Theileria parva: taking control of host cell proliferation and survival mechanisms.

The intracellular parasite Theileria parva infects and transforms bovine T-cells, inducing their uncontrolled proliferation and spread in non-lymphoid as well as lymphoid tissues. This parasite-induced transformation is the predominant factor contributing to the pathogenesis of a lymphoproliferative disease, called East Coast fever. T. parva-transformed cells become independent of antigenic stimulation or exogenous growth factors. A dissection of the signalling pathways that are activated in T. parva-infected cells shows that the parasite bypasses signalling pathways that normally emanate from the T-cell antigen receptor to induce continuous proliferation. This review concentrates on the influence of the parasite on the state of activation of the mitogen-activated protein kinase (MAPK), NF-kappaB and phosphoinositide-3-kinase (PI3-K) pathways in the host cell. Of the MAPKs, JNK, but not ERK or p38, is active, inducing constitutive activation of the transcription factors AP-1 and ATF-2. A crucial step in the transformation process is the persistent activation of the transcription factor NF-kappaB, which protects T. parva-transformed cells from spontaneous apoptosis accompanying the transformation process. Inhibitor studies also suggest an important role for the lipid kinase, PI-3K, in the continuous proliferation of T. parva-transformed lymphocytes.

Characterization of recombinant class I MHC alloantigen action upon the survival of disparate heart allografts

Previous studies have led to the development of allochimeric class I MHC proteins as agents that effectively induce donor-specific transplantation tolerance in a rat system with or without additional immunosuppression. Within the α1-helical region of RT1.Au, an epitope that conferred immunologic tolerance was discovered. Studies presented herein were designed to test our central hypothesis that allochimeric proteins onfer tolerance in a mouse model. To test this hypothesis, portal vein (PV) injection of wild-type H2Kd and H2Dd proteins were produced in a bacterial expression system and found to specifically prolong the survival of BALB/c (H2d) heart allografts in C57BL/10 (H2b) recipients. Although a single PV injection of 50 μg α1–α 3 H2Kd alone was ineffective, 50 μg α1 –α3 alone slightly prolonged BALB/c heart allograft survivals. In contrast, the combination of 25 μg α1–α 3 H2Kd and 25 μg α1–α 3 H2Dd proteins prolonged BALB/c graft survivals to 20.2 ± 6.4 days (p < 0.004). The effect was donor-specific, since a combination of 25 μg α1–α3 H2Kd and 25 μg α1–α3 H2Dd proteins failed to affect survivals of third-party C3H (H2k k) heart allografts, namely 9.0 ± 0.0 days in treated versus 7.8 ± 0.5 days in untreated hosts. Thus, the combination of two H2K d and H2Dd proteins is more effective in prolonging allograft survival than a single protein produced in a bacterial expression system. A single PV injection (day 0) of 25 μg α1–α 2 H2Kd and 25 μg α1–α 2 H2Dd proteins to C57BL/10 mice prolonged the survival of BALB/c heart allografts to 22.4 ± 4.5 days. Within a WF to ACI rat heart allograft system, a single PV injection of 20 μg 70–77 u-RT1.Aa induced specific tolerance of allografts. This therapy could be combined with CsA to induce transplantation tolerance. However, combination of 70–77u-RT1.Aa with CTLA4Ig, rapamycin, or AG-490 effectively blocked the induction of transplantation tolerance. Tolerance generated by allochimeric protein could be adoptively transferred to naive recipients. Intragraft cytokine mRNA levels showed a bias towards a Th2-type phenotype. Additionally, studies of cytokine signaling and activation of transcription factors revealed a requirement that these pathways remain available for signaling in order for transplantation tolerance to occur. These studies suggest that the generation of regulatory cells are required for the induction of transplantation tolerance through the use of allochimeric proteins. ^

A study in the molecular and cellular functions of GSK3beta in prostate adenocarcinoma

