ExperimentalGerontology,Voi. 27, pp. 455--459, 1992
0531-5565/92 S5.00 + .00 Copyright © 1992 Pergamon Press Ltd.
Printed in the USA. All rights reserved.
CLONAL ATI'ENUATION AND CELL SENESCENCE: T H E N E X T 30 Y E A R S
GEORGE M. M A R T I N Departments of Pathology and Genetics and The Alzheimer's Disease Research Center, University of Washington, Seattle, Washington 98195
Key Words: cell senescence, physiological processes, cell injury
IN DEALINGwith this awesome assignment, I have made two reasonably safe assumptions: (1) Research in fields of great relevance to cellular gerontology and to our understanding of how cell aging sets the stage for major age-related diseases will enjoy continued robust growth, but (2) the rate of technology transfer will be too slow to keep my own somatic cells alive long enough to hear the answers in the year 2021! I can thus feel free to make unrestrained and erroneous predictionsma precious asset that I hope we scientists will never have to give up in order to maintain funding. Figure 1 gives an example (one of many), from the field of research on polymerases, supporting the first assumption above. It also demonstrates the early and continuous influence of basic research on nonmammalian model organisms, in this case Escherichia coli. Research on such models is another precious asset I hope we never have to give up in order to maintain funding. In 199 l, the core problem in biogerontology is the dilemma of attempting to reconcile two powerful and seemingly contradictory lines of evidence germane to the related questions of (1) the degree of complexity of underlying molecular and cellular mechanisms of senescence and (2) the degree of uniformity of such mechanisms in the biosphere. On the one hand, there is the evidence of evolutionary biologists (Rose, 1991) arguing forcefully for the existence of a potentially very large number of genetic loci for which allelic or mutational variation can arise and can contribute to the complex phenotype of senescence (an epiphenomenon not subject to stringent genetic selection). Since the maintenance of such alleles is attributable to their relative contributions to reproductive fitness, one can imagine an incredible diversity of such genes and, hence, a potentially vast diversity of mechanisms of senescence. Moreover, there would seem little reason, a priori, to expect the nature of the gene actions to be identical among mammals, let alone among unrelated organisms, although some types of gene action could be expected to be homologous. On the other hand, we have (1) the single gene mutation in Caenorhabditis elegansthat is associated with decreased age-specific mortality rates (Johnson, 1990); (2) the enhancement of life span of a specific strain of Drosophila melanogaster via transfection of additional copies of an expressed gene functioning in an elongation step of protein synthesis 455
G.M. MARTIN
456
No. citations
No. including coil 50O
5000 + 13ol
4000-
. .400
3000-
- 300
2000-
- 200
1000-
1966
i~t
1970
1974
1978 Year
100
1982
1986
1990
FIG. 1. An example of the substantial rate of growth of the biomedical literature in fields of high relevance to clonal attenuation and cell aging. The figure also shows evidence of the early and continued importance of research on simple model organisms (in this case, Escherichia coli). The Medline database of the National Library of Medicine was queried on line for hits of the text words polymerase or polyrnerases (in titles and/or abstracts) and for that subset in which the term coli appeared (in titles and/or abstracts).
