Biology Reference
In-Depth Information
regardless of p53 status.
186
However, a detailed mecha-
nism was not fully elucidated for the role of p73 in
apoptosis.
187
Prior data revealed that p73
a
was induced
and stabilized during MMR-dependent apoptosis after
cisplatin exposure.
101
Furthermore, studies from other
groups defined a physical interaction between hPMS2
(a binding partner of hMLH1) and p73
a
.
188,189
Since
MMR-dependent p73-
a
and -
b
stabilization was noted
in response to MNNG treatment, shRNA silencing of
p73
a
was used to elucidate the function of p73
a
in
MMR-dependent cellular death responses.
90,91
Interest-
ingly, while p73
a
knockdown partially abrogated
MMR-dependent apoptosis and spared long-term
survival, MMR-dependent G
2
cell cycle arrest responses
were not altered in response to MNNG.
90,91
Further
elucidation of the signaling responses demonstrated
that MMR/c-Abl-dependent activation of Inhibitor of
growth 2 (ING2)/p73
a
signaling regulated cell death
triggered by MNNG.
190
Taken together, these data
strongly suggest that p73
a
was a key mediator of
MMR-dependent apoptosis (see
Figure 9.2
).
was the first to demonstrate MMR-dependent induc-
tion/stabilization of p53 protein levels after 6-TG, IR,
FdUrd, or MNNG exposures.
90,92
e
94,97,106
We noted
that p53 stabilization was the result of MMR-dependent
ser-15 phosphorylation of this tumor suppressor
protein. We also noted MMR-dependent elevation of
Bax, a known downstream gene regulated by p53 in
response to MNNG treatment.
90
Although other labora-
tories have reported that MMR-dependent apoptosis in
human colon cancer cells following treatment with
MNNG or MNU was dependent on p53, studies using
isogenic cells varying in p53 function were not
explored.
177
e
179
In addition, TMZ-induced apoptotic
responses were reported to be dependent on p53,
180
although responses were not examined for MMR-
dependency. Collectively, these data highly suggested
that p53 was an important candidate mediating MMR-
dependent apoptosis, in processes similar to non-
MMR-dependent cell death responses after other
chemotherapeutic agents.
However, our data strongly suggest that p53 does not
play a role in MMR-dependent apoptotic responses. We
utilized several different approaches to abrogate p53
expression and function in MMR-competent RKO7 or
HCT116 3-6 cells. These alterations in p53 function did
not affect MMR-dependent apoptotic responses in
response to various agents that specifically stimulate
MMR-dependent lethality, including 5-FU, 6-TG,
and MNNG.
90
e
93,181
Similarly, Topping et al.,
95
also
found that functional p53 was not a mediator of MMR-
dependent apoptotic signaling pathways in response to
cisplatin exposures, even though the same MMR-depen-
dent stabilization of p53 was noted. Thus, all data to date
strongly suggest that p53 is not a mediator of MMR-
dependent G
2
arrest or apoptotic signaling. Although
ATR and ATM are typical kinases that stabilize p53 after
DNA damage and appear activated after MNNG, 6-TG
or other agents known to stimulate MMR, activation of
these kinases does not play a functional role in G
2
arrest
responses or apoptotic cell death signaling.
Role of c-Abl in MMR-Dependent Apoptosis
It was previously reported that p73 (
a
and
b
both)
was phosphorylated and stabilized in a c-Abl-depen-
dent manner in response to various DNA damaging
agents.
101,184,185
Numerous studies have attempted to
clarify the role of c-Abl in MMR-dependent cellular
responses.
101,191,192
Although it has not been fully
defined, direct physical interaction between c-Abl and
MMR has been reported.
101,184,185
Yi et al.,
193
defined
a physical and functional interaction between hMSH5
and c-Abl in response to IR-induced DNA damage.
Kim et al.,
152
further clarified the enhanced physical
interaction between hMLH1 or hPMS2 and c-Abl. Our
laboratory was able to abrogate MMR-dependent
apoptosis, as well as G
2
cell cycle arrest and lethality,
after MNNG exposures by pre-treating cells with the c-
Abl inhibitor, STI571, or by introducing an shRNA
specific for c-Abl knockdown
90,91
(see
Figure 9.2
). A
recent study from Wang et al.,
194
suggested that c-Abl
was bound to chromatin and their data suggest an
enhanced interaction with ATM and ATR in response
to DNA damage. They demonstrated that c-Abl can
phosphorylate ATR at tyrosines Y291 and Y310, and
have suggested that these phosphorylation events may
play a positive role in ATR activation under genotoxic
stress. Taken together, c-Abl is a key player in genotoxic
stress-induced apoptosis, cell cycle progression, and
DNA repair.
Role of p73 in MMR-Dependent Apoptosis
After excluding a role for p53 in MMR-dependent
apoptotic responses, we examined the role(s) of other
p53 family members in the apoptotic responses. The
p53 homologue, p73
a
has previously shown to be acti-
vated in response to a variety of genotoxic
agents.
101,182
e
185
Three seminal articles have demon-
strated the key role of c-Abl in the activation and stabi-
lization of p73
a
.
101,184,185
Blocking p73
a
function using
various methods, such as expression of a dominant-
negative p73
a
mutant form, siRNA-mediated knock-
down of p73
a
levels, or knockout by homologous
recombination, led to chemoresistance of tumor cells
Role of GADD45
a
in MMR-Dependent Apoptosis
Numerous studies have shown that GADD45 gene
family members play a major role in the regulation of
