(CANCER RESEARCH 26 Part 1, 1980-1993, September 1986]

Neoplastic Transformation1

HILARY KOPROWSKI, FRED JENSEN, ANTHONY GIRARDI AND IRENA KOPROWSKA

The Wistar Institute of Anatomy and Biology, and Hahnemann Medical College (I. K.), Philadelphia, Pennsylvania

Summary

"Scientific controversies constantly resolve themselves intodifferences about the meaning of words" (Schuster, cited in Ref.18). The meaning of the word "transformation" has plagued

scientists for quite some time since it apparently means onething to bacterial geneticists and another thing to oncologists.It is proposed in this review to use the term "conversion" for

description of events which take place within a defined set ofcircumstances after animal cells have been exposed to tumorvirus. These events can then be observed at the single cell level.It is further proposed that the term "transformation" be applied

to the other types of tumor-virus-animal cell interactions, withthe understanding that these interactions can be described onlyat cell population levels and that many events associated withtransformation may occur sporadically; thus, they cannot be observed with the precision that is characteristic of the "conversion"phenomenon. Various criteria proposed for "neoplastic transformation" are examined in light of experimental facts and re

alities.

The term transformation, in the biologic sense, was first usedabout 20 years ago by bacterial geneticists to describe the transfer by a DXA molecule of genetically recognizable traits from onebacterium to the other (1). Virologists and oncologists later borrowed the word to extend its use to the virus-host relationship inneoplasia, apparently ignoring the original meaning. So now wedeal with 2 definitions of the same word, having nothing whatsoever in common but operating with 2 separate sets of referents.

The term conversion, in the biologic sense, was first coined inthe field of bacterial viruses, meaning the transfer of toxigenicityfrom one strain of Corynebaclerium diphtheriae to another (12)by means of a specific phage (20). The hereditary nature of thenewly acquired traits implies changes in genotype of the host cellthrough the addition of new genetic material to the existingbackground (20). It has been shown that conversion can be induced by a bacterial virus in both its vegetative and in its pro-phage forms, whether or not the cell response to the infection isof lytic or of lysogenic type (26). However, in the case of non-lysogenic response, the newly acquired traits are lost after a fewgenerations.

Since the term conversion has not yet been as misused as transformation has by oncologists, it can still be redefined by sub

stituting carcinogenic agents for phages and animal cells forbacteria.

On Table 1 are listed postulates for neoplastic conversion remodeled after its original definition: the referents are self-explanatory. Animal cells exposed to carcinogenic agents must beunaffected by the agent as they are in the case of lysogenic infection. Most animal cells should be susceptible to conversion inorder to insure that the phenomenon is observed on the single celllevel and not on the level of cell population. In order to eliminatean indirect effect of the carcinogenic agent, conversion should bedetected shortly after the cells are exposed to the agent. If we aredealing with viruses, changes in morphology should be a characteristic of a genotype of a tumor virus or of a particular propertyof the carcinogenic agent. The expressions of the new cell genotypes may be recognized by the appearance of specific markers.Finally, the converted cells should be neoplastic for the speciesof their origin, and the acquisition of these tumorigenic propertiescan be expected to occur early in the process.

Conversion: Facts and Realities

Now let us analyze the definition of neoplastic conversion inconfrontation with the facts and the realities of scientific experimentation.

In Table 2, the list of referents to conversion is juxtaposed tothe events following the exposure of animal cells to 2 types ofcarcinogenic viruses: the RNA viruses, and the other DNA viruses. You will notice that the DNA virus-host cell relationshipfalls short of satisfying the criterion of conversion. Conversely,the virus-host cell relationship, in the case of RNA viruses andparticularly RSV2 almost fulfills the points of references estab

lished for the definition of neoplastic conversion.We would like to discuss in greater detail the sequence of

events that follows infection of avian cells with RSV, since thisis the only way to understand how closely this process approaches all citeria for conversion.

As shown in Table 3, synthesis of cell DNA continues for about4-5 hr after absorption and penetration of RSV, and stops just

about the time the synthesis of viral DNA starts. Cell divisiontakes place following the synthesis of virus-dependent DNA (22).The virus is then synthesized, and changes characterized by anincreased rate of glycolysis and increased production of hyal-uronic acid synthetase (Temin, personal communication) are

1This work was supported, in part, by USPHS Research GrantCA-04534 and contract PH-43-G2-157 from the National CancerInstitute, and the American Cancer Society, Grant E-89 andUSPHS Grant C-Y-3651.

