Introduction

Chronic lymphocytic leukemia is the most frequent leukemia in the Western world, accounting for an estimated 8100 new cases annually in the United States.¹ Accumulation of CD5+ mature B-lymphocytes seems to be caused by defective programmed cell death, although a proliferating pool of cells might contribute to disease progression.^2,3

Chemotherapy with alkylating agents, nucleoside analogues or monoclonal antibodies (mAb) can induce remissions in CLL, but few patients are cured even with high-dose chemotherapy and stem cell support.^4,5,6 B-CLL cells are characterized by high expression of a variety of antiapoptotic proteins.⁷ Although the underlying defect in apoptosis remains undefined, antiapoptotic molecules of the Bcl-2 family including BCL-2 and MCL-1 seem to be involved.^8,9 Higher levels of the antiapoptotic protein MCL-1⁷ and increased ratios of Bcl-2 or Mcl-1 relative to the proapoptotic antagonist Bax have been correlated with refractory disease.^10,11 Recently, it has been recognized that B-CLL consists of at least two different biologic subgroups, characterized by the presence or absence of somatic mutations in specific immunoglobulin heavy-chain variable region genes.¹² With the use of comparative gene expression profiling, the tyrosine kinase zap-70 has been found to be selectively expressed in the unmutated group with a poor outcome¹³ and has been identified as a powerful prognostic factor.¹⁴ New drugs should therefore be tested in both biological subgroups.

In the peripheral blood, B-CLL cells express high levels of the cyclin-dependent kinase (cdk) inhibitor p27 and are arrested in the G0 phase of the cell cycle.¹⁵ Surprisingly, these resting cells have been shown to be susceptible to Flavopiridol, a cell cycle inhibitor, although Flavopiridol was equally cytotoxic towards normal human lymphocytes.¹⁶ Flavopiridol and 7-hydroxy-staurosporine induced apoptosis and downregulated antiapoptotic proteins in B-CLL cells.¹⁷ Clinical trials of Flavopiridol in B-CLL are currently ongoing and modest activity as a single agent has been reported in mantle-cell lymphoma.¹⁸

Roscovitine is a purine analog that competes with ATP for its binding site on CDKs^19,20 and has demonstrated antitumor activity in a variety of cancer cell lines.^21,22 While Roscovitine is quite specific for cdk2 inhibition at doses up to 10 M, effects on other kinases (like cdk 1 and cdk7) have also been shown.²³ It has been reported that Roscovitine does not only cause cell cycle arrest but also apoptosis in cancer cells,^21,22 and the induction of cell death occurred from all compartments of the cell cycle.²⁴ Nuclear accumulation of p53 was reported to be involved in apoptosis induction, although the precise mechanism remains to be determined.^25,26

Recently, Cyc202 (R-Roscovitine) has been demonstrated to be highly effective in a panel of 19 human tumor cell lines and a human tumor xenograft model. This drug is currently entering clinical phase I studies.²⁴

In the present study, we have demonstrated that Roscovitine induced apoptosis in a dose-dependent manner in B-CLL cells that was accompanied by caspase activation and modulation of Bcl-2 family proteins. Importantly, Roscovitine was not capable of inducing cell death in normal mononuclear cells or B cells at doses that were toxic for B-CLL cells. Therefore, our results suggest a possible therapeutic role for Roscovitine in the treatment of patients with B-CLL.

Materials and methods

Cell samples

After informed consent, peripheral blood was obtained from patients with a diagnosis of B-CLL according to clinical and immunophenotypic criteria. Patients were either untreated or had not received chemotherapy for a period of at least 2 months prior to investigation. At the time of analysis, all patients were clinically stable, free from infectious complications and undergoing routine clinical outpatient review. Cells were immediately purified as described below and either cultured or carefully frozen using standard procedures. Frozen samples were used after careful thawing and after Ficoll–Hypaque (Biochrom, Berlin, Germany) density-gradient centrifugation, resulting in a viability of more than 95%.

Reagents and antibodies

Ab specific for Bax, Bak and retinoblastoma (RB) were purchased from BD-Pharmingen (San Diego, CA, USA); MCL-1 and p53 were from Santa Cruz Biotechnology (CA, USA); XIAP, Bcl-2 and zap-70 were from BD Biosciences Transduction lab (San Diego, CA, USA); and mAb specific for actin was obtained from Sigma (Deisenhofen, Germany). Mouse monoclonal anti-Bax antibody (clone YTH-6A7) was from Trevigen, Gaithersburg, MD, USA). Roscovitine was obtained from Sigma. The pancaspase inhibitor zVAD.fmk was purchased from Calbiochem (Schwalbach, Germany).

