Debate Gerontology 23: 211-218 (1977)
Possible Molecular Mechanisms of Ageing Jean-Claude Dreyfus, Henriette Rubinson, Fanny Schapira, Annette Weber, Joelle Marie and Axel Kahn Institut de Pathologie Moléculaire, INSERM U 129 et LA 85 CNRS, Paris and Centre de Recherches sur les Enzymopathies, INSERM U 24 et ERA 573 du CNRS, Hôpital Beaujon, Clichy
Key Words. Error theory • Posttranslational modification Abstract. While the error theory of ageing has attracted most interest in recent times it cannot yet be regarded as being demonstrated. Posttranslational modifications of proteins explain many observed facts, but do not at present constitute a theory of ageing. The genetic theory appears logical but has little in vivo evidence to prove it. Basic mechanisms of ageing probably involve the interaction of several processes.
Received: December 2, 1975.
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The first contributions to the debate (Baird et al., 1975;Holliday, 1975; Gershon and Gershon, 1976) have centered the discussions on Orgel’s hypo thesis. While most other hypotheses remain theoretical, Orgel’s hypothesis has been chosen especially because experiments can be devised to test it. Discus sions, however, are often obscured by confusing several levels at which ageing phenomena may act. At least three levels could be distinguished: (a) molecular ageing, i.e. the posttranslational events undergone by a molecule between its synthesis and its degradation, and their relationships with molecular turnover; (b) cellular ageing of nondividing cells (for example cells of the nematode, Turbatrix aceti, and muscle or nerve cells in higher animals), and (c) ageing of cell lines, which may reach the limit of their mitotic activity, either in vitro or in vivo. These phenomena are probably all involved to different extents in the ageing of the whole organism. Conclusions pertaining to this ultimate level are still complicated by circulatory (vascular insufficiency), hormonal (Adelman et al., 1972) or immunological factors, which will not be discussed here. In vivo and in vitro techniques have been used to approach these various problems. Techniques using in vitro replication of fibroblasts (Hayflick and
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Moorhead, 1961) are very convenient. Relationships between behavior of cells in vitro and in vivo, however, have been questioned by many authors. In addition, in vitro techniques deal only with one aspect, the mitotic capacity of the cells, which in many cases is not exhausted at the death of the organism. The 'Error Catastrophe’ Theory (Orgel, 1963) This theory is advocated by Holliday (1975), from experiments on fibro blasts in culture, and neurospora mutants possessing a modified leucyl-t-RNA synthetase (Lewis and Tarrant, 1972). In favor of the error theory we could also quote a decrease of the discriminating index for amino acid analogues (Ogrodnik et aL, 1975) and preliminary results on infidelity of a DNA polymerase in aged mouse liver (Barton et aL, 1975). By contrast, many results have been negative or could be interpreted in another way. Holland et aL (1973) have shown that viral replication takes place on senescent as well as in young cells. Danot et aL (1975) found no alterations in enzyme molecules in senescent mouse fibroblasts in culture. We ourselves have found no modifications in the isoelectric focussing pattern of glucose-6-phosphate dehydrogenase (Leibovich and Dreyfus, unpubl.) and no immunological cross-reacting material (CRM) for four enzymes in human liver-derived senescent cells in culture (Kahn et aL, in preparation). In vivo, we have measured the molecular specific activity (ratio of enzyme activity to anti genic reactivity) of five cytoplasmic and two lysosomal enzymes in granulocytes of newborn babies, young adults and people over 80 (Rubinson et al., 1976). No difference could be found. This work seems to indicate that the protein synthe sis machinery of young cells from old organisms is intact. Finally, Schapira et al. (1975) have shown that the CRM for lactic dehydrogenase which is found in the liver of old rats decreases during post-hepatectomy regeneration. These results give evidence against faulty transcription or translation in liver cells of old rats and favor the interpretation of CRM as resulting from postsynthetic changes in the molecule.
Most abnormalities found in proteins of aged cells (CRM, heat lability, electrophoretic modifications) could be ascribed to alterations of the protein molecule occurring after its biosynthesis; this mechanism has been well em phasized by the Gershons contribution, and also by other authors (Reiss and Rothstein, 1975). It seems that this explanation may account for most examples of altered proteins, but its importance varies very much as a function of the various cells and tissues.
