Resistance to TRAIL-induced apoptosis: role of IG20 splice variants

Tumor necrosis factor receptor–related apoptosis-inducing ligand (TRAIL) can induce apoptosis primarily in cancer cells with little or no effect on normal cells; therefore, it has the potential for use in cancer therapy. TRAIL binding to death receptors DR4 and DR5 triggers the death-inducing signal complex formation and activation of procaspase-8, which in turn activates caspase-3, leading to cell death. Like FasL, TRAIL can trigger type 1 (caspase-8 caspase-3) or type 2 (caspase-8 Bid cleavage capsase-9 caspase-3) apoptotic pathways depending on the cell type. Some cancers are resistant to TRAIL treatment because most molecules in the TRAIL signaling pathway, including FLIPs and IAPs, can contribute to resistance. In addition, we have identified an essential role for splice variants of the IG20 gene in TRAIL resistance

Reactivity of the18 Neutralizing HmAbs with SARS CoV 12-510-S1 proteins.

<p>Medisorp ELISA plates were coated with 100 ng/well of Urbani and RBD mutant 12-510S1-IgG proteins and 2.5 µg/ml of each HmAb was used as the primary antibody. Anti-human IgG2 HRP mouse monoclonal antibody was used as secondary antibody. OD was measured at 450 nm. Error bars represent SD of a representative experiment performed in triplicates. (A) Urbani versus Sin845 mutant. (B) Urbani versus GD01 mutant. (C) Urbani versus GZ0402 mutant. (D) Urbani versus GZ-C mutant.</p

HmAbs to HR1 and HR2 can efficiently neutralize surrogate clinical isolates.

a<p>Likely binding region of antibodies.</p>b<p>S glycoprotein ectodomain.</p>c<p>Anti-SARS-S protein polyclonal antibody.</p

Combinations of HmAbs more efficiently inhibit the entry of SARS-CoV RBD surrogate clinical isolates.

<p>Neutralizing HmAbs binding to different regions of S protein 4D4 (S1), 1F8 (HR1), 5E9 (HR2)) were tested for their ability to neutralize pseudoviruses in different combinations as well as individually at a concentration of 6.25 µg/ml each. The virus/Ab mixture was incubated for 1 hr at 37°C then added to 293/ACE2 stable cell line. Seventy two hours later, the virus entry was determined by luciferase expression. The percentage entry inhibitions by individual antibodies as well as combinations of antibodies were calculated. Error bars represent SD of representative experiment performed in triplicates. Statistical analysis was done using Student-t test, significant differences are indicated by asterisks,<i>* p<0.05.</i></p

<i>In vitro</i> pseudovirus neutralization assay.

<p>Eighteen neutralizing HmAbs were tested against different mutant as well as Urbani pseudoviruses. Pseudoviruses equivalent to 10 ng of HIVp24 were incubated for 1 hr with 25 µg/ml of each of the HmAbs at 37°C. The virus/Ab mixtures were then added to 293/ACE2 stable cell line. Seventy two hours later, the virus entry was determined by luciferase expression. The percentage entry inhibitions obtained with Abs were calculated and normalized to HIV/Urbani-S inhibitions (A) HIV/GZ-C and HIV/Sin845 inhibitions (B) HIV/GZ0402 and HIV/GD01 inhibitions. Polyclonal rabbit immune serum (PolyAb) was used as a positive control. Error bars represent SD of a representative experiment performed in triplicates.</p

Reactivity of Urbani SARS-CoV-S protein antibodies with Urbani S1 protein and mutant S1 proteins.

