title: A newly established GDL1 cell line from gpt delta mice well reflects the in vivo mutation spectra induced by mitomycin C.
abstract: In order to create a novel in vitro test system for detection of large deletions and point mutations, we developed an immortalized cell line. A SV40 large T antigen expression unit was introduced into fibroblasts derived from gpt delta mouse lung tissue and a selected clone was established as the gpt delta L1 (GDL1) cell line. The novel GDL1 cells were examined for mutant frequencies (MFs) and for molecular characterization of mutations induced by mitomycin C (MMC). The GDL1 cells were treated with MMC at doses of 0.025, 0.05, and 0.1 microg/mL for 24h and mutations were detected by Spi- and 6-thioguanine (6-TG) selections. The MFs of the MMC-treated cells increased up to 3.4-fold with Spi- selection and 3.5-fold with 6-TG selection compared to MFs of untreated cells. In the Spi- mutants, the number of large (up to 76 kilo base pair (kbp)) deletion mutations increased. A majority of the large deletion mutations had 1-4 base pairs (bp) of microhomology in the deletion junctions. A number of the rearranged deletion mutations were accompanied with deletions and insertions of up to 1.1 kbp. In the gpt mutants obtained from 6-TG selection, single base substitutions of G:C to T:A, tandem base substitutions occurring at the 5'-GG-3' or 5'-CG-3' sequence, and deletion mutations larger than 2 bp were increased. We compared the spectrum of MMC-induced mutations observed in vitro to that of in vivo using gpt delta mice, which we reported previously. Although a slight difference was observed in MMC-induced mutation spectra between in vitro and in vivo, the mutations detected in vitro included all of the types of mutations observed in vivo. The present study demonstrates that the newly established GDL1 cell line is a useful tool to detect and analyze various mutations including large deletions in mammalian cells.
authors.affiliation: Fuji Gotemba Research Laboratories, Chugai Pharmaceutical Co. Ltd., 1-135 Komakado, Gotemba, Shizuoka 412-8513, Japan. takeiriakr@chugai-pharm.co.jp
authors.forename: Akira
authors.initials: A
authors.lastname: Takeiri
authors.forename: Masayuki
authors.initials: M
authors.lastname: Mishima
authors.forename: Kenji
authors.initials: K
authors.lastname: Tanaka
authors.forename: Akifumi
authors.initials: A
authors.lastname: Shioda
authors.forename: Asako
authors.initials: A
authors.lastname: Harada
authors.forename: Kazuto
authors.initials: K
authors.lastname: Watanabe
authors.forename: Ken-Ichi
authors.initials: K
authors.lastname: Masumura
authors.forename: Takehiko
authors.initials: T
authors.lastname: Nohmi
mesh_terms: D000477:Alkylating Agents / Q000633:toxicity; D000818:Animals; D010582:Bacteriophage lambda / Q000235:genetics; D001483:Base Sequence; D002461:Cell Line, Transformed; D029968:Escherichia coli Proteins / Q000235:genetics*; D005814:Genes, Viral / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D016685:Mitomycin / Q000633:toxicity*; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional / Q000187:drug effects / Q000235:genetics; D009152:Mutagenicity Tests / Q000379:methods; D009154:Mutation / Q000187:drug effects* / Q000235:genetics; D010430:Pentosyltransferases / Q000235:genetics*; D015203:Reproducibility of Results; D017384:Sequence Deletion / Q000187:drug effects / Q000235:genetics
chemical_list: D000477:Alkylating Agents; D029968:Escherichia coli Proteins; D016685:Mitomycin; D010430:Pentosyltransferases; C071962:Gpt protein, E coli