INTRODUCTION

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Murine leukemia viruses (MLVs) with simple genomes require a relatively long latency for the development of thymic lymphoma. In AKR mice, thymic tumors begin to arise when the animals are about 6 to 7 months old (6, 29a). It is thought that one of the reasons for this long latency in AKR mice is the requirement for the generation of recombinant MLVs, i.e., mink cell focus-forming (MCF) viruses, which are the proximal etiologic agent of disease (6, 34). The oncogenic class I MCF MLVs are recombinant retroviruses with an ecotropic MLV backbone and nonecotropic viral sequences at the N-terminal portion of the SU protein and the C terminus of TM and the U3 region of the long terminal repeat (12, 28, 51). Class I MCF viruses are detectable in the thymus of AKR mice beginning at about 4 to 5 months of age (12, 47). Compared with the time course for the appearance of spontaneous thymic tumors in AKR mice, MCF13 inoculation into AKR neonates results in accelerated leukemogenesis which begins at about 10 weeks postinoculation (54). Although extensive studies have examined the later events of MCF MLV tumorigenesis (8, 9, 36), less work has been done to study the earlier preleukemic period. It has been observed that efficient virus replication in the thymus during the preleukemic period is essential for thymus development (21, 34, 35). We have recently reported that the sequences between the enhancer and promoter (DEN) in the long terminal repeat of the MCF13 MLV regulate virus replication in the thymus during the early stages of lymphomagenesis (59).

As a part of this study we identified the thymic subpopulations which were infected by MCF13 MLV. We observed that the subpopulation in which the highest level of virus replication occurred during the preleukemic period was the immature CD3⁺ CD4⁺ CD8⁺ (59). These cells accounted for 70 to 80% of virus-infected cells. The subpopulation with the next highest percentage (10 to 18%) of virus-infected cells was CD3 CD4⁺ CD8⁺, which are the precursors to the CD3⁺ CD4⁺ CD8⁺ cells (14, 41, 44). The effect of MLV infection on CD4⁺ CD8⁺ cells has also been observed for the SL3-3 virus, which produces thymic lymphoma similar to MCF13 (18, 27). It has been shown that the normal development of these cells is affected during the maturation of CD4 CD8 cells when they were isolated from SL3-3 virus-inoculated mice and either cultured with fetal thymic stroma in vitro or inoculated intrathymically into mice (16, 17). The idea that thymic lymphocytes which have a CD4⁺ CD8⁺ phenotype may be the major targets for cellular transformation is further supported by the detection of this phenotype for cells present in the majority of MLV-induced thymic tumors by ourselves and other investigators (18, 26, 59).

The CD4⁺ CD8⁺ thymic subpopulation in which MCF13 MLV replication predominantly occurs (59) is the same subset in which both positive and negative selection of thymic T lymphocytes normally occurs (14, 41). It has been observed that the negative selection process involves the apoptosis of CD4⁺ CD8⁺ cells which recognize self-antigens. Because in our previous study we noticed that one of the effects of MCF13 MLV infection was the reduction of thymus size, we explored the effect of virus replication on the dynamic cellular processes of this organ, and particularly on the CD4⁺ CD8⁺ subpopulation during the early stages of tumorigenesis. This report summarizes the results of this study.

	MATERIALS AND METHODS

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Virus inoculation and isolation of thymic lymphocytes. Neonatal AKR mice were injected intraperitoneally with 50 µl of inoculum containing 10⁶ infectious units (IFUs) of MCF13 MLV. Control mice were inoculated with tissue culture medium. Mice were euthanized by CO₂ inhalation and cervical dislocation, and thymuses were removed into cold RPMI 1640 containing 2% inactivated fetal calf serum (IFCS). Single-cell suspensions were made by pressing the thymus tissue through a wire mesh into RPMI plus 2% IFCS. Cells were washed once with medium without serum. The viability of thymic lymphocytes in suspension, which was determined by trypan blue dye exclusion, was routinely greater than 90%.

In vitro culture of thymic cells. Single-cell suspensions of thymic lymphocytes were placed into 75-cm² tissue culture flask with RPMI 1640 containing 10% IFCS and 100 µg of penicillin-streptomycin per ml at a density of 10⁷ cells per ml. Cells were harvested at 6, 24, and 48 h after incubation. Cell viability was determined at each time point.

