Nanotechnology Driven Cancer Chemoradiation: Exploiting the Full Potential of Radiotherapy with a Unique Combination of Gold Nanoparticles and Bleomycin.

One of the major issues in current radiotherapy (RT) is the associated normal tissue toxicity. Enhancement of the RT effect with novel radiosensitizers can address this need. In this study, gold nanoparticles (GNPs) and bleomycin (BLM) were used as a unique combination of radiosensitizers. GNPs offer a two-fold promise as a delivery vehicle for BLM and as a radiosensitizing agent. In this study, GNPs were functionalized and complexed with BLM using a gold-thiol bond (denoted GNP-BLM). Our results show that there was a 40% and 10% decrease in cell growth with GNP-BLM vs. free BLM for the MIA PaCa-2 and PC-3 cell lines, respectively. Testing the GNP-BLM platform with RT showed an 84% and 13% reduction in cell growth in MIA PaCa-2 cells treated with GNP-BLM and GNPs, respectively. Similar results were seen with PC-3 cells. The efficacy of this approach was verified by mapping DNA double-strand breaks (DSBs) as well. Therefore, this proposed incorporation of nanomedicine with RT is promising in achieving a significantly higher therapeutic ratio which is necessary to make a paradigm change to the current clinical approach.

Cytoplasmic switch of ARS2 isoforms promotes nonsense-mediated mRNA decay and arsenic sensitivity.

The life of RNA polymerase II (RNAPII) transcripts is shaped by the dynamic formation of mutually exclusive ribonucleoprotein complexes (RNPs) that direct transcript biogenesis and turnover. A key regulator of RNA metabolism in the nucleus is the scaffold protein ARS2 (arsenic resistance protein 2), bound to the cap binding complex (CBC). We report here that alternative splicing of ARS2's intron 5, generates cytoplasmic isoforms that lack 270 amino acids from the N-terminal of the protein and are functionally distinct from nuclear ARS2. Switching of ARS2 isoforms within the CBC in the cytoplasm has dramatic functional consequences, changing ARS2 from a NMD inhibitor to a NMD promoter that enhances the binding of UPF1 to NCBP1 and ERF1, favouring SURF complex formation, SMG7 recruitment and transcript degradation. ARS2 isoform exchange is also relevant during arsenic stress, where cytoplasmic ARS2 promotes a global response to arsenic in a CBC-independent manner. We propose that ARS2 isoform switching promotes the proper recruitment of RNP complexes during NMD and the cellular response to arsenic stress. The existence of non-redundant ARS2 isoforms is relevant for cell homeostasis, and stress response.

Docetaxel-Mediated Uptake and Retention of Gold Nanoparticles in Tumor Cells and in Cancer-Associated Fibroblasts.

Due to recent advances in nanotechnology, the application of nanoparticles (NPs) in cancer therapy has become a leading area in cancer research. Despite the importance of cancer-associated fibroblasts (CAFs) in creating an optimal niche for tumor cells to grow extensively, most of the work has been focused on tumor cells. Therefore, to effectively use NPs for therapeutic purposes, it is important to elucidate the extent of NP uptake and retention in tumor cells and CAFs. Three tumor cell lines and three CAF cell lines were studied using gold NPs (GNPs) as a model NP system. We found a seven-fold increase in NP uptake in CAFs compared to tumor cells. The retention percentage of NPs was three-fold higher in tumor cells as compared to CAFs. Furthermore, NP uptake and retention were significantly enhanced using a 50 nM concentration of docetaxel (DTX). NP uptake was improved by a factor of three in tumor cells and a factor of two in CAFs, while the retention of NPs was two-fold higher in tumor cells compared to CAFs, 72 h post-treatment with DTX. However, the quantity of NPs in CAFs was still three-fold higher compared to tumor cells. Our quantitative data were supported by qualitative imaging data. We believe that targeting of NPs in the presence of DTX is a very promising approach to accumulate a higher percentage of NPs and maintain a longer retention in both tumor cells and CAFs for achieving the full therapeutic potential of cancer nanotechnology.

Three-Dimensional Tumor Spheroids as a Tool for Reliable Investigation of Combined Gold Nanoparticle and Docetaxel Treatment.

