Duane Michals, The Return of the ProdigalSon, 1982

BEHOLD DOLLY! BORN JULY 5, 1996, THEwoolly icon of cloning emerged, wet andgangly, from the womb more than a yearago. But how old is Dolly really?

Her nuclear mother-the ewe that supplied the mam­mary-cell DNA from which Dolly grew-was six years oldwhen the mammary cell was removed and placed in frozenstorage in Ian Wilmut's laboratory. Therefore, is Dolly,as she appears, a one-year-old sheep-or isshe, perhaps, a six­year-old clone? DidDolly at birth alreadycarry the worn, sec­ondhand DNA of anolder sheep, like a childborn with progeria, amedical condition thatdooms infants to pre­mature aging? Noteven Dolly's clonersknow for sure.

What biologists doknow is that the an­swer could help pin­point which of thetwo general theoriesofaging is correct: thetheory of programmed aging or that of random damage.Programmed aging holds that the breakdown of cells andtheir functioning follows a preset genetic recipe, much asa computer program moves inescapably from one prede­termined step to the next. In programmed aging the pro­gression from conception to birth to puberty to bifocalsand beyond reflects a series of appropriately timed, andinescapable, life events. The theory ofrandom damage, onthe other hand, proposes that aging results from the buildupof haphazard chemical damage to an organism's biomole­cules. Ifinvestigators could sort out which is the more accu­rate theory, that knowledge would help them better learnhow to stave off the disease and decay of old age.

But Dolly may have something much stranger to teach.What if appearances are not deceiving? What if somehowDolly turns out, against many scientists' expectations, to bea genuine yearling, no different from any other sheep recent-

ly sprung from its mother's womb? Then would one haveto conclude that the cloning process has somehow renderedDolly's secondhand DNA as good as new-that the cyto­plasmic fluid of the egg, which baptized the nuclear moth­er's DNA, has somehow restored aging genetic material to astate ofinfant grace? Or does a lessfanciful explanation exist?Can the most recent discoveries regarding aging explain whyDolly survived in the first place, and why she stands a mod-

erately good chance ofdeveloping into a nor­mal adult?

THE IM­

portanceof the

studies by Wilmut andhis colleagues at theRoslin Institute not­withstanding, theyrepresent only the nextstep in a long line ofexperiments aimed atlearning how animalsdevelop. AsJ.B. Gur­don, Marie A. Di Ber­ardino and Robert G.McKinnell recount thematter in their articles

in this issue [see pages 26 and 32], in 1952 Robert Briggsand Thomas J. King first showed that blastula-cell nucleitransferred into frogs' eggs can direct the development ofcomplete tadpoles. Many other investigators later confirmedanother of Briggs and King's basic findings: the older theanimal from which the donor cell came, the greater the fail­ure rate and the earlier the developmental stage at whichthe new animal died. Those studies led to the hypothesisthat as an organism develops, its genome undergoes irre­versible changes-thereby making the cloning ofadult ani­mals or their organs impossible.

Asearlyasthe 1960s,however, that belief began to change,and by the 1980s it was in full retreat. In 1986 the embry­ologist Steen M. Willadsen, then at the Agricultural andFood Research Council Institute of Animal Physiology inCambridge, England, created sheep clones by transplantingnuclei from early-stage embryos, made up of eight or six-

September/October 1997 • THE SCIENCES 47

teen cells, into unfertilized sheep eggs. In 1996 the embry­ologist Keith H.S. Campbell and others from the RoslinInstitute showed that nuclei from embryonic cells grown inculture could also create viable cloned lambs, making large­scaleproduction ofgenetically engineered animals and organspossible for the first time. Finally, with Wilmut's resound­ing news announcement in February, it has become man­ifest to nearly everyone that the changes that take place inthe cell during differentiation are reversible.

But do the changes that seem to have rendered the DNAof a mature specialized cell totipotent-capable of givingrise to a complete, fertile organism-also turn back thesupposedly unstoppable clock of aging? To answer thatquestion one must look at the theories of how and whyorganisms get old, and the role of changed DNA in theagmg process.

