Tag Archives: adenoviruses

Frederick Li, p53, and the Li-Fraumeni syndrome

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).

Frederick Li and Joseph Fraumeni  in 1991

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.

References:

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.

5. Norkin, L.C. 2010. Virology: Molecular Biology and Pathogenesis, ASM Press.

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Maurice Hilleman: Unsung Giant of Vaccinology

In January 2005, more than 100 of the world’s most renowned biomedical researchers got together to pay tribute to the 85-year-old Maurice Hilleman. When it was Hilleman’s turn to address the gathering, he alluded to them as his “peers in the world of science.” Referring to Hilleman’s gracious comment, science journalist Alan Dove wrote: “By any objective measure, a gathering of Maurice Hilleman’s scientific peers would not fill a telephone booth.” (1)

Hilleman truly was a giant in the history of virology. But, if you have only a vague idea of who Hilleman was or of his achievements, you are not alone. Anthony Fauci, director of the U.S. National Institutes of Allergy and Infectious Diseases, who was present at the gathering, noted: “Very few people, even in the scientific community, are even remotely aware of the scope of what Maurice has contributed….I recently asked my post-docs whether they knew who had developed the measles, mumps, rubella, hepatitis B and chickenpox vaccines. They had no idea,” Fauci said. “When I told them that it was Maurice Hilleman, they said, ‘Oh, you mean that grumpy guy who comes to all of the AIDS meetings?’”

hillemanMaurice R. Hilleman: The greatest vaccinologist.

Consider this. Hilleman developed nine of the 14 vaccines routinely recommended in current vaccine schedules. These are the vaccines for the measles, mumps, rubella, hepatitis A, hepatitis B, and chickenpox viruses, and for meningococcal , pneumococcal, and Haemophilus influenzae bacteria. Moreover, he was the first to forecast the arrival of the 1957 Asian flu and, in response, led the development of a flu vaccine that may have saved hundreds of thousands or more lives worldwide (2). And, independently of Robert Huebner and Wallace Rowe, he discovered cold-producing adenoviruses, and developed an adenovirus vaccine. Overall, Hilleman invented nearly 40 vaccines. And, he was a discoverer of simian virus 40 (SV40). If the above accomplishments were not enough to ensure his fame, he also was the first researcher to purify interferon, and the first to demonstrate that its expression is induced by double-stranded RNA.

[Aside: I first became aware of Maurice Hilleman 44 years ago. It was in the context of his 1959 discovery of SV40, which I came across only because I was beginning my post-doctoral studies of the related murine polyomavirus. Bernice Eddy, at the U. S. National Institutes of Health (NIH), was probably the first to discover SV40, which she detected in early lots of the Salk polio vaccine (3). Hillman, then at Merck & Co, independently discovered the same virus in rhesus monkey kidney cell cultures, in which the polio vaccine was being produced. Hilleman gave SV40 its name. It was the 40th simian virus the Merck lab found in the monkey kidney cells. In 1961, both Eddy and Hilleman found that inoculating SV40 into hamsters causes tumors in the animals. Merck withdrew its polio vaccine from the market. But, by then, live SV40 had been unknowingly injected into hundreds of millions of people worldwide! More on this in a future posting.]

We begin our account of Hilleman’s achievements with his development of the mumps vaccine. In the days before the vaccine, mumps struck about 200,000 children in the United States, annually. Yet except in rare circumstances, the infection was mild, and was generally regarded as a childhood rite of passage. There is a sweetness to the story of the mumps vaccine that I hope you might enjoy.

The tale began at about 1:00 AM, on March 21, 1963, when 5-year-old Jeryl Lynn Hilleman ambled into her father’s bedroom complaining of a sore throat. Jeryl Lynn’s father felt his daughter’s swollen glands, and knew in a flash that it was mumps. And, while I suspect that many lay parents back in the day would also have recognized Jeryl Lynn’s symptoms, few would have done what her father did after first comforting his daughter. Although it was already past midnight, Maurice hopped into his car and drove the 20 minutes to his lab at Merck & Co. to pick up some cotton swabs and beef broth. Returning home, he then awakened Jeryl Lynn, gently swabbed her throat, and immersed the swabs in the nutrient broth. Next, he drove back to his lab and put the inoculated broth in a freezer.

Hilleman made the early A.M. dashes to his lab and back because he had to leave in the morning for a conference in South America, and his daughter’s infection might have cleared by the time he returned home from there. So, upon his return from South America, Hilleman, thawed the frozen sample from his daughter’s throat and inoculated it into chick embryos. Serial passage of the mumps virus in the chick embryos eventually generated attenuated mumps virus that in 1967 would serve as a live mumps vaccine.

