Establishment of lung fibroblastic cell lines from a non-human primate Tupaia belangeri and their use in a forward gene mutation assay at the hypoxanthing—guanine phosphoribosyl transferase locus
Taketomi, Masako; Nishi, Yoshisuke; Ohkawa, Yoshihiko; Inui, Naomichi; Section of Cell Biology and Cytogenetics, Biological Research CenterJapan Tobacco Inc., 23 Nakogi Hatano, Kanagawa 257, Japan
Журнал:
Mutagenesis
Дата:
1986
Аннотация:
The cells obtained from a lung of a new-born male Tupaia belangeri were maintained in mass culture for >400 days. After 55 population doubling levels (100 days in culture), three cell lines were separately established; these lines showed constant growth properties. One line, designated as T-23, was used for a mutation assay. The T-23 cells showed an absolute plating efficiency of 30–50%, and a population doubling time of 18–19 h in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. The cells had a modal chromosome number of 62 (pseudodiploid) with the loss of a chromosome and the gain of an unidentified one. T-23 cells, like human cells, were much more susceptible to ouabain than mouse cells but relatively less susceptible to 8-azaguanine, while, unlike human cells, they were less sensitive to 6-thioguanine (6TG). N-Methyl-N′-nitro-N-nitrosoguanidine (MNNG) was less, but 4-nitroquinoline-l-oxide (4NQO) was more toxic to T-23 cells than to human or mouse cells. Benzo[a]pyrene-induced toxicity was almost comparable among the cell types. For the mutation assay, we chose 6TG-resistance (100 μM) as a marker. The optimal expression time (8–13 days) and cell density at selection to eliminate metabolic cooperation (2 × 10<sup>4</sup> cells/ 60-mm dish) were determined. Some of the cells selected with 6TG showed <0.4% of the total incorporation of [<sup>14</sup>C]hypox-anthine into wild-type cells, suggesting the mutants under selection were affected at the hypoxanthine-guanine phos-phoribosyl transferase locus. Following the optimal protocol, we performed a quantitative mutation assay with simple alky-lating agents, MNNG, N-ethyl-N′-nitro-N-nitrosoguanidine, methyl methanesulphonate, ethyl methanesulphonate and 4NQO. All induced mutants in a dose-dependent fashion. The ability to induce mutants in T-23 cells by these mutagens was comparable with that in a conventional V79 assay. This is the first report of a gene mutation assay system using non-human primate cells.
671.3Кб