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(a) Young dividing cells, as stressed above, show little or no altered protein. (b) At the extreme opposite are cells which survive with no autonomous protein synthesis. The best example is the red blood cell. In old red cells modifi cations of many enzymes are known, which can be due to postsynthetic altera tions. Another example is the nucleus of the lens. (c) Between these two extreme situations are the cells which divide either very rarely (liver cells) or not at all (nerve or muscle cells). While the best model could be nerve cells, most of the work has been devoted to liver cells. Work with the nematode, Turbatrix aceti, can also enter into this category, since this organism only possesses nondividing cells (Gershon and Gershon, 1970). Ab normal proteins are often found in this type of cell. We agree in general with the Gershons conclusions, but we are at odds with them on the following points: (1) Accumulation of CRM does not seem to us to be constant. Even similar enzymes can behave in a very different way: an example is two enzymes of the purine salvage pathway, hypoxanthine — and adenine — phosphoribosyl trans ferases in the red cells (Yip etal., 1974). Different results have been found with aldolase in liver: presence of CRM in old mice (Gershon and Gershon, 1973), absence in old rats ( Weber and Schapira, to be publ.); presence of muscle-type lactic dehydrogenase in the liver of old rats (Schapira et al, 1975), absence in the muscle (Oliveira and Pfuderer, 1973). It has even been suggested that dietary changes end in the disappearance of heat-labile molecules of glucose-6-phosphate dehydrogenase in the liver of old rats (Wulfand Cutler, 1975). (2) Posttranslationally altered molecules are not necessarily, according to us, either entirely normal or completely inactivated. For red cell enzymes, many electrophoretic modifications have been described (Turner et al, 1975). We have been able to directly demonstrate (Kahn et al., 1974) that old red cells contain fractions of glucose-6-phosphate dehydrogenase with a lowered isoelectric point, and that these fractions have a specific activity lower than that of the enzyme from young cells. Analogous conclusions were reached by Reiss and Rothstein (1975) with isocitrate lyase from Turbatrix aceti. Besides, we do not see why either errors in synthesis or postsynthetic noncovalent modifications should necessarily induce a complete inactivation of the biological activity of a protein. The mechanism of posttranslational modifications is still hypothetical. Only one reaction is well defined (Robinson, 1974), e.g. a deamidation of asparagine, and perhaps glutamine residues. This mechanism has been demonstrated for aldolase, cytochrome c, a-crystalline from lens. This deamidation lowers the pHi of proteins, it is not proved that it decreases their activity. Intracellular processes might enhance postsynthetic alterations. We (Kahn et al, 1974) have demon strated the existence in white blood cells of a thermostable factor which is able to duplicate in vitro the modification of G6PD observed in ageing cells. This factor is probably a peptide (Kahn eta l, 1976). Finally, posttranslational modi
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fications are known to occur in collagen and elastin, due to the amino acid composition of these proteins (Gallop and Paz, 1975). Posttranslational modifi cations are observed in most cases in old cells. The reasons for their accumula tion are not clear. A possible explanation could be that the rate of protein synthesis and, therefore, of protein turnover, decreases in senescent tissues {Young et ai, 1975). Since posttranslational alterations are related to the age of the molecule they would be more apparent in older organisms. The importance of the rate of protein turnover (as well as the fact that each protein turns over at its proper rate) seem to have been underestimated. Posttranslational modifications do not, at present, constitute a theory of ageing. They might only be a transition step in the normal catabolism of pro teins. Two mechanisms could play a role in damaging a cell: (1) a considerable accumulation of biologically inactive proteins, such as described in Turbatrix aceti, might create a special kind of storage protein disease, and (2) ‘old’ mole cules might acquire a modified specificity which would result in altered metab olisms, and, if key enzymes are involved, in errors in synthesis of proteins which would be of nongenetic origin. Genetic Theory
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Many authors consider that ageing and death are the result of a genetic program. This theory has been clearly developed by Hayflick (1973). Ageing is programmed, either by ‘ageing genes’ which would come to operation late in life, or by shutting off adult genes. Arguments in favor of this theory come from two sets of evidence: in vitro the number of doublings of any strain of diploid fibroblasts is finite and relatively constant* for a given species. In vivo biological clocks do exist, as shown in physiology by puberty or menopause, and in pathology by the appearance only in adults of some genetic diseases like Huntington’s chorea. Of course, the difference of longevity between various species is also interpreted in the same way. It can also, to some extent account for the immortality of malignant cells, resulting from a complex disturbance in the genetic machinery. Direct evidence of the genetic theory, however, is difficult to provide. Furthermore such a theory deals with the limited mitotic capacity of cells and would have to be modified to account for ageing of non dividing cells. A variant of the genetic theory is the theory of the isozymic shifts (Krooth, 1974). New isozymes (or isoproteins) would be derepressed late in life. If their accuracy is less than that of the adult enzymes or proteins, a progressive damage will take place. This theory was forged in analogy to the shifts in some proteins observed during or after fetal life, especially the non-o-chains of hemoglobin. It can claim only little experimental support up to now (Kanungo, 1975).