Separation procedures

Enrichment of B-CLL cells by density-gradient centrifugation over a Ficoll–Hypaque layer (Biochrom, Berlin, Germany) and immunomagnetic depletion of T-lymphocytes and monocytes has been described.²⁷ Peripheral blood mononuclear cells (PBMNC) from normal controls were used after Ficoll–Hypaque density-gradient centrifugation without further separation procedures. B cells from normal controls were separated by positive selection using CD19-coated magnetic beads (Dynal Biotech, Oslo, Norway) and Detachabead (Dynal) according to the manufacturer's instructions to a purity of greater than 97%.

Culture conditions

Purified leukemic B cells, B cells from normal controls or PBMNC were cultured in RPMI 1640 medium (Biochrom) supplemented with 10% fetal calf serum (Biochrom), penicillin/streptomycin 50 IU/ml, Na-pyruvate 1 mM, L-glutamine 2 mM, L-asparagine 20 g/ml, 2-mercaptoethanol 0.05 mM, HEPES 10 mM and MEM nonessential amino acids 0.7 (Biochrom) at 37°C and 5% CO₂ in a fully humidified atmosphere.

Analysis of fragmented DNA and of membrane flipping

The amount of fragmented DNA was assessed by a TdT-mediated dUTP nick-end labeling (TUNEL) assay as reported.²⁸ Incorporation of fluorescein-labeled dUTP into DNA strand breaks was detected by flow cytometry. Exposure of phosphatidylserine (PS) on the outside of the plasma membrane was analyzed by flow cytometry as described.²⁸ In brief, cells were stained with FITC-labeled mAbs to Annexin V, and counterstained with propidium iodide (PI). The numbers of viable cells (VC), identified as being negative for both Annexin and PI, varied from one sample to another due to spontaneous apoptosis in culture.

Investigation of mitochondrial membrane potential

The mitochondrial membrane potential, _m, was studied by flow cytometric analysis of the fluorescence of 3,3'-dihexyloxacarbocyanine iodide (DiOC6(3)) as reported.²⁸

Analysis of caspase-3 activity

Cells were fixed and permeabilized using Fix and Perm kit (Caltag, Burlingame, CA, USA) for 15 min at room temperature. Activated caspase-3 was detected by flow cytometry with an FITC-conjugated mAb that fails to recognize the inactive enzyme (clone C92-605, BD Biosciences, San Diego, CA, USA).

Immunoblotting

A total of 2–4 10⁷ cells were lysed as described previously²⁸ in lysis buffer (10 mM Tris-HCl (pH 7.4), 5 mM EDTA, 130 mM NaCl, 1% Triton, 1 mM phenylmethylsulfonyl fluoride, 1 mM Na₃VO₄ and 10 mg/ml of each phenantroline, aprotinin, leupeptin and pepstatin) for 20 min at 4°C. Lysates were spun at 12 000 rpm for 20 min and the supernatant was collected. Protein concentration was assessed by the Bio-Rad assay method (Bio-Rad Laboratories, Munich, Germany). Total extracts (50 g/lane) were subjected to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and blotting was performed on PVDF membranes (Immobilon-P, Millipore GmbH. Bedford, MA, USA). Blots were developed using SuperSignal^® chemoluminescent substrates from Pierce Chemical Company (KMF GmbH, St Augustin, Germany).

For determination of zap-70 expression, Western blots included total cellular extracts from a mantle cell lymphoma cell line (NCEB) and a T-cell lymphoma line (Jurkat) as a negative and positive control, respectively.

Detection of nuclear accumulation of p53

Cytosolic and nuclear protein fractions were prepared as described elsewhere.²⁹ In brief, cells were lysed in lysis buffer (150 mM HEPES, pH 7.9, 150 mM KCl, 150 mM sucrose, 150 mM MgCl₂, 150 mM DTT, NP-40 0.15%, 15 mM PMSF and 10 mg/ml of each phenantroline, aprotinin, leupeptin and pepstatin) and spun at 13 200 rpm for 5 s. Protein concentration of the supernatant containing cytosolic proteins was determined and subjected to 7.5% SDS-PAGE. The nuclear pellet was resolved in a buffer containing glycerol and sonicated. Protein concentration of the supernatant containing nuclear proteins was determined and Western blots were performed using 20 g of protein/per lane.

Mutational analysis of the p53 gene

Mutational analysis of the p53 gene was performed as described previously.³⁰ The highly conserved exons 5–8 of p53, including the intron/exon boundaries, were amplified by PCR. The quality and correct size of the PCR products were checked on agarose gels. Prior to sequencing, the PCR products were purified using the Quiaquick PCR Purification Kit (Quiagen).

