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i NK19, 0126 0%/j 0 Biological Aspect® of Cancer Ileee;arclz's . c,.4aewas C. irrrte, &a., Diremr Bmeriua, Rosnue B. ,]adcson Mmarial La6oratory, Bar HarBor, Maine; 3ciu,u'fic Diroor, Tobaao lnduaay Reaeardr Conrmiaee The following discussion of cancer research is frankly from the biological point of view and is therefore limited in scope and application. Principles and processes of biological interest will be chiefly considered. No attempt will be made to oover in detail the vast amount of published data on experiments which contribute to-definition or evaluation of the various broader subdivisions of the field. If the reader is interested in a more detailed perusal of c-epporting experimental evidence for general etate- mentg, he oan find a partial bibliography in the writer'e booklet on Genetica, Btiotagieat Individualify, and Chvuer.^_ From this legimcing he can, by covering the literature since 1954 and by tracing back from tho given references, obtain some idea of the tremendous vigor of preaentrday reseaech in this field and the extent and divereity of the experimental work already accomplished. Bankgronnd Researoh The history of the development of the contemporary major action in the $eld of experimental biological research on aanew presents an intereat- ing parallel with the last great critical battle in the war between the Staft. At (Iettyeburg the first contacts of the opposing forqee was largely a "blind" and unexpected meeting of the cavalry "antennae" of both armies. Each had fittle or no idea where the other was until actual contact occurred. From and around t,hie initial center of entanglement there developed, by the assembly of more and more unite of infantry and artiltery, a major alignment and a fierce and final engagement of long duration and decisive significance. In biological cancer research early experiments on animele, largely with mice, rate, and fowl, were 9cattered and chiefly observational. The technique of tumor traneplantation was one of the most important tools and was being extensively employed by workers under the Imperial ~ WoUsA tapu6lloetlam eeptem6n!!, tYbf. • Dr. UtW.u IovlOed by tEe naefd er 6Alear m esprou Ele vlem mlmvet proElem• m oenaer. e 9lrolmd Ldvanlty Pnq ML 40 l.-d d fb Nnlwd drw lnden4 V.L {q l1w b ne..f 1189

I H KE01 26061 442 LI7TLE 1 Cancer Fund in Great Britain and at the Rockefeller Institute, the Cancer Commission of Harvard Unive.-eity, and the Crocker Institute in this country. Continental European research was largely aimed at the discovery of infectious agents and the possible development of immunity. This point of view also activated a number of British and American investigators. This was the stage comparable in many ways to the "cavalry" contacts with the enemy. There was similar limitation of the type of a-eapons which could be used and there was also the same sort of isolation and . importance of individual effort or the work of smell units. It is, of course, impossible to single out all the factors disclosed by research at this stage, which later became the major elements in leading to further developments. Some, however, can be safely mentioned because their basic connection with existing and future research is clear and functional. In presenting them there is no intent to indicate relative "imporsance" for, until the final solution of <uany problems that are still unsolved or even unformulated, any rating would be unwise if not impossible. Dtsoooery ond Isobcion cLf 111eenable Agewte From the pioneer work of Rous and his associates, the existence of a filterable agent (virus) was dearly identified as the chief etiological factor in producing psreomsa in the domestic fowl. This was an exciting and stimulating discovery. From it there developed a still active series of experiments (Claude, Duran-Reynals, Porter, and others) which gave important information on the morphology, reproduction, and modifiability of the virus and of other similar agenta. Shope described a papilloma-fonming virus in the rabbit and thereby demonstrated that the mammslian cell can include a virus which actually allows an increased rate of cell division. The staff of the Jackson Laboratory, stimulated by the recognition of a strong matensl influence in the etiology of mouse mammary adesooar- einomes (Murray, Little), published in 1933 evidence of the general esistr enoe of this phenomenon in several high-tumor strainA. In the eame laboratory, Bittner detenained that the agent which caused this maternal in9nence was ordinarily transmitted in milk during nursing by the young. The agent was later shown to be preeent in the blood and in various body tissues of both males and females of "high-tumor" etrains (Woolley, Little). - Its selective distribution was later demonstrated by Hummel who found that it was absent from the placenta. The fact that it apparently remained inactive in the intact meles was also highly significant and suggestive, which encourages the theory that other latent virmses may be factors in the etiology of other types of neopleem. The filterable nature of the mammary-tumor inciter and its immuno- logicel properties, together with some evidence of its identification under the electron microec+ope, have further etrengthened the belief that viruses are possible etiologieal agents of general importance in neopUaeia. The work of Gross aud others on salivary-gland tumora and on leukemia in mice also sse..el ur N. flW..d Cawen lrtltW I

I HY,801 26062 BIO100iCAL ABFEC'r8 OF CANCER RESEARCH 44H falla in with the theory that virus phenomena are a field of continuing promise for the investigator. In pure genetic research certain similarities between viroid and genic qualities and behavior keep active the interest of research v'vologlata and biologists in intracellular activity in relation to mitosis and molecu'.ar organization. The development of the initial and early contacts with the tumorigenie filterable agents into a full-scale, broad, and vigorous research "front" is one phase of the major "engagement" in which those iuves- tigating the oause and control of neoplesia are and will be concerned. Genetic Theory qf Tissue Traneplantation I The initial objectives of tumor transplantation in animals were: (a) to propagate the chosen tumor in order to obtain a larger supply of tissue for continued study, (b) to investigate the host reaction to subcutaneous or intraperitoneal implants of auto-, homo•, and heteroplastic tissues with a vim to detsrmining the nature of the factors which determine suscepti- bility and nonsueceptibility. These two objectives were, o[course, pursued at the same time, with the emphasis of earlier work ah.ifting somewhat whimeically as tme experimental results suggested. Since, in most cases, little or nothing was known s~.R•ut the genetic nature ot the annhals used as hosts and since these animals differed funda- mentaAg and invisibly from one another in their genetic characteristics, their responea to inoculation was varied. Succeai;ve groups of animals inoculated gave "t,akes'• of the tumor from 0 to 100 percent. T're British sehool' and some Americans explained this variation eq a response to changes in °virulenco" in the tumor itself. Extensive papers were published dealing with euch changes both in untreated animals and in those which bed, before or after inoculation, received treatment with varioo4 eupposedlJ+ "preventive" or °therapeutic" agents. To add to the confusion of the unpredictability and of the irregularity of consecutive results, it was obaerved that temporary pLTeietenee or even growth of the tumor implant was foIlowed, in some aninnale, by its regrrs- eion and dieappearance even in "untreated" animals. A quantitative phase was thus added to the already obscune problem This was the eituat.lon in the 6rat decade of the Twentieth Century when th-s work of Tyszer begge, to point the way to an opportuaity for progress toward a more definite and satiefaotory level of experimentation. This waas the phase of bringing up heavier units after the "cavalry•' aontaot. Tysaer had access to a etrain of Japanese waltzing mice which had been used by Yerkes in extensive behavior atudW, and which were being produced for research purposes by a Mrs. Lambert in a town outeide of Bost9n. The Japanese mice were probably descended from the Asiatic mouse Mue badris+HCa and were undoubtedl',p consh!irabl,y inbred having been derived from an originally very restricted nucleus of animsls. They were amall and delicate, and latsr experience with their eueceptibili,ty to 1.te4naa,H.miriia

