The immune response to a fungus in pancreatic cancer samples.

Pancreatic ductal adenocarcinoma (PDAC) is a poor prognosis cancer with an .aggressive growth profile that is often diagnosed at late stage and that has few curative or therapeutic options. PDAC growth has been linked to alterations in the pancreas microbiome, which could include the presence of the fungus Malassezia. We used RNA-sequencing to compare 14 paired tumor and normal (tumor adjacent) pancreatic cancer samples and found Malassezia RNA in both the PDAC and normal tissues. Although the presence of Malassezia was not correlated with tumor growth, a set of immune- and inflammatory-related genes were up-regulated in the PDAC compared to the normal samples, suggesting that they are involved in tumor progression. Gene set enrichment analysis suggests that activation of the complement cascade pathway and inflammation could be involved in pro PDAC growth.

GSI-I (Z-LLNle-CHO) inhibits γ-secretase and the proteosome to trigger cell death in precursor-B acute lymphoblastic leukemia.

Gamma secretase inhibitors (GSIs) comprise a growing class of compounds that interfere with the membrane-bound Notch signaling protein and its downstream intra-nuclear transcriptional targets. As GSI-I (Z-LLNle-CHO) is also a derivative of a widely used proteosome inhibitor MG-132, we hypothesized that this compound might be active in precursor-B acute lymphoblastic leukemia (ALL) cell lines and patient samples. We found that GSI-I treatment of precursor-B ALL blasts induced apoptotic cell death within 18-24 h. With confirmation using RNA and protein analyses, GSI-I blocked nuclear accumulation of cleaved Notch1 and Notch2, and inhibited Notch targets Hey2 and Myc. Microarray analyses of 207 children with high-risk precursor-B ALL demonstrate that Notch pathway expression is a common feature of these neoplasms. However, microarray studies also implicated additional transcriptional targets in GSI-I-dependent cell death, including genes in the unfolded protein response, nuclear factor-κB and p53 pathways. Z-LLNle-CHO blocks both γ-secretase and proteosome activity, inducing more robust cell death in precursor-B ALL cells than either proteosome-selective or γ-secretase-selective inhibitors alone. Using Z-LLNle-CHO in a nonobese diabetes/severe combined immunodeficiency (NOD/SCID) precursor-B ALL xenograft model, we found that GSI-I alone delayed or prevented engraftment of B-lymphoblasts in 50% of the animals comprising the experimental group, suggesting that this compound is worthy of additional testing.

Absolute myocardial blood flow determination using real-time myocardial contrast echocardiography during adenosine stress: comparison with single-photon emission computed tomography.

OBJECTIVE: To assess the feasibility and diagnostic accuracy of real-time myocardial contrast echocardiography (MCE)-derived absolute myocardial blood flow for detection of myocardial perfusion abnormalities compared with simultaneous technetium 99 m sestamibi single-photon emission computed tomography (SPECT). DESIGN: Prospective study. SETTING: Tertiary-care medical institution. PATIENTS: 79 patients with known or suspected coronary artery disease. INTERVENTIONS: Simultaneous SPECT and real-time MCE during adenosine stress. MAIN OUTCOME MEASURES: Absolute myocardial blood flow (MBF, ml/min/g), microbubble velocity (beta, min(-1)), and reserve values. Endpoints included sensitivity, specificity, positive likelihood ratio (LR+) or negative likelihood ratio (LR-) and area under the curve (AUC) of the receiver operating characteristic curve. RESULTS: Reserve measurements were feasible in 975 of 1343 segments (73%); of these, 130 segments (13%) were abnormal by SPECT. MCE perfusion parameters clearly distinguished abnormal from normal segments for beta reserve (1.13 (0.99) vs 2.22 (1.36), p<0.001) and MBF reserve (1.80 (2.29) vs 3.69 (2.79), p<0.001). The beta reserve cut-off of 1.60 provided the following: AUC, 0.787; sensitivity, 82%; specificity, 66%; LR+, 2.40; and LR-, 0.28. The MBF reserve cut-off of 1.90 provided the following: AUC, 0.779; sensitivity, 73%; specificity, 72%; LR+, 2.69; and LR-, 0.37. MBF reserve had an AUC of 0.773 for the left anterior descending coronary artery, 0.885 for the left circumflex coronary artery and 0.739 for the right coronary artery. CONCLUSIONS: Real-time MCE-derived absolute MBF, beta, and reserve values are feasible and accurate for detecting myocardial perfusion abnormalities as defined by SPECT.