Automated fluorescent sequencing in both directions was performed with the BigDye Terminator Cycle Sequencing kit (PE Applied Biosystems, Foster City, CA, USA) in an ABI Prism 377 automated sequencer (PE Applied Biosystems). Comparison with the germline sequences was performed with DNASIS 2.6 software (Hitachi Engineering).

Results

Roscovitine induces apoptosis in B-CLL cells in a dose-dependent manner

The effect of Roscovitine was investigated in purified B-CLL cells. Viability was determined after 24 h in vitro culture using the Annexin-V/PI assay as described in Materials and methods. One representative Annexin-V/PI stain of B-CLL cells cultured in medium alone or in the presence of increasing doses of Roscovitine is shown in Figure 1a. Figure 1b shows the means.e.m. (standard error of the mean) of VCs of 17 independent samples cultured in medium or Roscovitine, as indicated. Although Roscovitine significantly reduced the amount of VCs at 10 M, the effect was much more pronounced at doses of 20 M with less than 10% VCs in most samples when cells were treated with Roscovitine 50 M. Control experiments demonstrated that the amount of DMSO needed to dissolve Roscovitine did not induce apoptosis in B-CLL cells (data not shown).

Figure 1.

Dose-dependent induction of apoptosis in B-CLL cells by Roscovitine. Purified B-CLL cells were cultured with Roscovitine as indicated. Viability was assessed after 24 h of in vitro culture. One representative Annexin/PI staining is shown in (a). Cells negative for Annexin V and PI represent VCs. Viability of 17 independent B-CLL samples cultured in medium alone or with Roscovitine at 10, 20 and 50 M is presented as means.e.m. in (b).

Full figure and legend (101K)

Roscovitine toxicity in PBMNC and normal B cells

In contrast to B-CLL cells, apoptosis was not induced in PBMNC cultured in the presence of 10 M Roscovitine and only minor effects on cell viability were observed in the presence of 20 M Roscovitine. However, profound induction of cell death was detected in the presence of the highest concentration. One representative experiment out of three performed is depicted in Figure 2a. To further compare whether normal B cells were as sensitive towards Roscovitine treatment as B-CLL cells, we cultured highly purified B cells with Roscovitine at the indicated concentrations. Similar to PBMNC, only minor effects were observed with Roscovitine at 10 or 20 M, but cell death was strongly induced in the presence of Roscovitine at 50 M. One experiment is shown in Figure 2b with similar results in two additional samples. To rule out that differences between normal B cell and B-CLL cells were caused by differences in the separation method (positive and negative separation, respectively), we performed Annexin-V/PI assays in positively and negatively separated B-CLL cells and found no differences in apoptosis induction (data not shown).

Figure 2.

Effect of Roscovitine (Ros) in normal lymphocytes: PBMNC and purified normal B cells were cultured with or without Roscovitine as indicated. Representative Annexin/PI stains of PBMNC (a) and purified B cells (b) are shown. Experiments with PBMNC and purified B cells were repeated twice with similar results.

Full figure and legend (141K)

Roscovitine selectively kills B-CLL cells at 20 M: no correlation with zap-70 expression

We analyzed the effect of Roscovitine at 20 M in a total of 28 patient samples. Apoptosis was potently induced in 21 of 28 samples tested, while the response was only moderate in the remaining seven samples (<20% absolute reduction in VCs) (Table 1). All but three samples were cultured immediately after purification. The remaining samples were used after careful thawing and Ficoll–Hypaque density-gradient centrifugation. Additional experiments confirmed that Roscovitine was equally effective in fresh and frozen cells from individual samples (data not shown). Importantly, the difference between apoptosis induction (% absolute reduction) in B-CLL cells was significantly different (P=0.003) from apoptosis induction in nine different PBMNC samples, demonstrating that Roscovitine at 20 M selectively kills B-CLL cells (Figure 3).

Figure 3.

Toxicity of Roscovitine at 20 M in B-CLL cells and PBMNC: purified B-CLL cells from 28 independent samples and PBMNC from nine normal controls were cultured for 24 h with or without Roscovitine at 20 M. The percentage of VCss.e.m. is shown.

Full figure and legend (75K)

Table 1 - Patient characteristics.

Full table

B-CLL cells consist of at least two subgroups with different biology and prognosis, which can be easily separated by expression of the tyrosine kinase zap-70.¹⁴ Zap-70 expression was analyzed in 20 of 28 samples by Western blotting after T-cell depletion. Jurkat cells and NCEB cells (a mantle cell lymphoma cell line) were used as positive and negative controls, respectively (data not shown). No differences in terms of apoptosis induction were observed in 11 zap-70-negative samples as opposed to nine zap-70-positive samples when cells were exposed to 20 M Roscovitine (P=0.6) (Figure 4).

Figure 4.

