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

Dennis Fontijn, Auke D. Adema, Kishor K. Bhakat, Herbert M. Pinedo, Godefridus J. Peters, Epie Boven

Research output: Contribution to journalArticle

11 Citations (Scopus)

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 O 6-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 O 6-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.

Original languageEnglish (US)
Pages (from-to)2807-2815
Number of pages9
JournalMolecular cancer therapeutics
Volume6
Issue number10
DOIs
StatePublished - Oct 1 2007

Fingerprint

temozolomide
Methyltransferases
Fibroblast Growth Factor 2
Melanoma
DNA
Protein Isoforms
Clone Cells
DNA Mismatch Repair
docetaxel
Cisplatin
O(6)-Methylguanine-DNA Methyltransferase
Azacitidine
Proteins
O-(6)-methylguanine
Doxorubicin
Methylation

ASJC Scopus subject areas

  • Oncology
  • Cancer Research

Cite this

O6-Methylguanine-DNA-methyltransferase promoter demethylation is involved in basic fibroblast growth factor-induced resistance against temozolomide in human melanoma cells. / Fontijn, Dennis; Adema, Auke D.; Bhakat, Kishor K.; Pinedo, Herbert M.; Peters, Godefridus J.; Boven, Epie.

In: Molecular cancer therapeutics, Vol. 6, No. 10, 01.10.2007, p. 2807-2815.

Research output: Contribution to journalArticle

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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 O 6-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 O 6-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.",
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AU - Adema, Auke D.

AU - Bhakat, Kishor K.

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AU - Peters, Godefridus J.

AU - Boven, Epie

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AB - 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 O 6-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 O 6-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.

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