Mechanistic Interrogation of Cell Transformation In Vitro: The Transformics Assay as an Exemplar of Oncotransformation

Abstract

The Transformics Assay is an in vitro test which combines the BALB/c 3T3 Cell Transformation Assay (CTA) with microarray transcriptomics. It has been shown to improve upon the mechanistic understanding of the CTA, helping to identify mechanisms of action leading to chemical-induced transformation thanks to RNA extractions in specific time points along the process of in vitro transformation. In this study, the lowest transforming concentration of the carcinogenic benzo(a)pyrene (B(a)P) has been tested in order to find molecular signatures of initial events relevant for oncotransformation. Application of Enrichment Analysis (Metacore) to the analyses of the results facilitated key biological interpretations. After 72 h of exposure, as a consequence of the molecular initiating event of aryl hydrocarbon receptor (AhR) activation, there is a cascade of cellular events and microenvironment modification, and the immune and inflammatory responses are the main processes involved in cell response. Furthermore, pathways and processes related to cell cycle regulation, cytoskeletal adhesion and remodeling processes, cell differentiation and transformation were observed.

The Cell Transformation Assay: A Historical Assessment of Current Knowledge of Applications in an Integrated Approach to Testing and Assessment for Non-Genotoxic Carcinogens

“…With the advent of new molecular technologies and with a better understanding of the key events in the multistep carcinogenesis process, it has become possible to explore the complete pattern of molecular modifications characterizing the oncotransformation in vitro. This gives greater evidential support to the early observations on the role of genes related to cancer and clarifies some (apparent) discrepancies and incongruities from early studies of cellular and molecular mechanisms of cell transformation [ 61 , 67 , 68 , 69 , 74 ].…”

“…Only chromosomal changes are reported, including mutations, loss of heterozygosis (LOH) or gain of function. Each molecular change is then associated with dysregulation of key signal pathways, as described elsewhere for both human cancer [ 66 ] and 3T3 CTAs [ 67 , 68 , 69 ].…”

“…The application of transcriptomics tools has made it possible to elucidate the molecular events that mark the steps to cell transformation in vitro [ 67 , 68 , 69 , 85 ]. Results from these studies, performed using the three currently most used CTA models, show an excellent level of concordance and resemblance of the human carcinogenesis process ( Figure 3 and Figure 4 ).…”

“…Cytoskeleton remodeling is one of the first events highlighted by the analysis of gene modulation induced by different chemicals, recognized as carcinogens via genotoxic and non-genotoxic mechanisms [ 67 , 68 , 69 , 85 ]. The cytoskeleton is involved in the innate response to danger signals from xenobiotics, leading to an adaptive response [ 86 ].…”

Analyses of Transcriptomics Cell Signalling for Pre-Screening Applications in the Integrated Approach for Testing and Assessment of Non-Genotoxic Carcinogens

“…In essence, this study demonstrates the added value of combining the CTA with gene expression analysis, for obtaining insight into likely non-genotoxic carcinogenic mechanisms of a chemical of interest, in early and late stages of cell transformation that occur in addition to the known chemical’s genotoxic properties. This approach has also been successfully applied both for mouse BALB/c 3T3 cells [ 31 ] and for the Bhas CTA [ 32 ].…”