In vitro activity of cyclin-dependent kinase inhibitor CYC202 (Seliciclib, R-roscovitine) in mantle cell lymphomas†
In vitro activity of cyclin-dependent kinase inhibitor CYC202 (Seliciclib, R-roscovitine) in mantle cell lymphomas
K. Lacrima 2
A. Valentini 2
C. Lambertini 2
M. Taborelli 2
A. Rinaldi 2
E. Zucca 1
C. Catapano 2
F. Cavalli 1 2
A. Gianella-Borradori 0
D. E. MacCallum 0
F. Bertoni 2 3
0 Cyclacel Ltd , Dundee, Scotland , UK
1 Lymphoma Unit, Oncology Institute of Southern Switzerland , Bellinzona , Switzerland
2 Experimental Oncology
3 Experimental Haematology, St Bartholomew's Hospital and The London Hospital , London , UK
Background: Mantle cell lymphoma (MCL) has the worst prognosis of all B-cell lymphomas and has poor response to conventional therapy. It is characterized by the presence of a chromosomal translocation t(11:14) (q13;q32) which results in deregulated cyclin D1 expression. Since defects in cell cycle regulation and apoptosis are primary events in MCL, small-molecule inhibitors of cdks - cyclins may play an important role in the therapy of this disorder. CYC202 (Seliciclib, R-roscovitine; Cyclacel Ltd., Dundee, UK) is a purine analogue and a selective inhibitor of the cdk2 - cyclin E as well as cdk7 - cyclin H and cdk9 - cyclin T. Materials and methods: The activity of CYC202 was tested in four human MCL cell lines: REC, Granta-519, JeKo-1 and NCEB-1. The effect of CYC202 on the cell cycle and on apoptosis-, cell-cycle- and transcription-regulation-related proteins was assessed. Results: The IC50 was 25 mM for REC, Granta-519 and JeKo-1 cells and 50 mM for NCEB-1 cells. CYC202 caused an accumulation of cells in the G2 - M phase of the cell cycle and apoptosis. CYC202 caused down-regulation of cyclin D1 and Mcl-1 protein levels, possibly because of the inhibition of transcription elongation. Conclusions: Our data suggest that CYC202 is an active agent in MCL. The concomitant decrease of the phosphorylated and total forms of RNA polymerase II suggests that this could be the main mechanism mediating the biological effects of CYC202 in MCL cells. The drug might represent a new therapeutic agent in this lymphoma subtype.
Mantle cell lymphoma (MCL) accounts for approximately 8%
of all non-Hodgkins lymphomas . Despite being previously
considered a low-grade indolent lymphoma, it appears to have
the worst characteristics of both low- and high-grade
lymphomas, i.e. incurability and rapid growth . The median time to
progression and survival are the shortest among all lymphoma
subtypes . The initiating events of MCL are thought to be
caused by the t(11;14) (q13;q32) chromosomal translocation
that places the cyclin D1 gene under the regulation of the
immunoglobulin heavy chain (IgH) gene promoter. Additional
K. Lacrima and A. Valentini contributed equally to this work.
molecular abnormalities mainly involving genes that regulate
the cell cycle, such as loss of p27 protein expression, are also
common. Abnormalities regulating apoptosis pathways, have
also been identified, including overexpression of Bcl-2 .
Currently, there is no convincing evidence that any
conventional chemotherapy regimen is curative . Thus there is a
need for new therapeutic modalities that would selectively
target those pathways that are deregulated . The karyotypic
abnormality and the gene expression profile characteristic of
MCL and involving mainly proliferation and cell-cycle-related
genes make this disease an attractive candidate for therapeutic
agents targeted to the cell cycle and apoptosis.
