On the Origins of Cellular Senescence
Originally described as gradual deterioration over time, senescence has taken on a much more intricate meaning in the past few decades. What used to be a hand-wavy term to describe the increased rate of mortality with age is now a term used often by molecular biologists to describe cellular dysfunction due to DNA damage and other environmental stressors. This radical shift came in 1961 when Leonard Hayflick, now a titan in the field of aging research, observed that senescence occurs on a cellular level, and not just on an organismal level as previously believed.
“Senescence is an inevitability. All we can do is try to strike the right balance between graceful acceptance and raging against a dying light.” — Marty Nemko, PhD.
Before Hayflick’s discovery, it was believed that cells in culture could grow indefinitely. This idea was fueled by work done from the 1910s to 1940s by Alexis Carrel, a Nobel-prize winning French surgeon, in which he observed that fibroblasts isolated from chicken hearts could grow in culture for over 30 years . This finding sent shockwaves through the scientific community, especially since chickens have a maximum lifespan of only ~10 years. This work led researchers in the field to believe that cells were essentially immortal in vitro and would only succumb to aging in an in vivo setting.
The end of immortality
In 1958, the Wistar Institute hired Hayflick as a cell culture expert, and he was tasked with generating large quantities of human cells for purposes such as vaccine development. At the time, lab technicians had been struggling, unable to successfully maintain a culture of healthy (non-cancerous) cells without these cultures degrading over time. Hayflick set out to resolve this problem using a technique known as serial passaging. With serial passaging, once the cells start outgrowing their culture dish and running out of food (called “culture media”), a small portion of cells is transferred to a clean dish with fresh media. Though he achieved success using a rigorous application of this method to grow healthy human cells for many months, there was still a limit: eventually, his cells too began to degrade and stop replicating.
Convinced that he had made an error at some point in the protocol, he conducted a series of experiments with Paul Moorhead, a skilled cytogeneticist, to investigate this issue . The duo conclusively determined that healthy cells indeed undergo a finite number of divisions — enough to undergo only about 40–60 passages — after which they will replicate no more. This finding represented a major break from Carrel’s earlier work, which was still widely accepted at the time. Hayflick and Moorehead’s observations suggested for the first time that aging occurs on a cellular level, as healthy cells eventually cease to divide and enter a state of cellular senescence.
It is now believed that the work originally done by Carrel was deeply flawed . There are a few theories as to how this may have occurred: one popular theory is that the original population of cells contained pluripotent stem cells; another theory is that new cells were being added to the original population with every addition of fresh cell culture media. Despite Carrel’s lauded “discovery” at the time, his experiments with cell division have been irreproducible to this day.
Hunting for an internal counter
The discovery that somatic cells undergo a finite number of cell divisions was replicated in numerous cell types across a plethora of species, and this constraint became known as the Hayflick Limit. Hayflick speculated that cells could somehow “count” or keep track of their population doublings and began investigating this hypothesis. He observed that cryogenically preserved cells retained a “memory” of past cell divisions and adhered to the Hayflick Limit even after being frozen for years. This finding suggested that the mechanism responsible for the Hayflick Limit was not based on the passage of time, but on an unknown biological phenomenon.
Dubbed the “replicometer”, Hayflick and his lab set out to find the mechanism responsible for restricting the maximum number of cell divisions. The first mystery to solve was where the cellular machinery underlying this mechanism was located, and from there they planned to decipher which organelles and molecules were involved. They postulated that there were two plausible scenarios: either the machinery was located in the cytoplasm or the nucleus. If located in the cytoplasm, the counting mechanism would most likely be due to the progressive accumulation of damaged organelles; if in the nucleus, then the explanation could be that senescence is a programmed genetic event.
In 1975, Woodring Wright, a graduate student in Hayflick’s lab, answered this question by fusing together cells from different rounds of population doublings . Wright took cells and removed their nuclei by treating them with cytochalasin B. This process leaves two distinct parts of biological material: cytoplasts (cells without nuclei) and nucleoplasts (free nuclei). For his experiments, he took nucleoplasts from young cells (10–15 rounds of population doublings) and inserted them into the cytoplasts of old cells (40–50 population doublings). He also did the reverse: he inserted nucleoplasts from old cells into the cytoplasts from young cells. Wright found that the number of population doublings exhibited by these hybrid cells depended entirely on the donor nucleus. In other words, cells with young nuclei exhibited many more population doublings before becoming senescent, while the cells with old nuclei reached senescence after far fewer doublings. This experiment showed that the internal counting mechanism takes place in the nucleus and not in the cytoplasm.
