Development of a novel mammalian display system for selection of antibodies against membrane proteins.

Reliable, specific polyclonal and monoclonal antibodies are important tools in research and medicine. However, the discovery of antibodies against their targets in their native forms is difficult. Here, we present a novel method for discovery of antibodies against membrane proteins in their native configuration in mammalian cells. The method involves the co-expression of an antibody library in a population of mammalian cells that express the target polypeptide within a natural membrane environment on the cell surface. Cells that secrete a single-chain fragment variable (scFv) that binds to the target membrane protein thereby become self-labeled, enabling enrichment and isolation by magnetic sorting and FRET-based flow sorting. Library sizes of up to 109 variants can be screened, thus allowing campaigns of naïve scFv libraries to be selected against membrane protein antigens in a Chinese hamster ovary cell system. We validate this method by screening a synthetic naïve human scFv library against Chinese hamster ovary cells expressing the oncogenic target epithelial cell adhesion molecule and identify a panel of three novel binders to this membrane protein, one with a dissociation constant (KD ) as low as 0.8 nm We further demonstrate that the identified antibodies have utility for killing epithelial cell adhesion molecule-positive cells when used as a targeting domain on chimeric antigen receptor T cells. Thus, we provide a new tool for identifying novel antibodies that act against membrane proteins, which could catalyze the discovery of new candidates for antibody-based therapies.

Helenalin bypasses Bcl-2-mediated cell death resistance by inhibiting NF-κB and promoting reactive oxygen species generation.

Evasion of cell death by overexpression of anti-apoptotic proteins, such as Bcl-2, is commonly observed in cancer cells leading to a lack of response to chemotherapy. Hence, there is a need to find new chemotherapeutic agents that are able to overcome chemoresistance mediated by Bcl-2 and to understand their mechanisms of action. Helenalin, a sesquiterpene lactone (STL), induces cell death and abrogates clonal survival in a highly apoptosis-resistant Bcl-2 overexpressing Jurkat cell line as well as in two other Bcl-2 overexpressing solid tumor cell lines (mammary MCF-7; pancreatic L6.3pl). This effect is not achieved by directly affecting the mitochondria-protective function of Bcl-2 in the intrinsic pathway of apoptosis since Bcl-2 overexpressing Jurkat cells do not show cytochrome c release and dissipation of mitochondrial membrane potential upon helenalin treatment. Moreover, helenalin induces an atypical form of cell death with necrotic features in Bcl-2 overexpressing cells, neither activating classical mediators of apoptosis (caspases, AIF, Omi/HtrA2, Apaf/apoptosome) nor ER-stress mediators (BiP/GRP78 and CHOP/GADD153), nor autophagy pathways (LC3 conversion). In contrast, helenalin was found to inhibit NF-κB activation that was considerably increased in Bcl-2 overexpressing Jurkat cells and promotes cell survival. Moreover, we identified reactive oxygen species (ROS) and free intracellular iron as mediators of helenalin-induced cell death whereas activation of JNK and abrogation of Akt activity did not contribute to helenalin-elicited cell death. Our results highlight the NF-κB inhibitor helenalin as a promising chemotherapeutic agent to overcome Bcl-2-induced cell death resistance.

The cephalostatin way of apoptosis.

The cephalostatins, bis-steroidal natural products from the marine tube worm Cephalodiscus gilchristi, were isolated by Dr. G. R. Pettit and his group. These compounds show a unique cytotoxicity profile in the in vitro screen of the National Cancer Institute, suggesting a novel mechanism of action. Indeed, cephalostatin 1 ( 1) is an extremely powerful agent that acts via an unusual apoptosis pathway. It induces selective Smac/DIABLO, but no cytochrome c release from mitochondria. Nevertheless, caspase-9 is required for apoptosis induction. Interestingly, caspase-9 is activated without the participation of the apoptosome, leading to the question of its mechanism of activation. We found that endoplasmic reticulum stress-associated caspase-4 contributes to nonclassical cephalostatin-mediated caspase-9 activation, additionally pointing out the unusual pathway used by this substance. Cephalostatin 1 ( 1), therefore, provides a very good tool to discover novel apoptotic pathways, which might be important in the understanding and treatment of chemo-resistant cancer.

Sesquiterpene lactones induce distinct forms of cell death that modulate human monocyte-derived macrophage responses.