;i K101 2Ei063 i i infections suggests that Mrs. Lambert possessed an unusual amount of skill and patience to produce them in the numbers that she did. • These nrica developed "spontaneously" a number of matamary adeno- careinomas and an occasional Sbrosarcoma. Tyzzer found that such tumors, on transplantation to other mice of the lesmbert strain, grew in practically 100 percent of the inoculated animals. The same tumors failed to grow in all mice of other strains. This presented Tyner with an opportunity to study the reaction of first- and second-generation hybrids between the 100 percent susceptible and the nonsuscaptible strains. The crosses were made and a mammary adenocarcinoma JWA was used for inoculation. Ald the first generation hybrids grew the tumor. Surprisingly none of the 70-odd second-generation hybrids grew it. At this point the writer took over the experiment and repeated the crosses. As before, all F, hybrids were susceptible. Of 183 seoond-generstion hybrids, 3 grew the tumor and 180 were noneusceptible. This reaultt wes confusing and unorthodox genetically. In 1814-10 the writer explained the experimental findings on the theory that the Japfinean we3"g mice introduced into the cross from 12 to 14 independent Mendelian genes which were p,e+eut ia att fi, animaFa, thus producing suaceptibility, to the Japanese waltzer tumor tissue. In the second generation the random aasortment and recombination of these genes would not reproduce simultaneously ;he total of 12 to 14 required genps, eacept rarely, for most F, animals would be derived from germ cells lacking one or more of the needed genes and these animale wnuld be nonsusceptible. A later tsperiment using a Japanese waltzer fibmsarcoma gave an F, ratio, suggesting that here 4 to 6 genes were involved. This wss the Geneti.3 Theory of Mransplantaitaon. Soon after, Strong began working with transplanted tumors of various cownon (nonwaltffing') mouse strains. He obtained 3-factor, 2-factor. and finally 1-faetor ratios. Backcroas generations confirmed the genetic theory and it became established and is generally accepted. By the use of this genetic theory various important lines of investigation were opened up. Among them the following may be mentioned as examples. 1) Changes in the genetic nature of tumora themselves were detated and analysed. These were always in the directiou; of simplification by the inactivation or by the disappearance of one or more genes. The changes occurred suddenly tuo irregularly as far as controllable faatom were concerned (Bittner, Strong, Cloudman). 2) By selective inbreeding, "single factor" lines were established and the nature o4 the genes uv anelfzed (Saell, Gorer). As a result, the genetics of "histooompatzbility" has been, and is being, determined with great aocaracy. 8) The genetio theory has led to the recognition of "enhancing" effects in tumor and in normal tissue growth following transplantation; these enhancing effects are of basic importance to our inLreaeing knowledge of immunologiaal prooesees. /aart .t ub R.N.d CoM. Irs~.

I H H101 26064 BIOLOpICAL A8P8CT8 OF CANCER 8F$EARCH 445 i 4) The ose of known and controllable genetic stocks and of tbeir tumors has provided the animal material for the established program bioassay of chemotberspeutic agents. 6) The fact that the host apimal can sometimes modify the biologicel nature of tho transplanted tumor has been recognized and is being investi- gated (Barrett). 6) The problem of temporary growth followed by regression of the tumor he.s been sufficiently defined to make it practical to study and control some of the quantitative aspects of that proeess. 7) The successful application of the genetic theory of transplantation to normal as well as to neoplestio tissue hes provided an experimental method of analyzing strain and individual differences in biochemical composition and in growth phenomena of various organs and tissues as well as the comparison of normal with neoplastic tissue. 8) The rate at which the individual develops its specificity in various tissues and in organ systems can be studied and analyzed by inoculation of alien tissues at various chronological nge8. Transplantation has thus eorue to bu snot,her bra:d and active 8eld in which biological research will continue to expand and to deepen with fruitful results. It is a major "front" in the war against cancer. Development of Inbred St.aina In t909 Johannsen, a Danish botanist, described and explained the production and development of genetically homogeneous "pure lines" of besns by the continued pronese of inbreeding through oelt-fertilitation. He bed previously (1903) seen the possibility of such a technique but did not prove it experimentally until latsr. In that same year the writer, working under Dr. W. E. Castle at Harvard, had, by recombination of coat-cofor genes, produced dl7ute-brown (DBA) and pink-eyed dilute- brown p)BAp) mice. Brother X eiater matings of several linee of each of these varieties was etsrted at once. One of the dilute-brown hpmoge- neous lines survived and is now the DBA/t strain. It ia eleo the progenitor of the homogeneous DBAJ2 eubstaain. The proeeea of producing homogeneous genetic strains by eliminating hetero$ygoeity can be sueoesefully osrried out by unbroken brother )( sister or parent X offspring matings. Although the "purification" proceeds more slowly than under self-fertilization, the end result is the eeme. Strong, by similar methods, produced the albino A strain and the very valuable C8H agouti strain. The writer eimilerf,v produced C67BL and turaed over C88BL to MacDowell who completed its inbreeding. The C57BR strains A, B, end C were isolated from descendsnte of a brown segreg4te in C67BL by J. M. Murray. These strains, together with BdLBk: STaLI, AKIi, and otbers, form the foundation for the great majority of medical reseanoh being carried on with inbred mice: Duuning and Curtis, at the GTocker Institute;Xing, at the Wietar Institute; and Castle, at Harvard, produced and studied inbred rat straine. Eaton and Wright did the same for guinea pigs. Castle and, later, 8nwin inbred a.u.% n..a.un..roa

H NI01 26065 I i I 446 r.rMe rabbits also, though not eo closely as was the case with smaller rodents. At about the same time that the earlier inbred strains of mice were being developed, Slye was publishing a eeries of papers emphasizing the effecL of beredity on the production of various types of neoplaems among the mice in bar laboratory. Although the pedigreea of her experimental animals were complicated, she established beyond any doubt: (a) that cancer in general occurred much more frequently in successive generations of some families than it did in others; (6) that tumors of the sama type often occurred within certain families much more frequently than within others. The creation of inbred strains definitely proved that the tendency to produce a given type of tumor could be fixed and maintained in successive generations. This was also evidence that at least some of the etiological factors in tumorigenesis are specific for tumor type and can be separated from one another by genetic selection. Within a genetically homogeneous strain, transplants of normal or of neoplastic tissue from animal to animal are treated by the host as auto- tranaplanta. F, animals produ^.ed by or-Asing two inbred strains can grow, either simultaneously or separately, normal or neoplastic tissue from bctA parent.etraina. Some of the important lines of research opened up by the formation and utilisstion of inbred strains are as follows: 1) By the appearance with a high degree of fidelity of the same general types of morphological and physiological development among the in- dividuals and among successive generations of an inbred strain, the inveatigator knows in advance with a far greater degree of accuracy what to expect than would be possible under any other circumstances or with any other type of material. 2) By exhibiting definite characteristic types and degrees of hormonal aution within thp inbred strain, experimental manipulation of hormonal balance can give a new method for anal.ysing the possible role of hor- monal action in tumortgenesie or in other biological prorease.s. 3) The high degree of genetic uniformity in an inbred strain provides a "constant" biological unit for quantitation in assaying extraneous agenta, or in experimental procedures introduced by the investigator. 4) The reverse technique can be used aleo with great effectiveness; a single substance or experimental procedure can be tested against a num- ber of different genetic backgrounde of known origin end natiu-e. Use of these opportunities bae already resulted in exciting and stimulat- ing advances in almost every phase of experimental cancer research. 1lere is every reason to believe that they will continue to be an easential ,lement In hastening and extending discovery of new knowledge and will also be the beat obtainable tool for repeatiag, extending, and evaluating experimental procedures in biological and in medical research. The inbred strains and their hybrids used in conjunction with the other methods ard tenhniques descibed is tbus an invaluable "weapon" for future re- eeareh. jCOIMW W do NatlOtld dumR ItldlYtY

