in the different phases of
the cell cycle. Three bands were observed: a nonphos-
phorylated form of Rb, which is the only band present in
starved cells; a hypophosphorylated form, with inter-

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

)6
M
BSM-I produced an increase in Rb expression
that represented 40% of the increase caused by 50 n
M
UCN-01 (Fig. 1D).
We also aimed to determine the levels of Sp1 protein in
UCN-01-treated cells. To achieve this, the blots used for
determining Rb protein levels were reprobed with a specific
antibody (PEP 2) against Sp1. Three bands of Sp1 were
detected in nuclear extracts from CHO K1 cells, as reported
previously [75]. The levels of this transcription factor
increased, in a dose-dependent manner, when K1 cells were
incubated for 48 h with UCN-01 (Fig. 1B), peaking at
5 · 10
)8
M
UCN-01.
Mechanisms explaining the increase in Rb and Sp1
protein caused by CDK inhibitors in CHO cells
The increase in total levels of Rb and Sp1 proteins caused by
UCN-01 may be a result of the enhanced transcription and
stability of Rb and Sp1.
To assess transcription, we determined Rb and Sp1
mRNA levels after incubation of CHO cells with 50 n
M
UCN-01 for different periods of time. The CDK inhibitor
increased Rb mRNA levels by 3.5-fold at 20 h of incubation
(Fig. 2A) and Sp1 mRNA levels by fivefold at 6 h (Fig. 2B).
In both cases, the signal obtained for APRT mRNA was

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

shared by other CDK inhibitors, we extended the analysis
to roscovitine and olomoucine. These inhibitors belong to
the C2,N6,N9-substituted adenine family and mainly
inhibit CDK2 activity, as their IC
50
values for CDK4/6
are  100 and 1000 times higher than for CDK2,
respectively. Cells were incubated with increasing concen-
trations of these two chemical inhibitors, and the levels of
Rb and Sp1 protein were determined. The range of
concentrations of olomoucine used was higher than for
roscovitine, given its higher IC
50
for CDK2. Both
inhibitors resulted in an increased total amount of the
proteins Rb and Sp1 (Fig. 4A,B).
We also tested the effect of overexpression of the
intrinsic CDK inhibitor p21 in pooled permanent trans-
fectants. CHO K1 cells were co-transfected with increas-
ing amounts of an expression vector for p21 together with
BPV-Neo and, upon selection with Geneticin, the pools
were used to determine the protein levels of Rb and Sp1.
The levels of both proteins increased, even in the
transfectants obtained with 1 lgofp21(Fig.4C).The
overexpression of p21 was confirmed in these transfect-
ants. (Fig. 4D).
These results extended the original observations for
UCN-01 and confirmed that the inhibition of CDK activity
increases the expression of Rb and Sp1.
Fig. 1. Effects of UCN-01 on the levels of Rb and Sp1 protein. (A), (B) Dose–response of the effect of UCN-01 on the levels of retinoblastoma gene

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

) or olomoucine
(500 n
M
) caused an increase in the expression of Rb
protein, both in nuclear and total extracts from K562
cells (Fig. 5A,C,E). Sp1 expression was also increased
by low concentrations of these three inhibitors in K562
cells (Fig. 5B,D). The changes in transcriptional activity
caused by UCN-01, roscovitine and olomoucine in K562
cells were also determined by using luciferase assays. The
three CDK inhibitors caused an increase in transcription
Fig. 2. Rb (A) and Sp1 (B) mRNA levels upon incubation with UCN-01.
CHO K1 cells (5000 cells) were incubated with 5 · 10
)8
M
UCN-01
for the indicated periods of time. The mRNA levels for Rb and Sp1
were then determined from cell lysates by RT-PCR in quantitative
conditions using specific primers. The top panels correspond to rep-
resentative autoradiographs of the amplified products and the quan-
tification of the bands is shown in the bottom panels. Rb and Sp1
mRNA levels were determined from the ratio of the intensities between
the Rb or Sp1 and the APRT signals, which was used as an internal
control of the reaction. Results are the mean ± SEM of three
experiments.
Fig.3. EffectofUCN-01onRbhalf-life.CHO K1 cells (1000 cells per
35 mm diameter dish) were incubated with 5 · 10
)8
M
UCN-01 for