ACENTRA L TENET OF THE THEORY OF PRO­

grammed aging is that the number of timesdifferentiated cells can reproduce themselves

is subject to what is known as the Hayflick limit.In 1961 the cell biologist Leonard Hayflick of the Schoolof Medicine at the University ofCalifornia, San Francisco,showed that whennormal human bodycells grow in culture,they divide only a cer­tain number of timesand then stop forever,dying soon afterward.Further work by Hay­flick and his colleaguesthroughout the 1960sand 1970s showed thatthe number of times acell divides in culturedepends on the age ofthe cell donor: the old­er the donor, the few­er the divisions. More­over, their additionalexperiments in whichthe nucleus of onebody cell was placed in the cytoplasm ofanother body cellshowed that the stifling effect ofage on cell replication is aproperty of the nucleus and its DNA, not of the cytoplasm.

The Hayflick limit has its limitations, however. For onething, germ cells-the cells that develop into sperm andeggs-have never been considered subject to programmedaging; otherwise each newborn organism would start outas DNA-exhausted as its parents. And long-standing obser­vations of how cells act in living organisms, not just inthe petri dish, have spurred biologists to question the pro­grammed aging theory. For example, not all kinds ofcellsdivide at the same rate; liver cells replicate much fasterthan, say, nerve cells. In addition, some cell types thatoccur in tissues that are continuously growing or beingreplaced-for example, hair follicles or the lining of theintestine-divide many more times than the Hayflick lim­it would allow.

48 THE SCIENCES • September/October 1997

ENTER THE TELOMERES. As EARLY AS THE 1930sinvestigators took note of pieces of noncod­ing DNA-DNA that does not give rise to pro-

teins-at the ends ofeach chromosome, which they calledtelomeres (from the Greek words for "end" and "part").When the differentiated cells of higher organisms under­go mitosis, the ordinary process ofcell division, not all theDNA in their nuclei is replicated. The enzyme that copiesDNA misses a small piece at the ends ofeach chromosome,and so the chromosomes get slightly shorter each time acell divides. As long as enough of each telomere remainsto buffer its chromosome against the shortening process,mitosis does not bite into any genes (remember that thetelomeres are noncoding, much like the leaders at the endsof a reel of film). Eventually, however, the telomeres getso short that they can no longer protect vital parts of thechromosome. At that point the cell usually stops dividingand dies.

That shortening process has been observed in many kindsofbody, or somatic, cells. In germ cells, however, the pro­cessis generally neutralized by a countervailing effect, whichenables each new fertilized egg to start life with a comple­ment of full-length telomeres. What accounts for that

effect? In 1989 work­ers isolated an enzymefrom human cancercells that can lengthenthe telomeres. Theenzyme, known as te­lomerase, confers oncancer cells a devastat­ing immortality: theycan keep dividing vir­tually forever. Investi­gators discovered aswell that telomerase isalso expressed in thegerm cells of theovaries and testes,though not in mostordinary somatic cells.

That discovery oftelomerase in germ

cells explained why each new embryo has a set ofcompletetelomeres. It also put the spotlight on telomerase as a poten­tial fountain of youth, because of the perceived power ofthe enzyme to make old cells young again.

In the past three years, however, several reports haveshown that many kinds of somatic cells also make telom­erase, and that the activity is highly regulated during celldivision. For example, the cells ofhair follicles, white bloodcells and cells from the lining of the gut all express telom­erase. But terminally differentiated fibroblasts, such as thoseHayflick studied in his cell-mortality experiments, do not.Hence the discovery of telomerase supplies a possiblemechanism for programmed aging, but it also suggests thatsome kinds of somatic cells may resist that kind of pro­grammed aging. (Other kinds ofprogrammed aging couldstill come into play.) Even more recently, investigators havenoted that the expression of telomerase itself is controlled

by proteins attached to the telomeres. Thus the absenceof telomerase in a cell does not necessarily imply that thecell will not produce it later.

THE ALTERNATE THEORY OF AGING, THE RAN­

dom damage theory, was championed by thephysician and chemist Denham Harman of

the University of Nebraska in Lincoln in 1981. The theoryturns on questions of DNA damage and repair. Biologistshave long known that changes take place constantly in DNAas it interacts with chemicals both inside and outside thebody. Those include chemicals in the environment, such ascertain components of cigarette smoke, as well as reactivechemicals inside the cell, such as hydroxyl radicals-wasteproducts that cells spew out during normal metabolism. Theresultant changes to the DNA molecule, called DNA adducts,distort its shape and block an accurate reading of the genet­ic information. DNA-repair enzymes can detect the distor­tions and fix them, but sometimes distortions surviveuntil the DNA replicates. At that point theycan cause mutations-errors in the sequence of base pairsthat make up the DNA code-which in turn cause unde­tectable and therefore irreversible changes in the genetic infor­mation. Those changesare passed on to subse­quent generations.

