Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1995 Sep 11;23(17):3508–3515. doi: 10.1093/nar/23.17.3508

Nitrogen mustard inhibits transcription and translation in a cell free system.

A Masta 1, P J Gray 1, D R Phillips 1
PMCID: PMC307231  PMID: 7567463

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 approximately 300 nM mustard (i.e. approximately 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 microM mustard. The data presented support the notion that DNA is a critical molecule in the mode of action of mustards.

Full text

PDF
3508

Images in this article

3510Image
on p.3510
3511Image
on p.3511
3512Image
on p.3512
3513Image
on p.3513

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bohr V. A. Gene specific DNA repair. Carcinogenesis. 1991 Nov;12(11):1983–1992. doi: 10.1093/carcin/12.11.1983. [DOI] [PubMed] [Google Scholar]
  2. Brookes P. The early history of the biological alkylating agents, 1918-1968. Mutat Res. 1990 Nov-Dec;233(1-2):3–14. doi: 10.1016/0027-5107(90)90145-t. [DOI] [PubMed] [Google Scholar]
  3. Carr F. J., Fox B. W. The effects of bifunctional alkylating agents on DNA synthesis in sensitive and resistant Yoshida cells. Mutat Res. 1982 Aug;95(2-3):441–456. doi: 10.1016/0027-5107(82)90277-9. [DOI] [PubMed] [Google Scholar]
  4. Crathorn A. R., Roberts J. J. Mechanism of the cytotoxic action of alkylating agents in mammalian cells and evidence for the removal of alkylated groups from deoxyribonucleic acid. Nature. 1966 Jul 9;211(5045):150–153. doi: 10.1038/211150a0. [DOI] [PubMed] [Google Scholar]
  5. Decker C. J., Parker R. A turnover pathway for both stable and unstable mRNAs in yeast: evidence for a requirement for deadenylation. Genes Dev. 1993 Aug;7(8):1632–1643. doi: 10.1101/gad.7.8.1632. [DOI] [PubMed] [Google Scholar]
  6. Gilman A., Philips F. S. The Biological Actions and Therapeutic Applications of the B-Chloroethyl Amines and Sulfides. Science. 1946 Apr 5;103(2675):409–436. doi: 10.1126/science.103.2675.409. [DOI] [PubMed] [Google Scholar]
  7. Gray P. J., Cullinane C., Phillips D. R. In vitro transcription analysis of DNA alkylation by nitrogen mustard. Biochemistry. 1991 Aug 13;30(32):8036–8040. doi: 10.1021/bi00246a022. [DOI] [PubMed] [Google Scholar]
  8. Gray P. J., Phillips D. R. Effect of alkylating agents on initiation and elongation of the lac UV5 promoter. Biochemistry. 1993 Nov 23;32(46):12471–12477. doi: 10.1021/bi00097a027. [DOI] [PubMed] [Google Scholar]
  9. Grunberger D., Pergolizzi R. G., Jones R. E. Translation of globin messenger RNA modified by benzo[a]pyrene 7,8-dihydrodiol 9,10-oxide in a wheat germ cell-free system. J Biol Chem. 1980 Jan 25;255(2):390–394. [PubMed] [Google Scholar]
  10. Haas R., Pulkrabek P., Takanami Y., Grunberger D. Translation of satellite tobacco necrosis virus RNA modified by (not equal to)-r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene is inhibited in a wheat germ cell-free system. Carcinogenesis. 1983;4(2):221–225. doi: 10.1093/carcin/4.2.221. [DOI] [PubMed] [Google Scholar]
  11. Hartley J. A., Berardini M. D., Souhami R. L. An agarose gel method for the determination of DNA interstrand crosslinking applicable to the measurement of the rate of total and "second-arm" crosslink reactions. Anal Biochem. 1991 Feb 15;193(1):131–134. doi: 10.1016/0003-2697(91)90052-u. [DOI] [PubMed] [Google Scholar]
  12. Hastie N. D., Held W. A. Analysis of mRNA populations by cDNA.mRNA hybrid-mediated inhibition of cell-free protein synthesis. Proc Natl Acad Sci U S A. 1978 Mar;75(3):1217–1221. doi: 10.1073/pnas.75.3.1217. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hemminki K., Kallama S. Reactions of nitrogen mustards with DNA. IARC Sci Publ. 1986;(78):55–70. [PubMed] [Google Scholar]
  14. Hemminki K., Ludlum D. B. Covalent modification of DNA by antineoplastic agents. J Natl Cancer Inst. 1984 Nov;73(5):1021–1028. [PubMed] [Google Scholar]
  15. Iserentant D., Fiers W. Secondary structure of mRNA and efficiency of translation initiation. Gene. 1980 Apr;9(1-2):1–12. doi: 10.1016/0378-1119(80)90163-8. [DOI] [PubMed] [Google Scholar]