1 NK1012606G BIOIA(iICAL ABPSC'1'8 OF CANCER RESEARCH 447 7yseue-Ceafture Deoe[opments Although interesting and important discoveries in cell physiology and morphology have already resulted from the use of tissue-culture techniques, there remains a great number of exciting twa~aibiiities as yet uninvesti- gated or not su9iciently explored. Among these is the potential value of synthetic culture medin in analysis of cell composition and physiology. There should be experimental variation in chemical components not only of adequate synthetic media but a]so of those media which may reveal selective and significant survival differences, in the response of various genetically controlled cells or tissues. The possibilities in this field of research would seem to be almost unlimited. More extensive use of exposure in vitro of various genetically controlled tissues to diS'ereotsuspected or known carcinogens should, if followed by transplantation into hosts of known but differ•ent genetic types and into hosts of different chronological ages, reveal many new facts of impor- tance. These are the eorta of techniques chat should help ce to bring isto direct obserroatiwa ohanges in tissue response which will in turn aid in analyzing and in cventually understanding tho activities of cells and tis- sues in different types of msmmaLsn individo.als tnhere direst obasasntion is sot g+eeaible. . IrrstHaeion ffects and Isotopes From the more directly biological point of view, the ui.ilisation of gene~.ically controlled animals, tissues, and cells offers great opportunities bot.L in quslit3 :md quantity r.i irradiation problems. The work of W. L. and L. B. Russell has alreaay shown the practicsl:ty and fruitfulness of this approxob. Much of the wor:; with tbese agents will, however, be in their biopbysia.l and biochemical relationship. The writer is not sufficiently inforu4ed in either of these fields to comment intelligently on their possible details of development. If full advantage is taken of the known techniques of transplantation, inbreeding, and t5ssue culture, either singly or in combination, thp venra- tility and effectiveness of naaly:ing and interpreting irradiation eSeotg and the significance of isotope pathways i„ ,u.d.e'ur.lio ond morphogenetic processes will be greatly incresaed. Since be'n- and psr,ma =;rraiaat+'or: has been well identaBed as hn-ring mutagenic and carcinogenic properties, the rolationship betaeen the exaerimentp3 responses of somatic and germ ce119 should be further ex- plained by experiments comparing such irradistion effects on fJto two types of celle. Up to now the effects of irradiation on the germ cells of mamtaals have been expreased in mutations which are usually either lethal or which produce morphological abnormalities involving dieproportion or other depsrtures from the symmetry and balance of normal development. It is interesting that there la little if any evidence of Increased unoon- trolled neoplastio growth in the emblyos or young nrodueed by the combination or participation of mutated germ cells. Ye1. !q Ne. 0. rn.1 19fs

i HKI0126067 I 448 zrrm On the other hand, there are in somatic cells many casea of induced neopkaiia, which often occur after a long latent period. The biological significance of such prolonged latent periods is not understood and is not receiving oearly so much attention and investigation as it deserves. Compared with the vast number of cells receiving irradiation in any given area, the number that exhibit a neoplastic change is infinitesimal. If a direct, simple, single process is involved in this change, why do not alt or at least the great majority of exposed cells show it? The evidence seems to favor a hypothesis of a series or chain of intracellular changes which, in the vast majority of cells, is broken or obstructed before the sequence reaches the tumorigenic threshold. What the trigger mecha- ni4ms may be for each step and for each type of tissue are the sort of problems that will require patient and prolonged investigation to match the duration and finality of the processes being studied. Cell Cheoaistrr and Funetion Modern methods of analyzing the chemical composition of cells give real promise of adding knowledge of the differences betweea cRlls at various stages of differentiation, of various specific tissues, an;f even at different stages of functional activity in the same tissue. It would, however, be well to remember that when a chemical is isolated fmm the cell and is measured or ageayed, the osll itself is no longer dynamic or an integrated living system. This, of course, is not in the nature of a atitieiem. It is merely a naturol technical Gmitation which is made necessary by the definition of the type of the investigation itself. The same limitation applies to pathology where it will always be impossible to state definitely what the "next stage" of future development would have been hed the cell or tissue been left in the living bod~ instead of being fixed, stained, and sectioned. Studies of cell ehemistiy and cell function based on isolated material should thoniore be integrated with and eupplemented by studies on grrups of living cells of krlown and uniform genetic composition in oioo or in oilro. Farpert.nental Cancinogeneais Since the initial discovery of the prooess of experimental oaroinogenesis on the ears of rabbits, there have been literally thousands of ezperiments with different challenging agents, dosages, sites, strains, species, and ages of animals. Agents have been used alone, in combination, or in soguenoe. The excellent and comprehensive assembly and tabulation of tbese ea- periments by Hartwell w8l con'vince anyone of several facts. i) There is a very large number of chemical agents that ean prove to be carcinogenic in one or more types of animal material. 2) There is every sown to believe that the number of auch substances will be steadily and eigni& cantl jr innressed as time goes on. 3) The action of these substances ie now being recorded and assayed under such scattered and uncoordinated conditions that no integrated or progressively analvyaable program of prooedure, approach, or reduction of variables essts. s.o.d a do r.uead c~ t..o.r .

I r i I HKI0126068 sloLGaICAL ABPSCTB OF cAMCBn a88BAaca 449 There is naturally a question as to when and bow far one can extrapolate from animal research to man under any given set of experimental condi- tions. Some general comments in this conneetion may be helpful. The germ cells of higher mssmmstls appear, as a group, to poesee~a most of their baeic types of organization in common. The proeeas of matura- tion, ovulation, ferta7ization, embryonic developmental sequence, im- plantation, and parturition aro essentially similar in primates, carnivores, ungulatee, and rodents, which are the four orders commonly studied. The internal organization of chromosomes and the behavior of genes in the four orders aro closely parallel. It would eeem likely, therefore, that a high degree of extrapolation f' om one order to another is permissible in the field of germ-ceII and early developmental studies. Hormonal origins and behavior are also very much alike in the four orders. Until new evidence of divergence is found, one may compare with oonsiderable scientifio justification, experimental results in endocrin- ology with expectation of fairly olose conformity in interorder behavior. The functions and baei a utructure of the great organ eyetsrss-nervoue, digestive, excretory, circulatory, respiratory, skeletal, and reproductive- are essentially the same, although proportional, regional tissue develop- ment may vary considerably within an organ or organ system. The outer epithelial tissues show a considerable variation in structure and response to environmental stimuli. The sldn, hair, nw1s, and teeth are higWy specialized; the3e vary greatly in form and function not only between orders but also betn eon species, strains, individuals, and even between different parts of the same individual. Esperieiental re:tponse of these structures to various challenges or environmental faetors may be expected to be as specific, characteristic, and varied as is the type of animal selected for study. It is very important to remember this fact, for the speed and convenient experimental response of many of these easily accessible and observable tissues sre tempting qualities for the avid and impatient investigator. There is real danger of overextension and unwise generalization from highly restricted and specialized data. This is the main reason why in order to use such dara to fJieir full limit of f ustt}/'iabre extrapol,aeion. the de- wlopmend of a tabfe of alandard Quantitotim raeponase of peneteeallyeontrofled animal teasues to known chaflenges or expoetaw is an absofute pragquieife. Motlr such a foundation available, the value of such tissues for assay may prove to be unique and of last3ng importance. R'itAove it, observa- tions :;,ll renkain isolated, fragmentary, e.nd of doubtful usefulness in transfer, extension, or applicability to other work with the same or with different species. It may prove to be impractical to integrats work of this sort; but until the possibility is considered and some concerted e,nd continuing effort at definition and coordination is made, the total of eaparimental results will remain a hodgepodge of disconnected observa- tions of minimum value as knowledge exchangeable or utilizable between investigetme. It may be well, while waiting for the development of new and more wL a4n.ur.nk 1060 I