As an organism ages,the irreversible DNAdamage builds up in itscells. That leads toaltered protein struc­tures, which in turnhobble the normalfunctioning of thecells. Those impair­ments can lead to thediseases characteristicofaging: atherosclero­sis, heart disease andcancer, for instance.The crippled func­tioning ofthe cells alsosteps up the rate ofDNA damage and reduces the efficiency of DNA repair.

The quality ofDNA repair naturally has a profound effecton the long-term survival of the organism. In 1974 one ofus (Hart), along with the biologist Richard B. Setlow ofBrookhaven National Laboratory in Upton, New York, dis­covered a strong correlation in mammals between the great­est achievable life span of a species and the ability of cellsfrom that species to repair DNA damage caused by expo­sure to ultraviolet light. In 1994 the biologists Kiyoji Tana­ka of Osaka University in Japan and Richard D. Wood ofthe Imperial Cancer Research Fund in London reportedthat defects in one or more ofthe various DNA-repair mech­anisms are associated with several hereditary human disor­ders, such asxeroderma pigmentosum, that have been linkedto an increased cancer risk or a faster rate ofaging-or both.DNA repair is so important that the genome even includesits own policeman, the p53 tumor-suppressor gene, which

temporarily stops the cell-division cycle after DNA damagehas taken place, to allow time for the damage to be fixedbefore the DNA gets copied.

DNA repair takes many forms: there is excision repair,in which misshapen, damaged DNA is removed andreplaced by DNA that codes for the correct gene sequence;strand-break repair, in which broken strands of DNA arejoined together again; bypass repair, in which damagedregions ofDNA are skipped during replication and the gapsin the gene sequence filled in later; and others. None ofthe mechanisms, however, is error-free, and so some DNAdamage builds up in all organisms as they age. Only thegerm cells appear relatively shielded from such damage untillate in life; otherwise it would be hard if not impossibleto maintain the level ofgenetic specification found in high­er animals [see "What Good Is Sex?" by Richard E.Michod, page 42].

How do germ cells escape unscathed? Biologists are notsure. Certainly, despite a masterful capacity for DNA repair,germ cells respond to external injury much the way somat­ic cells do; for instance, ionizing radiation is well knownto induce sterility and birth defects. But mammals seem tohave evolved strategies to protect their germ cells from inter-

nal, metabolic threats.In females, for in­stance, the egg cellsliedormant in the ovaryfrom the time theyform during fetaldevelopment untilshortly before ovula­tion. Since dormanteggs do not activelyreplicate, there is lesschance that any DNAdamage will becomepermanent. On theother hand, the malegerm cells replicatefrequently to providea continuous supply ofsperm-but manymammals keep their

testes inside the scrotum, at a lower temperature than therest of their bodies. Reduced temperature is thought todecrease the reactivity of free radicals.

ANOTHER SELECTIVE PROTECTION MECHANISM

is meiosis, the process whereby cells dividewithout replicating their chromosomes and

produce mature eggs and sperm. The chromosomes ofsomatic cells are present in pairs, so that ifa random muta­tion knocks out a gene on one chromosome, that gene hasa spare on the other chromosome that is likely to remainundamaged. In many cases the cell will get by withjust thatsingle copy. But during meiosis the pairs ofchromosomessplit so that only one of each pair goes into each of thegerm cells that is formed. The germ cell with the defectivegene will then be on its own, and will either die or becomea less hardy competitor for fertilization. That provides a

September/October 1997 • THE SCIENCES 49

way to erase even seemingly irreversible damage from thenext generation.

A similar series ofevents occurs in crippled somatic cells,through a process of programmed cell death called apop­tosis [see "Death Wish," by Martin C. Raff, July/August1996]. In a healthy organism the relative rates of cell divi­sion and apoptosis remain in balance; that equilibrium main­tains the size ofeach organ as well as the quality ofits cells.But if a tissue receives hormonal or other commands todecrease its size, apoptosis steps up. Mutated, damaged orinefficient cellsare usually the first to go. Sometimes, how­ever, mutations injure the genes that control the cell's sys­tem ofproliferation or differentiation, and then the man­gled cell may multiply out ofcontrol, resulting in a tumor.Clearly, cells with greatly damaged or mutated DNA wouldnot make successful templates for cloning.

