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Intratumoral hypoxia and Hypoxia Inducible Factor-1α (HIF-1α)-dependent CD39/CD73 ecto-enzymes may govern the accumulation of tumor-protecting extracellular adenosine and signaling through the A2A adenosine receptors (A2AR) in tumor microenvironments (TME). Here, we explored the conceptually novel motivation to use supplemental oxygen as a treatment to inhibit the hypoxia/HIF-1α-CD39/CD73-driven accumulation of extracellular adenosine in the TME in order to weaken the tumor protection. We report that hyperoxic breathing (60% O2) decreased the TME hypoxia, as well as levels of HIF-1α and downstream target proteins of HIF-1α in the TME according to proteomics studies in mice. Importantly, oxygenation also down-regulated the expression of adenosine-generating ecto-enzymes and significantly lowered levels of tumor-protecting extracellular adenosine in the TME. Using supplemental oxygen as a tool in studies of the TME, we also identified FHL-1 as a potentially useful marker for the conversion of hypoxic into normoxic TME. Hyperoxic breathing resulted in the up-regulation of antigen-presenting MHC-class I molecules on tumor cells and in the better recognition and increased susceptibility to killing by tumor-reactive cytotoxic T cells. Therapeutic breathing of 60% oxygen resulted in the significant inhibition of growth of established B16.F10 melanoma tumors and prolonged survival of mice. Taken together, the data presented here provide proof-of principle for the therapeutic potential of systemic oxygenation to convert the hypoxic, adenosine-rich and tumor-protecting TME into a normoxic and extracellular adenosine-poor TME that, in turn, may facilitate tumor regression. We propose to explore the combination of supplemental oxygen with existing immunotherapies of cancer.

Sex-dependent differences in adaptation to famine have long been appreciated, thought to hinge on female versus male preferences for fat versus protein sources, respectively. However, whether these differences can be reduced to neurons, independent of typical nutrient depots, such as adipose tissue, skeletal muscle, and liver, was heretofore unknown. A vital adaptation to starvation is autophagy, a mechanism for recycling amino acids from organelles and proteins. Here we show that segregated neurons from males in culture are more vulnerable to starvation than neurons from females. Nutrient deprivation decreased mitochondrial respiration, increased autophagosome formation, and produced cell death more profoundly in neurons from males versus females. Starvation-induced neuronal death was attenuated by 3-methyladenine, an inhibitor of autophagy; Atg7 knockdown using small interfering RNA; or L-carnitine, essential for transport of fatty acids into mitochondria, all more effective in neurons from males versus females. Relative tolerance to nutrient deprivation in neurons from females was associated with a marked increase in triglyceride and free fatty acid content and a cytosolic phospholipase A2-dependent increase in formation of lipid droplets. Similar sex differences in sensitivity to nutrient deprivation were seen in fibroblasts. However, although inhibition of autophagy using Atg7 small interfering RNA inhibited cell death during starvation in neurons, it increased cell death in fibroblasts, implying that the role of autophagy during starvation is both sex- and tissue-dependent. Thus, during starvation, neurons from males more readily undergo autophagy and die, whereas neurons from females mobilize fatty acids, accumulate triglycerides, form lipid droplets, and survive longer.