Thymus histology. Thymuses from euthanized MCF13-inoculated and control mice were rapidly removed and immediately fixed overnight in 10% formalin. After dehydration in graded ethanol and xylene, the tissues were embedded in paraffin. Serial 5-µm sections were prepared, and alternate sections were stained by Giemsa or hematoxylin and eosin stains.

Staining of thymic cells. Thymic lymphocytes (10⁶) were washed twice with phosphate-buffered saline (PBS) containing 1% bovine serum albumin and 0.02% sodium azide. Washed cells were incubated with 100 µl of monoclonal antibody (MAb) 514 on ice for 30 min, after which time 100 µl of the secondary antibody (fluorescein isothiocyanate-conjugated goat anti-mouse immunoglobulin G [IgG]) at a 1:100 dilution was added. After being washed twice with PBS, the cells were resuspended in 100 µl of PBS containing hamster anti-CD3 antibody conjugated to phycoerythrin, rat anti-CD4 antibody conjugated to Cy-Chrome, rat anti-CD8 antibody conjugated to biotin (PharMingen), and NeutraLite avidin conjugated to Cascade blue (Molecular Probes, Eugene, Oreg.) at dilutions previously determined by titration assays. Washed cells were resuspended in 0.7 ml of 0.5% paraformaldehyde and analyzed by flow cytometry.

Flow cytometric analysis. Prior to analysis, dead cells were gated out based on forward versus side scatter dot plots. Flow cytometry was performed on a FACS Vantage flow cytometer equipped with an HP 9000 computer running the LYSYS II software (Becton Dickinson Immunocytometry Systems, San Jose, Calif.). Fluorescein isothiocyanate, phycoerythrin, and Cy-Chrome were excited with an ILT 5500A argon ion laser (Ion Laser Technology, Salt Lake City, Utah). Cascade blue was excited with 50 mW of all lines of UV light (351 to 365 nm wavelength). Electronic compensation for spectral overlap of the fluorochromes was performed with single-color control samples prepared with the test samples. All data presented were based on analysis of 2 × 10⁴ cells with the Paint-A-Gate software (Becton Dickinson). Analysis gates were set on isotype controls.

7-AAD staining. Staining was performed with 0.0625 µg of 7-amino-actinomycin D (7-AAD) PharMingen added to 10⁵ cells in 100 µl of PBS. Cells were stained for 15 min at room temperature (RT) in the dark, then brought to a final volume of 500 µl of PBS. Samples were run within 1 h on the FACScan. Unstained cells were used for a negative control. Data on 2 × 10⁴ cells were analyzed using the CELLQuest software. Scattergrams were generated by combining forward light scatter with 7-AAD fluorescence. Regions of clear-cut populations having negative, dim, and bright fluorescence were selected.

DNA fragmentation assay. Cellular DNA was extracted from 2 × 10⁷ cells using the Apoptotic DNA Ladder kit (Boehringer Mannheim). Cells were lysed in 200 µl of lysis buffer (6 M guanidine-HCl, 10 mM Tris-HCl, 10 mM urea, 20% Triton X-100 [pH 4.4]) and incubated for 10 min at RT. Samples subsequently were mixed with 100 µl of isopropanol, filtered by centrifugation for 1 min at 8,000 rpm in an Eppendorf centrifuge, and washed twice. DNA was eluted into 100 µl of 10 mM Tris-HCl (pH 8.5) buffer, and 1.5 µg of DNA was electrophoresed through a 1.5% agarose gel for 1.5 h at 100 V in 1× TBE (81 mM Tris base, 81 mM boric acid, 1.8 mM EDTA). DNA was visualized by staining with ethidium bromide and examination under UV light.

Caspase-3 assay. Cells (2 × 10⁷) were collected at each time point, washed twice with PBS, and lysed in 200 µl of 50 mM Tris-HCl (pH 7.5) containing 0.03% Nonidet and 1 mM dithiothreitol. Nuclei were removed by centrifugation (1,200 × g) for 5 min, and 50 µl of the cytosolic fraction was incubated with 40 µM DEVD-amc-10 mM HEPES (pH 7.5)-50 mM NaCl-2.5 mM dithiothreitol in a total volume of 200 µl for 120 min at 37°C. Coumarin fluorescence, released by caspase cleavage of DEVD-amc, was measured using 360-nm excitation and 415-nm emission wavelengths. A charge-coupled device (Instaspec IV; Oriel, Stratford, Conn.) fitted with a monochromator was used to measure the fluorescence emission spectrum. The system was initially calibrated with known levels of aminomethylcoumarin. Protein amounts were measured using the bicinchoninic acid protein assay (Pierce). Units of enzyme activity are expressed as picomoles of substrate hydrolyzed per microgram of protein per hour.

