Biology Reference
In-Depth Information
The Cytotoxic Effect of O
6
meG Depends on the
Activities of both MGMT and Mismatch Repair
Machinery
In cells, the cytotoxic effect of O
6
meG is not only
dependent on MGMT but also other repair mechanisms.
Chemotherapeutic methylating agents such as DTIC
(dacarbazine) and temozolomide are used extensively
in the treatment of melanoma and glioma based on
a consequence of their ability to donate a methyl group
to the bases of DNA. O
6
meG, one of a dozen lesions
generated in DNA, is the principal toxic lesion. This
lesion is also mutagenic, causing not only point muta-
tions but also recombinations, and carcinogenic.
43
Therefore, the cellular events underlying the genotoxic
and cytotoxic effects of O
6
meG induced by methylating
agents have been the focus of attention for several
decades. It has been established that following exposure
to methylating agents, two rounds of DNA replication
and the post-replication mismatch repair (MMR) system
are absolute requirements for cell killing by O
6
meG.
As a result, DNA replicative polymerases will
generate O
6
meG:C or O
6
meG:T mispairs that apparently
undergo a variety of fates. If MGMT is active either
before or after DNA replication past the O
6
meG, its
action on the O
6
meG:C mispair will result in G:C,
whereas the O
6
meG:T, on the next round of replication,
will result in both a G to A transition mutation and
a newO
6
meG:Ton the opposite strand. Or MGMTrepair
will result in G:T, which is a substrate for thymine-DNA
glycosylase that restores G:C via base excision repair
(BER).
60,61
The processing of the O
6
meG:C or O
6
meG:T
can also occur through the function of mismatch repair
(MMR). If MMR is absent or ineffective, the mismatch
is resolved through an A:T transition mutation, the
mutation hallmark of the methylating agents. Then, cells
display a mutator phenotype that cells contain substan-
tial numbers of genetic abnormalities and tolerate the
killing effect by high doses of methylating agents.
30
It
has been demonstrated that tumors lacking any of the
MMR proteins are very resistant to methylating agent
temozolomide. In fact, loss of MMR appears to be domi-
nant in drug resistance to O
6
meG lesions since inactiva-
tion of MGMT by BG fails to potentiate TMZ-toxicity in
MMR-deficient tumor cells, indicating that even large
numbers of O
6
meG DNA adducts are present in DNA,
they are very mutagenic but not cytotoxic in the absence
of the MMR recognition.
Cellular response to O
6
meG in the presence of active
MMR is quite different. MMR proteins bind O
6
meG:T
mispairs and initiate a futile DNA repair cycle with
exonuclease removal of the newly synthesized strand
and polymerase reincorporation of the sequence with
the preferential insertion of T opposite the O
6
meG,
a process that appears to be repeated over and over,
a strong correlation between high MGMT expression
and tumor drug resistance.
45
e
47
Not surprisingly, high
tumor MGMT levels reduce tumor responses in patients
to alkylating therapeutic agents.
MGMT Inhibitors Sensitize Tumor Cells
to Alkylating Agents
Since the reaction of MGMT leads to its inactivation
and degradation through ubiquitination, MGMT is an
ideal target for depletion through the use of a substrate
that acts as an irreversible inhibitor. Small molecule pseu-
dosubstrates, without the oligonucleotide backbone,
have been shown to be effective inhibitors. O
6
-benzyl-
guanine (BG) was developed in 1990 based on the under-
standing of the reaction of bi-molecules displacement
between cysteine residue of MGMT and the alkyl-group
at O
6
position of guanine.
48
e
51
In a series of important
studies, BG has been identified as a potent inhibitor of
human MGMT. BG at non-toxic concentrations inacti-
vates human MGMT with an EC
50
of 0.2
M through
the binding to MGMT that transfers the benzyl moiety
to its active-site cysteine. BG is not incorporated into
DNA in living cells and reacts directly and rapidly with
both cytoplasmic and nuclear MGMT. In addition, the
studies also show that BG is extensively metabolized to
its 8-oxo-derivative.
52
The 8-oxo-derivative can be
formed by several enzymes, including aldehyde oxidase
and cytochromes CYP3A4 and CYP1A2. Among these,
cytochrome CYP1A2 probably plays the major role
in conversion of BG to the 8-oxo-O
6
benzyl guanine
(8-oxo-b
6
G). The 8-oxo-b
6
G is almost as active an inhib-
itor of MGMT as BG itself but has a longer half-life
in
vivo
and is therefore probably responsible for most of
the MGMT-inactivation occurring at later times. A more
soluble derivative, in clinical trials as an oral agent,
6-(4-bromo-2-thienyl)methoxypurin-2-amine (lomegua-
trib) has similar potency.
53
The therapeutic impact of the inactivation of MGMT
by BG and these related compounds has been evaluated
in basic, preclinical and clinical settings. The depletion
of MGMT activity by BG renders tumor cells sensitive
to alkylating agents by 2- to 14-fold
in vitro
and in xeno-
graft settings in a variety of tumors.
30,52
e
55
However,
often, drug doses, particularly of BCNU or temozolo-
mide, need to be reduced due to marrow toxicity. This
establishes the potential therapeutic impact of BG as
an enhancer of alkylating agents. Clinical trials estab-
lished the maximal tolerated dose of BG to achieve
biochemical inhibition of MGMT to be 120 mg/m
2
,
and the maximal does of BCNU to be 40 mg/m
2
and
temozolomide 470 mg/m
2
given with this dose of
BG.
56
e
58
Phase 2 trials with BG and lomeguatrib have
had marginal activity in glioma, myeloma and mela-
noma, often due to myelosuppression ADD.
59
m