Kinases are part of a complex network of signaling pathways that enable a cell to respond to changes in environmental conditions in a regulated and coordinated way. For example, Glycogen Synthase Kinase 3 beta (GSK3β) modulates conformational changes, protein-protein interaction, protein degradation, and activation of unique domains in proteins that transduce signals from the extracellular milieu to the nucleus. ^ In this project, I investigated the expression and function that GSK3β exhibits in prostate cells. The capacity of GSK3β to regulate two transcription factors (JUN and CREB), which are known to be inversely utilized in prostate tumor cells, was measured. JUN/AP1 is constitutively activated in PC-3 cells; whereas, CREB/CRE activity is ∼20 fold less than the former. GSK3β overexpression obliterates JUN/AP1 activity. With respect to CREB GSK3β increases CREB/CRE activity. Cellular levels of active GSK3β can determine whether JUN or CREB is preferentially active in the PC-3s. Theoretically, in response to a particular cellular context or stimulus, a cell may coordinate JUN and CREB function by regulating GSK3β.^ A comparison of various prostate cell lines showed that active GSK3β is less expressed in normal prostate epithelial cells than in tumor cells. Differentially expressed active (GSK3β) may correlate with progression of prostate carcinoma. If a known marker associated with carcinoma of the prostate could be shown to be regulated by GSK3β then, further study of GSK3β may lead to a better understanding of both possible prevention of the disease and improved therapy for advanced stages. ^ The androgen receptor (AR) is an intriguing phosphoprotein whose regulation is potentially determined by a variety of kinases. One of these is (GSK3β) I found that (GSK3β) is a regulator of the androgen receptor in both the unliganded and liganded states. It can inhibit AR function as measured by reporter assays. Also, GSK3β associates with the AR at the DNA binding domain because deletion constructs expressing either the n-terminus or the c-terminus (both having the DBD in common) immunoprecipitated with GSK3β. Increased understanding of how GSK3β functions in prostate cancer would provide clues into how (1) certain signal pathways are coordinated and (2) the androgen receptor may be regulated. ^

NFkappaB and STAT binding elements participate in regulation of MUC1 expression in normal and transformed mammary epithelial cells

The MUC1 gene encodes a transmembrane mucin glycoprotein that is overexpressed in several cancers of epithelial origin, including those of breast, pancreas, lung, ovary, and colon. Functions of MUC1 include protection of mucosal epithelium, modulation of cellular adhesion, and signal transduction. Aberrantly increased expression of MUC1 in cancer cells promotes tumor progression through adaptation of these functions. Some regulatory elements participating in MUC1 transcription have been described, but the mechanisms responsible for overexpression are largely unknown. A region of MUC1 5′ flanking sequence containing two conserved potential cytokine response elements, an NFκB site at −589/−580 and a STAT binding element (SBE) at −503/−495, has been implicated in high level expression in breast and pancreatic cancer cell lines. Persistent stimulation by proinflammatory cytokines may contribute to increased MUC1 transcription by tumor cells. ^ T47D breast cancer cells and normal human mammary epithelial cells (HMEC) were used to determine the roles of the κB site and SBE in basal and stimulated expression of MUC1. Treatment of T47D cells and HMEC with interferon-γ (IFNγ) alone enhanced MUC1 expression at the level of transcription, and the effect of IFNγ was further stimulated by tumor necrosis factor-α (TNFα). MUC1 responsiveness to these cytokines was modest in T47D cells but clearly evident in HMEC. Transient transfection of T47D cells with mutant MUC1 promoter constructs revealed that the κB site at −589/−580 and the SBE at −503/−495 and were required for cooperative stimulation by TNFα and IFNγ. Electrophoretic mobility shift assays (EMSA) revealed that the synergy was mediated not by cooperative binding of transcription factors but by the independent actions of STAT1α and NFκB p65 on their respective binding sites. Independent mutations in the κB site and SBE abrogated cytokine responsiveness and reduced basal MUC1 promoter activity by 45–50%. However, only the κB site appeared to be constitutively activated in T47D cells, in part by NFκB p65. These findings implicate two cytokine response elements in the 5 ′ flanking region of MUC1, specifically a κB site and a STAT binding element, in overexpression of MUC1 in breast cancer cells. ^

