Submitted August 1, 1997.
A brief history of cell culture as it pertains to aging research had its origins with the thoughts of Weismann and the work of Carrel. Until the early 1960's it was believed that normal cells had an unlimited capacity to replicate. Consequently, aging was thought to have little to do with intracellular events. In the early 1960's we overthrew this dogma after finding that normal cells do have a finite replicative capacity. We interpreted this phenomenon to be aging at the cellular level. In subsequent years the objective was to identify the putative cell division counting mechanism that had been postulated to exist. Efforts to achieve this goal have had a remarkable degree of success only in the last few years with the discovery of the shortening of telomeres at each round of DNA replication that occurs in normal cells both in vivo and in vitro. Immortal abnormal cell populations overcome telomere shortening by activating an enzyme, telomerase, that catalyzes the synthesis of the TTAGGG sequences that compose mammalian telomeres, thus maintaining their length constant. Telomere shortening in normal cells is not a chronometer because time is not measured but rounds of DNA replication are measured. I propose the term replicometer for the device that measures the loss of telomeric sequences in normal cells because the action is that of a meter, and it is counting DNA replications. Telomere shortening and the finite lifetime of normal cells is more likely to represent longevity determination than it is aging. The hundreds of biological changes that herald the loss of replicative capacity in normal cells are more likely age changes.
KEY WORDS: mortality, immortality, aging, longevity, cells, telomere, telomerase, replicometer.
Efforts to understand the role of the cell in the aging process began about one hundred years ago, a fact that was not appreciated until it was brought to light relatively recently .
In 1881 the great German biologist August Weismann speculated that the somatic cells of higher animals will be found to have a limited doubling potential. Although he provided no experimental evidence for his speculation, Weismann stated: "...death takes place because a worn-out tissue cannot forever renew itself, and because a capacity for increase by means of cell division is not everlasting but finite" .
An essential element in considering the role of the cell as the fundamental locus of age changes is whether or not the cells studied are normal. Age changes do not occur in abnormal, immortal cells which are a class of cells that have many of the properties of cancer cells . Cells in vitro must be normal if the are to yield useful information about the cell biology of aging in vivo.
There are at least two ways in which the mortality or immortality of animal cells can be determined. First, cells can be serially cultured in laboratory glassware and second, cells having specific markers allowing them to be distinguished from host animal cells, can be serially transplanted in isogeneic laboratory animals. The transplanted tissue is then regrafted to a younger host when the donor host becomes old. The goal that these in vitro and in vivo studies has been to answer this fundamental question: Can normal animal cells functioning and replicating under ideal conditions escape from the inevitability of aging and death that is obligatory for the animal from which the were derived?
The Finite Lifetime of Normal Cells in vitro
In cell culture studies done before 1960, one investigation stood out as the classic response to this question.
Early in this century, Alexis Carrel, a noted cell culturist, surgeon and Nobel Laureate, described experiments purporting to show that fibroblasts derived from chick heart tissue could be cultured serially indefinitely. The culture was voluntarily terminated after 34 years . This finding sparked intense interest world-wide not only in the scientific community but in the lay press as well .
The importance of the observation that cultured chick cells were immortal was clear to biogerontologists. If true, it strongly implied, if not proved, that cells released from in vivo control had the potential to divide and function normally for a period of time in excess of the lifespan of the species (the maximum recorded lifespan for the domestic chicken is twelve years ). Thus, either the types of cells cultured play no role in the aging process or aging results from changes similar to those that occur in the intercellular matrix or from changes that occur at higher levels of cell organization. That is, aging must result from physiological interactions between cells only when the are organized as tissues or organs. Carrel's results and their interpretation were of vital concern to biogerontologists because they strongly suggested that aging is not the result of events occurring within individual cells.
In the years that followed Carrel's observations, support for the interpretation of his experiments seemed to emerge from several laboratories where it was observed that cell populations from other species, including humans, also seemed to have the striking ability to replicate apparently indefinitely.
Immortal cell populations derived from a variety of human and animal tissues were reported to occur spontaneously in dozens of laboratories in the twenty year period from the early 1940's to the early l960's. These cell populations, now numbering in the hundreds, are best known by the prototype cell lines HeLa (derived from a human cervical carcinoma in 1952) and L cells (derived from mouse mesenchyme in 1943). They continue to flourish in cell culture laboratories throughout the world to this day.
Immortal cell populations still occasionally arise spontaneously from normal cell cultures by an unknown process. Today, however, they can be created purposely, albeit at low efficiency, by exposing normal, mortal cells to radiation, chemical carcinogens or certain oncogenic viruses.
