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20 4 4 be shown to have antibodies against the otherwise undetectable virus. If the investigators had not known that the tumors were due to a virus and, furthermore, had not had the virus at hand so as to make the antibody tests, there would be no evidence that the domestic rabbit papillomas were of viral origin. A very important finding, however, which colored all the other findings, was the early recognition, by Rous and Beard, that animals infected with the Shope papilloma virus often developed cancer. If the animals with papillomas were kept and observed for six months or more, 25 per cent of the wild rabbits and 75 per cent of the do- mestic rabbits developed cancers with proliferating tissue at the base of the horny growths. This process could be speeded by painting the rabbits' skin with chemical carcinogens. In such cases, the papilloma- to-cancer changes took place at the site where the carcinogen had been applied but the tumor was always of the same type as that induced by the virus alone. Although the cancers, in both cases, clearly de- rived from the virus-caused papillomas, none of the cancers, either in the domestic or in the cottontail, contained detectable virus. The question continues of where the virus goes when it disappears and whether or not the virus causes the cancer, or whether it just causes the papillomas and they undergo an independent and separate trans- formation. As Peyton Rous says, "the tumor problem is the last stronghold of metaphysics in medicine." In any case, a virus and cancer in mammals had for the first time been shown to have an intimate relationship. The third ma j or type of cancer virus to be discovered shook the entire cancer field. Cancer of the breast, in mice as in human beings, is one of the most common forms of the disease. Among the early classics of cancer research was a series of experiments, running con- currently with Rous' first reports on the chicken sarcoma virus, by Leo Loeb in which he showed that breast cancer developed only in the presence of the female hormone. Not long after, Dr. Maud Slye, working another vein of cancer investigation, showed that by selec- tive inbreeding, strains of mice can be produced in which qo per cent or more will develop certain types of cancer while other strains will be virtually cancer-free. The group at the Jackson Memorial Labora- tories in Bar Harbor, Maine, grew to be the center for the main- C) I j3q 40n11344 1.

i Aj-'c /9• 14wx.eL FRANK A. HOWARD, FRANK L. HORSFALL, JR., M.D., Chairman President It is a privilege to submit this latest statement of our progress against cancer to those whose financial support makes our program possible. We remain deeply grateful for your aid and hope you will derive continuing satisfaction from your share in this great enterprise. f SLOAN-KETTERING INSTITUTE FOR CANCER RESEARCH 01134 4b2 I

21 tenance and study of these purebred lines, which are still among the most valuable tools of cancer research. In attempts to pin down the nature of the genetic influence on cancer development, crossbreeding experiments were tried between high cancer and low cancer strains, similar to Mendel's oft-cited crosses between the smooth and wrinkled peas. Unlike Mendel's experiments, however, the genetic studies of mouse breast cancer refused to come out as expected. Actually Lath- rop and Loeb had glimpsed the crux of the problem many years before but had lacked the purebred strains with which to prove it. Contrary to all classic genetics, the family background of the mother mouse was obviously more important than the genealogy of the father. Working on a hunch, Dr. John J. Bittner, a member of the Bar Harbor group, took from their mother a whole litter of newborn mice of a high cancer strain and turned them over for foster nursing to a low breast cancer strain female. When the females of the litter matured, far fewer than the expected number developed breast cancer. Conversely, a litter of low cancer strain mice when given to a high cancer strain mouse to nurse and raise, developed far more than the expected number of breast cancers in later life. Bittner quickly traced this maternal influence to a filterable sub- stance present in the milk of the high cancer strain mothers, which he dubbed, with the characteristic reluctance of cancer investigators to use the word "virus," the mammary cancer milk agent. There were a number of special features about the mammary cancer milk agent that heavily influenced scientific thought about the relationship between cancer and viruses. First, the agent alone would not cause the cancer. Bittner maintained from the beginning, and all subsequent work has borne him out, that breast cancer in the mouse is due to an interplay between three factors: hormones, genetics and the virus. Actually it is now known that any two of these factors, if unusually strong, can lead to the characteristic mammary cancer in mice. Second, it was found that mice had to be removed from their mothers immediately after birth, before they had any opportunity to nurse. As little as o. i ml. of milk from a high cancer strain is sufficient 01134403

22 J a ~'J - i 7 - .: 1 'd G . . . ~ i ! i U 'i ._ 7 : . . : ' .. _ , _ A : , :j to infect a susceptible newborn mouse. After the first few hours, in- fection by the viral agent becomes increasingly difficult. This dis- covery was to influence profoundly the subsequent course of much virus work, including that in the tumor field, and to cost virus workers countless sleepless hours beside expectant murine mothers. Third, the Bittner virus was found often to coexist with its host for months and even an entire lifetime without making its presence known. During the long latent period-of 6 to 20 months-between the introduction of the virus and the appearance of the tumor, there are no signs of disease, although virus can be isolated from the milk (and hence passed on to newborns) and from tissues of the high cancer strain. Furthermore, an animal of low cancer strain can carry the virus all its life without ever developing cancer. Male mice of high cancer strains, although they never develop mammary cancer unless they are given female hormones, can pass the virus to females at mating. (This is apparently the only other natural route of spread of the virus, since contact infection does not seem to incur. ) Fourth, as a result of Dr. Bittner's discovery, the cancer-inducing viruses could no longer be considered as biological curiosities, or ex- ceptions that proved the "rules" of expected cancer or virus behavior. A virus had been shown to be responsible for the most common form of cancer in the most commonly used laboratory animal, and viruses were, from that time onward, in the cancer picture to stay. 01134404

