Global Regulation of Nucleotide Biosynthetic Genes by c-Myc

The c-Myc transcription factor is a master regulator and integrates cell proliferation, cell growth and metabolism through activating thousands of target genes. Our identification of direct c-Myc target genes by chromatin immunoprecipitation (ChIP) coupled with pair-end ditag sequencing analysis (ChIP-PET) revealed that nucleotide metabolic genes are enriched among c-Myc targets, but the role of Myc in regulating nucleotide metabolic genes has not been comprehensively delineated.Here, we report that the majority of genes in human purine and pyrimidine biosynthesis pathway were induced and directly bound by c-Myc in the P493-6 human Burkitt's lymphoma model cell line. The majority of these genes were also responsive to the ligand-activated Myc-estrogen receptor fusion protein, Myc-ER, in a Myc null rat fibroblast cell line, HO.15 MYC-ER. Furthermore, these targets are also responsive to Myc activation in transgenic mouse livers in vivo. To determine the functional significance of c-Myc regulation of nucleotide metabolism, we sought to determine the effect of loss of function of direct Myc targets inosine monophosphate dehydrogenases (IMPDH1 and IMPDH2) on c-Myc-induced cell growth and proliferation. In this regard, we used a specific IMPDH inhibitor mycophenolic acid (MPA) and found that MPA dramatically inhibits c-Myc-induced P493-6 cell proliferation through S-phase arrest and apoptosis.Taken together, these results demonstrate the direct induction of nucleotide metabolic genes by c-Myc in multiple systems. Our finding of an S-phase arrest in cells with diminished IMPDH activity suggests that nucleotide pool balance is essential for c-Myc's orchestration of DNA replication, such that uncoupling of these two processes create DNA replication stress and apoptosis

Probing the relationship between Universal Stress Protein C (UspC) and flagellar biosynthesis in Escherichia coli

UspC, like other members of the UspA superfamily, is induced in response to a variety of starvation conditions and environmental insults. The biochemistry and physiological role of UspC have remained largely uncharacterized. It has been established that strains of Escherichia coli deficient in uspC lack flagella. It was hypothesized that UspC is a positive regulator of the flagellar biosynthesis pathway. To test this hypothesis, several techniques were used. The motility of a uspC deficient strain of E. coli was rescued by introduction of flhDC, the master regulator of flagellar biosynthesis, on a plasmid under a constitutive promoter. Gene expression was compared in wild type and ∆uspC E. coli using microarrays. Finally, plasmids were constructed containing promoter regions from flagellar biosynthesis genes fliA and fliC transcriptionally fused to the lux operon. The results of these experiments suggest that UspC is indeed a positive regulator of the flagellar biosynthesis pathway and offer the opportunity for future experiments to better characterize the biochemistry of this enigmatic protein

Abstract A12: Establishing in vitro and in vivo models of pancreatic ductal adenocarcinoma (PDA) utilizing both acinar and ductal lineages

Abstract As it seems that both acinar and ductal cells can give rise to PDA and that the developmental pathways for these two lineages differ, it is essential that we have models to study the biology of these cells both in vitro and in vivo. To facilitate this, we have developed in vitro organoid models using both acinoductal and ductal cells. These models involve isolation of primary acinar or ductal cells from the mouse pancreas and subsequent 3D culture in Matrigel. Importantly, these models allow us to study both WT, KRAS transformed, or otherwise manipulated primary cells in a physiologically relevant context. We have gone on to perform orthotopic allografts using these cells to study their behavior in vivo. KRAS transformed acinoductal and ductal cells exhibit markedly different phenotypes within these allografts. Ductal cells form large cystic lesions, while acinoductal cells form small, well differentiated ductal lesions within the context of a residual Matrigel plug. Preliminary data suggest that these differences may be due to differential activation of pancreatic stellate cells (PSCs). Citation Format: Jesse Handler, Dafna Bar-Sagi. Establishing in vitro and in vivo models of pancreatic ductal adenocarcinoma (PDA) utilizing both acinar and ductal lineages. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A12.</jats:p

Stability Determination of an Extemporaneously Compounded Ambrisentan Suspension by High Performance Liquid Chromatography Analysis