Sesquiterpene lactones (SQTLs) are shown to possess anti-inflammatory as well as cytotoxic activity. No study, however, links both activities. We, therefore, hypothesized that SQTL-treated, dying cells might induce an anti-inflammatory response in cocultured THP-1 macrophages. Here we show that SQTLs bearing either an alpha,beta-unsaturated cyclopentenone or an alpha-methylene-gamma-lactone induce different forms of cell death. Whereas the cyclopentenone SQTL induced typical apoptosis, the alpha-methylene-gamma-lactone SQTLs-induced cell death lacked partly classical signs of apoptosis, such as DNA fragmentation. All SQTLs, however, activated caspases and the nuclear morphology of cell death was dependent on caspase activation. Most interestingly, alpha-methylene-gamma-lactone SQTLs induced a more pronounced phosphatidylserine (PS) exposure than the cyclopentenone SQTL. Especially, 7-hydroxycostunolide (HC), with an alpha-methylene-gamma-lactone substituted with a hydroxyl group, showed a striking fast and pronounced PS translocation. This result was in agreement with a strong activation of phagocytosis in cocultured THP-1 macrophages. Interestingly, HC-treated Jurkat cells led to an early (3.5 h) but transient increase in TNF-alpha levels in macrophage coculture. Release of TGF-beta remained unaffected after 18 h. We propose that this type of SQTL may influence local inflammation by transiently activating the immune system and help to clear cells by inducing a form of cell death that promotes phagocytosis.

The marine product cephalostatin 1 activates an endoplasmic reticulum stress-specific and apoptosome-independent apoptotic signaling pathway.

Cephalostatin 1, a bis-steroidal marine natural product, has been reported to induce apoptosis without the requirement of an active caspase-8 or mitochondrial cytochrome c release and apoptosome formation. Here we show that despite the absence of these events, caspase-9 activation is essential for cephalostatin 1-induced apoptosis. Cephalostatin 1 initiates a rapid endoplasmic reticulum stress response characterized by phosphorylation of eukaryotic initiation factor-2 alpha-subunit and increased expression of the chaperone immunoglobulin heavy chain-binding protein GRP78 as well as the transcription factor C/EBP homologous protein (CHOP)/GADD153. Cephalostatin 1 activates apoptosis signal-regulating kinase 1 and c-Jun N-terminal kinase (JNK). However, this pathway does not play a major role in cephalostatin 1-induced apoptosis, as assessed by stable expression of a dominant negative apoptosis signal-regulating kinase 1. Importantly, the endoplasmic reticulum-associated caspase-4 is required and as shown by biochemical and genetic inhibition experiments, acts upstream of caspase-9 in cephalostatin-induced apoptosis.

Cephalostatin 1 inactivates Bcl-2 by hyperphosphorylation independent of M-phase arrest and DNA damage.

Cephalostatin 1 is a marine product that induces a novel cytochrome c-independent apoptotic pathway in Jurkat leukemia T cells (Cancer Res 63:8869-8876, 2003). Here, we show that overexpression of the antiapoptotic protein Bcl-2 protects cells only partially against cephalostatin 1-induced apoptosis. The mechanism of Bcl-2 inactivation by cephalostatin 1 is based on hyperphosphorylation of Bcl-2 on Thr69 and Ser87 because Jurkat cells overexpressing a Bcl-2 protein with mutations on both phosphorylation sites were completely protected against cephalostatin 1. In search of the kinase responsible for Bcl-2 phosphorylation, c-Jun NH2-terminal kinase (JNK) was found to be activated by cephalostatin 1. Reduction of Bcl-2 phosphorylation by the specific JNK inhibitor (anthra(1,3-cd)pyrazol-6(2H)-one) SP600125 suggested a crucial role for JNK in this process. JNK activation was not a consequence of DNA damage, a known stimulus of JNK, because cephalostatin 1 did not induce DNA lesions as shown by the comet assay. Arrest in M-phase is also demonstrated to be associated with JNK activation. However, cephalostatin 1 does not evoke an arrest in M-phase as shown by flow cytometry. Together, cephalostatin 1 is shown to induce JNK activation with subsequent Bcl-2 phosphorylation and inactivation. Reported triggers, such as the induction of an M-phase arrest or DNA damage are not involved in this process, suggesting a novel mechanism for cephalostatin 1-mediated Bcl-2 hyperphosphorylation.