[CANCER RESEARCH 42, 2722-2728,
0008-5472/82/0042-OOOOS02.00
July 1982]
Morphological, Immunological, and Biochemical Analyses of Chicken
Spleen Cells Transformed in Vitro by Reticuloendotheliosis
Virus Strain T
Tsunefumi Shibuya, Irvin Chen, Allan Howatson, and Tak W. Mak
Ontario Cancer Institute and Department of Medical Biophysics and Institute of Medical Sciences, University of Toronto. Toronto, Canada M4X 1K9 ¡T.S., A. H.,
T. W M.¡.and McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, Wisconsin [I. C.I
ABSTRACT
Reticuloendotheliosis
virus strain T (REV-T) is a highly oncogenic avian retrovirus which causes a rapid neoplastic dis
ease of the lymphoreticular system. We derived six cell lines
(1-3, 1-5, 2-10,2-14,
2-16, and 2-20) from chicken spleen
cells infected with REV-T. These cells can produce both the
REV-T and its associated reticuloendotheliosis
helper virus,
REV-A. Histochemical analyses of these cells indicate that,
while they are not stained by benzidine, peroxidase, /8-glucuronidase or acid a-naphthyl acetate esterase, they contain a
high proportion (95%) of cells positive for acid phosphatase.
Light and electron microscopic studies of these cells also
revealed morphologies of lymphoblasts or activated lympho
cytes with irregular nuclei and dispersed chromatin. Immunochemical analyses indicate that essentially all (90 to 100%) of
the cells contain the surface marker la, but no cytoplasmic
immunoglobulin M and immunoglobulin G could be detected
by immunofluorescence staining. Results also show that some
of these cell lines contain a low level of terminal transferase
(0.02 to 0.17 unit/109 cells), and a proportion (3 to 35%) of
these cells can be stained by an antiserum directed against
chicken bursa cells. These results are consistent with the
conclusion that the cells transformed by the highly oncogenic
REV-T are lymphoid in nature. In addition, at least some of
these cell clones may contain features characteristic of acti
vated B-lymphocytes. Analysis of these cell clones indicates
that some cell lines contain an adherent and nonadherent
population with some differences in morphologies. In addition,
electron microscopic examination revealed that, while the nonadherent cells are actively producing type C viruses, type C
viruses are either absent or very rare in the adherent cell
populations. These results support the conclusion that some of
these cell lines are heterogeneous and contain subpopulations
of cells with differences in their ability to produce viruses.
INTRODUCTION
Avian and murine type C acute leukemia viruses are repli
cation defective and contain within their viral genomes se
quences not shared by their corresponding helper viruses (33).
These sequences, which are of host origin, have been found to
code for products presumed to be involved in the rapid trans
formation of yet to be identified target cells. Although the
mechanism of transformation by these viruses is not known,
the different isolates of acute leukemia viruses contain distinct
1 Supported by the Medical Research Council of Canada, the National Cancer
Institute, and the Public Health Service.
Received October 6, 1981; accepted January 28, 1982.
2722
and unique nucleotide substitutions (3, 28, 32). The leukemic
cells that were derived as a consequence of transformation by
each of these viruses have enzymatic and immunological prop
erties which are characteristic of specific lineages in the hemopoietic system (5, 11, 23, 29). In the hope of understanding
the nature of the rapidly transforming viruses and their inter
action with the host cells, much effort has been devoted to the
definition and characterization of these avian and murine leu
kemic cells.
REV-T2 is a highly oncogenic retrovirus that causes visceral
reticuloendotheliosis
and peripheral nerve lesions in several
species of fowl (8, 15, 36). Like other avian and murine acute
leukemia viruses, REV-T is replication defective and requires
a helper virus (REV-A) for replication. The helper virus REV-A
and a variant of REV-T have been recently molecularly cloned,
and the nature of the sequences specific for REV-T was char
acterized (7). These specific sequences, termed rei, are pres
ent in the DNA of normal uninfected avian cells, and sequences
homologous to these sequences are also present in DNA of
uninfected salmon, mouse, rat, and the human cell (7).
