The 2017 Lasker-DeBakey Prize for Clinical Research went to two virologists at the National Cancer Institute, Douglas Lowy, 75, and John Schiller, 64, for developing technologies that led to FDA-approved vaccines against human papillomavirus (HPV) strains that cause cervical carcinoma and other cancers. Lasker awards are considered the United States’ most prestigious biomedical research awards. They often precede a Nobel Prize in Physiology or Medicine. Thus, they are referred to as “America’s Nobels.” Eighty-seven Lasker awardees have gone on to win a Nobel.
Lowy and Schiller’s achievements were prompted by Harald zur Hausen’s 1983 discovery that two HPV subtypes, HPV-16 and HPV-18, together account for about 70% of all cervical cancers. Since more than 120 distinct HPV subtypes had been identified, the high degree of association of cervical carcinoma with only two of these subtypes provided compelling evidence for the viral etiology of cervical carcinoma. Later studies showed that HPV-31, HPV-33, HPV-45, HPV-52, and HPV-58 are responsible for another 20% of cervical cancers. Thus, an HPV infection can be detected in virtually all cervical carcinomas. Harald zur Hausen was awarded a share of the 2008 Nobel Prize in Physiology or Medicine for his discovery. [His story is told in Harald zur Hausen, Papillomaviruses, and Cervical Cancer, posted June 19, 2015.]
Lowy and Schiller did not begin their work on papillomaviruses with the intent to produce a vaccine. Instead, like many papillomavirus researchers at the time, they were investigating how papillomavirus oncogene products affected cell growth and replication (i.e., how they cause cancer). Toward that end, they were making use of bovine papilloma virus (BPV) in their studies, rather than HPV. BPV was easier to work with than HPV, because BPV, but not HPV, could be studied in standard cell cultures (see Aside 1).
[Aside 1: The replication cycle of HPV depends upon the differentiation states of the cells making up the layers of an intact, stratified epithelium. Details are as follow. Since the outer layer of the skin is comprised of dead cells, cutaneous HPV infection requires a break or puncture of the skin for the virus to access cells of the underlying germinal stratum of the epithelium. In the actively dividing basal cells, the viral genome replicates more frequently than the cellular genome, thus amplifying the viral genome copy number. However, because the viral genes that encode the capsid proteins are not expressed in these cells, progeny virus particles, which might induce an immune response, are not yet produced. As the basal cells differentiate and move up in the epithelium, the viral genomes replicate only once per cell cycle, on average, to maintain the viral genome copy number. Then, as the infected cells go through their final stages of differentiation in the outer layers of the epithelium, the virus life cycle switches to its productive phase. Capsid proteins are produced, and thousands of virus particles are generated from the each of the infected, terminally differentiated cells. Thus, the HPV life cycle is regulated by the differentiated state of the host cell within the stratified epithelium. Because virus production is restricted to the outermost layers of the epithelium, the virus can evade the immune system, such that the infection can persist, and be passed on for years. However, in most instances, the host appears to eventually mount a successful immune response, which clears the infection.
The development of so-called organotypic raft cultures eventually made it possible to study HPV in cell culture. But one could produce only very limited amounts of the virus in that system.]
Working with BPV, Lowy and Schiller developed protocols they would later use when they turned their attention towards an HPV vaccine. One of these protocols was for an assay to measure the titer of neutralizing antibodies against BPV. Importantly, they also discovered that they could generate “virus-like particles” (VLPs), comprised only of the major BPV coat protein (L1). The BPV L1 proteins (which were generated by a baculovirus vector in insect cells) self-assembled into VLPs that were morphologically like actual BPV particles. What’s more, using their assay to measure the titer of neutralizing serum antibodies, they found that the VLPs induced neutralizing antibodies in rabbits that were effective against the actual virus. Importantly, since the VLPs did not contain viral genes, they could not cause cancer.
Again, using their assay for measuring the titer of neutralizing antibodies against BPV, Lowy and Schiller compared the immunogenicity of BVP VLPs, to that of individual BPV proteins. The VLPs indeed are more immunogenic than individual viral proteins, since they induced higher levels of neutralizing antibodies than were induced by individual L1 proteins (see Aside 2).
[Aside 2: The activation of antibody-producing B-cells is triggered by the cross-linking of their antigen-binding B-cell receptors, which is facilitated by the multimeric VLPs, but not by individual viral proteins.]
The innovations resulting from their work with BPV would enable Lowy and Schiller to overcome the formidable challenges they faced when working to develop the HPV vaccine. One obstacle was that HPV cannot replicate in standard cell cultures. Thus, it was difficult to study HPV, and importantly, it also was difficult to propagate it. Being able to propagate substantial amounts of the virus would be necessary to produce a vaccine.
Another obstacle to an HPV vaccine was the potentially unacceptable risk of inoculating people with a virus (either attenuated or killed) that contains known oncogenes. Lowy and Schiller overcame this impediment, and the one noted above, by implementing protocols they previously developed while researching BPV. Specifically, they generated HPV VLPs that were comprised only of the HPV L1 capsid protein, and which induced an immune response that produced protective antibodies. [They used the L1 protein of HPV-16; the most carcinogenic strain of HPV.] In addition, they developed cell lines, which contained high copy numbers of the plasmid that encoded the HPV L1 protein; a step which enabled them to scale-up production of the VLPs.
Together, these breakthroughs made a compelling case for the feasibility of an HPV vaccine. So, Lowy and Schiller prevailed upon several pharmaceutical companies to produce a vaccine in commercial amounts, and to see the vaccine through the clinical trials process. Most companies remained skeptical about the ultimate success of the vaccine. But two companies, Merck and GlaxoSmithKline (which later bought Merck), accepted the challenge. Thus, Merck developed Gardasil, while GlaxoSmithKline developed Cervarix. [The VLPs in Gardasil are produced in yeast, whereas the VLPs from Cervarix are produced in insect cells, via a recombinant baculovirus.]
Clinical trials showed that the Merck and the GlaxoSmithKline vaccines induce significant antibody titers against high-risk HPVs. The US FDA approved the respective HPV vaccines in 2006 and 2009.
The HPV vaccines have had a substantial effect on human health. Consider the following: Cervical cancer is the second most common cause of death from cancer among women worldwide. HPV infection is the cause of virtually all cases of cervical cancer. HPVs also cause 95% of anal cancers, 70% of oropharyngeal cancers (more common in men than in women), 65% of vaginal cancers, 50% of vulvar cancers, and 35% of penile cancers. Next, consider that, since Gardasil and Cervarix were introduced, HPV infection rates have dropped by 50 percent among teen-age girls in U.S., even though only a third of teens between 13 to 17 years-old have received the full course (3 shots) of the vaccine (see Aside 3).
[Aside 3: Current CDC recommendations are as follows: “All kids who are 11 or 12 years old should get two shots of HPV vaccine six to twelve months apart. Adolescents who receive their two shots less than five months apart will require a third dose of HPV vaccine…If your teen hasn’t gotten the vaccine yet, talk to their doctor or nurse about getting it for them as soon as possible. If your child is older than 14 years, three shots will need to be given over 6 months. Also, three doses are still recommended for people with certain immunocompromising conditions aged 9 through 26 years.”]
