The Inhibitory Effect of ddC on Human Immunodeficiency Virus Replication Diminishes in Cells that are Chronically Exposed to the Drug

One possible explanation for the failure of human immunodeficiency virus type 1 (HIV-1) antiretroviral inhibitors to block the clinical progression of the infection may be a failure to maintain adequate drug levels at the site of viral replication. We have previously found that exposure of human monoblastoid cells (U937) for several months to a therapeutically relevant concentration (0.1 μM) of 2′,3′-dideoxycytidine (zalcitabine, ddC) allowed the isolation of a drug-resistant cell line characterized by a normal drug transport but a reduced ability to accumulate 2′,3′-dideoxycytidine 5′-triphosphate (the active antiretroviral form of the drug). In this paper we show that the drug-resistant cells were indistinguishable from normal cells in terms of surface CD4 receptors. The susceptibility of parental and ddC-resistant U937 cells to infection by HIV-1 was similar, as measured by proviral DNA formation. However, HIV-1 p24 production and the number of infectious virus particles produced were significantly lower in the drug-resistant compared to control cells. Addition of 0.1 μM ddC inhibited viral production by up to 92% in the control cells but had no effect on ddC-resistant cells. Thus, human cells exposed to therapeutically relevant ddC concentrations for several months show a reduced ddC anabolism and allow ddC-sensitive HIV-1 to replicate in the presence of inhibitory ddC concentrations

Seeking critical nodes in digraphs

The Critical Node Detection Problem (CNDP) consists in finding the set of nodes, defined critical, whose removal maximally degrades the graph. In this work we focus on finding the set of critical nodes whose removal minimizes the pairwise connectivity of a direct graph (digraph). Such problem has been proved to be NP-hard, thus we need efficient heuristics to detect critical nodes in real-world applications. We aim at understanding which is the best heuristic we can apply to identify critical nodes in practice, i.e., taking into account time constrains and real-world networks. We present an in-depth analysis of several heuristics we ran on both real-world and on synthetic graphs. We define and evaluate two different strategies for each heuristic: standard and iterative. Our main findings show that an algorithm recently proposed to solve the CNDP and that can be used as heuristic for the general case provides the best results in real-world graphs, and it is also the fastest. However, there are few exceptions that are thoroughly analyzed and discussed. We show that among the heuristics we analyzed, few of them cannot be applied to very large graphs, when the iterative strategy is used, due to their time complexity. Finally, we suggest possible directions to further improve the heuristic providing the best results

6-(7-Nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol, a specific glutathione S-transferase inhibitor, overcomes the multidrug resistance (MDR)-associated protein 1-mediated MDR in small cell lung cancer

In the present work, we have investigated the antitumor activity of 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol (NBDHEX) on aggressive small cell lung cancer. NBDHEX not only is cytotoxic toward the parental small cell lung cancer H69 cell line (LC50 of 2.3 +/- 0.6 mu mol/L) but also overcomes the multidrug resistance of its variant, H69AR, which overexpresses the ATP-binding cassette transporter multidrug resistance-associated protein 1 (MRP1; LC50 of 4.5 +/- 0.9 mu mol/L). Drug efflux experiments, done in the presence of a specific inhibitor of MRP1, confirmed that NBDHEX is not a substrate for this export pump. Interestingly, NBDHEX triggers two different types of cell death: a caspase-dependent apoptosis in the H69AR cells and a necrotic phenotype in the parental H69 cells. The apoptotic pathway triggered by NBDHEX in H69AR cells is associated with c-Jun NH2-terminal kinase and c-Jun activation, whereas glutathione oxidation and activation of p38(MAPK) is observed in the NBDHEX-treated H69 cells. In contrast to the parental cells, the higher propensity to die through apoptosis of the H69AR cell line may be related to the lower expression of the antiapoptotic protein Bcl-2. Therefore, down-regulation of a factor crucial for cell survival makes H69AR cells more sensitive to the cytotoxic action of NBDHEX, which is not a MRP1 substrate. We have previously shown that NBDHEX is cytotoxic toward P-glycoprotein-overexpressing tumor cell lines. Therefore, NBDHEX seems a very promising compound in the search for new molecules able to overcome the ATP-binding cassette family of proteins, one of the major mechanisms of multidrug resistance in cancer cells

A strong glutathione S-transferase inhibitor overcomes the P-glycoprotein-mediated resistance in tumor cells - 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol (NBDHEX) triggers a caspase-dependent apoptosis in MDR1-expressing leukemia cells

