No abstract

General activation of hypoxia-inducible factor (HIF) pathways is classically associated with adverse prognosis in cancer and has been proposed to contribute to oncogenic drive. In clear cell renal carcinoma (CCRC) HIF pathways are upregulated by inactivation of the von-Hippel-Lindau tumor suppressor. However HIF-1α and HIF-2α have contrasting effects on experimental tumor progression. To better understand this paradox we examined pan-genomic patterns of HIF DNA binding and associated gene expression in response to manipulation of HIF-1α and HIF-2α and related the findings to CCRC prognosis. Our findings reveal distinct pan-genomic organization of canonical and non-canonical HIF isoform-specific DNA binding at thousands of sites. Overall associations were observed between HIF-1α-specific binding, and genes associated with favorable prognosis and between HIF-2α-specific binding and adverse prognosis. However within each isoform-specific set, individual gene associations were heterogeneous in sign and magnitude, suggesting that activation of each HIF-α isoform contributes a highly complex mix of pro- and anti-tumorigenic effects.

Cancer progression and metastasis involves interactions between tumor cells and the tumor microenvironment (TME). We reported that mice deficient for cytosolic phospholipase A 2 (cPLA 2 -KO) are protected against the development of lung tumors. The goal of this study was to examine the role of cPLA 2 in the TME. Mouse lung cancer cells (CMT167 and Lewis lung carcinoma cells) injected directly into lungs of syngeneic mice formed a primary tumor, and then metastasized to other lobes of the lung and to the mediastinal lymph nodes. Identical cells injected into cPLA 2 -KO mice showed a dramatic decrease in the numbers of secondary metastatic tumors. This was associated with decreased macrophage staining surrounding the tumor. Wild-type mice transplanted with cPLA 2 -KO bone marrow had a marked survival advantage after inoculation with tumor cells compared with mice receiving wild-type (WT) bone marrow. In vitro, coculturing CMT167 cells with bone marrow-derived macrophages from WT mice increased production of interleukin 6 (IL-6) by cancer cells. This increase was blocked in cocultures using cPLA 2 -KO macrophages. Correspondingly, IL-6 staining was decreased in tumors grown in cPLA 2 -KO mice. These data suggest that stromal cPLA 2 plays a critical role in tumor progression by altering tumor-macrophage interactions and cytokine production.

Hypoxia‐inducible factor (HIF) is the major transcriptional regulator of cellular responses to hypoxia. The two principal HIF‐α isoforms, HIF‐1α and HIF‐2α, are progressively stabilized in response to hypoxia and form heterodimers with HIF‐1β to activate a broad range of transcriptional responses. Here, we report on the pan‐genomic distribution of isoform‐specific HIF binding in response to hypoxia of varying severity and duration, and in response to genetic ablation of each HIF‐α isoform. Our findings reveal that, despite an identical consensus recognition sequence in DNA, each HIF heterodimer loads progressively at a distinct repertoire of cell‐type‐specific sites across the genome, with little evidence of redistribution under any of the conditions examined. Marked biases towards promoter‐proximal binding of HIF‐1 and promoter‐distant binding of HIF‐2 were observed under all conditions and were consistent in multiple cell type. The findings imply that each HIF isoform has an inherent property that determines its binding distribution across the genome, which might be exploited to therapeutically target the specific transcriptional output of each isoform independently.

Polymorphic differences distinguishing MHC class I subtypes often permit the presentation of shared epitopes in conformationally identical formats but can affect T-cell repertoire selection, differentially impacting autoimmune susceptibilities and viral clearance in vivo. The molecular mechanisms underlying this effect are not well understood. We performed structural, thermodynamic, and functional analyses of a conserved T-cell receptor (TCR) which is frequently expanded in response to a HIV-1 epitope when presented by HLA-B*5701 but is not selected by HLA-B*5703, which differs from HLA-B*5701 by two concealed polymorphisms. Our findings illustrate that although both HLA-B*57 subtypes display the epitope in structurally conserved formats, the impact of their polymorphic differences occurs directly as a consequence of TCR ligation, primarily because of peptide adjustments required for TCR binding, which involves the interplay of polymorphic residues and water molecules. These minor differences culminate in subtype-specific differential TCR-binding kinetics and cellular function. Our data demonstrate a potential mechanism whereby the most subtle MHC class I micropolymorphisms can influence TCR use and highlight their implications for disease outcomes.

Background: Peptidylglycine α-Amidating Monooxygenase (PAM) is solely responsible for catalysis of amidation, a biologically important post-translational modification.Results: Modification-specific antibodies reveal that peptide substrate amidation is strikingly sensitive to the exposure of cells to moderate hypoxia.Conclusion: PAM-dependent amidation has the potential to signal oxygen levels in the same range as the hypoxia-inducible factor (HIF) system.Significance: Physiological effects of hypoxia may be PAM-dependent.

The antigen-presenting cell environment in patients with cGVHD, as represented by immature blood DCs, is of donor origin but distorted in terms of subset distribution.

SummaryMost (98%) of the fat in human milk is present as triglycerides. This paper describes the use of a clarification procedure that enables the level of human milk fat to be determined by measurement of glycerol released by enzymic hydrolysis of triglycerides. The method requires only 10–50 μl milk, thus presenting a possible technique for work with small mammals, and is suitable for use with autoanalysers, permitting rapid sample throughput.