(Shepherd et al., 1989); and (3) the huge literature on a simple environmental perturbation, dietary restriction, that results in a slowing of the age-specific mortality rates of comparatively short-lived mammals such as mice and rats (reviewed by Weindruch and Walford, 1988) and of short-lived invertebrates such as the bowl and doily spider (Austad, 1989). These lines of experiments argue for relatively few cellular and molecular mechanisms of senescence, and could be interpreted as support for major communal mechanisms of senescence in the biosphere. My own view is that, whenever there is a disagreement between cogent evolutionary theory and an experimental result, one should exercise extreme caution in generalizing from the experimental result. I predict that, especially for the case of Homo sapiens, the model of multiple mechanisms and of highly polygenic modulations will prevail. Even for the case of a single general mechanistic hypothesis, the free radical theory, numerous genetic loci can be invoked, including loci involved in the process ofclonal attenuation. Much more research in comparative biogerontology will be required to address these vital issues. We already know, however, that modulation of rates ofclonal attenuation cannot be of major significance in the senescence ofC. elegans or D. melanogaster, since their nongerminal somatic lineages are postreplicative. It will be increasingly important for us to define more precisely the elements of "cell aging" in the in vitro system of limited replicative potential pioneered by Hayrick and Moorhead 30 years ago. In 1974 and 1975 (Fig. 2), my colleagues and I offered a nomenclature of clonal senescence based upon two steps: (1) clonal attenuation, defined kinetically as the gradual loss of proliferative potential associated with the segregation of slower
457
CELL SENESCENCE
J STEM CELLS
I
clonal attenuation ----b-,--b
post-replicative senescence
TERMINALLY DIFFERENTIATED CELLS (normal "luxury" proteins)
SENESCENT CELLS (abnormal proteins)
FIo. 2. A two-stage model of the clonal senescence of cultured somatic cells, as presented by Martin et al. (1974, 1975). It was emphasized that, for the case of such in vitro systems, the phenotype of the putative terminally differentiated cell could be merely a caricature of the in vivo counterpart, which, for skin fibroblast-like cells, might be a type of soft-tissue histiocyte. A potentially important role for cell-cell interactions was also emphasized.
replicating and postreplicative cells, with the increasing loss of replicative potential of the best growing subset of cells within a developing clone, and (2) cell senescence, a series of steps leading to the increasing degree of senescence of postreplicative cells. We speculated that the former was a physiological process akin to terminal differentiation, involving differential expression of normal proteins, and that the latter involved the generation of abnormal proteins. (Today, one would specify abnormal proportions as well as qualitative abnormalities of proteins; Murano et al., 1991.) Thus, the system offers the opportunity to study more than the phenotype of limited proliferative potential. In the subsequent discussion, I shall attempt to differentiate between these two stages of clonal senescence. As regards clonal attenuation, the core problem is to decide, for several independent somatic cell types, the degree to which the process does indeed mimic (however unfaithfully) a comparable physiological process in vivo, be it terminal differentiation or something else. The alternative, of course, is that we are dealing with a substantive process of cell injury. The two may not be mutually exclusive. (Both, incidentally, are consistent with the stochastic nature of the loss of proliferative potential as a cell proceeds through the mitotic cell cycle.) Returning to evolutionary biology, it seems to me that the ultimate test of the physiological hypothesis is the degree to which clonal attenuation has been subject to natural selection. We have heard only one idea along this line, that it has adaptive value as a buffer against neoplasia (Sager, 1990). We need more such ideas, especially as the process may relate to more efficient tissue building and remodeling during development, normal cell turnover, and wound healing (Martin et al., 1974). The wonderful current progress in unraveling the biochemical genetic details of mammalian cell cycle regulation as cultures attenuate will surely continue with vigor and will include, to an increasing degree, the research on cell-free systems that is just beginning. We should have a greatly improved diagram showing positive and negative regulations at levels of replication, transcription, translation, posttranslational modification, and turnover, and will need a modern Rube Goldberg to illustrate the scheme for textbooks. It is my fondest hope that we shall be more vigorous in pursuing our research with various somatic cell types (especially including cells of the vascular wall) to the point where we can establish specific concordances and discordances of the phenotype as observed in vitro
