Biology Reference
In-Depth Information
cycle checkpoint responses, and inhibition of this
pathway resulted in resistance to TMZ in glioma cells.
In agreement with results from our laboratory, Hirose
et al.,
155
found that abrogation of G
2
arrest by inhibition
of p38
a
had no affect on the level of Chk1 phosphoryla-
tion after TMZ exposures. Their data suggested that
MMR-dependent G
2
arrest was dependent on p38
a
activation, and independent of Chk1. Later reports
from Hirose et al.,
156
demonstrated that both p38
a
and
Chk1 pathways contributed separately, but equally, to
MMR-dependent G
2
arrest after TMZ exposures in
glioma cells. However, over-expression of GADD45
a
activated both the p38
a
MAPK and JNK kinase path-
ways to induce G
2
/M arrest.
157
Taken together, the
data suggested that p38
a
MAPK kinase may affect c-
Abl or GADD45
a
signaling pathways. We proposed
the possibility that p38
a
MAPK kinase signaling could
possibly exist in a feedback loop with c-Abl/
GADD45
a
, even further regulating the c-Abl-mediated
G
2
arrest pathway. While, GADD45
a
appears to be
a strong downstream candidate for c-Abl-mediated
G
2
arrest, more detailed studies will be needed to deter-
mine if GADD45
a
actually has a role in feedback regu-
lating MMR-dependent G
2
arrest. These data further
supported our findings that c-Abl was a critical medi-
ator of MMR-dependent G
2
arrest in response to
MNNG treatments. Taken together, these findings
strongly suggest that GADD45
a
regulates G
2
arrest
responses after MNNG treatment, in direct response
to MMR-mediated c-Abl activation.
The other potential factor that appears to play a role
in regulating MNNG-induced G
2
cell cycle arrest
responses is breast cancer 1, early onset (BRCA1).
BRCA1 is a tumor suppressor gene that can be a major
hereditary factor for risk (with ~5 % risk in the popula-
tion) to breast and ovarian cancers.
158
e
160
BRCA1 can
activate ATM/Chk2 and ATR/Chk1 kinases after
various cell stresses.
161
e
164
For example, BRCA1-mutant
cells are more resistant to 6-TG than BRCA1-positive
cells in a clonogenic survival assay, and showed reduced
apoptosis (cell death). Additionally, mutated BRCA1
resulted in an almost complete loss of a G
2
-M cell cycle
checkpoint responses induced by 6-TG. Transfection of
specific siRNAs against MSH2, MLH1, ATR, or Chk1
in 6-TG-treated BRCA1-positive cells markedly reduced
G
2
-M checkpoint responses.
165
These data suggest that
BRCA1 has a role in the G
2
checkpoint responses to 6-
TG and are consistent with involvement of MMR
proteins in the BRCA1-associated genome surveillance
complex (BASC) previously reported by Wang et al.
166
blebbing, chromatin condensation and nuclear fragmen-
tation under the various physiological and pathological
situations. However, only a very few anticancer chemo-
therapeutic agents induce apoptosis to achieve cancer
cell killing.
167
Nevertheless, defects in apoptosis can
contribute to the growth of cancers in vivo, and can result
in resistance to chemotherapeutic agents during therapy.
Prior publications from other groups suggested that
cancer cells were killed through apoptosis after
MNNG exposure.
95,168
e
172
Apoptosis can be signaled
by either intrinsic pathways mediated by alterations in
mitochondrial membrane integrity or extrinsic path-
ways initiated at the outer plasma membrane upon acti-
vation of death receptors. Ultimately, activation of either
pathway leads to caspase activation. Intrinsic apoptosis
is highlighted by the initial release of cytochrome c from
the mitochondria into the cytosol so it can interact with
Apaf-1 and activates caspase-9.
173
Caspase-9 then acti-
vates downstream effector caspases (e.g., caspases -3,
-6, -7) and triggers apoptosis. In contrast, extrinsic
apoptotic pathways are initiated by stimulation of
membrane surface-bound death receptors, such as
the tumor necrosis factor (TNF) receptor super-
family (e.g., CD95 (APO-1/Fas) or TRAIL receptors.
These pathways, in turn, activate caspase-8 that then
propagates the apoptotic signal by direct cleavage
of downstream effecter caspases, such as caspases -9
and -3.
174,175
Although activated caspase-8 is fairly
specific to the extrinsic apoptotic pathway, once acti-
vated it will eventually trigger intrinsic apoptotic path-
ways, causing release of cytochrome c and caspase-9
activation.
176
Reciprocally, once caspase-9 is activated
it can stimulate caspase-8 activation. In either case,
a cascade of cysteine proteases are activated that ulti-
mately result in apoptotic cell death responses. Most
data suggested that MMR-dependent apoptosis was
solely mediated by the activation of intrinsic pathways
in response to MNNG exposures.
172
In agreement,
time-course analyses of the apoptotic pathways induced
by MMR-dependent recognition of specific DNA lesions
induced by MNNG, demonstrated that cleavage of
caspase-9 occurred 20 h early than caspase-8 activation
in MMR-dependent human colon cancer cells, suggest-
ing that the intrinsic apoptotic pathway was triggered
by MMR-dependent signaling after MNNG exposures.
90
More recently, MMR-dependent intrinsic apoptotic
responses in response to cisplatin exposures also sug-
gested the activation of intrinsic apoptotic responses
mediated cell death after this bifunctional alkylating
agent.
95
Role of p53 in MMR-Dependent Apoptosis
Early studies from our group and others clarified the
mechanism of MMR-dependent cell death after expo-
sure to specific chemotherapeutic agents. Our laboratory
MMR-Dependent Apoptosis
Apoptosis was initially described by its morpholog-
ical characteristics, such as cell shrinkage, membrane
