Pauling’s Final Years

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Pauling posing at lower campus, Oregon Agricultural College, ca. 1917.

[An examination of the end of Linus Pauling’s life, part 1 of 4]

In 1917, at sixteen years of age, Linus Pauling wrote in his personal diary that he was beginning a personal history. “My children and grandchildren will without doubt hear of the events in my life with the same relish with which I read the scattered fragments written by my granddad,” he considered.

By the time of his death, some seventy-seven years later, Pauling had more than fulfilled this prophecy. After an extraordinarily full life filled with political activism, scientific research, and persistent controversy, Pauling’s achievements were remembered not only by his children, grandchildren and many friends, but also by an untold legion of people whom Pauling himself never met.

Passing away on August 19th 1994 at the age of 93, Pauling’s name joined those of his wife and other family members at the Oswego Pioneer Cemetery in Oregon. What follows is an account of the final three years of his life.


 

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Linus Pauling, 1991.

In 1991, Pauling first learned of the cancer that would ultimately take his life. Having experiencing bouts of chronic intestinal pain, Pauling underwent a series of tests at Stanford Hospital that December. The diagnosis that he received was grim: he had cancer of the prostate, and the disease had spread to his rectum.

Between 1991 and 1992, Pauling underwent a series of surgeries, including the excision of a tumor by resection, a bilateral orchiectomy, and subsequent hormone treatments using a nonsteroidal antiandrogen called flutamide. During this time, Pauling also self-treated his illness with megadoses of vitamin C, a protocol that he favored not only for its perceived orthomolecular benefits, but also as a more humane form of treatment than chemotherapy or radiation therapy.

Pauling’s interest in nutrition dated to at least the early 1940s, when he had faced another life-threatening disease, this time a kidney affliction called glomerulonephritis. Absent the aid of contemporary treatments like renal dialysis – which was first put into use in 1943 – Pauling’s survival hinged upon a rigid diet prescribed by Stanford Medical School nephrologist, Dr. Thomas Addis.  At the time a radical approach to the treatment of this disease, Addis’ prescription that Pauling minimize stress on his kidneys by limiting his protein and salt intake, while also increasing the amount of water that he drank, saved Pauling’s life and led to his making a full recovery. Though his famous fascination with vitamin C would not emerge until a couple of decades later, Pauling’s nephritis scare instilled in him a belief that dietary control and optimal nutrition might effectively combat a myriad of diseases. This scientific mantra continued to guide Pauling’s self-treatment of his cancer until nearly the end of his life.

Pauling also believed that using vitamin C as a treatment would, as opposed to chemotherapy, allow him to die with dignity. Were his condition terminal and his outlook essentially hopeless, Pauling felt very strongly that he should be permitted to pass on without “unnecessary suffering.” Pauling’s wife, Ava Helen, had died of cancer in December 1981. She too had refused chemotherapy and other conventional approaches for much of her illness, a time period during which Linus Pauling had helped his wife the only way he knew how: by administering a treatment involving megadoses of vitamin C. This attempt ultimately failed and, by his own admission, Pauling never really recovered from his wife’s passing.

Nonetheless, Pauling continued to lead research efforts to substantiate the value of vitamin C as a preventive for cancer and heart disease in his capacity as chairman of the board of the Linus Pauling Institute of Science and Medicine (LPISM). By the time of his own diagnosis in 1991 however, the Institute was in a desperate financial situation, several hundred thousand dollars in debt and lacking the funds necessary to pay its staff.


 

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In 1992, while he recovered from his surgeries and managed his illness, Pauling continued to act as chairman of the board of the LPISM. No longer able to live entirely on his own, he split his time between his son Crellin’s home in Portola Valley, California, and his beloved Deer Flat Ranch at Big Sur. When at the ranch, Pauling was cared for in an unofficial capacity by his scientific colleague, Matthias Rath. Pauling was first visited by Rath, a physician, in 1989, having met him years earlier in Germany while on a peace tour. Rath was also interested in vitamin C, and Pauling took him on as a researcher at the Institute. There, the duo collaborated on investigations concerning the influence of lipoproteins and vitamin C on cardiovascular disease.

Not long after Pauling’s cancer diagnosis, a professor at UCLA, Dr. James Enstrom, published epidemiological studies showing that 500 mg doses of vitamin C could extend life by protecting against heart disease and also various cancers. This caused a resurgence of interest in orthomolecular medicine, and it seemed that Pauling and Rath’s vision for the future of the Institute was looking brighter.

As it happened, this bit of good news proved to be too little and too late. LPISM had already begun to disintegrate financially, its staff cut by a third. The Institute’s vice president, Richard Hicks, resigned his position, and Rath, as Pauling’s protégé, was appointed in his place. Following this, the outgoing president of LPISM, Emile Zuckerlandl, was succeeded by Pauling’s eldest son, Linus Pauling Jr. Finally Pauling, his health in decline, announced his retirement as chairman of the board and was named research director, with Steve Lawson appointed as executive officer to assist in the day-to-day management of what remained of the Institute.

One day prior to his retirement as board chairman, Pauling signed a document in which he requested that Rath carry on his “life’s work.” Linus Pauling Jr. and Steve Lawson, however, had become concerned about Rath’s role at the Institute, and particularly on the issue of a patent agreement that Rath had neglected to sign. Adhering to the patent document was a requirement for every employee at the Institute, including Linus Pauling himself. When pressed on the issue, Rath opted to resign his position, and was succeeded as vice president by Stephen Maddox, a fundraiser at LPISM.

After this transition, Pauling met with Linus Jr. to discuss the Institute’s dire straits. Pauling’s youngest son, Crellin, had also became more active with the Institute as his father’s illness progressed, in part because he had been assigned the role of executor of Pauling’s will. Together, Crellin, Linus Jr., and Steve Lawson struggled to identify a path forward for LPISM. Eventually it was decided that associating the Institute with a university, and focusing its research on orthomolecular medicine as a lasting legacy to Pauling’s work, would be the most viable avenue for keeping the Institute alive. The decision to associate the organization with Oregon State University, Pauling’s undergraduate alma mater, had not been made by the time that Pauling passed away.

Vitamin C and Cancer: Rays of Hope

 

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[Part 4 of 4]

Ridiculed by the medical profession for two decades, the tide began to shift for vitamin C and cancer starting in 1992. That year, the New York Academy of Sciences voted to discuss high-dose vitamins and nutrients at its annual meeting, devoting several sessions to the antioxidant properties of vitamin C and its potential value at higher-than-dietary amounts in preventing lung, stomach, colon, and rectal cancers.

Oddly, throughout the proceedings, one prominent name had been missing from the conversation, a point noted by a professor from Alabama who finally spoke up, saying,

For three days I have been listening to talks about the value of large intakes of vitamin C and other natural substances, and I have not heard a single mention of the name Linus Pauling. Has not the time come when we should admit that Linus Pauling was right all along?


Since 1996 the Linus Pauling Institute, relocated from California, has continued work on cancer from it’s new home at Oregon State University. Basing these contemporary orthomolecular studies on the hard sciences of cellular biology, molecular biology, and organic chemistry, the Institute continues to explore the cutting edge of health and nutrition research.

Working under Dr. Balz Frei, the current director of the Institute, as well as former LPI principal investigator Dr. Roderick Dashwood (now director of the Center for Epigenetics and Disease Prevention at Texas A&M University), OSU student Matt Kaiser has spent time analyzing the benefits of vitamin C treatment for colorectal cancer, which remains the third leading cause of cancer related deaths in the United States. The Pauling Blog has interviewed Kaiser in the past, and we met with him again recently to gain a better sense of trends in the community of researchers interested in vitamin C and cancer.


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One primary question that begs further exploration is, why didn’t earlier studies find evidence of the value of vitamin C?

As it turns out, the problem appears to have been primarily located in the way that vitamin C was being administered. The 1979 Mayo studies to which Pauling so strongly objected had assumed that, since vitamin C was filtered out of the body after a certain point of blood saturation, higher doses need not be examined. This assumption – that excess vitamin C could not be absorbed and was simply excreted in the urine – was one of the most basic issues of contention that Pauling was never able to get past with the medical community. However, it now appears that the assumption applies only if vitamin C is taken as an oral supplement, a breakthrough that was first identified by Mark Levine, a Senior Investigator at the National Institutes of Health.

Matt Kaiser explains

Mark Levine realized in the 1990s that the way drugs are distributed and function in the body [pharmacokinetics] can drastically change the amount of vitamin C entering blood plasma. Eating vitamin C you can only get about 250 micromolar [a measure of vitamin C, or ascorbate— to use its chemical name— that can be concentrated in the blood stream]. With intravenous injection, the levels are much larger: 200 times. One millimole is a thousand micromoles, so 30 millimolar [of ascorbate in blood plasma] is a huge difference!

