During mitosis, temporal release of separase from its inhibitor securin results in cohesin cleavage, thereby promoting anaphase. The developing fetal brain has rapidly proliferating neuronal cells whilst adult neurons no longer proliferate. When comparing 61 fetal and 12 adult human brain samples, we found significantly increased separase mRNA and protein throughout ontogeny and reduced securin expression in fetal brain compared with adult. Using MTT assays, we examined the effects of overexpressing separase and securin on neuronal precursor N-TERA2 (NT2) cell proliferation. Interestingly, overexpression of separase (0.3 micrograms DNA/well (96 well plate)) inhibited proliferation at 24 (n=3, p<0.001), 48 (n=3, p<0.001) and 72 (n=3, p<0.001) hours. Securin also significantly inhibited proliferation at all time points post transfection and at a range of doses (0.05 micrograms/well to 0.3 micrograms/well) (n=3, p<0.001). Further experiments employed a mutant separase (C2029A) incapable of cleaving cohesin and a Ken/Destruction Box (KEN/DB) securin mutant which cannot be ubiquinated to release separase. Overexpresssion of both wild-type and mutant separase and securin resulted in a highly significant inhibition of proliferation (n=3, p<0.001). C2029A demonstrated a greater inhibition of proliferation than wild-type separase (n=3, p<0.01). Conversely, wild-type securin was a less potent inhibitor than KEN/DB (n=3, p<0.05). Transient co-transfection of separase and securin mutants resulted in the greatest inhibition of neural cell proliferation (n=3, p<0.001). FACS analysis of asynchronous transfected cells showed no statistical differences in cell cycle progression, but instead revealed a significant increase in apoptosis in securin treated cells (n=6, p<0.01) which was negated by KEN/DB (n=6, p<0.05). Both separase and C2029A significantly promoted apoptosis (n=6, p<0.05). Co-transfection of separase and securin further induced apoptosis (n=6, p<0.05). In conclusion, overexpression of separase, as apparent in fetal brain, is not sufficient alone to enhance cell division, but rather acts with securin to affect neural cell proliferation and apoptosis.
|Publication status||Published - 1 Jan 2005|
|Event||24th Joint Meeting of British Endocrine Societies, Harrogate - |
Duration: 1 Jan 2005 → …
|Conference||24th Joint Meeting of British Endocrine Societies, Harrogate|
|Period||1/01/05 → …|