Viral Etiology of "Spontaneous" Mouse Leukemia
A Review*
LUDWIKGROSS
(Cancer Research Unit, Veterans Administration Hospital, Bronx, N.Y.)
It has long been suspected that leukemia is
caused by a transmissible virus. There were many
reasons fostering such an assumption. The course
of acute leukemia in humans is often quite sugges
tive of an infectious disease; fever, general symp
toms, exacerbations, and temporary remissions—all suggest a general sepsis-like infection.
There were considerable difficulties, however, in
accepting a viral concept of etiology of mammalian
leukemia. First, the fact that leukemia is actually
not contagious, and does not spread from host to
host, remained unchallenged. Only very recently
have we learned that, under certain conditions,
avian lymphomatosis can spread from host to host
among very young chicks (8). Similar observa
tions, however, have never been made in humans,
mice, rats, or cattle.
VERTICALTRANSMISSION
OF LEUKEMIA
A WORKINGHYPOTHESIS
When speaking of a communicable disease, we
are conditioned to think of a contagious disease,
spreading from one host to another within the
same generation. It is apparent, however, that
oncogenic viruses may be transmitted in an inac
tive form from one generation to another directly
through the germinal cells, causing only occasion
ally symptoms of disease, not unlike the temperate
phage in a lysogenic bacterial clone. The term
"vertical transmission" was suggested to designate
the transmission of potentially pathogenic agents
from one generation to another (29, 36).
When observing a disease appearing in several
descendants of successive generations within the
same family of hosts, a casual observer may con
sider such cases to be caused by some obscure, in
herited, genetic factors; yet, inheritance and verti
cal transmission are fundamentally different phe
nomena. Inheritance is conditioned by genetic fac* Aided, in part, by grants from the Damon Runyon Me
morial Fund and the American Cancer Society.
Received for publication January 17, 195S.
tors, transmitted in an orderly and foreseeable
manner, and is not acquired. Vertically trans
mitted diseases, on the other hand, are caused by
viral agents and represent a form of infection. The
potentially pathogenic agents had been most prob
ably at one time acquired, even if this might have
happened in individual families many thousand of
years ago; transmitted, since that time, from one
generation of their hosts to another, invisible and
completely adapted, such agents represent ex
amples of "perfect parasites," causing in most in
stances no harm to their hosts. Now and then only,
for obscure reasons, they may change into for
midable pathogens, causing then symptoms of
fatal disease and killing their hosts. Such "acci
dents" may, however, be separated by several gen
erations of healthy carrier-hosts.
It is quite possible that leukemia, not only in
chickens and mice but in other species also, is ac
tually transmitted in such a manner and that
agents of this disease are seeded in millions of ap
parently healthy hosts (36).
THE CHICKENLEUKOSISCOMPLEX
The discovery by Ellerman and Bang in 1908
that leukemia in chickens could be transmitted by
cell-free extracts (18) provided the first experi
mental support of the concept of the viral nature
of leukemia in the fowl and strengthened the suspi
cion that in other species also, including humans
and mice, leukemia may be caused by similar
filtrable agents.
There exist several forms of chicken leukosis. In
general, they can be divided into an intravascular
and extravascular group. The intravascular group
consists of myeloblastosis and erythroblastosis,
which are characterized by the presence of a very
large number of myeloblasts and erythroblasts in
the circulating blood. Both are readily transmitted
to normal birds of susceptible strains by filtrates of
plasma from diseased hosts. On electron micro
graphs, the viruses of myeloblastosis and erythro
blastosis appear identical, as spheroidal particles
371
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372
Cancer Research
averaging about 120-140 m/t in diameter; they
are, however, biologically distinct, though prob
ably related (2-4, 15-17). An outstanding prop
erty of the virus of avian myeloblastosis is its
enzymatic ability to dephosphorylate adenosinetriphosphate (47). Plasma of chickens with myelo
blastosis can thus be tested for this enzymatic ac
tivity and its content of myeloblastosis virus de
termined within a few minutes by a colorimetrie
method. Systematic studies provided substantial
evidence that this enzymatic activity is quantita
tively related to the virus particle and inseparable
from it by the methods employed. No similar
enzymatic activity, however, could be thus far es
tablished for the virus of erythroblastosis (6).
The second group of avian leukemias consists of
usually extravascular, essentially aleukemic forms,
in which primitive cells only occasionally reach the
circulating blood. The most frequent of them (54)
is visceral lymphomatosis, in which the lymphoid
cells infiltrate liver, lungs, and other visceral or
gans, causing considerable enlargement of liver
("big liver disease"). This form of chicken leuke
mia is responsible for a yearly loss exceeding
$60,000,000 to the U.S. poultry industry. Closely
associated but less frequent forms are those in
which the infiltration with lymphoid cells is par
ticularly prominent in the eyes (ocular lymphomatosis) or in the nerve trunks, causing then
paralysis (neurolymphomatosis). Another associ
ated form is osteopetrosis, involving the perios
teum and resulting in excessive deposition of bone.
The various forms of Ivmphosarcomas also belong
to this group of avian leukemias.