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Several modifications of the genetic material have been reported in old cells. These alterations, however, can be interpreted by any of the above theories. We quote them since they might be instrumental in the progressive deterioration of senescence. DNA and Nuclear Proteins Base composition of DNA could be randomly altered, giving rise to an increasing rate of errors in transcribed messenger RNA. This ‘somatic mutations theory’ (Curtis, 1964) has not been substantiated and is now disreputed. The physicochemical structure of DNA could be modified. Von Hahn (1966) described a greater heat stability of DNA and a greater difficulty to split histone from DNA in thymus of aged cattle. He hypothesized that sticking of histone to DNA could prevent local strand separation, creating new repressions. Gene expression is now thought to be modulated by nuclear acid non histone proteins. Stein (1975) has shown that among the proteins which bind to DNA in vitro one is greatly increased in senescent human fibroblasts. Enzymes Acting on DNA and RNA Synthesis of DNA in vitro by a low molecular weight DNA polymerase shows more errors when working with liver of mice over 2 years (Barton et al, 1975). Modification of DNA and RNA polymerase, as well as of elongation factor 1, have been found in Turbatrix aceti (,Bolla and Brot, 1975). Methylation of tRNA decreases in the liver of old rats (Wust and Rosen, 1972) because of a decrease of tRNA methyltransferase activity concomitant with an increase in the competing enzyme glycine N-methyltransferase (Mays et al., 1973). DNA repair: Epstein et al. (1974) and Mattem and Cerutti (1975) have reported that the rate of DNA repair is reduced in vitro in human senescent fibroblasts and in progeria. This phenomenon can be due to a genetic change in the repair enzymes, but also to translational or posttranslational modifications of these enzymes. It can therefore be either one of the causes or a consequence of senescence. All the above pieces of evidence point to functional disturbances of enzymes involved in the synthesis and regulation of nucleic acids. None of them is apparently able to account for the whole phenomenon of ageing.
