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[CANCER RESEARCH 44, 5463-5474, December 1984]

Cellular Biochemistry of the Stepwise Development of Cancer with Chemicals:G. H. A. Clowes Memorial Lecture1

Emmanuel Farber2

Departments of Pathology and Biochemistry, University of Toronto, Banting Institute, Toronto, Ontario, Canada M5G 1L5

ABSTRACT

The sequence of possible cellular, tissue, biochemical, andmolecular changes that are important during the development ofexperimental liver cancer with chemicals is reviewed from theviewpoint of the author's experience in carcinogenesis over the

past 25 years. The development of a new model for the analysisof liver carcinogenesis, the resistant hepatocyte model, is brieflydescribed, as are the known steps between exposure to aninitiating dose of a chemical carcinogen to the appearance ofhepatocellular carcinoma. These steps include: (a) the interactions with DNA; (o) the dependence on a round of cell proliferationfor initiation; (c) one type of initiated hepatocyte, a resistanthepatocyte; (d) the selection of these new hepatocytes, probablyby clonal expansion, to form synchronously the first type ofhepatocyte nodules, early nodules; (e) the election of the majorityof these nodules to undergo remodeling to normal-appearingliver by differentiation ("redifferentiation"); (f) the election of a

minority of early nodules to persist; (gr)the slow growth of thefew persistent nodules; and (/?) the precursor role of persistentnodules in the development of hepatocellular carcinoma. Theevidence for such a precursor role includes: (a) the commonoccurrence in persistent nodules of a subsequent precancerousstep, "nodules in nodules"; (o) the occurrence of metastasizing

cancer inside nodules without cancer elsewhere in the liver; and(c) the high rate of evolution to cancer of persistent nodules, butnot of early nodules, when transplanted to the spleen.

Based on the common architecture, organization, blood supply, and biochemical pattern of properties relating to the metabolism of xenobiotics in hepatocyte nodules in six different modelsof liver carcinogenesis and on the common occurrence of ahighly programmed redifferentiation pattern of carcinogen-in

duced hepatocyte nodules, it is concluded that heterogeneityand diversity seen in many phenotypic properties of cancers,including liver cancers, is preceded by a precursor populationthat is unusually homogeneous and uniform in phenotype. Thus,the heterogeneity and diversity of cancers are probably latemanifestations in carcinogenesis.

The available evidence is very suggestive that the hepatocytenodules are an expression of physiological adaptation to exposure to hazardous xenobiotics and not a form of aberration ormutation. The data also suggest that hepatocyte nodules are anadditional pattern of liver differentiation and that liver cancer, tobe understood, should be compared with this precursor new

1Based on the 24th Annual G. H. A. Clowes Memorial Lecture, presented in

May 1984 at the 75th Annual Meeting of the American Association for CancerResearch in Toronto, Ontario, Canada.

2The investigations of the author have been supported by grants from theNational Cancer Institute of Canada; the National Cancer Institute, NIH (CA-21157);and the Medical Research Council of Canada (MT-5994). To whom requests forreprints should be addressed, at Department of Pathology, University of Toronto,Banting Institute, 100 College Street, Toronto, Ontario, Canada M5G 1L5.

Received July 16,1984; accepted August 16,1984.

state rather than the conventional adult, fetal, or embryonicstates. This concept also throws doubt on the validity andfundamental usefulness of the suggested relation between theoncofetal states of differentiation and cancer.

These considerations generate a different perspective concerning the fundamental basis for the prolonged nature of thecarcinogenic process and emphasize the physiological nature ofat least some of the steps in the development of cancer withchemicals. The possible role, if any, of oncogenes in this newconceptual framework is discussed briefly.

I am deeply honored by having been chosen the 24th G. H. A.Clowes awardee. Although I am naturally flattered by the selection, I am much more pleased about the recognition this gives toour group for its research. I also welcome the opportunity andprivilege to share with you some of the enjoyment we have hadand are still having in our research.

Our research in chemical carcinogenesis is a cooperative andcollaborative effort with mainly biochemistry, molecular biology,cell biology, and cell physiology providing the approaches andpathology the synthesis. I would like to thank in particular mytwo more senior colleagues of some years, Drs. Sarma andRajalakshmi, who have contributed in major ways to our researchefforts. Of more recent vintage are Drs. Murray, Mediine, Rao,Ghoshal, and Roomi. Since coming to Toronto, we have had anunusually stimulating group of visiting scientists and fellows: theyinclude Drs. Ericksson, Hayes, and Nachtomi as well as Drs. R.Becker, Columbano, Denda, Enomoto, Laishes, Ledda, Naga-

mine, Ogawa, Rotstein, Rushmore, Tatematsu, Tsuda, andWard. Among our former students, I should mention Drs. Soft,Cameron, Ying, Cayama, and Makowka. Miriam Ahluwalia, Dr.Etzio Laconi, George Lee, Esther Roberts, Joel Rotstein, Jo AnnSpiewak, and Shanthi Vasudevan are currently major contributors as research assistants and graduate students. We havebeen exceedingly fortunate in having such a vigorous group ofvisitors and students.

In this paper, I would like to concentrate upon what weconsider to be the most basic and fundamental aspect of cancerâ€”how does a cancer develop? Given the wide heterogeneity

in and diversity of virtually all cancers; given the inability so farto dissect for analysis any cancer into its three minimal constituent variables, autonomous growth, ability to invade, and abilityto metastasize; and given the inordinately long time span between initiation with most types of agents and the first appearance of a malignant cell population, it appears to us that only byunderstanding the steps through which target cells and tissuesevolve during cancer development can we ever hope to developany real understanding of any cancer.

In human experience, we have known for decades that cancerin a variety of organs and tissues is preceded by many cellularand tissue changes during the 10, 20, or 30 years it takes for

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CANCER DEVELOPMENT AS A STEPWISE PROCESS

cancer to develop. In several systems, such as the cervix, skin,bronchial tree, and breast, to name but four of many, there areconvincing statistical and tissue data to implicate some of thesechanges, such as atypical hyperplasia and carcinoma in situ, ashighly probable steps in the carcinogenic process.