458
G.M. MARTIN
with what is seen with the homologous cell types in vivo. The cell culture systems will also become much more sophisticated in terms of emulating the milieu in vivo, and will include investigations of cell-cell interactions and more work with organoid cultures. Novel new systems will be developed, such as proliferating cultures of primary olfactory neurons and their differentiated progeny (Farbman, 1990; Buck and Axel, 1991). There will be a greater emphasis on the pathogenesis of specific aberrations in proliferative homeostasis that are very conspicuous in older human subjects (Martin, 1979), including various atrophies, benign and malignant neoplasms, atherosclerosis, osteoarthrosis, benign prostatic hyperplasia, and osteoporosis (clonal attenuation of osteoblasts?). I predict major new initiatives in the study of the aging ofpostreplicative cells in cell and tissue culture, both in terms of alterations as functions of time in culture and in terms of alterations as functions of age and genotype of donor. Of paramount importance will be the terminally differentiated cells of specific subsets of neuroblasts and proliferating, differentiating populations such as the olfactory neurons mentioned above. Similar studies will be carried out with oocytes, myocardial tissue, and with skeletal muscle from specific sites. The genetic paradigm will continue to dominate, with the utilization of both spontaneous mutations that affect aspects of clonal attenuation and cell senescence as well as targeted mutagenesis. The embryonal stem cell system for the synthesis oftransgenic mice will come to be routine and will be extended to other mammalian species, of which the rat and guinea pig may be of special interest because of their relative resistance to the spontaneous transformation of cultured primary cells to lines of indefinite growth potential. We shall see the development of a number oftransgenic models of mutations in man that lead to high susceptibility to specific patterns of accelerated clonal attenuation (Werner's syndrome) and cellular aging (several forms of familial Alzheimer's disease). Perhaps of greater interest will be the identification of alleles that lead to unusual resistance to altered proliferation and to cellular degenerative changes associated with aging, both as a result of transgenic experiments and of selective breeding; these could include, for example, the synthesis of antimutator strains and strains with enhanced efficiencies for the targeting and degradation of abnormal proteins. The field ofepigenetics (Holliday, 1990) will be greatly developed. Some brave souls will try to utilize this new knowledge to appropriately alter patterns of genomic imprinting (Haig and Graham, 1991) so as to clone a mouse using cultured somatic cells. (They will be investigated by a congressional committee.) The multipotency ofembryonal stem cells and of certain other cell types (including those of plant origins) (Dudits et al., 1991) and their unusual replicative potentials will be elucidated in some detail at the molecular level. The question of germ line immortality will be pursued at the molecular level, including investigations of the relationships between recombination and repair and of cell selection during meiosis. In terms of technology transfer to geriatric medicine, we shall see more instances ofphenotypic engineering than of genetic engineering, with beginning attempts to combine the two--for example, the utilization of neurotransplantation for the amelioration therapy of Parkinson's disease and Alzheimer's disease using transfected, cultivated neural cells (Rosenberg et al., 1988). "Differentiation therapy" (Warrell et al., 1991) for a number of neoplasms and preneoplastic proliferations will become established. Genetic probes will become available for the diagnosis of the carrier state for a number of late-onset degenerative and proliferative lesions. Ethical guidelines will become established for how such probes should be used in the absence of available preventive or therapeutic management.