CYC202 (Seliciclib, R-roscovitine) is a purine analogue
that competes with ATP for its binding site on cdks. CYC202
is selective towards cdk2 cyclin E, cdk7 cyclin H and
cdk9 cyclin T1, followed by cdk2 cyclin A and cdk1
Cyclin B [6 8]. CYC202 has cytotoxic activity against a range
of human cancer cell lines, as well as in tumour xenograft
models . Phase I clinical trials with an oral capsule
formulation have been completed in patients with solid tumours 
and phase II studies are ongoing for non-small-cell lung
cancer. The aim of this work is to assess antitumour activity of
CYC202 in MCL cells in vitro and to characterize the
mechanisms of action of CYC202 in this disease.
Materials and methods
Four established human MCL cell lines (NCEB-1, REC, Granta-519 and
JeKo-1) were used [10 13]. NCEB-1, JeKo-1 and REC cell lines were
cultured in RPMI-1640 (GIBCO Invitrogen, Basel, Switzerland), while
Granta-519 cells were cultured in Dulbeccos modified Eagles medium
(GIBCO Invitrogen). All media were supplemented with fetal calf serum
(10%), gentamycin (0.1%) and L-glutamine (1%). Classic and molecular
cytogenetic techniques were used to confirm the presence of the
rearrangement involving the cyclin D1 (11q13) and the IgH locus (14q32) in all
four cell lines (data not shown).
MTT cytotoxicity assay and cell growth inhibition
Cells were seeded into 96-well plates according to doubling time and
incubated overnight at 37 8C. CYC202 (Seliciclib, R-roscovitine; Cyclacel
Ltd., Dundee, UK) was dissolved in dimethyl sulphoxide (DMSO) and
serially diluted in tissue culture media, added to cells (in triplicate) and
incubated for 72 h at 37 8C. Control cells were treated with equal amounts
of DMSO. A 5 mg/ml stock solution of
3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) (Sigma, Buchs, Switzerland) was
prepared in cell media and filter sterilized. MTT solution was then added
at 50 ml per well and incubated in the dark for 4 h at 37 8C. Cells were
processed as described previously . Absorbance was read at 540 nm
on a Beckman Coulter-AD340 instrument.
For cell growth inhibition, cells were seeded in 24-well culture plates at
a density of 3 105 cells per well. After 24 h, CYC202 was added at the
corresponding IC50 concentration. Cell number and cell viability were
determined daily using a Coulter Counter (Beckman Coulter-Z2) and the
trypan blue dye exclusion test.
Cell cycle analysis
Cells were treated with DMSO or CYC202 added at the IC50
concentrations, harvested, washed once in phosphate-buffered saline (PBS) and
then fixed in 70% ethanol at 20 8C for at least 1 h. Cells were stained
with propidium iodide (PI 50 g/ml, Sigma) in PBS containing RNase-A
(75 kU/ml, Sigma) and analysed for DNA content using a FACScan flow
cytometer (Becton Dickinson, USA). The analysis of cell cycle and
apoptosis was performed using the ModFit LT (Verity Software House Inc.,
Topsham, ME, USA).
Cells were harvested and fixed in 4% paraformaldeyde for 45 min at room
temperature, after exposure to IC50 concentrations of CYC202 or DMSO
for 72 h. After rinsing with PBS, the cells were permeabilized in a solution
of 0.1% Triton X-100 and 0.1% sodium citrate for 2 min on ice. Samples
were washed with PBS and incubated in the TUNEL reaction mixture
(Boehringer Mannheim-Roche) for 1 h at 37 8C in the dark. After a final
wash with PBS samples were analysed using a FACScan flow cytometer
(Becton Dickinson, USA). The percentage of FITC-positive in the overall
cell population was determined using the Cell Quest software package
Western blotting analysis
Cells were solubilized in lysis buffer [10 mM Tris HCl pH 7.5, 144 mM
NaCl, 0.5% Nonidet P-40, 0.5% sodium dodecyl sulphate (SDS), 0.1%
aprotinin, 10 mg/ml leupeptin, 2 mM phenylmethylsulfonyl fluoride] and
sonicated for 10 s. The protein content in the different samples was
determined using the BCA protein assay (Pierce Chemical Co., Rockford, IL,
USA). Lysates (30 mg) were fractionated by SDS PAGE using 8 15%
polyacrylamide gels, based upon the expected molecular weight. The