At these high pharmacological— or even super physiological— doses, Levine found that cancer cell populations dropped significantly. To understand why, it is important to know a bit about how cancer works.

Human DNA can wrap up tight (heterochromatin) or unwind into a loose, more open configuration (euchromatin). When it is wrapped up tight, the genetic information on the DNA cannot be expressed. This is because transcription, which is the process by which a cell reads and expresses the genetic code, requires access to DNA.

There are very specific times when DNA should be wrapped tight to maintain optimum health, and other times when one’s body needs to be able to use the instructions for cellular function that are contained in DNA. When DNA needs to be unwound, molecules called histone acetyltransferases (HATs) help to unwind it. When it needs to be wound up tight, the process is aided by histone deacetylases (HDACs).

HDAC overexpression is a hallmark of cancer cells, and hyperactive HDAC cells lead to messy, knotted DNA winding. This biological circumstance hinders the cell from reading important instructions found in DNA, which in turn prevents the production of important tumor suppressor proteins. At the same time, it leaves certain sections of the genetic code open that should not be expressed.

“Basically,” says Kaiser, “You remove the break from the car, and then you also step on the gas. And that’s cancer.”


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Matthew Kaiser.

The prevailing theory of how vitamin C acts on tumors is that it functions as a “prodrug,” meaning that it stimulates biochemical processes that allow something else to kill the cancer cell, rather than acting on it directly. In this case, the active agent is hydrogen peroxide, which is produced in saturated tissues by excess vitamin C. “Vitamin C acts as the Trojan horse that allows hydrogen peroxide to enter the tumor site,” Kaiser explains. “You can’t inject it straight in; your body will react too strongly. Hydrogen peroxide is a reactive oxygen species…it tears cells apart.”

However, since working on the project, Kaiser has found that this consensus on how vitamin C fights cancer isn’t necessarily the whole story. Pharmacological levels of ascorbate appear to selectively reduce the presence of proteins that regulate reactive oxygen species, like hydrogen peroxide, in cancerous cells. Some of these same proteins also happen to promote cell growth, which is not something that one would wish for cancer cells to do. In addition to producing hydrogen peroxide, ascorbate actually inhibits the runaway HDAC production that makes cancer cells so dangerous.

“What makes it really hard, really complicated,” Kaiser laments, “is that this might not work the same way for different types of cancer cells in different locations. There’s still so much to understand about how vitamin C is having this protective effect…That’s what’s lacking and that’s why we need studies like this.”


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And indeed, more studies are coming. In keeping with it’s mission to extend and promote what it calls “healthspan,” LPI hosts a bi-annual Diet and Optimum Health Conference, bringing together experts from around the world to talk about topics in orthomolecular medicine, among other fields. This year the conference, which was held at OSU in September, featured several speakers discussing vitamin C and cancer. One of them was Dr. Mark Levine, the NIH scientist who first showed the value of intravenous ascorbate.

Margreet Vissers and Anita Carr, of the University of Otago in New Zealand, also described their own advances on the subject. Vissers found in her studies that levels of 50 micromolar ascorbate in blood plasma (average dietary levels are between 40 and 80) had little to no protective effect against cancer. Doubling the amount to 100 micromolar, however, boosted a patient to the lowest level of the protective range. It would seem, then, that Pauling was right to suggest that mega doses might be important for optimum health.

Vissers also explained that, in animal models, ascorbate injected intravenously will peak after about twenty hours in both healthy tissue and in tumors. However, unlike the healthy tissue, tumor tissues hold onto the vitamin C and do not return to a natural baseline. This detail is important because it allows high doses of ascorbate to build up in tumor tissue, and these doses disproportionately kill cancer cells instead of healthy tissues for reasons that are still not fully understood.

Conversely, the dangers of using vitamin C, even in high intravenous doses, appear to be small. While some people harbor an enzymatic deficiency that can cause a severe negative reaction, most individuals simply cannot overdose on vitamin C. Even in the blood plasma, vitamin C usually reaches a saturation point and is filtered from the body.

At the LPI conference, Dr. Carr pointed out that this form of treatment also dramatically improves the quality of life of cancer patients as compared to chemotherapy. For one, vitamin C treatments involve significantly less pain, mental and physical fatigue, nausea and insomnia. As of March 2015, three clinical trials involving pharmacological levels of ascorbate have been conducted, all of them showing that it is well tolerated by patients and reduces chemotherapy-related toxicity.

Additionally, vitamin C at high doses is known to aid cognitive function, and these positive benefits work together to aid in social satisfaction for the patient. As Pauling pointed out in the 1970s, it is not only the disease that the doctor should be concerned about treating, but the patient as well.


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Pauling in 1989 – an extraordinary life. Photo by Paolo M. Sutter.

So is Linus Pauling vindicated when it comes to vitamin C and cancer? The answer is complicated.

On the one hand, it would appear that vitamin C can serve as an important preventative and treatment for cancer. However, the method that Pauling advocated— taking large supplemental doses orally— is pretty clearly not an effective form of application. Rather, contemporary research indicates that the levels of ascorbate that are required to slow or stop tumor growth are far greater than that which can be achieved naturally by ingesting vitamin C; they can be accomplished only by intravenous injections of ascorbate. Furthermore, it is likely that this form of treatment will not replace, but instead will augment, existing protocols including chemotherapy.

But the broader trend is optimistic and, one might argue, validating. And with the Linus Pauling Institute and many others around the world continuing to investigate the potential for vitamin C and other nutrients to help people live longer and feel better, exciting new studies on optimum diet and effective treatments for diseases like cancer would appear to be on the near horizon.

Vitamin C and Cancer: Raising the Stakes

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Ewan Cameron, Ava Helen and Linus Pauling. Glasgow, Scotland, October 1976.

[Part 3 of 4]

By 1970, the year that Linus Pauling published Vitamin C and the Common Cold, the federal government’s “war on cancer” was soon to arrive. The National Cancer Act, passed in 1971, increased federal funding for treatment and prevention research, embracing cytotoxic treatment solutions like chemotherapy. That same year, Pauling began to push for investigations between nutrition and cancer, especially concerning vitamin C. Since the role of vitamin C in immune defense is arguably much less significant than Pauling supposed, the idea that intake of vitamin C should prevent or treat cancer seemed ludicrous to many physicians. Incredibly, evidence is now emerging that the opposite might be true.

In hindsight, there is a tendency for critics to see Pauling simply as a politically liberal proponent of alternative medicine; one who lashed out against a consumerist medical establishment that was firmly supported by conservative citizens, among others. However, proponents of alternative health and holism in the 1960s and 1970s prescribed to a broad range of political ideologies; Pauling was just one among many people who were searching for better preventative and alternative treatments.

In 1980, when Pauling was actively campaigning for a vitamin C treatment for cancer, Americans spent 13.1 billion dollars on cancer diagnosis and treatment. Five years later, a survey of over one-thousand individuals showed that a majority believed clinics using unorthodox cancer therapies should be permitted to operate in the U.S., and just over half said they would seek alternative treatment if seriously ill.

Pauling and his ideological positions are remembered now as having been central to the vitamin C “movement.” Perhaps this is because he was renowned in many arenas and easily attracted a great deal of media attention. Or perhaps, especially knowing his penchant for protesting against nuclear weapons testing and war, this was another issue on which Pauling was the most outspoken opponent of what he saw as a wrong to be made right.


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Table from “Ascorbic acid and cancer: a review”, co-authored by Pauling and Cameron, 1979.

For Pauling, the continuing suffering of cancer victims was unnecessary, since a useful treatment was already cheap and readily available. He argued that,

The involvement of ascorbic acid (vitamin C) in the natural defense mechanisms is now known to be so great that we hope that a really significant control of cancer might be achieved by the proper use of ascorbic acid.

Of the studies that Pauling found so convincing, none were as crucial as those conducted at the Vale of Leven Hospital, near Glasgow, Scotland. There, Dr. Ewan Cameron found that mega doses of vitamin C (10 grams daily or more) seemed to slow and even reverse cancerous growth in some patients. He wrote to Pauling in 1971, who eagerly responded that this “attack” on cancer was the most promising application of vitamin C that he knew of.  Pauling, who had been studying the role of dietary vitamin C in issues of orthomolecular psychiatry such as schizophrenia, now shifted his focus to cancer.

Far from being the flaky alternative health guru that many came to see him as, Pauling’s work with vitamin C— like all his research on the subject of orthomolecular medicine (a field that he spearheaded)— was consistent with a biomedical model of molecular disease. Since Pauling saw this work as fitting within the framework of molecular biology, it was frequently unclear to him why the medical community resisted what was, to him, a straightforward and significant scientific endeavor.

