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Methods for evaluating cell-specific, cell-internalizing RNA aptamers.
Hernandez L, Flenker K, Hernandez F, Klingelhutz A, McNamara J, Giangrande P (2013) Methods for evaluating cell-specific, cell-internalizing RNA aptamers. Pharmaceuticals (Basel) 6:295-319. doi: 10.3390/ph6030295
Objective: Isolate aptamers that internalize upon binding to their cognate receptor on the cell surface
Summary: Among the methods used to characterize aptamers that internalize is a way to monitor for cytoplasmic delivery using the ribosome inactivating protein-based (RNA-RIP) assay. Biotin-labeled A9g was conjugated to streptavidin-modified saporin (streptavidin-ZAP). First, it was verified that conjugation of biotinylated aptamer to Streptavidin-ZAP (A9g-SAP) did not affect the inhibitory effect of the aptamer. Next, the effect was examined of A9g-SAP on PC3(PSMA+) and PC3(PSMA-) cells. Cells were treated with varying amounts of aptamer-saporin conjugate for 72 h at 37°C and then an assay was performed to determine potential cytotoxicity of the conjugate. Results confirm that A9g is internalized preferentially into target cells and that A9g is efficiently accessing the cytoplasm of target cells possibly through a mechanism of endosomal escape, resulting in inhibition of protein synthesis and ultimate cell-death. FGF-SAP was used as a control.
Related Products: Streptavidin-ZAP (Cat. #IT-27), FGF-SAP (Cat. #IT-38)
Direct retino-raphe projection alters serotonergic tone and affective behavior.
Ren C, Luan L, Wui-Man Lau B, Huang X, Yang J, Zhou Y, Wu X, Gao J, Pickard GE, So KF, Pu M (2013) Direct retino-raphe projection alters serotonergic tone and affective behavior. Neuropsychopharmacology 38(7):1163-1175. doi: 10.1038/npp.2013.35
Summary: Although recent work has shown that some intrinsically photosensitive retinal ganglion cells (ipRGCs) are responsible for processing nonimage-forming visual functions, it is unclear whether the ipRGCs or conventional RGCs modulate affective behavior. The authors injected 2 μg of melanopsin-SAP (Cat. #IT-44) into each eye of gerbils, or biotinylated CTB monoclonal antibody coupled to Streptavidin-ZAP (Cat. #IT-27). The data suggest that retino-raphe signals modulate dorsal raphe nucleus serotonergic tone and affective behavior.
Related Products: Melanopsin-SAP (Cat. #IT-44), Streptavidin-ZAP (Cat. #IT-27)
Photochemical internalization (PCI) of HER2-targeted toxins: Synergy is dependent on the treatment sequence.
Berstad MB, Weyergang A, Berg K (2012) Photochemical internalization (PCI) of HER2-targeted toxins: Synergy is dependent on the treatment sequence. Biochim Biophys Acta 1820(12):1849-1858. doi: 10.1016/j.bbagen.2012.08.027
Summary: A majority of patients develop acquired resistance to trastuzumab, the monoclonal antibody recognizing HER2, coupled to a toxin as a breast cancer therapeutic. One of the modes of resistance is that the therapeutic molecule is trapped inside an endocytic vesicle. PCI is a technique that facilitates cytosolic release of molecules in vesicles. The authors investigated the potency of biotinylated trastuzumab combined with streptavidin-ZAP (Cat. #IT-27) on several cell lines.
Related Products: Streptavidin-ZAP (Cat. #IT-27)
Identification of a cKit(+) colonic crypt base secretory cell that supports Lgr5(+) stem cells in mice
Rothenberg ME, Nusse Y, Kalisky T, Lee JJ, Dalerba P, Scheeren F, Lobo N, Kulkarni S, Sim S, Qian D, Beachy PA, Pasricha PJ, Quake SR, Clarke MF (2012) Identification of a cKit(+) colonic crypt base secretory cell that supports Lgr5(+) stem cells in mice. Gastroenterology 142:1195-1205.e1196. doi: 10.1053/j.gastro.2012.02.006
Objective: To investigate the existence of colonic Paneth-like cells that have a distinct transcriptional signature and support Lgr5 stem cells.