Radiotherapy and chemotherapy are the gold standard for treating patients with cancer in the clinic but, despite modern advances, are limited by normal tissue toxicity. The use of nanomaterials, such as gold nanoparticles (GNPs), to improve radiosensitivity and act as drug delivery systems can mitigate toxicity while increasing deposited tumor dose. To expedite a quicker clinical translation, three-dimensional (3D) tumor spheroid models that can better approximate the tumor environment compared to a two-dimensional (2D) monolayer model have been used. We tested the uptake of 15 nm GNPs and 50 nm GNPs on a monolayer and on spheroids of two cancer cell lines, CAL-27 and HeLa, to evaluate the differences between a 2D and 3D model in similar conditions. The anticancer drug docetaxel (DTX) which can act as a radiosensitizer, was also utilized, informing future potential of GNP-mediated combined therapeutics. In the 2D monolayer model, the addition of DTX induced a small, non-significant increase of uptake of GNPs of between 13% and 24%, while in the 3D spheroid model, DTX increased uptake by between 47% and 186%, with CAL-27 having a much larger increase relative to HeLa. Further, the depth of penetration of 15 nm GNPs over 50 nm GNPs increased by 33% for CAL-27 spheroids and 17% for HeLa spheroids. These results highlight the necessity to optimize GNP treatment conditions in a more realistic tumor-life environment. A 3D spheroid model can capture important details, such as different packing densities from different cancer cell lines, which are absent from a simple 2D monolayer model.

Deciphering the Enigma of the Histone H2A.Z-1/H2A.Z-2 Isoforms: Novel Insights and Remaining Questions.

The replication independent (RI) histone H2A.Z is one of the more extensively studied variant members of the core histone H2A family, which consists of many replication dependent (RD) members. The protein has been shown to be indispensable for survival, and involved in multiple roles from DNA damage to chromosome segregation, replication, and transcription. However, its functional involvement in gene expression is controversial. Moreover, the variant in several groups of metazoan organisms consists of two main isoforms (H2A.Z-1 and H2A.Z-2) that differ in a few (3-6) amino acids. They comprise the main topic of this review, starting from the events that led to their identification, what is currently known about them, followed by further experimental, structural, and functional insight into their roles. Despite their structural differences, a direct correlation to their functional variability remains enigmatic. As all of this is being elucidated, it appears that a strong functional involvement of isoform variability may be connected to development.

ARS2 is required for retinal progenitor cell S-phase progression and Müller glial cell fate specification.

During a developmental period that extends postnatally in the mouse, proliferating multipotent retinal progenitor cells produce one of 7 major cell types (rod, cone, bipolar, horizontal, amacrine, ganglion, and Müller glial cells) as they exit the cell cycle in consecutive waves. Cell production in the retina is tightly regulated by intrinsic, extrinsic, spatial, and temporal cues, and is coupled to the timing of cell cycle exit. Arsenic-resistance protein 2 (ARS2, also known as SRRT) is a component of the nuclear cap-binding complex involved in RNA Polymerase II transcription, and is required for cell cycle progression. We show that postnatal retinal progenitor cells (RPCs) require ARS2 for proper progression through S phase, and ARS2 disruption leads to early exit from the cell cycle. Furthermore, we observe an increase in the proportion of cells expressing a rod photoreceptor marker, and a loss of Müller glia marker expression, indicating a role for ARS2 in regulating cell fate specification or differentiation. Knockdown of Flice Associated Huge protein (FLASH), which interacts with ARS2 and is required for cell cycle progression and 3'-end processing of replication-dependent histone transcripts, phenocopies ARS2 knockdown. These data implicate ARS2-FLASH-mediated histone mRNA processing in regulating RPC cell cycle kinetics and neuroglial cell fate specification during postnatal retinal development.

Modulation of nanoparticle uptake, intracellular distribution, and retention with docetaxel to enhance radiotherapy.

