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The Journal of Immunology, 2002, 169: 2866-2874.
Copyright © 2002 by The American Association of Immunologists

Enforced Expression of Runx2 Perturbs T Cell Development at a Stage Coincident with {beta}-Selection1

François Vaillant2,*, Karen Blyth*, Linda Andrew{dagger}, James C. Neil* and Ewan R. Cameron*

* Molecular Oncology Laboratory and {dagger} LRF Virus Center, Institute of Comparative Medicine, University of Glasgow Veterinary School, Glasgow, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The development of T cells in the thymus is regulated by a series of stage-specific transcription factors. Deregulated expression of these factors can lead to alterations in thymocyte development with the production of aberrant cell subsets and predispose to tumor formation. The three genes of the Runx family are multilineage regulators of differentiation that have been reported to be expressed in the T cell lineage. However, their roles in thymocyte development and T cell function are largely unknown. While the Runx2/Cbfa1/AML3/Pebp2{alpha}a gene plays a primary role in osteogenesis and regulates a number of key bone regulatory genes, we show here that Runx2 is also expressed during the earliest phase of thymic development, in the double-negative subset. Furthermore, enforced expression of Runx2 in transgenic mice under the CD2 promoter was found to affect T cell development at a stage coincident with {beta}-selection, resulting in an expansion of double-negative CD4 and CD8 immature single-positive cells. Unlike wild-type controls this preselection population (CD4-CD8+heat-stable Ag+TCR-) is in a nonproliferative state, but appears to be primed for further transformation events. Overall the data suggest that Runx2 accelerates development to the CD8 immature single-positive stage, but retards subsequent differentiation to the double-positive stage. Thus, Runx2 joins a small group of transcription factors that can interfere with early T cell development, cause an expansion of a specific subset, and predispose to lymphoma.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The core binding factor (CBF3/RUNX/AML) family comprises a set of heterodimeric ({alpha}- and {beta}-chains) regulatory proteins with vital roles in eukaryotic development. In mammals the {alpha} subunits are encoded by three distinct genes, Runx1, Runx2, and Runx3 (1, 2, 3, 4, 5), that share a common highly conserved DNA binding domain, runt. The ubiquitously expressed {beta}-subunit is encoded by one gene in mammals (CBFB) and, although lacking intrinsic DNA binding activity, increases the affinity of its {alpha}-chain partner for DNA (2, 6). The Runx1 and Runx2 genes are expressed in a restricted set of tissues and cell types (2, 7), while the Runx3 gene is more widely expressed (3, 8). Runx1 is essential at early stages of hemopoietic development and is expressed in a wide range of myeloid and lymphoid tissues (9, 10). Runx2 is essential for osteoblast differentiation and the development of hypertrophic cartilage (11, 12). However, until gene inactivation demonstrated its critical role in bone development (11, 13), the expression of this gene had been described only in T cells and non-lymphoid cells such as 3T3 and Buffalo rat liver cells (2, 7, 14).

The Runx gene family exemplifies the intimate relationship between development and neoplastic disease. Two genes from this family, RUNX1 and CBFB, are targets for a number of chromosomal translocations associated with specific human leukemias. The striking feature of these translocations is the production of novel chimeric oncoproteins in which all or part of the CBF protein is fused to a heterologous partner. However, it is possible that the oncogenic activity of RUNX is not restricted to the products of chromosomal translocations. In addition to recent reports of RUNX gene amplification in leukemia (15, 16, 17, 18) we have previously shown that enforced expression of the normal form of Runx2 promotes lymphoma development in mice (19, 20). In this current study we have explored the possibility that in addition to its oncogenic properties Runx2 may have a role in T cell development.

During T cell development precursor cells pass through a series of developmental stages before being exported to the periphery (21, 22). This process is tightly controlled through the timely expression of an array of transcription factors (21, 23). During early thymocyte development the most immature CD4 and CD8 double-negative (DN) cells can be further divided into four developmental stages using CD44 and CD25: DN1 (CD44+CD25-), DN2 (CD44+CD25+), DN3 (CD44-CD25+), and finally DN4 (CD44-CD25-). An essential developmental checkpoint, known as {beta}-selection, occurs at the transition from the DN3 to the DN4 stage. At this point, {beta}-chain rearrangements occur and successfully rearranged {beta}-chain associates with the pre-TCR{alpha} (pT{alpha}) and CD3 {gamma}-, {delta}-, {epsilon}-, and {zeta}-chains to form the pre-TCR complex. Signaling from the complex leads to survival, differentiation from DN3 to DN4, proliferation, and allelic exclusion (24). The absence of signaling due to the lack of functionally rearranged {beta}-chain leads to cell death and a developmental block at the DN3 stage.

We have generated transgenic mice that express the full-length Runx2 gene under control of the CD2 promoter (19). These mice develop T cell lymphoma preceded by a preneoplastic expansion of CD8 single-positive (SP) thymocytes (19, 20). In this report we have investigated this phenotype and show that Runx2 perturbs thymocyte differentiation at a stage coincidental with {beta}-selection. Combined with our observation that Runx2 expression is normally restricted to the most immature thymocytes and a subset of CD8 SP cells these data suggest a role for Runx2 in the regulation of early T cell development.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Transgenic animals

The CD2-Runx2 transgenic mice were generated as described previously (19). Genotypes of mice were identified by Southern blot hybridization analysis on DNA extracted after tail biopsy. The transgene sequence was detected using a 800-bp til-1E PCR fragment (25). Runx2-/-, TCR{alpha}-/-, MHC class I (MHC I)-/-, p53-/-, Runx1+/- mice have been previously described (11, 26, 27, 28, 29). Genotypes were identified by Southern blotting using a specific probe (30) or by PCR analysis (31) (http://lena.jax.org/resources/documents/imr/protocols/B2 m_KO.html). Probes were radiolabeled by random priming using [{alpha}-32P]dCTP (3000 Ci/mmol; Amersham Pharmacia Biotech, Arlington Heights, IL) to sp. act. 5 x 108 cpm/µg.