Sheldrake (1974) proposed that cells die because of the accumulation of toxic substances in the course of their ageing. Cells could divide unequally, between cells which would live and be regenerated, and cells which would die
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Theory o f Intoxication
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prematurely. The only known example in animals is the meiotic division of the egg cell. The theory can be applied to other cell divisions, including the can cerous cells. No conclusive evidence, however, can be produced to support this theory. Moreover, Wright and Hayflick (1975) by enucleating human old lung fibroblasts and by hybridizing them with young cells have shown that nuclear and not cytoplasmic factors limit the proliferative capacity of cultured cells. Consequently, this accumulation ought to be in the nucleus, if it was the cause of senescence. Conclusions No hypothesis appears to account for the observed facts at present. Hayflick’s genetic theory appears logical but there is little experimental evidence to prove it. The error theory can claim only few nondisputable facts to support it, since most observations can be better explained by posttranslational changes, but still deserves credit as being liable to experimental testing. Postsynthetic modifications certainly exist, and their mechanisms of production can now be investigated; but they do not seem to be universal, and pending the demonstra tion of their physiological importance, they cannot yet be evaluated. So the basic mechanism of ageing processes remains mysterious; it is probable that it involves exhaustion of mitotic potential, interactions of cells and of tissues and organs in the whole organism. References
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Adelman, R.C.; Freeman, C, and Cohen, B.S.: Enzymes adaptation as a biochemical probe of development and ageing. Adv. Enzyme Regul. 10: 365-382 (1972). Baird, M.B.: Samis, H. V.; Massie, H.R., and Zimmerman, J.A.: A brief argument in opposi tion to the Orgel hypothesis. Gerontologia 21: 57-63 (1975). Barton, R.W.; Waters, L.C., and Yang, W.K.: In vitro DNA synthesis by low molecular weight DNA polymerase. Increased infidelity associated with ageing. Fed. Proc. Fed. Am. Socs exp. Biol. 1975 abstract No. 1102. Bolla, R. and Brot. N.: Age dependent changes in enzymes involved in macromolecular synthesis in Turbatrix aceti. Archs Biochem. Biophys. 169: 227-236 (1975). Curtix, H.J.: Cellular processes involved in ageing. Fed. Proc. Fed. Am. Socs exp. Biol. 23: 662-667 (1975). Danot, M.: Gershon, H., and Gershon, D.: The lack of altered enzyme molecules in senes cent mouse embryo fibroblasts in culture; quoted by Gershon and Gershon (1975) (in press). Epstein, J.; Williams, J.R., and Little, J.B.: Rate of DNA repair in progeric and normal human fibroblasts. Biochem. biophys. Res. Commun. 59: 850-857 (1974). Gallop, P.M. and Paz, M.A.: Posttranslational protein modifications with special attention to collagen and elastin. Physiol. Rev. 55: 418-487 (1975).
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Gershon, H. and Gershon, D.: Detection of inactive enzyme molecules in aging organisms. Nature, Lond.227: 1214-1217 (1970). Gershon, H. and Gershon, D.: Inactive enzyme molecules in aging mice: liver aldolase. Proc. natn. Acad. Sci. USA 70: 909-913 (1973). Gershon, D. and Gershon, H.: An evaluation of the 'error catastrophe’ theory of ageing in light of recent experimental results. Gerontology 22: 212-219 (1976). Hayflick, L.: The biology of human aging. Am. J. med. Sci. 265: 433-445 (1973). HayfUck, L. and Moorhead, P.S.: The serial cultivation of human diploid cell strains. Expl. Cell Res. 25: 585-621 (1961). Holland, J.J.; Kohne, D., and Doyle, M.V.: Analysis of virus replication in ageing human fibroblast culture. Nature, Lond. 245: 316 (1973). Holliday, L.: Testing the protein error theory of ageing: a reply to Baird, Samis, Massie and Zimmerman. Gerontologia 21: 64-68 (1975). Kahn, A.; Boivin, P.: Rubinson, H.; Cottreau, D.; Marie, J., and Dreyfus, J.C.: Modifications of purified glucose-6-phosphate dehydrogenase and other enzymes by a factor of low molecular weight abundant in some leukemic cells. Proc. natn. Acad. Sci. USA 73: 77-81 (1976). Kahn, A.; Boivin, P: Vibert, M.; Cottreau, D., and Dreyfus, J.C.: Posttranslational modifications of human glucose-6-phosphate dehydrogenase. Biochimie 56: 1395-1407 (1974). Kanungo, M.S.: A model for