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Purpose: Regulatory T cell (Treg) frequency and activity are increased in cancer patients and play a major role in tumor escape. Although disease progression is favored by the presence of Treg, mechanisms used by Treg to suppress antitumor immunity are unknown. The ectonucleotidases CD39 and CD73 are expressed in Treg and convert ATP into immunosuppressive adenosine. In this study, the involvement of the adenosinergic pathway in Treg-mediated suppression in head and neck squamous cell carcinoma (HNSCC) patients was evaluated. Experimental Design: HNSCC patients with an active disease (n = 19) and patients with no evident disease after therapy (n = 14) were studied. Ectonucleotidase expression on CD4 + T cells and CD4 + CD25high Treg was evaluated by flow cytometry and compared with normal controls. Ectonucleotidase activity was also compared within these three groups. The data were analyzed for associations of ectonucleotidase expression/function with disease stage. Results: The percentages and expression levels of CD39 and CD73 in CD4 + T cells and Treg were greater in HNSCC than in normal controls and highest in patients with no evident disease. Patients' Treg hydrolyzed ATP at higher rates and produced higher levels of adenosine than normal controls' Treg. The increased frequency and enzymatic activity of CD4 + CD39 + cells corresponded to increased adenosine-mediated suppression of effector T cells, which was partly inhibited by ARL67156, an ectonucleotidase inhibitor, and by ZM241385, a selective A 2a /A 2b receptor antagonist. Conclusions: CD39+ Treg frequency and adenosine-mediated suppression are significantly increased in HNSCC patients. The adenosinergic pathway is involved in Treg-mediated immunosuppression in cancer and its attenuation could be a promising immunotherapeutic strategy for patients with HNSCC. ( Head and neck squamous cell carcinoma (HNSCC), arising from the mucosal epithelium, is the fifth most common type of cancer worldwide and the sixth most common cause of cancerrelated mortality (1, 2). Although advances in surgical methods, chemotherapy, and radiation have improved patients' treatment and quality of life, the overall outcome and survival rate among patients with this disease have not notably improved (3). Therefore, novel therapeutic strategies are necessary for the treatment of HNSCC. Recent studies have focused on the dynamic interplay and coevolution of antitumor immune responses and tumor progression in HNSCC (4, 5). These studies have shown that T lymphocytes play a major albeit contradictory role in local tumor expansion, metastasis, and neoangiogenesis. In most human cancers, regulatory T cells (Treg), a small subset of CD4 + T cells, are significantly increased in the peripheral blood as well as in the tumor microenvironment (6-8). Treg are phenotypically defined as CD4 + CD25high FOXP3 + and modulate immune responses by suppressing functions of other T cells. To date, at least two types of Treg are known to exist in man: (a) naturally occurring Treg, which devel...

We discovered that renal injury releases 2,3-cAMP (positional isomer of 3,5-cAMP) into the interstitium. This finding motivated a novel hypothesis: renal injury leads to activation of an extracellular 2,3-cAMP-adenosine pathway (i.e. metabolism of extracellular 2,3-cAMP to 3-AMP and 2-AMP, which are metabolized to adenosine, a retaliatory metabolite). In isolated rat kidneys, arterial infusions of 2,3-cAMP (30 mol/ liter) increased the mean venous secretion of 3-AMP (3,400-fold), 2-AMP (26,000-fold), adenosine (53-fold), and inosine (adenosine metabolite, 30-fold). Renal injury with metabolic inhibitors increased the mean secretion of 2,3-cAMP (29-fold), 3-AMP (16-fold), 2-AMP (10-fold), adenosine (4.2-fold), and inosine (6.1-fold) while slightly increasing 5-AMP (2.4-fold). Arterial infusions of 2-AMP and 3-AMP increased secretion of adenosine and inosine similar to that achieved by 5-AMP. Renal artery infusions of 2,3-cAMP in vivo increased urinary excretion of 2-AMP, 3-AMP and adenosine, and infusions of 2-AMP and 3-AMP increased urinary excretion of adenosine as efficiently as 5-AMP. The implications are that 1) in intact organs, 2-AMP and 3-AMP are converted to adenosine as efficiently as 5-AMP (previously considered the most important adenosine precursor) and 2) because 2,3-cAMP opens mitochondrial permeability transition pores, a pro-apoptotic/pronecrotic process, conversion of 2,3-cAMP to adenosine by the extracellular 2,3-cAMP-adenosine pathway would protect tissues by reducing a pro-death factor (2,3-cAMP) while increasing a retaliatory metabolite (adenosine).Our past work supports the existence of a biochemical mechanism of adenosine biosynthesis that involves 3Ј,5Ј-cAMP and that contributes to the extracellular levels of adenosine, i.e. the extracellular 3Ј,5Ј-cAMP-adenosine pathway (Fig. 1, left side) (1-20). In this regard, we propose that the extracellular 3Ј,5Ј-cAMP-adenosine pathway involves four sequential steps that are spatially linked and occur on a quasi-solid surface (i.e. the membrane surface). The four sequential steps are: 1) receptormediated intracellular production of 3Ј,5Ј-cAMP; 2) active transport of 3Ј,5Ј-cAMP from the intracellular compartment to the cell surface; 3) extracellular metabolism of 3Ј,5Ј-cAMP to 5Ј-AMP; and 4) extracellular conversion of 5Ј-AMP to adenosine. Independent laboratories now confirm the existence of the extracellular 3Ј,5Ј-cAMP-adenosine pathway in pial microvessels (21) and more recently in skeletal muscle (22) and the gastrointestinal tract (23). Moreover, Müller et al. (24) report the existence of a 3Ј,5Ј-cAMP-adenosine pathway in which the sequential conversion of 3Ј,5Ј-cAMP to 5Ј-AMP and 5Ј-AMP to adenosine occurs on the outward facing membrane surface of lipid droplets within adipocytes.To study in more detail the role of 3Ј,5Ј-cAMP-adenosine pathway in the kidney, we recently developed a novel assay to measure 3Ј,5Ј-cAMP and other purines in the renal venous effluent from isolated, perfused kidneys (25). Unlike commercially available kits for 3Ј,5...

