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Finally, the most apparent drawback is the Whether or not nucleic acid computers 175–179 (2000). 3. Faulhammer, D., Cukras, A., Lipton, R. J. & Landweber, L. time required for each computation. ultimately prove feasible, they have already F. Molecular computation: RNA solutions to chess Whereas a simple desktop computer can contributed to multi-disciplinary science by problems. Proc. Natl Acad. Sci. USA 97, 1385–1389 (2000). solve the seven-city instance of the Travelling causing us to question the nature of comput- 4. Ouyang, Q., Kaplan, P. D., Liu, S. & Libchaber, A. DNA Salesman Problem in less than a second, ing and to forge new links between the biolog- solution of the maximal clique problem. Science 278, 1 446–449 (1997). Adleman took seven days . The use of DNA ical and computational sciences. For example, 5. Henegariu, O., Heerema, N. A., Dlouhy, S. R., Vance, G. H. chips2 or other approaches may eventually it has led us to focus on the nature of biologi- & Vogt, P. H. Multiplex PCR: Critical parameters and step- by-step protocol. Biotechniques 23, 504–511 (1997). lead to automation, which would save con- cal DNA computations, such as the assembly 6. Karp, G. Cell and Molecular Biology: Concepts and siderable amounts of time, but fundamental of modern genes from encrypted building- Experiments 2nd edn (John Wiley & Sons, New York, 1999). DNA computing technology needs to blocks in the genomes of some single-celled 7. Seife, C. RNA works out knight moves. Science 287, advance far beyond its current bounds before ciliates (FIG. 5)14. After all, our bodies already 1182–1183 (2000). 8. Condon, A. & Rozenberg, G. (eds) Prelim. Proc. 6th Int. it can be made practical. contain millions of complicated, efficient, Meet. DNA Based Computers (Leiden Univ., The DNA computing has its advantages, evolved molecular computers called cells. Netherlands, 2000). 9. Meller, A. et al. Rapid nanopore discrimination between though. One is its massive parallelism — that Adam J. Ruben and Laura F. Landweber are in the single polynucleotide molecules. Proc. Natl Acad. Sci. is, brute-force algorithms can search through Department of Ecology and Evolutionary Biology, USA 97, 1079–1084 (2000). 10. Sakamoto, K. et al. Molecular computation by DNA quadrillions of molecules at the same time Princeton University, Princeton, hairpin formation. Science 288, 1223–1226 (2000). and find a correct solution, akin to in vitro New Jersey 08544, USA. 11. Seeman, N. C. DNA engineering and its application to biotechnology. Trends Biotechnol. 17, 437–443 (2000). selection3. Another is miniaturization. And e-mail: [email protected] Links 12. Winfree, E. et al. in DNA Based Computers II. DIMACS once the procedures are under control, the Series in Discrete Mathematics and Theoretical Computer Science Vol. 44 (eds Landweber, L. F. & Baum, raw materials cost less too.“Here’s nature’s FURTHER INFORMATION DNA computing: a E. B.) (American Mathematical Soc., Providence, Rhode toolbox,”commented Adleman7,“a bunch of primer | Laura Landweber’s homepage Island, 1999). 13. Winfree, E., Liu, F., Wenzler, L. A. & Seeman, N. C. little tools that are dirt cheap; you can buy a Design and self-assembly of two-dimensional DNA DNA strand for 100 femtocents.” crystals. Nature 394, 539–544 (1998). 1. Adleman, L. Molecular computation of solutions to 14. Landweber, L. F., Kuo, T.-C. & Curtis, E. A. Evolution and combinatorial problems. Science 266, 1021–1023 (1994). assembly of an extremely scrambled gene. Proc. Natl The near future 2. Liu, Q. et al. DNA computing on surfaces. Nature 403, Acad. Sci. USA 97, 3298–3303 (2000). Now is an exciting time in the field of DNA computing, as there is so much that has not been tried. In June, over 120 molecular biolo- gists, computer scientists, mathematicians TIMELINE and chemists from around the world gath- ered in Leiden8 to discuss the latest in DNA computing technology. Hayflick, his limit, and cellular Clearly a next step is automation. McCaskill and colleagues in Germany have constructed a ‘microflow reactor’ on which Jerry W. Shay and Woodring E. Wright they propose to solve a 20-bit satisfiability problem in an hour and a half 8. One could also construct a microfluidic device consist- Almost 40 years ago, in culture are immortal, and that the lack of ing of gated channels so small that only one discovered that cultured normal cells continuous cell replication was due to igno- molecule can pass through at a time9, vastly have limited capacity to divide, after which rance on how best to cultivate the cells. improving readout8. And a team led by they become senescent — a phenomenon