Biology Reference
In-Depth Information
(CIN) phenotypes. Such responses were predicted using
isogenic cell culture models of MMR-competent and
non-competent cells responding to exposures of 5-FU or
5-fluorodeoxyuridine.
92
e
94
The reaction is initiated by transfer of the alkyl group
from DNA onto the cysteine residue (Cys
145
)of
MGMT. This reaction is irreversible, resulting in the
inactivation of MGMT, which is subsequently ubiquiti-
nated
107
and degraded by the proteasome.
108
Compared
to other DNA repair systems, MGMT is unique in that
it acts as a single protein with no other factors reported
to date. Although the methyl group on the DNA is
a preferred substrate, MGMT is also capable of
repairing longer alkylation groups on O
6
-guanine-
DNA, such as ethyl, n-propyl, n-butyl, 2-chloroethyl,
2-hydroxyetyl, iso-propyl, and iso-butyl substitutions.
MGMT-mediated O
6
-meG repair is very fast, but since
the reaction is a suicide enzymatic action, functional
MGMT levels can be depleted in situations of excess
O
6
-meG DNA lesions.
109
Indeed, early reports found
that MGMT-deficient cells were unable to repair
O
6
-meG DNA lesions, and were, thereby, more sensitive
to specific alkylating agents than MGMT-proficient
cells.
110
Unlike MMR, repair of DNA lesions by
MGMT does not lead to a lethal event in cells. Indeed,
the DNA repair reaction mediated by MGMT is without
major DNA processing (i.e., no DNA incision, excision,
synthesis or DNA ligation), since it involves a simple
transfer of the alkylated substitution on O
6
-guanine,
creating a hypomethlylated, and therefore, intact
O
6
-guanine-DNA. Consequently, high levels of MGMT
activity in tumor tissues are associated with resistance
to alkylating agents.
111
Cells that do not express detect-
able levels of AGT (MGMT), or that are treated with
a specific AGT inhibitor (such as O
6
-benzylguanine),
are very sensitive to treatment with methylating
agents.
112
In concert with functional MMR, BER, and
NER, MGMT has overlapping roles to the protection
of the genome from mutagenic and carcinogenic DNA
lesions. NER (reviewed elsewhere) is an elaborate
repair system that can remove bulky and/or non-bulky
lesions in DNA, such as apurinic/apyrimidinic sites
(AP sites) or O
6
-meG residues. NER also plays a backup
role for other repair systems.
113,114
CELLULAR RESPONSES TO
ALKYLATING AGENTS
DNA Lesions and Repair
MMR
As mentioned above, loss of MMR confers resistance
to a variety of chemotherapeutic agents, all of which
produce DNA lesions that do not necessarily cause
lethality in the cell. In contrast, many of these agents
result in DNA lesions (at least in part) whose actual
repair results in lethal DNA damage that mediates cell
death via apoptotic signaling.
101
e
103
Most agents that
ultimately lead to damage tolerance in MMR-deficient
cells are alkylating agents. Monofunctional alkylating
agents can be separated into two major classes of DNA
damaging agents, S
N
1andS
N
2. S
N
2-type alkylating
agents include methylmethane sulphonate (MMS) and
dimethylsulphate. S
N
1-type alkylating agents include
TMZ, MNNG, and MNU, which are cytotoxic to cancer
cells at lower concentrations than S
N
2-type agents. As
a result, few S
N
1-type compounds have been used
successfully. Treatment of cancer cells with TMZ (the
clinical form of MNNG), results in N
7
-methylguanine
(N
7
-meG), N
3
-methyladenine (N
3
-meA), O
4
-methylthy-
mine (O
4
-meT), and O
6
-methylguanine (O
6
-meG) DNA
lesions. These lesions are detected and repaired by
several specific DNA repair pathways. N
7
-meG and
N
3
-meA DNA lesions are removed by base excision
repair (BER). Although O
6
-meG can be repaired by
both O
6
-methylguanine-DNA methyltransferase
(MGMT) and nucleotide excision repair (NER) path-
ways, these DNA lesions are also good targets for
MMR.
104
Hallmarks of MMR-mediated restoration of
these lesions are prolonged G
2
cell cycle arrest and selec-
tive MMR-induced lethality highlighted by “damage
tolerance” responses observed in MMR-deficient
cells.
105,106
Specific mechanisms involved in signaling
G
2
arrest and apoptosis in response to these lesions are
discussed below. Understanding these signaling and
cell death responses are important for restoring proper
responses in MMR-deficient cells, ultimately restoring
chemosensitivity to these agents and preventing survival
of highly mutagenic cells that can ultimately progress to
metastatic disease.
BER
DNA adducts generated from oxidative and DNA
alkylating agents can also be processed by BER path-
ways.
115
BER activity is initiated by removal of abnormal
or inappropriate bases through the action of DNA glyco-
sylases that have separate, but overlapping affinities for
specific DNA lesions. Methylpurine DNA glycosylase
(MPG) and 8-oxo-guanine-DNA glycosylase-1 (OGG1)
are two representative human DNA glycosylases. MPG
excises N
3
-meA, N
7
-meG, and other minor forms of alky-
lated bases, while OGG1 excises 8-oxo-guanine (8-
oxoG).
116
These DNA lesion-specific glycosylases remove
damaged bases and create AP sites that are then recog-
nized and cleaved by AP endonuclease 1 (APE1), leaving
MGMT
Most O
6
-meG DNA lesions are repaired through
a one-step alkyl transfer reaction mediated by MGMT.