Toxicity of Roscovitine in zap-70-positive and -negative B-CLL samples: zap-70 expression in B-CLL cells was analyzed by Western blot in purified B-CLL cells (data not shown). Zap-70-positive (n=9) and zap-70-negative (n=11) samples were cultured with or without Roscovitine at 20 M. Results are depicted as means.e.m.

Full figure and legend (77K)

Induction of apoptosis by Roscovitine is caspase dependent

Cell death in B-CLL cells might be executed by different mechanisms. To investigate whether Roscovitine-induced cell death was dependent on caspase activity, we cultured B-CLL cells for 24 h with or without Roscovitine 20 M in the presence or absence of the pancaspase inhibitor zVAD.fmk. Treatment with zVAD.fmk resulted in a small increase in VCs in cells cultured in medium alone. Inhibition of caspase activity resulted in a complete disappearance of DNA strand brakes in a TUNEL assay. Caspase-3 cleavage, which occurred to a small extent in medium cultured cells, was also inhibited (Figure 5).

Figure 5.

Roscovitine-induced apoptosis in B-CLL cells involves loss of the mitochondrial membrane potential and caspase activation. CLL cells were cultured for 24 h in the presence of Roscovtine 20 M or medium with or without the pancaspase inhibitor zVAD.fmk. After staining with fluorescent compounds, cells were analyzed by flow cytometry. Cells were stained for Annexin V and PI and DNA strand breaks were assessed with a TUNEL assay. For analysis of the mitochondrial membrane potential _m, cells were incubated with DiOC6(3) for 30 min. Intracytoplasmatic staining with an mAb against active caspase-3 was performed. The results from one representative experiment B-CLL are displayed. One additional experiment gave similar results.

Full figure and legend (159K)

Treatment with Roscovitine resulted in a decrease of VCs, which was accompanied by a loss in the mitochondrial membrane potential. In addition, DNA fragmentation and active caspase-3 strongly increased upon treatment with Roscovitine. Cotreatment with zVAD.fmk prevented PS externalization, DNA fragmentation and appearance of active caspase-3. The mitochondrial membrane potential was partially restored in the presence of zVAD.fmk, suggesting that caspase-dependent and -independent pathways are involved in the perturbation of mitochondria (Figure 5).

Roscovitine modulates expression of pro- and antiapoptotic proteins without evidence of increased nuclear accumulation of p53

Western blot analyses were performed in total cellular lysates of purified B-CLL cells cultured for 24 and 48 h with or without Roscovitine 20 M. The antiapoptotic protein MCL-1 was downregulated in cells cultured in medium alone as compared to MCL-1 expression in freshly isolated B-CLL cells. However, the expression of MCL-1 was further reduced in the presence of Roscovitine, and MCL-1 was no longer detectable after 48 h of culture. While the expression of bcl-2 remained unchanged, XIAP was also downregulated in response to Roscovitine. Expression of the antiapototic proteins bcl-xl and survivin was not analyzed because these proteins are not expressed in resting B-CLL cells.^31,7

Bak and Bax are proapoptotic members of the Bcl-2 family and the expression of Bak was increased in Roscovitine-treated cells, while the expression of full-length Bax remained unchanged. However, an 18 kDa cleavage product of Bax became detectable in Roscovitine-treated cells. Staining with mAk specific for actin confirmed that the observed differences in protein expression were not due to differences in the amount of protein per lane. The results of one experiment are shown in Figure 6a, and similar results were obtained in an additional patient sample. Intracellular flow cytometry with a Bax antibody directed against the NH2-terminal region of Bax (clone YTH-6A7) was performed to detect Bax conformational changes. This region is not available for binding by Bax NH2-terminal epitope-specific antibodies in intact cells. A strong increase of fluorescence intensity was noted in Roscovitine-treated cells as compared to medium controls. Figure 6b shows one experiment, and similar results were obtained in an independent sample.

Figure 6.

Roscovitine modulates the expression and conformation of pro- and antiapoptotic proteins. (a) Purified B-CLL cells were cultured in the presence or absence of Roscovitine at 20 M for 24 and 48 h, respectively. MCL-1 was revealed by immunoblotting with mAb in total cell lysates (50 M). The same membrane was sequentially stripped and probed with anti-XIAP, anti-Bcl-2, anti-Bak and anti-Bax. Protein concentrations were normalized by the Bio-Rad assay method and blots were reprobed with antiactin antibody to control for equal protein loading. In two independent experiments, intracellular FACS staining was performed with the conformation-specific Bax antibody YTH-6A7 (shaded histogram) and a corresponding isotype control. One experiment is shown in (b); the other one gave very similar results.