Immunohistochemistry and immunocytochemistry

Thymi were fixed overnight in 10% neutral buffered formalin. Sections were pressure-cooked for 1.5 min in 0.01 M sodium citrate, pH 6.0. Following washing in Tris-HCl, pH 7.5, sections were treated for 30 min with 1.5% hydrogen peroxide in methanol to block endogenous peroxidases, followed by blocking with 1.5% swine serum. The primary Ab anti-Runx2 (25) or anti-Runx1 (raised in rabbits using the last 68 C-terminal amino acid residues) was applied overnight at a dilution of 1/10,000. Following washes the secondary Ab (swine anti-rabbit IgG; 1/200 dilution) was added for 30 min. Staining was revealed using StreptABComplexHRP kit (Dako, Copenhagen, Denmark) and diaminobenzidine tablets (Sigma) according to the manufacturer’s instructions. Sections were counterstained with hematoxylin. Alternatively staining was revealed using nickel enhancing solution (63 mM (NH4)2Ni(SO4)2, 1.3 mM diaminobenzidine, 11 mM glucose, 7.5 mM NH4Cl, and 1.700 U glucose oxidase in 0.1 M acetate buffer, pH 6). The time of incubation was controlled microscopically. Samples were counterstained with 0.1% safranin. Thymocytes were sorted according to their cell surface expression of CD4 and CD8 (see below) directly onto poly-L-lysine-coated slides. Following evaporation of the buffer, cells were fixed in acetone for 5 min. Cells were air-dried, washed in water, and treated for 30 min with 1.5% hydrogen peroxide in methanol, followed by blocking with 1.5% swine serum. Anti-Runx2 Ab was applied for 1 h (1/100 dilution). After the washes the secondary Ab (swine anti-rabbit IgG; 1/200 dilution) was added for 30 min. Staining was revealed as described above.

Flow cytometric analysis of isolated thymocytes

Flow cytometry of thymic cells was performed as previously described (19). The following Abs were used: PE-conjugated anti-CD4 (Serotec, Oxford, U.K.), FITC-conjugated anti-CD8 (Serotec), CyChrome-conjugated anti-CD4 (BD Biosciences, Mountain View, CA), CyChrome-conjugated anti-CD8 (BD Biosciences), PE-conjugated anti-CD25 (Serotec), FITC-conjugated anti-CD44 (Serotec), PE-conjugated anti-TCR{alpha}{beta} (BD Biosciences), biotinylated anti-heat-stable Ag (HSA; BD Biosciences), and allophycocyanin-conjugated avidin (BD Biosciences). When required, the thymocyte population was depleted of CD4-expressing cells using magnetic beads (anti-mouse CD4; catalog no. 114.05; Dynabeads; Dynal, Oslo, Norway) according to the manufacturer’s instructions. Usually 1–2 x 107 cells were subjected to CD4 depletion. The purity of the resulting population was assessed by CD4 and CD8 analysis. To measure cell proliferation, a CD8 immature SP (ISP; CD3-CD4-CD8+) population was isolated by negative depletion of CD4+ cells from control and CD2-Runx2 cells on a TCR{alpha}-/- background. Cells were labeled with FITC-anti-CD8 and were fixed overnight in 70% ethanol. Following washing, cells were resuspended in sample buffer containing 50 µg/ml propidium iodide and 100 U/ml RNase and were analyzed at least 1 h later on a Coulter EPICS Elite (Hialeah, FL).

Anti-CD3 depletion of the thymocyte population

Six-week-old mice were injected i.p. with 200 µl PBS containing 50 µg anti-CD3 Ab (2C11) or isotype control. The mice were culled 2 days following injection, and the thymocytes were isolated. Cells were labeled with CyChrome-anti-CD4 and FITC-anti-CD8 before analysis by flow cytometry .

Measure of cell proliferation by [3H]thymidine incorporation

Thymocytes and splenocytes were isolated as previously described (19). Cells (2–5 x 104 cells/200 µl in 96-well plate) were cultured in RPMI medium containing 10% FCS, 2 mM glutamine, 50 µM 2-ME, and penicillin/streptomycin in the presence of Con A (2 µg/ml), IL-2 (1 ng/ml) or PMA (50 ng/ml), ionomycin (1 µM), and IL-2. When required, TGF-{beta} (1ng/ml) was added. After 48-h incubation [3H]thymidine (0.1 µCi) was added, and the culture was incubated for an additional 18 h. Cells were harvested on a filter plate (UnifilterTM-96; Packard Instrument, Downers Grove, IL), and the amount of radioactivity was measured using a microplate scintillation counter (TopCount; Packard Instrument).

Statistical methods

All data are expressed as the mean ± SD. Data were analyzed for significance using Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Runx2 is expressed in a small subset of thymocytes

We have previously reported that the Runx2 gene is implicated in T cell lymphoma as a target for retroviral insertional mutagenesis (25) and that enforced expression of Runx2 skews thymocyte populations (19). We therefore hypothesized that in addition to being a major bone regulatory gene, Runx2 may have a role in T cell development or homeostasis. Although Runx2 has previously been reported to be expressed in the murine thymus (7), we investigated this further by examining expression at the level of the single cell using an immunocytochemical approach. As shown in Fig. 1Goa, Runx2 is expressed in a small number of cells in the thymus. This is in contrast to the pattern obtained with the Runx1 Ab, suggesting that this family member is much more widely expressed throughout the thymocyte population, albeit at a lower level (Fig. 1Gob and data not shown). The Runx2 Ab shows a high degree of specificity, as thymus sections derived from Runx2-/- mice show no positive cells (Fig. 1Goc). This expression pattern appears to parallel that reported by Satake et al. (7) using in situ hybridization. However, while they reported weak staining in neonates that became prominent in 4-wk-old mice, our staining is strong in a minority of cells and does not increase significantly with age. Sections derived from TCR{alpha} -/- mice revealed that Runx2 could be expressed at a relatively early stage of thymocyte development, i.e., before selection events have taken place (Fig. 1God). Analysis of fetal thymi also revealed Runx2-positive cells in 16.5 days postcoitum (p.c.) fetal sections where, despite the presence of an emerging double-positive (DP) population, the majority of thymocytes are still largely DN cells (Fig. 1Goe). At this stage of development the proportion of Runx2-expressing cells appears considerably greater than that found in the adult thymus (Fig. 1Goa), although Runx1-staining cells are still in the majority (Fig. 1Gof).



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  		FIGURE 1. Runx 2 is expressed in a small number of cells in the thymus consistent with the DN subset. Immunohistochemical analysis of Runx2 expression was carried out on thymus sections using an anti-Runx2 Ab (a, c, d, e, and g–j) or anti-Runx1 (b and f). Thymi were obtained from the following mice: a and b, 6-wk-old control mouse; c, day 18.5 p.c. Runx2-/- mouse; d, young adult TCR{alpha}-/- mouse; and e and f, day 16.5 p.c. control mouse. Thymocytes from control mice were labeled with CyChrome anti-CD4 and FITC anti-CD8 and sorted according to their expression of CD4 and CD8 (g–j). Each cellular subset was then subjected to immunocytochemical staining with anti-Runx2 Ab. Inset, no primary Ab control. Bar = 20 µm.