In this paper, we describe the derivation of several tumor cell
lines from spleen cells infected with REV-T (REV-A). The mor
phological, immunological, and biochemical properties of these
cells were studied.
MATERIALS
AND METHODS
Cells and Viruses. REV-T (REV-A) was obtained from the BMC cell
line (8). To generate REV-T (REV-A)-transformed
cell lines, spleen
cells from 3-week-old chickens were infected in vitro by REV-T (REVA) as described before (15). Six cell lines were generated by this
method and are designated 1-3, 1-5, 2-10, 2-14, 2-16, and 2-20.
These cell lines together with the BMC cell line were cultured in medium
(31) supplemented with 10% fetal calf serum (Microbiological
Associ
ates, Walkersville, Md.).
Light and Electron Microscopic Studies. Cells were harvested,
washed twice in PBS, and resuspended in PBS containing 1% (w/v)
bovine serum albumin. For light microscopic studies, 105 cells in 0.1
ml were spun onto a glass slide using a cytocentrifuge
(Shandon
Scientific Company, London, England). The cells were then air-dried
and stained with Wright-Giemsa stain solution.
For electron microscopy, cell samples were centrifugea into pellets,
fixed overnight with 3.5% glutaraldehyde in cacodylate buffer, postfixed with 1% OsO4 in cacodylate buffer, dehydrated in acetone, and
embedded in Epon. Sections about 1 /¿mthick were cut, stained with
toluidine blue, and examined in a light microscope. Thin sections
2 The abbreviations used are: REV-T, reticuloendotheliosis
strain T; REV-A,
reticuloendotheliosis
helper virus; PBS, phosphate-buttered
saline [0.01 M so
dium phosphate (pH 7.2MD.1 M sodium chloride); slgG, surface IgG; slgM,
surface IgM; clgG, cytoplasmic IgG; clgM, cytoplasmic IgM; FITC, fluorescein
isothiocyanate; TdT, terminal deoxynucleotidyl transferase; TEMG, 0.05 M TrisHCI (pH 7.7M IHM EDTA-1 mw 2-mercaptoethanol-20%
glycerol.
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Analyses of Chicken Spleen Cells Transformed in Vitro
prepared in a Reichert Ultramicrotome were stained with uranyl acetate
and lead citrate and observed in a Siemens Elmiskop Model 1a electron
microscope.
Hlstochemical Staining. For histochemical staining, cells were spun
onto glass slides by cytocentrifuge as described above, dried in air,
and then fixed. Histochemical staining procedures were used as de
scribed previously: acid phosphatase (16); /î-glucuronidase (22); acid
o-naphthyl acetate esterase (25); peroxidase (17); periodic acid-Schiff
reaction (13); and benzidine. For hemoglobin staining, slides were
stained with 1% benzidine in ethanol and placed in 7.5% H2O2 in
ethanol. Chemicals and stains were obtained from Sigma Chemical Co.
(St. Louis, Mo.).
Immunofluorescence Staining. For immunofluorescence staining,
cells were harvested, washed, and stained for slgG, slgM, clgG, clgM,
la, bursa cell antigens, and chicken thymus cell antigens.
In all of these assays, the indirect immunofluorescence
technique
was used. For slg and clgG, rabbit anti-chicken IgG serum was used.
For slgM and clgM, goat anti-chicken IgM serum was used. These
antisera were obtained from Miles Biochemicals (Elkhart, Ind.). For
second antibody, FITC-labeled goat F(ab')? anti-rabbit F(ab')2 and
FITC-labeled rabbit F(ab')2 anti-goat F(ab')2 were used (Cappel Labo
ratories, Cochranville, Pa.).
For staining of la antigen, a monoclonal
mouse IgM anti-chicken
la
in ascitic fluid was used. This reagent was a generous gift of Dr. M.