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

higher concentrations, this effect persisted, whereby an
increase of cells in G2/M was observed (Fig. 5F).
Roscovitine produced a displacement of the distribution
of the cell cycle towards the G2/M phase that started at
the low concentration of 100 n
M
. Roscovitine and
olomoucine caused similar effects, and the results cor-
responding to roscovitine are shown in Fig. 5F. The
CDK-independent effect of UCN01 was studied upon
incubation with BSM-I, a PKC inhibitor. In K562 cells,
concentrations of BSM-I that have a similar PKC
inhibitory ability as UCN-01, based on their IC
50
values,
increased Rb and Sp1 expression to a lower level than
UCN-01 (Fig. 5A,B). These concentrations of BSM-I
increased Rb and Sp1 promoter activity in luciferase
assays (Fig. 6A,B) to  30% of the increase observed
with UCN-01.
Effects of UCN-01 on DHFR activity
Taking into account that Sp1 is a powerful activator of
DHFR transcription and that Rb stimulates Sp1 transcrip-
tional activity, we aimed to assess whether the increases in
Rb and Sp1 proteins caused by CDK inhibitors affected
DHFR expression. To achieve this, we determined the levels
of DHFR mRNA upon incubation of CHO K1 cells with
UCN-01, and DHFR activity in transient transfections after
incubation with UCN-01, roscovitine and olomoucine and
in co-transfections with p21. After incubation with 50 n

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,

depending on the amount of co-transfected p21 (Fig. 7E).
Discussion
We aimed to explore the effects of the inhibition of CDKs
on the expression of their natural substrate, retinoblastoma.
This has special interest given that some CDK inhibitors,
Fig. 5. Effect of UCN-01, roscovitine, olomoucine or bisindolylmaleimide I on Rb and Sp1 expression in human cells. (A), (B), (C), (D) K562 cells
(10
6
cells per 100-mm diameter dish) were incubated with the indicated concentrations of bisindolylmaleimide I, UCN-01, roscovitine (RSC) and
olomoucine (OLM) for 48 h. Nuclear extracts were prepared and resolved by electrophoresis on a 5% polyacrylamide/SDS gel. Western blots were
performed to detect Rb protein using G3-245 antibody against Rb (A,C), or Sp1 protein using PEP 2 antibody (B,D). The same blots were reprobed
with C-21 antibody against Oct-1 to normalize the results. Quantification of the signal is shown on the bottom panel. Results represent the
mean ± SEM of three experiments. (E) Cells were treated with the indicated concentrations of the CDK inhibitors and total extracts were prepared
as described in the Materials and methods. The levels of Rb protein were determined by Western blot using G3-245 antibody. (F) Cell cycle
distribution of K562 cells upon incubation with CDK inhibitors. Cells were treated with either UCN01 for 24 h or roscovitine for 48 h, at the
indicated concentrations, and the percentage of cells in each phase of the cell cycle was analyzed by flow cytometry.
4816 S. Pen
˜
uelas et al. (Eur. J. Biochem. 270) Ó FEBS 2003
like UCN-01 and roscovitine, are undergoing clinical trials
for use in anticancer treatment, as high concentrations of
CDK inhibitors have antiproliferative effects.
We used the chemical CDK inhibitors UCN-01, roscovi-
tine and olomoucine, as each shows a distinct specificity
towardCDKs.p21wasalsousedasanintrinsicCDK
inhibitor. UCN-01 inhibits CDK4/6 and CDK2 with IC
50
values of 0.032 l
M
and 0.030 l

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

(Fig. 1C) when used at high concentrations (10
)6
M
), in
agreement with previous observations [78]. The amount of
Rb protein was also increased by the CDK intrinsic
inhibitor, p21, with the same broad spectrum of action as
UCN-01, and by roscovitine and olomoucine, which
inhibit CDK2 activity.
UCN-01 was originally described as a PKC inhibitor and
shows a low IC
50
for this kinase (0.007 l
M
). Therefore, we
explored the possible contribution of the PKC inhibitiory
activity of UCN-01 to the increase in Rb expression, to
characterize the extent of the CDK-independent effect.
Indeed, inhibition of PKC by using BSM-I, a more selective
inhibitor of PKC, also triggered the expression of Rb
protein and Rb transcription. Therefore, the action of
Fig. 6. Transcriptional activity of the retinob-
lastoma gene product (Rb) and Sp1 upon
treatment with UCN-01 and bisindolylmalei-
mide I in human cells. prmRb-LUC (125 ng) or
prmSp1-LUC (250 ng) were transiently
transfectedintoK562cellsandassayedfor
luciferase activity after 48 h of treatment with
the indicated concentrations of bisindolylma-
leimide I or UCN-01 (A), (B) and roscovitine

