Evan P. Booy

Brevinin-2R1 semi-selectively kills cancer cells by a distinct mechanism, which involves the lysosomal-mitochondrial death pathway

Brevinin‐2R is a novel non‐hemolytic defensin that was isolated from the skin of the frog Rana ridibunda. It exhibits preferential cytotoxicity towards malignant cells, including Jurkat (T‐cell leukemia), BJAB (B‐cell lymphoma), HT29/219, SW742 (colon carcinomas), L929 (fibrosarcoma), MCF‐7 (breast adenocarcinoma), A549 (lung carcinoma), as compared to primary cells including peripheral blood mononuclear cells (PBMC), T cells and human lung fibroblasts. Jurkat and MCF‐7 cells overexpressing Bcl2, and L929 and MCF‐7 over‐expressing a dominant‐negative mutant of a pro‐apoptotic BNIP3 (ΔTM‐BNIP3) were largely resistant towards Brevinin‐2R treatment. The decrease in mitochondrial membrane potential (ΔΨm), or total cellular ATP levels, and increased reactive oxygen species (ROS) production, but not caspase activation or the release of apoptosis‐inducing factor (AIF) or endonuclease G (Endo G), were early indicators of Brevinin‐2R‐triggered death. Brevinin‐2R interacts with both early and late endosomes. Lysosomal membrane permeabilization inhibitors and inhibitors of cathepsin‐B and cathepsin‐L prevented Brevinin‐2R‐induced cell death. Autophagosomes have been detected upon Brevinin‐2R treatment. Our results show that Brevinin‐2R activates the lysosomalmitochondrial death pathway, and involves autophagy‐like cell death.

Serum cytochrome c indicates in vivo apoptosis and can serve as a prognostic marker during cancer therapy.

Despite significant progress in cancer therapy, the outcome of the treatment is often unfavorable. Better treatment monitoring would not only allow an individual more effective, patient‐adjusted therapy, but also it would eliminate some of the side effects. Using a cytochrome c ELISA that was modified to increase sensitivity, we demonstrate that serum cytochrome c is a sensitive apoptotic marker in vivo reflecting therapy‐induced cell death burden. Furthermore, increased serum cytochrome c level is a negative prognostic marker. Cancer patients whose serum cytochrome c level was normal 3 years ago have a twice as high probability to be still alive, as judged from sera samples collected for 3 years, analyzed recently and matched with survival data. Moreover, we show that serum cytochrome c and serum LDH‐activity reflect different stages and different forms of cell death. Cellular cytochrome c release is specific for apoptosis, whereas increased LDH activity is an indicator of (secondary) necrosis. Whereas serum LDH activity reflects the “global” degree of cell death over a period of time, the sensitive cytochrome c‐based method allows confirmation of the individual cancer therapy‐induced and spontaneous cell death events. The combination of cytochrome c with tissue‐specific markers may provide the foundation for precise monitoring of apoptosis in vivo, by “lab‐on‐the‐chip” technology.

Cancer-specific toxicity of apoptin is independent of death receptors but involves the loss of mitochondrial membrane potential and the release of mitochondrial cell-death mediators by a Nur77-dependent pathway

Apoptin, a small proline-rich protein derived from the chicken anaemia virus, induces cell death selectively in cancer cells. The signalling pathways of apoptin-induced, cancer cell-selective apoptosis are not well understood. Here, we demonstrate that apoptin triggers apoptosis by activating the mitochondrial/intrinsic pathway, and that it acts independently of the death receptor/extrinsic pathway. Jurkat cells deficient in either FADD or caspase-8 (which are both necessary for the extrinsic pathway) were equally as sensitive to apoptin as their parental clones. This demonstrates that apoptin is likely to act through the mitochondrial death pathway. Apoptin treatment causes a loss of mitochondrial membrane potential, and release of the mitochondrial proteins cytochrome c and apoptosis-inducing factor. Apoptin-induced cell death is counteracted by the anti-apoptotic Bcl-2 family members, Bcl-2 itself and Bcl-XL, as shown in Jurkat leukaemia cells. In addition, we describe the processing and activation of caspase-3. By contrast, cleavage of caspase-8, which is predominantly triggered by the death receptor pathway, is not observed. Furthermore, apoptin triggers the cytoplasmic translocation of Nur77, and the inhibition of Nur77 expression by siRNA significantly protects MCF7 cells from apoptin-triggered cell death. Thus, our data indicate that the apoptin death signal(s) ultimately converges at the mitochondria, and that it acts independently of the death receptor pathway.