Cooper (University of Alabama, Birmingham, Ala.). For the second
antibody, an FITC-conjugated goat anti-mouse IgM (Cappel Laborato
ries) was used.
Immunofluorescence
staining of chicken bursa and thymus antigen
was performed using rabbit anti-chicken bursa cell and rabbit antichicken thymus cell sera. For a second antibody, FITC-conjugated
goat anti-rabbit F(ab')? was used. The anti-chicken bursa and antichicken thymus sera were a generous gift of Dr. J. M. Sharma of the
Poultry Research Laboratory, Michigan State University, Lansing,
Mich.
Assay for TdT. Biochemical assay of TdT was performed essentially
as described before (19, 27). Phosphocellulose columns were used to
analyze the activities of the enzyme in different cell lines. This method
is necessary in order that a small amount of enzyme activity can be
detected (1, 19). Single-cell suspensions, containing 1 to 3 x 108
chicken thymus, bursa, or liver cells, or cell lines derived from REV-T
(REV-A)-transformed
spleen cells were washed twice in 0.05 M TrisHCI (pH 7.7)-1 mM EDTA-1 mw 2-mercaptoethanol.
The resuspended
cells were freeze-thawed 3 times in 0.5% Triton X-100-1.5 M KCI-4
mol phenylmethylsulfonyl
fluoride (Sigma) and centrifuged at 100,000
x g for 60 min. The supernatant was chromatographed in a phosphocellulose column (0.5- x 12-cm column) after dialysis against 100 times
volume of TEMG at a flow rate of 0.2 ml/min. After a 10-ml wash with
50 mw KCI in TEMG, a gradient (60 ml) of 0.05 to 1 M KCI in TEMG
was applied. One-mi fractions were collected. Sixty fil of the fractions
were assayed for TdT activity as described (27) except that [3H]dGTP
was added at 20 ¡ÕM
(5000 cpm/pmol).
Detection of IgM Synthesis by Immunoprecipitation. All cell lines
and normal chicken bursa cells were cultured at a concentration of 1
x 106/ml for 18 hr with 100 /iCi [35S]methionine per ml (Amersham/
Searle Radiochemicals, Mississauga, Ontario, Canada) in methioninefree «-medium supplemented with 10% heat-inactivated fetal calf se
rum.
After labeling, cells were washed twice with cold PBS and lysed in
cold PBS containing 0.5% Nonidet P-40 and 1 mM phenylmethylsul
fonyl fluoride. The lysed cells were then centrifuged at 100,000 x g
for 60 min at 4°.Then, supernatants were precleaned by incubation
with 20% fixed Staphylococcus aureus in 10% suspension. All samples
were divided into 2 groups, and 10% anti-chicken IgM goat serum or
normal goat serum was added. The antisera were then allowed to bind
for 1 hr at room temperature followed by further incubation for 18 hr at
4°. The S. aureus suspension was added again, and the precipitate
was sedimented with an Eppendorf centrifuge.
JULY
After washing five times
with PBS containing
0.5% Nonidet P-40, the bound immune complex
was removed by boiling, and released immune complexes were ana
lyzed by sodium dodecyl sulfate gel electrophoresis and subsequent
autoradiography.
RESULTS
Isolation of REV-T (REV-A)-transformed Cell Lines. An in
vitro transformation assay for REV-T (REV-A) has been de
scribed by Hoelzer ef al. (15). With the use of this assay and
REV-T (REV-A) produced by the BMC cell line (8), we have
infected the spleen cells from 3-week-old chickens in vitro. Six
transformed cell lines were generated, and they were desig
nated 1-3, 1-5, 2-10, 2-14, 2-16, and 2-20. All the cell lines
generated produced REV-A, as determined by the appearance
of cytopathic effects (35) and DNA polymerase activity follow
ing infection of chicken embryo fibroblast cells (24). In addition,
all cell lines also produced REV-T, as determined in the in vitro
spleen cell assay (15).
Morphology of the REV-T-transformed Spleen Cell Lines.