PARP cleavage assay. Cells (2 × 10⁷) were washed twice with PBS, lysed with buffer containing 2% sodium dodecyl sulfate (SDS), 0.5 M Tris-HCl (pH 6.8), and 20% glycerol and subsequently boiled for 5 min. Equal amounts of protein were loaded on a 8% polyacrylamide gel and separated by electrophoresis at 120 V for 2 h. Following transfer, nitrocellulose membranes were reacted with a poly(ADP-ribose) polymerase (PARP)-specific primary antibody (C-2-10; BIOMOL Research Laboratory) for 1 h. After being washed three times with 1× TTBS (0.2% Tween 20, 0.136 M NaCl, 2.7 mM KCl, 25 mM Tris-HCl [pH 7.4]; Sigma), the membrane was incubated with horseradish peroxidase-conjugated rabbit anti-mouse IgG secondary antibody for another hour at RT. Antigen was detected using the ECL detection system (Pierce) according to the manufacturer's instructions. Membranes were exposed to Kodak X-ray film.

	RESULTS

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Depletion of thymic T lymphocytes by MLV infection. In our studies of the pathogenic properties of the MCF13 MLV, we examined the effect of virus infection on thymus size. We detected a significant decrease in thymus weight at 8 weeks after MCF13 MLV inoculation of neonatal AKR/J mice. The mean weight of thymuses from 23 mice at 8 weeks postinoculation (p.i.) was 110.3 ± 20.4 mg compared with a mean weight of 128.2 ± 19.5 mg for 19 age-matched control thymuses. The difference in thymus weights for MCF13-inoculated and uninoculated mice was statistically significant, with a P value of 0.003 as determined by Student's t test. We picked 8 weeks to examine thymus weight because at this time point we had previously observed a maximum of 58% of virus-infected cells in the thymus before thymic lymphoma first began to appear at 10 weeks p.i. (54, 59).

Morphological lymphocyte depletion of thymic lymphocytes produced by MCF13 MLV infection. Because we detected a decrease in thymus cellularity due to MCF13 MLV infection of this organ, we examined thymic sections at 8 weeks p.i. by histological staining (Fig. 1). Sections from MCF13-inoculated mice showed an overall decrease in lymphocyte numbers throughout the thymus relative to control animals. At low magnification (e.g., Fig. 1), this is apparent as a decrease in the intensity of hematoxylin staining for the virus-infected thymus. This decrease in cellularity was most pronounced in the CD4⁺ CD8⁺-rich cortical regions.

View larger version (54K): [in this window] [in a new window]   FIG. 1.   Histological comparison of thymuses from MCF13-inoculated and control mice. (A) The thymus of an 8-week-old control mouse shows a thick zone of densely staining cortex (C) and a small zone of lightly staining medulla (M). (B) Lymphocyte depletion of the thymus is detectable 8 weeks after inoculation of MCF13 MLV.

Identification of thymic subpopulations which are depleted by MCF13 MLV infection. The thymus comprises a number of different subpopulations of T lymphocytes, which have been identified by their differential expression of the CD3, CD4, and CD8 cell surface antigens (14, 41, 44). To identify the subpopulation which was reduced in number in thymuses from mice inoculated with MCF13 MLV, we performed flow cytometric analysis of thymic lymphocytes stained with anti-CD3, anti-CD4, and anti-CD8 fluorescent-labeled antibodies. We examined the five predominant subpopulations in the thymus detectable with these antibodies, as presented in Table 2. These subpopulations were the immature CD3 CD4 CD8, CD3 CD4⁺ CD8⁺, and CD3⁺ CD4⁺ CD8⁺ cells and the more mature CD3⁺ CD4⁺ CD8 and CD3⁺ CD4 CD8⁺ cells. Although other investigators have identified additional subsets of thymic cells, i.e., CD3 CD4⁺ CD8, CD3 CD4 CD8⁺, and CD3⁺ CD4 CD8 (14, 23, 24, 44), their numbers were too few to be reproducibly detectable and hence they were not included in our analysis.

Thymic lymphocyte depletion involves apoptosis. To better understand the basis for the reduction in thymic cell number after MCF13 MLV infection, we assessed the percentages of live, apoptotic, and dead cells in thymuses at approximately 2 to 3 and 8 to 9 weeks after virus inoculation. These two time points were selected because they corresponded to times when we detected either no difference in total thymus cell number (3 weeks) or a significant decrease in cell number (8 weeks) as described above (Table 1).

