Beta1-integrins in part support adhesion and migration of NL CD34+ cells to BM stroma and fibronectin (FN). NL CD34+ cell adhesion /migration is also influenced by chemokines, such as SDF-1alpha. The molecular mechanism(s) underlying integrin and chemokine dependent adhesion and migration of NL CD34+ cells are not known. Preliminary studies from our lab suggest involvement of PI3-kinase and the GTPase Cdc42 in these processes. CML is a stem cell leukemia characterized clinically by premature circulation of a massively expanded malignant CD34+ cell population in the blood. CML is caused by the Bcr/Abl oncogene. Although numerous studies have described alterations in signal pathways induced by Bcr/Abl in cell lines, the mechanism(s) underlying the features characteristic for chronic phase CML are not understood. Our lab has shown that (a) CML CD34+ cells adhere less and migrate more over FN than NL CD34+ cells which is not caused by decreased beta1-integrin expression. (b) In CML, the cell cytoskeleton is constitutively activated which decreases the mobility of integrins in the cell membrane. (c) CML CD34+ cell adhesion nor migration is affected by chemokines, such as SDF-1alpha. (d) GTPases and PI3-K, thought to play a role in NL CD34+ cell adhesion and migration, may be constitutively activated in CML and contribute to the aberrant adhesion and migration of CML CD34+ cells. (e) In CML CD34+ cells we have detected abnormally spliced and potentially dominant negative, isoforms of the focal adhesion kinase Pyk2. (f) Suppression of Bcr/Abl with antisense ODNs reverses these abnormalities and transfer of Bcr/Abl cDNA in cell lines induces these anomalies, strongly suggesting that Bcr/Abl is responsible for decreased integrin mobility and affinity, adhesion and increased migration. These observations support the following hypotheses (l) Engagement of beta1-integrins and stimulation of CXCR4 via SDF-1alpha activates Pyk2- H. This leads to the activation of signal pathways such as PI3-K, which activates the GTPases Cdc42 and Rac2 responsible for adhesion and migration of CD34+ cells. (2) Constitutive phosphorylation and activation of some of these signal molecules as a result of Bcr/Abl in CML HPC underlies the aberrant adhesion and migration seen in CML. We will test these hypotheses in the following specific aims: SA l. Characterize expression and function of Pyk2-H in CD34+ HPC. SA 2. Examine role of Cdc42 and Rac1/2, PI3-K, and Pyk2 in beta1-integrin- and SDF-1alpha mediated adhesion and migration of CD34+ HPC. SA 3. Examine effect of p210-Bcr/Abl on the function and interactions of GTPases, PI3-K, and Pyk2 leading to the decreased adhesion and enhanced migration characteristic of CML.
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