Biology Reference
In-Depth Information
DNA helix into a binding pocket containing the cysteine
residue site. Such base flipping involves a rotation of the
entire nucleoside and requires the interaction between
DNA and amino acid residues of the MGMT protein to
bind and displace the base. Studies of MGMT mutation
screening
35,36
have identified that mutations occurred at
many conserved residues of MGMT protein, which play
a structural role in its reaction of alkyl transfer including
the base flipping, binding affinity, and protein stability,
can significantly affect MGMT repair activity.
To specifically remove the methyl/alkyl group from
the O
6
-position of guanine, MGMT mediates an unique
repair process compared to other repair pathways: (a) it
acts alone without relying on any other proteins or
cofactors; (b) it transfers the alkyl group to an internal
cysteine residue in the protein, acting as both a trans-
ferase and an acceptor of alkyl-group; (c) it inactivates
itself after receiving the alkyl-group from guanine,
thus, it is a suicide protein; (d) it does repair in a stoi-
chiometric fashion (
Figure 2.2
). One molecule of
MGMT only removes one molecule of alkyl group,
therefore, an excess of DNA adducts at O
6
-position
can completely deplete MGMT. These properties render
MGMT an important drug-resistant factor and an
ideal target for biochemical modulation of drug
resistance.
37
e
39
MGMT is also capable of repairing O
4
-methylthy-
mine, which is a very minor product of the reaction of
DNA with methylating agents. However, human
MGMTrepairs O
4
-methylthymine with a very slow reac-
tion rate
in vitro
assay,
40
e
42
which raises the question as
to whether it does
in vivo
play a useful role in the repair
of this adduct. In addition to repairing methyl groups,
MGMT is known to repair longer alkyl groups including
n
-propyl-,
n
-butyl-, 2-chloroethyl, 2-hydroxyethl,
iso
-
propyl and
iso
-butyl. The capacity of MGMT removal
of these large alkyl substrates suggests the importance
of MGMT in protecting cells against DNA-damaging
stress. For instance, repairing 2-chloroethyl or the
pyridyloxobutyl adducts can protect cells from chloro-
ethylating therapeutic agents and tobacco-derived
carcinogens.
39
MGMT Expression Protects Cytotoxicity from
Alkylating Agents
The clinical effectiveness of BCNU (1,3-bis(2-
chloroethyl)-1-nitrosourea) and related methylating
compounds, procarbazine, streptozotocin, or TMZ
(temozolomide) is attributed, in part, to the potentially
cytotoxic DNA lesions, O
6
meG and O
6
-chloroethylgua-
nine. It has been known for several decades that
O
6
-chloroethylguanine lesions induce cell death by
forming G
C interstrand crosslinks that are able to
inhibit DNA replication and transcription, eventually
leading to DNA double-strand breaks and
apoptosis.
43
e
44
O
6
meG has been known as a pro-muta-
genic and carcinogenic DNA lesion. More recently,
O
6
meG-directed cytotoxicity has been revealed through
the lethal interaction between persistent O
6
meG adducts
and the DNA mismatch repair (MMR) pathway. The
mispairing of O
6
meG with thymine during the next
cycle of DNA replication initiates futile cycles of DNA
MMR and leads to the activation of apoptotic cell death.
Therefore, MGMT-mediated repair of O
6
-alkylguanine
protects cells from the cytotoxic effects of these DNA
lesions and thus contributes to drug resistance in cancer
cells treated with alkylating agents. MGMT expressing
tumor cell lines are 4- to 10-fold more resistant to
BCNU, temozolomide and related compounds than cells
expressing low MGMT. While tumor cells have many
mechanisms of resistance to alkylating agents, there is
e
FIGURE 2.2
Mechanism of MGMT repair. In this figure, an
example of methylating agents, TMZ, is used to induce a methyl
adduct on DNA. MGMT recognizes the adduct, and binds to the
DNA. Amino acid with the adduct is then flipped out, and the methyl
group is transferred to a cysteine residue on MGMT active site. The
transfer is permanent, and MGMT protein is then destined for
degradation. DNA is repaired. (
Please refer to color plate section
).
Cysteine residue (145)
G C O
6
-Methylguanine
Temozolomide
A T
Degradation
CH
3
MGMT
CH
3
MGMT
CH
3
MGMT
MGMT mediated repair
Repaired DNA