  16. Kozak M. Influence of mRNA secondary structure on binding and migration of 40S ribosomal subunits. Cell. 1980 Jan;19(1):79–90. doi: 10.1016/0092-8674(80)90390-6. [DOI] [PubMed] [Google Scholar]
  17. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  18. Lawley P. D., Brookes P. Molecular mechanism of the cytotoxic action of difunctional alkylating agents and of resistance to this action. Nature. 1965 May 1;206(983):480–483. doi: 10.1038/206480a0. [DOI] [PubMed] [Google Scholar]
  19. Liebhaber S. A., Cash F., Eshleman S. S. Translation inhibition by an mRNA coding region secondary structure is determined by its proximity to the AUG initiation codon. J Mol Biol. 1992 Aug 5;226(3):609–621. doi: 10.1016/0022-2836(92)90619-u. [DOI] [PubMed] [Google Scholar]
  20. Masta A., Gray P. J., Phillips D. R. Molecular basis of nitrogen mustard effects on transcription processes: role of depurination. Nucleic Acids Res. 1994 Sep 25;22(19):3880–3886. doi: 10.1093/nar/22.19.3880. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Mattes W. B., Hartley J. A., Kohn K. W. DNA sequence selectivity of guanine-N7 alkylation by nitrogen mustards. Nucleic Acids Res. 1986 Apr 11;14(7):2971–2987. doi: 10.1093/nar/14.7.2971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Munroe D., Jacobson A. Tales of poly(A): a review. Gene. 1990 Jul 16;91(2):151–158. doi: 10.1016/0378-1119(90)90082-3. [DOI] [PubMed] [Google Scholar]
  23. Murnane J. P., Byfield J. E., Ward J. F., Calabro-Jones P. Effects of methylated xanthines on mammalian cells treated with bifunctional alkylating agents. Nature. 1980 May 29;285(5763):326–329. doi: 10.1038/285326a0. [DOI] [PubMed] [Google Scholar]
  24. Pieper R. O., Futscher B. W., Erickson L. C. Transcription-terminating lesions induced by bifunctional alkylating agents in vitro. Carcinogenesis. 1989 Jul;10(7):1307–1314. doi: 10.1093/carcin/10.7.1307. [DOI] [PubMed] [Google Scholar]
  25. Pullman A., Pullman B. Molecular electrostatic potential of the nucleic acids. Q Rev Biophys. 1981 Aug;14(3):289–380. doi: 10.1017/s0033583500002341. [DOI] [PubMed] [Google Scholar]
  26. Roberts J. J., Kotsaki-Kovatsi V. P. Potentiation of sulphur mustard or cisplatin-induced toxicity by caffeine in Chinese hamster cells correlates with formation of DNA double-strand breaks during replication on a damaged template. Mutat Res. 1986 May;165(3):207–220. doi: 10.1016/0167-8817(86)90056-8. [DOI] [PubMed] [Google Scholar]
  27. Rubin H. N., Halim M. N. Why, when and how does the poly(A) tail shorten during mRNA translation? Int J Biochem. 1993 Mar;25(3):287–295. doi: 10.1016/0020-711x(93)90615-l. [DOI] [PubMed] [Google Scholar]
  28. Saini K. S., Summerhayes I. C., Thomas P. Molecular events regulating messenger RNA stability in eukaryotes. Mol Cell Biochem. 1990 Jul 17;96(1):15–23. doi: 10.1007/BF00228449. [DOI] [PubMed] [Google Scholar]
  29. Shooter K. V., Edwards P. A., Lawley P. D. The action of mono- and di-functional sulphur mustards on the ribonucleic acid-containing bacteriophage mu2. Biochem J. 1971 Dec;125(3):829–840. doi: 10.1042/bj1250829. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Tan K. B., Mattern M. R., Boyce R. A., Schein P. S. Elevated DNA topoisomerase II activity in nitrogen mustard-resistant human cells. Proc Natl Acad Sci U S A. 1987 Nov;84(21):7668–7671. doi: 10.1073/pnas.84.21.7668. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Wang P., Bauer G. B., Bennett R. A., Povirk L. F. Thermolabile adenine adducts and A.T base pair substitutions induced by nitrogen mustard analogues in an SV40-based shuttle plasmid. Biochemistry. 1991 Dec 10;30(49):11515–11521. doi: 10.1021/bi00113a005. [DOI] [PubMed] [Google Scholar]
  32. Zhou W., Doetsch P. W. Effects of abasic sites and DNA single-strand breaks on prokaryotic RNA polymerases. Proc Natl Acad Sci U S A. 1993 Jul 15;90(14):6601–6605. doi: 10.1073/pnas.90.14.6601. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES