IN SEARCH OF THE SECRETS OF AGING
Is there a maximum biological limit to the human life
span of somewhere around 120 years?
Could we live
much longer, given the right conditions?
these and other fundamental questions about aging
may now be within reach.
IS THERE AN AGE LIMIT?
One hundred and twenty years, as far as we know, is the longest that anyone has ever lived. A man in Japan, Shirechiyo Izumi, reached the age of 120 years, 237 days in 1986, according to documents that most experts think are authentic. He died after developing pneumonia.
Long lives always make us wonder: What is the secret? Does it lie in the genes? Is it where people live or the way they live -- something they do or do not do? Eat or do not eat? Most of the scientists who study aging, gerontologists, say the secret probably lies in all of the above -- heredity, environment, and lifestyle.
But gerontologists also ask other and more difficult questions. For example, if the 120-year-old had not finally succumbed to illness, could he have lived on and on? Or was he approaching some built-in, biological limit? Is there a maximum human life span beyond which we cannot live no matter how optimal our environment or favorable our genes?
Whether or not there is such a limit, what happens as we age? What are the dynamics of this process and how do they make life spans short, average, or long? Once we understand these dynamics, could they be used to extend everyone's life span to 120 or even, as some scientists speculate, to much greater ages?
And finally for all of us, the most important question: How can insights into longevity be used to fight the diseases and disabilities associated with old age to make sure this period of life is healthy, active, and independent?
In Search of the Secrets of Aging describes what we know so far about the answers to these questions and what we want to know. It gives an overview of research on aging and longevity, showing the major puzzle pieces already in place and, to the extent possible, the shapes of those that are missing.
WHAT ARE THE REASONS FOR AGING?
The Genetic Connection
In laboratories around the country, scientists are isolating specific genes, cloning them, mapping them to chromosomes, and studying their products to learn what they do and how they influence aging and longevity.
Humans seem to have a maximum life span of about 120 years, but for tortoises it's 150 and for dogs, about 20. What underlies these differences among species are genes, the coded segments of DNA (deoxyribonucleic acid) strung like beads along the chromosomes of nearly every living cell. In humans, the nucleus of each cell holds 23 pairs of chromosomes, and together these chromosomes contain about 100,000 genes.
The link between genes and life span is unquestioned. The simple observation that some species live longer than others -- humans longer than dogs, tortoises longer than mice -- is one convincing piece of evidence. Another comes from recent, dramatic laboratory studies in which researchers, through selective breeding or genetic engineering, have been able to raise animals with extended life spans. For example, fruit flies bred selectively have lived nearly twice as long as average.
By demonstrating that genes are linked to life span, the long-lived fruit flies have set the stage for more questions.
What specific genes are involved? What activates them? How do they influence aging and longevity? In numerous laboratories, the search for answers is on.
Some leads are coming from yeast cells in which researchers have found evidence of 14 genes that seem to be related to aging (see Tracking Down a Longevity Gene, page 10). Longevity-related genes have also been found in tiny worms called nematodes and in fruit flies. Like yeast, nematodes and fruit flies have short life spans and their genes, which are known and do not vary greatly, are relatively easy to study.
In the Lab of the Long-Lived Fruit Flies
A laboratory at the University of California, Irvine, is the home of thousands of Drosophila melanogaster or fruit flies that routinely live for 70 or 80 days, nearly twice the average Drosophila life span. Here evolutionary biologist Michael Rose has bred the long-lived stocks by selecting and mating flies late in life.
To begin the process of genetic selection, Rose first collected eggs laid by middle-aged fruit flies and let them hatch in isolation. The progeny were then transferred to a communal Plexiglas cage to eat, grow, and breed under conditions ideal for mating. Once they had reached advanced ages, the eggs laid by older females (and fertilized by older males) were again collected and removed to individual hatching vials. The cycle was repeated, but with succeeding generations, the day on which the eggs were collected was progressively postponed. After two years and 15 generations, the laboratory had stocks of Drosophila with longer life spans.
The next question is what genes and what gene products are involved? Since the first experiments, Rose has bred longer life spans into fruit flies by selecting for other characteristics, such as ability to resist starvation, so the flies' long life spans are not necessarily tied to their fertility late in life.