Full figure and legend (165K)

To analyze whether Roscovitine induced nuclear accumulation of p53 in B-CLL cells, cytosolic and nuclear fractions of proteins were prepared as described in Materials and methods. Western blotting revealed that p53 showed a nuclear accumulation in B-CLL cells cultured in medium alone that was not increased in the presence of Roscovitine. Successful separation of nuclear and cytosolic protein fractions was confirmed by staining with mAK against the RB protein (Figure 7). A mutational analysis of the p53 gene demonstrated the presence of wild-type p53 in the sample shown. One additional experiment gave similar results.

Figure 7.

Roscovitine treatment does not result in nuclear p53 accumulation. Nuclear (n) and cytosolic (c) protein fractions were prepared as described in Materials and methods. P53 was revealed by immunoblotting. The same membrane was sequentially stripped and reprobed with RB antibody to control for the successful separation of nuclear and cytosolic fractions, respectively. Cells were cultured in medium alone (a) or in Roscovitine at 20 M (b).

Full figure and legend (91K)

Discussion

Cyclin-dependent kinase inhibitors are a new class of anticancer agents that are extensively analyzed in vitro and are in early clinical trials in patients with non-Hodgkin's lymphoma.^32,33 Although B-CLL is characterized by the accumulation of small resting malignant B cells in the peripheral blood, two cdk inhibitors, Flavopiridol and UCN-01, have been shown to induce apoptosis in vitro in these G0 arrested cells.^16,17 The relevance of these findings has been questioned because Flavopiridol was equally cytotoxic towards mononuclear cells from normal controls.¹⁶

Roscovitine, an olomoucine-related purine has been developed as a cdk inhibitor that competes with ATP for its binding site on CDKs.^19,20 While Roscovitine was thought to inhibit cdk (especially cdk2) activity specifically, Roscovitine treatment was not only accompanied by cell cycle blockade but also induced apoptosis from all compartments of the cell cycle in different tumor models.^24,26

In our present study, we show for the first time that the cdk inhibitor Roscovitine induces apoptosis in B-CLL cells in a dose-dependent manner. Importantly, Roscovitine was much more cytotoxic at 20 M in B-CLL cells than in mononuclear cells from normal donors (Figure 3). It is important from a clinical point of view that Roscovitine was equally effective in B-CLL cells expressing the tyrosine kinase zap-70, which is a powerful prognostic factor and separates two biologically distinct disease entities,¹⁴ and in zap-70 negative cells (Figure 4).

Experiments with purified normal B cells demonstrated that this was not a B-cell-specific effect as the B-cell viability of control cells was only reduced in the presence of 50 M Roscovitine.

Cell death in response to chemotherapeutic agents usually involves the mitochondrial pathway, which is associated with a permanent loss of the mitochondrial membrane potential. The release of proapoptotic factors activates effector caspases, which cause DNA fragmentation and apoptosis.³⁴ However, caspase-independent cell death has also been described in different tumor models and B-CLL.^34,35,36 Inhibition of caspase activity using the pancaspase inhibitor zVAD.fmk inhibited Roscovitine-induced DNA fragmentation, cleavage of caspase-3 and restored viability to baseline values, suggesting that Roscovitine-induced cell death depends on caspase activity. Roscovitine-induced apoptosis was also associated with a loss of the mitochondrial membrane potential, which was only partially restored upon treatment with the caspase inhibitor. These data indicate that the loss of the mitochondrial membrane potential was at least in part independent of caspase activity (Figure 5).

BCL-2 family proteins play a central role in controlling the mitochondrial pathway.³⁷ The antiapoptotic proteins BCL-2 and MCL-1 have been implicated in B-CLL pathophysiology,^8,9 and high expression seems to be associated with chemotherapy resistance.^10,11 While Bcl-2 levels remained unchanged in Roscovitine-treated cells, MCL-1 expression was markedly reduced. Similar results were reported in B-CLL cells and myeloma cells that were exposed to the cdk inhibitors UCN01 and Flavopiridol.^17,38 While expression levels of the proapoptotic BCL-2 family members Bax and Bak were not altered in Flavopiridol-treated cells,¹⁷ treatment with Roscovitine resulted in increased expression of Bak and the appearance of a prominent 18 kDa cleavage product of Bax (Figure 6). The appearance of an 18 kDa Bax product has been reported previously in apoptotic B-CLL cells^10,28 and has been demonstrated to be more potent in apoptosis induction than p21 Bax.³⁹

The presence of either Bax or Bak seems to be essential for apoptosis in most cell types.⁴⁰ Although both proteins are transcriptionally regulated, Bax and Bak are stringently controlled at the post-translational level and conformational changes have been reported to be necessary for apoptosis induction in experimental systems and B-CLL cells.^41,42,43,44 Bax translocation to the mitochondrial membrane involves a conformational change that exposes the NH2 terminus and the hydrophobic COOH terminus.⁴¹