 
These data suggested that Runx2 could be expressed at the earliest stages of thymocyte development, but it was not possible to definitely ascribe expression to a discrete subset. To clarify this issue, we analyzed Runx2 expression on sorted cell populations. Four cell subsets were isolated according to CD4 and CD8 cell surface expression (DN, double-positive (DP), CD4 SP, and CD8 SP) and were analyzed for Runx2 expression. Runx2 was strongly positive in the DN population, but was virtually absent in the DP and CD4 SP subsets, which showed a similar level of staining as control cells (Fig. 1Go, g–i). Some positive staining was observed in the CD8 SP population, but at a lower level than seen in DN cells (Fig. 1Goj). These results indicate that Runx2 is expressed in a small subset in the thymus and in a developmentally restricted fashion.

Enforced expression of Runx2 results in an expanded CD8 ISP population

We have generated transgenic mice that express the full-length Runx2 gene under control of the CD2 promoter (19). Previously, we reported that these tumor-prone mice display an expanded population of preneoplastic CD8 SP thymocytes and that this enlargement is mirrored in the tumor phenotype (19, 20). These results are confirmed here, and representative data are shown in Fig. 2Go. Overall CD2-Runx2 transgenic animals display a 3-fold increase in the CD8 SP population (16.2 ± 4.8%) compared with littermate controls (5.0 ± 2.0%). The difference between the two groups is highly significant (p < 0.001). The thymocyte phenotype is dose dependent, as mice homozygous for the CD2-Runx2 transgene display an enhanced CD8 skew (48.7 ± 8.7%), which is significantly greater (p < 0.001) than that observed in CD2-Runx2 heterozygote mice. It is unlikely that the observed phenotype arises as a result of integration site effects, as a similar CD8 skew is observed in an independently derived transgenic line (data not shown).



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  		FIGURE 2. The enlarged CD8 SP population in CD2-Runx2 occurs before selection in a population corresponding to the CD8 ISP thymocyte subset. a, Thymocytes from mice carrying the heterozygote CD2-Runx2 transgene (right column) or their littermate controls (left column) were analyzed for CD4 and CD8 expression on various backgrounds: wild type (top), TCR{alpha} -/- (middle), or MHC 1-/- (bottom). b, Thymocytes were depleted of CD4-expressing cells as described in Materials and Methods. CD4+ cell-depleted populations from littermate control (left) or CD2-Runx2 (right) mice were gated on CD8+ cells and analyzed for HSA and TCR expression.

 
Expansion of the CD8 population could result from perturbed development at two discrete stages of thymocyte differentiation: enlargement of the CD8 SP mature thymocytes or an increase in the CD8 immature SP population, a transient step that lies between the DN and DP stages (32). To distinguish between these two possibilities we made use of TCR{alpha}-/- mice, which are unable to generate mature CD4 SP and CD8 SP cells. Fig. 2Goa shows that despite the absence of positive selection in TCR{alpha}-/- mice, a sizeable population of CD8 SP thymocytes is observed in CD2-Runx2/TCR{alpha} -/- animals (20.1 ± 5.6%). This result suggests that the enlarged pool of CD8 SP cells can arise independently of TCR selection events. This finding was confirmed by crossing CD2-Runx2 mice onto {beta}2-microglobulin-null (MHC I-/-) animals. Again, the absence of functional MHC class I and the inability to select for mature CD8+ SP thymocytes did not appreciably diminish the number of CD8 SP cells (18.9 ± 5.5%) in the thymi of CD2-Runx2 animals.

These data suggest that the observed CD8 skew is due at least in part to an increase in the CD8 ISP population. However, a possible alternative explanation would be that Runx2 could allow cells to bypass the need for positive selection in the CD8 lineage. To distinguish between these possibilities we assessed maturity of the expanded CD8 SP population using Abs to HSA and TCR. Following depletion of CD4-positive cells, these markers can discriminate between the two populations, as CD8 ISP cells are HSA+TCR-, while the CD8 SP cells are HSA-TCR+. As shown in Fig. 2Gob the proportion of CD8 ISP cells is significantly increased in CD2-Runx2 mice compared with wild-type mice. Taken together these data show that Runx2 perturbs early T cell development, resulting in an apparent expansion of the transient CD8 ISP stage.

The presence of a consensus Runx binding site within the CD4 silencer element (33) suggested that the phenotypic changes could be due to delayed up-regulation of CD4 rather than a block in differentiation. To explore this possibility, we looked at independent expression of CD4 or CD8 in normal and CD2-Runx2 homozygous thymocytes. A reduced number of CD2-Runx2 thymocytes expressed CD4 compared with controls, consistent with the possibility of CD4 silencing. However, the analysis of CD8 expression showed that the number of cells expressing CD8 and the general intensity of expression were both increased in transgenic thymi (data not shown). It would be difficult to reconcile this finding simply with CD4 silencing, and while it does not exclude the existence of a silencing effect, it suggests a more profound developmental defect.

Runx2 perturbed development coincides with pre-TCR selection

Having established that perturbed development occurs around the time of pre-TCR selection, at or just after the late DN stage (DN4), we examined early stages of thymocyte development in greater detail. DN thymocytes can be divided into four stages depending on CD44 and CD25 staining. Analysis of the DN population revealed that CD2-Runx2 mice have a significant increase (p < 0.02) in DN4 (CD44-CD25-) cells. The effect is even more dramatic in CD2-Runx2 homozygote mice, which also show a significant reduction (p < 0.05) of the DN3 (CD44-CD25+) cell subset (Table IGo). Therefore, in addition to causing an accumulation of CD8 ISP cells Runx2 affects the transition from DN3 to DN4. It is possible that the reduction in DN3 cells and the expansion of DN4 cells reflect accelerated development through this checkpoint.


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  		Table I. DN subset distribution is perturbed in CD2-Runx2 micea

 
Thymocyte development is initiated during embryogenesis, and at the time of birth DN and DP cells are already present within the thymus. Shortly after birth, selection occurs, and CD4 SP and CD8 SP cells appear in the thymus, where equilibrium is established among entry of naive cells, apoptosis due to neglect or negative selection, and exit of mature cells. To circumvent the problems associated with examining a dynamic postnatal system, we investigated thymocyte ontogeny in developing CD2-Runx2 fetuses. In control mice the predominant population (DN) starts differentiating on day 16.5 with the appearance of DP cells (Fig. 3Goa and Table IIGo). On days 17.5 and 18.5 the proportion of DP cells increases to become the major population before birth.