Although he HPV vaccines have significantly reduced the incidence of cervical cancer in the developed world, the rates of cervical cancer in the United States are needlessly high, in comparison to the rates in other industrialized nations. The HPV vaccines have a loweracceptance rate than other childhood vaccines in the United States, perhaps because many American parents, some of whom associate with the religious right, have reservations about vaccinating their children against a sexually transmitted disease. Other individuals, liberals as well as conservatives, may oppose vaccines in general because they distrust pharmaceutical companies, or because they resent government interference in their lives. In any case, the CDC found no evidence of any increase in sexual activity among teenage girls who received the vaccine. Nor did it report any major ill effects]. See Aside 4.
[Aside 4: Since HPVs alone account for about 5% of all human cancers worldwide, we might ask what percentage of human cancers have a viral etiology. Hepatitis C virus, a flavivirus, and hepatitis B virus, a hepadnavirus, cause hepatocellular carcinoma; Epstein-Barr virus (EBV), a herpesvirus, causes Burkitt’s lymphoma and nasopharyngeal carcinoma; human herpesvirus 8 (HHV-8), causes Kaposi’s sarcoma, the most frequent cancer seen in AIDS patients; the human T-lymphotropic retrovirus I (HTLV-I) induces adult T-cell leukemia; and Merkel cell polyomavirus (MCV) causes its eponymous cancer. Together, viruses may account for as many as 20% of all human cancers, and a similar percentage of all deaths due to cancer!
As shown by the HPV vaccine, and earlier by vaccines against hepatitis B, cancers that have a viral etiology might be prevented by vaccination. Apropos hepatitis B, in the late 1980s, Merck and GlaxoSmithKline developed the respective hepatitis B vaccines, Recombivax and Engerix. Like, the HPV vaccines, they are based on VLPs, and they have significantly reduced the incidence of HBV-associated hepatoma; once one of the most lethal cancers.
Bacterial and parasitic infections too may lead to cancer. For example, Heliobacter pylori infections may lead to stomach cancer, and Schistosoma, Opisthorchis, and Clonorchis have been linked to rectum and bladder cancers in areas of Northern Africa and Southeast Asia, where those pathogens are prevalent.]
Lowy and Schiller’s achievement stands out as a superb example of basic research translating into very considerable public health benefits. Moreover, it serves as a strong endorsement for government support of basic research. To these points, Schiller noted that companies would not likely have carried out the necessary basic research and development necessary to produce the HPV vaccine, considering the seemingly small likelihood of success, as suggested by earlier failed attempts to develop a vaccine.
At a September 6, 2017 press conference announcing the Lasker-DeBakey Clinical Medical Research Award, Lowry related that he first learned about vaccines in 1955, when he went with his mother, a physician, to a talk by Jonas Salk about his then new polio vaccine. “I learned far more about polio virus and the vaccine than was probably appropriate for a 12-year-old boy.” Many years afterwards, Lowy began his “extraordinarily effective” collaboration with Schiller, which has endured for more than 30 years.
Schiller said that a high point in his career was taking his daughter to get the vaccine he helped to create. “We first came up with the idea of the vaccine when she was born and it became available when she was 13 years old (1).”
A Revolutionary Vaccine, New York Times, September 6, 2017.
George Klein, professor emeritus of tumor biology at the Karolinska Institute in Stockholm, where he worked with his wife Eva from the very beginning, passed away on December 10, 2016, at the age of 91. Klein was best known for discovering that Epstein-Barr virus (EBV)—the herpesvirus now known to cause infectious mononucleosis—causes two human cancers, Burkitt’s lymphoma and nasopharyngeal carcinoma. Moreover, Klein discovered that EBV triggers Burkitt’s lymphoma by facilitating a chromosomal translocation of the cellular c-myc oncogene, resulting in its constitutive expression. Klein also played pioneering roles in developing the concept of tumor-suppressor genes, and in opening the field of tumor immunology. Klein’s key discoveries are summarized below. But, first, Klein, like several other protagonists in these tales, was profoundly affected by events of the Second World War, and by the early days of the Cold War that followed.
George Klein’s Jewish family moved from Eastern Slovakia to Budapest in 1930. Nineteen-year-old George was working as an assistant secretary to the Jewish Council in Budapest when Nazi Germany began its occupation of Hungary in March 1944. Because George had been working for the Jewish Council, in April 1944 he chanced that to see the Vrba-Wetzler Report, known at the time as the “Auschwitz Report.” It was written by, and was secretly transmitted to the Jewish Council by Rudolf Vrba and Alfred Wetzler, two escapees from Auschwitz. It described firsthand the fate of Jews arriving at Auschwitz, and was meant to warn Hungary’s Jews, so that they might hide from, or rebel against their Nazi oppressors.
The Auschwitz report was not publicized in Hungary for reasons explained below. However, George’s supervisor at the Jewish Council gave him permission to tell his relatives and friends of what the report revealed. But they, like most Hungarian Jews, could not believe that such atrocities could actually be taking place. [During May, June, and July 1944, 437,000 Hungarian Jews were deported to Auschwitz; to be “resettled” according to the Nazis. But, in fact, most were murdered in the gas chambers.]
Klein was arrested and pressed into forced labor by the Nazis. Afterwards, since he knew the contents of the Auschwitz Report, he fled when he was about to be ordered to board one of the deportation trains to Auschwitz. Having escaped from almost certain death, he lived underground until January 1945, when the Russian Army liberated Budapest.
Forty-three years later, Klein was watching, Shoa, the monumental (nine-hour-long) French documentary film about the holocaust. Watching the movie, Klein chanced to see a man named Vrba (one of the six principal holocaust witnesses in the film) describe his experiences as a prisoner in Auschwitz. The events that Vrba recounted horrified Klein.
Later in the film, as Vrba described his escape from Auschwitz, Klein suddenly realized, “the report I had been given to read under a promise of secrecy in Budapest in May 1944—at the age of nineteen and at a time when deportations from the Hungarian countryside were at their peak—was identical to the Auschwitz Report of Vrba and Wetzler (1).”
Next in this remarkable tale, Klein decided to try to find Vrba, to “tell him of what enormous help his report had been to me. If I had not known what was awaiting me at the other end of the train trip, I would never have dared to risk an escape. It was not difficult to find Vrba, for it turned out that we were scientific colleagues. He is a professor of neuropharmacology in Vancouver, and I am now (in the Spring of 1987) sitting in a comfortable armchair in the faculty club at a Canadian university, talking with someone who, at first glance, seems quite ordinary. He impresses me as being relaxed and jovial. By now I have also read his book (Escape from Auschwitz, 1964), and I am aware that he has survived more death sentences than anyone else I have ever met (1).”
Vrba (1924–2006), was indeed a professor of pharmacology at the University of British Columbia; a position he held from 1976 until the early 1990s. Note that he and Wetzler were the first prisoners ever to escape from Auschwitz. Vrba’s real name was Walter Rosenberg. Rudolf Vrba was the nom de guerre he used after joining the resistance in his native Czechoslovakia. Afterwards, he made the change legal.
