(CANCER
RESEARCH
Neoplastic
26 Part 1, 1980-1993, September
1986]
Transformation1
HILARY KOPROWSKI,
FRED JENSEN, ANTHONY
GIRARDI
AND
IRENA KOPROWSKA
The Wistar Institute of Anatomy and Biology, and Hahnemann Medical College (I. K.), Philadelphia,
Summary
"Scientific controversies constantly resolve themselves into
differences 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 one
thing 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 of
circumstances after animal cells have been exposed to tumor
virus. 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, with
the understanding that these interactions can be described only
at cell population levels and that many events associated with
transformation may occur sporadically; thus, they cannot be ob
served with the precision that is characteristic of the "conversion"
phenomenon. Various criteria proposed for "neoplastic trans
formation" are examined in light of experimental facts and re
alities.
The term transformation, in the biologic sense, was first used
about 20 years ago by bacterial geneticists to describe the trans
fer by a DXA molecule of genetically recognizable traits from one
bacterium to the other (1). Virologists and oncologists later bor
rowed the word to extend its use to the virus-host relationship in
neoplasia, apparently ignoring the original meaning. So now we
deal with 2 definitions of the same word, having nothing what
soever in common but operating with 2 separate sets of referents.
The term conversion, in the biologic sense, was first coined in
the field of bacterial viruses, meaning the transfer of toxigenicity
from one strain of Corynebaclerium diphtheriae to another (12)
by means of a specific phage (20). The hereditary nature of the
newly acquired traits implies changes in genotype of the host cell
through the addition of new genetic material to the existing
background (20). It has been shown that conversion can be in
duced by a bacterial virus in both its vegetative and in its prophage forms, whether or not the cell response to the infection is
of lytic or of lysogenic type (26). However, in the case of nonlysogenic response, the newly acquired traits are lost after a few
generations.
Since the term conversion has not yet been as misused as trans
formation has by oncologists, it can still be redefined by sub
1This work was supported, in part, by USPHS Research Grant
CA-04534 and contract PH-43-G2-157 from the National Cancer
Institute,
and the American Cancer Society, Grant E-89 and
USPHS Grant C-Y-3651.
19SO
Pennsylvania
stituting carcinogenic agents for phages and animal cells for
bacteria.
On Table 1 are listed postulates for neoplastic conversion re
modeled after its original definition: the referents are self-explan
atory. Animal cells exposed to carcinogenic agents must be
unaffected by the agent as they are in the case of lysogenic in
fection. Most animal cells should be susceptible to conversion in
order to insure that the phenomenon is observed on the single cell
level and not on the level of cell population. In order to eliminate
an indirect effect of the carcinogenic agent, conversion should be
detected shortly after the cells are exposed to the agent. If we are
dealing with viruses, changes in morphology should be a charac
teristic of a genotype of a tumor virus or of a particular property
of the carcinogenic agent. The expressions of the new cell geno
types may be recognized by the appearance of specific markers.
Finally, the converted cells should be neoplastic for the species
of their origin, and the acquisition of these tumorigenic properties
can be expected to occur early in the process.
Conversion: Facts and Realities
Now let us analyze the definition of neoplastic conversion in
confrontation with the facts and the realities of scientific experi
mentation.
In Table 2, the list of referents to conversion is juxtaposed to
the events following the exposure of animal cells to 2 types of
carcinogenic viruses: the RNA viruses, and the other DNA vi
ruses. You will notice that the DNA virus-host cell relationship
falls short of satisfying the criterion of conversion. Conversely,
the virus-host cell relationship, in the case of RNA viruses and
particularly 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 this
is the only way to understand how closely this process ap
proaches all citeria for conversion.
As shown in Table 3, synthesis of cell DNA continues for about
4-5 hr after absorption and penetration of RSV, and stops just
about the time the synthesis of viral DNA starts. Cell division
takes place following the synthesis of virus-dependent DNA (22).
The virus is then synthesized, and changes characterized by an
increased rate of glycolysis and increased production of hyaluronic acid synthetase (Temin, personal communication) are
* The abbreviations used are: RSV, Rous sarcoma virus; GMK,
green monkey kidney; CPC, carcinogenic polycyclic hydrocar
bons; SV40, simian virus 40; IIDCS, human diploid cell strain;
ICFA, induced complement fixation antigen; RIF, avian leukosis
complex.
CANCER
RESEARCH
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1966 American Association for Cancer Research.
