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Research Articles: Therapeutics, Targets, and Development

O⁶-Methylguanine-DNA-methyltransferase promoter demethylation is involved in basic fibroblast growth factor–induced resistance against temozolomide in human melanoma cells

¹ Department of Medical Oncology, VU University medical center, Amsterdam, the Netherlands and ² Sealy Center for Molecular Science and Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, Texas

Requests for reprints: Epie Boven, Department of Medical Oncology, VU University medical center, De Boelelaan 1117, 1081 HV Amsterdam, the Netherlands. Phone: 31-20444-4336; Fax: 31-20444-4079. E-mail: e.boven{at}vumc.nl

Abstract

Basic fibroblast growth factor (bFGF) is a multifunctional protein and one of the most important growth factors in cutaneous melanoma development and progression. We hypothesized that high bFGF expression might be responsible for chemoresistance in advanced melanoma. M14 human melanoma cells expressing low levels of bFGF were successfully transfected with vectors encoding either the 18 kDa or all isoform proteins of bFGF. M14 cells and bFGF-overexpressing clones had a similar growth rate in vitro. Overexpression of 18 kDa or all isoform proteins of bFGF resulted in, respectively, 2.9- and 6.9-fold resistance against temozolomide. O⁶-Alkylguanine-DNA-alkyltransferase (AGT) protein levels were highly elevated. Specific inhibition of AGT with O⁶-benzylguanine completely reversed the resistance in the 18 kDa clone, and partially in the clone overexpressing all isoforms. A methylation-specific PCR showed that at least in the 18 kDa overexpressing clone, increased AGT expression was the result of demethylation of the O⁶-methylguanine-DNA-methyltransferase promoter. In parental M14 cells, the demethylating agent 5-azacytidine generated AGT expression resulting in temozolomide resistance. Overexpression of all isoform proteins of bFGF, but not the 18 kDa isoform alone, resulted in 2.9-fold resistance against cisplatin, which could not be reversed by O⁶-benzylguanine. The expression levels of the mismatch repair proteins MSH2, MSH6, and MLH1 were not decreased, which likely excludes a defective mismatch repair system as a cause for cisplatin resistance. There were no changes in sensitivity to docetaxel and doxorubicin. In conclusion, bFGF overexpression can result in resistance against temozolomide mediated by demethylation of the O⁶-methylguanine-DNA-methyltransferase promoter. [Mol Cancer Ther 2007;6(10):2807–15]

Grant support: Fritz Ahlqvist Foundation.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 1/19/07; revised 7/18/07; accepted 8/30/07.