[CANCER RESEARCH 32, 2803-2806,
December 1972]
Ultrastructural Characterization of Hamster Cells Transformed
Following Exposure to Ultraviolet-irradiated Herpes Simplex
Virus Type 21
Ronald Glaser, Ronald G. Duff, and Fred Rapp
Department of Microbiology. College of Medicine, The Milton S. Hershey Medical Center of The Pennsylvania State University, Hershev,
Pennsylvania I 7033
SUMMARY
Herpes simplex virus type 2 rendered defective by
ultraviolet irradiation transformed hamster embryo fibroblasts
in vitro. When the transformed cells were inoculated into
newborn hamsters, solid tumors developed and cell cultures
were established. The virus-transformed cells, the solid tumors,
and the tumor cell cultures were examined by electron
microscopy. The transformed cultures and tumors contained
cells with nuclei in which the chromatin had become
condensed and marginated; this picture resembled the
cytopathology
induced during lytic infection by herpes
simplex virus. These abnormalities were observed in some cell
culture lines established from the tumors. Virus-like particles,
similar in size and morphology to nonenveloped herpesvirus,
were found in the nuclei of a small number of degenerating
cells in some of the specimens examined.
INTRODUCTION
There are several lines of evidence connecting herpesviruses
and cancer. Of these, HSV-22 has been suggested as a possible
etiological agent for cervical carcinoma in humans. This
hypothesis has been based on seroepidemiological studies (3,
11, 12) and on the finding that antigens related to HSV-2 are
demonstrable by immunofluorescent studies (14) in cells from
patients with cervical carcinoma.
Recently, HEF's in vitro were transformed following
exposure to UV-irradiated HSV-2. The transformed cells, when
inoculated into newborn hamsters, produced solid tumors
(4—6).The work reported here is the 1st ultrastructural study
performed on the transformed hamster cells, the solid tumors
induced by them, and the cell cultures that were derived from
the solid tumors.
MATERIALS AND METHODS
In Vitro Transformation. The HSV-2 was a fresh human
isolate and was obtained from Dr. W. Rawls, Baylor College of
'This investigation was supported under Contract 70-2024 within
the Special Virus-Cancer Program of the National Cancer Institute,
NIH, USPHS.
'The abbreviations used are: HSV-1 and -2, herpes simplex virus
types 1 and 2; HEF, hamster embryo fibroblast.
Received August 10, 1972; accepted September 13,1972.
Medicine, Houston, Texas, and designated
HSV-2-333.
Transformation of the cells has been previously described (4,
5). Briefly, 2 ml of virus suspension were placed in a 60-mm
plastic Petri dish. The virus was irradiated for 480 sec and
adsorbed onto HEF cell monolayers for 1 hr at 37°at an input
multiplicity of about 1 original plaque-forming unit/cell. The
cells were trypsinized, grown in 8-oz prescription bottles, and
passed ad libitum. (4).
Oncogenicity of the Transformed Cells in Syrian Hamsters.
Newborn LSH hamsters were used for the in vivo study.
Within 24 hr after birth, each newborn hamster was given a s.c.
injection of approximately 2 X 10s viable transformed cells
(designated 333-8-9) at passage 21 (4).
Electron Microscopy. Cell monolayers maintained in glass
bottles were washed with phosphate-buffered saline, scraped
off the surface of the glass with a rubber policeman, and
washed once in phosphate buffer, pH 7.3. The cells were
pelleted in conical glass centrifuge tubes at 800 rpm for 8 min
and fixed overnight
in cold 1% phosphate-buffered
glutaraldehyde, pH 7.3, or in Karnovsky's fixative containing
glutaraldehyde and paraformaldehyde (7). Tumor specimens
were washed in phosphate buffer and fixed in cold 3%
phosphate-buffered glutaraldehyde.