Oncogenic mutations cause dramatic, qualitative changes in the transcriptional activity of c-Myb.

The v-Myb oncoprotein encoded by Avian Myeloblastosis Virus is highly oncogenic, induces leukemias in chickens and mice and transforms immature hematopoietic cells in vitro. The v-Myb protein is a mutated and truncated version of c-Myb, a DNA-binding transcription factor expressed in many cell types that is essential for normal hematopoiesis. Previous studies suggested that two types of differences, DNA binding domain mutations and the deletion of a C-terminal negative regulatory domain were important for increasing the transforming activity of v-Myb. Here, we combined structure-function studies of the v-Myb and c-Myb proteins with unbiased microarray-based transcription assays to compare the transcriptional specificities of the two proteins. In human cells, the v-Myb and c-Myb proteins displayed strikingly different activities and regulated overlapping, but largely distinct sets of target genes. Each type of mutation that distinguished v-Myb from c-Myb, including the N- and C-terminal deletions, DNA binding domain changes and mutations in the transcriptional activation domain, affected different sets of target genes and contributed to the different activities of c-Myb and v-Myb. The results suggest that v-Myb is not just a de-repressed version of c-Myb. Instead, it is a distinct transcriptional regulator with a unique set of activities.

The conserved DNA binding domain mediates similar regulatory interactions for A-Myb, B-Myb, and c-Myb transcription factors.

The vertebrate A-Myb, B-Myb, and c-Myb proteins comprise a family of related transcription factors that share a highly conserved DNA binding domain. Although all three proteins are capable of binding the same sites in DNA, they have distinct, but overlapping patterns of expression and are presumed to be regulated independently. Here we show that the transcriptional activity of all three vertebrate Myb proteins can be severely inhibited by coexpression of a dominant-negative allele of p100, a coactivator protein that interacts with Myb DNA binding domains. Thus, the conserved Myb domains mediate interactions with common sites in DNA, as well as common regulators, suggesting that the proteins provide alternative or complementary responses to common upstream signaling pathways.

The MHC class II-associated chicken invariant chain shares functional properties with its mammalian homologs.

The nucleotide sequence of chicken invariant chain (Ii) was determined, and the amino acid sequence similarity with human Ii is 61%. Certain regions important for the biological function of human Ii are highly conserved between chicken and mammals. The cytoplasmic tail of chicken Ii fused to the plasma membrane reporter molecule neuraminidase relocated the protein to endosomes. Moreover, like the mammalian orthologs, the cytoplasmic tail was found to contain two independent leucine-based endosomal sorting signals. Chicken Ii was found to interact with human Ii and crosslinking studies also indicate that chicken Ii assembles as a trimer. The chicken Ii can furthermore bind the human MHC class II (HLA-DR1). Many of the functional properties between the chicken Ii and its mammalian orthologs are thus maintained in spite of their sequence differences.

Myb binding proteins: regulators and cohorts in transformation.

The c-Myb and v-Myb proteins are transcription factors that regulate cell proliferation and differentiation. Both Myb proteins have been shown to interact with a number of cellular proteins, some of which are transcription factors that cooperate to activate specific promoters, while others regulate the transcriptional activity of Myb in specific contexts. By comparing and analysing the types of proteins that bind Myb, and the conserved domains of Myb that interact with other proteins, conclusions can be drawn regarding the role of specific partner proteins in the regulation of gene expression, cell proliferation and disease.

Pim-1 kinase and p100 cooperate to enhance c-Myb activity.

