MODE OF ACTION OF CARC1NOGKNS A. HaMa*

964

MODE OF ACTION OF CHEMICALCARCINOGENS

ALEXANDER HADDOW, M.D., D.Sc., Ph.D.Ouster Batty RtsearA Institute, Royal Cancer Hospital (.Fret), London

1 Physical properties2 Chemical relationships3 Substitution and addition reactions4 Energy states in the carcinogenic molecule5 Biological considerations6 Carcinogenesis and cellular nutrition7 Energetics of the malignant transformdtion8 Relation to the viruses of chicken sarcoma

References

Sufficient has already been said in a previous paper1

(Haddow & Kon, 1947) to indicate the nature, number andgreat variety of the chemical carcinogens. Several hundredsof these are at present known, and while the list is almostcertainly far from complete, it is natural that less attentionshould now be paid to descriptions of the carcinogensalone, and progressively more to their mode of action.~ • \BMB 9«T]

Hence, the problems of major importance which must nowconcern us are rather the nature of the structural rela-tionship between carcinogens of one chemical class andthose of another, the extent to which any such .com-munity of structure may signify a common principle ofaction, and finally, the nature of that action itself by whichthe biological change, from the normal to the malignantstate, is achieved. Before proceeding, it.may be nofcd howcomparatively little in the way of correlation has beenobtained, at least until very recently, by the use of purelyphysical methods, how fertile in contrast have been thesuggestions arising from the chemical and biochemicalattack, and how consonant these are with independentevidence from an exclusively physiological or biologicalapproach, which has in itself been able to establish a practicalworking hypothesis comprehensive enough to includecarcinogens of the most varied types, and apparentlysound enough to predict the activity of an entirely newclass—the aminostilbenes.

1. Physical Properties

So' far as physical properties are concerned, the greatmajority of all chemical carcinogens are virtually insoluble inwater, although soluble in lipoids and to some extent inbody-fluids such as serum and bile; but no relation exists,directly at any rate, between the greatly varying degrees ofsuch solubility and carcinogenic action. For the polycyclichydrocarbons, no specific differences have been disclosed

CARCINOGENIC HYDROCARBONSContinued from page 330Berenblum, 1. & Schoental, R. (1943a) Cancer Res. 3, 145Berenblum, I. & Schoental, R. (1943b) Brit. J. exp. Path.

24,232Berenblum, I. & Schoental, R. (1946) J. chem. Soc.p. 1017Berenblum, I., Schoental, R. & Holiday, E. R. (1943) Cancer

Res. 3, 686Berenblum, I., Schoental, R., Holiday, E. R. & Jope, E. M.

(1946) Cancer Res. 6, 699.Bowen, E. J. (1946) The chemical aspects of light, OxfordBoyland, E. (1932) Lancet, 2, 1108Boyland, E., Levi, A. A., Mawson, E. H. & Roe, E. (1941)

Biochem. J. 35, 184Boyland, E. & Weigert, F. (1947) Brit. med. Bull. 4, 354British Empire Cancer Campaign (1943) Annual report, p. 53Caspersson, T. (1940) J. R. micr. Sac. 50, 8Chalmers, J. G. (1934) Biochem. J. 28, 1214Chalmers, J. G. (1940) Biochem. J. 34, 678Chalmers, J. G. & Crowfoot, D. (1941) Biochem. J. 35, 1270Cook, J. W. (1931) / . chem. Soc. p. 2529Cook, J. W., Hewett, C. L. & Hieger, I. (1933) / . chem. Soc

p. 395Dobriner, K., Rhoads, C. P. & Lavin, G. I. (1939) Proc. Soe.

exp. Biol., N. Y. 41, 67Dobriner, K., Rhoads, C. P. &. Lavin, G. I. (1942) Cancer Res.

2,95Doniach, I., Mottram, J. C. & Weigert. F. (1943a) Brit. J. exp.

Path. 24, 1Doniach, I., Mottram J. C. & Weigert, F. (1943b) Brit. J. exp

Path. 24, 9Dnickrey, H. (1938) Arch. exp. Path. Pharmak. 190, 184Fieser, L. F., Fieser, M.. Hershberg, E. B., Newmann, M. S.

Seligman, A. M. & Shear, M. J. (1937) Arrter. J. Cancer,29, 260

Grafti, A. (1939) Z. Krebsforsch. 49, 477331

Hieger, I. (1930) Biochem. J. 24, 505Hieger, I. (1936) Amer. J. Cancer, 28, 522Hieger, I. (1937) Amer. J. Cancer, 29, 705Holiday, E. R. (1937a) / . sci. Instrum. 14, 166Holiday, E. R. (1937b) Biochem. J. 31, 1299Holiday, E. R. (1945) Biochem. J. 39, Proc. IxvJones, R. N. (1942) Cancer Res. 2, 237Jones, R. N. (1943) Chem. Rev. 32, 1Kon, G. A. R. & Roe, E. M. F. (1945) / . chem. Soc., p. T43Lorenz, E. &. Andervont, H. B. (1936) Amer. J. <Mncer, 26, 783Lorenz, E. & Shear, M J. (1936) Amer. J. Cancer, 26, 3.Lorenz, E. & Stewart, H. L. (1940) J. nat. Cancer Inst. 1,17Mayneord, W. V. (1927) unpublished, quoted by Cook et ai,

1933Mayneord, W. V. & Roe, E. M. F. (1935) Proc..roy. Soc. A,

152,299 •Mayneord, W. V. & Roe, E. M. F. (1937) Proc. roy. Soc. A,

158, 634Miller, J. A. & Baumann, C. A. (1943) Cancer Res: 3, 223Morton, R. A. & de Gouveia, A. J. A. (1934) J. chem. Soc,

p. 916Peacock, P. R. (1936) Brit. J. exp. Path. 17, 164Peacock, P. R. (1940) Amer. J. Cancer, 40, 251Peacock, P. R. (1944) Nature, Lond. 153, 136Simpson, W. L. & Cramer, W. (1943) Cancer Res. 3, 362Simpson, W. L. & Cramer, W. (1945) Cancer Res. 5, 449Stamer, S. (1945) Acta path, microbiol. scand. 22, 65Waterman, N. (1937) Acta brev. neerl. Physiol. 9, 143Waterman, N. (1939) C. R. Soc. Biol. Paris, 130, 1115Wawzonek, S. & Laitinen, N. A. (1942) J. Amer. chem. Soc.

64,2365Weigert, F. & Mottram, J. C. (1943) Biochem. J. 37, 497 ,Weigert, F. & Mottram, J. C. (1946) Cancer Res. 6, 97WeU-Malherbe, H. (1944) Cancer Res. 4, 102Winterstein, A. A. Schdn, K. (1934) Hoppe-Seyt. Z. 230, 146Winterstein, A., Schon,,K.. & Vetter, H. (1934) Hoppe-Seyt. Z.

230,158 . , .. • .Winterstein, A. & Vetter H. (1934) rJoppe-Sfy^Z. 230, 169

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MODE OF ACTION OF CARCINOGENS A.

between carcinogens and non-carcinogens, by means ofeither fluorescence or ultra-violet absorption spectroscopy.In the case of the aminostilbenes, the ultra-violet absorptionspectra of a considerable series are being investigated byDr. E. M. F. Roe, and will be reported upon later. For boththe carcinogenic hydrocarbons and aminostilbenes, it is tooearly to say whether developments may arise from theradically different type of information to be obtained by theapplication of infra-red spectroscopy. Other physical orphysico-chemical techniques, such as the interaction ofcarcinogens with monomolecular films of sterols, proteinsand other substances, have still been very inadequatelystudied, although Clowes and his collaborators (Clowes, 1943)suggested that this type of interaction might influence thenormal function of cholesterol.

