A few months later a letter from a group of Hayflick supporters appeared in the pages of Science, calling attention to the settlement. It is significant for the vehemence with which it takes the NIH to task for its treatment of Hayflick—and for the number of its signatories: eighty-five scientists, including some lesser-knowns but also some leading lights of U.S. biology like Lewis Thomas, the chancellor and immediate past president of the Memorial Sloan-Kettering Cancer Center in New York City, and the esteemed virologist Joseph Melnick.28
The letter writers claimed that the settlement “exonerated” Hayflick and wrote that many of them wished he had not settled but had continued in court to press his case against the government. Nonetheless, the settlement was a “happy outcome of Dr. Hayflick’s courageous, sometimes lonely, emotionally damaging, and professionally destructive ordeal.”
The signatories reflected the strong feeling in part of the biological community that Hayflick had been extremely poorly dealt with by the NIH—and it’s certainly clear that those driving the government’s investigation harbored a nasty, personal, take-no-prisoners approach to Hayflick. (Even after he had retired, the lead NIH investigator, Schriver, would personally intervene to try to derail Hayflick’s hiring at the University of Florida.) But like Hayflick in a letter he penned to Science several years earlier, defending himself and excoriating the government, the indignant biologists who wrote to the illustrious magazine did not acknowledge or address his sales of the WI-38 cells for tens of thousands of dollars.29 Even the sea changes in the law governing patent ownership did not in 1981, and do not today, allow university biologists to walk away from their campuses with unpatented cells developed with U.S. support and begin selling them.
CHAPTER TWENTY-FIVE
Hayflick’s Limit Explained
Stanford, California, 1973–Summer 1974
Berkeley, Boston, and Beyond, 1978–2009
It remains for us to discuss youth and age, and life and death. To come to a definite understanding about these matters would complete our course of study on animals.
—Aristotle, On Longevity and Shortness of Life, 350 BC
In the paper he wrote with Paul Moorhead, Hayflick had laid down a huge challenge to a half century of received wisdom that said cells in culture would keep dividing month after month and year after year, if only they were properly treated.1 He had gone even further in a 1965 paper by suggesting that normal cells’ lab-dish mortality might be related to the aging of whole organisms, including human beings.2
He met with plenty of push-back from the start. His findings were publicly challenged by Theodore Puck, the influential cell culturist whom the young Hayflick had stood up to question at that packed lecture in Atlantic City in 1961. “Sharp divisions were evident” between Puck and Hayflick about whether cells aged in culture, Science magazine reported from a 1965 meeting where Puck claimed that he had grown normal cells in culture through five hundred divisions without their dying.3 Like other noted biologists, Puck suggested that technical failures in Hayflick’s cell-culture methods were responsible for the cells’ deaths.4 Only time would definitively prove that Puck, whatever his eminence, was patently wrong. By 1974 the widely respected Nobel laureate Sir Frank Macfarlane Burnet allowed that “there is probably a majority opinion” that the lab-dish mortality that Hayflick had observed “measures an important biological quality of . . . cells which is significant for the interpretation of ageing.”5
Burnet coined the term “the Hayflick limit” to describe the number of replications that normal, noncancerous cells can undergo before they cease dividing. It took lasting root not only in science but also in popular culture. Today you can buy Hayflick limit T-shirts; the limit plays a role in the 2004 horror film Anacondas: The Hunt for the Blood Orchid; and it has inspired the naming of at least one garage band.
Hayflick’s findings opened a whole new field, the laboratory study of cellular aging. By the early 1970s the subject was enticing a growing number of biologists. In 1974 Congress created the National Institute on Aging as the newest among the cluster of research institutes that comprise the National Institutes of Health. (Congress launched the new institute in response to a relentless lobbying effort led by a wealthy Georgetown widow named Florence Mahoney. President Richard Nixon twice vetoed the bill establishing the new institute. He finally signed it when it was sent to his desk a third time, weeks before he was forced to resign in August 1974.)