The death of cells in culture had been observed since Ross Harrison prepared the first generally accepted tissue culture in 1907 . Largely because of Carrel's work, however, the belief developed that all cultured cells were inherently immortal and those cells that died did so because proper culture conditions were unknown. In subsequent years the belief that cultured cells were inherently immortal became dogma among cell culturists. And, as indicated earlier, it led the attention of biogerontologists away from the individual cell as a site for the genesis of age changes.
Nevertheless, what seemed in the 1950's to be incontrovertible evidence that the fate of all cultured cells was immortality, soon fell to new insights and a preponderance of opposing information.
Aging under Glass
In 1961 Paul Moorhead and I described the finite replicative capacity of normal human fibroblasts . For the first time, we ruled out artifacts or lack of knowledge of adequate culture conditions as the cause of normal cell death in culture. We interpreted the phenomenon to be aging at the cell level.
We showed that when normal human embryonic cells are grown under the most favorable conditions, aging and death is the inevitable consequence after about fifty population doublings. We called this the Phase III Phenomenon. We showed that the death of cultured normal human cells was not due to some trivial cause involving medium components or culture conditions but was an inherent property of the cells themselves. The observation has since been confirmed in hundreds of laboratories [9-12].
As indicated above, and at the time we made our observation, the paramount dogma in cell biology was that the failure of cells to proliferate indefinitely in vitro must be attributable to errors in the "art" required to keep cells dividing forever. That dogma was so well entrenched that our original manuscript  was rejected in 1960 by The Journal of Experimental Medicine with the statement that: "The largest fact to have come out from tissue culture in the last fifty years is that cells inherently capable of multiplying will do so indefinitely if supplied with the right milieu in vitro." As to our data on the exquisite virus sensitivity of our human diploid cell strains the letter continued that: "The observations on the effect of viruses on cultures of these (cells) seems extraneous." And, as to our suggestion that the finite lifespan of cultured normal cells might be aging at the cell level the letter writer commented: "The inference that death of the cells ... is due to "senescence at the cellular level" seems notably rash." This letter, rejecting our manuscript, was signed by Dr. Peyton Rous.
The paper was then submitted, unchanged, to Experimental Cell Research where it was accepted without revision. That paper was reported in Current Contents to be one of the 100 most cited papers of the two million biomedical papers published in the 1960's [13, 14].
The belief, stated in Rous's letter, that "cells inherently capable of multiplying will do so indefinitely if supplied with the right milieu in vitro" was, of course, a re-statement of the dogma we believed we had overturned. Yet, the dogma was so well entrenched that it represented grounds for rejection of the paper regardless of the incontrovertible evidence that we thought we had provided and that was ultimately confirmed in hundreds of laboratories.
The dogma was tantamount to the belief that, given the right milieu in vivo, human beings also will live forever. Ponce de Leon called the right milieu in vivo "The Fountain of Youth" and those who believe that cultured normal cells must be immortal if only the right medium can be found are, like Ponce de Leon, still searching for that fountain of youth.
Our suggestion that the Phase III Phenomenon was aging at the cell level was made because it was relatively simple to eliminate other explanations for our observations but we were unable to eliminate the possibility that the phenomenon was, indeed, aging at the cellular level. Our failure to eliminate aging as the cause of the event was not based on our inability to design the right experiment but because there were no accepted criteria for identifying biological aging--a problem that to this day remains substantially unresolved.
Nevertheless, and in spite of the failure to reach a consensus on this question biogerontologists, as individuals, do have operating definitions of aging. These definitions often satisfy personal biases and qualify biogerontologists to labor in the field with little concern because their peers suffer from the same disadvantage.
Our studies were done with fibroblasts because these cells have the greatest longevity in vitro. It is important to point out that other more highly differentiated normal cell types also have a finite replicative capacity in culture that is usually less than that for fibroblasts. Or, if the cultured cell type is inherently incapable of division, like neurons or muscle cells, then they too have a finite capacity to function in vitro. Replicative potential, like many other cell functions, declines with age. Thus, cultured normal cells with low replicative potential or inability to divide at all, show losses in function typical for that cell type while cells capable of division show loss of that function as well. Cell division capacity is a cell function and like most other cell functions it declines with age.
Mortal and Immortal Cells
Thirty years ago I put forth the notion of that there was a relationship between the biological properties of normal cells in vitro and normal cells in vivo that distinguished them from the biological properties of abnormal or cancer cells in vitro and transplanted cancer cells in vivo . I suggested that all normal cells both in vitro and in vivo are mortal and that abnormal cancer cells both in vitro and in vivo are immortal. This relationship, published in 1965, appeared as follows (see the table).
Alexis Carrel and the Myth of Immortal Normal Chick Cells
Our finding that cultured normal cells have a finite capacity to replicate has had important implications for gerontological theory. However, before these implications are considered, it will be necessary to discuss how Carrel was mislead into believing that he had successfully cultured chick heart fibroblasts for 34 years.