The horrors of the holocaust remained an obsession for Klein, although he was uncertain as to why that was so. “Was it to honor my murdered family, my murdered classmates? Or was it rather to steel myself against the darkest side of our human heritage?” In any case, Auschwitz and the holocaust were the main topics of conversation when Klein met with Vrba.
Vrba took Budapest’s Jewish Council to task for not widely broadcasting the warnings in the Auschwitz Report. He, and others, have alleged that Dr. Kastner, a well-known Zionist leader in Budapest, decided to keep the Report secret, in return for a promise from the Germans to allow sixteen-hundred people, as selected by Kastner, to safely emigrate from Hungary. Klein retorted that he knew Kastner from his work for the Jewish Council, and considered him to be a hero, because he had rescued many, while others tried to rescue only themselves or their own families. [In 1957, Kastner was murdered in Israel by a young man whose family was exterminated by the Nazis. Kastner remains a controversial figure to this day.]
Klein and Vrba next discussed whether dissemination of the Auschwitz Report might have caused Budapest’s Jews to revolt against the Nazi program of annihilation. Klein argued that of the dozen or so people that he warned, no one believed him. Vrba countered, “You were a mere boy. Why would anyone believe what you were saying? The Jews would certainly have believed their responsible leaders (1).” Nonetheless, Vrba conceded that even the prisoners at Auschwitz were in denial of what they could see with their own eyes: “…prisoners, who knew full well that no one ever returned from the gas chambers, repressed such knowledge as they themselves lined up for execution in front of the chamber doors.”
Klein asked Vrba how he is able to live and function in Vancouver, a pleasant and friendly place, where no one has the slightest concept of what he endured: “…you must go back constantly to those days. You are called in as a witness at trials of old Nazis or their followers, people who claim that the holocaust never happened. You try to describe something that cannot be described in any human language, you try to explain the incomprehensible, you want people to listen to something they do not want to hear (1).” Vrba, in fact, never did reveal his Auschwitz experience to his colleagues. Vrba explained: “What would have been the use? No one who has not experienced it can understand.” Their conversation went on for almost ten hours. Afterwards, they parted like old friends, despite any differences in their views.
In the Fall of that year, Klein was reunited with Vrba in Paris, together with another newfound friend, German scientist Benno Muller-Hill. In 1966, Muller-Hill was a graduate student in Walter Gilbert’s Harvard laboratory, when he purified the lac repressor; the first genetic control protein to be isolated. Muller-Hill then began a second career lecturing and writing about the role of Nazi doctors and scientists in the holocaust. Klein met Müller-Hill for the first time at a meeting at the Institute for Genetics in Cologne, and the two immediately developed a close friendship.
Muller-Hill was in Paris to visit colleagues at the Pasteur Institute, as well as to meet Vrba. Klein was visiting Paris after attending a scientific meeting in Lyon. Vrba was in Paris at the invitation from the French radio service to refute claims of the ultra-right French leader, Jean Marie Le Pen, that the Nazi gas chambers never existed, and that if the Nazis indeed had any intent to annihilate the Jews, it was merely one of many episodes of the war. [Marine Le Pen, currently a leader of France’s ultra-right National Front, and a candidate for the presidency of France, is Jean Marie’s daughter. She was recently taken to task for denying that French officials and police were complicit in the Nazi roundup of more than 13,000 French Jews in July 1942 (they were later deported to Auschwitz). Le Pen also calls for the deportation of all immigrants from France; a stance that mainly targets Muslims.]
Klein and his two companions ambled about Paris on a beautiful Fall afternoon. They strolled around the Luxembourg Gardens, then continued along the banks of the Seine, turned toward the Latin Quarter, and then stood before the façade of Notre Dame. Yet their minds were elsewhere. “Vrba suggested that we visit the holocaust memorial behind Notre Dame…That walk of only a few minutes took us from the noisy tourist crowd to the silence of the museum’s rooms, where you feel alone and isolated among the symbolic chains and barbed wire. A faint glow of sunlight came in through the narrow openings in the wall. We were surrounded by the voices of the victims…We were all completely speechless. Even Vrba’s macabre sense of humor and his sharp sarcasm had fallen silent for the moment (1).”
After they exited from the memorial, they sat down in a small bistro, where Klein asked his two companions whether German scientists and doctors were actual architects of the holocaust or, instead, merely passive followers. “Benno had concluded from his exhaustive documentation that, contrary to what many wanted so desperately to believe, the ‘euthanasia programs’…and the horrible human experiments… could not be ascribed to a small minority of madmen, opportunists, or charlatans. On the contrary, they had been carried out by quite ordinary and in some instances, eminent physicians and scientists… He (Verba) thought … that would not explain why so many apparently ordinary people took part in the murders without showing any signs of remorse, or how the annihilation program could have been carried out with such efficiency… The discussions between Benno and Vrba continued for several hours (1).”
The day became even more notable later, since Klein had arranged for the threesome to have dinner that evening with Francois Jacob. After a glass of sherry in Jacob’s Latin Quarter apartment, the foursome went to a small restaurant around the corner.
Francois Jacob, and fellow Pasteur Institute scientist Jacques Monod, were awarded Nobel Prizes for their work together on the regulation of lactose metabolism in E. coli (2). More apropos the current episode, Jacob and Monod each received France’s highest military honors for his service during the Second World War—Jacob for his heroism serving with the Free French forces, and Monod for his heroism in the Resistance (2). Yet Jacob’s harrowing escape from Nazi-occupied France at 19-years in age, and his wartime exploits as one of Charles De Gaul’s most highly decorated volunteers, were barely known to his three dinner companions.
At first, Klein was somewhat worried that his friends might not like each other. Jacob often found conversation to be difficult; partly because the thousands of pieces of shrapnel that he carried in his body from the war, made it hard for him to sit comfortably. [Jacob’s wartime wounds prematurely ended his surgical career, and led him to turn to a career in science (2).] But, the get-together didn’t go badly at all.
Conversation eventually turned to the issue of holocaust deniers, as well as to those who would put the past completely behind them. As they talked, the incongruity of the scene suddenly struck Klein. They were sitting in a “first-class Parisian restaurant, surrounded by elegant people, having a very nice dinner in the best French tradition.” “…why did the three of us, with Jacob listening, choose to spend that beautiful Saturday in Paris compulsively focusing our attention on the black birds? We were all citizens of free countries, living well in peaceful times. Were we haunted by feelings of guilt toward the dead? Were we afraid that the whole experience would recur if we let go? We knew that the wide and relentless river of history is rarely influenced by knowledge of the past. In no more than one or two generations, archives of extreme horror turn into scraps of faded paper, with no more influence than dried leaves. I suddenly felt that we were like a traveler with a fear of flying, forcing himself to stay awake and keep his seatbelt buckled during the entire flight, obsessed with the idea that the plane would surely crash if he were to fall asleep. But perhaps we had other motives. Perhaps we wanted to feel a solidarity with each other by selecting a more or less taboo subject for our conversation, one avoided by most others. Or did we try to perform a kind of autopsy, using our brains to understand what human minds are capable of at their worst? Have we appointed our brains to serve as the pathologist and the cadaver at the same time?”
