By Mary K. Miller
Elizabeth H. Blackburn, a pioneerin the study of telomeres—the ends of chromosomes, which play a role inaging and cancer—has always taken the unexpected path. Growing up in afamily of physicians in Tasmania, Australia, she chose to enter medicalresearch rather than medicine. Instead of studying the animals she hadloved as a gift, she became fascinated with the chemical machinery ofcells. At the University of Melbourne, where she lived in a women'sresidential college, she majored in biochemistry. For graduate school,she ventured abroad, in 1972, to the University of Cambridge. Whilethere, Blackburn immersed herself in genetics under the mentorship ofthe Nobel laureate biochemist Frederick Sanger.
After three years at Cambridge, Ph.D. inhand, Blackburn was bound for a postdoctoral appointment at theUniversity of California, San Francisco, to sequence viral DNA. But herfiancé, John W. Sedat, was headed for Yale. She switched projects andopted for Yale. Thus began a lifelong passion for telomeres.
The early-twentieth-century American geneticist Hermann J. Muller coined the term "telomere" from the Greek words telos (end) and meros(part). Muller and the American geneticist Barbara McClintockindependently theorized that telomeres must serve a protective functionfor chromosomes, somehow keeping them separated from one another (the"naked" ends of two long, string-like chromosomes would otherwise fuseend to end). "McClintock did an amazing thing in the 1930s," Blackburnnotes with admiration. No one knew about DNA at the time, butMcClintock "could see and study chromosomes under the light microscope.She correctly surmised that the chromosome ends somehow stabilized thestructure [of the chromosomes] during replication." Forty years afterMcClintock, when Blackburn decided to apply the DNA sequencing skillsshe had picked up at Cambridge, she was the only scientist studyingtelomeres. "I thought, 'Wow, I wonder what they're like?' Nobody knew.There was no hypothesis."
Blackburn's encounter with telomeres, andtheir associated biochemistry, began a life's work that has placed heramong the world's leading cell biologists today. Telomeres have turnedout to be a far more fascinating, and more important, line ofbiomedical research than even Blackburn originally suspected they wouldbe. In her three decades of research and more than 120 peer-reviewedpapers on the once-neglected subject, Blackburn has played a key partin major discoveries in the field of telomeres. Having joined thefaculty at the University of California, San Francisco, after afifteen-year delay in her original plans to go there, she is a mentorherself, to a number of young scientists. Together, Blackburn and herstudents, both former and current, have helped explain how telomeresact in protecting chromosomes from damage, in regulating cell divisionand cell death, and in such processes as aging and its associateddiseases.
For her innovative and groundbreaking work,Blackburn has been recognized by peers, and richly honored. She is amember of the National Academy of Sciences and an elected fellow of theRoyal Society of London, as well as the American Association for theAdvancement of Science. She served on the President's Council onBioethics during President George W. Bush's first administration butwas dismissed in 2003 for her vocal objections to reports on aging andon stem cell research, among others. The reports, she felt, wereneither balanced nor accurate reflections of the scientific fields fromwhich they purported to draw. This April she will receive the BenjaminFranklin Medal in Life Sciences, presented annually by the venerableFranklin Institute in Philadelphia; the award has become one of thenation's most prestigious honors conferred on a scientist. ManyFranklin Medal winners in science are also past or future recipients ofthe Nobel Prize.
For the first decade of her career, however,Blackburn toiled in relative obscurity. At her postdoctoral fellowshipat Yale she joined the laboratory of cell biologist Joseph G. Gall.Gall had seen the value of working with a model organism, Tetrahymena,a pond-dwelling, single-celled ciliated protozoan. Like all eukaryoticorganisms (organisms whose cells have a nucleus), including people, Tetrahymenahas linear chromosomes inside the cell nucleus. What sets ciliatedprotozoans apart, though, is the sheer number of their chromosomes: Tetrahymenahas as many as 40,000 in a single cell. (Each somatic cell of a humanbeing carries just forty-six chromosomes.) The abundance of chromosomeends makes Tetrahymena an ideal organism for the study of telomeres, and so Blackburn set about determining their genetic sequence.
