Human fibroblast replicative senescence can occur in the absence of extensive cell division and short telomeres

Abstract

Ectopic expression of telomerase blocks both telomeric attrition and senescence, suggesting that telomeric attrition is a mitotic counting mechanism that culminates in replicative senescence. By holding human ®broblast cultures con¯uent for up to 12 weeks at a time, we con®rmed previous observations and showed that telomeric attrition requires cell division and also, that senescence occurs at a constant average telomere length, not at a constant time point. However, on resuming cell division, these long-term con¯uent (LTC) cultures completed 15 ± 25 fewer mean population doublings (MPDs) than the controls prior to senescence. These lost divisions were mainly accounted for by slow cell turnover of the LTC cultures and by permanent cell cycle exit of 94% of the LTC cells, which resulted in many cell divisions being unmeasured by the MPD method. In the LTC cultures, p27 KIP1 accumulated and pRb became under-phosphorylated and under-expressed. Also, coincident with permanent cell cycle exit and before 1 MPD was completed, the LTC cultures upregulated the cell cycle inhibitors p21 WAF and p16 INK4A but not p14 ARF and developed other markers of senescence. We then tested the relationship between cell cycle re-entry and the cell cycle-inhibitory proteins following subculture of the LTC cultures. In these cultures, the downregulation of p27 KIP1 and the phosphorylation of pRb preceded the complete resumption of normal proliferation rate, which was accompanied by the down-regulation of p16 INK4A . Our results show that most normal human ®broblasts can accumulate p16 INK4A , p21 WAF and p27 KIP1 and senesce by cell division-independent mechanism(s). Furthermore, this form of senescence likely requires p16 INK4A and perhaps p27 KIP1 . Oncogene (2001) 20, 3541 ± 3552.

Senescing oral dysplasias are not immortalized by ectopic expression of hTERT alone without other molecular changes, such as loss of INK4A and/or retinoic acid receptor-β: but p53 mutations are not necessarily required

“…The high molecular weight smears in the D17 experiment (particularly lanes 3 and 7) are often seen in keratinocyte TRF blots (Gordon et al, 2002) and are probably due to small amounts of 3T3 DNA contamination that gave a disproportionately high signal because mouse TRFs are 10-25 more abundant than in our human oral cultures. TRF measurements were carried out as described by Munro et al (2001), except that the blot was hybridized with a radiolabelled oligonucleotide probe (TTAGGG) 3 at 421C and subsequently washed in 6 Â SSC/1% SDS at 421C Figure 2 Telomerase activity in D30 and D17 cultures before and after infection with the hTERT expression vector. D30 cells were infected with hTERT or empty vector retroviruses at 9 PDs and analysed as soon as sufficient cells were available; D17 cells were infected at 39 PDs and analysed at 58 PDs.…”

MnSOD activity regulates hydroxytyrosol-induced extension of chronological lifespan

“…The authors referred to this mode of cellular ageing as chronological lifespan (CLS). We and others have shown that quiescent human fibroblasts cultured for a long duration were unable to proliferate following replating at a lower cell density (Allsopp et al 1995;Munro et al 2001;Sarsour et al 2005;Sitte et al 1998). We observed that quiescent fibroblasts lost their proliferative capacity (measured as percentage of S phase at 48 h following replating of cells at a lower cell density) beginning from 30 days of quiescence; a maximal drop in S phase percentage was observed in cells replated from 60 days of quiescence (Sarsour et al 2005).…”

“…Although HT is an antioxidant, it can initially act as a prooxidant leading to the generation of superoxide and activation of MnSOD expression. Previous literature showed that long duration of quiescence resulted in a significant loss in the proliferative capacity of NHFs (Munro et al 2001;Sarsour et al 2005;Sitte et al 1998). We used contact-inhibited quiescent, but metabolically active cultures to study the effects of HT on CLS of NHFs.…”

“…c HT generates free radicals: in the presence of hydrogen peroxide, peroxidases oxidize HT to its semiquinone radical, which in turn leads to the formation of superoxide and quinone. (Munro et al 2001) where the authors report loss of 15-25 population doublings in long-term quiescent cultures of normal human fibroblasts. This loss in population doublings was associated with approximately 94% of the cells exiting the cell cycle permanently.…”

“…The decrease in S phase was not due to cell death or changes in telomerase activity. Mid-lifespan fibroblasts kept in quiescence for 7-12 weeks lost their proliferative capacity, yet there were no detectable shortening of telomere (Allsopp et al 1995;Munro et al 2001;Sitte et al 1998). Results from these previous studies suggest the presence of telomere-independent pathways that influence cellular lifespan.…”