* The abbreviations used are: RSV, Rous sarcoma virus; GMK,

green monkey kidney; CPC, carcinogenic polycyclic hydrocarbons; SV40, simian virus 40; IIDCS, human diploid cell strain;ICFA, induced complement fixation antigen; RIF, avian leukosiscomplex.

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Neoplastic Transformation

TABLE 1

REFERENTS FOR NEOPLASTIC CONVERSIONOF ANIMAL CELLS

Absence of cell-killing effect.All or most cells susceptible to conversion.Conversion occurs shortly after contact with carcinogenic agent.Morphologic changes characteristic for the genotype of the agent.Presence of markers expressing new genotype.Converted cells oncogenic for species of origin.

TABLE 2

NEOPLASTICCONVERSIONRELATEDTOTHE EVENTSACCOMPANYINGTUMOR VIRUS INFECTION OF ANIMAL CELLS

Referents forconversionCell

destructionUniform susceptibilityTime of conversionSpecific morphologic changesMarkers for new genotypeOncogenicityRNA

virusesShortDNA viruses±Long±

noted about 36 hr after infection. Approximately 3-4 days after

exposure to infection, the culture changes its morphologic appearance. It is important to note that the morphologic alterationsof the cells are specific for the strains of RSV used to infect thecultures (22). In other words, the genotype of the virus determines alteration in the morphology of the infected cells.

We would like to emphasize that only 1 round of cell andviral DNA synthesis is needed to change normal avian fibre-blasts into tumor cells within 72-96 hr after exposure to RSV.

The rapidity of this conversion process, and the efficiency withwhich most, if not all, cells of the culture change into morphologically distinct tumor cells, is unique among all tumor virusesunder study at present. The production of infectious virus andmorphologic conversion are 2 separate phenomena (Ref 22, andTemin, personal communication). It is important to note thatthe rapidity and efficiency of RSV conversion depends largelyon the conditions under which the experiments are conducted,with particular attention being given to the physiologic state ofthe culture. Morphologic and biochemical changes occur, asdescribed above, under culture conditions ideally adjusted tothis type of experimentation. Under other circumstances, andparticularly in other systems—such as mammalian cells—(as itwill be shown below) the RSV-cell interaction may lead to differ

ent results.

Transformation: Definition

Since the events following exposure of animal cells to DNAtumor viruses cannot be defined as conversion, the term transformation will be used in this paper, but not as a definition per se.Listed on Table 4 are experimental events which fall into thecategory of transformation. Here we are dealing with cell population phenomena and not with events observable on the level ofindividual cells. Thus, when we analyze the results of our investigations, we have to remember that most of our data refer to aculture, or to a tumor, or to a fraction of the cell population, butrarely refer to individual cells.

Degeneration and Proliferation

In contrast to conversion, in the case of transformation, celldegeneration and proliferation may run either simultaneously orsequentially. Except in the case of massive destruction of pol-yoma-exposed mouse cultures (see below), cytopathic changes

accompanying transformations are of mild intensity and maybe observed in rabbit, mouse, pig, and calf tissues exposed toSV40 (6).

If cytopathic interaction exists in hamster tissue exposed topolyoma virus, it cannot be detected either by morphologic orby biologic tests (9). In mouse tissue exposed to polyoma virus,extensive destruction of the cell population precedes the prolif-

erative processes observed in the surviving cells (9). GMK cultures destroyed by SV^o under normal conditions of cultivationmay transform (11), but then the physiologic conditions of theinitial interaction between virus and culture must be adjusted insuch a way that only a small fraction of the cell population willbe destroyed by the incoming virus (8).