What she discovered was very curious:Telomeric DNA is made up of short, simple, repeating sequences ofnucleic acids. (Much longer, more complex runs of nucleic acids make upthe DNA sequences that constitute genes.) Soon she and otherinvestigators found similar patterns of repeating DNA segments in thetelomere sequences of other species though the number of repeats variedfrom organism to organism. For instance, Tetrahymena has strings of TTGGGG repeated about 50 times, and humans have strings of TTAGGG repeated about 2,000 times. (T, A, and G stand for the nucleic acids thymine, adenine, and guanine, respectively.)
That evidence and some other results ledBlackburn to suspect that the simple sequences were performing a morecomplex function, and that something else in the cell was controllingthe telomeres. Her colleagues remained politely interested butunimpressed, even as she was working out how the sequences weremaintained over time. "After we described this work, I would go tomeetings and be the last speaker, in the last session of the day"Blackburn says.
From McClintock's era on,biologistshad simply accepted the idea that telomeres somehow cap the end of achromosome, just as plastic caps the ends of a shoelace and protects itfrom becoming unraveled. The cell is normally vigilant about detectingand repairing breaks in its chromosomes. In so doing, though, the cellcould mistake an unprotected chromosome end for a break and attempt tofuse it to another chromosome end. The telomeres prevent that fromhappening. Blackburn was determined to find out how, but it would bemany more years before she would have enough clues to sketch theprocess, and much about it remains unknown.
"We now think that there's something thathides the chromosome ends in plain sight," Blackburn says. "The cellsees the ends, but instead of hiding—and we still don't understand howthey do this—the cell turns the recognition of the ends into a responseappropriate to the telomeres. It's a very dynamic process, not like apassive shoelace end, and that was not expected at all."
Another cellular enigma was how telomeresmanage to maintain their length and, hence, their functionality. By theearly 1970s, biochemists realized that the normal process of DNAreplication could not copy a chromosome all the way to its end.Consequently, with every chromosome replication and cell division, thetelomeres should theoretically get shorter. Eventually, withoutanything to arrest the process, the telomeres would get so short thatany further chromosome replication would cut into the genes themselves,and the cell would die. Because bacterial cell lines can live anddivide for thousands of generations, chromosome shortening became aparadox known as the "end-replication problem."
There was much speculation about how thecells might solve the problem, but no empirical explanation. "Inbiology you can wave your hands and make up all these paper schemes"Blackburn says. "But the key thing was to show in the test tube thatthere really was a tangible mechanism." So in the mid-1980s Blackburn,who was running a laboratory at the University of California, Berkeley,and an especially determined graduate student named Carol W. Greiderwent back to Tetrahymena to figure out how cells preserve their telomeres. "We normally think of genetic material as sacred," Blackburn says. "But [Tetrahymena] chop up their somatic [non-germ line] chromosomes and add new repeat DNA [sequences] to the ends."
Blackburn had hypothesized that an undescribed enzyme within Tetrahymenacells was building new telomeric sequences. Another enzyme thatassembles strands of DNA, called DNA polymerase, was already known. ButDNA polymerase builds a new strand of DNA by using a single strand ofthe double helix that forms a chromosome as a copy template. The newstrand is a complementary copy of the original. (Nucleic acids thatmake up DNA always pair with their complement: adenine with thymine,and cytosine with guanine.)
Unlike DNA polymerase, Blackburn andGreider's mystery enzyme, which they called telomerase, would have tobuild telomere sequences from scratch, with no template. To find theenzyme, Greider mixed synthetic telomeres, created in the laboratory,with extracts of Tetrahymena cells. The synthetic telomeres, Greiderand Blackburn had reasoned, would be extended only if the Tetrahymenaextracts contained the hypothesized telomerase enzyme. To theirdelight, the synthetic telomeric DNA grew longer, proving the existenceof telomerase.
Their newly discovered enzyme turned out tobe a remarkable molecular complex. Like most enzymes, telomerasecontains protein. But the telomerase complex also includes a singlemolecule of RNA, a chemical cousin to DNA. "Telomerase is acollaboration between RNA and a protein" Blackburn explains. No oneunderstands exactly how the two work together, but what is known isthat the RNA codes for short segments of DNA that are added piece bypiece to the ends of telomeres. Thus telomerase restores bits oftelomere lost during cell division
The finding came as a surprise, Blackburnrecalls, because "people had thought that only bad things, like the HIVvirus, did this conversion of RNA to DNA. But here is a molecule thatdoes this, not for evil, but for a critical function necessary forcontinued life." She now suspects that telomerase is an ancientmolecule, a relic from a prebiotic world dominated by RNA reactions,rather than by proteins and DNA.
