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Human Molecular Genetics Advance Access originally published online on November 9, 2006
Human Molecular Genetics 2006 15(24):3485-3497; doi:10.1093/hmg/ddl425
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Published by Oxford University Press

Itch genetically interacts with Notch1 in a mouse autoimmune disease model

Lydia E. Matesic1,*, Diana C. Haines2, Neal G. Copeland1 and Nancy A. Jenkins1

1 Mouse Cancer Genetics Program, Center for Cancer Research, National Cancer Institute, Frederick, MD 21702, USA and 2 Pathology/Histotechnology Laboratory, SAIC-Frederick, Frederick, MD 21702, USA

* To whom correspondence should be addressed at: Department of Biological Sciences, CLS 703, University of South Carolina, Columbia, SC 29208, USA. Tel: +1 8037772520; Fax: +1 8037774002; Email: lmatesic{at}biol.sc.edu

Received September 11, 2006; Revised October 16, 2006; Accepted October 26, 2006


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Homozygous itchy mice develop a fatal, late-onset autoimmune-like disease due to a loss of function mutation in an ubiquitin protein ligase. Phylogenetic and in vitro analyses suggest that Itch is a negative regulator of Notch signaling. Since Notch proteins have many important functions in the immune system, we determined whether Itch regulates Notch signaling in vivo. This was accomplished by breeding homozygous itch mice to mice carrying an activated Notch1 transgene that was specifically overexpressed in developing thymocytes. Interestingly, all itch mice carrying this transgene were smaller than their littermates and died by 12 weeks of age. These mice had a similar autoimmune disease to that seen in itch animals. However, the lesions were more severe with a much earlier age of onset, supporting the assertion that these mutations genetically interact. In addition, the combination of these mutations produced novel phenotypes including a perturbation in T cell development, with a reduction in the number of double-positive (DP) and an increase in the number of double-negative and single-positive T cells. TUNEL staining showed reduced apoptosis in the thymus of itch animals that carry the Notch1 transgene. Antibody staining displayed increased levels of full-length Notch1 and phospho-AKT specifically in DP thymocytes but no change in other signaling pathways including MAPK, p38 and JNK. These results provide the first direct demonstration that increased AKT-mediated Notch1 signaling results in autoimmunity and may provide insight into the treatment of a group of diseases that affect a significant proportion of the population.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Autoimmune diseases are a collection of more than 80 individual diseases that are estimated to affect upwards of 3% of the U.S. population (1). Underlying this diverse group of diseases is one common pathology: the malfunction of the immune system, resulting in the destruction of self-tissue. The etiology of autoimmune disease is thought to have both genetic and environmental contribution. A monogenic mouse model of autoimmune disease could aid in dissecting key signaling pathways common to the genesis of many types of autoimmune diseases in a controlled environmental context, but very few such models are available. One of the notable exceptions to this is the itchy mutant (itch–/–) mouse. On a C57BL/6J background, itch–/– mice develop a progressive autoimmune-like disease characterized by lymphoproliferation in the spleen, lymph nodes and medulla of the thymus, dermatitis with alopecia (especially prevalent in the head and neck region), and fatal pulmonary interstitial inflammation with alveolar proteinosis at 6 to 8 months of age (2). The molecular lesion responsible for this phenotype was determined to be a radiation-induced inversion on distal chromosome 2 that caused the loss of function of Itch, a HECT E3 ubiquitin ligase (3). In addition to the enzymatic HECT domain, Itch putatively contains a C2 domain, which is thought to mediate association with the phospholipid membrane in response to increases in levels of intracellular Ca2+ (4), and four WW domains, which have been implicated in protein–protein interactions (5).

Ubiquitination is a post-translational modification that commonly affects protein half-life, function or subcellular localization, thus playing a vital role in diverse cellular processes, such as signal transduction, regulation of transcription, DNA repair, cell cycle progression, antigen presentation and apoptosis (6). Ubiquitination of a protein is carried out by a sequential series of enzymatic reactions. First, the ubiquitin (Ub) moiety is activated in an ATP-dependent manner by an Ub-activating enzyme (E1). The activated Ub is then transferred to a Ub-conjugating enzyme (E2), which then directly or in association with a Ub-ligase (E3) mediates the transfer of the Ub to the target protein. This process can be repeated to form a polyubiquitin chain or can be terminated after the addition of a single Ub unit. Polyubiquitination of a chain of four or more Ub usually signals for degradation by the 26S proteasome, whereas monoubiquitination is most often associated with receptor internalization (7). The E3 is thought to confer substrate specificity in ubiquitination by serving as an adaptor between the Ub-protein conjugation machinery and the target molecule.

Itch targets potentially relevant to autoimmune disease have been identified. These include JunB; without proper regulation of this transcription factor, itch–/– mice develop a Th2 bias in T cell differentiation, which is hypothesized to account for the allergic response aspect of the phenotype (i.e. IgG1 and IgE antibody production as well as eosinophil activation) (8). Itch also plays an important role in T cell anergy by ubiquitinating PLC-{gamma}1 and PKC-{theta}, two key molecules induced by Ca2+/calcineurin signaling, which destabilizes the immunological synapse and induces T cell unresponsiveness after T cell receptor (TCR) engagement in response to restimulation with antigen plus antigen-presenting cells (9). The failure to induce anergy may account for the inability to establish peripheral tolerance and the development of autoimmune disease in itch–/– mice.

