Viral Carcinogenesis: Phenomena of Special Significance in ... · viral etiology in human cancers,...

[CANCER RESEARCH 28, 1849-1858, September 1968]

Viral Carcinogenesis: Phenomena of Special Significance in theSearch for a Viral Etiology in Human Cancers1

Albert B. SabinThe Children's Hospital Research Foundation, University oj Cincinnati College oj Medicine, Cincinnati, Ohio

IntroductionHabel's (12) review of the biology of viral carcinogenesis

provides an excellent critical evaluation of our current knowledge of some aspects of this problem. My purpose in this communication is to synthesize that part of our knowledge of viralcarcinogenesis that has a special bearing on the search for aviral etiology in human cancers, and to indicate what has beenlearned thus far and what is currently being done to determinewhether any human malignancy may be caused by a virus.

Despite many illuminating studies on the mechanism andphenomena of viral carcinogenesis in recent years, it is still notpossible to say that any human malignancy is or is not causedby a virus. The establishment of the viral etiology of certainnaturally occurring malignancies of lower animals, such as certain leukemias and sarcomas of chickens and mice and of mammary carcinoma of mice, was achieved by the old, classicalvirologie technics of serial transmission with cell-free extractsin appropriate hosts of the same species. Since this approach,for a variety of reasons, cannot be used in the study of humanmalignancy, it is of special interest to examine the data, whichhave provided in vitro methods for identifying the causativeviruses of the naturally occurring malignancies of lower animalsand the phenomena associated with the malignancies that areproduced experimentally by certain viruses which permit identification of the causative virus in the absence of infectious virusin the tumor.

Categories of Oncogenic Viruses in Relation to Their BiologicBehavior

I find it helpful to think of the oncogenic viruses in threecategories: (a) the DNA viruses responsible for naturallyoccurring benign tumors, e.g., papillomas of man and lower animals, including the Shope papilloma of rabbits, (o) the DNAviruses which produce malignant transformation in vivo andin vitro and thus far have not been proved to be the cause ofany naturally occurring malignancy in either lower animals orman, and (c) the RNA viruses responsible for the naturallyoccurring leukemia-sarcoma complexes of chickens and miceand of the hormone-dependent mammary carcinoma of mice.

Behavior of DNA Viruses Responsible for Benign Tumors

In the naturally occurring benign tumors, the causative DNA

1 Personal studies mentioned in this paper were aided by research Grant CA-04557 from the National Cancer Institute,USPHS.

virus reproduces itself to the infectious state, and the conditionis transmitted from host to host in the same way as other infectious viral diseases. When fully infectious virus is not formed,as in the case when the Shope papilloma is transmitted experimentally to certain domestic rabbits, there is nevertheless synthesis of infectious DNA (23) and certain structural antigensof the virus (35).

It is noteworthy that the DNA viruses responsible for thebenign tumors have not been found to produce cytopathiceffects in any cell cultures tested thus far. Recent studies withShope papilloma virus which is not cytopathic for rabbit kidney cells (13) indicated that in monolayer of fetal rabbit skinthe virus adsorbed to the cells, but there was no evidence ofviral replication or morphologic transformation of the cellsin vitro. Yet when the cells were transplanted to the hamstercheek pouch, typical papillomas developed (27). In this connection it is of special interest that fluorescent antibody studies(35) and, subsequently, also electron microscopic studies (56)indicated that viral antigens as well as fully formed virus particles do not occur in the basal proliferating cells, but ratherin the keratohyaline and keratinized cells of the Shope papilloma of wild cottontail rabbits. There is at this time no evidence for the presence of viral genetic material in the â€¢pro

liferating cells of the Shope papilloma and the alternative hypothesis that the virus acts indirectly to bring about proliferation of the basal cells of the epidermis cannot now be disproved.

The emergence of carcinomas in the experimentally producedpapillomas of domestic rabbits (43), as well as in the naturallyoccurring papillomas of wild cottontail rabbits (57), stimulatedmany studies on the question of the role of the Shope papilloma virus in the production of the carcinomas. In extensivestudies on many Shope papilloma-associated epidermoid carcinomas in wild cottontail rabbits and on metastatic tumorsderived from them, Syverton (57) found no evidence of persistence of virus or of immunizing antigens of the virus andconcluded that "it seems unlikely that papilloma virus functions as the actuating carcinogenic agent." One transplantable

carcinoma (Vx2) yielded evidence of persistent productionof viral antigen in the first 22 transplant passages (25) but notin the fifty-first or later passages (32, 44). On the other hand,another transplantable carcinoma (Vx7) still produced viralantigen after more than 80 transplant passages, as measuredby production of neutralizing antibodies for the virus and resistance to challenge with the Shope papilloma virus, and alsoyielded nucleic acid capable of producing papillomas (24).

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Albert B. Sabin

There is, however, a possibility that the Vx7 tumor does notrepresent a homogeneous cell population (24, 32), a portionof the tumor perhaps being made up of benign papilloma cellsâ€”especially since the fluorescent antibody studies detectedantigen in only a small proportion of the tumor cells (32).