By the late l960's it became apparent to us and others that Carrel's claim to have cultured chick fibroblast cells for 34 years was spurious [15, 16]. It had to be assumed that Carrel's chick cultures consisted of normal cells because no one has ever reported a spontaneously arising transformed chicken cell strain free of oncogenic viruses. Several reports of alleged immortal chick cell lines have appeared, but all have been associated with oncogenic viruses, chemical carcinogens or radiation [17, 18]. In 1984, Ogura, Fujiwara, and Namba reported their development of two immortal chick cell lines . One was produced by exposure of the cells to the carcinogen N-methyl-N´-nitro-N´-nitrosoguanidine and the other arose spontaneously. However, both the spontaneously transformed cell line and the chemically induced transformed cells were shown to be abnormal and to produce the avian leukosis retrovirus.
It has been fifty years since the voluntary termination of Carrel's alleged immortal chick fibroblasts and no one, other than Ogura's group, have reported a spontaneous transformation of normal chick fibroblasts. But, even the finding of Ogura et al. is clouded by the presence of a retrovirus in the cells.
The rarity of spontaneous immortalization of chicken cells can be appreciated when one considers that chick tissues have been one of the most frequently cultured tissues in the past fifty years. Thus, the likelihood that Carrel had an immortal population is remote especially because all attempts to confirm his findings have failed except for the possibility noted above. Furthermore, even if Carrel's observation was legitimate, his "immortal" cells must have been abnormal since there is no known exception to this rule. Thus, Carrel's findings cannot be used as evidence that normal cells have escaped the inevitability of the Phase III Phenomenon.
The alleged immortal chick heart cell culture was fed chick embryo extract prepared fresh daily. We have proposed the preparation of this material, which included a primitive centrifugation procedure and crude removal of the nutrient containing supernatant, permitted the addition of new living cells to the so-called immortal culture at each feeding.
Even if this explanation is invalid it is correct to say that no one has ever confirmed Carrel's work even to the extent of keeping continuously proliferating normal chick cells alive for as short a period of time as two or three years. Since confirmation is lacking and the scientific method demands this, Carrel's studies are invalid. Others have attributed more sinister reasons for Carrel's alleged success [23-25].
Properties of Mortal Cells
1. If derived from human embryos, cell strains undergo about 50 population doublings. The potential cell yield from 50 population doublings is about 20 million metric tons.
2. Human diploid cells undergo a number of population doublings inversely proportional to donor age. This suggested to us that the finite replicative capacity of cultured normal cells is an expression of aging at the cell level. This notion received considerable experimental support in subsequent years and became the basis for the field of cell aging that we named cytogerontology .
3. If derived from normal tissue, cell strains have the diploid karyotype and unlike immortal cell lines, normal cell strains are incapable of replication in suspension culture. This property is now called anchorage dependence.
4. Human cell strains will not produce tumors when inoculated into the hamster cheek pouch or even when directly inoculated into terminal human cancer patients.
5. Human diploid cell strains can be cryogenically preserved. When, for example, our first widely distributed normal human diploid cell strain WI-38, developed in 1962, is preserved at a particular doubling level and then reconstituted, the number of doublings remaining is equivalent to 50 minus the number of doublings spent prior to preservation. The cells have an extraordinary memory and "remember" at what doubling level the were preserved even after 35 years of continuous storage in liquid nitrogen. WI-38 has been cryogenically preserved longer than any other normal human or animal cell population.
1. Primary cell cultures or cells derived from intact tissue and undergoing no sub-cultivations.
2. Cell strains which: a) have a finite capacity to replicate; b) do not produce tumors when inoculated into experimental animals; c) have the karyology of the tissue of origin; and d) are anchorage dependent.
3. Cell lines which: a) are a population of immortal cells; b) may produce tumors when inoculated into laboratory animals; c) do not have the karyology of the tissue of origin; and d) are usually anchorage independent.
Is Transformation Interesting?
Our definitions were not universally accepted nor has anyone else been able to bring order out of the current terminological chaos, including heroic efforts by several nomenclature committees established by the Tissue Culture Association. Because of this, today, the scientific literature in this field is virtually unintelligible . I will use the terms "cell line" and "cell strain" as I just defined them throughout this essay (see the table) in an effort to avoid the chaos.
