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BOOK REVIEW |
Emeritus Professor of Physiology University of Texas Health Science Center San Antonio, TX 78229-3900
Cells, Aging, and Human Disease, by Michael B. Fossel. Oxford University Press, New York, 2004, 489 pp., $69.95 (cloth).
The "Hayflick Hypothesis"
More than 40 years ago, Hayflick and Moorhead (1961) reported that human fibroblasts in culture exhibit a limited proliferative capacity. It was a surprising finding because the dogma of that time, based on the work of Alexis Carrel and colleagues, was that cells in culture have the potential for unlimited proliferation (Carrel & Ebling, 1921). Leonard Hayflick hypothesized that his and Moorhead's finding has gerontologic significance. His view of the significance is summarized in the following two quotations from Hayflick's writings.
Consequently, we have proposed that the finite lifetime of diploid cell strains in vitro may be the cellular expression of senescence so well known at the level of the whole animal (Hayflick, 1970, p. 295).
It follows, therefore, that an understanding of the mechanism by which cultured normal cells lose their capacity to replicate could provide insights into the causes of the decrements in other functional properties that are characteristic of nondividing cells and that may be even more direct causes of biological aging (Hayflick, 1976, p. 1,306).
For the sake of simplicity, the views in these two quotations will be referred to as the "Hayflick hypothesis" in this review essay.
Those of us who were trained as mammalian physiologists found this hypothesis to be outlandish and not to be taken seriously. We felt that it was highly unlikely that cells of a specific lineage in an artificial medium would undergo senescence in a manner similar to that of cells residing in organisms, which are influenced by neighboring cells of various types and by the modulating actions of the nervous, endocrine, and other systems. Surprisingly, however, the hypothesis was enthusiastically embraced, at first by cell biologists and later by molecular biologists. Indeed, studies on the loss of proliferative ability of cells in culture rapidly became a major focus of research in biological gerontology. Moreover, the term "cell senescence," which one might expect to describe the vast array of age-associated cellular deterioration occurring in an organism, has come to denote this permanent loss of proliferative ability. Thus, Cell Senescence, in the title of this review essay, is used in this restricted way and does not broadly refer to age-associated cellular deterioration.
It was soon reported that the maximum amount of proliferation undergone by human cells in culture correlates inversely with the age of the donor. This finding was viewed as an important piece of evidence in support of the "Hayflick hypothesis." Also, a direct correlation was reported for the replicative capacity in culture of the cells of different species and the maximum life span of the species. Given these findings, a spectrum of studies was launched, using the tools of biochemistry, genetics, molecular biology, and other disciplines. It is not within the scope of this review essay to present even a small fraction of the multitude of findings; rather, only those that are directly related to Michael Fossel's book will be considered.
Probably the most important are the findings by Harley, Futcher, and Greider (1990) that telomere shortening takes place as cells undergo mitotic division in culture, and that there is permanent arrest of cell proliferation (i.e., cell senescence) when a given decrease in the length of the telomeres is reached. At this juncture, a brief overview of telomeres seems in order. Telomeres are structures at the end of chromosomes; they are composed of a tandem repeat of a specific DNA base pair sequence that extends for several thousand bases, ending with a guanine-rich single strand. The DNA of the telomere is associated with an array of specialized proteins. The function of telomeres is to cap and stabilize the physical ends of the chromosomes.
The germ cells also undergo cell division during adult life, but they do not exhibit shortening of the telomeres because, unlike somatic cells, germ cells are rich in the telomerase enzyme. Human telomerase contains a protein component (hTERT), which functions as a reverse transcriptase; it also has an RNA component (hTR), which serves as a template complementary to the tandem repeat DNA. This enzyme catalyzes the lengthening of telomere DNA, and its activity is regulated so that the length of the telomere in germ cells is not shortened when the cell undergoes mitotic divisions. It is interesting to note that most cancer cells contain telomerase; stem cells do as well, but not enough to totally prevent telomere shortening.
