Download Transformation of Hamster Embryo Fibroblasts

[CANCER RESEARCH 33, 1527-1534, June 1973]
Transformation of Hamster Embryo Fibroblasts by Herpes Simplex
Viruses Type 1 and Type 21
Fred Rapp2 and Ronald Duff
Department of Microbiology. College of Medicine, The Milton S. Hershev Medical Center, The Pennsylvania Slate University, Hershev, Pennsylvania
17033
Summary
Hamster embryo fibroblast cells can be transformed after
infection by ultraviolet light-irradiated herpes simplex
viruses type 1 (HSV-1) or type 2 (HSV-2). Two ofl2 strains
of HSV-1 and 7 of 15 strains of HSV-2 induced cell
transformation. Transformed cells from two strains of
HSV-2 and one strain of HSV-1 have been tested for
oncogenicity in newborn Syrian hamsters. One cell line
transformed by HSV-2 strain 333 was found to be oncogenic. The remaining two cell lines were not oncogenic.
Herpesvirus antigens were found in the cytoplasm and on
the surface of the transformed cells. Neutralizing antibodies
were produced in hamsters developing tumors after the
injection of the HSV-2-transformed cells. Weanling ham
sters that were preimmunized by injection of HSV-1 or
HSV-2 did not develop transplantation immunity to the
HSV-2-transformed cells as a result of this preimmunization. HSV-2-transformed cells metastasized after injection
into hamsters. The number of métastaseswas not inhibited
by HSV-1 or HSV-2 immunization but was actually
enhanced. Immunization of hamsters with simian virus 40
inhibited métastasesbut did not reduce the rate of primary
tumor development.
Introduction
Four of the major groups of viruses containing DNA
induce in vitro transformation and in vivo induction of
tumors (2, 9). The most likely candidates for viruses
oncogenic in humans appear to be members of the herpesvirus group. Several lines of evidence have been documented
that link these viruses to cancer in both lower animals and
man. The best documented case of the expression of
oncogenic potential by a herpesvirus is found in a disease of
chickens, Marek's disease (4, 22). The infection of a young
chicken with a herpesvirus induces a highly proliferative
lymphosarcoma that ultimately results in the death of the
animal. In addition to the direct demonstration by virus
injection that a herpesvirus is responsible for this disease,
further evidence that Marek's disease is caused by a
' Presented at the American Cancer Society Conference on Herpesvirus
and Cervical Cancer, December 8 to 10, 1972, Key Biscayne, Fla. This
work was conducted under Contract 70-2024 within the Special VirusCancer Program of the National Cancer Institute, NIH, USPHS.
2Presented by.
JUNE 1973
herpesvirus comes from the observation that the disease can
be prevented by the injection of a similar attenuated virus
that does not induce the neoplasia (23).
More recently a herpesvirus has been implicated in
lymphomas that can be induced in monkeys (16 19, 29).
This virus, Herpesvirus saimirÃ-, can be isolated from
squirrel monkeys (SaimirÃ-sciureus) without a tumor; how
ever, it induces lymphomas or lymphocytic leukemia in
marmosets (Saguinus oedipus, Saguinus fusciollis, and
Saguinus nigricollis), owl monkeys (Aotus albifrons), and
cinnamon ringtail monkeys (Cebus albifrons). In addition to
these demonstrations of herpesvirus oncogenicity in ani
mals, the Epstein-Barr virus of humans has been associated
with Burkitt's lymphoma. Cells derived from Burkitt lym
phomas contain virus antigens that can be detected by
immunofluorescence and herpes-like particles that can be
observed with the electron microscope (10, 13). The virus
that can be isolated is not highly infectious but has been
shown to induce the in vitro transformation of leukocytes
(15). A very similar, and probably identical, virus has been
shown to be associated with infectious mononucleosis and is
now thought to be the causative agent ofthat disease (14).
A 2nd human herpesvirus which has been associated with
human neoplasia is HSV-2.3 The original observation that
implicated this virus in cancer was the result of seroepidemiological studies. It was demonstrated that women with
cervical carcinoma have circulating antibodies directed
against the HSV-2 more often than women without the
disease (21, 24). HSV-2 has also been isolated from cervical
carcinoma cells and HSV-2 antigens can be demonstrated in
similar cells by immunofluorescent techniques (1, 27).
