Kidney Tumors of the Leopard Frog: A Review'
KEEN A. RAFFERTY,JR.
(Department of Anatomy,
The Johns Hopkins
University School of Medicine, Baltimore, Maryland)
BIOLOGY OF THE LÃœCKE TUMOR
Kidney tumors of the leopard frog (Rana pipiens) were
first correctly described in 1934 by Balduin Lücke,of the
Pathology Department,
University of Pennsylvania
School of Medicine (27). Previously, Smalhvood (65)
and Downs (10) had described the tumors but believed
their origin to be adrenal and intestinal, respectively.
Lückeexamined this material and was the first to recog
nize their true nature as epithelial tumors of the kidney
(27). He described the tumor as metastasizing adenocarcinoma and devoted much of the remaining 20 years of
his life to its study (27-36; 62). In the process he ex
amined more than 10,000 frogs and determined that 2.7
per cent bore spontaneous tumors, with a frequency
about twice as high in males as in females (32). The
tumors are of particular interest today because of the
likelihood that they are of viral origin and naturally
transmissible. Since the system seems to be unusually
complex, it is of considerable general interest and justifies
an attempt to marshall in one place the significant in
formation now available.
Pathology.—The tumors are invasive, unencapsulated
adenocarcinoma (Fig. 1), with extreme forms resembling
adenoma (Fig. 2) to anaplastic cell carcinoma (Fig. 3;
[30]). Although it is customary to refer to all epithelial
malignancies of the kidney as Lücke
tumors, it is important
to note that a graded series of types actually occurs.
Several other kinds of tumor have been reported in the
kidneys and other organs of frogs (see review by Balls
[2]), but it is clear that the vast majority of kidney tumors
properly belong to the group described by Lücke. In a
series of subpalpable tumors representative of field popula
tions, 913 of 1,429 were bilateral in distribution, with
neither kidney favored in unilateral cases (32). Since
growth appears always to be progressive, the unilateral
condition is undoubtedly referable in most cases to recent
origin. In a few cases, however, one kidney may remain
grossly free of carcinoma even after a tumor in the other
has grown to quite a large size. Much more rarely,
metastasis to the liver and lungs is seen before establish
ment in the contralateral kidney. Similarly, one com
monly finds tabs of normal kidney attached to very large
tumors. Thus, the conversion of adjacent normal cells
seems to be controlled in some cases by local factors.
That the tumors grow in part by inducing transformation
of adjacent cells is well established by the studies of
Duryee (13, 14) and Tweedell (69) in which precancerous
transformation was followed in such areas.
* Aided by Grant no. CA-06008(S1) from the U.S. National
Cancer Institute.
Received for publication August 9, 1963.
Some tumore grow relatively slowly. Although in a
few tumors regression is strongly suggested on histological grounds (27), no cases of complete remission are on
record. At death, all normal kidney tissue may be
destroyed, in which case terminal edema is usually ob
served. In other cases, however, no normal tissue is seen
in afflicted frogs killed in apparently good general health.
Most tumors occur (when small) as multicentric foci,
implying simultaneous malignant transformation
at
several sites. Duryee (13) has pointed out that occasional
'replica' tumors retain the shape and anatomical relation
ships of the kidneys until large, giving the impression of
simultaneous transformation of all of the kidney tissue.
Formation of replica tumors is not necessarily associated
with injection of tumor extracts. An example of a
replica tumor is illustrated in Figure 4.
Metastasis is not usually seen in tumor-bearing frogs
taken in the field, but occurs frequently when they are kept
in the laboratory for a few months. Lückeet al. (36)
showed that both high temperature and copious feeding,
examined as independent factors, greatly increased the
incidence. The lung is most frequently involved, with
the liver a close second. Other common sites are bladder,
mesentery, peritoneum, pancreas, intestine, ovary, and
orbit.
THE TUMOR CELL
Cells of origin.—Nearlyall workers agree that the tumor
cells probably arise in the proximal tubules, since many
possess a brush border (13, 32), seen as microvilli in the
electron microscope (19). Fawcett (19) also finds that
membrane profiles and desmosomes are similar to those of
normal proximal tubule cells. The present writer's
experience is in accord with this idea, except for a few
instances of apparent early malignancy in the transverse
collecting ducts or in the Wolffian duct1 (Fig. 5). Figure
6 illustrates the junction between normal and neoplastic
cells in the proximal convoluted portion of a single nephron.
Inclusions.—Consideration of inclusions has led to much
confusion, partly because of problems in definition. Thus,
various workers disagree whether organelles present in
normal cells can be considered to be inclusions when
present in some abnormal form in diseased cells (7).
This has been the case in the study of Lücketumor cells.
Lücke described large, lightly acidophilic nuclear
inclusions as present in at least some cells of half the
tumors he examined (32). These inclusions were restudied by Cowdry (7) and by Fawcett (19), both of whom
described them as type A in Cowdry's now classical
terminology, and noted that they are similar to those seen
Unpublished
observations
by the writer.
169
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Cancer Research
in numerous virus diseases, such as herpes simplex.
Figures 7 and 8 represent tumor cells with and without
type A inclusions.
Using the electron microscope, Fawcett found particles
characteristic of mature virus and virus precursors present
in cells bearing type A inclusions. He described three
types of inclusion commonly found in some of the cells of
'inclusion' tumors:
1. Nuclear inclusions, Cowdry type A; large, lightly
eosinophilic. Chromatin marginated, mitosis rare, cells
appear inviable. Nucleoli frequently not seen hi light
micrographs, but present in electron micrographs as
small but essentially normal structures embedded in the
marginated chromatin. Inclusion often contains nu
merous empty vesicles 100 m/¿
in diameter (presumed virus
precursor forms) and a few vesicles with dense nucleoids
of 30-40 mp (presumed mature virus).
2. Filamentous cytoplasmic inclusions with which are
associated considerable numbers of virus particles of the
mature type. These are probably seen in light micro
graphs as irregular particles which stain with hematoxylin.
3. Intracytoplasmic clusters of very large, seemingly
empty, membranous vacuoles. Only occasional viras
particles are seen in association, and this is thought to be
incidental; possibly correspond to eosinophilic cytoplasmic
inclusions described on the basis of light microscope
studies of cultured cells, and believed to be derived from
extruded nucleoli (12; 14-18; 24, 25; 39, 40).
Type A nuclear inclusions: their origin and significance.—
In the present writer's experience, type A nuclear inclu
sions as seen in sectioned tumor tissue are distinct and
characteristic structures.