One possibility is that the anti-oxidant enzyme, superoxide dismutase (SOD), is involved. In another laboratory at Irvine, the late Robert Tyler discovered that the longer-lived flies had a somewhat different form of the SOD gene, which was more active than its counterpart in the flies with average life spans. This finding has given a boost to the hypothesis that anti-oxidant enzymes like SOD are linked to aging or longevity.
Some of the genes found in yeast and fruit flies seem to promote longevity. But others may shorten life span. One such "death gene" has been isolated in nematodes by researchers at the University of Colorado in Boulder, who found that mutation of a certain gene more than doubles the nematode's normal 3-week life span. Thomas Johnson's laboratory in Boulder has also uncovered evidence that the mutant may extend life span by overproducing superoxide dismutase (SOD) and catalase, two anti-oxidant enzymes that have been linked to longevity in other studies.
The genes isolated so far are only a few of what scientists think may be dozens, perhaps hundreds, of longevity- and aging-related genes. Tracking them down in organisms like nematodes and yeast is just the beginning. The next big question for many gerontologists is whether there are counterparts in people -- human homologs -- of the genes found in laboratory animals.
Other unanswered questions concern the roles played by these genes. What exactly do they do? On one level, all genes function by transcribing their "codes" -- actually DNA base sequences -- into another nucleic acid called messenger ribonucleic acid or mRNA. Messenger RNA is then translated into proteins. Transcription and translation together constitute the process known as gene expression.
The proteins expressed by genes carry out a multitude of functions in each cell and tissue in the body, and some of these functions are related to aging. So when we ask what longevity- or aging-related genes do, we are actually asking what their protein products do at the cellular and tissue levels. Increasingly, gerontologists are also asking how alterations in the process of gene expression itself may affect aging.
Some proteins, such as anti-oxidants, appear to prevent damage to cells, and others may repair damaged DNA or help cells respond to stress; more about these comes later. Other gene products are thought to control cell senescence, a process that could prove to be a key piece in the puzzle of aging and longevity.
Picture a cell: the threadlike pairs of chromosomes inhabit a nucleus that floats in a sea of cytoplasm along with other tiny organelles that do the cell's work, the whole surrounded by a membrane at the surface of which the cell sends and receives messages from other cells. Then picture the chromosomes, condensing into rod-like structures that divide in two, the nucleus disappearing, the chromosomes migrating to opposite sides of the cell where other nuclei are formed, and after that the entire cell following the chromosomes' lead, pulling apart and forming two identical daughter cells.
This, the process of mitosis, or asexual cell division, takes place in nearly all of the 100 trillion or so cells that make up the human body. But it does not go on indefinitely. About the middle of this century, researchers learned that cells have finite life spans, at least when studied in test tubes -- in vitro.
A built-in limit on cell division may help explain the aging process.
After a certain number of divisions, they enter a state of cell senescence, in which they do not divide or proliferate and DNA synthesis is blocked. For example, young human fibroblasts -- collagen-producing cells frequently used in this branch of aging research -- divide about 50 times and then stop. This phenomenon has become known as the Hayflick limit, after Leonard Hayflick, who with Paul Moorhead first described it while at the Wistar Institute in Philadelphia.
Intrigued by the possibility that the Hayflick limit might help explain some aspects of bodily aging, gerontologists have looked for and found links between senescence and human life spans. Fibroblasts taken from 75-year-olds, for example, have fewer divisions remaining than cells from a child. Moreover, the longer a species' life span, the higher its Hayflick limit; human fibroblasts have higher Hayflick limits than mice fibroblasts.
Searching for explanations of proliferation and senescence, scientists have found certain genes that appear to trigger cell proliferation. One example of such a proliferative gene is c-fos, which encodes a short-lived protein that is thought to regulate the expression of other genes important in cell division.
But c-fos and others of its kind are countered by anti-proliferative genes, which seem to interfere with division. The first evidence of an anti-proliferative gene came from an eye tumor called retinoblastoma.