Further complicating matters was the fact that Stanford University, Pauling’s academic home at the time, rejected his request for additional lab space to pursue cancer research. Now the target of regular media pummelings, Pauling’s ideas were becoming a potential source of bad press for the university. Refusing to take no for an answer, Pauling and his young lab assistant, Arthur Robinson, solicited private funding to continue their work on vitamin C outside of the university setting. Raising $50,000 in donations from wealthy supporters of vitamin therapies, the duo helped to found the Institute for Orthomolecular Medicine in 1973, subsequently renamed the Linus Pauling Institute of Science and Medicine (LPISM) one year later.

From 1973 to 1976, Pauling published co-authored articles with Cameron, who continued to study the effects of vitamin C on cancer from his base in Glasgow. And in 1975, Pauling and Robinson secured additional funds to begin their own animal testing. Two years later, the collaborators began reporting their results in the Institute’s newsletter.  In 1979 Cameron and Pauling likewise published an extensive review article in Cancer Research that cited previous studies corroborating their own conclusions. The duo published their book, Cancer and Vitamin C, that same year.


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A sample of Pauling’s notes compiled in response to the Mayo Clinic trials, 1979.

Cameron and Pauling’s data seemed to show that vitamin C would be especially valuable for cancer patients. Whereas a daily intake of 10 g of vitamin C in a healthy individual would bring the vitamin C level in the blood to a saturation point that could not be exceeded by increasing or prolonging intake, cancer patients showed a different pattern. Known already to have abnormally low blood levels of vitamin C, the patients in fact achieved just over half the same level of vitamin C blood saturation found in healthy individuals subscribing to a daily intake of 10 grams. For those afflicted with cancer, it was seen as necessary to take 10 grams a day just to reach the normal level of vitamin C found in healthy individuals who did not take supplements at all.

To Pauling, this alone justified continued research on the matter. After persistently stating his case to Dr. Vincent De Vita, director of the National Cancer Institute, two rounds of trials were conducted through the Mayo Clinic to solve what the medical community perceived to be problems in Cameron’s studies. When the trials indeed failed to produce anything like Cameron’s results, funding effectively dried up for vitamin C research – a significant blow to LPISM’s functional well-being.

In response, Pauling and his supporters argued that the Mayo Clinic was missing the point. The Mayo trials had attempted to measure the effectiveness of vitamin C in a manner similar to drug treatments, because the advent of chemotherapy and antibiotics, and the biases of the pharmaceutical industry, had placed primary medical emphasis on the disease, and not on the patient. Pauling saw the results of the Mayo studies not as a definitive defeat, but as the triumph of a complex of interdependent federal and private organizations that held a vested interest in supporting the chemotherapy status quo.


Pauling had claimed that, with vitamin C, lifespan could be increased, tumors could regress, and even full recovery was possible. For many in the medical community, these were not only foolish assertions, they were dangerous as well.

Dr. Charles Moertel, chairman of the Department of Oncology at the Mayo Clinic, was particularly vocal in his rebuke, stating that

For such a message to be conveyed to desperate and dying people, with the endorsement of a Nobel laureate, the presumption must be that it is based on impeccable scientific methodology.

Moertel’s implication, of course, was that Pauling’s argument was instead based on unsound science and certainly lacked the scientific basis to challenge the use of chemotherapy.

Yet vitamin C retained a broad appeal because many saw the prevailing treatment, and its manifold side effects, as inhumane. John Cairn, head of the Mill Hill Laboratory of the British Imperial Cancer Research Fund, provided a voice to the other side the coin by calling out the survivorship data. To wit: in 1986, 200,000 patients were receiving chemotherapy and, by 1991, five year survival rates for colon cancer remained at just 53%. Cairn spoke for many in suggesting that, when it came to the prevailing course of treatment, “the benefit for most categories of patients has yet to be established.”


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Ava Helen Pauling, June 1981.

For Pauling, the debate turned from the public to the personal when, at the height of his study of vitamin C, his wife Ava Helen was diagnosed with stomach cancer. Following Ewan Cameron’s advice, she took 10 grams of vitamin C daily, and did not receive chemotherapy.  Throughout her treatment, Linus clung to the belief that mega doses of vitamin C would work for Ava Helen, just as it had for Cameron’s success stories in Scotland.

“Daddy was convinced that he was going to save her,” remembered Linus and Ava Helen’s daughter, Linda. “And that was, I think, the only reason he was able to survive… He said to me after she died that until five days before, he thought he was going to be able to save her.”

Ava Helen Pauling passed away in December of 1981. And though he was badly shaken by his wife’s death, belief in the value of vitamin C in the fight against cancer did not fade from Pauling’s mind. Suffice it to say, the medical community remained whole-heartedly unconvinced.

Continuing Work on Vitamin C and Cancer: An Interview with Matthew Kaiser

Matthew Kaiser.

Matthew Kaiser.

The blog recently had the opportunity to sit down with Matthew Kaiser, an Oregon State University undergraduate senior in microbiology from Salem, Oregon.  Kaiser, who hopes to pursue a career as an MD/Ph.D., has led an exciting research project on the potential treatment of cancer using intravenous vitamin C.  He also recently delivered a talk titled “Is Humanity Ready for an Upgrade?” at a recent TEDx symposium hosted by OSU.

What follows below is an edited excerpt of our interview with Kaiser in which he discusses the roots of his project, its potential application, and his experience of conducting and presenting high level research at a very young age.

The Roots of the Research Project 

The beginnings of this research project were more or less like most undergraduate project tend to start. Not all, but some tend to be these big black box projects, we call them, in that there are a lot of unknowns. It’s almost like, “we really don’t know a lot about this but hey, we’ll give it to an undergraduate to take a stab at it. Because even that way if it doesn’t work out, if we find out that there really is no story here, they get the research experience and then we don’t necessarily waste a graduate student’s time or post-doc’s time on a project that didn’t end up being published.”

But where this project started was, of course, back in the days of Linus Pauling who was among the first to suggest that high doses of Vitamin C could have an anti-cancer effect. But following his initial studies with Vitamin C, or ascorbate, there were studies that came out by the Mayo Clinic and other labs that showed that Vitamin C did not have a protective or anticancer effect. And so it was largely abandoned by the medical community for several years but it continued to be researched in kind of an alternative medicine environment. Through that, as our understanding of how Vitamin C is metabolized by the body developed, we were able to understand that if Vitamin C was delivered orally, it was completely different than how Vitamin C could be regulated if it was administered through an IV, because if you administer it through an IV you’re able to bypass all the digestive control and renal reabsorption in your small intestine. That normally would limit the amount of Vitamin C that gets into your bloodstream and then becomes vitally available.

So this project started kind of on the cusp of these exciting studies looking at the pharmacokinetics and, again, looking at the bioavailability of Vitamin C. And just to put it in perspective: so if you go home and eat fifty oranges, like all my friends like to try and do because they know I work on Vitamin C, they’re like “oh, Vitamin C and cancer, I can eat fifty oranges, right? And I can prevent cancer or cure myself or colon cancer?” And what we’re looking at in this project are doses that can only be achieved by IV because if you eat these fifty oranges, the maximum you can saturate your blood plasma level is about 220 micromolar. To put it in perspective, so if you can saturate your blood to a level of about 200 micromolar following oral ascorbate, if you go home and had an IV or you went to a clinic and you had an infusion of IV ascorbate, you can saturate blood plasma up to 30 millimolar. And there’s a thousand micromolars in one millimolar. So, extremely different doses can be achieved by these two different routes.

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The Discovery of Human Plastin at the Pauling Institute

Milestones in Plastin Research

[Guest post written by John Leavitt, Ph.D., Nerac, Inc., Tolland, CT.]

In 1985 my lab at the Linus Pauling Institute of Science and Medicine (LPISM) in Palo Alto, California started to work on an abundant protein of white blood cells (lymphocytes, macrophages, etc.) that mysteriously appeared in human tumor-derived cells of solid tissues (carcinomas, fibrosarcomas, melanomas, etc). I had noticed this phenomenon a few years earlier while at the National Institutes of Health. I also noticed that this protein appeared in oncogenic virus-transformed (SV40 virus) human fibroblasts, but the protein was not expressed in the normal fibrolasts.

I was intrigued by the fact that a major protein of circulating blood cells would be induced during solid tumor cell development because it is well known that solid tumor cells become more anchorage-independent and can circulate like white blood cells to metastasize to other organs. My colleague, David Goldstein, took the lead in examining the expression of this mysterious protein in different cell types of fractionated white blood cells. At the time this protein was assigned only a number (p219/p220) corresponding to its position in two-dimensional protein profiles. We found that this protein was abundantly expressed in all normal white blood cell types that we examined but it was not expressed in normal cells of solid tissues (Goldstein et al, 1985).