Beginning in the late 1930s, it was shown also experimentallythat cancer induction in the skin of rabbits or mice with chemicals,especially with small doses, requires two separate external manipulations. From these studies evolved the paradigm of "two-stage carcinogenesis," initiation and promotion. This was ex

tended to several organs or tissues since 1971 beginning withthe pioneer work of Peraino and colleagues (95). Clearly, thedesignation "two stage" is merely operational, a colloquialism,

and is not a valid interpretation of the many steps that cells andtissues go through during their evolution to cancer (35, 42).These will have to be identified and characterized in differentsystems if "initiation and promotion" are to be understood me

chanistically.I began my formal and voluntary entrÃ©einto cancer research

in 1955 after the report of Popper and his colleagues (102) thatethionine induced tumors in the livers of rats. Because of ourinterest in this methionine analogue and the possibilities oflearning more about how methionine might be related to cancerand more about molecular mechanisms in cancer induction, webegan our first overture into carcinogenesis.

Almost from the beginning, our research involved two majorfacets: (a) the interactions of chemical carcinogens with DNA(and RNA), the nature of the resultant changes in DNA and theirrepair; and (b) the tissue and cellular changes, especially biochemical, that constitute the carcinogenic process. As PeterMagee outlined during his Clowes Award lecture in 1983, weproposed in 1958 that cellular nucleic acids should be majortarget molecules for carcinogens. Our collaborative work showedthat dimethylnitrosamine and ethionine altered the DNA and/orRNA in a target organ for these carcinogens, the liver, in highlyreproductive ways (36). Somewhat later, 2-AAF3 and N-hydroxy-

2-AAF were added to the early list of carcinogens studied (78).

DNA damage and repair was a major focus in our research forseveral years (118).

However, I propose to concentrate exclusively on the secondfacet, the stepwise development of cancer, its characterization,and its possible meaning. At the beginning of our studies, wefollowed what can be seen in the liver as cancer develops, as aprelude to the analysis of the underlying biochemical mechanisms. It became evident very quickly that ethionine, despite itsrelation to a natural amino acid, methionine, and its relativelyunreactive nature, induced changes in the liver that appeared tobe similar to those reported with several other, already "classical"

liver carcinogens, such as azo dyes and an aromatic amine, 2-

AAF (26). The two most prominent tissue responses were: (a)proliferation of ductular cells and perhaps other cells close by togenerate what we called "oval cell proliferation"; and (b) liver cellor hepatocyte nodules, so-called "hyperplastic nodules." Al

though there are some differences in the tissue responses todifferent carcinogens, we became increasingly impressed withthe many similarities (26, 28).

This initial work stimulated an increasing interest in understanding what the cellular and tissue changes mean in cancer

3The abbreviations used are: 2-AAF, 2-acetylammofluorene; DENA. diethylm-

trosanrane; RH. resistant hepatocyte; PH, partial hepatectomy; PB, phÃ©nobarbital.

development. We wondered more and more about the role ofthe hepatocyte nodule in carcinogenesis and the basis for someof the common features. We ultimately realized that we wereapproaching what has become in my view the critical questionin cancer development in most systems. Why does it take some15 to 20 years or more to develop cancer in several sites inhumans and one-third to two-thirds of the life span in many

experimental animals, despite the fact that many biochemicaland anatomical changes in cells and cell populations can be seenlong before the first appearance of malignant neoplasia? If wehad an inkling of why this is the dominant pattern, we wouldhave made a major advance towards understanding carcinogenesis, especially with chemicals, radiation, and at least someviruses.

Before we can even hope to discuss this intelligently, it appearsto me that we must know much more about the genesis, nature,and properties of at least some of the steps that appear to beinvolved in the development of cancer. The skin in the mouse,the mammary gland in the mouse and in the rat, and the liver inthe rat have received most of the attention in this area. Sincethe liver has been the organ that has received the lion's share of

our efforts, I would like to outline briefly the current state of theart concerning chemical carcinogenesis in this organ, as we seeit. Then I shall discuss some implications of our current views ofliver carcinogenesis for cancer development in general and thenreturn to the key fundamental question in carcinogenesis, "Whythe long multistep patterns in cancer development?"

Sequences in the Stepwise Development of Liver Cancerwith Chemicals

The First Sequenceâ€”the Genesis of Hepatocyte Nodules.

In our view, the first step of crucial importance to liver cancerdevelopment is the appearance of nodules of hepatocytes. Bynodules, we mean focal proliferations of hepatocytes visible tothe naked eye and measuring at least 2 to 3 mm in diameter.

Initiation. Several steps are involved in the genesis of noduleswith chemicals (Charts 1 and 2). The past 15 years or so haveseen the phenomenal growth of knowledge concerning the metabolism of carcinogens, the nature of their active forms, the

CARCINOGENMETABOLISM

Chart 1. The two-step nature of the initiation of carcinogenesis in the liver. At

least two options are available after the first step: (a) repair without initiation; or(b) initiation. The option elected depends upon whether or not a round of cellproliferation occurs before the repair process is complete.

Chart 2. The genesis of hepatocyte nodules from initiated hepatocytes (liver III).Microscopic foci (or islands) are a step in this process. 21 Kd. M, 21,000.


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interactions of such derivatives with DMA and other cellularconstituents, the resultant alterations in structure and functionof DNA, and the repair of such alterations, many in some relationship to initiation. Such studies have told us and shouldcontinue to tell us much about the physicochemical changes thatcould be a basis for the appropriate change in phenotype ininitiated cells.

However, work in our laboratory (9, 13, 143, 151) and byScherer and Emmelot (20, 122-124) and Pitot and colleagues

(100) have clearly established that, while the interactions withcellular DNA may be essential, they are not sufficient, since around of cell proliferation is required for initiation with chemicals.This offers one possible explanation for the known dependenceof liver cancer generation with a single or brief exposure to acarcinogen on cell proliferation (see Ref. 16 for referencestherein). It may also explain why so many carcinogens caninteract with liver DNA and yet so few are liver carcinogens.Unless a carcinogen is a mitogen or more commonly is a cyto-

toxic agent and can induce cell death and reparative cell proliferation (152), such an agent cannot initiate liver carcinogenesisin the adult animal. This has possible broad implications forhumans in the genesis of cancer in organs that are quiescent,such as the liver, pancreas, kidney, urinary bladder, brain, salivary glands, thyroid, etc. Cell damage with cell death may playa key role as a major rate-limiting step in the initiation of cancer

development in some organs. Parenthetically, the exact mechanistic role of cell proliferation in the initiation process remainsunknown, although fixation of some chemical change in DNAduring DNA replication is one possibility (72,111).

