[CANCER
RESEARCH
Antigenicity
27 Tart 1, 962-967, May 1967]
of a Virus-induced
A. FEFER,1'- J. L. McCOY;'
AND
Murine Sarcoma (Moloney)
J. P. GLYNN2
Drug Evaluation Branch, Cancer Chemotherapy, National Service Center, NIH, Belhesda, Maryland 20014
SUMMARY
Moloney sarcoma virus rapidly induced sarcomas in newborn
BALB/c and CBF (BALB/c x C57BL/6) mice. A significant
incidence of regressions of primary tumors was noted. Moloney
sarcoma virus release from a transplantable Moloney sarcoma
was suggested by the rapid induction of tumor in newborn mice
inoculated with lethally irradiated cells (15,000 R). Specific
transplantation resistance to Moloney sarcoma was induced by
pretreatment of histocompatible hosts with Moloney sarcoma
virus preparation or X-irradiated Moloney sarcoma cells. Crossresistance was demonstrated between Moloney sarcoma and
Friend, Moloney, and Rauscher (FMR) leukemia cells, but not
with two long-transplanted lymphomas of non-FMR origin.
Mice immunized with Moloney sarcoma cells produced humoral
antibodies which reacted with FMR leukemia cells by the indi
rect fluorescence test. Sera from mice immunized with FMR
leukemia cells or Moloney sarcoma cells, mixed with Moloney
sarcoma virus in vitro neutralized the oncogenic activity of the
Moloney sarcoma virus preparation. The results may be ex
plained either by assuming antigenic similarity between FMR
leukemia cells and Moloney sarcoma cells, or their causative
agents, or by postulating a coexistence of Moloney lymphoma
virus and Moloney sarcoma virus in our Moloney sarcoma virus
preparation.
INTRODUCTION
Generally, leukemias induced by a given virus have a common
antigenic specificity. However, they may share antigens with
leukemias induced by other viruses. Thus, immunologie studies
in vitro (7) and in vivo (J. P. Glynn, J. L. McCoy, and A. Fefer,
unpublished data) have demonstrated that leukemia cells in
duced by Friend, Moloney, and Rauscher viruses have antigens
in common.
Moloney (6) recently noted the development of sarcomas in
newborn BALB/c mice inoculated with a Moloney lymphoma
virus preparation. Cell-free extracts of the sarcomas induced
rhabdomyosarcoma at the site of inoculation in virtually all
newborn mice (6, 8, 9). No leukemias were detected in the ex
perimental mice over an extended period of observation (6).
The sarcomas contained particles morphologically indistinguish
able from the murine leukemia viruses (1). This report concerns
1United States Public Health Corps.
2Drug Evaluation Branch, National Cancer Institute.
3Microbiological Associates, Inc., Bethesda, Md.
Received October 27, 196f>;accepted January 6, 1967.
9(V_>
studies to explore the antigenic relationship between FMR4
leukemia cells and the Moloney sarcoma.
MATERIALS AND METHODS
Mice. Male and female BALB/c, C57BL/6, and CBF
(BALB/c 9 x C57BL/6 d") mice, 7-12 weeks old, were used.
For any given experiment, mice from only one source were used.
All mice were obtained from the production colonies of Micro
biological Associates, Batelle Memorial Institute, and Texas
Inbred Mice.
Tumors. Tumors induced by FMR viruses, Moloney sarcoma
virus, radiation, and chemicals were studied. Basic data on the
transplantable tumors are presented in Table 1. The tumors
were maintained by transplantation
subcutaneously (solid
tumor) or intraperitoneally (ascites form) into normal histocom
patible adult recipients.
The Moloney sarcoma studied, the SV-122-TR4 (8), was ob
tained from Dr. J. B. Moloney. It had developed in a newborn
BALB/c mouse 3-5 days following intramuscular inoculation
of Moloney sarcoma virus. Ten days later, the tumor was excised
and transplanted for 22 passages as a brei inoculated subcu
taneously into 4-week-old BALB/c mice. We have since then
canned the tumor for 12 generations by excising the tumor when
it attains a size of 12-15 mm and implanting trocar fragments
into 7- to 12-week-old BALB/c mice.