The question remains, however, whether papilloma-derivedcarcinomas contain specific virus-induced, nonvirion antigenssuch as have now been demonstrated in the malignant tumorsexperimentally produced by the DNA viruses of the secondcategory (i.e., SV40, polyoma, and adenoviruses). Friedewaldand Kidd (7, 8) reported that rabbits bearing the virus-freetransplanted Vx2 carcinomas developed complement-fixing(CF) antibodies that reacted with extracts of the transplantedcarcinomas but not with extracts of Shope papillomas thatwere rich in viral antigens. Subsequently, however, Kidd (personal communication) found that such rabbit sera also failedto react with extracts of many primary papilloma-derived carcinomas and concluded that the earlier observations were probably the result of transplantations in rabbits that were not inbred. This conclusion was further reenforced by later studiesof Friedewald and Kidd (8), who showed that, after transplantation of the Vx2 carcinoma, rabbits developed antibodiesnot only for the transplanted carcinoma but, depending on theindividual rabbit used, also for normal spleen, or liver, or kidney, or for the Brown-Pearce carcinoma, or for all of these(see their Table III). These investigators (8) also found thatthe sera from ten domestic rabbits carrying virus papillomasand squamous cell cancers deriving from them for manymonths, as well as the sera from three wild cottontails carryingvirus papillomas and derivative cancers, all failed to react withextracts of the multiply transplanted Vx2 carcinoma, extractsof normal tissues, or extracts of the Brown-Pearce carcinoma(see their Table VII). It is thus evident that the type of specific tumor antigen that has been found in the experimentalmalignancies produced by various DNA viruses could not bedemonstrated in the Shope papilloma virus-derived carcinoma,and there is therefore no evidence at this time that genetic material of the papilloma virus is present in the derivative cancers.

At the present time, therefore, there are no data from themany interesting studies on the benign DNA tumor viruseswhich provide approaches to the study of the human cancerproblem.

Behavior and Properties of DNA Viruses Involved in Experi

mental Carcinogenesis

The majority of DNA viruses have thus far not been foundto be oncogenic. In addition to the polyoma virus of mice andSV40 of Asiatic monkeys, only certain members of the adenoviruses of man and lower animals have been found to possessthe capacity for experimental malignant transformation. Thecomparatively small number of DNA viruses that have beenshown experimentally to produce malignant transformation,unlike the benign DNA tumor viruses, all have the capacityto produce cytopathic, cytocidal infections in some cells. It isevident, however, that in the cells which are transformed tomalignancy there is ordinarily replication neither of infectiousvirus nor of infectious DNA. However, there is very good evidence now that either all of the viral genome in a partly re

pressed state or varying portions of the genome remain in themalignant cells in a form which permits its replication duringcell division and its function for the production of a variety ofvirus-specific "fingerprints."

The evidence for the continued transmission of the completegenome in a partly repressed state was supplied by the demonstration that, with certain SV40 hamster tumor lines, the progeny of single or very small numbers of tumor cells possess thecapacity of being "derepressed" with a very low spontaneous

frequency (as in some lysogenic bacteria) and with a greaterfrequency under the influence of certain factors (53, 54). Themost efficient method of "derepression" leading to the forma

tion of small amounts of fully infectious virus was found to beby association of live tumor cells (free of infectious virus) withvirus-susceptible, normal African monkey kidney cells underspecial conditions of cultivation (46, 47, 54).

The evidence for the continuing transmission of functionalincomplete portions of viral genome in all DNA virus-inducedmalignant tumors, without reference to their capacity for de-repression, is based on the following three findings: (a) Inthe case of the adenovirus type 12 tumors (and thus far onlywith this one type of adenovirus, enough type-specific structural viral antigen is synthesized in tumors free of infectiousvirus to produce neutralizing and CF antibodies in many ofthe tumor-bearing animals, but not enough to be directly demonstrable in the tumor cells (22). (b) There is a synthesis inall other DNA virus-free tumor cells of virus-specific antigensthat are not part of the structure of the virus particle but arealso found in normal cells during the early stages of cytocidalinfection (18, 55) and, in the case of SV40, all of the specificCF antibody in the sera of tumor-bearing hamsters for the antigens in the tumor cells can be absorbed by the early antigensin the cytocidally infected normal cells (55). (c) The virus-freetumors or in vitro transformed cells contain messenger RNAthat hybridizes specifically with the DNA of the inducing virus(3, 9).

All of these phenomena provide methods by which one mayattempt to answer the question whether any of the knownDNA viruses are involved in certain human cancers.

Habel (12) has critically evaluated the interesting work (11)that seemed to suggest that the guanine + cytosine (G + C)content of a DNA virus might provide a biochemical indication of its malignant potential and concluded that the overallbase composition of a nucleic acid was too crude a characterization. Initially it appeared that there was a good correlationbetween the 47 to 49% G + C content of the viruses possessing the highest potential for malignant transformation (i.e.,polyoma, SV40, human adenoviruses types 12, 18, and 31) andthe 43% G + C content of mammalian cell DNA; the humanadenovirus types 3, 7, 14, 16, and 21 with a lesser capacity formalignant transformation were found to have a G + C contentof 50 to 53%, while the nononcogenic human adenovirus types,which included until recently type 2, had the highest G + Ccontent of 56-60%. Since DNA viruses causing the benign papillomas have a G + C content in the range of 41%, it wasalready evident that this criterion could not serve as a guideto malignancy. When the human type 2 adenovirus (G + Ccontent of 56%) was recently found to be capable of trans-

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Viral Etiology in Human Cancers

forming rat embryo cells when media of very low calcium concentration were used (6), Green (personal communication)found that the G + C content of different portions of the DNAmolecule of type 2 adenovirus varied, some being in the rangeof mammalian cell DNA and some much higher. Perhaps thesame may turn out to be the case for the highly oncogenicsimian adenovirus (SV7), for which Habel (12) found a G + Ccontent of 58%. It is obvious from this that further studies onbiochemical "fingerprints" that might differentiate the DNA

viruses with malignant potential from those that produce benign tumors and those that are not experimentally oncogenicare highly desirable.