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

in human cells both at the level of protein expression and
transcriptional activity. The changes in the cell cycle
distribution in human K562, produced by the low concen-
trations of these compounds, show an effect on CDK
activity, and it is precisely at these submaximal concentra-
tions where the clearest effects on RB and Sp1 expression
are seen.
Given that Rb has various phosphorylation states
depending on the phase of the cell cycle, two mechanisms
may explain the increase in the total amount of Rb upon
CDK inhibition. Rb expression may be enhanced by a
negative effect caused by the hyperphosphorylated form,
by a positive effect caused by accumulation of the
hypophosphorylated form or by a combination of
the two mechanisms, in keeping with the self-regulation
of the Rb gene by its own gene product, as proposed
elsewhere. On the one hand, Hamel et al. [79], using
transfections of Rb in differentiated P19 cells, thus
overexpressing nonphosphorylated Rb, and Gill et al.
Fig. 7. Effect of CDK inhibitors on mRNA levels and dhfr activity. (A) Dihydrofolate reductase (DHFR) mRNA levels after incubation of K1 cells
with UCN-01. One thousand K1 cells were plated in 35-mm diameter dishes and treated with 5 · 10
)8
M
UCN-01, for the indicated periods of time,
on the following day. Cells were then harvested and lysed, and DHFR mRNA levels were determined by quantitative RT-PCR using specific
primers in exonic sequences of the dhfr and aprt genes. The top panel of the figure corresponds to a representative autoradiograph of the amplified
products, and quantification of the bands is shown in the bottom panel. DHFR mRNA levels were determined from the ratio of the intensities
between the DHFR and APRT signals. Results represent the mean ± SEM of three experiments. (B), (C), (D) Effect of UCN-01, roscovitine and
olomoucine on DHFR activity upon transient transfection with a DHFR minigene. DG44 cells (225 000 cells per 35 mm diameter dish) were plated
and, 18 h later, transfected with 1 lg of plasmid p410 (dhfr minigene) using the calcium phosphate method. Immediately after the transfection,

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

hyperphosphorylated RB. Thus, a partial increase of the
hypophosphorylated form may increase Rb transcription,
and a decrease of the hyperphosphorylated form may
serve as a signal to restart Rb transcription.
From the experiments performed in UCN-01 treated
cells, we conclude that the increased level of total Rb protein
is caused by two mechanisms. On the one hand, there is
an increase in transcription, as shown by the luciferase
experiments and in the levels of Rb mRNA; and, on the
other hand, the stability of Rb protein is increased.
As stated in the Introduction, Rb interacts with a variety
of proteins. We selected Sp1 to study the possible effect of
CDK inhibitors for the following reasons, namely that (a)
thepresenceofGC-boxesintheRbpromotermayallow
regulation through Sp1, (b) Sp1-mediated transcription is
stimulated by Rb through a physical complex between the
two proteins, (c) Sp1 can be phosphorylated by a cyclinA/
CDK complex and (d) Sp1 is degraded by SPase, a protease
that is also active on Rb and regulates this protein in the cell
cycle.
There are remarkable similarities in the protein expres-
sion of Sp1 and Rb in response to low concentrations of
CDK inhibitors. First, the expression of Sp1 strongly
increases with the chemical inhibitors UCN-01, roscovitine
and olomoucine, and with the overexpression of the intrinsic
inhibitor p21. Second, the levels of Sp1 mRNA increase
upon incubation with UCN-01, reflecting an elevated
transcription, as demonstrated in the transient transfection
with Sp1-luc in human cells. However, there are also some
differences, namely (a) in the case of Rb, there is an increase

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

CDK natural substrate, retinoblastoma, and the Rb-inter-
acting protein, the transcription factor Sp1. These results
uncover intriguing aspects of Rb regulation, e.g. that Rb
may be self-regulated through its phosphorylation status in
combination with Sp1, also subjected to changes in phos-
phorylation. Additionally, these findings emphasize the
need for caution when adjusting the dose of CDK inhibitors
to be used in anticancer treatments, alone or in combination
with other chemotherapeutic agents. Lower than optimal
concentrations of CDK inhibitors can increase the activity
of potential target proteins, such as DHFR, which is
inhibited by treatment with methotrexate. Moreover, over-
expression of Rb decreases the susceptibility of cells to
therapy with agents that increase apoptosis [83]. Thus, the
dose adjustment of potential chemotherapy combinations is
required. In this regard, the results presented in this study
may prove useful.
Acknowledgements
This work was supported by grant SAF99-0120, SAF02-0363 (from
Comisio
´
n Interdepartamental de Ciencia y Tecnologı
´
a, Spain) and
2001SGR00141 (from Direccio
´
General de Recerca, Catalunya). We
thank Mr Robin Rycroft (from the Language Advisory Service) for
correcting the English manuscript.
References

