Mechanisms of Aging: Cellular Clock

Bacteria are immortal, at least in principle. If a bacterial cell is placed in favorable conditions it will grow and divide indefinitely. What about the cells of higher organisms like birds or mammals? Until mid twentieth century, it was thought that cells from higher species were also immortal, i.e. capable of dividing indefinitely under proper conditions. In 1912, Alexis Carrel, then a researcher at the Rockefeller Institute, started an experiment designed to test how long chicken fibroblasts could divide. Fibroblasts are connective tissue cells whose job is to build the resilient three-dimensional framework that supports other cells. Carrel fed the fibroblasts with a special broth derived from chicken embryos, supplying fresh broth every few days. Excess cells were periodically discarded. The fibroblasts kept dividing year after year, without any signs of slowing down until, after Carrel's death some 30 years later, the experiment was stopped. It seemed that in a proper environment the cells of higher organisms were as immortal as bacteria.

In the early 1960s, Leonard Hayflick made an observation that directly contradicted Carrel's results. Hayflick found that human fibroblasts in culture would divide about fifty times and then stop. Who was right, Carrel or Hayflick? It appears that Carrel's experimental technique had a flaw. The nourishing broth he used to feed the chicken fibroblasts is likely to have contained a small number of chicken fibroblasts. So, Carrel inadvertently added new cells every few days. The conflict was resolved in the favor of Hayflick, and the maximal number of divisions a cell could undergo in culture came to be known as the Hayflick limit.

It was suggested that the Hayflick limit is a genetic program that prevents cell division after a certain number of cycles. Why would our cells need to have such a program? The current view is that a built-in limit on the number of possible cell divisions reduces the risk of uncontrolled cell growth resulting in cancer. Indeed, studies indicate that in cancer cells this genetic clock is broken, allowing them to grow indefinitely.

Eventually, the molecular mechanism behind the Hayflick limit was discovered. In the cells of higher organisms, chromosomes are capped with special DNA structures called telomeres. The main role of telomeres is to protect the ends of chromosomes from degradation. During cell division the chromosomes are duplicated through the process of DNA replication. However, due to the nature of this process, the very ends of the telomeres cannot be copied. Imagine a road machine that moves along the road and makes a new layer of asphalt behind it. The machine's design is such that it can move only when it is on the road. When it has reached the end of the road, there is a new layer of asphalt over the entire road except for the spot under the machine. To be able to cover the remaining spot, the machine must move past it but it cannot because it is already at the end of the road. Similar scenario occurs during the replication of chromosomes. Thus, the very end of each telomere never gets copied, which leads to progressive shortening of telomeres after each cell division. It appears that after its telomeres have shortened beyond a certain point, the cell loses the ability to divide. (This doesn't occur in bacteria because their chromosomes are circular and do not have telomeres.) When fibroblasts approach fifty divisions not only they lose the ability to divide but also begin to look and behave "old": their metabolic activity decreases, they increase in size and accumulate lipofuscin, the pigment responsible for age spots. This state of is called cellular senescence.

Could it be that aging is the result of the cells' inability to divide once they have reached the Hayflick limit? There is no straight answer to that at present. It appears that in some tissues, such as the skin and the lining of blood vessels, the Hayflick limit may be an important part of the aging process. For instance, the accelerated progression of vascular diseases with age may, in part, be caused by the reduced ability of vascular epithelial cells to divide. Hayflick limit may also play a role in age-related skin changes as more dermal fibroblasts reach the state of senescence. On the other hand, the cells in the brain, retina, nerves and muscles normally do not divide, and probably never even approach the Hayflick limit.

Not all cells are subject to the Hayflick limit. Germ cells (the cells that develop into sperm or ova) and cancer cells are obviously immortal. Embryonic stem cells (and perhaps some adult stem cells) may also be potentially very long-lived or immortal. However, whether a stock of immortal stem cells exists in a tissues (such as skin) or not, the accumulation dysfunctional cell that reached the Hayflick limit appears to be a problem. Most cells do not die when they hit the Hayflick limit. Instead they enlarge, lose their useful functions, slow down and just sit there lazily interfering with younger cells. It was found that the skin of older individuals has about three times as much senescent fibroblasts as a young skin. In fact, the loss of capacity due to the accumulation senescent may affect a wide range of tissues in the body.

Can the Hayflick limit be overridden? Indeed it can be. Some of the mutations seen in cancer cells do exactly that. Also, some viruses, notably the papilloma virus, immortalize the cells they infect. At least one cellular mechanism for overriding the Hayflick limit has actually been found. All cells appear to have the gene that encodes an enzyme (called telomerase) capable of restoring shortened telomeres. The cells in which telomerase is active seem to be immortal. In most normal cells, however, the activity of telomerase is somehow suppressed, so they cannot divide beyond the limit. Interestingly, researchers found that in embryonic stem cells the immortality mechanism is switched on via the so-called Nanog gene. Another gene implicated in cellular senescence is INKa. It encodes the protein P16 whose role appears to be to prevent cancer by inducing cellular senescence. Mice lacking p16 were shown to have much less senescent cell than regular mice; their tissues did not loose regenerative capacity with age. However, p16-deficient mice had a shorter lifespan due to dramatically increased incidence of cancer.

What does this all mean in terms of life extension? Firstly, the existence of Hayflick limit may contribute to age-related changes and diseases in some tissues, such as skin and blood vessels. Secondly, even if it were possible, it may be dangerous to completely abolish the Hayflick limit because of the increased likelihood of cancer. Thirdly, it appears that the number of divisions before a cell reaches the limit is not carved in stone. Different factors in the environment may accelerate or slow down the cellular clock. Increased free radical formation was shown to shorten the Hayflick limit, and some agents were found to extend the limit in certain cell types. For example, a group of Dutch scientists demonstrated that garlic extract extended the Hayflick limit and improved the function of skin fibroblasts in culture. In another study, retinoic acid extended Hayflick limit of fibroblasts in culture by almost 50 percent. An even more intriguing result was obtained by scientists at Geron, a biotechnology company, who inserted a working copy of telomerase gene in fibroblasts. As a result, the cells have gone beyond the Hayflick limit without showing any signs of senescence, remaining seemingly young and robust.

What can we do to minimize the contribution of the cellular clock to the overall aging process? In the near future, it may be possible, using genetic engineering, to change the program that is responsible for the Hayflick limit - although this may have hidden dangers, such as an increased risk of cancer. Also, scientists may find a way to remove senescent cells from tissues without harming active, healthy cells. The best we can do for now is to avoid unnecessary cell divisions and, possibly, extend the limit by improving the internal environment of our bodies. Avoiding unnecessary cell divisions means minimizing the exposure to factors that promote it. All types of tissue damage and cellular stress can promote cell division. In particular, free radicals, inflammation, mutagens, some toxins and UV-radiation were shown to stimulate cell division. Antioxidants, on the other hand, appear to have the opposite effect. Some substances, such as garlic extract, might be able to modestly extend the Hayflick limit, although clinical studies are needed to determine their practical benefits.