The Hayflick limit[Note 1] (or Hayflick phenomenon) is the number of times a normal human cell population will divide until cell division stops. Empirical evidence shows that the telomeres associated with each cell's DNA will get slightly shorter with each new cell division until they shorten to a critical length.
The concept of the Hayflick limit was advanced by Leonard Hayflick in 1961, at the Wistar Institute in Philadelphia. Hayflick demonstrated that a population of normal human fetal cells in a cell culture will divide between 40 and 60 times. The population will then enter a senescence phase, which refutes the contention by Nobel laureate Alexis Carrel that normal cells are immortal. Each mitosis slightly shortens each of the telomeres on the DNA of the cells. Telomere shortening in humans eventually makes cell division impossible, and this aging of the cell population appears to correlate with the overall physical aging of the human body. This mechanism also appears to prevent genomic instability. Telomere shortening may also prevent the development of cancer in human aged cells by limiting the number of cell divisions. However, shortened telomeres impair immune function that might also increase cancer susceptibility.
Belief of cell immortality
Prior to Hayflick's discovery, it was believed that vertebrate cells had an unlimited potential to replicate. Alexis Carrel, a Nobel prize-winning surgeon, had stated "that all cells explanted in culture are immortal, and that the lack of continuous cell replication was due to ignorance on how best to cultivate the cells". He supported this hypothesis by having cultivated fibroblasts from chick hearts, and to have kept the culture growing for 34 years. This indicated that cells of vertebrates could continue to divide indefinitely in a culture. However, other scientists have been unable to repeat Carrel's result.
Carrel's result is suspected to be due to an error in experimental procedure. To provide required nutrients, chick embryonic stem cells may have been readded to the culture daily. This would have easily allowed the cultivation of new, fresh cells in the culture, so there was not an infinite reproduction of the original cells. If this is true, it has been speculated that Carrel knew about the error, but he never admitted it.
Also, it has been theorized that the cells Carrel used were young enough to contain pluripotent stem cells, which, if supplied with a supporting telomerase-activation nutrient, would have been capable of staving off replicative senescence, or even possibly reversing it. Cultures not containing telomerase-active pluripotent stem cells would have been populated with telomerase-inactive cells, which would have been subject to the 50–60 mitosis cycles until apoptosis occurs as described in Leonard Hayflick's findings.
Experiment and discovery
Dr. Leonard Hayflick first became suspicious of Carrel's theory while working in a lab at the Wistar Institute. Hayflick was preparing normal human cells to be exposed to extracts of cancer cells when he noticed the normal cells had stopped proliferating. At first he thought that he had made a technical error in preparing the experiment, but later he began to think that the cell division processes had a counting mechanism. Working with Paul Moorhead, a cytogeneticist, he designed an experiment to test Carrel's theory of cell division.
The experiment proceeded as follows. Hayflick and Moorhead mixed equal numbers of normal human male fibroblasts that had divided many times (cells at the 40th population doubling) with female fibroblasts that had divided only a few times (cells at the 10th population doubling). Unmixed cell populations were kept as controls. When the male control culture stopped dividing, the mixed culture was examined and only female cells were found. This showed that the old male cells remembered they were old, even when surrounded by young cells, and that technical errors or contaminating viruses were unlikely explanations as to why only the male cell component had died. The cells had stopped dividing and had become senescent based purely upon how many times the cell had divided.
These results disproved the immortality theory of Carrel and established the Hayflick limit[Note 1] as a credible biological theory that, unlike Carrel's experiment, has been repeated by other scientists.
Hayflick describes three phases in the life of a cell. At the start of his experiment he named the primary culture "phase one". Phase two is defined as the period when cells are proliferating – Hayflick called it the time of "luxuriant growth". After months of doubling the cells eventually reach phase three, a phenomenon of senescence – cell growth diminishes and then cell division stops altogether.
The Hayflick limit has been found to correlate with the length of the telomere region at the end of a strand of DNA. During the process of DNA replication, small segments of DNA at each end of the DNA strand (telomeres) are unable to be copied and are lost after each time DNA is duplicated. The telomere region of DNA does not code for any protein; it is simply a repeated code on the end region of DNA that is lost. After many divisions, the telomeres become depleted and the cell begins apoptosis. This is a mechanism that prevents replication error that would cause mutations in DNA. Once the telomeres are depleted, due to the cell dividing many times, it will no longer divide having reached its Hayflick limit.
This process does not take place in cancer cells due to an enzyme called telomerase. This enzyme maintains telomere length, which results in the telomeres of cancer cells never shortening. This gives these cells infinite replicative potential. A proposed treatment for cancer is the usage of telomerase inhibitors that would prevent the restoration of the telomere, allowing the cell to die like other body cells. On the other hand, telomerase activators might repair or extend the telomeres of healthy cells, thus extending their Hayflick limit. Telomerase activation might also lengthen the telomeres of immune system cells enough to prevent cancerous cells from developing from cells with very short telomeres.
