Searching anew for the origins of cancer.
Peter Duesberg of U.C. Berkeley has recently pointed out that virtually all solid malignant tumors have the wrong number of chromosomes; usually too many. Sometimes they may have several copies of a given chromosome, Cells with the wrong number of chromosomes are said to be "aneuploid"; those with the right number are "diploid." This is not a new discovery. When researchers such as Theodor Boveri studied cancer cells under the microscope a century ago, the first thing they noticed was the abundance of chromosomes. Amazingly, the correct number-46 in humans-was not determined until 1956, three years after Watson and Crick had described the much smaller, less visible double helix of DNA.
Boveri predicted in 1914 that aneuploidy would turn out to be the cause of cancer, and today Duesberg believes he was right. A recent article in the Economist about his cancer theories noted: "Dr. Duesberg achieved notoriety more than ten years ago for his hypothesis that the human immunodeficiency virus, HIV, does not cause AIDS. In terms of challenging conventional wisdom, that was a hard act to follow. The aneuploidy hypothesis, however, appears a worthy successor." Nonetheless, "the ubiquity of aneuploidy in cancers does need explaining."
Boveri's discovery was forgotten by the mid-1970's. The new theory was that gene mutations cause cancer. Michael Bishop and Harold Varmus won the Nobel Prize for discovering cellular (as opposed to viral) "oncogenes," but their ability to cause cancer is still in doubt. Later, Varmus became the director of the National Institutes of Health. For the last ao years or so the vast bulk of research funds at the disposal of the National Cancer Institute and other agencies has been devoted to the hunt for cancer genes.
Last summer, Robert A. Weinberg and William Hahn of MIT announced that the mutation of three genes was "sufficient to transform a normal human cell to one capable of producing a tumor." The New York Times duly came through with a page-one article-one of many the newspaper has published in the past 15 years about new cancer genes. But it was not reassuring to learn, in the commentary in Nature, that the most recent find had come "after more than 15 years of trying." What about all those earlier cancer genes?
When Duesberg's lab at Berkeley asked to see the cell lines generated by Weinberg's "three genes," they turned out to be aneuploid. This finding was published in the Proceedings of the National Academy of Sciences (March 28), with sample cells from each (of two) cell lines depicted. One had 78 chromosomes, another 82-almost double the right number. The significance of aneuploidy is that each chromosome carries anything from several hundred to thousands of genes, so that if a cell has two or three, or worse, 30 or 40 extra chromosomes, the number of genes at work in the cell has been greatly increased. Their added output is probably sufficient to override the control mechanisms that ensure the normal cell doesn't take on a life of its own, as cancer cells do. If a cell has extra chromosomes, then, and out of its thousands of extra genes a handful are mutated, how can we be sure that it is the few mutations, and not the huge DNA increment, that causes the cancer? Diploid cancer cells are what is needed to prove Duesberg wrong. (We are not talking about leukemia, where diploid cells are common.)
I phoned Weinberg's lab, and he picked up himself-a very confident voice. I asked him, first, if it is true that most solid cancers are aneuploid. His books on cancer research have not mentioned aneuploidy.
"Yes it is," he said. "But there are exceptions, and it is not true that aneuploidy is an inevitable prerequisite for making a tumor, as Duesberg would imply."
The cell lines sent to Duesberg's lab were apparently aneuploid, I said.
"That's what he says," Weinberg replied, "but we analyzed the same ones more carefully, and one of the cell lines we sent him is perfectly diploid." So there is a "disagreement in fact." Weinberg's lab had sent the work out, and a more detailed analysis, "much to our surprise, showed that one of the cell lines was diploid."
The normally phlegmatic Ruhong Li, who had examined the cell lines in Berkeley, became quite agitated when he heard this contradictory claim. Cell lines are grown in vitro, shipped in a bottle, and can be tricky to analyze, because tumors always contain some normal cells. Therefore, much depends on which cells are examined. Ruhong had studied 60, and of these 70 percent were found to be aneuploid. Moreover, two weeks before I spoke to Weinberg, the lead author of his Nature paper, William C. Hahn, had given a talk at a meeting of the American Association for Cancer Research in San Francisco. At the end of his remarks, he was asked a single question. Had he looked at the chromosomes? Yes, he said. They were 70 percent aneuploid.
A perhaps more serious problem with Weinberg's three-gene claim is that one of them was not a human gene at all. It was a monkey virus, SV40, whose ability to "aneuploidize" cells without further assistance has been known for decades. Weinberg could have achieved the same result with this single item. Duesberg surmises that Weinberg added the virus out of frustration, because he couldn't get anything else to work. This simian virus injected in vivo, into humans, might cause a small wart that would soon disappear, but it would not cause cancer, because the immune system would quickly wipe it out. In the petri dish, of course, there is no immune system.