With their molecule in hand,Blackburn and others could start to tease out how telomerase works inthe cell. From work in the 1960s it was known that human cells grownoutside the body, unlike the ceils of single-celled organisms, have alimited life span. After some twenty to fifty divisions (a numberthought to be highly dependent on cell type), human cells stop dividingand enter a static phase known as senescence. Could telomeres befunctioning as a clock that tells cells when they have reached the endof their line?
Greider left Blackburn's laboratory inBerkeley in 1988, for a postdoctoral fellowship at Cold Spring HarborLaboratory, in Long Island, New York. There she discovered that thetelomeres in laboratory-grown human skin cells get shorter with everycell division. The idea took hold that shortened telomeres could be asignal to the cell that its genetic material is getting old and is atrisk of losing its integrity--in short, the shortened telomeres becomethe canaries in the coal mine that tell a cell it is dangerous tocontinue dividing.
Greider's finding led to speculation that thetelomerase gene is turned off in normal cells; that telomerase remainsactive only in other actively dividing cells, such as immune cells andgerm cells. "We know now that there's a smidgen of telomerase in justabout all cells, and that it is protecting telomeres," says Blackburn."But there's not enough telomerase to keep up with the shortening. Withtime, she adds, "the telomeres will gradually run down."
Intriguingly, human telomeres vary in lengthfrom individual to individual. Telomeres in centenarians, for instance,are longer than one would expect. Could longer telomeres be protectinglonglived people? After all, centenarians live longer in part becausethey don't die from the diseases that kill most of their age cohorts.Perhaps robust telomeres and extra telomerase are helping protect themagainst heart disease and other diseases.
An important link between telomerase,disease, and aging was identified in 2001, with the discovery of agenetic mutation responsible for a rare disease called dyskeratosiscongenita. People with the condition are born with only one functioninggene for telomerase, and as a result, their telomeres shorten rapidly.They show some signs of premature aging, such as gray hair in theirteenage years, but the most dire effect is that they usually die inearly adulthood or middle age from bone marrow failure and a resultinginability to fight infections. "It's a striking reminder that we need alot of self-renewal and telomerase in immune cells," says Blackburn.Immune cells have to multiply rapidly when they meet an antigen.Without sufficient telomerase, those cells cannot survive enough celldivisions to overcome the invader.
Once it became clear that telomere shorteningmight have a role in cell aging and, conversely, that long telomeresmight somehow contribute to human longevity, Blackburn's colleaguesbegan to take notice. The once-quiet field exploded, and the cumulativecitations for "telomerase" in medical and biological journalsskyrocketed. As others began working on telomeres and telomerase, newinsights into disease and aging have come to light. With them has comethe potential for developing new treatments against some of humanity'smost intractable killers.
One recent discovery is that shortenedtelomeres do not necessarily spell imminent cell death, or even loss ofvitality; the more important factor is whether enough telomerase isavailable in the cell nucleus to rescue and protect the remainingtelomere ends. Remarkably, available telomerase turns out to be atleast one key to the ability of cancer cells to circumvent the geneticsafeguards of normal cell senescence.
In a malignant tumor, cancer cells divide andmultiply indefinitely, becoming immortal, runaway tissue that consumesall the resources that would otherwise go to healthy tissue. In theearly 1990s Greider and others found that the telomerase concentrationin cancer cells is 100 times higher than it is in normal cells. Theelevated telomerase occurs both in cancer-cell lines grown in thelaboratory and in ovarian tumors growing in the body.
Somehow, then, on the roadto becoming malignant, cancer cells switch on the telomerase genebefore the telomeres become too short for cell division. Surprisingly,the telomeres in cancer cells are often much shorter than the telomeresin the cells of surrounding tissue—evidence that the cancer cells hadalready begun to replicate (and their telomeres had begun to shorten)at breakneck speed, before the telomerase came back on the scene toperform its vital function.If telomerase could somehow be inactivated,malignancies would presumably stop before they could spread to otherparts of the body, establish new malignancies, and do their extensivedamage. (Blackburn suspects, nonetheless, that cancer cells may be ableto subvert telomere shrinkage in other ways as well.) Hence, blockingthe production of telomerase has become an attractive target for cancertherapies, particularly if they can home in on specific tissue andavoid cells, such as immune cells, that depend on telomerase to keepthe body healthy. For the investigators in Blackburn's lab, as well asfor geneticists at other universities and within the biotech industry,telomerase blockers have become an important, emerging line of research.