Nature of Initiated Cell-resistant Hepatocytes. A key ques

tion in all chemical carcinogenesis is the physiological and biochemical nature of the initiated cells. This is the question weposed early in the 1970s that led us into our present research.In 1973, we suggested that the phenotype of one type of initiatedliver cell might be an hepatocyte that has acquired resistance toinhibition of cell proliferation by carcinogens (28). This was basedon a considerable number of published studies, beginning withHaddow in 1938 (48), that showed that different types of carcinogens inhibited cell proliferation in several normal tissues including the liver (see Refs. 28 and 30 for references). With continuousor intermittent exposure to carcinogens leading to liver cancer,resistant cells would have to appear at some time in the process.Since hepatocyte nodules are the first step involving the expansion by proliferation of cells altered during initiation, it appearedreasonable to think that the first measurable change in phenotype, induction of initiated cells, might be an appropriate step forthe acquisition of resistance. With a healthy input of luck, Soltand I were fortunate in being able to devise an assay for resistanthepatocytes (132). Solt, Tsuda, and Lee showed subsequentlythat some 40 different chemical carcinogens of varying structureand properties each induced resistant hepatocytes during initiation (132,143). We used brief exposure to 2-AAF plus a mitogen

to select for resistant hepatocytes (Chart 3). Ito and his colleagues showed that several other carcinogens could be usedeffectively in place of the 2-AAF (57,138). We call this model the

resistant hepatocyte (RH) model for obvious reasons.With the RH model, it now becomes possible to follow day by

day the origin, growth, biological behavior, and biochemistry ofnodules, since they develop rapidly within a few days afterbeginning their selection and most importantly they develop

Carcinogen

I

PHorCCI4 Nodules

0.02W.2AAF diet I

Chart 3. Schematic representation of assay for resistant hepatocytes inducedby a carcinogen during initiation. The animal is given a diet containing 2-AAF for 2weeks, and at the midway point cell proliferation is induced by PH or by a necrogenicdose of CCU (143). Small nodules can be seen within 1 week to 10 days.

HEPATOCYTENODULESi

VERY SHALL\SUBSETÃŒ\PERSISTENTHEPATOCYTE

NODULES

ENLARGINGSLOÂ»CELL PERSISTENT

PROLIFERATION HEPATOCYTENODULES

Chart 4. The early hepatocyte nodule-to-persistent hepatocyte nodule sequence in liver carcinogenesis. The remodeling, via reditferentiation of nodulehepatocytes back to normal-appearing liver, is indicated for the majority (over 90%)of the early nodules with the persistence of only a small subset. Trie latter undergoslow progressive growth and show some defect in their constituent hepatocytesto "turn off" or "shut down" cell proliferation after a discrete mitogenic stimulus is

applied.

RARE EVENT

Chart 5. Schematic portrayal of the persistent nodule to metastasizing carcinoma sequence. A prominent feature is the appearance of "nodules in nodules."

synchronously. They arise from hepatocytes randomly throughout the liver and not from any special region or zone of theorgan, lobule, or acinus. Also, they clearly arise from hepatocytesand not from oval cells or ductular cells. The selection pressureis so intense that focal proliferation begins within 24 hr afterimposing the mitogenic stimulus (PH or CCU) during the periodof exposure to the 2-AAF and continues for at least 2 to 3 weeks.

By this time, the nodules measure about 0.5 cm in diameter andare quite uniform in size.

The nodules generated in the RH model are similar in appearance to those generated in several other models of liver carcinogenesis. I shall return to a comparison between the differentmodels a little later.

Biology of Hepatocyte Nodulesâ€”the Nodule-to-Cancer Se

quence. Because the generation of hepatocyte nodules is synchronous, it became possible to observe the properties, behavior, and fates of nodules with considerable confidence (Charts 4and 5). When they appear asynchronously, as in most other livermodels, it is impossible to be certain of "what" precedes "what"

in any proposed sequence.The most obvious property of the nodules is their disappear

ance within a few weeks. At 5 to 6 weeks postinitiation, the livermay contain up to 1000 nodules or so. Within 2 weeks thereafter,the majority of nodules, over 90%, begin to regress by remodeling and by 8 to 10 weeks have virtually disappeared grossly(Chart 4) (21,83,91,92,145). Tatematsu ef al. (139) have shown


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that this remodeling process is actually one of redifferentiationback to normal-appearing liver.

A small minority of nodules, perhaps 1 to 3%, do not showthis remodeling but rather persist as such (21, 83, 91, 92,139,145,146), and these persistent nodules are the sites of a furthersequence of changes leading to liver cancer (Chart 5) (91, 92,134).

These persistent nodules enlarge slowly (21), show an elevated labeling index (21), and are sites for the development ofnew hepatocyte populations that generate what Popper calledsome years ago "nodules in nodules" (103). Occasionally, a frank

hepatocellular carcinoma can be seen arising entirely within anodule (134).

In the RH model, the nodule-to-cancer sequence is entirely"self-generating," requiring no known manipulation. However, it

can be modulated by hormones (53, 82) and some drugs (18,109), including known liver cancer promoters. This critical sequence is now receiving major attention in our laboratory andothers. In studies begun by Leonard Makowka and currentlybeing led by George Lee, it has been shown that the earlypremodeled nodules and the persistent nodules differ considerably in their behavior on transplantation to the spleens of normalsyngeneic rats. The early nodules, like normal liver, grow slowlyin the spleen more or less diffusely and gradually replace thesplenic "pulp" over many months (41,73). In studies so far lasting

up to 2 years, no nodular growths or cancers have been seen.In contrast, persistent nodules grow as nodules in the spleen;

they become quite large, up to 2 to 3 cm in diameter; and allgenerate hepatocellular carcinoma within 18 months (74). Noneof the nodules at the time of their transplantation into the spleenhad any changes suggestive of hepatocellular carcinoma.