2. Ownfrnl Relationships'1

The possibility has already been discussed in a previouspaper (Haddow & Kon, 1947) that certain azo compounds,exemplified say by 2 : 2'-azonaphthalene, may be re-arrangedand transformed into carbazoles in the liver (in this case into3 : 4 : 5 : 6-dibenzcarbazole) and there produce tumours ofthat organ. Although no proof has so far been obtained thatsuch a process is effected in vivo, it seems a legitimate questionwhether such chemical transformations, even if they do notrepresent the mechanism itself, should be regarded assignificant and important affinities if only in the theoreticalsense. In the case of 4-aminostilbene, which producestumours in the rat of a type and distribution similar to thoseproduced by 2-acetylaminofluorene, a similar suggestion wasmade to the writer by Dr. F. L. Rose, of a conversion to3-aminofluorene or to 2-aminoanthracene. Here again, evenif such a mechanism is regarded as an extremely unlikelyexplanation of the action of 4-aminostilbene in the body, itschemical feasibility has perhaps some underlying meaningwhich we should not ignore.

Whatever may be the ultimate prospect of deriving logicalrelationships between them, it still remains a fact that com-pounds of widely different features in the purely chemicalsense (as anthracenes and fluorenes, aminostilbenes, acridinesand carbazoles) may none the less show an importantbiological property in common. Some of these cases ledCook (Barry, Cook, Haslewood, Hewett, Hicger &Kennaway, 1935) to indicate the significance, in themselves,of general molecular shape and dimensions, and a somewhatsimilar opinion was held by F. Bergm&nn (1942), whoconceived the molecule as functioning as a whole, and itsactivity as being determined by shape and size. Bergmannsuggested that all the carcinogenic hydrocarbons might beabsorbed by. a single receptor, and that all such compoundsmight be regarded as parts of an " ideal" carcinogenicstructure. According to this view, geometrical conformityof carcinogen and receptor is a necessary condition foractivity, although not a sufficient one. Pursuing thesenotions, it is a logical step to consider if the wide range ofcompounds already examined, or some class among them,possesses any structural characters In common, such asmight indicate the essential nature of the carcinogen-receptorrelation. It may be said at once that this is almost certainlynot so, but the writer has been impressed by a curious relationin certain pairs of isomers, one of which is carcinogenic andthe other inactive or only feebly active, and according towhich the active molecule alone might appear to incorporate

the skeletal structure of a /nwj-diaryl " ethylene " ; two suchpain are shown (I-IV) contrasting the carcinogens 3 :4-benz-pyrene and 1 : 2 : 7 : 8-dibenzfluorene with the inactive orfeebly active 1 :2-benzpyrene and the non-carcinogenic3 : 4 : 5 : 6-dibenznuorene.

3 :4-b«nzpyr«ne I : 2-b«nipyr«nt

IV

I : 2 : 7 : 8-dlbenzfluorene

•I .<•* '•••... :••• '

3 : 4 : 5 : 6-dlbenzfluorene

If such a relation were to represent more than merecoincidence—and Pacault (1946) has adduced evidence forthe existence of "ethylenic" structures in such aromaticnuclei—one might anticipate a marked distinction in carcino-genicity between the homologues of 1 :2-benzacridine (V),which could not incorporate the /raw-diaryl "ethylene",and of 3 :4-benzacridine (VI) ; it is of interest that Lacas-sagne and his colleagues (Lacassagne, Rudali, Buu-Hol &Lecocq, 1945 ; Lacassagne, 1946) have, in fact, describeda strong contrast in carcinogenicity between the two series,but in the opposite sense to the above! In many otherexamples, too, e.g. the carcinogenic alkyl-substituted3 :4-benzphenanthrenes, it is clear that no such simplerelation could possibly hold.

VI

I : 2-benzacrldlne 3 :4-benzacridine

Notwithstanding the absence of any very obvious clue, theextent of the existing data on effects of alkyl and othersubstitution in 1 :2-benzanthracene alone, makes it verydesirable that these data should soon be re-examined in afurther attempt to generalize the relation between chemicalconstitution and carcinogenic action. Some time ago thewriter obtained, with the help of Dr. H. O. Hartley, partialevidence that the carcinogenic derivatives of benzanthracenemay be members of a series, so far as their geometry alone isconcerned. The matter still requires very much closer scrutiny,and one would therefore hesitate to advance even the most

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MODE OF ACTION OF CARCINOGENS A. HadJom

tentative hypothesis. But it appears to be not beyond thebounds of possibility to derive in the future, by purelynumerical or geometric methods, an expression which wouldgeneralize all the information we already have in so manyindividual cases concerning the effects of substitution on thecarcinogenic activity of this group at least. Such an expres-sion would clearly represent simply a comprehensive state-ment, in the smallest number of terms, of what we alreadyknow ; but one gathers, assuming it could be fully verified,that it might also indicate some relationship not so apparentfrom the individual data themselves, and that it might reflect,without giving any direct indication of its nature, some typeof reactivity necessary for this kind of biological action.

3. Substitution and Addition Reaction*

Although as a class the carcinogenic hydrocarbons must beregarded as comparatively inert, they are neverthelesssusceptible to oxidation, eventually yielding non-carcinogenicquinones. Since anthracene is known to inhibit the auto-oxidation of benzaldehyde, it has been suggested that theactivity of the carcinogenic hydrocarbons may be due to somesimilar inhibition of the normal oxidative processes of the cell.Fieser (1941) laid great stress on the fact that the most potentof the carcinogens are endowed with a remarkable suscepti-bility to substitution reactions, and surpass all other knownaromatic hydrocarbons in this type of reactivity. Studyingthis phenomenon with the reactions of diazo coupling, leadtetra-acetate oxidation, oxidation with perbenzoic acid, andcondensation of the hydrocarbons with sulphur mono-chloride, he found in general that the order of reactivity wasessentially the same for a given group of compounds, anddeclined in the following sequence : methykholanthrene

> 3 :4-benzpyrene > 10-methyl-l : 2-benzanthracene> 1:2:5:6-dibenzanthracene. Again, as for everycorrelation yet attempted, there are exceptions, especially inthe case of certain anthracenes equally susceptible tooxidation, but these did not appear to Fieser to invalidatethe notion of a causal association between chemical reactivityand carcinogenesis, provided that other essential require-ments, such as solubility, absorbability and molecular size,were also satisfied by a given compound.

Such considerations suggested that the carcinogenicmolecule undergoes a substitution reaction as the first stagein the production of its biological effects. The earliestpossibility to be considered was introduction of a hydroxyl,sulphydryl or basic group, leading to a functional derivativewhich might then enter into other changes possibly involvingconjugation with constituents of the cell. Later, a moredirect interaction was suggested by the condensation ofsulphur monochloride with carcinogenic hydrocarbons;with the most potent and reactive hydrocarbons, introductionof a sulphur substituent occurs at room-temperature withoutcatalyst, and Fieser suggested that possibly the carcinogensimilarly combines with cell proteins by the opening of anS-S linkage as indicated for 3 : 4-benzpyrene in VII and VIII.