r HK10126069 wM$ "o refined methods, to review the commonly used methods for bioassay of chemical carcinogens and to attempt to determine and to define the variables encounteied in the present experimental approach to this problem. SwJaee paintdng is one of the more important and widely used methods of application. lnaedion is another. Inhalation (which is often ingestion) is a third. The following discussion applies primarily to the first two methods but also, at least in some of its phases, to experiments using inhalation or aerosol techniques. The first series (I) of six factore, noted below, involves pertinent controllable attributes of the animal or tissue used for assay. The second series (Q) of three variables deals with methodology to be considered in broad techniques of application. I. Host Reaponar A) The age factor should be evaluated, both by observation of the total animal and by study of the relative aging of the speciHcally challenged tissue. This can be done at prenatal, neonatal, and postnatal levels up to snd including senescence. Phyeiological and chronological age do not always develop pard paesu. B) The gen,eEic atn.dae of animals used should be considered as an aid in the analysis of the carcinogenic effects. Strain response to chailenge should be correlated with the known and reoorded morphological and physiological characteristics of the strain. C) The effects of application of the oxperimental :.gent should be studied on various areae of the akia or on selected target tissues and organs which develop in different ways atrudura[lp or at different rates (according to degree of differentiation, physiological or mitotic activity, repair rate, etc.). Such studies shonld lead to additionat information on host reaction not only in relation to laiatologdodl changes dure+ig mor- phogsnest,s, but in relation to certain genet+c influences which may predictably modify the development of the challenged structures (genes for brachyury, polydactyliem, short-eare, hairlessnese, et.o.). D) The evaluation of the factors of dispersion and of localisation of the agent used should a]eo be considered. This involves applica- tion to a defined structure (tail, ear, digit), which oan later be removed by surgery or isolated in part or sn toto by physical (temperature, radiation) or chemical (anesthesia, neutralization) means. E) Experimental manipulation of hormonal level should be intro- duced as another means of modifying internal balance and resulting host response. Such studies will oontribute not only to our knowledge of the generaC processes of earainogenesla but also to the possible anal,ysis of recognized ees (gonad, pituitary, adrenal) or developmental differences (thymus, thyroid, and above-mentioned glande), and the detection of new ones. F) Manipulation of vitamins and other ratttritional elementa as modifiers of the tissues obs]lenged should be a field of investigation. It is t.o.r .r H. n.d..d Ga... 1.ulue. I i r

I N 1iE01 260; U HIOLOOICAL ABPE:CTe OF CANCER EEBEARCH 451 remarkable how little such studies have been used in their broad analytical implications. II. Techn4quea o,f Applitalion A) Possible interreactions of ehallenging agente should be studied by application of individual agents alone, in separate coinciderelal or otherwise-timed sequential application, and in prealazed com6araa- tione of various relative proportions of the oomponent agents that are being compared. B) Dosage should be studied and analyzed with t5e following con- trollable variables in mind: total amoun.t of agent; concentration in aolvent or other vehicle; numLer of applieations; tirne dntera¢ls between applications. G*) All of these variables csn be studied in d.eo and, ae the technique of tissuo culture and the control nf synthetic culture media are developed, they can also be investigated in nitm. In this way the broad program of experimental contsctcarcanogenesis and of tiseue-culture research can be coordinated snd used to supplement one another. It may also be pointed out that any successful efforts to define and agree upon various elements in biosessS of pos°ibie cercinogens wi1_1 have a double value: 1) They will gradually build up a table of standards of reaction for known carcinogens with which assay of unknowns can be quantitatively compared with some prospect of repeatability and contiauing significance. 2) They will make possible much greater direct eachange value between investigators of the results which they obtain, thus accumulating at the maximum rate under our present levels of knowledge classifiable and coordinated information concerning csrcinogenmis. H) They will contribute directly to our fund of information eoneerning the faators within the organism which establish and maintain the internal balances on which health depends sod concerning the factors that, by threat to or upset of these balances, predispose to or originate the "coa- stitutionsl" diseases. Basis for Future Frogrese It would seem that a great deal of progress can be made indefinitely into the future by making deliberate efforts to plan research on the over- lapping border problems of the various fields which have been mentioned and to train research personnel who are fam'liar with two or more of them. For example, virologists who are also trained in genetics, or tissue oulture, or irradiation, or isotopes, or cytochemistry, or eaperi- mentsl eareinogenesis would find many chances to discover neW facts. So would those skilled in any combinations of tbese techniquea. It would seem that a knowledge of genetics at the strain, individual, tissue, and cell level would probably tdd to the other dieaplines the oommon basic element of which they were all most in need. V.L Sp Na 0. tlm1 1960

t HKE01 2607 1 452 LITTLE Leadera in orga n-zing borderline reeesrcb are today few and far between. They have arisen sporadically as a result of their own vision and ter.acity in obtaining the necessary training There should and wiil continue to be a certain number of them who will appear in the future without special effort or planning and they will continue to be of just as great value as have those who preceded them. Their number, however, will be pathetically inadequate to take full advantage of the opportunities tp expand, deepen, and hasten the increase of our knowledge of the processes underlying the genesis and progress of neoplasia. The existence of these opportunities is realized by only the relatively small group intimately in contact with them. Many of this group are actively and enthusiastically engaged in their own research, jealous of the all too fast passage of time, and painfully aware of the need for expansion of resources to give them the additional "eyes and hand that would multiply the effectiveness of their efforts. Few of the active group have the inclination to become missionaries to attempt to convert the powers that hold and distribute sources of increased support. Few can themselves initiate or develop the opportunity for intimate exchange of esperiences, views, and plans for the future. A oom- pany attracted by the more immediate, practical, and the more glamorous appeal of the clinical or clinically pertinent phases of education and investigation holds the stage and fails to understand that when its r+epeti toire reaches a certain point, unless a supply of new basic researcb imowl- edge is available, it will have to depend upon "revivab" perhaps with new "orchestration" or "stage settings" to keep the bex-0fdee receipts of financial support at a satisfactory level. Wise patrons of any creativa: activity, including cancer research, recog- nize the unavoidable and basic truth that unless the sources of new ideas ate developed the evolutionary pracess of any art or science will cease. The way in whioh such "46ise" patrons are developed is by bringing them into direct or indirect contact with those who are creating. As yet the east- ance of adequate opportunity for contacts of this sort, in the relationship of the biological sciences to research on both normal and abnormal growth, is conspicuous by its absence or whimeiaal uncertainty. The recent demise of the National Research Council's Commitfee on Growth and its replacement by more centralized control of even the reoommendatory phases of research support by the American Cancer Society appears to many to be an unfortunate retrograde etep. The flowera of polite "ra;ognition" placed on the corpse merely served to emphasize the tragedy of the failure to recognize the potentiality of its value during its life. Present indications are that the rate of progress of the biological age in aanaer research will be "on foot" in the immediate future unless pome financial "station wagon" with room to hold the h{toLh3ft "faW" of basic research stops and "picks it up" from the roadside. This eeeme unlikely for the road on which the "traffic" of prof eot research is buzzing along is broad and level and driving Is oomfortable. Other hitobhitcere #..=d .r u. UwMtl o,.w. d.rrr.

