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


METHODS
Immunoblot 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.


RESULTS
We 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.


CONCLUSIONS
NEU 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.
Satish
S
Bodakuntla
Anandi V
AV
Libi
Surojit
S
Sural
Prasad
P
Trivedi
Indian Institute of Science Education and Research, Pune, Maharashtra 411008, India. mayurika.lahiri@iiserpune.ac.in.
Mayurika
M
Lahiri
D064007:Ataxia Telangiectasia Mutated Proteins / Q000235:genetics / Q000378:metabolism*; D059447:Cell Cycle Checkpoints / Q000187:drug effects; D002471:Cell Transformation, Neoplastic / Q000187:drug effects* / Q000235:genetics; D000071877:Checkpoint Kinase 1; D064447:Checkpoint Kinase 2 / Q000096:biosynthesis* / Q000378:metabolism; D004249:DNA Damage / Q000187:drug effects; D004260:DNA Repair / Q000187:drug effects; D005038:Ethylnitrosourea / Q000494:pharmacology; D006801:Humans; D010766:Phosphorylation; D011494:Protein Kinases / Q000096:biosynthesis* / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects
D011494:Protein Kinases; D064447:Checkpoint Kinase 2; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C000605867:CHEK1 protein, human; C578634:CHEK2 protein, human; D000071877:Checkpoint Kinase 1; D005038:Ethylnitrosourea