1. Grana, X., De Luca, A., Sang, N., Fu, Y., Claudio, P.P.,
Rosenblatt,J.,Morgan,D.O.&Giordano,A.(1994)PITALRE,
a nuclear CDC2-related protein kinase that phosphorylates the
retinoblastoma protein in vitro. Proc. Natl Acad. Sci. USA 91,
3834–3838.
2. Fisher, P.M. & Gianella-Borradori, A. (2003) CDK inhibitors in
clinical development for the treatment of cancer. Expert Opin.
Investig. Drugs 12, 955–970.
3. MacLachlan, T.K., Sang, N. & Giordano, A. (1995) Cyclins,
cyclin-dependent kinases and cdk inhibitors: implications in cell
cycle control and cancer. Crit. Rev. Eukaryot. Gene Expr. 5,127–
156.
4. Edwards, M.C., Wong, C. & Elledge, S.J. (1998) Human cyclin K,
a novel RNA polymerase II-associated cyclin possessing both
carboxy-terminal domain kinase and Cdk-activating kinase
activity. Mol. Cell. Biol. 18, 4291–4300.
5. Coqueret, O. (2002) Linking cyclins to transcriptional control.
Gene 299,35–55.
Ó FEBS 2003 Rb and Sp1 regulation by CDK inhibitors (Eur. J. Biochem. 270) 4819
6. Jackman, M.R. & Pines, J.N. (1997) Cyclins an the G2/M trans-
ition. Cancer Surv. 29, 47–73.
7. Shuttleworth, J. (1995) The regulation and functions of cdk7.
Prog.CellCycleRes.1, 229–240.
8. Tang,D.&Wang,J.H.(1996)Cyclin-dependentkinase5(Cdk5)
and neuron-specific Cdk5 activators. Prog. Cell Cycle Res. 2,
205–216.
9. Lee, W.H., Bookstein, R., Hong, F., Young, L.J., Shew, J.Y. &
Lee, E.Y. (1987) Human retinoblastoma susceptibility gene:
cloning, identification, and sequence. Science 235, 1394–1399.
10. Tassan, J.P., Schultz, S.J., Bartek, J. & Nigg, E.A. (1994) Cell cycle

analysis of the activity, subcellular localization, and subunit
composition of human CAK (CDK-activating kinase). J. Cell.
Biol. 127, 467–478.
11. Kaldis, P., Russo, A.A., Chou, H.S., Pavletich, N.P. & Solomon,
M.J. (1998) Human and yeast Cdk-activatin kinases (CAKs) dis-
play distinct substrate specificities. Mol. Biol. Cell 9, 2545–2560.
12. Chen, P L., Scully, P., Shew, J Y., Wang, J.Y.J. & Lee, W H.
(1989) Phosphorylation of the retinoblastoma gene product is
modulated during the cell cycle and cellular differentiation. Cell
58, 1193–1198.
13. DeCaprio, J.A., Ludlow, J.W., Lynch, D., Furukawa, Y., Griffin,
J., Piwnica-Worms, H., Huang, C M. & Livingston, D.M. (1989)
The product of the retinoblastoma susceptibility gene has prop-
erties of a cell cycle regulatory element. Cell 58, 1085–1095.
14. Chellappan, S.P., Hiebert, S., Mudryj, M., Horowitz, J.M. &
Nevins, J.R. (1991) The E2F transcription factor is a cellular target
for the Rb protein. Cell 65, 1053–1061.
15.Weintraub,S.J.,Prater,C.A.&Dean,D.C.(1992)Retino-
blastoma protein switches the E2F site from positive to negative
element. Nature 358, 259–261.
16. Hiebert, S.W. (1993) Regions of the retinoblastoma gene product
required for its interaction with the E2F transcription factor are
necessary for E2 promoter repression and pRb-mediated growth
suppression. Mol. Cell. Biol. 13, 3384–3391.
17. Blake, M.C. & Azizkhan, J.C. (1989) Transcription factor E2F is
required for efficient expression of the hamster dihydrofolate
reductase gene in vitro and in vivo. Mol. Cell. Biol. 9, 4994–5002.
18. Slansky, J.E. & Farnham, P.J. (1996) Transcriptional regulation of
the dihydrofolate reductase gene. Bioassays 18, 55–62.
19. Pearson, B.E., Nasheuer, H.P. & Wang, T.S. (1991) Human