resolved proteins were blotted to a nitrocellulose membrane by semi-dry
electric transfer, and the membranes were blocked for 1 h in TBS buffer
(20 mM Tris HCl pH 7.6, 137 mM NaCl) containing 5% blotting-grade
non-fat milk. Membranes were incubated with primary antibodies diluted
in milk with 0.1% Tween 20 overnight. The following antibodies were
used: anti-cyclin D1 (clone G124 326, PharMingen, San Diego, CA,
USA), anti-cdk4 (clone H-22, Santa Cruz Biotechnology, CA, USA),
anticyclin H (clone G301 1, PharMingen), anti-cdk7 (clone 17, PharMingen),
anti-cyclin B1 (SC-245, Santa Cruz Biotechnology), anti-Bcl-2 (clone
N-19, Santa Cruz Biotechnology) anti-bax (Cell Signaling Technology,
Beverly MA, USA), anti-Mcl-1 (clone-22, PharMingen), anti-PARP
(clone F21-852, PharMingen), anti-E2F1 (clone KH95, Santa Cruz),
antiRNA polymerase II (clone 8WG16, Covance Research Groups, Berkeley,
CA, USA), anti-RNA polymerase II phosphoserine 2 5 (clone H5,
Covance Research Groups), XIAP (clone K4H, Santa Cruz), and
anti-atubulin (Ab-1 Oncogene, Darmstadt, Germany). Membranes were washed
three times in TBS for 5 min each and then incubated in TBS containing
the appropriate horseradish peroxidase conjugated anti-mouse or
antirabbit secondary antibodies (Amersham Life Science, Arlington Heights,
IL, USA) for 1 h. The membranes were washed three times for 5 min each
in TBS with 0.1% Tween 20 and then processed for enhanced
chemiluminescence detection according to the manufacturers instructions
(Amersham Life Science). Equal loading of samples was confirmed by
probing for a-tubulin.
Groups of data were compared using a paired two-sample Students t-test.
The effect of CYC202 on viability and growth
Granta-519, NCEB-1, REC and JeKo-1 cells were assayed for
cell viability after treatment with increasing concentrations of
Figure 1. Cytotoxic effect of CYC202 on MCL cells after 72 h exposure
determined by MTT assay.
CYC202 using the MTT assay. The IC50 dose for each cell
lines was determined after incubation with the drug for 72 h.
CYC202 caused a dose-dependent decrease in cell viability in
the four MCL cell lines (Figure 1). An IC50 of 25 mM was
calculated for Granta-519, REC and JeKo-1, and 50 mM for
NCEB-1. The anti-proliferative effect of CYC202 was
evaluated by measuring the growth rates of MCL cells seeded at
low density and treated with a drug dose corresponding to the
IC50 for each cell line (Figure 2). Treatment with CYC202
caused a time-dependent inhibition of cell growth in
accordance with the cell viability assay.
CYC202-induced changes in the cell cycle
To determine the effects of CYC202 on the cell cycle profile
of MCL, the four cell lines were treated with CYC202 for
24 h and 48 h with the corresponding IC50 drug concentrations.
After 24 h of treatment with the IC50 dose of CYC202, an
accumulation of cells in the G2 M phase was detected in
Granta-519, NCEB-1 and REC-1 cells and by 48 h in JeKo-1
cells compared to controls (Figure 3). After 24 and 48 h of
treatment with CYC202, an increase of the sub-G1 peak,
indicative of apoptosis, was evident in all the cells with the
exception of Granta-519.
CYC202-induced apoptosis in MCL cells
The cell cycle profiles showed an increase in cells with
sub-G1 DNA content after CYC202 treatment, suggestive of
induction of apoptosis. To address this question directly, we
measured the induction of apoptosis using more sensitive
techniques, the TUNEL assay and western blotting with antibodies
specific for the cleaved form of PARP. Figure 4 shows the
TUNEL assay results after 72 h of treatment with CYC202;
apoptosis was present in all the cell lines, with the highest
percentage of apoptotic cells in JeKo-1 cells and the lowest in
Granta-519 cells. These data were confirmed by western
blotting for the detection of the cleaved form of PARP, which
showed an increase of cleaved PARP at 24 h and 48 h in all
cell lines (Figure 5). Induction of PARP cleavage was
particularly prominent in JeKo-1 cells after 24 h of drug treatment.
