5-Bromodeoxyuridineを用いる細胞老化誘導系における核内タンパク質のプロテオーム解析

抄録

5-Bromdeoxyuridine (BrdU) immediately and clearly induces a phenomenon similar to premature senescence in any type of mammalian cells. To understand cellular senescence, characterization of genes is not sufficient because protein levels markedly fluctuate during cellular senescence and ageing. Since BrdU seems to target chromatin structure to affect expression of senescence-associated genes, we made an attempt to characterize nuclear proteins in HeLa cells undergoing senescence with BrdU by 2D DIGE.
We purified nuclei from HeLa cells cultured with BrdU for 4 days and control cells and prepared samples of soluble nuclear proteins. These samples were subjected to DIGE and the data were processed to find protein spots altered upon addition of BrdU. Sevral spots were found to alter reproducibly. Of such spots, lamin A and C were markedly increased and members of the hnRNA family markedly decreased. Levels of mRNA for these genes did not significantly change upon addition of BrdU. The changes in the lamin proteins seem to agree with the observation that nuclei enormously swell during cellular senescence. Recently, it is also shown that Hutchinson-Gilford progeria syndrome is caused by a mutation in the LMNA gene encoding lamin A/C. An abnormal lamin A protein is found to lead to nuclear swelling and a defect of nuclear lamina in cells from patients. We thus performed Western blot analysis to characterize lamin A/C in Hela cells using specific antibody. Interestingly, an abnormal lamin A protein was found to accumulate in BrdU-treated cells. The hnRNA family proteins are involved in various functions. We found that a member of the hnRNP family is responsible for stress response that leads to senescence.
Based on close similarity between BrdU-mediated senescence in immortal cells and replicative senescence in normal human cells, it is suggested that lamin A/C and hnRNPs play a central role in cellular senescence.