Molecular characterization of mitomycin C-induced large deletions and tandem-base substitutions in the bone marrow of gpt delta transgenic mice.
Deletion mutations constitute an important class of mutations that may result in a variety of human diseases, including cancer. Although many chemicals and ionizing radiations induce deletions, this class of mutation has been poorly characterized at the molecular level, particularly in vivo. Here we report the molecular nature of deletions as well as base substitutions induced by antitumor antibiotic mitomycin C (MMC) in the bone marrow using a novel transgenic mouse, gpt delta. In this mouse model, deletions and point mutations in lambda DNA integrated in the chromosome are individually selected as Spi(-) (sensitive to P2 interference) phages and 6-thioguanine-resistant bacterial colonies, respectively. The mice were treated with MMC (1 mg/kg/day) for five consecutive days. One week after the last treatment, lambda phage was rescued from the genomic DNA of the bone marrow by in vitro packaging reactions and subjected to Spi(-) and 6-thioguanine selections. The mutant frequency of Spi(-) with large deletions increased more than 20-fold over that of the control. Molecular sizes of the large deletions were mostly more than 2,000 base pairs. The large deletions frequently occurred between two short direct repeat sequences from 2 to 6 base pairs, suggesting that they are generated during the end-joining repair of double-strand breaks induced by interstrand cross-links in DNA. In 6-thioguanine selection, tandem-base substitutions, such as 5'-GG-3' to 5'-AT-3', were induced. It highlights the relevance of intrastrand cross-links as genotoxic lesions. Previous in vitro studies report the induction of single-base substitutions and single-base deletions by MMC. However, no such mutations were identified in vivo. Thus, our results strongly caution that in vitro mutation spectra do not necessarily reflect genotoxic events in vivo and emphasize the importance of transgenic rodent genotoxicity assays to examine the roles of DNA adducts in mutagenesis and carcinogenesis.
Takeiri
Akira
A
Fuji Gotemba Research Laboratories, Chugai Pharmaceutical Co., Ltd., 1-135 Komakado, Gotemba-shi, Shizuoka 412-8513, Japan.
Mishima
Masayuki
M
Tanaka
Kenji
K
Shioda
Akihumi
A
Ueda
Otoya
O
Suzuki
Hiroshi
H
Inoue
Makoto
M
Masumura
Ken-ichi
K
Nohmi
Takehiko
T
D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000633:toxicity*; D010582:Bacteriophage lambda / Q000235:genetics; D020137:Base Pair Mismatch; D001483:Base Sequence; D001853:Bone Marrow / Q000187:drug effects*; D004247:DNA / Q000235:genetics*; D005260:Female; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D016685:Mitomycin / Q000633:toxicity*; D008969:Molecular Sequence Data; D009152:Mutagenicity Tests; D009153:Mutagens / Q000633:toxicity; D017384:Sequence Deletion
D000903:Antibiotics, Antineoplastic; D009153:Mutagens; D016685:Mitomycin; D004247:DNA