In the case of a particular
tumor, it is a simple matter to decide whether some of the
cells have, or do not have, such inclusions. In addition
to the intrinsic distinctiveness of the large, pale inclusion
itself, other characteristics, such as margination of
chromatin, a tendency for large numbers of inclusion
cells to occur in certain areas, and the absence or near
absence of mitotic figures (30, 32) all combine to render
determination rapid and convincing (compare Figures 1
and 2, and 7 and 8). Because particles resembling
mature viruses have been seen in electron micrographs
only in connection with cells bearing type A inclusions, the
inclusion assumes an even greater significance as a distinct
entity and becomes especially important in view of linger
ing uncertainty concerning the origin of the Lücketumor.
For this reason the present discussion will center on the
type A inclusions.
Reports of other studies of inclusions in Lücketumor
cells (referred to below) have been based almost exclusively
on the study of cultured cells. However, it should be
stated at the outset that type A inclusions as originally
described in the tumor cells (29, 30) either do not occur in
tissue cultures of inclusion-bearing tumors (31) or else
occur in occasional individual cells without regard to the
presence or absence of the inclusions in the donor tumor.
The present writer has studied monolayer cultures of
some twelve Lücketumors and has been struck by the
conspicuous and uniform absence of type A inclusions.
A typical tumor cell culture is illustrated in Figure 9 and
may be compared with a culture derived from normal frog
Vol. 24, February 1964
kidney cells (Fig. 10). Although the two are readily
distinguishable, type A inclusions are seen in neither.
There is obviously much uncertainty as to how tumor
cultures might be screened visually for the possible
presence of mature infectious virus, and there is the
additionally indicated possibility that tumor cells cultured
by available technics produce little or no virus of the kind
visualized in the electron microscope. From the patho
logical appearance of cells with type A inclusions one
might expect that rapid replication of the associated agent
in cultures could lead to cytopathogenic, rather than
transformation, effects.
On the basis of light-microscopic studies of cultured
tumor cells, Duryee (12-18), Kopac et al. (24,25), Mateyko
et al. (39, 40), and Tweedeil (69-71) have described a
variety of inclusions which they considered to be derived
from originally normal organelles as a result of malignant
disease. These structures consist of nucleoli which are
enlarged, vacuolated, or contorted into fantastic and
bizarre forms, plus clumps of chromatin associated with
nucleoli or with the nucleolar-organizer loci of chromo
somes; large, lightly acidophilic cytoplasmic bodies;
intensely basophilic Feulgen-positive and Feulgen-negative
cytoplasmic structures; and cytoplasmic vacuoles. Many
structures, including Feulgen-positive bodies, are extruded
from the nuclei of malignant cells and have been observed
eventually to enter the nuclei of normal frog kidney cells
in mixed cultures (14). The process is followed by
transformation.
Duryee considers that
this DNAcontaining material is infectious and is produced in
association with the nucleolus. According to this view,
the viral genome is integrated into that of the host at or
near the nucleolar-organizing center. Malignancy may
thus be considered a 'nucleolar disease,' acting to cause
hyperactivity of the nucleolus with a resulting over
production of RNA and protein and consequent stimula
tion of cell division (14,16,18).
The degree of malignancy
and of nucleolar hyperactivity are well correlated (16).
Lunger's study with the electron microscope appears to
indicate that virus particles of the mature type first
appear in close relationship with the marginated chro
matin, but not in any particular association with those
areas containing embedded nucleoli.2 However, Lunger's
study deals with the first appearance of the viral nucleoid
and not with the possible production of infectious nucleic
acid, which could occur elsewhere at an earlier stage.
Similarly, it gives no information concerning the proximal
site (or sites) of cell damage. Thus, both sets of findings
could be correct with respect to these points.
There is no doubt from the cited studies of Duryee,
Kopac et al., and Mateyko et al. that nucleoli sometimes
give rise to structures which resemble type A inclusions.
Duryee has observed that it is possible to construct a
sequence of transformation from normal nucleoli to
structures indistinguishable from type A inclusions (14,
15). Nevertheless, these transformations seem to be
culture phenomena related to the degree of malignancy of
the cell, not to the presence of type A inclusions (and of
virus) in the donor tumor: they are found in cells derived
2Personal communication: Philip Lunger, Rockefeller Institute, New York 21, New York.
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RAFFERTY—KidneyTumors of Leopard Frog
from a variety of malignant tumors of various animals,
including the frog (12, 13), and are not, like type A inclu
sions, generally associated with the presence of demon
strable virus. Moreover, they do not occur in large
numbers in localized areas but are seen sporadically in
isolated cells in vitro.
Additionally, there is doubt whether the type A inclu
sions of in vivo tumors are in fact derived from nucleoli.
Electron micrographs of such material show the presence
of apparently normal nucleoli2 (19), even though they are
not usually visible in similar preparations sectioned and
stained for light microscopy. An example is illustrated in
Figure 11. Fawcett noted in the cited work that most of
the virus-like particles seen were embedded within the
type A inclusions and were not associated with the nucleoli.
The latter were always seen peripherally, in association
with the marginated chromatin. These observations have
been confirmed by Lunger.2
Finally, the acidophilic nuclear structures of cultured
malignant cells are not associated with margination of
chromatin (16, 18, 24), whereas margination is a char
acteristic feature of nuclei containing type A inclusions in
vivo (7).
Tweedell, in observations of cultured Lücketumor cells
stained with fluorescent dyes, also noted marked nucleolar
enlargement but was unable to follow the process to the
stage of unequivocal type A inclusions (69-71). Flewett,
however, demonstrated the evolution of nucleoli to
structures apparently somewhat similar to type A inclu
sions after infection of cultured cells with fowl plague
virus (20).
There seems no doubt that the type A nuclear inclusions
of Lücke,Cowdry, and Fawcett are characteristic and
possibly unique in their association with production of
large amounts of a mature virus. In addition, the
inclusions do not appear to be derived from nucleoli but
could instead be the product of viral replication, and hence
new structures.
In all probability their presence, as
determined by conventional light microscopy, is the most
convenient available guide to the presence of virus.
It hardly needs saying, however, that the nature of the
virus (causative or passenger disease agent) is unknown.
FACTORS INVOLVED IN THE FORMATION
OF TYPE A INCLUSIONS
Environmental temperature.—Study of the literature
makes it abundantly clear that type A inclusions are seen
only in some material. Thus, Duryee,3 Rafferty,1 and
Tweedell (69-71) have encountered very few examples in a
total of more than 500 tumors examined, compared with
Lucké'sincidence of 50 per cent among some 900 tumors
(32) and similar incidences found by Fawcett (19),
Lunger,2 and Granoff4 in smaller series. Since the inclu
sions are striking, failure of recognition is not a factor.
On the other hand, analysis of the details of the acquisition
and husbandry of tumor frogs by various workers strongly
suggests prolonged low temperature as the principal factor
1 Personal communication: W. R. Duryee, George Washington
Univ. Sch. Med., Washington D.C.