Viruses. Moloney sarcoma virus, Lot Nos. SV-15B and SV216-RP No. 45, and Moloney lymphoma virus, Lot No. MV-2,
were obtained from Dr. J. B. Moloney. The SV-15B lot of
Moloney sarcoma virus constituted a one-gram-equivalent of
tumor tissue derived from BALB/c mice. Lot No. SV-216-RP
No. 45 was prepared from an original SV-36B virus lot which
had been neutralized 4 times with serum from a rabbit immu
nized with Moloney lymphoma virus. The Lot No. SV-216-RP
No. 45 constituted the 45th passage of the SV-36B in 4-week-old
BALB/c mice, and contained a one-gram-equivalent tumor ex
tract. The Moloney lymphoma virus, Lot MV-2, consisted of a
ten-gram-equivalent of plasma from BALB/c mice infected
with Moloney lymphoma virus as newborns.
Preparation of Cell Suspensions. Solid lymphomas were
forced through an 18-gage needle into 2 volumes of Hanks'
balanced salt solution. Uniform cell suspensions were obtained
by filtering the brei through 4-6 layers of cotton gauze. Ascites
lines were prepared by serially diluting ascites fluid in Hanks'
4The following abbreviations are used. FMR, Friend-MoloneyRauscher; Anti-FL, anti-ML, anti-RL, and anti-MS, serum from
mice immunized with the respective Friend, Moloney, or Rauscher
leukemia cells or with Moloney Sarcoma cells.
CANCER RESEARCH VOL. 27
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Antigenicity of Virus-induced Murine Sarcoma
TABLE 1
Basic Data on the Transplantable Neoplasms
Used in the Present Study
DesignationSV-122-TR4LSTRAMBL-1RBL-3FBL-3EL-4L18464EtiologyMoloney
of transplant
use30298322515Longgenerations prior to
virusMoloney
sarcoma
virusMoloney
lymphoma
virusRauscher
lymphoma
virusFriend
virus7,12-Dimethyl
tedLong-transplanted
transplan
benzanthraceneRadiationGenotypeBALB/cBALB/cC57BL/6C57BL/6C57BL/6C57BL/6C57BL/6FormSolidAscitesSolidAscitesA
balanced salt solution. The sarcoma was quite firm and was
therefore prepared for transplantation
by cutting the solid
tumor into 1- to 2-mm fragments and implanting them with a
trocar. Occasionally, a tumor brei was prepared by passing the
sarcoma through a stainless steel tissue press and diluting in the
desired volume of Hanks' balanced salt solution.
Immunization
for Transplantation
Studies. Adult mice
were immunized either with a single intraperitoneal
inoculation
of infectious virus, or with two intraperitoneal
injections of
X-irradiated cells 2 weeks apart. The mice were challenged with
viable tumor cells 6-9 weeks after virus inoculation or 1-2 weeks
after the second cell immunization. Leukemia cells for challenge
were injected as graded doses of cells subcutaneously
into the
right inguinal region. Sarcoma cells for challenge were usually
injected subcutaneously
as trocar fragments or tumor brei.
Mice were palpated on alternate days for the development of
local tumors.
X-Irradiation.
Under the following conditions, 400 R of
whole-body X-irradiation
were administered
to mice: 200 kv,
15 ma, 0.25 mm Cu and 0.55 mm Al filtration; target distance,
54 cm; dose rate 122 R/min. Tumor cells were X-irradiated in
Hanks' balanced salt solution under the same conditions, but
with a target distance of 25 cm and a dose rate of 520 R/min.
Leukemia cells routinely received 5000 R, while Moloney sar
coma cells received 15,000 R.
Preparation
of Antisera.
