[CANCER RESEARCH 28, 1344-1350, July 1968]

Production of Embryonal Serum «-Globulin by Hepatomas:

Review of Experimental and Clinical Data

G. I. AbelevLaboratory of Tumor Immunochemistry, Department of Tumor Virology and Immunology, N. F. Gamaleya Institute oj Epidemiologyand Microbiology, USSR Academy of Medical Sciences, Moscow, USSR

INTRODUCTION

It was demonstrated in 1963 that transplantable mouse andrat hepatomas synthesize and secrete into the blood a specifica-globulin of embryonic sera, «(-globulin (1, 6, 7). It wasshown further that the same phenomenon of elaboration ofembryonic serum a-globulin by hepatomas could be obtainedby primary liver tumors of rats (20), mice (2, 23), and human beings (33, 34).

An important aspect of the problem is the possibility ofusing a [-globulin appearance in the blood of patients for diagnosis of hepatomas in the clinic.

The experimental data obtained in mice did not suggestsuch a possibility, since a rglobulin was revealed only in 25%of chemically induced primary liver tumors and this proteincould be also demonstrated by liver regeneration.

The detection of a {-globulin in clinical cases might, however,be of certain value in differential diagnosis of hepatocellularcarcinoma. Thus, Tatarinov (33), who was the first to findthis globulin in cases of human hepatoma, was unable to detectit either in cases of cholangioma and secondary localizationof other tumors in liver or in noncancerous diseases accompanied by liver regeneration, such as cirrhosis and hepatitis. Heregularly revealed «(-globulin in the blood of patients withhepatocellular liver cancer. Immunochemical diagnosis, as distinct from clinical, was in full coincidence with autopsy data.

By 1966 Tatarinov et al. (33, 34, 36, 37) described 6 casesof arglobulin appearance in primary hepatomas on the background of numerous negative controls. One additional case wasdescribed by Czech investigators (25). This suggests that thearglobulin test is both more regular and more specific in human hepatomas than in experimentally induced tumors, atleast in mice.

The main findings reported by Tatarinov have been confirmed and extended by 2 separate groups: ours, in collaboration with the Institute of Experimental and Clinical Oncologyof the USSR Academy of Medical Sciences (3, 10, 36), andby Grabar's group in Villejuif and Dakar (38). The results

independently obtained by the 3 groups testify to a diagnosticvalue of theo,-globulin test. In view of this, a more thoroughand wide investigation of the problem both clinically and experimentally appears necessary.

In this paper we shall give a short analysis of the experimental and clinical data and shall try to formulate possible

hypotheses for the causes of «(-globulin production by malignant liver cells.

EXPERIMENTAL MODELS

Two problems will be considered in this part—adequacy ofexperimental and clinical systems and analysis of the phenomenon by means of experimental models.

Adequacy of Experimental and Clinical Systems

At present, it may be considered that the protein structureof mammalian sera, including human, undergoes typical evolution in ontogenesis. One of the most characteristic features ofsuch an evolution is the presence of an embryo-specific a-globulin in the fetal sera. This a-globulin (fetuin, postalbumin,«rfetoprotein, or arglobulin) was first revealed by Pedersenin calf sera in 1944 (30). at-Globulin type proteins were demonstrated recently in 12 animal species investigated and in human beings (see review, Ref. 19). It is the main componentof the fetal serum in the earliest stages of ontogenesis. Itsquantity in this period greatly exceeds the level of albuminand transferrin which are the predominant proteins in thesera of adult animals. The high level of a,-globulin is maintained during the whole part of the intrauterine period formice and rats or during the greater part of the period forhuman beings (7, 15, 20, 27, 34).