4 Personal communication:
Allan Granoff, St. Jude Hospital,
Memphis, Tenn.
171
favoring inclusion formation and, presumably also, virus
production. Lückenoted that the inclusions are more
frequently seen in winter and spring than at other seasons,
although he gave no data dealing with seasonal occurrence
(30). Both Duryee and Rafferty, who seldom find such
inclusions, have worked for the most part with large
tumors arising in laboratory frogs which are maintained
at temperatures of 15°C.or higher. Indeed, the fact
that a tumor is large is in itself an indication that type A
inclusions are less likely to be present, since mitosis is
relatively infrequent in such tumors. Lückereported a
case of a tumor which arose after injection of an inclusion
tumor extract as reaching palpable size and containing an
even higher proportion of inclusion cells than the original
tumor (28). One may assume, however, that this in
dividual, like most others in Lucké'sexperiments, was
maintained in a cold vivarium which was partly open to
the outdoors (30). It seems highly significant that
workers who have regularly found inclusions have with
few exceptions worked with comparatively small tumors
taken from frogs recently arrived in the laboratory.
Furthermore, most such tumors have been found during,
or shortly after the end of, the hibernating season. The
workers referred to are Fawcett, Lücke,Granoff,4 and
Lunger.2 Roberts6 and Rafferty1 have observed inclusions
only in such frogs. The former attempted to induce
formation of inclusions by keeping frogs bearing large
tumors at 4°C.for 30 days, but without conclusive results.6
The possible role of hibernation.—It is suggested that a
type A inclusion stage, accompanied by the production of a
prolonged burst of mature virus, occurs as a phase in the
development of at least some tumors, probably in response
to hibernation. Later, the tumor may enter a phase of
rapid growth as the environmental temperature increases.
At this time, the cells with large type A inclusions die, and
the tumor is repopulated by cells lacking them. During
this phase the tumors are presumably deficient in mature
virus, although from Duryee's results (13) it is clear that
they may produce infectious nucleic acid which is extractable. Fawcett's (19) and Lunger's2 observations
showed that considerable numbers of mature particles are
present in intercellular spaces, but unfortunately there has
been no direct demonstration that virus is present in the
urine during the inclusion phase. Tumors which arise in
the laboratory either do not pass through a nuclear
inclusion phase or else do so fleetingly at very early
stages of development. It should again be noted that,
although inclusion tumors grow well in tissue cultures
(maintained at relatively high temperatures),
their
progeny lack the type A inclusions (31).
THE STATUS OF EXPERIMENTAL
TRANSMISSION
Experimental evidence of viral etiology.—Study of the
Lücketumor has followed a somewhat unusual course.
In general, infectiousness is suspected on biological or
epidemiological grounds and then confirmed experi
mentally. From the first the Lücketumor was suspected
of being viral in etiology, and by 1952 viral causation was
6 Personal communication:
Maria Roberts,
Univ. Massachusetts, Amherst, Mass.
c/o L. Roberts,
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considered to have been established (32). In 1963,
virtually all workers would undoubtedly agree that the
cause is probably viral, but part of the earlier basis for
confidence in that conclusion has been mitigated by the
recent establishment of high spontaneous rate under some
conditions and the consequent need to re-interpret the
results of transmission experiments. Indeed, retro
spection leads one to conclude that results of transmission
experiments have seldom been comfortably clear.
Four groups of workers have published the results of
attempted transmission involving injection of tumor
extracts. These are Lückeand co-worker (30, 32),
Duryee (13), Roberts (51, 52), and Rafferty (48). Of
particular interest is a detailed look at the data of Lücke,
brought together in his final publication in 1952 (32).
Table 1 is a summary of those data. 'Cell-free' extracts
were prepared either as filtrates or as extracts of cells
rendered inviable by desiccation, freezing, or storage in
Vol. 24, February 1964
formed by Duryee (13), who was concerned with the
transformation process and not with establishing filtrability. Duryee observed a somewhat higher tumor
incidence in experimental animals, probably related to his
route of injection through the kidney or to the method of
preparing extracts. After 1.4 months, the experimental
incidence amounted to 17 per cent (nine of 54 injected
frogs). For the reasons given, the experiment was not
controlled on an animal-for-animal basis, and so a com
parative incidence was not available in this case. How
ever, although Duryee has observed tumor formation in
frogs not given injections and kept in the laboratory
(13-15), the rate is low in his experience.6
Occurrence of spontaneous tumors.—Roberts (52), on the
other hand, was unable to find significant differences
between injected and uninjected frogs, but both incidences,
occurring in frogs observed for only 4 months, were
relatively high at about 17 per cent. Rafferty (49)
TABLE 1
EFFECTS OF INOCULATIONOF TUMOR MATERIALS IN FROGS
Taken from Lücke,Ann. N.Y. Acad. Sci., 64: 1093-1109, 1952.
INOCULATION0-3
AFTER
OÕODP'Cell-free'
TREATMENT
months8/351*
extracts
Transplants
in kidney
ControlMONTHS
months12/104
2.3f
2/31
16/683
6.5
2.34-6
months46/222
6
11.5
7/19
10/166
36.8
7/18
6.0Over 7/104
* Numerator: number of frogs which developed carcinoma;
f Per cent positive.
glycerol; controls received either no injections or injections
of normal tissue extracts. A variety of injection routes
was used. Since neither route of injection nor method of
preparation seemed to have much influence on the out
come, results have been lumped in the table presented
here. During observation, the spontaneous incidence
rose from 2.3 per cent (roughly the incidence seen in field
frogs) to 6.7 per cent in control animals kept for 6 months
or longer. After 3 months, the incidence in frogs given
injections of cell-free materials did not change with
respect to the controls, but the ratio doubled in the second
3-month period and eventually tripled. In the case of
living cells transplanted to the kidney or environs, the
relative incidence doubled after the first 3 months, then
reached a level 6 times higher than that of the correspond
ing controls within the next 3 months. The level did not
seem to be continuing to rise in frogs of this group which
were kept for more than 6 months but may have been
continuing to increase somewhat in the controls. In
conclusion, the injection of cell-free extracts eventually
tripled the incidence, relative to that of controls. This
result is not reassuring with respect to the efficacy of
cell-free materials, particularly since extraneous factors can
influence the rate of tumor formation in known virus
tumor systems (22, 37).
The second group of injection experiments was per-
20.7
denominator:
38.9
9.5
16/68
6.7TOTALS66/677
33/953
23.5
3.5
total number.
reported that 17.3 per cent of injected frogs (29 of 168)
developed tumors within 2-4 months, compared with 6.9
per cent (five of 72) for the corresponding controls, which
received no injections. However, similar frogs observed
for from 5 to 11 months showed incidences of 25.2 and
27.8 per cent, respectively (28 of 111, and 32 of 115 frogs).