The above recounts only a small sampling of Klein’s conversations with Vrba, Muller-Hill, and Jacob, during their day together in Paris. For more, see reference 1.
In January 1945, 19-year-old George Klein emerged from the Budapest cellar where had been hiding during the last weeks of the German occupation. He gazed on the dead soldiers, civilians, and horses that were frozen in the snow, and was struck by the thought that he had survived, despite the likelihood that he would have ended his 19 years in a Nazi gas chamber or a slave labor camp. However, with the city now in Russian hands, George faced a new threat to his freedom; the Russian patrols that were exporting young Hungarians to labor camps in Russia.
Mindful of the danger on the streets, George was yet eager to begin his medical studies. So, he cautiously dodged the Russian patrols as he made his way to Budapest’s medical school, only to find war-torn deserted buildings and dead soldiers there.
Undeterred by the situation in Budapest, George and a friend set out to Szeged, with the hope of attending the medical school there. The journey of 160 miles took the pair five days, by way of a variety of vehicles, including a Russian military truck. In any case, they were admitted to the Szeged university on the same day that they arrived. And while the school was a shadow of its former self, with all the professors having fled to the West, to George, it was a “previously forbidden paradise (3).”
George spent two years in Szeged, and then returned to Budapest when the University reopened there. Back in Budapest, George fell “desperately” in love with Eva Fisher, a fellow medical student. [George describes their whirlwind romance in reference 3.] George now faced a dilemma. Before he met Eva, he finalized plans to visit Stockholm (under the sponsorship of the Jewish Student Club there). But going to Stockholm would mean leaving Eva behind, under conditions in which travel back into Hungary could be risky. Nonetheless, George went to Stockholm, with Eva believing she would never see him again. Yet the trip would be a defining experience for George and, eventually, would be important for Eva too. In Stockholm, George would learn of, and be riveted by the research of renowned cell biologist Torbjörn Caspersson, at Stockholm’s Karolinska Institute.
Caspersson’s research so enthralled George that he diligently pressed Caspersson for a junior research assistantship in his laboratory. But once George had been accepted by Caspersson, he viewed his situation with a “mixture of ecstatic happiness and enormous anxiety.” “I knew virtually nothing…I was halfway through my medical studies…I was desperately in love with a girl whom I had only known during a summer vacation of eight days and who was on the other side of an increasingly forbidding political barrier (3).”
Despite these misgivings, George knew that his future lay in Sweden, rather than Hungary. He had been accepted into Caspersson’s laboratory, and Hungary was falling increasingly under totalitarian Soviet domination. But Eva was still in communist Hungary. So, George risked returning there with one goal; to marry Eva, and then to leave Hungary for good. “The reunion with Eva confirmed what we both already knew: we wanted to live and work together (3).”
But George and Eva didn’t have the necessary documents to get married, nor did Eva have a passport to leave Hungry. Moreover, communist bureaucrats made it increasingly difficult to obtain these documents. In some instances, up to six weeks might be needed. However, George and Eva were daring and resourceful. When told by a police officer that it would take at least three weeks to obtain a marriage license, George suddenly acted on impulse: “I had always heard others tell of such things but I myself had neither seen nor done it. I pulled a fairly modest bill out of my pocket and put it in the policeman’s hand. ‘Pardon me, how much time was it you said?’ ‘I’ll go get it at once,’ he answered (3).”
With similar persistence and ingenuity, George and Eva obtained all their necessary documents, and they were married that very day! One document, a certificate asserting that neither George nor Eva had a venereal disease, would normally require a three-week lab test. But they beseeched an older colleague, now a doctor at a children’s hospital, to write the certificate for them. Their colleague did so, on his Children’s Hospital stationary. George and Eva then went to the prefecture to be married, only to find a disagreeable marriage official, who was determined to leave work for the day. But, when the official leafed through their papers, and saw the venereal disease certificate written on Children’s Hospital stationary: “He laughed until tears ran down his cheeks. This was the funniest thing he had seen during his whole time in service.” He then gladly married the couple.
As the Iron Curtain descended about Hungary, George and Eva left for Sweden, where they would now continue their medical studies. What’s more, Eva joined George in Caspersson’s laboratory at the Karolinska Institute. The couple would work together at the Karolinska until George’s death at the age of 91. [Eva was born to Jewish parents in Budapest in 1925. In 1944 and 1945, she and several members of her family hid from the Nazis at the Histology Institute of the University of Budapest. Encouraged by Caspersson, Eva had an independent research career, while also collaborating with George. She is best known for discovering natural killer cells, and for generating the Burkitt’s lymphoma cell lines, which she and George studied together (see below).]
We conclude with a brief review of some of George Klein’s contributions to virology and to cancer research.
Tumor immunology: In 1960, George and Eva used methylcholanthrene to induce tumors in mice. Next, they surgically removed the tumors, killed them with irradiation, and inoculated them back into genetically compatible mice. Next, they challenged these mice with cells from a variety of different tumors, and showed that the immune systems of the inoculated mice rejected only those cancer cells that came from the original tumor. Thus, there are tumor-specific antigens that can be recognized by the immune system. See Aside 1.
[Aside 1: Importantly, the tumor resistance seen in these experiments did not arise spontaneously in the original tumor-bearing animals. Instead, it developed in the test mice, in response to sensitization with killed tumor cells. Thus, these experiments per se do not point towards an immune mechanism of tumor surveillance. Nonetheless, harnessing such a mechanism is currently a promising means of cancer therapy, and was a major theme in Klein’s thinking.]
The following year, Klein’s group showed that polyoma virus-induced tumors share a common antigen. Importantly, polyoma virus-induced tumors, and polyoma virus-transformed cells, were rejected irrespective of whether they released virus. Thus, antiviral immunity as such was neither necessary nor sufficient for tumor rejection. This was the first demonstration that tumors caused by a virus might share a common antigen. The Kleins, and others, later found similar “group-specific” transplantation antigens on other virus-induced tumors, including retrovirus-induced lymphomas.
Burkitt’s lymphoma: “Sometime in the mid-1960s, Eva suggested that we should use our experience on virus-induced murine lymphomas to examine a human lymphoma with a presumptive viral etiology. Could we detect group specific antibody responses that might be helpful in tracing a virus? Burkitt’s lymphoma (BL) was the obvious choice (3).” [Burkitt’s lymphoma, originally described by Dennis Burkitt in 1958, is a malignant B-cell lymphoma that is most prevalent in tropical Africa and New Guinea. It is the most common childhood cancer in equatorial Africa. Burkitt first proposed that the lymphoma might have a viral etiology, since its geographic distribution is like that of yellow fever, which is caused by a flavivirus. In 1964, Tony Epstein and Yvonne Barr, by means of electron microscopy, discovered a virus in cells which they cultured from BL tissue, thereby giving credence to Burkitt’s premise.]