OBJECTIVE: One of the major issues in current radiotherapy (RT) is the normal tissue toxicity. A smart combination of agents within the tumor would allow lowering the RT dose required while minimizing the damage to healthy tissue surrounding the tumor. We chose gold nanoparticles (GNPs) and docetaxel (DTX) as our choice of two radiosensitizing agents. They have a different mechanism of action which could lead to a synergistic effect. Our first goal was to assess the variation in GNP uptake, distribution, and retention in the presence of DTX. Our second goal was to assess the therapeutic results of the triple combination, RT/GNPs/DTX. METHODS: We used HeLa and MDA-MB-231 cells for our study. Cells were incubated with GNPs (0.2 nM) in the absence and presence of DTX (50 nM) for 24 h to determine uptake, distribution, and retention of NPs. For RT experiments, treated cells were given a 2 Gy dose of 6 MV photons using a linear accelerator. RESULTS: Concurrent treatment of DTX and GNPs resulted in over 85% retention of GNPs in tumor cells. DTX treatment also forced GNPs to be closer to the most important target, the nucleus, resulting in a decrease in cell survival and increase in DNA damage with the triple combination of RT/ GNPs/DTX vs RT/DTX. Our experimental therapeutic results were supported by Monte Carlo simulations. CONCLUSION: The ability to not only trap GNPs at clinically feasible doses but also to retain them within the cells could lead to meaningful fractionated treatments in future combined cancer therapy. Furthermore, the suggested triple combination of RT/GNPs/DTX may allow lowering the RT dose to spare surrounding healthy tissue. ADVANCES IN KNOWLEDGE: This is the first study to show intracellular GNP transport disruption by DTX, and its advantage in radiosensitization.

Mapping the Pax6 3' untranslated region microRNA regulatory landscape.

BACKGROUND: PAX6 is a homeodomain transcription factor that acts in a highly dosage-sensitive manner to regulate the development and function of the eyes, nose, central nervous system, gut, and endocrine pancreas. Several individual microRNAs (miRNA) have been implicated in regulating PAX6 in different cellular contexts, but a more general view of how they contribute to the fine-tuning and homeostasis of PAX6 is poorly understood. RESULTS: Here, a comprehensive analysis of the Pax6 3' untranslated region was performed to map potential miRNA recognition elements and served as a backdrop for miRNA expression profiling experiments to identify potential cell/tissue-specific miRNA codes. Pax6 3'UTR pull-down studies identified a cohort of miRNA interactors in pancreatic αTC1-6 cells that, based on the spacing of their recognition sites in the Pax6 3'UTR, revealed 3 clusters where cooperative miRNA regulation may occur. Some of these interacting miRNAs have been implicated in α cell function but have not previously been linked to Pax6 function and may therefore represent novel PAX6 regulators. CONCLUSIONS: These findings reveal a regulatory landscape upon which miRNAs may participate in the developmental control, fine-tuning and/or homeostasis of PAX6 levels.

Protecting Pax6 3' UTR from MicroRNA-7 Partially Restores PAX6 in Islets from an Aniridia Mouse Model.

Aniridia is a rare congenital syndrome that is associated with reduced visual acuity and progressive loss of vision. Aniridia patients may also develop systemic health issues associated with defects in the pancreas, digestive, and central nervous systems. The spectrum of symptoms associated with aniridia is due to haploinsufficiency of the paired box 6 gene (PAX6) and its role in the development and maintenance of the affected tissues. Here, we isolated pancreatic islets from mice heterozygous for Pax6 to test whether a Pax6-specific miRNA suppression (target protector) strategy can restore PAX6 protein levels. We show that miR-7 and miR-375 target specific sites within the Pax6 3' UTR in a mouse pancreatic ß-insulinoma cell line. Tough decoys (Tuds) against miR-7 and miR-375 increase expression of a mouse Pax6 3' UTR luciferase reporter and increase PAX6 protein levels in these cells. Finally, we demonstrate that the shielding of the miR-7 binding site with a target protector restores PAX6 protein levels in the Pax6 heterozygous islets. The data presented here represent a proof of concept for RNA-based therapy for the progressive defects associated with aniridia and suggest the target protector approach may be a useful therapeutic strategy for other haploinsufficiency diseases.

The prolyl isomerase FKBP25 regulates microtubule polymerization impacting cell cycle progression and genomic stability.

FK506 binding proteins (FKBPs) catalyze the interconversion of cis-trans proline conformers in proteins. Importantly, FK506 drugs have anti-cancer and neuroprotective properties, but the effectors and mechanisms underpinning these properties are not well understood because the cellular function(s) of most FKBP proteins are unclear. FKBP25 is a nuclear prolyl isomerase that interacts directly with nucleic acids and is associated with several DNA/RNA binding proteins. Here, we show the catalytic FKBP domain binds microtubules (MTs) directly to promote their polymerization and stabilize the MT network. Furthermore, FKBP25 associates with the mitotic spindle and regulates entry into mitosis. This interaction is important for mitotic spindle dynamics, as we observe increased chromosome instability in FKBP25 knockdown cells. Finally, we provide evidence that FKBP25 association with chromatin is cell-cycle regulated by Protein Kinase C phosphorylation. This disrupts FKBP25-DNA contacts during mitosis while maintaining its interaction with the spindle apparatus. Collectively, these data support a model where FKBP25 association with chromatin and MTs is carefully choreographed to ensure faithful genome duplication. Additionally, they highlight that FKBP25 is a MT-associated FK506 receptor and potential therapeutic target in MT-associated diseases.