Under proper experimental conditions, erythro
blastosis, myeloblastosis, as well as visceral lym
phomatosis and osteopetrosis and at least some of
the lymphosarcomas, can be transmitted by in
oculation of filtrates into susceptible hosts. The
susceptibility to inoculation of filtrates, relative to
the age and breed of the recipient hosts, varies for
the different forms of avian leukemias. Thus, 3day-old chicks were found to be 40 times more sus
ceptible than 21-day-old birds to the inoculation
of myeloblastosis virus. On the other hand, 2-6week-old chicks were found most suitable for in
oculation with the virus of erythroblastosis (2-5,
15-17). One-day-old chicks were found susceptible
to visceral lymphomatosis filtrates; either visceral
lymphomatosis, or, in some instances, osteopetro
sis resulted from such inoculations (7, 9).
A marked difference in susceptibility of the
recipient hosts, according to the breed used, could
be observed in studies on cell-free transmission of
avian leukemias. Under otherwise identical experi
mental conditions, White Leghorn chickens of the
Vol. 18, May, 1958
inbred Line 15 (developed at the Regional Poultry
Research Laboratory in East Lansing, Mich.)
were found highly susceptible, whereas White
Plymouth Rock, New Hampshire chicks, and
other strains of White Leghorns purchased on the
market were considerably more resistant to the
inoculation of leukemic filtrates (17).
SPONTANEOUS
ANDINDUCEDLEUKEMIA
IN MICE
Leukemia occurs as a spontaneous disease in
mice of various strains. In some strains, such as
C3H or C57BR/cd, it develops only occasionally.
In other strains it is more frequent, and in some,
such as Ak or C58, it develops in over 85 per cent
of animals. Actually, it would be very difficult, if
at all possible, to find a strain of mice entirely free
from spontaneous leukemia. Now and then, par
ticularly in old mice, leukemia or reticular tumors,
such as lymphosarcomas, develop in most of the
strains known, though the incidence is generally
low. Even in strains having a very low incidence of
spontaneous leukemia, however, thymic lympho
sarcomas or generalized leukemia can be induced
in a relatively high number of animals by totalbody x-radiation, or by application of estrogenic
hormones or certain carcinogens, such as methylcholanthrene.
TRANSPLANTED
LEUKEMIAS
Leukemia which developed spontaneously or
was induced by irradiation or with a carcinogenic
chemical can be transplanted, by inoculating a cell
suspension prepared from the leukemic donor into
another host oÃ-the same, or a genetically related,
inbred line. Transplantation of leukemic cells is
also possible to genetically unrelated hosts if new
born mice are used for inoculation; thus, Ak or
C 58 leukemic cells can be transplanted into new
born mice of the C3H or C57BL inbred lines, etc.
(28).
Transplanted leukemias, i.e., leukemias carried
by cell graft in certain inbred strains of mice, can
be divided into the following three main groups:
(a) those which originated as a spontaneous leuke
mia in a host of a high-leukemic line, such as Ak or
C58, and have been subsequently carried by cell
transplantation, in susceptible mice, usually of the
same or of a related strain; (6) those which origi
nated as a spontaneous leukemia in a host of a
low-leukemic strain, and have then been carried
by cell-graft in susceptible mice, usually of the
same or of a related line; and finally (c) those
which originated from a leukemia that resulted
from application of either ionizing radiation, or a
carcinogen, in an animal of a usually low-leuke-
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GROSS—
Viral Etiology of "Spontaneous" Mouse Leukemia
mie line and have then been carried by cell trans
plantation in susceptible mice of the same or of a
related strain.
THE ETIOLOGYOP SPONTANEOUS,
TRANS
PLANTED,ANDINDUCEDLEUKEMIA
The etiology of the first two groups of trans
planted leukemias could be discussed together
with that of spontaneous mouse leukemia in gen
eral; whether initially spontaneous, or spontaneous
first and then carried by graft in other hosts, its
fundamental etiology would be essentially the
same. On the other hand, the etiology of leukemia
first induced by radiation or carcinogens in an ani
mal of an otherwise low-leukemic strain, and then
carried on by cell transplantations, could be dis
cussed together with the etiology of primary leuke
mia that was induced by ionizing radiation or with
carcinogens.
Leukemia developing spontaneously in certain
strains of mice, such as C58 or Ak, was found to be
caused by a filtrable, transmissible virus. On the
other hand, leukemias induced in mice by the ac
tion of ionizing radiation, hormones, or chemical
carcinogens have not yet been adequately studied;
additional experimental studies are needed to de
termine their nature. This does not necessarily im
ply, however, that these two types of leukemia
have a different etiology. Both may be caused by
fundamentally similar factors.
This review deals with considerations of the
etiology of leukemia developing spontaneously in
certain inbred strains of mice, such as C58 or Ak.
SPONTANEOUS
LEUKEMIAIN STRAINS
C58 ANDAK
Those interested in leukemia research are in
debted to the early work of MacDowell and
Richter (46) and subsequent work of Furth (26)
and his associates, who developed, by inbreeding,
two outstanding high-leukemic lines of mice,
namely strains C58 and Ak, in both of which a
very high incidence of spontaneous leukemia, usu
ally of the lymphatic type, occurred.
MacDowell and Richter observed accidentally
that mice of a strain designated by the symbol
C58 and bred in their laboratory died with large
spleens; they soon realized that they had been
breeding a strain of mice developing a high inci
dence of leukemia. On the other hand, Furth and
his associates purposely developed a high-leukemic
inbred line of mice, designated by the symbol Ak,
by breeding selectively relatives of leukemic mice
and continuing such selective inbreeding through
a number of successive generations.