Like others, Hayflick was drawn to the study of cellular aging and the raft of questions cracked open by his discovery. Was cellular aging in a lab dish simply the microequivalent of aging in human beings? What caused it? Was some essential factor that was needed for cell replication being depleted over time? Did cells age in lockstep, or were there individual differences between them? Were genes involved? How was it that the frozen fetal cells in his original experiments “remembered” their ages when they were thawed? Was there a different Hayflick limit for every species, and was it the number that one would expect, given the animal’s natural life span? The questions were endless.
Hayflick had tackled one of the most obvious and tantalizing of them in a 1965 paper that appeared in Experimental Cell Research, the same journal that had published his groundbreaking 1961 report. Did cells taken from adult humans—as opposed to fetuses—divide fewer times before reaching the Hayflick limit? From a colleague at the Mayo Clinic in Rochester, Minnesota, Hayflick obtained lung cells from the cadavers of eight people, ranging in age from a twenty-six-year-old who was killed in a car crash to an eighty-seven-year-old who died of heart failure. He found that they replicated far fewer times before growing old and ceasing to divide than had the thirteen lung-cell strains he had derived over time from human fetuses. The fetal lung cells ceased dividing, on average, after forty-eight replications; the eight adult strains petered out after an average of twenty divisions.6
It made intuitive sense, and yet his data showed that our cells don’t simply tick away toward the Hayflick limit on a predictable path as we age. The lung cells from the young auto crash victim doubled just twenty times in culture, while those from the eighty-seven-year-old who died of heart failure replicated themselves twenty-nine times before hitting the Hayflick wall.7 These cells from the most elderly lungs in fact doubled more times than the lung cells from any of the other seven cadavers, whose donors were aged mostly in their fifties and sixties when they died.
“There appears to be no exact correlation between the age of the [adult] donor and the doubling potential of the derived strain,” Hayflick wrote. Or if such a relationship did exist, “it cannot be detected by the present crude methods.” What was clear, he wrote, was the general observation that adult lung cells doubled far fewer times than their fetal counterparts before they ground to a halt in their cultures. Papers by other scientists over the coming fifteen years would establish definitively that a cell’s doubling potential did vary inversely with donor age, and that this was true for cell types as various as skin, liver, and the smooth muscle in the walls of arteries.8
Adding more complexity, a paper published in 1980 in Science showed that even which cells happened to have been randomly sliced from the lungs of those eight cadavers doubtless influenced Hayflick’s findings. That report, published by biologists James R. Smith and Ronald G. Whitney of the W. Alton Jones Cell Science Center in Lake Placid, New York, blew away any notion that cells within a tissue are identical, preprogrammed entities all marching rigidly toward a predetermined finish line.9 Smith and Whitney took a single lung cell from an aborted human fetus and, as it multiplied in culture, extracted one hundred or two hundred cells now and then. Then they tested each cell within that subgroup to see how many divisions it had left in it. They found a wide variation in the remaining doublings.
Digging deeper, they next took the two daughter cells that arose from the division of a single fetal lung cell and counted how many times the population of cells arising
from each daughter was able to double itself. They did this over and over, beginning with many single cells. To their amazement, they found that the proliferative potential of the two daughter cells of each single cell was not identical and, in fact, varied by as many as eight population doublings.10 When it came to theories about what caused the Hayflick limit, “clearly, all current hypotheses should be reexamined in light of our data,” they concluded.
In the midst of the puzzlement, at least one thing had become clear, thanks to the efforts of Hayflick’s red-bearded graduate student, Woody Wright, the young biologist who was also completing an MD at Stanford’s medical school. In an elegant series of experiments under Hayflick’s oversight, Wright used a chemical called cytochalasin B, which he applied to WI-38 cells in culture. At high doses the chemical ejected the nuclei from the WI-38 cells, resulting in WI-38 cells dubbed cytoplasts, full of cytoplasm but without nuclei.11 Separately, Wright treated normal WI-38 cells with a different pair of chemicals that inactivated a broad range of components in their cytoplasm. Then he fused the nucleus-free cytoplasts—which still had functioning cytoplasm—with the WI-38 cells that still had nuclei.
