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Journal of Virology, March 1999, p. 2434-2441, Vol. 73, No. 3
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Moloney Murine Leukemia Virus-Induced Preleukemic
Thymic Atrophy and Enhanced Thymocyte Apoptosis Correlate with
Disease Pathogenicity
Christine
Bonzon and
Hung
Fan*
Department of Molecular Biology and
Biochemistry and Cancer Research Institute, University of
California, Irvine, California 92697
Received 2 July 1998/Accepted 24 November 1998
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ABSTRACT |
Moloney murine leukemia virus (M-MuLV) is a replication-competent,
simple retrovirus that induces T-cell lymphoma with a mean latency of 3 to 4 months. During the preleukemic period (4 to 10 weeks
postinoculation) a marked decrease in thymic size is apparent for
M-MuLV-inoculated mice in comparison to age-matched uninoculated mice.
We were interested in studying whether the thymic regression was due to
an increased rate of thymocyte apoptosis in the thymi of
M-MuLV-inoculated mice. Neonatal NIH/Swiss mice were inoculated
subcutaneously (s.c.) with wild-type M-MuLV (approximately 105 XC PFU). Mice were sacrificed at 4 to 11 weeks
postinoculation. Thymic single-cell suspensions were prepared and
tested for apoptosis by two-parameter flow cytometry. Indications of
apoptosis included changes in cell size and staining with
7-aminoactinomycin D or annexin V. The levels of thymocyte apoptosis
were significantly higher in M-MuLV-inoculated mice than in
uninoculated control animals, and the levels of apoptosis were
correlated with thymic atrophy. To test the relevance of enhanced
thymocyte apoptosis to leukemogenesis, mice were inoculated with the
Mo+PyF101 enhancer variant of M-MuLV. When inoculated
intraperitoneally, a route that results in wild-type M-MuLV
leukemogenesis, mice displayed levels of enhanced thymocyte apoptosis
comparable to those seen with wild-type M-MuLV. However, in mice
inoculated s.c., a route that results in attenuated leukemogenesis,
significantly lower levels of apoptosis were observed. This supported a
role for higher levels of thymocyte apoptosis in M-MuLV leukemogenesis.
To examine the possible role of mink cell focus-forming (MCF)
recombinant virus in raising levels of thymocyte apoptosis,
MCF-specific focal immunofluorescence assays were performed on
thymocytes from preleukemic mice inoculated with M-MuLV and Mo+PyF101
M-MuLV. The results indicated that infection of thymocytes by MCF virus
recombinants is not required for the increased level of apoptosis and
thymic atrophy.
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INTRODUCTION |
Moloney murine leukemia virus
(M-MuLV) is a replication-competent, simple retrovirus. When inoculated
into neonatal mice, it induces T lymphomas with a mean latency period
of 3 to 4 months in 100% of the animals (13).
Phenotypically, tumor cells can be classified as immature T
lymphocytes. Because of its predictable time course for disease
induction, M-MuLV provides a model system in which the changes that
occur before the onset of leukemia can be examined.
Generally, M-MuLV leukemogenesis involves both early and late events.
Late events include long terminal repeat activation of proto-oncogenes
(8, 13, 14). Overexpression of these genes presumably leads
to uncontrolled proliferation of the cells and subsequent
transformation. Although these insertional activation events are
essential for tumor formation, other, earlier events establish a
preleukemic state within the animal that is required for efficient
disease induction (11). These early changes include defects
in bone marrow hematopoiesis, splenomegaly, and thymic atrophy.
A valuable tool in the study of M-MuLV pathogenesis is an enhancer
variant of M-MuLV, Mo+PyF101 M-MuLV (10). Mo+PyF101 M-MuLV is similar to wild-type M-MuLV, except that it contains the enhancer elements of the F101 strain of murine polyomavirus inserted into the U3
region of the long terminal repeat, just downstream of the wild-type
M-MuLV enhancer sequences. Both viruses, however, encode exactly the
same viral proteins. When inoculated subcutaneously (s.c.) into
neonatal mice, Mo+PyF101 M-MuLV displays attenuated pathogenicity
(4, 10). Thus, comparisons between mice inoculated s.c. with
wild-type M-MuLV and those inoculated with Mo+PyF101 M-MuLV have
provided insights into the viral and physiological events leading to
leukemogenesis. It was later observed that if the route of inoculation
of Mo+PyF101 M-MuLV was switched to the intraperitoneal (i.p.) route,
animals developed disease with wild-type-like kinetics (2).