Specific anti-ML, anti-RL, and
anti-FL sera were obtained by intraj>eritoneal inoculation of
adult C57BL/6 mice with X-irradiated,
histoeompatible
FMR
leukemia cells, followed by progressively increasing doses of
viable cells. The mice were bled from the retroorbital sinus 7-10
days after each immunization.
The anti-FMR sera used were
obtained after 6-12 immunizations
with viable tumor cells.
The anti-MS serum used was obtained by pretreating adult
CBF mice with two injections (2 weeks apart) of X-irradiated
SV-122-TR4 and bleeding two weeks after the second injection.
The sera were distributed into 0.5-ml aliquota and stored at less
than -70°C.
The Indirect Fluorescent
Antibody Technic. The technic
developed by Möller (5) using suspensions of viable cells was
employed. Trypan blue unstained cells (2 X IO6) in a volume
of 0.05 ml of Hanks' balanced salt solution were added to 0.05
ml of undiluted serum or to serum serially diluted in Hanks'
balanced salt solution. Following a 20-minute incubation at
37°C,the cells were washed four times, and the resulting jjellet
was mixed and incubated for 20 minutes at 37°Cwith 0.05 ml
of fluorescein-conjugated
goat anti-mouse
globulin
(Hyland's)
diluted 1:5. After four more washings, the cells were examined
under the fluorescence microscope. Samples were read blind,
and 100-180 cells per sample were counted. Cells manifesting
diffuse fluorescence of the type that Möller has demonstrated
to be indicative of dead cells were omitted from calculations.
This was not a significant problem as the viability of our cell
preparations usually was greater than 90%. Viable cells were
counted and classified as stained or unstained. All cells exhibiting
bright green granular or sectorial fluorescence—or any other
staining pattern except that characteristic
of dead cells, were
counted as positive. The result of each sample, therefore, was
expressed as the number of stained viable cells X 100, divided
by the total number of viable cells examined.
Absorption of Sera. Tumor cells were prepared as described
above, washed three times, and centrifuged at 1800 rpm (4°C)
for 10 minutes. Packed cells were mixed with an equal volume of
suitably diluted serum. The mixture was incubated at room tem
perature for 45 minutes and then at 4°Cfor 45 minutes. The
unabsorbed serum control was kept at the same tem|>erature for
the same lengths of time. After incubation, the mixtures were
centrifuged at 1800 rpm at 4°Cfor 10 minutes. The absorbed
and unabsorbed sera were tested by the indirect fluorescent
antibody technic.
Neutralization
of Moloney Sarcoma Virus. Dilutions of
the Moloney sarcoma virus preparation
(10~l, 10~2, or 10~3)
were mixed with an equal volume of undiluted anti-ML, antiFL. and anti-MS serum, normal C57BL/6 serum, or with Hanks'
balanced salt solution. The mixtures were thoroughly stirred,
incubated at room temperature for 90 minutes, and inoculated
(0.05 ml intramuscularly)
into BALB/c mice less than 7 days
old. The mice were observed for mortality daily and for palpable
tumors on alternate days. Mice dying without palpable tumors
were often examined postmortem for systemic disease.
RESULTS
Induction
of Sarcomas in Newborn Mice. Representative
data regarding tumor induction in newborn mice are shown in
Table 2. In this experiment, BALB/c mice, 2-7 days old, and
CBF mice less than 24 hours old were inoculated intramuscu
larly with Moloney sarcoma virus. With rare exceptions, all
mice develo))ed palpable local tumors. No significant differ
ence was evident in the susceptibility of the two strains tested
to the oncogenic property of the virus, as expressed by the fre-
MAY 1967
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963
A. Fefer, J. L. McCoy, and J. P. Glynn
quency and latency period of tumor induction. A high incidence
of regression of primary tumors was noted. More deaths and
fewer regressions were observed in the BALB/c mice.