When David’s paper was submitted to Cancer Research, the reviews came back positive and the paper was accepted for publication, but one reviewer asked that we give the protein a name. I was thrilled by the thought of naming a protein and its gene which would immortalize our work, so I took on the serious task of coming up with a name that had lasting meaning. My theory was that this cancer marker contributed in some then-unknown way to the plasticity of the cytoplasm in solid tumor cells because of its normal presence in circulating white blood cells. Also, I had seen the great movie, The Graduate, with Dustin Hoffman and recalled that amusing scene depicted in the picture included below. So I named the protein “plastin” – the greatest new thing since sliced bread. 🙂

The Graduate

That same year, I met Steve Kent from Caltech at a meeting in Heidelberg, Germany. After hearing my talk, Steve suggested that we collaborate. He mentioned that a postdoctoral fellow in Leroy Hood’s lab, Dr. Ruedi Aebersold, was trying to develop a more sensitive protein sequencing method for purposes of determining snippets of amino acid sequences from small amounts of unknown proteins eluted from two-dimensional gels (protein profiles) like the gels that we used to characterize plastin in David’s paper. If we could get an accurate partial sequence of plastin, we could devise a nucleic acid probe based on the genetic code that could be used to clone a plastin “copy DNA” from a cDNA library. If the plastin cDNA was cloned, we could then define the protein and perhaps its function by determining the nucleic acid coding sequence in the clone.

Madhu Varma.

Madhu Varma.

I gave Dr. Madhu Varma at LPISM the arduous task of isolating the plastin polypeptide “spot” for sequencing. Madhu cut out the stained spot from 140 two-dimensional gels, in effect purifying enough protein for sequencing by Ruedi at Caltech. Madhu succeeded and Ruedi produced eight short peptide sequences that could be used to develop short nucleic acid probes that would hybridize to the plastin cDNA clone isolated from a tumorigenic human fibroblast cDNA library.

Ching Lin.

Ching Lin.

Dr. Ching Lin at LPISM took one of the nucleic acid probes and immediately attempted to screen a tumorigenic fibroblast cDNA library. If we identified any clones that bound this probe, then we would perform a quick test to determine that we had cloned the plastin coding sequence. But science is full of surprises and we found that the first clone he isolated detected a gene product that was not in lymphocytes but only in normal human fibroblasts – in other words, it failed the test. This is where Ching’s brilliance took over. He was convinced that this first clone he had isolated was indeed a plastin coding sequence so he used this clonal DNA as a new probe against the tumorigenic fibroblast cDNA library. He isolated a new clone that passed the test and detected a gene that was expressed in lymphocytes and tumorigenic fibroblasts but not in normal human fibroblasts.

We performed other experiments that proved that we had cloned two different isoforms of plastin: L-plastin, expressed in lymphocytes and solid tumor-derived cells, and T-plastin that was expressed in normal solid tissues and co-expressed with L-plastin in tumor cells from solid tissues (Lin et al, 1988; Lin et al, 1990). Ultimately this work led to the complete characterization of the human plastin multigene family and verification that both isoforms were aberrantly expressed in various types of human tumors.

The figure at the top of this post maps the progression of discovery that followed our research, which began at the Pauling Institute in 1985. Our publications are shown in red in the graph and research published by other labs is shown in the blue bars.

Here are several plastin milestones discovered by other researchers:

  • T-plastin is abundantly induced in Sezary lymphomas, a lethal T-lymphocyte cancer (Su et al, 2003);
  • L-plastin induction in solid tumors contributes to invasive cancer growth and metastasis (Klemke et al, 2007);
  • Mutations in T-plastin play a role in the genetic disease Spinal Muscular Atrophy (Oprea et al, 2008); and
  • Most recently mutations in both L- and T-plastin promote re-growth of colon carcinomas following surgical resection of these tumors and chemotherapy (Ning et al, 2014).

These developments are more or less typical of the way science works. Progress in understanding complex phenomena like human cancer is the work of many scientists that builds on the observations of other scientists. This is just one example of the productive contributions in biomedical research that came about through early discovery research at LPISM in the 1980s.

Research Completed at LPISM in 1988 – Reproduced and Extended in 2014

The author in his laboratory at the Linus Pauling Institute of Science and Medicine. Originally published in Science Digest, June 1986.

The author in his laboratory at the Linus Pauling Institute of Science and Medicine. Originally published in Science Digest, June 1986.

[Guest post written by John Leavitt, Ph.D., Nerac, Inc., Tolland, CT.]

In 1987, my colleagues at the Pauling Institute in Palo Alto, colleagues at Stanford and I published a paper that clearly demonstrated that expression of a charge-altered mutant human beta-actin (glycine to aspartic acid substitution at amino acid 245; G245D) caused non-tumorigenic, immortalized human fibroblasts to form aggressive tumors in nude mice (Leavitt et al, 1987a). When these tumor-derived cells were examined, we discovered that they exhibited further elevated expression of the mutant beta-actin and these tumor-derived cells formed tumors even more rapidly – observations that were consistent with the role of this mutation in the tumorigenic phenotype. Furthermore, over-expression of mutant beta-actin was associated with down-regulation of three abundant tropomyosin isoforms in a well-documented transformation-sensitive manner (Leavitt et al, 1986; Leavitt et al, 1987a and Ng et al, 1988). These final papers were the culmination of research conducted at the Linus Pauling Institute of Science and Medicine (LPISM) from December 1981 to March 1988.

Normally when a scientific observation is never repeated it is usually not worth remembering. In this case, twenty-six years after our 1987 publication, a study was published by Schoenenberger et al. at the Biozentrum in Basel, Switzerland, that reproduced our findings in a different cell system, a rat fibroblast model (provided to them by LPISM in 1986). Furthermore, these investigators extended our findings by characterizing new aspects of abnormal behavior of the mutant beta-actin and cells that express this aberrant protein, which help to explain its potential role in cancer such as enhancement of tumor cell motility and invasiveness.

In addition to enhancement of tumor growth and alteration of cell shape, the Swiss investigators presented the following findings to clarify and support the oncogenicity of this mutation:

  1. The mutant actin stimulated formation of ruffles at the cell periphery as shown by staining of cells with an antibody that bound specifically to the mutant epitope of the mutant beta-actin (left image below)
  2. The mutant actin concentrated primarily in these ruffles (palloidin staining reveals the location of filamentous actin in stress fibers; right image below)
  3. The expression of mutant actin inhibited the tropomysin binding to filamentous actin and tropomysin did not accumulate in the ruffles
  4. Mutant actin colocalized with Rac1 (a GTPase mediator of membrane ruffling) and beta1-integrin (adhesion protein) in the ruffles

ruffles

Back-tracking several years, the discovery of this actin mutation was made in a mutagenized cell line isolated by Takeo Kakunaga at the National Cancer Institute (NCI) in 1978. During the month that his paper was published, I walked over to NCI from my lab across the street at the Bureau of Biologics (FDA) to have a chat with Takeo about using his in vitro transformed Syrian Hamster cells as a model system to identify changes in protein expression that correlated with neoplastic transformation. After describing what I wanted to do, he seemed agreeable but then casually mentioned that he had succeed in transforming human fibroblasts into tumor forming cells. I nearly fell off my chair because human cells had never been transformed in vitro before, a major problem for cancer researchers at that time.

I blurted out that we should do the work that I had proposed in his human cell model system, comparing protein expression by the transformed neoplastic cells with their normal precursor cells. My hypothesis was that this comparison would allow identification of proteins that were turned on or turned off in expression by comparing protein profiles of the most abundant 1,000 proteins expressed in these cells and resolved by high-resolution 2-D gel separation (protein profiling). My plan was to look for charge-altering mutations in proteins that might govern neoplastic transformation and tumorigenesis. A fall-back goal was to define the pattern of qualitative and quantitative changes in protein synthesis to try and get a handle on the mysterious mechanism of human cancer development. A summary of the global changes in gene expression of neoplastic human fibroblasts was published from LPISM in 1982 (Leavitt et al, 1982).

Within two weeks, in May of 1978, I was metabolically labeling the total cellular proteins (with the amino acid S-35 methionine) of the normal fibroblasts and three strains of cell lines derived from the normal culture which were immortalized, only one of which formed subcutaneous tumors in nude mice. After four hours of labeling, I prepared extracts of S-35 methionine labeled proteins from each of the four cultures and loaded 25-microliter aliquots of each sample onto the top of clear noodle-like isoelectric focusing gels (7-inch long urea-polyacrylamide gels with the thickness of thin spaghetti) which separated the complex mixture of total cellular proteins by their net charge (isoelectric point). These gels were subjected to isoelectric electrofocusing of the proteins overnight. The next morning I harvested the spaghetti-like gels, and incubated them in a detergent that would bind to the proteins to help separate them by their molecular weights in a second dimension. So, these proteins were first denatured and separated by their net charge and then, in a second dimension, separated by their size on a thin rectangular slab gel.