Moreover, i.p. inoculation with Mo+PyF101 M-MuLV induces the
preleukemic states seen in wild-type M-MuLV-inoculated mice, whereas
s.c. inoculation of Mo+PyF101 M-MuLV generally does not induce these changes.
One of the characteristics of an M-MuLV infection is the generation of
mink cell focus-forming (MCF) virus recombinants (18). During the course of an M-MuLV infection, the input ecotropic virus
undergoes an in vivo recombinational event with endogenous polytropic
proviral sequences. The resulting MCF virus differs from the input
virus in its envelope gene sequences. Several lines of evidence suggest
important roles for MCF viruses in both early and late steps of
M-MuLV-induced leukemogenesis (reviewed in reference 13). Most importantly for this investigation, MCF
virus recombinants have been detected in mice inoculated by the i.p.
route with Mo+PyF101 M-MuLV (wild-type-like pathogenicity)
(3), whereas MCF virus recombinants have not been detected
in mice inoculated s.c. with Mo+PyF101 M-MuLV (attenuated
pathogenicity) (4).
The focus of this study was to examine the role of thymic atrophy as a
preleukemic event in M-MuLV-induced leukemogenesis. Thymic size in
preleukemic M-MuLV-inoculated mice is typically reduced in comparison
to that of uninoculated, age-matched control mice (12). The
thymus is an organ that supports T-lymphocyte maturation and
differentiation through interactions with stromal elements
(21). As thymocytes undergo differentiation, they receive signals from the stromal cells (i.e., cortical and medullary epithelial cells, bone marrow-derived macrophages, and dendritic cells) that result in positive and negative selection (23). The result
of either a lack of a positive selection signal or the presence of a
negative one is the elimination of the thymocyte by the apoptotic death
pathway (29).
Due to the dynamic nature of the thymus, it seemed possible that M-MuLV
infection was interfering with positive and/or negative selection
processes, resulting in aberrant apoptosis of thymocytes and accounting
for the size differentials seen between M-MuLV-inoculated and
uninoculated mice. In order to test this hypothesis and the relevance
of thymic atrophy to disease, we examined the levels of thymocyte
apoptosis in preleukemic mice inoculated with M-MuLV and Mo+PyF101
M-MuLV (s.c. versus i.p.). The role of MCF virus recombinants in thymic
atrophy was also investigated. The results of those studies are
reported here.
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MATERIALS AND METHODS |
Viruses and inoculation of mice.
Viral stocks were cell
culture supernatants derived from NIH 3T3 cells confluently infected
with either M-MuLV or Mo+PyF101 M-MuLV (10). Viral titers
were determined by the UV/XC plaque assay (24). Neonatal
NIH/Swiss mice were inoculated s.c. with 0.2 ml of wild-type M-MuLV
viral stock (approximately 105 XC PFU) or either s.c. or
i.p. with 0.2 ml of Mo+PyF101 M-MuLV viral stock (approximately
105 XC PFU). Mice were sacrificed by cervical dislocation
at 4 to 12 weeks postinoculation. Thymi were dissected, removed, rinsed briefly in ice-cold phosphate-buffered saline (PBS), and immediately placed on ice. Thymic single-cell suspensions were prepared by gently
teasing the organ apart in PBS and passing it through a 94-µm
(pore-size) wire mesh (Bellco Glass), allowing thymocytes to flow
through, with stromal components remaining on the mesh. Thymocytes were
then washed twice in ice-cold PBS and counted with a hemacytometer.
Flow cytometric analyses for apoptosis.
Approximately 2 × 106 thymocytes were incubated with 1 µg of
7-aminoactinomycin D (7-AAD) per ml in ice-cold PBS for 20 min on ice,
in the dark. Thymocytes treated with 7-AAD were then analyzed simultaneously by flow cytometry (FACSCalibur; Becton Dickinson) for
forward angle scatter (FSC), as a measure of cell size, and 7-AAD
fluorescence. Alternatively, thymocyte single-cell suspensions were
incubated with annexin V-fluorescein isothiocyanate (FITC) and
propidium iodide (PI) (with an apoptosis detection kit; R & D Systems)
per the manufacturer's protocol and were subjected to flow cytometric
analysis. In all cases, cell debris was gated out on the basis of
forward scatter and side scatter analysis.