These results appear to show that the injection of tumor
extracts accelerates tumor development somewhat for the
first few months but does not change the final incidence,
provided observations are continued for a sufficiently long
period.
Factors influencing spontaneous tumor formation.—
Investigation of conditions which influence the develop
ment of spontaneous tumors has permitted a satisfactory
explanation for the differences in spontaneous incidence
found by various workers in their control groups. These
influencing factors were termed 'promoting conditions'
(47-50) and consist of:
1. Temperature: Formation of tumors is drastically
retarded at environmental temperatures of 13.5°C.,
whereas temperatures of 23°or 26°C.yield spontaneous
tumors at the incidence of 25-50 per cent (47).
2. Body length: Frogs of medium size seldom develop
tumors, and juvenile (first-summer) frogs probably almost
never do. The spontaneous incidence increased sharply
in males of 70 mm. or more in body length and in females
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RAFFERTY—KidneyTumors of Leopard Frog
in excess of 74 mm. (48). It would seem a reasonable
guess, based on casual field observation,1 that these are
third-summer frogs or older: growth rate slows after the
first 2 years, and it may be impossible to set upper limits
on age.
3. Time: An incidence of 25 per cent or thereabouts is
not usually reached until after about 8 months of observa
tion (46) under high temperature conditions.
These parameters of tumor formation have been used to
explain why Duryee and Lückeboth observed low spon
taneous rates, since both workers had by chance utilized
conditions of environmental temperature,
age, and
observation time in various combinations which discourage
tumor development (see "Discussion" in [48]). The
results of Duryee's inoculated groups appear to have been
highly significant, since a 17 per cent incidence was realized
in injected frogs (13) under conditions sharply inimical to
development of spontaneous tumors (48).
Acceleration effect.—The accelerating effect apparently
associated with injection of tumor extracts is of some
interest, although more data concerning its magnitude
and regularity would be desirable. Rafferty observed the
effect consistently, even in small groups of animals. A
similar effect obtained by Roberts (52) was not large
enough to be statistically significant, however. If the
accelerating effect can tentatively be accepted as real in
some situations, it was suggested that the phenomenon
may be analogous to the dose-response effect studied by
Bryan (5), who showed that the time required for develop
ment of Rous sarcoma is related to size of the initiating
dose within rather wide limits. Furthermore, length of
the developmental period is a better measure of the
amount of initiating virus present because of considerable
variation in susceptibility of the host chickens. There is
no doubt that frogs are at least as variable in suscepti
bility, since about half appear to be completely refractory.
It was also suggested that the nature of the accelerating
effect may be one of adding to a pool of virus already
present and perhaps acquired by natural routes in young
adult or larval stages, or else in ovo (49, 50). A possibly
similar accelerating effect has been observed in the case
of mouse lymphatic leukemia (63), the agent of which is
known to be transmitted early in life.
Other possible influencing factors.—In addition to those
discussed, other factors must be considered as possibly
involved in tumor formation. Hormonal factors are
obviously concerned, since the tumors are partly sexdependent (32).
Another such possibility is nutritional condition. This
factor is difficult to test, because the formation of spon
taneous tumors at a high incidence requires maintenance of
frogs at room temperature for prolonged periods, and
feeding is necessary to prevent starvation.
However, a
recent report indicates that frogs which were emaciated at
the end of 8 months' observation were as likely to produce
tumors as were normal animals (50). This is in interesting
contrast to Lucké'sfinding (36) that both elevated tem
perature and feeding, examined independently, promoted
the development of metastasis in frogs already bearing
tumors. Hence, factors which influence the development
173
of formed tumore are not necessarily those involved in
their origin.
Parasitism has been suggested as an influencing factor
by several workers (see "Discussion" following the paper
cited in [46]), although helminth infestation is high in both
susceptible and refractory races. Duiyee (17) and Lücke
(27) observed several cases in which worms were found
adjacent to small tumore. Moreover, it is evident that
most tumore examined have been large specimens in which
any possible parasite association might well have been
lost. Lückestated that trematode and myxosporidian
parasites of the kidney were more common in frogs from
areas in which the tumor is commonly found than in
areas where it is rare or nonexistent; however, he did not
regard them as of etiological importance (27). Other
areas are now known in which infestation is high but in
which Luckétumore apparently do not occur (38). In
recent observations of uninoculated frogs helminthic
infestation of lungs and coelom was roughly the same in
normal and tumor-bearing animals (50). Kidney hel
minths were seldom seen on gross examination, and when
small worms were discovered on section there did not
seem to be any consistent tendency toward pronounced
hyperplasia of nearby nephrons,1 as was observed by
Duryee (17).
Although parasitism may be a factor in the development
of the tumors (possibly encouraging activation of latent
virus through chronic irritation), no simple or obvious
relationship has been shown. In studies of flatworm
infestations of the kidneys and lungs of leopard frogs and
of bullfrogs (Rana catesbiana), Duryee often observes
advanced hyperplasia in association with the worm.3
Occasionally, striking transformations are seen in situa
tions where parasitic involvement is unequivocal, as in
apparently neoplastic areas of lung epithelium which
serve as the attachment point and which project into the
buccal apparatus.
From inspection of this material the
present writer agrees that many such areas are morpho
logically indistinguishable from adenoma, or, in other
cases, from adenocarcinoma. A few specimens show
apparently
adenocarcinomatous
transformations
con
tinuous with normal or hyperplastic epithelium elsewhere,
and it is doubtful that these growths can be distinguished
from renal tumors. Most examples of transformed
pulmonary epithelium are seen in frogs already bearing
grossly detectable kidney tumors, but their occurrence on
epithelial surfaces militates against their origin as mé
tastases. It was pointed out, in fact, that some of the
kidney tumore could be secondary to primaries in the lung:
a few cases of small adenocarcinomatous growths have been
seen confined within Bowman's capsules in kidneys
apparently otherwise free of tumor. Although such
emboli could also be of renal origin from small undetected
sites elsewhere, these findings emphasize the possibility
that an unknown proportion of apparently endogenous
renal tumore may in fact be derived elsewhere from
parasite-induced primaries. It was suggested that kidney
tumors in which adjacent tubule epithelium does not show
evidence of transformation might be of such origin. An
objection to this interpretation is the fact that no large
primary tumors of the lung have been seen, but there are
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well-known examples from human pathology in which
large distant métastasesare seen while the primary is still
quite small. Although these results are fragmentary at
present, they caution against the assumption that all
tumors of the kidney are of endogenous origin.
THE QUESTION OF NATURAL
TRANSMISSION
Field transmission is implied by high spontaneous
incidence and by the likelihood that virus-like particles
appear in the urine of frogs with inclusion tumors. Direct
tests for transmission in the laboratory via natural routes
were made by Lücke(32). Although some test groups
seemed to be affected, others were not, and Lückecon
cluded that transmission was not demonstrated.
