Methionine Labeling Research Articles

Chemoselective Methionine Labelling of Recombinant Trastuzumab Shows High In Vitro and In Vivo Tumour Targeting.

A highly effective 2-step system for site-specific antibody modification and conjugation of the monoclonal antibody Herceptin (commercially available under Trastuzumab) in a cysteine-independent manner was used to generate labelled antibodies for in vivo imaging. The first step contains redox-activated chemical tagging (ReACT) of thioethers via engineered methionine residues to introduce specific alkyne moieties, thereby offering a novel easy way to fundamentally change the process of antibody bioconjugation. The second step involves modification of the introduced alkyne via azide-alkyne cycloaddition 'click' conjugation. The versatility of this 2-step approach is demonstrated here by the selective incorporation of a fluorescent dye but can also be applied to a wide variety of different conjugation partners depending on the desired application in a facile manner. Methionine-modified antibodies were characterised in vitro, and the diagnostic potential of the most promising variant was further analysed in an in vivo xenograft animal model using a fluorescence imaging modality. This study demonstrates how methionine-mediated antibody conjugation offers an orthogonal and versatile route to the generation of tailored antibody conjugates with in vivo applicability.

Heparanase expression and activity are increased in platelets during clinical sepsis

BackgroundHeparanase (HPSE) is the only known mammalian enzyme that can degrade heparan sulfate. Heparan sulfate proteoglycans are essential components of the glycocalyx, and maintain physiological barriers between the blood and endothelial cells. HPSE increases during sepsis, which contributes to injurious glyocalyx degradation, loss of endothelial barrier function, and mortality. ObjectivesAs platelets are one of the most abundant cellular sources of HPSE, we sought to determine whether HPSE expression and activity increases in human platelets during clinical sepsis. We also examined associations between platelet HPSE expression and clinical outcomes. Patients/MethodsExpression and activity of HPSE was determined in platelets isolated from septic patients (n = 59) and, for comparison, sex‐matched healthy donors (n = 46) using complementary transcriptomic, proteomic, and functional enzymatic assays. Septic patients were followed for the primary outcome of mortality, and clinical data were captured prospectively for septic patients. ResultsThe mRNA expression of HPSE was significantly increased in platelets isolated from septic patients. Ribosomal footprint profiling, followed by [S35] methionine labeling assays, demonstrated that HPSE mRNA translation and HPSE protein synthesis were significantly upregulated in platelets during sepsis. While both the pro‐ and active forms of HPSE protein increased in platelets during sepsis, only the active form of HPSE protein significantly correlated with sepsis‐associated mortality. Consistent with transcriptomic and proteomic upregulation, HPSE enzymatic activity was also increased in platelets during sepsis. ConclusionsDuring clinical sepsis HPSE, translation, and enzymatic activity are increased in platelets. Increased expression of the active form of HPSE protein is associated with sepsis‐associated mortality.

An Activity-Based Methionine Bioconjugation Approach To Developing Proximity-Activated Imaging Reporters.

Chemical probes that report on protein activity, rather than protein abundance, with spatial and temporal resolution can enable studies of their native function in biological contexts as well as provide opportunities for developing new types of biochemical reporters. Here we present a sensing platform, termed proximity-activated imaging reporter (PAIR), which combines activity-based methionine bioconjugation and antibody labeling with proximity-dependent oligonucleotide-based amplification to monitor dynamic changes of a given analyte in cells and animals through context-dependent methionine labeling of specific protein targets. We establish this PAIR method to develop sensors for imaging reactive oxygen species (ROS) and calcium ions through oxaziridine-directed labeling of reactive methionine residues on β-actin and calmodulin (CaM), respectively, where the extent of methionine bioconjugation on these protein targets can serve as an indicator of oxidative stress or calcium status. In particular, application of PAIR to activity-based CaM detection provides a method for imaging integrated calcium activity in both in vitro cell and in vivo zebrafish models. By relying on native protein biochemistry, PAIR enables redox and metal imaging without introduction of external small molecules or genetically encoded indicators that can potentially buffer the natural/existing pools. This approach can be potentially generalized to target a broader range of analytes by pairing appropriate activity-based protein probes with protein detection reagents in a proximity-driven manner, providing a starting point not only for designing new sensors but also for monitoring endogenous activity of specific protein targets in biological specimens with spatial and temporal fidelity.