One example of the seriousness of this problem can be given by asking: What is a Chinese Hamster Ovary (CHO) cell culture? There are at least three possible answers. A CHO cell could be: 1) a primary culture of Chinese hamster ovary cells; 2) a cell strain, that is, an anchorage dependent culture of normal cells with a finite capacity for replication and having a diploid karyotype; or 3) an immortal cell line with an abnormal karyotype and the possibility of producing tumors. The term "CHO cell" is used in the literature to mean any of these three fundamentally different cell populations. This results in enormous opportunities for misunderstandings and confusion. But, the matter of clarity of expression doesn't end there.
We suggested (Hayflick and Moorhead, 1961) that the phenomenon by which a cell strain acquires the properties of a cell line be called an "alteration" because the term transformation had a specific meaning in the world of microbiology . It meant the acquisition of new genetic properties by a microorganism (e.g., the pneumococcus) when exposed to the DNA of another pneumococcal type. We believed that the term "transformation" should be reserved in the event that transformation, similar to pneumococcal transformation, would some day be discovered in eukaryotic cells. Our suggestion was ignored and some years ago transformation, similar to pneumococcal transformation, was described. Now, what is called transformation in bacteriology is called transfection in cell biology.
Today even the word transformation is rarely used as originally defined. The original meaning is extremely important because, after all, it defines one of the most important research areas in all of cell biology--the acquisition by a population of normal cells of the characteristics of abnormal cells that are often cancer cell properties. This is the basis for a substantial part of the world's biological research yet, more often than not, cells reported to be transformed are abnormal even before the experiments began (e.g., C3H 10T 1/2, CHO lines, NIH 3T3, BHK21, etc.). No animal has in its anatomy a normal, or even an abnormal, cell population similar to these cells.
The word transformation is so abused that the only possible way to know what it means is to ask its user, who often defines it vaguely or in one of a dozen possible ways. When used in a publication, "transformation" probably means something quite different to the reader than it does to the author. It's my belief that if one would substitute the word "interesting" for the word transformation in the world's biological literature today, nothing substantive would be lost .
Lewis Carroll in "Through the Looking Glass" had it right when he wrote: "When I use a word -- Humpty Dumpty said in a rather scornful tone -- it means just what I choose it to mean, neither more nor less." "The question is -- said Alice -- whether you can make words mean so many different things." "The question is -- said Humpty Dumpty -- which is to be master, that's all!"
The Finite Lifetime of Normal Cells in vivo
If all cell types were continually renewed without loss of function or capacity for self-renewal, organs composed of such cells would be expected to function normally indefinitely. Their host would live forever. As Weismann opined, renewal cell populations do not occur in most tissues, and when the do, cell proliferation is not indefinite.
In vitro experiments yield similar results. Serially cultured normal cells are mortal and reveal age changes before they die.
Is it possible, then, to circumvent the death of normal animal cells that results from the death of the "host" by transferring marked cells to younger animals seriatim? Such experiments would provide an in vivo counterpart to the in vitro experiments. If the analogy is accurate we would predict that all cells transplanted serially to proper inbred hosts would, like their in vitro counterparts, age. Such experiments would largely rule out objections to in vitro studies that are based on the artificiality of the in vitro environment. The question could be answered by serial orthotopic transplantation of normal somatic tissue to new, young, inbred hosts each time the recipient approaches old age.
Data reported from many different laboratories in which rodent mammary tissue , skin  and hematopoietic cells [30-35] were employed demonstrate that normal cells, serially transplanted to inbred hosts, do not proliferate or survive indefinitely.
The trauma of transplantation does not appear to influence the results and, in heterochronic transplants, survival time is related to the age of the grafted tissue . Cancer cells, on the other hand, frequently can be transplanted indefinitely. Thus, the immortality of cancer cells in vitro is also expressed in vivo.
Many grafts of normal tissue transplanted in vivo have been found to survive much longer than the lifespan of the host or donor species before aging and dying. This fact has been erroneously interpreted by some to mean that normal cells can replicate continuously for periods of time in excess of the species' known lifespan. However, grafted tissue behaves quite differently from cultured cells. Cultured cells contain a large component of dividing cells and, in addition, are usually kept in a state of continuous proliferation. The dividing cells in grafted normal tissue represent a small component of the total number of cells in the tissue and, furthermore, they may not be in a state of continuous replication.
For example, if fibroblasts in grafted tissue replicated to the extent that comparable cells do in vitro, the graft would quickly weigh more than its host. Thus, it is important to appreciate that long survival time is not equivalent to proliferation time or rounds of division. Cells in grafts have a very low reproductive turnover rate. This is analogous to holding normal cell cultures at room temperature, a condition that extends calendar time for cell survival but does not result in increased population doublings. In fact, one could mimic, in vitro, the survival of grafted tissue beyond the lifespan of some donor species by holding normal cultured cells at low temperature for times exceeding the lifespan of the donor species. But, this should not imply, as it should not for grafted tissue, that the cultured cells had escaped from aging.
