Malignant Progression of Human CML Clone Demonstrates a Unique Developmental Pathway Characterized by Altered Expression Pattern of Cancer, Adhesion and Drug Resistance Genes.

The K562 cell line is a clone derived from a patient with CML in blast crisis and is widely used as a meaningful model for experimental and clinical studies. Similarly to primary CML blasts, K562 grow mostly in suspension in the absence of supportive bone marrow stromal cells or plate-coating of adhesion molecules. However, under standard culture conditions a relatively small cell-fraction (2–3%) adheres to the plastic dish. In this study, we investigated whether the plastic-adhesive sub-population is a side population of clonal origin and whether it has unique malignant properties. For these reasons, we have established and characterized a new cellular model system of plastic-adhesive variants of K562 (termed K562/Adh and K562/sAdh). These adhesive-variants retain self-renewal ability with indications of increased malignant properties as increased colony-forming ability in soft agar, impairment of contact inhibition and loss of serum-dependent attenuation of adhesion. cDNA microarray analyses that were validated by quantitative RT-PCR analyses have shown altered expression levels of adhesion related genes in the adhesive variants relatively to the parental cells. This included 4.0-, 3.1- and 2.5- fold up-regulation of the KiSS-1, THBS3, ITGB5 genes, respectively, and 1.7- fold down-regulation of the FN1 gene. Moreover, up-regulation of the BCR-ABL fusion gene (3.1- fold) and of the AP1 transcription factors genes, c-JUN (4.5-fold) and c-FOS (2.8-fold), was found in the adhesive variants relatively to their parental cells. Other transcription factors from the AP1 family (FRA1, JUN-B and JUN-D) similarly expressed in all K562 variants. Interestingly, both adhesive cell variants also demonstrated a de-novo expression of the multidrug resistance, MDR1 gene. Our data strongly suggest that the adhesive-variants of K562 cells are an outcome of a unique pathway of malignant progression that not only involves novel adhesion properties. Cellular cloning experiments and molecular gene-expression analyses of various clones indicated that this malignancy progression pathway is a global inherited property of K562 cells. Although the progeny of any parental K562 have the potential to transform into adherent cells, only a confined fraction (3.0±0.5 %) of progeny cells undergo such transformation in apparently stochastic manner. The factors involved in the induction of this transformation process in a particular progeny cell are yet unknown. However, growing K562 cells in pre-conditioned medium from cultures of K562/Adh or K562/sAdh did not change the size of adhered cell fraction, suggesting that their adhesion is independent of molecules that are specifically released or secreted by the adhesive variants of K562. Neither growing K562/Adh nor K562/sAdh in pre-conditioned medium from cultures of the parental K562 affected their adhesion properties. In conclusion, we demonstrated existence of a unique unidirectional, adhesion-based transformation pathway of CML blasts. This pathway is also characterized by altered pattern of gene expression that is associated with increased malignant properties. As the resultant transformed cells can be discriminated by adhesion to plastic, this system may serve as a model to investigate the clinical and therapeutic implications of such transformation in CML progression, drug-resistance and minimal residual disease.