Functional analysis of BMP4 signaling in mouse neural development

During early mouse neural development, bone morphogenetic protein (BMP) signaling patterns the dorsal neural tube and defines distinct neural progenitor cell domains along the dorsoventral axis. Unlike the ventral signaling molecule Sonic hedgehog, which has long-range activity by establishing a concentration gradient in the ventral neural tube, these dorsally expressed BMPs appear to have a limited domain of action. This raises questions as to how BMP activity is restricted locally and how restricted BMP signaling directs dorsal neural patterning and differentiation. I hypothesize that BMPs are restricted in the dorsal neural tube for correct dorsoventral patterning. ^ Previous studies have shown that the positively charged basic amino acids located at the N-terminus of several BMPs are essential for heparin binding and diffusion. This provides a novel tool to address these questions. Here I adapted a UAS/GAL4 bigenic mouse system to control the ectopic expression of BMP4 and a mutant form of BMP4 that lacks a subset of the N-terminal basic amino acids. The target genes, UAS-Bmp4 and UAS-mBmp4 , were introduced into the Hprt locus by gene targeting in mouse embryonic stem cells. The expression of the GAL4 transactivator was driven by a roof plate specific Wnt1 promoter. ^ The bigenic mouse embryos exhibit phenotype variations, ranging from mid/hindbrain defects, hemorrhage, and eye abnormalities to vasculture formation. Embryonic death starts around E11.5 because of severe hemorrhage. The different expression levels of the activated transgene may account for the phenotype variation. Further marker analysis reveals that mutant BMP4 induces ectopic expression of the dorsal markers MSX1/2 and PAX7 in the ventral neural tube. In addition, the expression of the ventral neural marker NKX2.2 is affected by the expanded BMP4 activity, indicating that ectopic BMP signaling can antagonize ventral signaling. Comparison of the phenotypes of the Wnt1/ Bmp4 and Wnt1/mBmp4 bigenic embryos that express transgenes at the same level, respectively, shows that mutant BMP4 causes the expansion of dorsal neural fates ventrally while wild type BMP4 does not, suggesting that mutant BMP4 acts farther than wild type BMP4. Together, these data suggest that the N-terminus basic amino acid core controls BMP4 long-range activity in neural development, and that BMP signaling patterns the dorsal neural tube through a secondary signaling pathway that involves homeodomain transcription factors MSX1/2 and PAX7. ^

Molecular mechanisms for the insulin sensitizing activity of rexinoids in skeletal muscle of diabetic (Db/Db) mice

Rexinoids are synthetic agonists for the retinoid X receptors (RXRs), a member of the nuclear receptor family of ligand-activated transcription factors. Rexinoids have been shown to lower serum glucose and insulin levels in animal models of type 2 diabetes. However the mechanisms that are responsible for the insulin-sensitizing action of rexinoids are largely unknown. Skeletal muscle accounts for the majority of insulin-regulated whole-body glucose disposal and impaired insulin action in muscle is an important contributor to the pathophysiology of type 2 diabetes. Glucose transport is a rate-limiting step in glucose utilization. The goal of these studies is to examine the mechanisms of the anti-diabetic activity of rexinoids in skeletal muscle of diabetic db/db mice. The results we have obtained showed that treatment of db/db mice with rexinoids for two weeks resulted in a significant increase in insulin-stimulated glucose transport activity in skeletal muscle. Insulin stimulates glucose transport in muscle via the regulation of both the insulin receptor substrate-1 (IRS-1)/Akt pathway and the Cbl-associated protein (CAP)/Cbl pathway. Rexinoids increased the insulin-stimulated IRS-1 tyrosine phosphorylation and Akt phosphorylation without effects on the activity of the CAP/Cbl pathway. The effects of rexinoids on the IRS-1/Akt pathway were associated with a decrease in the level of IRS-1 Serine 307 phosphorylation as well as qualitative and quantitative alterations in the fatty acyl-CoAs present within the muscle cells. In addition, rexinoids increased the expression of uncoupling protein 3 (UCP3) and activation of AMPK in diabetic muscle. This effect may also enhance the IRS-1/Akt signaling. We believe that it is the concerted activation of the IRS-1/Akt and AMPK signaling systems, a pharmacological mechanism that as far as we know, is unique to rexinoids, that results in the anti-diabetic effects of these drugs. Our results also suggest that the glucose-lowering mechanism of rexinoids is distinct from that of the thiazolidinediones (TZDs), peroxisome proliferator-activated receptor γ (PPARγ) agonists with well-characterized anti-diabetic activity. Rexinoids appear to represent a novel class of insulin sensitizers, with potential applications for the treatment of type 2 diabetes. ^

