DNA damage responses after exposure to DNA-based products.
BACKGROUND
The development of DNA-based therapies holds great promise for the treatment of diseases that remain difficult to manage using conventional pharmaceuticals. Whilst there are considerable data regarding chemical-induced DNA damage, there are limited reports published studying the potential of exogenous DNA to damage genomic DNA.


METHODS
To investigate this problem, the differential gene expression (DGE) of DNA repair genes was examined to identify biomarkers, based on the hypothesis that DNA damage, including double-strand breaks (DSBs) and insertional mutagenesis, would be expected to induce biological pathways associated with repair. Human HepG2 cells were exposed to the chemical genotoxins, etoposide (ETOP) and methylmethanesulphonate (MMS), as positive controls, or biological agents (i.e. exogenous DNA with and without the use of transfection complexes or via various viral vectors). Following transfection (6-72 h) the cells were harvested for RNA and DGE was determined by quantitative real-time polymerase chain reaction (qRT-PCR).


RESULTS
The expression of genes involved in the repair of DSBs were significantly increased after treatment with ETOP (>4-fold) or MMS (>5-fold). Transfection using Effectene and ExGen 500 resulted in no significant changes; however, transfection with ExGen 500 resulted in an increase in the expression levels of GADD45 mRNA, consistent with global cellular stress. Viral vectors increased (3-6-fold) expression of genes associated with DSBs and cellular stress responses and, as expected, the effect was the most marked with the retroviral vector.


CONCLUSIONS
The DGE profiles observed in HepG2 cells following transduction/transfection suggest that a subset of DNA repair genes may provide novel biomarkers to rapidly detect DNA damage induced by DNA products at the level of the genome, rather than at selected genes.
Smith
Catherine C
CC
Biological Chemistry (Molecular Toxicology), Faculty of Medicine, Imperial College London, Sir Alexander Fleming Building, London SW7 2AZ, UK.
Aylott
Michael C
MC
Fisher
Krishna J
KJ
Lynch
Anthony M
AM
Gooderham
Nigel J
NJ
D002460:Cell Line; D004247:DNA / Q000502:physiology*; D004249:DNA Damage / Q000502:physiology*; D045643:DNA Repair Enzymes / Q000502:physiology; D000229:Dependovirus; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000009:adverse effects*; D005822:Genetic Vectors; D006801:Humans; D009152:Mutagenicity Tests; D010957:Plasmids; D014161:Transduction, Genetic; D014162:Transfection
D004247:DNA; D045643:DNA Repair Enzymes