VOL. 26
Neoplastic Transformation
TABLE 1
REFERENTS FOR NEOPLASTIC CONVERSIONOF ANIMAL CELLS
Degeneration and Proliferation
In contrast to conversion, in the case of transformation,
cell
degeneration and proliferation may run either simultaneously or
sequentially. Except in the case of massive destruction of polyoma-exposed mouse cultures (see below), cytopathic changes
accompanying
transformations
are of mild intensity and may
be observed in rabbit, mouse, pig, and calf tissues exposed to
SV40 (6).
If cytopathic interaction exists in hamster tissue exposed to
polyoma virus, it cannot be detected either by morphologic or
TABLE 2
NEOPLASTICCONVERSIONRELATEDTOTHE EVENTS ACCOMPANYING by biologic tests (9). In mouse tissue exposed to polyoma virus,
extensive destruction of the cell population precedes the prolifTUMOR VIRUS INFECTION OF ANIMAL CELLS
erative processes observed in the surviving cells (9). GMK cul
conversionCell
Referents for
virusesShortDNA
viruses±Long± tures destroyed by SV^o under normal conditions of cultivation
may transform (11), but then the physiologic conditions of the
destruction
initial interaction between virus and culture must be adjusted in
Uniform susceptibility
such a way that only a small fraction of the cell population will
Time of conversion
be destroyed by the incoming virus (8).
Specific morphologic changes
An interesting case is presented when human fibroblasts are
Markers for new genotype
exposed
in culture to SV40. A slight destruction of cellular ele
OncogenicityRNA
ments is noted just before the culture undergoes morphologic and
karyologic transformation
(19). Weeks later, when the entire cul
noted about 36 hr after infection. Approximately 3-4 days after
ture is morphologically and karyologically altered, a massive cell
loss takes place. This stage, referred to as crisis, has been ob
exposure to infection, the culture changes its morphologic ap
served in every human culture transformed by SV4o. Very few
pearance. It is important to note that the morphologic alterations
of the cells are specific for the strains of RSV used to infect the
cultures (22). In other words, the genotype of the virus deter
TABLE 3
mines alteration in the morphology of the infected cells.
SEQUENCE OF EVENTS IF SYNCHRONIZEDCELLS ARE INFECTED
We would like to emphasize that only 1 round of cell and
WITH RSV (AFTER TEMIN)
viral DNA synthesis is needed to change normal avian fibreTime (hr)
Event
blasts into tumor cells within 72-96 hr after exposure to RSV.
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.
The rapidity of this conversion process, and the efficiency with
which most, if not all, cells of the culture change into morpho
logically distinct tumor cells, is unique among all tumor viruses
under study at present. The production of infectious virus and
morphologic conversion are 2 separate phenomena (Ref 22, and
Temin, personal communication).
It is important to note that
the rapidity and efficiency of RSV conversion depends largely
on the conditions under which the experiments are conducted,
with particular attention being given to the physiologic state of
the culture. Morphologic and biochemical changes occur, as
described above, under culture conditions ideally adjusted to
this type of experimentation.
Under other circumstances,
and
particularly in other systems—such as mammalian cells—(as it
will be shown below) the RSV-cell interaction may lead to differ
ent results.
Transformation: Definition
Since the events following exposure of animal cells to DNA
tumor viruses cannot be defined as conversion, the term trans
formation will be used in this paper, but not as a definition per se.
Listed on Table 4 are experimental events which fall into the
category of transformation. Here we are dealing with cell popula
tion phenomena and not with events observable on the level of
individual cells. Thus, when we analyze the results of our inves
tigations, we have to remember that most of our data refer to a
culture, or to a tumor, or to a fraction of the cell population, but
rarely refer to individual cells.
SEPTEMBER
011
cellExposure
of
synthesisRSVcell
toSynthesis
2I<6-816-8i8-1538i74-98Start
ofSynthesis
stopsviral
DNA
ofCell
(provirus)new
DNA
divisionSynthesis
ofBiochemicalMorphologicDNA
virusexpressionschanges
TABLE 4
TRANSFORMATIONOF ANIMAL CELLS BY TUMOR VIRUSES
Population phenomena:
Cell destruction and proliferation.
Few cells susceptible to transformation.
Replication of cells necessary for expression of transformation.
Morphologic and karyologic changes not characteristic
for a
given virus.
Absence of markers other than possibly new antigens for ex
pression of new genotype.
Transformed culture may or may not be oncogenic.
1906
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1966 American Association for Cancer Research.
