Identification of T-cell receptor a-chain genes in the chicken

T-cell receptor (TCR) -chain (TCR) and ß-chain (TCRß) genes are well characterized in mammals, while only TCRß genes have been identified in other vertebrates. To identify avian TCR genes, we used monoclonal anti-CD3 antibodies to isolate chicken TCR for peptide sequence analysis. Degenerate oligonucleotide probes were then used to isolate a candidate TCR cDNA clone that hybridized with a 1.7-kb mRNA species present only in ß T cells and in tissues populated by these cells. Southern blot analysis revealed gene rearrangement in thymocytes and ß T-cell lines. The TCR cDNA candidate encoded an openreading frame of 275 amino acids, the predicted variable (V)-, joining (J)-, and constant (C)-region amino acid sequences of which shared 40%, 60%, and 25% homology with corresponding mammalian sequences. A single C gene and 25 V genes were identified by using region-specific probes. The V cDNA probe isolated from a Vß1+ cell line reacted with transcripts from one of five Vß2+ cell lines, suggesting shared use of V genes by Vß1+ and Vß2+ T cells and the existence of other V gene families. A genomic V sequence was flanked by classical recombination signal sequences but, unlike previously defined V genes, the leader and V region were encoded by a single exon. The data indicate evolutionary conservation of the basic TCR gene structure in birds and mammal

Prevalence of Necrosis in C 2 -Ceramide-Induced Cytotoxicity in NB16 Neuroblastoma Cells

ABSTRACT The mechanism of cell death triggered by C 2 -ceramide was investigated using the NB16 neuroblastoma cell line. Treatment of NB16 cells with 20 M C 2 -ceramide for 20 h resulted in approximately 75% loss of cell viability, but only 25% of cells were scored as apoptotic based on terminal deoxynucleotidyl transferase nick-end labeling. Ultrastructural analysis revealed early development of necrotic cytoplasmic vacuolization. After 20 h of treatment with C 2 -ceramide, the majority of cells possessed necrotic morphology with pronounced cytoplasmic vacuolization and without any nuclear changes, although a quarter of the cell population also exhibited clear perinuclear chromatin condensation characteristic of apoptosis. Flow cytometric analysis of cells labeled with both annexin V and propidium iodide showed the rapid accumulation of C 2 -ceramide-treated cells in the necrotic/ late apoptotic fraction. In contrast, cells treated with tumor necrosis factor ␣ plus cycloheximide (TNF␣ ϩ CHX) first appeared in the early apoptotic fraction and then accumulated in the necrotic/late apoptotic fraction. Both C 2 -ceramide and TNF␣ ϩ CHX increased caspase 8-and 3-like activities in cytosolic extracts; however, treatment of cells with the broad-spectrum caspase inhibitor Nbenzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone protected NB16 cells from TNF␣ ϩ CHX-induced cell death but did not prevent C 2 -ceramide cytotoxicity. Although C 2 -ceramide triggered apoptosis in a fraction of the cells, cell death in the population was primarily caused by necrosis. Thus, C

Th-MYCN Mice with Caspase-8 Deficiency Develop Advanced Neuroblastoma with Bone Marrow Metastasis