WHAT ABOUT DOLLY, THEN, WHOSE DNAcame from a somatic cell ofa six-year-oldsheep? A six-year-old sheep is the equiv-

alent ofa human being in her early thirties. Although someirreversible DNA damage is likely in even a healthy sheepofthat age, the animal can still generally give birth to healthyoffspring, and most ofits somatic cells stillwork normally.

One must also con­sider what happened toDolly's nuclear DNA asit sat in the petri dishawaiting transplanta­tion. For cloning towork, the DNA fromthe donor cellmust firstbe dedifferentiated.During cellular differ­entiation, genes notrequired by the cell inquestion are perma­nently switched off,while others that thecell will need are pro­grammed to respondto specific regulatory stimuli. Those processes do not involvechanges to the DNA sequences themselves; rather, the basesthat make up the DNA are chemically modified by the addi­tion ofmethyl groups, and the protein environment withinthe chromosomes is altered. During the dedifferentiation ofDolly's nuclear DNA, those chemical and structure modifi­cations would have been reversed.

The turnaround probably took place during the cell-cul­ture stage, when Wilmut and his team were growing cellsto find suitable candidates for nuclear transfer. During thatstage the donor udder cells were grown in dishes withabnormally low concentrations of the serum componentofthe cell-culture medium. Udder cells need the hormonesprolactin and estrogen to grow and specialize, and the serumcomponent of the medium supplies them. In the absenceof those hormones, many cells would be expected to diethrough apoptosis, while others would lose their differen-

50 THE SCIENCES· September/October 1997

tiated functions and enter the"quiescent" stage of the cellcycle, in which the cell stops dividing. Under those con­ditions, Wilmut and his colleagues were able to glean acohort of relatively undamaged undifferentiated cells.

Starving the cells may have had other, hidden, effects.Intriguingly, investigators showed recently that when cellsfrom a rat's prostate gland go through a similar process afterthe removal ofthe hormone testosterone, they make telom­erase. Perhaps the udder cells in Wilmut's experiment alsomade that enzyme, thus enabling their shortened telomeresto be replaced. Because the donor egg cytoplasm wouldalso have been making telomerase, Dolly's telomeres wouldhave had ample opportunity to grow back during thecloning process.

The very fact that Dolly has survived fetal development,birth and her first year oflife implies that her donor DNAcarried no lethally damaged genes. Whether she will con­tinue to develop and age normally, of course, remains to beseen. If aging is programmed and if it is controlled primar­ily through the activity of telomerase, Dolly will age in arelatively normal way. If programmed aging is controlledby other factors that reside exclusively in the nuclear DNA,or indeed, if aging is largely the effect of random change

that is normally erasedfrom germ cellsduringfertilization, then Dol­ly is already consider­ably older than herchronological age, andher lifeexpectancy willbe abnormally low. Ineither case, Dolly isat risk of develop­ing inherited defectscaused by age-depen­dent DNA damagebuilt up in the genomeof her nuclear donorcell; so although itappears that she hasmade it through herfirst year relativelyunscathed, the possi­

bility exists that genetic damage has caused at least somenonlethal mutations that may show up as diseases or pre­mature aging later in her life.

THE EFFECTS OF THE RECENT CLONING BREAK­

throughs on human aging research could beimmense-particularly when one combines

the promise of cloning with recent successes in organ andtissue transplantation [see "Cell Block," by Burkhard Bil­ger, page 17]. For more than two decades investigators havebeen trying to grow living replacements for body parts intissue culture: skin, heart valves, parts of the urinary sys­tem. But successful culturing ofliving stand-ins for worn­out parts remains the province ofthe science-fiction writer.

There is one organ, however, whose properties couldserve as a model for the replacement of other organ sys­tems: the liver. Medical investigators have replaced genet-

ically defective livers in experimental animals with culturedliver cells, and they are now testing the technique in peo­ple. Unlike the more structurally complex organs of thebody, the liver is made up ofa relatively few kinds ofcells.In the past decade workers have repeatedly shown that ifhepatocytes-the kind ofcells that make up most liver tis­sue-are grown in tissue culture and then placed in theblood vessels that lead to a diseased liver, they will mergeinto the organ and replace its defective hepatocytes.