Thus, the RH model has enabled us to generate severaldiscrete new hepatocyte populations as steps in the carcinogenicprocess (Chart 6). This model now makes available in vivo adiscrete reproducible population of hepatocytes in which earlyseemingly autonomous growth is available for study without thecomplications of invasion and metastatic spread. This might beconsidered to be truly an example of "minimal deviation," a term

suggested originally by Potter (104,105). Recent studies by JoelHolstein already indicate that this population may have a dis-

Inltiatton

cÂ»u| ItoUboDc

Activation Prallt. Promotion

PÂ«rÂ»iÂ«t.nt' NoduleÂ»

CancerProgression (Growth+

Invasion

Cancer+ Metastasis

Charts. Schematic representation of several key steps in the sequence ofsteps between exposure to a carcinogen and the appearance of advanced cancer.Some of the steps in initiation, promotion, and progression are portrayed. Thecircle beside promotion represents physiological redifferentiation of nodules tonormal-appearing adult liver.

turbance in its ability to terminate or shut off cell proliferationafter a discrete mitogenic stimulus is applied (117). It nowbecomes possible to plan meaningful biochemical studies of theearly steps in altered growth control with a view to mechanisms.Also, this offers an attractive new model for the study of thepossible genesis and role of growth factors produced by newcells in the premalignant steps in carcinogenesis.

This model has suggested several new ideas about carcinogenesis and the development of cancer. I would like now todiscuss a few of these, especially since they may well havebroader implications for cancer development in general, not onlyfor the liver.

Heterogeneity and Diversity versus Commonality in ProbableCancer Precursors

It is now widely appreciated that the majority of if not allcancers of any cell type in humans and in animals are heterogeneous and diverse in their phenotypic expressions. This pertainsnot only to their histolÃ³gica! appearance but also to their kary-otype, expression of surface antigens, immunogenicity, contentof hormone receptors, responsiveness to the host, capacity formetastatic spread, and many biochemical properties (40, 51, 84,106,150).

Given the metabolic generation from most carcinogens ofreactive metabolites that interact widely with DMA and with othercellular constituents, given the mutagenic activity of many oftheir reactive metabolites, and given the stepwise nature ofcancer development with new cell populations at several steps,are the diversity and heterogeneity seen in cancers an earlyphenomenon inherent in the carcinogenic process or do theyappear only relatively late with advanced stages of progression?The scientific strategy toward the metabolic and mechanisticanalysis of cancer and its development can be radically differentdepending upon which of these general patterns seems to predominate.

The evidence we now have from the liver models indicatesthat some of the early steps in the carcinogenic process, asepitomized by hepatocyte nodules, show a commonality, not agreat diversity or heterogeneity.

Grossly, the nodules are grayish white and quite sharplydemarcated from the surrounding reddish brown liver. They arereadily distinguished from normal adult liver and from liver cancer.Unlike normal liver with its predominant architectural pattern assingle-cell plates (113), the hepatocytes in nodules are arranged

in plates usually two or three cells thick or in a glandular arrangement (29, 91, 92). The blood supply is also different in thatnodules show a relative decrease in portal blood supply and anormal or increased arterial blood supply (14,15,133). Althoughthe individual hepatocytes in the early-developing nodules resem

ble hepatocytes in regenerating adult liver, they begin to acquiredistinctive cytoplasmic and nuclear changes with proliferation ofsmooth endoplasmic reticulum and an apparent increase in eu-chromatin and decrease in heterochromatin. These nuclearchanges suggest a "more open," "more available" genome for

transcription. Also, such an alteration in DNA organization orarchitecture could be related to the "heritable fragile sites" pro

posed recently by LeBeau and Rowley for many cancers (71).In composite, the nodules anatomically do not resemble liver

at any stage of normal development or maturation (91,92). They


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are not similar to embryonic, fetal, neonatal, or regenerating liverbut show a unique phenotype.

These special architectural and structural features are accompanied by a special biochemical pattern that again is not similarto any developmental phase (Table 1). The nodules show largedecreases (75 to 90%) in total microsomal cytochromes P-450and cytochrome bs and in several mixed-function oxygenaseactivities (4, 6-8, 23, 25, 34, 38, 47, 60, 94, 116). These

decreases in Phase I enzymes and components are accompaniedby relatively large increases (2- to 15-fold) in glutathione (re

duced, oxidized, and bound) (1,19,39,67,116,120), in severalcytosolic glutathione S-transferases and DT-diaphorase, in microsomal epoxide hydrdase and UDP-glucuronyltransferase 1,and in membrane 7-glutamyltransferase (1, 4, 6-8, 19, 23, 25,

34, 38, 39, 47, 60, 66, 67, 90, 94, 116, 120, 121). All of thesecomponents play a role in the detoxification of several types ofxenobiotics and their metabolic derivatives including the generation of mercapturic acids (10,23,58,61,76,80,135,136,147,148). In addition, the nodules show a special polypeptide with amolecular weight of 21,000 in the cytosol (24).

These patterns of architecture, cytological appearance, andbiochemistry are seen in nodules in six different models of livercarcinogenesis (Models 1 to 6 in Table 2) and in nodules fromthe RH model 16 months after they have been transplanted toand are growing in the spleens of normal syngeneic animals (23,34,116). Thus, the phenotype is clearly constitutive, not induced,and appears to represent a new state of liver differentiation. Thedifferent models studied involved short- or long-term continuousor intermittent exposure to 2-AAF or 3'-methyl-4-dimethylami-

noazobenzene; initiation with DENA, 2-AAF, or dimethylhydra-zine or one of over 40 other carcinogens; and promotion with 2-AAF plus PH or CCIÂ«,PB, choline-deficient, low-methionine diet,or dietary orotic acid (Chart 7). Thus, under quite diverse exper-

TabtelBiochemical pattern of Phase I, Phase II, and some other components seen in

hepatocyte nodules In six different models of liver carcinogenesis

Phase II Cytochromes P-450

i Cytochrome b*14 mixed-function oxygenases

PhasenÃŽUDP-glucuronyltransferase If Glutathione S-transferasest Glutathionef 7-Glutamyltransferasei Sulfotransferase

Other| Epoxide hydrolasef DT-diaphorase (quinone reducÃase)t M, 21,000 polypeptide

Table 2

Currently available models of hepatocarcinogenesis

1. Long-term continuous exposure to a chemical (since 1933) (see Ref. 33)2. Intermittent chronic exposure [Reuber (114); Epstein et al. (22); Teebor and

Becker (141)]3. Chronic enzyme induction models [Peraino et al. (95); Pilot ef al. (99); Schulte-

Hermannefa/. (127))4. Resistant hepatocyte model [Farber and Cameron (35); Soft ef al. (132,134)]5. Choline-methionine-deficient model [Lombardi and Shinozuka (130,131)]6. Orotic acid model [Sarma, Rao, Rajalakshmi et al. (12, 70,72,112)]7. Choline-deficient, low-methionine diet; no added carcinogen |Mikol ef al. (81);

Poirier ef al. (101 ); Ghoshal and Farber (45)]

Â¡mentalconditions, a new cell population, the hepatocyte nodules, shows an unusual degree of commonality in several aspectsof their phenotype.