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  		FIGURE 3. Thymocyte maturation is delayed in CD2-Runx2 fetal mice. Thymi from fetuses at various ages were harvested, and cells were analyzed for CD4 and CD8 expression. Results are expressed graphically either in combination (large profile; middle) or as a single histogram (CD4 on the left; CD8 on the right). a, control mice; b, CD2-Runx2 homozygote mice. The position of the DP population in the adult mouse is indicated (ellipse) as well as that of CD4+ and CD8+ cells in the relevant histogram (line).

 

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  		Table II. Cell subset distribution in fetal thymia

 
CD2-Runx2 transgenic fetuses show marked differences in thymocyte ontogeny, such that DP development is significantly delayed. In CD2-Runx2 fetuses, the DN cells represent the major subset from days 14.5–17.5 (Fig. 3Gob and Table IIGo). The onset of CD8 expression is slightly delayed compared with that in controls (day 17.5 in CD2-Runx2 fetuses compared with day 16.5 in control mice; percentages of CD8-expressing cells in control and CD2-Runx2 mice on day 17.5, 60.0 ± 10.9 and 36.2 ± 0.3%, respectively), but still reaches maximum expression on day 18.5 (64.7 ± 0.4%). Another major difference between control and transgenic fetal thymi is the sustained presence of an enlarged CD8 ISP population in the latter. For example, by day 17.5 about one-third of the CD2-Runx2 cells are CD4-CD8+. Even on day 18.5 the major population remains the CD8 ISP cell, with only a minor population of the cells being DP (Table IIGo). These results indicate that early thymocyte ontogeny is markedly perturbed in CD2-Runx2 mice. In summary, this aberrant development is characterized by a general delay in transition from the DN stage, followed by a block at the intermediate CD8 ISP stage, resulting in abnormal accumulation of these cells. It is of interest that a CD2-driven transgene can exert such a profound effect at early stages of thymocyte ontogeny, but these results are consistent with transgene expression, which can be detected by immunocytochemistry as early as day 15.5 (data not shown). An additional and rather striking observation was the marked reduction in thymus cellularity. As shown in Fig. 3Gob, the number of cells analyzed is very low due to the difficulty of obtaining enough cells from CD2-Runx2 thymi. For example, on day 14.5 the number of cells recovered in control and CD2-Runx2 fetal thymi were, respectively, 5 x 104 ± 0.5 cells and 1.5 x 104 ± 0.1 cells/thymus (p < 0.02).

The presence of an enlarged cell subset could be due to accelerated development to that stage, a partial block in development to the subsequent stage, or a change in the proliferative status of that particular subpopulation. We therefore examined the cell cycle characteristics of the CD8 ISP subset of control and CD2-Runx2 transgenic mice. To analyze the CD8 ISP population we again made use of TCR{alpha}-/- mice. The thymocyte population was depleted of CD4+ cells, and the DNA content of CD8+ cells was assayed. In control mice the CD8 ISP is an actively dividing population, as shown by the high percentage of cells in S/G2/M phase (Table IIIGo). The relatively high proliferative index for this population has been reported previously (32, 34). Surprisingly, the proliferative index of the enlarged CD8 ISP population is sharply decreased in CD2-Runx2 mice (Table IIIGo). This result, which suggests that these cells may harbor a defect in proliferation, may help to explain the paucity of thymocytes in the developing fetus. Alternatively, the low cell numbers found in developing thymi might be due to CD2-Runx2 thymocytes being more sensitive to proapoptotic stimuli.


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  		Table III. Proliferation is reduced in CD2-Runx2 thymocytesa

 
Despite the apparently reduced capacity for cell division and/or survival, CD2-Runx2-expressing thymocytes are predisposed to transformation. Therefore, it is possible that before neoplastic conversion can occur additional events must counteract these aspects of the Runx2 phenotype. In this regard we (35) have reported that CD2-Runx2 mice null for trp53 rapidly develop T cell lymphomas at a relatively young age. It is possible that the acellularity observed during fetal development may be dependent on functional trp53. To test this we examined the phenotype of trp53-null CD2-Runx2 thymocytes from adult animals and 16.5 days p.c. fetuses. The absence of trp53 did not appreciably alter the phenotype in either fetal or mature thymi, indicating that these changes are not trp53 dependent (Fig. 4Go).



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  		FIGURE 4. The absence of trp53 does not alter the CD2-Runx2 phenotype. Flow cytometric analysis was performed for CD4 and CD8 surface expression of thymocytes from adult (top) or fetal (bottom) mice of various genotypes as indicated (heterozygote CD2-Runx2). Cells were isolated and labeled with CyChrome-anti-CD4 and FITC-anti-CD8 Abs before analysis.

 
Runx2 can affect thymocyte survival and proliferation

To further investigate the effect of Runx2 on cell death we treated control and CD2-Runx2 mice with anti-CD3 Ab. Such injection leads to massive depletion of the DP compartment in the thymus and is thought to mimic negative selection. Analysis following anti-CD3 treatment revealed that the residual population of DP cells was significantly greater in CD2-Runx2 transgenic mice than in littermate controls (p < 0.01). This result suggests that a small subpopulation of cells in this compartment may be capable of resisting the apoptotic effect of anti-CD3 Ab. Although a similar finding has been reported for the related Runx1 product in hybridoma cells (36), this is the first demonstration of protection in vivo.

We also investigated the basis for the reduced proliferative capacity of CD2-Runx2 thymocytes. To this end we explored the effect of TGF-{beta}, as previous reports have pointed to a role for TGF-{beta} in T cell proliferation (37), CD8 induction (38), and progression from DN to CD4-CD8low cells (34). Moreover, Runx gene products have been associated with Smads (39, 40, 41), and we have previously reported that some biological effects of TGF-{beta} are accentuated in CD2-Runx2 thymocytes (19). We therefore analyzed the effect of TGF-{beta} on the proliferation of CD2-Runx2 thymocytes following mitogenic stimuli. Although TGF-{beta} reduces the proliferation of control cells following mitogen treatment, the anti-proliferative effects of TGF-{beta} were significantly (p < 0.01) augmented in CD2-Runx2 thymocytes, although no significant change was observed in splenocytes (Fig. 5Go). These data indicate that CD2-Runx2 cells have a greatly heightened sensitivity to the effects of TGF-{beta} and suggest that signaling via this pathway may be amplified in cells overexpressing Runx2.



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  		FIGURE 5. Runx2 increases the sensitivity of lymphoid cells to TGF-{beta}. Thymocytes and splenocytes were cultured in the presence of the indicated mitogens (Con, Con A; iono, ionomycin). Proliferation was measured by the incorporation of [3H]thymidine over 18 h. Data are the results of four experiments (six replicates each) and are presented as a percentage of the value in untreated (no TGF-{beta} added) controls.