In our laboratory, approximately 85 per cent of
373
mice of the C58 Une develop leukemia spontane
ously at an average age of 9 and 10.8 months for
females and males, respectively (40). Most of our
studies, however, have been carried out with Ak
leukemia. Spontaneous leukemia develops in Ak
mice of both sexes. Our experience referring to
spontaneous Ak leukemia has been, with only few
exceptions, limited to female mice. In some in
stances we have observed leukemia also developing
in Ak males kept for a sufficiently long period of
time in the breeding colony of this strain in our
laboratory. We have not kept large numbers of
Ak males for prolonged periods of tune, however,
because Ak males, when kept in cages together,
fight ferociously. The incidence of spontaneous
leukemia in a sample of 500 Ak females was, in
our laboratory, 94 per cent at 8.6 months average
age. Spontaneous leukemia, usually of the lym
phatic type, begins to develop in Ak females
approximately at 4 months of age; this incidence
rises rapidly, reaching its peak at 7-9 months,
then declines gradually.
THE SEARCHFORA LEUKEMICAGENT
The development of spontaneous leukemia in
over 85 per cent of mice in certain inbred lines,
such as C58 or Ak, immediately raised the ques
tion whether a virus transmitted from one genera
tion to another may not be responsible for the de
velopment of this disease in C58 or Ak mice. The
possible transmission of such a hypothetical agent
from nursing mothers to their offspring through
milk was investigated in 1935 by MacDowell and
Richter on C58 mice (46), and later on by Barnes,
Furth, and their associates (1, 24) on Ak mice. In
subsequent experiments, Ak mice removed from
the womb of their mother by Caesarean section
(20, 32) or born from transferred Ak ova in the
ingenious experiments of Fekete and Otis (20)
were raised by low-leukemic foster mothers.
Although in some experiments the incidence of
leukemia was at first lower in the foster-nursed
mice (1, 24, 46), it increased in the next generation
(1, 24). In other experiments (20, 32), the inci
dence of leukemia was immediately as high in the
first, foster-nursed generation as in the original
stock. Reciprocal nursing by foster mothers of a
high-leukemic strain failed to induce leukemia in
foster-nursed mice of a low-leukemic line (1, 24).
It was apparent, therefore, that a hypothetical
leukemic agent was not transmitted from mothers
to their offspring through milk. If such an agent
existed and was responsible for the development of
leukemia in mice of successive generations of the
high-leukemic Ak and C58 lines, it must have been
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374
Cancer Research
Vol. 18, May, 1958
transmitted from parents to offspring in a different
manner.
The existence of a leukemic agent, though long
suspected, was not established, however, until the
experimental conditions were recognized under
which mouse leukemia could be transmitted by
filtrates.
Since chicken leukosis could be transmitted
without difficulty by filtered extracts, it appeared
logical to assume that in mice also leukemia is
caused by a transmissible virus. All attempts, how
ever, to transmit mouse leukemia by filtered ex
tracts failed prior to 1951. Transmission of leuke
mia in mice appeared possible only when live cells
were transplanted from a leukemic mouse to an
other host usually of the same, or of a genetically
related, inbred line. Filtrates prepared from leuke
mic C58 or Ak donors and inoculated into adult
mice of the C58 or Ak strains, respectively, did not
induce the development of leukemia (19, 26, 46).
In a few instances, when centrifuged (but not
filtered) extracts were prepared from Ak leukemia
and inoculated into young adult Ak mice, some of
the mice developed leukemia (19). It was difficult,
however, to exclude the possibility that in those
few instances a few leukemic cells might have re
mained floating in the supernate; moreover, Ak
mice develop leukemia spontaneously anyway,
and some of those inoculated with the supernate
might have developed spontaneous leukemia,
though perhaps earlier than expected. Since Furth
has shown that a single leukemic cell may be suf
ficient to transmit leukemia to a susceptible donor
(25), it was apparent that only filtrates, in care
fully controlled experiments, could prove cell-free
transmission of mouse leukemia.
TRANSMISSION
OF "SPONTANEOUS"MOUSE
LEUKEMIABYFILTRATESINOCULATED
INTONEWBORNMICE
In 1951, it was unexpectedly found by Gross
that naturally occurring "spontaneous" mouse
incidence of less than 1 per cent among 166 littermate controls, either not treated or inoculated
simultaneously with heated (65°-68°
C., | hr.) leu
kemic extracts and surviving to an average age of
16 months (42).
Leukemia developing in C3H mice following in
oculation of the leukemic filtrates was usually of
the lymphatic type and resembled that which de
velops spontaneously in mice of the Ak strain. In
fully developed cases, there was a large thymic
lymphosarcoma, a large white, oblong, mesenteric
tumor, large tumorous peripheral lymph nodes,
and a large spleen and liver. In some instances, the
thymic lymphosarcoma developed first, filling out
gradually most of the chest cavity and killing the
mouse before other leukemic tumors might have
had an opportunity to develop elsewhere to a con
siderable degree. In most instances, however, the
disease was generalized: on microscopic examina
tion, liver, kidneys, and other organs showed in
filtration with leukemic cells. The peripheral blood
picture did not always reflect advanced pathology,
though abnormal cells, such as lymphoblasts and
particularly smudge cells, could often be seen; the
white blood count was in most instances not ele
vated, but anemia was usually present, and the
bone marrow showed the presence of large num
bers of lymphoblasts and other immature cells,
probably of the lymphatic series.