In our first attempts at examining the levels of thymocyte apoptosis in
these mice, we used a higher concentration of 7-AAD (20 µg/ml)
(25). However, this concentration did not effectively discriminate among live, early-stage apoptotic, and late-stage apoptotic murine thymocytes. Other studies using the higher
concentration of 7-AAD focused on human thymocytes (25),
which seem to possess somewhat different flow cytometric properties
than murine thymocytes (9, 15, 20). Apoptotic murine
thymocytes generally show a distinct downshift in forward scatter
characteristics (15, 30, 32), indicative of the early
shrinkage in cell size which occurs before later-stage DNA cleavage and
membrane alterations (26, 30, 32). In two-dimensional flow
cytometry for FSC and 7-AAD staining, apoptotic cells were defined as
those falling within two gates. The R4 gate identified cells that were
smaller by FSC with no increases in 7-AAD staining; they were
considered early-stage apoptotic cells. The R3 gate identified smaller
cells that also showed increases in 7-AAD staining; they were
considered intermediate and late-stage apoptotic.
Focal immunofluorescence assay (FIA).
The monoclonal
antibody specific for the MCF virus envelope glycoprotein used was MAb
514 (5). To assay for thymocytes that express infectious MCF
virus (28), serial dilutions of thymocytes from inoculated
and uninoculated mice were prepared and overlaid onto NIH 3T3 cells
(8 × 104 cells/5-cm-diameter dish seeded the previous
day). Cells were cocultured overnight in the presence of 8 µg of
Polybrene per ml. Thymocytes were washed off twice with cold PBS, and
the NIH 3T3 cells were refed and allowed to grow to confluency
(approximately 4 to 5 days). Upon reaching confluency, the NIH 3T3
cultures were incubated with MAb 514, rinsed, and subsequently
incubated with an FITC-conjugated goat anti-mouse immunoglobulin G
(IgG), IgA, and IgM secondary antibody diluted 1:200. Foci of infected
cells were visualized by fluorescence microscopy and counted.
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RESULTS |
M-MuLV-induced thymic atrophy correlates with enhanced thymocyte
apoptosis.
In order to test whether the decreases in thymic size
observed in preleukemic M-MuLV-inoculated mice were due to enhanced levels of thymocyte apoptosis, M-MuLV-inoculated mice and uninoculated age-matched controls were analyzed by flow cytometry for two hallmarks of apoptotic death
shrinkage in cell size and the cleavage of genomic
DNA at internucleosomal sites (6, 27). FSC, as a measure of
cell size (30), and 7-AAD fluorescence were therefore used
as indicators of apoptosis (15, 25, 27). 7-AAD is a DNA
intercalator that will fluoresce when stimulated by 488-nm-wavelength light. In normal live cells, 7-AAD is unable to cross the cell membrane
and is therefore unable to intercalate into the DNA. However, in cells
that are in the late stages of apoptosis, when cell membrane
permeability is compromised and the DNA has been extensively cleaved,
7-AAD can enter the cell and intercalate into the DNA much more
efficiently. Since apoptotic thymocytes decrease in size relatively
early in the apoptotic death pathway, two-dimensional analysis for cell
size and 7-AAD staining provides an excellent discrimination of cells
in early stages of apoptosis from those in later stages of apoptosis.
Apoptotic thymocytes will have one of two characteristics: (i) small
size with low 7-AAD fluorescence, indicative of early-stage apoptosis,
and (ii) small size with high 7-AAD fluorescence, indicative of
late-stage apoptosis.
Thymocyte single-cell suspensions were prepared from mice and subjected
to flow cytometric analyses. Typical results from
this type of
two-parameter analysis are shown in Fig.
1A. Thymocytes
from an uninoculated
animal showed 0.84% of cells in the R4 region
(early apoptotic, i.e.,
small size with low 7-AAD fluorescence)
and 13.91% of cells in the R3
region (intermediate and late apoptotic,
i.e., small size with elevated
7-AAD fluorescence). In contrast,
an M-MuLV-inoculated animal showed
2.56% of cells in the R4 range,
more than twice as many as the
uninoculated animal, and 22.77%
of cells in the R3 region. Thus, the
increase in apoptotic cells
in M-MuLV-inoculated mice involved both
early and late stages
of apoptosis. Increases in the percentages of
cells in the R3
and R4 regions in M-MuLV-inoculated mice generally
correlated
with thymic atrophy (see below).
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FIG. 1.
Detection of apoptosis in thymocytes. (A) Thymocytes
from 6-week-old uninoculated and M-MuLV-inoculated mice were subjected
to two-parameter flow cytometric analysis for FSC and 7-AAD
fluorescence. 7-AAD fluorescence is plotted on a logarithmic scale.