ZNF384-Fusion Proteins Have High Affinity to EP300, Which Increases Their Transcriptional Activities

ZNF384 fusion (Z-fusion) genes are recently identified recurrent fusion genes of B-acute lymphoblastic leukemia (ALL) and cause differentiation block of B-cells; however, its molecular mechanisms have yet to be clarified. Common structural character of Z-fusion proteins is that fusion partners are fused to the N-terminal end of full-length ZNF384 (Figure 1A), suggesting that protein-fusions confer specific transcriptional targets on ZNF384. We searched Z-fusion-specific transcriptional targets that could cause differentiation block of B-cells by analyzing the data of gene expression profile of 54 primary B-ALL samples containing 9 Z-fusion positive ALL. We selected ID2 and SALL4 as potential targets. Both genes were expressed markedly higher in Z-fusion-positive ALL. ID2 acts as an inhibitor of E2A, B cell differentiation regulator, and SALL4 plays essential roles in maintaining pluripotency of embryonic stem cells. In the luciferase assays, EP300-ZNF384 (E-Z) and SYNRG-ZNF384 (S-Z) showed stronger transcriptional activities on the promoters of these genes than wild-type ZNF384 (Wild-Z). The introduction of E-Z or S-Z into 293T cells and THP-1 cells induced mRNA expression of these genes more strongly than that of Wild-Z (Figure 1B). We identified Z-fusion binding sites in the promoters of these genes. DNA binding abilities of Z-fusions to these sites were not stronger than that of Wild-Z in electro mobility shift assay. GST-pull down assay showed that E-Z associated with EP300 more strongly than Wild-Z (Figure 1C). Consistent with this, co-expression of EP300 enhanced the transcriptional activity of E-Z better than that of Wild-Z (Figure 1D). These results indicated that ID2 and SALL4 were Z-fusion-specific transcriptional targets and that the high affinity to EP300 was responsible for the strong transcriptional activity of Z-fusions. Our results shed a new insight into the molecular mechanisms of leukemia development by Z-fusions.Figure legendsFigure 1. A. Schematic presentation of structures of Wild-Z and Z-fusion proteins. B. Introduction of Z-fusion genes enhanced the mRNA expression of ID2 and SALL4. Wild-Z and Z-fusion genes were introduced to THP-1 cells by nucleofection. Twenty-four hours after gene introduction, the mRNA expression of ID2 and SALL4 were quantified by RQ-PCR and potted on bar charts as relative values to the mRNA expression in the control cells. The expression of ID2 and SALL4 were shown in the left and right bar charts, respectively. C. E-Z associated with HAT more strongly than Wild-Z. Glutathione beads attached with Glutathione S transferase (GST) or GST-fused HAT were incubated with Wild-Z or E-Z synthesized in vitro with [35S]-Methionine labeling. Wild-Z or E-Z associated with GST-HAT were visualized with autoradiography (left panel), quantified, and plotted on the bar charts (right panel). Of note, the quantified association were adjusted for the ratio of the number of methionine containing in Wild-Z and E-Z, 12 to 45, and plotted on the bar charts. D. Co-expression of EP300 enhanced the transcriptional activity of E-Z better than that of Wild-Z. Luciferase assay were performed with or without co-expression of EP300 and the relative value to the control were plotted on the bar charts. [Display omitted] DisclosuresNaoe:Astellas Pharma Inc.: Research Funding; Fujifilm Corporation: Patents & Royalties, Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Pfizer Japan Inc.: Research Funding; Toyama Chemical Co., Ltd.: Research Funding. Kiyoi:FUJIFILM Corporation: Research Funding; Eisai Co., Ltd.: Research Funding; Astellas Pharma Inc.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Zenyaku Kogyo Co., Ltd.: Research Funding; Novartis Pharma K.K.: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Celgene Corporation: Research Funding; Bristol-Myers Squibb: Honoraria; Takeda Pharmaceutical Co., Ltd.: Research Funding; Phizer Japan Inc.: Research Funding; Sanofi K.K.: Research Funding.

Solution NMR spectroscopy of GPCRs: Residue-specific labeling strategies with a focus on 13C-methyl methionine labeling of the atypical chemokine receptor ACKR3.