Surprisingly, among the inoculated C3H mice,
some developed, instead of leukemia, bilateral
parotid gland carcinomas, and a few developed
subcutaneous fibrosarcomas (34). When in subse
quent experiments newborn mice of the C57BR/cd
inbred line were used for the inoculations of the
cell-free leukemic extracts, only leukemia resulted;
parotid tumors and subcutaneous sarcomas failed
to appear (35).
leukemia could be transmitted by filtered extracts,
provided that certain specific experimental condi
tions were met (30, 31). Thus, newborn (less than
16 hours old) mice of a low-leukemic but suscep
tible strain, namely, the C3H inbred line, could be
successfully inoculated with filtered extracts pre
pared from spontaneous Ak leukemia (32, 33).
Later, it was realized that the activity of the indi
vidual extracts prepared from different leukemic
Ak donors varied to a considerable degree. Of a
total of 328 newborn C3H mice inoculated with
filtrates, 28 per cent developed leukemia at an
average age of 10 months. This compared with an
The leukemic agent is transmitted directly
through the embryos from one generation to an
other in mice of the C58 and Ak inbred lines. This
was demonstrated in a series of experiments in
which cell-free extracts were prepared from nor
mal C58 and Ak embryos and inoculated into new
born C3H mice. Leukemia and, in a few instances,
parotid tumors could be induced with such ex
tracts (30, 38, 40). It is therefore apparent that the
agent of mouse leukemia is transmitted in certain
families of mice, such as the Ak or C58 inbred lines,
from one generation to another directly through
the embryos (30, 38). In this respect, mouse leuke
mia would be essentially similar to chicken lym-
PRESENCEOF THE LEUKEMICAGENTIN NORMAL
EMBRYOSOF MICE OF THE C58 ANDAK
LINES
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GROSS—ViralEtiology of "Spontaneous" Mouse Leukemia
375
2-12 days old, some of them developed leukemia
but not before 14^ months of age (31). The poten
cy of the leukemic virus has now been considerably
VARIATIONSIN POTENCYOP EXTRACTS
increased, however, through cell-free passage. Fil
ANDDIFFERENCESIN STRAIN
trates prepared from this passage virus can now be
inoculated into suckling C3H mice 1-14 days old,
SUSCEPTIBILITY
inducing leukemia in up to 100 per cent of the in
There were some difficulties in initial attempts
to reproduce the cell-free transmission of mouse oculated mice after a latency of only 3-4 months
leukemia in other laboratories. When repeated at (43).
the National Cancer Institute, the induction of
It is interesting that leukemic filtrates inocu
parotid gland tumors following the inoculation of lated into C3H mice less than 16 hours old induced
the cell-free leukemic extracts into newborn C3H leukemia, and in some instances parotid tumors,
mice was readily confirmed; however, only very whereas the same filtrates inoculated into suckling
few of the inoculated mice developed leukemia C3H mice 1-14 days old induced leukemia, but not
(45, 51, 52). One of the difficulties was due to the parotid tumors. It appears, therefore, that, in or
lower susceptibility of mice of the National Cancer der to induce parotid tumors, newborn, less than
Institute substrain of the C3H line to the leukemo- 16 hours old, C3H mice have to be inoculated with
genic action of the Ak leukemic agent. C3H mice the leukemic extracts.
of the Bittner substrain, used in our laboratory,
Previous attempts to transmit Ak leukemia by
were found to be more susceptible (37). Further
inoculation of filtrates into adult C3H mice were
more, it soon became evident that the results of seldom successful. After the potency of the leuke
individual experiments may vary to a considerable mic agent has been increased through serial celldegree, depending on the extracts used. Of 70 leu
free passage, it is now also possible, however, to
kemic filtrates tested in our laboratory, each pre
induce leukemia by inoculating passage A leuke
pared from a different donor, eighteen were found mic filtrates into adult C3H mice; the incidence of
to be completely inactive on inoculation tests (42). induced leukemia is approximately 37 per cent
Woolley was the first to confirm the cell-free after a latency of 7 months. Suckling, 1-14-day-old
transmission of mouse leukemia, using either Ak C3H mice are, however, substantially more sus
or C58 mice as donors and newborn C3H mice for ceptible to the inoculation of the leukemic filtrates
the inoculation of the centrifuged or filtered leuke
than are adult hosts (43).
mic extracts (56). Some of the C3H mice inocu
SOME OF THE EXPERIMENTALCONDITIONSRE
lated with the leukemic filtrates developed parotid
QUIREDIN STUDIESONCELL-FREETRANSMIS
tumors or subcutaneous sarcomas. Subsequent
SIONOF SPONTANEOUS
MOUSELEUKEMIA
confirmations of the cell-free transmission of mouse
Donors.—Mice
with
either
spontaneous, trans
leukemia were reported by Furth (23), Dulaney
(14), Hays (44), Kassel,1 and their associates.
planted, or virus-induced leukemia could be used
as donors. Younger donors with spontaneous Ak or
DEVELOPMENT
OFA HIGHLYPOTENTLEUKEMIC C58 leukemia were more suitable for the prepara
AGENTBY SERIALCELL-FREEPASSAGE
tion of active extracts than old leukemic mice;
Selecting a particularly potent extract originally donors with spontaneous Ak or C58 leukemia were
obtained from spontaneous Ak leukemia and then more suitable than those with transplanted leuke
passing the agent serially through several succes
mia. The best donors were C3H mice in which
sive inoculations of newborn hosts made it recently leukemia resulted from inoculation, shortly after
possible to develop a very potent strain of a fil- birth, of cell-free, passage A, leukemic extracts
trable mouse leukemia agent. This agent now in
(41, 42).