Summary: cKit marks small intestinal Paneth cells and a subset of colonic goblet cells that are regulated by Notch signaling and support Lgr5+ stem cells.
Usage: For saporin experiments, small intestinal organoids with crypt buds and visible Paneth cells were passaged as single cells. Before embedding in Matrigel, dissociated cells were incubated with Anti-cKit-SAP (biotinylated-2B8 mixed with Streptavidin-ZAP). Treatment led to a marked decrease in organoid formation.
Related Products: Streptavidin-ZAP (Cat. #IT-27)
Strongly amphiphilic photosensitizers are not substrates of the cancer stem cell marker ABCG2 and provides specific and efficient light-triggered drug delivery of an EGFR-targeted cytotoxic drug.
Selbo PK, Weyergang A, Eng MS, Bostad M, Maelandsmo GM, Hogset A, Berg K (2012) Strongly amphiphilic photosensitizers are not substrates of the cancer stem cell marker ABCG2 and provides specific and efficient light-triggered drug delivery of an EGFR-targeted cytotoxic drug. J Control Release 159(2):197-203. doi: 10.1016/j.jconrel.2012.02.003
Summary: Many anti-cancer drugs are substrates of the ATP-binding cassette transporter ABCG2. Unfortunately ABCG2 is also thought to play an important role in multi-drug resistance and the protection of cancer stem cells against chemotherapeutics and photodynamic therapy. This paper examined whether photosensitizers used in photochemical internalization (PCI) are substrates for ABCG2. Streptavidin-ZAP (Cat. #IT-27) was combined with biotinylated EGF and applied to cells in culture; saporin (Cat. #PR-01) was used as a control. The data show that PCI with the EGF-saporin toxin did not utilize ABCG2 to enter cells.
Related Products: Streptavidin-ZAP (Cat. #IT-27), Saporin (Cat. #PR-01)
Tumour hypoxia promotes tolerance and angiogenesis via CCL28 and Treg cells
Facciabene A, Peng X, Hagemann IS, Balint K, Barchetti A, Wang L-P, Gimotty PA, Gilks CB, Lal P, Zhang L, Coukos G (2011) Tumour hypoxia promotes tolerance and angiogenesis via CCL28 and Treg cells. Nature 475:226-230. doi: 10.1038/nature10169 PMID: 21753853
Objective: To investigate whether a direct link between tumor hypoxia and tolerance occurs through the recruitment of regulatory cells.
Summary: Tumor hypoxia promotes the recruitment of regulatory T (Treg) cells through induction of expression of the chemokine CC-chemokine ligand 28 (CCL28), which, in turn, promotes tumor tolerance and angiogenesis.
Usage: In vivo depletion of CD4+ CD25+ cells was achieved by intraperitoneal administration of anti-CD25 or an immunotoxin consisting of anti-mouse CCR10 or anti-mouse CCR3 antibody conjugated at an equimolar ratio to Streptavidin–ZAP. Anti-CCR10–SAP depleted 90% of CCR101 or CCR31 cells. Anti-CCR10–SAP suppressed tumour growth and abrogated the effects of CCL28 overexpression, whereas anti-CCR3–SAP had no effect on tumor growth.
Related Products: Streptavidin-ZAP (Cat. #IT-27)
Sezary syndrome cells overexpress syndecan-4 bearing distinct heparan sulfate moieties that suppress T-cell activation by binding DC-HIL and trapping TGF-beta on the cell surface.
