R‐roscovitine (Seliciclib) affects CLL cells more strongly than combinations of fludarabine or cladribine with cyclophosphamide: Inhibition of CDK7 sensitizes leukemic cells to caspase‐dependent apoptosis

Abstract

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of malignant, apoptosis-resistant B CD19(+)/CD5(+) cells. Populations of CLL cells are heterogeneous and consist primarily of quiescent cells with a minor subset of dividing cells. In this study the efficacy of a first-line in vivo therapy was compared with treatment by R-roscovitine (ROSC) alone or by purine analogues (cladribine and fludarabine) combined with maphosphamide for 14 CLL patients under ex vivo conditions. ROSC induced the highest reduction in numbers of living B-cells, coinciding with an increased rate of apoptosis. After 24 h the percentage of apoptotic cells in ROSC-treated cultures was markedly higher than in untreated controls. ROSC also induced strong activation of the apoptosome and effector caspases in CLL cells. During progression of apoptosis the plasma membrane became permeable, resulting in the release of activated caspases into the culture medium. Leukemic cells were more sensitive to ROSC than normal mononuclear cells. Treatment with ROSC did not affect the activating phosphorylation of CDK2 or CDK1. However, ROSC decreased phosphorylation of survivin, CDK7, and RNA-Pol II, resulting in inhibition of transcription elongation and subsequent down-regulation of levels of anti-apoptotic factors, thereby facilitating apoptosis. Unlike ROSC, two other purine analogues barely affected the cellular levels of anti-apoptotic proteins and more weakly activated effector caspases. In addition, the efficacies of in vivo and ex vivo therapies were found to be correlated. Marked between-patient differences in expression patterns of apoptosis-regulating factors in CLL cells were observed, explaining the variations in patients' sensitivity to therapy.

Personalized therapy tests for the monitoring of chronic lymphocytic leukemia development

“…The leukemic PBMCs were subsequently incubated with anticancer agents. Cells were incubated with CM, FM, RCM, Rit or RK as described previously (25,26). Cladribine was supplemented in dose of 50 µg/ml, mafosfamide at 1 µg/ml, Rit at 20 µg/ml, fludarabine at 20 µM and kinetin riboside at 20 µM.…”

“…Following a 48 h incubation period, the control and drug-treated CLL cells were rinsed with PBS and homogenized in isotonic sucrose solution [5 mM MgCl 2 , 0.5% Triton X-100, 50 mM Tris-HCl (pH 7.4), protease inhibitors] as described previously (26). The homogenate was then centrifuged at 1,000 × g for 10 min to obtain the nuclear fraction.…”

“…The homogenate was then centrifuged at 1,000 × g for 10 min to obtain the nuclear fraction. The leukemic cells were subsequently lysed in a buffer containing 10 mM Tris-HCl (pH 7.5), 300 mM NaCl, 1% Triton X-100, 2 mM MgCl 2 , 0.1 M dithiothreitol and a mixture of protease inhibitors, as described previously (26). The protein content was measured by standard Lowry method.…”

Toward personalized therapy for chronic lymphocytic leukemia

“…Protein expression (Mcl-1, Bcl-2, procaspase-3 and Noxa) were analyzed using western blot technique as reported previously. 18,33 Total RNA was extracted from cells using RiboPure Blood Kit (Ambion) according to the manufacturer's instructions. cDNA was synthesized from total RNA using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) by priming with random hexamers (0.5 μg of RNA was transcribed to cDNA).…”

Interference with ER‐α enhances the therapeutic efficacy of the selective CDK inhibitor roscovitine towards ER‐positive breast cancer cells

“…Thus, the inhibition of CDK7 phosphorylation by ROSC results in a global transcriptional block [Bres et al, 2008;Core and Lis, 2008]. Abolishing the specific phosphorylation of CDK7 and RNA Pol II reduces cellular levels of important inhibitors of apoptosis and thus promotes programmed cell death in tumor cells [Hahntow et al, 2004;Alvi et al, 2005;Wesierska-Gadek et al, 2009a;Rogalinska et al, 2010]. These varied facets of responses to ROSC indicate that it has great potential in the clinical treatment of ER þ breast cancer.…”