Transplaiitahility
of Moloiiey Sarcoma Cells. In order
to obtain transplantable tumors, Moloney sarcoma virus prepa
ration was inoculated into BALB/c and C57BL/6 mice 1-14
days old. The primary tumors were transplanted as trocar frag
ments or brei into normal and X-irradiated histocompatible
hosts. No consistent or predictable growth pattern was ob
served, and no tumors were successfully carried for more than
three generations. Therefore, for these studies, the transplantable
SV-122-TR4 obtained from Dr. Moloney was used.
SV-122-TR4 has been carried in this laboratory for 12 genera
tions in adult BALB/c mice. Trocar or brei implantation of the
Moloney sarcoma resulted in the development of local tumors in
over 90% of such mice within 6-10 days. Approximately 5070% of the tumors eventually regressed, while the rest grew
progressively and killed the hosts. Tumor growth was more
difficult to obtain in CBF mice. For example, in adult BALB/c
and CBF mice inoculated with comparable tumor fragments,
tumors were obtained in 14/15 and 16/47 mice, respectively.
This pattern of growth resixmse is similar to that reported by
McCoy and Glynn (4) for BALB/c Rauscher lymphoma cells in
mice of the same strains.
Moloney Sarcoma Virus Release from Moloney Sarcoma
Cells. SV-122-TR4, a Moloney sarcoma of BALB/c origin, was
X-irradiated with 15,000 R and inoculated into BALB/c mice
24-48 hours old and into C57BL/6 mice 4-6 days old. Eighteen
out of 19 mice developed local tumors within 10 days (Table 3).
Induction of Specific Transplantation
Resistance to
FMR Leukemia Cells and Moloney Sarcoma Cells. To
demonstrate the antigenicity of Moloney sarcoma in histocom
patible hosts, adult mice were pretreated with two injections of
X-irradiated Moloney sarcoma cells and, two weeks after the
second injection, were inoculated with graded doses of histo
compatible Moloney sarcoma brei. Half the pretreated and un
treated recipients were X-irradiated with 400 R one day prior
to challenge. Table 4 shows that preimmunized mice were re
sistant to the transplantation of Moloney sarcoma cells. The
resistance was observed even in X-irradiated recipients, suggest
ing that the pretreatment exerted a s]>ecificimmunizing effect,
and not merely a nonspecific stimulation of host resistance.
TABLE 2
Development of Primary Tumors in Newborn BALB/c and CBF
Mice Inoculated with Moloney Sarcoma Virus"
of mice
tumors'*Total
developing
strainBALB/c
Mouse
CBF\o.
inoculated36/37
11/11Xo.
" BALB/c newborns were inoculated with
10~2 dilution of Moloney sarcoma virus (Lot
45) intramuscularly
when 2-6 days old. CBF
24 hours old) were inoculated with 0.05 ml
of complete
regressionsTotal
tumors8/36
10/11
0.05 ml of a 10~' or
No. SV-216-RP No.
newborns (less than
of a 10~' dilution of
Moloney sarcoma virus intramuscularly.
6 All tumors developed by Day 16. A mean maximal tumor size
of 15 mm was attained in BALB/c mice (range, 12-21 days) and
of 17 mm in CBF mice (range, 11-14 days). Tumors that did not
regress grew progressively and ultimately killed the host.
964
TABLE 3
Induction
of Moloney Sarcoma in Newborn Mice Inoculated with
SV-128-TR4 X-irradiated with 15,000 R
of mice
developing
of mice
tumors"No.inoculated10/10
dying with
tumor10
strainBALB/c Mouse
(24-48 hours old)
C57BL/6 (4-6 days old)N'o.
8/9'No.
time to
death (range
days)19
in
(14-23)
6Mean 37 (21-68)
" Mean latency period to development of palpable tumor was
less than 10 days for both strains.
6When last observed (Day 90), 3 mice were alive, 2 without
palpable tumors.