Regions R3 and R4 represent thymocytes that are in the
intermediate-to-late and early stages of apoptosis, respectively.
Percentages of cells in both these regions are shown. (B)
Dexamethasone-induced apoptosis. Primary thymocytes from an
uninoculated mouse were cultured in vitro for 19 h in the presence
(+DEX) or absence ( DEX) of 0.1 mM dexamethasone. The cells were then
incubated with 7-AAD and subjected to two-parameter flow cytometric
analysis to determine the percentages of cells in the R3 and R4 regions
of late-to-intermediate- and early-stage apoptosis. Percentages of
cells in both these regions are shown.
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As a further test that cells in the R3 and R4 regions were apoptotic,
primary thymocytes from an uninoculated mouse were cultured
in vitro in
the presence of the glucocorticoid dexamethasone to
induce apoptosis
(
22,
25). The primary thymocytes were cultured
for 19 h
in the presence or absence of 0.1 mM dexamethasone, incubated
with
7-AAD, and then subjected to two-parameter flow cytometric
analysis.
The data in Fig.
1B show that dexamethasone treatment
led to higher
percentages of cells in the R3 and R4 regions, consistent
with these
regions representing apoptotic cells. It should be
noted that the
profile of the control thymocytes incubated in
the absence of
dexamethasone for 19 h showed substantially higher
levels of
apoptosis than the thymocytes analyzed directly after
removal from the
animal (Fig.
1). This was likely due to the fact
that thymocytes are
extremely sensitive to their environment and
will spontaneously undergo
apoptosis when placed into culture
(
7), presumably stemming
from loss of contact with thymic stromal
elements that provide survival
signals. It was necessary to slightly
adjust the R3 and R4 gates for
the in vitro-incubated thymocytes
so that early- and late-stage
apoptotic cells fell into these
regions.
As an independent test for apoptosis, cells were stained with annexin
V-FITC and PI. Annexin V is a calcium and phospholipid
binding protein
that selectively binds to negatively charged phospholipids
(
16). Under specific salt and calcium concentrations,
annexin
V preferentially binds phosphatidylserine (PS) (
1).
Very early
in the apoptotic pathway, cells rearrange their membrane
asymmetry
with respect to PS by translocating PS from the inner leaflet
of the cell membrane to the outer side. Therefore, cells that
are
undergoing apoptosis can bind annexin V. This property can
be used to
detect apoptotic cells by flow cytometry (
31). Figure
2 shows the results of a two-parameter
flow cytometric analysis,
of the same 6-week-old uninoculated and
M-MuLV-inoculated mice
studied to produce the data in Fig.
1A, for
staining by annexin
V-FITC and PI (which binds DNA). The comparison of
the percentages
of annexin V-positive cells (upper and lower right
quadrants)
for the uninoculated animal (19.19 and 5.61%, respectively)
with
those for the M-MuLV-inoculated animal (29.74 and 9.12%,
respectively)
also indicated that there was an enhanced level of
apoptosis in
the inoculated animal. Moreover, it was possible to
distinguish
early-stage apoptotic cells from late-stage apoptotic
cells: the
early ones were annexin V positive and PI negative (lower
right
quadrant), and the late ones were positive for both annexin V
and
PI (upper right quadrant). Thus, by this independent analysis,
the
thymocytes from M-MuLV-inoculated mice showed higher levels
of
apoptosis than those from uninoculated mice. It should be noted
that
the annexin V-FITC and PI staining gave somewhat higher percentages
of
apoptotic cells than procedures described in Fig.
1. This may
reflect
the ability of annexin V to detect cells at an earlier
stage of
apoptosis than is evident from a change in cell size,
as well as a
somewhat greater overall ability to detect either
early- or late-stage
apoptotic cells.
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FIG. 2.
Annexin V-PI staining. Thymocytes from the same
6-week-old animals for which results are shown in Fig. 1 were incubated
with FITC-conjugated annexin V and PI, and flow cytometric analysis was
performed. Percentages of late-stage apoptotic cells (upper right
quadrant) and early-stage apoptotic cells (lower right quadrant) are
shown. Both FITC-conjugated annexin V and PI fluorescence are plotted
on logarithmic scales.
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To test the generality of the increased apoptosis in thymi from
M-MuLV-inoculated mice, multiple animals were examined over
a time
course of 4 to 11 weeks postinoculation by the sort of
analysis shown
in Fig.
