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M. TERESA GÓMEZ-CASARES Y COLS. AN. R. ACAD. NAC. FARM.

medium (Invitrogen) containing 8% fetal calf serum and gentamycin
(80 mg/mL). For proliferation assays, exponentially growing cells
at a concentration of 250,000 cells per mL were treated with the
indicated concentrations of hydroxyurea (Sigma), IFNa (kindly
provided by Roche Farma, Madrid, Spain), busulfan (Sigma) and
imatinib (kindly provided by Novartis, Basel, Switzerland).

Cell proliferation and apoptosis assays

Cells were counted in a hemocytometer. When appropriate, cell
viability was scored with trypan blue vital staining. For thymidine
incorporation assays, cells were incubated with 1 µCi/mL of 3H-
thymidine for 2 h, harvested onto glass wool filters and the
radioactivity was counted by liquid scintillation. Each experiment was
repeated at least three times. Cells with apoptotic morphology were
analyzed by May-Grünwald-Giemsa staining of cytocentrifuge
preparations, and scored by light microscopy. Binding of annexin V
to cell surface was carried out by flow cytometry with annexin V-FITC
following the manufacturer’s instructions (Genzyme Diagnostics).

Northern analysis

RNAs were prepared by the guanidine thiocyanate method. RNAs
(15 µg of total RNA per lane) were separated by electrophoresis
through agarose-formaldehyde gel and transferred to nitrocellulose.
Probe labeling with [a-32P]dCTP and filter hybridization were carried
out according to standard procedures. Probe for human BCL2 was a
1.8 kb fragment from human cDNA.

RESULTS AND DISCUSSION

In order to investigate a possible effect of Bcl2 and BclX on the
antiproliferative activity of imatinib , we compared the growth rates
of K562 (parental cells), Kbcl2v and KbclX cells in response to
imatinib at two concentrations of the drug (0.5 µM and 2.5 µM).
The results are shown in Fig. 1A, and demonstrate that imatinib

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