Carrel’s view was based on his and Albert Adleman recently solved a 6-variable, 11- now known as the ‘Hayflick limit’. Hayflick’s Ebeling’s work, done at the Rockefeller clause satisfiability problem using a ‘dry’ com- findings were strongly challenged at the Institute in New York City, in which they puter consisting of thin, gel-filled glass tubes8. time, and continue to be questioned in a few claimed that chick heart fibroblasts grew con- As for DNA chips, their future in DNA circles, but his achievements have enabled computing looks bright as well, because ‘uni- others to make considerable progress versal’ DNA chips could contain every possi- towards understanding and manipulating the ble DNA sequence of a given length (probably molecular mechanisms of ageing. about 8–12 base pairs). Hagiya and colleagues in Tokyo are finding creative uses for single- To set Hayflick’s discoveries in context, we stranded DNA molecules that fold into intra- need to go back to 1881 (TIMELINE, overleaf), strand ‘hairpins’8,10. Winfree, Seeman and col- when the German biologist August leagues — responsible for construction of Weismann1 speculated that “ takes place beautiful assemblies with DNA, such as a because a worn-out tissue cannot forever DNA nano-cube11 — have proposed the renew itself, and because a capacity for assembly of even more ordered structures that increase by means of is not ever- show patterned algorithmic supramolecular lasting but finite”.This concept, which was self-assembly8,11–13. Even a handful of mathe- almost entirely forgotten by the time Hayflick maticians have lent a hand, proposing faster began his work, was later challenged by the and more efficient algorithms tailored to the French Nobel-prize-winning surgeon Alexis Figure 1 | Leonard Hayflick in 1988. needs of DNA computing8. Carrel, who suggested that all cells explanted (Photograph: Peter Argentine.)

72 | OCTOBER 2000 | VOLUME 1 www.nature.com/reviews/molcellbio PERSPECTIVES tinuously for 34 years2. This led to the general normal cells3,4. The experiment with mixed idea that all vertebrate cells could divide indef- “The largest fact to have cells further assured Hayflick and Moorhead initely in . However, Carrel’s origi- that culture artefacts could not explain their nal observations could not be reproduced by come from in observations. They submitted a paper other scientists3,4, and may have been due to the last fifty years is that describing their findings to the Journal of an experimental error4. The cells were fed with Experimental Medicine but Peyton Rous, one a daily extract of chick embryo tissue extract- cells inherently capable of of the journal’s editors, was not easily per- ed under conditions that permitted the addi- multiplying will do so suaded. After the paper had been peer- tion of fresh living cells to the culture at each indefinitely if supplied with reviewed, Rous included the following state- feeding3. It has been suggested that Carrel ment in his covering letter:“The largest fact to knew about this error but never admitted it5,6, the right milieu in vitro.” have come from tissue culture in the last fifty but even if this explanation is untrue, no one years is that cells inherently capable of multi- has ever confirmed Carrel’s work. all, it had been 60 years since Ross Harrison plying will do so indefinitely if supplied with The Carrel experiments were of great had started the field of cell culture, and nor- the right milieu in vitro.”The article was not importance because, if valid, they meant that mal cultured cells were thought to be immor- accepted. Fortunately, the editors of normal cells freed from in vivo control mecha- tal. For Hayflick to propose that a cell-division Experimental Cell Research, where the paper nisms could function normally and, apparent- counting mechanism could be involved in was published3 in 1961, were less swayed by ly, forever. However, reports were beginning to ageing was a completely new idea. But the dogma of the day. This work and subse- emerge of difficulties in long-term cell culture Hayflick was young and ambitious, and a quent studies (TIMELINE) changed the tenor of when Leonard Hayflick (FIG. 1) and Paul series of carefully conducted experiments over research, eventually leading Sir Macfarlane Moorhead entered the field. They brilliantly about three years convinced him that the fail- Burnett, Nobel laureate from Australia, to got to the heart of the matter, demonstrating ure of his normal cells to replicate indefinitely coin the phrase “the Hayflick limit” for the finite replicative capacity of normal human was not due to technical errors. first time in his book Intrinsic Mutagenesis, fibroblasts