So far as addition reactions are concerned, Fieser indicatedthat the carcinogens enter into such reactions only sluggishly,and are attacked at points other than the reactive centresindicated by substitutions. The principal reaction studiedwas catalytic hydrogenation which, as he pointed out, bearssome analogy to a biochemical transformation proceedingunder the influence of an enzyme. Fieser & Hershberg(1938) found that methylcholanthrene and 10-methyl-

VII

protald*

VIII

proteMe

H S -

1 :2-benzanthracene tended to add hydrogen at positions(e.g. the 6 : 7 double bond of methylcholanthrene) other thanthe meso centres or other centres hindered by neighbouringrings or by the presence of substituents (DC, X).

Boyland & Weigert (1947)* have already indicated how acarcinogenic hydrocarbon may first of all combine with an

IX

enzyme, or with other tissue-constituents such as ascorbicacid or purines, through the positions of prime reactivity,and have shown the way in which a hydrocarbon can bealtered in the body, to give neutral water-soluble substances,by addition of the elements of hydrogen peroxide at positionsof only secondary reactivity. From the data available on thefate of carcinogens in vivo, it therefore appears that themetabolism-reactions involve attack at centres other than,those specially liable to substitution, and Fieser regarded thissituation as comprehensible on the hypothesis that thecarcinogen is subject to two reactions proceeding by differentmechanisms, one responsible for the induction of malignantgrowth an'd the other leading to detoxification :

" . . . the assumption that the metabolic change follows afundamentally different reaction mechanism from the postulatedsubstitution reaction of carcinogenesis seems necessary inorder to account for the fact that the attack occurs at a partof the molecule different from that involved in substitutions."That it may in fact be possible to dissociate detoxication

mechanisms from those which are specifically concerned inthe process of carcinogenesis, is supported in evidence of adifferent kind described by Crabtree (1947)'. Crabtreeshowed that the usual course of carcinogenesis might beimpeded first by certain chlorine compounds which he

• [BMB MS]• IBMB 966]

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-MODU OP ACTION OF CARClNOt.l.NS I. //«/<*»

suggested could act through condensation with SH-containingcomponents so as to impair enzyme systems dependent onintact SH groups ; by bromobenzene ; by another classtypified by the unsaturated maleic and citraconk acids, whichform addition products with SH-containing compounds anddisturb the sulphur metabolism of mouse skin by fixation ofglutathione ; and by naphthalene, anthracene and phenan-threne, which are excreted as mercapturates. Since thesefour groups appear to possess only one common feature(their action upon sulphur metabolism), and since there is noevidence that sulphur is involved in the detoxication ofcarcinogens, Crabtree suggests that it may be some inter-ference -with sulphur metabolism, by preferential combina-tion, which tends specifically to inhibit the mechanism ofcarcinogenesis.

In the attempt to define the essential structural require-ments for carcinogenicity, Robinson (1946) has recentlysuggested that the weight of evidence indicates the possibilityof reaction at an activated phenanthrene-type bridge in thegreat majority of cases. But again there are certain apparentexceptions, more than one mechanism may be involved, andRobinson made clear his unwillingness to advance even aprovisional hypothesis until more facts had been gathered.There are. equally, however, several examples not merelyconsistent with such a view, but remarkably striking andsuggestive in the relationships they show. The extremecarcinogenicity of 9 : 10-dimethyl-l : 2-benzanthracene (XI)is a clear illustration that unsubstituted meso positions arenot essential for activity, and it is of great interest in thepresent connexion that 9 :10-dimethyl-l : 2 : 7 : 8-dibenz-anthracene (XII) has now been found by Berenblum (1946)to possess marked carcinogenicity, in contrast with 9 : 10-dimsthyl-1 : 2 : 3 : 4-dibenzanthracene (XIIT) which is com-pletely devoid of such activity.

XIV XV XVI

XI XII XIII

9 : 10-dimeth/l- 9 : 10-dlmethyl-9 : 10-dlmethyl- 1 : 2 : 7 : 8 - 1 : 2 : 3 : 4 -

I : 2-benzanthracene dJbenzanthracene dlbenzamhracene

These examples are in keeping with the view that anessential requirement may be the phenanthrene double bond,and that the 9- and 10-positions of phenanthrene (shown inXI and XII by *) must be unsubstituted ; it also appearsthat the reactivity of these positions may be enhanced (orcompetitive reactivity reduced) by appropriate substitutionelsewhere. Another critical case, also in agreement, hasrecently been described by Harris & Bradsher (1946) in anastonishing contrast between the high carcinogenic activityof 1:2:3:4-dibenzphenanthrene (XIV) and the totalabsence of activity in cither the 9-methyl (XV) or 10-methyl(XVI) derivatives.

One further method of attack, applied by Robinson, hasbeen to test substances analogous with known carcinogensin that a benzene ring is replaced by the isosteric thiophene

I : 2 : 3 : 4- 9-methyl-l : 2 : 3 : 4- 10-methyl-l : 2 : 3 :4-dlbtnzpheninthrene dlbenzphenanthrene dlbenzphenanthrene

nucleus. In 9 : 10-dimethyl-l : 2-benzanthracene itself thereare three benzene nuclei which might be replaced in this way,and in two of these cases (XVII, XVIII; Fieser, 1941) theproducts are already known to be carcinogenic. The thirdisomeride, which is of greater significance in that the phenan-threne bridge is here replaced by sulphur (XIX), has nowbeen prepared by B. Tilak (1946), and it is certainly of interestnot merely that this compound is non-carcinogenic bysubcutaneous injection in mice, and seems to be only weaklyactive on painting, but that high potency again emerges inthe benzo-derivative (XX), where the phenanthrene deublebond is once again a feature.

XVII XVIII

XIX XX

The apparent importance of the phenanthrene doublebond, even if it is not completely determining in its influenceupon carcinogenic activity, finds a parallel in the ethylenebridge of the aminostilbenes (infra). Between thesestructures there is a high degree of similarity not merelyon purely chemical grounds, but also in the way inwhich substitution, modification or total replacement,leads to annulment of the biological activity of the moleculeas a whole. So far as the growth-inhibitory effects of theaminostilbenes are concerned, such activity disappears whenthe ethylene bridge is extended to three or four carbon atoms,when it is reduced, when either or both hydrogen atoms aresubstituted by an alkyl group, when either =CH-group isreplaced by a nitrogen atom, or when the whole bridge isreplaced by oxygen or sulphur.

It is still too early to say whether the same strict rulesgovern the carcinogenicity of the aminostilbenes, even though

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M O D E O F ACTION O F CARCINCXIHNS . 1 .

these were discovered as the direct outcome of a study oftheir inhibitory properties ; to the extent however that ispermitted by the results obtained so far, it can be said thatthe correspondence is complete. In the aminostilbenes itwould seem to be a necessary condition, although clearly nota sufficient one, that the molecule should be capable ofassuming a quinonoid disposition, with an electron-donatinggroup in one ring conferring a high degree of negative polarityin the other, and the outstanding feature being the necessityfor unsubstituted hydrogen atoms in the.ethylene bridge andat the p' position. Such a system would appear not merelyto contain a structure in the ethylcne bridge which isanalogous with the phenanthrene double bond in the cyclichydrocarbons, but to possess electronic characters which areproduced in condensed systems of benzene rings by ratherdifferent means. Further, the amino-diarylethylenes presentcertain advantages in their comparative simplicity and in therelative ease with which one may hope to establish a,connexion between constitution and activity. Finally, thechanges they are likely to undergo in the body—a few ofwhich are indicated in XXI—XXIII for 4-dimethylamino-stilbene in comparison with known steps in the oxidation ofp-aminopheriol (XXIV-XXVII)—are again such as might beexpected to interfere with the oxidative mechanisms ofthe ccU.