This allowed for a series of mix-and-match experiments in which Wright created cells with young nuclei but old cytoplasm; young nuclei and young cytoplasm; old nuclei and young cytoplasm; and old nuclei and old cytoplasm. With each kind of hybrid he watched to see how many more divisions occurred before it hit the Hayflick limit and stopped dividing. If the control seat of cellular aging was in fact in the cytoplasm, then the hybrid WI-38 cells with “young” cytoplasm should live much longer than those with “old” cytoplasm, regardless of the age of the nucleus. But if the control seat of cellular aging was in the nucleus, then the cells with younger nuclei should live longest, regardless of the age of the cytoplasm. The latter is what Wright found.
His experiment was published, with Hayflick as coauthor, in 1975, as Wright completed his MD and PhD and launched a long career pursuing the mysteries of aging.*12
He had aptly demonstrated that whatever was controlling the number of cell doublings in normal diploid cells like WI-38 was located in the nucleus and not in the cytoplasm. Years later Hayflick would dub this whatever-it-was a “replicometer.”13 As distinct from a clock, which measures time, the replicometer measured replications. When a cell wasn’t replicating—for instance, when it was in the freezer—the replicometer stopped chalking up replications.
That the replication-counting mechanism resided in the nucleus wasn’t surprising. The nucleus was known to govern myriad important functions of cellular life. But locating the mechanism and understanding it were two vastly different projects. How it was teased out is one of the great stories of late-twentieth-century biology, because it shows how profound findings with huge implications for human health can emerge from the unlikeliest of organisms, the depths of basic biology, the powerful engine of scientific curiosity, and the cross-pollination that happens when scientists share ideas. It also connected Hayflick’s iconoclastic observation in the early 1960s with the discoveries that would lead to a Nobel Prize for others nearly fifty years later.
• • •
In 1938 the Nobel Prize–winning American geneticist Hermann Muller described what seemed like protective caps of DNA located at the ends of chromosomes, something like the bits of plastic banding that cap the ends of shoelaces to keep them from fraying. He named them “telomeres,” from the Greek telos + meros, meaning “end part.”14 Three years later another great American geneticist, Barbara McClintock, who would also win a Nobel, described a crucial role for telomeres in maintaining the integrity of chromosomes.15
Both biologists worked with broken chromosomes, and both noted that chromosomes in their natural state had ends that were somehow protected from the abnormalities that occurred at unnatural chromosome breaks induced by radiation or rupture. Their tantalizing observations sat unexplained for most of the next forty years. There simply weren’t the tools available to probe them further.
The revelation of the molecular structure of DNA by James Watson and Francis Crick in 1953 began the stepwise process that would produce those tools. It was soon followed by the discovery of a vital enzyme by Arthur Kornberg—one of the “Bergs” whose reputations drew Hayflick to Stanford. Kornberg described DNA polymerase—the enzyme that duplicates a cell’s DNA in preparation for cell division—and teased out how this essential enzyme works, by attaching itself to a long strand of DNA and moving along its length, transcribing the letters of the DNA alphabet as it goes. Kornberg won a Nobel Prize in 1959 for his discovery.
One autumn evening in 1966, a young Russian biologist had a flash of insight that linked Kornberg’s all-important enzyme to the Hayflick limit. Alexey Olovnikov was a postdoctoral student at the Gamelaya Institute of the Academy of Sciences of the USSR. He was on his way home from a lecture at Moscow University on the radical new findings of the American scientist Leonard Hayflick. “I was simply thunder-struck by the novelty and beauty of the Hayflick Limit,” he later wrote.16
Mulling what he had heard, Olovnikov descended into the subway. As he stood on the platform and heard the roar of an approaching train, he thought in a flash that the tracks made an apt analogy to DNA and the train running along them to DNA polymerase, the enzyme that copies DNA in preparation for cell division by traveling along the length of the DNA in a chromosome, making a new copy as it passes. But just as there was a “dead zone” between the front of the train and the first passenger door—“dead” because the whole purpose of the train was to carry passengers—perhaps there was a dead zone at the very tip of the chromosome that DNA polymerase couldn’t copy, although the enzyme’s entire purpose was to copy DNA. If this was true, a cell’s genome—its instruction book for a life—would be slowly shortened through the multiple cell divisions of a lifetime, with DNA at one end of its chromosomes getting lost bit by bit, copy by copy. A railway that kept losing track was untenable.