Solving the end replication problem
Though the location of this internal counter had been identified, the molecular mechanism remained a mystery. Alexey Olovnikov, a Russian theoretical biologist studying DNA replication, proposed an answer . He knew that each time a cell divides, it first duplicates its DNA so as to pass on one copy of its genome to each of the two daughter cells. He also knew that DNA replication machinery was unable to access the very end of the DNA strand, known as the 3’ end. Thus, he postulated that with each cell division, the DNA strands must become shorter and shorter; given enough of these divisions, the cell will eventually lose important genetic information, leading to dysfunction or death. This is known as the end replication problem.
To get a sense of how this occurs, imagine a construction worker standing on a brick wall. His job is to lay down bricks while walking on the wall in a single direction. He places one brick down, then takes one step back, then places another brick down, takes another step back, etc. He is able to successfully carry out his job for almost the eternity of the wall, except he is unable to lay the very last brick because he has nothing left to stand on, and so he falls off, leaving the wall incomplete. This is the end replication problem in a nutshell, wherein the new layer of bricks is the new DNA strand, and the construction worker is the DNA replication machinery.
Both the end replication problem and the cellular senescence phenomenon raised important questions about the nature of life itself. Namely, if the DNA in cells continues to shrink with each generation, then would all life not eventually die out? Similarly, if cells are capable of a finite number of cell divisions before becoming senescent, then how does life continue to persist? Since life has been thriving on our planet for over 4 billion years, Olovinikov hypothesized in 1971 that there must be mechanisms responsible for protecting genetic information as well as lengthening DNA. Despite these thought-provoking hypotheses, Olovinikov never conducted the laboratory experiments necessary to support this idea.
Fortunately, scientists in the United States were making significant progress to support the existence of both a mechanism to protect genetic information and a mechanism to lengthen DNA. In 1978, while conducting experiments on tetrahymena, a single-cell organism, Elizabeth Blackburn and Joseph Gall noticed something peculiar about its DNA. They observed that the ends of each DNA strand had repeated sequences of six nucleotides, TTGGGG. These redundant regions of the DNA, known as telomeres, repeat 20–70 times . This finding provided evidence that telomeres act as buffers to protect important genetic information from being eliminated. Telomeres, or some variant of them, exist in many different types of organisms ranging from fungi to animals.
In 1985, a mechanism for telomere lengthening was co-discovered by Elizabeth Blackburn and Carol Greider when they identified the enzyme telomerase, which is responsible for adding telomeric repeats onto the ends of DNA . Telomerase is active in germline and stem cells, but not somatic cells, which is why Hayflick and others observed that cells like dermal fibroblasts have finite replicative lifespans. In 1990, Blackburn and Greider demonstrated that telomeric DNA length decreases with each cell division, both in vitro and during natural aging in vivo . Finally in 1998, Woodring Wright, now running his own laboratory, showed that telomere shortening was sufficient for cells to enter senescence . Thus, the mechanism responsible for cells reaching the Hayflick Limit was finally elucidated. Elizabeth Blackburn would go on to win the 2009 Nobel Prize in Physiology or Medicine and share it with Carol Greider and Jack Szostak for these findings.
These seminal discoveries raised important questions in the field of cellular biology. What is the purpose of cellular senescence? Is senescence a mechanism that has evolved to confer a survival advantage to a species, or is it merely an accident that evolution has failed to eradicate? In this article, I have described the initial discovery of cellular senescence, the mechanism behind replicative senescence, and the great minds responsible for these findings. Still, this is only a small fraction of the vast array of captivating insights and outstanding questions within the field of cellular senescence. To learn more about cellular senescence, including the relationship between senescence, aging, and longevity, stay tuned for future articles in this series.
Timeline graphic created using BioRender.com.
Note: This article was updated on Oct 5, 2021 to more precisely describe Hayflick’s motivations and observations.
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