and interpreting the phenomenon In 1961, working with the talented cytoge- published8 in 1974. as ageing at the cellular level3,4. These initial neticist Paul Moorhead, Hayflick did a series observations sparked Leonard Hayflick’s pas- of experiments that challenged Carrel’s views. Hayflick’s enduring impact sion — which has lasted his entire career — to Hayflick and Moorhead showed that popula- The durability and importance of Hayflick’s overturn the central dogma that all vertebrate tions of cultured normal human fibroblasts work are reflected in its citation history. cells grown in culture are immortal. But even doubled a finite number of times, after which Between 1961 and 1999 this paper was cited today, there are sceptics. One is Harry Rubin7, the cells stopped dividing and entered what about 3,000 times. Of the roughly 70 million who stated:“The concept of a genetically pre- Hayflick termed the phase III phenomenon3. scientific papers published since 1945, only determined number of human fibroblast He called the primary culture phase I; the ten one in every 135,000 has been cited as many replications, and its implied extension to other or so months of luxuriant growth, phase II; times or more than this paper. Eugene cells, is based on an artefact resulting from the and the period when cell replication dimin- Garfield9, editor of Current Contents, stated damage accumulated by the explanted cells ished and ultimately stopped, phase III (FIG. 3). during their replication in the radically foreign These initial experiments showed that the pre- environment of cell culture.” Rubin is not vious interpretation — that all cells are alone in his opinion, and perhaps the truth lies immortal — was incorrect. The principle somewhere in between. Nevertheless, the behind these experiments was simple: Hayflick limit is now generally accepted. Hayflick and Moorhead mixed equal numbers of normal human male fibroblasts that had How Hayflick found his limit divided many times (cells at the fortieth popu- After obtaining his Ph.D. in 1956 from the lation doubling) with female fibroblasts that University of Pennsylvania, Hayflick spent two had divided only a few times (cells at the tenth years with one of the leading personalities in population doubling). Unmixed cell popula- tissue culture at that time, Charles M. tions were kept as controls. When the male Pomerat, at the University of Texas in ‘control’ culture stopped dividing, the mixed Galveston. In 1958, Hayflick was recruited to culture was examined and only female cells run the Wistar Institute’s cell-culture laborato- were found. This showed that the old cells ry and also to initiate research on the possible ‘remembered’ they were old, even when sur- viral aetiology of human cancer. He intended rounded by young cells, and that technical to expose normal human embryonic cells to errors or contaminating were unlikely cancer-cell extracts, in the hope of observing explanations as to why only the male cell com- cancer-like changes in normal cells. When the ponent had died3. normal cells no longer grew (FIG. 2), Hayflick Hayflick was convinced that normal cells thought he might have made a mistake in have a finite capacity to replicate, and appreci- preparing the culture medium or washing ated that their behaviour differed profoundly glassware, or made some other technical over- from that of cultured cancer cells (for exam- Figure 2 | Young and old human diploid cells sight. He was assuming that Carrel was cor- ple, HeLa cells) and transplantable tumours, (strain WI-38). a | Young cells in phase II at rect, and that cells could propagate indefinitely which are immortal. It was this insight that population doubling 20. b | Old cells in phase III at if provided with appropriate conditions. After originated the concept of immortalization of population doubling 55.

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Woodring Wright Timeline | Hayflick and his limit shows that the replicometer is August Weismann Hayflick recognizes that a direct located in the proposes that worn-out Hayflick and Moorhead discover the relationship may exist between the nucleus while tissues occur because Hayflick receives his Ph.D. finite lifetime of cultured normal population-doubling potential of studying for a cell division is finite and refutes in medical microbiology and human cells and interpret this cultured cells and the maximum Ph.D. in Leonard this leads to decline in Weismann’s chemistry from the finding as a manifestation of human lifespan of species from which Hayflick’s organ performance1. model2. University of Pennsylvania. ageing at the cellular level3. they are taken39. laboratory12.