1. Figure
3 shows the cumulative
data for
all animals, as a function of age, with the sum of the
percentages
of cells in the R3 and R4 regions as the measure of
thymocyte
apoptosis. Each data point represents one animal. The level
of
thymocyte apoptosis in uninoculated mice remained relatively
constant
between 5 and 10 weeks of age. In contrast, M-MuLV-inoculated
mice showed higher levels of thymocyte apoptosis, particularly
from 6 to 10.5 weeks of age. Statistical analysis of the data
with a
two-sample
t test resulted in a calculated
t
score (
tcalc)
of 4.19 (
t < 0.001, one-tailed test), demonstrating a statistically
significant
difference between the two groups.
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FIG. 3.
Thymocyte apoptosis in M-MuLV-inoculated and
uninoculated mice. Thymocytes from uninoculated (diamonds) and
M-MuLV-inoculated (squares) mice were analyzed for apoptosis,
calculated as the sum of the percentages of cells in the R3 and R4
regions of intermediate-to-late- and early-stage apoptosis (see Fig.
1A). Mice were sacrificed between 4 and 11 weeks postinoculation. All
data points represent individual animals.
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The initial hypothesis for these experiments was that the
accelerated thymic atrophy in M-MuLV-inoculated mice was due to
enhanced levels of thymocyte apoptosis. To address this, the
relationship
between apoptotic value and visible thymic atrophy was
analyzed,
as shown in Fig.
4. Inoculated
animals were scored according to
the extent of thymic atrophy at the
time of necropsy, determined
by thymic weight, prior to flow cytometry.
Only animals of 7 to
10.5 weeks of age were used for this analysis, as
normal thymic
regression became evident in uninoculated animals after
11 weeks.
As shown in Fig.
4A, uninoculated mice with a mean thymic
weight
of 97.47 mg had low apoptotic values (<30%). For
M-MuLV-inoculated
mice, some animals exhibited normal thymic weight
while others
showed reduced weight (i.e., accelerated thymic
regression). For
the M-MuLV-inoculated mice there was a general
negative correlation
between thymic weight and apoptotic value. In Fig.
4B, the M-MuLV-inoculated
animals were divided into two groups: those
with no atrophy (weights
within 1 standard deviation of the mean thymic
weight of the control
group) and those with atrophy (weights below 1 standard deviation).
Uninoculated mice showed an average of 15.4%
apoptosis and an
average thymic weight of 97.47 mg. M-MuLV-inoculated
mice showing
no atrophy had an average of 22.1% apoptosis, while those
displaying
signs of atrophy had an average of 30.47% apoptosis. Thus,
the
degree of thymic atrophy in the M-MuLV-inoculated mice was
generally
consistent with the levels of thymocyte apoptosis. These
results
support the hypothesis that M-MuLV-induced thymic atrophy is
related
to enhanced levels of thymocyte apoptosis.
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FIG. 4.
Thymic atrophy correlates with elevated levels of
thymocyte apoptosis. At the time of sacrifice, thymi were dissected,
removed, and weighed, as a measure of thymic atrophy. (A) A comparison
of the levels of thymocyte apoptosis (calculated as the sum of the
percentages of cells in regions R3 and R4) with thymic weights (in
milligrams) in M-MuLV-inoculated (squares) and uninoculated
(diamonds) mice is shown. All data points represent individual animals.
(B) Thymi from M-MuLV-inoculated mice were scored as having either no
thymic atrophy (thymic weight was within 1 standard deviation of the
mean thymic weight of uninoculated mice) or thymic atrophy (thymic
weight was more than 1 standard deviation below the mean thymic weight
of uninoculated mice). A comparison of the mean levels of thymocyte
apoptosis observed in these mice and uninoculated mice is shown. Black
bar, uninoculated mice (mean weight, 97.47 mg); gray bar, no atrophy
(mean weight, 96.36 mg); hatched bar, atrophy (mean weight, 63.74 mg).
Average thymic weights of all three categories are displayed, and error
bars are based on standard error values.
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Relevance of enhanced levels of thymocyte apoptosis to
M-MuLV-induced leukemogenesis.
We tested whether the higher levels
of thymocyte apoptosis in preleukemic M-MuLV-inoculated mice were
related to disease induction. To do so, we employed the Mo+PyF101
M-MuLV enhancer variant. As described in the introduction, Mo+PyF101
M-MuLV has greatly attenuated leukemogenicity when inoculated s.c. On
the other hand, when inoculated i.p., the virus shows the same
leukemogenicity as wild-type M-MuLV. The rate of leukemogenesis for
wild-type M-MuLV is the same for either route of inoculation (mean
latency of 3 to 4 months in 100% of the animals).