When one of the genes from retinoblastoma cells -- later called the RB gene -- became inactive, the cells went on dividing indefinitely and produced a tumor. But when the RB gene product was activated, the cells stopped dividing. This gene's product, in other words, appeared to suppress proliferation.
Senescence is the norm in the world of cells. In some cases, however, a cell somehow escapes this control mechanism and goes on dividing, becoming, in the terms of cell biology, immortal. And because immortal cells eventually form tumors, this is one area in which aging research and cancer research intersect. Investigators theorize that a failure of anti-proliferative genes (also known as tumor suppressor genes) is the first step in a complex process that leads to development of a tumor. Senescence, according to this view, may have evolved because it protected against cancer,.
Still a mystery is how these genes' products function to promote and suppress cell proliferation. There are indications that a multi layer control system is at work, involving probably a host of intricate mechanisms that interact to maintain a balance between the two kinds of genes. Many gerontologists are now involved in unraveling these intricacies, studying both the genes and their products to learn which ones influence senescence and how.
Tracking Down a Longevity Gene
Investigators are finding clues to aging and longevity in yeast, one-celled organisms that have some intriguing genetic similarities to human cells. In a laboratory at Louisiana State University Medical Center in New Orleans, Michal Jazwinski has found genes that seem to promote longevity in these rapidly dividing, easy-to-study organisms.
Yeast normally have about 21 cell divisions or generations. Jazwinski observed that over the course of that "life span," certain genes in the yeast are more active or less active as the cells age; in the language of molecular biology, they are differentially expressed. So far, Jazwinski has found 14 such genes in yeast.
Selecting one of these genes, Jazwinski tried two different experiments. First, he introduced the gene into yeast cells in a form that allowed him to control its activity. When the gene was activated to a greater degree than normal, or overexpressed, some of the yeast cells went on dividing for 27 or 28 generations; their period of activity was extended by 30 percent.
In his second experiment, Jazwinski mutated the gene. When he introduced this non-working version into a group of yeast cells, they had only about 12 divisions.
The two experiments made it clear that the gene, now called LAG-1, influences the number of divisions in yeast or, according to some researchers' ways of thinking, its longevity. (LAG-1 is short for longevity assurance gene.) But how it works is still a mystery. One small clue lies in its sequence of DNA bases -- its genetic code -- which suggests that it produces a protein found in cell membranes. One next step is to study the function of that protein. Similar sequences have been found in human DNA, so a second investigative path is to clone the human gene and study its function. If there turns out to be a human LAG-1 counterpart, new insights into aging may be uncovered.
In the meantime, scientists are finding more clues to senescence in the architecture of DNA. Every chromosome, they have discovered, has tails at the ends that get shorter as a cell divides. Named telomeres, the tails all have the same, short sequence of DNA bases repeated thousands of times. The repetitive structure stabilizes the chromosomes, forming a tight bond between the two strands of the DNA.
Each time a cell divides, the telomeres shed a number of bases, so telomere length gives some indication of how many divisions the cell has already undergone and how many remain before it becomes senescent.
This apparent counting mechanism, almost like an abacus keeping track of the cell's age, has led to speculation that telomeres do serve as molecular meters of cell division. But they may play a more active role, and telomere researchers are exploring the possibility that these chromosome ends regulate cellular life span in some way. The repeated DNA bases in telomeres form tight bonds that help stabilize chromosomes. About 50 bases are lost from each telomere every time a normal cell divides.
Telomere research is another territory where cancer and aging research merge. In immortal cancer cells, telomeres act abnormally -- they stop shrinking with each cell division. In the search for clues to this phenomenon, researchers have zeroed in on an enzyme called telomerase. Normally absent in adult cells, telomerase seems to swing into action in advanced cancers, enabling the telomeres to replace lost sequences and divide indefinitely. This finding has led to speculation that if a drug could be developed to block telomerase activity, it might aid in cancer treatment.
Whether cell senescence is explained by abnormal gene products, telomere shortening, or other factors, the question of what senescence has to do with the aging of organisms remains and continues to be the focus of intense study.
In the meantime, gerontologists are also studying proteins in the body that may play a role in aging and longevity. Genes hold the codes to these proteins, but what substances turn the genes on and off? And once activated, how do their products interact with the products of other genes? What is their effect on cells and tissues? The biochemistry of aging holds some of the answers.