The past decade has witnessed remarkable progress in the determination of G protein-coupled receptor (GPCR) structures, profoundly expanding our understanding of how GPCRs recognize ligands, become activated, and interact with intracellular signaling components. In recent years, numerous studies have used solution nuclear magnetic resonance (NMR) spectroscopy to investigate GPCRs, providing fundamental insights into GPCR conformational changes, allostery, dynamics, and other facets of GPCR function are challenging to study using other structural techniques. Despite these advantages, NMR-based studies of GPCRs are few relative to the number of published structures, due in part to the challenges and limitations of NMR for the characterization of large membrane proteins. Several studies have circumvented these challenges using a variety of isotopic labeling strategies, including side chain derivatization and metabolic incorporation of NMR-active nuclei. In this chapter, we provide an overview of different isotopic labeling strategies and describe an in-depth protocol for the expression, purification, and NMR studies of the chemokine GPCR atypical chemokine receptor 3 (ACKR3) via 13CH3-methionine incorporation. The goal of this chapter is to provide a resource to the GPCR community for those interested in pursuing NMR studies of GPCRs.

Prostaglandin A1 triggers Mayaro virus inhibition and heat shock protein 70 expression in an epithelial cell model.

The Mayaro virus (MAYV), which is an arbovirus closely related to the Chikungunya virus, causes a dengue-like acute illness that is endemic to Central and South America. We investigated the anti-MAYV activity of prostaglandin A1 (PGA1), a hormone which exhibits antiviral activity against both ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) viruses. Further, we examined the effects of inducting the stress protein HSP70 following PGA1 treatment. Hep-2 cells infected with MAYV were treated with PGA1 (0.1-6μg/ml) 12h before infection and for different periods post-infection. Inhibition of viral replication inhibition was analyzed via viral titer determination, whereas the effect of PGA1 on viral morphogenesis was examined via transmission electron microscopy (TEM). Autoradiography (with 35S methionine labeling) and western blotting were used to assess the effect of PGA1 treatment on viral and cellular protein synthesis, and on HSP70 induction, respectively. PGA1 strongly reduced viral replication in Hep-2 cells, particularly when added during the early stages of viral replication. Although PGA1 treatment inhibited viral replication by 95% at 24 hours post-infection (hpi), viral structural protein synthesis was inhibited only by 15%. TEM analysis suggested that PGA1 inhibited replication before viral morphogenesis. Western blot and densitometry analyses showed that PGA1 treatment increased HSP70 protein levels, although this was not detectable via autoradiography. PGA1 inhibits MAYV replication in Hep-2 cells at early stages of viral replication, prior to production of viral structural proteins, possibly via HSP70 induction.

Chasing Medium for [35S]Methionine Labeling

Chasing Medium for [³⁵S]Methionine Labeling

Cost-effective large-scale expression of proteins for NMR studies.

We present protocols for high-level expression of isotope-labelled proteins in E. coli in cost-effective ways. This includes production of large amounts of unlabeled proteins and 13C-methyl methionine labeling in rich media, where yields of up to a gram of soluble protein per liter of culture are reached. Procedures for uniform isotope labeling of 2H, 13C and 15N using auto-induction or isopropyl-β-D-1-thiogalactopyranoside-induction are described, with primary focus on minimal isotope consumption and high reproducibility of protein expression. These protocols are based on high cell-density fermentation, but the key procedures are easily transferred to shake flask cultures.

Optimization and 13CH3 methionine labeling of a signaling competent neurotensin receptor 1 variant for NMR studies

Neurotensin is a 13-residue peptide that acts as a neuromodulator of classical neurotransmitters such as dopamine and glutamate in the mammalian central nervous system, mainly by activating the G protein-coupled receptor (GPCR), neurotensin receptor 1 (NTS1). Agonist binding to GPCRs shifts the conformational equilibrium of the transmembrane helices towards distinct, thermodynamically favorable conformations that favor effector protein interactions and promotes cell signaling. The introduction of site specific labels for NMR spectroscopy has proven useful for investigating this dynamic process, but the low expression levels and poor stability of GPCRs is a hindrance to solution NMR experiments. Several thermostabilized mutants of NTS1 have been engineered to circumvent this, with the crystal structures of four of these published. The conformational dynamics of NTS1 however, has not been thoroughly investigated with NMR. It is generally accepted that stabilized GPCRs exhibit attenuated signaling, thus we thoroughly characterized the signaling characteristics of several thermostabilized NTS1 variants to identify an optimal variant for protein NMR studies. A variant termed enNTS1 exhibited the best combination of signaling capability and stability upon solubilization with detergents. enNTS1 was subsequently labeled with 13CH3-methionine in E. coli and purified to homogeneity in the absence of bound ligands. Using solution NMR spectroscopy we observed several well dispersed 13CH3-methionine resonances, many of which exhibited chemical shift changes upon the addition of the high affinity agonist peptide, NT8-13. Thus, enNTS1 represents a novel tool for investigating ligand induced conformational changes in NTS1 to gain insights into the molecular mechanisms underlying neurotensin signaling.

