A mechanistic evaluation of the Syrian hamster embryo cell transformation assay (pH 6.7) and molecular events leading to senescence bypass in SHE cells

• Published in: Mutation research. Genetic toxicology and environmental mutagenesis Vol. 802; pp. 50 - 58
• Main Authors: Pickles, Jessica C., Pant, Kamala, Mcginty, Lisa A., Yasaei, Hemad, Roberts, Terry, Scott, Andrew D., Newbold, Robert F.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.05.2016; Elsevier BV; Elsevier
• Subjects: Aging; Animals; Bioassays; Carcinogens - toxicity; Cell Line; Cell transformation assay; Cell Transformation, Neoplastic - genetics; Cells; Cricetinae; Cyclin-Dependent Kinase Inhibitor p16 - genetics; DNA Methylation - drug effects; DNA Methylation - genetics; Mesocricetus; Molecular biology; Morphological transformation; p16/CDKN2A; Rodents; Senescence bypass; Syrian hamster; SHE; CTA; Cell transformation assay; SH; Morphological transformation; Syrian hamster; p16/CDKN2A; Senescence bypass
• ISSN: 1383-5718; 1879-3592
• DOI: 10.1016/j.mrgentox.2016.04.002

•Mechanistic evaluation of SHE CTA, specific to B(a)P.•Applicability of the Syrian hamster as a relevant model of carcinogenesis.•Molecular analysis of immortal lines derived from benzo(a)pyrene-induced MT clones.•Morphological transformation (MT) does not guarantee senescence bypass.•Secondary events to MT are necessary for cellular immortalisation. The implementation of the Syrian hamster embryo cell transformation assay (SHE CTA) into test batteries and its relevance in predicting carcinogenicity has been long debated. Despite prevalidation studies to ensure reproducibility and minimise the subjective nature of the assay’s endpoint, an underlying mechanistic and molecular basis supporting morphological transformation (MT) as an indicator of carcinogenesis is still missing. We found that only 20% of benzo(a)pyrene-induced MT clones immortalised suggesting that, alone, the MT phenotype is insufficient for senescence bypass. From a total of 12 B(a)P- immortalised MT lines, inactivating p53 mutations were identified in 30% of clones, and the majority of these were consistent with the potent carcinogen’s mode of action. Expression of p16 was commonly silenced or markedly reduced with extensive promoter methylation observed in 45% of MT clones, while Bmi1 was strongly upregulated in 25% of clones. In instances where secondary events to MT appeared necessary for senescence bypass, as evidenced by a transient cellular crisis, clonal growth correlated with monoallelic deletion of the CDKN2A/B locus. The findings further implicate the importance of p16 and p53 pathways in regulating senescence while providing a molecular evaluation of SHE CTA −derived variant MT clones induced by benzo(a)pyrene.