An interesting case is presented when human fibroblasts areexposed in culture to SV40. A slight destruction of cellular elements is noted just before the culture undergoes morphologic andkaryologic transformation (19). Weeks later, when the entire culture is morphologically and karyologically altered, a massive cellloss takes place. This stage, referred to as crisis, has been observed in every human culture transformed by SV4o. Very few

TABLE 3

SEQUENCE OF EVENTS IF SYNCHRONIZEDCELLS ARE INFECTEDWITH RSV (AFTER TEMIN)

Time (hr) Event

011

2I

Hilary Koprowski, Fred Jensen, Anthony Girardi, and Irena Koprowska

111z

J

5 IO 15 20 22.6 25 30 35 40 45

WEEKS AFTER ONSET OF TRANSFORMATION'RECOVERY FROM AUTOLOGOUSIMPLANTATION

CHART1. Time from onset of transformation to loss of viability of 19 cultures of human fibroblasta infected with SVt«as Phase II cells.

time in vitro, the infected cultures and their normal controlcounterparts being followed until crisis or Phase III. The controlcultures reached their Phase III after 43 passages, and the crisisof the transformed cultures set in 9-10 weeks later. All 4 cul

tures entered the crisis phase at the same time, regardless of thepassage level at which they were exposed to the virus.

Thus, it seems likely that the transformed SVw cell populationretains the same built-in mechanism of the finite lifetime characteristic that the normal cell cultures do, but the SV40 cell'slifespan is extended for a predictable period of 9-10 weeks.

We can postulate (Chart 4) that the fraction of a cell population which reaches transformation early will die out 9 weeks later,even though this subcrisis may not be easily detected amongother proliferating cells. This 1st wave of transformation, proliferation, and death of the transformed population may be followed by superimposed waves of transformation, proliferation,and minor subcrises affecting a fraction of the cell populationeach time until the entire culture comes to a standstill.

At this stage the remaining viable cells can be rescued and theygive rise to colonies of rapidly proliferating cells which can thenbe propagated indefinitely. No infectious SV«was demonstratedin such cultures. Rescue operations are facilitated if, in 5-10passages prior to the expected crisis, cultures are maintainedwithout subcultivation with occasional changes of nutrientmedia. At present, colonies have been rescued from every transformed culture to form a permanent line.

The nature of crisis remains obscure. We may hypothesizethat the larger fraction of the nonhomogenous cell population,which transforms early, dies out because of the formation of aforbidden genetic combination (13) (Fig. 1). The other fraction,metabolically less active and consisting of a small number of cells,does not perish but becomes a stem line for the abnormal pernia-

WEEKS AFTER ONSET

10

OF

15

TRANSFORMATION

20

CHAKT 2. Time from onset of transformation to crisis in 12cultures of human fibroblasts infected with SV«>as Phase IIIcells.

cells survive the crisis stage, and when rescued, the cells giverise to an autonomous permanent line (Fig. 1).

Crisis occurs about 22 weeks after transformed cells appear inhuman culture (13) (Phase II) (Chart 1). If, however, human cultures were infected in Phase III, i.e., at the end of their in vitrolifetime (14), crisis occurred 9 weeks after the transformation.Adherence to this time schedule was observed in every culturestudied (13) (Chart 2). Might we consider crisis in human cultureas a parallel phenomenon to the "dying out" of normal human

cells, whose lifespan was extended? Or should crisis be thought ofas a characteristic of neoplastic transformation by SV40?In theexperiment shown in Chart 3, human cultures were exposed toSV40at the 12th, 20th, 30th, and 40th passages during their life

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Neoplastic Transformation

nent culture. The reason why these cells stay dormant and bywhat mechanism they then take off to form a line is not known.However, a certain parallel may be drawn between the eventsobserved in vitro with culture of human fibroblasts undergoingtransformation and the neoplastic transformation in vivo.

For instance, an involvement of 2 types of cells may also occurin the transformation process of the epithelium of human uterinecervix from dysplasia to carcinoma in situ.

The cervical dysplasia is an epithelial lesion involving differentiated and undifferentiated cell populations (Fig. 2). The smallundifferentiated cells, "basal" or "reserve" cells, are adjacent to

the basal membrane. During reproductive years, in uterine cervix under physiologic conditions, these cells remain inconspicuous, giving rise to more differentiated cell populations increasingin size towards the surface. The largest cells die off and desquamate.