Olovnikov spent the next four years thinking about the Hayflick limit and developing his theory as he rode the Moscow subway to and from work, waiting for the approaching train in the same subway station where he had his epiphany. He finally published his theory in Russian in 1971.17 He proposed that DNA polymerase could not copy the DNA at one end of each chromosome, so that chromosomes were inexorably shortened with each cell division. But he added that the DNA at the ends—the telomeres, that is—might be expendable, not spelling out any information that was vital for the life of the cell. If so, the DNA of the telomeres could be sacrificed, chopped down bit by bit, replication by replication, while the important DNA—the DNA that directed the life of the cell—survived intact.18 Telomeres, in other words, played a buffering role. But they were finite buffers. Buffers that could be shortened and shortened until, when the last bit of telomeric DNA was reached, so was the Hayflick limit. Because the cell at this point could no longer replicate without eating into its life-directing DNA, it would stop dividing. Olovnikov also conjectured, presciently, that cancer cells might have a mechanism allowing them to escape this telomere shortening, making them immortal.19
In the Western-dominated world of science, Olovnikov’s theory remained all but invisible, even after an English translation was published in 1973.20 However, the theory that the copying enzyme, DNA polymerase, could not replicate that troublesome end bit of the chromosome was separately described in 1972 by James Watson, the American scientist who had twenty years earlier discovered the structure of DNA with Francis Crick.21 While Watson did not make the connections to the Hayflick limit and cancer that Olovnikov did, his paper nonetheless put on the map the phenomenon that became widely known as the “end replication problem.”
Olovnikov’s ideas were, in any event, theoretical when he published them. They were not corroborated by any experimental evidence. The quest that would produce that evidence—and a Nobel Prize almost four decades later—was led by a trio of American s
cientists: an ambitious Californian graduate student with dyslexia; an exuberant, cerebral daughter of Australian doctors; and a competition-averse Canadian migrant to Boston.
• • •
Elizabeth Blackburn had been fascinated with the natural world from her earliest days growing up as the child of physicians in Tasmania, when she used to hold and even sing to jellyfish that washed up on the beach.22 In the mid-1970s, as a postdoctoral student at Yale University, she began working with Tetrahymena thermophila, a single-celled, pond-dwelling organism that is covered with fine, hairlike projections called cilia. Tetrahymena are a favorite of research scientists because they are cheap, they grow rapidly, and they engage in many of the cellular processes common in more complicated organisms, like humans. Most important for Blackburn, Tetrahymena have a multitude of mini chromosomes. This provided plenty of material for her studies of telomeres, whose function had mystified scientists for decades, and which also captivated Blackburn.
She set out to determine the letter-by-letter sequence of Tetrahymena telomeres. While gene sequencing was a new tool at the time, Blackburn was ideally placed to use it—she had learned the technique from its pioneer, Nobel Prize winner Frederick Sanger, with whom she completed her PhD at Cambridge University. The paper she produced was coauthored by Joseph Gall, her mentor at Yale—although it was published in 1978, the year that Blackburn became an associate professor of molecular biology at the University of California at Berkeley. It showed that the ends of Tetrahymena chromosomes contained a short sequence of six letters of DNA code—CCCCAA—that was repeated over and over, from twenty to seventy times.23 Her discovery was groundbreaking. No one had described the molecular structure of the chromosome tips in any species. Soon scientists were sequencing the telomeres of other simple organisms and finding that these too consisted of short DNA sequences repeated many times over at chromosome ends.
The Vaccine Race Page 42