ageing. J. theor. Biol. 53: 253-262 (1975). Krooth, R.S.: A deterministic mechanism for cellular and organismic ageing. J. theor. Biol. 46: 501-507 (1974). Lewis, C.M. and Holliday, R.: Mistranslation and ageing in neurospora. Nature, Lond. 228: 877-880 (1970). Lewis, CM. and Tarrant, G.M.: Error theory and ageing in human diploid fibroblasts. Nature, Lond. 239: 316-318 (1972). Mattern, M.R. and Cerutti, P.A.: Age dependant excision repair of damaged thymine from 7-irradiated DNA by isolated nuclei from human fibroblasts. Nature, Lond. 254: 4 50 452 (1975). Mays, L.L.; Borek, E., and Finch, C.E.: Glycine N-methyltransferase is a regulatory enzyme which increases in ageing animals. Nature, Lond. 243: 411-413 (1973). Ogrodnik, J.P.; Wulf J.H., and Cutler, R.G.: Altered protein hypothesis of mammalian ageing processes. Discrimination ratio of methionine versus ethionine in the synthesis of ribosomal protein and RNA. Expl. Gerontol. 10: 119-136 (1975). Olivera, R.J. and Pfuderer, P : Test for missynthesis of lactate dehydrogenase in aging mice by use of a monospecific antibody. Expl Gerontol. 8: 193-198 (1973). Orgel, L.E.: The maintenance of the accuracy of protein synthesis and its relevance to ageing. Proc. natn. Acad. Sci. USA 49: 517-521 (1963). Reiss, V. and Rothstein, M.: Age related changes in isocitrate lyase from the free-living nematode, Turbatrix aceti. J. biol. Chem. 250: 826-830 (1975). Robinson, A.: Evolution and the distribution of glutaminyl and asparaginyl residues in proteins. Proc. natn. Acad. Sci. USA 71: 885-888 (1974). Rubinson, H.; Kahn, A.; Boivin, P.: Schapira, F.; Gregori, C, and Dreyfus, J.C.: Aging and accuracy of protein synthesis in man: search for inactive enzymatic cross reacting material in granulocytes of aged people. Gerontology (in press). Schapira, F.; Weber, A. et Gregori, C: Vieillisement de la lacticodeshydrogénase hépatique du rat et renouvellement cellulaire. C. r. hebd. Séanc. Acad. Sci., Paris 280: 1161 — 1163 (1975). Sheldrake, A.R.: The ageing, growth and death of cells. Nature, Lond. 250: 381-384 (1974).
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Stein, G.H.: DNA binding proteins in young and senescent normal human fibroblasts. Expl Cell Res. 90: 237-248 (1975). Turner, B.M.; Fisher, R.A., and Harris, H.: Posttranslational alterations of human erythro cyte enzymes; in Isozymes. I. Molecular structure, pp. 781-795 (Academic Press, New York 1975). Von Hahn, H.P.: A model of regulatory aging of the cell at the gene level. J. Geront. 21: 291-294 (1966). Wright, W.E. and Hayflick, L.: Contributions of cytoplasmic factors to in vitro cellular senescence. Fed. Proc. Fed. Am. Socsexp. Biol. 34: 76-79 (1975). Wulf, J.H. and Cutler, R.G.: Altered protein hypothesis of mammalian ageing processes. Thermal stability of glucose-6-phosphate dehydrogenase in C57BL/GJ mouse tissue. Expl Gerontol. 10: 101-117 (1975). Wust, C.J. and Rosen, L.: Amino acylation and methylation of tRNA as a function of age in the rat. Expl Gerontol. 7: 331-343 (1972). Yip, L.C.; Dancis, J.; Mathieson, R., and Balis, M.E.: Age induced changes in adenosine monophosphate pyrophosphate phosphoribosyl transferase and inosine mono phosphate: pyrophosphate phosphoribosyltransferase from normal and Lesch-Nyhan erythrocytes. Biochemistry 13: 2558-2561 (1974). Young, V.R.; Steffee, W.R.; Pencharz, P.B.; Wintere, J.C., and Scrimshaw, N.S.: Total human body protein synthesis in relation to protein requirements at various ages. Nature, Lond.253: 192-194 (1975).
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Dr. Jean-Claude Dreyfus, Institut de Pathologie Moléculaire, INSERM U 129 et LA 85 CNRS, 24, rue du Faubourg-St-Jacques, F -75014 Paris (France)
Possible Molecular Mechanisms of Ageing Jean-Claude Dreyfus, Henriette Rubinson, Fanny Schapira, Annette Weber, Joelle Marie and Axel Kahn Institut de Pathologie Moléculaire, INSERM U 129 et LA 85 CNRS, Paris and Centre de Recherches sur les Enzymopathies, INSERM U 24 et ERA 573 du CNRS, Hôpital Beaujon, Clichy
Key Words. Error theory • Posttranslational modification Abstract. While the error theory of ageing has attracted most interest in recent times it cannot yet be regarded as being demonstrated. Posttranslational modifications of proteins explain many observed facts, but do not at present constitute a theory of ageing. The genetic theory appears logical but has little in vivo evidence to prove it. Basic mechanisms of ageing probably involve the interaction of several processes.