DNA polymerase alpha gene: sequences controlling expression
in cycling and serum stimulated cells. Mol. Cell. Biol. 11, 2081–
2095.
20. Dou, Q.P., Fridovich Keil, J.L. & Pardee, A.B. (1991) Inducible
proteins binding to the murine thymidine kinase promoter in late
G1/S phase. Proc.NatlAcad.Sci.USA88, 1157–1161.
21. Dou,Q.P.,Zhao,S.,Levin,A.H.,Wang,J.,Helin,K.&Pardee,
A.B. (1994) G1/S regulated E2F-containing protein complexes
bind to the mouse thymidine kinase gene promoter. J. Biol. Chem.
269, 1306–1313.
22. Oswald, F., Dobner, T. & Lipp, M. (1996) The E2F transcription
factor activates a replication-dependent human H2A gene in early
Sphaseofthecellcycle.Mol. Cell. Biol. 16, 1889–1895.
23. Lee, H.H., Chiang, W.H., Chiang, S.H., Liu, Y.C., Hwang, J. &
Ng, S.Y. (1995) Regulation of cyclin D1, DNA topoisomerase I,
and proliferating cell nuclear antigen promoters during the cell
cycle. Gene Expr. 4, 95–109.
24. Lam,E.W.,Morris,J.D.,Davies,R.,Crook,T.,Watson,R.J.&
Vousden, K.H. (1994) HPV16 E7 oncoprotein deregulates B-myb
expression: correlation with targeting of p107/E2F complexes.
EMBO J. 13, 871–878.
25. Zwicker, J., Liu, N., Engeland, K., Lucibello, F.C. & Mu
¨
ller, R.
(1996) Cell cycle regulation of E2F site occupation in vivo. Science
271, 1595–1597.
26. Herber,B.,Truss,M.,Beato,M.&Mu
¨
ller, R. (1994) Inducible
regulatory elements in the human cyclin D1 promoter. Oncogene

9, 1295–1304.
27. Mu
¨
ller, H., Lukas, J., Schneider, A., Warthoe, P., Bartek, J.,
Eilers, M. & Strauss, M. (1994) Cyclin D1 expression is regulated
by the retinoblastoma protein. Proc. Natl Acad. Sci. USA 91,
2945–2949.
28. Botz, J., Zerfass Thome, K., Spitkovsky, D., Delius, H., Vogt, B.,
Eilers, M., Hatzigeorgiou, A. & Jansen Durr, P. (1996) Cell cycle
regulation of the murine cyclin E gene depends on an E2F binding
site in the promoter. Mol. Cell. Biol. 16, 3401–3409.
29. Henglein, B., Chenivesse, X., Wang, J., Eick, D. & Brechot, C.
(1994) Structure and cell cycle-regulated transcription of the
human cyclin A gene. Proc. Natl Acad. Sci. USA 91, 5490–5494.
30. Schulze, A., Zerfass, K., Spitkovsky, D., Middendorp, S., Berges,
J.,Helin,K.,JansenDurr,P.&Henglein,B.(1995)Cellcycle
regulation of the cyclin A gene promoter is mediated by a variant
E2F site. Proc. Natl Acad. Sci. USA 92, 11264–11268.
31. Dalton, S. (1992) Cell cycle regulation of the human cdc2 gene.
EMBO J. 11, 1797–1804.
32. Furukawa, Y., Terui, Y., Sakoe, K., Ohta, M. & Saito, M. (1994)
The role of cellular transcription factor E2F in the regulation of
cdc2 mRNA expression and cell cycle control of human hema-
topoietic cells. J. Biol. Chem. 269, 26249–26258.
33. Sherr, C.J. & Roberts, J.M. (1990) CDK inhibitors: positive and
negative regulators of G1-phase progression. Genes Dev. 13, 1501–
1512.
34.LaBaer,J.,Garrett,M.D.,Stevenson,L.F.,Slingerland,J.M.,
Sandhu, C., Chou, H.S., Fattaey, A. & Harlow, E. (1997) New
functional activities for the p21 family of CDK inhibitors. Genes