The cell cultures and tumors were postfixed in Dalton's
chrome-osmium (2) for 2 hr and embedded in Mollenhauer's
Araldite plastic (9) or Spurr's low-viscosity plastic. Sections
were placed on naked copper grids, stained with 0.5% uranyl
acetate in 50% methanol and Reynolds' lead citrate (13), and
examined with a Hitachi HU-12 electron microscope at 75 kV.
RESULTS
In
Vitro
Transformation
and Tumor
Induction.
Transformed foci were observed 30 days after infection of
HEF with UV-irradiated-HSV-2. Each focus was isolated and
grown out, and a cell line resulting from 1 focus was
designated 333-8-9. Palpable tumors were detected 10 to 16
weeks after injection of newborn LSH hamsters with 5 X 10s
cells from passage 21 of the 333-8-9 cell line (4, 5).
Electron Microscopy. We have examined 3 groups of
specimens in this study. They include HEF's transformed in
vitro following exposure to the inactivated virus, solid tumors
from hamsters which had been inoculated
with the
transformed HEF, and, finally, cell cultures derived from the
solid tumors.
DECEMBER 1972
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2803
Ronald G. Glaser, Ronald G. Duff, and Fred Rapp
A majority of the sections of the virus-transformed HEF
Examination of specimens from solid tumors revealed a
cells observed contained nuclei with abnormalities that were large number of cells with similar ultrastructural changes as
similar to those seen in cells infected with HSV-1 or HSV-2. observed in the HSV-2-transformed cells. The chromatin was
The herpes-like cytopathology
included condensation of highly condensed and, in many instances, limited to the
chromatin material and chromatin margination along the periphery of the nuclei. Bizarre-shaped nucleoli were present
periphery of the nuclear membrane (1) (Fig. 1). In many in many nuclei with condensed and marginated chromatin.
instances, structurally abnormal nucleoli and a lack of cellular Bedoya et al. (1) have described similar abnormalities in
organe lies were also observed. Herpes-like virus particles were HSV-infected lymphoblastoid cells. Virus-like particles were
observed in a small number of cells (Fig. 2).
observed in a small number of cells. The cell cultures prepared
Examination of specimens from solid tumors revealed a from solid tumors subcultured
several times in vitro
large number of cells with similar ultrastructural changes as demonstrated a rare cell with incomplete herpes-like particles.
observed in the HSV-2-transformed
cells (Fig. 3). The
The morphology of the cells examined from 3 out of 5
chromatin was highly condensed and, in many instances, tumor cell lines appeared "normal." The chromatin was evenly
limited to the periphery of the nuclei. Bizarre-shaped nucleoli distributed in the nucleus and the nucleoli had no anomalies.
were present in almost every nucleus with condensed and The remaining 2 cell lines contained many cells with
marginated
chromatin.
On 50 grids examined so far, abnormalities similar to those already described. In addition,
incomplete herpes-like virus particles were found in 2 nuclei giant mononucleated and multinucleated cells were commonly
(Fig. 3, inset; Fig. 4). The particles had dense cores and were observed in all the tumor cell lines examined.
Incomplete herpes-like virus particles were found in the
approximately 100 nm in diameter. There was no evidence of
enveloped
particles. Cells with more normal nuclear nuclei of a few cells. The particles had dense cores and were
morphology were also found in the tumor specimens.
approximately 90 to 100 nm in diameter. There was no
When cell cultures which had been derived from solid tumor evidence of enveloped particles. The morphological alterations
specimens were examined, the nuclear changes already observed in the specimens were comparable in material fixed
the
phosphate-buffered
glutaraldehyde
and
described for the transformed cells and the tumors were in both
observed in some but not all cell lines. As a rule, in the Karnovsky's fixative as well as specimens embedded in the
cultures not exhibiting abnormalities, the chromatin was high- and low-viscosity plastic, suggesting the alterations were
evenly distributed in the nucleus and the nucleoli had a more not artifacts. In most instances in which incomplete virus-like
normal morphology (Fig. 5). Herpes-like virus particles have particles were found, the morphology of the particles was
also been observed in tumor cell cultures. In every instance, poor. This may have been due to the degenerating cells in
when virus-like particles were observed in any of the which they were located and the subsequent problems of
specimens, the cells were degenerating and the structural
fixation for electron microscopy of these cells or, most likely,
integrity of the cells was deteriorating.