He did
not, however, state whether frogs bearing inclusion
tumors were used as the 'infecting' source. Although the
environmental conditions under which these experiments
were performed were not detailed, one may conclude that
these experiments, like others from Lucké'slaboratory,
were conducted under conditions unfavorable for the
appearance of spontaneous tumors. Roberts found that 4
months' individual isolation had no influence on the high
tumor rate in frogs kept under promoting conditions (52)
but utilized animals which had been shipped by a dealer in
crowded containers. Rafferty (50) observed frogs isolated
from the moment of capture in the field and then kept for
8 months under promoting conditions. The incidence
was the same as in the crowded controls, however, with 24
per cent developing tumors in both groups. It is evident
that natural infection takes place, if at all, during an
earlier stage of development.
TRANSPLANTATION
STUDIES
Transplants to adults.—Homologous transplants of
tumor cells to adults do not normally succeed, although
Lückedemonstrated that many tumors take and grow
well in a "privileged site"—the anterior eye chamber
(33-35). Frogs bearing such growths often develop
tumors of the kidney, although the significance of tumor
formation is not always clear, since conditions which
favor growth of the transplant are also those which favor
formation of spontaneous tumors. At any rate, more is
involved than a simple seeding of cells into the circulation,
since tumors always arise first in the kidneys, rather than
in the common metastatic sites (33).
The anterior chambers of various species of Rana
support a growth of 65 per cent or more of tumors trans
planted, whereas fewer takes are seen in species of Bufo
and none in goldfish and alligators (34,66,67).
Regardless
of the degree to which the primary transplant prospers in
the anterior chamber, however, autochthonous kidney
tumors appear only in Lake Champlain Rana pipiens
and not in frogs of the same species taken in Wisconsin,
Illinois, or Kentucky (55, 67). Transplants typically grow
well for a time, and then regress. In a proportion of
cases, regrowth may then appear, sometimes followed by
a second regression and regrowth. These are referred to
by Tweedell as primary and secondary regrowths and
tend to be successively more malignant in character:
regrowing tumore may fill and destroy the entire eye (66,
Vol. 24, February 1964
67). In some cases, destruction of the lens occurs during
first growth, and the lens may then regrow (an abnormal
occurrence in anurans) after regression of the transplant
(68).
Although, as noted, frogs of the Wisconsin race did not
develop tumors even after secondary regrowth of a tumor
transplant, tumors did appear in the kidneys when tissue
from the primary or secondary regrowths were re-trans
planted to the eyes of Wisconsin frogs. Furthermore, sec
ondary regrowths induced a poxlike histolytic disease in
those hosts which failed to develop tumors in the kidneys
(66).
There does not seem to be a change in malignancy asso
ciated with serial subculture of primary growths, how
ever. In strains carried in the anterior chamber for
fourteen generations (62) and nine generations6 (see also
9, 23), no such increase was observed. In the latter case
karotype analysis of the starting tumor, plus generations 2,
4, 6, and 8 showed the chromosome number to be essen
tially diploid in all cases, with only a slight tendency
toward development of chromosomal abnormalities and
polyploidy in the sixth and eighth generations (8, 9).
Transplants to embryos.—Transplants of tumor tissue to
tadpole tails, coelom, or trunk mesenchyme often grow
for a while but regress before or during metamorphosis
(4, 41). Interestingly, tumors then develop in the kidneys
of some of the juvenile adults soon after metamorphosis,
an event which seldom or never occurs normally. In all
such cases of one series the original transplant had been
made to the vicinity of the kidney (4), and it is difficult
to rule out the possibility that the later tumors could have
represented regrown transplants
in immunologically
tolerant adults. In the second series, however, trans
plants were made to the tail and resorbed well before
metamorphosis; furthermore, the host frogs were of the
Wisconsin race (41). These observations plus those
noted concerning the behavior of transplants to the eye
suggest that the genetic resistance of Wisconsin frogs may
operate through a more rapidly acquired immune response.
An interesting observation is that the infiltrative host
reaction is much more pronounced when Vermont tumore
are transplanted to Wisconsin frogs' eyes than when
transplanted either to Vermont frogs' eyes or to those of
alien species (66).
The claim has also been made that tumor tissue grows
on the chorioallantoic membrane of hens' eggs, provided
the incubation temperature is lowered to 35°C. (45).
Membrane nodules were sectioned to confirm the presence
of viable tumor tissue. As many as twenty serial mem
brane transfers were made, after which the egg material
grew in anterior chambers, accompanied by formation of
tumors after 4-6 months. These interesting findings
deserve restudy. At least one attempt to confirm the
findings has failed.7
All other transplant work has involved homologous or
xenoplastic transfer to nonprivileged sites, which results
at best in slow growth for a time (54, 55). Rose et al.
(52) stated that such transplants may revert to normal
•Personal communication:
R. McKinnell, Newcomb
New Orleans, La.
7 S. Silver and K. Rafferty, unpublished observations.
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College,
RAFFERTY—Kidney Tumors of Leopard Frog
175
tissue under the influence of a regeneration field within the Tweedell's) might have been caused by activation of
salamander limb. In this case it was possible to identify unrelated passenger tumor agents (15, 16). In view of
particular cells by means of size and other differences the fact that enhanced growth of transplanted fragments
between frog and salamander nuclei; tumor cells had of altered kidney tumors was a concomitant feature in the
apparently given rise to muscle, cartilage, and fibroblasts. appearance of histolytic disease, and that histolytic
Mizell (41) has restudied this effect, utilizing tadpole tails disease tended to appear in frogs that failed to develop
kidney tumors (67), the interpretation that the agents of
as the host site and amputating through temporarily
all these conditions are closely related seems more likely.
established but slowly growing implants. He subse
quently noted breakdown and disappearance of those Alternatively, the agent may have some polyoma-like
characteristics.
portions of the tumor which resided in the field of regenera
tion, whereas more proximal portions continued to survive.
TISSUE CULTURE STUDIES
Although an adequate marker was not available in this
The
question
of transformation in vitro.—Both tumor
case, study of longitudinal sections suggested that some
and normal kidney cells are readily amenable to primary
tumor cells may have reverted to normal and were partici
culture by conventional technics utilizing diluted mam
pating in formation of the regeneration blastema. Pre
malian media (1, 13, 31, 37, 71, 72) or a modified Earle's
liminary results by the same worker, utilizing Tritiumlabeled tumor cells, tend to confirm this finding.8
medium (65), but neither has been successfully subculRuben (57-61) found that tumor transplants in regener
tured for more than a few generations. Tumor and
normal cells are readily distinguished either in monoating salamander limbs can result in formation of super
layer cultures established from dissociated cells (Figs.
numerary limbs; however, it was subsequently shown that
normal kidney is even more effective in this respect (60). 9, 10), or in organ cultures. Four groups of workers have
King et al. (23) showed that replacement of the nucleus of exposed cultures of normal adult frog kidney cells to
a normal unfertilized frog egg by ten to twenty Lücke tumor extracts without observing cytopathogenic effects.
