To analyze this, we carried out confocal microscopy and sub-cellular fractionation of HepG2 cells to determine the intracellular trafficking of the two proteins

To analyze this, we carried out confocal microscopy and sub-cellular fractionation of HepG2 cells to determine the intracellular trafficking of the two proteins. analyzed for their translation inhibitory activity using the translation assay. Here, rabbit reticulocyte lysate was treated with different concentrations (10 pM to 1 1 nM) of rABRa-A or rABRa-A (R167L) in a cocktail containing luciferase mRNA. The extent of luciferase synthesized by the lysate, in presence of the protein, was analyzed by adding luciferase substrate and determining the extent of luminescence produced. C: Construction and purification of immunotoxin: MAb F1G4 was conjugated to rABRa-A using SMPT as the crosslinker. a: The conjugate, purified on Cibacron blue 3GA affinity column was tested for purity on a 7.5% polyacrylamide SDS-gel under non-reducing conditions and immunoblotted with mAb D6F10-biotin. Lanes: 15 g mAb F1G4-rABRa-A; 25 g rABRa-A; 31 g mAb F1G4. b: The purified conjugate, obtained from Cibacron blue column, was re-purified using protein A affinity column to remove any remaining free A chain. The purity of the samples was tested on a 7.5% polyacrylamide SDS gel under non-reducing conditions and immunoblotted with mAb D6F10. Lanes: 1: Load; 2: Flow through; 3?4: Washes; 5?7: elution fractions.(TIF) pone.0058304.s002.tif (2.6M) GUID:?3ED5247B-84ED-40D9-BC16-25821EBB0838 Figure S3: MCF-7 cells are more sensitive than MCF-10A to mAb F1G4-rABRa-A induced toxicity. MCF-7 and MCF-10A cells (1106/ml) were cultured in the presence of different concentrations of F1G4-IT and assayed for protein synthesis as described earlier. The incorporated radioactivity for each sample was plotted as % of that for the control cells. Each lane represents a mean of at least three different experiments, with each treatment carried out in duplicates.(TIF) pone.0058304.s003.tif (229K) GUID:?F8A67695-84FF-44F6-ACC1-62FC1F5B4D9B Figure S4: FACScan profiles of HepG2 cells treated with abrin, F1G4-IT or F1G4-IT(R167L). HepG2 cells (1106/ml) were treated with 19.2 nM of either one IOX4 of the immunoconjugates: F1G4-IT or F1G4-ITR167L, or abrin (51.25 pM) IOX4 for different time intervals. The cells were harvested, fixed with 70% ethanol at ?20C, stained with staining solution (20 g/ml propidium iodide and 50 g/ml RNase A in PBS) and analyzed by flow cytometry. The samples were analyzed by WinMDI v2.9. The X-axis is the mean fluorescence intensity of PI and the Y-axis, the cell number, as events. Each profile indicates the statistics of cells in sub-G0/G1 stage, as M1, which indicates the extent of DNA fragmentation, a direct correlation to cells undergoing cell death. a: Cells treated with abrin; b: Cells IOX4 treated with F1G4-IT; c: Cells treated with F1G4-IT(R167L).(TIF) pone.0058304.s004.tif (1.1M) GUID:?E9B90DD8-5A8B-44B2-BB31-39EFEBFA54A6 Abstract Background Immunotherapy is fast emerging as one of the leading modes of treatment of cancer, in combination with chemotherapy and radiation. Use of immunotoxins, proteins bearing a cell-surface receptor-specific antibody conjugated to a toxin, enhances the efficacy of cancer treatment. The toxin Abrin, isolated from the plant, is a type II ribosome inactivating protein, has a catalytic efficiency higher than Rabbit polyclonal to PNPLA2 any other toxin belonging to this class of proteins but has not been exploited much for use in targeted therapy. Methods Protein synthesis assay using 3[H] L-leucine incorporation; construction and purification of immunotoxin; study of cell death using flow cytometry; confocal scanning microscopy and sub-cellular fractionation with immunoblot analysis of localization of proteins. Results We used the recombinant A chain of abrin to conjugate to antibodies raised against the human gonadotropin releasing hormone receptor. The conjugate inhibited protein synthesis and also induced cell death specifically in cells expressing the receptor. The conjugate exhibited differences in the kinetics of inhibition of protein synthesis, in comparison to abrin, and this was attributed to differences in internalization and trafficking of the conjugate within the cells. Moreover, observations of sequestration of the A chain into the nucleus of cells treated with abrin but not in cells treated with the conjugate reveal a novel pathway for the movement of the conjugate in the cells. Conclusions This is one of the first reports on nuclear localization of abrin, a type II RIP. The immunotoxin mAb F1G4-rABRa-A, generated in our laboratory, inhibits protein synthesis specifically on cells expressing the gonadotropin releasing hormone receptor and the pathway of internalization of the protein is distinct from that seen for abrin. Introduction Chemotherapy is the most common modes of treatment.