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

The retardation in growth rate of Kbcl2v and KbclX provoked
by imatinib could be explained by reduced cell proliferation or
by increased apoptosis. To investigate this point we determined the
DNA synthesis rate of DNA by the 3H-thymidine incorporation
method in the three cell lines treated with 0.5 µM and 2.5 µM
imatinib for 48 and 72 h. The results indicate that DNA synthesis
in parental K562 (as well as cells transfected with the empty vector,
KXLSN, not shown) was almost completely suppressed after 72 h
of treatment with 0.5 and 2.5 µM imatinib. Imatinib also decreased
the DNA synthesis in Kbcl2v and KbclX in a time- and dose-
dependent manner (Fig. 2A). However, DNA synthesis rate in Kbcl2v
and KbclX was not suppressed but dramatically reduced (20% and
30% respectively with respect to controls). These differences in DNA
synthesis were roughly consistent with the growth curves of the
distinct cell lines (Fig. 1A).

Next we compared the effect of imatinib with the other three
drugs used in CML treatment (hydroxyurea, busulfan and IFNa). We
chose drug concentrations slightly above the minimal cytostatic
concentrations for the three cell lines: 2000 UI of IFNa per mL, 0.5
mM hydroxyurea and 0.5 mM busulfan (data not shown). K562,
Kbcl2v and KbclX cells were treated with the drugs at the indicated
concentrations for 48 h and the 3H-thymidine incorporation was
measured. The results (Fig. 2B) indicate that neither Bcl-2 nor Bcl-
XL conferred resistance to the antiproliferative effect of busulfan,
IFNa and hydroxyurea in K562, in accordance with the absence of
cells showing apoptotic morphology (not shown).

Next we analysed whether Bcl2v and BclX were capable to
antagonize the apoptosis elicited by imatinib in K562. We measured
the apoptosis extent by determining the binding of annexin V to cell
surfaces. This protein binds to phosphatidyl serine, which is exposed
to the outer layer of plasma membrane during apoptosis. K562,
Kbcl2v and KbclX cells were treated with annexin V-FITC and
binding determined by flow cytometry. The results (Fig. 3A) indicate
that imatinib-mediated apoptosis was dramatically reduced in Kbcl2v
and KbclX. Similar result was observed by scoring cells with
apoptotic morphology after Giemsa staining of the cells. Cytospin
preparations of K562 cells treated with imatinib demonstrated a

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