Duryee (14, 16-18), Auclair (1) and Smith9 have observed
tumor cell nuclei could support gastrulation and neurulation in a few instances but that no embiyos survived until changes which seemed to represent a slow or incomplete
metamorphosis.
transformation of normal cells after exposure to tumor
Induced transformation of an agent.-—Ofparticular filtrates, but it is doubtful that this system is presently
significance concerning the etiology of the tumor is the practical for virus assay. In unpublished studies Rafferty
finding of Rose et al. (55, 56) that the sojourn of tumor seldom found evidence of transformation, and possible
tissue in the limb of immature salamanders (efts), or in positive cases were vague in character, resembling normal
adult salamanders during limb regeneration, leads to cells much more closely than neoplastic ones. Possibly
abnormal cartilaginous growths in the host periosteum.
of great significance, however, is the apparent fact that
Moreover, transplantation of these induced growths to no published studies have involved extracts intentionally
the eye of the relatively refractory Wisconsin race of frogs made from type A inclusion tumors.
led to the development of autochthonous renal tumors, an
Auclair determined that cultures of exposed normal
event which did not occur following the direct establish
cells could grow upon transfer to anterior chambers of
ment of original Vermont frogs' tumors in Wisconsin intact adult frogs (1), a situation in which normal kidney
frogs' eyes (66, 67). Retransplantation of one of the tissue is merely maintained or, more often, rapidly re
tumors thus induced led to the formation of both periosteal gresses (1, 31). The character of the eye chamber growth
cartilaginous growths and additional renal tumors. When was not determined histologically.
the growths of frog cartilage were in turn transplanted to
As previously noted, Lückefound that tumors with
other Wisconsin frogs' eyes, generalized histolytic disease inclusion-bearing cells could be cultured but that nuclear
resulted (55, 56). Thus, tissue and host specificity were inclusions did not occur in the cultured cells (31). Find
broadened by residence in salamander tissues. It is ings of the extensive culture experiments of Duryee
difficult to explain these findings without invoking a (12; 14-18), Kopac et al. (24, 25), and Mateyko et al.
causative virus, and this work now constitutes one of the (39, 40) have been discussed.
principal bases for the belief that the Vermont tumor is
BIOCHEMISTRY
OF THE TUMOR
indeed viral in origin.
Duryee's observations that Feulgen-positive materials
Rose et al. (56) explain these results in terms of altera
tion of the original kidney tumor agent as a result of extruded from cultured tumor cells appear to be infectious
interaction between the agent and cells of a foreign host. (14, 16, 18) invites a tentative inference that the causative
Dulbecco has pointed out that directed selection of agent is of the DNA type. On the other hand, Leuchtenspontaneous variants of the agent cannot be ruled out berger et al. (26) have shown by spectrophotometric
(11), but this alternate interpretation does not change the analysis that the amount of DNA in the tumor cells is
significance of the findings with respect to the etiology of not much greater than that of normal cells, an observation
the tumor. Duryee, however, has noted occasional consistent with the kaiyotype analysis of DiBerardino
sarcoma-like growths in the digits of Vermont frogs (8). However, the Leuchtenbergers listed the presumed
bearing spontaneous kidney tumors, and on this basis has agent as an RNA virus, apparently on the basis of their
suggested that Rose's results (and presumably also hypothesis that infection with DNA viruses always leads
»Personal communication:
New Orleans, La.
M.
Mizell,
Tulane
University,
9 Personal communication:
W. Smith, Dept. Pathobiology,
Johns Hopkins Univ. School of Hygiene, Baltimore 5, Md.
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Vol. 24, February 1964
Cancer Research
176
to large increases in total DNA content (26). Since this
hypothesis is not established, the claim of RNA composi
tion should be treated with skepticism. Furthermore,
it is evident that even a slight increase in the total DNA
content of the cell could represent a great deal of DNA
virus. A further point is that the cells studied apparently
lacked type A inclusions and hence were presumably defi
cient in virus-like particles.
Catalase activity is reported to be sharply reduced in
tumor tissue as compared with normal kidney (32). How
ever, it was not indicated whether erythrocytes were
eliminated as a source of contamination, and unless this
were done apparent differences in activity of parenchymal
cells would be insignificant in view of the fact that the
tumors possess a far poorer vascular supply. More
over, our experience has been that such differences tend
to disappear when catalase activity is assayed in primary
tissue cultures and computed on a cell-for-cell basis:
under these conditions, the apparent difference is less than
twofold, with both cell types showing low activity.1 Even
this difference could be accounted for by entrapment of
red cells within cell clumps of the primary cultures or by
the greater volume of the tumor cells. Of potentially
great interest, however, is Lucké'sobservation (32) that
the injection of tumor homogenates into the coelom of
normal frogs causes a prompt reduction in liver catalase
activity. Such a system might be adaptable as a screening
device or as an assay method for an agent.
Finally, phosphatase activity (tested between pH 4 and
11) is stated in the same paper to be reduced in tumor
tissue as compared with normal kidney.
Immunology.—Comparative studies of the antigens of
tumor and normal cells have been made by the geldiffusion technic (3) and by this method in combination
with immunoelectrophoresis and fluorescent antibody
technics (43, 44). Barch found a distinctive kidney
antigen which was missing from tumor but was unable to
identify any antigens unique to the tumor. Nace et al.
recognized an antigen 'X' which was absent from tumor
but regularly found in both adult frog cells and in tadpole
cells. Antigen X was subsequently found to occur in
normal cell membranes; it was identified as a lysozyme,
for which claims of an anti-viral activity have been made,
according to Nace. Immunoelectrophoresis has revealed
an isozyme of glucose 6-phosphate dehydrogenase peculiar
to the tumor; other protein moieties were found in tumor
and tadpole cells but not in normal kidney (44). At
present their significance is unknown.
EVIDENCE
THAT THE LÃœCKE TUMOR IS
VIRUS-CAUSED
The following list summarizes the evidence for viral
etiology.
1. Mature-type virus-like particles are present in some
tumors. From the general findings of electron microscopy
there can be little doubt that these are virus particles,
but whether a causative or passenger virus is unknown.
2. Injection of tumor extracts may accelerate develop
ment of tumors or, under low temperature conditions,
may make a nearly absolute difference.
3. Tumors may be altered by transplantation in such a
way that they can induce kidney tumor formation in a
relatively resistant geographical race. In addition, such
material can induce the formation of other types of tumore.
