cytochalasin-d and Leukemia--Myelogenous--Chronic--BCR-ABL-Positive

Abnormal circulation and unregulated proliferation of chronic myelogenous leukemia (CML) progenitors is related, at least in part, to BCR/ABL induced abnormalities in beta1 integrin-mediated adhesion and signaling. The BCR/ABL oncogene has several potential interactions with cytoskeletal elements that are important for normal integrin signaling. In the present study, we evaluated whether abnormalities in beta1 integrin-cytoskeletal interactions were present in primary CML progenitors and contributed to defective integrin function. beta1 integrin-cytoskeletal interactions were studied in CML and normal CD34+ primary hematopoietic progenitors as well as BCR/ABL-transfected or mock-transfected M07e cells. In normal CD34+ progenitors, antibody-mediated cross-linking of beta1 integrins resulted in their redistribution into caps via a process requiring receptor-cytoskeletal interactions. CML CD34+ cells demonstrated significantly impaired beta1 integrin capping. This defect was related to the presence of the BCR/ABL gene, because capping also was impaired in BCR/ABL-transfected M07e cells. Defective receptor capping was not seen for non-integrin receptors. In addition, CML CD34- and M07eBCR/ABL cells also demonstrated increased actin polymerization and altered actin cytoskeletal organization. Further studies suggested that impaired beta1 integrin capping and defective integrin-mediated adhesion and proliferation inhibition in CML cells were related to abnormally enhanced integrin-cytoskeletal association and restricted receptor mobility. Finally, interferon alpha, which restores integrin-mediated adhesion and signaling in CML progenitors, also enhanced integrin capping in CD34+ cells. These studies suggest that p210BCR/ABL induces abnormal association of integrin receptors with the cytoskeleton and restricted receptor mobility and provide new insights into mechanisms underlying abnormal integrin function in CML progenitors.

Topics: Actins; Cell Adhesion; Cell Division; Cell Membrane; Cells, Cultured; Cytochalasin D; Cytoskeleton; Fusion Proteins, bcr-abl; Hematopoietic Stem Cells; Humans; Integrin beta1; Interferon alpha-2; Interferon-alpha; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Neoplastic Stem Cells; Receptor Aggregation; Recombinant Proteins; Transfection; Tumor Cells, Cultured

Focal adhesion kinase (p125FAK; FAK) is a protein tyrosine kinase that is tyrosine-phosphorylated in response to v-src-mediated transformation, cell adhesion, and stimulation with neuropeptides. To elucidate a possible functional relationship between FAK and BCR-ABL oncoprotein detected in Philadelphia chromosome-positive (Ph+) leukemias, we investigated the tyrosine phosphorylation state of FAK in a murine growth factor-dependent cell line and in its stable human bcr-abl cDNA transfectant. In interleukin-3 (IL-3)-dependent NFS/N1.H7 cells, tyrosine phosphorylation of FAK was not detected after stimulation with either IL-3 or Steel factor (SLF), both of which involve Ras-mediated signaling pathways. However, stable gene transfection with p210bcr-abl cDNA into H7 cells made these cells growth factor-independent for proliferation and resulted in constitutive tyrosine phosphorylation and kinase activation of FAK. Constitutive phosphorylation and activation of FAK was also observed in all Ph+ leukemia cell lines examined--that is, K562, TS9;22, and YS9;22, which express p210BCR-ABL, and NALM-21 and OM9;22, which express p185BCR-ABL. Ph-negative (Ph-) cell lines, such as MO7e and JM, did not show any detectable tyrosine phosphorylation of FAK. FAK phosphorylation in BCR-ABL-expressing cells was inhibited in a dose-dependent manner by cytochalasin D, a reagent that disrupts the intracellular network of actin filaments. However, no suppression of kinase activity or protein expression of BCR-ABL was observed after treatment with cytochalasin D. A physical association between BCR-ABL and FAK was not apparent. These data suggest that BCR-ABL may be involved in the activation of FAK. Moreover, FAK may be distinct from components in Ras-mediated signaling cascades that are activated by stimulation of myeloid cells with various cytokines.

Topics: Animals; Cell Adhesion Molecules; Cell Division; Cell Line; Cytochalasin D; Enzyme Activation; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Fusion Proteins, bcr-abl; Gene Expression; Humans; Interleukin-3; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Mice; Phosphorylation; Phosphotyrosine; Protein-Tyrosine Kinases; Proto-Oncogene Proteins c-abl; Proto-Oncogenes; Recombinant Proteins; Stem Cell Factor; Transfection; Tumor Cells, Cultured

The BCR/ABL oncogene causes chronic myelogenous leukemia (CML) in humans and induces growth factor independence of hematopoietic cell lines in tissue culture. p210BCR/ABL is localized at least in part to the cytoskeleton, and has been shown to interact directly with actin filaments through an actin binding domain located in the C-terminus of ABL. CML cells have reduced adhesion to some extracellular matrix components but the mechanism of this phenomenon is unknown. In this study we examined tyrosine phosphorylation of focal adhesion proteins in cells expressing p210BCR/ABL. An interleukin-3 (IL-3)-dependent cell line, 32Dc13, was transformed with a BCR/ABL cDNA, and the patterns of localization, expression, and tyrosine phosphorylation of focal adhesion proteins were compared among untransformed 32Dc13 cells with and without IL-3 stimulation and BCR/ABL-transformed 32Dc13 cells. Of the focal adhesion proteins examined, only paxillin exhibited tyrosine phosphorylation in response to IL-3; while in cells transformed by p210BCR/ABL, paxillin, vinculin, p125FAK, talin and tensin were constitutively tyrosine phosphorylated. IL-3 induced a transient association between paxillin and vinculin, while in BCR/ABL-transformed cells, several proteins coimmunoprecipitated with paxillin, including vinculin, p125FAK, talin and tensin. Pseudopodia enriched in focal adhesion proteins were transiently detected in 32Dc13 cells in response to IL-3, but constitutively detected in cells expressing p210BCR/ABL. p210BCR/ABL protein was also found concentrated in punctate structures adjacent to the cell membrane in myeloid cell lines, which often contained vinculin and paxillin.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Cell Adhesion Molecules; Cell Line; Cytochalasin D; Cytoskeletal Proteins; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Fusion Proteins, bcr-abl; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Mice; Microfilament Proteins; Paxillin; Phosphoproteins; Phosphorylation; Protein-Tyrosine Kinases; Rabbits; Talin; Tensins; Tyrosine; Vinculin