Conditional Toxin Splicing Using a Split Intein System.

Protein toxin splicing mediated by split inteins can be used as a strategy for conditional cell ablation. The approach requires artificial fragmentation of a potent protein toxin and tethering each toxin fragment to a split intein fragment. The toxin-intein fragments are, in turn, fused to dimerization domains, such that addition of a dimerizing agent reconstitutes the split intein. These chimeric toxin-intein fusions remain nontoxic until the dimerizer is added, resulting in activation of intein splicing and ligation of toxin fragments to form an active toxin. Considerations for the engineering and implementation of conditional toxin splicing (CTS) systems include: choice of toxin split site, split site (extein) chemistry, and temperature sensitivity. The following method outlines design criteria and implementation notes for CTS using a previously engineered system for splicing a toxin called sarcin, as well as for developing alternative CTS systems.

ImiRP: a computational approach to microRNA target site mutation.

BACKGROUND: MicroRNAs (miRNAs) are small ~22 nucleotide non-coding RNAs that function as post-transcriptional regulators of messenger RNA (mRNA) through base-pairing to 6-8 nucleotide long target sites, usually located within the mRNA 3' untranslated region. A common approach to validate and probe microRNA-mRNA interactions is to mutate predicted target sites within the mRNA and determine whether it affects miRNA-mediated activity. The introduction of miRNA target site mutations, however, is potentially problematic as it may generate new, "illegitimate sites" target sites for other miRNAs, which may affect the experimental outcome. While it is possible to manually generate and check single miRNA target site mutations, this process can be time consuming, and becomes particularly onerous and error prone when multiple sites are to be mutated simultaneously. We have developed a modular Java-based system called ImiRP (Illegitimate miRNA Predictor) to solve this problem and to facilitate miRNA target site mutagenesis. RESULTS: The ImiRP interface allows users to input a sequence of interest, specify the locations of multiple predicted target sites to mutate, and set parameters such as species, mutation strategy, and disallowed illegitimate target site types. As mutant sequences are generated, ImiRP utilizes the miRBase high confidence miRNA dataset to identify illegitimate target sites in each mutant sequence by comparing target site predictions between input and mutant sequences. ImiRP then assembles a final mutant sequence in which all specified target sites have been mutated. CONCLUSIONS: ImiRP is a mutation generator program that enables selective disruption of specified miRNA target sites while ensuring predicted target sites for other miRNAs are not inadvertently created. ImiRP supports mutagenesis of single and multiple miRNA target sites within a given sequence, including sites that overlap. This software will be particularly useful for studies looking at microRNA cooperativity, where mutagenesis of multiple microRNA target sites may be desired. The software is available at imirp.org and is available open source for download through GitHub ( https://github.com/imirp ).

Mutagenesis of ARS2 Domains To Assess Possible Roles in Cell Cycle Progression and MicroRNA and Replication-Dependent Histone mRNA Biogenesis.

ARS2 is a regulator of RNA polymerase II transcript processing through its role in the maturation of distinct nuclear cap-binding complex (CBC)-controlled RNA families. In this study, we examined ARS2 domain function in transcript processing. Structural modeling based on the plant ARS2 orthologue, SERRATE, revealed 2 previously uncharacterized domains in mammalian ARS2: an N-terminal domain of unknown function (DUF3546), which is also present in SERRATE, and an RNA recognition motif (RRM) that is present in metazoan ARS2 but not in plants. Both the DUF3546 and zinc finger domain (ZnF) were required for association with microRNA and replication-dependent histone mRNA. Mutations in the ZnF disrupted interaction with FLASH, a key component in histone pre-mRNA processing. Mutations targeting the Mid domain implicated it in DROSHA interaction and microRNA biogenesis. The unstructured C terminus was required for interaction with the CBC protein CBP20, while the RRM was required for cell cycle progression and for binding to FLASH. Together, our results support a bridging model in which ARS2 plays a central role in RNA recognition and processing through multiple protein and RNA interactions.