The pim-1 oncogene is regulated by hematopoietic cytokine receptors, encodes a serine/threonine protein kinase, and cooperates with c-myc in lymphoid cell transformation. Using a yeast two-hybrid screen, we found that Pim-1 protein binds to p100, a transcriptional coactivator that interacts with the c-Myb transcription factor. Pim-1 phosphorylated p100 in vitro, formed a stable complex with p100 in animal cells, and functioned downstream of Ras to stimulate c-Myb transcriptional activity in a p100-dependent manner. Thus, Pim-1 and p100 appear to be components of a novel signal transduction pathway affecting c-Myb activity, linking all three to the cytokine-regulated control of hematopoietic cell growth, differentiation, and apoptosis.

Point mutations in v-Myb disrupt a cyclophilin-catalyzed negative regulatory mechanism.

The c-Myb protein is controlled by intramolecular interactions, and point mutations can enhance its oncogenic activity. We tested whether conformational changes regulate c-Myb and found that Cyp-40, a widely distributed cyclophilin and peptidyl-prolyl isomerase, could inhibit c-Myb DNA binding activity. Inhibition by Cyp-40 required both its C-terminal protein-interaction domain, which bound specifically to c-Myb, and its N-terminal catalytic domain and was blocked by the competitive inhibitor cyclosporin A. Cyp-40 failed to bind or inhibit the oncogenic derivative v-Myb, which has a mutated Cyp-40 binding site. These results suggest that mutations in v-Myb allow it to evade a negative regulatory mechanism mediated by enzymes such as Cyp-40, and implicate peptidyl-prolyl isomerases in the regulation of transcription, transformation, and differentiation.

The EVES motif mediates both intermolecular and intramolecular regulation of c-Myb.

The c-Myb transcription factor is a proto-oncoprotein whose latent transforming activity can be unmasked by truncation of either terminus. Because both ends of Myb are involved in negative regulation, we tested whether they could associate in a two-hybrid assay and identified a carboxy-terminal motif that interacts with the amino-terminal DNA-binding domain. The EVES motif is highly conserved in vertebrate c-Myb proteins and contains a known site of phosphorylation previously implicated in the negative regulation of c-Myb. Interestingly, a related EVES motif is present in p100, a ubiquitously expressed transcriptional coactivator found in diverse species. We show that p100 interacts with and influences the activity of c-Myb, implicating it in the regulation of c-Myb, differentiation, and cell growth. Our results suggest that Myb is regulated by a novel mechanism in which intramolecular interactions and conformational changes control the intermolecular associations among Myb, p100, and the transcriptional apparatus.

Vintage reds and whites: combinatorial transcription factor utilization in hematopoietic differentiation.

Pluripotent hematopoietic stem cells can differentiate into a number of distinct specialized cell types; however, no single lineage-specific master regulators have been identified that can activate individual patterns of gene expression. Recent evidence suggests that such lineage determination is regulated by a combinatorial matrix of regulatory proteins with overlapping tissue specificities which cooperate to define individual cell types.

C/EBP beta regulation of the tumor necrosis factor alpha gene.

Activated macrophages contribute to chronic inflammation by the secretion of cytokines and proteinases. Tumor necrosis factor alpha (TNF alpha) is particularly important in this process because of its ability to regulate other inflammatory mediators in an autocrine and paracrine fashion. The mechanism(s) responsible for the cell type-specific regulation of TNF alpha is not known. We present data to show that the expression of TNF alpha is regulated by the transcription factor C/EBP beta (NF-IL6). C/EBP beta activated the TNF alpha gene promoter in cotransfection assays and bound to it at a site which failed to bind the closely related protein C/EBP alpha. Finally, a dominant-negative version of C/EBP beta blocked TNF alpha promoter activation in myeloid cells. Our results implicate C/EBP beta as an important regulator of TNF alpha by myelomonocytic cells.

Myb and NF-M: combinatorial activators of myeloid genes in heterologous cell types.