 
The phenotype of CD2-Runx2 mice is not due to antagonism of Runx1 function

Potentially, enforced expression of Runx2 could inhibit the action of other family members due to competition for DNA binding sites and transcriptional cofactors. Previous reports have indicated that Runx1 has the greatest binding activity in the thymus, and we postulated that it was possible that the phenotype observed in our transgenic models may be due at least in part to diminished Runx1 function. To address this we made use of mice heterozygous for Runx1. The reduced gene dosage in these animals has previously been shown to affect the maturation of mature CD4 and CD8 SP thymocytes (42). Although the two phenotypes appear quite distinct, we investigated the effect of reduced Runx1 function in CD2-Runx2 animals. We hypothesized that if Runx2 was antagonizing the function of Runx1, then a haploinsufficient reduction of Runx1 would result in an accentuated phenotype, similar to that observed in CD2-Runx2 homozygous animals. However, as shown in Table IVGo, the phenotype of CD2-Runx2 transgenics is neither exaggerated nor ameliorated when Runx1 is diminished. This result suggests that Runx2 is exerting its effects independently of Runx1 in these animals.


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  		Table IV. CD2-Runx2 phenotype is independent of Runx1 reduced dosagea

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Although knockout studies have aligned Runx function with specific lineages, these genes are expressed widely, and it is likely that multiple functions will emerge for this family of transcription factors. In addition to its role in bone formation, Runx2 can be expressed in a range of tissues, including those of lymphoid origin (7, 14). Here we demonstrate that Runx2 protein is restricted to a specific thymocyte subpopulation. Detailed analysis revealed that Runx2 is expressed principally in the DN population and is absent from the DP and CD4 SP populations. This suggests that the expression of Runx2 is developmentally regulated and points to a role for this gene during early thymocyte development. This hypothesis is strongly supported by the phenotype observed in CD2-Runx2 transgenic mice, which manifests itself primarily as disturbance of early differentiation. Although Runx1 appears to be pre-eminent in terms of expression and activity (7, 14), it is possible that the other Runx genes could be involved in distinct phases of thymocyte development and function (43). Certainly Runx1 and Runx2 do not appear to have completely redundant functions in this tissue compartment. The phenotype of transgenic mice overexpressing Runx1 in the T cell compartment involves a defect in postselection maturation (44) and is therefore quite distinct from the effects of Runx2 described here. Furthermore, it does not appear that Runx2 is mediating its biological effects by simply antagonizing Runx1 function, as reducing Runx1 gene dosage in CD2-Runx2 transgenics does not amplify the phenotype.

We initially identified the phenotypical defect in CD2-Runx2 transgenic mice as an enlargement of the CD8 SP subset (19). However, in this report we demonstrate that enforced expression of Runx2 in fetal thymi induces a marked delay in early thymocyte differentiation at a developmental stage coincident with {beta}-selection (45). The most obvious manifestation of this phenotype is the presence of a greatly expanded DN4/CD8 ISP population that persists into adulthood. This observation combined with a reduction in the DN3 population in CD2-Runx2 homozygous mice have led us to suggest that Runx2 expression may augment development from the DN3 stage to the DN4/CD8 ISP stage, but retard further differentiation to the DP stage.

Successful expression of a functional TCR {beta}-chain and subsequent assembly of the pre-TCR result in a number of distinct biological events collectively known as {beta} selection. These include termination of TCR{beta} locus rearrangements and survival of selected thymocytes with resultant proliferation and differentiation to the DP stage. The pre-TCR complex is required for the transition from the DN3 to the DN4 stage, and when essential components of the pre-TCR complex are absent, thymocyte differentiation is blocked at the DN3 stage. The level of pT{alpha} may be rate limiting at this point in development, as DN3 to DN4 differentiation is potentiated, but subsequent maturation to DP is impaired in transgenic models constitutively expressing pT{alpha} (46). This finding is similar to the alterations reported in this manuscript. However, in common with a number of other transgenic and knockout models that manifest an expanded CD8 ISP population (47, 48, 49, 50, 51, 52, 53, 54), the Runx2 phenotype points to a two-step transition following successful pre-TCR signaling. It appears therefore that Runx2 can permit and even augment some of the outcomes associated with {beta} selection, but inhibit later stages of the development. In this respect, Runx2 appears to mimic the phenotypic changes seen following treatment of immature thymocytes with Ab to the TCR {beta}-chain. Such treatment accentuates progression from the DN to the CD8 ISP stage, but retards onward development to the DP stage, perhaps because sustained signaling through the pre-TCR inhibits subsequent differentiation (55, 56). It may be that the inability to extinguish Runx2 expression in our transgenic model results in this partial block in development.

Although the CD8 ISP population is greatly expanded in CD2-Runx2 mice, in sharp contrast to their wild-type counterparts these cells are largely noncycling. Therefore, the wave of proliferation normally associated with {beta} selection appears to be reduced in CD2-Runx2 transgenics. Whether this is due to an effect on pre-TCR signaling or reflects a more direct effect on the cell cycle is not known at present. However, the general acellularity of CD2-Runx2 fetal thymi and the increased sensitivity to the anti-proliferative effects of TGF-{beta} suggest that fundamental aspects of the growth regulatory program are being engaged. It is possible that the developmental block observed here is secondary to a defect in proliferation, as there is evidence that this is an essential part of the differentiation process (34, 57).

Basic helix-loop-helix transcription factors, such as E2A and HeLa E-box binding protein (HEB), are important in the regulation of early thymocyte differentiation and control of pT{alpha} expression. The phenotype of mice with reduced E2A-HEB function has interesting parallels with that observed in CD2-Runx2 animals. Loss of HEB or overexpression of genes that negatively regulate HEB-E2A function, such as stem cell leukemia and LIM-only protein 1, is characterized by arrested development at the CD8 ISP stage. These phenotypic changes might be mediated at least in part by decreased pre-TCR function, as there is evidence that HEB-E2A positively regulates pT{alpha} expression. It has been proposed that E2A-HEB can act as a tumor suppressor, because mice null for E2A subsequently develop thymic lymphoma, as do stem cell leukemia/LIM-only protein 1 transgenic mice (58, 59). Therefore, not only do these models have a similar preneoplastic phenotype to that in Runx2 animals, but they also share a tendency to develop lymphoid tumors. The relationship between perturbed development and the oncogenic process has yet to be established, and it remains to be seen whether a common mechanism unifies these different models. However, whatever the basis of their common pathogenic manifestations, a number of diverse studies have now implicated aberrant pre-TCR function in T cell neoplasia (59, 60). Determining whether this is the direct result of distorted pre-TCR signaling or is secondary to a differentiation block in a population of cells exquisitely sensitive to transformation events remains a goal of future studies.