TABLE 4
Resistance to Transplantation of Moloney Sarcoma in Normal and
Irradiated CBF Mice Pretreated with X-Irradiated Moloney
Sarcoma Cells"
/TotalUntreatedtakes6
Recipients
X-irradiated
with400
dose
(dilution of
tumor
brei)1/101/501/101/50Tumor
mice5/55/5Total
mice
(dilution
oftumor
brei)0/50/40/91/50/51/10
RNoneChallenge
10/104/51/5Total
5/10Immunized
" Mice were pretreated on Days 0 and 14 with X-irradiated
(15,000 R) SV-122-TR4 tumor cells. On Day 24, one-half of the
recipients were X-irradiated, and all mice were challenged with
viable SV-122-TR4 tumor cells.
bMean maximal tumor size of mice developing tumor = IGmm.
Old et al. (7) demonstrated by the cytotoxicity test that
FMR leukemia cells had antigens in common. Furthermore
(J. P. Glynn, J. L. McCoy and A. Fefer, unpublished observa
tions), it has been demonstrated that immunization with any
one of the FMR leukemia viruses induces resistance to the trans
plantation of tumors induced by all three viruses. Likewise, pre
treatment of recipients with leukemia cells induced by any one
of the FMR leukemia viruses conferred immunity to the trans
plantation of tumors induced by all three viruses. By analogy
with such experiments, we attempted to induce transplantation
resistance to FMR leukemia cells and Moloney sarcoma cells.
Adult mice were pretreated with a single injection of Moloney
sarcoma virus, and 8-9 weeks later were challenged with graded
doses of FMR leukemia cells and Moloney sarcoma cells from
histocompatible mice. Tables 5 and 6 show that Moloney sar
coma virus immunized against the transplantation of FMR
leukemia cells and Moloney sarcoma cells. The s]>ecificity of the
resistance is evidenced by the inability of Moloney sarcoma virus
to induce resistance to long-transplanted tumors of non-FMR
origin (EL-4 and L18464). Further evidence for the antigenic
relationship between Moloney lymphoma and Moloney sarcoma
CANCER RESEARCH VOL. 27
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Antigenicily of Virus-induced Murine Sarcoma
TABLE 5
Resistance to Transplantation
of Friend-Moloney or Rauscher
Leukemia Cells and Moloney Sarcoma Cells in Adult
Hislocompalible CBF Mice Pretreated with Moloney
Sarcoma Virus Preparation"
Takes /Total
Tumor challenge
Cell dose
Untreated
mice
Treated
mice
SV-122-TR4
Trocar
6/7"
0/7
RBL-3
1 X 10'
1 X 10»
7/10
2/10
2/10
1/10
9/20
3/20
9/9
8/10
0/10
0/10
17/19
0/20
9/9
0/10
0/10
1/10
9/19
1/20
10/10
2/10
10/10
2/10
Total
1 X 10'
1 X 10«
FBL-3
virus in Hanks' balanced salt solution or normal serum dcvcloi>ed
tumors. With decreasing
virus there was an increase
velopment. Mice receiving
anti-ML serum developed
concentration
of Moloney sarcoma
in the latency period to tumor de
Moloney sarcoma virus treated with
no tumors. There was a moderate
TABLE 6
Resistance
to Transplantation
of C67BL/6
Friend-Moloney
or Rauscher Leukemia Cells in Adult C67IÃŒL/6Mice Pre
treated with Moloney Sarcoma Virus Preparation"
challengeFBL-3RBL-3MBL-1L18464Cell
Tumor
dose5
mice10/10
X IO5
5 X 10*
IO34
5 X
mice1/101/9
7/10
1/103/295/10
0/10Total
17/3010/10
Total
LSTRA
5 X 10'
5 X 10'
Total
1 X 10«
1 X 10s
EL-4
Total
12/20
X IO6
4 X IO6
IO43
4 X
2/10
5/10Total
0/10
1/106/302/10
17/3010/10
X IO5
3 X IO1
IO318
3 X
9/10
8/10Total
0/10
0/102/3010/10
12/20
27/3010/10
•
Moloney sarcoma virus Lot No. SV-216-RP No. 45—0.1ml of
a 10~' dilution inoculated intraperitoneally
eight weeks prior to
X 10"
18 X 10*
9/10
18 X IO3Takes/TotalUntreated
4/10Total
tumor challenge.