An especially interesting observation that cell lines transformed by the polyoma or SV40 viruses multiplied at 41Â°C,while those transformed by chemical carcinogens or X-ray andnormal embryonic cells did not (36), raises the possibility ofdifferentiation between malignancies caused by DNA virusesand by chemicals or radiation. Work carried out in Sachs' labo

ratory indicated that RNA virus-induced malignant cell linesalso failed to multiply at 41Â°C(personal communication). The

validity of these observations needs to be confirmed and extended because, if it can be established as a valid phenomenon,it could prove most helpful in studies on the role of viruses inhuman cancer.

In response to the question whether any naturally occurringmalignant tumor of lower animals has been proved to be causedby a DNA virus, the carcinoma in the leopard frog reportedby LÃ¼cke(29) is often mentioned. Evidence has been presentedthat the intranuclear inclusion producing virus found in manyof the frog tumors is a DNA virus (62), but many recentstudies on a variety of frog viruses strongly suggest that theintranuclear inclusion virus is probably a "passenger" in, rather

than the cause of, the carcinoma (37, 38).

Behavior and Properties of RNA Viruses Responsible forNaturally Occurring Malignancies of Lower Animals

First of all, it is important to emphasize that the naturallyoccurring malignancies of proved viral etiology of lower animalsare caused by RNA viruses, and that, unlike the DNA virusesresponsible for the experimental malignancies, the RNA oncogenic viruses are replicated to the fully infectious state in thenaturally occurring tumors they produce. Also, unlike the DNAviruses just mentioned, none of the RNA oncogenic viruseshave as yet been found to produce a cytopathic effect in tissue cultures. Moreover, none of the many cytopathic RNAviruses have thus far been found to produce experimental malignant transformation in animals or tissue cultures. Recentstudies suggest that the RNA's of all RNA tumor viruses are

single stranded and have molecular weight values around IO7,which is significantly higher than that of the single-strandedRNA of the cytopathic viruses (42). Another point of interestis that the synthesis of oncogenic viral RNA (specifically thatof Rous sarcoma virus) is prevented by inhibitors of DNAsynthesis which have no effect on the synthesis of nononco-genic viral DNA, suggesting an RNA to DNA to RNA type ofreplication for RNA oncogenic viruses (58).

In the search for phenomena that might be applicable to asearch for RNA oncogenic viruses in human beings, it is helpful

to consider the RNA virus etiologically associated with malignancy of lower animals in two categories: (a) the virus ofmammary carcinoma of mice, which is hormone dependent forits oncogenic expression and which thus far cannot be biologically identified in vitro (28, 48), and (o) the sarcoma-leukemia complexes of chickens and mice, for which a variety ofmethods are now available for identification in vitro (19, 20).

Sarcoma-Leukemia Complex of Chickens

The viruses of this group belong to the family of myxo-viruses and measure 80-120 m/t. The internal protein capsidcontains a CF antigen common to all members of this group.The outer lipid-containing envelope has antigens which determine the type-specificity of the virus, and at least three an-tigenic subgroups are known. The outer envelope of some sarcoma viruses contains antigens of widely prevalent leukemiaviruses. The viruses of this group can be transmitted not onlyto very young but also to mature members of the avianspecies. Natural transmission occurs through the egg ("vertical") and also by contact. In "vertical" transmission the in

cidence of the disease is high, there is permanent virus shedding,and no antibody formation. In the infection transmitted bycontact, there is a transitory viremia in young and adults, alower incidence of disease, and antibody develops after bothclinically manifest or subclinical infection.

The viruses of this group can be identified in vitro by tissue culture and serologie technics, but it is important to beaware of the intricate conditions that are required for success.While the original demonstration of Manaker and GroupÃ©(31)in 1956 that Rous sarcoma virus can be recognized in vitro bythe formation of discrete foci of altered cells in chicken embryotissue cultures opened the door to a tremendous increase inour knowledge of this group of viruses, it is now realized thatthis identification can be accomplished only in cultures fromchicken embryos that are free of certain naturally occurringleukemia viruses and are of a genetic constitution that willpermit the viruses to replicate (45, 59, 60). The sarcomaviruses replicate to the fully infectious state in the transformed cells, and so-called "nonproducer" cells, in which no

virus was demonstrable by the usual methods, were recentlyshown by Vogt (59) to produce virus with an envelope ofsomewhat different composition that can attach only to chickencells of a special genetic constitution or to certain other aviancells.

Leukemia viruses multiply in leukemia-free chick embryofibroblasts of appropriate genetic constitution. With one exception, the avian myeloblastosis virus, they produce no recognizable morphologic change in cells, but replication can berecognized by (19): (a) interference with focal tumor formation in chick embryo fibroblast cultures by a sarcoma virus ofthe appropriate group, i.e., one possessing envelope antigensand receptors similar to those of the leukemia virus (this isknown as the RIF test) ; (fe) a CF test for the group-specificantigen, using appropriate sera from hamsters carrying trans-plantable sarcomas induced by avian sarcoma virus (this isknown as the COFAL test) ; and (c) the fluorescent antibody-test for type-specific antigens using appropriate chicken sera.

