N-nitroso-N-ethylurea activates DNA damage surveillance pathways and induces transformation in mammalian cells

Abstract

BackgroundThe DNA damage checkpoint signalling cascade sense damaged DNA and coordinates cell cycle arrest, DNA repair, and/or apoptosis. However, it is still not well understood how the signalling system differentiates between different kinds of DNA damage. N-nitroso-N-ethylurea (NEU), a DNA ethylating agent induces both transversions and transition mutations.MethodsImmunoblot and comet assays were performed to detect DNA breaks and activation of the canonical checkpoint signalling kinases following NEU damage upto 2 hours. To investigate whether mismatch repair played a role in checkpoint activation, knock-down studies were performed while flow cytometry analysis was done to understand whether the activation of the checkpoint kinases was cell cycle phase specific. Finally, breast epithelial cells were grown as 3-dimensional spheroid cultures to study whether NEU can induce upregulation of vimentin as well as disrupt cell polarity of the breast acini, thus causing transformation of epithelial cells in culture.ResultsWe report a novel finding that NEU causes activation of major checkpoint signalling kinases, Chk1 and Chk2. This activation is temporally controlled with Chk2 activation preceding Chk1 phosphorylation, and absence of cross talk between the two parallel signalling pathways, ATM and ATR. Damage caused by NEU leads to the temporal formation of both double strand and single strand breaks. Activation of checkpoints following NEU damage is cell cycle phase dependent wherein Chk2 is primarily activated during G2-M phase whilst in S phase, there is immediate Chk1 phosphorylation and delayed Chk2 response. Surprisingly, the mismatch repair system does not play a role in checkpoint activation, at doses and duration of NEU used in the experiments. Interestingly, NEU caused disruption of the well-formed polarised spheroid archithecture and upregulation of vimentin in three-dimensional breast acini cultures of non-malignant breast epithelial cells upon NEU treatment indicating NEU to have the potential to cause early transformation in the cells.ConclusionNEU causes damage in mammalian cells in the form of double strand and single strand breaks that temporally activate the major checkpoint signalling kinases without the occurrence of cross-talk between the pathways. NEU also appear to cause transformation in three-dimensional spheroid cultures.

The Role of Genetic Polymorphisms as Related to One-Carbon Metabolism, Vitamin B6, and Gene–Nutrient Interactions in Maintaining Genomic Stability and Cell Viability in Chinese Breast Cancer Patients

“…Anyhow, the cells will not arrive at the mitotic telophase when MNBNs are marked in BN cells. The responses to DNA damage are serious and can decrease cell viability, and they can further induce the activation of surveillance systems, which either postpones cell cycle progression until the damaged DNA is repaired or eliminated, or induces apoptosis in case the damage is irreparable [ 32 ]. Additionally, we inspected whether the B6 deficiency-induced MNBNs from 9 day would be accompanied by a reduction in NVC and the NDI of the breast cancer cases and controls.…”

ATR facilitates the degradation of Api5 through the ubiquitin-proteasome pathway via FBXW2 to regulate apoptosis upon DNA damage

“…Immunoprecipitations were performed as described earlier (27) using Api5, Myc, GFP and ATR specific antibodies. Western blotting was performed with Api5, GFP, ATR, Ubiquitin, pSerine and GAPDH antibodies as previously mentioned (28). Immunoblots include all the experimental lanes of a said experiment.…”

Interplay between p300 and HDAC1 regulate acetylation and stability of Api5 to regulate cell proliferation

“…Sense sequences of the siRNA are: Api5, 5′-GACCUAGAACAGACCUUCAUU-3′, LacZ, 5′-CGUACGCGGAAUA CUUCGAdTdT-3′. siRNA against LacZ and Api5 were transfected as described earlier [62]. Api5 K251 mutants which were generated by site directed mutagenesis were transfected 24 hrs post knockdown of Api5.…”

“…Immunoprecipitations were performed as described [63] using GFP, RFP, p300, or HDAC1 specific antibodies. Western blotting was performed with Api5, GFP, acetyl lysine and GAPDH antibodies as previously mentioned [62].…”