Purpose  

The DNA repair protein, O ⁶-alkylguanine-DNA alkyltransferase (AGT), is a primary source of tumor resistance to agents such as temozolomide and chloroethylnitrosoureas that form DNA lesions at the O ⁶-position of guanines. To increase the efficacy of these drugs, pseudosubstrate inactivators of AGT such as O ⁶-benzylguanine have been developed. A novel inactivator of AGT, O ⁴-benzylfolic acid (O⁴-BFA), has been reported which is more potent and water soluble than O ⁶-benzylguanine. Previous studies have suggested that uptake of O⁴-BFA is mediated by the folate receptor (FR), and, thus, its use may be limited to cells expressing FR.

Methods  

We measured AGT activity in cell extracts from a panel of brain tumor cells exposed to O⁴-BFA. Inactivation of AGT by O⁴-BFA was measured in cells grown without folic acid as well as in cells grown in folic acid-containing media. Competitive binding studies were performed using purified FR to determine its affinity for O⁴-BFA.

Results  

The observed IC₅₀ for O⁴-BFA in brain tumor cell lines ranged from 0.2 to 1.3 μM for cells grown in media containing 2.3 μM folic acid. At this concentration, folic acid would saturate the FR and the FR would be unable to take up O⁴-BFA. When cells were grown in folic acid free media, there was at most a 50% decrease in the observed IC₅₀s, indicating that the FR was not essential for O⁴-BFA uptake. Competitive binding studies using purified FR confirmed that the IC₅₀ for O⁴-BFA is ∼180 times greater than folic acid, i.e., it has a very weak affinity for FR.

Conclusion  

These results indicate that O⁴-BFA has potentially broad use as an inactivator of AGT as its use is not limited to tumors expressing high levels of FR.