The effects of Gleevec upon K-562 cell differentiation and adherence properties

Chronic myelogenous leukemia (CML) is a hematological stem cell malignancy characterized by excessive myeloid proliferation. K-562 cells, developed from a CML patient in blast crisis, provide an excellent cell model with which to study multi-lineage leukemia cell differentiation and pathology. Virtually all patients with CML express the BCR-ABL protein. This chimeric protein is the result of a reciprocal (9;22) chromosomal translocation. The fusion gene generates a constitutively active tyrosine kinase, which plays a fundamental role in the regulation of cell proliferation, growth, and function. Gleevec, a small molecule that selectively inhibits the BCR-ABL tyrosine kinase by competitively binding to the ATP binding site, was recently approved by the FDA. This thesis provides evidence that Gleevec induces de novo morphological changes and adherence characteristics in K-562 cells. We characterize some of the features of initial treatment with Gleevec and the progression towards resistance. K-562 cells co-cultured with Gleevec showed morphologic changes along the dendritic pathway. These induced changes were consistent for three K-562 sublines as examined by sequential temporal observation. Gleevec-treated cells displayed altered adhesive characteristics, which included homotypic aggregation and adherence to plastic. Brighter fluorescence at points of cell-cell contact suggests that f-actin was involved at these adhesive junctions. Additionally, induced morphological alterations, including dendritic hairs, pseudopodia and filopodia, were all directly associated with factin staining. Resistance to Gleevec has been a clinical occurrence, especially with CML patients in blast crisis. We demonstrated that K-562 cells are capable of becoming resistant to concentrations of Gleevec as high as 3.0µM. Resistant cell lines were shown to have increased BCR-ABL mRNA and BCR-ABL protein expression as compared to the controls. We also determined that the over-amplification of the BCRABL gene in K-562 cells makes it difficult to conclude quantifiable results from FISH analysis. This study provides further insight regarding the complexities of altered adhesion in BCR-ABL positive cells. We show that K-562 cells and Gleevec can be used as a model system for the in vitro analysis of differentiation-inducible modification of blastic leukemia cells