Erythropoietin Receptor Expression Is a Potential Prognostic Factor in Human Lung Adenocarcinoma

Recombinant human erythropoietins (rHuEPOs) are used to treat cancer-related anemia. Recent preclinical studies and clinical trials, however, have raised concerns about the potential tumor-promoting effects of these drugs. Because the clinical significance of erythropoietin receptor (EPOR) signaling in human non-small cell lung cancer (NSCLC) also remains controversial, our aim was to study whether EPO treatment modifies tumor growth and if EPOR expression has an impact on the clinical behavior of this malignancy. A total of 43 patients with stage III–IV adenocarcinoma (ADC) and complete clinicopathological data were included. EPOR expression in human ADC samples and cell lines was measured by quantitative real-time polymerase chain reaction. Effects of exogenous rHuEPOα were studied on human lung ADC cell lines in vitro. In vivo growth of human ADC xenografts treated with rHuEPOα with or without chemotherapy was also assessed. In vivo tumor and endothelial cell (EC) proliferation was determined by 5-bromo-2’-deoxy-uridine (BrdU) incorporation and immunofluorescent labeling. Although EPOR mRNA was expressed in all of the three investigated ADC cell lines, rHuEPOα treatment (either alone or in combination with gemcitabine) did not alter ADC cell proliferation in vitro. However, rHuEPOα significantly decreased tumor cell proliferation and growth of human H1975 lung ADC xenografts. At the same time, rHuEPOα treatment of H1975 tumors resulted in accelerated tumor endothelial cell proliferation. Moreover, in patients with advanced stage lung ADC, high intratumoral EPOR mRNA levels were associated with significantly increased overall survival. This study reveals high EPOR level as a potential novel positive prognostic marker in human lung ADC.     

Epithelial-Mesenchymal Transition Markers and HER3 Expression Are Predictors of Elisidepsin Treatment Response in Breast and Pancreatic Cancer Cell Lines

Elisidepsin (elisidepsin trifluoroacetate, Irvalec®, PM02734) is a new synthetic depsipeptide, a result of the PharmaMar Development Program that seeks synthetic products of marine origin-derived compounds. Elisidepsin is a drug with antiproliferative activity in a wide range of tumors. In the present work we studied and characterized the mechanisms associated with sensitivity and resistance to elisidepsin treatment in a broad panel of tumor cell lines from breast and pancreas carcinomas, focusing on different factors involved in epithelial-mesenchymal transition (EMT) and the use of HER family receptors in predicting the in vitro drug response. Interestingly, we observed that the basal protein expression levels of EMT markers show a significant correlation with cell viability in response to elisidepsin treatment in a panel of 12 different breast and pancreatic cancer cell lines. In addition, we generated three elisidepsin treatment-resistant cell lines (MCF-7, HPAC and AsPC-1) and analyzed the pattern of expression of different EMT markers in these cells, confirming that acquired resistance to elisidepsin is associated with a switch to the EMT state. Furthermore, a direct correlation between basal HER3 expression and sensitivity to elisidepsin was observed; moreover, modulation of HER3 expression levels in different cancer cell lines alter their sensitivities to the drug, making them more resistant when HER3 expression is downregulated by a HER3-specific short hairpin RNA and more sensitive when the receptor is overexpressed. These results show that HER3 expression is an important marker of sensitivity to elisidepsin treatment.     

Neuroimmunomodulation in human autoimmune liver disease

Bidirectional interaction between immune and nervous systems is considered an important biological process in health and disease. However, little is known about the mechanisms involved in their interaction in the human liver. This study examines the distribution of intrahepatic NPY, SP immunoreactive (IR) nerve fibers and their antomical relationship with immunocells containing tumor necrosis factor-α (TNF-α) and nuclear factor κB (NF-κB) in patients with autoimmune hepatitis. Liver specimens were obtained from control liver and autoimmune hepatitis patients. The immunoreactivity was determined by immunohisto- and immunocytochemistry and confocal laser microscopy. In hepatitis, the number of NPY-IR and SP-IR nerve fibers increased significantly. These IR nerve fibers were in very close contact with the lymphocytes. In healthy controls, no NPY-IR, SP-IR or NF-κB IR lymphocytes and only a few TNF-α positive cells, were observed. In hepatitis, some of the lymphocytes showed immunoreactivity for SP and NPY in the portal area. Fluorescent double-labeled immunostaining revealed that in these cells NPY did not colocalize with TNF-α or NF-κB. However, some of the SP fluorescence-positive immune cells exhibited immunostaining for p65 of NF-κB, where their labeling was detected in the nuclei. Under the electronmicroscope, these cells could be identified (lymphocytes, plasmacells and mast cells). The gap between the IR nerve fibers and immunocells was 1 μm or even less. Overexpression of SP in lymphocytes may amplify local inflammation, while NPY may contribute to liver homeostasis in hepatitis. Neural immunomodulation (SP antagonists and NPY) might be a novel therapeutic concept in the management of liver inflammation.     