The level of this protein in newborn mice and rats corresponds to 50% of its concentration in fetal sera. It disappearscompletely after the 4th week of postnatal life (7, 20). Newborn human beings contain only small amounts of «(-globulin,about 1-3% of its concentration in the sera of 14- to 22-week-old fetuses (15, 34). Neither biochemical nor immunologiemethods in the blood of adult animals and human beings hasrevealed «(-globulin.«,-Globulin could be detected in differentfetal sera by paper electrophoresis (9, 13, 16), agar electro-phoresis (6, 7), immunofiltration (2), and electrophoresis oncellulose-acetate (15, 19), and starch gel (29, 39). The lasttechnic seems to be more adequate.

In human fetal sera this protein could be identified accordingto its characteristic position on paper electropherogram between the zones of albumin and arglobulin.

Immunologie methods of a,-globulin detection are mostsimple and specific (1-3, 6, 7, 15, 20, 27, 31-33). Immuneserum against pool of fetal sera, having been absorbed by theserum of adult individuals of the same species, continues to

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react in immunoelectrophoresis with 1-2 antigens of fetalserum in the «-globulinregion. «,-Globulin can be easily identified by the 3 following features: (a) It is the most abundantfraction of embryo-specific proteins in the electropherogram.It considerably exceeds the level of other embryo-specific «-globulins, if any, in rats for example (20, 31, 32). (6) It showsthe highest electrophoretic mobility as distinct from so-called"slow" a„-globulin, which is present in the serum of ratfetuses, (c) It is absent from the serum of adult animals andanimals with inflammation or which are sham-operated (asdistinct from slow «2-globulins).

Thus, af-globulin can be defined as the predominant component of the«-globulin fraction of fetal serum, which is absentfrom the sera of adult individuals and is different from theslow «„-globulins.Human «(-globulin does not differ, in thisrespect, from analogous animal proteins, besides species specificity. Its molecular weight (70,000) (15) is near to that ofthe rat arglobulin (65,000) (24) but is different from calffetuin, which has a molecular weight of 45,000-50,000. It doesnot differ significantly from mouse and rat a f-globulin in its

electrophoretic mobility.During ontogenesis in human beings, the concentration of

«,-gIobulinfalls gradually, reaching a negligible value at birth(15, 34). A simlar occurrence is observed in mice and rats, butthe fall in concentration takes place only in the first month ofpostnatal life (7, 20).

Thus, there is every reason for regarding human and animal«,-globulin as species-specific representatives of the same typeof the protein (15), as it has been accepted for albumins ory-globulins of different species. Immunologie cross-reactionsof human and animal «rglobulin give additional evidence tothis suggestion. «(-Globulin from different species, like otherserum proteins, cross-react immunologically with each other.Thus, mouse «(-globulin cross-reacts with its rat analog (2,31), bovine with sheep (15, 35), and so on.

According to Tatarinov and Afanasyeva (35) and Gitlin andBoesman (15), human «(-globulin cross-reacts with 6 out of11 species-specific «(-globulins. Because human «(-globulincross-reacts with sheep a(-globulin (19) and the latter hascommon determinants with calf fetuin (35), the human af-globulin seems to be related to Pedersen fetuin.

Mouse and rat «{-globulinsdo not reveal immunologie cross-reactivity with «(-globulins of human origin when immunodif-fusion tests are used. In experiments with Drs. Tsvetkov andElhort, we obtained such cross-reactivity using an immuno-radioautography technic (131I).af-Globulin of human origin wasadded to nonlabeled newborn mouse serum. Mouse «(-globulinwas precipitated in agar with corresponding monospecific antisera. 131I was revealed in the precipitin line after radio-

autography of the agar plates. No radioactivity was revealedwhen 131I serum of adult man was used. The cross-reactionwas inhibited by absorption of anti-mouse af-globulin serumwith human a£-globulin-containing materials (8).

Finally, the similar appearance of «(-globulin in the sera of

human beings affected by hepatomas and its occurrence in thesera of experimental animals with induced hepatomas areother evidences in favor of the suggestion that the appearance

of this protein in human beings, in rats, and in mice involvesa common mechanism.