The virus in the vaccine was dubbed the Jeryl Lynn strain, in honor of its source. Years later, an adult Jeryl Lynn Hilleman noted that her father had a need to be “of use to people, of use to humanity.” She added: “All I did was get sick at the right time, with the right virus, with the right father.”

We’ll have a bit more to say about the mumps vaccine shortly. But first, a few words about measles and rubella.

If mumps was not a major killer, measles certainly was. Before Hilleman and his colleagues introduced their measles vaccine (Rubeovax) in 1962, there were 7 to 8 million measles fatalities worldwide each year, and virtually all of the victims were children. Hilleman developed his attenuated measles vaccine from a measles strain isolated earlier by John Enders. Hilleman attenuated the Enders isolate by putting it through 80 serial passages in different cell types.

[Aside: In a previous posting, we noted that Enders, together with colleagues Thomas Weller and Frederick Robbins, shared a Nobel Prize in Physiology or Medicine for growing poliovirus in non-nervous tissue (3). Apropos the current story, bear in mind that Salk and Sabin developed polio vaccines that have nearly rid the world of this once dread virus. Nevertheless, the Nobel award to Enders, Weller, and Robbins was the only Nobel award ever given in recognition of polio research!]

Rubeovax was somewhat tainted by its side effects; mainly fever and rash. While these reactions were successfully dealt with by combining Rubeovax with a dose of gamma globulin, in 1968 Hilleman’s group developed a new, more attenuated measles strain by passage of the Rubeovax virus 40 more times through animal tissues. Hilleman dubbed the new measles strain “Moraten,” for “More Attenuated Enders.” The new measles vaccine, Attenuvax, was administered without any need for gamma globulin.

Our chronicle continues with the rubella vaccine. Rubella poses its greatest danger to fetuses of non-immune pregnant woman, particularly during the first trimester of pregnancy. In up to 85% of these women, infection will result in a miscarriage or a baby born with severe congenital abnormalities. An outbreak of rubella began in Europe in the spring of 1963, and quickly spread worldwide. In the United States, the 1963 rubella outbreak resulted in the deaths of 11,000 fetuses, and an additional 20,000 others born with birth defects (e.g., deafness, heart disease, cataracts).

Hilleman had been working on a rubella vaccine at the time of the 1963 outbreak. But, he was persuaded to drop his own vaccine and, instead, refine a vaccine (based on a Division of Biologics Standards’ rubella strain) that was at the time too toxic to inoculate into people. By 1969 Hilleman was able to attenuate the DBS strain sufficiently for the vaccine to be approved by the FDA.

Next, and importantly, Hilleman combined the mumps, measles, and rubella vaccines into the single trivalent MMR vaccine, making vaccination and, hence, compliance vastly easier. Thus, MMR was a development that should have been well received by many small children and their mothers, as well as by public health officials.

In 1978 Hilleman found that another rubella vaccine was better than the one in the trivalent vaccine. Its designer, Stanley Plotkin (then at the Wistar Institute), was said to be speechless when asked by Hilleman if his (Plotkin’s) vaccine could be used in the MMR. Merck officials may also have been speechless, considering their loss in revenues. But for Hilleman, it was simply the correct thing to do.

Like Jonas Salk and Albert Sabin before him (3), Maurice Hilleman was never awarded a Nobel Prize. There is no obvious reason for the slight in any of these three instances. In Salk’s case, it may have been because Alfred Nobel, in his will, specified that the award for Physiology or Medicine shall be for a discovery per se; not for applied research, irrespective of its benefits to humanity. But, Max Theiler received the Nobel Prize for producing a yellow fever vaccine. What’s more, the Nobel committee seemed to equivocate regarding the discovery that might have been involved in that instance. Regardless, the Nobel award to Theiler was the only Nobel Prize ever awarded for a vaccine! [A more complete accounting of the development of Theiler’s yellow fever vaccine can be found in The Struggle Against Yellow Fever: Featuring Walter Reed and Max Theiler, now on the blog.]

Sabin had done basic research that perhaps merited a Nobel Prize (3). But, the Nobel committee may have felt uneasy about giving the award to Sabin, without also recognizing Salk. Or, perhaps the continual back-and-forth carping between supporters of Salk and Sabin may have reduced enthusiasm in Stockholm for both of them.