<p>(A) Different dilutions of a rabbit anti-Urbani SARS-CoV-S protein immune serum were tested in an ELISA against Urbani as well as mutant S1-IgG proteins. Anti-rabbit donkey polyclonal HRP antibody was used as the secondary antibody. (B) Competitive ELISA assay: Different protein concentrations of Urbani or GZ-C proteins were pre-incubated with 5A7 antibody then the protein/Ab mixtures were tested for binding to the other protein by ELISA. OD was measured at 450 nm.</p

Down-Modulation of Expression, or Dephosphorylation, of IG20/MADD in Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand–Resistant Thyroid Cancer Cells Makes Them Susceptible to Treatment with This Ligand

Background: The IG20/MADD gene is overexpressed in thyroid cancer tissues and cell lines, and can contribute to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) resistance. The ability of the MADD protein to resist TRAIL-induced apoptosis is dependent upon its phosphorylation by Akt. Interestingly, while TRAIL induces a significant reduction in the levels of phospho-Akt (pAkt) and phospho-MADD (pMADD) in TRAIL-sensitive cells, it fails to do so in TRAIL-resistant cells. In this study, we investigated if MADD phosphorylation by Akt was contributing to TRAIL resistance in thyroid cancer cells. Methods: We determined the susceptibility of different thyroid cancer cell lines to TRAIL-induced apoptosis by fluorescence-activated cell sorting (FACS) analysis. We tested for various TRAIL resistance factors by FACS analyses or for IG20/MADD expression by quantitative reverse transcription-polymerase chain reaction. We determined the levels of pAkt and pMADD upon TRAIL treatment in thyroid cancer cells by Western blotting. We tested if down-modulation of IG20/MADD gene expression using shRNA or phosphorylation using a dominant negative Akt (DN-Akt) or pretreatment with LY294002, a PI3 kinase inhibitor, could help overcome TRAIL resistance. Result: BCPAP and TPC1 cells were susceptible, while KTC1 and FTC133 cells were resistant, to TRAIL-induced apoptosis. The differential susceptibility to TRAIL was not related to the levels of expression of death receptors, decoy receptors, or TRAIL. KTC1 and FTC133 cells showed higher levels of IG20/MADD expression relative to BCPAP and TPC1, and were rendered susceptible to TRAIL treatment upon IG20/MADD knockdown. Interestingly, upon TRAIL treatment, the pAkt and pMADD levels were reduced in TRAIL-sensitive BCPAP and TPC1 cells, while they remained unchanged in the resistant KTC1 and FTC133 cells. While expression of a constitutively active Akt in BCPAP and TPC1 cells rendered them resistant to TRAIL, pretreating KTC1 and FTC133 cells with LY294002 rendered them TRAIL-sensitive. Moreover, expression of a DN-Akt in KTC1 and FTC133 cells reduced the levels of pAkt and pMADD and sensitized them to TRAIL-induced apoptosis. Conclusion: Our results show that pMADD is an important TRAIL resistance factor in certain thyroid cancer cells and suggest that down-modulation of either IG20/MADD expression or phosphorylation can render TRAIL-resistant thyroid cancer cells sensitive to TRAIL

Knockdown of MADD and c-FLIP overcomes resistance to TRAIL-induced apoptosis in ovarian cancer cells

Objective The clinical utility of tumor necrosis factor–related apoptosis–inducing ligand (TRAIL) in the treatment of established human malignancies is limited by the development of resistance to TRAIL. We hypothesized that knockdown of map-kinase activating death domain containing protein (MADD), a TRAIL-resistance factor, may overcome TRAIL resistance in ovarian cancer cells. Study Design MADD expression in resected ovarian cancer specimens and cell lines was quantified with the use of polymerase chain reaction. Sensitivity of ovarian cancer cell lines to TRAIL, with or without MADD knockdown, was assessed. Results MADD is expressed at relatively higher levels in human malignant ovarian cancer tissues and cell lines, compared with normal ovarian tissues. The cell lines OVCA429 and OVCAR3 were susceptible, and cell lines CAOV-3 and SKOV-3 were resistant to TRAIL. MADD knockdown in CAOV-3 cells, but not in SKOV-3 cells, conferred TRAIL sensitivity. Knockdown of cellular Fas-associated death domain–like interleukin-1 beta-converting enzyme–inhibitory protein (c-FLIP) in SKOV-3 cells increased spontaneous and TRAIL-induced apoptosis, which was further increased on MADD knockdown. Conclusion MADD/c-FLIPL knockdown can render TRAIL-resistant ovarian cancer cells susceptible to TRAIL