- Hayflick L, Moorhead PS (1961). "The serial cultivation of human diploid cell strains". Exp Cell Res 25 (3): 585–621. doi:10.1016/0014-4827(61)90192-6. PMID 13905658.
- Hayflick L. (1965). "The limited in vitro lifetime of human diploid cell strains". Exp. Cell Res. 37 (3): 614–636. doi:10.1016/0014-4827(65)90211-9. PMID 14315085.
- Eisenberg DTA (2011). "An evolutionary review of human telomere biology: The thrifty telomere hypothesis and notes on potential adaptive paternal effects". American Journal of Human Biology 23 (2): 149–167. doi:10.1002/ajhb.21127. PMID 21319244.
- Shay JW, Wright WE; Wright (2000). "Hayflick, his limit, and cellular ageing" (PDF). Nature Reviews Molecular Cell Biology 1 (1): 72–76. doi:10.1038/35036093. PMID 11413492.
- Carrel A, Ebeling AH (1921). "Age and multiplication of fibroblasts". J. Exp. Med. 34 (6): 599–606. doi:10.1084/jem.34.6.599.
- Witkowski JA (1985). "The myth of cell immortality". Trends Biochem. Sci. 10 (7): 258–260. doi:10.1016/0968-0004(85)90076-3.
- Witkowski JA (1980). "Dr. Carrel's immortal cells". Med. Hist. 24 (2): 129–142. doi:10.1017/S0025727300040126. PMC 1082700. PMID 6990125.
- Watson JD (1972). "Origin of concatemeric T7 DNA". Nature New Biol. 239 (94): 197–201. PMID 4507727.
- Olovnikov AM (1996). "Telomeres, telomerase and aging: Origin of the theory". Exp. Gerontol. 31 (4): 443–448. doi:10.1016/0531-5565(96)00005-8. PMID 9415101.
- Olovnikov, A. M. (1971). "Принцип маргинотомии в матричном синтезе полинуклеотидов" [Principles of marginotomy in template synthesis of polynucleotides]. Doklady Akademii Nauk SSSR 201: 1496–1499.
- Feng F et al. (1995). "The RNA component of human telomerase". Science 269 (5228): 1236–1241. doi:10.1126/science.7544491. PMID 7544491.
- Wright WE, Shay JW (2000). "Telomere dynamics in cancer progression and prevention: Fundamental differences in human and mouse telomere biology". Nature Medicine 6 (8): 849–851. doi:10.1038/78592. PMID 10932210.
- McFarlan GA, Holliday R (1994). "Retardation of the senescence of cultured human fibroblasts by carnosine". Exp. Cell Res. 212 (2): 167–175. doi:10.1006/excr.1994.1132. PMID 8187813.
- Shao L; Li QH, Tan Z (2004). "L-carnosine reduces telomere damage and shortening rate in cultured normal fibroblasts". Biochem Biophys Res Commun. 324 (2): 931–936. doi:10.1016/j.bbrc.2004.09.136. PMID 15474517.
- Watts, Geoff (2011). "Leonard Hayflick and the limits of ageing". The Lancet 377 (9783): 2075. doi:10.1016/S0140-6736(11)60908-2.
- Harley, Calvin B.; Futcher, A. Bruce; Greider, Carol W. (1990). "Telomeres shorten during ageing of human fibroblasts". Nature 345 (6274): 458–60. doi:10.1038/345458a0. PMID 2342578.
- Gavrilov LA, Gavrilova NS (1991). The Biology of Life Span: A Quantitative Approach. New York: Harwood Academic Publisher. ISBN 3-7186-4983-7.
- Gavrilov LA, Gavrilova NS (1993). "How many cell divisions in 'old' cells?". Int. J. Geriatric Psychiatry 8 (6): 528–528.
- Wang, Richard C.; Smogorzewska, Agata; De Lange, Titia (2004). "Homologous Recombination Generates T-Loop-Sized Deletions at Human Telomeres". Cell 119 (3): 355–68. doi:10.1016/j.cell.2004.10.011. PMID 15507207.
- Watson, J. M.; Shippen, D. E. (2006). "Telomere Rapid Deletion Regulates Telomere Length in Arabidopsis thaliana". Molecular and Cellular Biology 27 (5): 1706–15. doi:10.1128/MCB.02059-06. PMC 1820464. PMID 17189431.
- Historical review of studies on cell division limit
- Cell immortality and cancer
- WNYC RadioLab Episode on Mortality