Klein’s group identified a membrane antigen (MA) that was expressed in some BL-derived cell cultures. Werner and Gertrude Henle had previously discovered that the MA antigen is a structural protein from a newly discovered herpesvirus—the virus that Epstein and Barr first saw in 1964. Klein decided to call that virus the Epstein Barr virus (EBV). The MA antigen is now known to be one of the EBV envelope glycoproteins. Klein and collaborators later identified complement receptor type 2 (CR2), also known as the complement C3d receptor, as the cell surface attachment protein for the viral MA glycoprotein. CR2 receptors on B cells play a role in enabling the complement system to activate B cells.
By 1970, Klein’s group, in collaboration with Harald zur Hausen, found that the subset of BL-derived cell lines that express MA are, in fact, those that produce EBV. However, more than 90% of the BL cell lines, and all nasopharyngeal carcinomas, were found to contain multiple EBV genomes per cell, irrespective of whether they produced virus. Thus, only a subset of BL and nasopharyngeal carcinoma cells that harbor EBV genomes, actually produce the virus. During this time, the Henles discovered that EBV is the cause of infectious mononucleosis, and that EBV could immortalize normal B cells in culture.
Oncogene activation by chromosomal translocation: A sero-epidemiological study, begun in Uganda in 1971 by Geser and de-The, showed that children with a high EBV load are more likely to develop BL than are children with a low EBV load. Thus, the presence of EBV genomes in a B cell increases the likelihood of it turning into a BL. “But this is still not a satisfactory explanation; some essential element is obviously missing (3).”
What then is the missing event that gives rise to BL? A 1972 study by Manolov and Manolova, Bulgarian scientists working with the Kleins, found that a particular chromosomal marker, 14q+, was present in about 80% of BL tumors. After the Manolovs returned to Bulgaria, the Kleins, in collaboration with Lore Zech, used the chromosomal banding technique recently developed by Caspersson and Zech to examine the BL-cell chromosomes more precisely. They showed that the 14q+ marker was derived from chromosome 8, which broke at the same site (8q24) and underwent a reciprocal translocation with the short arm of either chromosome 2 or chromosome 22. All BLs carried one of the translocations.
Meanwhile, another research group found that carcinogen-induced mouse plasmacytomas are associated with an almost homologous chromosomal translocation. Thus, a common mechanism seemed to underlie two distinct types of tumors, in two distinct species. In each instance, a putative oncogene was translocated to an immunoglobulin locus, which might then have caused the oncogene to be constitutive expressed. A somewhat similar mechanism was reported earlier for the induction of bursal lymphomas in chickens by the avian leukosis virus (ALV) . In that instance, the cellular c-myc gene came under the control of the ALV provirus promotor. What’s more, Michael Cole’s group identified the transposed gene in BL, and in the mouse plasmacytomas, as c-myc. It is not yet clear how EBV infection promotes the chromosomal translocation.
Tumor suppressor genes: In the early 1970s, Klein, and collaborator Henry Harris, played a pioneering role in developing the concept of tumor suppressor genes. They found that when highly malignant mouse cells are fused with normal mouse cells, the hybrid cells are non-malignant when inoculated into genetically compatible mice. That is, tumorgenicity is suppressed by fusion with normal cells. However, tumorgenicity reappears after some apparently important chromosomes, contributed by the normal cell, are lost from the hybrid cells.
Frederick Li passed away on June 12 of this year. In 1969, Li and Joseph Fraumeni, working together at the U.S. National Cancer Institute, discovered a familial (inherited) cancer syndrome, known as the Li-Fraumeni syndrome. Members of Li-Fraumeni syndrome families have a greatly increased risk of developing several types of cancer; particularly breast cancer, but also brain tumors, leukemias, and other cancers as well (1).
In 1990, Li and Fraumeni, in collaboration with Stephen Friend and coworkers at the Massachusetts General Hospital Cancer Center, discovered that all Li-Fraumeni syndrome families harbor germ line mutations in TP53; the gene which encodes the cellular p53 protein (2). This report was the first to document that a mutation in TP53 can be inherited. What’s more, the 1990 paper proved to a previously skeptical medical community that heredity can play a major role in some cancers. [Fraumeni says that environmental factors such as air pollution, occupational exposures, diet, and even viruses were, at the time, considered far more likely causes of cancer than genetic mutation (3).]
Although Li’s research focus concerned genetic mutations that might cause cancer, rather than virology, his story is relevant to the blog because p53 is a key factor in the life cycles of the DNA tumor viruses (i.e., the polyomaviruses, papillomaviruses, and adenoviruses). Moreover, p53 was discovered by virologists. So, we begin with a brief review of the discovery of p53 and its mode of action.
In 1979, the p53 protein was discovered independently by several research groups. The discovery happened when David Lane and Lionel Crawford at the Imperial Cancer Research Fund, and Daniel Linzer and Arnold Levine at Princeton University, unexpectedly discovered a non-viral protein of molecular mass around 53 in association with immunoprecipitates of the SV40 LT protein in SV40-transformed cells.
Importantly, the SV40 LT protein was already known to be a key factor in the ability of that virus to induce neoplastic transformation. What’s more, the papillomavirus E6 gene product likewise interacts with p53, as do the adenovirus E1B proteins. Furthermore, each of these viral proteins promotes transformation, and each does so by either inactivating p53 or by facilitating its degradation. Taken together, these facts strongly implied a role for p53 in transformation. See Aside 1.
[Aside 1: Our last posting featured Harald zur Hausen and his discovery that cervical cancer is caused by papillomaviruses (4). Recall that papillomavirus genomes are integrated into the cellular DNA of cervical cancer cells. Harald zur Hausen and coworkers found that while these integrated viral genomes often contain deletions, two papillomavirus genes, E6 and E7, are present and transcribed in all cervical cancer cells; a finding which implied that these viral genes act to initiate and maintain the neoplastic state. And especially germane to the current tale, Peter Howley and coworkers demonstrated that the interaction of the papillomavirus E6 gene product with p53 results in the degradation of p53.]
At the time TP53 was discovered, it was thought to act like the oncogenes carried by the retroviral RNA tumor viruses. Retrovirus oncogenes are actually captured cellular genes, which promote cancer when they are inappropriately expressed under control of viral promoter elements. However, clues eventually emerged which pointed to a very different understanding of p53’s function. The p53 protein is actually a tumor suppressor. Evidence in that regard included the mid 1980s findings of David Wolf and Varda Rotter at the Weizmann Institute, and others as well, who showed that cell lines derived from a number of sporadic (nonfamilial) cancers have TP53 genes that are dysfunctional by mutation. Importantly, it is the loss of p53 function, rather than its expression, which may lead to cancer. [Retroviral oncogenes act dominantly when introduced into non-malignant cells, whereas mutations in tumor suppressor genes are recessive to their wild-type alleles.]