1881 1907 1921 1928 1956 1958 1961 1965 1973 1974 1975 1978

Ross Harrison Leonard Hayflick is After a post-doctoral fellowship at Hayflick describes memory in cultured Macfarlane Burnett coins the Elizabeth describes the ability to born on 20 May 1928 the University of Texas, normal human cells: cells reconstituted phrase “the Hayflick limit” to Blackburn maintain cells in in Philadelphia. Galveston, Hayflick returns to from the frozen state remember at what describe Hayflick’s discovery that discovers the culture. Philadelphia, where he spends population doubling level they were normal cells have finite capacity sequence ten years as an associate frozen and undergo further doublings to replicate as opposed to cancer of the member of the Wistar Institute. only up to a predetermined cells, which usually become Tetrahymena 19 maximum4,10. immortal8. .

in 1983: “By studying accelerated ageing doublings, and second, that cryogenically pre- and under glass, as Hayflick calls it, we can learn a served cells can ‘remember’ how many times In the early 1970s it was realized that the prop- great deal about changes in ageing cells that they have divided when they have frozen10. erties of DNA replication prevent the cells could contribute to functional losses Although this mechanism has been referred to from fully copying the ends of linear DNA, throughout our bodies. Therefore, it is not as a clock or timing mechanism, the replicative called telomeres. Because of the nature of lag- surprising that research on tissue culture in limit of normal cells is actually related to ging-strand synthesis, DNA polymerase can- ageing research is one of the most active age- rounds of DNA replication, and not to the pas- not completely replicate the 3′ end of linear ing research fronts.” sage of time. Hayflick suggested the term duplex DNA. This was referred to as the end- “replicometer”be used to designate the puta- replication problem (FIG. 4) in 1972 by one of A cellular counting mechanism tive molecular event counter11. So what is the the discoverers of the double helix, James The existence of a counting mechanism is molecular basis of the replicometer? In 1975, a Watson13. At around the same time, Alexey implied by two of Hayflick’s observations: doctoral student in Hayflick’s laboratory, Olovnikov, a Russian theoretical biologist, first, that normal cultured human fetal cells Woodring Wright, showed that the replicome- had heard a lecture in which Hayflick’s work only undergo a specific number of population ter was located in the nucleus12. was discussed. Olovnikov entered a Moscow subway station while wondering how normal cells might have a limited capacity to repli- Box 1 | Hayflick’s achievements cate, and, as the train stopped, he had a flash During his distinguished career, Hayflick has made several fundamental observations and is often of insight. Olovnikov saw an analogy between credited with starting the field of cellular gerontology — the study of ageing at the cellular level. the train representing the DNA polymerase Hayflick, who is now a professor of anatomy on the faculty of the University of California, San and the track representing the DNA. If the Francisco, was Editor-in-Chief of Experimental Gerontology for 13 years, president of the train replicated the DNA track underneath Gerontological Society of America, chairman of the Scientific Review Board of the American the car, the first segment of DNA would not Federation for Aging Research, and a founding member and chairman of the executive committee be replicated because it was underneath the of the Council of the National Institute on Aging, NIH. He has received more than 25 major engine at the start14. This was analogous to awards, is a fellow of the American Association for the Advancement of Science, an honorary the end-replication problem described by member of the Tissue Culture Association, and author of over 225 scientific papers, reviews and Watson. Olovnikov realized that this repeated the popular book How and Why We Age (Ballantine Books, New York, 1995). shortening of the DNA molecule at each Hayflick’s achievements extend beyond cellular gerontology: he is also an accomplished round of DNA replication might explain microbiologist and was appointed Professor of Medical Microbiology at the Stanford University Hayflick’s finding that normal cells can repli- School of Medicine, Stanford, California, in 1968. He developed the first normal human diploid cate only a specific number of times. fibroblast cell strains. One of these, called WI-38, is still the most widely used and highly Although published in both Russian and characterized normal human cell strain in the world10. He described the extraordinary sensitivity English15,16, Olovnikov’s ideas languished in of cultured normal human fibroblasts to human