The morphology of the spleen cells transformed in vitro by
REV-T (REV-A) was examined by light and electron micros
copy. As observed in an inverted phase-contrast microscope,
these cell lines grew as single-cell suspensions or in small
aggregates and had a round or fusiform appearance with hairy
projections from their surface (Fig. 1a). Some cells in cell lines
2-14, 2-16, and 2-20 showed a tendency to adhere to the
culture dish, thus forming an adherent and a nonadherent
population.
Cytospin preparations of these cell lines stained with WrightGiemsa stain solution had the morphological characteristics of
immature lymphoid or erythroid cells. The cytoplasm was
strongly basophilic and contained some vacuoles. The nucleocytoplasmic ratio was increased in the transformed cells, and
a high frequency of mitotic cells was observed (Fig. 10).
The morphology of the cells was also examined by electron
microscopy. Populations of adherent and nonadherent cells
from line 2-20 were processed and examined separately to
determine and compare their structural features. In the adher
ent cell preparations, the nuclei were generally irregular in
outline and showed deep indentation and sometimes separated
portions as seen in section. The chromatin was dispersed with
some peripheral condensation, and nucleoli were prominent
(Fig. 2a). The cytoplasm was extensive and contained numer
ous mitochondria and smooth-surfaced vesicles. Free ribosomes and polyribosomes were plentiful, but there was little or
no rough endoplasmic reticulum. Type C virus particles were
observed but only very rarely. In their fine structure, these cells
had the characteristics of lymphoblasts or activated lympho
cytes.
The nonadherent cells resembled in thier general features
the adherent cell population, but there were some marked
differences. The nuclei were less irregular in outline, and many
had large nucleoli. The cytoplasm was less extensive and
contained many densely packed ribosomes. Mitochondria were
fewer, and smooth-surfaced
vesicles were relatively rare.
These cells were actively producing type C virus particles, as
evidenced by budding activity and numerous extracellular ag
gregates of immature and mature type C viruses (Fig. 2b). The
fine structure of these cells is consistent with their being of
lymphoid type.
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2723
T. Shibuya et al.
Histochemical Staining of REV-T (REV-A)-transformed Cell
Lines. In an effort to examine more closely the nature of these
cells, the histochemical staining patterns of these cells were
examined. Virtually all the cells from every REV-T (REV-A)transformed cell line are stained by acid phosphatase, indicat
ing that the cells have histochemical staining patterns charac
teristic of lymphocytes, granulocytes, or macrophages. How
ever, these cells do not contain cytoplasmic granules and are
negative for the stains /S-glucuronidase, acid a-naphthyl ace
tate esterase, peroxidase, and periodic acid-Schiff reaction
(for which stains granulocytic cells and macrophages are pos
itive), suggesting that the transformed cell lines are lymphocytic
in nature. This conclusion is consistent with the results of the
morphological studies described above, which indicated that
the cells were of lymphoid type.
Immunofluorescence
Staining of Immunological Markers.
The above studies indicate that the REV-T (REV-A)-transformed chicken spleen cells have the characteristics of imma
ture lymphocytic cells. To further define the nature of these
cells with respect to ¡mmunological markers associated with
lymphocytes, several surface and cytoplasmic marker charac
teristics of B- and T-lymphocytes were examined. These in
cluded the cell surface markers la-like antigen, surface anti
gens specific for bursa cells and thymic cells, cell slgM and
slgG, and clgM and clgG. These markers were all examined by
indirect immunofluorescence.
The results of these investiga
tions are summarized in Table 1.
The data show that a large percentage (88 to 100%) of the
cells from every cell line examined was positive for la-like
antigen on their surface, indicating that they may be B-lymphocytes or activated T-lymphocytes. Staining of cell surface an
tigens specific for bursa and thymus cells indicated that 3 to
35% of the cells from these REV-T (REV-A) cell lines were
stained by antisera directed against bursa cells, while the cells
were stained by the thymus-specific antisera only at back
ground level. Controls using chicken bursa and thymus cells
also indicate that these antisera are specific for their respective
tissues (Table 1). To prove that the determinants recognized
on these cells were directed against bursa cells, absorption
experiments using bursa and thymus cells were performed.
