JAEA-Review-2010-065.pdf:15.99MB - 日本原子力研究開発機構

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Ion Beam Irradiation Has Different Influences on the
Expression of p53 in Cultured Human Retinal
Vascular Endothelial Cells Exposed to L-dopa among
20 Ne, 12 C and 4 He
K. Akeo a,b) , T. Funayama c) , N. Hamada d) , Y. Kobayashi c) and Y. Akeo a)
a) Akeo Eye Clinic, b) Department of Ophthalmology, Keio University School of Medicine,
c) Radiation-Applied Biology Division, QuBS JAEA,
d) Radiation Safety Research Center, Central Research Institute of Electric Power Industry
Deficiency of L-dopa causes degeneration of the
substantia nigra in the brain and L-dopa is used in the
treatment of Parkinson’s disease. We proved that L-dopa
produced NO and superoxide, and had the cytotoxic effects
on the retinal pigment epithelial cells 1) . L-dopa injected
into the vitreous of the rats dilated the vena in the ciliary
body 2) . We wondered if RE cells could be exposed to
oxidative stress by administrated L-dopa. We already
3)
reported that oxidative stress such as hyperoxia augmented
the cytotoxicity of the aortic endothelial cells by L-dopa.
Glutathione peroxidase (GPX), a selenium-dependent
and lipid peroxide-scavenging enzyme that effectively
reduces lipid peroxides with the concomitant oxidation of
glutathione is distributed in mitochondria 4) . Faucher et al.
measured the expression of two bcl-2 family members, bax
and bcl-2, in a human endothelial like cell-line
overexpressing the organic hydroperoxide-scavenging
enzyme GPX, in the absence of any apoptotic/oxidant
stimulus, and showed that overexpressing an antioxidant
gene such as GPX in endothelial cells is able to change the
basal mRNA and protein bax levels without affecting those
of p53 and bcl-2. This phenomenon could be useful to
antiatherogenic therapies which use antioxidants with the
aim of protecting the vascular wall against oxidative stress
injury 5) .
Exposure to L-dopa inhibited the expression of GPX in
RE cells. Ion beam irradiations both of 4 He and 12 C
decreased the expression more remarkably than 20 Ne. The
expression of GPX in RE cells incubated with L-dopa
decreased significantly after 8 h of exposure to 4 He, and
after 4 and 24 h of exposure to 12 C. On the contrary, ion
beam irradiation of 20 Ne increased the expression of GPX in
RE cells incubated with L-dopa after 4 h of the exposure of
the irradiation significantly. We considered the different
accumulation of the energy irradiated at a point by various
ions could be concerned with the effects on the expression
GPX in RE cells incubated with L-dopa.
Established human RE cells in vitro incubated with
L-dopa (250 µM) for 2 h were exposed to ionization
radiation that is induced by acceleration of the ionizing atom
of 350 MeV 20 Ne, 220 MeV 12 C, and 50 MeV 4 He. We
obtained the RE cells after 0, 4, 8, 24 h of the irradiation and
extracted total cellular RNA to synthesize cDNA. We used
the Primer3 website to design the primers for RT-PCR
JAEA-Review 2010-065
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amplification of the cDNA of p53 and 18S RNA. The
reactions were carried out at the following temperature:
95 ºC, for denaturation; 60 ºC, for annealing; and 72 ºC, for
extension for 17–27 cycles. After mixing the cDNA,
primer, and SYBR green, the expression of 18S RNA and
p53 was measured using the LightCycler system. The
technology of this system is extremely innovative and
enables rapid and simultaneous evaluation PCR experiments.
Fluorometric analysis of the formed PCR products was
performed as a real-time measurement either continuously
or at specifically defined time points during each PCR cycle.
The expression of p53 in RE cells significantly was not
influenced by only exposure to L-dopa, but increased just
after the irradiation both of 350 MeV 20 Ne and 50 MeV 4 He
in those cells incubated with L-dopa. Ion beam irradiation
caused the induction of apoptosis in RE cells damaged by
lipid peroxidation with L-dopa.
References
1) K. Akeo et al., Pigment Cell Res. 13 (2000) 80.
2) S. A. Amaki et al., Pigment Cell Res. 14 (2001) 256.
3) K. Akeo et al., Exp. Eye Res. 49 (1989) 335.
4) K. Watanabe, Tran. Soc. Pathol. Jpn. 76 (1986) 39.
5) K. Faucher et al., Mol. Cell Biochem. 277 (2005) 81.