Expression of cell-cycle-related proteins
Owing to the perturbation of the cell cycle induced by
CYC202, we analysed by western blotting the expression of
proteins involved in the progression of the cell cycle in cells
exposed to IC50 doses of the drug. First, we analysed the
expression of cyclin D1 that regulates cdk4 activity and
controls progression through the G1 phase. Cyclin D1 is
constitutively expressed in MCL and it is believed to play a
continuing role in the growth of MCL cells. Cyclin D1 protein
was down-regulated after treatment with CYC202, most
dramatically in the NCEB-1 cells (Figure 6). No change was
observed in the cdk4 level.
CYC202 shows activity against cdk7 cyclin H in vitro.
Therefore we analysed the effect of CYC202 treatment on
the expression of cyclin H and cdk7 in the MCL cell lines. In
the Granta-519, NCEB-1 and JeKo-1 cell lines, cyclin H was
down-regulated after 48 h of exposure to the CYC202
(Figure 6). No change in the levels of cyclin H was seen in
REC cells. The expression of cdk7 did not change in any of
the cell lines.
To investigate the observed G2 M accumulation (Figure 3)
further, cells were treated with the corresponding IC50 dose of
CYC202 for 48 h and cyclin B1 protein expression was
analysed. Up-regulation of cyclin B1 levels was detected in
Granta-519, NCEB-1 and REC compared with the untreated
cells (Figure 6). No change in the expression of cyclin B1 was
detected in JeKo-1 cells, consistent with the reduced G2 M
accumulation detected by flow cytometry in these cells.
Figure 4. Effect of CYC202 on the induction of apoptosis in MCL cells.
Apoptotic cells were detected by TUNEL assay and flow cytometry after
48 h exposure to the IC50 dose of CYC202. Dotted line, negative control
incubated in the absence of terminal transferase; black line, untreated
control cells (DMSO only); grey line, cells treated with CYC202. The
percentage of FITC-positive cells in CYC202-treated samples is reported in each
Expression of apoptosis-related proteins
Flow cytometry, TUNEL assay and PARP cleavage western
blotting each demonstrated that CYC202 was able to induce
apoptosis in all MCL cells. Thus we examined the expression
level of proteins that regulate cell survival and apoptosis in
cells treated with the drug. No changes were observed in the
expression of the anti-apoptotic proteins Bcl-2 and XIAP
(BIRC4) or the pro-apoptotic protein Bax were observed
(Figure 7). The level of the anti-apoptotic protein Mcl-1 was
considerably down-regulated by treatment with CYC202 in all
four cell lines.
Expression of transcription regulatory proteins
To understand the mechanism of cyclin D1 and Mcl-1
downregulation we looked at the effects of CYC202 on the levels
of total and phosphorylated RNA polymerase II and of the
transcription factor E2F-1, which is known to regulate Mcl-1
expression negatively. Both RNA polymerase II and E2F1 can
be affected by cdk inhibitors. Phosphorylation of the
COOHterminal domain of RNA polymerase II is required for
transcription elongation and is regulated by cdk7 cyclin H and
cdk9 cyclin T. The levels of both phosphorylated RNA
polymerase II and total RNA polymerase II of the protein were
decreased after treatment with CYC202 in all the MCL cells
(Figure 8). E2F-1 was expressed in all four cell lines
(Figure 8). Its level was increased in NCEB-1.
Downregulation of E2F-1 was observed in Granta, Jeko-1 and REC.