The slow development of a tumor is to be expected with aneuploidy. Everyone seems to agree that cancer starts in a single cell. It spawns others like it, and they never seem to stop dividing, eventually forming a tumor. The obvious place to look for the initiating event is mitosis, as cell division is called. The chromosomes double up, and then something called the spindle apparatus "segregates" the chromosomes into opposite halves of the cell, which then divides. Carcinogens upset the spindle, so that daughter cells have either too many or too few chromosomes. Since the unbalanced chromosome array is itself unstable, they are likely to go on recombining in abnormal ways as the cells divide. Most of these recombinations will prove fatal to the cell, especially those that lack chromosomes. But eventually a combination is reached that keeps on going. The small chance of this happening, and the many cell deaths en route, explains the long hiatus between exposure to a carcinogen and the appearance of a tumor. Finding just the right combination may take years, or it may never happen.
This resolves one of the mysteries of cancer research. If Boveri saw aneuploidy right away in the microscope, why was it not taken more seriously? The answer is that later investigators looked for a characteristic chromosomal pattern (comparable to the triple chromosome ar found in Down's syndrome) as the cellular signature of the disease. But they couldn't find it. Instead they kept finding different chromosome disarrays in different cancers, and instability in a single tumor. So they couldn't find what they were looking for. Meanwhile, the gene mutation theory began to dominate cancer research.
In 1985, the Environmental Protection Agency funded a four-day conference on aneuploidy, driven by concern about "exposure to environmental agents that cause aneuploidy." The proceedings were published in a 56o-page volume, and guess which word isn't in the index? Cancer. That's how strong the faith in mutation was (still is). One problem with the carcinogens-aremutagens theory is that the underlying research, by Bruce Ames of U.C. Berkeley, was done on bacteria. They are convenient-they multiply quickly and show mutations easily. But bacteria are singlecelled organisms, they have one chromosome, and they do not get cancer.
Where aneuploidy is recognized at all in cancer, it is usually considered to be an effect, not the precursor. A leading cancer textbook, Fundamentals of Oncology, refers to the "dilemma" as to whether aneuploidy is "the result or the primary cause of neoplasia." An article in Science last year said aneuploidy is "usually viewed as a consequence." Investigating this while on sabbatical at the University of Heidelberg, Duesberg and assistants induced tumors in hamsters by inoculating them with carcinogens. After six months on average, 14. of the 23 animals developed tumors. The long latency allows plenty of time to look at the chromosomes. Aneuploidy was indeed observed months before the tumors appeared, and in all of the tumors.
Even as he was writing this up and delving into back-issues of journals, Duesberg found ample confirmation in decades-old experiments. "It all gets lost in the huge literature on cancer," he told me. A book from the prestigious Cold Spring Harbor Laboratory Press has now retrieved some of this history. In The Cells of the Body: A History of Somatic Cell Genetics, Henry Harris of Oxford University's school of pathology summarizes experiments back to the ig5o's showing that aneuploidy appeared "long before the emergence of tumors." Nonetheless, one worker at the National Cancer Institute assured me by e-mail: "No one disagrees with the fact that aneuploidy is evident in lots of solid tumors. The fact that they are such a chromosomal mess is a result of final loss of any normal controls over cell growth and division. However, the aneuploidy is not a cause, but an effect, and a late-stage one at that " But one of the leading cancer-research labs in the country disputes this, as we shall see.
I sent an e-mail to Harold Varmus, now president of the Memorial Sloan Kettering Cancer Center. His reply to my question about cancer and aneuploidy came back within four hours:
Aneuploidy and other manifestations of chromosomal instability are major manifestations of many cancers and many labs have been working on them-with difficulty, it must be said-for many years. The functional significance of these abnormalities, however, is not well established in most cases, and, more importantly, any role they play will not diminish the crucial roles of mutant proto-oncogenes and tumor suppressor genes. Indeed it is likely that the gross chromosomal changes contribute to cancer by altering those genes. Carcinogenesis is a multi-step process!
But why wouldn't it "diminish" the role of mutated genes, if it could be shown that aneuploidy appears without benefit of mutation, and is present in every solid tumor? I sent him another e-mail, saying that he seemed to be implying "that the role of mutant oncogenes in causing cancer has been firmly established." If he knew of a case in which mutated genes had transformed ordinary cells into cancer cells, could he "give a reference to the experiment where this was done?"
This time there was no answer.
