Protection of mammalian cells against chloroethylating agent toxicity by an O6-benzylguanine-resistant mutant of human O6-alkylguanine-DNA alkyltransferase.
Fairbairn, Leslie J
Dexter, T Michael
Margison, Geoffrey P
Rafferty, Joseph A
AffiliationCRC Department of Experimental Haematology, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester, UK.
MetadataShow full item record
AbstractLow levels of expression in haemopoietic cells of the DNA repair protein O6-alkylguanine-DNA alkyltransferase (A Tase), is associated with the dose-limiting sensitivity of these cells to the chemotherapeutic chloroethylating and related methylating agents. Thus, the use of agents which deplete ATase such as O6-benzylguanine (O6-beG), as a tumour sensitisation strategy is likely further to potentiate collateral toxicity in bone marrow. In order to address this problem, we have engineered two mutants of human ATase (hAT) for resistance to O6-beG and characterised the in vitro properties of the proteins. In one mutant protein (hATPA), the proline at position 140 was changed to an alanine, whilst in the other (hATPA/GA) an additional mutation (glycine 156 to alanine) was also introduced. The I50 values for O6-beG of hAT, hATPA and hATPA/GA are 0.16, 2.5 and > 500 microM respectively, indicating that hATPA is resistant and hATPA/GA effectively refractory to O6-beG inactivation. Both mutant proteins retain comparable methyl transfer kinetics to those of nonmutant hAT and although they are thermally less stable in vitro than the wild-type protein, both can be substantially stabilised by DNA. Expression of either hAT or hATPA/GA following gene transfer into RJKO cells, raised the D37 value for mitozolomide from 0.35 microgram/ml for control cells to 10 micrograms/ml in the absence of O6-beG. However, whilst hAT-mediated protection was ablated by 20 microM O6-beG, the hATPA/GA protein provided protection against mitozolomide under the same conditions. Similar observations were made with chlorozotocin. The data suggest that transfer and expression of O6-beG resistant ATase in normal progenitor cells, should be a useful therapeutic strategy to protect the cells from the cytotoxic effects of the O6-alkylating agents even when used in combination with tumour sensitising agents such as O6-beG.
CitationProtection of mammalian cells against chloroethylating agent toxicity by an O6-benzylguanine-resistant mutant of human O6-alkylguanine-DNA alkyltransferase. 1996, 3 (10):868-77 Gene Ther.
- Chemoprotective gene transfer I: transduction of human haemopoietic progenitors with O6-benzylguanine-resistant O6-alkylguanine-DNA alkyltransferase attenuates the toxic effects of O6-alkylating agents in vitro.
- Authors: Hickson I, Fairbairn LJ, Chinnasamy N, Lashford LS, Thatcher N, Margison GP, Dexter TM, Rafferty JA
- Issue date: 1998 Jun
- Chemoprotective gene transfer II: multilineage in vivo protection of haemopoiesis against the effects of an antitumour agent by expression of a mutant human O6-alkylguanine-DNA alkyltransferase.
- Authors: Chinnasamy N, Rafferty JA, Hickson I, Lashford LS, Longhurst SJ, Thatcher N, Margison GP, Dexter TM, Fairbairn LJ
- Issue date: 1998 Jun
- Enhancing hemopoietic drug resistance: a rationale for reconsidering the clinical use of mitozolomide.
- Authors: Fairbairn LJ, Chinnasamy N, Lashford LS, Chinnasamy D, Rafferty JA
- Issue date: 2000 Feb
- O6-Benzylguanine potentiates BCNU but not busulfan toxicity in hematopoietic stem cells.
- Authors: Westerhof GR, Down JD, Blokland I, Wood M, Boudewijn A, Watson AJ, McGown AT, Ploemacher RE, Margison GP
- Issue date: 2001 May
- Modulation of O6-alkylating agent induced clastogenicity by enhanced DNA repair capacity of bone marrow cells.
- Authors: Chinnasamy N, Fairbairn LJ, Laher J, Willington MA, Rafferty JA
- Issue date: 1998 Aug 7