MCL is an incurable disorder with a median overall survival
of < 2 years. The main genetic event underlying MCL
pathogenesis is the presence of the t(11;14) (q13;q32)
chromosomal translocation which causes deregulated expression of
In this study we have characterized the effects of the cdk
inhibitor CYC202 in human cell lines derived from MCL
patients. CY202 is a synthetic cdk inhibitor with most potent
activity against the cdk2 cyclin E, cdk7 cyclin H and cdk9
cyclin T complexes and with anticancer activity demonstrated
in solid and haematological tumours [6 8, 15, 16]. All the
MCL cell lines treated showed sensitivity to the compound at
doses that are achievable in patients , and it was able to
induce apoptosis and caused a slight accumulation of cells in
the G2 M phase of the cell cycle. We observed a reduction in
Mcl-1 and cyclin D1 levels after treatment with CYC202 in all
four cell lines. Mcl-1 is an anti-apoptotic protein, often
overexpressed in MCL . It is known to be down-regulated by
cdk inhibitors, such as flavopiridol and roscovitine/CYC202
[15, 18 25]. Other anti-apoptotic molecules (Bcl-2 and XIAP)
were not affected by CYC202 treatment. This corroborates the
notion that apoptosis induced by some cdk inhibitors might be
mediated mainly by changes in Mcl-1 levels and that this
can occur despite high levels of Bcl-2 [18, 23]. However,
the exact mechanism of apoptosis is not clear yet. Flavopiridol
and Roscovitine induce Mcl-1 down-regulation following
decreased transcription [23, 26 30]. Inhibition of cdk7 and
cdk9 would ultimately induce the down-regulation of the
transcription. An additional mechanism for Mcl-1 down-regulation
could be mediated by up-regulation of E2F-1, which directly
represses Mcl-1 expression [25, 31]. In our MCL model, only
NCEB-1 showed a moderate increase of E2F-1, whilst all the
cell lines had a marked reduction of both total and
phosphorylated forms of RNA polymerase II levels. E2F-1 might
contribute to the apoptotic effect of the drug but, since MCL cells
have constitutively high levels of E2F-1 , they might be
less sensitive to E2F-1-mediated apoptosis than other cell
types. The down-regulation of RNA polymerase II activity
and level with consequent inhibition of transcription
elongation seems to be the main mechanism for induction of
apoptosis in our MCL model. Indeed, we also observed the
down-regulation of cyclin D1. Both flavopiridol and CYC202
have been shown to decrease the level of cyclin D1 in other
cell types [23, 26, 29, 33]. However, our data in MCL are
very interesting. The cyclin D1 expression is constitutive in
MCL owing to the juxtaposition of the gene to the
immunoglobulin heavy-chain genes that are always transcriptionally
active in B cells. The ability of CYC202 to down-regulate
cyclin D1 expression in this context is very promising for
the treatment of MCL patients with the drug. RNA polymerase
II has recently been shown to be constitutively bound to both
cyclin D1 promoter and 30 IgH regulatory regions in MCL
cells . The ability of CYC202 to down-regulate RNA
polymerase II activity might explain its marked effect on
growth and viability on MCL cells.
In conclusion, our in vitro data indicate that CYC202 is an
active compound in MCL with the potential to improve the
outcome of patients with this disease. A phase II study that
will test the activity of CYC202 in patients with MCL is
We thank our colleagues Catherine Thieblemont (France) and
Eisaku Kondo (Japan) for providing two of the cell lines. This
work was partially supported by Swiss Clinical Cancer
1. Non-Hodgkin 's Lymphoma Classification Project. A clinical evaluation of the International Lymphoma Study Group classification of non-Hodgkin's lymphoma . Blood 1997 ; 89 : 3909 - 3918 .
2. Weisenburger DD , Armitage JO . Mantle cell lymphoma . In Hancock BW, Selby PJ , MacLennan K , Armitage JO (eds): Malignant Lymphoma . London: Arnold : 2000 ; 27 - 41 .