Labeling Medium for [35S]Methionine Labeling

Labeling Medium for [35S]Methionine Labeling

Starvation Medium for [35S]Methionine Labeling

Starvation Medium for [35S]Methionine Labeling

Redox-based reagents for chemoselective methionine bioconjugation.

Cysteine can be specifically functionalized by a myriad of acid-base conjugation strategies for applications ranging from probing protein function to antibody-drug conjugates and proteomics. In contrast, selective ligation to the other sulfur-containing amino acid, methionine, has been precluded by its intrinsically weaker nucleophilicity. Here, we report a strategy for chemoselective methionine bioconjugation through redox reactivity, using oxaziridine-based reagents to achieve highly selective, rapid, and robust methionine labeling under a range of biocompatible reaction conditions. We highlight the broad utility of this conjugation method to enable precise addition of payloads to proteins, synthesis of antibody-drug conjugates, and identification of hyperreactive methionine residues in whole proteomes.

Pim1 kinase regulates c-Kit gene translation.

BackgroundReceptor tyrosine kinase, c-Kit (CD117) plays a pivotal role in the maintenance and expansion of hematopoietic stem/progenitor cells (HSPCs). Additionally, over-expression and/or mutational activation of c-Kit have been implicated in numerous malignant diseases including acute myeloid leukemia. However, the translational regulation of c-Kit expression remains largely unknown.Methods and resultsWe demonstrated that loss of Pim1 led to specific down-regulation of c-Kit expression in HSPCs of Pim1−/− mice and Pim1−/−2−/−3−/− triple knockout (TKO) mice, and resulted in attenuated ERK and STAT3 signaling in response to stimulation with stem cell factor. Transduction of c-Kit restored the defects in colony forming capacity seen in HSPCs from Pim1−/− and TKO mice. Pharmacologic inhibition and genetic modification studies using human megakaryoblastic leukemia cells confirmed the regulation of c-Kit expression by Pim1 kinase: i.e., Pim1-specific shRNA knockdown down-regulated the expression of c-Kit whereas overexpression of Pim1 up-regulated the expression of c-Kit. Mechanistically, inhibition or knockout of Pim1 kinase did not affect the transcription of c-Kit gene. Pim1 kinase enhanced c-Kit 35S methionine labeling and increased the incorporation of c-Kit mRNAs into the polysomes and monosomes, demonstrating that Pim1 kinase regulates c-Kit expression at the translational level.ConclusionsOur study provides the first evidence that Pim1 regulates c-Kit gene translation and has important implications in hematopoietic stem cell transplantation and cancer treatment.

Characterization of proteins by in-cell NMR spectroscopy in cultured mammalian cells.

In-cell NMR spectroscopy is a unique tool for characterizing biological macromolecules in their physiological environment at atomic resolution. Recent progress in NMR instruments and sample preparation methods allows functional processes, such as metal uptake, disulfide-bond formation and protein folding, to be analyzed by NMR in living, cultured human cells. This protocol describes the necessary steps to overexpress one or more proteins of interest inside human embryonic kidney 293T (HEK293T) cells, and it explains how to set up in-cell NMR experiments. The cDNA is transiently transfected as a complex with a cationic polymer (DNA:PEI (polyethylenimine)), and protein expression is carried on for 2-3 d, after which the NMR sample is prepared. (1)H and (1)H-(15)N correlation NMR experiments (for example, using band-selective optimized flip-angle short-transient heteronuclear multiple quantum coherence (SOFAST-HMQC)) can be carried out in <2 h, ensuring cell viability. Uniform (15)N labeling and amino-acid-specific (e.g., cysteine, methionine) labeling schemes are possible. The entire procedure takes 4 d from cell culture seeding to NMR data collection.

