annual report annual report annual report annual report 2005

RADIOBIOLOGY 103
calf serum, 2.5% PHA (phytohaemagglutinin),
anibiotic solution and incubated for 72 h at 37 o C
and 5% CO 2 . Lymphocyte preparations for micronuclei
analysis were prepared according to the
standard method of Fenech [2].
As shown in Fig., temperature of pre-irradiation
incubation as well as that during exposure
exerts an effect on the frequency of radiation-induced
micronuclei in human peripheral blood lymphocytes.
The highest frequency of micronuclei is
observed for blood samples incubated at 37 o C before
and during irradiation in vitro with doses of 2
and 2.7 Gy. Further work is aimed at elucidation
of the mechanism of this effect.
The work was supported by the Polish Ministry
of Scientific Research and Information Technology
statutory grant for the INCT.
References
[1]. Bajerska A., Liniecki J.: Int. J. Radiat. Biol., 16, 483-493
(1969).
[2]. Fenech M.: Mutat. Res., 285, 35-44 (1993).
EGF RECEPTOR KINASE ACTIVITY IS REQUIRED
FOR EFFICIENT DOUBLE STRAND BREAK REJOINING
IN X-IRRADIATED HUMAN GLIOMA M059 CELLS
Iwona Grądzka, Barbara Sochanowicz, Irena Szumiel
tive to X-radiation due to the lack of a catalytic subunit
of DNA-dependent protein kinase (DNA-PK cs
).
Tyrphostin AG 1478 was used as a specific inhibitor
to block EGFR tyrosine kinase activity.
We found that the total cellular level of EGFR
is 3-fold higher in M059 J than in M059 K cells.
This correlates with higher resistance of M059 J
to tyrphostin AG 1478. Addition of EGF to the
cell cultures results both in the increase of phosphorylation
status of EGFR (at the position Y1068)
and in the receptor translocation to the nuclei. In
HeLa cells the EGFR autophosphorylation is the
main effect of the stimulation with the ligand. On
the contrary, in M059 K and J cells, EGFR translocation
to the nuclei prevails over autophosphorylation.
Tyrphostin AG 1478 prevents the EGF-induced
receptor accumulation in the nuclei of M059
K and J cells (not shown). Thus, the kinase activity
of EGFR enables the receptor translocation to
the nuclei in the gliomas. In accordance with the
results of Dittmann et al. [4], in M059 K cells X-irradiation
induces EGFR translocation to the nuclei.
This process can be prevented by tyrphostin AG 1478
(Fig.1A). In M059 J cells no accumulation of EGFR
in the nuclei is observed following X-irradiation
(Fig.1B). The data indicate that the kinase activity
of EGFR and the presence of DNA-PK cs
, lacking
in M059 J cells, are required for EGFR translocation
to the nuclei following the DNA damage.
Double strand break (DSB) rejoining after X-irradiation
is faster in M059 K than in M059 J cells
because of a defect in a nonhomologous DNA end-
-joining (NHEJ) in the latter cell line (lack of an
Fig.1. Accumulation of EGFR in the nuclei of M059 K (A) and M059 J (B) cells after X-irradiation with 10 Gy and the
effect of tyrphostin AG 1478 (T) on this process.
The epidermal growth factor (EGF) receptor
(EGFR) is a mediator of both proliferative and
survival signals in mammalian cells (reviewed in [1]).
Nuclear translocation of EGFR was observed after
the ligand binding as well as following the ligand-independent
activation. The latter was always linked
to exposure to genotoxic stress and it was speculated
that it is important for regulation of DNA repair
processes [2]. We investigated the effect of EGF
and X-irradiation on EGFR activation and translocation
to the nuclei in two related human glioma
cell lines M059 K and M059 J, the latter highly sensi-
important NHEJ component – DNA-PK cs ) [3]. Tyrphostin
AG 1478 slows down the DSB rejoining in
M059 K and has no effect in M059 J cells. This
suggests a direct link between EGFR kinase activity
and DNA-PK-dependent DSB rejoining and is
consistent with observation of other authors [3].
The work was supported by the Polish Ministry
of Scientific Research and Information Technology
– grant No. 3 P05A 013 23.
References
[1]. Amorino G.P., Hamilton V.M., Valerie K., Dent P.,
Lammering G., Schmidt-Ulrich R.K.: Mol. Biol. Cell,
13, 2233-2244 (2002).
[2]. Harari P.M., Huang S.M.: Semin. Radiat. Oncol., 11,
281-289 (2001).
[3]. Anderson C.W., Dunn J.J., Freimuth P.I., Galloway
A.M., Allalunis-Turner M.J.: Radiat. Res., 156, 2-9
(2001).
[4]. Dittmann K., Mayer C., Fehrenbacher B., Schaller M.,
Raju U., Milas L., Chen D.J., Kehlbach R., Rodemann
H.P.: J. Biol. Chem., 280, 31182-31189 (2005).