duces acute generalized leukemia after only 2§-3|
Preparation of extracts.—Partsof liver, spleens,
months in 40-100 per cent of the inoculated C3H mediastinal and mesenteric tumors, and peripheral
mice (Bittner substrain) (41).
lymph nodes were ground with physiological saline
In previous experiments filtered extracts pre
solution. The cell suspensions, of 20 per cent and
pared from Ak leukemic donors had to be inocu
occasionally 10 per cent concentration, were cen
lated into newborn, less than 16 hours old, C3H trifuged at 0°C., first at 3,000 r.p.m. (1400 X g)
mice in order to induce disease. When such fil for 15 minutes, then at 9,500 r.p.m. (7000 X g) for
trates were inoculated into suckling C3H mice 5 minutes. The final supernate was then filtered
under vacuum pressure of approximately 20-25
1R. Kassel and A. Rottino, Problems in Production of
mm.
mercury. Seitz filters were found not suitable
Leukemia with Cell-free Extracts. Proc. Am. Asaoc. Cancer
for filtration, since they retained most of the virus.
Research, 2:313, 1958.
phomatosis (10): apparently,
virus diseases.
both are egg-borne
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376
Cancer Research
Berkefeld N and particularly
Selas microporous
porcelain filter candles, porosity 03 or 02, were found
satisfactory, retaining E. coli but permitting the
leukemic agent (34) to pass through.
Centrifuged
(7,000 X g) extracts were found
more potent than filtrates, the filtration procedure
apparently resulting in some loss of the virus (43).
Most of the extracts, kept at 0°C., were used
within a few hours after preparation,
none later
than after 48 hours. The activity of the extracts
could be preserved, however, for several months,
with no apparent change in their potency when
kept in sealed glass ampoules at —70°C. in COs
dry ice (41).
Mice used far inoculation.—When filtered ex
tracts prepared from spontaneous
Ak leukemia
were used for inoculation, newborn (less than 16
hours old) C3H mice of the Bittner substrain (37)
were found most susceptible. When filtrates pre
pared from the potent passage A leukemic strain
were used, suckling C3H mice (Bittner substrain)
1-14 days old were found at least as susceptible as
those less than 16 hours old; however, leukemia
only, but not parotid tumors, developed when
suckling mice more than 1 day old were inoculated
with the filtrates (43).
In general, C3H mice of the Bittner substrain
were found more susceptible than those of the
Andervont
subline (37). C57BR/cd
mice were
also found susceptible if given inoculations when
less than 16 hours old; however, only leukemia,
but not parotid tumors, could be induced with the
filtrates in mice of this inbred line (35).
Among the C3H mice of the Bittner substrain,
those free from the mammary tumor agent (by
foster nursing) were more susceptible to the leukemogenic action of the filtrates than C3H mice
carrying the Bittner mammary carcinoma virus;
furthermore,
females appeared more susceptible
than males (37, 42).
Inoculation of the leukemic filtrates was more
effective by intraperitoneal
than by the subcutane
ous routes (43).
Sedimentation
of the leukemic agent in ultracentrifuge.—The leukemic agent could be sedimented in Spinco Model L Ultracentrifuge
after
centrifugation
at 40,000 r.p.m. (average, 125,000 X fif) in swinging bucket rotor SW 39 for 30
minutes. After centrifugation
for 15 minutes at
maximum speed, biological particles of 70 m/i in
diameter should be sedimented. After centrifuga
tion for 30 minutes at maximum speed, with the
same SW 39 motor, biological particles of approxi
mately 50 jQfi should be sedimented. Since, after
15 minutes of centrifugation,
the supernate was
still active but had no activity after centrifugation
Vol. 18, May, 1958
of 30 minutes, it appears that the size of the leukemogenic particles should be placed somewhere be
tween 50 and 70 m/i (42).
ELECTRON MICROSCOPICSTUDIES OF THE
MOUSE LEUKEMIA AGENT
Electron microscopic examination of the leuke
mic filtrates in drop preparations
on collodion
membranes, shadowed with chromium, revealed
the presence of innumerable
spherical particles,
varying in diameters from 30 to 75 m/u, most of
them averaging between 60 and 70 m^ (40). Not
all extracts, however, contained sufficient numbers
of particles to be detected on routine electron mi
croscopic examination.
Furthermore,
it was not
possible thus far to correlate the leukemogenic ac
tivity of the extracts with the numbers of par
ticles observed on electron microscopic examina
tions.
More detailed studies were possible on thin sec
tions of the leukemic tissues. In collaboration with
Dmochowski and Grey (11, 12), sections were pre
pared from thymus, cervical glands, and livers
from C3H mice in which leukemia was induced by
inoculation of the leukemic filtrates. These sec
tions were then studied by Dmochowski (11,12) in
the electron microscope. Characteristic,
spherical,
virus-like particles, most of them averaging ap
proximately
100 m/i and showing dense centers,
were found in large numbers in the cytoplasm and
in extracellular
spaces. Examination
of normal
mouse tissues did not reveal the presence of similar
virus-like particles; additional studies are neces
sary, however, to further clarify this point.
Whether the particles observed in drop prepara
tion as well as those found on thin sections actually
represent the leukemic agent remains to be deter
mined. If so, the difference in size between the par
ticles in drop preparation, as compared with those
observed on sections, would have to be explained.
