Abstract 5359: TGFβ promotes the DNA damage response and repair of DNA damage induced by benzo[a]pyrene

Abstract

Abstract Transforming growth factor beta (TGFβ) is involved in coordinating a number of cellular processes including proliferation, differentiation and apoptosis, and as such, dysregulation of TGFβ signalling has significant implications for cancer development. TGFβ is rapidly and persistently activated in response to irradiation-induced DNA damage (Barcellos-Hoff et al, J Clin Invest 1994; 93:892-9) and has been implicated in regulation of the irradiation-induced DNA damage response (Kirshner et al, Cancer Res 2006; 66:10861-9). We have recently published two studies investigating the activation of DNA damage signalling in cells after exposure to two highly potent polycyclic aromatic hydrocarbons (PAHs), benzo[a]pyrene (BP) and dibenzo[a,l]pyrene (DBP) (Niziolek-Kierecka et al, Chem Res Toxicol 2012; 25:862-72 and Jarvis et al, Toxicol Appl Pharmacol 2013; 266: 408-18). The aim of this study was therefore to investigate a potential role for TGFβ in the response to PAH-induced DNA damage. Using human hepatocellular carcinoma cells (HepG2) we studied activation of DNA damage signalling by Western blotting. Formation of γH2AX and ATM foci was assessed by immunofluorescence and DNA damage levels were quantified by Comet assay. HepG2 cells were exposed to non-cytotoxic concentrations of BP and DBP (confirmed by MTT assay). Exposure to BP or DBP caused a concentration- and time-dependent increase in DNA damage signalling assessed by phosphorylation of Chk1, H2AX and p53. DBP was approximately 60-fold more potent at inducing prolonged DNA damage signalling than BP. Prolonged γH2AX and ATM foci were observed after exposure to 1 µM BP or 50 nM DBP for 48 hours consistent with persistent DNA damage whereas exposure to 0.1 µM BP or 5 nM DBP caused a transient increase in foci up to 24h and lower levels at 48h suggesting repair. To investigate the role of TGFβ signalling in repair of PAH-induced DNA damage, exogenous TGFβ (1 ng/ml) was added during incubation with BP and DBP. Addition of TGFβ significantly decreased the number of γH2AX and ATM foci in cells exposed to either BP or DBP and this was consistent with a decrease in DNA damage. Levels of both damage foci and Comet tail moments were similar to control levels suggesting activation of DNA repair in response to addition of TGFβ. Exposure to BP or DBP with or without TGFβ caused an increase in Smad2 phosphorylation confirming activation of TGFβ signalling. Smad2 activation was more pronounced after exposure to BP compared to DBP and RNA interference experiments are ongoing to further elucidate the mechanism of PAH induction of TGFβ signalling. Taken together our data suggest an important role for TGFβ signalling in repair of PAH-induced DNA damage. Furthermore, our data suggest a possible link between compromised TGFβ signalling and persistent DNA damage that might have important implications in understanding PAH-induced carcinogenesis. Citation Format: Ian WH Jarvis, Kristian Dreij, Ulla Stenius. TGFβ promotes the DNA damage response and repair of DNA damage induced by benzo[a]pyrene. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5359. doi:10.1158/1538-7445.AM2014-5359