Thus, the data available suggest that rat and mouse «,-globulin could be regarded as an adequate model for study ofhuman «rglobulin under normal and pathologic conditions. Itis expected that the basic mechanisms ofarglobulin productionand regulation would be similar in both cases. On the otherhand, obvious species differences in its behavior in ontogenesisand in different liver pathologies could not be neglected.

«(-Globulin as a Specific Product of Liver Parenchyma Cells

To understand the nature of the phenomenon in question,it is necessary to know whether «(-globulin synthesis reallytakes place in normal or malignant liver cells. Evidence forthis is given below.

Marked «(-globulin synthesis takes place in surviving livertissues of both newborn and 14-day-old rats (2, 4, 40). Surviving tissue slices actively incorporated amino acids14C into«(-globulin. The incorporation was registered by radioautog-raphy of a rglobulin precipitin lines, obtained in Ouchterlonyplates. The spleen, lungs, and kidney (4), as well as placenta(40) and adult liver, did not synthesize«rglobulin under identical conditions.

Since the liver of embryos and newborns is the site of hemo-poiesis and since it is rich in hemopoietic cells, it was necessaryto exclude the possibility of their participation in the«f-glob-ulin synthesis. Such a possibility was excluded for two reasons.In the first place, 14-day-old rat liver, which is practically deprived of hemopoietic elements, synthesized af-globulin in thesame degree as the liver of newborn animals (4), half of whichconsists of hemopoietic tissue (26, 39). Secondly, organ culturesof embryonic mouse liver actively synthesized albumin and«(-globulinin vitro, while the synthesis of neither of these proteins could be detected in cultures of bone marrow (E. Luria,R. Bakirov, G. Abelev, and A. Fridenstein. Organ Cultures ofMouse Embryonal and Adult Liver and Their Synthesis ofSerum Proteins. Exptl. Cell Res., submitted for publication).

Finally, more evidence is based on the fact that, in mousehepatoma, the malignant cells themselves synthesize «(-globulin. This has been shown in the experiments on heterotrans-plantation of mouse hepatoma into cortisone-treated rats. Thegrowth of tumors was accompanied by the appearance ofmouse «(-globulin in the rat sera (6, 7). Moreover, in experiments with mouse hepatoma grown in vitro, the accumulationof «(-globulin was obtained in cultural fluids during severalweeks (6, 7, 22).

Therefore, it may be concluded that «(-globulin synthesistakes place in embryonic or malignant liver cells. The fact that«(-globulinsynthesis has never been obtained in rat and humancholangiomas (3, 20, 36, 37) suggests that liver parenchymacells are the site of«(-globulin synthesis.

«(-Globulin in Liver Regeneration

Study of the conditions and causes of «,-globulin synthesisin liver regeneration is of great importance experimentally andclinically.

At first, this phenomenon was demonstrated in mice afterpartial hepatectomy (1, 6, 7). «(-Globulin appearance was

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usually observed on the 2nd day after the operation. The antigen level varied considerably in individual animals and wasless than 1% from its level in the sera of newborn mice.

Appearance of «(-globulinwas a specific response to hepa-tectomy, since a sham operation did not lead to its synthesis.

It was shown further that the synthesis of «(-globulin canalso be induced in adult mice by CC14 vapor. Fifteen totwenty minutes' exposure of adult animals to an atmosphere

containing 0.025 or 0.05 ml of CC14 in a volume of 3.51 gave aclear and uniform effect : all the animals treated responded witha marked production of «(-globulinon the 2nd day, reaching1.5-3% of its level in the serum of newborns on the 3rd dayfollowing treatment. By the 8th-10th day the antigen haddisappeared from the blood. Synthesis of at-globulin could berepeatedly induced by further treatment with CC14 (11).