Results indicate that the determinants recognized on the cell
lines can be completely eliminated by absorption with bursa
cells. These data indicate that the REV-T (REV-A)-transformed
cell lines share some surface determinants with bursa cells,
suggesting that they may have characteristics of B-cells. In an
Immunofluorescence
staining for immunological
attempt to define further the nature of these lymphocytes, the
possible presence of surface and cytoplasmic immunoglobulins
was examined by immunofluorescence staining. The data, also
shown in Table 1, indicate that all the cell lines examined were
negative for staining of slgG and slgM and clgG and clgM.
Level of Terminal Transferase in REV-T (REV-A)-transformed Cells. Studies of hemopoietic cells transformed by
Abelson leukemia virus indicate that these tumor cell lines
contain a low level of TdT (1, 19). To determine if the REV-T
(REV-A)-transformed cell lines also contain TdT, 1 to 3 x 108
cells of cell lines 1-3, 1-5, 2-10, 2-14, 2-16, 2-20, and
BMC were harvested, and their level of TdT was measured with
phosphocellulose
columns as described in "Materials and
Methods." This method of analysis is capable of detecting a
very low level of TdT (1). The levels of this enzyme in chicken
thymus, bursa, and liver cells were also measured as controls.
Table 2 summarizes the data from such experiments.
Results indicate that all the cell lines examined contained a
low level of TdT ranging from 0.02 to 0.17 unit/109 cells. A
low level of TdT (0.05 unit/10 cells) was also found in the
bursa cells. This is in contrast to a high level of TdT (4.2 units/
109 cells) found in thymus cells and a background of 0.005
unit/109 cells from chicken liver. These data indicate that REVT (REV-A)-transformed chicken spleen cells, like the Abelson
leukemia virus-transformed tumor cells, contain a low level of
TdT.
Synthesis of IgM in REV-T (REV-A)-transformed Cell Lines.
The above data indicate that the REV-T (REV-A)-transformed
spleen cells have morphological, histochemical, and immunological properties consistent with those of immature B-lymphocytes. Furthermore, they contain a low level of the differentia
tion-related enzyme TdT. The low level of TdT in Abelson
leukemia virus-transformed cells was found in what was pre
sumed to be pre-B-lymphocytes,
which were also found to
synthesize a low level of intracytoplasmic IgM. To determine if
REV-T (REV-A)-transformed
cell lines also synthesize a low
level of IgM, the transformed cell lines were incubated with
[35S]methionine, and synthesis of intracytoplasmic
IgM was
examined by immunoprecipitation with a goat anti-chicken IgM
serum. The synthesis of IgM was monitored by immunoprecip
itation and autoradiography.
Data showed that altogether a
band corresponding to molecular weight of the ¡ichain of
chicken IgM can be precipitated from chicken bursa cells with
the anti-lgM antiserum and not by nonimmune serum. No
precipitation corresponding to /nchain of IgM could be detected
Table 1
markers on chicken cells transformed by REV-T (REV-A)
For immunofluorescence staining, cells were harvested, washed, and stained for immunological markers
as described in "Materials and Methods." More than 200 individual cells were examined in each case.
% of cells stained
Cells1-31-52-102-142-162-20BMCThymusBursaslgG0000000NDaNDclgG0000000NDNDslgM0000000NDNDclgM0000000ND19la999799998896100
" ND, not determined.
