[CANCERRESEARCH30, 288—293,February 1970]
Virus-like Particles in Bowen's Diseases
Robert E. Nordquist, Robert L. Olson, Mark A. Everett, and Paul T. Condit
Cancer Section, Oklahoma Medical Research Foundation and Departments of Dermatology and Medicine, University of Oklahoma School of
Medicine, Oklahoma City, Oklahoma 73104
Biopsies from seven cases of Bowen's disease were studied
with the electron microscope. Virus-like particles were seen
10% neutral formalin, embedded, sectioned, and examined
histologically to assure that the specimens were Bowen's
lesions (16).
Tissue for electron microscopy was fixed for 2 hr in 2%
in six of the seven specimens.
glutaraldehyde
SUMMARY
(buffered
with
0.1 M cacodylate
7.2) and stored in 0.1 M cacodylate
INTRODUCTION
subsequent
Characteristically,
Bowen's disease presents as a slowly
enlarging, slightly inflammatory,
scaling plaque upon the
skin. The lesions usually spread peripherally by intraepi
dermal extension for many years without invasion of deeper
tissues, but occasionally transform into invasive squamous
cell carcinoma (I 6). In patients with Bowen's disease, the
incidence of systemic cancer is greater than in control groups
(15). As observed by light microscopy the epidermis in
Bowen's disease is thickened by the presence of anaplastic
cells, deeper tissue invasion is lacking, and the basement
membrane remains intact. The epidermal cells are pleomor
phic and lack the usual orderly structural arrangement.
Individual cell keratinization and epithelial giant cell forma
tion is prominent throughout the epidermis. Major electron
microscopic fmdings in Bowen's disease recently reported
(17) were cytoplasmic projections of basal cells extending
through the basement membrane into the dermis, loss of
attachment
sites
lesions, abnormal
presence
(desmosomes)
between
cells
mitoses within dyskeratotic
of a virus-like
particle
measuring
within
the
cells, and the
800
to 1 100 A
within the basal and spinous cells. This paper reports further
studies of the virus-like particles.
MATERIALS AND METHODS
Clinical Material. Clinically characteristic specimens 2 to 3
cm in diameter (wet tissue weight approximately 0.2 g) were
removed from Bowen's lesions under local anesthesia. The
specimens were weighed and divided with a scalpel into 2
equal parts for light microscopy and for electron micros
copy.
Microscopy. The tissue for light microscopy was placed in
1Supported by Grant E-450 from the American Cancer Society and
by NIH General Research Support Grant 5-S01-FR-5338-06.
Received January 9, 1969; accepted July 16, 1969.
288
Storage
in buffer
buffer,
pH
pH 7.2, for
for periods
up to
1 week produced no morphological alterations. The specimen
was then transferred to 2% osmium tetroxide, in 0.1 M
cacodylate buffer, pH 7.2. After 2 ha, the specimen was
dehydrated in graded alcohols and embedded in Cargille's
epoxy (Araldite 6005 obtained from R. P. Cargille's Labora
tones, Inc., Cedar Grove, N. J.). After polymerization
of the
resin for 24 hr at 61° the blocks were thin sectioned and
stained with acetic-uranyl acetate and lead citrate. Photo
micrographs were taken at various magnifIcations (3,000 to
100,000 X) on an Hitachi HU-l lB electron microscope.
RESULTS
Human
Tumors.
In 6 of 7 lesions of Bowen's
disease the
presence of a cytoplasmic particle measuring approximately
800 to 1 100 A in diameter was demonstrated in basal and
spinous
layers (Fig.
1). These virus-like
particles
seem to be
formed by budding from either the plasma membrane (Fig.
14 to D) or a plasma membrane-like surface within the
cytoplasm. Some particles also apparently budded from
tubular structures adjacent to the plasma membrane . The
first sign of a possible
budding
sequence
was an increase
in
the electron density and number of laminations of the
plasma membrane. This area began to evaginate, and the
membrane increased in complexity and density. As budding
continued
Tumors from 7 different patients were examined with the
electron microscope.
examination.
buffer,
the final structure
became
more evident,
and the
plasma membrane pinched off behind the emerging particle.
The particle was then extruded into the intercellular space
(Fig. 14 to D).
In each of the lesions studied the predominant particle was
extracellular, showing an outer shell consisting of alternating
light and dense layers (Fig. 3A to D), with a less dense
nucleoid apparently continuous with the innermost mem
brane. Within the nucleoid were small, electron-dense
particles
approximately
100 A (Figs.
2C to D and 3A, C,
and D) in diameter. Two or more of these small particles
were seen within each nucleoid. The extracellular particles
frequently were seen and were found to be the predominant
type of particle in the examined lesions.
