In vivo and ex vivo responses of CLL cells to purine analogs combined with alkylating agent.
BACKGROUND
The heterogeneity of chronic lymphocytic leukemia (CLL) is thought to be due to differences in the expression of factors that regulate apoptosis and cell cycle, giving rise to diverse apoptotic disturbances and tumor properties. Therefore, the primary goal in CLL treatment is to overcome resistance to apoptosis and efficiently trigger this process in leukemic cells.


METHODS
Mononuclear cells were obtained from the blood of CLL patients by Histopaque-1077 sedimentation. CLL cell samples from the blood of drug treated patients, (cladribine or fludarabine with cyclophosphamide; CC or FC), as well as the cell samples of untreated patients exposed to the used drug combinations (CM, FM) or mafosfamide alone for 48 h were fractionated into nuclear and cytoplasmic fractions or were lysed. DNA fragmentation was evaluated by agarose electrophoresis and also cytometrically as sub-G1 population. The expression of apoptosis related proteins and H1.2 histone translocation were evaluated in lysates and nuclear and cytoplasmic fractions, respectively with appropriate antibodies.


RESULTS
Cladribine (C) and fludarabine (F) combined with cyclophosphamide/mafosfamide in vivo, as well as ex vivo trigger apoptosis in CLL cells. These drug combinations (CC; FC/CM; FM) induce leukemic cell apoptosis confirmed by DNA fragmentation, sub-G1 cell number, down-regulation of anti-apoptotic proteins (Mcl-1, Bcl-2), and H1.2 histone translocation in comparison with appropriate control cells, however, to a different degree.


CONCLUSIONS
The kinetics and rate of drug-induced apoptosis in leukemic cells under ex vivo experiments differ between patients, mirroring the differences noticed during in vivo treatment. Individual model cell samples indicate comparable susceptibility to the used drug combinations under in vivo and ex vivo conditions.