Photo catalogue for the classification of foci in the BALB/c 3T3 cell transformation assay

Sasaki, Kiyoshi; Bohnenberger, Susanne; Hayashi, Kumiko; Kunkelmann, Thorsten; Muramatsu, Dai; Poth, Albrecht; Sakai, Ayako; Salovaara, Susan; Tanaka, Noriho; Thomas, B. Claire; Umeda, Makoto

doi:10.1016/j.mrgentox.2012.01.009

Cited by 34 publications

(29 citation statements)

References 3 publications

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“…When Bhas 42 cells alone were cultured and treated repeatedly with a given tumor promoter for an extended period of time (more than one week), quantifiable transformed foci were formed on a contact-inhibited monolayer (promotion assay). Since the transformed foci show similar morphology to those of BALB/c 3T3 cells, they are scored according to the criteria used for BALB/c 3T3 cells (basophilic, spindle-shaped, crisscross, piling up, and invasive) (Figure 4) [2,47]. Based on the cell growth pattern in the cytotoxicity test, Ohmori and colleagues [48] of chemicals must have different mechanism of promotion activity from the growth enhancement.…”

Section: Single-culture Transformation Assay For Tumor Promotersmentioning

confidence: 99%

Transformation Assay in Bhas 42 Cells: A Model Using Initiated Cells to Study Mechanisms of Carcinogenesis and Predict Carcinogenic Potential of Chemicals

Sasaki

Umeda

Sakai

et al. 2015

Journal of Environmental Science and Health, Part C

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Transformation assays using cultured cells have been applied to the study of carcinogenesis. Although various cell systems exist, few cell types such as BALB/c 3T3 subclones and Syrian hamster embryo cells have been used to study chemically induced two-stage carcinogenesis. Bhas 42 cells were established as a clone by the transfection with the v-Ha-ras gene into mouse BALB/c 3T3 A31-1-1 cells and their subsequent selection based on their sensitivity to 12-O-tetradecanoylphorbol-13-acetate. Using Bhas 42 cells, transformed foci were induced by the treatment with nongenotoxic carcinogens, most of which act as tumor promoters. Therefore, Bhas 42 cells were considered to be a model of initiated cells. Subsequently, not only nongenotoxic carcinogens but also genotoxic carcinogens, most of which act as tumor initiators, were found to induce transformed foci by the modification of the protocol. Furthermore, transformation of Bhas 42 cells was induced by the transfection with genes of oncogenic potential. We interpret this high sensitivity of Bhas 42 cells to various types of carcinogenic stimuli to be related to the multistage model of carcinogenesis, as the transfection of v-Ha-ras gene further advances the parental BALB/c 3T3 A31-1-1 cells toward higher transforming potential. Thus, we propose that Bhas 42 cells are a novel and sensitive cell line for the analysis of carcinogenesis and can be used for the detection of not only carcinogenic substances but also gene alterations related to oncogenesis. This review will address characteristics of Bhas 42 cells, the transformation assay protocol, validation studies, and the various chemicals tested in this assay.

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Section: Single-culture Transformation Assay For Tumor Promotersmentioning

confidence: 99%

Transformation Assay in Bhas 42 Cells: A Model Using Initiated Cells to Study Mechanisms of Carcinogenesis and Predict Carcinogenic Potential of Chemicals

Sasaki

Umeda

Sakai

et al. 2015

Journal of Environmental Science and Health, Part C

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“…After treatment with the tumor initiator MCA (treated on day 1 to 4) followed by the tumor promoter TPA (day 8–21), transformed cells started to grow over the normal monolayer and piled up to cell foci. The morphological aberrant foci appear blue colored after Giemsa staining and can be distinguished from the monolayer according to recommended criteria46. Figure 1c displays the result of a BALB-CTA cultured with MCA/TPA and in addition with different concentrations of OSI-906 given from day 1 to 42.…”

Section: Resultsmentioning

confidence: 99%

“…Type 3 foci were scored on the basis of the recommended photo catalogue46 and the following criteria: (i) deep basophilic staining, (ii) spindle-shaped cells, (iii) multilayer growth, (iv) random cell orientation and (v) invasive growth of cells at the edge of foci. Foci with a diameter less than 2 mm were not included into the scoring.…”

Section: Methodsmentioning

confidence: 99%

Insulin-IGF signaling affects cell transformation in the BALB/c 3T3 cell model

Poburski

Leovsky

Boerner

et al. 2016

Sci Rep

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The increased cancer mortality of diabetes type 2 patients is most likely an evidence of the tight connection between tumor development and energy metabolism. A major focus of today’s research is still the identification of key proteins of both diseases and the development of corresponding inhibitors. In this study we combined the two-stage BALB/c-3T3 cell transformation assay (BALB-CTA) with the IR/IGF-1R inhibitor OSI-906 (linsitinib) and analyzed alterations in protein activity and energy parameters in non-transformed as well as transformed cells. OSI-906 successfully inhibited the phosphorylation of IR/IGF-1R and decreased cell growth in non-transformed cells. In the BALB-CTA, a permanent treatment with OSI-906 reduced cellular transformation dose-dependently, whereas a temporary treatment gave evidence for a preventive effect in the promotion phase. Furthermore, even though several key proteins were affected, it was possible to show that the phosphorylation of GSK3, Erk 1/2 and the S6 protein are not crucial for the cell foci reducing effect of OSI-906. Taken together, the BALB-CTA confirmed results of OSI-906 from animal studies and enhanced the knowledge of its mode of action. Therefore, the BALB-CTA offers the opportunity to analyze alterations in the transformation process more precisely and will be helpful to identify effective cancer treatments.

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“…In addition, the photo catalogue for the classification of foci in the BALB/c 3T3 cell transformation assay by Sasaki et al 2012a [23] and Urani et al 2013 [7] were used as a visual aid for the identification of Type III foci.…”

Section: Foci Scoringmentioning

confidence: 99%

Analysis on the Performance of the Balb/c 3T3 Two-stage Assay in a Miniature Culture Area and in a Perfusion Culture Model

Teofilo¹,

Li²,

Liu³

et al. 2016

Proceedings of the 2016 International Conference on Biological Sciences and Technology

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Abstract. The Balb/c 3T3 cell transformation assay (CTA) is presently undergoing extensive pre-validation testing but its potential for routine chemical screening remains to be fully exploited due it being more costly and more time consuming than other CTAs. To downscale the assay 24-well plates were used to score foci and then compared to the standard T25 culture bottles. Furthermore, it was also attempted an adaptation to a perfusion cell culture model in order to achieve a closer equivalent to the in vivo carcinogenic processes. The miniaturized approach was successfully correlated to the standard technique. A reproduction in smaller culture plates can provide an alternative for a faster and more practicable handling by smaller laboratories. The assay failed to produce positive results in the fluidic model and further testing is necessary to determine the role medium flow in the in vitro carcinogenesis process.

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Photo catalogue for the classification of foci in the BALB/c 3T3 cell transformation assay

Cited by 34 publications

References 3 publications

Transformation Assay in Bhas 42 Cells: A Model Using Initiated Cells to Study Mechanisms of Carcinogenesis and Predict Carcinogenic Potential of Chemicals

Transformation Assay in Bhas 42 Cells: A Model Using Initiated Cells to Study Mechanisms of Carcinogenesis and Predict Carcinogenic Potential of Chemicals

Insulin-IGF signaling affects cell transformation in the BALB/c 3T3 cell model

Analysis on the Performance of the Balb/c 3T3 Two-stage Assay in a Miniature Culture Area and in a Perfusion Culture Model

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