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Chen, Ying (2006): In vivo detection of the BCR/ABL1 protein: towards a new therapeutic strategy for fusion protein associated leukemias. Dissertation, LMU München: Faculty of Medicine
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Abstract

The BCR/ABL1 fusion protein is found in virtually all cases chronic myeloid leukemia (CML) and a large proportion of acute lymphoblastic leukemia (ALL). The fact that the BCR/ABL1 fusion protein is crucial for the development of leukemia makes this fusion protein an attractive target for therapy development. We have developed a strategy for the in vivo detection of the BCR/ABL1 fusion protein, in which the presence of the BCR/ABL1 fusion protein is detected intracellularly and if the fusion protein is present an arbitrary action is initiated in the cell (e.g. mark the cells or selectively kill the cells). Our BCR/ABL1 detection strategy is based on protein-protein interactions. Two detection proteins are expressed in the cells: 1) protein A, a GAL4-DNA binding domain/BCR interacting protein fusion protein (GAL4DBD-BAP-1) and 2) protein B, a GAL4-activation domain/ABL interacting protein fusion protein (GAL4AD-CRKL). Only when BCR/ABL1 is present in the cell, do protein A, protein B, and BCR/ABL1 form a trimeric complex which activates the transcription of reporter genes under the control of GAL4-upstream activating sequence (UAS). A proof of principle for the strategy was implemented in the yeast system. We did not use full length BAP-1 or CRKL but only those portions of the proteins that directly interacted with BCR or ABL, respectively. We showed in the yeast two hybrid system, that the C-terminus of BAP-1(amino acids 617-879) binds to full length BCR. The site of interaction of CRKL and ABL was confirmed to be the N-terminal SH3 domain (SH3n) of CRKL as described in the literature. Yeast cells (strain CG1945) transformed with a protein A expressing plasmid (pGBT9-BAP), a protein B expressing plasmid (pGAD424-CRKLSH3n), and a BCR/ABL expressing plasmid (pES1/BCR-ABL) showed expression of the reporter genes HIS3 and LACZ. The expression of the HIS3 reporter gene was assayed by growth of the yeast cells on medium lacking histidine. The expression of the LACZ gene was verified by a beta-galactosidase filter assay. Yeast cells that were transformed with the pES1 plasmid without the BCR/ABL1 coding region did not show activation of the reporter genes. Several other negative controls demonstrated the specificity of the assay. Thus the method was able to clearly distinguish between BCR/ABL expressing cells and cells did not express BCR/ABL1. We then adapted this system for use in mammalian cells. The open-reading frames encoding the proteins A and B were recloned into mammalian expression vectors. The human embryonal kidney cell line HEK293 and the murine myeloid progenitor cell line 32D which had been stably transfected with a BCR/ABL expressing plasmid were tested. The firefly luciferase gene and the yellow fluorescent protein (eYFP) were used to evaluate the whole cell population and single cell, respectively. Unfortunately, the system failed to work in the mammalian cell lines tested. Even though the detection system did not work in mammalian cells, most likely due to the cytoplasmic localization of the BCR/ABL1 fusion protein, it should still be a viable strategy for the detection of leukemia-associated fusion protein, which localize to the nucleus (i.e AML-ETO). This strategy could be adapted for purging the bone marrow of leukemia patients using therapeutically more useful effector genes like suicide genes, which encode pro-drug converting enzymes (e.g. HSV thymidine kinase), or markers that can easily be assayed (e.g. YFP).