Two main clinical techniques take advantage of that in­triguing property. In one technique, called allogenic hepa­tocyte transplantation, cells from a healthy donor liver areincorporated, sometimes repeatedly, into the patient's dis­eased liver. The disadvantage of the approach is that thepatient's immune system usually rejects and destroys thereplacement liver cells, so that without the use ofpowerfulimmunosuppressive drugs the benefits are fleeting. The sec­ond technique, called autologous hepatocyte transplanta­tion, makes use of the patient's own liver cells. A segmentofthe patient's liver is surgically removed, and liver cells arecultured from it. The cells are genetically modified to repairthe defective gene, then infused back into the patient's liv­er. The disadvantage of the technique is that in many cas­es the patient may betoo sick for the safeharvesting ofthe orig­inalliver cells.

The lesson of Wil­mut's experiment withDolly-that differenti­ated cells of one kindcan be reprogrammedto produce every kindof cell necessary for acomplete organism­opens up profoundmedical possibilities.White blood cells, forinstance, could be har­vested and repro­grammed into what­ever cell type wasrequired. Or cellscould be taken from a young and healthy person and storedfor the future production ofreplacement organs, in the eventthat that person needed them because of illness or old age.So do we now revisit the science-fiction tale of using ourown tissues to grow replacement organs for our worn-outparts? If society-ours or someone else's-deems the ideaacceptable, should there be ethical or moral restrictions onthe conditions under which such activities may take place?

Whatever one thinks ofsuch schemes in principle, in prac­tice, old or sick people who need tissue and organ replace­ment-and who have not taken the precaution of storingtheir youthful tissues-may not be able to get much bene­fit from cloned organs. Such people generally carry muchmore damaged DNA in their cells than Dolly's donors did.The reason is that many diseasescause inflammation, whichcreates DNA damage in the affected cellsand stunts their nat­ural rate of apoptosis as they try to heal. That may make it

hard to find healthy donor cells for tissue replacement.One way one might find useful donor cells in old or ill

people is to cut down on their food intake before harvest­ing the cells, Dietary restriction has been shown repeated­ly in many species to make the animals live longer and tolower the incidence of degenerative disease. Work donein our laboratories and by others has shown that cuttingcaloric intake can increase DNA repair activity and spurapoptosis, while reducing inflammation. Moreover, cut­ting calories can also inhibit cell division and, in some tis­sues, increase the number of cells in the quiescent stage.Such conditions seem to mimic the low-serum culture envi­ronment imposed on the donor cells that produced Dolly.

AN O T HER HURDLE FOR NEW METHODS OF

organ and tissue replacement is the need toconvert undifferentiated cells to fully differ-

entiated cells within the laboratory, before they are trans­planted back into the patient. Certainly, recent advancesin molecular biology and protein chemistry have identi­fied a plethora ofhormonal and nutritional factors that con­trol growth and differentiation. But the conversion ofunspecialized cultured cells or embryos into individual spe-

cialized tissues has yetto be achieved. Andeven if embryo cellsare someday coaxedinto differentiating,the task of creating,in an artificial envi­ronment, the three­dimensional, hetero­geneous and multi­cellular structure ofanorgan such as a heartor kidney would bedaunting.

Nevertheless, suchan approach, howev­er difficult and futur­istic, is far preferable tothe idea of growingsuch organs within

viable embryos-for reasons not only ofethics but ofcom­patibility and size. A fetal heart used to replace an adult onewould be much too small to work successfully, which makesthe ethical question moot.

Undoubtedly, the contributions of Wilmut and hiscoworkers will spark further research into the question ofaging. And if, eventually, it becomes possible to mimic insomatic cells the same pristine genomic state that seems toprevail in germ-line cells, then scientists will have donenothing less than realize one ofthe most tantalizing dreamsof humankind since the days of Methuselah.•

RONAW HART isa biogerontologist and distinguished scientist in res­idence, ANGELO T'uRTURRO isabiologist andbiophysicist andJuUANLEAKEYisabiochemist attheNational Centerfor Toxicological ResearchinJiffersoH, Atkansas. Theirpapers on diet andaging have appeared ina recent volume if the Annals if the New York Academy of Sciencestitled THE AGING CLOCK, and in many other scholarly publications.

September/October 1997 • THE SCIENCES 51