Conditional protein splicing of α-sarcin in live cells.

Protein splicing technology harnesses the ability of inteins to ligate protein fragments, forming a mature protein. This report describes our effort to engineer rapamycin-dependent protein splicing of a ribotoxin, called α-sarcin. Engineering this system required the investigation of important splicing parameters, including extein context and splicing temperature. We show α-sarcin splicing is dependent on rapamycin, is inducible with rapid kinetics, and triggers apoptosis in HeLa cells. These findings establish a proof-of-concept for a conditional cell ablation strategy.

Live imaging and analysis of postnatal mouse retinal development.

BACKGROUND: The explanted, developing rodent retina provides an efficient and accessible preparation for use in gene transfer and pharmacological experimentation. Many of the features of normal development are retained in the explanted retina, including retinal progenitor cell proliferation, heterochronic cell production, interkinetic nuclear migration, and connectivity. To date, live imaging in the developing retina has been reported in non-mammalian and mammalian whole-mount samples. An integrated approach to rodent retinal culture/transfection, live imaging, cell tracking, and analysis in structurally intact explants greatly improves our ability to assess the kinetics of cell production. RESULTS: In this report, we describe the assembly and maintenance of an in vitro, CO2-independent, live mouse retinal preparation that is accessible by both upright and inverted, 2-photon or confocal microscopes. The optics of this preparation permit high-quality and multi-channel imaging of retinal cells expressing fluorescent reporters for up to 48h. Tracking of interkinetic nuclear migration within individual cells, and changes in retinal progenitor cell morphology are described. Follow-up, hierarchical cluster screening revealed that several different dependent variable measures can be used to identify and group movement kinetics in experimental and control samples. CONCLUSIONS: Collectively, these methods provide a robust approach to assay multiple features of rodent retinal development using live imaging.

Role of NF-κB in hepatocarcinogenesis and its potential inhibition by dietary antioxidants.

In this review, the role of nuclear factor-κB (NF-κB) in hepatocarcinogenesis is examined. The administration of several hepatic tumor promoters leads to the activation of NF-κB in the liver of rats and mice. Many studies support the hypothesis that the activation of NF-κB in the liver is inhibited by antioxidants. The role of NF-κB in hepatocarcinogenesis has been examined using NF-κB overexpression and knockout models. The role of NF-κB in liver carcinogenesis is complex; some models show that NF-κB contributes to carcinogenesis whereas others see no effect or an inhibition. Overall, although hepatic tumor promoting agents can activate NF-κB and this activation can be inhibited by antioxidants, the significance of this activation is unclear.

Soluble ephrin a1 is necessary for the growth of HeLa and SK-BR3 cells.

BACKGROUND: Ephrin A1 (EFNA1) is a member of the A-type ephrin family of cell surface proteins that function as ligands for the A-type Eph receptor tyrosine kinase family. In malignancy, the precise role of EFNA1 and its preferred receptor, EPHA2, is controversial. Several studies have found that EFNA1 may suppress EPHA2-mediated oncogenesis, or enhance it, depending on cell type and context. However, little is known about the conditions that influence whether EFNA1 promotes or suppresses tumorigenicity. EFNA1 exists in a soluble form as well as a glycophosphatidylinositol (GPI) membrane attached form. We investigated whether the contradictory roles of EFNA1 in malignancy might in part be related to the existence of both soluble and membrane attached forms of EFNA1 and potential differences in the manner in which they interact with EPHA2. RESULTS: Using a RNAi strategy to reduce the expression of endogenous EFNA1 and EPHA2, we found that both EFNA1 and EPHA2 are required for growth of HeLa and SK-BR3 cells. The growth defects could be rescued by conditioned media from cells overexpressing soluble EFNA1. Interestingly, we found that overexpression of the membrane attached form of EFNA1 suppresses growth of HeLa cells in 3D but not 2D. Knockdown of endogenous EFNA1, or overexpression of full-length EFNA1, resulted in relocalization of EPHA2 from the cell surface to sites of cell-cell contact. Overexpression of soluble EFNA1 however resulted in more EPHA2 distributed on the cell surface, away from cell-cell contacts, and promoted the growth of HeLa cells. CONCLUSIONS: We conclude that soluble EFNA1 is necessary for the transformation of HeLa and SK-BR3 cells and participates in the relocalization of EPHA2 away from sites of cell-cell contact during transformation.