View larger version (41K): [in this window] [in a new window]   FIG. 2.   Detection of live, apoptotic, and dead cells by 7-AAD staining and flow cytometry. Thymic lymphocytes isolated from the thymus of a 2-week-old AKR/J mouse and placed in culture for 24 h were stained with 7-AAD. Stained cells (2 × 104) were analyzed by a FACScan flow cytometer. The dot plot was generated with the CELLQuest software. Selected regions are live (R1), apoptotic (R2), and late apoptotic/dead (R3) cells. The percentage of cells in each population is indicated.

Additional assays for apoptosis of thymic lymphocytes from MCF13-inoculated mice. We performed additional independent assays for apoptosis to confirm the results from our 7-AAD analysis of freshly isolated thymic lymphocytes as well as those that were placed in culture. To determine whether the increase in apoptosis that we observed for the cultured cells may be due to apoptotic events which occur before 24 h, we included a 6-h time point in these studies. Assays for apoptosis included assessments of DNA fragmentation as well as cleavage of the proenzymes PARP and procaspase-3. All three cellular events are hallmarks of apoptosis in various types of cells, including lymphocytes (1, 7, 11). Again, we analyzed thymic lymphocytes which were isolated from mice at 8 weeks after virus inoculation.

View larger version (127K): [in this window] [in a new window]   FIG. 3.   DNA fragmentation of thymic lymphocytes. Cellular DNA (1.5 µg) isolated from thymic lymphocytes with the Apoptotic DNA Ladder kit (Boehringer Mannheim) was electrophoresed in each well of a 1.5% agarose gel at 100 V for 1.5 h. DNA was extracted from pooled lymphocytes from thymuses of three mice which were either control (C) or inoculated with MCF13 MLV for 8 weeks (MLV). Thymic cells were placed in culture for 0, 6, 24, and 48 h before DNA extraction. Lane M, 100-bp DNA ladder (Gibco-BRL). Three independent assays which were performed with different pools of thymic lymphocytes yielded similar results.

View larger version (28K): [in this window] [in a new window]   FIG. 4.   Assay of PARP cleavage. Equal amounts of cellular protein were electrophoresed through an SDS-8% polyacrylamide gel for 2 h at 120 V. The uncleaved 116-kDa and 85-kDa cleavage products of PARP were detected by immunoblotting with the C-2-10 antibody (BIOMOL Research Laboratory). Cellular protein was extracted from pooled lymphocytes from thymuses of three mice which were either control (C) or inoculated with MCF13 MLV for 8 weeks (MLV). Thymic cells were placed in culture for 0, 6, and 24 h before protein extraction. This immunoblot is representative of three independent assays performed at different times.

Increase in apoptosis of thymic lymphocytes correlates with MCF13 MLV infection. To better determine whether MCF13 virus infection of thymic cells affects their ability to undergo cell death caused by apoptosis, we stained cells with 7-AAD and MAb 514 (13), which specifically detects MCF envelope glycoproteins. By flow cytometric analysis we selected cells expressing MCF glycoprotein (gp70⁺) and those which were negative for glycoprotein expression (gp70). We then assessed the percentage of live, apoptotic, and dead cells in both of these populations of cells. Because we did not detect any difference between thymic lymphocytes isolated from virus-inoculated and control mice at 2 weeks p.i., we performed this analysis only at 8 weeks p.i. Furthermore, we analyzed only freshly isolated thymic lymphocytes because we observed that cells in culture were sensitive to the additional manipulations required for gp70 detection, which resulted in additional cell death for reasons that are not understood. As shown in Table 4, we observed significant differences between the gp70⁺ and gp70 cells in all cell populations examined. The percentages of apoptotic and dead cells were significantly higher, as determined by statistical analysis, for the gp70⁺ cells compared with the gp70 population. We also detected a concomitant decrease in the percentage of live gp70⁺ cells. Thus, MCF13 MLV infection of thymic lymphocytes leads to an increase in their ability to undergo cell death.