It is possible, however, that the particles visual
ized in drop preparations represent the dense cen
ters of the larger particles observed on the sec
tions. Technical differences in the manner of pre
paring the drop preparations,
as compared with
ultrathin sections, may also account, at least in
part, for the difference in size of the particles.
LEUKEMIA, PAROTID CARCINOMAS,AND
SUBCUTANEOUSFIBROSARCOMAS
The development of leukemia in some of the
mice inoculated with the filtrates, and that of
parotid carcinomas or subcutaneous
fibromyxosarcomas in others, immediately raised the funda
mental question whether a single agent may be
able to cause different types of tumors, or whether
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GROSS—Viral Etiology of "Spontaneous"
Mouse Leukemia
377
proved a source of unexpected findings also in
several distinct agents were present in the leuke
other laboratories.
mia extracts (34, 42).
Thus, Graffi and his co-workers (27) prepared
Mouse leukemia transplanted from one host to
filtered extracts from Ehrlich ascites mouse car
another by the conventional method of cell trans
cinoma and from several other mouse tumors.
fer would reproduce only leukemia. When, how
These extracts were then inoculated into newborn
ever, filtered extracts were prepared and inocu
lated into newborn hosts of a susceptible strain, mice of a low-leukemic inbred strain Agnes-Bluhm
(having an incidence of spontaneous leukemia less
either leukemia, carcinoma, or fibrosarcoma re
sulted. Transplantation of such tumors by the than 1 per cent) ; newborn noninbred mice, desig
conventional method of cell graft reproduced iden
nated by the symbols M and W, were also given
tical tumors—that is, transplantation of carcino
injections. As a result, 34-75 per cent of the in
ma resulted in the growth of a carcinoma, and oculated mice developed leukemia, almost exclu
transplantation of fibrosarcoma resulted in the sively of the myelogenous form, after an average
growth of a similar sarcomatous tumor. When, latency varying from 113 to 224 days. Most of the
however, cell-free extracts were prepared from leukemias were characterized by green discolora
such tumors and inoculated into newborn mice of a tion of the lymphoid nodes (chloroleukemia). The
susceptible strain, again either leukemia, carcino
most potent extracts were those prepared from
mas, or fibrosarcomas resulted (40, 42).
Ehrlich carcinoma and from three closely related
Thus, when cell-free extracts, prepared from the mouse sarcomas. On the other hand, extracts pre
induced leukemias, parotid tumors, or sarcomas, pared from Sarcoma 37 (S-37), and from several
were inoculated into newborn C3H mice, their mouse tumors that had been induced with car
pathogenic potential was highest when prepared cinogenic chemicals, had only a very weak po
from induced leukemias (50per cent), substantially
tency, inducing at best less than 10 per cent of
lower when prepared from parotid tumors (33 per leukemias in the inoculated mice. Of 332 control
cent), and rather weak when made from the mice inoculated with either normal organ extracts
subcutaneous sarcomas (16 per cent). However, from mice, rats, chickens, or rabbits, or with rat
regardless of whether they were prepared from the tumor filtrates, less than 1 per cent developed
induced C3H leukemias, parotid tumors, or sub
leukemia (27).
cutaneous sarcomas, such cell-free extracts, when
Friend (21) inoculated cell-free, centrifuged ex
inoculated into newborn C3H mice (42), were tracts from Ehrlich ascites carcinoma into new
capable of inducing, although in different degree, born Swiss mice. After a latency of 14 months,
either leukemia (7-37 per cent), parotid tumors some of the inoculated mice developed a form of
(5-10 per cent), and/or fibromyxosarcomas (4-10 myelogenous leukemia. This leukemia was found
to be transmissible by injection of cell suspensions,
per cent).
One could explain this by assuming that one or by intraperitoneal inoculation of filtrates into
young adult Swiss mice, and also into mice of the
agent may be able to cause different types of tu
mors. It is also possible to speculate, however, that DBA line, inducing leukemia after a short latency
of only several weeks.
certain tumors, at least, may carry, either acci
Actually, the initial results reported by Graffi,
dentally or for a good reason, a number of distinct and later on by Friend, were essentially similar;
oncogenic agents. Under proper experimental con both authors observed the development of leuke
ditions, some of these hitherto latent agents may mia following inoculation into newborn mice of
assert themselves, inducing a wide spectrum of dif cell-free extracts prepared from Ehrlich mouse car
ferent tumors (42). Such a hypothesis would ex cinoma. Graffi, however, found that the leukemias
plain the puzzling results of experiments reported thus induced could be only occasionally trans
by Graffi (27), Schmidt (49), and Friend (21).
planted by cell graft (27) and did not report any
subsequent attempts to pass such leukemias seri
DEVELOPMENTOF LEUKEMIA, FOLLOWINGIN ally by filtrates in the recipient hosts. Friend, on
OCULATIONOF NEWBORN MICE WITH CELL- the other hand, passed the induced leukemias se
FREE EXTRACTSFROMTRANSPLANTED
MOUSE rially in mice of the recipient line and eventually
TUMORS.THE EXPERIMENTSOF GRAFFI AND developed a potent, filtrable strain of a rather
FRIEND
unusual form of myelogenous mouse leukemia
The use of newborn, less than 1-day-old, mice which is now readily transmissible, by inoculation
for the inoculation of oncogenic agents, so success of filtrates, into adult mice of a susceptible strain
ful in studies on cell-free transmission of mouse (21). This, however, is not a form of leukemia
leukemia (30-43), soon became a useful tool and usually seen to occur spontaneously in mice; leu-
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1958 American Association for Cancer Research.
