Quantitative analysis of gene expression changes in response to genotoxic compounds.
Techniques that quantify molecular endpoints sufficiently sensitive to identify and classify potentially toxic compounds have wide potential for high-throughput in vitro screening. Expression of three genes, RAD51C, TP53 and cystatin A (CSTA), in HEPG2 cells was measured by Q-PCR amplification. In parallel, we developed alternative assays for the same 3 gene signature based on an acridinium-ester chemiluminescent reporter molecule. HEPG2 cells were challenged with eighteen different compounds (n=18) chosen to represent compounds that are genotoxic (n=8), non-genotoxic non-carcinogenic (n=2) or have a less well defined mechanism of action with respect to genotoxicity (n=8). At least one of the three genes displayed dysregulated expression in the majority of compounds tested by Q-PCR and ten compounds changed the CSTA expression significantly. Acridinium-ester labelled probes for the three genes were synthesised and tested. Analytical sensitivity was characterised and suggested a limit of detection generally better than 0.1fmol but often 10-50 attomol. A linear amplification step was optimised and this quantitative method detected statistically significant increases in RAD51C and CSTA expression in agreement with the Q-PCR results, demonstrating the potential of this technology. The broad agreement of the amplified chemiluminescent method and Q-PCR in measuring gene expression suggests wider potential application for this chemiluminescent technology.
School of Biosciences, Cardiff University, Cardiff CF10 3AT, UK; School of Medicine, Cardiff University, Tenovus Building, Heath Park, Cardiff CF14 4XN, UK. Electronic address: morrisc10@cardiff.ac.uk.
Ceri A
CA
Morris
Cornea Research Chair, Department of Optometry, College of Applied Medical Sciences, King Saud University, P.O. BOX 10219, Riyadh 11433, Saudi Arabia.
Gamal A
GA
El-Hiti
School of Medicine, Cardiff University, Tenovus Building, Heath Park, Cardiff CF14 4XN, UK.
Ian
I
Weeks
Invitron, Wyastone Leys, Monmouth NP25 3SR, UK.
Stuart
S
Woodhead
School of Chemistry, Cardiff University, Main Building, Park Place, Cardiff CF10 3AT, UK.
Keith
K
Smith
School of Biosciences, Cardiff University, Cardiff CF10 3AT, UK.
Peter
P
Kille
D055312:Cystatin A / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics*; D015870:Gene Expression / Q000187:drug effects*; D056945:Hep G2 Cells; D006801:Humans; D008163:Luminescent Measurements; D009153:Mutagens / Q000633:toxicity*; D016133:Polymerase Chain Reaction / Q000379:methods; D016159:Tumor Suppressor Protein p53 / Q000235:genetics*
D055312:Cystatin A; D004268:DNA-Binding Proteins; D009153:Mutagens; C448305:RAD51C protein, human; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; C026407:CSTA protein, human
Acridinium-ester; Chemiluminescence; Cystatin A (CSTA); Genotoxic; RAD51C; Tumour suppressor p53 (TP53)