Effect of Mutational Inactivation of Tyrosine Kinase Activity on BCR/ABL-Induced Abnormalities in Cell Growth and Adhesion in Human Hematopoietic Progenitors

Abstract

Chronic myelogenous leukemia (CML) results from transformation of a primitive hematopoietic cell by the BCR/ABL gene. The specific BCR/ ABL signaling mechanisms responsible for transformation of primitive human hematopoietic cells are not well defined. Previous studies have suggested that constitutively activated tyrosine kinase activity plays an important role for in abnormal proliferation of CML progenitors but has not clearly defined its role in abnormal adhesion and migration. We established a human progenitor model of CML by ectopic expression of BCR/ABL in normal CD34؉ cells using retrovirus-mediated gene transfer. CD34؉ cells expressing BCR/ABL demonstrated several features characteristic of primary CML progenitors including increased proliferation in committed and primitive progenitor culture, reduced adhesion to fibronectin, and reduced chemotaxis to stroma-derived factor-1␣. We expressed a kinase-inactive BCR/ABL gene to directly investigate the role of kinase activity in abnormal progenitor function. Abnormalities in proliferation were completely reversed, whereas defects in adhesion and migration were significantly improved but not completely reversed in cells expressing a kinase-inactive BCR/ABL. Furthermore, the BCR/ABL kinase inhibitor imatinib mesylate markedly inhibited proliferation of BCR/ ABL-expressing progenitors but did not fully correct the adhesion and migration defects. Expression of BCR/ABL genes with deletions of either the COOH-terminal actin binding or proline-rich domains resulted in enhanced adhesion and chemotaxis compared with wild-type BCR/ABL but did not affect progenitor proliferation. We conclude that abnormal kinase activity is essential for abnormal proliferation and survival of CML progenitors but that abnormal adhesion and migration result from both kinase-dependent and -independent mechanisms.

BCR/ABL and IL-3 activate Rap1 to stimulate the B-Raf/MEK/Erk and Akt signaling pathways and to regulate proliferation, apoptosis, and adhesion

“…The increase in cell adhesion to fibronectin induced by BCR/ABL in this study is also in agreement with previous studies demonstrating the increased b1-integrin-mediated adhesion of various hematopoietic cell lines expressing BCR/ABL as well as of primary hematopoietic progenitor cells from CML patients (Bazzoni et al, 1996;Kramer et al, 1999;Wertheim et al, 2002). On the other hand, BCR/ABL has also been reported to decrease adhesion of primary CML progenitors as well as normal hematopoietic progenitor cells when ectopically expressed (Gordon et al, 1987;Verfaillie et al, 1992;Ramaraj et al, 2004). In these studies, however, adhesion was evaluated indirectly by counting the number of colonies initiated by adherent cells after several weeks of culture.…”

“…These observations are also in accordance with our hypothesis that Rap1 mediates inside-out signaling that regulates integrin-mediated cell adhesion downstream of both hematopoietic cytokines and BCR/ABL, because the constitutive activation of this pathway by BCR/ABL would elicit negative regulatory mechanisms that diminish the effect of cytokines. It should be also noted that, in the present study, as well as in previous studies (Wertheim et al, 2002;Ramaraj et al, 2004), BCR/ABL has been shown to disturb cell adhesion at least partly in a kinase activity-independent manner, which is most likely related to the abilities of BCR/ABL to bind and modify actin cytoskeleton (Wertheim et al, 2003). Effects of Rap1-mediated disturbance in cell adhesion on proliferation and survival of BCR/ABL-expressing cells need to be addressed in future studies.…”

Identification of genes involved in imatinib resistance in CML: a gene-expression profiling approach

“…A clear relation between imatinib resistance and the expression of genes involved in cell-adhesion inhibition has been observed. Chronic myeloid leukemia is characterized by abnormal trafficking of malignant hematopoietic progenitor cells in the peripheral blood, as a consequence of aberrant adhesive interactions with the bone marrow stroma, 22 caused by kinase-dependent and -independent mechanisms, 23 and linked to cytogenetic resistance to imatinib. 24 The low levels of expression of adhesion molecules detected in imatinib-resistant patients could be related to their inability to respond to imatinib.…”

Expression of DLK1 in hematopoietic cells results in inhibition of differentiation and proliferation

“…After confirmation of sequence, wild type and mutant DLK1 genes were subcloned into the EcoR1 site of the MIG-R1 retroviral vector (a gift from Dr Warren Pear, University of Pennsylvania, Philadelphia, PA, USA). Infectious virions were produced by transient transfection of 293 cells as previously described (Ramaraj et al, 2004), and used to transduce HL-60 cells. GFP-positive cells were selected by flow cytometry sorting after 48 h. DLK expression was confirmed by RT-PCR.…”