CELL SENESCENCE
459
Unanticipated applications to other important disease processes may well emerge from our collective efforts. An example may be trials of intervention to moderate the proliferative fibrotic component of cirrhosis of the liver (Castilla et al., 199 l). In summary, we can all agree with Sam Goldstein that the scientific exploitation of the "Hayflick Limit" has "come of age" (Goldstein, 1990). In addition to the expected mapping of mammalian cell cycle genes and the elucidation of their mechanisms of action, we shall see relatively more attention devoted to the second limb (Fig. 2) of the cell culture model--the aging of postreplicative cells, including comparative studies with various homologous cell types in vivo. We shall discover that there are many independent pathways leading to the senescent phenotypes of somatic cells, as predicted by evolutionary theory. REFERENCES AUSTAD, S.N. Life extension by dietary restriction in the bowl and doily spider, Frontinella pyramitela. Exp. Gerontol. 24, 83-92, 1989. BUCK, L. and AXEL, R. A novel muitigene family may encode odorant receptors: A molecular basis for odor recognition. Cell 65, 175-187, !99 I. CASTILLA, A., PRIETO, J., and FAUSTO, N. Transforming growth factors Bl and a in chronic liver disease: Effects of interferon alfa therapy. N. Engl. J. Med. 324, 933-940, !991. DUDITS, D., BOGRE, L., and GYORGYEY, J. Molecular and cellular approaches to the analysis of plant embryo development from somatic cells in vitro, J. Cell Sci. 99, 475-484, 1991. FARBMAN, A.I. Olfactory neurogenesis: Genetic or environmentalcontrols? Trends Neurosci. 13, 362-365, 1990. GOLDSTEIN, S. Replicative senescence: The human fibroblastscomes of age. Science 249, I 129- l 133, 1990. HAIG, D. and GRAHAM, C. Genomic imprintingand the strange case of the insulin-like growth factor li receptor. Cell64, 1045-1046, 1991. HAYFLICK, L. and MOORHEAD, P.S. The serial cultivation of human diploid cell strains. Exp. Cell Res. 25, 585-621, 1961. HOLLIDAY, R. Mechanisms for the control ofgene activity during development. Biol. Rev. 65, 431-471, 1990. JOHNSON, T.E. The increased life span ofage-I mutants in Caenorhabditis elegans results from lowering the Gompertz rate of aging. Science 249, 908-912, 1990. MARTIN, G.M. Proliferative homeostasis and its age-related aberrations. Mech. Ageing Dev. 9, 385-391, 1979. MARTIN, G.M., SPRAGUE, C.A., NORWOOD, T.H., and PENDERGRASS, W.R. Cional selection, attenuation and differentiation in an in vitro model of hyperplasia. Am. J. Pathol. 74, 137-154, 1974. MARTIN, G.M., SPRAGUE, C.A., NORWOOD, T.H., PENDERGRASS, W.R., BORNSTEIN, P., HOEHN, H., and AREND, W.P. Do hyperplastoid cell lines "differentiate themselves to death"? In: Cell Impairment in Aging and Development, Cristofalo, V.J. and Holeckova, E. (Editors), pp. 67-90, Plenum Press, New York, NY, 1975. MURANO, S., THWEATT, R., SHMOOKLER REIS, R.J., JONES, R.A., MOERMAN, E.J., and GOLDSTEIN, S. Diverse gene sequences are overexpressed in Werner syndrome fibroblasts undergoing premature replicative senescence. Mol. Cell. Biol. I I, 3905-3914, 199 I. ROSE, M.R. Evolutionary Biology of Aging. Oxford University Press, New York, NY, 1991. ROSENBERG, M.B., FRIEDMANN, T., ROBERTSON, R.C., TUSZYNSKI, M., WOLFF, J.A., BREAKEFIELD, X.O., and GAGE, F.H. Grafting genetically modified cells to the damaged brain: Restorative effects of NGF expression. Science 242, 1575-1578, 1988. SAGER, R. Genetic strategies of tumor suppression. Am. Rev. Respir. Dis. 142, 40-43, 1990. SHEPHERD, J.C., WALLDORF, U., HUG, P., and GEHRING, W.J. Fruit flies with additional expression of the elongation factor EF-I a live longer. Proc. Natl. Acad. Sci. U. S. A. 86, 7520-7521, 1989. WARRELL, R.P., JR., FRANKEL, S.R., MILLER, W.H., JR., SCHEINBERG, D.A., ITRI, L.M., HITTELMAN, W.N., VYAS, R., ANDREEFF, M., TAFURI, A., JAKUBOWSK1,A., GABRILOVE, J., GORDON, M.S., and DMITROVSKY, E. Differentiation therapy of acute promyelocytic leukemia with tretinoin (alltrans-retinoic acid). N. Engl. J. Med. 324, 1385-1393, 1991. WEINDRUCH, R. and WALFORD, R.L. The Retardation of Aging and Disease by Dietary Restriction. Charles C. Thomas, Springfield, IL, 1988.
