Chloroethylnitrosoureas (CENUs) are bifunctional alkylating agents widely used against cancer. They exert anticancer activity by inducing interstrand dG-dC crosslinks of DNA, which cause apoptosis of cancer cells by inhibiting DNA double strands separation during replication and transcription. However, the formation of dG-dC crosslinks can be interrupted by O6-alkylguanine DNA alkyltransferase (AGT). This enzyme repairs O6-(2-chloroethyl)-deoxyguanosine or N1,O6-ethano-deoxyguanosine by removing the alkyl group on the O6 position of guanine before the formation of crosslink, and finally results in drug resistance against CENUs. O6-benzylguanine (O6-BG), which could be accepted as a pseudosubstrate by AGT, was found to be capable of inactivating AGT and enhancing the antitumor effects of CENUs. In this work, human brain glioma SF767 cells were treated with 1-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-3-(2-chloroethyl)-3-nitrosourea (ACNU) (0.2 to 0.8 mM) in combination with 20 �M O6-BG. Using HPLC-ESI-MS/MS, the levels of dG-dC crosslink in the groups exposed to the combination chemotherapy and to ACNU alone were determined and compared to investigate the enhancement of the formation of dG-dC crosslink by O6-BG. The results indicated that the levels of dG-dC crosslink in the groups treated with ACNU in combination with O6-BG were 402 to 3208 fmol/mg DNA, which were 1.2 to 3.3 times higher than those without O6-BG (307 to 1276 fmol/mg DNA). By Pearson correlation analysis, a significant correlation (R=0.62, P<0.01) was observed between the levels of dG-dC crosslink and the cell death rates, indicating that the crosslinking level was correlated with the cytotoxicity of the combination chemotherapy. To sum up, this study provided direct evidence for the enhancement of dG-dC crosslinking level due to the inhibition of AGT by O6-BG, which suggested that dG-dC crosslink might be developed as a biomarker for evaluating the activity of novel O6-BG analogs as AGT inhibitors in the combination therapy with CENUs.

Quantification of DNA interstrand crosslinks in human glioma cells treated by combination chemotherapy of chloroethylnitrosoureas and O<sup>6</sup>-benzylguanine using HPLC-ESI-MS/MS

248th National Meeting (2014)

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Quantification of DNA interstrand crosslinks in human glioma cells treated by combination chemotherapy of chloroethylnitrosoureas and O6-benzylguanine using HPLC-ESI-MS/MS

Quantification of DNA interstrand crosslinks in human glioma cells treated by combination chemotherapy of chloroethylnitrosoureas and O⁶-benzylguanine using HPLC-ESI-MS/MS