In cervical dysplasia, the small undifferentiated cells becomemore conspicuous, forming several rows known as basal cellhyperplasia (Fig. 3). The large differentiated cells often shownuclear abnormalities (Fig. 4), but they die off and desquamate,as under physiologic conditions. On occasion, apparently thelesion totally disappears. However, when the entire lesion consists only of a small population of undifferentiated cells thatapparently lack the capacity to differentiate into the large cells,it is recognized as carcinoma in situ (Fig. 5). It may in turn remain contained for 10-15 years within the limits of the epithelium—perhaps because of inhibition when coming into contactwith the neighboring cells and structures. When this is overcome,or when other stimuli are provided, the uncontrolled proliferationof the small undifferentiated cells and invasion of the underlying

stroma may lead to the appearance of squamous cell carcinoma.The possibility cannot be excluded that events observed in theSV4transformation of human cells in cultures are not unlikethose described in vivo. Perhaps the small, apparently still viablecell, shown on Fig. 1 in the crisis stage, represents the undifferentiated cell which can no longer differentiate into the large,dying off cells but may start reproducing rapidly into other undifferentiated cells giving rise to a carcinoma in vitro.

Degenerative and proliferativc changes were also described inthe only study on transformation of cultures in vitro by CPC(4, 5). In this study a fraction of the culture is destroyed beforethe surviving cells give rise to colonies with supposedly abnormalproperties.

The effect of CPC has been thoroughly investigated at theWistar Institute for the past 3 years. So far, we have not obtainedany evidence of their capacity to transform normal cultures.However, it has been found (personal communication) that thetoxicity of these compounds for cells in vitro depends on the originof the cells. Primary rodent cells are easily destroyed by as littleas 0.5 fig/ml of the compounds, whereas normal primate andhuman cells are highly resistant to the toxicity of the CPC.

Neoplastic rodent cells are resistant to the action of CPC:cultures of rodent cells in the process of transformation by SV4obecome progressively more resistant to CPC, and when completely transformed are as resistant as cells originating fromtumor tissue. However, resistance in cultures transformed bypolyoma is much less pronounced and appears much later thanit would after SV4oinfection. In addition, cells grown in CPCseem to transform earlier after exposure to SV4o,but this may be

o/

N'on-inlected control cultures

12 20 30 409-10 Weeks-

Passage Number

CHART3. Crisis of human cultures exposed to SV40at different passage levels during their in vitro lifetime.

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Hilary Koprowski, Fred Jensen, Anthony Girardi, and Irena Koprowska

24

Weeks after exposure io SV4QCHART 4. Hypothetical scheme of "waves" of transformation, proliferation, and death of human diploid cells exposed to

a trompe l'oeil since the destruction of a large portion of the back

ground may account for this phenomenon.If we could confirm cell transÃ-ormation in culture by chemical

or physical carcinogens, it should be hailed as a great step forward in the development of easily accessible tools for the studyof neoplastic transformation.

Low Efficiency of Transformation

The transformation process of animal cells by DNA tumorviruses is characterized by its low efficiency. Studies of this typewere conducted more successfully in cultures that were tumori-genie but morphologically normal (21). In this case 5-8% of thecells transformed after exposure to polyoma at a multiplicity of1000 plaque-forming units/cell. The same low efficiency oftransformation was observed when the effect of SV«was studiedin an abnormal mouse culture line (25). These results are probably applicable to any tissue culture system exposed to tumorviruses and contrast rather sadly with the uniform susceptibilityof avian embryo fibroblasts to RSV. This fact would not be sodesperate if the few cells which are transformable could be recognized either before or in the course of early transformation.Unfortunately, studies are just beginning to be made (10) onthe nutritional requirements of neoplastic and normal cultures,and we have to wait until more data become available on specific

differences in the nutritional requirements between virus-transformed cells and their normal counterparts.

Replication of Cells Necessary for Expression of Transformation

As we have mentioned before, Temin's data (Ref. 22 and per

sonal communication) show that only 1 cell division of RSV-infected avian fibroblasts is necessary for the expression of conversion. In transformation it is impossible to state accurately howmany times cells should replicate before their new traits becomeknown. After the primary event, such as abortive tumor virusinfection (16) or the incorporation of chemical carcinogeninto cell components (15, 16), a long series of events may takeplace before a new phenotype can be differentiated from a normalpopulation of the same cells.

Morphologic and Karyologic Changes Are Not Characteristic for aGiven Virus

In contrast to conversion, morphologic characteristics of thetransformed cultures do not appear to reflect the genetic characteristics of the virus. Hamster cultures transformed by SV40mayperhaps be considered morphologically distinguishable from thosetransformed by polyoma (9); the same holds true for the abnormal mouse cell lines transformed by either 1 of the 2 viruses(25) or by the 2 viruses in sequence (24). Although the number of

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mutants of DNA tumor viruses available for the study of morphologic markers of transformation Lssmall, at present neithertumorigenesis in vivo nor transformation in vitro result in characteristic morphologic changes.