The new glutathione S-transferase inhibitor 6-(7-nitro-2,1,3-benzoxadiazol- 4-ylthio)hexanol (NBDHEX) is cytotoxic toward P-glycoprotein-overexpressing tumor cell lines, i.e. CEM-VBL10, CEM-VBL100, and U-2 OS/DX580. The mechanism of cell death triggered by NBDHEX has been deeply investigated in leukemia cell lines. Kinetic data indicate a similar NBDHEX membrane permeability between multidrug resistance cells and their sensitive counterpart revealing that NBDHEX is not a substrate of the P-glycoprotein export pump. Unexpectedly, this molecule promotes a caspase-dependent apoptosis that is unusual in the P-glycoprotein-overexpressing cells. The primary event of the apoptotic pathway is the dissociation of glutathione S-transferase P1-1 from the complex with c-Jun N-terminal kinase. Interestingly, leukemia MDR1-expressing cells show lower LC50 values and a higher degree of apoptosis and caspase-3 activity than their drug-sensitive counterparts. The increased susceptibility of the multidrug resistance cells toward the NBDHEX action may be related to a lower content of glutathione S-transferase P1-1. Given the low toxicity of NBDHEX in vivo, this compound may represent an attractive basis for the selective treatment of MDR1 P-glycoproteinpositive tumors

Proapoptotic activity of new glutathione S-transferase inhibitors

Selected 7-nitro-2,1,3-benzoxadiazole derivatives have been recently found very efficient inhibitors of glutathione S-transferase (GST) PI-1,(5) an enzyme which displays antiapoptotic activity and is also involved in the cellular resistance to anticancer drugs. These new inhibitors are not tripeptide glutathione-peptidomimetic molecules and display lipophylic properties suitable for crossing the plasma membrane. In the present work, we show the strong cytotoxic activity of these compounds in the following four different cell lines: K562 (human myeloid leukemia), HepG2 (human hepatic carcinoma), CCRF-CEM (human T-lymphoblastic leukemia), and GLC-4 (human small cell lung carcinoma). The LC50 values are in the micromolar/submicromolar range and are close to the ICs values obtained with GSTPI-1, suggesting that the target of these molecules inside the cell is indeed this enzyme. The cytotoxic mechanism of 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol, the most effective GSTPI-1 inhibitor, has been carefully investigated in leukemic CCRF-CEM and K562 cell lines. Western blot and immunoprecipitation analyzes have shown that 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanoI promotes in both cell lines the dissociation of the GSTPI-1 in a complex with c-jun NH2-terminal kinase (JNK). This process triggers a reactive oxygen species (ROS) -independent activation of the JNK-mediated pathway that results in a typical process of apoptosis. Besides this main pathway, in K562 cells, a ROS-mediated apoptosis partially occurs (about 30%) which involves the p38(MAPK) signal transduction pathway. The low concentration of this new compound needed to trigger cytotoxic effects on tumor cells and the low toxicity on mice indicate that the new 7-nitro-2,1,3-benzoxadiazole derivatives are promising anticancer agents

Identification of a panel of tumor-associated antigens from breast carcinoma cell lines, solid tumors and testis cDNA libraries displayed on lambda phage

BACKGROUND: Tumor-associated antigens recognized by humoral effectors of the immune system are a very attractive target for human cancer diagnostics and therapy. Recent advances in molecular techniques have led to molecular definition of immunogenic tumor proteins based on their reactivity with autologous patient sera (SEREX). METHODS: Several high complexity phage-displayed cDNA libraries from breast carcinomas, human testis and breast carcinoma cell lines MCF-7, MDA-MB-468 were constructed. The cDNAs were expressed in the libraries as fusion to bacteriophage lambda protein D. Lambda-displayed libraries were efficiently screened with sera from patients with breast cancer. RESULTS: A panel of 21 clones representing 18 different antigens, including eight proteins of unknown function, was identified. Three of these antigens (T7-1, T11-3 and T11-9) were found to be overexpressed in tumors as compared to normal breast. A serological analysis of the 21 different antigens revealed a strong cancer-related profile for at least five clones (T6-2, T6-7, T7-1, T9-21 and T9-27). CONCLUSIONS: Preliminary results indicate that patient serum reactivity against five of the antigens is associated with tumor disease. The novel T7-1 antigen, which is overexpressed in breast tumors and recognized specifically by breast cancer patient sera, is potentially useful in cancer diagnosis