The potency of Roscovitine to induce apoptosis in B-CLL cells might be further enhanced by its ability to downregulate XIAP expression as XIAP is a member of the inhibitors of caspase (IAP) family of proteins that inhibit the activity of executioner caspases.³⁴

Taken together, the effect of Roscovitine closely resembles the effect of Flavopiridol and UCN-01 with respect to MCL-1 and XIAP expression, and also affects the expression and conformation of the proapoptotic Bcl-2 family members Bax and Bak (Figure 6). As has been reported in Flavopiridol-treated B-CLL cells, Roscovitine-induced apoptosis was not dependent on nuclear p53 accumulation¹⁶ (Figure 7). Although cdk2 is expressed in resting B-CLL cells, no activity has been observed in in vitro kinase assays, making it unlikely that inhibition of cdk2 contributes to the observed effects in resting B-CLL cells.³¹ However, cdks are not the only targets of cdk inhibitors and interference with the mitogen activated protein (MAP)-kinase pathway has been recently reported in colon carcinoma cell lines treated with Roscovitine.^45,46 Cyc202 (R-Roscovitine) is currently in clinical phase I trials. It is readily orally bioavailable and high plasma levels in the range 10–20 M have been achieved with a well-tolerated dose of 2500 mg/day for 5 day q3 weeks. Vomiting, hypokalemia and skin rash were the dose-limiting toxicities at higher doses with no signs of significant hematotoxicity (Pierga JY et al. Proc of Am Soc Clin Oncol 2003; 22: 210 (abstract 840)).

B-CLL is still an incurable disease and new therapeutic strategies are needed. Roscovitine is an attractive candidate drug, given its potency to induce apoptosis in B-CLL cells but not in normal leukocytes. Further in vitro studies will deal with the effect of combination schedules of chemotherapeutic agents and Roscovitine on B-CLL cells.

