Biology Reference
In-Depth Information
activated, both Chk1 and Chk2 are known to phos-
phorylate Cdc25C on serine 216 (S216) and it is this
phosphorylation that leads to inactivation of
Cdc25C.
66,67
In addition, Chk1 has also been reported
to phosphorylate and increase the kinase activity of
Wee1 directly.
68-70
Thus either Cdc25C inactivation
and/or Wee1 activation may contribute to inducing
and maintaining G2 cell cycle arrest in response to
DNA damage.
Therefore, targeting Wee1-like kinase (Wee1) offers
an alternative approach by which to abrogate DNA
damage-induced checkpoints (
Figure 10.1
). Wee1, like
the checkpoint kinases, is a crucial component of the
checkpoint signaling network that prevents cells
from progressing into mitosis in the presence of
incomplete replication or DNA damage. Wee1 is
downstream of Chk1 and Chk2, and thus it is antici-
pated that Wee1 inhibitors will also increase the
activity of DNA damaging anticancer therapies, and
that this effect will be specific for cells that lack the
ability to arrest at the G1/S checkpoint, e.g. through
p53 mutation. These inhibitors therefore also share
the promise that they will bring an increase in anti-
tumor activity without a concomitant increase in side
effects or additional toxicity in normal tissues. This
hypothesis has been supported by data from preclin-
ical studies with both biological tools and small mole-
cule Wee1 inhibitors.
71
e
73
Wee1 inhibition was shown
to dramatically inhibit irradiation-induced phosphory-
lation of Cdc2 residues Tyr-15 and Thr-14 in seven of
seven cancer cell lines studied, and to abrogate irradi-
ation-induced G2 arrest as determined by both
biochemical markers and fluorescence-activated cell
sorter analysis to show a significant increase in the
mitotic cell population. The radiosensitizing activity
was also shown to be p53 dependent with a higher
efficacy in p53-inactive cells.
71
RNAi-mediated down-
regulation of Wee1 kinase has also been used to show
abrogation of doxorubicin-induced G2 arrest in human
cervical carcinoma (HeLa) cells, and to sensitize them
to doxorubicin-induced apoptosis. In contrast, Wee1
downregulation did not sensitize normal human
mammary epithelial (HMEC) cells that possess an
intact G1 checkpoint response to doxorubicin-induced
apoptosis.
72
Interestingly, in the same study it was
reported that downregulation of Chk1 proved lethal
to HeLa cells independent of the induction of DNA
damage although this was not the case in HMEC cells.
This possibly argues that Wee1 inhibitors may possess
advantages over Chk inhibitors as potentiating agents
for chemo- or radiotherapies.
It has also been shown that Wee1 inhibition leads to
increased efficacy in xenograft models when given in
combination with DNA-damaging agents such as gem-
citabine. The synergistic activity of the combination
treatment was achieved without a corresponding
increase in toxicity.
73
EARLY CHECKPOINT ABROGATORS
e
CAFFEINE AND UCN-01
The hypothesis that abrogation of DNA damage
checkpoints could lead to potentiation of the effects of
DNA-damaging agents was first proposed in the
1980s, with research conducted in the early 1990s
providing support for the theory.
74
e
78
Caffeine (
Figure 10.2
) was among the first agents found
to be able to abrogate the cell cycle checkpoints that are
activated in response to DNA damage, albeit at high
(millimolar) concentrations.
74,75
Caffeine was first shown
to potentiate the lethal effects of nitrogen mustard by
a mechanism that results in the abrogation of G2 arrest,
inducing cells to undergo mitosis prior to DNA repair.
Later studies demonstrated that the radiosensitization
caused by caffeine was specific for p53 mutant cells and
was dependent on abrogation of the G2 checkpoint,
further suggesting that p53 mutant tumors might be
selectively killed by limiting their ability to maintain
a robust G2 checkpoint arrest.
41,43,79
While it provided
a prototypical agent that could sensitize cells to DNA
damage, caffeine has a broad range of activity on cell cycle
checkpoint control, and recent studies have suggested
that the pleiotropic effects induced by caffeine are, at least
in part, due to direct effects on ATM and ATR.
43,44
UCN-01 (7-hydroxy staurosporine) (see
Figure 10.2
)
was originally isolated from the culture broth of Strepto-
myces species, and was at first thought to be a selective
inhibitor of protein kinase C (PKC). UCN-01 potently
inhibits the classical calcium-dependent isozymes of
PKC, but further studies demonstrated that the
observed antitumor activity (both
in vitro
and
in vivo
)
could not be attributed solely to the inhibition of PKC.
It was later determined that UCN-01 is also a potent
inhibitor of a number of additional kinases (including
Chk1, Chk2, Wee1, and CDKs 2, 4 and 6). Indeed,
because of this selectivity profile, UCN-01 shows an
unusual profile, in that, depending upon the dose and
cell type used, treatment can result in three different
phenotypic endpoints, namely, cell cycle arrest, induc-
tion of apoptosis or cell cycle checkpoint abrogation.
When administered as a single agent at higher doses
(
0.5
m
M), UCN-01 treatment typically results in either
a cell cycle delay or induction of apoptosis. UCN-01-
induced cell cycle arrest is dependent upon both the
cell type and aberrations in the Rb pathway.
80
UCN-01
was shown to directly inhibit the activity of CDKs 2, 4,
and 6 (IC
50
42, 32, and 58 nM respectively) in A549 cells
(human lung cancer line)
in vitro
, and to reduce the level
of phosphorylated Rb in living A549 cells in a dose
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