A clear difference in af-globulin response was found in different mouse strains. While C3H mice are excellent a (-globulin producers in response to CC14 treatment, C57BL/6 micesynthesized 10-15 times less of this protein under the same

experimental conditions.The system described opens definite advantages for studying

the control of «,-globulin production: first, because of theuniformity of response in comparison with hepatectomy;second, because of a rather high level of «,-globulin underthese conditions; and, finally, because of the fact that animalscan easily endure such a treatment. This system allows thedetection of the lifetime of messenger RNA for «(-globulinto reveal the af-globulin-synthesizing cells in the liver, and itpermits the study of hormonal effects on «(-globulininductionand synthesis. For instance, Bakirov and Abelev (12) observed that adrenalectomy in adult mice followed by CC14treatment significantly decreases the level of «(-globulinin theirsera; moreover, the uniformity of the response was greatlydisturbed.

Liver regeneration in adult rats, as distinct from that ofmice, did not lead to «(-globulin synthesis in hepatectomizedanimals (20, 31, 32) or in rats exposed to CC14 (11). Hepatectomy could lead to «(-globulin production only when performed during the fifth week following birth, a time which isvery close to the time of a (-globulin disappearance from therat serum (4th week) (31).

To understand the reason for the difference between off-globulin induction in adult mice and rats is one of the mostimportant points in the study of the phenomenon.

«(-Globulin in Experimental Hepatomas

The study of «(-globulin by means of experimental modelsreveals some regularities. It is not in every case that «(-globulin was found in the serum or tumor in primary mouse hepa-tomas; only in one out of the 15 cases was it found in thenodules of spontaneous primary tumors of 25-month-old DBAmice (V. Guelstein and N. Khramkova, unpublished observations). In primary tumors induced by orthoaminoazotoluene,«(-globulin appearance was more regular: it was noticed in7 out of 29 nodules examined (2, 23). At the same time, Of-globulin was often existent in transplanted hepatomas; it wasfound in first generations of tumors in 10 cases out of 15. Asfor stable transplanted strains, «f-globulin was detected in 6

out of 8 such strains studied (N. Khramkova and V. Guelstein,unpublished observations). Two «(-globulin-negative tumorswere slowly growing, highly differentiated, hepatocellular carcinomas. According to Grabar et al. (20), «(-globulin foundin primary rat hepatomas induced by dimethylaminoazobenzenein 52 out of 72 of the cases, while it appears in all the 8 strainsof transplanted hepatomas originating from «rglobulin-posi-tive nodules. «(-Globulin has never been found in rat cho-langiomas (0 out of 14 investigated).

As for its level in different hepatomas, we may generallyaccept that it correlates with the growth rate of tumors. Inmice the highest level of «¡.-globulinwas obtained with rapidly growing hepatomas of 22a type. In minimal, highly differentiated hepatomas, «(-globulin is, as a rule, revealed insmall amounts (2, 23).

In view of all these data, we may suppose that there is apositive correlation between the degree of malignancy of ahepatoma and the production of «(-globulinby this hepatoma.However, such a generalization may be no more than a generalimpression and, therefore, needs a special investigation.

Some observations in this respect are of importance. Threeorthoaminoazotoluene-induced hepatomas with a different setof organospecific antigens converted to one antigenic andbiologic type in the process of transplantation. After conversion, the transplanted tumors grew rapidly, and they displayedthe same characteristic set of organospecific antigens (highlyreduced), high «(-globulin levels being found in tumor extracts. This suggests that there may be a certain type of hepatoma, characterized by rapid growth, reduced and characteristic antigenic set, and high «(-globulin production.

It should be pointed out that until now, not a single caseof «(-globulinappearance in experimental tumors different fromhepatocellular cancers has been described. It is of interest toinvestigate experimental teratoblastomas, which are «(-globulin-positive in some cases with human patients.

Experimental data show that a hepatoma can lose its abilityto synthesize «(-globulinwithout losing its malignant properties.Thus, according to Irlin et al. (22), the «,-globulin-positive,stable ascites strain of 22a hepatoma became «(-globulin-negative after prolonged cultivation in vitro and retransplan-tation into animals. This was obtained with one culture fromthe 3 investigated. The fact that the tumor continued to behepatoma was proved by its ability to synthesize albumin andtransferrin i»vitro, as well as by the continued presence ofthe organospecific liver antigen which was found in the original tumor.

