Putative telomere-independent mechanisms of replicative aging reflect inadequate growth conditions

Abstract

Telomere shortening is the mechanism underlying replicative aging in fibroblasts. A variety of reports now claim that inactivation of the p16 INK4a/pRB pathway is required in addition to telomere maintenance for the immortalization of cells such as skin keratinocytes and breast epithelial cells. We here show that the premature growth arrest of these cell types can be explained by an inadequate culture environment. Providing mesenchymal/epithelial interactions by cultivating the telomerase-expressing cells on feeder layers avoids the growth arrest associated with increased p16 INK4a. These results do not support a telomere-independent mechanism of replicative aging. The ability to proliferate indefinitely in tissue culture is one of many differences between normal human cells and established tumor cell lines. For nearly forty years (Hayflick and Moorhead 1961), the contrast between the immortality of cancer cells and the senescence of normal cells has been controversial. The major source of skepticism rests on the knowledge that in vitro culture conditions represent a departure from the in vivo environment, and that the aneuploidy and altered growth controls accumulated by tumor cells might simply allow them to divide under conditions inadequate to support the long-term proliferation of normal cells. Although greatly diminished by the recent advances uncovering cellular mechanisms for counting cell divisions (see below), concerns that cellular senescence represents a tissue-culture artifact have continued (Rubin 1997(Rubin , 1998.The best understood mechanism for replicative aging involves telomere shortening. Telomerase, the enzyme responsible for maintaining telomere length (Greider and Blackburn 1985), is down-regulated or turned off in most human tissues early in development (Wright et al. 1996). The inability of the normal replication machinery to copy all the way to the end of a linear chromosome, combined with potential end-processing events (Wellinger et al. 1996;Huffman et al. 2000), results in the progressive shortening of telomeres with each subsequent cell division (Harley et al. 1990). The eventual growth arrest of cultured cells is thought to occur when some of the telomeres become sufficiently short to compromise their interaction with specific telomere-binding proteins such as TRF2 (van Steensel et al. 1998). The resulting failure of these proteins to mask the end of the chromosome from being recognized as a double-strand break induces a DNA-damage response that in turn produces the final growth arrest (Harley 1991). The ability of cDNAs encoding the catalytic subunit of human telomerase (hTERT) to produce telomerase activity, maintain telomere length, and immortalize normal human skin fibroblasts (Bodnar et al. 1998;Vaziri and Benchimol 1998), retinal pigment epithelial cells (Bodnar et al. 1998) and endothelial cells (Yang et al. 1999) has greatly strengthened this model.Replicative aging is thought to provide a barrier against the accumulation of mutations and the formation of cancer. If each m...

Elevated Bmi-1 expression is associated with dysplastic cell transformation during oral carcinogenesis and is required for cancer cell replication and survival

“…The intrinsic, telomere-dependent senescence is triggered by accumulation of shortened and dysfunctional telomeres with altered telomeric state (Harley et al, 1990;Karlseder et al, 2002). The extrinsic senescence is telomere-independent and triggered in cells after exposure to environmental factors, such as genotoxic stress, in vitro culture shock, and oncogenic stimuli (Serrano et al, 1997;Ramirez et al, 2001;Itahana et al, 2004;Chen et al, 2005;Dimri, 2005). The onset of extrinsic senescence is frequently associated with induction of p16 INK4A (Itahana et al, 2004).…”

Methylation of the p16INK4a promoter region in telomerase immortalized human keratinocytes co-cultured with feeder cells

“…When grown in co-culture with post-mitotic fibroblast feeder cells, human keratinocytes, exhibit a delay in passage dependent p16 INK4a (p16) expression and have an extended lifespan in culture (Ramirez et al, 2001;Rheinwald et al, 2002;Baek et al, 2003;Fu et al, 2003;Kang et al, 2003;Darbro et al, 2005). We have found that co-culture of keratinocytes with feeder cells delays the accumulation of p16 protein but does not prevent the eventual increase of p16 expression in late passage keratinocytes cultured with feeder cells .…”

“…Previous reports have suggested that human keratinocytes can be immortalized by telomerase activity alone when grown in co-culture with fibroblast feeder cells (Ramirez et al, 2001;Harada et al, 2003). Furthermore, these studies suggest that p16 expression in TERTimmortalized keratinocytes is not inactivated and remains inducible by either transfer to plastic culture conditions or exposure to UV irradiation.…”

“…The p16 INK4a /Rb pathway has been shown to be modulated by differences in culture conditions. There is evidence that suggests some epithelial cell types can be immortalized by TERT expression alone when cultured in the presence of feeder cells (Ramirez et al, 2001;Herbert et al, 2002;Harada et al, 2003). Furthermore, it has been suggested that the p16/Rb pathway is still intact in these immortalized epithelial cells and remains responsive to both UV irradiation and transfer to the plastic culture condition.…”

Immortalized cells as experimental models to study cancer

“…Furthermore, constitutive ectopic hTERT expression is insufficient to immortalize other cell types including keratinocytes, human mammary epithelial cells (HMECs), (Kiyono et al 1998), airway epithelial cells (Lundberg et al 2002), and preadipocytes (Darimont et al 2003) although in some cases, culture conditions permit cell immortalization with the expression of hTERT (Ramirez et al 2001). Immortal keratinocyte clones that eventually arise after the overexpression of hTERT show evidence of inactivation of the retinoblastoma (RB)/p16 INK4A tumor-suppressor pathway (Kiyono et al 1998;Dickson et al 2000).…”