4. Energy States in the Carcinogenic MoleculeThe above relationships gain further interest from recent

attempts to establish a connexion between electron distri-bution and chemical reactivity on the one side, and carcino-genicity on the other, in the condensed unsaturated hydro-carbons. Otto Schmidt (1938, 1939a, 1939b, 1941) was thefirst to compare the electronic density of the meso regions of

XXI

XXVIII

CH3

XXII

= C H

METABOLISM

IH = CH

rXXIII OXIDATION

HN " C H

XXIV XXV XXVI

OXIDATION OXIDATION

p-tminophenol

certain carcinogenic substances with that in related non-carcinogens, and on the basis of this comparison he postu-lated that it is a necessary condition of carcinogenic activitythat the*dcnsity of such regions should exceed 0.44e/A*; inparticular, he further suggested that the activity of a car-cinogen is due to the electro-affinity of its excited statefacilitating a quanta! change in neighbouring molecules.More recently, A. & B. Pullman (A. Pullman, 1945, 1946a,1946b ; B. Pullman, 1946 ; A. & B. Pullman, 1946) andMartin (1946) have obtained supporting evidence of thisconception, through a quantum mechanical treatment (withDaudel) which permits a calculation of the density of velectrons for a given structure (in chrysenes, benzanthracenesand benzacridines), by means of which it is claimed that arelation can be formulated between carcinogenicity and theelectronic density of the region K (XXVIII), the density-threshold below which a substance ceases to be carcinogenicbeing 1.292e.

The Pullmans also state that it is not the position ofthe'K region which is of first importance, but its specificcharacter, and that it may appear at other points of themolecule and still possess the same function; hence, while theirwork would seem to confirm in the main, andon a quantitative basis, Schmidt's theory of the

relation between the carcinogenic power of amolecule and the existence within this moleculeof a region rich in mobile electrons, it is less inagreement on points of detail such as the positionof the active region, and the influence of sub-stituents. PacauTt (1946), by measurements ofmagnetic susceptibility, also indicates certainelectromeric structures, the existence of whichappears to be correlated with the particularbiological property of carcinogenicity, andDaudel (1946a, 1946b), like Schmidt, hasenvisaged the molecular alterations which mightproceed from the proximity of a region rich inir electrons to certain regions of protein mole-cules. These views, that the concentration ofT electrons is the preponderant cause, if not theonly one, of the carcinogenic property, maypossibly represent an over-simplification, and arein any case subject to further scrutiny and test.But there is no doubt they are symptomatic ofthe need for a deeper understanding of themanner in which carcinogens operate, and thatthey would if confirmed, as Daudel says, effecta remarkable liaison between biological problemson the one hand and the most abstract regionsof pure science on the other.

5. Biological ConsiderationsTurning next to the biological plane, it may

immediately be claimed that purely physio-benzoquinone logical methods have been surprisingly successful

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MODE OF ACTION OF CARCINOGENS A. Haddow

in the attempt to decipher the mechanisms ofcarcinogenesis, and have at the least made a fullcontribution. It is of course upon biological methodsthat we must depend for the primary data of carcinogenesis.and these may briefly be reviewed before proceeding. In thefirst place, we have no reason to doubt the view, and everyreason to support it, that the process is one which is local inorigin, and is due to direct action of the carcinogen or itsproducts upon the affected cells. That chimney-sweep'scancer is due to local contamination of the skin with soot,and not to any " disease of the habit", was clearly recognizedby Pott in 1775, although the local or constitutional originof cancer in general continued to be debated a hundred yearslater, as at a famous meeting of the Pathological Society ofLondon in 1874, when Sir James Paget was still interpretingcancer as a disorder of the blood, in opposition to Sir WilliamGull, Arnott, Hutchinson, Moxon and others who maintainedits strictly local origin. We now know that, while consti-tutional and genetic factors can greatly influence suscepti-bility to cancer, and may even determine the site of itsspontaneous occurrence, the disease is one of the individualtell as a separate organism and with no relation to the needsDf the body as a whole. It is this which gives cancer itsunique position in pathology, accounts for its intractablenature, and explains its growth, in Paget's words " irrespectiveof the maintenance of the rest of the body, discordant fromits normal type, and with no seeming purpose " (Paget, 1853).

As to the nature of the transformation from the normal tothe malignant cell, and the manner in which the carcinogenseffect h, no interpretation can be sufficient which fails totake count of the great diversity of tumour-producing agents.Indeed, one of the most striking features of cancer is themultiplicity of its experimental causes, including not merelythe variety of chemical compounds already considered, butphysical agents such as ultra-violet rays, x rays, and theradiations emitted by radium and other radio-active elements.Such apparent lack of specificity may be merely superficial,and it has already been shown that many of the chemicalcarcinogens have more in common than appears. Further,since the pathological end-result in all cases is the same, it isprobable that the cellular processes involved are not com-pletely dissimilar and may in fact have an identical basis,the same general types of interference with function beinginduced by a wide range of causes. This view is supportedby the fact that different carcinogenic influences may beinterchanged (Berenblum, 1930) or summated (Hfeger, 1936),during the period of tumour-induction, a result which is tobe expected if cancer can result not only from a single chainpf abnormal events but from several which lead by differentroutes to a final common path. Foulds (1945) has comparedthe seeming non-specificity of carcinogenic agents with thenon-specificity of agents which lead to the liberation of theH substance and Sir Thomas Lewis's " triple response ", andthe writer (Haddow, 1938) also pointed out that the basis ofnon-specificity in cases of1 this kind is probably the extremelylimited number of possible physiological responses. Asregards division, for instance, a normal cell can react in onlyone of three ways : (a) by continuing growth at a temporarilyincreased or decreased rate ; (b) by ceasing growth ; or (c) byundergoing malignant change so as to divide at a perman-ently increased rate.

Although our central problem is the means by which somany carcinogens produce malignant transformation in

somatic cells, it is certain that this is only a special case ofthe origin of discontinuous cellular variation in general,e.g. in bacteria, protozoa, fungi and plants. It was thereforesuggested by teveral workers (Lacassagne, 1936 ; Haddow,1937)—and has been fully confirmed—that a study of thephysiology of unicellular organisms, and particularly ofbacteria, might be of unique value in deciphering the funda-mental principles of such variation. As the result of experi-ments along these lines the writer (Haddow, 1937) concludedthat the sources of discontinuous variation are mainlyenvironmental in origin, and that its induction in a givencharacter depends upon two main requirements : (i) a cellwhich is inherently capable of variation; (ii) a source ofenvironmental interference with the character in question.Special attention was given to the environmental conditionsgoverning the origin of variants with a permanently increasedgrowth-rate, when it was found that such variants areproduced not by any process of direct growth-stimulation,as might be expected, but appear as a sequel to a long-continued period of growth-repression:

" . . . it therefore seems that when the growth of a potentiallyvariable organiun is continuously inhibited by a process whichallows a sufficient proportion of the affected cells to survive,a relatively small number may undergo an irreversible changein their metabolic properties in virtue of which they are thenable to achieve active multiplication in an environment whichmake* thii difficult, or even impossible, for their parent cell."