References

1. Greenlee RT, Murray T, Bolden S, Wingo PA. Cancer statistics, 2000. CA Cancer J Clin 2000; 50: 7–33. | PubMed | ISI | ChemPort |
2. Caligaris-Cappio F, Hamblin TJ. B-cell chronic lymphocytic leukemia: a bird of a different feather. J Clin Oncol 1999; 17: 399–408. | PubMed | ISI | ChemPort |
3. Granziero L, Ghia P, Circosta P, Gottardi D, Strola G, Geuna M et al. Survivin is expressed on CD40 stimulation and interfaces proliferation and apoptosis in B-cell chronic lymphocytic leukemia. Blood 2001; 97: 2777–2783. | Article | PubMed | ISI | ChemPort |
4. Wendtner CM, Schmitt B, Wilhelm M, Dreger P, Montserrat E, Emmerich B et al. Redefining the therapeutic goals in chronic lymphocytic leukemia: towards an evidence-based, risk-adapted therapy. Ann Oncol 1999; 10: 505–509. | Article | PubMed |
5. Mavromatis B, Cheson BD. Monoclonal antibody therapy of chronic lymphocytic leukemia. J Clin Oncol 2003; 21: 1874–1881. | Article | PubMed | ISI | ChemPort |
6. Dreger P, Montserrat E. Autologous and allogeneic stem cell transplantation for chronic lymphocytic leukemia. Leukemia 2002; 16: 985–992. | Article | PubMed | ISI | ChemPort |
7. Kitada S, Andresen J, Akar S, Zapata JM, Takayama S, Krajewski S et al. Expression of apoptosis-regulating proteins in chronic lymphocytic leukemia: correlations with in vitro and in vivo chemoresponses. Blood 1998; 91: 3379–3389. | PubMed | ISI | ChemPort |
8. Schimmer AD, Munk-Pedersen I, Minden MD, Reed JC. Bcl-2 and apoptosis in chronic lymphocytic leukemia. Curr Treat Options Oncol 2003; 4: 211–218. | PubMed |
9. Pedersen IM, Kitada S, Leoni LM, Zapata JM, Karras JG, Tsukada N et al. Protection of CLL B cells by a follicular dendritic cell line is dependent on induction of Mcl-1. Blood 2002; 100: 1795–1801. | Article | PubMed | ISI | ChemPort |
10. Thomas A, El Rouby S, Reed JC, Krajewski S, Silber R, Potmesil M et al. Drug induced apoptosis in B-cell chronic lymphocytic leukemia: relationship between p53 gene. Oncogene 1996; 12: 1055–1062. | PubMed | ISI | ChemPort |
11. Bannerji R, Kitada S, Flinn IW, Pearson M, Young D, Reed JC et al. Apoptotic-regulatory complement-protecting protein expression in chronic lymphocytic leukemia: relationship to in vivo rituximab resistance. J Clin Oncol 2003; 21: 1466–1471. | Article | PubMed | ChemPort |
12. Hamblin TJ, Davis Z, Gardiner A, Oscier DG, Stevenson FK. Unmutated Ig V(H) genes are associated with a more aggressive form of chronic lymphocytic leukemia. Blood 1999; 94: 1848–1854. | PubMed | ISI | ChemPort |
13. Rosenwald A, Alizadeh AA, Widhopf G, Simon R, Davis RE, Yu X et al. Relation of gene expression phenotype to immunoglobulin mutation genotype in B cell chronic lymphocytic leukemia. J Exp Med 2001; 194: 1639–1647. | Article | PubMed | ISI | ChemPort |
14. Crespo M, Bosch F, Villamor N, Bellosillo B, Colomer D, Rozman M et al. ZAP-70 expression as a surrogate for immunglobulin-variable-region mutations in chronic lymphocytic leukemia. N Engl J Med 2003; 348: 1764–1765. | Article | PubMed | ISI | ChemPort |
15. Vrhovac R, Delmer A, Tang R, Marie JP, Zittuon R, Ajchenbaum-Cymbalista F. Prognostic significance of the cell cycle inhibitor p27 in chronic B-cell lymphocytic leukemia. Blood 1998; 91: 4694–4700. | PubMed | ISI | ChemPort |
16. Byrd JC, Shinn C, Waselenko JK, Fuchss EJ, Lehman TA, Nguyen PL et al. Flavopiridol induces apoptosis in chronic lymphocytic leukemia cells via activation of caspase 3 without evidence of bcl-2 modulation or dependence of functional p53. Blood 1998; 92: 3804–3816. | PubMed | ISI | ChemPort |
17. Kitada S, Zapata JM, Andreeff M, Reed JC. Protein kinase inhibitors flavopiridol and 7-hydroxy-staurosporine down-regulate antiapoptosis proteins in B-cell chronic lymphocytic leukemia. Blood 2000; 96: 393–397. | PubMed | ISI | ChemPort |
18. Kouroukis CT, Belch A, Crump M, Eisenhauer E, Gascoyne RD, Meyer R et al. National Cancer Institute of Canada. Flavopiridol in untreated or relapsed mantle-cell lymphoma: results of a phase II study of the National Cancer Institute of Canada Clinical Trials Group. J Clin Oncol 2003; 21: 1740–1745. | Article | PubMed | ISI | ChemPort |
19. Vesely J, Havlicek L, Strnad M, Blow JJ, Donella-Deana A, Pinna L et al. Inhibition of cyclin-dependent kinases by purine analogues. Eur J Biochem 1994; 224: 771–786. | Article | PubMed | ISI | ChemPort |
20. Meijer L, Borgne A, Mulner O, Chong JP, Blow JJ, Inagaki N 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. | Article | PubMed | ISI | ChemPort |
21. Mgbonyebi OP, Russo J, Russo IH. Roscovitine induces cell death and morphological changes indicative of apoptosis in MDA-MB-231 breast cancer cells. Cancer Res 1999; 15: 1903–1910.
22. Edamatsu H, Gau CL, Nemoto T, Guo L, Tamanoi F. Cdk inhibitors, Roscovitine and olomoucine, synergize with farnesyltransferase inhibitors (FTI) to induce efficient apoptosis of human cancer cell lines. Oncogene 2000; 19: 3059–3068. | Article | PubMed | ChemPort |
23. Bain J, McLauchlan H, Elliott M, Cohen P. The specificities of protein kinase inhibitors; an update. Biochem J 2003; 371: 199–204. | Article | PubMed | ISI | ChemPort |