This commonality refers to the phenotype of the hepatocytenodules as a group. Does this also apply to a population ofhepatocyte nodules in any individual animal or group of animals?Some studies (93,99,110), including one of our own, suggestedthat nodules may be diverse in some specific enzymatic properties such as glucose-6-phosphatase, nucleotide polyphospha-tase ("ATPase"), and 7-glutamyltransferase. However, it must

be emphasized that the liver hepatocytes themselves showconsiderable heterogeneity in their phenotype and diversity indifferent zones (113). With some biochemical properties, relatingto xenobiotic metabolism (glutathione, glutathione S-transferases, epoxide hydrolase, 7-glutamyltransferase, and DT-dia

phorase), well over 90% of nodules and the majority if not all ofpersistent nodules show a common pattern of elevated activitiesor concentrations. Also, nodules generated by each of the sixmodels used have been shown to have an unusually commoncytosolic protein or subunit of a protein with a molecular weightof 21,000. It is present also in hepatocellular carcinomas. Thisprotein has not been found in fetal, neonatal, or regeneratingliver or in liver after enzyme induction with several inducersincluding PB and some carcinogens. It appears to be a goodcandidate for a new biochemical phenotypic marker for hepatocyte nodules and for carcinogenesis in liver. This newly discovered protein may well be the same as the new glutathione S-

transferase very recently reported by Sato and his colleagues(65, 120). Thus, it would appear that the commonality relatesnot only to the hepatocyte nodules as a group or population butalso, to a considerable degree, to individual nodules. Also, themany common phenotypic properties associated with hepatocyte nodules apply equally to both the early nodules and thelater persistent nodules. No phenotypic marker is available asyet that can distinguish the small number of persistent nodulesfrom the large number of early nodules other than persistenceand behavior on splenic transplantation.

Thus, in six different models of liver carcinogenesis, hepatocyte nodules show a remarkably similar group of phenotypiccharacteristics at several levels of organization. What evidenceis there that nodules have some relevance to liver cancer?Conceivably, they could be a common response to xenobiotics

CARCINOGEN

INITIATION

1 RESISTANT HEPATOCYTE MODEL

SELECTION VIA CARCINOGENS â€žnnil+ CELL PHOLIF. *

2. CHRONIC ENZYME INDUCTION MODEL

PB. gHCH. PCBÂ«. â€¢NODULES -DDT. BHT. CPA.

3. CHOLINE-METHIONINE DEFICIENCY MODEL

CM D

â€¢â€¢â€¢CANCER

4 OROTIC ACID MODEL

OA

â€¢NODULES-

NODULES Â»CANCER

Chart 7. Diagramatic representation of four of the five models used to generatehepatocyte nodules in the study of their phenotypic properties. With these fourmodels, initiation can be induced with one of many different carcinogens (12, 22,70,95,100,112,127,130,131,134,141,143). oHCH, o-hexachlorocyclohexane;PCBs, polychlorinated biphenyls; DDT, 1,1,1-trichloro-2,2-bis(p-chlorophenyl)-ethane; BHT, 2,6-di-fert-butyl-4-methylphenol; CPA, cyproterone acetate; CUD,choline-deficient, low-methionine diet; OA. orotic acid.


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occurring in parallel with but unrelated to hepatocellular carcinoma. This is a major problem in every carcinogenic system.The most convincing evidence implicating hepatocyte nodulesas steps in cancer development is: (a) with five different carcinogens in different models, cancer has been shown to arise insidenodules (Table 3); (b) in studies of the nodules before cancerappears, often one can see "nodules in nodules" (33,103); and

(c) persistent nodules show a high rate of evolution to cancerwhen transplanted to the spleen (74). This latter evidence indicates that the hepatocyte nodule, especially the persistent nodule, is subject to a further evolution of cells that can be conceptually related to liver cell cancer. Since hepatocellular carcinomasshow diversity and heterogeneity as much as do other highlymalignant neoplasms, it is quite probable that the commonalityseen in early preneoplastic liver cell populations may be seen inother organs and tissues as well. The most attractive hypothesisthat could explain the commonality is that, at this nonneoplasticstage in carcinogenesis, with no expression as yet of any autonomy of growth, only certain types of cells can be selected by anorgan or tissue. Thus, the commonality could be an expressionof the limitations imposed by the tissue environment duringpromotion.

Based on observations in liver carcinogenesis, it is possibleand perhaps even probable that the diversity and heterogeneityshown by advanced malignant neoplasms may represent "noise"accompanying a common underlying "theme." This may also

relate to the expression of at least some oncogenes. Albino andcoworkers (2) have recently reported that their findings withsome ras oncogenes in patients with melanomas may indicatethat, under the conditions studied, the oncogene expressioncould be an accompaniment of the advanced heterogeneity incancer and not an integral component of the carcinogenic process. It may be argued that the increasing diversity in cancerphenotype in any single cancer is epiphenomenological. Whenwe understand the biochemical basis for increasing growth,invasion, and metastasis, we may be in a position to denote anyphenotypic variation either as part of one of the common themesor as "noise." This is not to say that the so-called "noise" may

not .have an important influence in cancer diagnosis or therapy,but once we understand the relevance of any phenotypic modulation, we can begin to assign it some importance.