Expression of the Ring Ligase PRAJA2 in Thyroid Cancer

Introduction: In thyroid cells, binding of TSH to its receptor increases cAMP levels, sustaining thyrocytes growth and hormone production. The main cAMP effector enzyme is protein kinase A (PKA). Praja2 is a widely expressed RING (Really Interesting New Gene) ligase, which degrades the regulatory subunits of PKA, thus controlling the strengthandduration ofPKAsignaling in response to cAMP. Differentiated thyroid cancer expresses a functional TSH receptor, and its growth and progression are positively regulated by TSH and cAMP signaling. Aim: We aimed to analyze the expression of praja2 in a group of 36 papillary thyroid cancer (PTC), 14 benign nodules, and six anaplastic thyroid cancers (ATC). Methods: We measured praja2 mRNA levels by quantitative RT-PCR and praja2 expression by Western blot and immunohistochemistry. Possible association between praja2 mRNA and the presence of known mutations was evaluated. Results:We foundastatistical significant increase ofmRNAlevels inPTCtissue samples,comparedwith benign nodules and ATC. In particular, mRNA levels were maximal in differentiated thyroid cancer (PTC), progressively decreasing in more aggressive tumors, ATC having the lowest amount of praja2 mRNA. Accordingly, higher levels of praja2 protein were detected in lysates from PTC, compared with ATC. By immunohistochemistry, in PTC sections we observed a marked increase of cytoplasmic praja2 signal, which significantly decreased in less differentiated thyroid tumors, completely disappearing in ATC. Studies in cultured cells stably expressing RET/PTC1 oncogene or mutant BRAF revealed a direct correlationbetweenpraja2mRNAlevelsandmalignant phenotype of transformed cells. Similar results were obtained using thyroid cancer tissues carrying the same mutations. Conclusions: praja2 is markedly overexpressed in differentiated thyroid cancer, and its levels inversely correlate with the malignant phenotype of the tumor. Thus, praja2 is a novel cancer-related gene whose expression is linked to the histotype and mutational status of the thyroid tumor

Age-dependent divergent effects of OX40L treatment on the development of diabetes in NOD mice

<p>Earlier, we have shown that GM-CSF derived bone marrow (BM) dendritic cells (G-BMDCs) can expand Foxp3⁺ regulatory T-cells (Tregs) through a TCR-independent, but IL-2 dependent mechanism that required OX40L/OX40 interaction. While some reports have shown suppression of autoimmunity upon treatment with an OX40 agonist, others have shown exacerbation of autoimmune disease instead. To better understand the basis for these differing outcomes, we compared the effects of OX40L treatment in 6-week-old pre-diabetic and 12-week-old near diabetic NOD mice. Upon treatment with OX40L, 6-week-old NOD mice remained normoglycemic and showed a significant increase in Tregs in their spleen and lymph nodes, while 12-week-old NOD mice very rapidly developed hyperglycemia and failed to show Treg increase in spleen or LN. Interestingly, OX40L treatment increased Tregs in the thymus of both age groups. However, it induced Foxp3⁺CD103⁺CD38⁻ stable-phenotype Tregs in the thymus and reduced the frequency of autoreactive Teff cells in 6-week-old mice; while it induced Foxp3⁺CD103⁻CD38⁺ labile-phenotype Tregs in the thymus and increased autoreactive CD4⁺ T cells in the periphery of 12-week-old mice. This increase in autoreactive CD4⁺ T cells was likely due to either a poor suppressive function or conversion of labile Tregs into Teff cells. Using <i>ex vivo</i> cultures, we found that the reduction in Treg numbers in 12-week-old mice was likely due to IL-2 deficit, and their numbers could be increased upon addition of exogenous IL-2. The observed divergent effects of OX40L treatment were likely due to differences in the ability of 6- and 12-week-old NOD mice to produce IL-2.</p