Induction of replicative senescence by 5-azacytidine: fundamental cell kinetic differences between human diploid fibroblasts and NIH-3T3 cells.
To analyse the putative role of methylation of cytosine residues in the nuclear DNA as a regulatory step during cellular ageing, we incubated ageing human amniotic fluid derived fibroblast-like cells and non-ageing NIH-3T3 cells with 5-azacytidine. BrdUrd/Hoechst and acridine orange (AO) flow cytometry was used to compare the effects of the base analogue on cell proliferation and cell differentiation. In NIH-3T3 cultures, 96h exposures to 4 microM 5-azacytidine caused diminished cell proliferation due to cell arrest in the G1 compartments of the second and third cell cycles of serum stimulated cells. The exit from the G0/G1 compartment was not affected. The 5-azacytidine induced cell kinetic disturbances were unstable in NIH-3T3 cultures, such that pre-treated cells reverted to normal cell cycle transit within 2-3 days after termination of treatment. In contrast, 5-azacytidine pre-treated amniotic fluid derived fibroblast-like cell cultures showed persistently elevated G2 phase arrests and delayed G0/G1 phase exit kinetics, which explain the premature cessation of proliferation observed in these primary cultures. In both cell systems, 5-azacytidine exposed cultures showed elevated numbers of G1 phase cells with increased RNA content as revealed by AO flow cytometry. Again, this effect was reversible in NIH-3T3 cells but not in amniotic fluid derived fibroblast-like cells. These contrasting responses to 5-azacytidine are likely to reflect intrinsic differences in methylation patterns or de novo methylase activity between ageing cell strains and non-ageing cell lines.
Weller
E M
EM
Department of Human Genetics, University of Würzburg, Germany.
Poot
M
M
Hoehn
H
H
D016475:3T3 Cells / Q000187:drug effects; D000165:Acridine Orange; D000653:Amniotic Fluid / Q000166:cytology; D000818:Animals; D001374:Azacitidine / Q000494:pharmacology*; D001973:Bromodeoxyuridine; D002455:Cell Division / Q000187:drug effects*; D002469:Cell Separation; D002478:Cells, Cultured / Q000187:drug effects; D016922:Cellular Senescence / Q000187:drug effects; D005434:Flow Cytometry; D016193:G1 Phase / Q000187:drug effects*; D016195:G2 Phase / Q000187:drug effects*; D006801:Humans; D008745:Methylation; D051379:Mice; D012313:RNA / Q000032:analysis
D012313:RNA; D000165:Acridine Orange; D001973:Bromodeoxyuridine; D001374:Azacitidine