The human antibody fragment DIATHIS1 specific for CEACAM1 enhances natural killer cell cytotoxicity against melanoma cell lines in vitro

Several lines of evidence show that de novo expression of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is strongly associated with reduced disease-free survival of patients affected by metastatic melanoma. Previously published investigations report that homophilic interactions between CEACAM1 expressed on natural killer (NK) cells and tumors inhibit the NK cell-mediated killing independently of major histocompatibility complex class I recognition. This biological property can be physiologically relevant in metastatic melanoma because of the increased CEACAM1 expression observed on NK cells from some patients. Moreover, this inhibitory mechanism in many cases might hinder the efficacy of immunotherapeutic treatments of CEACAM1 malignancies because of tumor evasion by activated effector cells. In the present study, we designed an in vitro experimental model showing that the human single-chain variable fragment (scFv) DIATHIS1 specific for CEACAM1 is able to enhance the lytic machinery of NK cells against CEACAM1 melanoma cells. The coincubation of the scFv DIATHIS1 with CEACAM1 melanoma cells and NK-92 cell line significantly increases the cell-mediated cytotoxicity. Moreover, pretreatment of melanoma cells with scFv DIATHIS1 promotes the activation and the degranulation capacity of in vitro-expanded NK cells from healthy donors. It is interesting to note that the melanoma cell line MelC and the primary melanoma cells STA that respond better to DIATHIS1 treatment, express higher relative levels of CEACAM1-3L and CEACAM1-3S splice variants isoforms compared with Mel501 cells that are less responsive to DIATHIS1-induced NK cell-mediated cytotoxicity. Taken together, our results suggest that the fully human antibody fragment DIATHIS1 originated by biopanning approach from a phage antibody library may represent a relevant biotechnological platform to design and develop completely human antimelanoma therapeutics of biological origin

Generation of human antibody fragments recognizing distinct epitopes of the nucleocapsid (N) SARS-CoV protein using a phage display approach

BACKGROUND: Severe acute respiratory syndrome (SARS)-CoV is a newly emerging virus that causes SARS with high mortality rate in infected people. Successful control of the global SARS epidemic will require rapid and sensitive diagnostic tests to monitor its spread, as well as, the development of vaccines and new antiviral compounds including neutralizing antibodies that effectively prevent or treat this disease. METHODS: The human synthetic single-chain fragment variable (scFv) ETH-2 phage antibody library was used for the isolation of scFvs against the nucleocapsid (N) protein of SARS-CoV using a bio panning-based strategy. The selected scFvs were characterized under genetics-molecular aspects and for SARS-CoV N protein detection in ELISA, western blotting and immunocytochemistry. RESULTS: Human scFv antibodies to N protein of SARS-CoV can be easily isolated by selecting the ETH-2 phage library on immunotubes coated with antigen. These in vitro selected human scFvs specifically recognize in ELISA and western blotting studies distinct epitopes in N protein domains and detect in immunohistochemistry investigations SARS-CoV particles in infected Vero cells. CONCLUSION: The human scFv antibodies isolated and described in this study represent useful reagents for rapid detection of N SARS-CoV protein and SARS virus particles in infected target cells

Parkour, Counter-Conducts and the Government of Difference in Post-industrial Turin

The following paper aims to offer a critical discussion of the unfolding politics of belonging and exclusion taking place in Turin's regenerating cityscape as a way to illuminate the paradoxes, tensions and daily negotiations of emerging forms of social and spatial restructuring in the post-industrial city. In developing this analysis, we engage with an integrated methodological approach that privileges the voices and experiences of about 30 young men, mostly of migrant origins and aged 16-21, practicing parkour in the city's public spaces. In addressing these issues, we focus on the participants' engagement with one of the symbols of Turin's (multi)cultural, community-oriented and creative renewal, the post-industrial urban park of Parco Dora in order to unpack the processes of inclusion/exclusion and the conduct of conduct (Rose 2000) enacted in the creation, management and use of the city's regenerating areas. Our discussion of the participants' ambivalent and contested practices in Turin's cityscape enabled us to address how these young men re-inscribe tensions, instabilities and fault-lines relational to the “selective story-telling” (Vanolo 2015, 2) characterizing Turin's narratives of consensual transformation, post-industrial renaissance and (multi)cultural vitality. In particular, by engaging with the participants' bodily and spatial negotiations in Turin's public spaces through the lens of counter-conduct (Foucault 2007[1978]), we highlight the significance of recognising and examining partial, but productive forms of urban contestation within contemporary, pacified scenarios of urban regeneration