~ M , In the course of the last 20 years, the term "virus" gradually became the inclusive catchall description for a large number of fleet- ing indispositions as well as achieving recognition in medical circles as the cause of more serious maladies, such as epidemic influenzas, many of the common infectious diseases of childhood, and crippling attacks of poliomyelitis. From the patient's point of view, a virus is "caught," usually by contact with an infected person, goes completely undercover to "incubate" for an often surprisingly predictable period of time, and then reappears to be recognized as it leads to fever, sneezes, rashes, or whatever the typical symptoms of the particular disease may be. After an almost equally predictable period of time, the infection commonly will mysteriously disappear and often bestow a long-lasting or even permanent immunity to further episodes due to the same agent. No drugs or other specific treatments significantly alter the course of viral infection once its symptoms and signs have appeared. However, certain important viral diseases, such as small- pox, yellow fever, influenza and poliomyelitis, can be prevented or ameliorated by various types of immunization procedures. By those interested in how viruses achieve their effects, it was recognized fairly early in the history of virology that all viral activity is a product of a parasitic virus-cell relationship and that the symp- toms and signs of the various viral diseases are caused not by viral products but by the abnormal functioning or disintegration of some 01134405

24 of the body's own cells. Although cell death, such as may occur in parts of the nervous system in poliomyelitis, is the most generally recognized effect of viral infection, enlargement and proliferation of cells also occurs frequently and is not at all an exclusive attribute of the so-called tumor viruses. If the cornea of a rabbit is infected with cowpox virus, for example, the cells of the cornea enlarge and proliferate until the surface is some ten cell layers thick, instead of the usual two or three, before any cellular breakdown begins. Some viruses such as mumps may not cause cell death at all, but merely temporary enlargement and dysfunction. Other viruses settle down for years in a comfortable symbiotic relationship with cells and may produce little or no evidence of infection. The most common example is the herpes simplex virus that may induce fever blisters and canker sores. This virus which is present in many persons for most of their lives, becomes manifest only when fever, sunburn, severe infection, or perhaps even an emotional or hormonal upset causes a temporary imbalance in the cell-virus equilibrium. Some viruses are even more gentle guests than herpes and may never produce any noticeable symptoms at all. Indeed, rabies is the only uniformly fatal viral dis- ease that is known in man. Much of the most elegant and rewarding work on virus-cell relationships has been carried out with bacteriophages, the viruses which infect bacterial cells. A number of scientists-Cohen, Del- bruck, Hershey, Jacob, Lederberg, Luria, Lwoff, Wollman, Zinder and many others working in laboratories throughout the United States and in various other countries-have contributed to the knowledge in this field. All concerned cautiously advise against assuming that just because bacterial viruses behave in certain ways, that mammalian viruses do so too, although most agree, on the basis of studies thus far, that there are many close similarities among them, and that principles established with one variety probably have broad applica- bility to others. Amongst the most intensively investigated of the bacteriophages is a family of tadpole or sperm-shaped viruses with hexagonal bodies and stubby tails, known as the T-even phages. The phage particle has been shown to consist of a protein coat surrounding an inner coil of nucleic acid which constitutes the genetic determi- nants of the virus. In the case of the T-even bacterial viruses, this 01134406 `,;

25 nucleic acid is of the type known as DNA (deoxyribonucleic acid) which is the same type of nucleic acid found in the chromosomes of all cells. The phage particle backs up to the bacterial cell and attaches itself by the tip of its tail; the makeup of the protein overcoat appears to determine the type of bacterium to which the phage can become attached. Then an enzyme in the tail of the phage digests a hole through the bacterial cell wall, and the phage seems to squeeze itself like a tiny syringe, injecting its DNA into the bacterium. At this point, except for the empty protein jacket abandoned outside the cell wall, the phage particle has completely disappeared, and even if the cell is broken open no trace of it can be found. The first measurable event that takes place within the infected bacterial cell is an increase in protein. This was at one time thought to represent viral protein, but now it is known to result from the cell's production, under the virus's command, of a number of new cell enzymes which the virus is going to need to make new viruses. Shortly after, synthesis of nucleic acid starts. 'I'he cell begins to pro- duce viral nucleic acid, not the usual cellular nucleic acid; this can be shown clearly in the case of the T-even phages, because phage DN A is not identical with cellular DNA and contains a compound known as hydroxymethylcytosine in place of the component cytosine generally found in cellular DN A. As viral DN A is synthesized, cellular DNA breaks down but the viral DNA is protected from the enzymes that rip apart the cellular DNA by virtue of the slightly different hydroxymethylcytosine. Some of the components of cellular DN A are tised for manufacture of new viral DNA. Assembly of viral protein begins next, and it then becomes possible to see partly formed viral particles inside the cell by means of the electron microscope. The bacterial cell finally bursts open and some i oo or more new viruses emerge, identical in every respect to the original virus that backed up to the bacterium and started this amazing process. This entire chain of events can take place in as little as twenty minutes. Sometimes, certain types of bacteriophage, apparently acci- dentally, pick up large functional pieces of nucleic acids from the DN A of the bacterium they have destroyed and carry it on with them, like baggage, to the next cell, where it is injected along with the viral DN A. This passenger piece of DN A particle then may 01134407