In addition, virus-transformed tissues have shown no characteristic karyotype, and no characteristic chromosomal aberrations have been correlated with a specific biologic event (9, 17).Even though karyologic analysis revealed that the frequency andintensity of changes in structure and changes in the number ofchromosomes is significantly higher in human and hamster cellstransformed by SV4o than in hamster cells exposed to polyoma(cited in Ref. 9), no "stem line" distinguished by a defined

karyotype has been maintained in serial passages.

Absence of Markers Other Than Possibly New Antigens for Expression of New Genotype

We could attain a milestone on the tortuous road to an understanding of neoplastic transformation, if cells undergoing transformation could be identified by their changed metabolism. Theonly available marker indirectly linked with virus infection is anew cell antigen as discussed by Dr. Habel.

Transformed Cells May or May Not Be Oncogenic

We are in proud possession of about a dozen viruses whichwhen injected into an animal host will ultimately produce tumors.Attempts have been made to distinguish "strong" oncogenicviruses from "weak" oncogenic viruses, but since we do not know

the mechanism of viral carcinogenesis in vivo, the distinctionsmay not be directly related to the property of the virus itselfbut may be related instead to the reaction of the animal host.

Transformation in vitro of normal cells into transplantableneoplastic cells would, of course, enable us to get a better insightinto the "oncogenic" properties of a given virus. Unfortunately,

the problem is not as simple as that. We still know very littleabout the role of adenoviruses in in vitro transformation or aboutthe neoplastic potential of cultures exposed to these viruses.

Morphologically transformed hamster cultures, shortly afterexposure to polyoma, are not transplantable. Similarly, trans-plantability of polyoma-induced primary fibrosarcomas ofhamsters Lspoor. In both cases, the capability of causing tumorsin animals is acquired at the late stage of transformation. The

Xeoplastic Transformation

same late acquisition of oncogenic properties seems to charac*

terize interaction between SV40and its culture systems. Trans-plan tability once acquired seems to be a permanent characteristicof a transformed cell population and does not seem to fluctuateas do other characteristics.

Oncogenic properties of a transformed culture may be reachedeither by numerous progressive steps towards neoplasia or as theresult of overgrowth of the culture by the few cells that becametransplantable immediately after exposure to a tumor virus (7).

One should be wary in identifying neoplastic properties withmorphologic transformation: it has been observed that althoughmorphologically normal cell cultures (prior to tumor virus exposure) resembled normal cells, they were found to have alreadybeen oncogenic (9).

Comparative studies on inactivation of infectivity versus"transÃ-ormability" of tumor viruses indicate that only i to $

part of the viral genome isneeded to transÃ-orniceli lines (Refs. 2, 3,and Latarjet, personal communication).

•••.Vor Good Red Herring

Although the rules of the "transformation game" are playedmore loosely than the rules of the "conversion game," conditions

under which the experiments are conducted play an equally important role in each situation. The physiologic state of a cultureto be infected with a tumor virus may decide the outcome of theexperiment.

As an example, let us cite the effect produced when the sameSV4oinoculum is used in 2 systems—in human fibroblasta and ingreen monkey kidney tissue. Both systems are infected in different phases of their growth in culture, and the results (Table 5)indicate that when HDCS's are exposed to SV40 during their

Phase II (proliferating growth), only 3% will develop ICFA; 1%will show presence of virus coat antigen, and they will yield about3 infectious virus particles/cell. These cultures will start transforming in 6-8 weeks; prior to that period, a slight cytopathiceffect may be observed. By contrast, exposure of the same cultures to SV4oat the end of their in vitro lifetime (Phase III) resultsin approximately 30% of the cells showing the presence of ICFA,a greater yield of infectious virus/cell, and as described before, amarked acceleration of all the stages of the transformation process. Incontrasi to the results, complete monolayers of GMK cul-