24. McClue SJ, Blake D, Clarke R, Cowan A, Cummings L, Fischer PM 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. | Article | PubMed | ISI | ChemPort |
25. 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. | PubMed |
26. David-Pfeuty T. Potent inhibitors of cyclin-dependent kinase 2 induce nuclear accumulation of wild type p53 and nucleolar fragmentation in human untransformed and tumor derived cells. Oncogene 1998; 18: 7409–7422. | Article |
27. Decker T, Schneller F, Sparwasser T, Tretter T, Lipford GB, Wagner H et al. Immunostimulatory CpG-oligonucleotides cause proliferation, cytokine secretion and an immunogenic phenotype in B-CLL patients. Blood 2000; 95: 999–1006. | PubMed | ISI | ChemPort |
28. Bogner C, Schneller F, Hipp S, Ringshausen I, Peschel C, Decker T. Cycling B-CLL cells are highly susceptible to inhibition of the proteasome: involvement of p27, early D-type cyclins, Bax and caspase dependent and independent pathways. Exp Hematol 2003; 31: 218–225. | Article | PubMed | ISI | ChemPort |
29. Mackman N, Brand K, Edgington TS. Lipopolysaccharide-mediated transcriptional activation of the human tissue factor gene in THP-1 monocytic cells requires both activator protein 1 and nuclear factor kappa B binding sites. J Exp Med 1991; 174: 1517–1526. | Article | PubMed | ISI | ChemPort |
30. Quintanilla-Martinez L, Kremer M, Keller G, Nathrath M, Gamboa-Dominguez A, Meneses A et al. p53 mutations in nasal NK/T-cell lymphoma from Mexico: association with large cell morphology and advanced disease. Am J Pathol 2001; 159: 2095–2105. | PubMed | ISI | ChemPort |
31. Decker T, Hipp S, Ringshausen I, Bogner C, Oelsner M, Schneller F et al. Rapamycin-induced G1 arrest in cycling B-CLL cells is associated with reduced expression of cyclin D3, cyclin E, cyclin A, and survivin. Blood 2003; 101: 278–285. | Article | PubMed | ISI | ChemPort |
32. Senderowicz AM, Sausville EA. Preclinical and clinical development of cyclin-dependent kinase modulators. J Natl Cancer Inst 2000; 92: 376–387. | Article | PubMed | ChemPort |
33. Senderowicz AM. Development of cyclin-dependent kinase modulators as novel therapeutic approaches for hematological malignancies. Leukemia 2001; 15: 1–9. | Article | PubMed | ISI | ChemPort |
34. Herr I, Debatin KM. Cellular stress response and apoptosis in cancer therapy. Blood 2001; 98: 2603–2614. | Article | PubMed | ISI | ChemPort |
35. Pettitt AR, Cawley JC. Caspases influence the mode but not the extent of cell death induced by purine analogues in chronic lymphocytic leukaemia. Br J Haematol 2000; 109: 800–804. | Article | PubMed | ChemPort |
36. Bellosillo B, Villamor N, Lopez-Guilermo A, Marce S, Esteve J, Campo E et al. Complement-mediated cell death induced by rituximab in B-CLL lymphoproliferative disorders is mediated in vitro by a caspase-independent mechanism involving the generation of reactive oxygen species. Blood 2001; 98: 2771–2777. | Article | PubMed | ChemPort |
37. Cory S, Adams JM. The Bcl2 family: regulators of the cellular life-or-death switch. Nat Rev Cancer 2002; 2: 647–666. | Article | PubMed | ISI | ChemPort |
38. 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. | PubMed | ISI | ChemPort |
39. Wood DE, Newcomb EN. Cleavage of Bax enhances its cell death function. Exp Cell Res 2000; 256: 375–382. | Article | PubMed | ISI | ChemPort |
40. Bouillet P, Strasser A. BH3-only proteins – evolutionarily conserved pro-apoptotic Bcl-2 family members essential for initiating programmed cell death. J Cell Sci 2002; 115: 1567–1574. | PubMed | ISI | ChemPort |
41. Gross A, Jockel J, Wie MC, Korsmeyer SJ. Enforced dimerization of Bax results in its translocation, mitochondrial dysfunction and apoptosis. EMBO J 2000; 17: 878–3885.
42. Griffiths GJ, Dubrez L, Morgan CP, Jones NA, Whitehouse J, Corfe BM et al. Cell damage-induced conformational changes of the pro-apoptotic protein Bak in vivo precede the onset of apoptosis. J Cell Biol 1999; 144: 903–914. | Article | PubMed | ISI | ChemPort |
43. Dewson G, Snowden RT, Almond JB, Dyer MJS, Cohen GM. Conformational change and mitochondrial translocation of Bax accompany proteasome inhibitor-induced apoptosis of chronic lymphocytic leukemic cells. Oncogene 2002; 22: 2643–2654. | Article |
44. Bellosillo B, Villamor N, Lopez-Guilermo A, Marce S, Bosch F, Campo E et al. Spontaneous and drug-induced apoptosis is mediated by conformational changes of Bax and Bak in B-cell chronic lymphocytic leukemia. Blood 2002; 100: 1810–1816. | Article | PubMed | ISI | ChemPort |
45. Fischer PM, Gianella-Borradori A. CDK inhibitors in clinical development for the treatment of cancer. Expert Opin Invest Drugs 2003; 12: 955–970. | Article |
46. Whittaker SR, Poele RT, Walton MI. Gene expression of the cyclin-dependent kinase inhibitor CYC202 (R-roscovitine). Proc Am Assoc Cancer Res 2002; 43 (abstr. 3302).

Acknowledgements

This work was supported by a research grant from the Technical University of Munich (KKF H30-97) and a grant from the Deutsche Forschungsgemeinschaft DE 771.