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Analyses of Chicken Spleen Cells Transformed in Vitro
Table 2
Level of TdT activities in REV-T-transformed
cell lines
The lymphocytic nature of these cells was also supported by
several other lines of evidence, (a) With the use of histochem-
Cells were harvested, washed, disrupted, and assayed for terminal transferase
activities by phosphocellulose columns as described in "Materialsand Methods."
ical stains, the cells were found to be stained only by acid
phosphatase but not by any of the other stains including
benzidine, /3-glucuronidase, acid a-naphthyl acetate esterase,
peroxidase, or periodic acid-Schiff reaction, (b) Virtually all the
of TdT activities
cell
lines isolated were found to have la-like antigen on the cell
(units/
cells)4.20.05<0.0050.170.0830.150.0680.060.10.02
10"
CellsThymusBursaLiver1-31-52-102-142-162-20BMCLevel
surface suggesting that they may be B-lymphocytes or acti
vated T-lymphocytes. (c) A significant percentage of these cells
contained cell surface antigens specific for bursa cells, indi
cating that they may share some surface determinants with Bcells. The conclusion that these transformed cells derived as a
result of in vitro transformation of chicken spleen cells by REVT (REV-A) may be B-lymphocytic in nature is consistent with a
report that a cell line derived from tumor as a consequence of
REV-T infection also has properties similar to B-lymphocytes
(18). This finding that REV-T (REV-A)-transformed
cells may
in any of the REV-T (REV-A)-transformed cell lines using the
possess more characteristics of B-lymphocytes than of T-lym
immune or nonimmune sera. These results indicate that either
phocytes is further stressed by the finding that they contain a
low level (0.02 to 0.17 unit/109 cells) of the marker enzyme
these REV-T (REV-A)-transformed chicken spleen cells do not
One unit of enzyme activity was defined as the amount catalyzing the incorpo
ration of 1 nmol of deoxynucleotide monophosphate into acid-insoluble material
per hr.
synthesize IgM or it is being synthesized at a level which cannot
be detected by this method.
DISCUSSION
Avian and murine acute leukemia virus-transformed hemopoietic cells are known to possess properties characteristic of
cells along specific lineages in the hemopoietic system. This
phenomenon is best illustrated by the murine acute leukemia
viruses, Friend (9, 23), Rauscher (12), and Abelson (5, 29,
30), and the avian acute leukemia virus, avian erythroblastosis,
avian myeloblastosis, and avian myelocytomatosis (2, 4, 11).
These viruses upon infection of hemopoietic cells in vitro or in
vivo can give rise to cells bearing properties resembling erythroid, macrophage, myelocytic, or lymphocytic cells.
In this paper, we examined the morphological, immunologicai, and biochemical nature of transformed cell lines derived
from chicken spleen cells infected by REV-T. Our results con
firmed the findings of Hoelzer et al. (15) that chicken spleen
cells can be transformed by REV-T (REV-A) in vitro. These cell
lines are all capable of synthesizing both components of vi
ruses, the defective transforming REV-T component as well as
the helper virus REV-A. Morphological, immunological, and
biochemical characterizations
all indicate that these trans
formed cell lines are lymphocytic in nature. In addition, they
contain some properties characteristic of B-lymphocytes.
The morphological studies showed that the cell lines had the
fine structural characteristics of lymphoid cells, notably abun
dant free ribosomes and little or no rough endoplasmic reticulum. Significant differences between adherent and nonadherent cell populations were observed, particularly the reduced
amount of cytoplasm and the denser packing of ribosomes of
the nonadherent cells. There was a striking difference also in
the extent of type C virus production, the nonadherent cells
being much more active than were the adherent cells. The
difference in the level of type C viruses produced by 2 subpopulations of the same clone of leukemic cells is of interest, for it
is possible that the production of these viral particles is con
trolled by the state of differentiation of these leukemic cells, a
phenomenon similar to that described for murine myeloid leu
kemic cells (20, 21).
TdT. The detection of this low level of TdT was made possible
by the use of a phosphocellulose column to quantitate the
enzyme (1). With the use of this method, we have also found
that bursa cells, which have been presumed to be negative for
TdT, also contain a low level of this enzyme. This provides
direct biochemical evidence to confirm the work of Sugimoto
and Bollum (34), who reported a low percentage of cells in the
bursa containing the enzyme terminal transferase.