TABLE 5COMPARATIVEEFFECTOF SV«0in "TRANSFORMABLE"AND"LvTic" TISSUE CULTURESYSTEMS

CULTUREHDCSGMKPhase

II6PhaseIIPReplicatingStationary%

CELLSSHOWING:ICFA3

Up to301-1080Virus

coat1N.T.N.T.75VllUS

YIELD/CELLy>3

Hilary Koprowski, Fred Jensen, Anthony Girardi, and Irena Koprowska

tures exposed to SV4o(last line of the table) produce large quantities of ICFA, viral coat antigens, and infectious virus before theirdestruction. Under this condition it is impossible to maintain thecultures after 1 cell transfer (8). However, when the GMK platecontains only sparse cellular population, the results of infectionwith SV4oresemble the results obtained with H DCS and not withGMK in the stationary phase of growth. Again, only a smallnumber of cells show the presence of ICFA with a low yieldof infectious virus. There Is only a slight cytopathic effect,whereby the cultures propagate indefinitely in vitro. Whetherthis process can be termed transformation is difficult to decidedue to the fact that the SV40"transformed" GMK line differs,

only slightly, morphologically from its normal counterpartwhich has undergone so-called "spontaneous transformation."

The only difference between the 2 systems lies in the fact thatthe SV4o-exposedcultures, being noninfectious show the presenceof SV4o ICFA, whereas "spontaneously transformed" cultures

do not. But is this transformation?Another example of incomplete fulfillment of the criteria for

transformation may be found in the resultant infection of GMKcultures when exposed to Adeno type 7. Following the initialpartial cytopathic effect, the surviving cells gave rise to a permanent line that contained a low concentration of complementfixation antigen for Adeno 7 but was slightly different morphologically, from the parent noninfected culture. Even morebizarre results were obtained when guinea pig cultures were exposed to SV4o:these cells, apparently at the end of their in vitrolifetime, received a new lease on life and gave rise to a permanentline. In contrast to GMK cultures, guinea pig cells were left withno imprint of virus whatsoever, and showed no morphologicchanges. They may differ from control cultures in their surfaceproperties vis à vis cells of other species in the same culture. Isthis transformation? Even if tumors are produced in animalhosts it cannot be ascertained that tumor production and otherproperties resulted from virus infection.

Rons Sarcoma Virus in Mammalian Cell Cultures

The rapid morphologic alterations observed in the RSV-avianfibroblast conversion system do not take place in mammalian cellsinfected with RSV. After exposure of GMK cultures to the Continental group of RSV (Schmidt-Ruppin, Carr-Zilber, andDiadkova) (16) the lesions are somewhat similar to those observed in the RSV-converted chick-embryo cells (Fig. 6). Theyappear as discrete foci of round cells somewhat resembling adividing fibroblast piled up 3-dimensionally on monolayers ofseemingly normal cells. After a monolayer has been established,these foci are discernible at each passage level and they can bemaintained indefinitely.

Morphologic changes observed after the exposure of GMKcultures to the Anglo-Saxon strains of RSV (Bryan and Harris)are of a different type than those found after infection with theContinental strains. The lesions represent an accumulation ofthree types of abnormal cells: elongated fibroblast-like cells thataccumulate in thick strands (Fig. 7), vacuolated giant cells(Fig. 8), and cells with characteristic rosette patterns of theirnuclei (Fig. 9). The latter pattern has been observed previouslyin sections of various animal neoplasms caused by tumor viruses(Chesterman, personal communication) and in chorioid plexuscultures infected with visna virus.

Morphologic changes of human cell cultures exposed to theContinental and Anglo-Saxon strain of RSV were different fromthose observed in GMK cultures. Vacuolated cells that characterize the lesion at each cell transfer (Fig. 10) may coalesce, producing a clearly defined plaque (Fig 11), if a culture Lsmaintainedwithout transfer.

The lifespan of RSV-exposed human culture is not increasedappreciably, but the infected cells remain viable longer than dothose of their normal counterparts.

Lesions produced by RSV in the chick-embryo fibroblastculture may be inhibited by a prior exposure to viruses belongingto the RIF. When human cells and GMK cells are exposed to astrain of RIF and challenged, after 1 cell transfer, 8 days later,a slight suppression of the RSV lesion can be observed. However,this inhibition becomes rapidly reversed and the RIF-exposed-RSV-challenged cultures show more pronounced morphologiclesions as evidenced by increase in number and size of foci. Thistakes place regardless of the strain of RSV used in the experiments (16).