4. Transplants to tadpoles, after apparently complete
regression, can lead to the formation of autochthonous
kidney tumors in juvenile adults a few months after
metamorphosis, whereas spontaneous tumors are rare or
nonexistent at this age. Growth of transplanted cells in
animals rendered immunologically tolerant seems inade
quate to account for this observation, because the tumors
occur only within the kidneys and not at the transplanta
tion site.
5. Incomplete transformation of cultured normal cells
has been observed after their exposure to tumor extracts.
DIFFICULTIES
IN THE WAY OF THE VIRUS
HYPOTHESIS
Because of the high spontaneous rate of tumor forma
tion, the effect of injecting extracts is quantitative rather
than qualitative. Hence, it is usually not possible to
distinguish in individual cases between spontaneous and
induced tumors, and a rigorous and direct demonstration
of a causative virus has not been possible.
A SUGGESTED
LIFE
CYCLE FOR THE LÃœCKE
TUMOR
Although it has been carefully pointed out that neither
viral causation nor natural transmission is definitely
established, the weight of evidence renders the first
probable and the second only a little less likely. If it is
tentatively accepted that both occur, then it is possible
to construct a life cycle scheme which is consistent with
all established findings. Such a scheme is hereby offered,
with the expectation that important details may later be
found to be incorrect. However, various features of the
suggested cycle are subject to test, and the scheme may
serve a useful purpose if it stimulates work bearing on
particular points. This scheme is summarized in Chart 1.
Frogs of the Lake Champlain area complete meta
morphosis in June and early July, lead an active but soli
tary life in the field during the warm months, and enter the
lake to hibernate on about November 1, as the weather
approaches freezing. Emergence and spawning occur
with the first melting of the ice at the edge of the lake, in
late March and in April.
It is probable that field tumors arise in the summer,
during the 3d year or later, and that a proportion of ani
mals (about 3 per cent) bear small tumors as they enter
hibernation. Inclusion formation probably starts soon
after the tumor is established, possibly in response to
cold weather, and at least half of the tumore of winter
frogs are of the inclusion type. During this phase, growth
of the tumor virtually ceases, but virus production in
creases. By the time of emergence, both tumor cells and
intercellular spaces contain considerable amounts of virus,
which finds its way into the urine and hence into the water
at the time of spawning. Infection of the young may then
occur at the moment of fertilization, or tadpoles could be
infected after hatching; presumably, postmetamorphic
animals are refractory. The original infection would then
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RAFFERTY—KidneyTumors of Leopard Frog
A SUGGESTED LIFE-CYCLE
177
OF THE
LÃœCKE"
TUMORVIRUS
RAPIDTUMORGROWTHAND
DEATHOF HOSTWITHLARGETUMOR
»I
THINA FEWMONTHS:
LITTLEOR NO VIRUSPRODUCTION
DISAPPEARANCE
OF INCLUSIONS
ANDVIRUS,
RESUMPTION
OF TUMORGROWTH
- iS
* . Of SPAWNING
WATERS
. 22
METAMORPHOSIS
DEVELOPMENT
OF
NON-INCLUSION
TUMOR
AFTER4 MONTHS
OR LONGER (255Õ)
\
SPRING/
LATENT
INFECTION
»
INTER,,*SUMMER
DEATHOF HOST
WITHLARGETUMOR
DURINGWARMMONTHS
CESSATION
OF TUMORGROWTH
F INCLUSION
BODIES
ACCUMULATION
URINEDURING
SEP.21
\
J
DEVELOPMENT
OF SMALLTUMOR.
LATERSUMMERINADULTS
65-70MM.(3*)
\
NORMALFROGSTAKEN
AT ANY TIMEOF YEAR
•(TUMOR
FORMATION
IN LABORATORY
FROGS)-**
CHARTl.-A suggested life cycle of the Lücketumor virus.
be a latent one, tumor formation beginning more than 2
years hence.
Inclusion cells within the tumors of recently emerged
adults appear to be inviable and probably die with the
onset of warm weather in the spring. However, growth
of the remaining cells then begins, and the afflicted frog
dies with a large, noninclusion tumor during the summer
or early fall.
It appears that tumors arising in frogs kept in a warm
laboratory follow a different course. In this case rapid
growth continues unchecked by hibernation, and the
inclusion stage is probably omitted entirely. It appears
that tumors which lack inclusions, whether of laboratory
origin or in field frogs taken during the summer, produce
little mature virus, and perhaps none.
TAXONOMY AND BIOLOGY OF THE HOST FROG:
AN ADDENDUM
Classification of susceptible frogs.—Certain features of
the life of frogs belonging to the tumor-susceptible species
are little known but of potentially great significance with
regard to the formation and distribution of the Lücke
tumors. Some of these are discussed, and some hopefully
pertinent suggestions are offered.
Although distribution of the tumor is sharply limited,
the Rana pipiens complex is not. Found virtually every-
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178
Vol. 24, February 1964
Cancer Research
where in North America except west of the Sierra Nevada
Range in the United States, it perhaps enjoys a wider
continental distribution than any other Amphibian.
However, it is unusual also in its geographical variability:
although most members of Amphibian species are similar
in different areas of their distribution, R. pipiens is noto
riously variable in size and markings from one region to
another (73). Four to six subspecies are provisionally
recognized by most taxonomists. Of particular interest
is the fact that these are not always interfertile, a charac
teristic which might justify their original classification as
separate species, except for basically similar morphology
(73) and complex infertility relationships of the subspecific
groups (42). Although most individuals from the Oshkosh
vs. Lake Champlain regions can be distinguished from
one another (with a little study) on the basis of slight
differences in markings, they are of the same subspecies
(R. p. pipiens) and strikingly different from leopard
frogs of different subspecies taken, for example, in Browns
ville, Texas, and northeastern Missouri (R. p. berlandieri
and R. p. sphenocephala, respectively).
Although virtually all the frogs in Lucké'sseries had
been obtained from dealers in Vermont, Lückestated that
similar tumors had also been observed in leopard frogs from
North Dakota, Indiana, and the Mississippi Valley (32)
but could not be induced by injection in New Jersey
ß.pipiens (probably subspecies sphenocephala). How
ever, the occurrence of tumor frogs in the last three areas,
as claimed by Lücke,remains, for the most part, uncon
firmed : kidney tumors are reported to be found repeatedly
only in a single subspecies of leopard frog (Rana pipiens
pipiens) collected in areas surrounding the northern half
of Lake Champlain in Vermont and Lake Winnebago in
Wisconsin. On the other hand, recent conversations
with frog collectors have led the writer to suspect that
tumor-bearing frogs may also be taken from Minnesota
and eastern South Dakota. Mateyko10 reports that
tumor-bearing frogs were taken by H. Schlumberger in
the Wenona area of New Jersey, but both sphenocephala
and pipiens subspecies are found nearby, and it is not
clear which is involved. It is suggested that tumorbearing frogs purportedly collected from other areas prob
ably originated in Vermont, having been re-sold through
dealer wholesale operations, which are common practice.
