The Prevention of Thymic Lymphomas in Transgenic Mice by Human O ⁶ -Alkylguanine-DNA Alkyltransferase

Abstract

Nitrosoureas form O6-alkylguanine-DNA adducts that are converted to G to A transitions, the mutation found in the activated ras oncogenes of nitrosourea-induced mouse lymphomas and rat mammary tumors. These adducts are removed by the DNA repair protein O6-alkylguanine-DNA alkyltransferase. Transgenic mice that express the human homolog of this protein in the thymus were found to be protected from developing thymic lymphomas after exposure to N-methyl-N-nitrosourea. Thus, transgenic expression of a single human DNA repair gene is sufficient to block chemical carcinogenesis. The transduction of DNA repair genes in vivo may unravel mechanisms of carcinogenesis and provide therapeutic protection from known carcinogens.

Mice over-expressing human O6alkylguanine-DNA alkyltransferase selectively reduce O6methylguanine mediated carcinogenic mutations to threshold levels after N-methyl-N-nitrosourea

“…Following a single i.p. injection of 80 mg/kg MNU, the incidence of thymic lymphomas in nontransgenic mice was 84% compared to 13% in hMGMT+ mice (Dumenco et al, 1993). Since our report, others have shown a protective e ect of AGT on dimethylnitrosamine induced hepatic tumors (Nakatsuru et al, 1993) and on skin tumors induced by TPA and MNU (Becker et al, 1996).…”

“…We have previously described transgenic mice which overexpress human [h]AGT (Dumenco et al, 1993). These mice express hAGT mainly in thymus and spleen, because the transgene contains the 2 kb locus control region from the CD2 gene.…”

“…AGT activity in the spleen of hMGMT+/Big Blue TM mice was 21.7+5.1 fmol/mg DNA, 20-fold higher than in Big Blue TM mice. There was no di erence in liver AGT because the transgene is not expressed in liver (Dumenco et al, 1993).…”

“…Table 2 shows that a G : C to A : T mutation in the second position of codon 12 (GGT to GAT) was present in 43 out of 59 lymphomas from mice lacking the hMGMT transgene; 38% of lymphomas from mice treated with 50 mg/kg MNU, 53% from mice treated at 80 mg/kg MNU and 69% from mice treated with The alkyltransferase assay has been published in detail (Liu et al, 1994). Brie¯y, activity was measured in sonicated tissue extracts by quantifying the amount of (Dumenco et al, 1993) were bred to heterozygous male Big Blue TM (lacI+/B6; Stratagene, La Jolla, CA, USA), (Kohler et al, 1991) mice. O spring were treated with 80 mg/kg MNU at 6 weeks of age and followed for 1 year.…”

“…All such mice had thymic lymphomas. Left, cohort of hMGMT+ and nontransgenic mice, taken from Dumenco et al (1993); Right, six Big Blue TM and eight Big Blue TM hMGMT+ mice (83) 3 (14) 16 (88) 6 (25) 5 (38%) ± 16 (53) 1 (33) 22 (69) 3 (50) Mutations in codon 12 at K-ras were determined as previously described (Zaidi et al, 1995b), by restriction fragment length polymorphism (RFLP) using nested primers which insert a restriction site sequence into either mutant GAT or wild type GGT codon 12. Polymerase chain reaction (PCR) followed by restriction enzyme digestion identi®ed mutations as either G to A or not G to A.…”