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do not involve chemical techniques. The relation of hereditary factors to susceptibility to cancer, the transplantability of cancers, the transmission of tumors by filterable agents, the growth of tumor cells in tissue culture, the influence of hormones and of external agents on susceptibility to and growth of tumors, the attempts to develop antibodies to tumors, all these and many other studies are essential contributions without which biochemists would have nothing to investigate. In the end, however, our objective must be to describe all of these biological phenomena in chemical terms.
The difficult problem is to know where to begin. The biochemical answer does not lie on the surface. The cancer cell puts up a good front of normalcy. It does not wear its chemical aberrations on its sleeve. A chemical analysis of the cell, the best analyses that it has been possible to make of the chemical reactions by which it lives and grows, have revealed little or nothing to account for its asocial behavior. Yet we know that there are, there must be, differences. That they have eluded us up till now is no cause for dismay. Our knowledge of the chemistry of normal cells is still fragmentary. We are only on the threshold of an understanding of the complexity of molecular organization which makes a single minute cell a going concern-a machine of incredible diversity of function so uniquely adapted to the activities that it sustains.
Until recent years our knowledge of cellular organization came largely from studies with the microscope. The chemist was able to give little help. The situation is, however, changing rapidly and the cytologist and the biochemist are coming close together. The refined techniques of the cytochemist, the penetrating eye of the electron microscope are now reaching down to levels of organization far beyond the visual microscope. At the same time the chemist, having sharpened his wits and refined his tools in the study of relatively simple molecules, is beginning to make real progress in the much more difficult task of describing the giant molecules which are the organized components of living cells. The chemist no longer conceives the cell as a protein jelly into which nutritive materials permeate and within which a turbulent riot of chemical reactions is in constant progress. He now recognizes what the biologist has long insinuated, that the cell is a well-organized factory comprising many types of subunits. These function as separate compartments dedicated to make specific chemical contributions to the overall economy of the cell.
The subunits of the cell include a variety of particulate structures such as the chromosomes of the nucleus and the mitochondria, microsomes and other particulate bodies of the cytoplasm. They have long been familiar to the microscopist. The chemist is just beginning to get to know them. Within recent years he has developed methods of separating them from cells and from one another without destroying some of their physiological activities. He is now busy studying the reactions of the isolated subunits in order that he may define their chemical functions in the life and growth of the cell. The power of a machine is in the organization of its parts. The power of a living cell, likewise, is in the organization of its components. Malignant cells may not differ strikingly from normal cells in gross chemical composition. It is a plausible hypothesis, therefore, that their abnormal behavior may reside in abnormalities in the way their components are organized into functional structures.
Students of growth are, naturally, concentrating attention on the chromosomes of the nucleus because these play so dominant a role in cell multiplication and in the transmission of the inherited characteristics of the cells that contain them. Chromosomes consist essentially of a combination of protein with an almost equally complex type of matter called nucleic acid. A great deal of contemporary work is being devoted to the chemical characterization of the nucleic acid of different types of cells, to the identification of the foodstuffs from which nucleic acid is manufactured in the cell, to measurements of the rates at which it is built up and broken down in the cells of different tissues and to the effects of chemotherapeutic agents on these rates of manufacture and destruction. All such information is manifestly pertinent to the problem of the control of the multiplication of cells.
Much of the particulate material of the cytoplasm also contains nucleic acid. The latter differs in characteristic ways from the nucleic acid of the chromosomes and the particulates in which it is present contribute differently, but significantly, to growth. There is increasing evidence that some of the cytoplasmic units share with the nucleus the control of the rate of protein synthesis in cells. In recent years, moreover, evidence has accumulated that some cytoplasmic particles share with the nucleus in the transmission of certain heritable qualities of cells.
Viruses are organic particles with the capacity to invade and multiply within cells for which they have a specific affinity. They, likewise, are composed largely of nucleoprotein. Indeed, viruses, the particulates of cytoplasm and the ultimate particulate units of chromosomes, known as genes, have so much in common, chemically and biologically, that there is a growing feeling that they may represent variants of a common pattern of organized vital material. This thought will be further developed in later sections of this report because it may have a profound influence on theories of normal and malignant growth and, therefore, on the future course of experimental work on growth. It is mentioned at this point only to emphasize the importance of the many studies of the chemistry and the metabolism of nucleic acid which are now in progress.
