But equally as important, viruses were proving a useful tool for studying how cancer transforms healthy cells into the out-of-control masses that eventually kill the people in which they grow. With their ability to cause cancer in lab animals such as mice, viruses provided a vehicle for observing cancer in a controlled research environment. At a time when people knew nothing about how or why cancer occurred, any possible inroad was worthy of attention. “The level of ignorance was such that anything was valued as a potential clue,” said Stephen Goff, a Columbia University virologist whose research, begun in the 1970s, was instrumental in moving the field forward from these early days.
Throughout the history of cancer research, knowledge has advanced in step with technology. In the 1950s, a virologist named Howard Temin set out to find a new and improved way to study how viruses—RSV, specifically—cause cancer. After all, Temin reasoned, here was a virus that was known to transmit cancer. If he had a way to watch that happen, to witness the event outside of chickens and mice, in the cold light of a petri dish, then he might be able to unravel the minute mechanisms at play. In 1958, Temin and his colleague, Harry Rubin, succeeded.
The technique, called a focus assay, used a background of normal cells to bring cancer cells into stark relief. After exposing a culture of cells to a cancer-causing virus, the cells are smeared onto a dish and left to replicate. Cells that have turned cancerous will replicate faster, piling up in clusters that look strikingly different from normal cells. Now, they could quantitatively measure the transformation—how fast it happened, how severely, how much virus was required to launch the change. With the focus assay, Temin made the startling finding that a single particle, or virion, of RSV was enough to turn a cell cancerous.
Having a way to watch the transformation of virus-infected cells in turn transformed the entire field of cancer research. “Every day that a new technique came on line meant whole new things were possible to do,” Goff said. The creation of that assay was like introducing push-button dialing into a rotary world. It sped up cancer research enormously and brought Temin worldwide recognition. It wasn’t long before the assay led to the first shocking observation: RSV was a virus made up of RNA, not DNA.
CONSIDERING ALL THE havoc they wreak, it’s surprising how tiny and simple viruses can be. After all, they aren’t even considered to be living things on their own because they can’t reproduce outside of a host. Viruses come in two basic varieties: DNA and RNA. Both types consist of genetic material bundled inside a coating made of protein, and sometimes also some fat molecules. They typically contain very few genes, often just four or five. In the early days of virology, DNA viruses were thought to be the ones most relevant to cancer. But thanks in large part to Temin’s focus assay, it gradually became clear that RNA viruses could also transform cells from healthy to cancerous.
The presence of RNA, rather than DNA, in the cancer-causing RSV was a puzzle. Every scientist of the day knew that the DNA synthesis at the heart of cell division—the process by which cells multiply, and which occurs continuously throughout our lives—followed a well-worn path: DNA is translated into RNA, and RNA is translated into proteins. In light of the variety of cells in a single body, let alone the diversity of life, DNA replication is strangely simple. The entire genetic code is spelled out in patterns of just four nucleotides, symbolized by the letters A, T, G, and C (for adenine, thymine, guanine, and cytosine), each paired with another (A with T and G with C) in complementary strands held together in a spiraling ladder. The major visible difference between RNA and DNA is that the former contains uracil instead of thymine. As the DNA helix unwinds during cell reproduction, a complementary strand of RNA translates its pattern of base pairs into proteins. DNA is the instruction, RNA delivers the message, and proteins carry out the instruction.
There were DNA viruses known to cause cancer in animals, and how that happened was mystery enough. But RNA viruses were another story. Because it’s the go-between and not the final form, the RNA inside such viruses should not be able to cause permanent damage to the human genome. Poliovirus, comprised of RNA, was a terrible disease but it didn’t become integrated into an individual’s DNA; when the virus was gone, it was gone. The same was true for influenza, another RNA virus. When we catch the flu, it doesn’t become part of our genetic makeup and leave us passed out for the rest of our lives because our DNA now contains flu virus genes. Eventually, the virus is eradicated from the body, leaving our genome intact.