That a general principle of this kind might well apply tothe induction of tumours was supported by the finding thatthe great majority of a long series of chemical cardnogetuproduced a rather characteristic inhibition of body-growthin the rat, and that such activity was mostly absent in asimilar series of related non-carcinogenic compounds(Haddow, Scott & Scott. 1937 ; Haddow & Robinson, 1937,1939 ; Badger, Elson, Haddow, Hewett & Robinson, 1942).The main feature of the inhibition brought about by thecarcinogenic hydrocarbons is its relative prolongation, andit is almost certainly significant that x rays and radium,which are also carcinogenic in certain circumstances, areequally capable of producing a similar inhibition under thesame circumstances, the most suitable conditions being(a) continuous exposure to a weak source of radiation(Ross, 1936), or (b) prolonged or repeated exposures. Fromthe writer's results there seems little doubt of a substantialcorrelation between carcinogenicity and growth-inhibitorypower, and it therefore seemed a real possibility, as in thecase dfx rays and radium, that the mode of action of chemicalcarcinogens might well be indirect, and that they could operateby retardation of the growth of normal cells, the lattereventually reacting to give a new race of cells with an increasedrate of fission. While the correlation on which this view isbased seems sufficiently strong to justify it, undoubtedexceptions occur which, if they are not enough to invalidatethe hypothesis as a whole, indicate that we should regard itas a general approximation. Additional confidence in thisview was, however, given by the discovery first of all thatvarious derivatives of 4-aminostilbene possess such inhibitoryproperties par excellence, and only later that these com-pounds are, in fact, endowed with carcinogenic propertiesof an exceptionally interesting kind—an association whichwould seem to be more than one of chance alone.

Many other workers from time to time have also regardedthe malignant cell as representing a new race or strain, andas a discontinuous and irreversible somatic variant (Hauser,1903; Men&rier, 1926).

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The main interest of the present argument is the hint itgives of the origin of such variants—that cancer cells ariseand commence their career of proliferation as an adaptationto conditions which impair the growth of normal cells. Hereagain the conception is by no means new, and it is remarkablehow one or other expression of it recurs again and againthroughout the whole literature. Thus Minot (1889), whowas among the first to study the progressive decline in therate and power of growth from the beginning to the end oflife, maintained that the retardation of growth accompanyingsenescence is the stimulus which inaugurates discontinuousgrowth or proliferation. If interference with growth is anessential prelude to tumour-formation, it is not surprisingthat tumours arise in growing tissues essentially, and onlycomparatively rarely from obsolete or obsolescent structures,or from highly specialized organs such as the heart and largevessels, voluntary muscles and nerves. This too has beenlong recognized, for example, by Roger Williams (1908):

" . . . in all such instances . . . proliferous cells are scanty orabsent: and it is to this peculiarity that their comparativeimmunity from tumour growth may be ascribed. . . . Thegrowth of cancers, like discontinuous growth in general, ofwhich it is but a particular case, is . . . distinctly related tothe decline of growth . . . of the body in general, andespecially of the particular local tissues. Hence, while theforces of growth, development and reproduction are in greatestactivity—during the period of pre-natal life, infancy, childhood,adolescence and even adult age—the tendency to this diseaseis comparatively small. In both sexes, it begins to be of greatfrequency in the post-meridian period. Thus the tendency. . . waxes, as the developmental and reproductive activitieswane . . ; when growth declines, . . . new centres ofdevelopment are apt to arise, and growth tends to becomediscontinuous."

Adaptive variation in relation to the origin of tumourshas also been described by many other authors (e.g. White,1913), and Bang (1928) was among the first to recognize that,since the cancer cell arises from the normal cell, its behaviourmust be considered in relation to its environment, pointingout that most carcinogenic agents probably act by injury.Lewis (1935) also suggested that malignant cells are newcellular types or species derived from normal cells which havebeen altered by environmental influences or agents of onesort or another, and Beclere (1934, 1936), discussing therole of x radiation in carcinogenesis, opined that it is a slowdestruction of the activities of the cell, rather than directexcitation of its growth-capacity, that terminates in malig-nancy. Wolbach (1936, 1937), attempting to ascertainwhat processes are initiated by carcinogenic hydrocarbonsprior to the development of tumours, apparently regardedthe. primary action as destructive :

" . . . no evidence was found to support a theory that any one ofthe- chemicals employed owes its carcinogenic property todirect stimulation of cell growth."

Similarly Witts (1936), in a discussion of neoplasia of theblood-forming organs in relation to toxic agents such asbenzene, x rays and, radium, referred to the question" whether arrest of the normal development of the cells doesnot sometimes lead to the uncontrolled proliferation ofleukaemia "

6. Cardnogeneds and Cellular Nutrition

Still deeper insight into the mechanism of carcinogenesishas been gained by the application of biochemical in contrastto purely physiological methods. Boyland (1932) was amongthe first to study the action of the carcinogenic hydrocarbons

and their oxidation products upon enzyme systems in vitro.and there is now a vast literature which in many wayssuggests that the damage produced by the carcinogen in thenormal cell may be correlated with enzyme poisoning, and thatthe resulting cell-adaptation, which we call malignant change,may be reflected in the appearance of newly developed" rogue " enzymes upon which the newly acquired growth-properties may possibly depend.

One of the most remarkable developments in cancerresearch in the past few years has been the recognition thatthe carcinogenic action of azo compounds, and particularlyof dimethylaminoazobenzene, is greatly influenced by thediet (Mori & Nakahara, 1940 ; Mori, 1941 ; Sugiura, 1944)and especially dependent upon its content of protein, and thegreat bulk of such enzyme studies has been carried out withthis exceptionally favourable material. Rhoads (1940,1942a, 1942b) and Rhoads & Kensler (1941), in a study of theinduction of hepatic cancer by administration of dimethyl-aminoazobenzene to rats taking a diet of brown rice andcarrots, found that supplementing this diet with yeast- orliver-extract in adequate amounts completely prevented thedevelopment of tumours, the protective factor being noneof the constituents of the vitamin-B complex then described.The effect of feeding the carcinogen to animals taking theunsupplemented diet was an inhibition of the activity of atleast two enzyme systems, cocarboxylase and cozymase, thusproducing what these authors considered a conditioned orsecondary deficiency disease. On the other hand, thedevelopment of the mutation which characterizes the malignantcell was accompanied by the appearance of an oxidizingsystem insusceptible to the inhibitory effect of the toxicmetabolic products of the carcinogen, and Rhoads andKensler looked on their results as the first demonstrationthat cancer tissue evoked by a chemical carcinogen possessesan oxidative system immune to the inhibitory action of thatcarcinogen or of its metabolic products.

