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O 4-benzylfolic acid inactivates O 6-alkylguanine-DNA alkyltransferase in brain tumor cell lines

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Abstract

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.

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Abbreviations

AGT:

O 6-alkylguanine-DNA-alkyltransferase

BCNU:

1,3-bis(2-chloroethyl)-1-nitrosourea

O6-BG:

O 6-benzylguanine

O4-BFA:

O 4-benzylfolic acid

FR:

Folate receptor

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Acknowledgment

This work was supported by NIH Grant 5P50CA108786 and the Pediatric Brain Tumor Foundation.

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Authors and Affiliations

  1. Department of Surgery, Duke University Medical Center, Box 2616, Durham, NC, 27710, USA

    Stewart P. Johnson & Henry S. Friedman

  2. Department of Pediatric Hematology/Oncology, Cancer Institute of New Jersey, UMDNJ/Robert Wood Johnson Medical School, New Brunswick, NJ, 08901, USA

    Barton A. Kamen

  3. Department of Pathology, Duke University Medical Center, Durham, NC, 27710, USA

    Darell D. Bigner

  4. Department of Medicine, Duke University Medical Center, Durham, NC, 27710, USA

    Henry S. Friedman

  5. Department of Pediatrics, Duke University Medical Center, Durham, NC, 27710, USA

    Henry S. Friedman

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  1. Stewart P. Johnson
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Correspondence to Stewart P. Johnson.

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Johnson, S.P., Kamen, B.A., Bigner, D.D. et al. O 4-benzylfolic acid inactivates O 6-alkylguanine-DNA alkyltransferase in brain tumor cell lines. Cancer Chemother Pharmacol 60, 883–889 (2007). https://doi.org/10.1007/s00280-007-0435-6

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