The bcr-abl c-terminus is required for bcr-abl mediated apoptotic suppression and drug resistance but not proliferation or adhesion defects in haemopoietic progenitor cell line, fdcp-mix

Abstract

The chimeric Bcr-Abl protein has constitutively activated Abl protein tyrosine kinase (PTK), a phenomenon deemed to be essential for the transforming ability of Bcr-Abl. The F- and G-actin binding region of the Bcr-Abl C-terminus is also thought to be important in Bcr-Abl mediated transformation. In order to investigate the importance of this domain a temperature-sensitive Bcr-Abl protein tyrosine kinase (tsBcr-Abl) and a tsBcr-Abl mutant deleted for 167 C-terminal amino acids were transfected into the mutipotent haemopoietic progenitor FDCP-Mix cell line. The expressed proteins have activated kinase function at 32°C but are inactive at 39°C. C-terminus truncation does not abrogate the temperature-sensitive PTK activity of the Bcr-Abl protein. Previous studies have shown that tsBcr-Abl transfected FDCP-Mix remain growth factor-dependent for survival and proliferation. However, at 32°C these cells display enhanced survival and proliferation in the absence or at low levels of growth factor (IL-3). In this investigation, viability assays and tritiated thymidine (3H) incorporation found that deletion of the C-terminus reversed both Bcr-Abl mediated apoptotic-suppression and resistance to cytotoxic drugs, cytosine arabinofuranoside and hydroxyurea, but had no effect on Bcr-Abl mediated proliferative synergy. Immunofluorescence studies found that full length Bcr-Abl localised to puntate areas within the cytoplasm. In contrast, C-terminus deleted Bcr-Abl displayed some diffuse, non-specific cytoplasmic staining while retaining a high level of nuclear staining similar to c-Abl. This may indicate an inability to bind to actin filaments. Furthermore, adhesion studies showed that both full-length and truncated Bcr-Abl mediated reduced adhesion to fibronectin, in the absence of IL-3. In conclusion, these results indicate that the Bcr-Abl C-Terminus has an important role in several aspects of Bcr-Abl mediated transformation of haemopoietic progenitor cells including apoptotic suppression and cytotoxic drug resistance.