Dev. 11, 847–862.
35. Cheng, M., Olivier, P., Diehl, J.A., Fero, M., Roussel, M.F. &
Roberts, J.M. (1999) The p21 (Cip) and p27 (Kip) CDK inhibitors
are essential activators of cyclin D-dependent kinases in murine
firoblasts. EMBO J. 18, 1571–1583.
36. Sausville, E.A., Zaharevitz, D., Gusio, R., Meijer, L., Louarn-
Leost, M., Kunick, C., Schultz, R., Lahusen, T., Headlee, D. et al.
(1999) Cyclin-dependent kinases: initial aproaches to exploit a
novel therapeutic target. Pharmacol. Ther. 82, 285–292.
37. Senderowicz, A.M. (2000) Small molecule modulators of cyclin-
dependent kinases for cancer therapy. Oncogene 19, 6600–6606.
38. Van den Heuvel, S. & Harlow, E. (1993) Distinct roles for cyclin-
dependent kinases in cell cycle control. Science 262, 2050–2054.
39. Meijer, L., Borgne, A., Mulner, O., Chong, J.P., Blow, J.J.,
Inagaki, N., Iganaki, M., Delcros, J.G. & Moulinoux, J.P. (1997)
Biochemical and cellular effects of roscovitine, a potent and
selective inhibitor of the cyclin-dependent kinases cdc2, cdk2 and
cdk5. Eur. J. Biochem. 243, 527–536.
40. Park, D.S., Farinelli, S.E. & Greene, L.A. (1996) Inhibitors of
cyclin-dependent kinases promote survival of post-mitotic
neuronally differentiated PC12 cells and sympathetic neurons.
J. Biol. Chem. 271, 8161–8169.
41. Parker, B.W., Kaur, G., Nieves-Neira, W., Taimi, M., Kolhagen,
G., Shimizu, T., Losiewicz, M.D., Pommier, Y., Sausville, E.A. &
Senderowicz, A.M. (1998) Early induction of apoptosis in hema-
topoietic cell lines after exposure to flavopiridol. Blood 91,
458–465.
42. Buquet-Fagot, C., Lallemand, F., Montagne, M.N. & Mester, J.
(1997) Effects of olomucine, a selective inhibitor of cyclin-depen-
dent kinases, on cell cycle progression in human cancer cell lines.

Anticancer Drugs 8, 623–631.
43. Lee,H.R.,Chang,T.H.,Tebalt,M.J.III,Senderowicz,A.M.&
Szabo E. (1999) Induction of differentiation accompanies inhibi-
tion of cdk2 in non-small cell lung cancer cell line. Int. J. Oncol. 15,
161–166.
4820 S. Pen
˜
uelas et al. (Eur. J. Biochem. 270) Ó FEBS 2003
44. Rosania,G.R.,Merlie,J.Jr,Gray,N.,Chang,Y.T.,Shultz,P.G.
& Heald R. (1999) A cyclin-dependent kinase inhibitor inducing
cancer cell differentiation: biochemical identification using Xeno-
pus egg extracts. Proc. Natl Acad. Sci. USA 96, 4797–4802.
45. Liu, M., Subramanyam, Y.V. & Baskaran, N. (1999) Preparation
and analysis of cDNA from a small number of hematopoietic cells.
Methods Enzymol. 303,45–55.
46. Melillo, G., Sausville, E.A., Cloud, K., Lahusen, T., Varresio, L.
& Senderowicz, A. (1999) Flavopiridol, a protein kinase inhibitor,
down-regulates hypoxic induction of vascular endothelial growth
factor expression in human monocytes. Cancer Res. 59, 5433–
5437.
47. Gray, N.S., Wodicka, L., Thunnissen, A.M., Norman, T.C.,
Kwon,S.,Espinoza,F.H.,Morgan,D.O.,Barnes,G.,LeClerc,
S., Meijer, L. et al. (1998) Exploiting chemical libraries, structure,
and genomics in the search for kinase inhibitors. Science 281,
533–538.
48. Singh, P., Coe, J. & Hong, W. (1995) A role for retinoblastoma
protein in potentiating transcriptional activation by the gluco-
corticoid receptor. Nature 6, 562–565.
49. Chen, P.L., Riley, D.J., Chen, Y. & Lee, W.H. (1996) Retino-
blastoma protein positively regulates terminal adipocyte differ-