The c-Myb transcription factor regulates the differentiation of immature erythroid, lymphoid, and myeloid cells, although only the latter cells become transformed by the v-myb oncogene. These are also the only cells that express the Myb-regulated gene mim-1, suggesting that Myb requires tissue-specific, cooperating factors to activate such genes. Here, we investigated the tissue-specific regulation of the mim-1 promoter and found that it not only contains binding sites for Myb but also for NF-M, a myeloid-specific transcription factor that probably corresponds to mammalian C/EBP beta. Both types of binding sites were found to be required for full activity of the promoter. Remarkably, ectopic coexpression of Myb and NF-M proteins in erythroid cells or fibroblasts was sufficient to induce endogenous markers of myeloid differentiation, like the mim-1 and lysozyme genes. Our results indicate that c-Myb and NF-M proteins act as a bipartite, combinatorial signal that regulates the expression of myeloid-specific genes, even in heterologous cell types.

The NF-M transcription factor is related to C/EBP beta and plays a role in signal transduction, differentiation and leukemogenesis of avian myelomonocytic cells.

Retroviral oncogenes encode nuclear regulators of gene expression or signal transduction molecules, such as protein kinases, which stimulate the activity of cellular transcription factors. Here we describe the cloning of NF-M, a myeloid-specific transcription factor related to C/EBP beta, which is a target of activated protein kinases. NF-M stimulates the expression of the gene encoding cMGF, a myeloid cell-specific growth factor, creating an autocrine growth loop crucial to oncogene transformation of myeloid cells. The NF-M protein bound directly to the cMGF gene promoter and activated its transcription, even in erythroid cells where the promoter is usually inactive. In addition, a truncated, dominant-negative form of NF-M inhibited cMGF expression in macrophages, indicating that NF-M is required for the normal activation of the gene. When multipotent hematopoietic progenitor cells were stimulated to differentiate, NF-M expression was induced at a very early stage, suggesting that the transcription factor plays a role in lineage commitment. The stimulation of transformed myelomonocytic cells or of normal peripheral blood macrophages with kinases or LPS or TPA respectively, led to the rapid redistribution of NF-M protein from the cell bodies to the nucleus, consistent with the notion that NF-M was directly affected by such treatments. Our data indicate that NF-M plays a key role in myelomonocytic differentiation, in signal transduction during macrophage activation and in the development of myelogenous leukemia.

Expression patterns of c-myb and of v-myb induced myeloid-1 (mim-1) gene during the development of the chick embryo.

The v-myb oncogene of the acute avian leukemia virus E26 encodes a transcription factor that directly regulates the promyelocyte-specific mim-1 gene (Ness, S.A., Marknell, A. and Graf, T. Cell, 59, 1115-1125). We have investigated the relationship between the c-myb proto-oncogene and the transcription of the mim-1 gene both in vitro and in vivo. We demonstrate that the c-myb protein can transactivate the transcription of mim-1 in a transient transfection assay. In the chick embryo, we confirm that mim-1 is specifically expressed during granulopoiesis and we show that the expression of c-myb and mim-1 are perfectly correlated in the granulocytic spleen and pancreas. However we suggest that mim-1 is efficiently transcribed in the absence of c-myb in the yolk sac and in the promyelocytes at the onset of the colonization of the bursa of Fabricius. On the other hand c-myb transcripts detected in the early hemopoietic progenitor cells, in lymphoid cells and in proliferative epithelia are never associated with mim-1 transcription. We conclude that the granulocyte-specific mim-1 gene is regulated by c-myb-dependent and c-myb-independent mechanisms depending upon the environment in which granulocytic precursor cells differentiate.

Proposed structure for the DNA-binding domain of the Myb oncoprotein based on model building and mutational analysis.

Myb-related proteins from plants to humans are characterized by a DNA-binding domain which contains two to three imperfect repeats of approximately 50 amino acids each. Based on the evolutionary conservation of specific residues, secondary structural predictions suggest an arrangement of alpha helices homologous to that seen in the homeodomains, members of the helix-turn-helix family of DNA-binding proteins. We have used molecular modelling in conjunction with site-directed mutagenesis to test the feasibility of this structure. We propose that each Myb repeat consists of three alpha helices packed over a hydrophobic core which is built around the three highly conserved tryptophan residues. The C-terminal helix forms part of the helix-turn-helix motif and can be positioned into the major groove of B-form DNA, allowing prediction of residues critical for specificity of interaction. Modelling also allowed positioning of adjacent repeats around the major groove over an 8 bp binding site.