viruses and suggested that these cells could be the literature until the golden era of molecu- used for isolation, identification and vaccine production. He was the first to produce a vaccine (oral polio vaccine) from these cells44. WI-38 cells, or similar human-cell strains, are used lar biology emerged in the late 1970s. today for the manufacture of most human virus vaccines throughout the world45, including rubella The presence of telomeres at the tips of and the Salk polio vaccine. Over 750 million virus vaccine doses have been produced on WI-38 or had been noted at least since a 17 similar diploid cell strains. Hayflick established international standards for the production of lecture given by Hermann Muller in 1938 18 human biologicals in passaged cells, which are still used today by the biotechnology industry46. and the work of Barbara McClintock . Hayflick is also known for discovering the cause of primary atypical pneumonia in . This However, the function of these structures in type of pneumonia was thought to be of viral origin, but Hayflick showed that it is caused by cell replication was unclear. There was evi- Mycoplasma pneumoniae, a member of the smallest free-living class of microorganisms47. dence that telomeres prevented the ends of Mycoplasma pneumoniae was first grown by Hayflick on a medium that he developed48. chromosomes from fusing to each other and

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cells propagated in culture have yet to be Roslin Institute scientists claim that shown to be directly relevant to ageing of Jerry Shay and Geron the cloned sheep, The future — pharmaceutical organisms. However, it is an attractive hypoth- scientists show that Dolly, has shorter companies are developing lead telomerase is present telomeres than an compounds for inhibiting telomerase esis that the replicative potential of human in all cancer-derived age-matched in cancer cells. Methods for cell lines and in 90% of control41. Is Dolly a modulating telomere length in normal cells with an intrinsic capacity for replacement Carol Greider discovers primary human sheep in lamb’s cells may have medical applications telomerase26. cancers28. clothing? for treating age-related disease43. may be set to allow for normal growth, devel- opment, repair and maintenance, but not to allow the many divisions needed for cancer. 1981 1985 1990 1994 1998 1999 2000 Many cells (even in tissues noted for division) are not completely senescent — even in cente- narians. But this does not contradict the role

Hayflick and Calvin Harley shows Woodring Wright and Advanced Cell Technology of senescent cells in ageing. Although cells can colleagues that telomeres Geron scientists show scientists report cows grow out of tissues obtained from elderly transform a shorten as cultured that ectopic expression derived by nuclear transfer normal human cell normal cells of telomerase in normal from populations of donors, this does not mean there are no senes- population to an approach the Hayflick fibroblasts and epithelial senescent donor somatic immortal cell line limit22. cells bypasses the cells42, and that the cent cells in that specimen. In fact, only a with a chemical Hayflick limit31, showing telomeres are restored to at carcinogen and that the telomeres are least normal lengths. minority of cells in any tissue are likely to be 40 radiation . the cellular replicometer. senescent. However, the presence of some senescent cells may interfere with the function of otherwise normal somatic tissues35,36. that they allowed ends to attach cancer cells. The solution originated again in Hayflick (BOX 1) proposes that telomere to the nuclear envelope in some species. Fast- studies with Tetrahymena by Carol Greider, a shortening may be the molecular equivalent forward to 1978, when Elizabeth Blackburn, graduate student in Elizabeth Blackburn’s of longevity determination37. Hundreds of working in Joseph Gall’s group, found that the laboratory26. Greider and Blackburn discov- physiological, molecular and behavioural telomeres of the ciliated protozoan, ered the — telomerase — that syn- changes in normal cultured human cells her- Tetrahymena thermophila, consisted of a sim- thesizes and elongates telomeres. Telomerase ald the approach of the Hayflick limit. These ple sequence of hexameric repeats of the was later found in extracts of immortal changes represent increasing molecular disor- nucleotides TTGGGG19. The telomeres in human cell lines27 and in most human der, and all compromise the internal milieu, human cells also consist of thousands of tumours28. Telomerase contains an RNA tem- leading to loss of cell function. Hayflick sug- repeats, but in mammals the sequence is plate on which the new telomeres are made. gests that the number of population dou- TTAGGG20. Once this sequence was known, This