Avian myeloblastosis virus, the only avian leukemia virus

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Albert B. Sabin

producing morphologically recognizable transformation in cultured cells, requires special target cells derived from chickembryo tissues with active hematopoiesis at the time of ex-plantationâ€”yolk sac, bone marrow, or bursa of FabricÃ¡isâ€”for expression of its transforming capacity (2). These specialmesenchymal target cells are transformed chiefly into myelo-blasts, and sometimes also into erythroblasts and osteoblasts.The transformed cells release new virus that reproduce avianmyeloblastosis in vivo and transformation in vitro. In ordinarychick embryo fibroblasts, avian myeloblastosis virus multiplieswithout transformation or other recognizable morphologicchange, but with production of identifiable group-specific andtype-specific antigens.

Avian Sarcoma Viruses in Mammals. The pertinent factscan briefly be summarized as follows:

1. Some strains (presumably those with appropriate receptors in their outer envelope for some mammalian cells) producetumors on inoculation into newborn rats, hamsters, rabbits,mice, guinea pigs, and monkeys.

2. These tumors are transplantable in inbred animals andcontain structural viral antigens and recognizable viral particles, but no infectious virus has thus far been recovered fromcell-free extracts or frozen and thawed cells by the usual invitro technics. However, the possibility that the compositionof the outer envelope of the virus particles formed in themammalian cells has been altered with a consequent need foravian cells of special genetic constitution for their detectionâ€”comparable to the phenomenon discovered by Vogt (59) for"nonproducer" avian cellsâ€”has to my knowledge not yet been

explored.3. However, infectious avian sarcoma virus can be obtained

when live mammalian tumor cells are inoculated in chickensor cocultivated with chick embryo fibroblasts, in the absenceof leukemia virus with some strains of sarcoma virus, and onlywith the help of leukemia virus with other strains.

Sarcoma-Leukemia Complex of Mice

Following the recent discovery of a sarcoma virus of mice(15, 34), it became apparent that many, not all, of the phenomena observed in the sarcoma-leukemia complex of chickenshave their counterparts in the sarcoma-leukemia complex ofmice. The pertinent facts can briefly be summarized as follows:

1. The viruses also belong to the family of myxoviruses andmeasure about 100 m^. The internal protein capsid containsan antigen that is common to all viruses of the sarcoma-leukemia complex of mice. The outer lipid-containing envelopehas antigens which determine the type specificity of the virus,and at least two major subgroups are known.

2. The outer envelope of the sarcoma virus is supplied bya coexisting leukemia virus.

3. The naturally occurring disease is transmitted verticallyonly, i.e., in utero,2 and naturally infected mice have no anti

body for the virus.4. Experimental transmission is optimum in newborn mice

2 Transmission by contact may perhaps also be obtained when

mice of the right genetic constitution are used (R. Huebner, personal communication).

of suitable genetic constitution. Most strains are also pathogenic for rats and the Moloney strain (both the leukemia andthe associated sarcoma) is also pathogenic for hamsters.

In Vitro Identification of Murine Leukemia Viruses. Theimportant factors for in vitro identification are: (a) the availability of leukemia-free mice of appropriate genetic constitution and (o) artificially produced antisera in inbred rats thatcontain type-specific antibodies for the outer envelope andgroup-specific antibodies for the internal protein capsid of theviruses. The pertinent facts can briefly be summarized asfollows:

1. Leukemia viruses multiply in leukemia-free mouse embryofibroblasts (MEF) of appropriate genetic constitution withoutrecognizable morphologic change.

2. The inapparent multiplication, which can be proved byinoculation into suitable newborn mice, can be measured invitro by the appearance in the MEF cultures of CF antigensthat are demonstrable by sera from rats, which: (a) carrytransplantable lymphosarcomas induced by the Gross or Rauscher leukemia viruses or (6) are immunized with various leukemia viruses grown in MEF cultures.

The above rat sera, which have little or no group-specificantibody, were found to be useful for identifying various laboratory-passaged strains, but were relatively insensitive indetecting naturally occurring leukemia viruses.

3. The in vitro isolation and identification of naturally occurring leukemia viruses from a variety of strains of mice withboth high and very low incidence of leukemia was made possible by: (a) the use of the NIH strain Swiss mouse embryocultures, which proved to be much more sensitive for the "field"

strains than the BALB/c strain embryos ordinarily used forthe available laboratory-passaged viruses, and (o) the use ofFisher inbred rats carrying transplanted mouse sarcomas,which yielded a high proportion of potent sera for the groupantigen and almost never reacted with control antigens.

4. Since the discovery that mouse sarcoma virus (MSV)can produce tumor foci on appropriate MEF cultures, itbecame possible to identify leukemia viruses possessing outerenvelope antigens similar to those on naturally occurring andartificially produced MSV by their interference with the invitro production of tumor foci (20).

Unique Phenomenon in Hamster Tumors Produced byMouse Sarcoma Virus. MSV produces tumors in rats andhamsters, but the hamster tumors are unique in that theyyield no infectious virus, no CF antigens, and no formedvirus particles. In this respect they differ not only from thehamster tumors produced by the avian sarcoma viruses, whichcontain structural viral antigens in the absence of "demonstrable" infectious virus, but also from those hamster tumors

experimentally produced by DNA viruses, which in the absenceof infectious virus as well as of structural viral antigens, nevertheless, contain specific tumor antigens coded by a portion ofthe viral DNA. And yet, there is good evidence that the "virus-free'' and "antigen-free" MSV hamster tumors contain theMSV genome in a "repressed" state because it can be "dere-pressed" or "rescued" under special conditions. The special

conditions involve bringing together live MSV tumor cellswith a mouse leukemia virus and MEF in which the leukemia





virus can multiply. Under these conditions of cultivation, infectious MSV emerges which forms tumor foci on the MEFand possesses the type-specific antigenic characteristics of theleukemia virus that was used to "rescue" it. Infectious MSV

does not emerge when: (a) the MSV hamster tumor cells arekilled by freezing and thawing or (6) no leukemia virus isadded to the culture of leukemia-free MEF culture. The "rescue" phenomenon can also be demonstrated in vivo by inoc

ulating MSV hamster tumor cells with leukemia virus intonewborn mice of leukemia-free strains or without leukemiavirus into newborn mice of high leukemia strains (20).