378
Cancer Research
kemia occurring commonly in mice such as those
of the Ak or C58 inbred lines is different and ap
parently requires newborn mice for initial trans
mission by filtrates (30-40). It is quite possible
that there exist different forms of mouse leukemia,
caused by distinct viruses and requiring different
experimental conditions for cell-free transmission.
Certain leukemic agents may require newborn
hosts for transmission, others may also infect
adult hosts of a suitable line. In this respect, mouse
leukemia would not be different from the various
forms of chicken leukosis.
It is, of course, of considerable interest that a
cell-free extract prepared from a mouse carcinoma
was able to induce leukemia. Actually, this obser
vation was similar to our previous findings in
which cell-free extracts prepared from parotid
gland carcinomas or fibrosarcomas, and inoculated
into newborn C3H mice, induced in some instances
typical leukemias (39, 42). Whether the Ehrlich
ascites carcinoma filtrates contain a single agent
able to induce, under proper experimental condi
tions, leukemia instead of carcinoma, or whether
such extracts contain a mixture of distinct oncogenic agents, remains to be determined. It is of
interest that recently Schmidt (49) observed the
development of either leukemia, parotid gland car
cinomas, subcutaneous fibrosarcomas, or certain
other tumors, following inoculation of cell-free
Ehrlich carcinoma extracts into newborn mice of
the Agnes-Bluhm line. These results again are
similar to our studies in which Ak leukemic fil
trates induced a similar array of neoplasms follow
ing inoculation into newborn C3H mice (42).
The fact that Friend, when passing her leukemic
agent by filtrates inoculated into adult Swiss mice,
failed to observe the development of carcinomas in
the injected mice, does not necessarily exclude the
possibility that such an agent may yet be carried
in the leukemic filtrates, even though no car
cinomas, but only leukemia, appeared on succes
sive passages. In our studies on cell-free transmis
sion of spontaneous Ak leukemia, we have ob
served that the development of the induced tu
mors depends to a large extent on the recipient
strain of mice used for inoculation of the cell-free
leukemic extracts. Whereas newborn C3H mice
may react to the inoculation of such extracts by
the development of either leukemia, parotid tu
mors, or subcutaneous fibrosarcomas, newborn
mice of the C57BR/cd line, inoculated under ap
parently identical experimental conditions, devel
oped leukemia only, but not other tumors (35).
It is apparent, therefore, that the induction of
different types of tumors requires the use of a suit
able recipient strain for inoculation of the leuke
Vol. 18, May, 1958
mic extracts. Moreover, the cell-free extracts must
be inoculated into newborn mice less than 16 hours
old, if the full array of the different types of tu
mors is to be induced. This was clearly demon
strated in a series of recent experiments (43) in
which leukemic passage virus filtrates were inocu
lated into two groups of C3H mice, one less than
16 hours old, the other 1-14 days old. When the
filtrates were inoculated into newborn C3H mice
less than 16 hours old, either leukemia or parotid
tumors resulted; when the same filtrates, however,
were inoculated into suckling C3H mice 1-14 days
old, only leukemia resulted, but not parotid car
cinomas.
It is, therefore, quite possible to speculate that,
under proper experimental conditions, a leukemic
agent derived from Ehrlich carcinoma may induce,
instead of leukemia, a different type of a tumor,
such as a carcinoma, provided that the filtrate is
inoculated into newborn mice only a few hours old,
and of a suitable strain, susceptible to the induc
tion of a variety of tumors.
It remains to be determined what types of po
tentially pathogenic oncogenic agents may be
present in the many transplanted tumors carried
for years in various laboratories. On routine cell
transplantations, these mouse or rat tumors are
monotonously similar when compared from one
graft to another, only rarely, if ever, changing their
morphological structure. What would result, how
ever, if filtrates prepared from such tumors would
be inoculated into newborn hosts of a susceptible
stram?
INDUCTIONOF PAROTIDTUMOKSWITH
NORMAL,ORGANEXTRACTS
Cell-free extracts prepared from normal organs
(such as livers, spleens, lungs, embryos, etc.) of
normal, healthy C3H or C57BR/cd mice, and in a
few instances also from normal guinea pigs, inocu
lated into newborn (less than 16 hours old) C3H
mice, induced parotid gland tumors (39, 40). Al
though the incidence of the induced parotid tu
mors was low, it was significant, since parotid
gland tumors do not develop spontaneously in
untreated C3H mice, except on very rare occa
sions; during the past 10 years we have seen only a
single case when parotid tumors developed spon
taneously in a C3H mouse, among at least several
thousand C3H mice observed in our laboratory
during that period of time (39).
It is possible, therefore, that a parotid tumor
agent, usually harmless for its carrier host, may be
widely disseminated and carried by many normal
and perfectly healthy mice. A blind transfer, con
sisting of the inoculation of normal organ extracts
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GROSS—Viral Etiology of "Spontaneous"
Mouse Leukemia
379
(containing such an agent) into newborn mice of a ment of parotid tumors and other neoplasms after
susceptible strain (such as C3H), may increase the a latency varying from 2.5 to 10 months. Tumors
agent's pathogenic potential sufficiently to cause failed to develop in control mice given injec
the development of parotid tumors in its new host. tions of fluids from uninoculated tissue cultures.