1981
Hilary Koprowski, Fred Jensen, Anthony Girardi, and Irena Koprowska
111
z
J
5
IO
15
20
WEEKS AFTER
22.6
25
30
35
40
45
ONSET OF TRANSFORMATION
'RECOVERY FROM AUTOLOGOUSIMPLANTATION
CHART1. Time from onset of transformation
10
WEEKS
AFTER
ONSET
OF
to loss of viability of 19 cultures of human fibroblasta infected with SVt«as Phase II cells.
15
20
TRANSFORMATION
CHAKT 2. Time from onset of transformation
to crisis in 12
cultures of human fibroblasts infected with SV«>as Phase III
cells.
cells survive the crisis stage, and when rescued, the cells give
rise to an autonomous permanent line (Fig. 1).
Crisis occurs about 22 weeks after transformed cells appear in
human culture (13) (Phase II) (Chart 1). If, however, human cul
tures were infected in Phase III, i.e., at the end of their in vitro
lifetime (14), crisis occurred 9 weeks after the transformation.
Adherence to this time schedule was observed in every culture
studied (13) (Chart 2). Might we consider crisis in human culture
as a parallel phenomenon to the "dying out" of normal human
cells, whose lifespan was extended? Or should crisis be thought of
as a characteristic of neoplastic transformation by SV40?In the
experiment shown in Chart 3, human cultures were exposed to
SV40at the 12th, 20th, 30th, and 40th passages during their life
1982
time in vitro, the infected cultures and their normal control
counterparts being followed until crisis or Phase III. The control
cultures reached their Phase III after 43 passages, and the crisis
of the transformed cultures set in 9-10 weeks later. All 4 cul
tures entered the crisis phase at the same time, regardless of the
passage level at which they were exposed to the virus.
Thus, it seems likely that the transformed SVw cell population
retains the same built-in mechanism of the finite lifetime char
acteristic that the normal cell cultures do, but the SV40 cell's
lifespan is extended for a predictable period of 9-10 weeks.
We can postulate (Chart 4) that the fraction of a cell popula
tion which reaches transformation early will die out 9 weeks later,
even though this subcrisis may not be easily detected among
other proliferating cells. This 1st wave of transformation, pro
liferation, and death of the transformed population may be fol
lowed by superimposed waves of transformation, proliferation,
and minor subcrises affecting a fraction of the cell population
each time until the entire culture comes to a standstill.
At this stage the remaining viable cells can be rescued and they
give rise to colonies of rapidly proliferating cells which can then
be propagated indefinitely. No infectious SV«was demonstrated
in such cultures. Rescue operations are facilitated if, in 5-10
passages prior to the expected crisis, cultures are maintained
without subcultivation with occasional changes of nutrient
media. At present, colonies have been rescued from every trans
formed culture to form a permanent line.
The nature of crisis remains obscure. We may hypothesize
that the larger fraction of the nonhomogenous cell population,
which transforms early, dies out because of the formation of a
forbidden 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 perniaCANCER
RESEARCH
VOL. 26
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1966 American Association for Cancer Research.
Neoplastic Transformation
nent culture. The reason why these cells stay dormant and by
what mechanism they then take off to form a line is not known.
However, a certain parallel may be drawn between the events
observed in vitro with culture of human fibroblasts undergoing
transformation and the neoplastic transformation in vivo.
For instance, an involvement of 2 types of cells may also occur
in the transformation process of the epithelium of human uterine
cervix from dysplasia to carcinoma in situ.
The cervical dysplasia is an epithelial lesion involving differ
entiated and undifferentiated cell populations (Fig. 2). The small
undifferentiated cells, "basal" or "reserve" cells, are adjacent to
the basal membrane. During reproductive years, in uterine cer
vix under physiologic conditions, these cells remain inconspic
uous, giving rise to more differentiated cell populations increasing
in size towards the surface. The largest cells die off and desqua
mate.
In cervical dysplasia, the small undifferentiated cells become
more conspicuous, forming several rows known as basal cell
hyperplasia (Fig. 3). The large differentiated cells often show
nuclear abnormalities (Fig. 4), but they die off and desquamate,
as under physiologic conditions. On occasion, apparently the
lesion totally disappears. However, when the entire lesion con
sists only of a small population of undifferentiated cells that
apparently lack the capacity to differentiate into the large cells,
it is recognized as carcinoma in situ (Fig. 5). It may in turn re
main contained for 10-15 years within the limits of the epithe
lium—perhaps because of inhibition when coming into contact
with the neighboring cells and structures. When this is overcome,
or when other stimuli are provided, the uncontrolled proliferation
of 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 the
SV4<>transformation of human cells in cultures are not unlike
those described in vivo. Perhaps the small, apparently still viable
cell, shown on Fig. 1 in the crisis stage, represents the undiffer
entiated cell which can no longer differentiate into the large,
dying off cells but may start reproducing rapidly into other un
differentiated cells giving rise to a carcinoma in vitro.