After about five hours of separation in the second dimension, I was soon to learn that I had separated more than 1,000 denatured protein subunits (polypeptides) by their differing charges and molecular weights. The final step before autoradiography, which revealed the full protein profile, was to fix and stain the gels to get a glimpse of the resolution of these peptide patterns. The staining of these rectangular gels revealed only the most abundant architectural cellular proteins, the largest number of which were cytoplasmic beta- and gamma-actin, at a ratio of about 2:1 in abundance, respectively. The figure below shows what quickly appeared as the gels were de-stained. In the one tumorigenic cell line, instead of seeing a 2:1 ratio of beta- to gamma-actin, a new abundant protein at about one unit charge more negatively charged (more acidic), and half of the normal beta-actin was lost. The pixilation of these three radioactive “spots” immediately suggested to me that one of the two functional genes (alleles) encoding beta-actin had mutated, possibly due to the replacement of a neutral amino acid with a negatively charged amino acid. This prediction was no mystery to me as I had demonstrated this type of electrophoretic shift in another protein a year earlier at Johns Hopkins.

mutant actin further annotated

A number of experiments were done to build the case for the beta-actin mutation, and then I wrote a letter to Klaus Weber at the Max-Planck Institute in Goettingen, Germany, asking for his help in sequencing these actins. His lab was the only one in the world sequencing actins, e.g. the four muscle forms of actins. It only took Klaus two weeks to respond affirmatively, an indication to me that he was eager. I provided him with the actin proteins from this cell line and it took a postdoctoral fellow, Joel Vandekerhkove, and Klaus a little over a year to determine the complete amino acid sequences of the mutant beta-actin and both the wildtype beta- and gamma-actins, to define the mutation that had occurred. We published the result shown above in the top journal Cell in December 1980. Four years later, my colleagues at Stanford and I published my cloning of the mutant and wildtype human beta-actin gene, and the experiments that formally proved the mutation at the level of the gene (Leavitt et al, 1984). Three years after that, we published the experiments that demonstrated the tumorigenic effect of this mutation in immortalized human fibroblasts.

The dramatic nature of this discovery was never fully appreciated, perhaps, because no other actin mutations had been reported and it took Scheonenberger, et al. twenty-six years to complete the work published in September 2013. In another recent related development, Lohr et al. reported reoccurring beta-actin mutations in a panel of tumor cell samples from patients with diffuse large B-cell lymphoma.

One interesting piece of information that came out of our initial sequencing of these actins was the degree of evolutionary conservation of human beta- and gamma-actin. These two actins differ by only four amino acids at the N-terminus, whereas the four muscle-specific human isoforms are more divergent. Comparing the sequence of actin cloned from Saccharomyces cerevisiae (yeast) with these human sequences (sequences stored at the National Center for Biotechnology Information; NCBI) reveals that yeast and human cytoplasmic actins are 92% identical in their sequences (differing by only 31 amino acids out of 375) and most of these amino acid exchanges are conservative replacements both structurally and thermodynamically. This makes these actins the most highly conserved proteins (on a par with histones H3 and H4) among the 20,000 or so known human protein sequences. This fact presents an argument for the fundamental importance of non-muscle cytoplasmic actins in eukaryotic life. It turns out that among actin sequences of all species, no replacement of the Glycine 245 has ever been documented as a result of species divergence or mammalian isoform divergence.

When we introduced the mutant beta-actin gene into a non-tumorigenic immortalized fibroblast strain by gene transfer (Leavitt et al, 1987a), we isolated a transfected clone in which the ratio of exogenous mutant beta-actin to wildtype beta- + gamma-actin was 0.88 – a 76% higher level of expression than the mutant actin in the original mutated cell line in which the mutation arose (0.5 ratio). When we isolated and cultured the cells from a tumor formed by this cell line, the ratio of exogenous mutant beta-actin to wildtype beta- + gamma-actin had increased to 1.95, indicating that about 64% of the total cytoplasmic actin was the mutated beta-actin. Whereas the initial transfectant cell line produced visible tumors at about six weeks, the tumor-derived transfectant cells expressing 64% mutant actin formed visible tumors at about 1.5 weeks. Thus, expression of this mutation was not inhibitory to cell growth.

The other surprising finding was that cell lines expressing the transfected mutant actin gene did not have higher levels of cytoplasmic actins in them because the two endogenous wildtype beta- and gamma-actin genes were coordinately down-regulated (auto-regulated) so that the relative rates of total actin synthesis remained around 30% compared to S-35 methionine incorporation into 600 surrounding non-actin polypeptides in the protein profile (Leavitt et al, 1987b). This auto-regulation phenomenon was reproduced by Minamide et al. (1997) ten years later.

Cytoskeletal rearrangement of actin microfilaments, as well as changes in composition of tropomyosin isoforms and other actin-binding proteins, have long been associated with neoplastic transformation. However, before our study, causal mutations in a cytoplasmic actin had apparently not been considered. It is perhaps consistent then that Ning et al. (2014) have recently described genetically inherited polymorphisms in the actin-bundling protein, plastin (also discovered and cloned at LPISM), that significantly affect the time of tumor recurrence in colorectal cancer after resection and chemotherapy.

During my tenure at the Pauling Institute, I felt that Dr. Pauling understood and appreciated this work and its relevance to the fundamental nature of cancer development. Progress can be slow, but ultimately true understanding of cancer will emerge from this type of research…and I predict that cytoplasmic actins and actin-binding proteins that regulate actin organization and function in the cytoskeleton will be understood to play a central role in the manifestation of the tumorigenic phenotype.

Irwin Stone’s Impact on Pauling

Linus Pauling and Irwin Stone, 1977.

Linus Pauling and Irwin Stone, 1977.

[Part 2 of 2]

Four years after Irwin Stone first convinced Linus Pauling to start taking megadoses of vitamin C, Pauling decided to share with the world the successes that he had observed in his own improved mental and physical health.

In 1970 Pauling began to work on a book, Vitamin C and the Common Cold, and he wrote to Stone asking permission to dedicate it to him. He also sent Stone a copy of the manuscript to review. Stone wrote back praising the work.

The book is excellent and should go far to eliminate this thoroughly unnecessary and annoying condition, at least among your readers. The audience will increase over the years, especially if Medicine can eventually see the light.

Stone continued to encounter difficulty getting his own scientific articles about ascorbic acid published and he certainly did not have the funding to run his own clinical trials. Partly as a result, he too was writing a book about vitamin C and all of the many diseases that he thought were related to hypoascorbemia. A  major thrust of the book was its plea for large scale research on the topic. Stone hoped to get popular opinion on board with his ideas in order to place pressure on physicians and nutritionists to do research in this area.

Pauling’s Vitamin C and the Common Cold was a popular success. Many readers around the world were persuaded by his ideas and began to take vitamin C supplements to prevent and treat colds. Some of his acclaim rubbed off on Irwin Stone, who wrote to Pauling telling him that he too was finally receiving recognition from popular media sources, including NBC.

In 1971 Stone retired to San Jose, California and devoted the rest of his life to researching and promoting the need for high consumption of vitamin C by humans. That same year he finished his book, The Healing Factor: Vitamin C Against Disease, and asked that Pauling write a foreword for it. Pauling was glad to do so, calling it “an outstanding contribution to knowledge.”

Stone's inscription to Pauling in a first edition of The Healing Factor, 1972.

Stone’s inscription to Pauling in a first edition of The Healing Factor, 1972.

Despite their popular appeal, Pauling and Stone continued to encounter problems convincing medical practitioners and researchers to take their ideas about ascorbic acid seriously. Stone believed that this was so because vitamin C would be a much more inexpensive cure than the current treatments of the time, causing pharmaceutical companies and doctors to lose money.

One medical doctor, Ewan Cameron, did believe in the effectiveness of vitamin C against cancer and was treating his terminal cancer patients with megadoses of it in Glasgow, Scotland. He formed a trans-Atlantic research partnership with Pauling in 1971 and they began to collaborate on papers discussing the use of vitamin C against cancer, eventually publishing ten articles together.

Through his partnership with Pauling, Cameron also began to correspond with Stone about the implementation of vitamin C against cancer and their shared difficulties getting the medical community to accept their hypotheses.