The failure of grafted normal cells to replicate indefinitely in vivo means that, given the "perfect" conditions of nutrition and environment that an isologous animal represents, transplanted normal cells are still incapable of immortality. This leads to the further conclusion that a search for perfect in vitro conditions in which normal cells might reveal immortality is futile.
The finitude of normal cell replication has been substantially demonstrated to occur both in vivo and in vitro.
There Are No Immortal Cells
As a result of our demonstration that normal cells are mortal, the focus of attention in the field of aging began to be directed to the cell itself, and the dogma of fifty years duration began to die a slow death. But, is it really true that cell immortality exists in vitro? Is HeLa, for example, immortal? In fact, it has never been proven.
There are two kinds of immortality that are frequently confused or misunderstood. The two are immortality of individuals and immortality of populations. The immortality of a population depends upon a periodic exchange or rearrangement of genetic information. The immortality of individual organisms or cells, however, depends on demonstrating that exchange of genetic information has not occurred. That seems self evident but it is frequently ignored. For example, humans as individuals are mortal but humans as a population are immortal. That concept applies equally well to all animals and most plants. We frequently see it stated that bacteria and protozoa are immortal. That is not so. Like human populations, unicellular populations are immortal and like individual humans unicellular organisms are mortal .
No one has ever demonstrated that an allegedly immortal cell line, like HeLa, is actually immortal, that is that individual HeLa cells undergo continuous cell divisions without periodic exchange or reorganization of genetic information. The importance of this point is that immortalization or transformation of a cell population may be dependent upon a gene, virus, chemical, or radiation source that confers upon normal cells the ability to exchange or rearrange genetic information which appears to be the prerequisite for population immortality.
Advances in Cytogerontology
When we first suggested that the finite lifetime of normal cultured cells might be an expression of aging at the cellular level we expected that this idea, like others that we eliminated, would soon be proved wrong. To my surprise our notion was not quickly disproved. I think it is fair to say that our suggestion that the Phase III Phenomenon is aging at the cell level is still a tenable hypothesis even after thirty years of study. That is not to say that it has been proven unequivocally to be correct. This has not happened because there are no universally accepted criteria for defining biological aging.
The following represents a selection of some of the main observations that have been made in the past thirty years that have provided insights into the concept that the Phase III Phenomenon is telling us something about aging.
1. Inverse Relationship between Donor Age and Population Doublings. In 1965 we reported that cultured fibroblasts derived from older humans replicate less than those derived from embryos . Because the technique for determining population doublings at that time was crude, we were unable to establish a direct relationship between donor age and population doubling potential. Subsequently, studies done by others not only confirmed this observation but extended it significantly.
Martin et al.  derived cultures from human donors ranging from fetal to ninety years of age. Although the data reveals considerable scatter, they observed a regression coefficient, the first to the ninth decade, of -0.2 population doublings per year of life with a standard deviation of 0.05 and a correlation coefficient of -0.50. The scatter found is not unlike that reported to occur with virtually an other age related change that is measured cross-sectionally and not longitudinally.
Nevertheless, at least fifteen more studies have confirmed the finding that the number of population doublings of cultured human cells is inversely proportional to donor age. The inverse relationship between donor age and population doubling potential has now been shown to occur in normal human cells derived from such diverse tissue as lung [3, 8], skin [37-39], liver , arterial smooth muscle [41, 42], lens [43, 44], vascular endothelium , keratinocytes , myoblasts  and T-lymphocytes [48, 49].
2. Changes That Precede Phase III.More than two hundred changes in biological activity have been shown to occur in cultured normal human fibroblasts as they age in vitro . Of great importance is that many of these changes are identical to the changes recognized as characteristic of aging in intact humans.
We speculate that it is these changes, when occurring in vivo, that increase the vulnerability of intact humans and domestic animals to the age associated diseases that ultimately cause their demise. It is for this reason that we do not believe that aging results from loss of cell division potential. The maximum number of population doublings may simply represent a longevity limit that can be demonstrated in vitro but is never achieved in vivo because the accumulated physiological deficits predispose the animal to death well before the limit of divisions is reached. The important distinction to be made between longevity determination and aging will be discussed later.
3. Direct Relationship between Species Lifespan and Population Doublings of Their Cultured Normal Cells. Several years ago we suggested that the population doubling potential of cultured fibroblasts from several animal species revealed a surprisingly good direct correlation with maximum species lifespan . In the years that followed, several reports have appeared that have added substantially to this idea, especially the work of Rohme . One report, in which several marsupial species were studied, does not support this finding, however, the authors did not determine population doublings by conventional means nor are the maximum lifespans of the species they studied known . We have suggested that there may be a direct proportionality between the maximum lifespans of ten different vertebrate species and the population doubling potential of their cultured fibroblasts . These species range in diversity from mouse to humans to the Galapagos tortoise.