The gene mutation theory still lacks proof. Any given mutated gene is found in only a small minority of cancers, and when injected into experimental animals, these genes do not cause tumors. In one ingenious and devastating experiment, researchers succeeded in implanting "cancer genes" into the fertilized egg of a mouse, so that the off spring "onco-mice" had these genes in every cell of their bodies. Still they lived, admittedly with a higher risk of cancer. "Some further accidental change...is apparently required for the development of cancer," according to The Cell, a leading textbook by Bruce Alberts et al.
I called the National Cancer Institute press office and relayed a general question to the director, Richard Klausner, about aneuploidy and cancer. A few days later the press lady got back to me with word that I should call Christoph Lengauer, an assistant to Bert Vogelstein at the Johns Hopkins Oncology Center in Baltimore. "Beyond that I can't help," she said.
I did go and see Thomas Ried, a senior investigator in the Department of Genetics at the NCI. This is on the NIH "campus" in Bethesda, Maryland. Huge new buildings were nearing completion, named after generous donors-Stokes of Ohio and Hat field of Oregon were just two that I saw. A Heidelberg man, Ried has been here for six years, and for the last 18 months at NCI. He has written technical articles about the machinery of cell division, and knew all about Duesberg's cancer work. He had even presented Duesberg's papers at an NCI "journal club." He was familiar with the historical background, and had managed to hunt down an old copy of Boveri's book. Not even the National Library of Medicine, on the NIH campus, has a copy.
Ried was both encouraging and politic-obviously conscious of the minefields. "I don't find it entirely helpful to generate such a dichotomy between mutation and aneuploidy," he said. Couldn't it be both? Nonetheless, he said, "it is entirely conceivable" that an early change "could be a chromosome segregation error, which might then trigger the malignant transformation. This is basically what Duesberg is saying." In "the euphoria of sequencing the genome," he added, "mutations have gained too much importance." Now, NCI researchers are trying to put extra chromosomes into normal cells, to see whether this will lead to transformation. "That would be a proof of Peter's hypothesis." He politely inquired, had I spoken to Vogelstein?
Bert Vogelstein is a near-legend among cancer researchers. He runs what I heard referred to as the "Vogelstein empire." His graduate students have been known to take turns trying to reach the lab before his pre-dawn arrival, but according to legend, no one has yet succeeded. Every so often, the New York Times will run a front-page story about another cancer gene that his team has isolated. He has been anointed "hottest scientist" of the year-most cited journal articles and so on. Even Duesberg, whose comments on fellow scientists have been known to be acerbic, speaks of Vogelstein in tones that verge on the friendly.
I called, and was told I should e-mail him. His 6 a.m. reply: "We think chromosomal instability (resulting in aneuploidy) is extremely important for tumorigenesis. At least 90% of human cancers are aneuploid, with the others diploid or neardiploid." To pursue the matter further, look up the articles "with both Lengauer and me as co-authors," he advised. "Won't have time to talk, but hope the above helps."
At least 90 percent! That's so much higher than the figure for any known cancer gene. And near-diploid counts as aneuploid, in Duesberg's book. Maybe tumors really are all aneuploid.
Lengauer turned out to be another Heidelberg man-he worked in the same lab as Ried for a while. Germany's NCI-equivalent is at Heidelberg. Lengauer said that aneuploidy may well be "the feature that is most commonly associated with cancer." Interest in it is growing rapidly, and the Vogelstein lab has been on the case. One question they have looked at is whether aneuploidy is the cause or consequence of cancer. What did they find?
"I think we could exclude the possibility that it is a consequence," he said. "With our experiments, we found that it is a very early event in tumorigenesis."
He marveled that Boveri's "revolutionary thought" was arrived at "without knowing about genes or DNA." (A blessing, maybe?) Aneuploidy was then forgotten, "I think mainly because everybody believed, now that we had genes, all this cytogenetic stuff is old fashioned." Cytogenetics refers to the genetics of the whole cell, not just the DNA segments called genes.
"Many people believed that aneuploidy was a consequence of cancer," Lengauer said. "And that was the major mistake the scientific community made." But over the last three years there has been a change, and if you were to poll the researchers now, he thought, "almost nobody would doubt any more that aneuploidy is causative."
As one might expect, the Vogelstein lab is looking for a gene mutation-one that interferes with cell mitosis. They have found one, too, but it is present in "just about 5 to 10 percent of tumors:' Function has been demonstrated. Put it in a diploid cell, "let it express, form a protein, and all of a sudden that cell becomes aneuploid."
Then again, Lengauer allowed, aneuploidy could also be caused by "some catastrophic event that happens once in a while in a cell:' A cell divides, "makes a mistake," and that error is propagated in future divisions. So, just maybe, gene mutation isn't needed. And that "would fit into Duesberg's picture, which believes that mutations are irrelevant."
TOM BETHELL IS TAS's Washington correspondent. His latest book is The Noblest Triumph (St. Martin's Press).…