Nitrogen mustard inhibits transcription and translation in a cell free system
- 11 September 1995
- journal article
- research article
- Published by Oxford University Press (OUP) in Nucleic Acids Research
- Vol. 23 (17), 3508-3515
- https://doi.org/10.1093/nar/23.17.3508
Abstract
Nitrogen mustard and its derivatives such as cyclophosphamide, chlorambucil and melphalan are widely used anti-cancer agents, despite their non-specific reaction mechanism. In this study, the effect of alkylation by nitrogen mustard of DNA and RNA (coding for a single protein) was investigated using both a translation system and a coupled transcription/translation system. When alkylated DNA was used as the template for coupled transcription and translation, a single translation product corresponding to the 62 kDa luciferase protein was synthesised. Production of the translated product encoded by this template was inhibited by mustard concentrations as low as 10 nM, and 50% inhibition occurred with 30 nM mustard. A primer extension assay employed to verify alkylation 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, the amount of the 62 kDa luciferase protein decreased with increasing mustard concentration and a range of truncated polypeptides was synthesised. Under these conditions 50% inhibition of translation occurred with ∼300 nM mustard (i.e. ∼10 times that required for similar inhibition using an alkylated DNA template). Furthermore, a gel mobility shift assay revealed that mustard alkylation of the RNA template results in the formation of a more stable retarded RNA complex. The functional activity of the luciferase protein decreased with alkylation 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 μM mustard. The data presented support the notion that DNA is a critical molecule in the mode of action of mustards.Keywords
This publication has 29 references indexed in Scilit:
- Why, when and how does the poly(A) tail shorten during mRNA translation?International Journal of Biochemistry, 1993
- The early history of the biological alkylating agents, 1918–1968Mutation Research, 1990
- Tales of poly(A): a reviewGene, 1990
- Molecular events regulating messenger RNA stability in eukaryotesMolecular and Cellular Biochemistry, 1990
- Covalent Modification of DNA by Antineoplastic Agents5JNCI Journal of the National Cancer Institute, 1984
- Translation of satellite tobacco necrosis virus RNA modified by (±)-r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene is inhibited in a wheat germ cell-free systemCarcinogenesis: Integrative Cancer Research, 1983
- Secondary structure of mRNA and efficiency of translation initiationGene, 1980
- Influence of mRNA secondary structure on binding and migration of 40S ribosomal subunitsCell, 1980
- The action of mono- and di-functional sulphur mustards on the ribonucleic acid-containing bacteriophage μ2Biochemical Journal, 1971
- Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4Nature, 1970