Received: December 2, 1975.
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/6/2018 8:12:58 AM
The first contributions to the debate (Baird et al., 1975;Holliday, 1975; Gershon and Gershon, 1976) have centered the discussions on Orgel’s hypo thesis. While most other hypotheses remain theoretical, Orgel’s hypothesis has been chosen especially because experiments can be devised to test it. Discus sions, however, are often obscured by confusing several levels at which ageing phenomena may act. At least three levels could be distinguished: (a) molecular ageing, i.e. the posttranslational events undergone by a molecule between its synthesis and its degradation, and their relationships with molecular turnover; (b) cellular ageing of nondividing cells (for example cells of the nematode, Turbatrix aceti, and muscle or nerve cells in higher animals), and (c) ageing of cell lines, which may reach the limit of their mitotic activity, either in vitro or in vivo. These phenomena are probably all involved to different extents in the ageing of the whole organism. Conclusions pertaining to this ultimate level are still complicated by circulatory (vascular insufficiency), hormonal (Adelman et al., 1972) or immunological factors, which will not be discussed here. In vivo and in vitro techniques have been used to approach these various problems. Techniques using in vitro replication of fibroblasts (Hayflick and
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Moorhead, 1961) are very convenient. Relationships between behavior of cells in vitro and in vivo, however, have been questioned by many authors. In addition, in vitro techniques deal only with one aspect, the mitotic capacity of the cells, which in many cases is not exhausted at the death of the organism. The 'Error Catastrophe’ Theory (Orgel, 1963) This theory is advocated by Holliday (1975), from experiments on fibro blasts in culture, and neurospora mutants possessing a modified leucyl-t-RNA synthetase (Lewis and Tarrant, 1972). In favor of the error theory we could also quote a decrease of the discriminating index for amino acid analogues (Ogrodnik et aL, 1975) and preliminary results on infidelity of a DNA polymerase in aged mouse liver (Barton et aL, 1975). By contrast, many results have been negative or could be interpreted in another way. Holland et aL (1973) have shown that viral replication takes place on senescent as well as in young cells. Danot et aL (1975) found no alterations in enzyme molecules in senescent mouse fibroblasts in culture. We ourselves have found no modifications in the isoelectric focussing pattern of glucose-6-phosphate dehydrogenase (Leibovich and Dreyfus, unpubl.) and no immunological cross-reacting material (CRM) for four enzymes in human liver-derived senescent cells in culture (Kahn et aL, in preparation). In vivo, we have measured the molecular specific activity (ratio of enzyme activity to anti genic reactivity) of five cytoplasmic and two lysosomal enzymes in granulocytes of newborn babies, young adults and people over 80 (Rubinson et al., 1976). No difference could be found. This work seems to indicate that the protein synthe sis machinery of young cells from old organisms is intact. Finally, Schapira et al. (1975) have shown that the CRM for lactic dehydrogenase which is found in the liver of old rats decreases during post-hepatectomy regeneration. These results give evidence against faulty transcription or translation in liver cells of old rats and favor the interpretation of CRM as resulting from postsynthetic changes in the molecule.
Most abnormalities found in proteins of aged cells (CRM, heat lability, electrophoretic modifications) could be ascribed to alterations of the protein molecule occurring after its biosynthesis; this mechanism has been well em phasized by the Gershons contribution, and also by other authors (Reiss and Rothstein, 1975). It seems that this explanation may account for most examples of altered proteins, but its importance varies very much as a function of the various cells and tissues.
