Biology Reference
In-Depth Information
c-Abl. However, as the dose of alkylating agents is
elevated, ATR and Ataxia telangiectasia mutated (ATM)
may be activated, but, these responses are not MMR-
specific, or result from DSBs that are consequently
and not causally made (see below). Great care in the
specific doses of agents used in each study and
responses examined is required when attempting to
analyze the data in order to delineate which signaling
pathways are predominantly stimulated by MMR.
There is abundant evidence that G
2
cell cycle checkpoint
arrest can be mediated by the activation of ATM and/or
ATR
133
e
135
in response to DNA damage. These kinases
can phosphorylate, and thereby activate, p53 as well as
the downstream checkpoint kinases, Chk1 and Chk2,
respectively. The main targets of activated ATR/Chk1
or ATM/Chk2 in promoting G
2
arrest signaling
pathway are cyclin B1 and Cdc25C respectively. These
kinases can phosphorylate Cdc25C phosphatase at
Ser,
216
and thereby inhibit Cdc25C activity by cyto-
plasmic sequestration via 14-3-3
s
. This prevents
Cdc25C from de-phosphorylating cdc2 on Tyr,
15
which
is thought to be the main factor preventing initiation of
mitosis in mammalian cells after most DNA damaging
agents. The phosphorylation-mediated stabilization of
the p53 tumor suppressor by ATM/Chk2 and/or
ATR/Chk1 can also lead to its transcriptional regulation
of several downstream genes involved in cell cycle
checkpoint arrest, including p21, growth arrest and
DNA damage-inducible gene/protein (GADD45)
a
, and
14-3-3
s
.
91
However, as stated above, our data support
the notion that p53 has little functional role in MMR-
mediated G
2
arrest or apoptotic responses.
90,91,95
While some groups reported that MMR-dependent
G
2
arrest and cell death are a result of DSBs caused
by the repair pathway itself, data using low doses of
DNA damaging agents support the notion of direct
signaling through MMR-c-Abl-p73
a
-GADD45
a
signa-
ling (
Figures 9.1, 9.2
). Expression of GADD45
a
,in
particular, has been demonstrated by our laboratory
to be induced in a MMR-dependent manner after 5-flu-
oro-2
0
-deoxyuridine (FdUrd).
94
Others have shown that
GADD45
a
can mediate a G
2
checkpoint arrest by
directly binding to, and inhibiting, Cdc2.
136,137
Several
groups proposed that ATR/Chk1 or ATM/Chk2 path-
ways are preferentially stimulated to initiate MMR-
dependent G
2
arrest after exposure to methylating
agents, such as MNNG or TMZ.
138
e
142
However, these
responses are primarily noted after relatively high
doses of DNA damaging agents that can stimulate
ATR, but not necessarily in a MMR-dependent manner
(
Figure 9.3
). Indeed, we hypothesize that ATM/ATR
activation is stimulated in a MMR-independent
manner, but only potentially mediated by MMR after
high doses of agents.
91
In contrast, we found that
although the ATR/Chk1 pathway was activated after
FIGURE 9.1
MMR-independent and MMR-dependent G
2
cell
cycle checkpoint arrest responses. Accumulated evidence shows that
G
2
arrest in response to specific alkylating agent damage (e.g., from
MNNG, cisplatin or TMZ) can arise independent of MMR processing
through a transient ATR/Chk1 transient response, but prolonged and
sustained MMR-dependent G
2
cell cycle checkpoint arrest responses
are mediated by c-Abl/GADD45
a
signaling. The MMR-independent
responses mediated by ATR/Chk1 are a result of replication stalling
and the creation of DNA double-strand breaks. In contrast, MMR-
dependent detection of initial alkylation-damaged DNA lesions
appears to require replication, creating mismatched DNA lesions,
which triggers c-Abl activation. In turn, activated c-Abl stimulates
GADD45
a
pathway for G
2
cell cycle checkpoint arrest responses. DN-
ATR: dominant negative ATR.
high MNNG exposures, its phosphorylation and
activity did not dependent on functional MMR,
91
and
this pathway was not responsible for either G
2
arrest
or cell death responses noted in MMR-deficient cells.
Although exposure of MMR-deficient cells to high-
dose MNNG activated ATR and caused G
2
arrest,
similar responses were not noted in these cells after
FIGURE 9.2
hMLH1/c-Abl/p73
a
/GADD45
a
signaling controls
apoptosis. Shown is a signal transduction flow diagram beginning
with the MMR detection of O
6
-meG DNA lesions created after MNNG
or TMZ exposures. MMR complexes then process the damage for
repair, also stimulate downstream c-Abl activation that leads to
increased ING-2p73
a
and GADD45
a
expression. MMR-dependent
c-Abl/ING-2p73
a
and c-Abl/GADD45
a
signaling cascades, in turn,
direct cell death (apoptotic) response.
