cell transformation assay

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An Inter-laboratory Collaborative Study by the Non- Genotoxic Carcinogen Study Group in Japan, on a Cell Transformation Assay for Tumour Promoters Using Bhas 42 cells

Kiyomi Ohmori, Makoto Umeda, Noriho Tanaka, Hiroki Takagi, Isao Yoshimura, Kiyoshi Sasaki, Shin Asada, Ayako Sakai, Harumi Araki, Masumi Asakura, Hiroshi Baba, Yuichi Fushiwaki, Shuichi Hamada, Nobuko Kitou, Tetsu Nakamura, Yoshiyuki Nakamura, Hidetoshi Oishi, Satoshi Sasaki, Sawako Shimada, Toshiyuki Tsuchiya, Yoshifumi Uno, Masataka Washizuka, Satoshi Yajima, Yasuhito Yamamoto, Eiji Yamamura and Tomoko Yatsushiro

The Bhas promotion assay is a cell culture transformation assay designed as a sensitive and economical method for detecting the tumour-promoting activities of chemicals. In order to validate the transferability and applicability of this assay, an inter-laboratory collaborative study was conducted with the participation of 14 laboratories. After confirmation that these laboratories could obtain positive results with two tumour promoters, 12-O-tetradecanoylphorbol-13-acetate (TPA) and lithocholic acid (LCA), 12 coded chemicals were assayed. Each chemical was tested in four laboratories. For eight chemicals, all four laboratories obtained consistent results, and for two of the other four chemicals, only one of the four laboratories showed inconsistent results. Thus, the rate of consistency was high. During the study, several issues were raised, each of which were analysed step-by-step, leading to revision of the protocol of the original assay. Among these issues were the importance of careful maintenance of mother cultures and the adoption of test concentrations for toxic chemicals. In addition, it is suggested that three different types of chemicals show positive promoting activity in the assay. Those designated as T-type induced extreme growth enhancement, and included TPA, mezerein, PDD and insulin. LCA and okadaic acid belonged to the L-type category, in which transformed foci were induced at concentrations showing growth-inhibition. In contrast, M-type chemicals, progesterone, catechol and sodium saccharin, induced foci at concentrations with little or slight growth inhibition. The fact that different types of chemicals similarly induce transformed foci in the Bhas promotion assay may provide clues for elucidating mechanisms of tumour promotion.
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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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The Cell Transformation Assay: Toward a Statistical Classification of Mixed and Intermediate Foci Images

Claudio Procaccianti, Federico M. Stefanini and Chiara Urani

The human carcinogenicity evaluation of chemicals has a great impact on public health. In vitro methods, such as the cell transformation assay (CTA), allow for a fast and reliable assessment of the carcinogenic potential of a chemical compound in comparison with the standard two-year bioassay. The scoring and classification of foci in selected cell lines is performed, after staining, by light microscopy. Foci can be separated into three classes: type I, which are scored as non-transformed, and types II and III that are considered to include fully transformed foci. However, in a number of cases, even an expert is uncertain about the attribution of a focus to a given class, due to its mixed or intermediate nature. Here, we suggest a simple approach to classifying mixed or intermediate foci by exploiting the quantitative information available from images, which is captured by statistical descriptors. A quantitative index is proposed, to describe the degree of dissimilarity of mixed and intermediate images to the three well-distinguished classes.
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Cell Transformation Assays: Are We Barking Up the Wrong Tree?

Robert D. Combes

There has been a current resurgence of interest in the use of cell transformation for predicting carcinogenicity, which is based mainly on rodent carcinogenicity data. In view of this renewed interest, this paper critically reviews the published literature concerning the ability of the available assays to detect IARC Group 1 agents (known human carcinogens) and Group 2A agents (probable human carcinogens). The predictivity of the available assays for human and rodent non-genotoxic carcinogens (NGCs), in comparison with standard and supplementary in vitro and in vivo genotoxicity tests, is also discussed. The principal finding is that a surprising number of human carcinogens have not been tested for cell transformation across the three main assays (SHE, Balb/c 3T3 and C3H10T1/2), confounding comparative assessment of these methods for detecting human carcinogens. This issue is not being addressed in the ongoing validation studies for the first two of these assays, despite the lack of any serious logistical issues associated with the use of most of these chemicals. In addition, there seem to be no plans for using exogenous bio-transformation systems for the metabolic activation of pro-carcinogens, as recommended in an ECVAM workshop held in 1999. To address these important issues, it is strongly recommended that consideration be given to the inclusion of more human carcinogens and an exogenous source of xenobiotic metabolism, such as an S9 fraction, in ongoing and future validation studies. While cell transformation systems detect a high level of NGCs, it is considered premature to rely only on this endpoint for screening for such chemicals, as recently suggested. This is particularly important, in view of the fact that there is still doubt as to the relevance of morphological transformation to tumorigenesis in vivo, and the wide diversity of potential mechanisms by which NGCs are known to act. Recent progress with regard to increasing the objectivity of scoring the transformed phenotype, and prospects for developing human cell-based transformation assays, are reviewed.
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