Systems Synthesizing «(-globulin in Vitro

Systems synthesizing «(-globulin in vitro are of great valuefor experimental studies of the nature of the phenomenon inquestion. At present, 3 such systems are described: survivingmouse and rat liver tissues, organ cultures of mouse embryonicliver, and monolayer cultures of ascitic 22a hepatoma.

The first system presents interest for "acute" biochemical

experiments. Small bits of the liver of newborn or younganimals actively incorporated amino acids-14C into «(-globulin,in the Eagle's medium containing 20% calf serum and aminoacids-14C. Label incorporation is easily registered by radio-

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autography of the antigen precipitin line in agar plates. Atechnic for the semiquantitative evaluation of the rate of at-globulin synthesis in vitro has been devised (2, 4).

Organ cultures of embryonic mouse liver retaining the ability to synthesize arglobulin were recently obtained by Luriaet al. (Organ Cultures of Mouse Embryonal and Adult Liverand Their Synthesis of Serum Proteins. Exptl. Cell Res., submitted for publication). Cultures of 18-day-old embryos weregrown on Millipore filters layered on nutrient medium; intensive hemopoiesis and liver cell proliferation were obtained insuch cultures. Active synthesis of albumin and «,-globulinwasobserved during a 10- to 12-day period. In the Luria et al.investigation, it is possible to obtain even more prolonged cultures of embryonic liver. This system makes possible the studyof differentiation of liver cells out of their correlative connections within the organism.

Monolayer cultures of hepatomas described by Irlin et al.(2, 22) present special interest. In such cultures there was apeculiar incidence of «(-globulinproduction. The synthesis of 3serum proteins—albumin, transferrin, and «(-globulin—wasobserved in the primary cultures and in their first passages.After obtaining stable transplantable cultures, «(-globulinsynthesis ceased, while the synthesis of the remaining 2 proteinscontinued. The cultures did not change during a 3-year periodof work with them.

at-Globulin appearance was again observed after retrans-plantation of the cultures into mice and tumor growth. Thesame is referred to the clones of cells which were isolated fromthese cultures. This phenomenon has not yet been interpreted.The system is of great interest as it "induces" a (-globulin in

tumor cells under controlled conditions.

af-GLOBULIN IN CANCER PATIENTS

As mentioned previously, clinical investigations were begunby Tatarinov. Then they were principally confirmed and extended by two groups—in Moscow, and in Villejuif and Dakar,as well as in further work of Tatarinov's group in Astrakhan.

All the 3 groups examined patients suspected to have livercancer. Data of «(-globulin detection in their sera were compared with the autopsy results. Sera of patients with cancersof non-liver localization, of those with metastatic liver cancer,and of those with liver diseases of noncancerous origin wereused as control material. A summary of Tatarinov's published

data is presented in Table 1. It is seen from these data thata (-globulin was found only in cases of hepatocellular tumors,being absent in cholangiomas and metastatic liver tumors.Tatarinov could not find or(-globulin in patients with liverdiseases of noncancer nature, and he did not report a case ofhepatocellular tumor unaccompanied by or(-globulin.

Table 2 presents data from the Moscow group. It is obviousthat they confirm generally, but not fully, the above conclusions. Most positive results were obtained with hepatocellulartumors, while pure cholangiomas were «(-globulin-negative.(It must be noted that, in some cases, the level of arglobulinin patient sera reached 1/1000 titer. This level is almost thesame as in sera of the early fetus, and never has been obtained in animal hepatomas.) However, 5 out of 24 histo-

Table 1

DiagnosisNumberof cases

orf-GIobulin-positive

cases

Hepatocellular carcinomaCholangiocellular carcinomaMétastasesin liver of

non-liver tumorsNormal individuals

64

21200

60

00

«(-Globulinin cancer patients, according to Tatarinov's group

(33,34,36,37).