Salt-resistant blood pressure and salt-sensitive renal autoregulation in chronic streptozotocin diabetes.

Hyperfiltration occurs in early type 1 diabetes mellitus in both rats and humans. It results from afferent vasodilation and thus may impair stabilization of glomerular capillary pressure by autoregulation. It is inversely related to dietary salt intake, the "salt paradox." Restoration of normal glomerular filtration rate (GFR) involves increased preglomerular resistance, probably mediated by tubuloglomerular feedback (TGF). To begin to test whether the salt paradox has pathogenic significance, we compared intact vs. diabetic (streptozotocin) Long-Evans rats with normal and increased salt intake, 1 and approximately 3% by weight of food eaten, respectively. Weekly 24-h blood pressure records were acquired by telemetry before and during diabetes. Blood glucose was maintained at approximately 20 mmol/l by insulin implants. GFR was significantly elevated only in diabetic rats on normal salt intake, confirming diabetic hyperfiltration and the salt paradox. Renal blood flow dynamics show strong contributions to autoregulation by both TGF and the myogenic mechanism and were not impaired by diabetes or by increased salt intake. Separately, systolic pressure was not elevated in diabetic rats at any time during 12 wk with normal or high salt intake. Autoregulation was effective in all groups, and the diabetic-normal salt group showed significantly improved autoregulation at low perfusion pressures. Histological examination revealed very minor glomerulosclerosis and modest mesangial expansion, although neither was diagnostic of diabetes. Periodic acid-Schiff-positive droplets found in distal tubules and collecting duct segments were diagnostic of diabetic kidneys. Biologically significant effects attributable to increased salt intake were abrogation of hyperfiltration and of the left shift in autoregulation in diabetic rats.

A Francisella novicida pdpA mutant exhibits limited intracellular replication and remains associated with the lysosomal marker LAMP-1.

Several genes contained in the Francisella pathogenicity island (FPI) encode proteins needed for intracellular growth and virulence of Francisella tularensis. The pdpA gene is the first cistron in the larger of the two operons found in the FPI. In this work we studied the intracellular growth phenotype of a Francisella novicida mutant in the pdpA gene. The DeltapdpA strain was capable of a small amount of intracellular replication but, unlike wild-type F. novicida, remained associated with the lysosomal marker LAMP-1, suggesting that PdpA is necessary for progression from the early phagosome phase of infection. Strains with in cis complementation of the DeltapdpA lesion showed a restoration of intracellular growth to wild-type levels. Infection of macrophages with the DeltapdpA mutant generated a host-cell mRNA profile distinct from that generated by infection with wild-type F. novicida. The transcriptional response of the host macrophage indicates that PdpA functions directly or indirectly to suppress macrophage ability to signal via growth factors, cytokines and adhesion ligands.

Alpha-sarcin catalytic activity is not required for cytotoxicity.

BACKGROUND: Alpha-sarcin is a protein toxin produced by Aspergillus giganteus. It belongs to a family of cytotoxic ribonucleases that inactivate the ribosome and inhibit protein synthesis. alpha-Sarcin cleaves a single phosphodiester bond within the RNA backbone of the large ribosomal subunit, which makes the ribosome unrecognizable to elongation factors and, in turn, blocks protein synthesis. Although it is widely held that the protein synthesis inhibition caused by the toxin leads to cell death, it has not been directly shown that catalytically inactive mutants of alpha-sarcin are non-toxic when expressed directly within the cytoplasm of cells. This is important since recent studies have cast doubt on whether protein synthesis inhibition is sufficient to initiate apoptosis. RESULTS: In this report, we assay alpha-sarcin cytotoxicity and ability to inhibit protein synthesis by direct cytoplasmic expression. We show that mutations in alpha-sarcin, which impair alpha-sarcin's ability to inhibit protein synthesis, do not affect its cytotoxicity. The mutants are unable to activate JNK, confirming that the sarcin-ricin loop remains intact and that the alpha-sarcin mutants are catalytically inactive. In addition, both mutant and wildtype variants of alpha-sarcin localize to the nucleus and cytoplasm, where they co-localize with ribosomal marker RPS6. CONCLUSION: We conclude that although protein synthesis inhibition likely contributes to cell death, it is not required. Thus, our results suggest that alpha-sarcin can promote cell death through a previously unappreciated mechanism that is independent of rRNA cleavage and JNK activation.