Monoclonal and bispecific antibodies as novel therapeutics

Abstract.Gene amplification, over-expression, and mutation of growth factors, or the receptors themselves, causes increased signaling through receptor kinases, which has been implicated in many human cancers and is associated with poor prognosis. Tumor growth has been shown to be decreased by interrupting this process of extensive growth factor-mediated signaling by directly targeting either the surface receptor or the ligand and thereby preventing cell survival and promoting apoptosis. Monoclonal antibodies have long been eyed as a potential new class of therapeutics targeting cancer and other diseases. Antibody-based therapy initially entered clinical practice when trastuzumab/Herceptin became the first clinically approved drug against an oncogene product as a well-established blocking reagent for tumors with hyperactivity of epidermal growth factor signaling pathways. In the first part of this review we explain basic terms related to the development of antibody-based drugs, give a brief historic perspective of the field, and also touch on topics such as the “humanization of antibodie” or creation of hybrid antibodies. The second part of the review gives an overview of the clinical usage of bispecific antibodies and antibodies “armed” with cytotoxic agents or enzymes. Further within this section, cancer-specific, site-specific, or signaling pathway-specific therapies are discussed in detail. Among other antibody-based therapeutic products, we discuss: Avastin (bevacizumab), CG76030, Theragyn (pemtumomab), daclizumab (Zenapax), TriAb, MDX-210, Herceptin (trastuzumab), panitumumab (ABX-EGF), mastuzimab (EMD-72000), Erbitux (certuximab, IMC225), Panorex (edrecolomab), STI571, CeaVac, Campath (alemtuizumab), Mylotarg (gemtuzumab, ozogamicin), and many others. The end of the review deliberates upon potential problems associated with cancer immunotherapy.

The RNA helicase RHAU (DHX36) unwinds a G4-quadruplex in human telomerase RNA and promotes the formation of the P1 helix template boundary

Human telomerase RNA (hTR) contains several guanine tracts at its 5′-end that can form a G4-quadruplex structure. Previous evidence suggests that a G4-quadruplex within this region disrupts the formation of an important structure within hTR known as the P1 helix, a critical element in defining the template boundary for reverse transcription. RNA associated with AU-rich element (RHAU) is an RNA helicase that has specificity for DNA and RNA G4-quadruplexes. Two recent studies identify a specific interaction between hTR and RHAU. Herein, we confirm this interaction and identify the minimally interacting RNA fragments. We demonstrate the existence of multiple quadruplex structures within the 5′ region of hTR and find that these regions parallel the minimal sequences capable of RHAU interaction. We confirm the importance of the RHAU-specific motif in the interaction with hTR and demonstrate that the helicase activity of RHAU is sufficient to unwind the quadruplex and promote an interaction with 25 internal nucleotides to form a stable P1 helix. Furthermore, we have found that a 5′-terminal quadruplex persists following P1 helix formation that retains affinity for RHAU. Finally, we have investigated the functional implications of this interaction and demonstrated a reduction in average telomere length following RHAU knockdown by small interfering RNA (siRNA).

Binding of G-quadruplexes to the N-terminal recognition domain of the RNA helicase associated with AU-rich element (RHAU).

Background: The helicase RHAU requires an N-terminal extension to bind quadruplex structures. Results: This extension adopts an elongated shape and interacts with the guanine tetrad face of quadruplexes. Conclusion: We provide a basis for the understanding of quadruplex binding by the N-terminal domain. Significance: The N-terminal region does not require the 2′-OH of the ribose to mediate the protein-quadruplex interaction. Polynucleotides containing consecutive tracts of guanines can adopt an intramolecular G-quadruplex structure where multiple planar tetrads of hydrogen-bound guanines stack on top of each other. Remodeling of G-quadruplexes impacts numerous aspects of nucleotide biology including transcriptional and translational control. RNA helicase associated with AU-rich element (RHAU), a member of the ATP-dependent DEX(H/D) family of RNA helicases, has been established as a major cellular quadruplex resolvase. RHAU contains a core helicase domain responsible for ATP binding/hydrolysis/helicase activity and is flanked on either side by N- and C-terminal extensions. The N-terminal extension is required for quadruplex recognition, and we have previously demonstrated complex formation between this domain and a quadruplex from human telomerase RNA. Here we used an integrated approach that includes small angle x-ray scattering, nuclear magnetic resonance spectroscopy, circular dichroism, and dynamic light scattering methods to demonstrate the recognition of G-quadruplexes by the N-terminal domain of RHAU. Based on our results, we conclude that (i) quadruplex from the human telomerase RNA and its DNA analog both adopt a disc shape in solution, (ii) RHAU53–105 adopts a defined and extended conformation in solution, and (iii) the N-terminal domain mediates an interaction with a guanine tetrad face of quadruplexes. Together, these data form the foundation for understanding the recognition of quadruplexes by the N-terminal domain of RHAU.

Selected technologies to control genes and their products for experimental and clinical purposes

Abstract.“On-demand” regulation of gene expression is a powerful tool to elucidate the functions of proteins and biologically-active RNAs. We describe here three different approaches to the regulation of expression or activity of genes or proteins. Promoter-based regulation of gene expression was among the most rapidly developing techniques in the 1980s and 1990s. Here we provide basic information and also some characteristics of the metallothionein-promoter-based system, the tet-off system, Muristerone-A-regulated expression through the ecdysone response element, RheoSwitch®, coumermycin/novobiocin-regulated gene expression, chemical dimerizer-based promoter activation systems, the “Dual Drug Control” system, “constitutive androstane receptor” based regulation of gene expression, and RU486/mifepristone-driven regulation of promoter activity. A large part of the review concentrates on the principles and usage of various RNA interference techniques (RNAi: siRNA, shRNA, and miRNA-based methods). Finally, the last part of the review deals with historically the oldest, but still widely used, methods of temperature-dependent regulation of enzymatic activity or protein stability (temperature-sensitive mutants). Due to space limitations we do not describe in detail but just mention the tet-regulated systems and also fusion-protein-based regulation of protein activity, such as estrogen-receptor fusion proteins. The information provided below is aimed to assist researchers in choosing the most appropriate method for the planned development of experimental systems with regulated expression or activity of studied proteins.