Retinoic receptor gamma in squamous epithelial tumorigenesis

Retinoids, important modulators of squamous epithelial differentiation and proliferation, are effective in the treatment and prevention of squamous epithelial cancers, including squamous cell carcinomas (SCCs) of the skin. However, the mechanism is not well understood. Retinoids exert their effects primarily through two nuclear receptor families, retinoic acid receptors (RARα, β and γ) and retinoid X receptors (RXR(α, β and γ), ligand-dependent DNA-binding transcription factors that are members of the steroid hormone receptor superfamily. Retinoid receptor loss has been correlated with squamous epithelial malignancy. This has lead to the hypothesis that reduced RARγ expression and the resulting suppression of retinoid signaling contributes to squamous epithelial malignancy. To test this hypothesis, I attempted to reduce or abolish expression of RARγ, the predominant RAR in squamous epithelia, in several nontumorigenic human squamous epithelial cell lines. The most useful of these cell lines has been SqCCY1, the human head and neck squamous cell carcinoma cell line, along with several subclones stably transfected with RARγ sense and antisense expression constructs. By several criteria, we observed an overall suppression of squamous differentiation in RARγ sense transfectants and an enhancement in RARγ antisense transfectants, relative to parental SqCCY1 cells. We also observed that both sense and antisense cells could form tumors in athymic mice in vivo, while parental SqCCY1 cells could not. Although these results appear contradictory, several conclusions can be drawn. First, loss of RARγ contributes to squamous epithelial tumorigenesis. Second, overexpression of RARγ leads to tumor formation, suppressing differentiation and promoting proliferation, possibly due to a competitive inhibition of limiting concentrations of RXRα, a common heterodimeric partner for many nuclear receptors in addition to RARs, representing a mechanism for RARγ to modulate squamous epithelial homeostasis. The cause for tumorigenesis in the two conditions is likely due to different mechanisms/roles of RARγ in the cell, with the former as a retinoid signaling regulator; and the latter as an RXRα concentration modulator. Finally, High level of RARγ expression sensitizes cells to environmental RA, enhancing RARγ/RXRα-mediated RA signaling. Therefore, RA should be used in skin lesions with suppressed RARγ expression levels, not in skin lesions with overexpressed RARγ levels. ^

Antitumor activity of an Ets protein, PEA3, in breast cancer cell lines MDA-MB-361DYT2 and BT474M1

Polyomavirus enhancer activator 3 (PEA3) is a member of the Ets family of transcription factors. We demonstrated in a previous study that, through down-regulating the HER-2/neu oncogene at the transcriptional level, PEA3 can inhibit the growth and tumor development of HER-2/neu-overexpressing ovarian cancer cells. Here, we established stable clones of the human breast cancer cell line MDA-MB-361DYT2 that express PEA3 under the control of a tetracycline-inducible promoter. The expression of PEA3 in this cell line inhibited cell growth and resulted in cell cycle delay in the G1 phase independently of the HER-2/neu down-regulation. In an orthotopic breast cancer model, we showed that expression of PEA3 inhibited tumor growth and prolonged the survival of tumor-bearing mice. In a parallel experiment in another breast cancer cell line, BT474M1, we were unable to obtain stable PEA3-inducible transfectants, which suggests that PEA3 possessed a strong growth inhibitory effect in this cell line. Indeed, PEA3 coupled with the liposome SN2 demonstrated therapeutic effects in mice bearing tumors induced by BT474M1. These results provide evidence that the PEA3 gene could function as an antitumor and gene therapy agent for human breast cancers. ^