Itch also modulates Notch signaling. Notch proteins are evolutionarily conserved transmembrane receptors that play important roles in cellular differentiation, proliferation and apoptosis (10,11). In mammals, there are four Notch proteins that are essential for the development and function of various tissues and organ systems, including the immune system where the level of Notch signaling influences hematopoiesis, B and T cell development, as well as the function of peripheral T cells (reviewed in 12). Since small differences in Notch signaling can have vast phenotypic consequences, the levels of Notch proteins are tightly regulated (reviewed in 13). Notch1, the most extensively studied mammalian Notch homolog, is an integral membrane protein that exists at the cell surface as a functional heterodimer, resulting from a processing event in the trans Golgi. Upon binding of a transmembrane ligand presented by a neighboring cell, Notch undergoes two proteolytic cleavage events. The extracellular one is mediated by ADAM17, whereas the intracellular one is mediated by {gamma}-secretase (the catalytic site of which is thought to be contained in presenilin subunits). These cleavages release the intracellular fragment of Notch (ICN), which translocates to the nucleus and functions as a transcriptional coactivator for target genes like HES1. ICN is ubiquinated in the nucleus by FBXW7 and rapidly degraded. Studies in Drosophila have identified two additional E3’s, suppressor of deltex [Su(dx)] and DNedd4, which regulate the level of endogenous Notch. Specifically, these C2-WW-HECT E3's ubiquitinate full-length (FL), unactivated Notch in the endosome to target it for proteolysis. In the absence of these E3's, more FL Notch is present in the cell and can either be spuriously activated in the endosome by {gamma}-secretase or recycled back to the plasma membrane, thus effectively lowering the threshold for Notch signaling (14,15). It was also demonstrated in vitro that Itch can ubiquitinate the intracellular domain of Notch1 and target it for proteolysis (16). In studies described here, we evaluated itch's role in Notch signaling in vivo by breeding itch–/– mice to transgenic mice that overexpress the Notch1 ICN in T cells under the control of the lck proximal promoter (17). Our studies show for the first time that increased Notch1 signaling results in autoimmunity and suggest that Itch and Notch act in the same AKT-mediated pathway in the genesis of autoimmune disease. This could provide insight into the treatment of a group of diseases that affect a significant proportion of the population.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Itch and Su(dx)
Phylogenic analysis suggests that Itch is the mouse ortholog of Drosophila Su(dx) (Fig. 1). In Drosophila, a class of gain-of-function Notch alleles (AxE2) is enhanced by a loss-of-function Su(dx) mutation (18), suggesting that Su(dx) is a negative regulator of Notch signaling. To determine whether Itch also regulates Notch signaling in mice, itch–/– mice were bred to lck-Notch1 transgenic mice, which overexpress the Notch1 ICN in developing T cells. Although the numbers of thymocytes in Notch1 transgenics are equivalent to wild-type (WT) littermates, they contain increased numbers of CD8 single-positive (SP) and lower number of CD4 SP cells. These CD8 cells mature in the absence of MHC class I but not in the absence of both MHC class I and class II molecules (17). However, these SP CD8 cells are thought to be abnormal and are not found in the periphery where normal CD4 to CD8 ratios are instead observed (19). Previous studies have shown that ~20% of these transgenics will develop T cell lymphoma with a latency on the order of 5 months (20,21). Since Itch is carried on the C57BL/6J background, we bred the lck-Notch1 transgene onto the C57BL/6J background and then reassessed the phenotype to determine whether it was at all similar to itchy.


Figure 4251
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  		Figure 1. Itch is the ortholog of suppressor of deltex. Phylogenetic analysis was performed on all mouse (black and red type), S. cerevesiae (green), and Drosophila melanogaster (blue) C2-WW-HECT E3 ligases, using Rsp5p as an outgroup. The tree was constructed using the Vector NTI suite that bases its analysis on the Neighbor Joining method. The results indicate that Itch (in red) is the most closely related mouse protein of this family to Drosophila Su(dx).

 
lck-Notch1 tg+ mice develop lymphoma and autoimmune disease
In observing 306 transgenic mice in our colony over a 9 month window, we found that 37% of these animals died of undetermined causes. Further, there seemed to be three distinct clusters of disease onset, with the earliest cohort developing disease around 10 weeks of age, a second peak occurring around 5 months of age, and the last group displaying severe signs of illness around 7 months of age. In order to determine whether each of these clusters reflected different pathologies, 11 transgenic mice (five males and six females) that developed hunched posture, rough hair coat, reduced activity, labored breathing and weight loss were assessed by full body pathology. Five of these animals (two males, three females) had aggressive lymphoblastic T cell lymphoma (an immature T cell, based on morphology) with an average latency of 78 days. In its most advanced form (118 days), dissemination was present in every tissue examined. From these results, we estimate that 17% of animals that carry the ICN transgene on the C57BL/6J background will develop T cell lymphoma around 10 weeks of age.

Three mice (one male, two females) displayed splenomegaly, lymphadenopathy and atrophy of the thymus upon gross examination. Microscopic and immunohistochemical evaluation of these lesions revealed chronic inflammation of an admixture of lymphocytes and histiocytes in the lungs, heart, kidneys and liver. There was increased extramedullary hematopoiesis in the spleen and liver, glomerulonephopathy with interstitial inflammation, and pulmonary alveolar proteinosis with eosinophilic debris. This phenotype appeared with an average latency of 131 days. Therefore, we estimate that ~10% of transgenic animals will develop an autoimmune-like pathology around 5 months of age.

Finally, the last cohort (two males, one female) that developed disease around 216 days of age showed an unusual T cell lymphoma of a mature T cell origin with limited dissemination consistently found in the kidneys, liver and spleen. Neoplastic infiltrates were also occasionally found in the choroid plexus of the brain, lungs, heart, bladder, stomach, duodenum, thymus, mesenteric lymph node and the bone marrow. Superimposed on this was an autoimmune-like pathology in that these mice had kidney involvement with glomerulonephropathy and interstitial inflammation consisting of mononuclear cells and eosinophils. Additionally, there was cortical atrophy of the thymus and chronic inflammation in the heart and lungs. Based on these numbers, we estimate that ~10% of the transgenic animals will develop mature T cell lymphoma with kidney involvement and auto-immune-like pathology.

The autoimmune disease in lck-Notch1 transgenic mice is similar to that observed in itch–/– mice
Since the autoimmune disease observed in ~10% of lck-Notch1 tg+ animals was nearly identical to that observed in itch–/– animals with approximately the same age of onset, we did a direct comparison of the pathologies of these two diseases. At the level of gross observation, both autoimmune diseases yield splenomegaly and lymphadenopathy with a decrease in the size of the thymus. Histological analysis of diseased animals identified a similar profile of chronic inflammation with atrophy of the thymic cortical layer and lymphoplasmacytic infiltration in the lungs with acidophilic macrophage pneumonia; however, the latter lesion was more marked in itch–/– diseased animals (Fig. 2). The other difference observed between the two types of autoimmune-like disease was that the kidney involvement was much greater in the lck-Notch1 transgenics with membranoproliferative glomerulonephropathy and interstitial inflammation. Immunohistochemical analysis of the infiltrates in the thymus, lungs and kidneys revealed that most of the mononuclear infiltrate in the thymus and the lungs was B220+, whereas the infiltrate in the kidneys was almost exclusively CD3+. Both classes of diseased animals displayed a progressive deposition of IgG complexes in the glomeruli (Fig. 3) and the production of anti-nuclear antibodies (see Fig. 1S).