Table 2

DiagnosisPrimary

livercancerHepatocellularcarcinomaCholangiocellularcarcinomaMixed

type of primarylivercarcinomaAdenocarcinomaUnclassifiedBenign

liver tumor(adenoma)MetastaticlivertumorDifferent

tumors of non-liveroriginTesticulartumorsLiver

diseases other thantumors(cirrhosis,infectious hepatitis,etc.)«(-Globulin

in patients, according toNumberof

cases372444233701005668the

MoscowThistable includes additional data obtained bytheuntilNovember 1967.fff-Globulin-positivecases22190210000130group

(3,10).Moscowgroup

Table 3

HistopathologicdiagnosisTeratoblastoma

with the featuresofembryonalcarcinomaTeratoblastoma

with the featuresofseminomaandchorionepitheliomaTeratoblastomaSeminomaChorionepitheliomaTumor

of the stroma cellsNumber

oteases18861563af-Globulin-positive

cases814000

af-Globulin in patients with testicular tumors, according to theMoscow group (3, 10). This table includes some additional dataobtained in cooperation with Drs. Astrakhan and Monul.

logically confirmed hepatomas were also negative. Mixed hepa-tocholangiomas were positive in half of the cases. Out of fourpatients with mixed hepatocholangiocellular tumors, twoshowed the presence of «(-globulin. Tables 2 and 3 show that«(-globulin is revealed not only in liver tumors but also intesticle tumors, particularly in teratoblastomas with elementsof embryonic cancer. This important fact should be consideredin the diagnostic evaluation of an «(-globulin test. Moreover,it may be of interest for development of an immunologie testfor differential diagnosis of testicular tumors and for controlof embryocytoma growth in the course of chemotherapy. This

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Table 4

Diagnosis

of-GIobulin-Number positiveof cases cases Comments

Primary liver tumor

Noncancerous liverdiseases (cirrhoses,hepatitis, liverabscesses)

Tumors of non-liverorigin

Healthy donors

47

34

284

60

2993

37 Confirmed byautopsy, biopsy, orscintography

27 Only clinical diagnosis

af-Globulin in cancer patients, according to the Ville juif-Dakargroup (38).

fact also needs further investigation and adequate interpretation.

It is of considerable importance that cirrhoses and cholecis-tites, accompanied by liver regeneration, were negative in thea f-globulin test.

Table 4 represents the recent data of Grabar's group [Uriel

et al. (38)]. Among 81 patients with primary liver cancer, 64were af-globulin-positive. A large group of liver diseases otherthan cancer was investigated, always giving negative results.

Serologie diagnosis appeared to be more exact than clinical.As distinct from the latter, it did not give hyperdiagnostics.At the same time, the a rglobulin test may be negative in thepresence of hepatocellular tumor, at least in 20% of cases.

At present, the International Agency of Cancer in Lyon isorganizing an international experiment with the participationof European and African laboratories to evaluate the clinicalsignificance of «rglobulin test.

In our opinion, the following three questions are the mostimportant with respect to the clinical application of the test:

(a) Arearglobulin-negative hepatomas qualitative nonpro-ducers, or do they differ from positive one only in the level ofa(-globulin? (6) Is there any correlation between «,-globulin

production and a certain type of hepatocellular tumor? (c) Isit possible to reveal hepatoma by means of the arglobulin testbefore clinical symptoms will take place? Work is in progressin these directions.

DISCUSSION OF THE HYPOTHESES OF THENATURE OF THE PHENOMENON

The mechanism of the regulation of af-globulin productionin ontogenesis and the causes of its synthesis in hepatomasare completely obscure.

According to the first hypothesis, a,-globulin synthesis takesplace only in proliferating liver cells, being repressed in restingcells (6, 7). In most simple form it suggests that arglobulinis synthesized only in the S or G2 periods of cell cycle. Thishypothesis explained the changes in the production of «rglobulin observed during ontogenesis, the synthesis of «,-

globulin in regenerating mouse liver, and the correlation between the growth rate of hepatomas with the concentration ofcirculating arglobulin. However, further investigations showedthat this hypothesis could be rejected.

