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
Trang 1The 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 hyperphoshypophos-phorylated 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: cciudad@farmacia.far.ub.es
Abbreviations: APRT, adenine phosphoribosyltransferase; BSM-I,
bisindolylmaleimide I; CDK, cyclin-dependent kinase; CHO, Chinese
hamster ovary; DHFR, dihydrofolate reductase; IC 50 , 5 0% 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)
Trang 2CDK 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 (IC50) for CDK2 and
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 37C in a humidified 5% (v/v) CO2 -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 stained with 25 lgÆmL)1propidium 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 35mm-diameter dishes and, after several periods of incubation with 5· 10)8MUCN-01, they were collected in
500 lL of F-12 medium The cells were then centrifuged and washed with NaCl/Pi, and the final pellet was resuspended
in 11.25 lL of diethylpyrocarbonate-treated water The cells were lysed at 80C for 5min cDNA was synthesized in
20 lL of reaction mixture containing 125ng of random hexamers (Roche), 10 mM dithiothreitol, 20 U of RNasin (Promega), 0.5mM dNTPs (AppliChem), 4 lL of 5· RT buffer, 200 U of M-MLV reverse transcriptase (BRL-Gibco) and the cell lysate The reaction mixture was incubated at 37C 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 lL of 10· PCR buffer (Mg2+-free), 1.5mM
MgCl, 0.2 mM dNTPs, 2.5 lCi of [32P]dATP[aP]
Trang 3(3000 CiÆmmol)1; Amersham Ibe´rica), 1.5U 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 58–60C;
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 MgCl2, the dNTPs,
the [32P]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 94C Each cycle consisted of
dena-turation at 92C for 30 s; primer annealing at 59 C for
1 min for DHFR and Sp1, and at 55C for 1 min for Rb;
and primer extension at 72C 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-1D, 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 N2and stored at)80 C
Total extracts
Whole extracts were obtained from K1 or K562 cells
according to the method of Kraus et al [72] Cells were
collected in ice-cold F-12 medium and centrifuged at 1000 g
for 5min The cell pellet was gently resuspended in 5mL of
hypotonic buffer (15mM NaCl, 60 mM KCl, 0.5mM
EDTA, 1 mM phenylmethanesulfonyl fluoride, 1 mM
2-mercaptoethanol, 15mMTris/HCl, pH 8) After
centri-fugation (1000 g, 5min), the cell pellet was resuspended in
100 lL of a buffer containing deoxycholate (100 mMNaCl,
10 mM NaH2PO4, 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 4C
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 N2and stored at
)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 treated with UCN-01, 5· 10)8Mfor 48 h, were incubated with 50 lgÆmL)1cycloheximide 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:
TCC-3¢ (630 nt); Rbprm-rev, 5¢-cagtgctgcctcgagGACGC CTTTCGCGGCGGGAGC-3¢ (1 nt) The PCR product was sequenced using Big Dye v2.0 (PE Biosystems) After digestion with NheI and XhoI, the PCR fragment from the
Rb promoter was cloned into the NheI/XhoI sites of 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· 104 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
Trang 4FuGENE6 (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 5min The
mixture was added to 125ng of the Rb promoter
(prmRB-luc) or 250 ng of Sp1 promoter (prmSp1-(prmRB-luc) constructs
The DNA–lipid mixture was incubated at room
tempera-ture for 15min The mixtempera-ture 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 lL of the
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 lg of BPV-Neo, by using
the calcium phosphate method [73] After 24 h of
expres-sion, selection with Geneticin (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 [5 1]
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 nM) 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)8M(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)6M) (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 nM 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 (IC50 ¼ 7 · 10)9M) we investigated the different effects of UCN by using the PKC inhibitor, BSM-I (IC50 10)8M) In CHO cells,
10)6M BSM-I produced an increase in Rb expression that represented 40% of the increase caused by 50 nM
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)8MUCN-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 nM
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 nM 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)
Trang 5Therefore, 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 IC50 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 IC50 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 lg of p21 (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 (106cells 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-15antibody 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)6M 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-15antibody in CHO K1 cells.
Trang 6Effects 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 nM), roscovitine (100 nM) or olomoucine (500 nM) 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)8M 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 Effect of UCN-01 on Rb half-life CHO K1 cells (1000 cells per 35mm diameter dish) were incubated with 5· 10)8M UCN-01 for
48 h, followed by the addition of 50 lgÆmL)1cycloheximide 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.
Trang 7upon 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 nM, 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 nM 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 IC50values,
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 nM
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 nM 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 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-15antibody 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-15antibody 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.
Trang 8500 nM 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 (106cells 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 G3-245antibody 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 G3-245antibody (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.
Trang 9like 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
toward CDKs p21 was also used as an intrinsic CDK
inhibitor UCN-01 inhibits CDK4/6 and CDK2 with IC50
values of 0.032 lM and 0.030 lM, respectively However,
roscovitine and olomoucine have an IC50for CDK4/6 that
is 100-fold higher than for CDK2 (0.7 lMfor CDK2 and
> 100 lM for CDK4/6, for roscovitine, and 7 lM for
CDK2 and > 1000 lM for CDK4/6, for olomoucine,
respectively) [76,77] Thus, CDK2 can be selectively
inhi-bited by using low concentrations of these inhibitors,
according to their IC50values
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 nM, 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)6M), 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 IC50for this kinase (0.007 lM) 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 (125ng) or
prmSp1-LUC (250 ng) were transiently
transfected into K5 62 cells and assayed for
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
performed in duplicate, and the results
repre-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.
Trang 10UCN01 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 nM roscovitine and 500 nM olomoucine,
which increase Rb expression, do not inhibit PKC (IC50
values of roscovitine and olomoucine for PKC are
> 100 lMand > 1000 lM, 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)8M 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 (225000 cells per 35mm 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 (225000 cells per 35mm diameter dish) were co-transfected with 1 lg of the dhfr 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.