Figure 4252
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  		Figure 2. The autoimmune disease observed in lck-Notch1 tg+ mice is very similar to that in itch–/–mice. Representative histological and immunohistochemical analysis of thymus, lungs and kidneys from lck-Notch1 tg+ and from itch–/– animals affected by autoimmune disease as compared to age-matched WT mice reveals cortical atrophy with B220+ (brown) infiltrates in the thymus, lymphoplasmacytic infiltration (mostly B220+) in the lungs with acidophilic macrophage pneumonia, and membranoproliferative glomerulonephropathy and interstitial inflammation (CD3+) in the kidneys. Magnification for all photographs is 20x; M, medulla; C, cortex.

 


Figure 4253
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  		Figure 3. itch–/– and lck-Notch1 tg+ animals show progressive deposition of IgG complexes in the glomeruli. Kidneys were embedded in OCT, sectioned at 10 µm, and stained with Cy2-conjugated anti-mouse IgG antibodies (green). Nuclei are counterstained with DAPI (blue). Both genotypic classes show increased accumulation of auto-IgG complexes in the glomeruli over a 20 week period relative to age- and gender-matched WT control animals. This figure is representative of at least two animals examined for each time point. Magnification for all panels is 40x.

 
Genetic interaction of the itch–/– and lck-Notch1 tg+ mutations
Since the autoimmune disease of lck-Notch1 tg+ mice was so similar to that observed in itch–/– mice and since Itch had been shown to physically interact with Notch1 in vitro (16), we hypothesized that these two alleles may be genetically interacting (i.e. the combination of these mutations would result in the modification of the severity and range of the autoimmune disease that had been observed in either mutant alone and perhaps in the appearance of new phenotypic features). Genetic interaction in combination with physical interaction data often indicates that two genes function in the same or overlapping pathways. To determine whether this was the case, we bred itch–/– mice to lck-Notch1 tg+ mice and found that all itch–/–; lck-Notch1 tg+ mice were considerably smaller than their littermates (Fig. 4A). By 7 weeks of age, 100% of the itch–/–; lck-Notch1 tg+ mice were moribund, and all died between 8 and 12 weeks of age. When itch–/–; lck-Notch1 tg+ animals were examined grossly and histologically, numerous pathological lesions were noted including splenomegaly, hepatomegaly, lymphadenopathy and reduced thymic size due mainly to cortical atrophy. As with lck-Notch1 tg+ mice, some membranoproliferative glomerulonephropathy was noted in the kidneys. Most notable, however, was the presence of massive amounts of chronic, active inflammation with eosinophils in the lamina propria and to a lesser extent the submucosa of the entire digestive tract, in the dermal layer of the skin, as well as in the stroma of male sex and accessory sex organs (Fig. 4B). The liver and lungs displayed a similar infiltrate with a more vascular orientation, although the lungs were additionally affected by acidophilic macrophage pneumonia. Finally, the lymphoid organs, particularly the medulla of the thymus, showed inflammation consisting of mononuclear cells with admixed eosinophils. Immunohistochemical staining revealed that the majority of the infiltrate in the medulla of the thymus and lungs was B220+ (Fig. 4C). Consistent with the autoimmune-like disease aspect of the phenotype, the sera of itch–/–; lck-Notch1 tg+ mice contained anti-nuclear antibodies (see Fig. 1S) and more IgG deposition was detected in the glomeruli of 8-week-old itch–/–; lck-Notch1 tg+ mice when compared to age- and gender-matched WT, itch–/–, or lck-Notch1 tg+ animals (Fig. 5). Thus, itch–/–; lck-Notch1 tg+ animals develop a similar autoimmune-like disease as itch–/– or lck-Notch1 tg+ mice but with more severe lesions and a much earlier age of onset. The fact that the mutations in concert yielded severe early-onset disease, which was not seen in either mutation alone, supports the hypothesis that these alleles genetically interact.


Figure 4254
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  		Figure 4. itch–/–; lck-Notch1 tg+ mice develop a severe, early-onset autoimmune disease. Eight-week-old male littermates are compared (A). From left to right, the genotypes of the animals are itch+/-; lck-Notch1 tg+, itch–/– and itch–/–; lck-Notch1 tg+. Only the mouse that is itch–/–; lck-Notch1 tg+ shows smaller overall size, hunched posture, reduced activity and labored breathing. Histological evaluation of 8-week-old itch–/–; lck-Notch1 tg+ mice demonstrated chronic, active inflammation with eosinophils (arrow heads) in the lamina propria of the stomach, in the dermis of the thin skin of the neck, as well as in the stroma of the epididymus (B). The thymus from itch–/–; lck-Notch1 tg+ animals was reduced in size, showed cortical atrophy, and was infiltrated with B220+ cells and some eosinophils (C). Similarly, the lungs from these animals displayed acidophilic macrophage pneumonia and mononuclear infiltrate that was predominantly B220+ (C). Magnification for all panels is 20x.

 


Figure 4255
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  		Figure 5. itch–/–; lck-Notch1 tg+ mice have increased deposition of IgG complexes in the glomeruli by 8 weeks. Consistent with the suggestive autoimmune pathology, there is significant deposition of IgG complexes in the kidneys of 8-week-old itch–/–; lck-Notch1 tg+ mice, relative to age- and gender-matched controls of the indicated genotypes, as assessed by Cy2 (green) staining of 10 µm thick kidney cryosections. Magnification for all panels is 40x.

 
itch–/–; lck-Notch1 tg+ mice have abnormal T cell development and thymic architecture
Because the Notch1 transgene is expressed only in developing T cells and this was sufficient to cause disease pathology, this cellular compartment was characterized in greater detail. At 4 weeks of age, the thymi from all genotypic classes were comparable in overall size and cellularity. When examined by light microscope, a loss of the sharp boundary between the cortex and medulla was noted as well as a degree of lymphoproliferation in the medulla of only the itch–/–; lck-Notch1 tg+ animals (Fig. 6). The reason for this change was elucidated when thymocytes from 4-week-old mice were stained for the T cell developmental markers CD4 and CD8. This staining revealed increased percentage and absolute numbers of the most immature CD4–CD8– double-negative (DN) cells and of the most mature CD4 SP and CD8 SP cells, while the percentage and absolute numbers of CD4+CD8+ double-positive (DP) cells were decreased (Fig. 6). Since SP cells are usually found in the medulla, the increase of SP cells as revealed by cell staining correlates with the observed lymphoproliferation in the medulla.