First of all, liver regeneration in adult rats has never beenaccompanied by arglobulin synthesis.

Second, stable, proliferating, transplantable hepatoma cultures, synthesizing a£-globulin, reversibly lose this ability inspite of active cell divisions. Finally, detailed study of therate of «(-globulinsynthesis in the early postnatal developmentin rats showed that it ceased long before the end of the livercell proliferation (5). The liver growth in the early postnatalperiod has a peculiar curve (26, 28). During the first weekthe liver did not increase in weight and did not proliferate.Then, during the second and the third weeks, there is maximalproliferation and weight increase, after which the liver continues to grow but with a gradually lowering rate. The levelof af-globulin synthesis (according to gIycine-14C incorporation) is high during the first week, it increases further in thesecond, and drops sharply at the end of the third week. On the25th day after birth, the synthesis practically stops in spite ofthe continuing liver growth, high mitotic index, and weightincrease.

Thus, in all the three cases mentioned, proliferation of liveror hepatoma cells was not accompanied by «(-globulin synthesis, and, therefore, the transition of cells from a resting toproliferative state could not be the only factor that induces«f-globulinsynthesis. The experiments mentioned above on thedynamics of «,-synthesis in early postnatal life have led us tothe consideration that «rglobulin production characterizes anearly stage of liver cell differentiation which may be calledstage of hepatoblast, as distinct from the definitive liver cell,hepatocyte (17).

In the simplest form one can imagine that differentiation ofa liver cell is a function of a number of mitoses it underwent.As the number of mitoses increases, the synthesis of «,-globulindecreases, while the rate of synthesis of albumin, transferrin,and other proteins characteristic of an adult animal plasmaincrease. Based on this assumption, different hepatomas couldcorrespond to different stages of liver cell maturation. Somefacts are clearly elucidated by this hypothesis. It explains thepositive correlation of «(-globulin synthesis with the growthrate of different hepatomas. Less-differentiated hepatomas("hepatoblastomas") should evidently possess higher growth

rate and higher synthetic activity of «(-globulin than more-differentiated "hepatocytomas."

The hypothesis does not exclude the possibility of liver tissueproliferation, which is not accompanied by «(-globulinsynthesis(liver growth in young rats and liver regeneration in adultrats).

The hypothesis considers the loss of «(-globulin synthesisin hepatoma cultures in vitro as their peculiar differentiation,transition to the stage of hepatocyte and reproduction in thisstate (22). However, to explain some other facts, it is necessary to make an additional and not very probable assumption that hepatocytes are able to reverse back to the hepatoblaststate. This is necessary for the explanation of the inductionof «f-globulinsynthesis in adult animals by hepatectomy and

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exposure to CC14 in mice and for the explanation of the resumption of at-globulin synthesis after transplantation in vitroof «(-globulin-negative, "differentiated" hepatoma cultures.

The same explanation apparently may be given to the transition of at-globulin weakly positive hepatomas to arglobulinsharply positive, which are accompanied by the appearance ofa typical and definite pattern of liver-specific antigens and bythe growth-rate increase-(see above).

A third hypothesis which explains the known facts withoutthe assumption that mature hepatocyte is able to develop backto hepatoblast could be proposed. We may suppose that thecells, synthesizing «(-globulin (hepatoblasts), and those producing albumin, transferrin, and some other proteins of "adult"

plasma (hepatocytes), are the terminal elements of two different branches of differentiation arising from a common progenitor liver stem cells. In liver the proportion of hepatocytesto hepatoblasts and, consequently, its capacity to produce«(-globulin, depend upon the stage of development of theanimal in the course of which hormonal factors or other in-ducers can operate and stimulate one or other branches ofdifferentiation. From this point of view, hepatoma consists ofstem cells able to differentiate either toward the hepatoblast orthe hepatocyte in a proportion which depends upon thetype of tumor (18) as well as external conditions. It may bereversibly changed by such factors as conditions of cultivationin vitro, hormonal treatment, and so on. Hepatomas with highlevel of arglobulin must approach hepatoblast in their biologicand antigenic properties, while «(-globulin-negative tumors arecloser to the hepatocyte type.