Cameron maintained a unique viewpoint on the treatment of cancer and how ascorbic acid might fit into a clinical regimen. In December 1974, he explained his views to Stone.

It is completely contrary to all contemporary medical thought to even suggest that such a mundane substance as ascorbic acid could have any value in such a complicated disease as cancer. This is because cancer research is concentrating all its energies in searching for more and more sophisticated ways of selectively destroying cancer cells. The research is becoming so complex and so unproductive, that it is natural to assume that ‘the answer’ must be extraordinarily complex and almost beyond human comprehension….We would make much more progress if we accept that cancer cells are normal cells that merely happen to be behaving in an abnormal way. We would then accept that cancer cells have an equal right to live, and concentrate our energies in suppressing the abnormal behavior pattern.

Throughout their correspondence, Cameron described his successes treating cancer with ascorbic acid. But he also noted that a number of patients showed no improvement from it or, at best, their cancer was brought to a standstill. He was disappointed that his primary successes were mostly by way of increasing patients’ survival time, not in curing them. Cameron thought that the greatest success would be in prophylaxis – taking megadoses of ascorbic acid throughout one’s life in order to prevent cancer.


In 1978 Stone wrote a letter to the editor of Nutrition Today in response to the publication’s recent issue focusing on ascorbic acid. His letter shows how fervently he believed in hypoascorbemia.

I regard our most serious medical problem to be the dangerous complacency that the orthodox medical establishment exhibits toward Chronic Subclinical Scurvy and its refusal to do anything to correct and alleviate this potentially-fatal human birth defect. Chronic Subclinical Scurvy has killed more human victims, caused more disease and misery among Mankind than any other single factor in the past and is continuing this evil record in the present. I’m worried about the future, because that is where I’m spending the rest of my life.

Meanwhile, Stone and Pauling’s relationship continued to flourish. In 1977 Pauling invited Stone to become a member of the Board of Associates of the Linus Pauling Institute of Science, an offer that was accepted. Pauling also attended Stone’s surprise 70th birthday party that year. In 1981 Stone was unable to make it to Pauling’s 80th birthday, but he did pass along a message.

You will recall the promise I made you in 1966 of 50 more healthy years of life with Megascorbics. You thought I was exaggerating and said you would be satisfied with 15 years. Well the 15th year is now and I am looking forward to attending your 115th birthday party in 2016. Megascorbics makes you practically indestructible.

In response, Pauling wrote, “I am glad to express my thanks to you for having written to me in 1966. Your letter and the reprints of your papers changed my life.” While Pauling did not make it to 2016, he did live until 1994, passing away at 93 years of age.

The last letter that Pauling wrote to Stone concerned a joint award from the Academy of Orthomolecular Psychiatry and the Orthomolecular Medical Society that Stone was to receive. The Linus Pauling Institute of Science and Medicine was also going to surprise him with a second award. Pauling wrote,

For many years you have been an inspiration to me, because of your devotion to vitamin C and your conviction that a high intake of vitamin C has great value in improving the health of human beings. You have rendered a great service to the people of the world through your continued study of vitamin C over a period of fifty years.

Unfortunately, Dr. Irwin Stone died on May 4, 1984, at the age of 77, while in Los Angeles to receive the award. He died by choking on regurgitated food, the result of a constricted esophagus that had plagued him ever since his car accident many years prior.

Irwin Stone received two honorary doctorates, many additional awards, and 26 patents. He also published over 120 scientific papers throughout his life (at least 50 were about vitamin C) and wrote one book, The Healing Factor, published in 1972. He was father to one son, Steven, and was married to his wife Barbara for over 50 years.

In December 1986, two years after his death, Barbara Stone sent Pauling a card congratulating him on the publication of his latest book, How to Live Longer and Feel Better. She wrote “Irwin would have enjoyed reading it and noting the many references to him and other colleagues.” Pauling hadn’t exaggerated in his 1981 letter: Irwin Stone really did change his life and made a profound impact on the scientific legacy that Pauling leaves behind today.

Pioneering the Field of Proteomics

John Leavitt, 1982.

John Leavitt, 1982.

[Guest post written by John Leavitt, Ph.D., Nerac, Inc., Tolland, CT.]

In the fall of 1985, I went to a small meeting in Heidelberg, Germany, with Steve Burbeck from the Linus Pauling Institute of Science and Medicine, who had helped me by developing computerized microdensitometry to analyze two-dimensional protein profiles. At this meeting I described our protein profiling work and the discovery of the mutant beta-actins and another interesting protein which I named “plastin.”

Steve Kent, head of the protein sequencing facility in Leroy Hood’s lab at Caltech, heard my talk. We sat across from each other at dinner and he proposed a collaboration to develop methods of sequencing minute amounts of protein leached from spots in high resolution protein profiles. Lee Hood was well known for developing state-of-the-art protein and nucleic acid sequencing methods and machines, and was a founder of Applied Biosystems in Foster City, CA.

After I returned from Heidelberg, Ruedi Aebersold called me from Caltech and we began collaborating on microsequencing of pure nanomolar quantities of unknown proteins of interest eluted out of my protein profiles. In this work we essentially started the field of proteomics, which was eventually named ten years later by Jim Garrells, a protein profiler at Cold Spring Harbor. Proteomics is the search for and definition of proteins that could serve as diagnostic markers and drug targets for diagnosis and treatment of diseases, in our case cancer.

In 1987 we published a landmark paper in PNAS on the microsequencing technique that Ruedi developed. This paper would eventually be cited in references by more than 1,000 other research papers.

Ruedi

I gave a postdoctoral fellow, Mahdu Varma, the task of isolating the cancer-specific leukocyte isoform of plastin (L-plastin) from 140 protein profiles. This protein has been implicated in metastases in both melanoma and prostate cancer as well as in other aspects of cancer. The L-plastin spot was easily recognized and those spots on a nitrocellulose filter were “snipped out,” removing all the other proteins of the cell. We sent Ruedi a plastic tube containing the 140 “spots” of L-plastin. He had figured out a way to solubilize the protein from the nitrocellulose and was successful in determining the sequence of eight oligopeptides of between eight and sixteen amino acids derived by digestion of L-plastin with a proteolytic enzyme.

The peptide sequences he determined turned out to be perfectly accurate internal amino acid sequences of plastin when we decoded the sequence of the plastin gene (cDNA) clone, a reverse transcript of the messenger RNA. This was the first time that anyone had done this and it opened up the field of proteomics and led to the discovery of other diagnostic and drug targets.

plastin 1

We had chosen L-plastin, normally only expressed in white blood cells, because I had reported for years that it was a cancer marker in tumors that arose in solid tissues (identified in the image above by the two upward arrows). After we received the oligopeptide sequences from Ruedi, we made short DNA antisense probes that would hybridize to DNA sequences encoding these peptides in the human genome to fish out the full-length DNA clones that carried the sequence of the L- plastin gene.

Ching Lin and I, along with Reudi, published the sequences of the human L- and newly discovered T-plastin proteins, based upon sequencing of cDNAs, in Molecular and Cellular Biology. The discovery of a second isoform of plastin (T-plastin named for tissue plastin as opposed to L-plastin from leukocytes) was a surprise. We now had two genes to characterize at the genomic level. Today, T-plastin is a well recognized marker for cutaneous T-cell lymphoma (Sezary Lymphoma) and L-plastin, inappropriately expressed in solid tumor cells (carcinomas, fibrosarcomas, etc.), is understood to be a contributor to metastasis.

The Linus Pauling Institute was not all work and no play in the 1980s

We worked hard at the Institute and Linus Pauling was always there and visible.

We put together a softball team with Jim Fleming, Dan McQueeny, Zelek Herman, myself, and others at the Institute and played departmental teams at Stanford. I think we were called the “Pauling squeeze.” After these games we would often go dancing at the Class Reunion on El Camino Real near the corner of Page Mill Road.

We were fortunate to have on staff a first rate fundraiser in Richard (Rick) Hicks who arranged wonderful parties on Nob Hill at the Stanford Court. The most memorable of these parties occurred in late November 1986, when we honored Japanese billionaire Ryoichi Sasakawa with the annual Linus Pauling Medal. Another year Carl Sagan and Ann Druyan, who helped Carl put together the Cosmos series, likewise took part. We often saw Dr. Pauling’s sons, Linus Pauling Jr., Peter, and Crellin as well.

Here we are at the Stanford Court that night with postdoctoral fellows, Dr. Karin Sturm from Heidelberg, Germany, on the left and Dr. Madhu Varma from Madras, India, on the right. My wife, Becki, is in the middle. I recall that Dr. Pauling enjoyed this night as well.

