8.6 The Hayflick Limit

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The Hayflick Limit in Relation to Cancer and Senescence

 

The Hayflick limit was discovered by Leonard Hayflick. To demonstrate the mortality of cell lines in culture, Hayflick combined female fibroblasts that had undergone approximately 10 population doublings with an equal number of male fibroblasts that had undergone approximately 40 population doublings (2). He provided unmixed populations of each as controls and found only female cells existing in the mixed culture when the male control cells had stopped dividing, revealing an ability of older cells to somehow remember being older, even when surrounded by the relatively younger cells (2). Hayflick proposed that an intrinsic counting mechanism of cell divisions was responsible for his results, and not chronological differences, based on two findings: interrupting growth with cryopreservation showed senescence after the same number of cell divisions as control cells, and that normal human fetal cells all showed the same specific number of population doublings (1,2). These results were later published in Experimental Cell Research, and the total finite number of population doublings a cell can produce was coined the ‘Hayflick Limit’ (2).

 

The Hayflick Limit coincides with the cellular event of senescence, since cell growth and division arrest once it is reached - senescence describes the state of cells after reaching their Hayflick Limit (3). Hayflick proposed three phases from his observations; Phase I was the primary culture, Phase II was the luxurious growth and cumulative population doublings, and Phase III was senescence (Figure 8.6.1) (4). Hayflick’s finding remains true in vivo, seen through several lines of evidence (1). For example, epithelial cells of elderly donors have a highly increased frequency of an unusual form of β-galactosidase, which is also seen in senesced epithelial culture cells (1). β-galactosidase is typically active at pH 4, but as cells approach senescence the percentage of active β-galactosidase at pH 6 drastically increases (11). Therefore, a commonly used marker for senescent cells is senescence associated β-galactosidase activity (11).

Figure 8.6.1. Hayflick's three phases, leading to senescence.

 

What is responsible for the Hayflick Limit, and why does it differ between cells? The discovery of telomeres answers both of these questions (5). At the ends of chromosomes, telomeres offer protection, but are not replicated in full at each cell division – termed the ‘End Replication Problem’ – and when sufficiently short they result in growth arrest via similar pathways such as DNA damage (3, 5). It has been shown in mice that division limits are due to the shortest telomere, and not average telomere length, supporting the notion of DNA damage pathway utilization upon reaching a critical length (4). Although accepted as the main mechanism determining the Hayflick Limit and senescence, Intra-clonal variability is apparent, and therefore other contributions must exist (3, 6). Somatic and mitochondrial mutations, and methylation events have been noted to contribute to Hayflick Limit determinism, but best studied is the contribution of oxidative stress (6). Introduction of reactive oxidative species to cells increases the rate of telomere loss, resulting in a lower Hayflick Limit, and therefore earlier senescence (3, 6). Other molecules, like pro-inflammatory cytokines have shown similar results, since inflammation can promote the generation of oxidative species and mutation (4). Oxidative species are well established causes of DNA damage and mutation, and telomeric ends are known to be less efficiently repaired compared to more euchromatic regions (10). This results in an increased presence of unrepaired nucleotides and base damage due to chronic oxidative stress, which are thought to disrupt the replication fork during mitosis (10). This disruption causes an increased proportion of the telomeric ends to not be replicated, resulting in accelerated telomeric loss, and senescence (10). Additionally, oxidative species often heavily contribute to cancer simply through their ability to mutate DNA in exonic regions, as they are known to substantially promote guanine to thymine transversions, which can result in the activation of oncogenes or inactivation of tumor suppressor genes (10).

 

Some cells, however, appear to lack a Hayflick Limit, such as germ cells, pluripotent stem cells, and cancerous cells (1). Their common feature is the presence of telomerase – an enzyme responsible for elongating the ends of telomeres, although some cancerous cells show immortalization in the absence of telomerase (Refer to Chapter 1 for more information on immortalization) (1, 3). One interesting study revealed that fusion of a normal cell with an immortal tumor cell results in a cell possessing a Hayflick Limit, demonstrating it as a dominant trait (1). This finding led to the proposition that the Hayflick Limit evolved as a tumor suppressor mechanism by suppressing telomerase expression (1). This proposition is supported by the fact that senescence is often activated through known tumor suppressor gene pathways, such as those involving p53 and RB (12).

 

The Hayflick Limit’s role in human aging and disease is still not clear, but many insights do exist. If pushed past their Hayflick Limit though p53 and RB inactivation, the cells’ telomeres become too short, leading to genetic instability, and eventually a massive cell death event known as ‘crisis’ in vitro (7). During this period of genetic instability, cells may acquire mutations that lead to immortalization. The immortalized cells survive crisis, resulting in selection for the precancerous cells (7). Within a more natural setting, this is known to be a cancer causing mechanism in vivo as well (7). As T-cell lymphocytes reach their Hayflick Limit, they show decreased expression of CD28, decreased responsiveness to antigenic stimulation, and an overall increased response to apoptotic stimuli (1,8). This helps explain the elderly’s heightened susceptibility to disease, as they would possess fewer and less responsive immune cell populations (8). The diminishing capabilities of the immune system would further contribute to the progression of cancer, since the likelihood of cancerous or precancerous cells being eliminated would decrease (8).  The Hayflick Limit also suggests that, in the absence of disease, organisms have a finite age, which is known today as death by natural causes (9).