This hypothesis opens new possibilities of experimentalwork. It suggests that arglobulin and albumin synthesis takesplace in different cells which develop under the control of external factors (most probably hormonal). The hypothesis alsosuggests the presence of both cell types in hepatomas and thepossibility of changing their proportions by experimentalmeans. It requires a limited variability in the antigenic structure of hepatomas with their approximation either to one orthe other of the two extreme variants. Furthermore, it posesthe question whether the lifetime of two cell types is markedlydifferent, e.g., very short for hepatoblasts and much longer forhepatocytes.

Some other hypotheses were suggested for the explanationof af-globulin synthesis. Tatarinov contends that 2 geneticallydifferent cell populations exist in the liver, one «(-globulinsynthesizing and the other nonsynthesizing. Substitution ofcells which synthesize «(-globulin for non-arglobulin-pro-ducing cells takes place in ontogenesis. Hepatomas appear from«(-globulin-positive cells. In our opinion, this hypothesis doesnot explain such facts as «(-globulinsynthesis in liver regeneration, reversible loss of «(-globulin in vitro, and existence ofarglobulin-negative tumors.

The hypotheses mentioned above are based on the assumption that af-globulin synthesis is related to histogenèsesin theliver by analogy with the processes occurring in lymphoidtissue during immunogenesis. However, it cannot be entirelyexcluded that «(-globulin synthesis and its repression are different functional states of the same liver cells which are conditioned by external factors, such as inducers or inhibitors.

From this point of view, the synthesis of «(-globulin in hepatomas could be the result of disruption of a hypotheticalrepresser mechanism and is a random event (20). This supposition easily explains the presence of arglobulin-positiveand -negative hepatomas but does not explain the correlationbetween hepatoma growth rate and «,-globulin production, aswell as the dynamics of «(-globulin synthesis by hepatoma intissue culture.

At present it is impossible to give preference to any of theabove assumptions. However, their comparison allows the formulation of a number of questions, the solution of which willthrow more light on the nature of the phenomenon. It isnecessary to find out whether «(-globulinsynthesis takes placein all embryonic or regenerating liver cells or in some limitedareas of liver tissue; whether the decrease of «(-globulin synthesis in ontogenesis is due to the decrease of its productionby all the cells or to the decrease in the number of «,-globulin-positive cells in the liver; and whether the synthesis of «,-globulin, albumin, transferrin, and other serum proteins takesplace in the same or in different liver cells. The answer tothese questions could apparently be obtained by the Coons'

method; however, up to now, the attempts to use this methodfor the investigation of the problem have been unsuccessful.

In addition, it is important to investigate whether it is possible to induce «(-globulinsynthesis or to observe its decreaseunder the suppression of cell proliferation, i.e., with the exclusion of histogenèses.From this point of view, it is of interestto find out how the «(-globulinsynthesis would proceed in hypo-physectomized young animals or in those with irradiated liver;it would also be helpful to ascertain whether o,-globulin synthesis could be induced in adult animals in which liver regeneration is suppressed. In case of success, such experimentswould help to choose between the "histogenetic" and "physiologic state" hypotheses.

ACKNOWLEDGMENTS

The valuable help of Drs. S. D. Perova, N. I. Perevodchikova,and I. V. Assecritova in preparation of the paper is greatly appreciated. We would also like to thank Dr. B. Henrices for his helpin the correction of the English.

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1350 CANCER RESEARCH VOL. 28

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1968;28:1344-1350. Cancer Res   G. I. Abelev  Review of Experimental and Clinical Data

-Globulin by Hepatomas:αProduction of Embryonal Serum

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