0531-5565/92 S5.00 + .00 Copyright © 1992 Pergamon Press Ltd.
Printed in the USA. All rights reserved.
CLONAL ATI'ENUATION AND CELL SENESCENCE: T H E N E X T 30 Y E A R S
GEORGE M. M A R T I N Departments of Pathology and Genetics and The Alzheimer's Disease Research Center, University of Washington, Seattle, Washington 98195
Key Words: cell senescence, physiological processes, cell injury
IN DEALINGwith this awesome assignment, I have made two reasonably safe assumptions: (1) Research in fields of great relevance to cellular gerontology and to our understanding of how cell aging sets the stage for major age-related diseases will enjoy continued robust growth, but (2) the rate of technology transfer will be too slow to keep my own somatic cells alive long enough to hear the answers in the year 2021! I can thus feel free to make unrestrained and erroneous predictionsma precious asset that I hope we scientists will never have to give up in order to maintain funding. Figure 1 gives an example (one of many), from the field of research on polymerases, supporting the first assumption above. It also demonstrates the early and continuous influence of basic research on nonmammalian model organisms, in this case Escherichia coli. Research on such models is another precious asset I hope we never have to give up in order to maintain funding. In 199 l, the core problem in biogerontology is the dilemma of attempting to reconcile two powerful and seemingly contradictory lines of evidence germane to the related questions of (1) the degree of complexity of underlying molecular and cellular mechanisms of senescence and (2) the degree of uniformity of such mechanisms in the biosphere. On the one hand, there is the evidence of evolutionary biologists (Rose, 1991) arguing forcefully for the existence of a potentially very large number of genetic loci for which allelic or mutational variation can arise and can contribute to the complex phenotype of senescence (an epiphenomenon not subject to stringent genetic selection). Since the maintenance of such alleles is attributable to their relative contributions to reproductive fitness, one can imagine an incredible diversity of such genes and, hence, a potentially vast diversity of mechanisms of senescence. Moreover, there would seem little reason, a priori, to expect the nature of the gene actions to be identical among mammals, let alone among unrelated organisms, although some types of gene action could be expected to be homologous. On the other hand, we have (1) the single gene mutation in Caenorhabditis elegansthat is associated with decreased age-specific mortality rates (Johnson, 1990); (2) the enhancement of life span of a specific strain of Drosophila melanogaster via transfection of additional copies of an expressed gene functioning in an elongation step of protein synthesis 455
G.M. MARTIN
456
No. citations
No. including coil 50O
5000 + 13ol
4000-
. .400
3000-
- 300
2000-
- 200
1000-
1966
i~t
1970
1974
1978 Year
100
1982
1986
1990
FIG. 1. An example of the substantial rate of growth of the biomedical literature in fields of high relevance to clonal attenuation and cell aging. The figure also shows evidence of the early and continued importance of research on simple model organisms (in this case, Escherichia coli). The Medline database of the National Library of Medicine was queried on line for hits of the text words polymerase or polyrnerases (in titles and/or abstracts) and for that subset in which the term coli appeared (in titles and/or abstracts).
