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

Abstract

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.

In vitro cell transformation induced by synthetic amorphous silica nanoparticles

“…In the present study, we used an in vitro model of cell transformation; the transgenic Bhas 42 cells that are particularly sensitive to tumor-promoter substances but also being sensitive to tumor-initiator substances [16]. A validation study covering 98 chemicals established an 84% specificity of the Bhas 42 CTA towards carcinogens and non-carcinogens [21].…”

“…The principle of this assay is the following [15,16]: the transgenic Bhas 42 cell line, established from Balb/c 3T3 cells, contains several copies of the v-Ha-ras gene. Similar to the parental Balb/c 3T3 cells, untransformed Bhas 42 cells grow to confluence forming a density-dependent contact-inhibited monolayer.…”

Report of the Workshop on a Framework for the Development and Use Of Integrated Approaches to Testing and Assessment

“…Cell transformation assays (CTAs) were developed several decades ago to study chemical carcinogens (e.g., DiPaolo et al, 1969). The CTAs, particularly the Syrian Hamster Embryo or SHE (e.g., Isfort and LeBoeuf, 1995;Vasseur and Lasne, 2012;Benigni et al 2015) and Bhas 42 (Sakai et al, 2011;Sasaki et al, 2015) assays, have been used to predict carcinogenic potential of substances as well as for mechanistic studies. The SHE cells are primary cells that can mimic all the three stage of carcinogenesis process whereas Bhas 42 cells are non-transformed BALB/c 3T3 cells that have been transfected with human ras oncogene and can act as initiated cells.…”

Relationship between increasing concentrations of two carcinogens and statistical image descriptors offocimorphology in the cell transformation assay

“…In particular, work is underway to respond to concerns that have been raised, such as the need for an objective scoring of transformed foci . This was addressed in the prevalidation and validation studies co‐ordinated by the European Reference Laboratory on Alternative Methods (EURL ECVAM) and the Japanese Centre for the Validation of Alternative Methods (JaCVAM), which led to the publication of standardized protocols in combination with photo‐catalogues to support the researchers during the scoring procedure (Bohnenberger et al ., ; Maire et al ., ; OECD, Annex 2; Sasaki et al ., , ). In the framework of this study, a new statistical method to improve the analysis of BALB/c 3T3 CTA data was developed (Hoffmann et al ., ).…”