3. Bertoni F , Zucca E , Cotter FE. Molecular basis of mantle cell lymphoma . Br J Haematol 2004 ; 124 : 130 - 140 .
4. Barista I , Romaguera JE , Cabanillas F. Mantle-cell lymphoma. Lancet Oncol 2001 ; 2 : 141 - 148 .
5. Bertoni F , Ghielmini M , Cavalli F et al. Mantle cell lymphoma: new treatments targeted to the biology . Clin Lymphoma 2002 ; 3 : 90 - 96 .
6. Meijer L , Borgne A , Mulner O et al. Biochemical and cellular effects of roscovitine, a potent and selective inhibitor of the cyclin-dependent kinases cdc2, cdk2 and cdk5 . Eur J Biochem 1997 ; 243 : 527 - 536 .
7. McClue S J , Blake D , Clarke R et al. In vitro and in vivo antitumor properties of the cyclin dependent kinase inhibitor CYC202 (R-roscovitine) . Int J Cancer 2002 ; 102 : 463 - 468 .
8. Fischer P M , Gianella-Borradori A. CDK inhibitors in clinical development for the treatment of cancer . Expert Opin Investig Drugs 2003 ; 12 : 955 - 970 .
9. Pierga JY , Faivre, Faivre S et al. A phase I and pharmacokinetic (PK) trial of CYC202, a novel oral cyclin-dependent kinase (CDK) inhibitor, in patients (pts) with advanced solid tumors . Proc Am Soc Clin Oncol 2003 ; 22 : 210 .
10. Saltman DL , Cachia PG , Dewar AE et al. Characterization of a new non-Hodgkin's lymphoma cell line (NCEB-1) with a chromosomal (11:14) translocation [t(11:14) (q13;q32)] . Blood 1988 ; 72 : 2026 - 2030 .
11. Raynaud SD , Bekri S , Leroux D et al. Expanded range of 11q13 breakpoints with differing patterns of cyclin D1 expression in B-cell malignancies . Genes Chromosomes Cancer 1993 ; 8 : 80 - 87 .
12. Jeon HJ , Kim CW , Yoshino T et al. Establishment and characterization of a mantle cell lymphoma cell line . Br J Haematol 1998 ; 102 : 1323 - 1326 .
13. Jadayel DM , Lukas J , Nacheva E et al. Potential role for concurrent abnormalities of the cyclin D1, p16CDKN2 and p15CDKN2B genes in certain B cell non-Hodgkin's lymphomas . Functional studies in a cell line (Granta 519). Leukemia 1997 ; 11 : 64 - 72 .
14. Catapano CV , Carbone GM , Pisani F et al. Arrest of replication fork progression at sites of topoisomerase II-mediated DNA cleavage in human leukemia CEM cells incubated with VM-26 . Biochemistry 1997 ; 36 : 5739 - 5748 .
15. Hahntow IN , Schneller F , Oelsner M et al. Cyclin-dependent kinase inhibitor Roscovitine induces apoptosis in chronic lymphocytic leukemia cells . Leukemia 2004 ; 18 : 747 - 755 .
16. Decker T , Hipp S , Hahntow I et al. Expression of cyclin E in resting and activated B-chronic lymphocytic leukaemia cells: cyclin E/cdk2 as a potential therapeutic target . Br J Haematol 2004 ; 125 : 141 - 148 .
17. Khoury JD , Medeiros LJ , Rassidakis GZ et al. Expression of Mcl-1 in mantle cell lymphoma is associated with high-grade morphology, a high proliferative state, and p53 overexpression . J Pathol 2003 ; 199 : 90 - 97 .
18. Achenbach TV , Muller R , Slater EP . Bcl-2 independence of flavopiridol-induced apoptosis. Mitochondrial depolarization in the absence of cytochrome c release . J Biol Chem 2000 ; 275 : 32089 - 32097 .
19. Mohapatra S , Chu B , Wei S et al. Roscovitine inhibits STAT5 activity and induces apoptosis in the human leukemia virus type 1-transformed cell line MT-2. Cancer Res 2003 ; 63 : 8523 - 8530 .
