Objective: The overexpression of N-methylpurine DNA glycosylase (MPG) may imbalance the DNA base excision repair (BER) to sensitize tumor cells to current DNA damage chemotherapy. In an effort to improve the efficacy of cancer chemotherapy, we have constructed adenoviral vector of MPG, to study its ability to sensitize human osteosarcoma cell HOS to DNA damage agents.
Methods: The adenoviral infection and MPG expression, as well as enzyme activity were determined by flow cytometry, Western blot, and HEX labeled oligonucleotide-based assay respectively. The cell survival/proliferation was measured using MTS, SRB, and [(3)H] thymidine incorporation assay. Apoptosis cell death was assayed by flow cytometry after treatment using phycoerythin (PE)-conjugated Annexin V and 7-amino-actinomycin (7-AAD).
Results: A 10 MOI of recombinant nonreplicating adenovirus was found to infect more than 90% of HOS cells within 24 hours by EGFP fluorescence, in which the MPG overexpression and MPG enzyme activity were also detected. The MPG overexpression HOS cells were significantly more sensitive to the DNA damage agents, including MMS, MNNG, and TMZ, with changes in the IC50 of 6.0, 4.5, and 2.5 fold respectively.
Conclusions: These data establish transient MPG overexpression as a potential therapeutic approach for increasing HOS cellular sensitivity to DNA damage agent chemotherapy.