Telomere shortening seems to provide the mechanism for the "Hayflick limit" (i.e., the number of cell doublings in culture before a permanent loss of proliferative ability). However, this does not address the question of relevance to organismal aging. An important step in addressing this issue would be to determine whether senescent cells accumulate in the organism with advancing age. It was found that when cells in culture are no longer able to proliferate, they stain for a particular ß–galactosidase (SA-ß–gal). Thus, it appeared that a biomarker for the detection of such cells in the intact organism was at hand. Indeed, based on this biomarker, senescent fibroblasts and keratinocytes were identified in human skin (Dimri et al., 1995). Subsequently, in fact, such cells have been detected in various tissues of humans and other species.
In these studies, however, senescent cells were found to comprise only a small fraction of the cells in the tissues of elderly people and old animals. Thus, it seemed unlikely that their number is of sufficient magnitude to significantly alter the functioning of tissues. However, Judith Campisi pointed out that this would be the case if the senescent cells were inert, but it would not be the case if the senescent cells generated an unfavorable microenvironment for their neighbors. Recently, Campisi (2005) published a review article entitled "Senescent Cells, Tumor Suppression and Aging," which elaborates on this possibility. She suggests that proteins secreted by senescent cells, such as metalloproteinases, epithelial growth factor, and inflammatory cytokines, can alter the microenvironment of the tissue so as to adversely affect all cells in the tissue. Although this is an intriguing concept, there is as yet little evidence in its support. Nevertheless, a body of evidence has accumulated that favors what I call Brilliant Insight.
Significantly, however, the results of studies published during the past few years have challenged some of the key evidence in support of the "Hayflick hypothesis." Cristofalo, Allen, Pignolo, Martin, and Beck (1998) studied cultures of fibroblasts from healthy donors and found no relationship between the age of the donor and the replicative ability of the fibroblasts. They suggest that the finding of an inverse relationship, as reported in earlier studies, could have been due to the inclusion of subjects suffering from diseases. In addition, Lorenzini, Tresini, Austad, and Cristofalo (2005) have shown that the body size of a species, rather than its longevity, is the primary correlate of proliferative potential of its cells in culture. Finally, there is growing evidence that SA-ß–gal staining is not a reliable biomarker of cell senescence, and that its use for this purpose may potentially lead to spurious conclusions.
Fossel's Extension of the "Hayflick Hypothesis"
I suspect that Michael Fossel wrote Cells, Aging, and Human Disease, at least in part, in response to the findings that challenge cell senescence in culture systems as a valid model of organismal aging. Fossel is a long-time proponent of the "Hayflick hypothesis." Although he may be an advocate, he is to be commended for the absence of definitive claims in his book. When the evidence is not clearly in support of his view, he uses the verb "may" rather than "is," and "may" is used much more frequently than "is" throughout the book. My concern, however, is that the coverage gives much more attention to information that supports the "Hayflick hypothesis" than to evidence that indicates it may be incorrect.
In this work, Michael Fossel extends the "Hayflick hypothesis" and provides some novel views about biological aging. The following quote is an example of the latter: "Aging is arthritis, angina, strokes, dementias, incontinence, and infections" (p. 14). This is strikingly different from the current dogma of biological gerontology that aging is not a disease. The clear statement that age-associated disease is an integral part of aging is most welcome. In my opinion, divorcing age-associated disease from aging has impeded research on the biology of aging.
Returning to cell senescence, Fossel stresses that it is not the absolute length of the telomere that relates to cell senescence, but rather the change in length that occurs in response to mitotic activity. He further proposes that an alteration in the cell's gene expression accompanies the decrease in telomere length, and that this change is the significant factor in cell senescence. Fossel believes that senescence and accumulating damage result from the altered gene expression, which is not confined to cells that have permanently lost proliferative ability, but rather occurs progressively as cells undergo mitotic activity. The involvement of cell senescence in organismal aging is viewed as follows: The change in gene expression leads to the secretion of substances (such as those suggested by Campisi), which results in the acceleration of senescence by increasing the cell division activity of neighboring cells capable of mitotic activity. It also leads to the damage of nondividing cells (e.g., damage of myocardial cells due to a reduced blood supply as a result of the senescence of the mitotically active endothelial cells of the vasculature). Fossel proposes that this overall process constitutes organismal aging, but he follows this reasonable statement with the not-so-reasonable remark: "Cell senescence doesn't cause aging so much as it is aging" (p. 59).