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/6/2018 8:12:58 AM
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(a) Young dividing cells, as stressed above, show little or no altered protein. (b) At the extreme opposite are cells which survive with no autonomous protein synthesis. The best example is the red blood cell. In old red cells modifi cations of many enzymes are known, which can be due to postsynthetic altera tions. Another example is the nucleus of the lens. (c) Between these two extreme situations are the cells which divide either very rarely (liver cells) or not at all (nerve or muscle cells). While the best model could be nerve cells, most of the work has been devoted to liver cells. Work with the nematode, Turbatrix aceti, can also enter into this category, since this organism only possesses nondividing cells (Gershon and Gershon, 1970). Ab normal proteins are often found in this type of cell. We agree in general with the Gershons conclusions, but we are at odds with them on the following points: (1) Accumulation of CRM does not seem to us to be constant. Even similar enzymes can behave in a very different way: an example is two enzymes of the purine salvage pathway, hypoxanthine — and adenine — phosphoribosyl trans ferases in the red cells (Yip etal., 1974). Different results have been found with aldolase in liver: presence of CRM in old mice (Gershon and Gershon, 1973), absence in old rats ( Weber and Schapira, to be publ.); presence of muscle-type lactic dehydrogenase in the liver of old rats (Schapira et al, 1975), absence in the muscle (Oliveira and Pfuderer, 1973). It has even been suggested that dietary changes end in the disappearance of heat-labile molecules of glucose-6-phosphate dehydrogenase in the liver of old rats (Wulfand Cutler, 1975). (2) Posttranslationally altered molecules are not necessarily, according to us, either entirely normal or completely inactivated. For red cell enzymes, many electrophoretic modifications have been described (Turner et al, 1975). We have been able to directly demonstrate (Kahn et al., 1974) that old red cells contain fractions of glucose-6-phosphate dehydrogenase with a lowered isoelectric point, and that these fractions have a specific activity lower than that of the enzyme from young cells. Analogous conclusions were reached by Reiss and Rothstein (1975) with isocitrate lyase from Turbatrix aceti. Besides, we do not see why either errors in synthesis or postsynthetic noncovalent modifications should necessarily induce a complete inactivation of the biological activity of a protein. The mechanism of posttranslational modifications is still hypothetical. Only one reaction is well defined (Robinson, 1974), e.g. a deamidation of asparagine, and perhaps glutamine residues. This mechanism has been demonstrated for aldolase, cytochrome c, a-crystalline from lens. This deamidation lowers the pHi of proteins, it is not proved that it decreases their activity. Intracellular processes might enhance postsynthetic alterations. We (Kahn et al, 1974) have demon strated the existence in white blood cells of a thermostable factor which is able to duplicate in vitro the modification of G6PD observed in ageing cells. This factor is probably a peptide (Kahn eta l, 1976). Finally, posttranslational modi
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/6/2018 8:12:58 AM
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fications are known to occur in collagen and elastin, due to the amino acid composition of these proteins (Gallop and Paz, 1975). Posttranslational modifi cations are observed in most cases in old cells. The reasons for their accumula tion are not clear. A possible explanation could be that the rate of protein synthesis and, therefore, of protein turnover, decreases in senescent tissues {Young et ai, 1975). Since posttranslational alterations are related to the age of the molecule they would be more apparent in older organisms. The importance of the rate of protein turnover (as well as the fact that each protein turns over at its proper rate) seem to have been underestimated. Posttranslational modifications do not, at present, constitute a theory of ageing. They might only be a transition step in the normal catabolism of pro teins. Two mechanisms could play a role in damaging a cell: (1) a considerable accumulation of biologically inactive proteins, such as described in Turbatrix aceti, might create a special kind of storage protein disease, and (2) ‘old’ mole cules might acquire a modified specificity which would result in altered metab olisms, and, if key enzymes are involved, in errors in synthesis of proteins which would be of nongenetic origin. Genetic Theory
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/6/2018 8:12:58 AM
Many authors consider that ageing and death are the result of a genetic program. This theory has been clearly developed by Hayflick (1973). Ageing is programmed, either by ‘ageing genes’ which would come to operation late in life, or by shutting off adult genes. Arguments in favor of this theory come from two sets of evidence: in vitro the number of doublings of any strain of diploid fibroblasts is finite and relatively constant* for a given species. In vivo biological clocks do exist, as shown in physiology by puberty or menopause, and in pathology by the appearance only in adults of some genetic diseases like Huntington’s chorea. Of course, the difference of longevity between various species is also interpreted in the same way. It can also, to some extent account for the immortality of malignant cells, resulting from a complex disturbance in the genetic machinery. Direct evidence of the genetic theory, however, is difficult to provide. Furthermore such a theory deals with the limited mitotic capacity of cells and would have to be modified to account for ageing of non dividing cells. A variant of the genetic theory is the theory of the isozymic shifts (Krooth, 1974). New isozymes (or isoproteins) would be derepressed late in life. If their accuracy is less than that of the adult enzymes or proteins, a progressive damage will take place. This theory was forged in analogy to the shifts in some proteins observed during or after fetal life, especially the non-o-chains of hemoglobin. It can claim only little experimental support up to now (Kanungo, 1975).