Chung JS, Shiue LH, Duvic M, Pandya A, Cruz PDJ, Ariizumi K (2011) Sezary syndrome cells overexpress syndecan-4 bearing distinct heparan sulfate moieties that suppress T-cell activation by binding DC-HIL and trapping TGF-beta on the cell surface. Blood 117(12):3382-3390. doi: 10.1182/blood-2010-08-302034
Summary: Syndecan-4 (SD-4) is a transmembrane heparan sulfate proteoglycan. The Sézary syndrome (SS) subset of cutaneous T-cell lymphoma overexpresses distinct heparan sulfate moieties, giving the authors a specific target for these cells. Biotinylated DC-HIL- Fc (the extracelluar domain of dendritic cell- associated heparan sulfate proteoglycan- integrin ligand fused to Fc of mouse IgG) was combined at a 1:1 molar ratio with streptavidin-ZAP (Cat. #IT-27). In vitro, this targeted toxin eliminated SS cells, preventing their proliferation and suggesting a method for SS treatment.
Related Products: Streptavidin-ZAP (Cat. #IT-27)
The cell surface structure of tumor endothelial marker 8 (TEM8) is regulated by the actin cytoskeleton.
Yang MY, Chaudhary A, Seaman S, Dunty J, Stevens J, Elzarrad MK, Frankel AE, St Croix B (2011) The cell surface structure of tumor endothelial marker 8 (TEM8) is regulated by the actin cytoskeleton. Biochim Biophys Acta 1813(1):39-49. doi: 10.1016/j.bbamcr.2010.11.013
Summary: Tumor endothelial marker 8 (TEM8) is a cell surface protein that is up-regulated on tumor blood vessels. Overexpression of this protein, however, produces a form that is not recognized by the SB5 monoclonal antibody used to bind TEM8. While cells expressing normal levels of TEM8 were killed by an application of biotinylated SB5 plus either 1 nM or 10 nM streptavidin-ZAP (Cat. #IT-27), cells overexpressing the protein did not bind the immunotoxin. Understanding the structural differences between the two forms of TEM8 will help in the design of therapeutic antibodies against these tumor cells.
Related Products: Streptavidin-ZAP (Cat. #IT-27)
Saporin toxin-conjugated monoclonal antibody targeting prostate-specific membrane antigen has potent anticancer activity.
Kuroda K, Liu H, Kim S, Guo M, Navarro V, Bander NH (2010) Saporin toxin-conjugated monoclonal antibody targeting prostate-specific membrane antigen has potent anticancer activity. Prostate 70(12):1286-1294. doi: 10.1002/pros.21164
Summary: Current treatments for prostate cancer are only moderately effective. In this work the authors examined the cytotoxic efficacy of an anti-prostate-specific membrane antigen (PMSA) antibody conjugated to saporin on PMSA-positive cell lines. hJ591, a humanized anti-PMSA antibody, was biotinylated and combined with streptavidin-ZAP (Cat. #IT-27). The hJ591-streptavidin-ZAP complex was specifically cytotoxic to PMSA-positive cell lines, and had anti-cancer activity in a xenograft model. This work demonstrates the anti-cancer potential of targeting PMSA.
Related Products: Streptavidin-ZAP (Cat. #IT-27)
Toxin-coupled MHC class I tetramers can specifically ablate autoreactive CD8+ T cells and delay diabetes in nonobese diabetic mice.
Vincent BG, Young EF, Buntzman AS, Stevens R, Kepler TB, Tisch RM, Frelinger JA, Hess PR (2010) Toxin-coupled MHC class I tetramers can specifically ablate autoreactive CD8+ T cells and delay diabetes in nonobese diabetic mice. J Immunol 184(8):4196-4204. doi: 10.4049/jimmunol.0903931
Summary: MHC class I tetramers have been used to identify antigen-specific cells. In this work the authors used a biotinylated tetramer in conjunction with streptavidin-ZAP (Cat. #IT-27) to eliminate a specific subset of reactive T cells associated with islets in vivo. NOD mice received three 4.36 µg intravenous injections of the tetramer/saporin complex over 12 days. The onset of type I diabetes in the treated mice was significantly delayed.
Related Products: Streptavidin-ZAP (Cat. #IT-27)