Figure 4256
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  		Figure 6. Defects in T cell development and thymic architecture are seen in itch–/–; lck-Notch1 tg+ mice. Representative hematoxylin and eosin-stained thymus sections derived from 4- or 8-week-old mice of the given genotypes are pictured. Starting at 4-weeks, the itch–/–; lck-Notch1 tg+ thymus shows loss of the sharp demarcation between the medulla (M) and cortex (C) and lymphoproliferation in the medulla. At 8 weeks, the thymus of itch–/–; lck-Notch1 tg+ animals is greatly atrophied with a near complete loss of the cortical layer and interstitial inflammation. Corresponding scatter plots for PE-CD4 versus FITC-CD8 staining of thymocytes are presented next to the thymus sections. At 4 weeks, thymi derived from all four genotypes showed comparable cellularity (average of 2x108 for WT, 1.9x108 for itch–/–, 1.1x108 for lck-Notch1 tg+ and 1.5x108 for itch–/–; lck-Notch1 tg+), whereas thymi from 8-week-old itch–/–; lck-Notch1 tg+ animals showed reduced cellularity (1.9x107 versus 7.2x107 for WT, 7.0x107 for itch–/– and 6.4x107 for lck-Notch1 tg+). At both time points, there were increased numbers and percentages of DN and SP populations, and decreased numbers and percentages of DP cells (percentages shown in quadrants to the right of corresponding scatter plot). The increased numbers of DN cells in these animals were determined to be B220+. All data are representative of three to six independent experiments. Magnification for all panels is 5x.

 
These changes were greatly amplified by 8 weeks when the itch–/–; lck-Notch1 tg+ animals were moribund. At that point of time, the thymic architecture was completely destroyed and there was considerable inflammation in the organ. The thymi from 8-week-old itch–/–; lck-Notch1 tg+ mice were reduced in overall cellularity, consistent with what had been observed grossly, had increased percentage and absolute numbers of DN and SP cells, whereas the percentage and absolute numbers of DP cells were dramatically decreased (Fig. 6). When the DN population was gated and analyzed for the presence of the B220 B-cell marker, it was noted that there was an increase in the B220+ population in both itch–/– (20% of the DN population and 0.5% overall) and in itch–/–; lck-Notch1 tg+-derived thymocytes (25% of the DN population and 4% overall) relative to WT (10% of the DN population and 0.2% overall). Because immunohistochemical staining had also indicated there was an increase in B220+ cells in the thymus, this population was further characterized by flow cytometry. The thymus-derived B220+ cells also stained for the B cell marker CD19 but not for the T cell marker TCRß, indicating they were not the same abnormal T cells involved in the lymphadenopathy of Faslpr/Faslpr mice (data not shown). The majority of B220+ cells were IgM+ with an overall distribution similar to what is seen in the spleen, suggestive of lymphoid neogenesis (similar to what has been described in certain human autoimmune diseases) or of an expansion of the normal thymic B cell population (Fig. 2S). There was also a small percentage of IgA+ cells in the thymic B220+ population (Fig. 2S). The near total loss of the DP population at 8 weeks corresponded to the atrophy of the cortical layer, as that is where DP cells are normally found. Further, the cellular infiltrate observed at 8 weeks is likely composed mostly of DN B220+ cells. The consistent increase in SP cells suggests that some aspect of the DP to SP transition (like positive or negative selection) may be compromised in these animals, whereas changes in the thymic architecture could be secondary to the inflammation.

Increase in canonical Notch signaling does not correlate with severity of autoimmune-like disease
Canonical Notch signaling posits ligand-induced cleavages of the Notch receptor that releases the ICN, which translocates to the nucleus and functions as a transcriptional coactivator for target genes like HES1 (reviewed in 13). In order to assess the effects of increased Notch signaling in mice that carried the Notch1 transgene, some known downstream transcriptional targets of canonical Notch1 were examined by both northern blot and by quantitative PCR (Fig. 3S) and the results summarized in Figure 7. Hes1 showed an increased expression in thymi derived from lck-Notch1 tg+, and itch–/–; lck-Notch1 tg+ animals but not in itch–/– thymi (Fig. 7A). In the thymus of lck-Notch1 tg+ animals, Hes1 was upregulated 4.4-fold relative to WT, whereas in thymus derived from itch–/– lck-Notch1 tg+ animals, Hes1 was only upregulated 2.2-fold. No change in levels of Hes1 was detected in the brains of any of the genotypic classes examined. Similarly, levels of Notch1 and Notch3 message were increased in the thymus of lck-Notch1 tg+ and to a lesser extent in itch–/–; lck-Notch1 tg+ animals but not in itch–/–-derived thymus or in the brains of these animals (Fig. 7A). This is consistent with previous reports in Drosophila (22), in C2C12 cells (23) and in thymocytes derived from a different Notch1 transgenic mouse (24): Notch1 ICN activates its own expression in a positive feedback loop. These same studies also showed an upregulation of Notch3 by Notch1 ICN. Comparison of transcript levels by Northern analysis detected no change in the expression of Notch2, and the expression of Notch4 proved too weak to detect by Northern analysis. Since a previous report had also shown that Notch1 could antagonize the function of Nr4a1 (25), we determined whether there was any change in the transcription of this gene. The level of Nr4a1 was slightly down regulated in lck-Notch1 tg+ and in itch–/–; lck-Notch1 tg+ animals but unchanged in itch–/–-derived thymus. Taken together, these results show that although targets of canonical Notch signaling are upregulated in lck-Notch1 tg+ animals, the transcription of those genes is not increased in itch–/– animals and is not further upregulated in itch–/–; lck-Notch1 tg+ animals. Thus, the output of canonical Notch1 signaling does not correlate with the severity of the observed autoimmune disease, suggesting that this phenotype arises from a different mechanism.