No particles
of the types
samples of normal
described
have been found
in 44
skin or 12 samples of other epidermal
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Virus-like Particles in Bowen ‘s
Disease
diseases studied as controls during the course of this inves
tigation. No virus-like particles were found in the 1 case of
Bowen's disease in which there was a history of arsenic
intake (Table 1).
DISCUSSION
The role of viruses in animal cancer has been under
investigation for many years. The viral etiology of a number
of solid tumors and leukemias in fowls and mammals has
apparently been established (3, 4, 6, 7, 9). Although many
biological agents have been reported in association with
human leukemias and lymphomas (2—5, 8, 10, 11 , 13,
18—21), no virus has been demonstrated to be causative
in human
cancer,
with the possible
exception
of Burkitt's
lymphoma. The studies with Burkitt's lymphoma illustrate
the difficulties in the investigation of human tumors of
suggested viral etiology. Bell (1) pointed out in a recent
review of this tumor that numerous agents have been
isolated from these tumors, but the Bunyamuera virus, the
unidentified herpes virus, and reovirus type 3 are the most
likely etiological agents. The presence of multiple biological
agents which has been reported in Burkitt's lymphoma (12)
indicates the hazards involved in premature conclusions.
Many neoplastic tissues contain viruses that may not be
directly involved with the etiology of the disease.
Huebner (14) has suggested criteria for characterization of
suspected particles and proof of the identity, by which a
suspected biological agent could be unequivocally defmed.
However, human tumor viruses are not as readily studied as
animal
tumor
viruses,
and only
the human
papilloma
virus
has a proven etiological role in solid tumors of humans. This
virus (verruca) contains a DNA center and is found in the
upper strata of the human epidermis, and not in the basal
and lower spinous layers. The lesions produced by the virus
is the benign, common wart which involves only the
epidermis.
Dalton et a!. (4) described a thick-walled particle con
taming several subparticles in the nucleoid in sections of
plasma pellets from a patient with chronic lymphocytic
leukemia; these subparticles closely resemble the extracellular
particles described above (Fig. 3A to D). They also
published micrographs of a sequence of the formation of
C-type particles by budding from the plasma membrane in
murine leukemia induced by the Rauscher virus, similar to
that reported above in Bowen's disease (Fig. 14 to D).
This investigation of Bowen's disease has revealed virus-like
particles in 6 of 7 tumors studied. The viral nature of the
particles was assessed by distinct virus-like morphology of
the particles in the lesions. Furthermore
no virus-like
particles of these types have been in normal skin (44 cases
studied) or other lesions of the epidermis (12 cases studied).
The only lesion in which virus-like particles were not seen
occurred in a patient who had ingested considerable amounts
of arsenic over a long period of time. Arsenic is known to be
an etiological
factor
in skin cancer,
and some investigators
have found significant increases in cancers similar to Bowen's
disease in areas where drinking water contains large amounts
of arsenic (12).
ACKNOWLEDGMENTS
We acknowledge the valuable technical assistance of Linda VanAls
tine and James R. Gaylor. The help and advice of Dr. John A. Sykes
is gratefully appreciated.
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(seenDiagnosisNo.
microscopy)Actinic
of patientswith
electron
degeneration2.‘-“Actinic
keratosis4—Actinic
damage1-Seborrheic
keratosis1—Basal
cell nevus syndrome (palmar pit)
2—Bowen's
Basal
cell
carcinoma1
disease7b.gi@Nonnaiskin44a._, no virus observed.
b@e case in this group had a history of high arsenic intake.
c+, virus present (85.9% of specimensexamined).
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1970
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Fig. 1. Bowen's disease,human skin. Scanningviewoflower spinous layers. Note the extracellula.rparticles within the circles. X 15,000.
Fig. 2. liowen's disease, human skin. Possible sequence of events during the budding of a virus-likeparticle, X 220,000. A , thickening of plasma
membrane; B, evagination of thickened area; C, pinching off of complete particle; D, fmal extrusion of completed particle. Note the dense
subparticles in the core.
Fig. 3. Bowen's disease, human skin. A series of extracellular (mature) particles, some of which contain dense subparticles within the core (@).
A, X550,000;BtoD,X220,000.
290
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293
Virus-like Particles in Bowen's Disease
Robert E. Nordquist, Robert L. Olson, Mark A. Everett, et al.
Cancer Res 1970;30:288-293.
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