Although, at the moment, nucleic acid is much in the public eye its rate of synthesis is not necessarily the controlling factor in the rate of growth of cells. The major component of protoplasm is protein and, quantitatively speaking, the rate at which growth can occur is limited by the rate at which the cell synthesizes protein. Currently, there is wide interest in this problem. It is one of the many problems of metabolism which was scarcely open to experimental attack until the availability of isotopes made possible the use of tracer techniques. Even so, progress will be slow because proteins are so complex and so specific in structure that their total synthesis by cells must be conceived to be a most elaborate process. On the other hand, results of significance to cancer may well arise at any stage in the elucidation of the problem. As soon as any single step in the process has been established methods of blocking that step with chemical agents will suggest themselves. These may well open new vistas for cancer chemotherapy. The building-up (anabolism) of protoplasmic components requires a supply of chemical energy. The breakdown (catabolism) of foodstuffs or of protoplasm releases energy. A cell can grow because it is able to use energy released by the oxidation of some of its food to supply the energy required to convert the rest of the food into protoplasm. The great mystery of the chemistry of the cell has always been the manner in which the cell has achieved this energy-coupling process. The difficulty is this. When chemical energy is released it has a habit of degenerating into heat. Now heat is a useless form of energy to an organism that operates at constant temperature-at least it is useful only to keep him at constant temperature. Evidently the cell has some method of storing the energy of catabolism as chemical energy until it is needed, for synthetic processes. The biochemist is beginning to learn how the trick is turned. It appears that this energy is stored in certain labile phosphate compounds in the cell. The layman who scans the current literature of biochemistry will be intrigued by the frequent recurrence of such hieroglyphics as ATP, ADP, DPM, and TPN. These are symbols representing various phosphate compounds which form the transmission belt of energy in the cell. They are molecular storage batteries that are charged up when food is oxidized. They can then be switched into circuit with synthetic reactions and discharged to provide the energy required by the latter processes. Since growth is dependent on this power supply as well as on the supply of raw materials it should be possible to control growth by controlling the energycoupling process which has been described: If this argument be accepted, then it will have to be conceded that the student of growth is just as much concerned with catabolic reactions as with anabolic processes. The latter are dependent on the former. More than this, he dare not restrict his interest to the metabolism of proteins and nucleic acids. The oxidations of sugars and of fats are the chief sources of phosphate bond energy. He who would control growth may have to take into consideration the whole panorama of chemical reactions comprised in the metabolism of sugars, fats, proteins and nucleic acids. No aspect of metabolism can, a priori, be dismissed as having no pretense to the problem of cancer. So sweeping an assertion may, perhaps, be overstating the case. the other hand, the view is widely held that we do not yet know enough about the chemistry of cancer to delimit the field. We cannot afford to close any doors.
Until recently we knew few of the individual steps in the chain of conversion of raw materials into the end products of their metabolism. We knew what went into the organism and what came out. We could strike a balance sheet of these. What series of chemical changes went on within the cell was very much a matter of enlightened surmise. All this is now changed. The most revolutionary advance in the field of metabolism in recent years has been the almost universal adoption of isotopes as tracers of metabolic pathways. The principle of tracer studies has been described in many places and need not be repeated here. Suffice to say that it does make possible the ultimate mapping out of a complete flow
sheet of the fate of a given substance in the body. Already information is accumulating at an intoxicating pace. The biochemist, at last, has the tool which will enable him to probe the inner recesses of living processes and he is making the most of it.
Those who assert that medicine is slow in bringing new tools to its service should ponder the story of the isotopes. Seven years ago isotopes were available to a privileged few. In the last 5 years the supply to medical laboratories has increased 20-fold. The bill for isotopes for cancer research alone now amounts to $1 million a year. This development was not accomplished without a profound and concerted effort. Instruments for the manipulation and measurement of isotopes had to be designed and produced in quantity. Investigators had to learn how to use these instruments and how to adapt them to their particular needs. They had to train themselves and, then, to train their students in the quite novel techniques required for the safe and proper handling and measurement of radioactive materials. A profound reorientation in laboratory customs and habits was imposed upon them. The rate of growth of isotopic studies is a tribute to the versatility, awareness and willingness to discard the old for the new on the part of investigators in medicine and the medical sciences. In the pages that follow an attempt will be made to show that the biochemist and the biophysicist have now refined their techniques to the point that they are ready to join hands with the geneticist, the cytologist, the microbiologist, and the endocrinologist. When groups of investigators using different vocabularies reach common ground and begin to use a common language several worlds merge into one. There have been dramatic advances in the field of cancer in the past decade. None, however, have been so signficant and so encouraging as the drawing together of the biologist, the chemist, and the physicist in the unified attacks on the same problem.