entiation through direct interaction with C/EBPs. Genes Dev. 10,
2794–2804.
50. Batsche, E., Muchardt, C., Behrens, J., Hurst, H.C. & Cremisi, C.
(1998) RB and c-Myc activate expression of the E-cadherin gene in
epithelial cells through interaction with transcription factor AP-2.
Mol. Cell. Biol. 18, 3647–3658.
51. Noe
´
, V., Alemany, C., Chasin, L.A. & Ciudad, C.J. (1998) Retino-
blastoma protein associates with SP1 and activates the hamster
dihydrofolate reductase promoter. Oncogene 16, 1931–1938.
52. Kim, S.J., Lee, H.D., Robbins, P.D., Busam, K., Sporn, M.B. &
Roberts, A.B. (1991) Regulation of transforming growth factor
beta 1 gene expression by the product of the retinoblastoma-
susceptibility gene. Proc. Natl Acad. Sci. USA 88, 3052–3056.
53. Udvadia, A.J., Rogers, K.T., Higgins, P.D., Murata, Y., Martin,
K.H., Humphrey, P.A. & Horowitz, J.M. (1993) Sp-1 binds pro-
moter elements regulated by the RB protein and Sp-1-mediated
transcription is stimulated by RB coexpression. Proc.NatlAcad.
Sci. USA 90, 3265–3269.
54. Kim, S.J., Wagner, S., Liu, F., O’Reilly, M.A., Robbins, P.D. &
Green, M.R. (1992) Retinoblastoma gene product activates
expression of the human TGF-beta 2 gene through transcription
factor ATF-2. Nature 358, 331–334.
55. Chen,L.I.,Nishinaka,T.,Kwan,K.,Kitabayashi,I.,Yokoyama,K.,
Fu,Y.H.,Grunwald,S.&Chiu,R.(1994)The retinoblastoma gene
product RB stimulates Sp1-mediated transcription by liberating
Sp1 from a negative regulator. Mol. Cell. Biol. 14, 4380–4389.
56. Leggett, R.W., Armstrong, S.A., Barry, D. & Mueller, C.R. (1995)
Sp1 is phosphorylated and its DNA binding activity down-regu-

lated upon terminal differentiation of the liver. J. Biol. Chem. 27,
25879–25884.
57. Vin
˜
als,F.,Fandos,C.,Santalucia,T.,Ferre,J.,Testar,X.,Palacı
´
n,
M. & Zorzano, A. (1997) Myogenesis and MyoD down-regulate
Sp1. A mechanism for the repression of GLUT1 during muscle cell
differentiation. J. Biol. Chem. 20, 12913–12921.
58. Noe
´
,V.,Chen,C.,Alemany,C.,Nicolas,M.,Caragol,I.,Chasin,
L.A. & Ciudad, C.J. (1997) Cell-growth regulation of the hamster
dihydrofolate reductase gene promoter by transcription factor
Sp1. Eur. J. Biochem. 249, 13–20.
59. Black, A.R., Jensen, D., Lin, S.Y. & Azizkhan, J.C. (1999)
Growth/cell cycle regulation of Sp1 phosphorylation. J. Biol.
Chem. 15, 1207–1215.
60. Saffer, J.D., Jackson, S.P. & Annarella, M.B. (1991) Develop-
mental expression of Sp1 in the mouse. Mol. Cell. Biol. 4, 2189–
2199.
61. Wang, C.H., Tsao, Y.P., Chen, H.J., Chen, H.L., Wang, H.W. &
Chen, S.L. (2000) Transcriptional repression of p21 (Waf 1/Cip1/
Sdi1) gene by c-jun through Sp1 site. Biochem. Biophys. Res.
Commun. 2, 303–310.
62. Decesse, J.T., Medjkane, S., Datto, M.B. & Cremisi, C.E. (2001)
RB regulates transcription of the p21/WAF1/CIP1 gene. Onco-
gene 20, 962–971.
63. Fojas de Borja, P., Du Collins, N.K.P., Azizkhan-Cliffork, J. &

Mudryj, M. (2001) Cyclin A-CDK phosphorylates Sp1 and
enhances Sp1-mediated transcription. EMBO J. 20, 5737–5747.
64. Haidweger, E., Novy, M. & Rotheneder, H. (2001) Modulation
of Sp1 activity by a Cyclin A/CDK complex. J. Mol. Biol. 306,
201–212.
65. Nishinaka, T., Fu, Y.H., Chen, L.I., Yokoyama, K. & Chiu, R.
(1997) A unique cathepsin-like protease isolated from CV-1 cells is
involved in rapid degradation of retinoblastoma susceptibility
gene product, RB, and transcription factor Sp1. Biochem. Biophys.
Acta 1351, 274–286.
66. Fu, Y.H., Nichinaka, T., Yokoyama, K. & Chiu, R. (1998) A
retinoblastoma susceptibility gene product, RB, targeting protease
is regulated through the cell cycle. FEBS Lett. 421, 89–93.
67. Urlaub, G., McDowell, J. & Chasin, L.A. (1985) Use of fluores-
cence-activated cell sorter to isolate mutant mammalian cells
deficient in an internal protein, dihydrofolate reductase. Somat.
Cell Mol. Genet. 11, 71–77.
68. Urlaub, G., Mitchell, P.J., Kas, E., Funanage, V.L., Myoda, T.T.
& Hamlin, J.L. (1986) Effect of gamma rays at the dihydrofolate
reductase locus: deletions and inversions. Somat. Cell Mol. Genet.
12, 555–566.
69. Noe
´
, V., Ciudad, C.J. & Chasin, L.A. (1999) Effect of differential
polyadenylation and cell growth phase on dihydrofolate reductase
mRNA stability. J. Biol. Chem. 274, 27807–27814.
70. Ciudad, C.J., Morris, A.E., Jeng, C. & Chasin, L.A. (1992) Point
mutational analysis of the hamster dihydrofolate reductase mini-
mum promoter. J. Biol. Chem. 267, 3650–3656.
71. Bradford, M.M. (1976) A rapid and sensitive method for the

