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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