Genetic mechanisms of Na+, K+-ATPase regulation in hypertension

The spontaneously hypertensive rat (SHR) is a model of essential hypertension. During the early development of hypertension, the SHR demonstrates increased proximal tubule (PT) Na+ reabsorption. I hypothesized that the increased PT Na+ reabsorption exhibited by the young SHR was due to altered sub-cellular distribution of Na+, K +-ATPase compared to the normotensive Wistar Kyoto (WKY). The hypothesis is supported, herein, by observations of greater Na+, K +-ATPase α 1 abundance in PT plasma membrane and lower abundance in late endosomes of 4wk SHR despite no difference in total PT α 1 abundance. There is a greater amount of Ser-18 unphosphorylated α 1 in the 4wk SHR PT. Total PT Na+, K+-ATPase γ abundance is greater in SHR at 4wk and 16wk but γ abundance in plasma membrane is greater only at 4wk. The phosphatase, calcineurin, was chosen for study because it is involved in the stimulation of Na+, K +-ATPase. No difference in calcineurin coding sequence, expression, or activity was observed in SHR. Gene expression arrays were next used to find candidate genes involved in the regulation of Na+, K +-ATPase. The first candidate analyzed was soluble epoxide hydrolase (sEH). The gene encoding sEH (EPHX2) showed lower expression in SHR. There was also a reduction in sEH protein abundance but there was no correlation between protein abundance and blood pressure in F2 progeny. Two EPHX2 alleles were identified, an ancestral allele and a variant allele containing four polymorphisms. sEH activity was greater in animals carrying the variant allele but the inheritance of the variant allele did not correlate with blood pressure. Gene expression arrays also led to the examination of genes involved in redox balance/Na+, K+-ATPase regulation. A pattern of lower expression of genes involved in reactive radical detoxification in SHR was discerned. Six transcription factor binding sites were identified that occurred more often in these genes. Three transcription factors that bind to the HNF1 site were expressed at lower levels in SHR. This points to the HNF1 transcriptional complex as an important trans-acting regulator of a wide range of genes involved in altered redox balance in SHR. ^

STAT3 activation by the ErbB2 receptor and development of peptide-based STAT3 inhibitors

Stats (s&barbelow;ignal t&barbelow;ransducer and a&barbelow;ctivator of t&barbelow;ranscription) are latent transcription factors that translocate from the cytoplasm to nucleus. Constitutive activation of Stat3α by upstream oncoproteins and receptor tyrosine kinases has been found in many human tumors and tumor-derived cell lines and it is often correlated with the activation of ErbB-2. In order to explore the involvement of ErbB-2 in the activation of Stat3 and the mechanisms underlying this event, an erbB-2 point mutant was used as a model of a constitutively activated receptor. Phenylalanine mutations (Y-F) were made in the receptor's autophosphorylation sites and their ability to activate Stat3α was evaluated. Our results suggest that Stat3α and Janus tyrosine kinase 2 associates with ErbB-2 prior to tyrosine phosphorylation of the receptor and that full activation of Stat3α by ErbB-2 requires the participation of other non-receptor tyrosine kinases. Both Src and Jak2 kinases contribute to the activation of Stat3α while only Src binds to ErbB-2 only when the receptor is tyrosine phosphorylated. Our results also suggest that tyrosine 1139 may be important for Src SH2 domain association since a mutant lacking this tyrosine reduces the ability of the Src SH2 domain to bind to ErbB-2 and significantly decreases its ability to activate Stat3α. ^ In order to disrupt aberrant STAT3α activation which contributes to tumorigenesis, we sought small molecules which can specifically bind to the STAT3 SH2 domain, thereby abolishing its ability of being recruited into receptors, and also blocking the dimer formation required for STAT3α activation. A phosphopeptide derived from gp130 was found to have a high affinity to STAT3 SH2 domain, and we decided to use this peptide as the base for further modifications. A series of peptide based compounds were designed and tested using electrophoretic mobility shift assay and fluorescence polarization assay to evaluate their affinity to the STAT3 SH2 domain. Two promising compounds, DRIV-73C and BisPOM, were used for blocking STAT3α activity in cell culture. Either can successfully impair STAT3α activation induced by IL-6 stimulation in HepG2 cells. BisPOM proved to be the more effective in blocking STAT3α tyrosine phosphorylation in induced cells and tumor cell lines, and was the more potent in inhibiting STAT3 dependent cell growth. ^