If this relationship can be extended and confirmed, it suggests the presence of an event counter within normal cells that limits their capacity to function over time.
Terminal Differentiation Versus Aging
Presently, the only alternative explanation of the limited proliferative capacity of normal cells that competes with the notion that it represents aging at the cellular level is the conjecture that it might represent terminal differentiation. The definition of differentiation is: "The act or process of acquiring completely individual characters, such as occurs in the progressive diversification of cells and tissue of the embryo" (Dorlands Medical Dictionary).
Cells aging in vitro do, indeed, acquire hundreds of incremental and decremental physiological changes as the approach the end of their in vitro lifespan. Yet, none of these changes raise the cell to a higher level of differentiation or specialization. To call the Phase III Phenomenon differentiation seems entirely inappropriate. Indeed, cells approaching Phase III do "acquire completely individual characters" as the definition of differentiation requires. However, because the new characters represent biological deficits then the deficits of old age that precede death in whole animals also should be called differentiation. Clearly, that is absurd.
The process of differentiation implies that a primitive cell type undergoes a progression of changes that results in its capacity to perform more specialized activities than the cell was previously capable of performing. That does not happen in cells approaching Phase III or in cells as they age in vivo.
The adjective "terminal" when used with "differentiation" is redundant. All differentiations are continuing processes that reach an end point or termination. Presumably, that end point is where the cell has reached the maximum level of function determined by the process of differentiation. Yet all normal cells, differentiated or not, eventually become "terminal".
Although all normal cells age in vitro, the cell most studied is the fibroblast. The replicating normal fibroblast in vitro does not acquire more specialized properties in the sense that the cell undergoes modifications permitting it to perform some new physiological activity for which there is an in vivo counterpart. In fact, the changes that do occur in these cells, as the approach the end of their in vitro lifespan, are changes that herald losses in physiological function that leads ultimately to their death. Surely, the acquisition of characteristics that herald approaching cell death, and absent the appearance of more specialized properties, can hardly be called differentiation. Indeed, losses in physiological capacity are more reasonably characterized as aging than differentiation. It is for this reason that we refer to humans in their eighties as being old and not being terminally differentiated. Similarly, the finite lifespan of cultured normal cells is more characteristic of the phenomenon of aging than it is of differentiation.
Since our first tentative suggestion that the finite lifespan of cultured normal human cells was a manifestation of aging at the cellular level, an enormous fund of knowledge has been acquired about the nature of the phenomenon. For reviews, see references [9-12] and . Few if any of the experimental results that have been reported are completely incompatible with an interpretation that the finite lifetime of cultured normal cells is aging at the cell level.
The Telomere Replicometer
From the time of our report on the finitude of normal cell replicative capacity, the "holy grail" in cytogerontology has been to locate and understand the mechanism of the putative counting mechanism that governs this limit and how immortal cells circumvent the process. It was clear from an enormous amount of data that an intracellular counting mechanism must be present. These data included: 1) the Phase III Phenomenon itself; 2) our observation that cryogenically preserved cells "remember" at what doubling level they were preserved [3, 8]; and 3) the enormous literature describing hundreds of biological changes occurring in normal cells well before they lose their replicative capacity . Because cell mortality and immortality are inextricably linked to aging and cancer, the importance of this goal would be difficult to exaggerate.
The sought-after mechanism should not be called a clock or chronometer because these are devices used for the measurement of the passage of time. Because the replicative limit of normal cells is not directly the result of the passage of time but the number of DNA replications, the putative mechanism should be more properly referred to as an event counter. A device that counts is called a meter, which would justify the suggestion that the term "replicometer" be used to designate the putative molecular event counter.
In early efforts to determine the location of the replicometer, early experiments in which the nuclei of old and young cultured cells were fused to the enucleated cytoplasm of opposite aged cytoplasts revealed that the replicometer was located in the nucleus [54, 55].
But, more progress has been made in finding the replicometer in the last five years than was made in the previous thirty years thanks to a happy confluence of observations made in several diverse fields.
The recent observations of telomere shortening as normal cells divide provides the first evidence for the putative replicometer. This, in combination with the discovery of the enzyme, telomerase, has gone very far in explaining why most normal somatic cells have a finite capacity to replicate in vivo and in vitro and how immortal cells might circumvent this inevitability.
Telomeres are structures found at the ends of linear chromosomes and consist of repetitive DNA sequences. Telomeres apparent prevent recombination and allow the attachment of chromosome ends to the nuclear envelope.