(Shepherd et al., 1989); and (3) the huge literature on a simple environmental perturbation, dietary restriction, that results in a slowing of the age-specific mortality rates of comparatively short-lived mammals such as mice and rats (reviewed by Weindruch and Walford, 1988) and of short-lived invertebrates such as the bowl and doily spider (Austad, 1989). These lines of experiments argue for relatively few cellular and molecular mechanisms of senescence, and could be interpreted as support for major communal mechanisms of senescence in the biosphere. My own view is that, whenever there is a disagreement between cogent evolutionary theory and an experimental result, one should exercise extreme caution in generalizing from the experimental result. I predict that, especially for the case of Homo sapiens, the model of multiple mechanisms and of highly polygenic modulations will prevail. Even for the case of a single general mechanistic hypothesis, the free radical theory, numerous genetic loci can be invoked, including loci involved in the process ofclonal attenuation. Much more research in comparative biogerontology will be required to address these vital issues. We already know, however, that modulation of rates ofclonal attenuation cannot be of major significance in the senescence ofC. elegans or D. melanogaster, since their nongerminal somatic lineages are postreplicative. It will be increasingly important for us to define more precisely the elements of "cell aging" in the in vitro system of limited replicative potential pioneered by Hayrick and Moorhead 30 years ago. In 1974 and 1975 (Fig. 2), my colleagues and I offered a nomenclature of clonal senescence based upon two steps: (1) clonal attenuation, defined kinetically as the gradual loss of proliferative potential associated with the segregation of slower
457
CELL SENESCENCE
J STEM CELLS
I
clonal attenuation ----b-,--b
post-replicative senescence
TERMINALLY DIFFERENTIATED CELLS (normal "luxury" proteins)
SENESCENT CELLS (abnormal proteins)
FIo. 2. A two-stage model of the clonal senescence of cultured somatic cells, as presented by Martin et al. (1974, 1975). It was emphasized that, for the case of such in vitro systems, the phenotype of the putative terminally differentiated cell could be merely a caricature of the in vivo counterpart, which, for skin fibroblast-like cells, might be a type of soft-tissue histiocyte. A potentially important role for cell-cell interactions was also emphasized.
replicating and postreplicative cells, with the increasing loss of replicative potential of the best growing subset of cells within a developing clone, and (2) cell senescence, a series of steps leading to the increasing degree of senescence of postreplicative cells. We speculated that the former was a physiological process akin to terminal differentiation, involving differential expression of normal proteins, and that the latter involved the generation of abnormal proteins. (Today, one would specify abnormal proportions as well as qualitative abnormalities of proteins; Murano et al., 1991.) Thus, the system offers the opportunity to study more than the phenotype of limited proliferative potential. In the subsequent discussion, I shall attempt to differentiate between these two stages of clonal senescence. As regards clonal attenuation, the core problem is to decide, for several independent somatic cell types, the degree to which the process does indeed mimic (however unfaithfully) a comparable physiological process in vivo, be it terminal differentiation or something else. The alternative, of course, is that we are dealing with a substantive process of cell injury. The two may not be mutually exclusive. (Both, incidentally, are consistent with the stochastic nature of the loss of proliferative potential as a cell proceeds through the mitotic cell cycle.) Returning to evolutionary biology, it seems to me that the ultimate test of the physiological hypothesis is the degree to which clonal attenuation has been subject to natural selection. We have heard only one idea along this line, that it has adaptive value as a buffer against neoplasia (Sager, 1990). We need more such ideas, especially as the process may relate to more efficient tissue building and remodeling during development, normal cell turnover, and wound healing (Martin et al., 1974). The wonderful current progress in unraveling the biochemical genetic details of mammalian cell cycle regulation as cultures attenuate will surely continue with vigor and will include, to an increasing degree, the research on cell-free systems that is just beginning. We should have a greatly improved diagram showing positive and negative regulations at levels of replication, transcription, translation, posttranslational modification, and turnover, and will need a modern Rube Goldberg to illustrate the scheme for textbooks. It is my fondest hope that we shall be more vigorous in pursuing our research with various somatic cell types (especially including cells of the vascular wall) to the point where we can establish specific concordances and discordances of the phenotype as observed in vitro