We have already seen that the emergence of a nyQigrmnttumour has many analogies with discontinuous variation inbacteria, and there is little doubt that the phenomenon—probably on account of its fundamental and general nature—has features in common with such processes as smooth-t-rough variation in bacteria, and with acquired drug-fastnessin bacteria and protozoa. A similar conception has latelybeen formulated by Lederberg (1946), on the basis of thestudies by Beadle, Tatum and others of the genetic controlof biosynthetic reactions in the fungus Neurospora. Sincefield strains of Neurospora will grow on media containing onlysugar, salts and biotin* it is concluded that the fungusis capable of synthesizing all other essential metabolites.As the result of mutation of single genes, the capacity forsynthesis of different compounds (e.g. leudne) may howevrnbe lost—a process which possibly accounts for the differenti-ation and nutritional requirements of higher forms : it isobvious that the growth of the " leucineless" modification ofNeurospora would necessarily be regulated by the concentra-tion of leucine available to it externally. Exceptionally,however, a gene-mutation may once again lead to the capacityto synthesize such a metabolite, and Lederberg thereforecorrelates normal tissue cells with a culture of " Uucineless "Neurospora^ and the malignant cell with a variant having anewly acquired or re-acquired capacity to synthesize anessential metabolite otherwise only available in regulatoryamounts.

The present writer has very recently encountered a remark-

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able effect of the naturally-occurring pigment xanthopterin(syn. uropterin) upon the kidney of the rat, which is extremelyreminiscent of a similar mechanism. Administration of thiscompound induces an increase in kidney-size due very largely,if not entirely, to a direct stimulation of mitotic activityand cell-division in the tubule epithelium. The effect appearshighly specific and is automatically reversible—almostcertainly when the administered xanthopterin has beenexcreted. Although a great deal of necessary investigationhas still to be completed, this situation is highly suggestive ofa device by which the normal growth and occasional hyper-trophy of the kidney may be completely regulated andcontrolled, by its dependence upon an essential factor whichthe organ is unable to synthesize and which must be suppliedfrom elsewhere. If this mechanism should be fully sub-stantiated, it is clear that two further questions will arise,one affecting its general validity, and the second concerningits implications for the origin of tumours.

Is it possible that the co-ordinated growth of all the normaltissues similarly depends upon the supply of essential andspecific growth-factors, the chemical variety of which mayreflect the chemical basis of cellular differentiation ? If so,may the chemical carcinogens first of all impede the growthand protein synthesis of normal cells by restricting theutilization or access of such essential metabolites ? In sucha case, we may yet be able to correlate the new and adaptivegrowth-properties of the cancer cell, and especially its powerof continued and unregulated growth, with the acquisition of agenetic property to synthesize an essential growth-factor, orfactors, previously supplied from an external source : on thisview, the transformation from normal to malignant wouldrepresent a change from a fastidious or comparatively-exacting cell to a variant which is unexacting and self-sufficient, and the autonomy of the cancer cell, which haslong been known as its chief attribute on purely biologicalgrounds, would receive its explanation in terms of cellularnutrition.

The past fifteen years have seen enormous advances in ourunderstanding of the specific nutritional requirements ofbacteria, protozoa and fungi, of the extent to which they aredependent upon inherent synthetic capacities on the one hand,and the supply of preformed metabolites on the other, andof the ways in which these properties may be altered byenvironmental changes and induced mutation. An indi-cation has already been given that these principles have arelevance for biology far transcending their application tounicellular and freeliving organisms alone, and we have seenhow they are not merely suggestive but of practical value inincreasing our comprehension of the analogous situation inthe origin of cancer. They also point the way to the nextstage, namely, systematic investigation of the specific growth-requirements of malignant cells, in comparison with those oftheir normal prototypes, in vitro. If such an investigationis bound to be more arduous on technical grounds, it iscertain to gain immeasurably from the experience alreadyaccumulated from nutritional studies in protists and fungi.

7. Energetics of the Malignant Transformation

Possibly the most striking single feature of the cancer cellis its high stability, as shown by the manner in which itsnewly-acquired properties are transmitted and maintained,quite iadefinitely and with no sign of reversion, in the courseof serial transplantation. From this it is clear that, once a

given carcinogen has effected the malignant transformation,its continued presence is no longer necessary for the subse-quent autonomous growth of the tumour. In many waysthe processes of cell division are reminiscent of a chainreaction or self-propagating mechanism which varies in thedegree of control to which it is subject, and it may be thatthe new configuration of properties which distinguishes themalignant cell from its normal precursor can be expressedin terms of a different energy-level. Highly suggestive ofsuch a relation is the ease with which the normal cell may beconverted into a malignant form, in contrast with theimpossibility (thus far) of effecting a change in the reversedirection. It is also of the greatest significance that, whilethe normal and malignant cell-types have their own levelsof stability, and arc therefore discontinuous in their properties,transition from the former to the latter is by way of inter-mediate forms of high instability. Berenblum (1947)4 andothers have shown how carcinogenesis in the skin proceedsfrom the normal epithelium first to an early non-specifichyperplasia, secondly to a specific pre-neoplastic hyperplasia,and then to the emergence of papillomata, and how laterstages can be recognized in the progressive growth of suchpapillomata, their conversion into carcinoma, and theuncontrolled growth of the latter. While the earlier stagesin this sequence may be reversible (even in some cases to theextent of regression and disappearance of warts wljen thecarcinogen is withdrawn), the later steps are quite irreversible,and take place equally well whether exposure to the carcinogenis continued or not.

Considerable interest in the energy relations underlyingsuch changes has been evoked by Schrodingei's recentaccount (1944) of quantum theory in relation to biology andgenetics. Schrodinger was led, from a study of the highdegree of permanence and durability of the gene material onone hand and its discontinuous mutation on the other, tosuggest that such phenomena may be explained in terms ofquantum theory, and that mutations may in fact be due toquantum jumps in the gene molecule. The high degree ofpermanence of the gene material has a corollary in the com-parative rarity of mutation, but the so-called natural ratecan be increased to a high multiple by irradiation withx rays or gamma rays, the mutations produced differing inno way, save their number, from those that occur spon-taneously. And there is an energy relation :

" . . . the experiments on X-ray-produced mutations give theimpression that every particular ' transition', say from thenormal individual to a particular mutant, or conversely, hasits individual ' X-ray coefficient', indicating the percentage ofthe offspring which turns out to have mutated in that particularway, when a unit dosage of X-ray has been applied . . . ."

Schrodinger then recapitulated the laws governing theinduced mutation rate : that the increase is exactly propor-tional to the dosage of radiation, and that the coefficient ofincrease remains unaffected by wavelength provided thatthe same dosage, in r units, is applied. Mutation appearsto be due to a single event, represented by an ionization orsimilar process, occurring within some critical volume of thecell. For the size of this critical volume, Schrodingerfollowed DelbrQck (DelbrOck, Timofeeff & Zimmer, 1935),who arrived at an approximate size of only ten averageatomic distances cubed, comprising therefore 1,000 atoms.That such a group of atoms is much too small to displayexact statistical laws, and so conform to statistical and

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(RATE OFGROWTH)

(DEGREE OFDIFFERENTIATION)

FIG. I

Probable energy-barrier (X) Inth« transition from the normal tothe malignant state (see text, andSdirodlnger, p. 55). A = Minimumenergy required to effect the changeI (normal) —> II (malignant).

classical physics, is in contrast with the known permanenceand stability of the gene material, but in Schrodinger's viewthe explanation is wholly supplied by quantum theory.While a body on the large scale changes its energy con-tinuously, a sufficiently small system is bound by its verynature to possess only certain discrete amounts of energy(its peculiar energy-levels), and the event of transition fromone slate to another is the quantum jump. If the second stateor configuration has the greater energy (" is a higher level").the system must be supplied from outside with at least thedifference of the two energies to make the transition possible.To a lower level it may change spontaneously, emitting thesurplus of energy in radiation.

