Repair of
Mitomycin-C Induced DNA Interstrand Crosslink in Mammalian Cells
Zheng H1, Wang X2, Warren
A2, Hu Y1, Li L2
1Beijing Children’s Hospital, Beijing,
China
2UT MD Anderson Cancer Center,
Houston, USA
Objective: Study on the DNA repair mechanisms of interstrand
crosslink in mammalian cells. Mitomycin-C
(MMC) exerts its cytotoxic effects by
forming DNA interstrand crosslinks (ICLs) and preventing DNA replication
and transcription. Recent
clinical studies indicate that DNA repair capacity is strongly implicated
in both inherent tumor sensitivity and acquired drug resistance. For normal
cells, the ability of DNA repair reflects the density of drug toxicity.
Therefore, a detailed understanding of the cellular mechanisms to
eliminating the critical ICLs is very important.
Methods: The repair of MMC-induced ICLs was studied by using a
gene reactivation assay. A single MMC crosslink was introduced into a
luciferase reporter plasmid to block transcription of the reporter gene.
Consequently, luciferase can only be expressed after removal of the ICL in
the mammalian cells. A series of mammalian repair-proficient and NER mutant
cell lines were transfected with the ICL plasmid to test whether genetic
defects in the NER pathway affect the ICL repair-mediated luciferase
reactivation.
Results: The repair-proficient cells could repair the MMC
crosslink in the absence of undamaged homologous sequences, indicating the
existence of an ICL repair pathway independent of homologous recombination.
NER mutant cell lines were highly defective in the
recombination-independent repair of ICLs. Rescue and sequencing of repaired
plasmids indicated frequent base substitutions at or near the position of
the MMC crosslink, suggesting that NER involve in the repair with an
error-prone mechanism. Conclusion: We
have demonstrated that mammalian cells can remove MMC ICLs in the absence
of homologous sequences, and NER is required in the
recombination-independent repair of ICLs.