When the RIF-RSV foci were separated from the monolayersand cultured, their cell progeny was found to be morphologically distinct from normal GMK cells. After several cell transfers, a permanent, transformed culture line was established.Whether this culture of GMK cells can be considered as "transformed" by infection with the RIF-RSV complex is a question

which cannot be answered at the present time. The culture apparently has unlimited growth potential originating from thefoci of cells which, in comparison to avian embryo fibroblasta,should represent cells transformed by RSV. These cells differmorphologically from their non-infected counterpart, but thedifference does not parallel the easily recognizable alterationsseen in avian embryo fibroblasts. Moreover, it is doubtfulwhether these cells are tumorigenic in monkeys. The presence ofRSV, or its imprint, would help the matter considerably but thedetection of both poses a special problem.

Where, Oh Where is the RSV in the Mammalian Cell Cultures?

Baby chicks or cultures of chick embryo fibroblasts, inoculateddirectly with human and GMK cultures infected with RSV orRIF, showed no presence of a transmissible agent.

However, RSV lesions of human fibroblasts can be transferredon GMK cultures. The agent could not be further transmitted tochick tissue. Yet, baby chicks occasionally develop tumors wheninoculated with cells from mixed culture of RSV-infected GMKand normal chick-embryo fibroblasts.

The difficulties in the recovery of transmissible RSV are characteristic for other mammalian cell RSV systems. For instance,virus cannot be Isolated from extracts of RSV-induced rat andhamster tumors as well as from tissue cultures originating fromthese tumors. However, if intact cells obtained from the sametissues are implanted into susceptible chicks, tumors constitutedof chicken cells will grow.

As stated by Dr. Habel, tumor viruses may leave their imprintsin the transformed tissue. One such imprint is a complementfixation antigen present in avian tissue infected with the RSV-avian leukosis group of viruses. Unfortunately, in RSV-exposedmammalian tissue, presence of this antigen was demonstratedonly on rare occasions. Thus, we are left with a situation where

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Neoplastic Transformation

neither a transmissible agent nor its imprints can be detected inmorphologically transformed tissue.

There is a charming Greek myth about the prophetic priest-king of Délos,Anius, and his 3 daughters, Elais, Spermo, andOeno—whowere called the Winegrowers. Anius, himself a priestof Apollo, decided that 1 household god wasn't enough, so he

dedicated the Winegrowers to the god Dionysus. Dionysus,touched by such devotion, bestowed the power of transformation upon the 3 girls. Elais could turn whatever shetouched into oil; whatever Spermo touched was transÃ-ormedintocorn; whatever Oeno touched was transformed into wine.

Would it not be wonderful if, today, we could invoke Anius'

daughters, along with their powers of transformation, so that wemight transform or convert cells under the effect of various agentsinto malignant cells so that this transformation or conversionwould be a simple 1-step process which would add the missingpieces to the puzzle of the origin of neoplasia?

On the other hand, it might take us longer to come up with aformula to invoke King Anius' daughters—the transformationtrio—than it would to discover a way to deprive malignant tissue of all of the properties and traits which are characteristic formalignancy. This is the most important problem of all.

References

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2. Basilico, C., and di Mayorca, G. Radiation Target Size of theLytic and the Transforming Ability of Polyoma Virus. Proc.Nati. Acad. Sci. U. S., 54: 125-27, 19G5.

3. Benjamin, T. L. Relative Target Sizes for the Inactivation ofthe Transforming and Reproductive Abilities of PolyomaVirus. Ibid., 64: 121-24, 1965.

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C. Black, P. H., and Rowe, W. P. SV40 Induced Proliferation ofTissue Culture Cells of Rabbit, Mouse, and Porcine Origin.Proc. Soc. Exptl. Biol. Med., 114: 721 27, 1963.

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8. Carp, R. I., and Gilden, R. V. A Comparison of the Replicat ion Cycles of Simian Virus 40 in Human Diploid and AfricanGreen Monkey Kidney Cells. Virology, 28: 150-62,1966.

9. Defendi, V. Transformation in Vitro of Mammalian Cells byPolyoma and Simian 40 Viruses. Prog. Exptl. Tumor Res., 8:135-72, 1965.

10. Eagle, H. Metabolic Controls in Cultured Mammalian CellsScience, 145:42-51, 1905.

11. Fernandez, M. V., and Moorhead, P. Transformation ofAfrican Green Monkey Kidney Cultures Infected with SimianVacuolating Virus (SV40). Texas Kept. Biol. Med., 2S: 242-58, 1965.

