Cell Transformation Assay Based on the Bhas 42 Cell Line

The in vitro CTA using Bhas 42 cell line is aimed at detecting the carcinogenic potential of chemicals. The assay is based on the change of the phenotypic features of cells undergoing the first steps of the conversion from normal cells to neoplastic-like cell foci with oncogenic properties. Carcinogenesis is a complex multistage process by which normal cells are transformed into cancer cells characterised by an accumulation of changes at the cellular, genetic and epigenetic level. In vitro CTAs such as the Bhas 42 CTA have been shown to recapitulate a multistage process that closely models some stages of in vivo carcinogenesis. Cell transformation in all the systems studied to-date involves changes at each stage in order for progression to the next stage in the transformation process to occur. A minimum of four phenotypic stages appears to be involved in cell transformation. These include (from primary to fully malignant cells):

1. a block in cellular differentiation,
2. acquisition of immortality, characterised by unlimited lifespan, an aneuploidy karyotype and a decreased genetic stability;
3. acquisition of tumorigenicity, which is closely associated with the in vitro phenotypes of foci formation, anchorage independent growth in semi-solid agar and autocrine growth factor production;
4. full malignancy, including metastasis when the cells are injected into a suitable host, supporting the biological relevance of in vitro transformation to in vivo carcinogenicity.

The Bhas 42 cell line was developed from BALB/c 3T3 cells through transfection with a Harvey rat sarcoma viral mutated oncogene homolog (v-Ha-ras). Since Ha-ras is strongly related to multi-step carcinogenesis, Bhas 42 cells are more predisposed to transformation than the original cells BALB/c 3T3. The assay was first developed by Ohmori et al. (2004) to identify tumour promoters. Subsequently, Asada et al. (2005) modified the Bhas 42 CTA protocol such that it was capable of detecting tumour initiating activity, as well as tumour promoting activity of chemicals.

The current protocols consist of two assay components, the initiation assay and the promotion assay to detect the tumour-initiating and tumour-promoting activity of chemicals, respectively. Two variants of the protocol have been validated, a 6-well method and a 96-well method.