The existence of RNA genome in a completely "repressed"

state that is continuously transmitted during each cell divisionhas to my knowledge not previously been demonstrated, andthe virus-free and antigen-free MSV-induccd hamster tumorprovides a unique model system in which the mechanism ofgenetic transmission of the RNA viral genome can be studied.Since studies on the replication of avian Rous sarcoma virusindicated a possible RNA to DNA to RNA cycle (58), onehypothesis that might be considered is that the "repressed"

RNA viral genome may be attached to the DNA of the hamster tumor cell and may reproduce itself along with the DNAduring cell division.

The in vitro procedure for "rescue" of the "repressed" MSV

genome from the virus-free and antigen-free hamster tumorcells has obvious implications for the search of a possible sarcoma-leukemia complex of viruses in human beings.

Search For Phenomena of Viral Oncogenesis in Human Malignancy

At this time there is no evidence that any human malignancyis caused by a virus, but not enough work has been done thusfar to permit one to say that viruses are not implicated incertain human malignancies.

Problems in Attempting to Apply the Technics that Permit in Vitro Identification of the Avian and Murine Leukemia-Sarcoma Viruses to Human Leukemias and Sarcomas.A critical examination of the newer knowledge of the leukemia-sarcoma viruses of chickens and mice shows that the availability of inbred, leukemia-free stocks of chickens and micewas crucial in obtaining the embryonic tissue cultures in whichthe sarcoma viruses produced tumor foci. Interference withthe production of such tumor foci then became a tool for thedetection and identification of leukemia viruses that multipliedin such cultures without recognizable morphologic change. Ifonly random-bred chickens and mice of unknown leukemicstatus were available for the study of the avian and murineleukemia viruses, whose existence was first established byclassical animal transmission tests, identification by in vitroprocedures might still have been at the same stage as thelimited studies on human sarcoma and leukemia material arenow. The excellent serologie methods developed by Huebnerand his associates (14, 19, 20) for the identification of the avianand murine leukemia-sarcoma viruses depended on the selection of specially reactive sera from inbred hamsters carryingtransplantable tumors, induced by avian sarcoma viruses, forthe avian virus group, and from inbred rats carrying trans

plantable tumors, induced especially by the mouse sarcomavirus, for the murine virus group. The serologie methods, ofcourse, are dependent on the availability of known leukemia-free cultures of tissues of appropriate genetic constitution inwhich the leukemia viruses can multiply. The italicized wordsin the preceding sentences point to the key factors that wouldhave to be duplicated for the study of human leukemia andsarcoma along the lines thus far elucidated by the studies onthe animal models. Let us examine what has already been doneand what might be done in the future with material from human sarcoma and leukemia in relation to individual phenomena established for the avian and murine sarcoma andleukemia viruses.

Phenomenon: Tumor Foci Produced in Vitro by Sarcoma Viruses. It is, of course, possible to obtain human embryonic fibroblasts, but there is no way of knowing whetherthey are either leukemia-free or of the appropriate geneticconstitution. The negative results obtained thus far in sporadictests with extracts of human sarcomas are not significant.If many human sarcomas were tested in fibroblast cell linesderived from 100 or more human beings, it is possible that byaccident one might encounter a cell line that would meet therequirements of being both leukemia-free and of the appropriategenetic constitution and that tumor foci might be obtained. Although a few serial passages would be possible in cultures fromthe original cell line preserved in the frozen state, reproducibilityof the phenomenon and the further work that would be indicated if a human sarcoma virus were accidentally discoveredwould be greatly hampered by the difficulty of finding anotherleukemia-free and genetically appropriate cell line from tissuesof randomly bred people. It seems to me that the chances ofmeeting these requirements might be greatly increased bysearching out highly inbred human population groups. Culturesderived from either aborted or miscarried fetuses or from thecircumcised foreskin of newborn infants of highly inbred humangroups would be of special interest for tests with extracts froma large variety of human sarcomas.

Phenomenon: Serologie Identification of Inapparent Multiplication in Suitable Tissue Cultures. As was pointed outpreviously, the serologie approach, along the lines of the avianand murine leukemia-sarcoma viruses, is predicated on findinga suitable tissue culture in which inapparent multiplication ofa leukemia virus might be recognized by its interference withproduction of tumor foci by a human sarcoma virus. If humanleukemia is transmitted "vertically" as in all the known murine

viral leukemias and many of the avian viral leukemias, onewould not expect to find viral antibodies in the blood of patients. Although attempts to transmit human leukemia to subhuman primates has to my knowledge thus far yielded negative results, it seems to me desirable to concentrate more onthe production of solid tumors in newborn monkeys using human lymphomas, lymphosarcomas, and a variety of humansarcomas. Since transplantability of any tumor that mightappear is of the essence, it would be desirable to select for suchwork monkeys that are as closely related as possible, as determined by the procedures that are used for finding compatibledonors for human kidney transplants. It is possible, however,that the desirable results might be achieved only in highly

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Albert B. Sabin

inbred monkeys, and attempts at inbreeding at the variousprimate centers should, therefore, be encouraged.

