Abstract

Melphalan, a DNA crosslinker, is one of the most widely used and effective drugs in the treatment of multiple myeloma (MM). Interstrand cross-links (ICL) are amongst the most toxic types of DNA damage; therefore, DNA cross-linking agents are important drugs in cancer treatment. Unfortunately, although most patients respond to standard and high dose melphalan therapy, eventually patients acquire drug resistance. Acquired melphalan resistance has been associated with reduced DNA crosslinks, elevated levels of glutathione and increased radiation survival. However, mechanisms associated with resistance are not well understood.

Evidence has accumulated to suggest that ICL repair contributes to the melphalan resistance. In this study, we compared the gene expression profile (GEP) of the melphalan-resistant myeloma cell line, 8226/LR5 to the 8226/S drug sensitive cell line, and found genes involved in FANC/BRCA DNA cross-link repair pathway had increased expression in drug resistant cells. The aim of our study was to determine whether FANC-BRCA pathway affects the DNA cross link repair capacity and accounts for acquired melphalan-resistance.

Using real time RT-PCR and Western Blotting, we examined the expression levels of FANC/BRCA pathway genes in two different drug sensitive and resistant cell lines: 8226/S 8226/LR5, and U266/S and U266/LR6. The results showed that increased expression of FANC/BRCA pathway genes correlated with the melphalan resistance. The formation of ICL at 5 hours after a 2 hour melphalan exposure was reduced in the LR5 compared to the drug sensitive cell 8226 using single cell comet assay. Using siRNA to knock down FANCL or FANCF in melphalan-resistant cell line LR5 reversed the drug resistance. Conversely, overexpression of FANCL or FANCF in the 8226/S drug sensitive cell line enhanced cell survival. These data show that enhanced DNA repair via Fanconi anemia/BRCA pathway is involved in melphalan-resistant myeloma cells.

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