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Blood, 1 November 2000, Vol. 96, No. 9, pp. 3294-3295
CORRESPONDENCE
To the editor:
The ABL kinase inhibitor STI571 does not affect survival of
hematopoietic cells after ionizing radiation
The c-Abl gene encodes a widely expressed
tyrosine kinase that is important for development, particularly of the
nervous system.1 But the signaling pathways that employ
c-Abl in normal cells are not well defined. Abl protein is located in
both the nucleus and the cytoplasm, and recent studies suggest that a
substantial component of Abl resides in the actin cytoskeleton and
mediates signaling related to integrin activation.2 Other
studies have linked Abl to the cellular response to DNA damage,
including cell cycle arrest, DNA repair, and apoptosis.3-7
Abl tyrosine kinase is activated in response to ionizing radiation, and
fibroblasts from Abl knockout mice have been reported to have reduced
sensitivity to ionizing radiation.7 Abl kinase activity is activated in the Bcr/Abl and
Tel/Abl oncogene products, resulting in leukemia. STI571 is
a novel tyrosine kinase inhibitor (Novartis, Basel, Switzerland) that
inactivates Bcr/Abl, c-Abl, platelet-derived growth factor
receptor, and c-kit, but not members of the Src or Jak families of
tyrosine kinases.8 STI571 inhibits growth of cell lines
transformed by Bcr/Abl or primary cells from patients with chronic
myeloid leukemia (CML) and induces apoptosis of CML cells in tissue
culture studies.9 Importantly, STI571 inhibits Abl kinase
activity at concentrations that are otherwise nontoxic to normal
hematopoietic cells, and has shown impressive activity in early-phase
clinical trials in patients with CML.10 Because ionizing radiation activates Abl kinase and loss of the
Abl gene has been associated with reduced sensitivity to
ionizing radiation, it is possible that inhibition of Abl by STI571
would reduce sensitivity to ionizing radiation. To test this
hypothesis, normal bone marrow cells from BALB/c mice exposed
to 0, 0.1, or 1 µmol/L STI571 for 24 hours before being exposed to
0-6 Gy of gamma radiation, using a Gammacell 1000 (Atomic Energy of
Canada, Mississagua, Canada). One µmol/L STI571 is sufficient
to completely inhibit Bcr/Abl and c-Abl tyrosine kinase activity when
assayed by the sensitive technique of immune complex kinase assay
(Uemura, unpublished data, 1999). After irradiation,
granulocyte/macrophage colony-forming cells (CFU-GMs) were
measured. The methylcellulose medium contained STI571 at the same
concentration as pretreatment (Figure
1A). The survival of CFU-GMs at each dose
of radiation was the same with or without STI571.
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| Figure 1.
Effects of STI571 on survival after ionizing
radiation.
Bone marrow cells from BALB/c mice were treated with 0, 0.1, or 1 µmol/L STI571 for 24 hours before being exposed to 0-6 Gy
gamma irradiation. After irradiation, cells (5 × 104 to
2 × 105 cells) were plated in triplicate in 1 mL Iscove
modified Dulbecco medium containing 1% methylcellulose, 20% fetal
bovine serum, 5% WEHI-3B conditioned medium as a source of murine
IL-3, 20 ng/mL recombinant human G-CSF, and STI571 (at the same
concentration as pretreatment). (A) CFU-GMs (> 50 cells) were
enumerated on day 8 to determine radiation response. The results are
presented as the percentage of surviving colonies relative to an
unirradiated control. (B) U937 cells were treated with 0, 0.1, or 1 µmol/L STI571 for 24 hours before being exposed to 0- 6 Gy gamma
irradiation. After irradiation, cells (1 × 103 cells)
were plated in 1 mL of RPMI 1640 containing 1% methylcellulose, 20%
FCS, and STI571 (the same concentrations as pretreatment). Colonies
were counted on day 8. Shown here is 1 of 2 independent experiments.
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The responses to genotoxic stresses are believed to be mediated by both
p53-dependent and -independent pathways. The possibility that Abl
kinase activity contributes to radiation sensitivity in cell lines in
which p53 is mutated was, therefore, considered. The
p53 mutant hematopoietic cell line U937 was exposed to
ionizing radiation (0-6 Gy), and the fraction of surviving cells was
again measured by colony formation. Again, the addition of STI571 did not alter the surviving fraction or size of surviving colonies, after
radiation (Figure 1B). Overall, our results suggest that, with the
techniques used here, inhibition of c-Abl tyrosine kinase with STI571
at concentrations in culture of up to 1 µmol/L does not affect
radiation response where survival is the endpoint. In a previous study, Dan et al treated U937 cells with etoposide and
found that 10 µmol/L STI571 (previously known as CGP57148B) reduced
apoptosis of U937 cells without interfering with JNK1/SAPK activation.11 It was concluded that c-Abl acts downstream
of caspases during the development of p53- independent apoptosis. Our
study differs from that of Dan et al by the use of a lower dose of
STI571. We found that 1 µmol/L STI571 was sufficient to essentially
completely inhibit Bcr/Abl, Tel/Abl, or c-Abl kinase activity but,
unlike 10 µmol/L STI571, was not associated with any nonspecific
toxicity either on normal murine marrow cells or on U937 cells. It is
also possible that Abl kinase is necessary for DNA repair responses to
etoposide, but not to ionizing radiation. Whereas c-Abl seems to be activated in response to ionizing radiation,
the requirement for Abl tyrosine kinase activity to mediate cellular
responses to DNA damage is less clear. For example, while Abl  /
fibroblasts from mice have been reported to have increased survival
after radiation, Abl / avian cells had a normal survival
curve.7,12 Also, the role of Abl in DNA-damage-induced G1
arrest is controversial.1,6,12 In the present study, we
found no evidence that pharmacologic kinase inhibition of c-Abl with
STI571 altered radiation sensitivity in either primary murine hematopoietic cells or in a human p53 mutant cell line.
However, some functions of c-Abl are
kinase-independent,13,14 and it is possible that kinase
activity is not required for some cellular responses to radiation. In
any event, these results suggest that the effects of ionizing radiation
in patients should not be altered significantly by STI571 treatment.
Naoki Uemura and James D. Griffin
Dana-Farber Cancer Institute Boston, MA
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