The properties of DNA polymerase prevent it from full replicating the linear ends of DNA [56-58]. This has been called the "end-replication problem." The problem is the inability of DNA polymerase to completely replicate the 3´ end of linear duplex DNA. It was discovered that cells of many species contain highly conserved repeats of the sequence TTAGGG in their telomeres . Many unicellular organisms and viruses have evolved a special mechanism to circumvent the problem of termini. In these organisms, the chromosomes are circular, or the genome produces circular replicative intermediates that simply lack ends so that the problem encountered in linear chromosomes does not exist.
In 1971, Olovnikov suggested, on theoretical grounds, that a cell might become senescent through the loss of repetitive telomeric DNA that may take place because of DNA end underreplication [56, 57, 60]. The problem of DNA end underreplication was also addressed by Watson in 1972 . The notion that repetitive copies of functional genes might govern, or trigger, the aging process had been suggested earlier by Medvedev . It was not until recently, however, that Olovnikov's remarkably insightful speculation was proven to be correct experimentally. For recent reviews of this rapidly developing field see [62-65].
In brief, it has been found that eukaryotic cells have evolved a novel solution to the end replication problem in which the specialized chromosome end structures, or telomeres, contain repetitive sequences, some of which are lost at each round of replication. The loss of these sequences, which lack the information contained in downstream genes, acts as a buffer protecting those genes from loss during each round of DNA replication.
The telomeres in human cells consist of thousands of repeats of the sequence TTAGGG. This sequence is remarkably conserved from primitive organisms to humans [66-68]. The sequence is found in all invertebrates and in some trypanosomes and slime molds.
Harley et al.  observed that the mean telomere length decreased by 2 to 3 kilobase pairs (kbp) during the serial passage of several strains of normal human diploid fibroblasts. The decrease was found to be progressive and averaged 50 base pairs for each population doubling . The telomere shortening seen in aging normal human fibroblasts also occurs in vivo in skin epidermal cells , peripheral blood leukocytes and colon mucosa epithelia .
As pointed out by Goldstein , the finding that telomere shortening occurs as cells age in vitro is of great interest because the EST-1 yeast mutant contains a defect in telomere elongation which also leads to the senescent phenotype. There is no immediate loss of viability in the yeast cells but a slow progressive death occurs as it does in aging normal human fibroblasts .
Levy et al.  have modelled and analyzed the telomere shortening discovery in aging normal human cells and have reached the following conclusions. First, mean telomere length decreases. Second, if cells senesce after 80 population doublings, the mean telomere length decreases about 4000 base pairs but one or more telomeres in each cell will lose significantly more telomeric DNA. Third, variation in telomere length predicted by their model is consistent with the abrupt decline in dividing cells at senescence. Finally, variation in length of terminal restriction fragments is not fully explained by incomplete replication, suggesting significant intrachromosomal variation in the length of telomeric or subtelomeric repeats. Levy et al.  concluded that telomere loss could explain why senescent cultured normal cells age and lose their capacity to replicate.
Allsopp et al.  reported that after analyzing the cultured normal fibroblasts from 31 human donors, aged 0 to 93 years, a striking correlation, valid over the entire age range, was found between proliferative capacity and initial telomere length. Thus, cell strains with shorter telomeres underwent significantly fewer doublings than those with longer telomeres. The authors suggest that telomere length is a biomarker of somatic cell aging in humans and that this is consistent with a causal role for telomere loss in aging. They also reported that fibroblasts from Hutchinson--Gilford progeria donors had short telomeres consistent with their reduced division potential in vitro. Telomeres from sperm DNA did not decrease with donor age suggesting that a mechanism for maintaining telomere length such as telomerase expression, may be active in the germ line.
In a report by Vaziri et al. , studies are described in an effort to determine whether accelerated telomere loss is associated with the premature immunosenescence of lymphocytes in individuals with Down's syndrome (DS) and whether telomeric DNA is also lost during aging of lymphocytes in vitro. Genomic DNA was isolated from peripheral blood lymphocytes of 140 individuals ranging in age from 0 to 107 years including 21 DS patients. The DS patients showed a significantly higher rate of telomere loss with donor age (133 ± 15 bp/year) compared with age-matched controls (41 ± 7.7 bp/year) suggesting that telomere loss is a biomarker for premature immunosenescence in DS patients and that it may play a role in this process. In addition, telomere loss during aging in vitro was calculated for lymphocytes from four normal individuals grown in culture for 10-30 population doublings. The rate of telomere loss was ~120 bp per population doubling, comparable to that seen in other somatic cells. Also, telomere lengths of lymphocytes from centenarians and from older DS patients were similar to those of senescent lymphocytes in culture, which suggests that replicative senescence could partially account for aging of the immune system in DS patients and in elderly individuals.
