Histone H3 and heat shock protein GRP78 are selectively cross-linked to DNA by photoactivated gilvocarcin V in human fibroblasts

Abstract

Gilvocarcin V (GV) is an antitumor antibiotic with a coumarin-based aromatic structure that promotes protein-DNA cross-linking when photoactivated by near-UV light. We have now identified several proteins that are selectively cross-linked to DNA in human fibroblasts by photoactivated GV, using NH2-terminal amino acid sequencing and Western blot analysis of the purified cross-linked proteins. The selectively cross-linked proteins are histone H3 and GRP78, a heat shock protein belonging to the heat shock protein-70 family. The hydrophobic leader sequence is missing from the cross-linked GRP78, suggesting that only the processed form of the protein is cross-linked to DNA. It is primarily the phosphorylated form of histone H3 that is cross-linked to DNA. Gel retardation analysis from four different GV-treated human fibroblast cell lines revealed two distinct shifted bands, and subsequent immunoblotting confirmed in situ that the slower and the faster bands, respectively, contained GRP78 and histone H3 cross-linked to DNA. The selective cross-linking of these particular proteins is dependent on UV irradiation in the presence of GV, which may help to clarify the unique molecular mechanism of this potent antitumor agent.