Role of entorhinal cortical plasticity in spatial and contextual memory

It is well accepted that the hippocampus (HIP) is important for spatial and contextual memories, however, it is not clear if the entorhinal cortex (EC), the main input/output structure for the hippocampus, is also necessary for memory storage. Damage to the EC in humans results in memory deficits. However, animal studies report conflicting results on whether the EC is necessary for spatial and contextual memory. Memory consolidation requires gene expression and protein synthesis, mediated by signaling cascades and transcription factors. Extracellular-signal regulated kinase (ERK) cascade activity is necessary for long-term memory in several tasks, including those that test spatial and contextual memory. In this work, we explore the role of ERK-mediated plasticity in the EC on spatial and contextual memory. ^ To evaluate this role, post-training infusions of reversible pharmacological inhibitors specific for the ERK cascade that do not affect normal neuronal activity were targeted directly to the EC of awake, behaving animals. This technique provides spatial and temporal control over the inhibition of the ERK cascade without affecting performance during training or testing. Using the Morris water maze to study spatial memory, we found that ERK inhibition in the EC resulted in long-term memory deficits consistent with a loss of spatial strategy information. When animals were allowed to learn and consolidate a spatial strategy for solving the task prior to training and ERK inhibition, the deficit was alleviated. To study contextual memory, we trained animals in a cued fear-conditioning task and saw an increase in the activation of ERK in the EC 90 minutes following training. ERK inhibition in the EC over this time point, but not at an earlier time point, resulted in increased freezing to the context, but not to the tone, during a 48-hour retention test. In addition, animals froze maximally at the time the shock was given during training; similar to naïve animals receiving additional training, suggesting that ERK-mediated plasticity in the EC normally suppresses the temporal nature of the freezing response. These findings demonstrate that plasticity in the EC is necessary for both spatial and contextual memory, specifically in the retention of behavioral strategies. ^

Novel molecular insights into human tooth agenesis

The development of dentition is a fascinating process that involves a complex series of epithelial-mesenchymel signaling interactions. That such a precise process frequently goes awry is not surprising. Indeed, tooth agenesis is one of the most commonly inherited disorders in humans that affects up to twenty percent of the population and imposes significant functional, emotional and financial burdens on patients. Mutations in the paired box domain containing transcription factor PAX9 result in autosomal dominant tooth agenesis that primarily involves posterior dentition. Despite these advances, little is known about how PAX9 mediates key signaling actions in tooth development and how aberrations in PAX9 functions lead to tooth agenesis. As an initial step towards providing evidence for the pathogenic role of mutant PAX9 proteins, I performed a series of molecular genetic analyses aimed at resolving the structural and functional defects produced by a number of PAX9 mutations causing non-syndromic posterior tooth agenesis. It is likely that the pathogenic mechanism underlying tooth agenesis for the first two mutations studied (219InsG and IIe87Phe) is haploinsufficiency. For the six paired domain missense mutations studied, the lack of functional defects observed for three of the mutant proteins suggests that these mutations altered PAX9 function through alternate mechanisms. Next, I explored further the nature of the partnership between Pax9 and the Msx1 homeoprotein and their role in the expression of a downstream effector molecule, Bmp4. When viewed in the context of events occurring in dental mesenchyme, the results of these studies indicate that the Pax9-Msx1 protein interaction involves the localized up-regulation of Bmp4 activity that is mediated by synergistic interactions between the two transcription factors. Importantly, these assays corroborate in vivo data from mouse genetic studies and support reports of Pax9-dependent expression of Bmp4 in dental mesenchyme. Taken together, these results suggest that PAX9 mutations cause an early developmental defect due to an inability to maintain the inductive potential of dental mesenchyme through involvement in a pathway involving Msx1 and Bmp4. ^

The role of deregulated E2F1 expression in brain tumors

Mutations disabling the retinoblastoma (Rb) pathway are among the most common in human cancers, including brain cancer. These mutations promote tumor development through deregulated control of the E2F family of transcription factors. E2F1 belongs to a class of E2F's identified as transcriptional activators and involved in the G1/S phase transition of the cell. However, E2F-1 presents with a paradox as it is considered to have membership in two gene classes, functioning as both an oncogene and a tumor suppressor. This unusual trait generates a degree of uncertainty on the role that E2F1 plays in the development or maintenance of any given tumor. Here we show that E2F1 functions as an oncogene in brain tumors through the generation of mice engineered to overexpress E2F1 specifically within glial cells and neuronal progenitors as directed by the GFAP promoter. Mice carrying the transgene develop with high penetrance a phenotype characterized by neurological deficits including paresia, ataxia, head tilt and seizures. MRI imagining of the tgE2F1 mice reveals a low incidence of mild hydrocephalus, and most notably, histological analysis demonstrates that 25% of tgE2F1 mice present with the spontaneous formation of malignant brain tumors. Overall these neoplasms show histological features from a wide range of aggressive brain cancers including medulloblastoma, choroid plexus carcinoma, primary neuroectodermic tumor and malignant gliomas. Isolation and characterization of astrocytes from the tgE2F1 animal reveals a highly proliferative population of cells with 55% ± 2.5 of the tgE2F1astrocytes, 35% ± 3.4 normal mouse astrocytes in S-phase and the acquired capacity to grow in anchorage independent conditions. Additionally tgE2F1 astrocytes show an aberrant phenotype with random chromosomal fusions and nearly all cells demonstrating polyploidy. Taken together, this model forces a comparison to human brain tumor formation. Mouse age as related to tumoral mimics the human scenario with juvenile tgE2F1 mice presenting embryonal tumors typically identified in children, and older tgE2F1 mice demonstrating gliomas. In this regard, this study suggests a global role for E2F1 in the formation and maintenance of multilineage brain tumors, irrefutably establishing E2F1 as an oncogene in the brain. ^