The significance of this level of TdT in these lymphocytes
which also contain some B-cell characteristics is not known,
but a similar level of the enzyme can also be found in tumor
cells transformed by Abelson leukemia virus (30). It has been
suggested that these Abelson tumor cells are pre-B-cells and
that a low level of TdT could be a property characteristic of the
state of differentiation of these cells (30). However, IgG and
IgM were not detected in these REV-T (REV-A)-transformed
cells as they were in Abelson tumor cells.
In view of the similarities in biological properties between the
REV-T (REV-A)-transformed cells and Abelson tumor cells, it is
possible that the REV-T genome may be related to that of the
Abelson leukemia virus. Recent biochemical studies, using
REV-T-specific
complementary
DMA (38) and molecularly
cloned REV-T-specific sequences (7), indicate, however, that
there appear to be no homologies between the sequences of
these 2 viral genomes. Thus, transformation by these 2 viruses
may be the result of 2 different mechanisms.
The possibility that different mechanisms may be involved in
viral transformation of hemopoietic cells with apparently similar
properties is also strengthened by the recent studies on the
mechanism of transformation by the weakly oncogenic avian
leukemia virus, avian leukosis virus (10, 14), and reticuloendotheliosis virus (26). These results show that the viral ge
nomes of these slowly transforming viruses, which are also
known to transform chicken cells with B-cell characteristics
(37), can be found integrated adjacent to and activating a
gene, c-myc, with sequence homology to MC29 virus (10, 14,
26). This gene, which is presumed to be involved in the acti
vation of the transformation process in these chicken cells,
also appears to be distinct from sequences rei found on REVT (7, 32, 38) and sequences found on Abelson leukemia virus.
Finally, it is of interest to compare this REV-T-induced retic-
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2725
T. Shibuya et al.
uloendotheliosis disease of chicken with human hairy cell leu
kemia (6) or leukemic reticuloendotheliosis.
These 2 diseases
show many pathological and immunological similarities. For
example, both diseases cause fibrosis and lymphoid infiltration
in the bone marrow, spleen, and lymph nodes; tumor cells from
the avian and human diseases have characteristics of B-cells;
hairy projections are present on the surface of both types of
tumor cells; and finally, tartrate-resistant
acid phosphatase
(data not shown)-positive cells are found in both diseases.
These similarities are of interest because of the apparent
differences in the etiologies of these 2 diseases.
ACKNOWLEDGMENTS
Part of this work was carried out in the laboratory of Prof. H. Temin, to whom
we are grateful.
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CANCER
RESEARCH
VOL.
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42
Analyses of Chicken Spleen Cells Transformed in Vitro
la
Fig. 1. a, phase-contrast microscopy of REV-T-transformed spleen cell line 2-20; b. light micrograph of REVT-transformed spleen cell line 2-20 stained with Wright's
stain solution.
1b
JULY 1982
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2727
T. Shibuya et al.
V
> •
,v
2b
Fig. 2. a. electron micrograph of adherent cells from REV-T-transformed cell line 2-20. General view of adherent cell showing deeply indented nucleus with 2
prominent nucleoli and cytoplasm containing mitochondria, numerous free ribosomes, and smooth-surfaced vesicles, with some containing membranes and particulate
material. Bar, 1 ion. b, electron micrograph of nonadherent cells from REV-T-transformed cell line 2-20. Adjacent nonadherent cell. The cytoplasm contains tightly
packed ribosomes, a few smooth-surfaced vesicles, and swollen mitochondria. Aggregates of type C virus particles (V) are present between the cells.
2728
CANCER
RESEARCH
VOL. 42
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Morphological, Immunological, and Biochemical Analyses of
Chicken Spleen Cells Transformed in Vitro by
Reticuloendotheliosis Virus Strain T
Tsunefumi Shibuya, Irvin Chen, Allan Howatson, et al.
Cancer Res 1982;42:2722-2728.
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