DNA Damage Signaling In Cells Research Articles

Cornelia\xa0de Lange syndrome-associated mutations cause a DNA damage signalling and repair defect

Cornelia de Lange syndrome is a multisystem developmental disorder typically caused by mutations in the gene encoding the cohesin loader NIPBL. The associated phenotype is generally assumed to be the consequence of aberrant transcriptional regulation. Recently, we identified a missense mutation in BRD4 associated with a Cornelia de Lange-like syndrome that reduces BRD4 binding to acetylated histones. Here we show that, although this mutation reduces BRD4-occupancy at enhancers it does not affect transcription of the pluripotency network in mouse embryonic stem cells. Rather, it delays the cell cycle, increases DNA damage signalling, and perturbs regulation of DNA repair in mutant cells. This uncovers a role for BRD4 in DNA repair pathway choice. Furthermore, we find evidence of a similar increase in DNA damage signalling in cells derived from NIPBL-deficient individuals, suggesting that defective DNA damage signalling and repair is also a feature of typical Cornelia de Lange syndrome.

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

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

Abstract 5379: Mechanism of fludarabine and oxaliplatin combined activity depends on the activity of XPF endonuclease

Abstract We set out to determine the mechanism by which fludarabine and oxaliplatin exert more than additive cell killing in chronic lymphocytic leukemia (CLL). We postulate that enhanced activity of oxaliplatin and fludarabine combination is based on termination by fludarabine of DNA re-synthesis associated with nucleotide excision repair (NER) following removal of oxaliplatin adducts. The blocked repair would result in single-strand and double-strand DNA breaks which would activate cell DNA damage signaling and apoptosis. More than additive apoptotic killing was observed in 49% (±22) of CLL cells after exposure to oxaliplatin and fludarabine in vitro, although single agents showed minimal increase in cell death (3% ±9 for oxaliplatin, 13% ±14 for fludarabine), as demonstrated by Annexin V and PI staining at 24 hr (expected vs. observed p=0.0003; n=10). Oxaliplatin induced a 2-fold increase in DNA synthesis in CLL cells, which was inhibited by fludarabine, as determined by thymidine incorporation (n=5). Knockdown of XPF, the NER 5′ endonuclease, in CLL patient samples (n=3) eliminated oxaliplatin induced DNA synthesis (3-fold increase in thymidine incorporation with scramble siRNA compared to 1.4-fold increase with XPF siRNA). Wild type CHO (AA8) cells showed more than additive killing after combination treatment similar to the observation with CLL patient samples (predicted survival 50%; observed 21%). In contrast, only additive killing was observed in CHO cells lacking XPF (UV41) with equitoxic doses of oxaliplatin and fludarabine (predicted survival 56%; observed 54%) suggesting that XPF function is essential for the cooperative interaction of the combination. Alkaline assays demonstrated that inhibition of repair by fludarabine was accompanied by DNA strand break formation within 12 hr (n=4) in CLL patients samples. The increase in Olive tail moment ranged from 2- to 5-fold, confirming induction of single-strand breaks. The double-strand break repair proteins ATM, SMC1, H2AX and DNA-PKcs were phosphorylated as early as 2 hr post-treatment with the combination and persisted up to 24 hr (3-fold, 24-fold, 10-fold and 10-fold increase, respectively; n=4). This response suggests that DNA damage generated by fludarabine and oxaliplatin exposure was processed to double-strand DNA breaks in CLL cells. Our work provides evidence that the combination of oxaliplatin and fludarabine results in the formation of toxic DNA damage and that their combined activity depends on a functional NER pathway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5379.

Chk1 Instability Is Coupled to Mitotic Cell Death of p53-deficient Cells in Response to Virus-induced DNA Damage Signaling

Adeno-associated virus (AAV) DNA, by mimicking a stalled replication fork, provokes a DNA damage response that can arrest cells in the G2/M phase of the cell-cycle. This response depends strictly on DNA damage signaling kinases ATR and Chk1. Here, we used AAV to study long-term effects of DNA damage signaling in cells with altered p53 status. In HCT116 cells, in response to damage signaling, p53 represses transcription of the genes encoding mitotic regulators Cdc25C, cyclin B1, and Plk1 to establish a firm G2 arrest. Isogenic cells lacking p53 maintain these three proteins at constant levels yet can still arrest initially in G2 because Chk1 signaling inhibits their enzymatic activities. Unexpectedly, the levels of Chk1 fall abruptly in a proteasome-dependent manner after two days of arrest in G2. In p53-deficient cells, this Chk1 instability is coupled to recovery of the phosphatase activity of Cdc25C and in the kinase activities of Plk1 and Cdk1/cyclin B1. Consequently, the p53-deficient cells enter lethal mitosis. Thus, the Chk1-mediated arrest is transient: it initially causes cells to accumulate in G2 until p53-dependent transcriptional repression of mitotic proteins takes over. p53-deficient cells cannot maintain the DNA damage signaling-induced G2 arrest after Chk1 has disappeared, and continue into catastrophic mitosis. Restoring Chk1 prevents the cells from entering such mitosis. These results reveal a mechanism based on Chk1 stability that regulates mitotic entry after DNA damage and elucidate the controversial phenomenon of p53-promoted cell survival in the face of damage signaling.