6 Mean maximal tumor size = 13 mm. Tumors failed to grow in
untreated mice inoculated with lower levels of SV-122-TH4.
9/10
7/1026/30
23/30Test
is presented in Tables 7 and 8. Pretreatment
with Moloney
lymphoma virus or X-irradiated
Moloney lymphoma cells in
duced resistance to the transplantation
of Moloney lymphoma
and Moloney sarcoma. It is noteworthy that in all transplan
tation resistance studies almost all Moloney sarcoma tumors
which ¡crewultimately regressed in treated and untreated re
cipients.
Although the demonstration
of transplantation
resistance to
Moloney sarcoma was hamjiered by the low frequency of Mo
loney sarcoma tumor takes in unimmunized recipients, cumula
tive data (Tables 4-8) show 34/77 tumor takes in control mice,
compared with 1/75 takes in immunized recipients challenged
with equivalent doses of Moloney sarcoma.
Neutralization
of the Oncogenic
Property of Moloney
Sarcoma Virus by Anti-FMR and Anti-MS Sera. To inves
tigate the serologie relationship between the FM R viruses and
Moloney sarcoma virus, newborn lì
ALB/c mice were inoculated
with graded doses of Moloney sarcoma virus with and without
prior reaction with an equal volume of antiserum obtained from
adult mice preimmunized with irradiated FMR leukemia cells
or Moloney sarcoma cells. The mice were examined on alternate
days for tumor size and daily for mortality. The results are sum
marized in Table 9.
In the course of an observation |)eriod of 95-130 days, 53 out
of 55 mice receiving 10~l to 10~3 dilutions of Moloney sarcoma
" Moloney sarcoma virus Lot No. SV-15B—0.1 ml of a 10~l
dilution inoculated intraperitoneally
nine weeks prior to tumor
challenge.
TABLE 7
Resistance to Transplantation of Moloney Lymphoma and Moloney
Sarcoma Cells in Adult CBF Mice P retreated with Moloney
Lymphoma Virus"
challengeMBL-1
Tumor
dose5
mice7/7
mice0/101/81/180/300/30
X IO7
IO6TrocarTakes/TotalUntreated
5 X
1/7Total
8/146/30Total
SV-122-TR4Cell
6/306Treated
" Moloney
lymphoma
virus Lot No. MV-2—0.1 ml of a 10~'
dilution inoculated intraperitoneally
six weeks prior to tumor
challenge.
6 Mean maximal tumor size = 8 mm.
MAY 1967
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960
A. Fefer, J. L. McCoy, and J. P. Glynn
neutralization of Moloney sarcoma virus by anti-RL, anti-FL,
and anti-MS scrum. The neutralization was manifested by a
decreased frequency of tumor development and an increased
latency jjeriod.
Serologie Reactivity
of Anti-MS
Cells as Detected by the Indirect
TABLE 10
Serologie Activity of Anti-MS
Serum against Friend, Moloney,
and Rauscher Leukemia Cells as Tested by the Indirect
Fluorescent Antibody Technic
Serum against FMR
Fluorescent
Antibody
stained"FBL-35817RBL-.ÃŽ4820241819LSTRA52351239
TABLE 8
Resistance to Transplantation of Moloney Lymphoma and Moloney
Sarcoma cells in Adult CBF Mice Preireated with X-irradiated
Moloney Lymphoma Cells"
Anti-MSNormal
C57 SerumUndiluted1:21:41:8Undiluted%
challengeMBL-l
Tumor
dose7
mice8/8
X 10"
IO5Trocar
7 X
7/10Total
mice3/10
" In each sample, 100-180 cells were counted.
performed blind.
