Biology Reference
In-Depth Information
resulting in long single-strand patches, disruption of
DNA synthesis, and triggers of apoptosis. Additionally,
double-strand breaks are formed and repair by homolo-
gous recombination (HR) and non-homologous end
joining (NHEJ).
Thus, the cellular response to this single lesion,
O
6
meG, is complex. The killing effect of O
6
meG in
tumors will depend on MGMT, the levels of expression
of MMR proteins, the effectiveness of HR/NHEJ, the
presence of BER, and the length of the cell cycle.
TMZ or BCNU (one-third of effective doses of agent
used alone) showed significant antitumor effect without
toxic death. Lack of synergistic cell death in normal cells
is likely due to lower levels of glycolsylases initiating
BER and topoisomerase II, particularly in marrow
progenitors.
66
In MMR proficient cells, BG and MX act
synergistically in the presence of TMZ.
65
Thus, modula-
tion of MGMT, but not of BER, requires a MMR profi-
cient background and the simultaneous targeting of
MGMT and BER is a promising strategy to improve
the therapeutic efficacy of alkylating agents in MMR-
deficient and proficient tumors.
Inactivation of MGMT Coupled with
Interruption of BER Enhance Therapeutic
Efficacy of Alkylating Agents
As introduced above, the electrophilic nature of alky-
lating agents and the inherent nucleophilicity of nucleic
acid, dictate that several sites within DNA are suscep-
tible to alkylation. However, the presence of elaborate
mechanisms of DNA repair is a major obstacle to effec-
tive treatment with alkylating agents in cancer. For
instance, methylating therapeutic agents, TMZ forms
O
6
meG (about 6
MGMT Promoter Methylation: Mechanism,
Impact, and Tumor Prognosis
The remarkable finding reported in 1992 by Brent
et al.
67
that specific methylation patterns of the MGMT
gene would give rise to loss of expression began the effort
to understand the mechanisms behind this observation.
Brent went on to identify that it was the promoter region
that underwent CpG island methylation that was respon-
sible for loss of expression of the MGMT gene.
68
His
group identified that in the cell lines that did not express
MGMT, after demethylation treatment with 5-azacytidine
would re-express MGMT and provide drug resistance
in a process that correlated with loss of methylation of
the promoter region.
69
The group also identified that
immortalization of normal cells either with EBV or SV
40 large T antigen often resulted in MGMT promoter
methylation and loss of MGMT expression.
70,71
The process by which cells regulate MGMT expres-
sion in this on or off switch through promoter methyla-
tion is indeed complex. It is infrequently observed in
normal cells and tissues but commonly observed as
part of gene remodeling and heterochromatin changes
in malignant transformed cells, cell lines and tumors.
The only certain regions of the MGMT promoter are
susceptible to CpG methylation and correlate with
heterochromatin changes with loss of MGMT transcrip-
tion.
72
An extensive study by Herman and his collabora-
tors found that CpG island promoter methylation of
MGMT resulted in loss of gene expression not only in
cell lines but in approximately 25% of tumors of many
different types, including non-small-cell carcinoma of
the lung, lymphoma, head and neck cancers, and up to
40% of glioma and colorectal tumors.
73
In most
instances, the promoter methylation was associated
not only with loss of the MGMT protein but lack of
immunohistochemistry detection of the protein as
well. This led to a number of additional studies by
many laboratories around the world with additional
studies of primary tumors to assess overall MGMT
expression in these tumors. Some studies have sug-
gested an impact on drug sensitivity while others have
8% of adducts formed), 7-methylgua-
nine (70% of N
7
meG), and 3-methyladenine (9% of
N
3
meA) DNA adducts. These DNA adducts are repaired
by at least two mechanisms. The O
6
meG DNA adduct,
a cytotoxic and genotoxic lesion, is repaired by MGMT,
hence, MGMT becomes a major mechanism of resistance
to methylating agents. Meanwhile, cell death caused by
O
6
meG adducts is promoted by the MMR pathway
and, conversely, deficiency in MMR is associated with
pronounced resistance to methylating agents. N
7
meG
and N
3
meA DNA adducts are removed by the BER
pathway. Efficient BER repair minimizes the impact of
these lesions in normal and tumor cells. When BER is
disrupted, these abundant N-methylated DNA adducts
become highly cytotoxic. Most importantly, BER disrup-
tion is able to bypass other drug resistant factors such as
MMR defects and high MGMT activity: it acts as the
dominant pathway of cytotoxicity.
Several previous studies have demonstrated
improved therapeutic efficacy of alkylating agents
62
e
65
when blocking BER with methoxyamine (MX). MX binds
the aldehyde glycolytic group at the post-glycosyilase
AP site, an intermediate produced through BER process-
ing N
7
meG and N
3
meA DNA adducts. The complex of
MX-AP sites prevent AP site from repair by AP-endonu-
clease (APE), with subsequent recognition by topoiso-
merase II, forming cleavable complexes, double-strand
breaks, signaling apoptosis.
66
When combined with
TMZ or BCNU, MX potentiated therapeutic efficacy in
colon cancer and malignant glioma xenografts. Impor-
tantly, no additive systemic toxicity was noted in mouse
after receiving of treatment with TMZ or BCNU and
MX. Combinations of BG-BG, MX and a lower dose
e