Here we are at the Stanford Court that night with postdoctoral fellows, Dr. Karin Sturm from Heidelberg, Germany, on the left and Dr. Madhu Varma from Madras, India, on the right. My wife, Becki, is in the middle. I recall that Dr. Pauling enjoyed this night as well.

In 1988 I moved on to a new position in San Jose and then became Director of Research at Adeza Biomedical. Since we continued to live in Palo Alto, we continued to interact and party with the Linus Pauling Institute staff into the 1990s.

The 1980s at the Linus Pauling Institute – A Wonderful Place to Be

John Leavitt

John Leavitt

[Ed Note: This is part one of a two part series of guest posts written by John Leavitt, Ph.D., Nerac, Inc., Tolland, CT.]

There was an article about Linus Pauling in Time magazine in early 1981 about the fact that at the age of 80 he was still seeking a grant from the National Institutes of Health (NIH) to fund his research on ascorbic acid for treating diseases. This news caught my attention and I looked into the possibility of joining Dr. Pauling’s institute. Toward the end of the summer I was invited to visit the Pauling Institute in Palo Alto, CA to give a seminar on my research at NIH.

In late August Koloman Laki, an aging scientist at NIH, called me up and invited me over to his lab in NIH Building 10, a short walk across the campus from my lab in NIH Building 37. He was interested in talking to me about my recent discovery of mutations in human non-muscle cytoskeletal actin that was published in Cell in late 1980. This protein is the major architectural protein of all eukaryotic cells and we had shown that it was the most highly conserved protein in evolution of the species from yeast to humans. This fact made these mutations even more interesting.

Koloman was a protege of the Hungarian Nobel Prize winner Albert Szent-Györgyi who, I later learned, was much admired by Dr. Pauling because he had discovered both vitamin C and actin. Koloman described how Szent-Györgyi discovered muscle actin. When I mentioned that I was to visit the Linus Pauling Institute in late September, he told me about Emile Zuckerkandl’s and Dr. Pauling’s work on the ‘biological clock,’ which provided evidence in support of Charles Darwin’s theory on divergence of the species.

In the last week of September I flew to Oakland, CA and was picked up at the airport by Emile who was President of the Linus Pauling Institute of Science and Medicine. The next morning I stood up in front of Dr. Pauling and the institute staff to tell them about my discovery of a mutant human beta-actin and my speculation on its involvement in neoplastic transformation. The evidence suggested that I had actually discovered at least two mutations in the same gene, each of which caused a progression to a higher malignant state.

Linus Pauling was in the front row and was all smiles. He asked me if I knew who discovered actin. I was prepared to answer that question thanks to Koloman Laki. In the afternoon I met with Emile who offered me a Senior Scientist position at the Institute, which I accepted. At the time it would be me and Dr. Pauling with separate research interests. Nevertheless, Dr. Pauling could appreciate my discovery as, 32 years earlier, he had described the molecular basis for sickle cell anemia, which predicted that mutations in hemogloblin governed the sickled shape of red blood cells which caused the disease, sickle cell anemia. Likewise, human cancer cells exhibit altered shapes.

So I resigned my secure job-for-life at NIH and moved to Palo Alto to join the struggling Linus Pauling Institute. My technician, Patti Porecca, hired from Bob Gallo’s lab at NIH, would follow me to the Pauling Institute.

Cloning of the Human Beta-Actin Gene

After I arrived at the Pauling Institute, two of my colleagues at NIH and I published a comprehensive study of the changes in protein expression between normal and neoplastic cells in Carcinogenesis using high-resolution computerized microdensitometry to analyze the complex protein patterns (my first paper from the Pauling Institute). This was the first time that such a study had been published, e.g. the comparative profiling of expression of a large number of proteins in neoplastic cells. It was a study of the 1,000 most abundant proteins in normal and neoplastic human cells which revealed potential biomarkers and causative genetic events for human cancer. At the time it was staggering to view these patterns but perfect for my dyslexic brain and mind’s eye. In addition, we published another paper in Cell that described, for the first time, the progression of a neoplastic human cell to a higher malignant cell following a second mutation in the same beta-actin gene. Early in 1982, Steve Burbeck and Jerry Latter at the Institute set up the same computerized microdensitometry platform I had exploited at NIH.

Jerry Latter gave a stirring talk at Argonne Labs in Chicago demonstrating that computerized microdensitometry of protein profiles could be used to determine the identities of unknown proteins based upon determining their amino acid compositions in situ in protein profiles. This paper was published in Clinical Chemistry in 1984. At the same meeting, Steve Burbeck described a truly innovative invention that could measure beta-particles emitted from radioactive protein profiles to produce a direct image of the protein profile pattern. As a group we had entered an exciting period of discovery and innovation at the Linus Pauling Institute.

When I got to Palo Alto in December 1981, I called Professor Larry Kedes at Stanford and we embarked on a collaboration to clone the human beta-actin gene. His impressive postdoctoral fellow, Peter Gunning, taught me some basic recombinant DNA techniques, and I was off to the races. The difficulty was to identify the functional gene in a sea of actin pseudogenes (sometimes referred to as junk DNA). I used an elegant method of homologous recombination developed in Tom Maniatis’ lab at Harvard that had never been used before to clone a novel gene (In fact, cloning of human genes was just getting started at the time). This was smart because Professor Maniatis would be the chairman of the NIH study section that reviewed my first grant proposal submitted from the Pauling Institute. I did not know it at the time but within a month or two I had cloned the functional beta-actin gene a week before Christmas in 1982.

I developed a scheme to identify the correct gene among 300-400 clones of pseudogenes that Patti and I had cloned and the strategy worked. We gave Dr. Sun-Yu Ng the task of sequencing the DNA clone that we were betting on. Rather quickly we determined that we had cloned the functional human beta-actin gene because the DNA sequence that Sun-Yu determined from our candidate clone accurately encoded the amino acid sequence of human beta-actin protein that I had published in Cell in 1980 (with Klaus Weber). Quite coincidentally another lab discovered a rat oncogene that was a fusion of part of an actin gene with a tyrosine kinase gene. I sent this information off to the study section that was reviewing my grant in January 1984 as added evidence that the actin gene was in some way relevant to neoplasia.

My colleagues and I at the Pauling Institute and Stanford published our successful isolation of both the mutant and wildtype human beta-actin genes in Molecular and Cellular Biology in October 1984. As shown below, we had given Armand Hammer’s name to our cancer research program because of his generosity in helping to fund the Linus Pauling Institute.

actin cloning

In January 1984 I was awarded a grant of about $110,000 a year for two years from the American Cancer Society…what a relief. Later in the spring I received word from Professor Maniatis’ NIH study section that our program would also be funded in June by a grant of about $150,000 a year for 3.5 years from the National Cancer Institute for the same work. I was able to hire Dr. Ching Lin from Iowa State University and Dr. Ng (Sun-Yu) from Kedes’ lab. By 1985 Sun-Yu finished the complete DNA sequencing of the human beta-acid gene and Ching sequenced the copy of the beta-actin gene that had two mutations to formally prove the mutations at the level of the gene. Everything that we had learned about the genetic code and amino acid sequences of proteins made our findings predictable. I had learned from my own research how Darwin’s theory of evolution and natural selection worked.

This was the year I finally successfully transferred in recombinant gene inside a cell in culture. I transferred the mutant human actin gene into a rat fibroblast cell line to show that I had cloned the functional gene which could abundantly express its protein the way the natural endogenous beta-actin gene worked (shown in a protein profile below).

mutant actin annotated

At this point I had a brief meeting arranged by Emile with Alex Zafferoni, founder and CEO of Alza Corporation, a block away on Page Mill Road. Zafferoni recommended Bert Roland as a patent attorney. I arranged a meeting with Roland, also a block away, for that afternoon to discuss patenting the human beta-actin gene promoter because of its strong constitutive nature (the engine of the gene that drives its expression). I told Bert that this was a collaboration with Peter Gunning and Larry Kedes at Stanford. Roland was famous for filing Boyer’s and Cohen’s genetic engineering patent which created Genentech and eventually funded Stanford with hundreds of millions of dollars.

We published Sun-Yu’s work on the sequence, structure, and chromosomal location (chromosome 7) of the human beta-actin gene in Molecular and Cellular Biology and we published Ching’s work locating three mutations in this gene in the Proceedings of the National Academy of Sciences, sponsored by Linus Pauling. A patent was filed on the beta-actin promoter and over the years it was licensed to about 15 biotech companies by Stanford University. This patent was prosecuted for the full 17 years (the life of a patent) but never issued. The Institute’s first royalty check was about $10,000 in 1986, but most of the royalties were earned by Stanford’s patent attorneys.