quantitation of microgram quantities of protein utilizing the
principle of protein-dye binding. Anal. Biochem. 72, 248–254.
72.Kraus,M.H.,Popescu,N.C.,Amsbaugh,S.C.&King,C.R.
(1987) Overexpression of the EGF receptor-related proto-onco-
gene erbB-2 in human mammary tumor cell lines by different
molecular mechanisms. EMBO J. 6, 605–610.
73. Wigler, M., Pellicer, A., Silverstein, S., Axel, A., Urlaub, G. &
Chasin, L.A. (1979) Transformation of the aprt locus in mam-
malian cells. Proc. Natl Acad. Sci. USA 76, 1373–1376.
74. Ciudad, C.J., Urlaub, G. & Chasin, L.A. (1988) Deletion analysis
of the Chinese Hamster dihydrofolate reductase gene promoter.
J. Biol. Chem. 263, 16274–16282.
75. Noe
´
, V., Alemany, C., Nicola
´
s, M. & Ciudad, C.J. (2001) Sp1
involvement in the 4b-phorbol 12-myristate 13-acetate (TPA)-
mediated increase in resistance to methotrexate in Chinese hamster
ovary cells. Eur. J. Biochem. 268, 3163–3173.
76. Meijer, L. (1996) Chemical inhibitors of cyclin-dependent kinases.
Cell Biol. 6, 303–307.
77. Kawakami,K.,Futami,H.,Takahara,J.&Yamaguchi,K.(1996)
UCN-01, 7-hydroxyl-staurosporine, inhibits kinase activity of
cyclin-dependent kinases and reduces the phosphorylation of
the retinoblastoma susceptibility gene product in A549
human lung cancer cell line. Biochem. Biophys. Res. Commun. 219,
778–783.
78. Shimizu, E., Zhao, M.R., Nkanishi, H., Yamamoto, A., Yoshida,
S.,Takada,M.,Ogura,T.&Sone,S.(1996)Differingeffectsof

staurosporine and UCN-01 on RB protein phosphorylation and
expression of lung cancer cell lines. Oncology 53, 494–504.
79. Hamel, P.A., Gill, R.M., Phillips, R.A. & Gallie, B.L. (1992)
Transcriptional repression of the E2-containing promoters EIIaE,
Ó FEBS 2003 Rb and Sp1 regulation by CDK inhibitors (Eur. J. Biochem. 270) 4821
c-myc, and RB1 by the product of the RB1 gene. Cell Biol. 12,
3431–3438.
80. Gill, R.M., Hamel, P.A., Jiang, Z., Zacksenhaus, E., Gallie, B.L.,
& Phillips, R.A. (1994) Characterization of the human RB1 pro-
moter and of elements involved in transcriptional regulation. Cell
Growth Differ. 5, 467–474.
81. Park, K., Choe, J., Osifchin, N.E., Templeton, D.J., Robbins,
P.D. & Kim, S J. (1994) The human retinoblastoma susceptibility
gene promoter is positively autoregulated by its own product.
J. Biol. Chem. 8, 6083–6088.
82. Sandig, V., Brand, K., Herwin, S., Lukas, J., Bartek, J. & Strauss,
M. (1997) Adenovirally transferred p16 INK4/CDKN2 and p53
genes cooperate to induce apoptotic tumor cell death. Nat. Med. 3,
313–319.
83.Plath,T.,Peters,M.,Detjen,K.,Welzel,M.,Marschall,Z.,
Radke, C., Wiedenmann, B. & Rosewicz, S. (2002) Overex-
pression of pRB in human pancreatic carcinoma cells: function in
chemotherapy-induced apoptosis. J. Natl Cancer Inst. 94, 12942.
4822 S. Pen
˜
uelas et al. (Eur. J. Biochem. 270) Ó FEBS 2003