How ever, such compounds are still poorly developed. TFs activate transcription

In our experiments, Western blot analysis showed that anti GD2 [You must be registered and logged in to see this link.] antibodies 14G2a could bind to certain protein with a molecular weight of 105 115 kDa from lysate of EL 4 cells. At the same time anti GD2 antibodies ME361 did not react with any protein from the same EL 4 cell lysate, Although 14G2a antibodies reacted with the protein that has a molecular weight similar to ALCAM, these results do not provide ultimate evidence We further analyzed the mitochondria involvement in the cell death induced by anti GD2 mAb using two specific sen sitive fluorescent probes JC 1 and DiOC6. Flow cytometry analysis of mitochondrial membrane potential of AVD and 7 AAD negative EL 4 cells was performed and the results are shown in Figure 6C, D.

Using JC 1 and DiOC6 probes, we found that treatment of cells with anti GD2 mAb 14G2a for 2 h resulted in a significant increase in Ψm as determined by increased ratio of FL2 FL1 fluores cence for JC 1 and increase in MFI of green fluorescence in 14G2a treated cells for DiOC6 when compared [You must be registered and logged in to see this link.] with Ψm of intact cells. At the same time, staurosporine induced depolarization of that 14G2 antibodies react with ALCAM, but not with other proteins of the similar weight. Moreover, even if such interaction of 14G2 antibodies with ALCAM is con firmed, it does not necessarily indicate that 14G2a mAb specifically interacts with extracellular part of ALCAM molecule.

To assess [You must be registered and logged in to see this link.] the possibility of interaction of 14G2a with extracellular part of ALCAM molecule, we have selected several cell lines that expressed ALCAM and, at the same time, were negative for GD2, Using specific antibodies that recognize extracellular C terminus of the ALCAM molecule we demonstrated that GD2 positive cell line and two GD2 negative cell lines expressed ALCAM on their surface, At the same time, staining of Jurkat and L1210 cells with anti GD2 antibodies 14G2a demonstrated that these antibodies did not bind to these ALCAM positive cells, We concluded from these experiments that 14G2a antibodies did not bind the extracellular region of ALCAM on the surface of ALCAM positive cell lines. Due to similar structure of various types of gangliosides, it was also important to evaluate the ability of anti GD2 mAbs 14G2a and ME361 to cross react with other gangliosides. We evaluated binding properties of both monoclonal anti bodies 14G2a and ME361 to immobilized gangliosides by ELISA.

BODIPY FL C5 labeled gangliosides were used to check amounts of gangliosides adsorbed to the plate to ensure equal amount of gangliosides in each well for further ELISA analysis, This assay allowed us to conduct a quantitative comparison of binding patterns of anti GD2 mAbs 14G2a and ME361 to various gangliosides. Our analysis of cross reactivity of anti GD2 mAbs is presented in Figure 8A, B. The ME361 antibody displayed a weak cross reactivity with ganglioside GD3 and GD1b, while 14G2a anti bodies showed no significant cross reactivity with the gangliosides GM2, GD1b and GD3, Conse quently, the cytotoxic effects of ME361 antibodies could be also mediated by interaction with not only GD2, but also with gangliosides GD1b and GD3.