Induction of replicative senescence by 5‐azacytidine: fundamental cell kinetic differences between human diploid fibroblasts and NIH‐3T3 cells

Abstract

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.

Gene Expression and Protein Degradation

“…A loss of 5mC residues from the genome of human fibroblasts is also observed with increasing passage number (433). More importantly, several investigators (88,144,427) have shown that incubating human fibroblasts with 5-azacytidine, a compound that inhibits the methylation of cytosine in DNA and results in the hypomethylation of DNA, shortens the in vitro life span of human fibroblasts; that is, the number of population doublings was reduced after 5-azacytidine treatment.…”

Fluorescence enhancement of DNA-bound TO-PRO-3 by incorporation of bromodeoxyuridine to monitor cell cycle kinetics

“…Simultaneous staining of DNA with a BrdUrd-nonsensitive fluorochrome such as PI or ethidium bromide (EB) allows analysis of cells in G1-, S-, and G2/M-phases of several cell cycles. The continuous BrdUrd labeling technique, apply-ing the BrdUrd/Hoechst quenching effect, has been used for cell kinetic analysis of initially synchronous cell populations (4)(5)(6)(7)(8)(9)(10)(11)(12) and asynchronously dividing cells (13)(14)(15), resolving the complete history of cell proliferation and representing the heterogeneity of the proliferative status of cell populations. The BrdUrd/Hoechst quenching effect has also been applied for detecting DNA-replicating cells after pulse labeling with BrdUrd by staining cells with HO 33342 and mitramycin (MI), a dye whose fluorescence is enhanced by the incorporation of BrdUrd into DNA (16,17).…”

Role of p14ARF in Replicative and Induced Senescence of Human Fibroblasts

“…DNA damage (57), oxidative stress (14), histone deacetylase inhibitors (48), methylation inhibitors (71), and expression of some activated oncogenes, such as Ras, Raf, or MEK (59,75). Some conditions, such as prolonged culture under hyperoxic conditions, can accelerate the rate of telomere attrition (73), but most of the "premature senescence" states occur rapidly and appear to be independent of telomere length (45,69).…”