2/105/200/9
TABLE 11
Absorption of Fluorescent Activity of Anti-Friend, Moloney, and
Rauscher Sera by Friend, Moloney, and Rauscher Leukemia
Cells and Moloney Sarcoma Cells
15/184/10
SV-122-TR4Cell
6 X 10sTakes/TotalUntreated
3/10Total
All counts were
0/100/19
Serum from micea
dilution
stained84365782746858721195
withFBL-3"RBL-3MBL-lSerum
inoculated
in1:41:21:2Absorbed withFBL-3MSEL-4US"RBL-3MSEL-4USLSTRAMSEL-4U
7/20»Treated
0 CBF mice were immunized with X-irradiated (5000R) MBL-l
cells on Days 0 and 14 and were challenged on Day 28.
*Mean maximal tumor size = 8 mm.
TABLE 9
Neutralization
of Moloney Sarcoma Virus" Oncogenicily by Anti-FMRk and Anli-MS Sera
No tumors developed in mice inoculated with 10~4to 10~7dilu
tions of Moloney sarcoma virus preparation in Hanks' balanced
salt solution. Observation period above the line = 130 days; ob
servation period below the line = 95 days.
virusio-110/10"8/87/7 of Moloney sarcoma
" Aliquots from same pools of antisera as were used to neutral
ize Moloney sarcoma virus oncogenicity (Table 9). All counts
were performed blind.
6See Table 1 for the etiology of these designations.
" US, unabsorbed sera.
SerumHanks'
bal
saltsolutionAnti-FL
anced
Anti-RL
Anti-MLNormal
Technic. Aliquots from the same [X)olof anti-MS serum which
3/7
was used to neutralize the oncogenic property of the Moloney
0/78/88/8MLP)(days122026714io-a10/108/92/9
0/108/91/10MLP(days)2747692450
C57serumAnti-FL
Anti-RLAnti-MSDilution
4/9
42
2/10MLP(days)10953
1710-'9/93/7
- Lot No. SV-216-RP No. 45.
6Anti-FMR, collective term for anti-FL, anti-ML, and anti-RL;
MLP, mean latency period to development of palpable tumor;
anti-FL, anti-ML, and anti-RL, serum from mice immunized
with the respective Friend, Moloney, and Rauscher leukemia
cells; anti-MS, serum from mice immunized with Moloney sar
coma cells.
c Number of mice developing tumors/number inoculated.
966
sarcoma virus were tested by the indirect fluorescent antibody
technic against FMR leukemia cells. The results are summarized
in Table 10. Normal control sera stained 9-19% of the cells,
usually as a small number of barely perceptible dots on the cell
membrane. With anti-MS serum the staining was more intense
and involved larger sectors of the cell membrane. The anti-MS
serum reacted weakly with all three FMR leukemia cells, but
not with EL-4.
Absorption of Anti-FMR Activity by Moloney Sarcoma
Cells. Attempts to prepare a suspension of viable Moloney sar
coma cells adequate for use as target cells were unsuccessful.
Accordingly, the ability of Moloney sarcoma cells to absorb
anti-FMR serologie activity was tested. Aliquots from antiFMR serum pools used for neutralization (Table 9) were abCANCER RESEARCH VOL. 27
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Antigenicity of Virus-induced Murine Sarcoma
sorbed with equal volumes of packed cells—homologous leuke
mia cells, Moloney sarcoma cells, and EL-4. Each absorbed
serum was then tested by the fluorescence technic against its
homologous leukemia. Table 11 shows that Moloney sarcoma
cells absorbed appreciable amounts of anti-FMR activity. The
specificity of the reaction is attested to by the lack of absorption
by EL-4.
DISCUSSION
Moloney sarcoma virus is a potent oncogenic agent in new
born mice, which appears to have a marked affinity for muscle
tissue regardless of the route of inoculation (9). The primary
local tumors have been designated as rhabdomyosarcomas (8).
