Characterization of K562 cells: uncovering novel chromosomes, assessing transferrin receptor expression, and probing pharmacological therapies

Abstract

Human erythroleukemic K562 cells represent the prototypical cell culture model of chronic myeloid leukemia (CML). The cells are pseudo-triploid and positive for the Philadelphia chromosome. Therefore, K562 cells have been widely used for investigating the BCR/ABL1 oncogene and the tyrosine kinase inhibitor, imatinib-mesylate. Further, K562 cells overexpress transferrin receptors (TfR) and have been used as a model for targeting cytotoxic therapies, via receptor-mediated endocytosis. Here, we have characterized K562 cells focusing on the karyotype of cells in prolonged culture, regulation of expression of TfR in wildtype (WT) and doxorubicin-resistant cells, and responses to histone deacetylase inhibition (HDACi). Karyotype analysis indicates novel chromosomes and gene expression analysis suggests a shift of cultured K562 cells away from patient-derived leukemic cells. We confirm the high expression of TfR on K562 cells using immunofluorescence and cell-surface receptor binding radioassays. Importantly, high TfR expression is observed in patient-derived cells, and we highlight the persistent expression of TfR following doxorubicin acquired resistance. Epigenetic analysis indicates that permissive histone acetylation and methylation at the promoter region regulates the transcription of TfR in K562 cells. Finally, we show relatively high expression of HDAC enzymes in K562 cells and demonstrate the chemotoxic effects of HDACi, using the FDA-approved hydroxamic acid, vorinostat. Together with a description of morphology, infrared spectral analysis, and examination of metabolic properties, we provide a comprehensive characterization of K562 cells. Overall, K562 cell culture systems remain widely used for the investigation of novel therapeutics for CML, which is particularly important in cases of imatinib-mesylate resistance.

Keywords: BCR-ABL; Chronic myeloid leukemia; Doxorubicin; Histone deacetylase inhibitors; K562 cells; Philadelphia chromosome; Transferrin receptors.

Fig. 1

The importance of the Philadelphia chromosome-positive human erythroleukemic K562 cell line in CML research. A The BCR/ABL1 fusion gene formed by reciprocal translocation of chromosome 9 and chromosome 22. B Molecular model of binding of imatinib (STI-571) and myristate to the kinase domain (PDB 1OPJ, transparent red) of c-ABL. The activation loop (yellow), Rad9-derived peptide substrate (blue; PDB 1K2M) and associated phospho-tyrosine (spheres) are highlighted. The approximate substrate binding position is based on a tyrosine kinase-peptide co-crystal structure (PDB 1IR3). C Close-up of the imatinib binding pocket with nearby amino acids highlighted. Residues numbered according to PDB 1OPJ

Fig. 2

The karyotype of the erythroleukemic K562 cell line. A G-banded karyotype, and B M-FISH karyotype. Clonal abnormalities present in other subclones but not in this subclone are shown with the G-banded image on the left and M-FISH image on the right: C der(5)t(4;5), D del(11)(p12), and E der(18)t(1;18)(p32;q21). F Allele-specific FISH using the BCR/ABL1 Plus translocation dual fusion probe. Multiple ABL1/BCR fusion signals are present on the add(22q) and the der(22)t(18;22)add(22q) while a single ABL1/BCR fusion is present on the distal long arm of the der(2). One red ABL1 (9q34) signal is present on the del(9p). Two signals are present on the der(9)t(9;9): one on each arm. The intact chromosome 22 and the der(22)t(21;22) each have a single green BCR (22q11.2) signal. The single color gallery created from M-FISH experiments included: G Chromosome 22 (green, red, aqua) but not chromosome 9 (orange, near infrared) material identified on the distal long arm of the derivative chromosome 2, H Alternating regions of diminished green fluorescence intensity present on the long arm of the der(22)t(18;22)add(22q). This is consistent with previously described 13q (red, aqua) amplification on this chromosome, I Material from chromosome 16 (green, near infrared, aqua) inserted into the short arm of chromosome 6

Fig. 3

Functional gene expression profile of K562 cells. A Differential gene expression results conducted on microarray gene expression data for K562 cells (red symbols) and normal PBMC (blue symbols). Principal component analysis (PCA) was based on the variance observed in the overall expression profiles between K562 samples and normal PBMC (i). A total of 4476 genes were found to be significantly differentially expressed between the two sample groups: 2772 upregulated, and 1704 downregulated (ii). Hierarchical clustering of select genes which are known to be aberrantly expressed in the K562 cell line validates gene expression profile (iii). B The signature of gene expression in the K562 cell line compared to normal PBMC associated with BCR-ABL mediated leukemogenesis in CML (KEGG pathway; hsa05220). C Gene set enrichment analysis (GSEA) was utilized to characterize the K562 cell line at a functional level using available KEGG and GO biological pathway maps. Categorised based on common cellular function, enriched KEGG and GO gene sets with a FDR q-value of 0.25 were considered significant. D The addition of microarray gene expression data from primary CD34 + hematopoietic stem cell samples was used to further characterize the K562 cell line in the context of clinically relevant disease. PCA results based on the variance observed in the overall profiles between K562 and normal PBMC, as well as CD34 + cell samples from blast crisis (BC) CML (gold symbols), chronic phase (CP) CML (green symbols), and normal individuals (purple symbols) (i). Hierarchical clustering of differentially expressed in the K562 cell line compared to normal PBMC, with the addition of signals from primary samples allows for the visualization of the variance between the immortalized cell line, normal cells and primary samples collected from CML patients (ii). E Select genes which are differentially expressed between malignant and normal cell types, and have the capacity to be therapeutic targeted. Representative box plots of normalized probe intensity signals from each sample group for well known targets, ABL1 and MYC (i). Significant genes differentially expressed in malignant cells compared to normal controls were selected for analysis due to their involvement in the survival and function of the CML progenitor cell population, independent of BCR/ABL1 (ii)