Summary The QR regressor tumour (QR-32), a fibrosarcoma which is unable to grow progressively in normal syngeneic C57BL/6 mice, was able to grow progressively in 13 out of 22 mice (59%) when it was subcutaneously coimplanted with gelatin sponge. We established four culture tumour lines from the resultant tumours (QRsP tumour lines). These QRsP tumour lines were able to grow progressively in mice even in the absence of gelatin sponge. The ability of QRsP tumour cells to colonise the lungs after intravenous injection and to produce high amounts of prostaglandin E2 (PGE2) during in vitro cell culture was much greater than that of parent QR-32 cells. These biological characteristics of QR-32 cells and QRsP tumour cells were found to be stable for at least 6 months when they were maintained in culture.We also observed that QR-32 cells were able to grow progressively in five out of 12 (42%) mice after coimplantation with plastic non-adherent peritoneal cells obtained from mice which had been intraperitoneally implanted with gelatin sponge. These host cells reactive to gelatin sponge increased the production of high amounts of PGE2 by QR-32 cells during 48 h coculture. Preliminary in vitro studies implicated the involvement of hydrogen peroxide and hydroxyl radical as some of the factors necessary to induce QR-32 cells to produce high amounts of PGE2 and to accelerate tumour progression.

The RNA-dependent protein kinase (PKR) is activated by binding to double-stranded RNA (dsRNA). Activation of PKR by short-interfering RNAs (siRNAs) and stimulation of the innate immune response has been suggested to explain certain off-target effects in some RNA interference experiments. Here we show that PKR's kinase activity is stimulated in vitro 3- to 5-fold by siRNA duplexes with 19 bp and 2 nt 3′-overhangs, whereas the maximum activation observed for poly(I)•poly(C) was 17-fold over background under the same conditions. Directed hydroxyl radical cleavage experiments indicated that siRNA duplexes have at least four different binding sites for PKR's dsRNA binding motifs (dsRBMs). The location of these binding sites suggested specific nucleotide positions in the siRNA sense strand that could be modified with a corresponding loss of PKR binding. Modification at these sites with N2-benzyl-2′-deoxyguanosine (BndG) blocked interaction with PKR's dsRBMs and inhibited activation of PKR by the siRNA. Importantly, modification of an siRNA duplex that greatly reduced PKR activation did not prevent the duplex from lowering mRNA levels of a targeted message by RNA interference in HeLa cells. Thus, these studies demonstrate that specific positions in an siRNA can be rationally modified to prevent interaction with components of cellular dsRNA-regulated pathways.