Degenerative and proliferativc changes were also described in
the only study on transformation of cultures in vitro by CPC
(4, 5). In this study a fraction of the culture is destroyed before
the surviving cells give rise to colonies with supposedly abnormal
properties.
The effect of CPC has been thoroughly investigated at the
Wistar Institute for the past 3 years. So far, we have not obtained
any evidence of their capacity to transform normal cultures.
However, it has been found (personal communication) that the
toxicity of these compounds for cells in vitro depends on the origin
of the cells. Primary rodent cells are easily destroyed by as little
as 0.5 fig/ml of the compounds, whereas normal primate and
human 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 SV4o
become progressively more resistant to CPC, and when com
pletely transformed are as resistant as cells originating from
tumor tissue. However, resistance in cultures transformed by
polyoma is much less pronounced and appears much later than
it would after SV4oinfection. In addition, cells grown in CPC
seem to transform earlier after exposure to SV4o,but this may be
o/
N'on-inlected control cultures
12
20
30
Passage
40
9-10 Weeks-
Number
CHART3. Crisis of human cultures exposed to SV40at different passage levels during their in vitro lifetime.
SEPTEMBER 1966
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1966 American Association for Cancer Research.
1983
Hilary Koprowski, Fred Jensen, Anthony Girardi, and Irena Koprowska
24
CHART 4. Hypothetical
scheme of "waves"
Weeks after exposure
of transformation,
proliferation,
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 for
ward in the development of easily accessible tools for the study
of neoplastic transformation.
Low Efficiency of Transformation
The transformation process of animal cells by DNA tumor
viruses is characterized by its low efficiency. Studies of this type
were conducted more successfully in cultures that were tumorigenie but morphologically normal (21). In this case 5-8% of the
cells transformed after exposure to polyoma at a multiplicity of
1000 plaque-forming units/cell. The same low efficiency of
transformation was observed when the effect of SV«was studied
in an abnormal mouse culture line (25). These results are prob
ably applicable to any tissue culture system exposed to tumor
viruses and contrast rather sadly with the uniform susceptibility
of avian embryo fibroblasts to RSV. This fact would not be so
desperate if the few cells which are transformable could be recog
nized either before or in the course of early transformation.
Unfortunately, studies are just beginning to be made (10) on
the nutritional requirements of neoplastic and normal cultures,
and we have to wait until more data become available on specific
19N4
io SV4Q
and death of human diploid
cells exposed
to
differences in the nutritional requirements between virus-trans
formed 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 RSVinfected avian fibroblasts is necessary for the expression of con
version. In transformation it is impossible to state accurately how
many times cells should replicate before their new traits become
known. After the primary event, such as abortive tumor virus
infection (16) or the incorporation of chemical carcinogen
into cell components (15, 16), a long series of events may take
place before a new phenotype can be differentiated from a normal
population of the same cells.
Morphologic and Karyologic Changes Are Not Characteristic for a
Given Virus
In contrast to conversion, morphologic characteristics of the
transformed cultures do not appear to reflect the genetic charac
teristics of the virus. Hamster cultures transformed by SV40may
perhaps be considered morphologically distinguishable from those
transformed by polyoma (9); the same holds true for the ab
normal mouse cell lines transformed by either 1 of the 2 viruses
(25) or by the 2 viruses in sequence (24). Although the number of
CANCER RESEARCH VOL. 26
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1966 American Association for Cancer Research.
Xeoplastic Transformation
mutants of DNA tumor viruses available for the study of mor
phologic markers of transformation Lssmall, at present neither
tumorigenesis in vivo nor transformation in vitro result in charac
teristic morphologic changes.
In addition, virus-transformed tissues have shown no charac
teristic karyotype, and no characteristic chromosomal aber
rations have been correlated with a specific biologic event (9, 17).
Even though karyologic analysis revealed that the frequency and
intensity of changes in structure and changes in the number of
chromosomes is significantly higher in human and hamster cells
transformed 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 Expres
sion of New Genotype
We could attain a milestone on the tortuous road to an under
standing of neoplastic transformation, if cells undergoing trans
formation could be identified by their changed metabolism. The
only available marker indirectly linked with virus infection is a
new 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 which
when injected into an animal host will ultimately produce tumors.