Why, we ask, do the polyomaviruses, papillomaviruses, and adenoviruses inactivate p53? The answer reveals a key tumor suppressor function of p53. Basically, it is because these DNA viruses require the cellular DNA replication enzymes and substrates to support their own DNA replication. Since these cellular enzymes and substrates are available only in dividing cells, these viruses induce cells to bypass the complex circuits that regulate exit from the G0 or “resting” phase of the cell cycle. They do this by freeing the cellular E2F transcription factor from the blocking activities of the pRb family of tumor suppressor proteins, in that way enabling cells to enter into S phase. [The multifunctional SV40 LT protein, the papillomavirus E7 gene product, and the adenovirus E1A protein carry out this function for their respective viruses.] However, p53 remains as a crucial component of a cellular surveillance mechanism that prevents cells from undergoing unscheduled and potentially disastrous cell divisions. If the cell should enter an inappropriate S phase, p53 triggers apoptosis; a cell death program that can be activated by a variety of signals from within and outside the cell. [From the point of view of the host, cell suicide by p53-mediated apoptosis is preferable to the generation of rampant daughter cells that might produce full-blown tumors.] Consequently, the clever DNA tumor viruses (which comprise three unrelated virus families) undermine the normal regulatory functions of p53, as well as those of pRb. See reference 5 for details on these mechanisms.
Li, Fraumeni, and collaborator, Stephen Friend knew that they could not identify the genetic mutation underlying the Li-Fraumeni syndrome by conventional linkage analysis. That was so because Li-Fraumeni syndrome families are quite rare and, moreover, the cancer death rate among affected family members is high (nearly all individuals who carry the mutation develop cancer). So, their strategy was to investigate plausible candidate genes. They chose TP53 because, in their words: “Inactivating mutations of p53 have been associated with sporadic osteosarcomas, soft tissue sarcomas, brain tumors, leukemias, and carcinomas of the lung and breast. Together, these tumors also account for more than half of the cancers in selected series of LFS families (2).” Furthermore, evidence was emerging that TP53 actually encodes a tumor suppressor protein.
The finding by Li, Fraumeni, Friend, and their coworkers, that the TP53 mutation is present in the normal cells of Li-Fraumeni syndrome individuals, as well as in their tumor cells, proved that the mutation is passed down through the germ line. Yet these findings raise the following interesting question. If the TP53 mutation is present in all cells of an affected individual, why does that individual have “only” one or a few tumors? The reason, at least in part, is that progression to full blown cancer requires additional genetic changes. [Apropos that, Li helped discover that people with the Li-Fraumeni syndrome are particularly prone to developing additional tumors when given radiation therapy to treat their cancers.]
Li and his collaborators closed their 1990 paper as follows: “In conclusion, we have shown that alterations of the p53 gene occur not only as somatic mutations in human cancers, but also as germ line mutations in some cancer-prone families (3).” With that paper, the three researchers, and their collaborators, became the first to demonstrate a genetic condition in which a predisposition to cancer is passed from one generation to the next.
Li was born in Canton, China, in 1940. His father was a general in the Chinese Army (Kuomintang), who fought against the Japanese domination of China during the Second World War. The Li family immigrated to the United States in 1947, and opened a Chinese restaurant in White Plains, N.Y.
At 16 years of age, Li matriculated at NYU, where he majored in physics. He earned his MD from the University of Rochester.
Li joined the NCI in 1967, but spent the last 30 years of his career at the Dana-Farber Cancer Institute in Boston, where he also held appointments as a professor at Harvard Medical School and at Harvard’s School of Public Health. In 1991, he was appointed head of Dana-Farber’s Division of Cancer Epidemiology and Control. David G. Nathan, a former president of Dana-Farber, said that Li had been recruited to Dana-Farber to bring more scientific rigor to cancer research there (3). In 1996, Li was appointed to the NCI’s National Cancer Advisory Board by President Bill Clinton.
Li founded a clinic for immigrants in Boston’s Chinatown, where he frequently treated patients at night for free. He retired from his medical activities in 2008 because of dementia resulting from Alzheimer’s disease.
In July 2012, Joseph Fraumeni celebrated his 50th anniversary as a scientist at the NCI. He observed the occasion by stepping down as the NCI’s Director of the Division of Cancer Epidemiology & Genetics. He continues to serve as a senior investigator and adviser at the NCI and NIH, where his major research contributions concerned the environmental and genetic determinants of cancer. Fraumeni is an elected member of the US National Academy of Sciences.
Stephen Friend was on the Harvard Medical School faculty from 1987 until 1995, when he joined the Fred Hutchinson Cancer Research Center as chairman of Pharmacology. His research focused on genomic analysis of large patterns of gene expression. In 1997, Friend and Leroy Hood co-founded the company, Rosetta Inpharmatics, which specialized in genomic approaches to drug discovery. When Rosetta was acquired by Merck in 2001, Friend served as a Merck Senior Vice President and led the parent company’s Oncology Early Discovery and Development Divisions. Friend left Merck in 2009 to advocate for and promote open access biomedical research. Earlier, in 1986, Friend cloned the gene encoding pRB; the first tumor suppressor gene to be isolated.
1. Li, F.P., and Fraumeni J.F. Jr. 1969. Soft-tissue sarcomas, breast cancer, and other neoplasms. A familial syndrome? Annals of Internal Mededicine. 71:747-752.
2. Malkin, D., F.P. Li, L.C. Strong, J.F. Fraumeni Jr, C.E. Nelson, D.H. Kim, J. Kassel, M.A. Gryka, F.Z. Bischoff, M.A. Tainsky, and S.H. Friend. 1990. Germ line p53 mutations in a familial syndrome of breast cancer, sarcomas, and other neoplasms. Science 250:1233-1238.
3. Grady, D., Frederick P. Li, Who Proved a Genetic Cancer Link, Dies at 75, N.Y. Times, June 21, 2015.
4. Harald zur Hausen, Papillomaviruses, and Cervical Cancer, Posted on the blog June 19, 2015.
Harald zur Hausen (1936- ) was awarded a share of the 2008 Nobel Prize in Physiology or Medicine for discovering that papillomaviruses cause cervical cancer. He received the award jointly with Luc Montagnier and Françoise Barré-Sinoussi, who were given their portion for discovering HIV (1). Before getting on with zur Hausen’s story per se, we begin with bit of earlier history.
Harald zur Hausen in 2008
Genital warts are benign epithelial tumors that have been known and associated with sexual promiscuity since the time of the ancient Greeks. In 1907 these lesions were unequivocally proven to be an infectious disease by Italian researcher, G. Ciuffo, who showed that they can be transmitted by filtered extracts of wart tissue; a finding which also implied that the etiologic agent is a virus. Ciuffo inoculated himself to advance his case.
Ciuffo’s finding is relevant to our story since members of the papillomavirus family of DNA viruses are the cause of warts. What’s more, and importantly, some papillomaviruses also cause malignant cervical carcinomas.
In 1933 Richard Shope, at the Rockefeller Institute, became the first researcher to isolate a papillomavirus, the cottontail rabbit papillomavirus. Shope went on to show that this virus is the cause of skin papillomas in its rabbit host. This finding by Shope was the first to demonstrate that a DNA virus can be tumorigenic.
Years earlier, in 1911, Peyton Rous discovered that an RNA virus—the Rous sarcoma virus (the prototype retrovirus)—causes solid tumors in chickens. Peyton Rous was Richard Shope’s friend and colleague at the Rockefeller Institute. In 1934 Shope asked Rous to characterize the warts that the rabbit papillomavirus induces in jackrabbits. Rous found those warts to be benign tumors that could progress to malignant carcinomas.