However, HSV's when injected into experimental animals
rarely, if ever, induce tumors (20, 25). Recently, in our
laboratory we have demonstrated that HEF cells can be
transformed by UV-irradiated HSV-2 and that these cells
are oncogenic when injected into newborn hamsters (8, 9).
Transformation of Hamster Embryo Cells
HSV-1 and HSV-2 are extremely cytopathic in tissue
culture cells that have been infected by the virus. Any
potential oncogenic transformation would therefore be
masked by the cell death that results from HSV infection.
3The abbreviations used are: HSV-2. herpes simplex virus type 2; HSV.
herpes simplex virus; HEF, hamster embryo fibroblast; HSV-1, herpes
simplex virus type 1; SV40, simian virus 40.
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Fred Rapp and Ronald Duff
The elimination of cell death induced by HSV-1 and HSV-2
was accomplished by UV irradiation'(42 ergs/sec/sq cm) of
the virus before infection of the host cell. Inactivated HSV-1
and HSV-2 were absorbed onto HEF cells in vitro while the
cells and virus were in suspension. Three to 4 weeks after the
HSV infection, transformed cells appeared in the culture
monolayer. These cells could then be isolated from the
monolayer and grown in culture into cell lines. Under the
conditions used, both HSV-1 and HSV-2 have transformed
HEF in vitro (Table 1). Seven of 15 HSV-2 strains induced
HEF cellular transformation and 2 of 12 HSV-1 strains also
transformed HEF cells. The morphology of the transformed
hamster embryo cells has been constant depending upon the
type of HSV used to transform the cells. Cells transformed
by HSV-2 were usually of mixed morphology (Fig. 1). Three
types of cellular morphology were generally seen: (a) a
fibroblastic cell, (b) a giant cell, and (c) a rather primitive
mesenchymal-like cell. In contrast to this type of transfor
mation induced by HSV-2, HSV-1-transformed cells usu
ally exhibited an epithelial-like morphology (Fig. 2). In
addition to the epithelial cells, many giant cells were also
observed in HSV-1-transformed HEF cells.
Antigenic Characteristics of HSV-transformed
Table 2
HSV-specific antigens on the surface of HSV-2-iransformed hamster
embrvo cells
SerumAmi
of cells reacting
type333-8-9HEF333-8-9HEF333-8-9
at surface60.912.260.39.37.41
HSV-1Ami
HSV-2Weanling
hamsterCell
HEF%
Table 3
Induction of tumors in newborn hamsters by the injection of hamster
cells after in vitro transformation by U V-irradialed HS V-2
strain 333
hamsterswith
of
(wk)10
period
Cell
line333-8-9333-2-20333-2-21333-2-26333-2-29%
tumors36100KM)10098Latent
162
42-42-42-7
Cells
Cells that have been transformed by either DNA or RNA
oncogenic viruses usually contain virus-specific antigens.
For example, the transformation of a cell by the SV40
usually results in the induction of the SV40 T or tumor
antigen in the nucleus and in the induction of the SV40 S
antigen on the surface of the cell (3, 24, 28). These antigens
are usually demonstrated by the use of immunofluorescence
techniques. Cells that have been transformed after infection
by HSV-1 or HSV-2 were tested by indirect immunofluores
cence techniques for the presence of HSV-specific antigens.
When anti-HSV serum that had been prepared in weanling
hamsters was absorbed to acetone-fixed HSV-transformed
cells, a cytoplasmic fluorescence was detected within these
cells (Fig. 3). The diffuse fluorescence observed throughout
the cytoplasm was seen in 5 to 25% of the transformed HEF
cells.
antigen reacted with both antiHSV-1The
and cytoplasmic
anti-HSV-2 sera.^
serum stained only a small percentage of the transformed
cells exposed to this reagent.
