Identification of spontaneous mutations within the long-range limb-specific Sonic hedgehog enhancer (ZRS) that alter Sonic hedgehog expression in the chicken limb mutants oligozeugodactyly and silkie breed.

The evolutionarily conserved, non-coding ~800-base-pair (bp) zone of polarizing activity (ZPA) regulatory sequence (ZRS) controls Shh expression in the posterior limb. We report that the chicken mutant oligozeugodactyly (ozd), which lacks limb Shh expression, has a large deletion within the ZRS. Furthermore, the preaxial polydactylous, Silkie Breed chicken, which develops ectopic anterior limb Shh expression, has a single bp change within the ZRS. Using an in vivo reporter assay to examine enhancer function in the chick limb, we demonstrate that the wild-type ZRS drives ß-galactosidase reporter expression in the ZPA of both wild-type and ozd limbs. The Silkie ZRS drives ß-galactosidase in both posterior and anterior Shh domains in wild-type limb buds. These results support the hypothesis that the ZRS integrates positive and negative prepatterned regulatory inputs in the chicken model system and demonstrate the utility of the chicken limb as an efficient genetic system for gene regulatory studies.

ARTEMIS stabilizes the genome and modulates proliferative responses in multipotent mesenchymal cells.

BACKGROUND: Unrepaired DNA double-stranded breaks (DSBs) cause chromosomal rearrangements, loss of genetic information, neoplastic transformation or cell death. The nonhomologous end joining (NHEJ) pathway, catalyzing sequence-independent direct rejoining of DSBs, is a crucial mechanism for repairing both stochastically occurring and developmentally programmed DSBs. In lymphocytes, NHEJ is critical for both development and genome stability. NHEJ defects lead to severe combined immunodeficiency (SCID) and lymphoid cancer predisposition in both mice and humans. While NHEJ has been thoroughly investigated in lymphocytes, the importance of NHEJ in other cell types, especially with regard to tumor suppression, is less well documented. We previously reported evidence that the NHEJ pathway functions to suppress a range of nonlymphoid tumor types, including various classes of sarcomas, by unknown mechanisms. RESULTS: Here we investigate roles for the NHEJ factor ARTEMIS in multipotent mesenchymal stem/progenitor cells (MSCs), as putative sarcomagenic cells of origin. We demonstrate a key role for ARTEMIS in sarcoma suppression in a sensitized mouse tumor model. In this context, we found that ARTEMIS deficiency led to chromosomal damage but, paradoxically, enhanced resistance and proliferative potential in primary MSCs subjected to various stresses. Gene expression analysis revealed abnormally regulated stress response, cell proliferation, and signal transduction pathways in ARTEMIS-defective MSCs. Finally, we identified candidate regulatory genes that may, in part, mediate a stress-resistant, hyperproliferative phenotype in preneoplastic ARTEMIS-deficient MSCs. CONCLUSIONS: Our discoveries suggest that Art prevents genome damage and restrains proliferation in MSCs exposed to various stress stimuli. We propose that deficiency leads to a preneoplastic state in primary MSCs and is associated with aberrant proliferative control and cellular stress resistance. Thus, our data reveal surprising new roles for ARTEMIS and the NHEJ pathway in normal MSC function and fitness relevant to tumor suppression in mesenchymal tissues.

Colocalization of somatic and meiotic double strand breaks near the Myc oncogene on mouse chromosome 15.

Both somatic and meiotic recombinations involve the repair of DNA double strand breaks (DSBs) that occur at preferred locations in the genome. Improper repair of DSBs during either mitosis or meiosis can lead to mutations, chromosomal aberration such as translocations, cancer, and/or cell death. Currently, no model exists that explains the locations of either spontaneous somatic DSBs or programmed meiotic DSBs or relates them to each other. One common class of tumorigenic translocations arising from DSBs is chromosomal rearrangements near the Myc oncogene. Myc translocations have been associated with Burkitt lymphoma in humans, plasmacytoma in mice, and immunocytoma in rats. Comparing the locations of somatic and meiotic DSBs near the mouse Myc oncogene, we demonstrated that the placement of these DSBs is not random and that both events clustered in the same short discrete region of the genome. Our work shows that both somatic and meiotic DSBs tend to occur in proximity to each other within the Myc region, suggesting that they share common originating features. It is likely that some regions of the genome are more susceptible to both somatic and meiotic DSBs, and the locations of meiotic hotspots may be an indicator of genomic regions more susceptible to DNA damage.

Complex oncogenic translocations with gene amplification are initiated by specific DNA breaks in lymphocytes.

Chromosomal instability is a hallmark of many tumor types. Complex chromosomal rearrangements with associated gene amplification, known as complicons, characterize many hematologic and solid cancers. Whereas chromosomal aberrations, including complicons, are useful diagnostic and prognostic cancer markers, their molecular origins are not known. Although accumulating evidence has implicated DNA double-strand break repair in suppression of oncogenic genome instability, the genomic elements required for chromosome rearrangements, especially complex lesions, have not been elucidated. Using a mouse model of B-lineage lymphoma, characterized by complicon formation involving the immunoglobulin heavy chain (Igh) locus and the c-myc oncogene, we have now investigated the requirement for specific genomic segments as donors for complex rearrangements. We now show that specific DNA double-strand breaks, occurring within a narrow segment of Igh, are necessary to initiate complicon formation. By contrast, neither specific DNA breaks nor the powerful intronic enhancer Emu are required for complicon-independent oncogenesis. This study is the first to delineate mechanisms of complex versus simple instability and the first to identify specific chromosomal elements required for complex chromosomal aberrations. These findings will illuminate genomic cancer susceptibility and risk factors.