Scientists of the day knew that cancer was somehow connected to change at the genetic level, but the assertion was based more on logic than evidence. The thinking went that if a disease proliferated across generations of cells—that is, the disease is reproduced in each new cell, indicating that the cells were irrevocably changed, as they are in cancer—that some permanent genetic alteration had to be at play. But the idea of an RNA-containing virus causing such a change seemed preposterous.
With nothing to back up his assertion, Temin pronounced that RNA viruses made DNA, and that this DNA would be present in the tumor genome. This thinking went so far against the grain—everyone knew that DNA made RNA, not the other way around—that it prompted outright ridicule of Temin. “[He] was absolutely considered nuts for many years,” said Goff, who, though a boy at the time, would soon enough be immersing himself in RNA virology.
Among the few who did not consider Temin to be insane was David Baltimore, a rising-star virologist at MIT, who joined Temin in his effort to find the secret ingredient that enabled RNA to convert to DNA. Temin had hypothesized that the virus contained an enzyme—a protein that often operates as an assistant to various cellular processes, helping to speed them up and run smoothly—that enabled the reverse process of RNA’s conversion into DNA. And, in 1970, that was exactly what they both found. They named the enzyme “reverse transcriptase,” and RNA viruses were later renamed “retroviruses” because of their retrograde approach to reproduction. RNA viruses that cause cancer came to be called “oncogenic retroviruses.” Baltimore and Temin shared the Nobel Prize in Physiology or Medicine in 1975 (Temin was also recognized for his focus assay with Renato Dulbecco, a pioneering virologist at the Salk Institute who had been Temin’s mentor).
Virologists around the world continued to study RSV along with other so-called transforming viruses with the aim of understanding how a viral infection could cause cancer. A scientist named Hidesaburo Hanafusa, then at the University of California—Berkeley, made a crucial finding when he figured out, through a series of painstaking experiments, that some transforming viruses were actually mixtures of two viruses (though Rous’s virus was not this type). When detangled, one of the viruses would induce cancer transformation but couldn’t replicate, and the other could replicate but wouldn’t induce cancer transformation. Hanafusa knew that the only difference between the two viral strands was a single gene. If one strand lost the ability to cause cancer when it was separated from the other strand, then clearly there had to be some single gene responsible for triggering cancer.
The next breakthrough came from the study of mutant versions of RSV that were sensitive to changes in temperature. In each version, a different gene in the viral genome was rendered defunct by a change in temperature: The gene was induced to operate normally at a lower temperature but not at a higher temperature. By switching off individual segments of the RSV RNA, scientists could discern their various functions (although RSVs hold their genetic information in the form of RNA and not DNA, the information still encodes distinct genes). When the temperature was raised, what was different about the virus? Those differences helped pinpoint the nature of the temperature-sensitive gene. It was like trying to determine the window through which an intruder had entered. Each temperature-sensitive mutant provided clues about which one may have been broken into.
In 1970, G. Steven Martin, working in the lab of Harry Rubin—Temin’s old focus assay colleague—isolated a mutant strain of RSV that induced cancer transformation at 35° Celsius b
ut not at 41° Celsius. The virus did continue to replicate at the higher temperature. Martin now knew that the portion of RNA in the virus that had been made temperature sensitive in that strain was necessary for tumor formation and tumor formation alone. That was where the break-in had occurred.
The gene became known as src, pronounced “sark,” for the sarcoma it caused in chickens like the hen that Peyton Rous had examined sixty years earlier. “It was the most exciting idea at the time in cancer research,” said Ray Erikson, a Wisconsin farm boy who was about to enter the RSV fray. “Here you had apparently a single gene in a virus that, when it was turned on, could transform a cell population and cause tumors in chickens.” Very soon, studies of src were going to shake cancer research to its foundations and lead to sweeping changes in the research and treatment of this disease, beginning with CML. But first, someone had to figure out what, exactly, src did. How did this one tiny strand of DNA turn perfectly healthy cells into chicken-killing cancer? What was the exact chain of events inside the cell that led to tumor formation, and how was this gene involved?