Peter, Larry and I published a paper in PNAS on the use of the human beta-actin gene promoter for expression of other genes. This vector was distributed to anyone who asked for it – and many did – and to those companies that licensed the invention. At last count this paper had more than 1,000 reference citations.

Our paper popularized the actin promoter as a strong constitutive promoter of foreign gene expression. Soon the rice actin promoter would be used to make Round-up Ready crops by DeKalb Genetics and Monsanto, and giant tilapia fish would be engineered with growth hormone under the control of the fish beta-actin promoter. There were even fluorescent mice running around in Japan created with firefly luciferase expressed by the beta-actin promoter (which I called “the cat’s meow”). Since cytoplasmic actins are the most abundant proteins in most cells you could use the promoter to abundantly express foreign genes in most cells of any animal.

In 1987 we also published the culmination of my research on the mutant beta-actin gene in Molecular and Cellular Biology. When I introduced this gene into non-tumor forming immortalized human fibroblasts they became tumorigenic. The results showed that the more abundant the expression of the mutant beta-actin, the more tumorigenic the non-tumorigenic cells became and the cells that came out of the tumors were enhanced further in the level of mutant beta-actin expression. This was a sensational finding that was the goal of research which began with the discovery of the mutant beta-actin in 1978 at NIH.

The Departure of Art Robinson and Fallout from the First Mayo Clinic Study

Art Robinson, 1974.

Art Robinson, 1974.

[A history of the Linus Pauling Institute of Science and Medicine, Part 3 of 8]

By late 1978, the Linus Pauling Institute of Science and Medicine had reformed its fundraising strategy, an action which proved to be quite successful. As a result, for the first time in its five years of existence, LPISM was not struggling to keep its head above water.

This wave of good fortune carried with it unforeseen negative consequences. In particular, Rick Hicks and Art Robinson began to come into conflict over the best way to invest this sudden surplus. Robinson suggested that LPISM move to Oregon – which had recently announced “Linus Pauling Day” in honor of its native son – and build a campus of its own. The idea was not popular with many staff, most of whom did not want to leave the Bay Area.

At the same time, Robinson began cultivating ties with the Orthomolecular Research Institute in Santa Cruz, California, which was headed by Arnold Hunsberger. Linus Pauling was not pleased with this idea, as he felt Hunsberger’s research hypotheses to be off the mark. Pauling had also met Hunsberger and had said that his impression was “not a very favorable one.”

Robinson continued to press for closer ties between LPISM and ORI, a source of growing tension between him and Pauling. In particular, Pauling was angered when he learned that Robinson had begun to tailor experiments in accordance with Hunsberger’s ideas without first consulting Pauling. When confronted, Robinson defended his decision and redoubled his arguments for collaboration. Their relationship continued to sour and morale at LPISM plummeted as the tension between Pauling and Robinson mounted.

In June 1978, Pauling issued a memorandum to Robinson, ordering him to consult the Executive Committee – comprised of Pauling, Robinson, and Hicks – before making “any important decisions.” Robinson responded by immediately firing Hicks. Pauling responded in turn by overruling the termination and demanding Robinson’s resignation within thirty days. He then proceeded to issue a memorandum informing Institute staff that he had stripped Robinson of his position, and that the staff was to disregard all further instructions from Robinson. The next day, the staff arrived at work to find a second memorandum from Robinson, declaring that he was still the president, that neither Pauling nor Hicks had the authority to relieve him of his duties, and that he would not resign.

Pauling memorandum of July 10, 1978.

Pauling memorandum of July 10, 1978.


The Board of Trustees met in mid-July to try and settle the dispute. They decided to place Robinson on a thirty day leave of absence, empowered Pauling with all executive authority and told him to resolve the issue. On August 15, with Robinson’s leave expired, Pauling was elected President and Director of LPISM. On August 16, Pauling promptly informed Robinson that he was taking over all of Robinson’s research, Emile Zuckerkandl was being appointed Vice-Director, and that Robinson was fired.

Now that Robinson was gone, LPISM attempted to consolidate and return to normal. Pauling asked Steve Lawson to assume a portion of Robinson’s research agenda, a request to which Lawson consented. Over the course of 1978, Lawson had steadily become less involved with the financial arm of LPISM and more involved with its scientific work. Zuckerkandl also tasked Lawson with setting up a cell culture facility where the two would conduct research on the differences between primary and metastic cancer cells, as revealed by protein profiling. Lawson worked closely with UC-San Diego, University of Colorado, and SRI International. He was later joined on that project by Stewart McGuire, Eddy Metz, and Mark Peck, all fellow employees at LPISM.

Robinson, however, did not take his firing lightly and on August 25, LPISM was informed that Robinson was suing the organization for $25.5 million, alleging a breach of contract and unlawful termination among other charges. LPISM’s lawyers began gearing up for a serious legal battle, standing firm in their conviction that the Institute had done nothing wrong.

Meanwhile, the Institute’s vitamin C research continued on despite the added burden of the Robinson lawsuit. In early October 1978, Pauling convinced Ewan Cameron to accept a one-year appointment to LPISM while the two worked on a book about vitamin C and cancer. Additionally, Pauling, Cameron, Lawson, and their coworker Alan Sheets began an experiment to determine the effects of vitamin C on chemotherapeutic drugs. The research took the form of a toxicology experiment in which multiple groups of fish were subjected to chemotherapeutic agents in their water, after which various groups were given different amounts of vitamin C while the research team observed the results.


The year 1979 started with good news. LPISM was informed by Hoffmann-LaRoche, the world’s largest producer of vitamin C, that they had seen sales more than double during the 1970s, and they fully recognized that Pauling was the cause. As a thank you, they had decided to donate $100,000 a year to the Institute.

The happy days were not to last long. In April, LPISM received an advanced release of the results of the major Mayo Clinic study on the treatment of cancer with ascorbic acid. Its primary investigator, Charles Moertel, had concluded that vitamin C did absolutely nothing to help cancer patients. Pauling was stunned and immediately began writing to Moertel to discuss the study in detail.

Then, over the summer, Art Robinson filed six more charges against LPISM and Pauling, bringing the total number of suits to eight and the total requested damages to $67.4 million. The year-long and highly publicized suit was greatly hurting LPISM’s reputation, and the Institute noticed a subsequent decrease in the donor funds flowing their way.

"Vitamin C Fails as a Cancer Cure," New York Times, September 30, 1979.

“Vitamin C Fails as a Cancer Cure,” New York Times, September 30, 1979.

Things then went from bad to worse when, on September 27, the New York Times published the Mayo Clinic study, definitively stating its conclusion that vitamin C was useless in treating cancer. Pauling immediately responded by pointing out that the patients involved in the test were undergoing cytotoxic chemotherapy, which he felt crippled their immune system. He also asserted that the trial was not conducted for long enough to develop accurate results.

Pauling's response to the New York Times article, October 24, 1979.

Pauling’s response to the New York Times article, October 24, 1979.

Charles Moertel returned fire, defending his results and questioning Pauling, implying that he was fanatical in his zeal for vitamin C and refused to acknowledge the truth. Pauling and Moertel began exchanging volleys in public, writing articles and giving interviews that attacked the research and competence of the other. Unfortunately for Pauling, he took the worst of it, as many people began to agree with Moertel, thinking Pauling to be too enamored with vitamin C to see any negatives. Funding plummeted as donations shrank and LPISM began finding large numbers of grants rejected outright with no chance for an appeal.

Pauling refused to give up. Shortly after the New York Times article was released, he and Cameron published their book, Cancer and Vitamin C. Pauling personally bought 16,000 copies of the publication and mailed them to every member of Congress and to countless other physicians and researchers. This action helped Pauling’s cause significantly as many of the recipients read the book, or at least glanced through it. And even those recipients who didn’t read the text were made more aware of Pauling and his research. Likewise, in the marketplace the book sold well despite the bad reception it received from professional reviewers – the public seemed interested in Pauling and Cameron’s ideas.

In light of this, National Cancer Institute head Vincent DeVita agreed to a second round of trials. However, in doing so DeVita once again chose the Mayo Clinic to host the trials and chose Moertel to lead them. Pauling was furious with these decisions, an understandable point of view considering that he and Moertel had spent the past few months publicly accusing one other of being incompetent.  Pauling was also now without his co-author: their book completed, Ewan Cameron returned to Scotland to fulfill his duties at Vale of Leven Hospital. Before leaving, he was appointed a Research Professor at LPISM for a period of five years.

With a new decade approaching, the easier times of the mid-1970s were clearly gone and by early 1980 the future was once again uncertain. While the tensions evident during the Art Robinson era were now history, his lawsuits and the Mayo Clinic trials severely detracted from the future prospects of LPISM. Unfortunately for the Institute and Linus Pauling, their immediate future was not going to be a happy one.