to the fact that the virus genome contained in the cells is
incapable of complete expression because of the heavy
irradiation applied to the original virus. However, appearance
in a rare cell of defective HSV-like particles coincides with
demonstration of HSV-specific antigens in a few nuclei in the
DISCUSSION
cell cultures as described by Duff and Rapp (5) and may
represent derepression of the virus genome resulting in
HEF cell cultures were transformed following exposure to destruction of the cell, a phenomenon also observed with
UV-irradiated HSV-2. When cells from these cultures were cultures harboring Epstein-Barr virus (8, 15).
inoculated into newborn hamsters, solid tumors developed.
Type C virus particles were not observed in any of the
Continuous
cell cultures of the tumor specimens were specimens examined by electron microscopy. In addition,
established, and an electron microscopic study was performed
there was no evidence of type C specific viral RNA or gs
on the specimens to determine whether herpesvirus particles antigens in HSV-2-transformed HEF even after treatment of
were present and to characterize the morphology of the cells. cells by 5-bromodeoxyuridine, as determined by radioisotope
Between 50 and 100 grids of each specimen have been and immunofluorescence tests (6, 10).
Two explanations that might account for the "normal"
examined. A majority of the sections examined of the
virus-transformed
HEF observed contained
nuclei with appearance of tumor cell cultures are: (a) there has been a
selection of the more normal-appearing cell types as the cell
abnormalities that were similar to those seen in cells infected
with HSV-1 or HSV-2. The herpes-like cytopathology included cultures were being subcultured; (b) the viral genome has
condensation
of chromatin
material
and chromatin
become totally integrated in the host cell genome and no
margination along the periphery of the nuclear membrane. In longer expresses itself, as demonstrated by the lack of
many instances, structurally abnormal nucleoli and a lack of cytopathology.
cellular organdÃ-es were also observed. There was no evidence
of virus particles in the cell line 333-8-9 at passage levels up to
about passage 42. However, virus-like particles have been ACKNOWLEDGMENTS
found in passage 42. The particles were 90 to 100 nm in size
and were found in the nucleus. No enveloped particles were
We thank Miss Patricia Nelson and Mr. Ross Farrugia for their
technical assistance.
observed.
2804
CANCER RESEARCH VOL. 32
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Hamster Cells Exposed to UV-irradiated HSV-2
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Fig. 1. Electron micrograph of a UV-iiradiated-HSV-2-transformed cell. Note marginated chromatin in the nucleus. Approximately X 6,000.
Fig. 2. Nucleus of a UV-irradiated-HSV-2-transformed cell containing an incomplete herpes-like particle. Approximately X 40,000.
Fig. 3. Electron micrograph of a tumor cell. Note condensation and margination of chromatin in the nucleus. Approximately X 8,000. Inset,
incomplete herpes-like particles. Approximately X 41,000.
Fig. 4. Electron micrograph of part of a nucleus of a tumor cell containing an incomplete herpes-like particle. Approximately X 40,000.
Fig. 5. Electron micrograph of a giant mononucleated tumor culture cell in which the chromatin appears normally dispersed. Approximately X
2,500.
DECEMBER 1972
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2805
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CANCER RESEARCH VOL. 32
2806
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1972 American Association for Cancer Research.
Ultrastructural Characterization of Hamster Cells Transformed
Following Exposure to Ultraviolet-irradiated Herpes Simplex
Virus Type 2
Ronald Glaser, Ronald G. Duff and Fred Rapp
Cancer Res 1972;32:2803-2806.
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