RNA component was cloned a few years blings that a normal cell can undergo may be the length of human telomeres could be mea- later29 and subsequently the catalytic portion the in vitro expression of maximum potential sured. The first hints that human telomeres of the enzyme was cloned30. longevity. This is never reached in vivo owing might shorten appeared in 1986, when it was However, the idea that telomere shorten- to the hundreds of molecular disorders that, shown that telomere lengths are not the same ing causes cell has only recently in vitro, mark the approaching loss of replica- in all tissues21. These studies culminated in the been demonstrated31. Introduction of the tive capacity, and, in vivo, increase vulnerabili- demonstration that telomeres shorten as nor- telomerase catalytic protein component into ty to disease and death11. mal human fibroblasts divide in culture22. normal human cells resulted in telomerase These initial observations and others23–25 sup- activity31. Normal human cells stably Future challenges ported the concept that telomere attrition expressing transfected telomerase can main- Hayflick’s initial observations on cellular limits normal cell proliferation in culture. tain the length of their telomeres, and exceed replicative senescence have focused attention If short telomeres limit the rate of cell their maximum lifespan by more than five- 3' 5' growth, there had to be a solution to the fold. So the normal longevity-determination 5' 3' telomere problem in immortal organisms, in mechanism of telomere shortening in Leading strand the germline cells of higher organisms and in human cells can be circumvented — evi- 5' 3' dence for the role of telomere shortening in 3' 5' Lagging strand 60 cell senescence and that of telomerase No DNA for (Okazaki fragments) expression in cell immortality. another 50 Phase III priming event (senescence) This discovery has profound theoretical 40 and practical implications that include the Figure 4 | The end-replication problem. During 30 Phase II immortalization of normal human cells for the DNA replication the leading strand is synthesized as a continuous molecule that can potentially 20 production of commercially important pro- Phase I 32 replicate all the way to the end of a linear 10 teins . As there are sensitive methods for template. The lagging strand is made as a detecting telomerase in a single cell, the telom- 0 discontinuous set of short Okazaki fragments, Accumulated population doublings 10 50 90 130 170 210 250 290 erase assay is a potential diagnostic tool for each requiring a new primer to be laid down on Days in culture the detection of cancer cells in clinical speci- the template, that are then ligated to make a Figure 3 | Hayflick’s three phases of cell mens33. Telomerase inhibitors might be found continuous strand. The lagging strand cannot culture. Phase I is the primary culture; phase II that could, perhaps, be used for treating replicate all the way to the end of a linear chromosome, as there is no DNA beyond the end represents subcultivated cells during the period of cancer34. exponential replication. Phase III represents the for a priming event to fill in the gap between the period when cell replication ceases but last Okazaki fragment and the terminus. This metabolism continues. Cells may remain in this From cells to the ageing organism leaves a 3′ overhang. The leading strand is also state for at least one year before death occurs. These observations on telomere biology in probably processed to leave a 3′ overhang.

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Olovnikov, A. M. Principles of marginotomy in template 33. Shay, J. W. & Gazdar, A. F. Telomerase in the early sis challenges us to determine whether this is synthesis of polynucleotides. Dokl. Akad. Nauk S.S.S.R. detection of cancer. J. Clin. Path. 50, 106–109 (1997). 201, 1496–1499 (1971). 34. Herbert, B.-S. et al. Inhibition of telomerase leads to true in all multicellular organisms and, if so, to 16. Olovnikov, A. M. A theory of marginotomy: The incomplete eroded telomeres, reduced proliferation, and . what extent. For example, it is well established copying of template margin in enzyme synthesis of Proc. Natl Acad. Sci. USA 96, 14276–14281 (1999). polynucleotides and biological significance of the problem. 35. Dimri, G. P. et al. A biomarker that identifies senescent that short-lived organisms such as the inbred J. Theor. Biol. 41, 181–190 (1973). human cells in culture and in ageing skin in vivo. Proc. mouse have much longer telomeres and a 17. Muller, H. 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ENCYCLOPEDIA OF LIFE SCIENCES Ageing | Cell OPINION senesence in vitro FURTHER INFORMATION Jerry Shay’s homepage Cancer: looking outside the genome

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