Attempts have been made to distinguish "strong" oncogenic
viruses from "weak" oncogenic viruses, but since we do not know
the mechanism of viral carcinogenesis in vivo, the distinctions
may not be directly related to the property of the virus itself
but may be related instead to the reaction of the animal host.
Transformation in vitro of normal cells into transplantable
neoplastic cells would, of course, enable us to get a better insight
into the "oncogenic" properties of a given virus. Unfortunately,
the problem is not as simple as that. We still know very little
about the role of adenoviruses in in vitro transformation or about
the neoplastic potential of cultures exposed to these viruses.
Morphologically transformed hamster cultures, shortly after
exposure to polyoma, are not transplantable. Similarly, transplantability of polyoma-induced primary fibrosarcomas of
hamsters Lspoor. In both cases, the capability of causing tumors
in animals is acquired at the late stage of transformation. The
same late acquisition of oncogenic properties seems to charac*
terize interaction between SV40and its culture systems. Transplan tability once acquired seems to be a permanent characteristic
of a transformed cell population and does not seem to fluctuate
as do other characteristics.
Oncogenic properties of a transformed culture may be reached
either by numerous progressive steps towards neoplasia or as the
result of overgrowth of the culture by the few cells that became
transplantable immediately after exposure to a tumor virus (7).
One should be wary in identifying neoplastic properties with
morphologic transformation: it has been observed that although
morphologically normal cell cultures (prior to tumor virus ex
posure) resembled normal cells, they were found to have already
been oncogenic (9).
Comparative studies on inactivation of infectivity versus
"transÃ-ormability" of tumor viruses indicate that only i to $
part of the viral genome is needed to transÃ-orniceli lines (Refs. 2, 3,
and Latarjet, personal communication).
•
•
•
.Vor Good Red Herring
Although the rules of the "transformation game" are played
more loosely than the rules of the "conversion game," conditions
under which the experiments are conducted play an equally im
portant role in each situation. The physiologic state of a culture
to be infected with a tumor virus may decide the outcome of the
experiment.
As an example, let us cite the effect produced when the same
SV4oinoculum is used in 2 systems—in human fibroblasta and in
green monkey kidney tissue. Both systems are infected in differ
ent 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 about
3 infectious virus particles/cell. These cultures will start trans
forming in 6-8 weeks; prior to that period, a slight cytopathic
effect may be observed. By contrast, exposure of the same cul
tures to SV4oat the end of their in vitro lifetime (Phase III) results
in approximately 30% of the cells showing the presence of ICFA,
a greater yield of infectious virus/cell, and as described before, a
marked acceleration of all the stages of the transformation proc
ess. Incontrasi to the results, complete monolayers of GMK cul-
TABLE 5
COMPARATIVE
EFFECTOF SV«0in "TRANSFORMABLE"
AND"LvTic" TISSUE CULTURESYSTEMS
SHOWING:ICFA3
CELLS
CULTUREHDCSGMKPhase
YIELD/CELLy>3<250-100CPE
(%)Up
coat1N.T.N.T.75VllUS
II6
Phase
IIPReplicatingStationary%
Up301-1080Virus
to
10?1-1000-90CONTINUOUSPASSAGEUnlimited
to
UnlimitedUnlimited
None
a Abbreviations: SVio,simian virus 40; ICFA, induced complement fixation antigen; CPE, cytopathic
effect; HDCS, human diploid cell strain; GMK, green monkey kidney; N. T., not tested.
6 Stationary culture of the proliferation growth phase.
' Culture at the end of its life in vitro.
dWhen all cells register as infectious centers.
SEPTEMBER 190G
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1966 American Association for Cancer Research.
1985
Hilary Koprowski, Fred Jensen, Anthony Girardi, and Irena Koprowska
tures exposed to SV4o(last line of the table) produce large quanti
ties of ICFA, viral coat antigens, and infectious virus before their
destruction. Under this condition it is impossible to maintain the
cultures after 1 cell transfer (8). However, when the GMK plate
contains only sparse cellular population, the results of infection
with SV4oresemble the results obtained with H DCS and not with
GMK in the stationary phase of growth. Again, only a small
number of cells show the presence of ICFA with a low yield
of infectious virus. There Is only a slight cytopathic effect,
whereby the cultures propagate indefinitely in vitro. Whether
this process can be termed transformation is difficult to decide
due to the fact that the SV40"transformed" GMK line differs,
only slightly, morphologically from its normal counterpart
which has undergone so-called "spontaneous transformation."