Characteristics of HSV-transformed
Cells in Vivo
The transformed cells (2 x IO5 cells/hamster) were
injected into weanling and newborn Syrian hamsters. No
tumors were induced in weanling hamsters by any of the 7
cell lines tested. All hamster cell lines transformed by the
HSV-2 strain 333 were oncogenic after injection into
newborn hamsters. However, cell lines that had been
established following transformation by this strain of
HSV-2 differed with respect to their relative oncogenic
potential. As shown in Table 3, 1 cell line (333-8-9) was of
relatively low oncogenic potential after injection into new
born hamsters. However, the remaining 4 cell lines that
were transformed by strain 333 (333-2-20, 333-2-21,
333-2-26, and 333-2-29) induced tumors with greater fre
The HSV-2 transformed cells were also examined for the quency and with shorter latent periods than did the 333-8-9
presence of herpesvirus-specific antigens on the surface by cells. In contrast to the lines transformed by strain 333, cells
that were transformed by HSV-2 strain 332 and HSV-1
utilizing indirect immunofulorescence techniques and un
fixed cells. As shown in Table 2, significant numbers of strain KOS were not oncogenic when injected into newborn
these transformed cells demonstrated fluorescence on their hamsters. From these results we have now classified HSVsurface after treatment with anti-HSV serum. Normal cells transformed cells into 3 types with regard to their oncogenic
reacted in very low numbers and normal weanling hamster potential: high, low, and nononcogenic.
Sera from tumor-bearing hamsters were tested for anti
bodies against HSV's. It was found that a significant
Table I
number of the tumor-bearing hamsters developed neutraliz
Transforma/ion of HEF by isolates of HSV-1 and HSV-2
ing antibodies directed against HSV-2. Sera that neutral
isolateswith
of
ized HSV-2 did not neutralize HSV-1 as efficiently in most
ofisolatestested1215No.detectabletransformationpotential27
cases (Chart 1). It was also found that the development of
neutralizing antibodies was inversely proportional to the
Virus
typeHSV-1HSV-2No.
latent period of tumor development in the host hamster. The
cell lines transformed by HSV-2 strain 333 that had a
shorter latent period in the newborn hamsters did not induce
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Cellular Transformation by HSV
Transplantation Immunity Studies with HSV-2-transformed
Cells
100.
to
OC
O
en
D
in
o
oc
O
HSV
333-8-9
(LSH-1)
TYPE
SERUM
21
21
A
21
B
21
C
21
D
E
Chart 1. Neutralization
of HSV-2 and HSV-1 by sera obtained from
hamsters with tumors induced by an s.c. injection of HSV-2-transformed
cells (333-8-9, LSH-I). All hamsters were given injections of the trans
formed cells at 3 to 4 weeks of age. The results are expressed as the
percentage of virus survivors after incubation of 1000 plaque-forming units
of HSV with 1 ml of a 1/10 serum dilution.
neutralizing antibodies with as great a frequency as did the
cell line with the longer latent period (Table 4). The location
of the antigens that induced the neutralizing antibody
response in the tumor-bearing hamsters is not clear at the
present time. The surface or cytoplasmic antigens which
have been observed may be the antigens that induce this
immune response. However, in a few of the tumor cells
immature HSV-like particles have been observed and this
low-level production of presumably noninfectious HSV may
result in the induction of HSV-2-neutralizing antibodies
(ID-
Neutralization
of HSV-1
The study of the characteristics of transplantation im
munity was not possible with the primary transformed cell
lines because they failed to induce tumors in weanling
animals. For determination of whether weanling hamsters
could be used, transformed cells that had been passed 1time
in a newborn hamster were tested for their oncogenic
potential in weanling hamsters (Chart 2). After 1 passage in
a newborn hamster the 333-8-9 cell line was found to have
increased oncogenicity to the point where it induced tumors
when small numbers of cells were injected into weanling
hamsters. A 2nd transformed cell line (333-2-26) was also
tested for its oncogenic potential in weanling hamsters after
the cell line had been passed once in a newborn hamster. As
previously shown (Table 3), this cell line was much more
oncogenic when injected into newborn hamsters than was
the 333-8-9 cell line. The 333-2-26 cell line also showed
increased oncogenic potential after 1 animal passage and
induced tumors in weanling hamsters (Chart 2). However, it
was not as oncogenic as was the 333-8-9 cell line after
passage in a newborn hamster. It can be postulated from
this result that the longer in vivo latent period of the 333-8-9
cell line resulted in the selection of cells with a higher
oncogenic potential than did the shorter latent period of the
333-2-26 cells. Both cell lines induced metastatic tumors in
the lungs of animals after s.c. injection of the cells (Fig. 4).