   Acknowledgments
 
We thank Mike Owen for his generous gift of TCR{alpha}-/- mice and thymi from Runx2-/- embryos. The Runx1-/- mice were a generous gift from Nancy Speck and were kindly supplied by Andrew Thomson. We thank Margaret Bell and Alma Jenkins for their expert technical assistance.


   Footnotes
 
1 This work was supported by the Leukemia Research Fund and Cancer Research U.K. Back

2 Address correspondence and reprint requests to Dr. François Vaillant, Molecular Oncology Laboratory, Institute of Comparative Medicine, University of Glasgow Veterinary School, Glasgow, U.K. G61 1QH. E-mail address: f.vaillant{at}vet.gla.ac.uk Back

3 Abbreviations used in this paper: CBF, core binding factor; DN, double negative; DP, double positive; HEB, HeLa E-box binding protein; HSA, heat-stable Ag; ISP, immature single positive; p.c., postcoitum; pT{alpha}, pre-TCR {alpha}-chain; SP, single positive; MHC I, MHC class I. Back

Received for publication April 11, 2002. Accepted for publication July 5, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Miyoshi, H., T. Kozu, K. Shimizu, N. Maseki, Y. Kaneko, M. Ohki. 1991. t(8;21) breakpoints on chromosome 21 in acute myeloid leukemia are clustered within a limited region of a single gene, AML1. Proc. Natl. Acad. Sci. USA 88:10431.[Abstract/Free Full Text]
  2. Ogawa, E., M. Inuzuka, M. Maruyama, M. Satake, M. Naito-Fujimoto, Y. Ito, K. Shigesada. 1993. Molecular cloning and characterization of PEBP2{beta}, the heterodimeric partner of a novel Drosophila runt-related DNA binding protein PEBP2{alpha}. Virology 194:314.[Medline]
  3. Levanon, D., V. Negreanu, Y. Bernstein, I. Bar-Am, L. Avivi, Y. Groner. 1994. AML1, AML2, and AML3, the human members of the runt domain gene-family: cDNA structure, expression, and chromosomal localization. Genomics 23:425.[Medline]
  4. Bae, S. C., E. Takahashi, Y. W. Zhang, E. Ogawa, K. Shigesada, Y. Namba, M. Satake, Y. Ito. 1995. Cloning, mapping and expression of PEBP2{alpha}C, a third gene encoding the mammalian Runt domain. Gene 159:245.[Medline]
  5. Bae, S. C., Y. Yamaguchi-Iwai, E. Ogawa, M. Maruyama, M. Inuzuka, H. Kagoshima, K. Shigesada, M. Satake, Y. Ito. 1993. Isolation of PEBP2{alpha}B cDNA representing the mouse homolog of human acute myeloid leukemia gene, AML1. Oncogene 8:809.[Medline]
  6. Wang, S., Q. Wang, B. E. Crute, I. N. Melnikova, S. R. Keller, N. A. Speck. 1993. Cloning and characterization of subunits of the T-cell receptor and murine leukemia virus enhancer core-binding factor. Mol. Cell. Biol. 13:3324.[Abstract/Free Full Text]
  7. Satake, M., S. Nomura, Y. Yamaguchi-Iwai, Y. Takahama, Y. Hashimoto, M. Niki, Y. Kitamura, Y. Ito. 1995. Expression of the Runt domain-encoding PEBP2{alpha} genes in T cells during thymic development. Mol. Cell. Biol. 15:1662.[Abstract]
  8. Levanon, D., O. Brenner, V. Negreanu, D. Bettoun, E. Woolf, R. Eilam, J. Lotem, U. Gat, F. Otto, N. Speck, et al 2001. Spatial and temporal expression pattern of Runx3 (Aml2) and Runx1 (Aml1) indicates non-redundant functions during mouse embryogenesis. Mech. Dev. 109:413.[Medline]
  9. Okuda, T., J. van Deursen, S. W. Hiebert, G. Grosveld, J. R. Downing. 1996. AML1, the target of multiple chromosomal translocations in human leukemia, is essential for normal fetal liver hematopoiesis. Cell 84:321.[Medline]
  10. Speck, N. A., T. Stacy, Q. Wang, T. North, T. L. Gu, J. Miller, M. Binder, and M. Marin-Padilla. 1999. Core-binding factor: a central player in hematopoiesis and leukemia. Cancer Res. 59:1789.s.
  11. Otto, F., A. P. Thornell, T. Crompton, A. Denzel, K. C. Gilmour, I. R. Rosewell, G. W. Stamp, R. S. Beddington, S. Mundlos, B. R. Olsen, et al 1997. Cbfa1, a candidate gene for cleidocranial dysplasia syndrome, is essential for osteoblast differentiation and bone development. Cell 89:765.[Medline]
  12. Kim, I. S, F. Otto, B. U. Zabel, S. Mundlos. 1999. Regulation of chondrocyte differentiation by Cbfa1. Mech. Dev. 80:159.[Medline]
  13. Komori, T., H. Yagi, S. Nomura, A. Yamaguchi, K. Sasaki, K. Deguchi, Y. Shimizu, R. T. Bronson, Y. H. Gao, M. Inada, et al 1997. Targeted disruption of Cbfa1 results in a complete lack of bone formation owing to maturational arrest of osteoblasts. Cell 89:755.[Medline]
  14. Meyers, S., N. Lenny, W. Sun, S. W. Hiebert. 1996. AML-2 is a potential target for transcriptional regulation by the t(8;21) and t(12;21) fusion proteins in acute leukemia. Oncogene 13:303.[Medline]
  15. Busson-Le Coniat, M., F. Khac, M. T. Nguyen, O. A. Daniel, O. A. Bernard, R. Berger. 2001. Chromosome 21 abnormalities with AML1 amplification in acute lymphoblastic leukemia. Genes Chromosomes Cancer 2001 32:244.
  16. Mathew, S., P. H. Rao, J. Dalton, J. R. Downing, S. C. Raimondi. 2001. Multicolor spectral karyotyping identifies novel translocations in childhood acute lymphoblastic leukemia. Leukemia 15:468.[Medline]
  17. Streubel, B., P. Valent, K. Lechner, C. Fonatsch. 2001. Amplification of the AML1(CBFA2) gene on ring chromosomes in a patient with acute myeloid leukemia and a constitutional ring chromosome 21. Cancer Genet. Cytogenet. 124:42.[Medline]