Collections of leopard frogs were made by Mateyko (38)
east and west of the Rocky Mountains, in northwest
Montana; although several hundred frogs were collected,
none was found with a tumor. It is probably significant
that these collections were made largely or entirely outside
the distribution of subspecies pipiens.
It would be of interest to determine, by means of collec
tions in other areas of the pipiens subspecies distribution,
whether tumor susceptibility is limited exclusively to the
subspecies, and perhaps widespread within it. At any
rate, genetic factors are unquestionably of crucial im
portance in determining the rate of spontaneous tumor
formation within the subspecies.
The author has determined (unpublished) that adult
R. p. berlandieri from Brownsville, Texas, are refractory
10Personal communication:
G. Mateyko,
New York University, New York.
Dept.
of Biology,
to injected tumor extracts, and the same test is under way
with Maryland R. palustris, a closely related species.
Collections in other areas for similar tests are now being
made.
Biology of the host.—The two known areas of tumor
occurrence (in Wisconsin and in Vermont) both include
large lake systems and support extraordinarily dense
frog populations. This density may or may not be sig
nificant in the occurrence (and perhaps transmission) of
the kidney tumors. Although areas of equally dense
populations exist in the southern States, adults of the
native races are refractory to the tumor. If, as suggested,
warm climate promotes tumor growth at the expense of
virus production, selection for resistance to the tumor
would be more pronounced in such areas. In addition
virus production could be lessened to such an extent as to
prevent its establishment, and possible opportunities for
its adaptation, in the apparently refractory races of warm
climates. A further point is that tumor-bearing frogs
may survive for an extra winter (and an extra spawning
season) if they hibernate, but this possible factor is miti
gated by the fact that many southern groups of Rana
pipiens seem to spawn twice a year (73).
During the active months spent on land (approximately
May through October in Vermont), the leopard frog leads
an essentially solitary life. Late in the fall (about Novem
ber 1 in Alburg, Vermont), they migrate to the lake in
great numbers—near Alburg 35,000 have been collected
in a single night. After entering the water, the frogs lie
on the bottom for the winter. McKinnell,6 by cutting
through the ice, has exposed hundreds of frogs of the same
subspecies huddled togethei on the muddy bottom of
frozen ponds in Minnesota, and it would seem likely that
Lake Champlain frogs are similarly crowded during winter
hibernation. Although such crowding could be a factor
in possible transmission of the tumors, isolation experi
ments which were discussed tend to indicate that natural
transmission between active adults is not significant in
tumor development. In spite of opportunities afforded
by congregation for spring spawning and by wintertime
crowding, therefore, it appears that transmission occurs
if at all during ovarian, larval, or early adult stages. Cer
tainly tadpole populations are extremely congested during
late spring, as are newly metamorphosed frogs in summer
time marshes. Failure of tadpoles to form tumors by no
means precludes the possibility that active virus is present:
a possible parallel is seen in the agent of the avian leukosis
complex, found present in active form in otherwise normal
embryonated eggs (6), and in a similar situation which
exists in mouse embryos with respect to lymphatic leu
kemia virus (21).
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FIG. 1.—Lücke
tumor of the histológica! type most frequently
seen (adenocarcinoma
of moderate malignancy).
Found in an
untreated Vermont frog killed 2 days after arrival in the labora
tory on March 23, 1960. Almost all cells bear type A inclusions:
Fig. 7 is a higher magnification of the boxed area,
(n, normal
tubules.)
Giemsa, X 70.
FIG. 2.—Tumor resembling
adenoma.
Observed
as three
small foci involving both kidneys in an untreated Vermont frog
killed the day after arrival on Jan. 17, 1963. Region indicated
by arrows contains moderate numbers of type A nuclear inclusions.
H. & E., X 70.
FIG. 3.—Tumor with local areas of disorganized, anaplastic
appearance.
Tumor seen as a small focus in an untreated Wis
consin frog killed the day after arrival on May 10, 1959. Type A
nuclear inclusions are absent,
(n, region of normal tubules.)
Giemsa, X 70.
FIG. 4.—Whole view of a 'replica' tumor arising in an untreated
Vermont frog kept for several months at 26°C. No remnant of
normal kidney is visible. Weight, including ovary, is 15.4 gm.
Ovaries were being invaded at contact points (not shown), but
tumor was not grossly metastatic.
(Od, oviduct; Or, ovary; N,,
spinal nerves.)
X 1.3.
FIG. 5.—Adenocarcinoma of a Vermont frog apparently arising
in the transverse collecting duct and descending limbs of vertical
collecting ducts. Animal had been given an injection intra
venously of a kidney tumor brei 3 days previously.
(TT, trans
forming proximal convoluted tubule.)
Giemsa, X 200.
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5
181
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FIG. 6.—Junction between normal and neoplastic in a proximal
convoluted tubule in a Vermont frog kidney. The animal had
been given an injection intravenously 24 hours earlier of the
same tumor brei as that noted in Fig. 5. The tumor has some
cells with type A nuclear inclusions (not shown). (7',,, normal
portion of tubule; Tm, malignant portion of tubule.) Giemsa,
X 200.
FIG. 7.—Sametumor as Fig. 1, enlarged area. Most nuclei
with type A inclusions (/„). (Ar,cells lacking type A inclusions.)
X 200"
FIG. 8.—Similar high-power view of a 'typical' Lücketumor
lacking type A inclusions. Untreated Vermont frog killed 3
days after arrival in the laboratory on October 1, 1962. (M,
mitotic figure.) H & E, X 200.
FIG. 9.—Typical monolayer culture of a Lücketumor. No
type A inclusions are seen. Giemsa, X 000.
FIG. 10.—Similarculture of pooled normal kidney cells. The
tumor cells of Fig. 9 are larger and more variable in size. In
addition, the chromatin of the tumor cells tends to be more uni
formly dispersed than that of normal cells. Giemsa, X 000.
182
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183
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FIG. 11.—Electron micrograph of a tumor cell with a type A
nuclear inclusion. (G, granular inclusion matrix; V,, vesicles
presumably representing an early stage in virus synthesis; Vm,
virus-like particles containing dense nucleoids and often seen
in association with marginated chromatin; C, chromatin; N0,
nucleolus.) X 30,000. Photograph supplied by Dr. Philip
Lunger, The Rockefeller Institute, New York 21, N.Y.
184
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Kidney Tumors of the Leopard Frog: A Review
Keen A. Rafferty, Jr.
Cancer Res 1964;24:169-185.
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