HKI01260; 2 BIOLO6ICAL ASPECTS OF CANCER RE6EAnC$ 453 clad in appealing uniforms of "direct service to humanity" are as numerous as service men were at the height of World War II and they do not have the unwelcome "children" of ideas of unproved practical value along with them to require attention to growing and assertive demands for "food" and other resources. This situation, however, muet not discourage those concerned. It is merely evidence of a delay in recognition of true values. It does uat and mranot weaken, mar, or diminish those values. Patience and depction to ideals and the precious duty of guarding them are an essential part of the dutiea of those fitted by their nature and training to represent the frontier phases of experimental science. Having briefly considered some of the major fields of experimental aci- ence in which studies on growth are being conducted, we may next suit- ably discuss certain principles of biology which apply to the problems under investigation. Many of these are tacitly recognized but a summary and review of them may nevertheless be helpful! Latent Power of Growth It is the common custom to evaluate the nature of biological processes on evidence obtained by methods that satisfy our senses, with the least dis• turbance possible. The growth curve of the mauumal shows the moat rapid growth rvte in early embryology with a gradual and reaeonably steady decrease in rate until ti.c "adult" stage is reached and progressive growth has "eeased." The differentiated mamanalian cell is commonly considered to be a biologi. cal unit whicbh has lost at least the greater part of its earlier power of mitosis rate, which its antecedents demonstrated before differentiation. This point of view reached its high point in Cohnheim's thsor,v of "embry- onic" rests based on the hyp:,thesie that there were scattered throughout the body a limited and unpredictable number of ceJls which resisted the re- stricting process of differentiation and remained in physiological conceal- ment until an opportunity owurred for them to come out of hiding and to go beserk ne neoplasms. Any suoh conception of the loss of power of growth by "aormaP" nmam- malian cells overlooks a mass of evidence that indicates perhaps the univernal presence of enormous latent power of growth, which is available for use under a number of conditions which call for its espreseion. The primitive and most "normal" type of animal cell which hae, by an unbroken line of deecent, populated the earth with a,nimal life since that phenomenon began, still shows unabated power of growth and reproduc- tion. Studies of protozoa uniformly reveal such power of growth and incidentally but importantly also show that the different rafee of cell division are inherent functions of various aublines or clones. From the point of view of biological survival value, therefore, the primi- tive, rapidly reproducing animal cell ie the "normal" unit and the limited 4PQ.mssUmfl.eAhmWm ol uNp p*eiplof, unn.de I%a trdasrhte4 mftmeA m ILs t.ne L.otme~ gh® bl tse.rns m 1YE1(Blanhud IIaloeqV FreN. voL aa Ia, a, ao.f Ivi+

HKI01?6073 i 454 saTMs or reatricted differentiated cell is, to a certain degree, an "abnormal" prod- uct of aggregation of cella, of d;vision of labor, and of specialized function among them. Because in "higher" animals the vast majority of cells ordinarily obey the control of activity and funaion superimposed by the tissue, organ, or organism aa a whole, we have adopted very literally tbe evidence of our eyes and speak of "stimulation" when cells resume a power of growth similar to that of their less restricted antecedents. It would be more logical to consider increased growth to be the result of "release" of latent growth energy and to think of processes which are involved in maintaining lower growth rates as "control." Let us see what the body actually does when the internal environment of "control" is challenged by various circumstances. Regenerntion The striking response of a tremendous increase in rate of cell division by relatively inactive and "controlled" cells following mutilation by incision or amputation is a widespread phenomenon in many inve.lebrates and in the smphibia. It is hard to imagine a biological "etimuisot" as a result of a radicai trauma which cannot conceivably create an extrinsic ebemical with a etimulatars function. Io is also obvious that the cells which regenerate the amputated structure would not have revealed their great innate potentiality of increased cell division had they not been challetiged and had the balanced control of their environment remained intact. Repair The ability to release latent powers of cell division to repair minor t.-auma or unbalances is a basio quality in most nsammaLian tissues. It is the "rule" rather than the "exception" and again bears witness to a great latent power for growth which is so general and acnsensational that we are apt to overlook its great biological aignificance. Only when the "repair" process fails to cease when the original balance is regained do we recognize the potential energy that is involved The formation of keloids is a good example of such a situation. Racial, familial, and sex in8uenees, which affect this uncontrolled continuation of "repair',' by formation of relatively primitive fibrous tissue, give us further food for thought concerning its deep biological significance. Repboement An even Iea noticeable but perhaps more impressive type of expression of the continuing growth potential of the mandmalian cell is the steady process of replacement of worn out or dead cells by the production of new ones. It is a prooess that while regulated and orderly is continuous throughout the life span and revcals a silent and otanipresent ability to utilize a latent growth ability which is present in essentially all mammalian tissues. There are other atrildng examples of latent power of growth possessed by mammalian cells and ready to be used when circumstances require. journal .r w n.u.w a.»m L i

HItN0,Zb074 ,HiOLOOICAL ASPECTS 08 CANCER REBEARCH 455 i . i Ttcl.enong The ordinary course of development of the fertilized mammalian egg results in the amount of selective and controlled cell division necessary to produce a single complete individual. In some cases, however, a fertilized ovum of the normal size, structure, and chlnmosomal number will begin to develop into a abngle embryo which separates into two equal or approximately equal mesaes of embryonic tissue. Thereafter, each laalf develops into a complete individual which at the close of its period of continuing cell division, culminating at the adult stage, is as Iarge and has experienced just as miauy cell divisions as would a siragle normal product of fertilization. It is evident that in this case the original ferti- lized ovum demonstrated its latent ability to develop twice as much tissue by twice as much cell division. Parthenagemesis Ordinarily the development of vertebrates depends upon the addition of at least the nuclear material of the sperm to the unfertilized ovum. Eaperimente by Loeb and Bataillon showed, however, that a certain number of unfertilized frog ova merely pricked by a sterile needle develop into a complete embi yo. No chemical was added by this purely meeban- ical procedure and the logical conclusion is that it released a latent growth potential present but not ordina* used by the unfertilized ovum. Neopfasta This proeess may reasonably be considered as another form of released latent growth potential beginning fuaid'is uf a cell. It is a c1hauge which actually improves the cell as a biological unit, forit dividesmore frequmtly than do its neighboring celis and therefore produces more "descendaeta" in a given period of time. Internal Balance and Unbalanwe i The delicacy, accuracy, and persistence with which the animal cell maintains Its individual charaotaristias and reproduces them in its •de- aoendaute are reflections of an ama$ing internal balance in both form and function. The failure to reproduce exaet replicas iA a great rarity under any ordinary environment and any usual challenge. Asymoletrioal or unbalanced cells do not, as a rule, produce viable desonde.nte. This was clearly demonstrated aome 30 yeare ago by Bla[teslee and his aoworketg who induced ehromoeornal unbalanee in Detrer+a by cold and by other eaperimentaUy introduced factors. It was observed that plant9 with 2n, 8a, or 4n chromosomes were viable and fertile. Those, however, with 8s + 1, 8» + 1, or 4n + I wel+a weak and sterilP. It ia also the general experience with cancer that wGile many calls with extraordinary variations in chromosome number and eiee may be formed in a tumor, those that divide successfully and perpetuate themselves are Vo1. 2% ns. a, Y.eL I9SA smeso.-se-a