Closing the manufacturing process of dendritic cell vaccines transduced with adenovirus vectors.

Anticancer immunotherapy using dendritic cell (DC) based vaccines provides an adjuvant therapeutic strategy that is not cross reactive with conventional therapeutics. However, manufacturing of DC vaccines requires stringent adherence to Good Manufacturing Practice (GMP) methods and rigorous standardization. Optimally this includes a closed system for monocyte isolation, in combination with closed culture and washing systems and an effective vector transduction strategy. In this study, we used the Gambro Elutra to enrich monocytes from non-mobilized leukapheresis products collected from healthy donors. This approach enriched monocytes from an average frequency of 13.6+3.2% (mean+SEM), to an average frequency of 79.5+4.3% following enrichment with a yield of 79 to 100%. The monocytes were then cultured in a closed system using gas permeable Vuelife fluoroethylene propylene (FEP) bags and X-vivo-15 media containing 10 ng/ml granulocyte-macrophage colony-stimulation factor (GM-CSF) and 5 ng/ml Interleukin (IL) 4. The cultures were re-fed on days two and four, with a 25% media volume and cytokines. Following culture for seven days, the cells were harvested using a Cobe-2991 and concentrated using a bench centrifuge retrofitted with blocks to allow centrifugation of 72 ml bags and supernatant removed using a plasma extractor. This approach reduced the media volume to an average of 17.4 ml and an average DC concentration of 6.3+1.0x10(7) cells/ml, a viability of 93.8+2.2%, a purity of 88.9+3.3% and a total yield of 8.5+1.4x10(8) DCs. Based on the identification of DR+ cells as DCs we had an average yield of 46+8% using a calculation based on the number of monocytes in the apheresis product and the resulting DCs differentiated from monocytes. The use of DCs as a vaccine, required transduction with an adenovirus (Adv) vector with the tumor suppressor, p53 transgene (Adv5CMV-p53) as the antigen at a DC concentration of 9x10(6) DCs/ml at an Ad5CMV-p53: DC ratio of 20,000:1, and a 2 or 3 hour co-culture, followed by a 1:10 dilution with media and an additional 16-22 hour incubation. Following incubation, the DCs were washed twice and the supernatants removed using a plasma extractor. The average viability after infection with Ad5CMV-p53 was 87.9+/-2.6% with an average of 20.3+5.4% of the DCs expressing p53. The calculated yield of DCs following Ad5CMV-p53 transduction, based on the number of monocytes in the apheresis products, averaged 12.4+3.8%. We conclude that it is possible to efficiently manufacture Adv transduced DCs using a functionally closed system.

Dynamic clustered distribution of hemagglutinin resolved at 40 nm in living cell membranes discriminates between raft theories.

Organization in biological membranes spans many orders of magnitude in length scale, but limited resolution in far-field light microscopy has impeded distinction between numerous biomembrane models. One canonical example of a heterogeneously distributed membrane protein is hemagglutinin (HA) from influenza virus, which is associated with controversial cholesterol-rich lipid rafts. Using fluorescence photoactivation localization microscopy, we are able to image distributions of tens of thousands of HA molecules with subdiffraction resolution ( approximately 40 nm) in live and fixed fibroblasts. HA molecules form irregular clusters on length scales from approximately 40 nm up to many micrometers, consistent with results from electron microscopy. In live cells, the dynamics of HA molecules within clusters is observed and quantified to determine an effective diffusion coefficient. The results are interpreted in terms of several established models of biological membranes.

Single base pair change in the long-range Sonic hedgehog limb-specific enhancer is a genetic basis for preaxial polydactyly.

In most instances of preaxial polydactyly (PPD), Sonic Hedgehog (Shh), an essential limb patterning signal, is ectopically expressed in an anterior region of the developing limb in addition to the normal posterior domain. It is thought that this anterior Shh expression leads directly to the development of the extra digits. Recent reports have identified a conserved limb-specific Shh enhancer approximately 1 megabase upstream of the Shh transcription initiation site, and individual base pair changes within this region are associated with PPD. We report here that a single base pair change within this enhancer is sufficient to drive beta-galactosidase expression in both anterior and posterior limb domains, similar to Shh expression in animal PPD models, whereas a wild-type construct is expressed only in the posterior limb, similar to Shh expression in normal embryos. These findings provide the first direct evidence that a single base pair change within the limb-specific Shh enhancer acts as a genetic basis for PPD.

Isolation of the chicken Lmbr1 coding sequence and characterization of its role during chick limb development.

In the developing amniote limb, anteroposterior (A/P) patterning is controlled through secretion of the Sonic Hedgehog (SHH) protein by cells in the zone of polarizing activity (ZPA) located in the posterior mesoderm. In the chicken mutant oligozeugodactyly (ozd), Shh is expressed normally in the entire embryo with the exception that it is undetectable in the developing limbs; this results in the loss of specific bones in wings and legs. The ozd phenotype is similar to that of humans affected with acheiropodia (ACHR), and the ACHR mutation has been mapped to a deletion of exon 4 and portions of introns 3 and 4 in the LMBR1 gene. We have cloned the chick ortholog of LMBR1, Lmbr1, and report that, in chick, Lmbr1 is expressed within the ZPA. Although the ozd phenotype is similar to ACHR, the open reading frame of Lmbr1 is normal in ozd. Sequence analysis of Lmbr1 intron 3 demonstrated that this particular genomic region segregates with the ozd phenotype. In addition, overexpression of Lmbr1 throughout the developing limb mesoderm resulted in morphologically normal limbs. Collectively, these data suggest that the Lmbr1 coding sequence is not required for normal chick limb development. We propose that the ozd mutation is linked to the genomic region containing Shh and Lmbr1.