Despite the earlier findings of Ciuffo, Shope, and others, the notion that genital warts in humans is a sexually transmitted malady was slow to gain acceptance. Oddly, perhaps, recognition of that truth was prompted by a 1954 report that American servicemen, who had been serving in Korea, were transmitting genital warts to their wives upon returning to the U.S (T. J. Barrett, et al., J. Am. Med. Assoc. 154:333, 1954). [Sexually transmitted diseases were a long-standing problem in the military. Servicemen were most often infected by sex workers who frequented the vicinity of military quarters.]
The key discoveries of this tale are Harald zur Hausen’s 1983 and 1984 findings that two human papillomavirus subtypes, HPV-16 and HPV-18, together account for about 70% of all cervical cancers. Considering that more than 120 distinct HPV subtypes have been identified, the high degree of association of cervical carcinoma with only two of these subtypes provided compelling evidence for the viral etiology of this malignancy. Later studies showed that HPV-31, HPV-33, HPV-45, HPV-52, and HPV-58 are responsible for another 20% of cervical cancers. Indeed, an HPV infection is present in virtually all cervical carcinomas. See Aside 1.
[Aside 1: Cervical cancer was once the leading cause of cancer-related deaths in women in the United States. However, the number of cervical cancer deaths in the industrialized world decreased dramatically over the last 40 years, largely because of the Pap test, which can detect pre-cancer cervical lesions in their early stages. The CDC website reports 12,109 cervical cancer cases and 4,092 deaths from cervical cancer in the U.S. in 2011 (the most recent year for which data are available). Worldwide, cervical cancer was responsible for 275,000 deaths in 2008. About 88% of these deaths were in developing countries (J. Ferlay et al., Int. J. Cancer, 127:2893, 2010).]
Harald zur Hausen was a child in Germany during the Second World War, growing up in Gelsenkirchen-Buer, which was then a center for German coal production and oil refining and, consequently, a major target for allied bombing. [The city also contained a women’s sub-camp of the Buchenwald concentration camp. The Nazis used its prisoners for slave labor.] All members of zur Hausen’s family survived the war. However, zur Hausen’s primary education contained significant gaps because schools were closed during the allied bombing (2).
Despite the gaps in zur Hausen’s early education, he was keenly interested in biology and dreamed of becoming a scientist. Yet at the University of Bonn he opted to study medicine, rather than biology. After zur Hausen received his medical degree, he worked as a medical microbiologist at the University of Düsseldorf, where he enjoyed the opportunity that the University gave him to carry out research on virus-induced chromosomal aberrations.
Although zur Hausen was fascinated by his research, he was soon aware of the deficiencies in his scientific background. So, in 1966 he looked to enhance his proficiency as a scientist by securing a postdoctoral position in the laboratories of Gertrude and Werner Henle at the Children’s Hospital of Philadelphia.
The Henles were a German-born husband and wife research team, known for their work on flu vaccines. More apropos to our story, they are also known for demonstrating the link between the recently discovered Epstein-Barr virus (EBV; a herpesvirus) and infectious mononucleosis, as well as for showing that EBV is the etiologic agent of Burkitt’s lymphoma; a cancer found in parts of Africa. EBV was, in fact, the first virus associated with a cancer in humans. [Gertrude Henle’s mother was murdered by the Nazis in 1943.]
Although zur Hausen took part in the Henles’ experiments involving EBV, he did so grudgingly because he was intimidated by his inexperience in molecular biology. In his own words: “I urged Werner Henle to permit me to work with a different agent, namely adenovirus type 12, hoping that this relatively well established system would permit me to become acquainted with molecular methods. He reluctantly agreed. I started to work eagerly on the induction of specific chromosomal aberrations in adenovirus type 12-infected human cells…and, to please my mentor, I demonstrated electron microscopically the presence of EBV particles directly in… Burkitt’s lymphoma cells (2).”
In 1969 zur Hausen returned to Germany to take an appointment as an independent scientist at the University of Wurzburg. His research was now focused entirely on EBV. Specifically, he wanted to challenge the prevailing view that Burkitt’s lymphoma tumors are persistently infected with EBV (i.e., that the tumors continuously produce low levels of the virus).
I presume that zur Hausen was interested in this issue because it was reasonable to believe that EBV gene expression is necessary to maintain the neoplastic state of the Burkitt’s tumor cells. Persistent infection would be one means by which viral genes could be carried by the cells. But zur Hausen believed that EBV DNA might be maintained in Burkitt’s lymphoma cells, even if they did not produce EBV particles.
Werner Henle in Philadelphia (and also George Klein in Stockholm) sent zur Hausen a large number of Burkitt’s lymphoma cell lines and tumor biopsies to aid in his study. One of those cell lines, the Raji line of Burkitt’s lymphoma cells, did not produce EBV particles. Nevertheless, zur Hausen isolated sufficient EBV DNA from the Raji cells to prove that multiple copies of EBV DNA were maintained in them. This was the first time that tumor virus DNA was shown to be present in malignant human cells that were not producing virus. See Aside 2.
[Aside 2: In 1968 Renato Dulbecco and co-workers were the first to discover viral DNA integrated by covalent bonds into cellular DNA (J. Sambrook et al., Proc. Natl. Acad. Sci. U S A. 60:1288, 1968). They were studying cells transformed by the polyomavirus, SV40. Integration explained how SV40 genes could be stably maintained and expressed in transformed cells, in the absence of productive infection. Integration is now recognized as a key event in cell transformation by members of several virus families, including the polyomaviruses, papillomaviruses, and the oncogenic retroviruses.
The situation in the case of EBV, a herpesvirus, is different, as herpesviruses are able to enter into a latent state in host cells. In the latent state the viral genome is maintained as an episome, and only a subset of the viral genes (i.e., those concerned with latency) are expressed. The episomal viral genome is replicated by the cellular DNA replication machinery during the cell cycle S phase, and a viral gene product, EBNA-1, ensures that viral genomes are equally partitioned between the daughter cells. In 1978 George Klein and co-workers were the first to demonstrate episomal EBV DNA in a cell line derived from a Burkitt’s lymphoma biopsy (S. Koliais et al., J. Natl. Cancer. Inst. 60:991, 1978).]
In 1972, while zur Hausen’s attention was focused on EBV and Burkitt’s lymphoma, his research direction took a providential turn that would lead to his most important discoveries. It happened as follows.
Recent seroepidemiological evidence was suggesting a link between herpes simplex virus type 2 (HSV-2), a well known genital infection, and cervical cancer. Since HSV-2, like EBV, is a herpesvirus, and since zur Hausen had already demonstrated that EBV DNA is present in Burkitt’s lymphoma tumor cells, zur Hausen believed he was well positioned to search for HSV-2 DNA in cervical cancer biopsies. However, in this instance, all his repeated attempts failed.