  18. Niini, T., J. Kanerva, K. Vettenranta, U. M. Saarinen-Pihkala, S. Knuutila. 2000. AML1 gene amplification: a novel finding in childhood acute lymphoblastic leukemia. Haematologica 85:362.[Abstract/Free Full Text]
  19. Vaillant, F., K. Blyth, A. Terry, M. Bell, E. R. Cameron, J. Neil, M. Stewart. 1999. A full-length Cbfa1 gene product perturbs T-cell development and promotes lymphomagenesis in synergy with myc. Oncogene 18:7124.[Medline]
  20. Blyth, K., A. Terry, F. N Mackay, M. Vaillant, E. R. Bell, J. C. Neil Cameron, M. Stewart. 2000. Cbfa1/Runx2: a novel oncogenic pathway revealed by in vivo complementation and retroviral tagging. Oncogene 20:295.
  21. Farrar, M. A., P. Doerfler, K. Sauer. 1998. Signal transduction pathways regulating the development of {alpha}{beta} T cells. Biochim. Biophys. Acta 1377:F35.[Medline]
  22. Fehling, H. J., S. Gilfillan, R. Ceredig. 1999. {alpha}{beta}/{gamma}{delta} lineage commitment in the thymus of normal and genetically manipulated mice. Adv. Immunol. 71:1.[Medline]
  23. Kuo, C. T., J. M. Leiden. 1999. Transcriptional regulation of T lymphocyte development and function. Annu. Rev. Immunol. 17:149.[Medline]
  24. Kruisbeek, A. M., M. C. Haks, M. Carleton, A. M. Michie, J. C. Zuniga-Pflucker, D. L. Wiest. 2000. Branching out to gain control: how the pre-TCR is linked to multiple functions. Immunol. Today 21:637.[Medline]
  25. Stewart, M., A. Terry, M. Hu, M. O’Hara, K. Blyth, E. Baxter, E. Cameron, D. E. Onions, J. C. Neil. 1997. Proviral insertions induce the expression of bone-specific isoforms of PEBP2{alpha}A (CBFA1): evidence for a new myc collaborating oncogene. Proc. Natl. Acad. Sci. USA 94:8646.[Abstract/Free Full Text]
  26. Philpott, K. L., J. L. Viney, G. Kay, S. Rastan, E. M. Gardiner, S. Chae, A. C. Hayday, M. J. Owen. 1992. Lymphoid development in mice congenitally lacking T-cell receptor {alpha}/{beta}-expressing cells. Science 256:1448.[Abstract/Free Full Text]
  27. Zijlstra, M., M. Bix, N. E. Simister, J. M. Loring, D. H. Raulet, R. Jaenisch. 1990. {beta}2-microglobulin deficient mice lack CD4-8+ cytolytic T cells. Nature 344:742.[Medline]
  28. Donehower, L. A., M. Harvey, B. L. Slagle, M. J. McArthur, C. A. Montgomery, J. S. Butel, A. Bradley. 1992. Mice deficient for p53 are developmentally normal but susceptible to spontaneous tumours. Nature 356:215.[Medline]
  29. Wang, Q., T. Stacy, M. Binder, M. Marin-Padilla, A. H. Sharpe, N. A. Speck. 1996. Disruption of the Cbfa2 gene causes necrosis and hemorrhaging in the central nervous system and blocks definitive hematopoiesis. Proc. Natl. Acad. Sci. USA 93:3444.[Abstract/Free Full Text]
  30. Baxter, E. W., K. Blyth, L. A. Donehower, E. R. Cameron, D. E. Onions, J. C. Neil. 1996. Moloney murine leukaemia virus-induced lymphomas in p53 deficient mice: overlapping pathways in tumour development. J. Virol. 70:2095.[Abstract]
  31. Picard, J. K.. A PCR strategy to distinguish heterozygous and homozygous gene. "knockout" transgenic mice. Transgenics 1:241.
  32. MacDonald, H. R., R. C. Budd, R. C. Howe. 1988. A CD3- subset of CD4-8+ thymocytes: a rapidly cycling intermediate in the generation of CD4+8+ cells. Eur. J. Immunol. 18:519.[Medline]
  33. Ellmeier, W., S. Sawada, D. R. Littman. 1999. The regulation of CD4 and CD8 coreceptor gene expression during T cell development. Annu. Rev. Immunol. 17:523.[Medline]
  34. Takahama, Y., J. J. Letterio, H. Suzuki, A. G. Farr, A. Singer. 1994. Early progression of thymocytes along the CD4/CD8 developmental pathway is regulated by a subset of thymic epithelial cells expressing transforming growth factor {beta}. J. Exp. Med. 179:1495.[Abstract/Free Full Text]
  35. Blyth, K., A. Terry, N. Mackay, F. Vaillant, M. Bell, E. R. Cameron, J. C. Neil, M. Stewart. 2001. Runx2: a novel oncogenic effector revealed by in vivo complementation and retroviral tagging. Oncogene 20:295.[Medline]
  36. Fujii, M., K. Hayashi, M. Niki, N. Chiba, K. Meguro, K. Endo, J. Kameoka, S. Ito, K. Abe, et al 1998. Overexpression of AML1 renders a T hybridoma resistant to T cell receptor-mediated apoptosis. Oncogene 17:1813.[Medline]
  37. Letterio, J. J., A. B. Roberts. 1998. Regulation of immune responses by TGF-{beta}. Annu. Rev. Immunol. 16:137.[Medline]
  38. Inge, T. H., K. M. McCoy, B. M. Susskind, S. K. Barrett, G. Zhao, H. D. Bear. 1992. Immunomodulatory effects of transforming growth factor-{beta} on T lymphocytes: induction of CD8 expression in the CTLL-2 cell line and in normal thymocytes. J. Immunol. 148:3847.[Abstract]
  39. Lee, K.-S., H. J. Kim, Q.-L. Li, X.-Z. Chi, C. Ueta, T. Komori, J. M. Wozney, E.-G. Kim, J.-Y. Choi, H. M. Ryoo, et al 2000. Runx2 is a common target of transforming growth factor {beta}1 and bone morphogenetic protein 2, and cooperation between Runx2 and Smad 5 induces osteoblast-specific gene expression in the pluripotent mesenchymal precursor cell line C2C12. Mol. Cell. Biol. 20:8783.[Abstract/Free Full Text]
  40. Zhang, Y. W., N. Yasui, K. Ito, G. Huang, M. Fujii, J. Hanai, H. Nogami, T. Ochi, K. Miyazono, Y. Ito. 2000. A RUNX2/PEBP2{alpha}A/CBFA1 mutation displaying impaired transactivation and Smad interaction in cleidocranial dysplasia. Proc. Natl. Acad. Sci. USA 97:10549.[Abstract/Free Full Text]