OBJECTIVE Ambrisentan, an endothelin receptor antagonist FDA-approved for the treatment of pulmonary arterial hypertension in adult patients, lacks an acceptable pediatric dosage form. The objective of this investigation was to determine the stability of an extemporaneously compounded ambrisentan suspension. METHODS Ambrisentan suspension was compounded to a concentration of 1 mg/mL using commercially available suspending agents. The suspension was then evenly split into 2 plastic amber prescription bottles. One bottle was stored at room temperature and under continuous fluorescent light while the other bottle was stored under refrigeration and protection from light. A fast and selective reversed-phase high-performance liquid chromatography (HPLC) method was developed and validated for the analysis of ambrisentan. HPLC analysis was performed on samples withdrawn from the stock bottles at predetermined time intervals, up to 90 days. RESULTS The developed HPLC method enabled the elution and detection of ambrisentan peak at 4.4 minutes. HPLC analysis revealed that all samples from both storage conditions retained &amp;gt;90% potency throughout the study timeframe. There were no signs of any ambrisentan breakdown products on HPLC analysis. Color and odor of the final product was also consistent throughout the 90-day storage period. CONCLUSION Ambrisentan suspension, compounded to 1 mg/mL, is stable at room temperature or under refrigeration for up to 90 days. </jats:sec

Abstract B62: The role of B regulatory cells in pancreatic cancer

Abstract Background: Formation of pancreatic ductal adenocarcinoma (PDA) is accompanied by pronounced changes in stromal responses and immune surveillance programs, which are now recognized as some of the major drivers in PDA tumor evolution, and likely contribute to its notorious resistance to therapy. However, the cellular and molecular mechanisms that underlie immune response modulation in the context of pancreatic tumorigenesis remain poorly understood. We have observed that B lymphocytes accumulate at sites of pancreatic neoplasia and infiltrate lesion-adjacent stroma. The role of B-cell-mediated immune regulation in solid cancers has only recently begun to be appreciated, and virtually nothing is known about B cell function in pancreatic tumorigenesis. Our studies test the role of B cells in the initiation of pancreatic cancer and provide novel insight into functions of B regulatory cells in cancer pathogenesis. Experimental Design and Methods: In this study, we orthotopically grafted KRasG12D- pancreatic ductal epithelial cells (PDEC) into pancreata of recipient mice with normal or selectively immunocompromised immune responses. In particular, we investigated the role of B lymphocytes in pancreatic neoplasia by implanting KRasG12D-PDEC into the B cell deficient μMT strain, and evaluated the role of specific B cell subsets using adoptive lymphocyte transfer. Results: We demonstrate that B lymphocytes are specifically expanded in pancreatic neoplasia. We find that rather than being restricted to peri-pancreatic lymph node tissue, B cells infiltrate human PanIN lesions, as well as both LSL-KRasG12D; p48-Cre (KC) and pancreata orthotopically grafted with KRasG12D-PDEC. Such infiltration is closely correlated with expression of the B cell chemoattractant CXCL13 in the stromal compartment of the pancreas. Transplantation of KRasG12D-PDEC into pancreata of syngeneic B cell deficient μMT mice resulted in reduced growth of the cancer cells, indicating that B cells exert a tumor promoting effect. This defect in growth of KRasG12D-PDEC was accompanied by robust infiltration of macrophages. We found that pancreata-associated macrophages in μMT mice with KRasG12D-PDEC lesions expressed less of TAM-like CD206 and, correspondingly, more of M1-like CD86 on their cell surface. This observation suggested that absence of B cells prevents (or reverses) pro-tumorigenic macrophage polarization in pancreatic cancer towards a more anti-tumorigenic macrophage phenotype. After a more detailed analysis of B cell subtypes, we found that a regulatory subtype of B cells (Bregs, as defined by CD19+CD1dhighCD5+ surface phenotype and IL-10 expression) is specifically expanded in pancreata of KC and orthotopically grafted animals. Correspondingly, we find that a proportion of CD20+ B cells that infiltrate human PanIN lesions express IL-10. Reconstitution of μMT mice with B regulatory cells rescued the in vivo KRasG12D-PDEC growth defect. Conclusion: These results identify a novel mode of immunomodulation in pancreatic neoplasia and suggest that regulatory B cells may have an important role in promoting pro-tumorigenic immune responses. Citation Format: Yuliya Pylayeva-Gupta, Jesse S. Handler, Cristina Hajdu, Dafna Bar-Sagi. The role of B regulatory cells in pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr B62.</jats:p