Cancer is by no means the only cell-damagerassociated with telomere length. In 2004 Blackburn joined forces withElissa S. Epel, a psychiatrist and clinical colleague at the Universityof California, San Francisco, to test the role of psychological stressin aging at the cellular level. "We started with the observation [ofEpel's] that people look really old and drawn when they have chronicworries and stress in their lives," Blackburn explained. "But we had nohypothesis about whether we'd see an effect on telomeres in the cell.Nobody knew, so I said we should just look."
Blackburn, Epel, and Richard M. Cawthon, ageneticist at the University of Utah in Salt Lake City, conducted astudy of thirty-nine women, ages twenty to fifty, who had been caringfor a child suffering from a serious chronic illness, such as autism orcerebral palsy. Those women, presumably highly stressed, were comparedto a control group of nineteen mothers of healthy children. Stress wasquantified in part by the number of years each woman in the test grouphad been caring for an ill child. That number was combined with otherobjective measures of stress, including so-called oxidative stress(damage to DNA caused by "free radicals"), one of the major riskfactors for cardiovascular disease.
The investigators discovered a clearcorrelation between the number of years a woman had been caring for hersick child and shortening of telomeres. The stressed women also hadlower levels of telomerase in their white blood cells and higher levelsof oxidative stress. Moreover, the investigators found that theperceived stress in their test group, as measured by a subjectivebattery often questions called Cohen's Perceived Stress Scale, was alsocorrelated with shorter telomeres and lower telomerase levels in theblood cells. The finding held whether the mother had an ill child ornot. "We didn't expect to see such a clear relationship right acrossthe full range," Blackburn says. "Elissa crafted a beautiful studywhere she had a well-controlled group of individuals, and therelationship between stress and telomere length really held." In otherwords, a woman's perception of the level of her own stress iscorrelated with her body's cellular response. As far as Blackburn andEpel can determine, this result was the first time a mind-body linkthat reaches into the cell was established.
"Of course, now we want to Understand exactlyhow stress is affecting the cell," says Blackburn. "Stress is changinghormones in your blood and bathing the cells in something that'sdifferent. So that's what we're trying to do in the lab: figure outwhat things influence telomerase."
Blackburn credits much of her success tosupportive research environments and the resulting opportunity topursue curiosity-driven science. "Thank goodness I don't work inindustry," she says. "You can do really good research in industry, butyou have to stay on some kind of goal-directed line. [At universities]you're still goal directed, but you can be more creative."
She remains keenly aware ofthe importance of scientific mentors—in her case, Sanger at Cambridgeand Gall at Yale. "Sanger was supportive in a quiet way," she says. "Heloved being in the lab and liked talking about science. It wasimportant to feel that I could always converse with him." Gall wasequally supportive. "Joe Gall was famous for having a good proportionof women postdocs who had done well," Blackburn says. "He wouldannounce to the lab when one of his former students or postdocs gottenure. Joe realized it was important to be sending a positive message."
In her turn, Blackburn is quite serious abouther own role as a mentor, particularly for women in science. "CarolGreider said the fact that I had a child was encouraging to her,"Blackburn recalls. "It's important to show that you don't have to giveyour entire life over to science, that you can be successful by beingsmart and efficient and not always working long hours and weekends."
When Blackburn thinks about the futuredirection of her lab's work on telomerase, she has a two-prongedapproach. "I would like to go deep into the chromosome and reallyunderstand what is happening structurally and functionally around thetelomeres. This is a dynamic, robust system, like a buzzing bazaar withall sorts of molecules coming and going. I would love to understand thedynamics."
But it's also important to her that such deepknowledge be" applied to healing the body. What can knowledge add tothe understanding of how things can go wrong? How can it help treatcancer, chronic stress, and heart disease? "We want to exploitknowledge of telomerase and telomeres to develop therapies at acellular level," she says. Ambitious goals, to be sure. But consideringhow far Elizabeth Blackburn has already pushed the study of telomeres,such goals could well be within her grasp.
By Mary K. Miller