26 become part of the bacterial cell's DNA and confer upon the bac- terium some entirely new genetic character which is subsequently passed on to daughter bacteria as a permanent hereditary trait. Any bacterial "gene" may be carried in this way from one bacterial cell to another and, if the virus picks up a sufficiently large piece, two or even three genetic traits may be transported at the same time. This phenomenon, which is known as transduction, is of particular interest to geneticists since it demonstrates so directly the ability of one small segment of nucleic acid to carry one complete genetic command. Instead of viral multiplication ending with cellular disruption, which is the classical disease-inducing virus-cell relationship, an even more curious way of life between virus and cell may often result from viral infection. Bacteriologists many years ago noted that if some bacteria, which they termed lysogenic, were grown in cultures with other bacteria, known as the sensitives, the sensitive bacteria would disappear. It was discovered considerably later that the cause for the vanishing of the bacteria was the occasional release of viruses by the lysogenic strain of bacterial cells. From time to time an occa- sional one of the infected cells would break open and a score or more of new viral particles would emerge. These particles could then infect and destroy the sensitive bacteria, soon wiping out the entire culture. The most interesting facet of this discovery was the nature of the relationship between the lysogenic bacterium and its carried phage. The phage disappears as an infective agent. Virologists searched the cell for it and found that it no longer had a separate existence at all; apparently some of its DNA had actually become part of the cell, assuming a place in the cell's chromosome. The phage-gene, or "prophage" as it is called, "which once was an in- fective particle has become simply an inheritable ability of the cell to make phage." Such associations may also give the cell certain other new capabilities; the diphtheria bacillus, for example, produces diphtheria toxin and so becomes disease-inducing under the influence of a prophage. When the cell divides, the prophage or ability to pro- duce phage is passed along to the daughter cells. When bacterial cells reproduce by mating-that is by exchanging genetic material between cells-the ability to produce phage is passed along precisely 0113+1408 '; _

27 according to genetic laws. The carriage of prophage may also exert a protective effect on its host; a lysogenic bacterium cannot be in- fected by another phage of the same or a related strain. Another phage can by transduction introduce new genetic information to the lyso- genic cell, showing that the nucleic acid is actually injected into the bacterium, but the second phage itself disappears and does not reap- pear; apparently the carried prophage occupies a definite, precisely determined spot among the bacterial genes and if this spot is occupied there is no space for another prophage. It is these peculiar character- istics that have led Luria to characterize such viruses as "a bit of heredity in search of a chromosome." Prophage is apparently held in check by the presence in the cell of an inhibitor which reflects a genetic character. From time to time, occasional cells lose their ability to inhibit prophage expression, the balance between viral component and bacterium is then upset and the virus begins to multiply. This may happen spontaneously, or it may be induced by x-ray, ultraviolet light, or certain mutagenic chemicals. Investigators who are interested in such relationships, have noted that the agents that alter the benign relationship between phage and bacterium are also, in nearly every case, the same agents that are widely accepted as "causes" of cancer. 0113-1409

Many physicians have been struck with the suggestive resem- blance of certain types of cancer, especially acute leukemia and Hodgkin's disease, to an infectious process. Patients are often sys- temically ill, have spontaneous temporary improvements and re- lapses, run fevers, and even develop rashes and itching. Yet despite countless efforts, it was not until about ten years ago that a form of leukemia in a mammal was proved to be of viral origin although several malignant diseases of fowl similar to leukemia had been shown to be virus induced much earlier. Leukemia, like breast cancer, may develop in qo per cent of the adult animals in some laboratory strains, such as the AK mouse, but is rarely found in others such as the C3H strain, in which less than one half of one per cent of the animals develop the disease. In 195 I, Dr. Ludwik Gross removed the liver and spleen from the leukemic strain AK mice and prepared cell-free filtrates from them. These he inoculated, profiting from Bittner's earlier experience, into new- born mice of the C3H strain. Some four to nine months later, 28 per cent of the injected animals developed leukemia. At first Gross' reports were considered controversial due to previous failures to demonstrate a leukemia virus in mammals and to confusion engen- dered by uncertainties about various AK sublines that made it im- possible for others to duplicate his work exactly. But eventually other experimenters were able to produce the same results and soon Gross, 0113,141Q