Current data, like those of Perk and Moloney (8), show that
Moloney sarcoma virus induces tumors in newborn mice. How
ever, a significant incidence of regression of these primary tumors
was observed.
Transplantation experiments demonstrate that Moloney sar
coma cells are antigenic in histocompatible hosts. Moloney
sarcoma virus and Moloney sarcoma cells induced resistance to
the transplantation of Moloney sarcoma (Tables 4, 5). Preimmunization with X-irradiated Moloney sarcoma cells induced
resistance to the transplantation of Moloney sarcoma even in
mice X-irradiated with 400 R prior to the challenge inoculation,
indicating that the pretreatment exerted a specific immunizing
effect, and not merely a nonspecific potentiation of the host's
resistance (3).
Current studies suggest that Moloney sarcoma and FM R
leukemia cells (or their causative agents) possess similar anti
gens. This conclusion is based on the following transplantation
and serologie experiments:
1. Pretreatment of adult mice with Moloney sarcoma virus
induced transplantation resistance to histocompatible FM R
leukemia cells.
2. Immunization with Moloney lymphoma virus or Moloney
lymphoma cells induced resistance to the transplantation of
Moloney sarcoma cells.
3. Exposure of the Moloney sarcoma virus preparation in
vitro to sera obtained from mice immunized with FMR cells or
Moloney sarcoma cells resulted in a definite decrease in its onco
genic potency. Moloney sarcoma virus exposed to anti-ML
serum failed to induce any tumors when injected into 17 newborn
mice, whereas 35/36 mice receiving identical doses of Moloney
sarcoma virus exposed to normal serum or Hanks' balanced
salt solution developed tumors. The virus neutralization with
anti-MS, anti-FL, and anti-RL sera was of a lesser degree.
4. Sera obtained from mice preimmunized with X-irradiated
Moloney sarcoma cells reacted against FMR leukemia cells by
the indirect fluorescent antibody technic.
5. Moloney sarcoma cells absorbed appreciable amounts of
anti-FMR activity as measured by the fluorescence test.
The results do not differentiate cellular from viral antigens,
because FMR leukemia cells and Moloney sarcoma cells are
known to contain (1) virus particles. Inoculation of MS cells
irradiated with 15,000 R into newborn mice resulted in the de
velopment of progressively growing tumors and death in all
BALB/c mice (Table 3). The rapidity with which these tumors
appeared suggests that the Moloney sarcoma cells release appre
ciable quantities of Moloney sarcoma virus, since it is unlikely
that the tumors were the progeny of the inoculated cells.
In view of the marked antigenic similarity between leukemias
induced by the FMR agents, current transplantation and fluo
rescence results, which demonstrate that this antigenic similarity
extends to the Moloney sarcoma, may be explained by postu
lating a coexistence of Moloney lymphoma virus and Moloney
sarcoma virus in our Moloney sarcoma virus preparation. Such
a possibility cannot be completely ruled out. However, Moloney
(6) failed to detect Moloney lymphoma in newborn mice infected
with Moloney sarcoma virus, over an extended ]>eriodof obser
vation. Furthermore, we have demonstrated marked neutraliza
tion of the oncogenic property of our Moloney sarcoma virus
preparation by anti-ML serum. This suggests that either Mo
loney sarcoma virus or Moloney lymphoma virus have antigens
in common, or that Moloney lymphoma virus does coexist in
our Moloney sarcoma virus preparation and is essential for the
oncogenicity of the Moloney sarcoma virus. The Moloney
lymphoma virus would then be acting as a "helper virus" in
vivo, as it has been suggested to be operative in vitro (2).
ACKNOWLEDGMENTS
The excellent technical assistance of Mr. L. Turner and Mr. S.
Von Der Pool is gratefully acknowledged.
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967
Antigenicity of a Virus-induced Murine Sarcoma (Moloney)
A. Fefer, J. L. McCoy and J. P. Glynn
Cancer Res 1967;27:962-967.
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