| Summary: | 博士 === 國立臺灣大學 === 生化科學研究所 === 86 === An UV irradiation-induced A431 cell apoptosis system in
combination with an
in-gel kinase assay was employed to investigate the possible
kinase candidate
that might directly involve in the apoptotic-signaling
cascade. A 36-kDa myelin
basic protein(MBP)kinase was found.It could be activated during
the early stages
of UV irradiation-induced apoptosis and its activation
accompanied DNA
fragmentation in A431 cells.Immunoblot anslysis revealed that
this 36-kDa kinase
cound be recognized by an anti-PAK autibody against the C-
terminal regions of a
a family of p21Cdc42/Rac-activated kinases(PAKs). Using an anti-
PAK antibody and
a specific anti-PAK2 antibody against the N-terminal region of
PAK2 as studying
tool, we demonstrated that UV irradiation caused the cleavage
of PAK2 to produce
a 30-kDa N-terminal kinase fragment and a 36-kDa C-terminal
fragment that became
activated in A431 cells. Meanwhile, the caspase-3(cysteinyl
aspartate-directed
protease-3), but not caspase-1 and caspase-2, was
activated in UV-induced
apoptotic A431 cells induced by UV irradiation, when the
activation of caspase-3
was blocked by the specific tetrapeptidic caspase-3 inhibitors
(Ac-DEVD-cho and
Ac-YVAD-cmk). These rusults suggested that the cleavage of
PAK2 was possibly
causedby the activated caspase-3.
To further study the cleavage and activation of
PAK2, the role of
intracellular ReactiveOxygen Species (ROS) in the apoptotic
A431 cells was
investiged.The intracellar ROS was generated by exposure of
A431 cells preloaded
with the oxidation-senstitive dye 2',7'-dichlorofluorescin
diacetate (DCF-DA) to
UV irradiation. The flow cytometric analysis was used to
detect the production
of ROS. Curcumin(50 μM),a free-radical scavenger, was found
to be an effective
inhibitor to block the cleavage/activation of PAK2 and
the activation of
caspase-3 during the early stage of UV irradiation-induced
apoptosis in A431
cells. The protective effect of curcumin was significantly
attenuated by sodium
nitroprusside(SNP), a NO tenerator. The dexamethasone, an
inhibitor of inducible
NO synthase, could also effectively inhibit the
cleavage of PAK2 and the
activation of caspase-3 in the UV-induced apoptotic A431
cells. Therefore, we
proposed that the intracellular NO, which was produced
by UV irradiation
inductions,might be a mediator of caspase-3 activation during apoptosis.
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