The above paragraph provides the essence of Michael Fossel's argument, but it really doesn't do justice to the discussion in the book. A detailed presentation of the large literature base is used in support of his concepts, and the arguments favoring his view are carefully developed in a logical and thoughtful fashion. However, as stated above, much less attention is given than I would have to the evidence opposing the concept that cell senescence is aging. Moreover, as yet, empirical evidence in support of his views is scant. The author is aware of this problem, and considerable attention is devoted to how to address this deficit.
Throughout the book, Fossel suggests experiments to test his hypotheses. A few of these can be readily executed. However, most are technically challenging, and the feasibility of executing many of them must await future technological developments. His major focus in regard to empirical testing involves interventions that prevent or reverse age changes in physiology, as well as therapies for the prevention or alleviation of age-associated diseases. The titles of the following chapters indicate the scope of these suggestions: Cancer; Parasitic Disease; The Progerias; The Skin; Cardiovascular System; Orthopedic Systems; Hematopoetic and Immune Systems; Endocrine Systems; Nervous System; Kidneys; Muscle; Gastrointestinal System; and The Eye.
Most of the proposed interventions involve resetting gene expression in senescent cells by relengthening telomeres. Insertion of the hTERT gene into senescent cells is one suggested approach for accomplishing this. (As somatic cells have hRT, presumably the insertion of the hTERT gene will lead to a functional telomerase enzyme.) The author suggests that it is most feasible to accomplish this with cells ex vivo. For example, this could be done with lymphocytes and stem cells isolated from blood samples. After insertion of the hTERT gene, the cells would be reintroduced into the organism. The in vivo insertion of the hTERT gene is also envisioned, but it is recognized that there are major technical hurdles to overcome before this can be done successfully. Another approach involves inducing the expression of the hTERT gene because somatic cells have the gene but do not express it. However, the author offers no suggestions on how to achieve this.
Has this book convinced me to join the Brilliant Insight camp? No, I remain more comfortable in the Foolish Notion camp. However, the book has sensitized me to be open to persuasion by new empirical evidence. Indeed, it takes very strong evidence to make one give up long-held beliefs, and such is certainly the case in regard to the "Hayflick hypothesis." This is clearly shown in a recent article by Vincent Cristofalo and his colleagues (Cristofalo, Lorenzini, Allen, Torres, & Tresini, 2004). For many years, Cristofalo has been a leading proponent of the "Hayflick hypothesis," and his laboratory has executed a number of exceedingly careful studies on cell senescence in culture. However, as discussed above, during the past few years, studies conducted in Cristofalo's laboratory have challenged the validity of critical empirical evidence in support of the "Hayflick hypothesis." These findings are reviewed in Cristofalo et al. (2004) and the following is the final sentence of that review:
In fact, the human fibroblast model has already been valuable for explaining the cellular basis of some mechanisms underlying cellular aging changes observed in situ, also for testing hypotheses that address what may be common mechanisms underlying cell deterioration and loss of integrative function such as the effects of reactive oxygen species, overexpression and underexpression of signaling molecules and other modulations particularly important to mechanisms underlying aging. (p. 841)
All of this may be true, but it is hardly relevant to the "Hayflick hypothesis." Many in vitro systems provide insights into in vivo processes—the rat hind-limb preparation, brain slices from rodent models, and homogenates of liver, to name a few. Indeed, primary cultures of cells from humans and animal models of various ages have and likely will provide much more insight into organismal aging than studies on the proliferative aging of cells in culture. Clearly, Cristofalo has as much difficulty exiting the Brilliant Insight camp as I have entering it.
Would I recommend that others read Cells, Aging, and Human Disease? I believe that one requires a rudimentary understanding of molecular biology to profit from reading this book, and I therefore do not recommend it to those who lack this background. For those with such a background, the book provides a wealth of information on the "Hayflick hypothesis" and its further development by others in the field, albeit from a biased perspective. Certainly, such information can also be gained by the many review articles on this subject published in recent years in Science, Nature, Journal of Gerontology: Biological Sciences, Mechanisms of Ageing and Development, and Experimental Gerontology, to name a few; these articles would probably give a more balanced view, but at the cost of less convenience. The information on mammalian organismal physiology, although briefly presented by Fossel, is impressive and except for some minor points, it is quite accurate. Certainly, many cell and molecular biologists would gain much from this material; however, a textbook of medical physiology would be a better source, though with the loss of brevity.
References
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