If, as is the case with some avian viral Icukemias, somehuman Icukemias should be transmitted by contactâ€”and thereis no good epidemiologie evidence for it at this time (33)â€”remission sera from a variety of leukemic patients might alsobe a source of antibody if and when a way is found to identifya human leukemia virus in vitro.

Phenomenon: Rescue of Completely "Repressed" Sarcoma

Virus Genome as in Hamster Sarcomas Produca by MouseSarcoma Virus. This type of test could be duplicated withhuman sarcomas if we had (a) a known source of human fibro-blasts in which a human leukemia virus would replicate (whichwe do not have), (6) a known source of human leukemia virus(which we do not have), and (c) large numbers of live humansarcoma cells (which can be obtained). At the present time,the best one can do is to work with randomly obtained humanembryos and with a potential source of human leukemia virus(plasma or bone marrow from patients in the acute phase,preferably of the first clinical attack of leukemia, or purifiedvirus-like particles containing nucleic acid cores), and negativeresults under such conditions are not significant.

Phenomenon: Avian Myeloblastosis Virus Type of inVitro Transformation of Special Hematopoietic Target Cellsinto Myeloblasts. An attempt to duplicate this type of testwith material from human myeloblastosis was made in mylaboratory (52) using cultures prepared from spleen, liver,lymph nodes, and thymus of human fetuses of six- to seven-months' gestation, but with negative results. Here also the nega

tive results are not significant because the tissue cultures mighthave been genetically inappropriate and not leukemia-free.

Phenomenon: Virus-Like Particles in Leukemic Tissues orPlasma. Although many electron microscopic studies have beencarried out on human leukemic material, critically reviewedby me recently (52), they do not provide evidence for oragainst the involvement of viruses morphologically similarto the avian and murine leukemia variety in the human disease.Although virus particles are present in relatively large numbers in the plasma, tissues, or inapparently infected tissuecultures when the laboratory-passaged strains of the avian andmurine viruses are used, only small numbers are encounteredin the naturally occurring diseases of chickens and mice. Accordingly, the rarity with which virus-like particles of comparable morphology have been found in the human diseasecannot be taken as an indication that comparable viruses arenot involved in the majority of human cases of leukemia.

The many studies on the etiologic significance of the herpes-like particles found with such great frequency in cultures ofcells derived from Burkitt lymphomas (4) will not be reviewed.Comparable particles have been encountered in cultures froma variety of other malignant and normal tissues, although ina much lower frequency. These herpes-like particles apparentlyrepresent a hitherto unrecognized type of ubiquitous viruswhich recently has been shown to be regularly associated withinfectious mononucleosis (16). If this herpes-like virus particle were a mere passenger in the Burkitt lymphomas, as wellit might be, it is odd that it is not encountered with comparable frequency in other human lymphomas. These herpes-like

particles, which are very difficult or impossible to transmit andgrow in the usual virus-free cell lines, can, nevertheless, beobtained in fairly large amounts from certain of the Burkittlymphoma established cell lines. Unless these herpes-like particles can be shown to have the capacity specifically to transform normal cells (lymphocytes or certain cells from bonemarrow, etc.) from persons who have no antibody for thesevirus-like particles (to avoid the response of sensitized cellsto antigen) into continuously growing lymphoblasts or relatedcells, their etiologic relationship to the Burkitt lymphoma willremain in doubt.

Present Status of Search for Evidence Implicating DNAViruses as a Cause of Human Cancer

The previous summary of our knowledge about the phenomena encountered in the virus-free cancers experimentallyproduced by various oncogenic DNA viruses showed that the"fingerprints" of persistent viral genome in these tumors can

be detected by both biologic and biochemical procedures.Riologic Approach: "Derepression" or Rescue of "Re

pressed" Complete Provirus by Appropriate Association of

Live Tumor Cells with Normal Virus Susceptible Cells.Many malignant tumors of children, especially Wilms' ne-

phroma and neuroblastoma, were tested in my laboratory bygrowing many millions of either primary trypsinized tumorcells or cells derived from them by subsequent cultivation invitro, together with various indicator cell lines (including human embryonic kidney), but no virus was detected (49). Therecent use of inactivated Sendai virus for the formation ofheterokaryons between tumor cells and virus susceptible cells(10, 26, 61) provides still another method for carrying outsuch studies, but there is as yet no evidence that this methodcan induce synthesis of infectious virus in most of the virus-freeDXA virus tumors that have previously yielded negative results in cocultivation tests under optimum conditions (61).Moreover, many human epitheliomas cannot be tested in thisway because not enough primary cells can be obtained bytrypsinization or other methods of cell dispersion, and sub-cultivation either yields no growth or only fibroblast-like cellsthat bear no resemblance to the original tumor cells.