Neuroblastoma, the most common extracranial pediatric solid tumor, is responsible for 15% of all childhood cancer deaths. Patients frequently present at diagnosis with metastatic disease, particularly to the bone marrow (BM). Advances in therapy and understanding of the metastatic process have been limited due in part, to the lack of animal models harboring BM disease. The widely employed transgenic model, the Th-MYCN mouse, exhibits limited metastasis to this site. Here we establish the first genetic immunocompetent mouse model for metastatic neuroblastoma with enhanced secondary tumors in the BM. This model recapitulates two frequent alterations in metastatic neuroblasoma, over-expression of MYCN and loss of caspase-8 expression. Mouse caspase-8 gene was deleted in neural crest lineage cells by crossing a Th-Cre transgenic mouse with a caspase-8 conditional knockout mouse. This mouse was then crossed with the neuroblastoma prone Th-MYCN mouse. While over-expression of MYCN by itself rarely caused bone marrow metastasis, combining MYCN overexpression and caspase-8 deletion significantly enhanced BM metastasis (37% incidence). Microarray expression studies of the primary tumors mRNAs and microRNAs revealed extracellular matrix (ECM) structural changes, increased expression of genes involved in epithelial to mesenchymal transition, inflammation and down-regulation of miR-7a and miR-29b. These molecular changes have been shown to be associated with tumor progression and activation of the cytokine transforming growth factor beta (TGF-β) pathway in various tumor models. Cytokine TGF-β can preferentially promote single cell motility and blood borne metastasis and therefore activation of this pathway may explain the enhanced BM metastasis observed in this animal model.Fil: Teitz, Tal. St. Jude Children’s Research Hospital. Department of Tumor Cell Biology; Estados UnidosFil: Inoue, Madoka. St. Jude Children’s Research Hospital. Department of Tumor Cell Biology; Estados UnidosFil: Valentine, Marcus B.. St. Jude Children’s Research Hospital. Department of Tumor Cell Biology; Estados UnidosFil: Zhu, Kejin. St. Jude Children’s Research Hospital. Department of Tumor Cell Biology; Estados UnidosFil: Rehg, Jerold E.. St. Jude Children’s Research Hospital. Department of Pathology; Estados UnidosFil: Zhao, Wei. St. Jude Children’s Research Hospital. Department of Biostatistics; Estados UnidosFil: Finkelstein, David. St. Jude Children’s Research Hospital. Department of Computational Biology; Estados UnidosFil: Wang, Yong-Dong. St. Jude Children’s Research Hospital. Hartwell Center for Bioinformatics and Biotechnology; Estados UnidosFil: Johnson, Melissa D.. St. Jude Children’s Research Hospital. Animal Imaging Center; Estados UnidosFil: Calabrese, Christopher. St. Jude Children’s Research Hospital. Animal Imaging Center; Estados UnidosFil: Rubinstein, Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; ArgentinaFil: Hakem, Razqallah. University of Toronto. Ontario Cancer Institute. Department of Medical Biophysics; CanadáFil: Weiss, William A.. University of California. Departments of Neurology, Pediatrics and Neurological Surgery; Estados UnidosFil: Lahti, Jill M.. St. Jude Children’s Research Hospital. Department of Tumor Cell Biology; Estados Unido

CDK11 Promotes Cytokine-Induced Apoptosis in Pancreatic Beta Cells Independently of Glucose Concentration and Is Regulated by Inflammation in the NOD Mouse Model

Background: Pancreatic islets are exposed to strong pro-apoptotic stimuli: inflammation and hyperglycemia, during the progression of the autoimmune diabetes (T1D). We found that the Cdk11(Cyclin Dependent Kinase 11) is downregulated by inflammation in the T1D prone NOD (non-obese diabetic) mouse model. The aim of this study is to determine the role of CDK11 in the pathogenesis of T1D and to assess the hierarchical relationship between CDK11 and Cyclin D3 in beta cell viability, since Cyclin D3, a natural ligand for CDK11, promotes beta cell viability and fitness in front of glucose. Methods: We studied T1D pathogenesis in NOD mice hemideficient for CDK11 (N-HTZ), and, in N-HTZ deficient for Cyclin D3 (K11HTZ-D3KO), in comparison to their respective controls (N-WT and K11WT-D3KO). Moreover, we exposed pancreatic islets to either pro-inflammatory cytokines in the presence of increasing glucose concentrations, or Thapsigargin, an Endoplasmic Reticulum (ER)-stress inducing agent, and assessed apoptotic events. The expression of key ER-stress markers (Chop, Atf4 and Bip) was also determined. Results: N-HTZ mice were significantly protected against T1D, and NS-HTZ pancreatic islets exhibited an impaired sensitivity to cytokine-induced apoptosis, regardless of glucose concentration. However, thapsigargin-induced apoptosis was not altered. Furthermore, CDK11 hemideficiency did not attenuate the exacerbation of T1D caused by Cyclin D3 deficiency. Conclusions: This study is the first to report that CDK11 is repressed in T1D as a protection mechanism against inflammation-induced apoptosis and suggests that CDK11 lies upstream Cyclin D3 signaling. We unveil the CDK11/Cyclin D3 tandem as a new potential intervention target in T1D