Biochemistry and Aging
Proteins, in their myriad forms and functions, are the substances most responsible for the day-to-day functioning of living organisms. Some of these proteins seem to affect the way we age and how long we live.
Treacherous oxygen molecules, protective enzymes, hormones that seem to turn back the clock, and proteins that may speed it up: The biochemistry of aging is a rich territory with an expanding frontier. Major areas of exploration include oxygen radicals and glucose crosslinking of proteins, both of which damage cells; the substances that help prevent and repair damage; and the role of specific proteins, particularly heat shock proteins, hormones, and growth factors.
Demolishing proteins and damaging nucleic acids, oxygen radicals are thought to be the villains in the day-to-day life of cells. The free radical theory of aging, first proposed by Denham Harman at the University of Nebraska, holds that damage caused by oxygen radicals is responsible for many of the bodily changes that come with aging. Free radicals have been implicated not only in aging but also in degenerative disorders, including cancer, atherosclerosis, cataracts, and neurodegeneration.
They damage cells and may cause tissues and organs to age. A free radical is a molecule with an unpaired, highly reactive electron. An oxygen-free radical is a byproduct of normal metabolism, produced as cells turn food and oxygen into energy.
In need of a mate for its lone electron, the free radical takes an electron from another molecule, which in turn becomes unstable and combines readily with other molecules. A chain reaction can ensue, resulting in a series of compounds, some of which are harmful. They damage proteins, membranes, and nucleic acids, particularly DNA, including the DNA in mitochondria, the organelles within the cell that produce energy.
But free radicals do not go unchecked. Mounted against them is a multi layer defense system manned by anti-oxidants that react with and disarm these damaging molecules. Anti-oxidants include nutrients -- the familiar vitamins C and E and beta carotene -- as well as enzymes, such as superoxide dismutase (SOD), catalase, and glutathione peroxidase. They prevent most, but not all, oxidative damage. Little by little the damage mounts and contributes, so the theory goes, to deteriorating tissues and organs.
Support for the free radical theory comes from studies of anti-oxidants, particularly SOD. SOD converts oxygen radicals into the also harmful hydrogen peroxide, which is then degraded by another enzyme, catalase, to oxygen and water.
Anti-Oxidants and Aging Gerbils
A boost for the hypothesis that high levels of anti-oxidants can slow the aging process comes from a study of N-tert-butyl-alpha-phenylnitrone or PBN in gerbils. Although it does not occur naturally in the body, PBN works in much the same way as beta-carotene and other anti-oxidants by binding and neutralizing free radicals.
Older gerbils had been shown to have increased levels of oxidized protein in their brains by two researchers, Robert A. Floyd at the Oklahoma Medical Research Foundation and John M. Carney at the University of Kentucky. Curious about the effects of anti-oxidants in older animals, Floyd and Carney designed an experiment to learn whether PBN could lower oxidized protein levels in gerbils' brains. Over a period of 14 days they gave PBN to two groups of gerbils, one made up of young adults, the other of older adults.
As the older gerbils were treated with PBN, their levels of oxidized protein decreased until they were nearly comparable to levels found in the younger animals. After treatment ended, oxidized protein gradually returned to pretreatment levels. PBN had no effect on the young gerbils.
While it is only one study and more are needed, this investigation supports the idea that maintaining anti-oxidant defense levels may be critical during aging. It also suggests that an intervention such as PBN may someday provide the means.
At the National Institute on Aging (NIA), Richard Cutler has found that SOD levels are directly related to life span in 20 different species; longer-lived animals have higher levels of SOD, suggesting that the ability to fight free radicals has something to do with longer life spans. Levels of other anti-oxidants -- vitamin E and beta-carotene, for example -- have also been correlated with life span.
Other studies have shown that inserting extra copies of the SOD gene into fruit flies extends their average life span. In three different laboratories, researchers have reported that transgenic fruit flies, carrying extra copies of the gene for SOD, live 5 to 10 percent longer than average.