We therefore measured the levels of thymocyte apoptosis in Mo+PyF101
M-MuLV-inoculated mice. Thymic single-cell suspensions
from mice
inoculated with Mo+PyF101 M-MuLV by the i.p. route were
assayed for
apoptosis by flow cytometry, as described above. The
left panel of Fig.
5 shows the collective data for mice
inoculated
i.p. with Mo+PyF101 M-MuLV versus the data for uninoculated
animals.
The animals inoculated i.p. with Mo+PyF101 M-MuLV showed
elevated
levels of apoptosis at 6 to 12 weeks. Statistical analysis
using
a two-sample
t test resulted in a t
calc
score of 5.2 (
t < 0.001,
one-tailed test),
demonstrating a statistically significant difference
between the levels
of thymocyte apoptosis in uninoculated mice
and those in Mo+PyF101
M-MuLV-inoculated (i.p.) mice.
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FIG. 5.
Thymocyte apoptosis in mice inoculated i.p. with
Mo+PyF101 M-MuLV. (Left panel) Thymocytes from uninoculated mice
(diamonds) and mice inoculated i.p. with Mo+PyF101 M-MuLV (squares)
were analyzed for apoptosis, determined as the sum of the percentages
of cells in the R3 and R4 regions of intermediate-to-late- and
early-stage apoptosis. Mice were sacrificed between 4 and 12 weeks
postinoculation. All data points represent individual animals. (Right
panel) A comparison of the levels of thymocyte apoptosis in mice
inoculated s.c. with wild-type M-MuLV (diamonds) and mice inoculated
i.p. with Mo+PyF101 M-MuLV (squares) is shown.
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The increased level of apoptosis for the animals inoculated i.p. with
Mo+PyF101 resembled the level of apoptosis in mice inoculated
s.c. with
wild-type M-MuLV (Fig.
5, right panel). Thus, when Mo+PyF101
M-MuLV was
inoculated into mice under conditions where it showed
efficient
leukemogenicity, an increased level of apoptosis was
observed in
preleukemic
thymocytes.
The level of thymocyte apoptosis was also measured for mice inoculated
with Mo+PyF101 M-MuLV by the s.c. route (attenuated
leukemogenicity).
As shown in Fig.
6 (left panel), mice
inoculated
s.c. with Mo+PyF101 M-MuLV showed lower levels of thymocyte
apoptosis
than those inoculated i.p. Statistical analysis using a
two-sample
t test resulted in a t
calc score of
2.67 (
t < 0.005, one-tailed
test), demonstrating a
statistically significant difference between
the levels of thymocyte
apoptosis in mice inoculated i.p. with
Mo+PyF101 M-MuLV and those in
mice inoculated s.c. Hence, this
virus induced higher levels of
apoptosis when it was inoculated
into mice under conditions where it
efficiently induces disease.
On the other hand, the levels of apoptosis
were similar for mice
inoculated s.c. with Mo+PyF101 M-MuLV and
uninoculated mice (Fig.
6, right panel). The lower level of apoptosis
in mice inoculated
s.c. with Mo+PyF101 M-MuLV correlated with the
attenuated pathogenicity
of Mo+PyF101 M-MuLV when inoculated s.c. Thus,
studies with Mo+PyF101
M-MuLV supported the hypothesis that enhanced
levels of thymocyte
apoptosis are important for efficient
leukemogenesis by M-MuLV.
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FIG. 6.
Mice inoculated s.c. with Mo+PyF101 M-MuLV. (Left
panel) Thymocytes from mice inoculated s.c. with Mo+PyF101 M-MuLV were
analyzed for apoptosis, determined as the sum of the percentages
of cells in the R3 and R4 regions of intermediate-to-late- and
early-stage apoptosis. A comparison of the levels of thymocyte
apoptosis in mice inoculated i.p. with Mo+PyF101 M-MuLV (diamonds) and
those for mice inoculated s.c. (squares) is shown. (Right panel) A
comparison of the levels of thymocyte apoptosis for uninoculated mice
(diamonds) and those observed for mice inoculated s.c. with Mo+PyF101
M-MuLV (squares) is shown.
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Role of MCF recombinant virus in M-MuLV-induced thymocyte
apoptosis.
