Hayflick limit

The Hayflick limit (or Hayflick Phenomenon) is the number of times a normal cell population will divide before it stops, presumably because the telomeres reach a critical length.

The Hayflick limit was discovered by Leonard Hayflick in 1961,    at the Wistar Institute, Philadelphia, when Hayflick demonstrated that a population of normal human fetal cells in a cell culture divide between 40 and 60 times. It then enters a senescence phase (refuting the contention by Alexis Carrel that normal cells are immortal). Each mitosis shortens the telomeres on the DNA of the cell. Telomere shortening in humans eventually makes cell division impossible, and it correlates with aging. This mechanism appears to prevent genomic instability and the development of cancer.

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-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 claiming to have 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 were unable to reproduce Carrel's result.

In fact, Carrel's result was due to an error in his experimental procedure: chick embryonic stem cells were added to the culture daily. This allowed for the cultivation of new fresh cells in the culture, and not simply the infinite reproduction of the original cells present in the culture. It has been speculated that Carrel knew about the error, but he never admitted it.

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, he designed an experiment that showed the truth about normal 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 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 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 as accredited biological theory which, unlike the experiment of Carrel, has been reproduced by other scientists.

Cell phases
Hayflick describes three phases in the life of a cell. At the start of his experiment, he named the primary culture "phase one." In the period when cells were proliferating—or as Hayflick called it, the time of “luxuriant growth”—the cells were in "phase two." After so many months of doubling, eventually the cells reach what is called the “phase three phenomenon,” where cell growth is diminished and eventually stopped altogether.

Telomere length
This 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, the cell will no longer divide and the Hayflick limit has been reached.

This process errs in cancer cells. Cancer cells turn on an enzyme called telomerase which is able to restore telomere length. Thus the telomere of cancer cells is never shortened, giving these cells infinite replicative potential. A proposed treatment for cancer is a telomerase inhibitor 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 perhaps extend the telomeres, thus extending the Hayflick limit of healthy cells. This might strengthen the telomeres of immune system cells enough to prevent cancerous cells from developing from cells with very short telomeres.

Carnosine can increase the Hayflick limit in human fibroblasts,   and also appears to reduce the rate of telomere shortening.

Literature

 * Harley C, Futcher A & Greider C (1990) Telomeres shorten during ageing of human fibroblasts, Nature, 345, 458–460.
 * Leonid A. Gavrilov & Natalia S. Gavrilova (1991) The Biology of Life Span: A Quantitative Approach. New York: Harwood Academic Publisher, ISBN 3-7186-4983-7 (see section 5.6 there)
 * Gavrilov, L.A., Gavrilova, N.S. (1993). How many cell divisions in 'old' cells? Int. J. Geriatric Psychiatry, 8(6): 528-528.
 * Wang R, Smogorzewska A & Lange T (2004) Homologous Recombination Generates T-Loop-Sized Deletions at Human Telomeres, Cell, 119, 355–368.
 * Watson J & Shippen D (2007) Telomere Rapid Deletion Regulates Telomere Length in Arabidopsis thaliana, Molecular and Cellular Biology, 27(5), 1706-1715.