Abstract
Abstract 4720
Introduction:
Our efforts aim at the development of effective experimental vaccines against bcr-abl-transformed cells. For our study two mouse (Balb/c) bcr-abl-transformed cell lines were chosen, viz. Ba-p210 (B210) and 12B1. They express comparable amounts of p210bcr-abl protein and both are of early B cell lineage, but they differ in a number of properties, namely morphology, expression of MHC I molecules and some other surface markers (such as AA4.1, CD 19, CD10 and B7.H4) and oncogenic potential. Both induce leukemia after intravenous administration, but 12B1 cells are approximately 100x more oncogenic and, in addition, induce lymphoma-like solid tumours after subcutaneous administration (Sobotkova et al., 2005). Studies carried out in our laboratory with both B210 and 12B1 cells gene-engineered to secrete various immunostimulatory cytokines indicated that they were capable of inducing protection in immunization/challlenge experiments and, to a certain degree, had the potential to serve as therapeutic vaccines. Our results indicated that the fusion zone of the p210bcr-abl protein does not carry an epitope immunogenic for Balb/c mice, and that the two cell lines differ in their antigenic make-up. In general, it was much easier to induce immunity against the B210 cells than against the 12B1 cells. It has been the aim of the present undertaking to broaden the knowledge on the antigenic make-up of the two cell lines and, hopefully, to identify cell proteins carrying the immunodominant epitopes. Furthermore we hope that this investigation, which is still under way, may help us to understand the different in vivo behavior of B210 and 12B1 cells.
Methods:
Kinex™ antibody microarray monitoring the presence of 800 cell signaling proteins in cell lysates was used. Attempts were made to verify a portion of the microarray results by Western blotting (WB). Furthermore, using WB we have started a systematic search for the presence of proteins in the cell lysates, which were recognized to be overexpressed in human CML cells and found to be immunogenic in the patients.
Results:
Significant differences in the expression of the Kinex™-monitored proteins between the 12B1 and B210 were detected in the total of 31 instances. Most marked differences were the overexpression of integrin β1 in 12B1 cells and overexpression of pro-capase proteins in B210 cells. Eighteen of these proteins were selected for WB. These tests demonstrated overexpression of intergrin β 1 and calcium/calmodulin-dependent protein-serine kinase 2 (CaMK2b) in 12B1 cells and pro-caspase proteins 5 and 7, Hsp90a, mitogen-activated protein-serine kinase p38γ (MAKP12) and mitogen-activated protein-serine kinase p38α (MAPK14) in B210 cells. In the other cases there was no difference or the differences were quite small or the respective proteins were not detected at all. Till now no marked differences between the two lines were detected in the production of proteins, the analogues of which are overexpressed in human CML cells. Neither WT-1 nor Pr-3 proteins were detected in lysates of either 12B1 or B210 cells.
Conclusion:
Although the characterization of the two cell lines has not yet been completed, the present data reveal a number of differences in the protein composition of the two cell lines. Some of the differences revealed may be associated with the different oncogenic potential of the two cell lines. However, thus far no clear data are available which might explain the higher immunogenicity of B210 cells.
This work was supported by MZCR IGA NS 10634–3/2009 and by MZ0UHKT 2005.
Disclosures:
No relevant conflicts of interest to declare.
Comparative Analysis of Click Chemistry Mediated Activity-Based Protein Profiling in Cell Lysates