Preclinical Models for Neuroblastoma: Establishing a Baseline for Treatment

Preclinical models of pediatric cancers are essential for testing new chemotherapeutic combinations for clinical trials. The most widely used genetic model for preclinical testing of neuroblastoma is the TH-MYCN mouse. This neuroblastoma-prone mouse recapitulates many of the features of human neuroblastoma. Limitations of this model include the low frequency of bone marrow metastasis, the lack of information on whether the gene expression patterns in this system parallels human neuroblastomas, the relatively slow rate of tumor formation and variability in tumor penetrance on different genetic backgrounds. As an alternative, preclinical studies are frequently performed using human cell lines xenografted into immunocompromised mice, either as flank implant or orthtotopically. Drawbacks of this system include the use of cell lines that have been in culture for years, the inappropriate microenvironment of the flank or difficult, time consuming surgery for orthotopic transplants and the absence of an intact immune system.Here we characterize and optimize both systems to increase their utility for preclinical studies. We show that TH-MYCN mice develop tumors in the paraspinal ganglia, but not in the adrenal, with cellular and gene expression patterns similar to human NB. In addition, we present a new ultrasound guided, minimally invasive orthotopic xenograft method. This injection technique is rapid, provides accurate targeting of the injected cells and leads to efficient engraftment. We also demonstrate that tumors can be detected, monitored and quantified prior to visualization using ultrasound, MRI and bioluminescence. Finally we develop and test a "standard of care" chemotherapy regimen. This protocol, which is based on current treatments for neuroblastoma, provides a baseline for comparison of new therapeutic agents.The studies suggest that use of both the TH-NMYC model of neuroblastoma and the orthotopic xenograft model provide the optimal combination for testing new chemotherapies for this devastating childhood cancer

Two isoforms of the human cyclin C gene are expressed differentially suggesting that they may have distinct functions

Cyclin C is a highly conserved protein that is involved in divergent cellular processes. The exact roles of its isoforms are presently not very well defined and it is possible that there is a functional divergence amongst them. We therefore sought to assess the expression pattern of cyclin C1 and C2 isoforms in various human tissues and in cell cycle by using real-time PCR experiments. Our findings strongly suggest that the C2 isoform may play a presently unexplored and important role in mammalian testis and probably this isoform is the one that is mainly implicated in cell cycle regulation

Failure To Proliferate and Mitotic Arrest of CDK11(p110/p58)-Null Mutant Mice at the Blastocyst Stage of Embryonic Cell Development

The CDK11(p110) protein kinases are part of large-molecular-weight complexes that also contain RNA polymerase II, transcriptional elongation factors, and general pre-mRNA splicing factors. CDK11(p110) isoforms may therefore couple transcription and pre-mRNA splicing by their effect(s) on certain proteins required for these processes. The CDK11(p58) kinase isoform is generated from the CDK11(p110) mRNA through the use of an internal ribosome entry site in a mitosis-specific manner, suggesting that this kinase may regulate the cell cycle during mitosis. The in vivo role and necessity of CDK11(p110/p58) kinase function during mammalian development were examined by generating CDK11(p110/p58)-null mice through targeted disruption of the corresponding gene using homologous recombination. While heterozygous mice develop normally, disruption of both CDK11(p110/p58) alleles results in early embryonic lethality due to apoptosis of the blastocyst cells between 3.5 and 4 days postcoitus. Cells within these embryos exhibit both proliferative defect(s) and a mitotic arrest. These results are consistent with the proposed cellular functions of the CDK11(p110/p58) kinases and confirm that the CDK11(p110/p58) kinases are essential for cellular viability as well as normal early embryonic development