Figure 4257
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  		Figure 7. Increase in FL Notch1 in DP thymocytes correlates with the severity of autoimmune disease. (A) Representative analysis of transcriptional regulation of ICN targets from one set of age- and gender-matched animals. This analysis was reproduced by northern analysis with an independent set of animals and confirmed by quantitative PCR with a third set of animals. This analysis shows upregulation (normalized to Gapd for loading and then compared to WT) of the endogenous Notch1 message in lck-Notch1 tg+ (2.6-fold increase) and to a lesser extent in itch–/–; lck-Notch1 tg+-derived thymi (1.8-fold increase) with no change in the expression of Notch1 in itch–/– animals (0.88 relative to WT). These same genotypic classes also display an increase in the expression of Notch3 (2.1-fold for lck-Notch1 tg+ and 1.6-fold for itch–/–; lck-Notch1 tg+) and Hes1 (4.4-fold in lck-Notch1 tg+ and 2.2-fold for itch–/–; lck-Notch1 tg+) and a decrease in the expression of Nr4a1 (0.35-fold that of WT in lck-Notch1 tg+ and 0.45-fold that of WT in itch–/–; lck-Notch1 tg+). In contrast, increased amount of FL Notch1 protein as detected by PE-staining thymocytes derived from animals of the indicated genotypes was found in itch–/–, lck-Notch1 tg+ and itch–/–; lck-Notch1 tg+ animals (B). The mean fluorescence of these populations was 14.79, 16.41 and 25.25, respectively, relative to the WT level of 8.2. When thymocytes from WT or from itch–/–; lck-Notch1 tg+ mice were triply stained for CD4, CD8 and Notch1 and the DP population was gated, the presence of a Notch1hi population in itch–/–; lck-Notch1 tg+ animals was noted as early as 4 weeks of age and persisted and expanded in these mice through 8 weeks of age, despite the atrophy of the cortex and the concomitant decrease in the number of DP cells (C). Thus, the amount of FL Notch1 in DP thymocytes correlates with the severity of the observed autoimmune disease, whereas the transcription of canonical Notch signaling targets does not.

 
Increased FL Notch1 in itch–/–; lck-Notch1 tg+ mice
Our data suggest that the lck-Notch1 transgene activates expression of Notch1, resulting in the production of more FL Notch1 protein. This was confirmed by examining the amount of Notch1 protein in thymocytes and splenocytes. At the protein level, increased amounts of endogenous FL Notch1 could be detected (Fig. 7B) by staining thymocytes with an anti-Notch1 antibody whose epitope maps to the extracellular domain of Notch1 (i.e. an area not contained in the transgenic construct). Histograms representing the fluorescence (i.e. the amount of Notch1 protein) in the thymocytes derived from WT, itch–/–; lck-Notch1 tg+ and itch–/– lck-Notch1 tg+ animals were plotted and analyzed. Compared to WT, itch–/– and lck-Notch1 tg+ thymocytes showed increased average fluorescence for the population, with a right shift in both the median and geometric mean as well as a flattening and broadening of the peak towards the higher fluorescent values. This trend was even more pronounced in the itch–/–; lck-Notch1 tg+-derived thymocytes, suggesting that the genetic interaction between Itch and Notch1 manifests itself at the level of the FL Notch1 protein. In an attempt to identify which thymocytes were expressing the Notch1 antigen, we triply stained thymocytes derived from mice of all four genotypes for CD4, CD8 and Notch1 and identified a discrete population of Notch1hi cells in the DP population of itch–/–; lck-Notch1 tg+-derived cells (Fig. 7C). These cells were present as early as 4 weeks of age when the CD4/CD8 profile of these animals was not grossly altered and the cellularity of the thymus was comparable to the other genotypic classes. Further, these cells were still present in 8-week-old animals even though there was atrophy in the cortex and virtual ablation of the DP population. In contrast, when splenocytes were stained for FL Notch1, no difference in the amount of protein was detected among the four genotypic classes (data not shown). This suggests that the changes in Notch signaling important for the development of autoimmune disease occur in DP thymocytes. Further, the increased FL Notch1 does not seem to signal through Hes1.

Because FL Notch1 has been shown to co-immunoprecipitate with LCK and with PI3K to activate AKT signaling (26), we determined whether AKT signaling was activated in thymocytes with increased amounts of FL-Notch1. As illustrated in Figure 8A, thymocytes derived from itch–/–; lck-Notch1 tg+ animals had increased levels of AKT and of phospho-AKT (both the T308 and the S473 isoforms) relative to the other genotypic classes. Further, thymocytes derived from both itch–/– and from lck-Notch1 tg+ mice showed elevated levels of AKT and phospho-AKT (just the T308 specific form) as compared to WT thymocytes. The same pattern was seen in 6-week-old animals, but no significant difference in either AKT or phospho-AKT isoforms was seen among the genotypic classes at 4 weeks of age (data not shown). Since there is a distinct class of Notch1hi DP cells as early as 4 weeks with no difference in the levels of AKT or phospho-AKT isoforms, it is likely that the AKT signal lies downstream of Notch1. Moreover, since the amount of total and phospho-specific AKT in thymocytes parallels the severity of autoimmune disease in these mice as well as the amount of FL Notch1, our studies suggest that the autoimmune-like disease observed in the mice could result from increased levels of FL Notch1 that activates AKT signaling.


Figure 4258
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  		Figure 8. Increases in FL Notch1 result in specific and constitutive activation of the AKT signaling pathway. (A) Thymocytes derived from 8-week-old mice were PE-stained for total AKT and for phospho-specific (S473 or T308) AKT isoforms. Similar to what was seen with FL Notch1, there was increased amounts of total AKT in itch–/– and lck-Notch1 tg+ relative to WT and an even greater increase in itch–/–; lck-Notch1 tg+ (median fluorescence for WT was 21.10, for itch–/– was 49.14, for lck-Notch1 tg+ was 40.21, and for itch–/–; lck-Notch1 tg+ was 72.99). The same pattern held true for the phospho-specific T308 isoform (median fluorescence for WT was 6.04, for itch–/– was 9.56, for lck-Notch1 tg+ was 9.47, and for itch–/–; lck-Notch1 tg+ was 28.39), but for the S473 isoform, only itch–/–; lck-Notch1 tg+-derived thymocytes displayed increased amounts (median fluorescence for WT was 2.86, for itch–/– was 2.97, for lck-Notch1 tg+ was 3.40, and for itch–/–; lck-Notch1 tg+ was 10.18). In contrast, when other signaling pathways important in T cell development were examined (B), no difference could be detected among the four genotypic classes, suggesting that the increase in FL Notch1 constitutively and specifically activates AKT signaling.