The only difference between the 2 systems lies in the fact that
the SV4o-exposedcultures, being noninfectious show the presence
of 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 GMK
cultures when exposed to Adeno type 7. Following the initial
partial cytopathic effect, the surviving cells gave rise to a per
manent line that contained a low concentration of complement
fixation antigen for Adeno 7 but was slightly different morpho
logically, from the parent noninfected culture. Even more
bizarre results were obtained when guinea pig cultures were ex
posed to SV4o:these cells, apparently at the end of their in vitro
lifetime, received a new lease on life and gave rise to a permanent
line. In contrast to GMK cultures, guinea pig cells were left with
no imprint of virus whatsoever, and showed no morphologic
changes. They may differ from control cultures in their surface
properties vis à vis cells of other species in the same culture. Is
this transformation? Even if tumors are produced in animal
hosts it cannot be ascertained that tumor production and other
properties resulted from virus infection.
Morphologic changes of human cell cultures exposed to the
Continental and Anglo-Saxon strain of RSV were different from
those observed in GMK cultures. Vacuolated cells that charac
terize the lesion at each cell transfer (Fig. 10) may coalesce, pro
ducing a clearly defined plaque (Fig 11), if a culture Lsmaintained
without transfer.
The lifespan of RSV-exposed human culture is not increased
appreciably, but the infected cells remain viable longer than do
those of their normal counterparts.
Lesions produced by RSV in the chick-embryo fibroblast
culture may be inhibited by a prior exposure to viruses belonging
to the RIF. When human cells and GMK cells are exposed to a
strain 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-exposedRSV-challenged cultures show more pronounced morphologic
lesions as evidenced by increase in number and size of foci. This
takes place regardless of the strain of RSV used in the experi
ments (16).
When the RIF-RSV foci were separated from the monolayers
and cultured, their cell progeny was found to be morphologi
cally distinct from normal GMK cells. After several cell trans
fers, a permanent, transformed culture line was established.
Whether this culture of GMK cells can be considered as "trans
formed" by infection with the RIF-RSV complex is a question
which cannot be answered at the present time. The culture ap
parently has unlimited growth potential originating from the
foci of cells which, in comparison to avian embryo fibroblasta,
should represent cells transformed by RSV. These cells differ
morphologically from their non-infected counterpart, but the
difference does not parallel the easily recognizable alterations
seen in avian embryo fibroblasts. Moreover, it is doubtful
whether these cells are tumorigenic in monkeys. The presence of
RSV, or its imprint, would help the matter considerably but the
detection of both poses a special problem.
Rons Sarcoma Virus in Mammalian Cell Cultures
Where, Oh Where is the RSV in the Mammalian Cell Cultures?
The rapid morphologic alterations observed in the RSV-avian
fibroblast conversion system do not take place in mammalian cells
infected with RSV. After exposure of GMK cultures to the Con
tinental group of RSV (Schmidt-Ruppin, Carr-Zilber, and
Diadkova) (16) the lesions are somewhat similar to those ob
served in the RSV-converted chick-embryo cells (Fig. 6). They
appear as discrete foci of round cells somewhat resembling a
dividing fibroblast piled up 3-dimensionally on monolayers of
seemingly normal cells. After a monolayer has been established,
these foci are discernible at each passage level and they can be
maintained indefinitely.
Morphologic changes observed after the exposure of GMK
cultures to the Anglo-Saxon strains of RSV (Bryan and Harris)
are of a different type than those found after infection with the
Continental strains. The lesions represent an accumulation of
three types of abnormal cells: elongated fibroblast-like cells that
accumulate in thick strands (Fig. 7), vacuolated giant cells
(Fig. 8), and cells with characteristic rosette patterns of their
nuclei (Fig. 9). The latter pattern has been observed previously
in sections of various animal neoplasms caused by tumor viruses
(Chesterman, personal communication) and in chorioid plexus
cultures infected with visna virus.
Baby chicks or cultures of chick embryo fibroblasts, inoculated
directly with human and GMK cultures infected with RSV or
RIF, showed no presence of a transmissible agent.
However, RSV lesions of human fibroblasts can be transferred
on GMK cultures. The agent could not be further transmitted to
chick tissue. Yet, baby chicks occasionally develop tumors when
inoculated with cells from mixed culture of RSV-infected GMK
and normal chick-embryo fibroblasts.
The difficulties in the recovery of transmissible RSV are char
acteristic for other mammalian cell RSV systems. For instance,
virus cannot be Isolated from extracts of RSV-induced rat and
hamster tumors as well as from tissue cultures originating from
these tumors. However, if intact cells obtained from the same
tissues are implanted into susceptible chicks, tumors constituted
of chicken cells will grow.