P HKOO 12160715 456 i[Trts i usually the cells with a balanced and symmetrical chromosome count which allows successful mitosis, which in turn reflects functional internal balance. The problena of creating and me.intaining internal balsncs is an essential to orderly progress of life in the cell, tissue, and organ or organism. All of these structures have to utilize cyclic function of some sort. For some, such cyclic function is repeated at relatively uniform or predictable in- tervals. For others this function may be the response to an unusual or unpredictable challenge of some sort. There are a number of different levels at which internal balaace must be maintained in order to preserve normal function. These levels may be rougbly defined as follows: 1) Between components of the gene 2) Between genes Inthe chromosome Intraoelluler ................ S) Between ehromosomea 4) Between essential components of the oywplsem b) Between nucleus and cytoplasm lotercellutsr................ 8) Between oeIIs of any ttseue ~7) Between ttssuee of any organ 18) Between organe of aqy agnWem At any or sll of these levels various influences, either internal or external in origin, mc.y produce unbalance either temporary or penaanent. Examples of the establishment of new centers of balance at diffe•rent levels are: mutations upl/ain the gene, translocation, other new positional relationships of genes within the chromosome, polyploidy or nondisjuno- tional changes 6atweROm chromosomes, formation of cytasters in the •ylo- plasrn of anucleate cells, multinucleate cells or cells with a giant nucleus; nonconfortuing cells as in mouse mammary glands durisg the lactation oycle; hyperplesia of the cortical layer of the adrenal in mice and duplica- tion of orgade such as polydsotyTism. Ordinarily the gene, obmmosome, call, tissue, or organ adjusts itself to the challenge of such unbalance and regains equilibrium around the original center of balance. At times the unbalance is so great or so radical in nature that it impaus and destroys the function of the unbalanced structures or system. At other times the unbalanced structure regains equilibrium of function around a oem center of balance. There are there- fore tarre gencral types of possible response to the challenge of unbalance. The neoplastic process is undoubtedly related to and affected by un- balancing influences of various sorts. The fnereaseed rate of oeII division is one result, but more important is the independence of certain neoplenm fiom the control and balance ordinarily maintained between components of a tissue or between tissues within an organ. It has establaebed a new center ef balance with a degree uf differentiation or laok of it peculfarly its own. This is a basio reason why much mo:e extensive reeearcl' on the nature and function of controlling jnfluences in .wrmaW development and phyef- i lamml af ti. N.Carl cMev irtlau

I i I t H KI0126076 DIOLOGICAL A6PBC?8 OF CANCER $88EABCH 457 ology should continue to provide essential new knowledge of what the redrase from such control involves. A brief discuesion of unbalancing influences allowing neopleeia may help to give some idea of the great scope of this process. Hybrfdtaation and Ura6adance This is a problem of patvntsl dissimilarity. The results of combining dissimiler germ ceels by fertilization depend upon the degree of disaimi- larity between the celle combined. It the dissimilarity is very great no reaction occurs and the problem eliminates itself. F+om thie eatreme there is, with darvwing diasimilatity, a graded eeries of reactions, some stages of which mey be listed as follows: 1) Entrance of eperm-no nuclear fusion: a) no development; b) parthenogeneafo of egg nuoleue, no par- , tiaipatlon by sperm. 2) Entrance of eperm-nualear fusion: a) uneuooeestul attempts at mitosis; b) Impaired or abnormal mitasis; c) asymmetrieal, Impah+e3, or unsuccessful blestula or gestrula formatloa; Decreasing d) successful somatic development (eome- diselmtlsrity times witb Increased somatic vigor) and growtb, no fertilitq; e) same ae obove with impaired or eex- Ilmited fertility; J) eame ea above but with visible delete- rloue physiological effects or morpholog- ioal abnormalUfee, no 1n5uenoe on fertility; p) eame as above with no visible delete- rious effects on iertllfty and poeelble inereaee in somatic growth. It is in group Sf t.hst some estrnmely eigni6cant evidence of the effects of laek of edjuetment between different dev6lopmenlsl potentialities reveal tbemeelvea. Deacribed'tn order, in t,esme of the type of qffed, t.hese same levels may be liswd as follows: lo) no effect on releaee of latsae growth potential; lb) desuvotlon ad secondary "totel oell"--orge,niaation eontrol-followed by acUvity of letraoellulsr growth potential; 2a) destruction of "total cell" control and interferenoe by partial and in- oomplete deetruotioa of intsaoellular functional "meoho.nloe" control; 2b) a lesaer destruction and noerer approaoh to Ilberatlon from intaeoenukv meohanlae oontrol; Fo) destruction cd aocondary iaroroelluler, econtrols and orgenlsere oa the level of eariq latercellular oaasote; 2d) Impaired ability to control the usual amount of somatic mitoele, and failure to adjust to a point where the Internal orgselsstion af the aoo- deaeed potentlalhy of a balanced and funotlonst germ oell can be eoo- oompllahed; 9e) same as abore aith production of "nn.nooeeeful" type o/ germ eell= Vol. ao, *.. a. rntl laas t a.

i I r i HKI0126077 458 LI7TLE 2J) same as above with local failures to establish growth control in balanced and proportiooal quaUty or degree; 2p) all the proportion-maintaining controls function but at times leee rapidly eo that more mitosea otmur before the final control Is established. This delay is due to modified latraeellular activity, for the cells retain in- creesed latent growth potentiality or intreaeed power of "ineurgenoy" whioh later may express it8etf in renemed mitotic activity or neoplaele. The extensive work of Gordon with bybrid fishes has demonstrated that neoplaeia may be a predictable and direct result of unlike developmental patterne attempting to express themselves in the same individual. A similar striking increase in tumor formation over both parent strains was observed by the writer in a species croes in mice. The metabolic activity of two different genetic backgrounds attempting to adjust to one another may involve competing qualitative or quantitative variations in obemical components or in duration, speed, or sequence of chemical processes. It is therefore entirely logical to expect a greater risk of unbalance at all levels when dissimilar developmental patterns are in competition. A question that will naturally arise is why inbred strains with genetic homogeneity may show a very high incidence of neoplesia. 'Witbin such a strain the developmental pattern should be as uniform and predictable as one can espect in higher animals. The answer is that by selection of parents with characteristic genetic tendencies to produce unbalance in certain tissues or organs, these ten- dencies become genetically fixed and recur in the individuals of that partioular strain with a high degree of frequency. If the genetic unbalance occurs in a highly differentiated tissue or organ the localization of the neoplastic proem is more complete, as for example in mammary or pulmonary adenocarcinomaa. If the tissue is widespread and relatively undiffereutiated,,ae for example connective tiseue, the loca- tion of the neoplastic change is lea9 predictable, aa for example in f?bro- earcoma formation. Hormones and Unbalance The 6lst example of clear-cut e:perimnntal evidence of hormonal in- fluence on tumor formation was published by W. S. Murray (1927). The DBA etrain of mice with which he was working was regularly produo- rog about 80 parcent mammary tumdra in breeding females and from 8E to 40 percent in vitgins. No mammary tumora were produced by males. Recognizing the possibilities of eucceasful homologous transplantation within this inbred strain, Murray placed the obaries of sister animals in castrated males. The tumor incidence in these "feminized" molea was easentiail,y the same as that in virgin femnles. This demonstrated the influence of the intact ovary on memmtuy-tumor formation. Soon after Murray's work, I&caesagne, ut3liaing the follioular hormone and diethyletilbesterol, obtained results ttimilar to those resulting from traneplantation of the intact ovary. Later work by Strong and others at the Jackson Laboratory demon- etrated that different inbred strains had quantitatively distinct depees 1eam1 of a. fY.tle.d C...r trdtaa ,