  41. Leboy, P., G. Grasso-Knight, M. D’Angelo, S. W. Volk, J. V. Lian, H. Drissi, G. S. Stein, S. L. Adams. 2001. Smad-Runx interactions during chondrocyte maturation. J. Bone Joint Surg. Am. 83:S15.[Abstract/Free Full Text]
  42. Hayashi, K., W. Natsume, T. Watanabe, N. Abe, N. Iwai, H. Okada, Y. Ito, M. Asano, Y. Iwakura, S. Habu, et al 2000. Diminution of the AML1 transcription factor function causes differential effects on the fates of CD4 and CD8 single-positive T cells. J. Immunol. 165:6816.[Abstract/Free Full Text]
  43. Wijmenga, C., N. A. Speck, N. C. Dracopoli, M. H. Hofker, P. Liu, F. S. Collins. 1995. Identification of a new murine runt domain-containing gene, Cbfa3, and localization of the human homolog, CBFA3, to chromosome 1p35-pter. Genomics 26:611.[Medline]
  44. Hayashi, K., N. Abe, T. Watanabe, M. Obinata, M. Ito, T. Sato, S. Habu, M. Satake. 2001. Overexpression of AML1 transcription factor drives thymocytes into the CD8 single-positive lineage. J. Immunol. 167:4957.[Abstract/Free Full Text]
  45. Ogawa, S., H. Harada, M. Fujiwara, S. Tagashira, T. Katsumata, H. Takada. 2000. Cbfa1, an essential transcription factor for bone formation, is expressed in testis from the same promoter used in bone. DNA Res. 7:181.[Abstract]
  46. Lacorazza, H. D., C. Tucek-Szabo, L. V. Vasovic, K. Remus, J. Nikolich-Zugich. 2001. Premature TCR {alpha}{beta} expression and signaling in early thymocytes impair thymocyte expansion and partially block their development. J. Immunol. 166:3184.[Abstract/Free Full Text]
  47. Diehl, N. L., H. Enslen, K. A. Fortner, C. Merritt, N. Stetson, C. Charland, R. A. Flavell, R. J. Davis, M. Rincon. 2000. Activation of the p38 mitogen-activated protein kinase pathway arrests cell cycle progression and differentiation of immature thymocytes in vivo. J. Exp. Med. 191:321.[Abstract/Free Full Text]
  48. Barndt, R., M. F. Dai, Y. Zhuang. 1999. A novel role for HEB downstream or parallel to the pre-TCR signaling pathway during {alpha}{beta} thymopoiesis. J. Immunol. 163:3331.[Abstract/Free Full Text]
  49. Bain, G., I. Engel, E. C. Maandag-Robanus, H. P. te Riele, J. R. Voland, L. L. Sharp, J. Chun, B. Huey, D. Pinkel, et al 1997. E2A deficiency leads to abnormalities in {alpha}{beta} T-cell development and to rapid development of T-cell lymphomas. Mol. Cell. Biol. 17:4782.[Abstract]
  50. Verbeek, S., D. Izon, F. Hofhuis, E. Robanus-Maandag, H. te Riele, M. van de Wetering, M. Oosterwegel, A. Wilson, H. R. MacDonald, H. Clevers. 1995. An HMG-box-containing T-cell factor required for thymocyte differentiation. Nature 374:70.[Medline]
  51. Okamura, R. M., M. Sigvardsson, J. Galceran, S. Verbeek, H. Clevers, R. Grosschedl. 1998. Redundant regulation of T cell differentiation and TCR{alpha} gene expression by the transcription factors LEF-1 and TCF-1. Immunity 8:11.[Medline]
  52. Herblot, S., A. Steff, P. D. Aplan P Hugo, J. Hock. 2000. SCL and LMO1 alter thymocyte differentiation: inhibition of E2A-HEB function and pre-T {alpha} chain expression. Nat. Immunol. 1:138.[Medline]
  53. Miyazaki, T.. 1997. Two distinct steps during thymocyte maturation from CD4-CD8- to CD4+CD8+ distinguished in the early growth response (Egr)-1 transgenic mice with a recombinase-activating gene-deficient background. J. Exp. Med. 186:877.[Abstract/Free Full Text]
  54. Abraham, K. M., S. D. Levin, J. D. Marth, K. A. Forbush, R. M. Perlmutter. 1991. Delayed thymocyte development induced by augmented expression of p56lck. J. Exp. Med. 173:1421.[Abstract/Free Full Text]
  55. Hunig, T.. 1988. Cross-linking of the T cell antigen receptor interferes with the generation of CD4+8+ thymocytes from their immediate CD4-8+ precursors. Eur. J. Immunol. 18:2089.[Medline]
  56. Takahama, Y., K. Sugaya, S. Tsuda, T. Hasegawa, Y. Hashimoto. 1995. Regulation of early T cell development by the engagement of TCR-{beta} complex expressed on fetal thymocytes from TCR-{beta}-transgenic scid mice. J. Immunol. 154:5862.[Abstract]
  57. Falk, I., J. Biro, H. Kohler, K. Eichmann. 1996. Proliferation kinetics associated with T cell receptor-{beta} chain selection of fetal murine thymocytes. J. Exp. Med. 184:2327.[Abstract/Free Full Text]
  58. Chervinsky, D. S., X. F. Zhao, D. H. Lam, M. Ellsworth, K. W. Gross, P. D. Aplan. 1999. Disordered T-cell development and T-cell malignancies in SCL LMO1 double-transgenic mice: parallels with E2A-deficient mice. Mol. Cell. Biol. 19:5025.[Abstract/Free Full Text]
  59. Chervinsky, D. S., D. H. Lam, M. P. Melman, K. W. Gross, P. D. Aplan. 2001. scid thymocytes with TCR{beta} gene rearrangements are targets for the oncogenic effect of SCL and LMO1 transgenes. Cancer Res. 61:6382.[Abstract/Free Full Text]
  60. Bellavia, D., A. F. Campese, E. Alesse, A. Vacca, M. P. Felli, A. Balestri, A. Stoppacciaro, C. Tiveron, L. Tatangelo, M. Giovarelli, et al 2000. Constitutive activation of NF-{kappa}B and T-cell leukemia/lymphoma in Notch3 transgenic mice. EMBO J. 19:3337.[Medline]



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