As stated by Dr. Habel, tumor viruses may leave their imprints
in the transformed tissue. One such imprint is a complement
fixation antigen present in avian tissue infected with the RSVavian leukosis group of viruses. Unfortunately, in RSV-exposed
mammalian tissue, presence of this antigen was demonstrated
only on rare occasions. Thus, we are left with a situation where
1986
CANCER
RESEARCH
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1966 American Association for Cancer Research.
VOL. 26
Neoplastic
neither a transmissible agent nor its imprints can be detected in
morphologically transformed tissue.
There is a charming Greek myth about the prophetic priestking of Délos,Anius, and his 3 daughters, Elais, Spermo, and
Oeno—whowere called the Winegrowers. Anius, himself a priest
of 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 trans
formation upon the 3 girls. Elais could turn whatever she
touched into oil; whatever Spermo touched was transÃ-ormedinto
corn; 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 we
might transform or convert cells under the effect of various agents
into malignant cells so that this transformation or conversion
would be a simple 1-step process which would add the missing
pieces to the puzzle of the origin of neoplasia?
On the other hand, it might take us longer to come up with a
formula to invoke King Anius' daughters—the transformation
trio—than it would to discover a way to deprive malignant tis
sue of all of the properties and traits which are characteristic for
malignancy. This is the most important problem of all.
References
1. Avery, O. T., MacLeod, C. M., and McCarty, M. Studies on
the Chemical Nature of the Substance Inducing Transforma
tion of Pneumococcal Types. Induction of Transformation
by
a Desoxyribonucleic
Acid Fraction Isolated from Pneumococcus Type III. J. Exptl. Med., 79: 137-58, 1944.
2. Basilico, C., and di Mayorca, G. Radiation Target Size of the
Lytic 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 of
the Transforming
and Reproductive
Abilities of Polyoma
Virus. Ibid., 64: 121-24, 1965.
4. Berwald, Y., and Sachs, L. In Vitro Cell Transformation with
Chemical Carcinogens. Nature, 200: 1182-84, 1963.
5.
. The In vitro Transformation of Normal Cells to Tumor
Cells by Carcinogenic Hydrocarbons. J. Nati. Cancer Inst.,
35: 641-57, 1965.
C. Black, P. H., and Rowe, W. P. SV40 Induced Proliferation of
Tissue Culture Cells of Rabbit, Mouse, and Porcine Origin.
Proc. Soc. Exptl. Biol. Med., 114: 721 27, 1963.
7. Brookes, P., and Lawley, P. D. Reaction of Some Mutagenic
and Carcinogenic Compounds with Nucleic Acids. J Cell.
Comp. Physiol., 64 (Suppl. 1): 111-27, 1964.
8. Carp, R. I., and Gilden, R. V. A Comparison of the Replica
t ion Cycles of Simian Virus 40 in Human Diploid and African
Green Monkey Kidney Cells. Virology, 28: 150-62,1966.
Transformation
9. Defendi, V. Transformation
in Vitro of Mammalian Cells by
Polyoma and Simian 40 Viruses. Prog. Exptl. Tumor Res., 8:
135-72, 1965.
10. Eagle, H. Metabolic Controls in Cultured Mammalian Cells
Science, 145:42-51, 1905.
11. Fernandez, M. V., and Moorhead, P. Transformation
of
African Green Monkey Kidney Cultures Infected with Simian
Vacuolating Virus (SV40). Texas Kept. Biol. Med., 2S: 24258, 1965.
12. Freeman, V. J. Studies on the Virulence of BacteriophageInfected Strains of Corynebacterium
diphtheriae.
J. Bacteriol.,6/.-675-88,
1951.
13. Girardi, A. J., Jensen, F. C., and Koprowski, H. SV4o-Induced
Transformation 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 Life
time of Human Diploid Cell Strains. Symp. Int. Soc. Cell
Biol., 3: 155-73, 1964.
15. Heidelberger,
C. Studies on the Molecular Mechanism of
Hydrocarbon
Carcinogenesis.
J. Cell. Comp. Physiol., 64
(Suppl. 1): 129-48, 1964.
16. Koprowski, H. The Emperor's New Clothes or an Inquiry into
17.
18.
19.
20.
21.
22.
23.
24
the Present Status of Tumor Viruses and Virus Tumors.
Harvey Lecture, Series 60, Academic Press, New York, pp.
173-216.