458
G.M. MARTIN
with what is seen with the homologous cell types in vivo. The cell culture systems will also become much more sophisticated in terms of emulating the milieu in vivo, and will include investigations of cell-cell interactions and more work with organoid cultures. Novel new systems will be developed, such as proliferating cultures of primary olfactory neurons and their differentiated progeny (Farbman, 1990; Buck and Axel, 1991). There will be a greater emphasis on the pathogenesis of specific aberrations in proliferative homeostasis that are very conspicuous in older human subjects (Martin, 1979), including various atrophies, benign and malignant neoplasms, atherosclerosis, osteoarthrosis, benign prostatic hyperplasia, and osteoporosis (clonal attenuation of osteoblasts?). I predict major new initiatives in the study of the aging ofpostreplicative cells in cell and tissue culture, both in terms of alterations as functions of time in culture and in terms of alterations as functions of age and genotype of donor. Of paramount importance will be the terminally differentiated cells of specific subsets of neuroblasts and proliferating, differentiating populations such as the olfactory neurons mentioned above. Similar studies will be carried out with oocytes, myocardial tissue, and with skeletal muscle from specific sites. The genetic paradigm will continue to dominate, with the utilization of both spontaneous mutations that affect aspects of clonal attenuation and cell senescence as well as targeted mutagenesis. The embryonal stem cell system for the synthesis oftransgenic mice will come to be routine and will be extended to other mammalian species, of which the rat and guinea pig may be of special interest because of their relative resistance to the spontaneous transformation of cultured primary cells to lines of indefinite growth potential. We shall see the development of a number oftransgenic models of mutations in man that lead to high susceptibility to specific patterns of accelerated clonal attenuation (Werner's syndrome) and cellular aging (several forms of familial Alzheimer's disease). Perhaps of greater interest will be the identification of alleles that lead to unusual resistance to altered proliferation and to cellular degenerative changes associated with aging, both as a result of transgenic experiments and of selective breeding; these could include, for example, the synthesis of antimutator strains and strains with enhanced efficiencies for the targeting and degradation of abnormal proteins. The field ofepigenetics (Holliday, 1990) will be greatly developed. Some brave souls will try to utilize this new knowledge to appropriately alter patterns of genomic imprinting (Haig and Graham, 1991) so as to clone a mouse using cultured somatic cells. (They will be investigated by a congressional committee.) The multipotency ofembryonal stem cells and of certain other cell types (including those of plant origins) (Dudits et al., 1991) and their unusual replicative potentials will be elucidated in some detail at the molecular level. The question of germ line immortality will be pursued at the molecular level, including investigations of the relationships between recombination and repair and of cell selection during meiosis. In terms of technology transfer to geriatric medicine, we shall see more instances ofphenotypic engineering than of genetic engineering, with beginning attempts to combine the two--for example, the utilization of neurotransplantation for the amelioration therapy of Parkinson's disease and Alzheimer's disease using transfected, cultivated neural cells (Rosenberg et al., 1988). "Differentiation therapy" (Warrell et al., 1991) for a number of neoplasms and preneoplastic proliferations will become established. Genetic probes will become available for the diagnosis of the carrier state for a number of late-onset degenerative and proliferative lesions. Ethical guidelines will become established for how such probes should be used in the absence of available preventive or therapeutic management.