This conception requires to be amended for those cases inwhich free passage between two levels may be obstructed,even from a higher to a lower level. There are no spontaneoustransitions from one state towards another, when the twoconfigurations are not neighbouring, and transition can takeplace only over intervening configurations that have agreater energy than either of them. Most significant, such" isomeric " transitions are those of the greatest Importancefor biology :

" Transition* with no threshold interposed between the initialand the final state are entirely uninteresting, and that not onlyin our biological application. They have actually nothing tocontribute to the chemical stability of the molecule . . .They have no lasting effect, they remain unnoticed. For, whenthey occur,- they are almost immediately followed by a relapseinto the initial state, since nothing prevents their return."These relations at once recall the familiar and similar

distinctions between fluctuating and permanent biologicalmodifications, between variants that are irreversible andthose that return to the parent form with greater or lesserease. For all practical purposes, the origin of cancer is theoutstanding example of an irreversible cell variation. It doesnot however follow that this is necessarily so in an absolutesense, and Fig. 1 shows in schematic fashion, afterSchrodinger, the energy-barrier (X) interposed between thestable states I (normal) and II (malignant), with the minimumenergy (A) required to effect the change from energy-level Ito level II; it is obvious that amounts of energy less thanthis minimum will suffice only to produce impermanentchanges which revert to the normal. It would also appear.

theoretically at any rate, that reverse transition frommalignant to normal may be achieved by the applicationof a minimum amount of energy, greater than that requiredto change from the higher to the lower level; as to how muchgreater, nothing is known. In addition, the transition fromnormal to malignant is frequently marked not only by anincreased growth-rate, but also by loss of cellular differen-tiation. This inverse relation is also indicated in the figure,although it is far from being either simple or invariable.

An interpretation along these lines has almost certainlya close connexion with other features in the mode of actionof carcinogens : for instance, recent work on the metabolismof 3 :4-benzpyrene (Boyland & Weigert, 1947)5 hints thatit is not the hydrocarbon itself, or even one of its metabolites,which is the proximate carcinogenic agent, but rather theenergy released during the transformation from one meta-bolite to another. Here, too, may possibly lie the truesignificance of the allegedly characteristic electronic proper-ties of the carcinogenic molecule. Finally, it may provide alink between the carcinogenic action of x rays, gamma rays,ultra-violet radiation and chemical compounds, in' that allthese agents may be, as Daudel has suggested (1946b), sourcesor carriers of energy in such a form as can readily interferewith the normal growth of the cell. A similar argument hasrecently been used by Latarjet (1946), from a comparisonbetween ionization and molecular activation in the primaryaction of radiations on micro-organisms. Highly-ionizingradiations on the one hand, and ultra-violet radiation on theother, are found to produce lesions so similar that it is impos-sible to decide, from mere inspection, the nature of thestimulus which provoked them, and it is suggested that whatthey perform in common is to deliver to the appropriatelocation in the cell an amount of energy sufficient to producean identical primary effect:

" . . . les processus de liberation de cette energie serakntdifferents, mais l'effet priraaire serait le mfime . . . En d'autrestermes, l'ionisation et l'activation moleculaire induisent la mememodification chimique initiate."

From all these varied developments it would appear we artgradually approaching an understanding of cell variationin terms of the energies involved. Although an immense

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amount remains to be done, there can be no doubt of theprofound significance of any such achievement, not only forthe cancer problem but for biology as a whole.

8. Relation to the Viruses of Chicken-sarcoma

Any complete solution of the mode of action, such as wehope to achieve in the future, must make clear the relationbetween chemical carcinogens and the tumour-producingviruses. The beginning of modern experimental research incancer, in the latter part of the nineteenth century, coincidedwith the rise of bacteriology. From that day until this, andvery largely for that, reason, there has always existed a schoolof thought which seeks to interpret the origin of cancer asdue to a process of specific infection. In its first phase, thisbelief was based not on evidence but on intuition, and wasonly later followed by deliberate attempts to isolate andidentify the causal agent which it was felt—and which inmany quarters it is still felt—must be present Betweenforty and sixty years ago, this work merely resulted in adepressing catalogue of allegedly specific pathogens—bacteria, protozoa, yeasts and fungi—and Schaudinn stig-matized this period as the most melancholy chapter in thewhole history of the subject. Nevertheless, the logicalpossibility still remains, and must therefore very willinglybe admitted, that the etiology of cancer may in some way bebound up with processes of infection, and particularly withvirus infection.

Nowadays, however, the general thesis rests on evidenceof a very different kind, dealing particularly with the filterableagent of sarcomata in fowls, of which the virus of the Rouschicken-sarcoma is the best known example, with the milkfactor involved in the causation of breast cancer in mice, andwith Shope's papilloma virus in the rabbit.

On the other hand, and opposed to any general infectivetheory of causation, is the school which senses that theneoplastic change has in its very nature something funda-mental in biology, that it is a kind of change to which almostevery type of cell in nature is liable in appropriate circum-stances, and that it is far more profound than could dependupon infection in the bacteriological meaning, by independentand specific parasitic micro-organisms. Thus, the great massof fact shows that processes of infection in the natural historyof cancer are exceptional (that is, so far as we know), and notlikely to be an indispensable feature of the induction oftumours. Contrariwise, we already have the certain meansof producing a great variety of experimental tumours at will,by.the chemical carcinogens, in circumstances which assuredlydo not require us to introduce the conception of a ubiquitousvirus.

The fascination of the present situation is that there is nodifference of opinion on the facts themselves. There is nodebate concerning the remarkable properties of the chemicalcarcinogens, there is no question that an agent is transmittedin the milk, in mice of high liability to mammary cancer,which largely determines the incidence of the disease insubsequent generations, there is no denying the causation ofthe Rous tumour* by a virus-like agent Differences ariseonly over the interpretation of these phenomena, which tosome seem mutually incomprehensible, and to others evenirreconcilable. But no one of these facts can invalidate anyother, and it must therefore be our purpose not to foster anantithesis between one school and another, but to seek asynthesis.

The filterable agents of the avian sarcomata are submicro-scopic particles extractable from the cells of such tumours,the Rous agent being the most familiar example. Tumoursarising after inoculation of this and similar agents are derivedfrom infection of the corresponding normal cells of therecipient host, and the salient features of this action are,first, that conversion of the normal cell to the malignant formmay take place at once (in contrast to the action of thechemical carcinogens), and, secondly, that the new tumoursso induced usually conform in the minutest detail with thegrowth from which the agent was obtained, and usuallycontinue in their turn to produce further large amounts ofthe specific virus. From immunologkal experiments (Amies,Carr & Ledingham, 1940) the Rous agent appears to contain(in addition to a specific antigen) a second antigen which isalso present in normal fowl-tissue. This remarkable sero-logical property, coupled with intense specificity, and theapparent absence of any epidemiology whatever, havealways supported the conclusion that the Rous and alliedagents arise intrinsically.