Moorhead, P. S., and Saksela, E. Non-Random Chromosomal
Abberations
in SV4o-Transformed
Human Cells. J. Cell.
Comp. Physiol., 6e: 57-83, 1963.
Ogden, C. K., and Richards, I. A. The Meaning of Meaning.
London: Routledge & Kegan Paul, Ltd., 1950.
Pontén,J., Jensen, F., and Koprowski, H. Morphological and
Virological Investigation
of Human Tissue Cultures Trans
formed with SV10. J. Cell. Comp. Physiol., 61: 145-63, 1963.
Stent, G. S. Molecular Biology of Bacterial Viruses. San Fran
cisco and London: W. H. Freeman and Co., 1903.
Stoker, M. Mechanism of Viral Carcinogenesis. Can. Cancer
Conf., 6: 357-68, 1966.
Temin, H. M. Nature of the Provirus of Rous Sarcoma. Inter
national Conference on Avian Tumors. Nati. Cancer Inst.
Monograph, No. 17: 557-70, 1904.
Todaro, G. J., and Green, H. An Assay for Cellular Trans
formation of SV40. Virology, 2$: 117-19, 1964.
—. Successive Transformation
of an Established
Cell
Line by Polyoma Virus and SV40.Science, 147: 513-14, 1905.
25 Todaro, G. J., Green, H., and Goldberg, B. D. Transformation
of Properties of an Established Cell Line by SV«and Polyoma
Virus. Proc. Nati. Acad. Sei. U. S., 51: 66-73, 1904.
26 Uetake, H., Luria, S. E., and Burrous, J. W. Conversion of
Somatic Antigens in Salmonella by Phage Infection Leading to
Lysis or Lysogeny. Virology, 5: 08-91, 1958.
27 Winocour, E., and Sachs, L. Cell-Virus Interactions with the
Polyoma Virus. I. Studies on the Lytic Interaction in the
Mouse Embryo System. Ibid., 11: 099-721, 1960.
SEPTEMBER 1966
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1966 American Association for Cancer Research.
1987
Hilary Koprowski, Fred Jensen, Anthony Girardi, and Irena Koprowska
. i ,
W]
-,
••
v
•»,
••
' 'v Ia
%
'<>
V-
-l
Vi
^
x
.
»
«
v
L
•-
FIG. 1. Sequence of transformation process in human cells. Left: transformation. Note change in morphology of cell and loss of con
tact inhibition which is characteristic in the early stages of transformation.
Middle: crisis. After approximately 22 weeks, SV40-transformed 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
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1966 American Association for Cancer Research.
VOL. 26
Xeoplaslic
Transformation
*'
FIG. 3. Cervical dysplasia showing undifTorentiated cells which become more conspicuous and form several rows known as basal cell
hyperplasia.
FIG. 4. Cervical dysplasia indicating the large differentiated cells which show nuclear abnormalities.
SEPTEMBER
1966
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1966 American Association for Cancer Research.
1989
•â-v*v"-•'•
Â¥3s
€¢
'
^
'S
s»
ft
•Ã-¿^-
&
Ã
Fia. 5. Carcinoma ¿nsÃ-Ã-«.
FIG. 6. Foci oÃ-RSV-infected
1'J'JO
cells (Schmidt-Ruppin
strain) of GMK cultures.
CANCER
RESEARCH
VOL. 26
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1966 American Association for Cancer Research.
l •"~~~-
9
-, •:,»:••:.•}•-â
v:.-.v;r..;.
•'v^i-îi-'-'-.s:v?-i?¿A
Ã-w'*-:
-.v-^vv-r?ü/~>
.-••/
^ : t--"
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.
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1966 American Association for Cancer Research.
'»•'"'
'*' /; -' -i•
i.'',.''/*•"'-»'
^1<
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.
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1966 American Association for Cancer Research.
v f//
^
^
^
*
>- -
\
«*
•^ v
FIG. 11. Plaques observed in human diploid cell strain infected with RSV.
FIG. 12. Control GMK cells.
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1966 American Association for Cancer Research.
Neoplastic Transformation
Hilary Koprowski, Fred Jensen, Anthony Girardi, et al.
Cancer Res 1966;26:1980-1993.
Updated version
E-mail alerts
Reprints and
Subscriptions
Permissions
Access the most recent version of this article at:
http://cancerres.aacrjournals.org/content/26/9_Part_1/1980
Sign up to receive free email-alerts related to this article or journal.
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Department at [email protected].
To request permission to re-use all or part of this article, contact the AACR Publications
Department at [email protected].
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1966 American Association for Cancer Research.