The ectonucleotidases CD39 and CD73 hydrolyze extracellular adenosine triphosphate (ATP) and adenosine diphosphate (ADP) to generate adenosine, which binds to adenosine receptors and inhibits T-cell and natural killer (NK)–cell responses, thereby suppressing the immune system. The generation of adenosine via the CD39/CD73 pathway is recognized as a major mechanism of regulatory T cell (Treg) immunosuppressive function. The number of CD39+ Tregs is increased in some human cancers, and the importance of CD39+ Tregs in promoting tumor growth and metastasis has been demonstrated using several in vivo models. Here, we addressed whether CD39 is expressed by tumor cells and whether CD39+ tumor cells mediate immunosuppression via the adenosine pathway. Immunohistochemical staining of normal and tumor tissues revealed that CD39 expression is significantly higher in several types of human cancer than in normal tissues. In cancer specimens, CD39 is expressed by infiltrating lymphocytes, the tumor stroma, and tumor cells. Furthermore, the expression of CD39 at the cell surface of tumor cells was directly demonstrated via flow cytometry of human cancer cell lines. CD39 in cancer cells displays ATPase activity and, together with CD73, generates adenosine. CD39+CD73+ cancer cells inhibited the proliferation of CD4 and CD8 T cells and the generation of cytotoxic effector CD8 T cells (CTL) in a CD39- and adenosine-dependent manner. Treatment with a CD39 inhibitor or blocking antibody alleviated the tumor-induced inhibition of CD4 and CD8 T-cell proliferation and increased CTL- and NK cell–mediated cytotoxicity. In conclusion, interfering with the CD39–adenosine pathway may represent a novel immunotherapeutic strategy for inhibiting tumor cell–mediated immunosuppression. Cancer Immunol Res; 3(3); 254–65. ©2014 AACR.

Background: Colorectal cancer (CRC) is one of the most common causes of cancer death throughout the world. In this work our aim was to study the role of the phosphoserine aminotransferase PSAT1 in colorectal cancer development. Results: We first observed that PSAT1 is overexpressed in colon tumors. In addition, we showed that after drug treatment, PSAT1 expression level in hepatic metastases increased in non responder and decreased in responder patients. In experiments using human cell lines, we showed that ectopic PSAT1 overexpression in colon carcinoma SW480 cell line resulted in an increase in its growth rate and survival. In addition, SW480-PSAT1 cells presented a higher tumorigenic potential than SW480 control cells in xenografted mice. Moreover, the SW480-PSAT1 cell line was more resistant to oxaliplatin treatment than the non-transfected SW480 cell line. This resistance resulted from a decrease in the apoptotic response and in the mitotic catastrophes induced by the drug treatment. Conclusion: These results show that an enzyme playing a role in the L-serine biosynthesis could be implicated in colon cancer progression and chemoresistance and indicate that PSAT1 represents a new interesting target for CRC therapy.

The reaction of reduced NO synthase (NOS) with molecular oxygen was studied at −30 °C. In the absence of substrate, the complex formed between ferrous NOS and O₂ was sufficiently long lived for a precise spectroscopic characterization. This complex displayed similar spectral characteristics as the oxyferrous complex of cytochrome P450 (λ_max = 416.5 nm). It then decomposed to the ferric state. The oxidation of the flavin components was much slower and could be observed only at temperatures higher than −20 °C. In the presence of substrate (l-arginine), another, 12-nm blue-shifted, intermediate spectrum was formed. The breakdown of the latter species resulted in the production ofN ^ω-hydroxy-l-arginine in a stoichiometry of maximally 52% per NOS heme. This product formation took place also in the absence of the reductase domain of NOS. Both formation of the blue-shifted intermediate and ofN ^ω-hydroxy-l-arginine required the presence of tetrahydrobiopterin (BH₄). We propose that the blue-shifted intermediate is the result of reductive activation of the oxygenated complex, and the electron is provided by BH₄. These observations suggest that the reduction of the oxyferroheme complex may be the main function of BH₄ in NOS catalysis.

Background: Intrabodies are defined as antibody molecules which are ectopically expressed inside the cell. Such intrabodies can be used to visualize or inhibit the targeted antigen in living cells. However, most antibody fragments cannot be used as intrabodies because they do not fold under the reducing conditions of the cell cytosol and nucleus.Results: We describe the construction and validation of a large synthetic human single chain antibody fragment library based on a unique framework and optimized for cytoplasmic expression. Focusing the library by mimicking the natural diversity of CDR3 loops ensured that the scFvs were fully human and functional. We show that the library is highly diverse and functional since it has been possible to isolate by phage-display several strong binders against the five proteins tested in this study, the Syk and Aurora-A protein kinases, the αβ tubulin dimer, the papillomavirus E6 protein and the core histones. Some of the selected scFvs are expressed at an exceptional high level in the bacterial cytoplasm, allowing the purification of 1 mg of active scFv from only 20 ml of culture. Finally, we show that after three rounds of selection against core histones, more than half of the selected scFvs were active when expressedin vivoin human cells since they were essentially localized in the nucleus.Conclusion: This new library is a promising tool not only for an easy and large-scale selection of functional intrabodies but also for the isolation of highly expressed scFvs that could be used in numerous biotechnological and therapeutic applications.