A novel automethylation reaction in the Aspergillus nidulans LaeA protein generates S‐methylmethionine (555.2)

The filamentous fungi in the genus Aspergillus are opportunistic plant and animal pathogens that produce secondary metabolites including lovastatin, penicillin, and aflatoxin. The synthesis of these small molecules is dependent on gene clusters that are globally regulated by the LaeA protein. Null mutants of LaeA show decreased virulence coupled with reduced secondary metabolism. Although LaeA contains the motifs characteristic of seven‐beta‐strand methyltransferases, a methyl‐accepting substrate of LaeA has not been identified. In this work, we did not find a methyl‐accepting substrate in A. nidulans with various assays including in vivo S‐adenosyl‐[methyl‐3H]methionine labeling, targeted in vitro methylation experiments using putative protein substrates, or in vitro methylation assays using whole cell extracts grown under different conditions. However, in each experiment LaeA was shown to self‐methylate. Amino acid hydrolysis of radioactively‐labeled LaeA followed by cation exchange and reverse phase chromatography identified methionine as the modified residue. Point mutations show that the major site of modification of LaeA is on methionine‐207. LaeA is the first protein to exhibit automethylation at a methionine residue. These findings not only indicate LaeA may perform novel chemistry with S‐adenosylmethionine, but also provide insights into the physiological function of LaeA.Grant Funding Source: Supported by Ruth L. Kirschstein National Research Service Award GM007185

Molecular Mechanism of 17-Allylamino-17-demethoxygeldanamycin (17-AAG)-induced AXL Receptor Tyrosine Kinase Degradation

The receptor tyrosine kinase AXL is overexpressed in many cancer types including thyroid carcinomas and has well established roles in tumor formation and progression. Proper folding, maturation, and activity of several oncogenic receptor tyrosine kinases require HSP90 chaperoning. HSP90 inhibition by the antibiotic geldanamycin or its derivative 17-allylamino-17-demethoxygeldanamycin (17-AAG) causes destabilization of its client proteins. Here we show that AXL is a novel client protein of HSP90. 17-AAG induced a time- and dose-dependent down-regulation of endogenous or ectopically expressed AXL protein, thereby inhibiting AXL-mediated signaling and biological activity. 17-AAG-induced AXL down-regulation specifically affected fully glycosylated mature receptor present on cell membrane. By using biotin and [(35)S]methionine labeling, we showed that 17-AAG caused depletion of membrane-localized AXL by mediating its degradation in the intracellular compartment, thus restricting its exposure on the cell surface. 17-AAG induced AXL polyubiquitination and subsequent proteasomal degradation; under basal conditions, AXL co-immunoprecipitated with HSP90. Upon 17-AAG treatment, AXL associated with the co-chaperone HSP70 and the ubiquitin E3 ligase carboxyl terminus of HSC70-interacting protein (CHIP). Overexpression of CHIP, but not of the inactive mutant CHIP K30A, induced accumulation of AXL polyubiquitinated species upon 17-AAG treatment. The sensitivity of AXL to 17-AAG required its intracellular domain because an AXL intracellular domain-deleted mutant was insensitive to the compound. Active AXL and kinase-dead AXL were similarly sensitive to 17-AAG, implying that 17-AAG sensitivity does not require receptor phosphorylation. Overall our data elucidate the molecular basis of AXL down-regulation by HSP90 inhibitors and suggest that HSP90 inhibition in anticancer therapy can exert its effect through inhibition of multiple kinases including AXL.

A Novel Automethylation Reaction in the Aspergillus nidulans LaeA Protein Generates S-Methylmethionine