Early Steps in Carcinogenesis as New Forms of PhysiologicalAdaptation to Exposure to Xenobiotics

The common patterns of special architecture and organization,cellular structure, blood supply, and biochemical composition ofhepatocyte nodules indicate that this phase of the carcinogenicprocess in the liver is a new type of physiological adaptation toa severe form of exposure to xenobiotics or environmentalhazards.

Table 3Hepatocyte nodule as one established precursor for hepatocellular carcinoma

Studies of hepatocarcmogenesis in which hepatocellular carcinoma has beenfound to arise within hepatocyte nodules.

AramiteEthtonine3 ' -MethyM-dimethylaminoazobenzene

2-AAFDENA

Popper et al. (103)Farber eia/. (22,27)Goldfarb et al. (46)GokJfarbera/. (110)Scherer and Emmelot (122)SoltÃ©/a/. (134)

This suggestion is greatly strengthened by recent work ofTatematsu (139) in our laboratory which established unequivocally that remodeling of hepatocyte nodules to normal-appearingliver in the RH model is due to redifferentiation of nodule hepa-

tocytes. This is a highly complex process in which changes inhepatocyte structure, organization, architecture, and biochemicalpatterns are secondary to a rearrangement of gene expressionand possibly to gene product reorganization. This is clearlygenetically programmed and therefore must be physiological,i.e., built into the genome. If a complex redifferentiation is builtinto the genomic inheritance as a physiological pattern, thenodule itself must be physiological. We perceive the hepatocytenodule as a physiological response to environmental perturbation.

This process of "regression" or remodeling is by no means

unique to the RH model. The process was seen in many studiessince the first liver model was described in 1935 (62,119). TheRM model has allowed us to establish that the fundamentalnature of remodeling is one of redifferentiation. I should add thatwe have observed remodeling by redifferentiation only in grosslyvisible nodules, 1 to 2 mm in diameter or larger and not inmicroscopic nodule precursors ("foci" or "islands").

If the hepatocyte nodule is fundamentally a physiological response to certain types of alterations induced by carcinogensduring initiation, what physiological role might it have? Since oneform of promotion or selection pressure for the genesis ofhepatocyte nodules is differential resistance to the inhibitoryeffects of 2-AAF and other carcinogens on cell proliferation (31),

several aspects of the phenotype of the nodules seen in the RHmodel are not surprising. The low levels of activating enzymecomponents in the microsomes and the high levels of DT-diaphorase (quinone reductase) and epoxide hydrolase, glutathi-one, glutathione S-transferases, and one UDP-glucuronyltrans-

ferase are all consistent with a decreased ability of the cells togenerate reactive moieties from xenobiotics and an increasedability to inactivate whatever becomes activated. This is manifested in vivo as: (a) a more efficient excretion of one carcinogen(2-AAF) by animals with many liver nodules, as shown by Jo Ann

Spiewak and Dr. Ericksson (135,136); (b) a resistance of isolatednodule hepatocytes to some hepatotoxins such as aflatoxin B,and 2-AAF (59, 69,115); (c) a resistance to the induction of cell

death in vivo by CCU and dimethylnitrosamine (37); and, mostimportantly, (d) an ability to grow in an environment that severelyinhibits cell proliferation of the surrounding liver (132,134). Also,nodules show a resistance to some cytotoxic effects of DENA(44).

However, the finding of a common pattern in nodules in severaldifferent models of liver carcinogenesis indicated that this newpopulation may have a more general significance in the adaptiveresponses of the liver. In one model, the orotic acid model, whichis being developed by Drs. Sarma, Rajalakshmi, and Rao in ourdepartment, selection of nodules by differential inhibition of cellproliferation seems to have been ruled out (112). What then isthe general physiological significance of the appearance of a newpopulation of hepatocytes with a distinctive phenotype? We havefound repeatedly that animals with many liver nodules are betterable to withstand toxic doses of xenobiotics and handle at leastone such agent, 2-AAF, more efficiently for excretion (135). We

therefore consider that this physiological adaptation has considerable survival value in a hostile environment. It is conceivable


5468




that, as in the case of another common reaction to many hazardous stimuli, the heat shock pattern (75,125), the new patternin the nodules may be under the control of a common gene orfamily of genes that are triggered during initiation. The rediffer-entiation of such a population allows a return to the base-line

mature liver when the environment becomes less hazardous.Studies leading to the ultimate identification and isolation of sucha hypothetical gene or gene family might become feasible in thenear future.

It becomes attractive to suggest that a regulatory gene orgenes concerned with such genetic expression may well be thecrucial target site for the first or early macromolecular events inthe initiation process. Either a structural or steric change at sucha target are likely possibilities for the initial events in chemical,radiation, and even some viral carcinogenesis.

It is noteworthy that Schulte-Hermann and colleagues (129)

have suggested recently that foci of altered hepatocytes seen ina PB or a-hexachlorocyclohexane model using A/-nitrosomorpho-

line to initiate show a highly programmed pattern of enzymesrelating to glycolysis and the metabolism of some xenobiotics.The similarities in principle between this suggestion and oursconcerning the hepatocyte nodules as a form of physiologicaladaptation are interesting.

I would like to highlight a recent development that I considerof great potential importance mechanistically in carcinogenesis.Lionel Poirier and his colleagues at NIH (81,101) and Dr. Ghoshalin our group (45), as well as Dr. Lombardi and his coworkers inPittsburgh have independently shown that hepatocyte nodules(100%), very similar to the nodules seen in the other models ofliver carcinogenesis and hepatocellular carcinoma (over 50%)can be induced in rats by feeding diets deficient in choline andmethionine without any added carcinogens. The lesions areprevented by adding choline to the diet. Poirier and his colleaguesused pure amino acids, and we and Lombardi used purifiedproteins. This rediscovery of the induction of liver cancer in ratsby these dietary deficiencies offers another possible approachto the mechanistic analysis of the sequence of steps in livercancer development and to possible physiological componentsin the process.

Agents and Processes

As I have emphasized elsewhere (32), toxic manifestations ofexposure to environmental agents are often the result of themutual interactions between the agent and the target organismor tissue. Chemicals act on tissues, tissues act on chemicals,and each in turn evoke responses by the tissues or cells that arethe true toxicological effects on the organism. This more holisticrather than reductionist viewpoint is seen in full array in carcinogenesis in response to chemicals.