Métastaseswere also found in the liver, gastrointestinal
tract, and kidneys of the injection-treated animals. The
development of métastasesby tumor-bearing hamsters
indicated that, at most, a very low level of tumor immunity
was induced by the cells in the host hamster. As a test of this
hypothesis, weanling hamsters were immunized by at
tenuated HSV-1 strain 35 (25). The injection of this strain of
HSV-1 did not result in the death of the hamster. In
addition to the injection of HSV-1 strain 35, hamsters were
also immunized by the DNA tumor virus SV40, UVirradiated 333-8-9 cells, and UV-irradiated HEF cells. Each
animal received 3 injections of an immunizing agent at
weekly intervals followed 1 week later by the injection of the
tumor cells. As sfÃ-ownin Chart 3 the preimmunization of
hamsters by HSV-1 strain 35, SV40, 333-8-9 cells, or HEF
cells did not inhibit the development of primary tumors.
Table 4
and HSV-2 by sera from hamsters
HSV-2-transformed
cells
bearing tumors induced by
Anti-HSV-1
sera"/total
Anti-HSV-2
sera"/total
positive6064.312.541.213.8Positive
positive10035.762.570.675.9
line333-8-9333-2-20333-2-21333-2-26333-2-29Positive
Cell
sera
tested3/59/142/167/174/29%
tested5/55/1410/1612/1722/29%
sera
" Positive sera are defined as the sera that neutralized
of HSV-2 at a I/10 serum dilution.
JUNE 1973
at least 50% of 1000 plaque-forming
units
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Fred Rapp and Ronald Duff
métastasesby the HSV-transformed
cells when animals
were immunized with 3 injections of this virus. Since it has
been previously shown that SV40-transformed cells contain
new surface embryonic antigens (5, 7), it can be postulated
that the inhibition of métastasesmight be a result of the
stimulation of an immune response against hamster embry
onic antigens. The enhancement of métastasesafter the
injection of HSV-1 is possibly due to the presence of
blocking antibodies that have been previously described in
other tumor systems (12). These results that demonstrate
the enhancement and inhibition of métastasesafter an s.c.
injection of 333-8-9 cells have been repeated utilizing a 2nd
HSV-2-transformed cell line, the 333-2-26 line (Table 5).
Although the frequency of métastaseswas somewhat less
with the 333-2-26 line, the pattern of métastaseswith re
gard to the inhibition by SV40 and the enhancement by
HSV-1 was essentially the same.
o
0-
Discussion
10
WEEKS
AFTER
11
12
CELL CHALLENGE
Charl 2. The rate of tumor induction in weanling Syrian hamsters after
the injection of HSV-2 transformed hamster cells. Hamsters received s.c.
injections of 333-8-9 cells (•)or 333-2-26 (O). TPD/50, 50% tumorproducing dose.
Since it was possible that the attenuation of HSV-1 strain 35
destroyed its potential for the induction of tumor immunity,
nonattenuated strains of HSV-1 and HSV-2 were also
injected after UV irradiation to destroy virulence. However,
the results using UV-inactivated HSV-1 and HSV-2 were
the same as the results obtained using the HSV-1 strain 35.
No detectable inhibition of the primary tumors was ob
served using UV-irradiated HSV-1 or HSV-2.
If extremely low levels of transplantation immunity were
induced by injections of HSV-1 or HSV-2 in weanling
hamsters, this low level of immunity might be detected by
inhibition of métastasesrather than by inhibition of the
primary tumor. To answer this question, animals were
immunized by HSV-1 strain 35, SV40, UV-irradiated
333-8-9 cells, and UV-irradiated HEF cells. The preimmunization of weanling hamsters by HSV-1 resulted in an
enhancement of métastasesin these animals (Table 5). The
immunization of weanling hamsters by UV-irradiated
333-8-9 cells and HEF cells resulted in a significant decrease
in the number of métastases.SV40 completely inhibited
1530
The results summarized in this communication provide
evidence for the association of HSV-2 as well as HSV-1
with the transformation of normal hamster cells into
morphologically altered cells. In some cases this morpho
logical transformation was accompanied by the conversion
of the normal cells into cells with oncogenic potential.
However, in all cases HSV antigens could be demonstrated
in the cytoplasm and on the surface of the cells. Recently,
the transformation of human embryonic lung cells has been
reported following abortive infection by HSV-2 (6). Neither
HSV-1 nor HSV-2 has been conclusively shown to trans
form human cells in our laboratory. However, experiments
now in progress are designed to demonstrate this.