The filamentous fungi in the genus Aspergillus are opportunistic plant and animal pathogens that can adapt to their environment by producing various secondary metabolites, including lovastatin, penicillin, and aflatoxin. The synthesis of these small molecules is dependent on gene clusters that are globally regulated by the LaeA protein. Null mutants of LaeA in all pathogenic fungi examined to date show decreased virulence coupled with reduced secondary metabolism. Although the amino acid sequence of LaeA contains the motifs characteristic of seven-β-strand methyltransferases, a methyl-accepting substrate of LaeA has not been identified. In this work we did not find a methyl-accepting substrate in Aspergillus nidulans with various assays, including in vivo S-adenosyl-[methyl-(3)H]methionine labeling, targeted in vitro methylation experiments using putative protein substrates, or in vitro methylation assays using whole cell extracts grown under different conditions. However, in each experiment LaeA was shown to self-methylate. Amino acid hydrolysis of radioactively labeled LaeA followed by cation exchange and reverse phase chromatography identified methionine as the modified residue. Point mutations show that the major site of modification of LaeA is on methionine 207. However, in vivo complementation showed that methionine 207 is not required for the biological function of LaeA. LaeA is the first protein to exhibit automethylation at a methionine residue. These findings not only indicate LaeA may perform novel chemistry with S-adenosylmethionine but also provide new insights into the physiological function of LaeA.

Isolation of Radiolabeled Poliovirus Particles from H1 HeLa Cells

The following protocol describes the isolation of radioactive viral and subviral particles from infected cells. This protocol has been written for isolation of poliovirus particles from H1 HeLa cells. Infection protocol and timing of [35S] methionine labeling and particle collection should be tailored to the virus of interest. Following isolation of the viral particles, the viral proteins present in these particles may be separated by gel electrophoresis and visualized by autoradiography.

Endoplasmic reticulum stress decreases intracellular thyroid hormone activation via an eIF2a-mediated decrease in type 2 deiodinase synthesis.

Cells respond rapidly to endoplasmic reticulum (ER) stress by blocking protein translation, increasing protein folding capacity, and accelerating degradation of unfolded proteins via ubiquitination and ER-associated degradation pathways. The ER resident type 2 deiodinase (D2) is normally ubiquitinated and degraded in the proteasome, a pathway that is accelerated by enzyme catalysis of T(4) to T(3). To test whether D2 is normally processed through ER-associated degradation, ER stress was induced in cells that endogenously express D2 by exposure to thapsigargin or tunicamycin. In all cell models, D2 activity was rapidly lost, to as low as of 30% of control activity, without affecting D2 mRNA levels; loss of about 40% of D2 activity and protein was also seen in human embryonic kidney 293 cells transiently expressing D2. In primary human airway cells with ER stress resulting from cystic fibrosis, D2 activity was absent. The rapid ER stress-induced loss of D2 resulted in decreased intracellular D2-mediated T(3) production. ER stress-induced loss of D2 was prevented in the absence of T(4), by blocking the proteasome with MG-132 or by treatment with chemical chaperones. Notably, ER stress did not alter D2 activity half-life but rather decreased D2 synthesis as assessed by induction of D2 mRNA and by [(35)S]methionine labeling. Remarkably, ER-stress-induced loss in D2 activity is prevented in cells transiently expressing an inactive eukaryotic initiation factor 2, indicating that this pathway mediates the loss of D2 activity. In conclusion, D2 is selectively lost during ER stress due to an eukaryotic initiation factor 2-mediated decrease in D2 synthesis and sustained proteasomal degradation. This explains the lack of D2 activity in primary human airway cells with ER stress resulting from cystic fibrosis.

Vesicular stomatitis virus as a treatment for colorectal cancer

M protein mutant vesicular stomatitis virus is an attractive candidate oncolytic virus for the treatment of metastatic colorectal cancer due to its ability to kill cancer cells that are defective in their antiviral responses. The oncolytic activity of recombinant wild-type and M protein mutant vesicular stomatitis viruses was determined in RKO, Hct116 and LoVo colorectal cancer cells, as well as in human fibroblast and hepatocyte primary cultures. RKO and Hct116 cells were sensitive to both viruses, whereas LoVo cells were resistant. [(35)S]methionine labeling experiments and viral plaque assays showed that sensitive and resistant colorectal cancer cells supported viral protein and progeny production after infection with either virus. Colorectal cancer cells were pretreated with β-interferon and infected with vesicular stomatitis virus to evaluate the extent to which interferon signaling is downregulated in colorectal cancer cells. Although colorectal cancer cells retained some degree of interferon signaling, this signaling did not negatively impact the oncolytic effects of either virus in sensitive cells. Murine xenografts of RKO cells were effectively treated by intratumoral injections with M protein mutant virus, whereas LoVo xenografts were resistant to treatment with this virus. These results suggest that M protein mutant vesicular stomatitis virus is a good candidate oncolytic virus for the treatment of selected metastatic colorectal cancers.