Other experimental evidence lends support to the free radical hypothesis. For example, higher levels of SOD and catalase have been found in long-lived nematodes. And in another important study, giving gerbils a synthetic anti-oxidant has reduced high levels of oxidized protein, a sign of aging, in their brains.
The discovery of anti-oxidants raised hopes that people could retard aging simply by adding them to the diet. Unfortunately taking SOD tablets has no effect on cellular aging; the enzyme is simply broken down in the body during digestion. And when anti-oxidant vitamins are added to cells, they compensate by halting production of their own anti-oxidants, leaving free radical levels unchanged.
Researchers have not abandoned all hope for dietary anti-oxidants, however. Current studies, for example, are exploring the possibility that vitamin C can reduce heart disease by blocking oxidation of low-density lipoproteins. Oxidation of these cholesterol-carrying proteins is thought to be a key element in hardening of the arteries. In addition, there is evidence that vitamin E in the diet may be linked to heart attacks, with low vitamin E intake appearing to increase the risk.
Another suspect in cellular deterioration is blood sugar or glucose. In a process called non-enzymatic glycosylation or glycation, glucose molecules attach themselves to proteins, setting in motion a chain of chemical reactions that ends in the proteins binding together or crosslinking, thus altering their biological and structural roles. The process is slow but increases with time.
Crosslinks, which have been termed advanced glycosylation end products (AGEs), seem to toughen tissues and may cause some of the deterioration associated with aging. AGEs have been linked to stiffening connective tissue (collagen), hardened arteries, clouded eyes, loss of nerve function, and less efficient kidneys.
These are deficiencies that often accompany aging. They also appear at younger ages in people with diabetes, who have high glucose levels. Diabetes, in fact, is sometimes considered an accelerated model of aging. Not only do its complications mimic the physiologic changes that can accompany old age, but its victims have shorter-than-average life expectancies. As a result, much research on crosslinking has focused on its relationship to diabetes as well as aging.
One happy finding is that the body has its own defense system against crosslinking. Just as it has anti-oxidants to fight free-radical damage, it has other guardians, immune system cells called macrophages, that combat glycation. Macrophages with special receptors for AGEs seek them out, engulf them, break them down, and eject them into the blood stream where they are filtered out by the kidneys and eliminated in urine.
Glucose, the fundamental source of energy, reacts with and crosslinks essential molecules.
The only apparent drawback to this defense system is that it is not complete and levels of AGEs increase steadily with age. One reason is that kidney function tends to decline with advancing age. Another is that macrophages, like certain other components of the immune system, become less active. Why is not known, but immunologists are beginning to learn more about how the immune system affects and is affected by aging. And in the meantime, diabetes researchers are investigating drugs that could supplement the body's natural defenses by blocking AGE formation.
Crosslinking interests gerontologists for several reasons. It is associated with disorders that are common among older people, such as diabetes; it progresses with age; and AGEs are potential targets for anti-aging drugs. In addition, crosslinking may play a role in damage to DNA, which has become another important focus for research on aging.
In the normal wear and tear of cellular life, DNA undergoes continual damage. Attacked by oxygen radicals, ultraviolet light, and other toxic agents, it suffers damage in the form of deletions, or destroyed sections, and mutations, or changes in the sequence of DNA bases that make up the genetic code.
Biologists theorize that this DNA damage, which gradually accumulates, leads to malfunctioning genes, proteins, cells, and, as the years go by, deteriorating tissues and organs. Not surprisingly, numerous enzyme systems in the cell have evolved to detect and repair damaged DNA. The repair process interests gerontologists. It is known that an animal's ability to repair certain types of DNA damage is directly related to the life span of its species. Humans repair DNA, for example, more quickly and efficiently than mice or other animals with shorter life spans. This suggests that DNA damage and repair are in some way part of the aging puzzle.
In addition, researchers have found defects in DNA repair in people with a genetic or familial susceptibility to cancer. If DNA repair processes decline with age while damage accumulates, as scientists hypothesize, it could help explain why cancer is so much more common among older people.
Gerontologists who study DNA damage and repair have begun to uncover numerous complexities. Even within a single organism, repair rates can vary among cells, with the most efficient repair going on in terms (sperm and egg) cell. Moreover, certain genes are repaired more quickly than others, including those that regulate cell proliferation.
