Hayflick limits

Figure 26.1 Immortalization of human cells Cells enter replicative senescence at mortality stage 1 (Ml Hayflick limit) after about 60 population doublings (PD). The protein p 16 accumulates in senescent cells. The simian virus 40 (SV40) large T antigen as well as the human papilloma virus (HPV) type 16-E6 and E7 proteins sequester the retinoblastoma protein (Rb) and/or p53 constitutively releases the transcription factor E2F. E2F induces expression proteins required for progression through Gl/S transition, thus the cells escape cell cycle arrest. At mortality stage 2 (M2), transformed cells must overcome senescence and crisis before they are immortalized. This is likely to involve the activation of telomerase either by the introduction of hTERT cDNA or by a genetic change that activates telomerase.

Beyond the Hayflick limit, cells that have escaped senescence and/or crisis must progress beyond mortality stage 2 (Figure 26.1). Recent studies have shown that human telomerase reverse transcriptase cDNA (hTERT) in combination with viral oncogenes also has the potential to immortalize human airway epithelial cells [15, 78-82], However, even though hTERT alone will cause enhanced growth potential of a cell line, with the exception of one study [81],... 

Cell tines offer several advantages over primary cell cultures, such as an unlimited life-span and the lack of time-consuming isolation procedures. Additionally once established, they are often more stable than primary cells which are usually in a continuous state of de-differentiation. Thus, the majority of in vitro nephrotoxicity studies have been performed on renal epithelial cell tines. In normal somatic cells, telomeres, the tandemly repeated hexamers at the end of mammalian chromosomes, act as the cellular replicative clock [43] and shorten at each cell division. Once telomeres have exceeded a certain critical length, the so called "Hayflick limit" [44], the cell enters replicative senescence and no longer proliferates. Until recently the most widely used renal cell tines were those which arose from spontaneously acquired immortalization in culture. These cell tines include LLC-PK (Hampshire pig) [45,46], JTC-12 (cynomolgus monkey) [47] and OK (American opossum) [48] cells, which exhibit biochemical and antigenic characteristics suggestive of proximal... 

These cells may be rejuvenated, however, by furnishing new instructions, even though these instructions may be different. For example, if exiled human cells are treated with horse serum, they become more horseUke. Or if a mutation takes place in one or another of the cells, furnishing new instructions, the new proliferation will then grow beyond the Hayflick limit, no longer being constrained by the old cellular restraints. The new cellular Une thus becomes cancerous. 

There are intriguing variations on this theme. Fibroblasts taken from short-lived species, such as mice, have a lower Hayflick limit than fibro-... 

The story of the Hayflick limit came to a head in 1990, when Cal Harley, founder of the Californian biotechnology company Geron Corporation, made a connection between the ability of cells to count and the length of their telomeres — the tips at the ends of individual chromosomes. Telomeres are often said to resemble the ends of a shoelace — their purpose is to prevent fraying in other words, to preserve the integrity of the chromosome. They are also said to be the secret of eternal life. They are not, as we shall see. 

Figure 11.4-1. Cellular senescence and immortalization. Telomere length is maintained by telomerase, and most human somatic cells have lower levels of telomerase. The cells are telomerase-negative and experience telomere shortening with each cell division. Shortened telomeres may start the cells to enter senescence at the Hayflick limit, or Ml. This proliferative checkpoint can be overcome by an inactivation of pRB/pl6 or p53, for example, by the use of SV40 or human papilloma virus oncoproteins. Snch cells continue to suffer telomere erosion and ultimately enter crisis, or M2, characterized by cell death. Quite a few surviving cells acquire stabilization of telomere length and unlimited proliferative potential, mostly due to activation of telomerase. (This figure is available in full color at ftp //ftp.wiley. com/public/sci tech med/pharmaceutical biotech/.)...

Telomeres play an important role in defining the replicative life span of cells, i.e., the maximal number of cell divisions or the so-called Hayflick limit. Normal human somatic cells, such as fibroblasts, after isolation ftom the body are only able to undergo a limited number of cell divisions, dependent on the age of the donor, before they stop cycling and go into the senescent state , which becomes manifest in phenotypic charges like cellular (lattenii and expression of a senescence-associated. alactosidase. Such cells ate arrested in G, which is different from quiescent cells, which arrest in Gq. After acquirir the senescent phenotype cells are still viable and can be maintained in culture for up to several months. The telomeres in fibroblasts shorten with each di ion due to the end-replication problem , because conventional DNA polymerases need a free 3 -hydroxyl toup lor DNA synthesis. This is usually provided by the activity of the polymerase o/ptimase complex, which synthesizes an initial RNA o%onucleotide primer. Durit strand synthesis, the most distally located primer... 

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