MCF virus recombinants can be found in almost all
M-MuLV-induced tumors, suggesting that they are advantageous for
tumorigenesis, although their exact role is not completely understood
(see the introduction). It has been previously suggested that MCF
viruses play a role in early preleukemic events (4). This
was based on the fact that s.c. inoculation with Mo+PyF101 M-MuLV does
not result in preleukemic physical changes, such as accelerated thymic atrophy (11, 13), and that at the same time mice
inoculated s.c. with this virus do not show development of MCF viruses
(4). Moreover, when the same virus is inoculated i.p., the
mice concomitantly show thymic atrophy and generate MCF virus
recombinants (3).
In order to address the role of MCF viruses in M-MuLV-induced enhanced
thymocyte apoptosis, we assayed thymocytes for the
production of
infectious MCF virus. To detect cells expressing
MCF virus, an FIA was
used (see Materials and Methods) (
28).
This assay quantifies
MCF virus-infected cells by employing a
monoclonal antibody (MAb 514)
that is specific for the MCF virus
envelope glycoprotein
(
5). In this method, an initial MCF virus-infected
cell acts
as an infectious center to create a focus of infected
cells that can be
visualized by immunofluorescent staining for
the polytropic envelope
glycoprotein. Figure
7 shows the rate
of
appearance of MCF virus recombinants in mice inoculated s.c.
with
wild-type M-MuLV. MCF virus recombinants could be detected
at high
levels in thymocytes from inoculated mice beginning at
4 weeks. The
appearance of MCF virus infection in these inoculated
animals
accompanied (or preceded) accelerated thymic atrophy and
enhanced
levels of apoptosis. This was consistent with the hypothesis
that MCF
virus recombinants are involved in establishing M-MuLV-induced
preleukemic events.
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FIG. 7.
MCF recombinant virus infection of thymocytes in
preleukemic mice. Thymocytes from mice inoculated s.c. with M-MuLV
(diamonds) and from mice inoculated i.p. with Mo+PyF101 M-MuLV
(squares) were tested for productive MCF virus infection by FIA. Titers
are shown on a logarithmic scale (FIU per 106
thymocytes).
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|
In contrast, when mice inoculated i.p. with Mo+PyF101 M-MuLV were
examined, 6 of 12 mice showed no detectable MCF virus recombinants
by
11 weeks of age, and the remaining six had very low levels
(<10
4 focal infectivity units [FIU] per 10
6
thymocytes), even though the great majority of them (9 of 12)
showed
thymic atrophy and enhanced levels of apoptosis. Thus,
despite the
positive correlation of MCF virus appearance and enhanced
levels of
thymocyte apoptosis observed in wild-type M-MuLV-inoculated
mice, the
results for the mice inoculated i.p. with Mo+PyF101
M-MuLV indicated
that MCF virus infection of thymocytes does not
play an obligate role
in enhancing the level of thymocyte apoptosis.
For the latter mice,
high-level MCF virus infection of thymocytes
did not precede or
accompany the onset of thymic atrophy and enhanced
levels of thymocyte
apoptosis.
| |
DISCUSSION |
In this paper, preleukemic changes in the thymi of
M-MuLV-inoculated mice were studied. It was found that enhanced levels of thymocyte apoptosis were evident beginning at 6 weeks
postinoculation. The thymocyte apoptosis was assessed by several
different flow cytometric criteria, including decreased cell size and
enhanced staining with 7-AAD or annexin V. The enhanced
levels of thymocyte apoptosis were also correlated with accelerated
thymic atrophy in M-MuLV-inoculated mice. This was plausible since the
thymus is a very dynamic organ, with a continual turnover of thymocytes undergoing positive and negative selection. Both of these selection processes involve apoptosis. Thus, enhanced rates of
thymocyte apoptosis without compensating increases in
thymocyte division would result in decreased numbers of thymocytes in
an M-MuLV-infected thymus.
Once increased preleukemic thymocyte apoptosis had been documented, we
investigated whether it was likely to be important for leukemogenesis
by M-MuLV. Experiments with the Mo+PyF101 M-MuLV variant were
supportive of a role for enhanced levels of thymocyte apoptosis in
leukemogenesis. When Mo+PyF101 M-MuLV was inoculated by the i.p. route
(whereby it efficiently induces leukemia), enhanced levels of thymocyte
apoptosis and thymic atrophy equivalent to those observed in wild-type
M-MuLV-inoculated animals were observed. On the other hand, when the
same virus was inoculated s.c. (by which leukemogenicity is
attenuated), substantially less thymocyte apoptosis and thymic atrophy
were observed. Hence, Mo+PyF101 M-MuLV-induced thymic atrophy and
enhanced levels of thymocyte apoptosis were positively correlated with
efficient leukemogenesis.