In approaching this matter, it seems that there is no likeli-hood whatever of success if we restrict ourselves to the tradi-tional methods of bacteriology or of cancer research alone.The net must be cast much wider, and advances are in factalready being made in general cell-physiology, usually withoutany direct reference to cancer or viruses, to which we mustpay great attention and which offer the prospect of elucidatingsome at least of these problems. These advances now con-stitute nothing short of a revolutionary view of the importanceof the cytoplasm, as distinct from the nucleus, in cellulargrowth and heredity : pointing directly to a conception ofgenetic determinants located in the cytoplasm, they are of thegreatest interest to students of cancer who had been inde-pendently considering the possibility, for other reasons, thatmalignancy is due to an alteration not necessarily restrictedto the nucleus, but affecting the cytoplasm as well (Haddow,1944).

Perhaps the most apposite case is that described by Sonne-born (1943), in what must be regarded as one of the mostsignificant biological papers of the past ten years. Sonnebornstudied two heritable characters known as " killer" and" sensitive " in different races of Paramedum aurelia. Thephysiological situation is that fluid in which the killer racehas lived kills individuals of the sensitive races, and that whenpure races of the two types are crossed, the killer clones arefound to derive their cytoplasm from the killer parent, andthe sensitive clones are those with cytoplasm derived from thesensitive parent This phenomenon was next shown bySonneborn to be not merely a case of cytoplasmic inheritance,but due to the continued production of a cytoplasmic sub-stance under the influence of a single gene known as K. Thelaw governing this relationship was defined as follows :

" Addition of the cytoplasmic determinant to an organismlacking the character dependent on it, but containing therequired gene, results in the continued production of thecytoplasmic substance, in the development of the characterdetermined by the combined presence of gene and cytoplasmicsubstance, and in the hereditary maintenance of the characterin successive generations."

The parallel between this relationship on the one hand, andthe propagation of the Rous sarcoma by virus on the other, iscertainly remarkable, on the assumption that the Rous agentmay indeed be a particle derived from or located in thecytoplasm—in other words, that it is a plasmagene. For

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this view there is in point of fact a good deal of support fromthe physical and chemical resemblances between the Rousagent and the sub-microscopic mkrosomes of Claude (1938a,1938b, 1939, 1943a, 1943b), which are normally found in thecytoplasm.

If then we admit that this may be a reasonable and sugges-tive hypothesis, Sonneborn's law may be paraphrased forthe special case of the Rous virus somewhat as follows:" Penetration of the mutant plasmagene into the cytoplasmof a susceptible normal cell—that is, a cell which lacks thecharacter of malignancy which is partly dependent on it, butwhich contains the required gene—results in the continuedproduction of the cytoplasmic substance, that is the virus,in the development of the character of malignancy determinedby the combined presence of gene and virus, and in thehereditary maintenance of malignancy in successive cellgenerations."

In other words, there seem to be no differences of funda-mental principle, between the activity of the Rous agent andthe behaviour of a mutant plasmagene, and this suggestion, ifcorrect, would account for the individual specificity of thefowl-sarcoma agents, by which each transmits to the new hostthe characters of that particular tumour alone from which itwas obtained.

Although our eventual aim must be a correlation, ascomplete as possible, between all the available facts, it is onlywithin the past year or two that we have had any inkling ofthe way in which this might be achieved. We have alreadyconsidered, firstly the association between the property ofcarcinogenicity and the capacity of chemical carcinogens toproduce a rather characteristic type of interference withnormal growth, and secondly, the working hypothesis whichthen emerged, that the chemical carcinogens might operateby producing an interference with growth, of a kind to whichthe cells could adapt themselves only by an irreversibledifferentiation, which would automatically confer upon thenew cell-strain (that is, the induced tumour) a permanently-increased rate of division.

Quite recently, Elson (Elson & Warren, in press) has foundthat this initial inhibitory action of the chemical carcinogenscan be greatly reduced, or even prevented, by a diot sufficientlyrich in protein. Particularly since it was already known thatthe experimental production of cancer of tne livci by variousazo compounds could be similarly deU/ed or again evenprevented, by administiation of a high proportion of proteinin the diet (v. supra), we are beginning to consider whetherthe initial action of the carcinogenic hydrocarbons and otherchemical carcinogens may not be to inhibit growth bydepleting the cellular protein, by rendering it unavailable, orpossibly by interfering with its normal synthesis, that theeventual emergence of the malignant cell may be achievedthrough an irreversible modification of protein synthesis,and that it is this re-orientation which underlies the unregu-lated and permanently-enhanced capacity for growth.

Starting from an entirely different approach, Crabtree(1947)* has also found it tempting to speculate that a dis-turbance of enzyme function could lead, through the forma-tion of a carcinogen-enzyme complex and by cellular adapt-ation, to the emergence of a new type of protein with auto-synthetic properties. Bearing in mind the physical andchemical similarities between the Rous agent and the non-infective particles, may it be that the filterable agent is achemical variant of a normally-occurring particle, produced

• [BMB 96*)

through the action of a chemical carcinogen ? Certainly,this view can provide no explanation of the comparativerarity of cell-free transmission of tumours generally, but thereis, on the other hand, a reasonable probability that elucidationof the nature of the Rous virus would furnish importantprinciples applicable to cancer as a whole. This wouldcertainly seem to be the opinion of Potter (1945), who frompurely biochemical evidence has suggested that the uncon-trolled growth of cancer may be the result of competitiveinterference between two autosyntbetic proteins—one anormal enzyme protein and the other a modified form whichcould arise spontaneously from mutation, be produced bythe action of carcinogenic chemicals, or be introducedpreformed as a virus.

In all these varied possibilities, we are constantly remindedof the point to which Stanley (1939) originally drew attentionin the case of the plant viruses, that the influence of suchviruses can be likened to that of normal agents alreadypresent In other words, the notion that the avian-tumourviruses are of intrinsic origin may be related with the viewthat many of the plant viruses are autocatalytic proteins ofultimate host-cell origin. There is a further aspect of thegeneral problem.which may prove of wider significance thanany so far mentioned, namely, the relation between plasma-genes and cellular differentiation (Rhoades, 1943). It haslong been one of the puzzles of biology that, while all the cellsof an organism presumably have the same genetic constitutionin their nuclei, they nevertheless show wide morphologicaland physiological differences. These differences might bedue to variations in tissue environment, but this is not thewhole explanation, since many cells retain their maincharacteristics, at any rate in part, when they are transportedor transplanted to another part of the organism. It is nowpossible to suggest that cellular differentiation of this kindmay be determined by genetical particles in the cytoplasm.The production of different characters in cells with the samenuclear genes would thus be brought about by differentialsegregation of those cytoplasmic determiners at cell division.If the differentiation and rate of growth of both normal andmalignant cells are controlled by paniculate determinantsin the cytoplasm, may it be that the Rous agent functions byimposing a decreased level of differentiation, and hence anincreased rate of growth, upon the normal cell which itattacks?

Nothing can be more remarkable than the way in whichthese different trends and developments, some arising fromcancer research itself and others from advances in biologygenerally, are gradually producing a unified picture in placeof the two contrasting interpretations of the genesis of cancer.The ultimate solution of the origin of tumour viruses willno doubt be expressed in terms of enzyme and proteinchemistry, in a new sort of taxonomy which will doubtlessrequire the powers of a chemical Linnaeus and a modemDarwin combined. But it already appears that all thesevaried findings are irreconcilable only on the narrow view,and that there is a real prospect of deeper understanding ifwe change our viewpoint, and especially if we broaden it.Only in this way can we hope to follow the sequence as awhole, and to piece together all our facts, from whereverthey come.

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MODE OP OF CARCINOGENS A.

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