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Several modifications of the genetic material have been reported in old cells. These alterations, however, can be interpreted by any of the above theories. We quote them since they might be instrumental in the progressive deterioration of senescence. DNA and Nuclear Proteins Base composition of DNA could be randomly altered, giving rise to an increasing rate of errors in transcribed messenger RNA. This ‘somatic mutations theory’ (Curtis, 1964) has not been substantiated and is now disreputed. The physicochemical structure of DNA could be modified. Von Hahn (1966) described a greater heat stability of DNA and a greater difficulty to split histone from DNA in thymus of aged cattle. He hypothesized that sticking of histone to DNA could prevent local strand separation, creating new repressions. Gene expression is now thought to be modulated by nuclear acid non histone proteins. Stein (1975) has shown that among the proteins which bind to DNA in vitro one is greatly increased in senescent human fibroblasts. Enzymes Acting on DNA and RNA Synthesis of DNA in vitro by a low molecular weight DNA polymerase shows more errors when working with liver of mice over 2 years (Barton et al, 1975). Modification of DNA and RNA polymerase, as well as of elongation factor 1, have been found in Turbatrix aceti (,Bolla and Brot, 1975). Methylation of tRNA decreases in the liver of old rats (Wust and Rosen, 1972) because of a decrease of tRNA methyltransferase activity concomitant with an increase in the competing enzyme glycine N-methyltransferase (Mays et al., 1973). DNA repair: Epstein et al. (1974) and Mattem and Cerutti (1975) have reported that the rate of DNA repair is reduced in vitro in human senescent fibroblasts and in progeria. This phenomenon can be due to a genetic change in the repair enzymes, but also to translational or posttranslational modifications of these enzymes. It can therefore be either one of the causes or a consequence of senescence. All the above pieces of evidence point to functional disturbances of enzymes involved in the synthesis and regulation of nucleic acids. None of them is apparently able to account for the whole phenomenon of ageing.
Sheldrake (1974) proposed that cells die because of the accumulation of toxic substances in the course of their ageing. Cells could divide unequally, between cells which would live and be regenerated, and cells which would die
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Theory o f Intoxication
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prematurely. The only known example in animals is the meiotic division of the egg cell. The theory can be applied to other cell divisions, including the can cerous cells. No conclusive evidence, however, can be produced to support this theory. Moreover, Wright and Hayflick (1975) by enucleating human old lung fibroblasts and by hybridizing them with young cells have shown that nuclear and not cytoplasmic factors limit the proliferative capacity of cultured cells. Consequently, this accumulation ought to be in the nucleus, if it was the cause of senescence. Conclusions No hypothesis appears to account for the observed facts at present. Hayflick’s genetic theory appears logical but there is little experimental evidence to prove it. The error theory can claim only few nondisputable facts to support it, since most observations can be better explained by posttranslational changes, but still deserves credit as being liable to experimental testing. Postsynthetic modifications certainly exist, and their mechanisms of production can now be investigated; but they do not seem to be universal, and pending the demonstra tion of their physiological importance, they cannot yet be evaluated. So the basic mechanism of ageing processes remains mysterious; it is probable that it involves exhaustion of mitotic potential, interactions of cells and of tissues and organs in the whole organism. References
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Dr. Jean-Claude Dreyfus, Institut de Pathologie Moléculaire, INSERM U 129 et LA 85 CNRS, 24, rue du Faubourg-St-Jacques, F -75014 Paris (France)