This is seen in its most elemental form in the varied responseof a tissue or organ to the same agent, depending upon thesetting (Chart 8). For example, since 1971, several chemicalssuch as PB, 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane, poly-chlorinated biphenyls, 3-fert-butyl-4-methoxyphenol(2-ferf-butyl-4-hydroxyanisole, 2,6-di-ferf-butyl-4-methylphenol, cyproteroneacetate, a-hexachlorocyclohexane, and orotic acid have been

shown to be effective promoting agents for liver cell cancer whenused after initiation is induced by one of several carcinogens (3,43, 52, 54, 56, 57, 63, 64, 68, 77, 79, 86-89, 95-98,126, 128,

(i)PHENOBARBITAL

(b)POLYCHLORINATEDBIPHENYLS

(c)ETC.

ICARCINOGENy'ASELECTION

\,OKAMODULI:->PROMOTION

(2nd CARCINOGEN)2-AAF

ANTI-CARCINOGENIC

ANTI-PROMOTER

ORANTI-SELECTOR

Chart 8. Schematic representation of the different effects of the same agent oncarcinogenesis depending upon when the agent is administered. The effects ofseveral known promoting agents for the liver can be pro-carcinogenic or anti-carcinogenic depending upon the circumstances.

137, 140, 142, 144, 149). Clearly under these conditions, theyare active promoters of liver cancer. However, when added tothe diets with an active carcinogen, such as 2-AAF, 3'-methyl-

4-dimethylaminoazobenzene, or DENA, they often prevent ordelay the development of cancer; i.e., they become anticarcino-

genic.In recent studies in our laboratory by Dr. Tony Hayes, Esther

Roberts, and George Lee, it is evident that neither PB, somepure polychlorinated biphenyls, nor 3-methylcholanthrene, another anti-liver carcinogen, inhibit initiation with 2-AAF or withDENA but are very strong inhibitors of selection by 2-AAF plus

PH (49, 50).A similar group of contradictions and confusions may be seen

,with antioxidants. As Ito and his colleagues (55) have recentlysummarized, the same antioxidant such as 3-fert-butyl-4-meth-oxyphenol or 2,6-di-fert-butyl-4-methoxyphenol may act as a

promoter of cancer development in one organ, may act as anantipromoter or inhibitor in another tissue, and may have noeffect on a third system. How does one categorize such agents?

A similar type of difficulty has been shown by Roberfroid andhis colleagues on the effects of agents on nodules generated inthe RH model (17, 18, 109). Here, for example, although PBinhibits nodule formation with 2-AAF plus PH, it enhances thenodule-to-cancer sequence.

These data raise another important issue. With multistepprocesses, one not infrequently finds that the same agent mayhave quite different if not opposing effects on different steps. PBand other enzyme inducers, hormones, antioxidants, and dietarycomponents have been shown to affect different steps in carcinogenesis in different ways. Any measurement of an overall effect,although useful in some contexts, may be impossible to interpretbecause of the differing concentration dependencies for differentsteps.

States of Differentiation, Dedifferentiation, "Oncofetal Considerations," and the Origin of Cancer

It has been stated often that cancer has a close relation toembryological or fetal states of differentiation, that cancers oftenshow "dedifferentiation," and that carcinogenesis is somehow

related to some disturbance in the normal differentiation pattern(3). These proposed generalizations are usually based on similarities in the phenotypic expression between cancers and some


5469




developmental state of differentiation.Our experience with liver carcinogenesis raises some serious

doubts concerning some of these generalizations. The liver, ofcourse, has been a favorite site for such considerations, since"oncofetal proteins" and arrested differentiation of liver are fre

quent topics of study in this organ.It is well established that the hepatocyte nodule is one frequent

site of origin for liver cell cancer. As already discussed, thehepatocyte nodule is not an "abnormal" or "pathological" lesion

in the liver but rather a new physiologically adapted state ofdifferentiation. The special architecture and organization, thespecial cytological appearance, and the special biochemical pattern, all common to six different models of liver carcinogenesis,coupled with the special programmed ^differentiation of thehepatocyte nodule phenotype back to normal-appearing liver,clearly point to this new cell population as a totally new "state"

of liver with a mixture of features, some of adult liver, some ofembryonic or fetal liver, and some special to this new state.

If this is a valid conclusion, it becomes evident that hepato-

cellular carcinoma, at least in some instances, has, as its origin,a new physiological population of liver cells, i.e., a special stateof differentiation. Any meaningful comparisons should then bemade between liver cell cancer and this new differentiation state.Thus, any direct comparison between liver cell cancer and embryonic, fetal, neonatal, or adult liver is not meaningful unlesseach is related to the new state of differentiation in hepatocytenodules.

In this context, a special feature of the persistent nodule, thehepatocyte nodule with special relevance to liver cell cancer,becomes some defect or alteration in its ability to redifferentiateto normal-appearing liver, i.e., a block in normal redifferentiation.

This resembles in principle, but not in detail, the block in normaldevelopment and maturation proposed by Potter (106,107).

Comparisons of Liver and Other Systems

The many similarities in principle between the genesis, behavior and possible role in cancer development between hepatocytenodules in liver cancer and other focal proliferations in othersystems are intriguing. Papillomas in the skin in mice, papillomasin the urinary bladder in humans and in rats, polyps in the colonin humans and some animals, polyps in the stomach in humans,and hyperplastic nodules in the mammary glands of mice andrats are but a few such systems that readily come to mind. Thenature of the precursor lesions for cancer, the multiple optionsavailable for these lesions including regression or redifferentiation, and the slow kinetics of the steps in the precursor to cancersequences are surprisingly similar.

One of the most intriguing comparisons is between rat livercancer and human melanoma. In unusual studies of the biologyof the development of some forms of melanoma in relation tonevi, Wallace Clark and his collaborators (11) at the Universityof Pennsylvania have proposed a sequence of steps involvingselection pressures relating to UV, different states of differentiation with redifferentiation, and a selective subset of nevi withpersistence of the special phenotype. This proposed sequenceof biological and cellular changes in the genesis of human melanoma from initiated melanocytes has many similarities in principleto the proposed development of liver cell cancer in the rat. Suchapparent commonalities in principle make one optimistic that a

mechanistic analysis of carcinogenesis may be in sight, once theappropriate model is identified or developed.