The exact mechanism by which M-MuLV-induced thymic atrophy and enhanced
levels of thymocyte apoptosis putatively facilitate leukemogenesis
remains to be determined. In AKR mice, potentially leukemic cells (PLC)
have been detected by transplantation experiments (17).
These PLC are initially found in the bone marrow and spleen but not in
the thymus; at later times they can be detected in the thymus as well.
Phenotypic characterization of the PLC indicated that they have the
properties of prothymocytes. By analogy, M-MuLV-enhanced levels of
apoptosis in the thymus of an inoculated mouse might result in enhanced
recruitment of preleukemic prothymocytes from the bone marrow or spleen
into the thymus, where development of the tumor ultimately occurs.
It also seems possible that M-MuLV-induced thymic atrophy and
thymocyte apoptosis affect cellular immunity in inoculated mice. M-MuLV-inoculated mice do not generally seem to display substantial immunodeficiency. This might reflect the fact that thymic physiology appears fairly normal in M-MuLV-inoculated animals up to 6 weeks of age
(see Results). Thus, the thymus would produce normal amounts of T
lymphocytes up to that time. However, a premature decrease in thymocyte
maturation might cause decreases in cellular immunity, particularly as
animals age. One consequence could be that inoculated animals might not
efficiently raise their cellular immune responses to neoantigens on
developing tumors. However, it is unclear whether such a putative
effect would play a role in M-MuLV leukemogenesis, since the tumors
appear at approximately 4 months.
The mechanism by which M-MuLV infection leads to enhanced levels of
thymocyte apoptosis is of considerable interest. Initially, our
hypothesis was that formation of MCF virus recombinants played a role.
Indeed, it has been shown previously that when Mo+PyF101 M-MuLV is
inoculated into mice by the s.c. route, the slowly appearing tumors
lack MCF viruses (4). On the other hand, i.p. inoculation with the same virus resulted in rapidly appearing tumors that contained
MCF proviruses (3). In addition, other experiments done on
in vitro bone marrow cultures from M-MuLV-inoculated mice indicated
that bone marrow stroma showed a quantitative defect for growth that
could be associated with MCF virus infection (19). The
results for mice inoculated with wild-type M-MuLV supported the
hypothesis, since high level MCF virus infection in thymocytes preceded
thymic atrophy. However, in mice inoculated i.p. with Mo+PyF101 M-MuLV,
little or no MCF virus was detected, even in animals that showed
accelerated thymic atrophy. Thus, enhanced levels of thymocyte
apoptosis could not be attributed to direct infection of these cells
with MCF recombinant virus.
One possible explanation currently under investigation is that MCF
recombinant virus infection in thymic stromal cells is important for
enhancing levels of apoptosis. As mentioned above, we previously
developed in vitro evidence for the role of MCF virus infection
in bone marrow stroma defects; these defects are associated with
decreased hematopoiesis (19). MCF virus infection of thymic
stroma might interfere with the ability of the stroma to support proper
thymocyte maturation (e.g., positive and negative selection), which
could result in increased thymocyte apoptosis. It should be noted that
the infectious center assays in Fig. 7 were carried out on nonadherent
thymocytes, which are largely T lymphoid. Thus, MCF virus infection of
stromal cells could have been missed. Experiments to assess the
infection state of thymic stromal cells are currently in progress.
If the involvement of MCF virus recombinants is ruled out as a factor
underlying increased thymocyte apoptosis, then infection of thymocytes
or thymic stroma with M-MuLV itself might be considered as the cause.
While this would be consistent with the fact that the preleukemic
changes are not observed in uninoculated animals, it has been shown
previously that the extent of thymocyte infection for animals
inoculated with Mo+PyF101 M-MuLV by either the i.p. or s.c. route was
the same (2). Any proposed mechanism for the increase of
thymic apoptosis must take this result into account.
| |
ACKNOWLEDGMENTS |
This work was supported by NIH grant CA32455. C.B. was
supported by NIH training grant 5T32-CA09054. The support of the Cancer Research Institute and the Optical Biology Core Facility of the Chao
Family Comprehensive Cancer Center is acknowledged as well.
We thank Chris Hughes for help with flow cytometry.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Molecular Biology and Biochemistry and Cancer Research Institute,
Biological Sciences II, Rm. 3221, University of California, Irvine, CA
92697. Phone: (949) 824-5554. Fax: (949) 824-4023. E-mail:
hyfan{at}uci.edu.
| |
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0022-538X/99/$04.00+0
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Top
Abstract
Introduction