GENETICS AND CANCER
Genetics has probably contributed more than any other field to advance our understanding of the causes of cancer. In this story the mouse has been cast in the leading part. There are good reasons for the choice. The mouse is a mammal, subject to many characteristic types of tumors. Its life span is short and it breeds rapidly. Large colonies of mice can be maintained under controlled conditions at reasonable cost and many generations can be produced and studied within the span of a few years. Nevertheless, 30 years of tedious effort have been devoted to the analysis of the hereditary factors affecting certain types of tumors in mice. The chemist who wishes to study the chemical reactions of selected substances with one another must begin by preparing pure compounds. The geneticist who would investigate the way in which certain traits of an organism are inherited must begin by preparing pure strains of the organism with clearly defined differences in the chosen traits. Only then is it possible to interpret with confidence the systematic programs of cross-breeding that form the core of his experiments. Pure strains are obtained by brother to
sister matings for 20 or 30 generations. The mills of heredity grind slowly. As a result of these sustained studies of the mouse several important conclusions are now clearly established. In the first place, different strains vary greatly in their susceptibility to a particular tumor. Some strains, for example, show a negligible incidence of mammary cancer. In other strains one may anticipate not only that every mouse that survives long enough will develop such a cancer, but may even predict the approximate time at which the tumor will become evident. Comparable strain specificities for tumors of the lungs and the liver and for leukemia have been established. In the second place, a strain with a high incidence of tumors of one type may have little or no susceptibility to other kinds of cancer. There is no escape from the conclusion that the incidence of several types of tumors in mice is under genetic control and that each type of tumor is a separate genetic problem and must be studied separately. Similar, though less comprehensive, studies have established the same broad conclusions with respect to a variety of malignant growths characteristic of other mammals, of birds, of fishes and, even, of plants.
Incidentally, acknowledgment should be made of the fact that the work of the geneticist has contributed not only to the understanding of the hereditary components of cancer but has been of inestimable benefit to students investigating malignant growths from quite different points of view. One of the great advances of the past 20 years has been the rendering available to all investigators who want them a variety of strains of mice with well-defined sensitivity or
resistance to particular types of tumors. Only with such pure strains, in which the incidence of tumors of various types is predictable, is it possible, for example, to obtain consistent results on the potencies of cancer-producing or cancerdestroying agents. General acceptance of the practice of using pure strains has resolved many of the contradictions found in earlier studies and has greatly speeded progress.
Overdosage of animals with X-rays, with radioactive emanations and with ultraviolet radiations results in the development of malignant growths of various sorts. It has been established that these radiations also increase the rate at which heritable changes in all kinds of characteristics of organisms. occur. Such spontaneous or induced changes are called mutations. Appropriate dosage of animals with certain chemical substances, commonly referred to as carcinogens, will also lead to tumors. There is evidence that a number of these chemicals will also increase mutation rates in general although no clear correlation between carcinogenic and mutagenic potency has been established. Nevertheless, the hypothesis that the cancer cell is a mutant cell is an appealing one and has found many adherents. The genetic point of view is so cogent to the main theme of this report that it merits further definition.
Body cells multiply by increase in size followed by division. The daughter cells are faithful reproductions of the parent so that growth may be described as a process of self-duplication of cells. Cell division appears to be initiated by a complex series of changes in the nucleus. In the course of these, a bundle of filamentous structures-the chromatin network-separates into individual filaments, called chromosomes, which proceed to split lengthwise. The duplicate halves separate and reassemble to form two new chromatin bundles at opposite ends of the cell. The cell as a whole then divides to form two new cells each of which carries a nucleus which, by inference, contains a complete replica of the chromosomes of the parent. Thus the nucleus and the individual chromosomes as well as the cell as a whole have this unique property of self-duplication.
All this can be seen under the microscope. Where vision ends the eye of the scientific mind takes over to project events into the invisible. Current ideas in genetics rest upon one of those magnificent flights of imagination that change the face of thought and create new worlds. The basic idea is the concept of the gene. Genes are conceived to be the ultimate units of heredity. They are assembled in the nucleus like strings of beads to form the chromosomes. Each gene differs from its neighbors and each occupies its own particular place in the chromosome. Like the ultimate particles of matter, genes are invisible. They are recognizable only by their effects. The observable effect of a gene is on some particular trait of the cell that contains it. Each heritable characteristic of the cell is assumed to be determined by the character of a single gene or of a group of genes acting together. The gene theory is the atomistic theory of cell personality.
When each chromosome duplicates itself, each gene is assumed to reproduce itself without alteration of its position in the chain. The daughter cells contain a faithful reproduction of the genic pattern as well as the genic composition of the parent. This, according to the theory, is the physical basis of the faithful reproduction of the heritable qualities of the cell.