Fig. 4

The upregulation of transferrin receptor expression on K562 cells. A Schematic representation of the transferrin receptor-mediated endocytosis pathways. Diferric transferrin binds to transferrin receptors in clathrin coated pits. The pits bud and pinch off from the membrane forming endocytotic vesicles which move into the cytoplasm. In the major transferrin pathway, which represents ~ 85–95% of the internalised transferrin/transferrin receptor complexes, the endocytotic vesicles fuse with early endosomes. At the low pH (~ 5.5) of the endosome the ferric ions dissociate from the transferrin peptide. Within this environment the receptor retains a high affinity for apo-transferrin which is returned to the cell surface bound to the transferrin receptor. At the neutral pH of the extracellular fluid the apo-ligand has a reduced affinity for the receptor and therefore, dissociates from the receptor. The alternative transferrin pathway which represents ~ 5–15% of the internalised transferrin/transferrin receptor complexes, involves trafficking through the trans-Golgi to the lysosomes leading to degradation of transferrin and transferrin receptors. B N-SIM super resolution microphotograph of the TfR1 protein (CD71) in a K562 cell. C Equilibrium binding of ¹²⁵I-transferrin to cell-surface transferrin receptors on K562 cells. D Scatchard presentation of the binding characteristics of ¹²⁵I-transferrin to cell-surface transferrin receptors on K562 cells to estimate equilibrium dissociation constants and number of binding sites. E Box plot of normalized probe intensity signals from microarray gene expression data of each sample group for the transferrin receptor (TFRC gene; 207332_s_at). F Chromatin modifications within the TFRC gene in K562 cells using ChIP-Seq data. Dotted lines on plot indicate the transcription start site (0 bp), the promoter region (2000 bp either side of TSS) and the transcription termination site (far left). G Comparison of chromatin modifications, H3K4me1 and H3K4me3, within the TFRC gene in K562 cells and normal PBMC

Fig. 5

Overview of the biological effects of doxorubicin in K562 cells. A K562 cells treated with 1 and 10 µM doxorubicin for 24 h showed a dose-dependent reduction in cell viability measured using the Cell Titer^® (Promega) assay kit (B) and increase in caspase 3/7 apoptosis measured using the Apo-one (Promega) assay kit. C K562 and PBMC cells treated with the indicated concentration of doxorubicin for 1 h, were washed and incubated in fresh media for a further 24 h before fixing and staining for sensitive marker of DNA-double strand breaks – γH2AX (green) and DAPI nuclear stain (blue) (i). The average number of γH2AX foci per cells were quantified in Image J and represented by the mean ± SD of five independent experiments (ii). D Representative FTIR absorbance spectra and PCA analysis from data collected of the K562 cell line following treatment with doxorubicin, in comparison to an untreated control. The averaged vector-normalised spectra calculated from 60 to 150 single K562 cells collected from each experimental group with variance in the major amide and DNA bands labelled (i). The second-order derivatives were also calculated from the corresponding spectra by a Savitzky-Golay algorithm (17 smoothing points) with changes in intensity and frequency labelled, including those in the protein and DNA regions (ii). PC-1/PC-2 scores plot with a largely delineated clustering of the experimental cell sets along PC-1 (iii). The PC-1 loadings plot labelled with major spectral components found to contribute to the variance observed in spectra corresponding to untreated or treated K562 cells (iv). E A doxorubicin-resistant K562 cell line denoted PW-15 was derived from the doxorubicin-sensitive cells by incremental exposure to doxorubicin over a period of 461 days (A—day 0, 2.6 µM, B—day 21, 3.5 µM, C—day 57, 4.3 µM, D—day 68, 5.2 µM, E—day 112, 6 µM, F—day 203, 6.9 µM, G—day 300, 7.8 µM, H—day 404, 8.6 µM). Cells were maintained at a drug concentration until their growth rate approached that of the untreated doxorubicin sensitive K562 cell line (i). The fluorescence intensity of the transferin receptor (Tfr) of PW-15 cells compared to K562 cells was imaged (ii) and quantified in Image J (iii) and showed expression of the Tfr remains a reliable target in the resistant cell line

Fig. 6

Expression of histone deacetylase enzymes and the cytotoxic effects of vorinostat in K562 and PBMC cells. A Histone deacetylase enzyme expression in K562 vs. PBMC cells was measured by immunofluorescence staining (images not shown) and quantified using Image J. The mean total fluorescence intensity represented as a heat map of 3 independent experiments is shown of all metal-dependent HDAC1-11 enzymes relative to each other and in both cell lines; green = less expression, red = greater expression. B Percentage of relative cell viability and C caspase 3/7 apoptosis in K562 and PBMC cells treated with the indicated concentrations of vorinostat for 24 h. D Vorinostat causes K562 cells to arrest in G2/M phase of the cell cycle. Cells treated with (ii) or without (i) 10 µM vorinostat for 24 h were fixed and stained with propidium iodide and measured using FACs. E 10 µM vorinostat causes hyperacetylation and hypermethylation of histones and methy-CpG islands in K562 cells. F Vorinostat augments doxorubicin -induced γH2AX foci in K562 cells but not PBMC cells