20. Kim EH , Kim SU , Shin DY et al. Roscovitine sensitizes glioma cells to TRAIL-mediated apoptosis by downregulation of survivin and XIAP. Oncogene 2004 ; 23 : 446 - 456 .
21. MacCallum DE , Melville J , Watt K et al. CYC202 (R-Roscovitine) induces apoptosis in multiple myeloma cells by down regulation of Mcl-1 . Proc Am Assoc Cancer Res 2004 ; 45 : Abstr 823.
22. Wittmann S , Bali P , Donapaty S et al. Flavopiridol down-regulates antiapoptotic proteins and sensitizes human breast cancer cells to epothilone B-induced apoptosis . Cancer Res 2003 ; 63 : 93 - 99 .
23. Gojo I , Zhang B , Fenton RG . The cyclin-dependent kinase inhibitor flavopiridol induces apoptosis in multiple myeloma cells through transcriptional repression and down-regulation of Mcl-1. Clin. Cancer Res 2002 ; 8 : 3527 - 3538 .
24. Dai Y , Rahmani M , Grant S. Proteasome inhibitors potentiate leukemic cell apoptosis induced by the cyclin-dependent kinase inhibitor flavopiridol through a SAPK/JNK- and NF-kappaB-dependent process . Oncogene 2003 ; 22 : 7108 - 7122 .
25. Ma Y , Cress WD , Haura EB . Flavopiridol-induced apoptosis is mediated through up-regulation of E2F1 and repression of Mcl-1 . Mol Cancer Ther 2003 ; 2 : 73 - 81 .
26. Carlson B , Lahusen T , Singh S et al. Down-regulation of cyclin D1 by transcriptional repression in MCF-7 human breast carcinoma cells induced by flavopiridol . Cancer Res 1999 ; 59 : 4634 - 4641 .
27. Lam LT , Pickeral OK , Peng AC et al. Genomic-scale measurement of mRNA turnover and the mechanisms of action of the anti-cancer drug flavopiridol . Genome Biol 2001 ; 2 : RESEARCH0041 .
28. Ljungman M , Paulsen MT . The cyclin-dependent kinase inhibitor roscovitine inhibits RNA synthesis and triggers nuclear accumulation of p53 that is unmodified at Ser15 and Lys382 . Mol Pharmacol 2001 ; 60 : 785 - 789 .
29. Whittaker SR , Walton MI , Garrett MD et al. The cyclin-dependent kinase inhibitor CYC202 (R-Roscovitine) Inhibits retinoblastoma protein phosphorylation, causes loss of cyclin D1, and activates the mitogen-activated protein kinase pathway . Cancer Res 2004 ; 64 : 262 - 272 .
30. Monaco EA 3rd, Beaman-Hall CM , Mathur A et al. Roscovitine , olomoucine, purvalanol: inducers of apoptosis in maturing cerebellar granule neurons . Biochem Pharmacol 2004 ; 67 : 1947 - 1964 .
31. Jiang J , Matranga CB , Cai D et al. Flavopiridol-induced apoptosis during S phase requires E2F-1 and inhibition of cyclin A-dependent kinase activity . Cancer Res 2003 ; 63 : 7410 - 7422 .
32. Korz C , Pscherer A , Benner A et al. Evidence for distinct pathomechanisms in B-cell chronic lymphocytic leukemia and mantle cell lymphoma by quantitative expression analysis of cell cycle and apoptosis-associated genes . Blood 2002 ; 99 : 4554 - 4561 .
33. Yu C , Rahmani M , Dai Y et al. The lethal effects of pharmacological cyclin-dependent kinase inhibitors in human leukemia cells proceed through a phosphatidylinositol 3-kinase/Akt-dependent process . Cancer Res 2003 ; 63 : 1822 - 1833 .
34. Liu H , Wang J , Epner EM . Cyclin D1 activation in B-cell malignancy: association with changes in histone acetylation, DNA methylation, and RNA polymerase II binding to both promoter and distal sequences . Blood 2004 ; 104 : 2505 - 2513 .