The Prolonged Nature of the Carcinogenic Process

I would like to return to the theme that I consider most basicin cancer research, "Why does it take one-third to two-thirds of

the life span of an organism to develop a malignant cell population, and what is the need for a multistep process of cellularevolution?" This pattern predominates in most systems in vivo

and in many systems in vitro with chemicals, radiation, and someviruses. Clearly, except in a number of retrovirus systems, cancerdevelopment has one and perhaps several highly improbablesteps. What influences overcome this or these barriers to changeand what is their nature?

In view of the increasing evidence for the physiological adaptive nature of the postinitiation phase of carcinogenesis, as brieflydiscussed, it is not at all surprising that the development ofcancer should take so long. The part of the process or reactionleading to cancer can be looked upon as an infrequent aberrationof adaptation. The "natural" or "physiological" adaptation to therelevant cellular biochemical changes induced by a "carcinogen"

is a new population that can undergo either expansion or redifferentiation, based upon the nature and intensity of the currentperturbations in the environment. Only an occasional focal collection of such cells, for reasons not yet apparent, fails toundergo the "normal" redifferentiation and thereby opens up

another biological option that may lead to cancer. The better thephysiological adaptation, the less likely is the option to cancerand therefore the longer the "carcinogenic process" lasts. If

cancer development is viewed in this perspective, a whole newset of questions can now be posed that may lead to new insightsinto the fundamental ways in which the living organism reacts topotential hazardous xenobiotics in the environment.

A commonly discussed possibility is immune surveillance, firstsuggested for cancer in the late 1950s. The evidence availableto date fails to implicate the immune system in many types ofcarcinogenesis with chemicals until very late in the process, aftermalignant cancer has appeared.

There are at least two sequences that appear to have rate-

limiting steps: (a) the expansion of the initiated hepatocytes togenerate nodules; and (b) the evolution of the persistent nodulesto cancer.

With respect to the first, we now have some insights into thissequence under some conditions. With carcinogens as promoters, a common phenomenon in the real world, selection ofappropriately altered target cells, such as resistant hepatocytes,by the creation of a differential inhibition, is a very likely possibility.The basis of promotion with noncarcinogens remains unknown.With respect to the second rate-limiting sequence, the nodule-to-cancer sequence, our understanding is largely incomplete.

In looking at several systems, both in humans and especiallyin experimental animals and in vitro as well as in vivo, some ormany of these later steps are self-generating (Chart 9). For

example, in the liver, most of the time taken for cancer development is self-generating.

Thus far, the experimental data are minimal or nonexistent,forcing a discourse on possibilities. In the nodule-to-cancer sequence, as in the papilloma-to-cancer and polyp-to-cancer sequences, there are two general possibilities, (a) external environ-


5470



CANCER DEVELOPMENT AS A STEPWISEPROCESS

HAKE EVENT il RAM EVENT12

SELECTION (PROMOTION)

RARE EVENT #5

RARE EVENT Â«4

?*--

ENVIRONMENTDCPENDENT

SELFGENERATING

Chart 9. Schematicportrayal of the initialsteps in carcinogenesis,which dependupon an appropriate manipulation from "the outside" ("environment-dependent")and the several subsequent steps which seem to be "self-generating" in that they

require no further manipulations by the investigator.

mental influences and (b) internal endogenous influences. In theformer, a general model that is receiving some attention is theso-called "multihit" one (108). This views the evolution to cancer

as largely an external environmental problem. In the latter, atleast two attractive considerations come to mind: (a) a genetic;and (b) a local endogenous environmental. For example, hepa-

tocyte nodules have been shown by Dr. Nachtomi (85) to havea considerable decrease in the activities of Superoxide dismutaseand glutathione peroxidase, enzymes that play a role in counteracting the possible damaging effects of free radicals. Conceivably, an imbalance between the rates of genesis and rates ofdestruction of active oxygen and other radicals could play animportant role in increasing the critical molecular damage incancer precursor cells, if such damage is important in cancerdevelopment.

Alternately, the prolonged alteration in nuclear organization,as may be seen by simply looking at the nuclei in persistenthepatocyte nodules, could favor gene rearrangements or othergenomic reorganizations. If the oncogenes play an active role inthe genesis of cancer, I would think that they would mostprobably do so in the nodule-to-cancer sequence. The onco

genes might play a role by one or more of the following possibilities: (a) gene activation via promoter insertion or promoteractivation; (b) gene rearrangement; or (c) somatic mutation. Thedevelopment of many models for the analysis of the nodule tocancer sequence would seem to be ever so important in theeventual analysis of the roles of oncogenes in cancer development.

The current availability of a system in liver to obtain discreteliver nodule hepatocytes at different steps in the nodule-to-

cancer sequence allows a more critical test for the intimateinvolvement of oncogenes in carcinogenesis (Chart 6). Early workin collaboration with Dr. Barbackj at NIH on transfection withDNA from about 20 frank hepatocellular carcinomas have so farbeen negative. The analysis of earlier steps in the nodule tocancer sequence is now being planned.

In summary, it is evident that liver cell cancer in the rat involvesa minimum of 7 or 8 steps, some of which are represented bydiscrete isolatable clean hepatocyte populations (Chart 6). Thesecan be analyzed in increasing depth for metabolic alterations thatmight be related to the early steps in neoplasia. They allow aready separation of the major biological variables, autonomy of

growth, invasion, and metastasis. Equally important, this studyis forcing us to look at the carcinogenic process with a totallydifferent perspective. This is both provocative and exciting. Inthis context, I am reminded of a favorite quotation from MarkTwain:

When I was a boy at 14, my father was so ignorant that I could hardlystand to have the old man around. But, when I got to be 21, I wasastonished by how much the old man learned in 7 years.

Acknowledgments

I would like to express my sincere gratitude to Dr. Sarma for many helpfulsuggestions in preparing this manuscript. Janet Jeffrey and Carolyn Quillet weremost helpful in the preparationof the manuscripts for the talk and for publication.

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1984;44:5463-5474. Cancer Res Emmanuel Farber with Chemicals: G. H. A. Clowes Memorial LectureCellular Biochemistry of the Stepwise Development of Cancer

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