 
In contrast, no such difference was seen among the four genotypic classes when other signaling pathways important in thymocyte development were examined. Specifically, there were no detectable differences in phospho-protein levels of MEK1/2, MAPK, p38 or JNK in thymocytes derived from 8-week-old animals (Fig. 8B, data not shown). This suggests that the increased Notch signaling associated with the autoimmune-like disease results in a specific and constitutive elevation in AKT-mediated signaling. Since one of the physiological consequences of increased AKT signaling is inhibition of apoptosis, we examined the degree of apoptosis in the thymus of all four genotypes at 4, 6 and 8 weeks of age by relative TUNEL staining. When the number of TUNEL+ cells was determined in five independent high power fields of both the cortex and medulla and then normalized to the number observed in an age- and gender-matched WT control, a statistically significant reduction of apoptosis was found only in the cortex of itch–/–; lck-Notch1 tg+ mice at 4, 6 and 8 weeks of age, consistent with the genetic interaction of these two mutations. Reduced TUNEL staining was also observed in the medulla of these animals, but this reduction was not statistically significant (Table 1). Interestingly, the level of apoptosis in the itch–/–; lck-Notch1 tg+ thymus was constant across the time period examined, despite large changes in the thymic architecture, especially in the cortex, suggesting a constitutive inhibition of apoptosis consistent with the increase in AKT signaling we observed.


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  		Table 1. Reduced apoptosis in itch–/–; lck-Notch1 tg+ mice. Five 40X fields were evaluated from the cortex and from the medulla for each genotype. The level of apoptosis is reported as a percentage relative to that seen for the age-matched controls for each timepoint. Significant reduction in apoptosis was seen in the cortex of itch–/–; lck-Notch1 tg+ mice at all timepoints. This genotype was the only class to display cortical atrophy and significant thymic architectural changes during the examined timeline. One to three animals were tested for each timepoint

 

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Here, we show that mice carrying an activated lck-Notch1 transgene on the C57BL/6J background can, in some cases, develop an autoimmune-like disease that is very similar to that observed in itch–/– mice, suggesting these genes act in the same or overlapping pathway. This is the first direct demonstration of the involvement of increased Notch1 signaling in autoimmune disease. In addition, other transgenic mice were identified that developed an unusual, mature T cell lymphoma with kidney involvement superimposed upon a profile of autoimmune-like pathology. This is interesting in light of the fact that there have been clinical associations of the progression of certain types of autoimmune disease to lymphoma states (27). When we bred itch–/– animals to lck-Notch1 tg+ animals, we found a genetic interaction in that the autoimmune phenotype was more severe with an earlier age of onset. The severity of autoimmune directly correlated with an increase in both FL Notch1 protein and in phospho-AKT but not in phospho-MEK1/2, MAPK, p38 or JNK. This increase in non-canonical Notch signaling resulted in a defect in T cell development with secondary changes in the architecture of the thymus and decreased apoptosis in the thymus to generate a systemic autoimmune disease that proves lethal within 12 weeks of birth.

A possible mechanism by which excessive signaling through the Notch1 receptor could account for the phenotypes we observed is shown in Figure 9. Notch1 has been shown to co-localize to the immunological synapse in activated T cells with the TCR (28,29). We have shown that there are increased amounts of FL Notch1 in lck-Notch1 tg+ mice, which likely results from a positive feedback loop whereby the transgene activates transcription of the endogenous Notch1 gene and produces more FL protein, effectively lowering the signaling threshold. In contrast, in itch–/– mice, unactivated Notch1 receptor cannot be ubiquitinated in the endosome by Itch and either spuriously signals from the endosome because of close proximity to other components of the signaling pathway, or recycles back to the plasma membrane, also lowering the effective threshold for Notch1 signaling. We base our assumption on the fact that Itch acts on recycling, unactivated Notch1 similar to what has been described for Su(dx), the Itch ortholog in Drosophila (14,15). It is, however, possible that Itch could ubiquitinate ligand-activated Notch1 that has not yet undergone the {gamma}-secretase cleavage event in the endosome since there is evidence suggesting presenilin activity is dependent on an acidic pH such as that found in the late endosome or lysosome (30).


Figure 4259
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  		Figure 9. Model for increased Notch signaling in autoimmune disease. Increased amounts of FL Notch1 from transcriptional upregulation in lck-Notch1 tg+ mice, from decreased ubiquitination in itch–/–mice, or from both in itch–/–; lck-Notch1 tg+ mice complexes with PI3K and with p56lck to generate a survival signal through phospho-AKT. This survival signal allows the persistence of pathological cells that can initiate either autoimmune disease or a lymphoma.