View this table: [in this window] [in a new window]   TABLE 4.   Analysis of gp70+ and gp70 thymic cellsa

	DISCUSSION

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We have shown that MCF13 MLV infection of thymic lymphocytes during the preleukemic period results in an increase in cellular apoptosis as measured by several independent assays. Our data suggest that this increase in apoptosis is responsible for the decrease in thymus cellularity which mainly affects the CD3 CD4⁺ CD8⁺ and CD3⁺ CD4⁺ CD8⁺ subpopulations of immature thymic lymphocytes. We have previously shown that these are the same two subpopulations which are predominantly infected by the MCF13 virus (59). A decrease in the percentage of CD4⁺ CD8⁺ cells has also been implicated in the development of thymic tumors by the SL3-3 MLV (16, 17). Our detection of an increase in apoptosis and dead cells for freshly isolated gp70⁺ cells compared with gp70 cells supports the notion that virus infection is directly responsible for cellular apoptosis and subsequent death. The receptor protein for the MCF glycoprotein is closely related to the yeast SYG1 protein (2, 49, 58), which induces transient cell cycle arrest in response to the yeast mating pheromone (46). Whether the mammalian homolog of the SYG1 protein is part of a signal transduction pathway that is involved in the induction of apoptosis of virus-infected cells remains to be determined.

It is interesting that the same subpopulations in the thymus which are depleted by MCF13 MLV infection are those which undergo positive and negative selection (15, 22, 30). The majority of cells in these subpopulations, which together comprise 80 to 90% of total thymic lymphocytes (14, 41), are eliminated by apoptosis within a few days of development in the thymus (29). The CD4⁺ CD8⁺ cells are mainly present in the cortical regions of the thymus. Our results of a histological examination of the thymus after MCF13 virus inoculation showed lymphocyte depletion predominantly of the cortical regions, which correlated with our detection of reduced numbers of CD4⁺ CD8⁺ cells in virus-infected animals. This observation is corroborated by an earlier study which showed that thymuses from 6-month-old AKR mice undergo cortical involution during the development of spontaneous tumors in these animals (33).

It has been shown that tumorigenesis in the AKR mouse is a complex phenomenon, which also involves a role of the thymic stroma. During the development of both spontaneous and SL3-3 MLV-induced disease, it has been shown that virus infection of thymic stromal cells affects T-lymphocyte maturation (10, 16, 17). Virus infection of the CD4⁺ CD8⁺ cells may contribute to an alteration in the interactions of thymic lymphocytes with stromal epithelial cells, thus possibly resulting in abnormal maturation of the virus-infected cells.

Although both the inhibition and induction of apoptosis have been implicated in tumorigenesis, it has been most commonly observed that the development of malignancies is dependent upon an initial inhibition of apoptosis (3, 31, 56). In contrast, we have demonstrated that the development of thymic tumors by MCF MLV infection results in an enhancement of apoptosis during the early stages of lymphomagenesis. Induction of apoptosis as an early event in cellular transformation has been observed for other oncogenic retroviruses as well. Fan and coworkers have shown that the Moloney MLV, which also generates thymic tumors, increased thymic cell apoptosis also during the preleukemic period (4). Their data furthermore demonstrated that the level of cellular apoptosis correlated with viral pathogenicity. Another example includes the subgroups B and D avian leukosis viruses, which cause high levels of cell death during the acute phase of infection (55). This viral cytopathicity has been attributed to the binding of the avian leukosis virus glycoprotein to the receptor molecule CAR1, which is a member of the tumor necrosis factor receptor family (5). Other examples of the induction of apoptosis during cellular transformation include the transformation of pre-B cells in vitro by the Abelson murine leukemia virus (38, 52) and of T cells by the feline leukemia virus subgroup C (40).

For the induction of thymic tumors by MCF13 MLV, we hypothesize that cells which survive the apoptotic crisis induced by virus infection are those which will evolve into a fully malignant cell. It is likely that the progression to a tumor cell requires the inheritance of multiple genetic mutations, some of which result in the inhibition of apoptosis. An example of such an event is proviral insertional mutagenesis of the c-myc protooncogene, which occurs at a significant frequency in thymic tumors induced by various murine leukemia viruses. It has been shown that overexpression of c-myc can rescue lymphocytes from apoptosis (19, 57). Additional genetic mutations involving other cellular proteins which are able to rescue cells from apoptosis, such as members of the Bcl-2 and NF-B families, may also play a role in the development of MLV-induced thymic tumors. Our detection of an increase in NF-B in MCF13-induced tumors by 50- to 200-fold compared with normal thymuses would support this idea (unpublished results). It is noteworthy that NF-B and c-Myc have synergistic roles in inhibiting apoptosis for certain cell types (42). Our results suggest that the induction of apoptosis as an early event in the development of lymphomas may be a common mechanism used by different transforming retroviruses.