Hl4()1260i8 1BtOLOO7CAL ASPECTS OF CANCER RESEARCH 459 I I of follicular hormonal activity. Thus while strain C3H produced the same tumor incidence (90t%) in breeding females and in virgins, strain DBA bad approximately 80 percent ± and 30± percent, respectively. Strain A, however, produced 80 percent ± and 5 percent. Hummel in studying the suitability of the A strain for Aschheim-Zondek preenancy tests found that its level of follicular hormone secretion was so low that there was an uncertain and unreliable response. Beginning in 1939 an even clearer and more specific series of results were obtained, by Woolley, Dickie, Fekete, and the author, on inter- hormonal action in relation to mammary and to adrenocortical neoplasia. When the gonads were removed from neonatal mice of three distinct inbred strains (C57BL, DBA, and CE) each strain gave a different char- acteristic and consistent response. The Cb7 mice behaved like the classical examples of castration effect. Relatively underdeveloped genitalia and secondary sex characters and no discernible interhormonal reaction by compensatory activity was the rule. In the DB9 mice, after a period of inactivity, there was evidence of resumed development of female secondary sex characters including mam- mary-tissue growth and the appearance of maramary neoplasms. Ex- ami.nataon of the adrensle showed hypertrophy and hyperplesia of the adrenal cortex. Irregular blunt "Hngers" of hypertr,rphic and hyper- plastic cortical cells grew down toward the medulla. These outgrowths histologically bore some resemblance to ovarian tiss-se and were undoubt- edly the source of the 'Yeminisiog" secretions. In the CE mice, again after an inactive period, distinct, "masculiniza- tion" effects were evident. These included renewed male-type growth of genitalia, attempted copulatory behavior, etc. Within a few months adrenal nodules were grossly palpable. Latsr histological examination showed in eeery ease, adrenocortical carcinoma with little or no discernible glandular structure. These carcinomas were readily transplantable into CE mice and when placed in castrated animals of either sex produced marked signs of mssoulinisstion. More recently, by early gonadectomy, Diekie has released, in certain genetic types of miee, activity of the pituitary resulting in hyperplasia and neoplasia. This response is prediotable and controllable. There is, therefore, overwhelming evidence that gonadal-adrenal-pitui- tary balance, which normally determines and regulates the hormonal growth-0ontrolling activity of these three glands, can be experimentally upset by "deprivation" technique; the results of the "upsets" are constant, predictable, and strilring. It is entirely logical to assume that if a major and critical unbalance can be produced by the "all-out" experimental deprivation procedures, there are minor strains and challenges toward unbalance produced by impairment or diminution of hormonal production or function under the eonditfons which occur "normally" in the body. There are many ea- amples of experimental evidence which bosr directly on this conclusion. vw.saMo.s.uoa waa

r 11KI01 2"'6079 , I I I 460 LIITLE A number of simple Mendelian genic mutants in mice and other mam- mals show established differences in hormonal activity which markedly affect general or local growth. Dwarfism and anemia are examples. Less simple but still clearly determined genetic differences appear in cyclic phenomena such as the reaction of mammary tissue to the estrus and pregnancy sequences. Hero Fekete has demonstrated ovulation and corpora lutea differences between intact C57BL and DBA mice. The histology of the mammary tissue during pregnancy is also characteristic of each strain. Many growth changes including hyperplasia and neoplasia have also beea recorded by various investigators in response to unbalances experi- mentally induced by excess or deprivation of hormones. It would appear that the infiuence of hormonal unbalance upon the Incidence of certain neoplasms is well established and that the future is likely to reveal that this relationship is widrepivad, varied, and highly aignificant. Yiroida andt Unbalance Reference has already been made to the work of Rous and others who demonstrated the importance of a virus in the etiology of avian sarcomas. Shope, Bittner, and Gross are among those who have observed the same general type of etiological factor in mammals. The important fact to remember is tnat the entrance into the cell of a growth-influencing viroid, produces an unbalance between nucleoid : nd cytoplasmic cellular components. It is also evident that this process is not pathogenic when neoplasia (increased mitosis) results. The "modi- fied" cells often retain, completely, the biochemical specificity of the organism in which they occur. At times the specificity is lost to a greater or lesser degree but such a losa is not a eine gua aon of neoplasis due to viroid-induced unbalance. The mitosie-in9uencing viroid is, therefore, "at home" in the cell and arouees no barmful disturbance of organization or function. In this respect it does not differ from plasmagenic type of activity. There have been a number of interesting and etimulating discussions of the parallelism and perhaps similarity between genes and viruses, and the field of viroid participation in neopla®ia will be a fruitlua contributor to the further elucidation of such relationship and of other basic biologicsl proceeee®• Unbafanoe q! IneracelG.far Components The facts that oyclio unbalance within the oall is the chief characteristic of the prooees of mll division and that the rate of mitosis and its control, or lack of it, are basic parts of the neoplastio proc,er indicate that eome- where within the boundaries of this field-widely defined---should be found the answers to or the major leads toward the explanation of the origin of cancers and other tumors. There are various levels of organiea- tion of aompcnents within the cell at which unbalance may occur. They oan be roughly ooneidered ae follows: a ~...t t4 &. wuow c..w l.ww. 5I

I H K101 26G30 BIOLOOICAL A6P8Ci8 OF CANCER RESEARCH 461 I I I Nuelear-eytoplasmis un,6alareu.-Reactions between nucleoid material and cytoplasm are, of courae, one of the essential and basic types of intracellular aetivity. The origin anJ function of plaemagenea, the degree of permanence of changes induced in the cytoplasm, macronucleus, and micronucleus and the spread of gene products through the cytoplasm and through oe11 membranes arA evidence of the complexity and orderliness of intraoallular function. Mechanical modification of the proportion of nuclear to cyWplaemic material has produced interesting and important changes in cell function reHected in the mitotic procem. Heat, cold, colchicine, X rays, radium, and chemical carcinogens are among the agents that have been used experimentally to modify existing "normal" nucleo-cytoplasmic balance within the cell. Various general and/or characteristic responses affecting type and rate of mitosis have been observed. lntraayeopfaemie aan6alarue.-Although comparatively few investiga- tions have been carried out in this field there Is evidence that it is an area of research worthy of far greater emphasis. Changes in the proportion of mitochondria in the cytoplasm are believed by some to be aseociated with anaphasia and the deV.ee of independence and gra :-th in neoplasia. Anucleate cells in Arlernia and in Triloa can divide successfully for at least a time. This indicates that the cytoplasm has, in its own right, latent potentialities for activity in cell division as yet littls understood, and of great scientific intereat. The development of a combination of microdiesection and tissue- culture techniquES qhould give new and interesting knowledge of intra- cellular processes affecting eell division and therefore bearing on neoplasia. lnbnreuedear unbalance.-The moat important and most studied intra- nuclea.r structures are, of course, the chromosomes. Their constancy in number, form, and genic structure mskes it possible to recognize and record divergencies from the normsl structural conditions and from normal function as well. Reterence has been made to the existence of changes in chromosome number, which may be balanced (tstraploidy, triplofdy, haploidy) or unba4noed. Boveri's theory of cancer origin considered the greatly increaeed numbors of chromosomes eeen in certain eelle of some neoplasms as uhe etiological basis for uncontrolled mitosis. The later observations that the fastest dividing and most "®ucceaaful" neoplastio cells often have the usual 2n chromosome count and tltae many cells with large numbers of extra chromosomes are not neoplestie would seem to invalidate the latter phenomenon as the ecuse of cancer. It is more likely that the exuberance of mitosis, released when neoplastio growth occurs, may produce unusual or new cell types of many eorfa including hypernormal chromosome oontent. bifterencas in relationship bstraeea chromosomes, euch as nondisjunction, fractionation, tranalocatSon, etc., are aell known. They may be htduced by experimental processes, euoh as cold, irradiation, or chemicals. They V.1. aa Rs a. Mafs IYaa