 
How might this increase in FL Notch1 result in autoimmune disease or in T cell lymphoma? Previous results have suggested a role for Itch in T cell differentiation (8) and in T cell anergy (9), whereas canonical Notch signaling has been shown to be important at various stages of T cell development as well as in the activation and differentiation of peripheral T cells (12,31). Transplantation of bone marrow derived from an itch–/– mouse into lethally irradiated WT recipients completely recapitulates the itchy phenotype, indicating that this phenotype is autonomous to a hematopoietic cell (L.E.M., M. Ortiz, J. Keller, N.G. Copeland and N.A.J., unpublished observations). It is unlikely that the Th2 bias in T cell differentiation and the resulting T cell hyperproliferation in response to increased interleukin 4 previously described in itch–/– mice solely accounts for the autoimmune phenotype observed here since, when itchy was moved onto a B10 background, the Th2 bias was eliminated but the autoimmune phenotype persisted (9). The role of Notch1 signaling in promoting Th1/Th2/Treg differentiation is more controversial, with reports supporting a role for Notch signaling in both Th1 bias (32) and in Th2 bias (33). Analysis of peripheral T cells derived from itch–/– mice show no difference in the percentage of CD4+CD25+ regulatory T cells relative to WT controls (X. Chen, L.E.M., N.G. Copeland, N.A.J., O.M.Z. Howard and J. Oppenheim, unpublished observations). Thus, it seems unlikely that loss of the Treg population is the cause of the autoimmune disease described in these mice. Itch has also been shown to play a role in T cell anergy by ubiquitinating PLC-{gamma}1 and PKC-{theta}, two key molecules induced by Ca2+/calcineurin signaling, destabilizing the immunological synapse and inducing T cell unresponsiveness after TCR engagement in response to restimulation with antigen plus antigen-presenting cells (9). The failure to induce anergy may account for the inability to establish peripheral tolerance and the development of autoimmune disease in itch–/– mice; however, this process may be independent of Notch signaling because the increases in the amount of FL-Notch1 were only observed in the thymus and not in the periphery (i.e. the spleen), although this possibility needs to be formally excluded.

It is interesting to note that animals heterozygous for a knockout allele of presenilin1 and homozygous for a knockout allele of presenilin2 develop seborrheic keratosis and autoimmune disease with aspects similar to the autoimmune disease described here, including IgG deposition in the kidneys, production of anti-nuclear antibodies, splenomegaly, and dermatitis consisting of a mixed inflammatory infiltrate that was predominantly B220+ (34). This was originally interpreted as resulting from a reduction in Notch signaling through the canonical pathway, which caused an excess of B lymphocytes and of CD4 T cells. Our studies suggest it is instead possible that this autoimmune phenotype results from the increased amount of FL Notch1-4 and non-canonical Notch signaling that would be present in the T cells of these mice since it has been reported that FL Notch and not processed Notch complexes with LCK and with PI3K to activate AKT signaling (26).

Interestingly, there are four different transgenic mouse models that express various forms of activated AKT in T cells that develop autoimmune disease and/or lymphomas, similar to what we have observed in the lck-Notch1 tg+ animals (35–37). This supports our findings that levels of increased FL Notch1 and of phospho-AKT correlate with the severity of autoimmune disease and may lay at the root of the etiology of the pathology. Recently, a role for Notch receptor-ligand interactions in promoting the survival and glucose uptake/metabolism of pre-T cells (at the point of ß selection) via AKT signaling has been demonstrated (38). AKT may inhibit apoptosis by phosphorylating Nr4a1 and suppressing Nr4a1-mediated cell death in T cells (39). Activation of the PI3K-AKT pathway (40,41) or suppression of Nr4a1-mediated signaling (42) is known to inhibit negative selection. Further, Nr4a1 has been shown to interact with Notch1 in vitro to confer protective effects on TCR-induced apoptosis (25). AKT can also phosphorylate and inactivate forkhead transcription factors that are capable of activating the transcription of the proapoptotic gene Bcl2l11 (43), a protein known to play an important role in negative selection (44).

The timing of the cell survival signal from the PI3K-AKT pathway may influence the nature of the phenotype (i.e. autoimmunity versus lymphoma). Overexpression of activated Lck under the control of the lck proximal promoter during the DN to DP transition has been shown to cause transformation and accumulation of immature thymocytes (45), whereas the overexpression of constitutively active Lck under the control of the lck distal promoter in mature thymocytes and mature T cells resulted in an acceleration of the DN to DP transition even in the absence of appropriate MHC engagement as well as elevated levels of cross-reactivity toward syngenic targets in vitro, indicating that negative selection may also be impaired in these mice (46). Similarly, when activated AKT is expressed under the control of the lck proximal promoter, lymphoblastic T cell lymphomas were observed (35). However, when activated AKT was expressed under the control of the CD2 promoter, transgenic animals exhibited lymphadenopathy and splenomegaly resulting from the accumulation of both hyperproliferative B and activated T cells; Ig deposition and lymphocytic infiltration in a variety of organs; and a significant number of transgenic animals developed lymphomas or thymomas (36). In an independent model that expressed activated AKT under the control of the entire lck promoter (i.e. proximal and distal) with the CD2 enhancer, transgenic animals displayed enlarged T cells with increased rates of glycolysis; T cells that were resistant to death-by-neglect in culture; T cells that were less dependent on co-stimulation by CD28; accumulation of activated T cells (particularly CD4) and B cells with increasing age; autoimmunity with increased glomerular deposition of IgG complexes; and increased incidence of lymphoblastic T cell lymphomas (37).

In the lck-Notch1 tg+ mice, overexpression of the ICN is driven by the lck proximal promoter. However, there may well be position effects for this construct and modifier genes in the C57BL/6J background that influence the precise timing of expression of the ICN. Accordingly, if expression of the ICN is initiated early enough in a cell that was fated to undergo death by neglect because of failure to meet the criteria of positive selection, this cell could be given an inappropriate survival signal by the Notch1-PI3K-LCK complex and expand, resulting ultimately in a T cell lymphoma. If the survival signal is delivered a little later in a cell destined to undergo apoptosis after negative selection, autoimmunity would develop. Finally, if the survival signal is delivered instead to a cell not fated to die because of appropriate MHC-TCR interactions, no phenotype would be noted. Indeed, this entire phenotypic spectrum is evidenced in the C57BL/6J-lck Notch1 tg+ mice. Since there is increased canonical Notch1 signaling in these mice, it is possible that the canonical pathway contributes to the development of lymphomas as well. Certainly the recent finding that the majority of human T-ALL-derived tumors have activating NOTCH1 mutations supports this hypothesis (47). The observation that itch–/– and itch–/–; lck-Notch1 tg+ mice develop only autoimmune disease suggests that Itch normally interacts with Notch1 during the DP to SP transition and that there is no functional redundancy with any other member of the C2-WW-HECT E3 family at this particular stage of T cell development. The increase in FL Notch1 and in phospho-AKT correlated with a decrease in apoptosis and a growth advantage to the cell, whereas the increase in canonical Notch signaling effectors like Hes1 did not. This suggests that genetic lesions in NOTCH1, ITCH, LCK or PI3K and downstream signaling effectors may underlie certain human autoimmune malignancies as well. Clearly, this pathway needs to be fully understood to allow for the design of better therapies for pathologies as diverse as autoimmune disease and cancer, which manifest themselves in the shadow of improper regulation of cell proliferation and apoptosis.


