1 Experimental Oncology and 2 Lymphoma Unit, Oncology Institute of Southern Switzerland, Bellinzona, Switzerland; 3 Cyclacel Ltd, Dundee, Scotland, UK; 4 Experimental Haematology, St Bartholomew's Hospital and The London Hospital, London, UK
*Correspondence to:Dr F. Bertoni, Experimental Oncology, Oncology Institute of Southern Switzerland, via Vincenzo Vela 6, 6500 Bellinzona, Switzerland. Tel: +41 (0)91 8200 367; Fax: +41 (0)91 8200 397; Email: frbertoni{at}mac.com
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Abstract |
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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 µM for REC, Granta-519 and JeKo-1 cells and 50 µM for NCEB-1 cells. CYC202 caused an accumulation of cells in the G2M 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.
Key words: apoptosis, cdk inhibitor, cyclin D1, lymphoma
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Introduction |
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CYC202 (Seliciclib, R-roscovitine) is a purine analogue that competes with ATP for its binding site on cdks. CYC202 is selective towards cdk2cyclin E, cdk7cyclin H and cdk9cyclin T1, followed by cdk2cyclin A and cdk1Cyclin B [68
]. CYC202 has cytotoxic activity against a range of human cancer cell lines, as well as in tumour xenograft models [7
]. Phase I clinical trials with an oral capsule formulation have been completed in patients with solid tumours [9
] 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.
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Materials and methods |
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MTT cytotoxicity assay and cell growth inhibition
Cells were seeded into 96-well plates according to doubling time and incubated overnight at 37 °C. 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 °C. 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 µl per well and incubated in the dark for 4 h at 37 °C. Cells were processed as described previously [14]. 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 x 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 °C 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).
TUNEL assay
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 °C 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 (Becton Dickinson).
Western blotting analysis
Cells were solubilized in lysis buffer [10 mM TrisHCl 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 µg) were fractionated by SDSPAGE using 815% 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 TrisHCl 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 G124326, PharMingen, San Diego, CA, USA), anti-cdk4 (clone H-22, Santa Cruz Biotechnology, CA, USA), anti-cyclin H (clone G3011, 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), anti-RNA polymerase II (clone 8WG16, Covance Research Groups, Berkeley, CA, USA), anti-RNA polymerase II phosphoserine 25 (clone H5, Covance Research Groups), XIAP (clone K4H, Santa Cruz), and anti--tubulin (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 anti-rabbit 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 manufacturer's instructions (Amersham Life Science). Equal loading of samples was confirmed by probing for
-tubulin.
Statistics
Groups of data were compared using a paired two-sample Student's t-test.
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Results |
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To investigate the observed G2M 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 G2M accumulation detected by flow cytometry in these cells.
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.
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Discussion |
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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 cdk2cyclin E, cdk7cyclin H and cdk9cyclin T complexes and with anticancer activity demonstrated in solid and haematological tumours [68
, 15
, 16
]. All the MCL cell lines treated showed sensitivity to the compound at doses that are achievable in patients [9
], and it was able to induce apoptosis and caused a slight accumulation of cells in the G2M 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 [17
]. 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 [32
], 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 3' IgH regulatory regions in MCL cells [34
]. 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 currently ongoing.
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Acknowledgements |
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Notes |
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Received for publication November 29, 2004. Revision received February 8, 2005. Accepted for publication February 8, 2005.
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References |
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