A novel role for neural crest cells in thymus organogenesis

Classical ablation studies have shown that neural crest cells (NCC) are critical for thymus organogenesis, though their role in this process has never been determined. We have used a mouse model deficient in NCC near the thymus rudiment to investigate the role of NCC in thymus organogenesis. Splotch mice exhibit a lack of NCC migration due to mutation in the gene encoding the transcription factor Pax 3. Homozygous mutants, designated Pax3Sp/Sp, display a range of phenotypes including spina bifida, cardiac outflow tract deformities, and craniofacial deformities. Pax3Sp/Sp, mice have also been reported to have hypoplastic and abnormal thymi, which is consistent with the expected result based on the classical ablation studies. However, in contrast to the dogma, we find that the thymus lobes in Pax3Sp/Sp, mice are even larger in size than those of littermate controls, although they fail to migrate and are therefore ectopic. Differentiation of the thymic epithelial compartments occurs normally, including the ability to import hematopoietic precursors, until the embryos die at embryonic day E13.0. We also investigated the patterning of the third pharyngeal pouch which gives rise to both the thymus and the parathyroid. Using RNA probes to detect expression of transcription factors exclusively expressed in the ventral, thymus- or dorsal, parathyroidfated domains of the E11.5 third pouch, we show that the parathyroid domain is restricted and the thymus-fated domain is expanded in Pax3Sp/Sp, embryos. Furthermore, mixing of the boundary between these domains occurs at E12.0. These results necessitate reconsideration of the previously accepted role for NCC in thymus organogenesis. NCC are not required for outgrowth of the thymus up to E13.0, and most strikingly, we have discovered a novel role for NCC in establishing parathyroid versus thymus fate boundaries in the third pharyngeal pouch. ^

Tp53-dependent repression of cell cycle target genes: Global and mechanistic analysis

Most studies of p53 function have focused on genes transactivated by p53. It is less widely appreciated that p53 can repress target genes to affect a particular cellular response. There is evidence that repression is important for p53-induced apoptosis and cell cycle arrest. It is less clear if repression is important for other p53 functions. A comprehensive knowledge of the genes repressed by p53 and the cellular processes they affect is currently lacking. We used an expression profiling strategy to identify p53-responsive genes following adenoviral p53 gene transfer (Ad-p53) in PC3 prostate cancer cells. A total of 111 genes represented on the Affymetrix U133A microarray were repressed more than two fold (p ≤ 0.05) by p53. An objective assessment of array data quality was carried out using RT-PCR of 20 randomly selected genes. We estimate a confirmation rate of >95.5% for the complete data set. Functional over-representation analysis was used to identify cellular processes potentially affected by p53-mediated repression. Cell cycle regulatory genes exhibited significant enrichment (p ≤ 5E-28) within the repressed targets. Several of these genes are repressed in a p53-dependent manner following DNA damage, but preceding cell cycle arrest. These findings identify novel p53-repressed targets and indicate that p53-induced cell cycle arrest is a function of not only the transactivation of cell cycle inhibitors (e.g., p21), but also the repression of targets that act at each phase of the cell cycle. The mechanism of repression of this set of p53 targets was investigated. Most of the repressed genes identified here do not harbor consensus p53 DNA binding sites but do contain binding sites for E2F transcription factors. We demonstrate a role for E2F/RB repressor complexes in our system. Importantly, p53 is found at the promoter of CDC25A. CDC25A protein is rapidly degraded in response to DNA damage. Our group has demonstrated for the first time that CDC25A is also repressed at the transcript level by p53. This work has important implications for understanding the DNA damage cell cycle checkpoint response and the link between E2F/RB complexes and p53 in the repression of target genes. ^