12. Freeman, V. J. Studies on the Virulence of Bacteriophage-Infected Strains of Corynebacterium diphtheriae. J. Bac-teriol.,6/.-675-88, 1951.

13. Girardi, A. J., Jensen, F. C., and Koprowski, H. SV4o-InducedTransformation of Human Diploid Cells: Crisis and Recovery.J. Cell. Comp. Physiol., 65: 09-83, 1965.

14. Hayflick, L., and Moorhead, P. S. The Limited in Vitro Lifetime of Human Diploid Cell Strains. Symp. Int. Soc. CellBiol., 3: 155-73, 1964.

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the Present Status of Tumor Viruses and Virus Tumors.Harvey Lecture, Series 60, Academic Press, New York, pp.173-216.

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18. Ogden, C. K., and Richards, I. A. The Meaning of Meaning.London: Routledge & Kegan Paul, Ltd., 1950.

19. Pontén,J., Jensen, F., and Koprowski, H. Morphological andVirological Investigation of Human Tissue Cultures Transformed with SV10. J. Cell. Comp. Physiol., 61: 145-63, 1963.

20. Stent, G. S. Molecular Biology of Bacterial Viruses. San Francisco and London: W. H. Freeman and Co., 1903.

21. Stoker, M. Mechanism of Viral Carcinogenesis. Can. CancerConf., 6: 357-68, 1966.

22. Temin, H. M. Nature of the Provirus of Rous Sarcoma. International Conference on Avian Tumors. Nati. Cancer Inst.Monograph, No. 17: 557-70, 1904.

23. Todaro, G. J., and Green, H. An Assay for Cellular Transformation of SV40. Virology, 2$: 117-19, 1964.

—. Successive Transformation of an Established Cell24

25

26

27

Line by Polyoma Virus and SV40.Science, 147: 513-14, 1905.Todaro, G. J., Green, H., and Goldberg, B. D. Transformationof Properties of an Established Cell Line by SV«and PolyomaVirus. Proc. Nati. Acad. Sei. U. S., 51: 66-73, 1904.Uetake, H., Luria, S. E., and Burrous, J. W. Conversion ofSomatic Antigens in Salmonella by Phage Infection Leading toLysis or Lysogeny. Virology, 5: 08-91, 1958.Winocour, E., and Sachs, L. Cell-Virus Interactions with thePolyoma Virus. I. Studies on the Lytic Interaction in theMouse Embryo System. Ibid., 11: 099-721, 1960.

SEPTEMBER 1966 1987

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Hilary Koprowski, Fred Jensen, Anthony Girardi, and Irena Koprowska

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FIG. 1. Sequence of transformation process in human cells. Left: transformation. Note change in morphology of cell and loss of contact inhibition which is characteristic in the early stages of transformation. Middle: crisis. After approximately 22 weeks, SV40-trans-formed human cells enter the stage of "crisis." Proliferation stops, nuclei show lobulation and there is presence of many multinucleated

cells. The period of crisis can take from a short period of 3 weeks up to 3 months, after which time a new proliferation (see right side)appears. Right: recovery. At this stage, cells lost infectious virus and all nuclei containing ICFA and became an "immortal" cell line.

FIG. 2. Cervical dysplasia of human uterus in an epithelial lesion involving differentiated and undifferentiated cell populations.Note: The small undifferentiated cells are "basal" or "reserve" cells.

CANCER RESEARCH VOL. 26

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Xeoplaslic Transformation

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FIG. 3. Cervical dysplasia showing undifTorentiated cells which become more conspicuous and form several rows known as basal cellhyperplasia.

FIG. 4. Cervical dysplasia indicating the large differentiated cells which show nuclear abnormalities.

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Fia. 5. Carcinoma ¿nsÃ-Ã-«.FIG. 6. Foci oÃ-RSV-infected cells (Schmidt-Ruppin strain) of GMK cultures.

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FIG. 7. Elongated fibroblast-like cells in GMK cultures infected with Bryan strain of RSV.FIG. 8. Vacuolated giant cells of GMK cultures infected with Bryan strain of RSV.

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FIG. 9. Rosette patterns of nuclei of GMK cultures infected with Bryan strain of RSV.FIG. 10. Vacuolated cells of human diploid cell strain infected with RSV.

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1966;26:1980-1993. Cancer Res Hilary Koprowski, Fred Jensen, Anthony Girardi, et al. Neoplastic Transformation

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