Occasionally the machinery of ordered duplication goes wrong. This is inferred from the observation that the progeny of a particular cell differ in some characteristic way from the parent and continue to produce offspring with the same modified character. A heritable variation-a mutation-has occurred. It follows, from the basic premise of the gene, that, sometime in the life of the parent cell, some physical or chemical change in one or more genes, or some change in the relative positions of the genes in the chromosomes has occurred. These modified genes, or gene patterns, reproduce themselves and a new strain is launched upon its course.
In recent years new horizons have been opened up by the demonstration that a mutation in a single gene may result in a single metabolic disability. That is to say, a particular gene is responsible for the control of one specific chemical reaction within the cell. These biochemical developments of genetics hold such great promise for the future that the type of experiment on which they are based merits illustration.
It will be assumed that the investigator has elaborated a nutrient medium of known composition on which a particular strain of organism will grow and reproduce. From the components of this medium the organism is able to synthesize all the constitutents of its protoplasm. Let one of the nutrients be A and let it be assumed that this is converted to the protoplasmic constituent Z. This
relation could readily be established by appropriate tracer studies. In the course of breeding the organism, a mutant strain is isolated which is unable to grow on the original medium. It will, however, grow when substance A in the medium is substituted by substance B. The original strain will also grow on the modified medium. Appropriate tests then show that B is converted to Z both by the original and by the mutant strain. The conclusion from all this is that the original strain is able to convert A to B and B to Z. The mutant organism also converts B to Z but is unable to produce B from A. The defeat in the mutant is narrowed down to an inability to conduct one single chemical reaction in the myriad reactions which comprise its total metabolism. The one missing link makes the difference between growth and no growth.
It is a short step from this conclusion to the demonstration that the mutant cells lack an enzyme (i. e., a specific catalyst) necessary for the conversion of A to B. Patient exploration leads to the isolation of other mutant strains which will not grow when A is substituted by B but will grow when C or D or E respectively is the substitute nutrient. The biochemist now assumes direction of the work. Bit by bit he pieces together the links in the metabolic chain and emerges with a convincing argument that the metabolic pathway of A is through B to C to D to E and so to Z. He demonstrates that each step requires its own specific enzyme and that each enzyme is under separate genetic control. The sweeping hypothesis develops that the genes control the complement of enzymes that a cell contains. The genes cease to be hypothetical units responsible for the biological characteristics of organisms. They become definite chemical entities with definite chemical relations to the life of the cell that contains them.
The biochemical developments of genetics were not undertaken in the name of cancer research. They emerged from basic investigations of the nutritional requirements of micro-organisms. They have been seized upon and are being vigorously exploited as new and powerful ways of exploring the chemical pathways of metabolism. They have also been recognized as having great significance to the problem of cancer. If the cancer cell is conceived as a mutant cell, it is reasonable to infer that it will differ from normal cells in its conduct of certain specific chemical reactions. If these anomalies can be uncovered, the logical point at which to attack the concer cell with chemical weapons will have been laid bare. This argument is justification enough for the patient, deliberate, and systematic search for nutrients on which malignant cells are more dependent than are normal cells and for drugs to which the cancer cells are specifically sensitive.
The foregoing discussion of genetics has emphasized the predominant part that is played by the nucleus in the transmission of heritable qualities. There is no doubt that this emphasis is correct. In recent years, however, evidence has been accumulating to indicate that the role of the cytoplasm is not entirely passive. This story centers around a large unicellular micro-organism known as paramecium. Paramecium has peculiar reproductive habits. These can be controlled experimentally in such a way as to produce, at will, daughter cells containing identical genes in different cytoplasms or those with different genes in the same cytoplasm. This is obviously an ideal situation in which to discriminate between nuclear and cytoplasmic contributions to inheritance.
The work which has been done on paramecium cannot be summarized in a few words. Suffice to say that the transmission of certain unique heritable characteristics of this organism appear to be under the control of the cytoplasm. Similar conclusions have been arrived at in studies of some plant species and yeasts. The results can best be interpreted by assuming that transmission is effected through self-duplicating entities located in the cytoplasm. Since these units are the vehicle for the transmission of heritable traits from generation to generation, since they have the capacity to multiply in the cells that contain them and since, as we shall see, they are prone to undergo mutations, they are much like the genes. Indeed, they are often referred to as plasmagenes. There is evidence that plasmagenes are located in some of the microscopically visible particles of the cytoplasm which have been referred to in an earlier section. If this should prove to be general these particulates would be the cytoplasmic counterparts of the chromosomes of the nucleus.
Plasmagenes are probably not able to function independently of the genes. Their ability to multiply seems to be dependent on genic factors so that genes and plasmagenes operate cooperatively in realizing the genetic potentialities of the cells that contain them.
The duplication of genes is always synchronized with cell division with the result that successive generations of cells contain the same numbers of genes. The plasmagenes appear to have a freedom of action denied to the genes in the