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Mice
lck-Notch1 tg+ mice were obtained from the Jackson Laboratories (Bar Harbor, ME, USA) and bred to C57BL/6J mice in our colony. All animals were treated in accordance with the guidelines provided by the Animal Care and Use Committee of the National Cancer Institute at Frederick (Maryland, USA).

Histology, immunohistochemistry and tunel staining
Organs were collected and fixed in 10% neutral buffered formalin. Tissues were then embedded in paraffin and sectioned at 5 µm. Slides were stained with hematoxylin and eosin and evaluated for pathology. Alternatively, slides were stained with biotin-conjugated anti-CD45R/B220, 1:200 (RA3-6B2 BD-Pharmingen, San Jose, CA, USA) or with purified anti-CD3 1:600 (Dako, Carpinteria, CA, USA) for B-cell or T-cell-specific immunostaining, respectively. Antigen retrieval for both B220 and for CD3 was carried out by microwaving in citrate buffer. Detection of B220 signal was performed using the Avidin-Biotinylated enzyme complex only (Vector Laboratories) with 3,3'-diaminobenzidine (Sigma) as a chromagen. Detection of CD3 was accomplished using the Rabbit Elite kit (Vector Laboratories) using 3,3'-diaminobenzidine as a chromagen. Slides were counterstained with hematoxylin to visualize nuclei.

Apoptotic cells were detected with the Apoptag kit (Serologicals Corporation) following the manufacturer's recommendations and counterstained with hematoxylin. Percentage of apoptosis was evaluated by counting the number of TUNEL+ cells in five 40X fields and calculating the average. This average was then divided by the average number of the TUNEL+ cells in the corresponding layer of age- and gender-matched C57BL/6J controls.

Glomerular IgG deposition
Kidneys were snap frozen in liquid nitrogen, embedded in OCT and cryosectioned at 10 µm. Tissue sections were fixed for 15 min in cold acetone (–20°C) then washed three times for 5 min in PBS with 0.005% sodium azide. Non-specific sites were blocked by incubating in 10% normal goat serum, 2% BSA and 0.1% Triton X-100 in a humidity chamber for 1 h at room temperature. After three 5 min washes in PBS with 0.005% sodium azide, a 1:50 dilution of Cy2-conjugated goat anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA, USA) was applied for 1 h at room temperature. Antibody was diluted in 1% normal goat serum, 0.1% Triton X-100 and DAPI for nuclear staining. Tissues were once again washed three times for 5 min in PBS containing 0.005% sodium azide and then coverslips were mounted with Gelmount. Immunofluorescence was evaluated on a Zeiss Axiophot microscope with the appropriate filters, and images were acquired with the OpenLab program attached to a Zeiss digital camera.

Flow cytometry
Antibodies used for these studies included PE-conjugated anti-mouse CD4 (L3T4, BD-Pharmingen) and CD45R/B220 (RA3-6B2, BD-Pharmingen); FITC-conjugated anti-mouse CD8a (Ly-2, BD-Pharmingen), IgA (C10-3, BD-Pharmingen), IgM (II/41, BD-Pharmingen) and active caspase-3 (CPP32, BD-Pharmingen); and purified anti-mouse Notch1 (A6, Abcam, Cambridge, MA, USA) that was detected using a secondary PE-conjugated AffiniPure F(ab’)2 donkey anti-mouse IgG (H+L) (Jackson Immunoresearch, West Grove, PA, USA), whereas purified anti-mouse Akt1, anti-phospho-AKT (S473 and T308 specific isoforms), anti-phospho- MEK1/2, anti-phospho-MAPK, anti-phospho-p38 and anti-phospho JNK (all from Cell Signaling Technology) were detected using a secondary PE-conjugated AffiniPure F(ab’) goat anti-rabbit IgG (H+L) from Jackson Immunoresearch. Staining was performed essentially as described (48) with the following modifications: for Notch1, cells were stained as above and then permeabilized with the Cytofix/Cytoperm kit (BD-Pharmingen) and stained again for Notch1 according to the kit specifications. The same kit was used to permeabilize and stain cells for AKT and phospho-specific proteins. Data were acquired on a FACSCalibur machine (Beckton-Dickinson) and analyzed using Cell Quest software (Beckton–Dickinson).

Expression analysis
Notch1, Notch2, Notch3, Hes1 and Nr4a1 expression levels in brain and thymus were determined by first isolating total mouse RNA from 8-week-old animals with STAT-60 RNAzol (Tel-Test, Friendswood, TX, USA) and then single-selecting poly(A)+ RNA with the microPoly(A) Pure Isolation kit from Ambion (Austin, TX, USA). Two micrograms of poly (A)+ RNA from each tissue was loaded and separated on a 1.2% agarose and 6.2% formaldehyde gel and then transferred to a nylon membrane (Amersham Pharmacia). Hybridization was performed exactly as described for Southern blotting (49). Signal intensity for the gene of interest was normalized to Gapd to adjust for loading differences using Image Quant software to analyze phosphorimages obtained with the Molecular Dynamics Storm 820 system (Amersham Pharmacia). For real-time RT-PCR, random hexamer-primed cDNA samples were prepared from poly A+ RNA using Superscript (Invitrogen), and quantitative-PCR analysis was performed using Syber Green Chemistry (Qiagen) according to the manufacture's instructions in 10 µl final volume in 384-well microtiter plates. Gene-specific primer sequences and thermocycling conditions are available on request.


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 ABSTRACT
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Supplementary Material is available at HMG Online.


   ACKNOWLEDGEMENTS
 
The authors wish to thank Deborah A. Swing and Joanne Dietz for their excellent assistance with mouse husbandry and photography as well as T. Norene O'Sullivan, Linda Cleveland, Kathleen Noer and Roberta Matthai for their invaluable technical assistance. This research was supported by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research and in part, by federal funds from the National Cancer Institute under contract NO1-CO-12400 to SAIC Frederick. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products or organizations imply endorsements by the U.S. Government.

Conflict of Interest Statement: The authors wish to note that they have no financial conflicts of interest.


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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 

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