Telomeric shortening, which occurs in several classes of dividing normal somatic cells, may be the replicometer that determines the number of times that a normal cell is able to divide. Once a critical or threshold number of telomeric TTAGGG repeats is reached, cells will then be unable to divide. An alternative explanation of how telomere shortening acts as a biological clock has been offered by Wright and Shay . Their telomere positional effect explanation of cell senescence is based on a novel two-stage model.
The essential remaining question in this fascinating story is this: How do the cells composing immortal populations avoid telomere shortening that, if it occurs, would lead to their demise?
The answer to this critical question originated in studies in Tetrahymena by Greider and Blackburn who discovered the ribonucleoprotein enzyme terminal transferase called telomerase . The found that telomeres are synthesized de novo by telomerase, a ribonucleoprotein enzyme that extends the 3´ end of telomeres and thus elongates them. This ribonucleoprotein complex contains a reverse transcriptase and RNA template for the synthesis of the repeated sequence . Telomerase was later found to occur in extracts of immortal human cells [79, 80].
Unlike normal mortal cultured cell strains, immortal cultured abnormal cell lines that do not senesce produce telomerase. Thus, the telomeres of immortal cells do not shorten with serial passage in vitro .
In recent years telomerase has also been found to be expressed in some classes of normal cells. These include fetal tissue, normal bone marrow stem cells, testes, peripheral blood lymphocytes, skin epidermis and intestinal crypt cells (for references, see reference ). All of these cells have high turnover rates or are in a continuously replicating pool of differentiating cells. It is important to note that the level of telomerase activity found in these normal cell populations is significantly less per cell than that found in cancer cell populations .
The observation that telomerase is qualitatively expressed only in cancer cells or is expressed quantitatively less per cell in differentiating cells has sparked interest in finding inhibitors of telomerase as a novel approach to cancer therapy . The presence and level of telomerase expression appears to be the single most specific characteristic that distinguishes cancer cells from normal cells. Because of this distinction, there is also interest in the development of tests for the early detection of cancer cells based on the exquisitely sensitive TRAP assay which is capable of detecting telomerase in a single cell .
Aging and Longevity Determination
I would like to suggest an alternative hypothesis for the role of telomeres in aging. I propose that the shortening of telomeres in normal dividing cells is not an expression of aging, but rather an expression of longevity determination. The distinction between longevity determination and aging is significant.
Species continuation depends on the survival of a sufficient number of members of that species to live long enough to reproduce and raise progeny to independence. This fundamental premise leads to the belief that the best way to insure that this occurs is for natural selection to favor those animals having greater physiological capacity in vital organs. Greater, or redundant physiological capacity increases the likelihood for animal survival to reproductive success just as redundant vital systems in complex machines, like space vehicles, better insures that they will achieve their goals. Once animals reach sexual maturation and raise progeny to independence, the excess physiological capacity, like that engineered into a space vehicle, allows each to continue beyond the vital goal. The further longevity of each is determined by the excess capacity present at the time the goal was reached. In animals that goal, of course, is reproductive success.
Thus, the forces of natural selection diminish after animals reach reproductive success because survival beyond that event has diminished value for the survival of the species. Energy is better spent on guaranteeing reproductive success than it is for increasing individual longevity. Thus, after reproductive success the forces of natural selection do not favor increased longevity. However, after reproductive success an animal has the potential to survive for a period of time determined by the level of excess physiological capacity reached at sexual maturation.
Clearly, the events that occurred leading up to the survival of animals to reproductive success are determined genetically. Therefore, the survival of animals beyond that point is determined only indirectly by the genome. The state of survival beyond reproductive success can be regarded as a period of "coasting" or "free-wheeling", that is, developmental processes have ended and the ability to maintain those systems declines. The length of this post reproductive period plus the time taken to reach sexual maturation can be viewed as the two components of an animals longevity.
The crucial suggestion is that the events that occur after reproductive success that fail to maintain the system and increase the likelihood of dying are called age changes. That is, there is an increase in molecular disorder for which repair processes increasingly fail to correct. The second law of thermodynamics applies, that is, entropy increases.
It is because of these considerations that I propose that telomere shortening may be the molecular equivalent of longevity determination, not aging, and that the increasing molecular disorder that is known to occur in normal cells as telomeres shorten is equivalent to age changes. As indicated earlier hundreds of biological changes occur in normal cells as the age in vitro representing increasing molecular disorder and all compromise the internal milieu that ultimately leads to the Phase III Phenomenon. Thus, the number of population doublings that a normal cell is capable of undergoing may be the in vitro demonstration of longevity determination. The molecular disorders that herald its approach are age changes. In vivo, these age changes lead to an increase in vulnerability to disease or pathology which results in death well before maximum longevity is reached.
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