Gerontology is headed toward a deeper understanding of aging in the search for ways to make it a healthier process.
New territory, unexplored or only sketchily mapped, lies ahead. As gerontologists isolate and characterize more and more longevity- and aging-related genes in laboratory animals, insights into genes and gene products important in human aging will emerge. Comparable human genes will be identified and mapped to chromosomes.
This information will be useful in designing both genetic and non-genetic interventions to slow or even reverse some aging-related changes. Already, for example, a study by Helen Blau of Stanford University has shown that muscle cells can be genetically modified and injected into muscle where they will produce and secrete human growth hormone. Non-genetic strategies will include the development of interventions to reduce damage to cellular components, such as proteins, nucleic acids, and lipids.
Normal aging will be more closely defined. For instance, at NIA's Gerontology Research Center, the behavior of the cells that line blood vessels during aging is now providing clues to the stiffening of blood vessels that occurs with age as well as insights into vascular disease. As key biomarkers of aging are identified, researchers will be able to use them to test interventions to slow aging. Studies will begin to delve more deeply into differences in aging between the sexes and among ethnic groups.
In short, gerontologists will be charting the paths and intersections of genetic, biochemical, and physiologic aging. What they find will reveal some of the secrets of aging. It may lead to extended life spans. It will very certainly contribute to better health, less disability, and more independence in the second fifty years of life.
Anti-oxidants - Compounds that neutralize oxygen radicals. Some are enzymes like SOD while others are nutrients such as vitamin C, vitamin E, and beta-carotene. High levels of anti-oxidants have been associated with longer life spans.
Anti-proliferative genes - Genes that inhibit cell division or proliferation; also known as tumor suppressor genes.
Average life span - The average number of years that members of a population live.
Biomarkers - Biological changes that characterize the aging process; because biomarkers are considered a better measure of aging than chronological time, studies are underway to identify biomarkers in cells, tissues, and organs.
Caloric restriction - An experimental approach to studying longevity in which life spans of laboratory animals have been extended by reducing calories while the necessary level of nutrients is maintained.
Cell senescence - The stage at which a cell has stopped dividing permanently.
Chromosomes - Structures in the cell's nucleus, made up of protein and DNA, that contain the genes.
DNA (deoxyribonucleic acid) - A large molecule that carries the genetic information necessary for all cellular functions, including the building of proteins. Damage to DNA and the rate at which this damage is repaired may help determine the rate of aging.
Free radicals - Molecules with unpaired electrons that react readily with other molecules. Oxygen-free radicals, produced during metabolism, damage cells and may be responsible for aging in tissues and organs.
Gene - A segment of DNA that contains the "code" for a specific protein or other product.
Gene expression - The process by which genes are transcribed and translated into proteins. Age-related changes in gene expression may account for some of the phenomena of aging.
Glycation - The process by which glucose links with proteins and causes them to bind together, thus stiffening tissues and leading to the complications of diabetes and perhaps some of the physiologic problems associated with aging.
Hayflick limit - The finite number of divisions of which at cell is capable.
Interleukins - Substances secreted by lymphocytes; their levels vary with age.
Lymphocytes - Small white blood cells that are important to the immune system. A decline in lymphocyte function with advancing age is being studied for insights into aging and disease.
Maximum life span - The greatest age reached by any member of a given species.
Mitochondria - Cell organelles that metabolize sugars into energy. Mitochondria also contain DNA, which is damaged by the high level of free radicals produced in the mitochondria.
Proliferative genes - Genes that promote cell division or proliferation; also known as oncogenes.
Photo-aging - The process initiated by sunlight through which the skin becomes drier and loses elasticity. Photo-aging is being studied for clues to aging because it has the same effect as normal aging on certain skin cells.
Proteins - Molecules make up of amino acids arranged in a specific order determined by the genetic code. Proteins are essential for all life processes. Certain ones, such as the enzymes that protect against free radicals and the lymphokines produced in the immune system, are being studied extensively by gerontologists.
Telomeres - Repeated DNA sequences found at the ends of chromosomes; telomeres shorten each time a cell divides.