Yet by virtually any measure, Hilleman’s achievements vastly exceeded those of Salk, Sabin, Theiler, and just about everyone else. His basic interferon work alone should have earned him the Prize. Hilleman’s group demonstrated that certain nucleic acids stimulate interferon production in many types of cells, and detailed interferon’s ability to impede or kill many viruses, and correctly predicted its efficacy in the treatment of viral infections (e.g., hepatitis B and C), cancers (e.g., certain leukemias and lymphomas), and chronic diseases (e.g., multiple sclerosis). What’s more, Hilleman developed procedures to mass-produce and purify interferon. And, regarding his unmatched achievements as a vaccinologist, he did more than merely emulate Pasteur’s procedures for developing attenuated viral vaccines. His hepatitis B vaccine was the first subunit vaccine produced in the United States. It was comprised of the hepatitis B surface antigen (HBsAg), which Hilleman purified from the blood of individuals who tended to be infected with hepatitis B virus (e.g., IV drug abusers). Subsequently, to avoid the potential danger of using human blood products in the vaccine, Hilleman developed recombinant yeast cells that produced the HBsAg. And, Hilleman’s meningococcal vaccine was the first vaccine to be based on polysaccharides, rather than on a whole pathogen or its protein subunits.

So, why then was Hilleman bypassed by the Nobel committee? John E. Calfree, in The American, wrote: “As the 80-plus-year-old Hilleman approached death, Offit and other academic scientists lobbied the Nobel committee to award Hilleman the Nobel Prize for Medicine, based partly on his vaccine work and partly on his contributions to the basic science of interferons. The committee made clear that it was not going to award the prize to an industry scientist.” (4) [Paul Offit, referred to here, is the co-developer of the rotavirus vaccine, Rotateq, and a biographer of Hilleman.]

Calfree also notes that Hilleman’s tendency towards self effacement, and his absence from the academic and public spotlight, may also have worked against him. And, unlike Salk, whose name was closely linked to his polio vaccine (3), Hilleman’s name was never associated with any of his nearly forty vaccines. [Yet in the case of Jonas Salk, his public acclaim is generally believed to have hurt him in the eyes of his colleagues and of the Nobel committee.]

Considering the enormity of Hilleman’s contributions, his anonymity was really quite remarkable. As Calfree relates: “In one of the most striking of the dozens of anecdotes told by Offit, Hilleman’s death was announced to a meeting of prominent public health officials, epidemiologists, and clinicians gathered to celebrate the 50th anniversary of the Salk polio vaccine. Not one of them recognized Hilleman’s name!”

With Hilleman’s public anonymity in mind, we conclude our account with the following anecdote. In 1998, a Dr. Andrew Wakefield became a celebrity and hero in the eyes of the public. How this happened, and its consequences are troubling for several reasons, one of which is that it brought undeserved suffering to the self-effacing and benevolent Maurice Hilleman. The Wakefield incident merits, and will have a full-length blog posting of its own. But for now, in 1998 Wakefield authored a report in the prestigious British journal The Lancet, in which he claimed that the MMR vaccine might cause autism in children. The story had a bizarre series of twists and turns, with Wakefield and co-authors eventually issuing a retraction. The immediate cause of the retraction was the disclosure that Wakefield, on behalf of parents of autistic children, had accepted funding to investigate a link between the MMR vaccine and autism. The purpose of the investigation was to determine whether a legal case against the vaccine manufacturer might have merit. In addition to the obvious conflict of interest, Wakefield’s paper had serious technical flaws as well. At any rate, a number of independent studies subsequently demonstrated that there is no causal link between the MMR vaccine and autism. And, in 2010 Wakefield was barred by the British Medical Society from the practice of medicine. But the harm had been done. Hilleman had become the recipient of hate mail and death threats. And, more important to Hilleman I expect, many worried parents, even today, prevent their children from receiving the MMR vaccine (5). Ironically, the very success of the MMR vaccine enabled people to forget just how devastating measles and rubella could be.  Maurice Hilleman succumbed to cancer on April 11, 2005.

1. Nature Medicine 11, S2 (2005)
2. Opening Pandora’s Box: Resurrecting the 1918 Influenza Pandemic Virus and Transmissible H5N1 Bird Flu  On the blog.
3. Jonas Salk and Albert Sabin: One of the Great Rivalries of Medical Science  On the blog
4. Calfree, J.E., Medicine’s Miracle Man , The American, January 23, 2009
5. Reference 4 contains a somewhat similar tale, in which a 1992 article in Rolling Stone attributed the emergence of HIV to Hillary Koprowski’s polio vaccine. It created a sensation but, as might be expected, there was no evidence to support its premise.