Mechanism of flexibility control for ATP access of hepatitis C virus NS3 helicase

Hepatitis C virus (HCV) NS3 helicase couples adenosine triphosphate (ATP) binding and hydrolysis to polynucleotide unwinding. Understanding the regulation mechanism of ATP binding will facilitate targeting of the ATP-binding site for potential therapeutic development for hepatitis C. T324, an amino acid residue connecting domains 1 and 2 of NS3 helicase, has been suggested as part of a flexible hinge for opening of the ATP-binding cleft, although the detailed mechanism remains largely unclear. We used computational simulation to examine the mutational effect of T324 on the dynamics of the ATP-binding site. A mutant model, T324A, of the NS3 helicase apo structure was created and energy was minimized. Molecular dynamics simulation was conducted for both wild type and the T324A mutant apo structures to compare their differences. For the mutant structure, histogram analysis of pairwise distances between residues in domains 1 and 2 (E291-Q460, K210-R464 and R467-T212) showed that separation between the two domains was reduced by ～10% and the standard deviation by ～33%. Root mean square fluctuation (RMSF) analysis demonstrated that residues in close proximity to residue 324 have at least 30% RMSF value reductions in the mutant structure. Solvent RMSF analysis showed that more water molecules were trapped near D290 and H293 in domain 1 to form an extensive interaction network constraining cleft opening. We also demonstrated that the T324A mutation established a new atomic interaction with V331, revealing that an atomic interaction cascade from T324 to residues in domains 1 and 2 controls the flexibility of the ATP-binding cleft.     

Phylogenetic Analyses of Teleki Grapevine Rootstocks Using Three Chloroplast DNA Markers

Teleki rootstocks are used in grapevine-producing countries all over the world. They represent one of the largest groups of available rootstocks but their origin is still in dispute although they have been regarded as Vitis berlandieri × V. riparia hybrids. To investigate their possible origin, we amplified and sequenced three chloroplast regions, two non-coding spacers (trnL-F, trnS-G) and the trnL group I intron in a core collection of Teleki rootstocks representing widespread accessions and related wild North American grape species (V. berlandieri, V. riparia and V. rupestris). Concatenated sequence data coupled with microstructural changes discovered in the chloroplast regions provided data to trace the maternal ancestry of the Teleki lines. All chloroplast regions showed both nucleotide and length variation. Length mutations in the non-coding regions represented mostly simple sequence repeats of poly-A and -T stretches. These indel characters exhibited additional diversity comparable with the nucleotide diversity and increased resolution of the phylogenetic trees. We found that a group of Teleki accessions position together with the wild grape species V. riparia. Another group of Teleki rootstocks formed a sister group to the other North American species V. berlandieri. These clades had moderate support values, and they do not share ancestry with other accessions of Teleki rootstocks resolved with high support value in the V. riparia clade. It seems that Teleki-Kober 5BB and 125 AA accessions might have a V. berlandieri maternal background. We also found great differences within putative clones of Teleki 5C and Teleki-Kober 5BB suggesting that the selection of these accessions was performed on heterogenous or mislabeled plant material collectively maintained under these names.     

The Highly Conserved Layer-3 Component of the HIV-1 gp120 Inner Domain Is Critical for CD4-Required Conformational Transitions

The trimeric envelope glycoprotein (Env) of human immunodeficiency virus type 1 (HIV-1) mediates virus entry into host cells. CD4 engagement with the gp120 exterior envelope glycoprotein subunit represents the first step during HIV-1 entry. CD4-induced conformational changes in the gp120 inner domain involve three potentially flexible topological layers (layers 1, 2, and 3). Structural rearrangements between layer 1 and layer 2 have been shown to facilitate the transition of the envelope glycoprotein trimer from the unliganded to the CD4-bound state and to stabilize gp120-CD4 interaction. However, our understanding of CD4-induced conformational changes in the gp120 inner domain remains incomplete. Here, we report that a highly conserved element of the gp120 inner domain, layer 3, plays a pivot-like role in these allosteric changes. In the unliganded state, layer 3 modulates the association of gp120 with the Env trimer, probably by influencing the relationship of the gp120 inner and outer domains. Importantly, layer 3 governs the efficiency of the initial gp120 interaction with CD4, a function that can also be fulfilled by filling the Phe43 cavity. This work defines the functional importance of layer 3 and completes a picture detailing the role of the gp120 inner domain in CD4-induced conformational transitions in the HIV-1 Env trimer.     

A Self-compartmentalizing Hexamer Serine Protease from Pyrococcus Horikoshii: SUBSTRATE SELECTION ACHIEVED THROUGH MULTIMERIZATION*

Oligopeptidases impose a size limitation on their substrates, the mechanism of which has long been under debate. Here we present the structure of a hexameric serine protease, an oligopeptidase from Pyrococcus horikoshii (PhAAP), revealing a complex, self-compartmentalized inner space, where substrates may access the monomer active sites passing through a double-gated “check-in” system, first passing through a pore on the hexamer surface and then turning to enter through an even smaller opening at the monomers'' domain interface. This substrate screening strategy is unique within the family. We found that among oligopeptidases, a residue of the catalytic apparatus is positioned near an amylogenic β-edge, which needs to be protected to prevent aggregation, and we found that different oligopeptidases use different strategies to achieve such an end. We propose that self-assembly within the family results in characteristically different substrate selection mechanisms coupled to different multimerization states.