In other instances, inoculation of foreign protein Hamsters were also found to be susceptible, devel
oping multiple neoplasms following inoculation of
extracts (prepared from guinea pigs in our experi
ments) into newborn mice of a susceptible strain the tissue culture extracts.2
may apparently activate a latent agent, also caus
OF THE DEVELOPMENTOF SPON
ing the development of tumors in the inoculated ACCELERATION
TANEOUS
LEUKEMIAIN
AK MICE, FOLLOWING
hosts (40). This concept would perhaps also ex
INOCULATION
O
F
LEUKEMIC
EXTRACTS
plain the results of experiments carried out by
Rudali, Duplan, and Latarjet observed that the
Woolley (55), in which prolonged treatment with
cortisone resulted in the development of parotid inoculation of cell-free leukemic extracts into new
gland tumors in some of the cortisone-treated
born Ak mice accelerated the development of
spontaneous leukemia (48). A similar observation
mice. In these instances cortisone might have ac
tivated a latent oncogenic agent. The action of was also made in our laboratory.
Schwartz and his associates reported (50) that
cortisone could therefore have a similar effect to
that of foreign protein (guinea pig) extracts in inoculation of filtrates prepared from brains of leu
jected into newborn mice (40). It is conceivable kemic Ak mice into young adult Ak mice also ac
that other oncogenic agents may also be carried in celerated the development of spontaneous leuke
mia in such animals. In our hands, however, leu
an inactive, latent form in many apparently
healthy carriers, and that some of such agents, kemic filtrates prepared from leukemic donors
were found to be considerably more potent when
prompted by inducing factors, may become patho
inoculated into newborn mice, and, furthermore,
genic, causing the development of tumors.
The parotid tumor agent seems to be more elu filtrates prepared from livers, spleens, and leuke
mic tumors were at least 18 times more potent than
sive than other known tumor viruses. Apparently
those prepared from brains of the leukemic donors.
highly adapted, it only rarely causes tumors, un
less its relationship to the carrier host is disturbed
VIRUSES,RADIATION,ANDCHEMICAL
or its potency increased by experimental passage
CARCINOGENS
through newborn hosts.
One of the great difficulties in the proper inter
Under proper experimental conditions, parotid
gland tumors could be induced also in mice of pretation of results obtained with experimental
transmission of tumors by means of filtrates has
strains that have never before shown the spon
taneous development of such tumors. Thus, in been the fact that in many instances morphologi
oculation into newborn mice of cell-free extracts cally similar tumors could be obtained with chemi
containing oncogenic agents induced parotid gland cal carcinogens, hormones, or ionizing radiation.
Theoretically, therefore, one might be inclined
tumors in either the C57BR/cd strain (40) or in
mice of the Agnes-Bluhm line (49). Dulaney ob
to classify the transmissible and filtrable leukemic
served the development of typical parotid gland agent as just another leukemia-inciting factor,
tumors following inoculation of newborn Ak mice comparable to ionizing radiation, hormones, or
with the cell-free leukemic Ak extracts (13). A carcinogens. Those who would favor such an in
terpretation, however, would have to reconsider
similar observation was also made in our labora
the generally accepted assumption that tumors
tory.
such as Rous sarcoma or mouse mammary car
MULTIPLICATION
OF THE PAROTIDTUMOK
cinoma are caused by specific, filtrable viruses. Al
AGENTIN TISSUE CULTUBE
though either of these tumors can be induced in a
In a series of recent experiments, Stewart, Eddy, susceptible host by inoculation of a specific fil
and associates at the National Cancer Institute
trable virus, sarcomas in chickens can also be in
(53) succeeded in demonstrating the survival, and duced with chemical carcinogens, and mammary
possibly also the multiplication, of mouse leuke
tumors in mice can be induced in female mice
mia-derived parotid tumor agent in tissue culture known to be free from the Bittner virus with
media. After the agent was passed through mon
either methylcholanthrene or estrogenic hormones.
key kidney or chicken chorion allantoic membrane
The fact that ionizing radiation, hormones, or
culture media, inoculation into newborn (less than carcinogenic chemicals may be able to induce tu12 hours old) (C3Hf X AKR)Fi hybrid mice in
»B.E. Eddy, S. E. Stewart, et al., 3. Nat. Cancer Inst, (in
duced in up to 51 per cent of the mice the develop
press).
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1958 American Association for Cancer Research.
380
Cancer Research
Vol. 18, May, 1958
mors and/or leukemia does not necessarily imply pathogenic agents, causing then the development
that these tumor-inducing factors are similar in of malignant tumors, including the various forms
their pathogenic action to oncogenic viruses.
of leukemias. Should this concept prove to be true,
Sarcomas, carcinomas, or leukemias induced by the fundamental question would have to be an
viruses, hormones, or chemical carcinogens may swered whether a small group of oncogenic agents
not necessarily represent identical results arrived could induce an endless variety of tumors, or
at with different means. There may exist a large whether there exists in nature, and within each
variety of tumors which, although similar in their species, a multitude of individually distinct types
morphology, may be individually distinct. They of oncogenic agents, each one responsible for a dif
all may be virus-caused; hormones, chemical car
ferent type of a new growth.
cinogens, and ionizing radiation may serve only as
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Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1958 American Association for Cancer Research.
Viral Etiology of ''Spontaneous'' Mouse Leukemia: A Review
Ludwik Gross
Cancer Res 1958;18:371-381.
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