Finding out how src could do such a thing meant figuring out the protein it encoded. The science at the time said that every gene coded for a specific product in the cell, a mechanism integral to survival. What was src’s product? “I regarded that question as probably the most important question that I could work on in my small laboratory at the University of Colorado,” said Erikson, who had left his family farm to pursue a career in science. Erikson and the members of his Denver lab set to work to try to find that single protein against a background of hundreds of thousands of other gene products in the host cell. It was the needle in a haystack taken to an outlandish extreme. Diving in to find that needle set in motion a second trajectory of research—the first being the Philadelphia chromosome—that would prove crucial to uncovering one of cancer’s most deadly schemes.
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RIGHT NUMBER, WRONG PLACE
As Erikson was getting under way with his search, reports from England about new techniques for coloring chromosomes surfaced. Up until 1969, Giemsa staining, a process using a dye originally created to test cells for the presence of malaria and other parasites, was the only available method. It was effective—chromosomes, derived from the Greek for colored body, had been named as such because they so readily soaked up the dye, enabling the otherwise clear structures to be seen. But Giemsa staining could only turn chromosomes into a single uniform color, which, as Nowell and Hungerford found out, was useful but had serious limitations. Now, there was chatter about new “banding” techniques that lit up chromosomes in a completely new way.
Chromosomes look like pairs of stubby worms belted together at their middles, or, in more graceful depictions, headless dancers. They float in a sea of jelly-like plasma within the center of every cell in the body. Composed entirely of genetic information, on a closer look each of these inky blobs appears as a tightly curled squiggle, a single DNA molecule and its associated proteins, compressed into a spiraling ladder. In humans, these tightly packed helixes contain about 20,500 genes altogether. (The Human Genome Project and the subsequent Encyclopedia of DNA Elements [ENCODE] pilot revealed that just 1.5 percent of the human genome encodes proteins and that, though the vast majority of the remaining genes are doing something, their exact biochemical function is still largely unknown.)
In the early years of genetic research, scientists often focused on other species to learn how chromosomes work. Methods for using blood or bone marrow hadn’t yet been developed, and finding volunteers willing to donate tissues that were most amenable to genetic research, such as the gonads, was not easy. The first key discoveries were made by observing sea urchins and horse roundworm. In the late nineteenth and early twentieth centuries, the German scientist Theodor Boveri proved three essential facts about chromosomes: that they are the carriers of heredity, that each chromosome contains different genetic information, and that every developing egg receives a full set of chromosomes from each parent—to make an embryo, the same story of life must be told twice. These three facts are the tripod supporting the entire field of genetics.
Boveri’s experiments also led him to believe that cancer is caused by genetic abnormalities, a theory that stands at the center of modern cancer research. In 2006, the National Cancer Institute launched the Cancer Genome Atlas, one of many worldwide efforts to catalog the genomes of all different types of cancer. Just as the Human Genome Project spelled out the nucleotide sequences for the 20,000-plus genes that make up human DNA, the atlas maps the sequences of A, C, T, and G in cancer, where the slightest changes in those patterns can alter the instruction issued by a gene. Switch an A for a T, and a gene that encoded a protein that helped program a cell to die at the right time could cease to operate, leaving the cell essentially immortal, a trait of many malignancies. Alterations in nucleotide sequences can be inconsequential to a cell, or they may disappear just as quickly as they arrived. But some are deadly. Spotting the crucial changes in gene sequences, the ones that are responsible for triggering tumor formation, growth, and survival, is a central goal of cancer research today.
