Balb/c 3T3 cells

/Tag:Balb/c 3T3 cells

An Interlaboratory Validation Study of the Improved Transformation Assay Employing Balb/c 3T3 Cells: Results of a Collaborative Study on the Two-stage Cell Transformation Assay by the Non-genotoxic Carcinogen Study Group

Toshiyuki Tsuchiya, Makoto Umeda, Hiroshi Nishiyama, Isao Yoshimura, Shozo Ajimi, Masumi Asakura, Hiroshi Baba, Yasuaki Dewa, Youji Ebe, Yuichi Fushiwaki, Shuichi Hamada, Tetsuo Hamamura, Makoto Hayashi, Yumiko Iwase, Yoshitsugu Kajiwara, Yasushi Kasahara, Masayoshi Kawabata, Emiko Kitada, Kinya Kubo, Kaori Mashiko, Daisaku Miura, Fukutaro Mizuhashi, Fumio Mizuno, Madoka Nakajima, Yoshiyuki Nakamura, Naoko Nobe, Hidetoshi Oishi, Erina Ota, Ayako Sakai, Miho Sato, Sawako Shimada, Toshie Sugiyama, Chitose Takahashi, Yuko Takeda, Noriho Tanaka, Chikako Toyoizumi, Takeki Tsutsui, Shinobu Wakuri, Satoshi Yajima and Nobuhiro Yajima

The Non-genotoxic Carcinogen Study Group of the Environmental Mutagen Society of Japan organised the first step of an interlaboratory validation study on an improved cell transformation assay employing Balb/c 3T3 A31-1-1 cells. Nineteen laboratories participated in this study. The modified transformation assay was evaluated for its responsiveness, its interlaboratory reproducibility and its transferability. In this study, a mixture of Dulbecco's modified Eagle's medium and nutrient mixture F12, supplemented with insulin"transferrin" ethanolamine-sodium selenite and 2% fetal bovine serum (FBS) was used during the period of expression of transformed foci, intead of the usual minimum essential medium with 10% FBS. 20-Methylcholanthrene (MCA) and 12-O-tetradecanoylphorbol-13-acetate (TPA) were selected as a prototype initiator and a tumour promoter, respectively. Two series of experiments were conducted. In the first series, the transformation activity of MCA was examined at various concentrations. In the absence of the promoting treatment with TPA, exposure to MCA only weakly induced transformed foci. In the presence of 0.1µg/ml TPA, all laboratories observed significant dose-dependent increases in the number of transformed foci with increasing MCA concentrations. In the second series of experiments, various concentrations of TPA were tested. In the absence of initiating treatment with MCA, exposure to TPA weakly induced transformed foci in about half of the laboratories. In the presence of 0.2µg/ml MCA, all the laboratories observed significant dose-dependent increases in the number of transformed foci with increasing TPA concentrations. The results from this study support the usefulness of this modified two-stage transformation assay with Balb/c 3T3 cells.
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Technical Modification of the Balb/c 3T3 Cell Transformation Assay: The Use of Serum-reduced Medium to Optimise the Practicability of the Protocol

Kumiko Hayashi, Kiyoshi Sasaki, Shin Asada, Toshiyuki Tsuchiya, Makoto Hayashi, Isao Yoshimura, Noriho Tanaka and Makoto Umeda

The two-stage Balb/c 3T3 model of cell transformation can mimic the two-stage carcinogenicity bioassay, and has been recognised as a screening method for detecting potential tumour initiators and promoters. A technical modification to the original protocol (which involved the use of M10F medium, consisting of MEM plus 10% fetal bovine serum [FBS]) has been previously proposed, in order to increase its efficacy, namely: the introduction of enriched, serum-reduced medium (DF2F medium, comprising DMEM/F12 plus 2% FBS and other supplements). The aim of this study was to further modify the protocol, so as to attain higher practicability for the assay. The protocol was further optimised by: a) reducing the number of plates required, through the use of larger plates; b) reducing the cost of the assay by retaining the reduced serum concentration and by using 2μg/ml insulin, rather than the more-complex insulin–transferrin–ethanolamine–sodium selenite (ITES) supplement (i.e. DF2F2I medium); and c) extending the culture period from 24–25 days to 31–32 days, resulting in clearer foci (the number of medium changes did not increase, as less-frequent medium changes were performed during the extended culture period). Growth curve construction revealed that variations in the saturation densities of the parental Balb/c 3T3 cell line and its three transformed clones were highest when M10F medium was replaced with DF2F2I medium just before cells reached confluence. We applied this newly-optimised protocol to the assessment of: a) the tumour initiating activity of 3-methylcholanthrene (MCA), N-methyl-N´-nitro-Nnitrosoguanidine, mitomycin C, methylmethane sulphonate, CdCl2 and phenacetin, combining a posttreatment of 100ng/ml 12-O-tetradecanoylphorbol-13-acetate at the promotion stage; and b) the tumour promoting activity of insulin, lithocholic acid, CdCl2 and phenobarbital, with pre-treatment of 0.2μg/ml MCA at the initiation stage. In the present study, only phenobarbital was negative when tested by using the modified protocol. Key words: Balb/c 3T3 cells
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