NITROGEN-MUSTARD INHIBITS TRANSCRIPTION AND TRANSLATION IN A CELL-FREE SYSTEM

Nitrogen mustard and its derivatives such as cyclophosphamide, chloram bucil and melphalan are widely used anti-cancer agents, despite their non-specific reaction mechanism. In this study, the effect of alkylati on by nitrogen mustard of DNA and RNA (coding for a single protein) wa s investigated using both a translation system and a coupled transcrip tion/translation system. When alkylated DNA was used as the template f or coupled transcription and translation, a single translation product corresponding to the 62 kDa luciferase protein was synthesised. Produ ction of the translated product encoded by this template was inhibited by mustard concentrations as low as 10 nM, and 50% inhibition occurre d with 30 nM mustard, A primer extension assay employed to verify alky lation sites on the DNA revealed that all guanine residues on the DNA template are susceptible to alkylation by nitrogen mustard, Similarly, when alkylated RNA was used as the template for protein synthesis, th e amount of the 62 kDa luciferase protein decreased with increasing mu stard concentration and a range of truncated polypeptides was synthesi sed, Under these conditions 50% inhibition of translation occurred wit h similar to 300 nM mustard (i.e. similar to 10 times that required fo r similar inhibition using an alkylated DNA template), Furthermore, a gel mobility shift assay revealed that mustard alkylation of the RNA t emplate results in the formation of a more stable retarded RNA complex , The functional activity of the luciferase protein decreased with alk ylation of both the DNA and RNA templates, with a half-life of loss of activity of 1.1 h for DNA exposed to 50 nM mustard, and 0.5 h for RNA exposed to 50 mu M mustard, The data presented support the notion tha t DNA is a critical molecule in the mode of action of mustards.