Boveri was onto this trail in 1914, though the idea would garner little, if any, traction among his peers. Boveri’s idea was like a visitor from the future that no one could understand. As little clue as Nowell and Hungerford had about why CML cells had a strangely small chromosome, the scientists of Boveri’s day had even less of a framework for understanding that cancer could be linked to genes. They also had no way to compare the chromosomes of cancer cells with those of normal cells. It would be decades before such a framework would be built, as the field of cytogenetics, the study of the connection between genes and disease, gradually came to life. First and foremost was figuring out how many chromosomes humans have. Without this number, all other inquiry was useless.
Early in the twentieth century, the effort to get the right count of human chromosomes was wild and haphazard. The numbers ranged from 15 to 115, with each researcher clamoring for his declared estimate to be accepted as correct. Finally, in 1921, Theophilus Painter, a world-famous zoologist at the University of Texas, pronounced what he considered to be the final word on the subject: Humans have forty-eight chromosomes.
It wasn’t a bad estimate considering the material he was working with. Painter had been counting the chromosomes inside the sperm of testicles taken from castrated mental patients. But the chromosomes inside of sperm cells are unreliable in their appearance. Some chromosomes appear as doubles, and their messy arrangement makes them hard to keep track of. It could also be that the samples he was working with were abnormal, considering they had come from people who were seriously ill. But regardless of how close to correct it was, forty-eight was still wrong.
The mistake would not have been so bad if scientists to follow did not feel beholden to confirm Painter’s finding instead of just reporting what they saw. Because Painter was such a renowned scientist and because he’d been so certain he was right, forty-eight became the dogma, and no one wanted to contradict it. Plus, technology was so rudimentary that whenever scientists did come up with an alternative number, they had little confidence in its accuracy. The loudest answer became the accepted answer, delaying genetics research by more than thirty years.
It wasn’t until 1955 that the mistake was corrected. It was Joe Hin Tjio, an Indonesian scientist working in a Swedish laboratory, who figured it out. Tjio had fled his home country following his imprisonment by the Japanese during World War II. He’d been tortured for offering medical help to his fellow inmates and had kept himself sane by knitting clothes and refusing to give in to despair. Before the war, Tjio had been making headway with his efforts to breed a disease-resistant potato, and upon his release the Spanish government offered him a position with its own plant-improvement project. During holidays and summer breaks, Tjio went to Sweden to work with Albert Levan, a famed geneticist who was doing pioneering work with cytogenetics.
/> In the winter of 1955, Tjio and Levan were studying lung tissue from human embryos. One late December night, his enthusiasm unaffected by the frigid weather and deep snowdrifts, Tjio trudged to the lab to prepare some slides for the microscope. Levan had discovered that a few drops of colchicine, a plant-derived toxin, caused mammal cells to halt right in the middle of dividing, a feature that had already been seen in plant cells. It was like stopping the music and telling the dancing children to freeze, providing the ideal moment to make a head count. Tjio dropped the colchicine onto the lung tissue cells, let the mixture sit for a few hours, and made a slide squash. He put the slide under the microscope, switched on the light, and focused the two small lenses on his target.
Tjio had no intention of counting chromosomes that night, but he couldn’t help himself. The colchicine technique had enabled Tjio to view the chromosomes more accurately than any scientist before him, including Painter. And there was no denying what he saw: forty-six chromosomes. Tjio made the bold move of reporting his results, risking the judgment and ridicule of peers who had cleaved to Painter’s count for so long. But as Tjio’s colleagues around the world examined their own cell samples anew, the only response was confirmation.
FIFTEEN YEARS LATER, Janet Rowley, a geneticist at the University of Chicago who knew well the peculiar history of chromosome research and was already an avid researcher of the connection between genes and cancer, heard about the new staining methods that had been created in England. Right away, she knew she had to learn them. Rowley was a scientific genius who’d graduated from college at age 19 and was the only woman in her class at medical school. Fortunately, 1971 was a sabbatical year for her, and, never wanting to be too far from a microscope, she decided to spend it with her husband, a scientist also on leave that year, in Oxford learning about the rumored new chromosome banding techniques.
The Philadelphia Chromosome Page 4
