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.