 

A mathematical aside

 

Fibroblasts from middle aged humans are able to perform another 20 to 40 population doublings (PD) before their Hayflick limit is reached (13). A cell that undergoes 40 PD would produce 240 cells, which is approximately equal to a 1kg tumour (13). Thus, some people argue that immortalization is not necessary for tumour formation because a tumour can theoretically be formed without surpassing a cell’s Hayflick limit (13). However, the realistic number of PD necessary to result in a 1kg tumour is significantly higher than 40 due to two major reasons (13).

 

The first reason is that the production of 240 cells after 40 PD does not account for the fact that cells die, especially at the centre of tumours where there is a lack of blood supply (13). The maximum number of cells that can be produced from x PD is 2x cells. If the fraction of cells that survive (SF for survival fraction) each PD is y where y is between 0 and 1, the number of cells after x PD becomes (2y)x. Assuming y is 0.75, 68 PD is required to generate a 1kg tumour (13). The second reason is that cells cannot inherently form a tumour, multiple genetic mutations must occur beforehand (13). If the mutation rate is 1 per 104 cells, a rate found in highly unstable genomes, a normal cell has to go through 13 PD to generate a cell with one mutation (13). To get a cell with n hits, 13n PD must occur (13). Factoring in cell death will increase this number and a SF of 0.75 requires 23 PD for each mutation (13). The cell with n hits must then proliferate into a 1kg tumour (13). Ignoring the presence of cells with <n hits to simplify the math, a cell with n hits needs another 40 PD for SF 1 or 68 PD for SF 0.75 (13). In total, with SF 0.75, a normal cell must go through 23n + 68 PD to generate a 1kg tumour (13). It is estimated that most tumours require at least five mutations, so 23(5) + 68 = 183 PD (13).

 

These PD calculations are highly dependent on the variable numbers for survival fraction, mutation rate, and number of mutations, but demonstrate that in most cases, immortalization is a requirement for tumour formation because the number of PD needed is way higher than the Hayflick limit (13). There are some situations though where immortalization might not be required (13). A tumour may form without immortalization if the cells still have many PD remaining until they reach the Hayflick limit, such as during childhood (13). This might also happen if the SF is quite high, such as in leukemia where blood supply is not a problem (13). Finally, immortalization might not be necessary if the tumour is caused by a virus, as a viral infection can significantly speed up mutation accumulation (13).

 

Table 8.6.1. The number of population doublings required to form a 1kg tumour based on survival fraction and number of mutations. Mutation rate is assumed to be 1 per 10000 cells.

 

Survival Fraction

1

0.9

0.8

0.7

0.6

Number of mutations

3

79

95

119

163

305

4

92

111

139

190

356

5

105

127

159

217

407

6

118

143

179

244

458

7

131

159

199

271

509

 


 

References

1. Effros, R.B., and Pawelec, G. (1997). Replicative senescence of T cells: does the Hayflick Limit lead to immune exhaustion? Immunology Today 18, 450–454.

2. Shay, J.W., and Wright, W.E. (2000). Hayflick, his limit, and cellular ageing. Nature Reviews. Molecular Cell Biology 1, 72–76.

3. von Zglinicki, T., Saretzki, G., Ladhoff, J., d’ Adda di Fagagna, F., and Jackson, S.P. (2005). Human cell senescence as a DNA damage response. Mechanisms of Ageing and Development 126, 111–117.

4. Toussaint, O., Remacle, J., Dierick, J.-F., Pascal, T., Frippiat, C., Zdanov, S., Magalhaes, J.P., Royer, V., and Chainiaux, F. (2002). From the Hayflick mosaic to the mosaics of ageing. Role of stress-induced premature senescence in human ageing. The International Journal of Biochemistry & Cell Biology 34, 1415–1429.

5. Greider, C.W. (1998). Telomeres and senescence: the history, the experiment, the future. Current Biology : CB 8, R178–81.

6. Sozou, P.D., and Kirkwood, T.B. (2001). A stochastic model of cell replicative senescence based on telomere shortening, oxidative stress, and somatic mutations in nuclear and mitochondrial DNA. Journal of Theoretical Biology 213, 573–586.

7. Maser, R.S., and DePinho, R.A. (2002). Connecting chromosomes, crisis, and cancer. Science (New York, N.Y.) 297, 565–569.

8. Effros, R.B. (1998). Replicative senescence in the immune system: impact of the Hayflick limit on T-cell function in the elderly. American Journal of Human Genetics 62, 1003–1007.

9. Juckett, D.A. (1987). Cellular aging (the Hayflick limit) and species longevity: a unification model based on clonal succession. Mechanisms of Ageing and Development 38, 49–71.

10. Von Zglinicki, T. (2002). Oxidative stress shortens telomeres. Trends in Biochemical Sciences 27, 339–344.

11. Pedro de Magalhães, J. (2004). From cells to ageing: a review of models and mechanisms of cellular senescence and their impact on human ageing. Experimental Cell Research, 300(1), 1-10.

12. Ben-Porath, I., and Weinberg, R.A. (2005). The signals and pathways activating cellular senescence. The International Journal of Biochemistry & Cell Biology 37, 961–976.

13. Reddel, R. (2000). The role of senescence and immortalization in carcinogenesis. Carcinogenesis 21, 477-484.