Harald zur Hausen then came across anecdotal reports of genital warts converting to squamous cell carcinomas. Importantly, those genital warts were known to contain typical papillomavirus particles. Taking these two points into account, zur Hausen considered the possibility that papillomaviruses, rather than herpesviruses, might be the cause of cervical carcinomas. Indeed, his initial thought was that the genital wart papillomavirus might also be the etiologic agent for cervical carcinomas.
Thus, Harald zur Hausen began his foray into papillomavirus research. His first experiments found papillomavirus particles in benign plantar (cutaneous) warts. His next experiments demonstrated that there are multiple papillomavirus subtypes. [In brief, zur Hausen used in vitro-transcribed plantar papillomavirus RNA as a hybridization probe against the DNA from various plantar and genital warts. Only some of the plantar wart DNAs, and none of the genital wart DNAs, reacted with his planter wart RNA probe. Restriction endonuclease patterns of a variety of human papillomavirus isolates confirmed that the HPVs comprise a heterogeneous virus family.]
Harald zur Hausen’s next experiments sought to detect papillomavirus DNA in cervical carcinoma biopsies. However, his initial trials in this crucial undertaking were unsuccessful. He was using DNA from HPV-6 (isolated from a genital wart) as a hybridization probe in those failed attempts. But zur Hausen and co-workers had at hand a number of additional HPV subtypes, from which they prepared other DNA probes. DNA from HPV-11 (from a laryngeal papilloma) indeed detected papillomavirus DNA in cervical carcinomas.
In 1983, two of Zur Hausen’s former students, Mathias Dürst and Michael Boshart, using HPV-11 DNA as a probe, isolated a new HPV subtype, designated HPV-16, from a cervical carcinoma biopsy. In the following year, they isolated HPV-18 from another cervical carcinoma sample. Harald zur Hausen’s group soon determined that HPV-16 is present in about 50% of cervical cancer biopsies, while HPV-18 is present in slightly more than 20%. [The famous HeLa line of cervical cancer cells contains HPV-18 DNA.]
Additional key discoveries took place during the next several years, including the finding that papillomavirus DNA is integrated into the cellular DNA of cervical carcinoma cells. This finding clarified how papillomavirus genes persist in the cancers, and also revealed that the cancers are clonal (see Aside 2, above). Moreover, while the integrated viral genomes often contain deletions, zur Hausen’s group found that two viral genes, E6 and E7, are present and transcribed in all cervical cancer cells. This finding implied that E6 and E7 play a role in initiating and maintaining the oncogenic state. [In 1990 Peter Howley and co-workers demonstrated that the interaction of the E6 gene product with the cellular tumor suppressor protein p53 results in the degradation of p53. In 1992 Ed Harlow and coworkers showed that the E7 gene product blocks the activity of the cellular tumor suppressor protein pRb. Reference 3 details the mechanisms of papillomavirus carcinogenesis.]
The above findings led to widespread acceptance that cervical carcinoma is caused by papillomaviruses. Yet acceptance was not immediate. The prevailing belief, that herpesviruses cause cervical carcinoma, was well-entrenched and slow to fade away. It was based on the observation that many women afflicted with cervical carcinoma also had a history of genital herpes. But, individuals infected with one sexually transmitted pathogen are often infected with others as well. Apropos that, genital warts were long thought to be associated with syphilis, and later with gonorrhea. In any case, in 1995 the World Health Organization officially accepted that HPV-16 and HPV-18 are oncogenic in humans.
Harald zur Hausen was awarded one half of the 2008 Nobel Prize for Medicine or Physiology for proving that cervical cancer is caused by human papillomaviruses. By the time of his award, his findings had led to key insights into the mechanism of HPV-mediated carcinogenesis and, importantly, to the development of a vaccine to prevent cervical cancer. See Aside 3.
[Aside 3: The first generation of Gardasil, made by Merck & Co., helped to prevent cervical cancer by immunizing against HPV types 16 and 18, which are responsible for an estimated 70% of cervical cancers. Moreover it also immunized against HPV types 6 and 11, which are responsible for an estimated 90% of genital warts cases. Apropos genital warts, there are 500,000 to one million new cases of genital warts (also known as condylomata acuminate) diagnosed each year in the United States alone.
The original vaccine was approved by the USFDA on June 8, 2006. An updated version of Gardasil, Gardasil 9, protects against the HPV strains covered by the first generation of the vaccine, as well as five additional HPV strains (HPV-31, HPV-33, HPV-45, HPV-52, and HPV-58), which are responsible for another 20% of cervical cancers. Gardasil 9 was approved by the USFDA in December 2014.]
Harald zur Hausen reviewed the overall contribution of viruses to human cancer in his 2008 Nobel lecture (4). Some of his key points are as follows. HPVs were discussed above with respect to cervical carcinoma. HPVs also are associated with squamous cell carcinomas of the vagina, anus, vulva, and oropharynx. What’s more, 40% of the 26,300 cases of penile cancer reported worldwide in 2002 could be attributed to HPV infection.
Epstein-Barr virus too was discussed above. This member of the herpesvirus virus family causes nasopharyngeal carcinoma, as well as Burkitt’s lymphoma. Another herpesvirus, human herpesvirus 8, causes Kaposi’s sarcoma; the most frequent cancer affecting AIDS patients. Hepatitis B virus (HBV, a hepadnavirus), as well as hepatitis C virus (HCV, a flavivirus), causes hepatocellular carcinoma. The human T-lymphotropic virus 1 (HTLV-1), a retrovirus, induces adult T-cell leukemia. And the recently discovered Merkel cell polyomavirus (MCPyV) is responsible for Merkel cell carcinoma.
Harald zur Hausen estimated that viruses directly cause about 20% of all human cancers, and a similar percentage of all deaths due to cancer! And while 20% might seem to be a remarkably high figure for the extent of viral involvement in human cancer, zur Hausen suggests that it is actually a minimal estimate. That is so because it is difficult to determine that a particular virus is actually the cause of a cancer. Consequently, it is likely that other examples of viral involvement in human cancer will be discovered.
Harald zur Hausen gave two principal reasons for why it is difficult to establish that an infectious agent is the cause of a cancer in humans. First: “… no human cancer arises as the acute consequence of infection. The latency periods between primary infection and cancer development are frequently in the range of 15 to 40 years…” Second: “Most of the infections linked to human cancers are common in human populations; they are ubiquitous… Yet only a small proportion of infected individuals develops the respective cancer type.”
Viruses also contribute to the human cancer burden in an indirect way. For instance, HIV types 1 and 2 play an indirect role in cancer via their immunosuppressive effect, which is the reason for the extraordinarily high prevalence and aggressiveness of Kaposi’s sarcoma in AIDS patients.
Bacterial infections also contribute to the cancer burden. For example, Helicobacter pylori infections may lead to stomach cancer. What’s more, the parasites Schistosoma, Opisthorchis, and Clonorchis have been linked to rectum and bladder cancers in parts of Northern Africa and Southeast Asia, where they are prevalent.
Obviously, but important enough to state anyway, knowing that a particular cancer is caused by a particular infectious agent opens the possibility of developing a rational strategy to prevent that cancer. Gardasil is an exmple. A vaccine against HBV is also available, and one against HCV is under development.
1. Who discovered HIV, Posted on the blog January 23, 2014.