The frequency of oncogenic transformation by HSV-1 or
HSV-2 still remains to be completely delineated. Cell lines
that have been transformed by 2 strains of HSV-2 and 1
strain of HSV-1 have been tested for oncogenic potential.
Only 1 group of 5 transformed cell lines that was trans
formed by the HSV-2 strain 333 was oncogenic after
injection into newborn hamsters. After further extensive
testing of many cell lines transformed by several virus
strains it can be determined whether this oncogenic conver
sion is a relatively rare characteristic of HSV-1 and HSV-2
strains or whether it occurs at a relatively high frequency.
The final observation described in this communication is
that HSV-2-transformed cells do not contain levels of
virus-specific transplantation antigens that are found in
most cell lines transformed by other DNA viruses. This
apparent lack coupled with failure to stimulate the immune
response of the host animal is probably responsible for the
high rate of métastasesthat is found in HSV-2 tumor-bear
ing animals. The procedure described to control enhance
ment and repression of these métastasessuggest that the
HSV-2-transformed cells may represent a model system for
the experimental control and characterization of métastases
in mammals.
CANCER RESEARCH VOL. 33
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Cellular Transformation by HSV
10*1
e io
10
WIIK1
«Ft« CHI
CHAUINOE
WIIKS
»ft«
CUI
CHAUINOI
Chart 3. The rate of tumor induction by HSV-2-transformed cells in weanling Syrian hamsters after immunization. Immunizing agents in Chart ÃŒA
were HSV-1 (O). SV40 (•),or Medium 199control (x). Immunizing agents in Chart 3B were UV-irradiated HEF cells (•).UV-irradiated 333-8-9 cells
(D), and Medium 199 control (x). Hamsters in each immunization group received 3 immunizing injections at 1-week intervals. The animals were
challenged with 333-8-9 cells I week after the final immunizing injection. TPD/50. 50% tumor-producing dose.
Table 5
Effect of immunization on the development of métastases¡nweanling hamsters
of
with lung
hamsters with
given injections27403840343540404039Hamsters
Cell
line333-8-9333-2-26Immunizing
agentHSV-1"SV40"333-8-9'HEF"Medium'
métastases13032760124%
métastases48.107.95.020.617.102.55.010.3
con
trolHSV-1-SV40*333-8-9'HEF"Medium'
con
trolTotalhamsters
" Hamsters in this group were immunized by 3 injections of IO6plaque-forming units of HSV-1
strain 35.
" Hamsters in this group were immunized by 3 injections of IO6plaque-forming units of SV40.
' Hamsters in this group were immunized by 3 injections of 10" 333-8-9 cells that were UVirradiated for 3 min (42 ergs/sec/sq cm).
" Hamsters in this group were immunized by 3 injections of 10e HEF cells that were UV-ir
radiated for 3 min (42 ergs/sec/sq cm).
' Hamsters in this group were given 3 injections of Medium 199as a control.
JUNE 1973
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Fred Rapp and Ronald Duff
Acknowledgments
The authors wish to thank Myron Katz and Doreen Lakatosh for their
valuable technical assistance.
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CANCER RESEARCH
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Cellular Transformation by HSV
Fig. I. Photomicrograph of hamster embryo cells transformed by HSV-2 (333-8-9). H & E, x 300.
Fig. 2. Photomicrograph of hamster embryo cells transformed by HSV-1 (KOS-6-9). H & E. x 300.
JUNE
1973
1533
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Fred Rapp and Ronald Duff
riv v
r^v
&7'i
t-¿
-A 2¿J
/tó-i/MT^
Fig. 3. Photomicrograph of HSV-2-specific cytoplasmic fluorescence in HSV-2-transformed cells (333-8-9 cells). The indirect immunofluorescence
technique was used with anti-HSV-2 serum prepared by the injection of weanling Syrian hamsters, x 300.
Fig. 4. Photomicrograph of a tumor cell metastasis in the lung of an adult hamster. The animal had received injections s.c. as a weanling with
HSV-2-transformed hamster cells, x 125.
1534
CANCER RESEARCH
VOL. 33
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1973 American Association for Cancer Research.
Transformation of Hamster Embryo Fibroblasts by Herpes
Simplex Viruses Type 1 and Type 2
Fred Rapp and Ronald Duff
Cancer Res 1973;33:1527-1534.
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Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1973 American Association for Cancer Research.