CELL SENESCENCE
459
Unanticipated applications to other important disease processes may well emerge from our collective efforts. An example may be trials of intervention to moderate the proliferative fibrotic component of cirrhosis of the liver (Castilla et al., 199 l). In summary, we can all agree with Sam Goldstein that the scientific exploitation of the "Hayflick Limit" has "come of age" (Goldstein, 1990). In addition to the expected mapping of mammalian cell cycle genes and the elucidation of their mechanisms of action, we shall see relatively more attention devoted to the second limb (Fig. 2) of the cell culture model--the aging of postreplicative cells, including comparative studies with various homologous cell types in vivo. We shall discover that there are many independent pathways leading to the senescent phenotypes of somatic cells, as predicted by evolutionary theory. REFERENCES AUSTAD, S.N. Life extension by dietary restriction in the bowl and doily spider, Frontinella pyramitela. Exp. Gerontol. 24, 83-92, 1989. BUCK, L. and AXEL, R. A novel muitigene family may encode odorant receptors: A molecular basis for odor recognition. Cell 65, 175-187, !99 I. CASTILLA, A., PRIETO, J., and FAUSTO, N. Transforming growth factors Bl and a in chronic liver disease: Effects of interferon alfa therapy. N. Engl. J. Med. 324, 933-940, !991. DUDITS, D., BOGRE, L., and GYORGYEY, J. Molecular and cellular approaches to the analysis of plant embryo development from somatic cells in vitro, J. Cell Sci. 99, 475-484, 1991. FARBMAN, A.I. Olfactory neurogenesis: Genetic or environmentalcontrols? Trends Neurosci. 13, 362-365, 1990. GOLDSTEIN, S. Replicative senescence: The human fibroblastscomes of age. Science 249, I 129- l 133, 1990. HAIG, D. and GRAHAM, C. Genomic imprintingand the strange case of the insulin-like growth factor li receptor. Cell64, 1045-1046, 1991. HAYFLICK, L. and MOORHEAD, P.S. The serial cultivation of human diploid cell strains. Exp. Cell Res. 25, 585-621, 1961. HOLLIDAY, R. Mechanisms for the control ofgene activity during development. Biol. Rev. 65, 431-471, 1990. JOHNSON, T.E. The increased life span ofage-I mutants in Caenorhabditis elegans results from lowering the Gompertz rate of aging. Science 249, 908-912, 1990. MARTIN, G.M. Proliferative homeostasis and its age-related aberrations. Mech. Ageing Dev. 9, 385-391, 1979. MARTIN, G.M., SPRAGUE, C.A., NORWOOD, T.H., and PENDERGRASS, W.R. Cional selection, attenuation and differentiation in an in vitro model of hyperplasia. Am. J. Pathol. 74, 137-154, 1974. MARTIN, G.M., SPRAGUE, C.A., NORWOOD, T.H., PENDERGRASS, W.R., BORNSTEIN, P., HOEHN, H., and AREND, W.P. Do hyperplastoid cell lines "differentiate themselves to death"? In: Cell Impairment in Aging and Development, Cristofalo, V.J. and Holeckova, E. (Editors), pp. 67-90, Plenum Press, New York, NY, 1975. MURANO, S., THWEATT, R., SHMOOKLER REIS, R.J., JONES, R.A., MOERMAN, E.J., and GOLDSTEIN, S. Diverse gene sequences are overexpressed in Werner syndrome fibroblasts undergoing premature replicative senescence. Mol. Cell. Biol. I I, 3905-3914, 199 I. ROSE, M.R. Evolutionary Biology of Aging. Oxford University Press, New York, NY, 1991. ROSENBERG, M.B., FRIEDMANN, T., ROBERTSON, R.C., TUSZYNSKI, M., WOLFF, J.A., BREAKEFIELD, X.O., and GAGE, F.H. Grafting genetically modified cells to the damaged brain: Restorative effects of NGF expression. Science 242, 1575-1578, 1988. SAGER, R. Genetic strategies of tumor suppression. Am. Rev. Respir. Dis. 142, 40-43, 1990. SHEPHERD, J.C., WALLDORF, U., HUG, P., and GEHRING, W.J. Fruit flies with additional expression of the elongation factor EF-I a live longer. Proc. Natl. Acad. Sci. U. S. A. 86, 7520-7521, 1989. WARRELL, R.P., JR., FRANKEL, S.R., MILLER, W.H., JR., SCHEINBERG, D.A., ITRI, L.M., HITTELMAN, W.N., VYAS, R., ANDREEFF, M., TAFURI, A., JAKUBOWSK1,A., GABRILOVE, J., GORDON, M.S., and DMITROVSKY, E. Differentiation therapy of acute promyelocytic leukemia with tretinoin (alltrans-retinoic acid). N. Engl. J. Med. 324, 1385-1393, 1991. WEINDRUCH, R. and WALFORD, R.L. The Retardation of Aging and Disease by Dietary Restriction. Charles C. Thomas, Springfield, IL, 1988.