1 N140126001 462 UrrLs are also subject, in some cases, to the influence of one or more genes. They are often followed by abnormalities in growth end/or structure. WitAin the chromosome the balanced relationships between genes can also be upset by various experimental procedures. This is a third level of intranuclear organization where the principle of balance can be investigated. Finally within the gene itself there esists a high degree of constancy in substance and organization. In the vast majority of its reproductive efforts the gene forms an exact replica of itself with amazing effectiveness. Occseaonally, however, a rearrangement of its molecular structure appears as a "mutation" and the "new" type of molecular organization then repro- duces itself with the same order or accuracy as did the old type that pre- ceded it. The fact that many of the known carcinogens have also the power to increase mutation rate has led to the considerable popularity of the mutation theory of cancer causation. While it is probable that in some cases intrachromosomal change (mutation) may be involved in changing the mitotic rate of the coII, it seems equally probable that other intrenuclear, cytoplaemic, or combined unbalances can result from variation in amount or in activity of other cellular components than the genes. - Thero is nothing mutuelly exclusive about the coLCert of coeaistence of many causal possibilities and circumstances in the origin of neoplasia. Cancer may arise in almost any body tissue and the tendency for it to appear in certain sites may be increased and maintained by genetic selec- tion. Tiqsues are balanced and controlled in many ways. Their cells are often in very different environmental relationships to one another. Release of control may well have many different origins and effects. It would indeed be surprising if this were not the case. Refoose of Controf In the establishment and maintenance of functional balance in any living unit it is obvious that the establishment of £wacellular organization must have been the first order of evolutionary progress. Only in this way could the sing.ecelled organisms have preceded and made possible multi- celled forms. Secondary balance betuagen cells must first have been of a type that main- tained cMetaed between individual cells. At the same time a high degree of independence and of complete functional capacity of the individual cell must have been necessary be&use of the "looseness" of affiliation. Later, more complicated controls of functional balance between tissues end organs which resulted in more complex and complete division of labor between cells and tissues and organs must have appeared in the evolution- ary chain. When the orgaanism is challenged by unbalancing agents for which it has no established defense, it is logical to expect that the more reasntly acquired and accessory types of "controls" would be the first to be impaired or de- J.ae.d .f 6. N.dond P..ee.r lnCmr

I HK1012 b0u? nrOLOOICAL ASPECTS OF CANCER RESEARCH 463 I I stroyed. The result of such impairment or destruction on intertiesue or interorgan "controls" would be the release of the latent powers of growth which these "controls" had held in check. It may well be that the "increased" growth following "whole-body" irradiation with small doses is the result of the desfrwdion of such more complex and vulnerable "secondary" intsrcellular'bontrols." This may give the misleading impression of °et,imulstion" of growth when actually only the release of the latent intracellular activity is involved by the destruction of intercellular restrictions. ' With increased exposure (doesge) the destructive process would affect the intrscellular balance first by upeetting the interrelations of cellular components, and then by destroying the basic phyeical-chemicsl moleculas and atomic structure. Thus, the series of effects of irradiation would first give the appearance of "stimulation" of growth by destruction of secondarj balance controls, thereby releasing the latent potential for growth. The continuation or inteneification of ita destructive activity would then break down primary controls and impair or destroy cell function and finally cell orgsnisation and structure. It would seem that a hypothesip or explanation of this type wos more logical, simple, and consistent than one that, believing all we see, accepted an dikwt atimudation tha increased growth activity of small doses of an unl,dmoirg ogeut, which in largar doees was admittsdls destructive and lethal. The question can fairly be asked, "what does the amall doss,ge of a physical agent, affecting molecular and atomic straeture and errange- ment, add as a etimulant to processes already perfected by evolutionary selection?" The proposed hypothesis also brings into a consistent and eimilsr relationship the effects of other chemical (carcinogens), biological kpbridi- estion), endocrine, viroid, and other growth-affecting iafluencea. In me.py ceses, light exposure to such egents may release growth poteatial- if any effect is noted-and in massive doses they can impair nr destroy life organization and function. Concluafon It is evident that if the origin of cancer involves deGcate intracellular changes we must achieve certain advances before we can hope to under etand either the changes themselves or the essential eteps which,lead up to them. The nature of the cell must be known and controlled in terms of genetic origin, nutritive requirements, and potentiality forr division. In order to analyze the pmoess we must be able to observe it and its descendants both in vitro and an si8n (by indireot appraissi of its fimctions), and by tmeplantstion in oioo into sites deliberately ohosen and into individuals of known genetic composition. The nutrient media of the cell must be capable of synthetic control ia oib+o and of measurable experimental modifiability in odvo. Any lesser degree of control will involve the existence of variables which may Vd. 0% i1w a, r...h iV6s

I 1 HKi01260~3 464 Lr1TLE prevent complete analysis and which will certainly limit and obscure the extent to which results can be applied in a general way. There are certain fields of biological research which must be far more completely organized and extensively developed before vve can complete the necessary foundation for continued and progressive increase in easen- tial knowledge. Among these may be mentioned the following: 1) Maintenance of a Iarge number of genetically known strains of normal and cancer cells and of a wide variety of neoplasms by traneplantatioa both in oBvo and in oitro. 2) Development of standard tables of quantitative potency relation- ships of known carcinogens for use in assay of tireue changes and of suspected agents. 3) Now and improved techniques of manipulating both unfertilized and fertilized msmmelian ova so that their development a,ner eaperi- mental challenge may be accurately and conveniently compared with the normal developmental processes. It is admitted that such a conclusion is not encouragin fi to those, and there are many, who allow ready optimism, impatience, or material expediency to lead them to underestimate the complexity of the problem •On the other hand, the honest :eeeognition of the extent of one's ignorance and of the scattered immaturity of at:r prasenL attar.h, in apito of i', increaeed volume and intensity, is the first step on the read to the acquisi- tion of lmowledge essential to the final solution. Continued exposition aad emphasis of this oomplex situation is an un- avoidable duty and perhaps the best contribution remaining to those of us who ha^e seen the early contacts with the "enemy" develop into the full-scale conflict from which final victory will be won by those who follow. hm.d .r rSe N.dqd Cirr leMlefs I

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