Search for Specific Antigens in Human Cancers UsingPatient's Serum against His Own Tumor. Studies on the pos

sible presence of CF antibodies in the serum of patients, takenboth before excision of the tumor and about two weeks thereafter, against their own tumor cells (mostly on cancers of earlychildhood) were carried out in my laboratory with negativeresults (17, 50). Comparable tests using the fluorescent antibody technic, as well as CF, mostly on cancers of later life byRapp et al. (40) also yielded negative results. These negativeresults do not exclude a possible role of DNA viruses in thehuman tumors that were tested because of the finding in certain of the experimental models that with some DNA virustumors a large proportion of the tumor-bearing hamsters mayfail to develop CF antibody even when their tumors containlarge amounts of antigen, or that some tumor lines contain toolittle antigen to be demonstrable by direct test, yet enoughto stimulate antibody in the tumor-bearing animal (49). However, even if positive results were obtained by this approach,




Virai Etiology in Human Cancers

they would not necessarily be indicative of a viral etiologybecause we have found that a hamster tumor (Fortner fibrosarcoma No. 3B) of no known viral etiology and without anydemonstrable passenger virus or mycoplasma also contains aspecific antigen for which the tumor-bearing hamsters develop

antibody (51).Antibody in Sera of Specially Selected Cancer Patients

vs. Nonvirion Antigens of Various DN V Viruses. One of thequestions that needs to be answered is whether any of theDNA viruses of the human heritageâ€”the 31 types of adeno-viruses, herpes simplex, varicella-zoster, and cytomegalovirusesâ€”or a DNA virus-like vaccinia, so widely administered to human beings, or DNA viruses of lower animals with whichhuman beings come in contact may be etiologically related tohuman cancer. All of these viruses must be considered becauseviruses that are not oncogenic in hamsters or other newbornexperimental animals may conceivably be oncogenic in manand vice versa.

The point about using sera from specially selected cancerpatients is influenced by a number of observations that havebeen made in detailed studies on hamsters bearing differenttumors experimentally produced by different DNA viruses:(a) generally hamsters bearing large tumors for long periodsof time are more likely to have antibody; (6) with some SV40tumors about 80 percent of hamsters can die bearing largetumors about a month after transplantation without developingantibody (51); and (c) hamsters bearing certain polyoma tumors develop very little antibody initially, but after excision ofthe tumor and subsequent inoculations with it, specific antibodies often appear in much higher concentration. Accordingly,when the National Cancer Institute set up a special workinggroup (Drs. R. Huebner, E. Lennette, J. Melnick, and A.Sabin) to concern itself with the problem of the possible roleof DNA viruses in human malignancy, the first step was toinitiate a program for the collection of sera from speciallyselected cancer patients and simultaneously from persons without malignancy matched for age, sex, and residence. Patientswere to be selected on the basis of having a malignant tumorfor a long period of time with extensive metastasis. This program called for obtaining and processing in a standard manner sera from patients with the following types of cancer:(a) respiratory tractâ€”lung and bronchus (at least 40 patients),(Â£>)digestive tractâ€”colon, stomach, etc. (at least 20), (c)uterus (at least 20), (d) childhood tumorsâ€”Wilms', neuro

blastoma, etc. (at least 20), (e) breast (at least 20), (/) bladder, liver, prostate, kidney, thyroid (at least 10 of each), (g)head and neckâ€”nonlymphoid types and lymphoid types (atleast 20 of each), and (/;) melanoma. Except for the absenceof sera for the childhood tumors, most of the other sera andmatching controls had been collected by the end of 1967.

For the DNA viruses that produce virus-free transformed

cells in vitro or transplantable tumors in experimental animals, one can use specific tumor antigens of known potencyas measured by appropriate sera of tumor-bearing hamsters.For the DNA viruses of unknown oncogenic potential, it isnecessary to attempt to prepare antigens that may be presentin greatest concentration early after infection of cells in whichthe virus replicates to the fully infectious stage, or in cells in

which it may produce only an abortive infection. This approach,of course, is based on the demonstration that in the case of theknown oncogenic viruses the specific nonvirion antigens thatare present in the malignant cells are also present in maximalconcentration early after productive infection in cells that arenot transformed, as measured by appropriate sera from tumor-bearing hamsters. The antigens that are needed for such testsmust be free of viral structural components, which would reactwith sera from many persons without cancer. To be meaningfulone must also have appropriate sera with which to identify,measure the concentration, and determine the properties ofany nonvirion antigen that might be found in cells productivelyinfected by the DNA viruses of unknown oncogenic potential.Since certain monkeys infected with SV40 virus have beenshown to develop specific CF antibodies for the nonvirion antigens in productively infected cells and in SV40 hamster tumors(49), one may perhaps find some recent convalescent phasesera which, after appropriate absorption with purified virusand internal viral antigens, would be able to detect nonvirionantigens in infected cells. However, on the basis of extensive,as yet unpublished, studies in my laboratory (in associationwith Dr. Giulio Tarro) with the virus of herpes simplex, themost promising approach appears to involve the immunizationof guinea pigs with guinea pig kidney tissue culture cells,harvested at 3 hours after infection with a high multiplicity ofherpes virus and used sonicated but uncentrifuged within a veryshort time after harvest. The sera obtained from some guineapigs immunized in this way, after absorption with concentratedsedimented virus and subcellular components obtained fromlate harvests, can be shown to contain an antibody that reactswith a highly labile, sedimentable antigen in the early harvested cells but not with concentrated virus or the "soluble"

antigens obtained from cells at the end of infection.On the basis of the observations with SV40 virus (39), it

was at first thought that it would be easy to prepare nonvirionantigens free of structural viral antigens by the use of certaininhibitors of DNA synthesis. However, in the case of herpesvirus, Dr. G. Tarro and I (unpublished studies) found thatnone of the DNA inhibitors in general use prevented the synthesis of structural viral antigens even though most of themprevented the production of infectious virus. In the case ofherpes virus, a variety of other procedures, including argininedeprivation and cultivation at low and high temperatures, allfailed to yield cells without structural viral antigens as determined by CF tests with human or guinea pig sera that reactedonly with washed virus particles but not with the "soluble"

antigens. DNA inhibitors were also ineffective in completelypreventing the synthesis of structural viral antigens in cellsinfected with some of the adenoviruses (21). Another complication that arose in connection with the adenoviruses is thatmany strains were found to be contaminated with so-called"adenovirus-associated" DNA viruses (1, 17). Since humanbeings have antibodies for these "adenovirus-associated"

viruses, it was important that the adenoviruses to be used inthis type of study be proved to be free of demonstrable extraneous agents.