Trichromacy and ultraviolet vision in a nocturnal marsupial

Abstract Color vision among mammals is diverse and complex, with many physiological and genetic factors affecting spectral sensitivity, the ability to perceive different wavelengths of light. In this study, the color vision of the sugar glider (Petaurus breviceps), a nocturnal, gliding mammal, was examined through a series of behavioral tests, genetic analyses, and immunohistochemistry. This is the first study to classify the color vision capabilities of this species. Sugar gliders demonstrated trichromacy and ultraviolet (UV) sensitivity, the latter of which was further supported by genetic analysis. Visualization of the sugar glider retina exhibited a rod-dominant retina that expresses rhodopsin, short-wavelength sensitive 1 opsin, and long/medium-wavelength sensitive opsin. Diurnal primates were thought to be the only mammals able to visualize trichromatically, however the results of this examination and evidence from a few other marsupial studies provide support for nocturnal trichromacy in Metatheria. Intriguingly, the genetic basis for the medium-wavelength sensitivity in marsupials has yet to be discovered. Our results are evidence of a fourth Australian marsupial that is UV-trichromatic, supporting complex spectral sensitivity and UV vision as benefits to survival in nocturnal environments. Given that Rh1 sensitivity at 501 nm explains the green sensitivity behaviorally, question arises how many other nocturnal ‘dichromatic’ species use rods for trichromatic vision in mesopic light

IL35-Producing B Cells Promote the Development of Pancreatic Neoplasia

Abstract A salient feature of pancreatic ductal adenocarcinoma (PDAC) is an abundant fibroinflammatory response characterized by the recruitment of immune and mesenchymal cells and the consequent establishment of a protumorigenic microenvironment. Here, we report the prominent presence of B cells in human pancreatic intraepithelial neoplasia and PDAC lesions as well as in oncogenic Kras-driven pancreatic neoplasms in the mouse. The growth of orthotopic pancreatic neoplasms harboring oncogenic Kras was significantly compromised in B-cell–deficient mice (μMT), and this growth deficiency could be rescued by the reconstitution of a CD1dhiCD5+ B-cell subset. The protumorigenic effect of B cells was mediated by their expression of IL35 through a mechanism involving IL35-mediated stimulation of tumor cell proliferation. Our results identify a previously unrecognized role for IL35-producing CD1dhiCD5+ B cells in the pathogenesis of pancreatic cancer and underscore the potential significance of a B-cell/IL35 axis as a therapeutic target. Significance: This study identifies a B-cell subpopulation that accumulates in the pancreatic parenchyma during early neoplasia and is required to support tumor cell growth. Our findings provide a rationale for exploring B-cell–based targeting approaches for the treatment of pancreatic cancer. Cancer Discov; 6(3); 247–55. ©2015 AACR. See related commentary by Roghanian et al., p. 230. See related article by Lee et al., p. 256. See related article by Gunderson et al., p. 270. This article is highlighted in the In This Issue feature, p. 217</jats:p

Supplementary Figures S1 - S8 from IL35-Producing B Cells Promote the Development of Pancreatic Neoplasia

Supplementary Figure S1. Treatment with anti-CXCL13 antibody reduces B cell accumulation and growth of pancreatic neoplasia. Supplementary Figure S2. B cells promote growth of KrasG12D-PDEC in vivo. Supplementary Figure S3. Engraftment of GFP- KRasG12D-PDEC or GFP-KPC cells in microMT mice is accompanied by a switch in the polarization of the macrophage population. Supplementary Figure S4. Systemic perturbation of B lymphocyte maturation and differentiation in mice with pancreatic neoplasia. Supplementary Figure S5. Adoptive transfer of CD1dhighCD5+ and CD1dlowCD5- B cell subsets into microMT mice. Supplementary Figure S6. Expression of IL-10 in B cells infiltrating pancreatic cancer. Supplementary Figure S7. Expression of p35, IL12b and IL27 in B cells infiltrating pancreatic neoplasia. Supplementary Figure S8. Adoptive transfer of wild-type and IL12a-/- B cells into microMT mice.</p

Supplementary Figure Legends from IL35-Producing B Cells Promote the Development of Pancreatic Neoplasia

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