The RNA helicase RHAU (DHX36) suppresses expression of the transcription factor PITX1

RNA Helicase associated with AU-rich element (RHAU) (DHX36) is a DEAH (Aspartic acid, Glumatic Acid, Alanine, Histidine)-box RNA helicase that can bind and unwind G4-quadruplexes in DNA and RNA. To detect novel RNA targets of RHAU, we performed an RNA co-immunoprecipitation screen and identified the PITX1 messenger RNA (mRNA) as specifically and highly enriched. PITX1 is a homeobox transcription factor with roles in both development and cancer. Primary sequence analysis identified three probable quadruplexes within the 3′-untranslated region of the PITX1 mRNA. Each of these sequences, when isolated, forms stable quadruplex structures that interact with RHAU. We provide evidence that these quadruplexes exist in the endogenous mRNA; however, we discovered that RHAU is tethered to the mRNA via an alternative non–quadruplex-forming region. RHAU knockdown by small interfering RNA results in significant increases in PITX1 protein levels with only marginal changes in mRNA, suggesting a role for RHAU in translational regulation. Involvement of components of the microRNA machinery is supported by similar and non-additive increases in PITX1 protein expression on Dicer and combined RHAU/Dicer knockdown. We also demonstrate a requirement of argonaute-2, a key RNA-induced silencing complex component, to mediate RHAU-dependent changes in PITX1 protein levels. These results demonstrate a novel role for RHAU in microRNA-mediated translational regulation at a quadruplex-containing 3′-untranslated region.

Activation of 2' 5'-oligoadenylate synthetase by stem loops at the 5'-end of the West Nile virus genome.

West Nile virus (WNV) has a positive sense RNA genome with conserved structural elements in the 5′ and 3′ -untranslated regions required for polyprotein production. Antiviral immunity to WNV is partially mediated through the production of a cluster of proteins known as the interferon stimulated genes (ISGs). The 2′ 5′-oligoadenylate synthetases (OAS) are key ISGs that help to amplify the innate immune response. Upon interaction with viral double stranded RNA, OAS enzymes become activated and enable the host cell to restrict viral propagation. Studies have linked mutations in the OAS1 gene to increased susceptibility to WNV infection, highlighting the importance of OAS1 enzyme. Here we report that the region at the 5′-end of the WNV genome comprising both the 5′-UTR and initial coding region is capable of OAS1 activation in vitro. This region contains three RNA stem loops (SLI, SLII, and SLIII), whose relative contribution to OAS1 binding affinity and activation were investigated using electrophoretic mobility shift assays and enzyme kinetics experiments. Stem loop I, comprising nucleotides 1-73, is dispensable for maximum OAS1 activation, as a construct containing only SLII and SLIII was capable of enzymatic activation. Mutations to the RNA binding site of OAS1 confirmed the specificity of the interaction. The purity, monodispersity and homogeneity of the 5′-end (SLI/II/III) and OAS1 were evaluated using dynamic light scattering and analytical ultra-centrifugation. Solution conformations of both the 5′-end RNA of WNV and OAS1 were then elucidated using small-angle x-ray scattering. In the context of purified components in vitro, these data demonstrate the recognition of conserved secondary structural elements of the WNV genome by a member of the interferon-mediated innate immune response.

RNA species generated in vaccinia virus infected cells activate cell type-specific MDA5 or RIG-I dependent interferon gene transcription and PKR dependent apoptosis

RNA species produced during virus replication are pathogen-associated molecular patterns (PAMPs) triggering cellular innate immune responses including induction of type I interferon expression and apoptosis. Pattern recognition receptors (PRRs) for these RNAs include the retinoic acid-inducible gene I (RIG-I) like receptors (RLRs) RIG-I and melanoma differentiation associated gene 5 (MDA5) and the dsRNA dependent protein kinase (PKR). Currently, poxvirus PAMPs and their associated PRRs are not well characterized. We report that RNA species generated in vaccinia infected cells can activate MDA5 or RIG-I dependent interferon-β (IFN-β) gene transcription in a cell type-specific manner. These RNA species also induce the activation of apoptosis in a PKR dependent, but MDA5 and RIG-I independent, manner. Collectively our results demonstrate that RNA species generated during vaccinia virus replication are major PAMPs activating apoptosis and IFN-β gene transcription. Moreover, our results delineate the signaling pathways involved in the recognition of RNA-based poxvirus PAMPs.