An important observation that considerably facilitates thework with the large number of human adenoviruses is that,

SEPTEMBER 1968 1855



Albert B. Sabin

by the CF test, the tumor (T) antigens produced by some ofthe oncogenic adenoviruscs in tumors, transformed cells, orproductively infected cells are very closely related (in someinstances perhaps even identical) to those produced by certainother types (21). Thus, the highly oncogenic types 12, IS,and 31 form one group (A), and the moderately oncogenictypes 3, 7, 14, 16, and 21, another group (B). Group B hamstersera also cross-react with antigens from cells infected withadcnovirus type 4, which is not oncogenic in hamsters, andadcnovirus type 11 of unknown status. Adenovirus types 2and 5, which have produced virus-free transformed rat embryocells in cultures with reduced calcium concentration, containT antigens or messenger RNA that cross-react with adenovirustypes 1 and 0 (6). Thus, by using antigens from only 3 adeno-viruses, one from each group, one can test the specially selected human cancer sera for the possible role of at least 14human types of adenovirus, types 1 to 7, 11, 12, 14, 16, 18, 21,and 31. The inclusion of types 1, 2, and 5 is of special interestbecause they are the types that infect children in early lifeand persist in the tonsils and adenoids and perhaps also othertissues (6).

Although nonvirion T antigens in tumors and productivelyinfected cells can also be demonstrated by the fluorescent antibody technic using appropriate sera from tumor-bearing hamsters, it has been found that hamster sera containing hightiters of tumor CF antibody (e.g., vs. adenovirus type 7) failedto detect homologous T antigens by the fluorescent antibodytechnic. Nevertheless, it is of special interest that adenovirus Tantigens have been demonstrated by the fluorescent antibodytechnic in simian cells abortively infected with human adeno-viruses (5, 30). Since structural viral antigens were not detectedby the fluorescent antibody technic in the abortively infectedcell with the sera that were employedâ€”despite the fact thatviral DNA was synthesized in these cells (40)â€”such cells canbe used for the detection of T antibody in the sera of cancerpatients. Although cross-reactions are observed by the fluorescent antibody technic that are not found by the CF technic(21, 40), such abortively infected simian cells provide stillanother way for searching for adenovirus tumor antibody inthe sera of cancer patients.

The adenovirus tumor collaborative group working in conjunction with the ad hoc committee on DNA tumor viruses ofthe National Cancer Institute recently completed a series ofCF and fluorescent antibody tests on the whole collection ofspecially selected sera from cancer patients and from controlsdescribed earlier, using representative members of the threegroups of adenoviruses mentioned above, and negative resultswere obtained (R. Huebner, personal communication). Although a critical analysis of these negative results must stillbe made, they strongly suggest that the human adenoviruses,of varying degrees of oncogenicity for newborn hamsters andcell-transforming capacity for rat cells in culture, are not etio-logieally involved in the wide variety of human malignanciesthat were testedâ€”a conclusion that is being checked by themessenger RNA-DNA hybridization technic.

Biochemical Approach. Based on the finding in the virus-free DNA virus hamster tumors of polyribosomal messengerRNA that specifically binds to the DNA of the virus (or in the

case of adenovirus, the group of viruses) that initially produced the experimental tumor (3, 9), the biochemical approachinvolves the isolation by similar methods of messenger RNAfrom a great variety of human cancers and testing for theirspecific binding capacity with a large array of DNA's from

suspect viruses. On the basis of the original procedure, it wouldbe necessary to label with uridine-3H live, metabolizing humantumor cells either from the original tumor or from malignantcell cultures derived from it, in order to have a sufficient quantity of radioactive messenger RNA to put on the membranescontaining the various unlabeled DNA's. Since this approach

has been found to be difficult because most human tumorseither yield insufficient numbers of cells or not enough of themtake up the minimum required amount of radioactive label andbecause it has not yet proved possible to establish most humanmalignant cells in culture, it will be necessary to approach theproblem in a different way (M. Green, personal communication). Dr. Maurice Green and his associates have developedhybridization-inhibition technics for the experimental tumors,in which the unlabeled messenger RNA is allowed to bind tothe DNA first and the extent to which this inhibits the bindingof known radioactive messenger RNA is measured (personalcommunication). Another approach would involve attempts tolabel the messenger RNA after it has been isolated from thecells and purified. This work is being actively pursued by Dr.Green and his associates, and the results are being awaitedwith great interest.

The question is sometimes asked how long one should continue with the search for a possible viral etiology in humanmalignancy in the face of repeated frustrations. The only answer I can give is that as long as there are reasonable questionsto ask and appropriate technics with which to attempt toanswer them, so long it is necessary to persist.

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1968;28:1849-1858. Cancer Res Albert B. Sabin Search for a Viral Etiology in Human CancersViral Carcinogenesis: Phenomena of Special Significance in the

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