Genomic and transcriptomic profiling of resistant CEM/ADR-5000 and sensitive CCRF-CEM leukaemia cells for unravelling the full complexity of multi-factorial multidrug resistance

Abstract

We systematically characterised multifactorial multidrug resistance (MDR) in CEM/ADR5000 cells, a doxorubicin-resistant sub-line derived from drug-sensitive, parental CCRF-CEM cells developed in vitro. RNA sequencing and network analyses (Ingenuity Pathway Analysis) were performed. Chromosomal aberrations were identified by array-comparative genomic hybridisation (aCGH) and multicolour fluorescence in situ hybridisation (mFISH). Fifteen ATP-binding cassette transporters and numerous new genes were overexpressed in CEM/ADR5000 cells. The basic karyotype in CCRF-CEM cells consisted of 47, XX, der(5)t(5;14) (q35.33;q32.3), del(9) (p14.1), +20. CEM/ADR5000 cells acquired additional aberrations, including X-chromosome loss, 4q and 14q deletion, chromosome 7 inversion, balanced and unbalanced two and three way translocations: t(3;10), der(3)t(3;13), der(5)t(18;5;14), t(10;16), der(18)t(7;18), der(18)t(21;18;5), der(21;21;18;5) and der(22)t(9;22). CCRF-CEM consisted of two and CEM/ADR5000 of five major sub-clones, indicating genetic tumor heterogeneity. Loss of 3q27.1 in CEM/ADR5000 caused down-regulation of ABCC5 and ABCF3 expression, Xq28 loss down-regulated ABCD1 expression. ABCB1, the most well-known MDR gene, was 448-fold up-regulated due to 7q21.12 amplification. In addition to well-known drug resistance genes, numerous novel genes and genomic aberrations were identified. Transcriptomics and genetics in CEM/AD5000 cells unravelled a range of MDR mechanisms, which is much more complex than estimated thus far. This may have important implications for future treatment strategies.

Figure 1. Gene networks influenced by ABCB1 and ABCG2 in CEM/ADR5000 cells.

IPA software was used to depict the networks. Genes that are labelled in green were down-regulated and genes that are labelled in red were up-regulated. The lower panel depicts ABCB1 and ABCG2 playing role in “cell death of leukaemia cell lines” and “apoptosis” inhibition as shown by blue dotted lines. ABCB1 up-regulation is predicted to activate “transport of cyclosporine A” as shown by the orange dotted line.

Figure 2

(A) Biological function of differentially expressed genes in CEM/ADR5000 cells in comparison to wild-type CCRF-CEM cells as determined by IPA software. The orange line depicts the statistical significance threshold (p = 0.05). (B) Signaling pathways of differentially expressed genes in CEM/ADR5000 cells in comparison to wild-type CCRF-CEM cells as determined by IPA software. The orange line depicts the statistical significance threshold (p = 0.05) and the orange chart depicts the ratio of deregulated genes in each pathway.

Figure 3. Protein expression of FOXO1 and NQO1 in CEM/ADR5000 and CCRF-CEM cells as determined by western blotting (cropped blots are displayed).

Figure 4. mFISH analysis of CCRF-CEM and CEM/ADR5000 cells.

Two clones detected in CCRF-CEM are depicted in (A,B). All derivative chromosomes present in clones 1 and 2 are highlighted by light-green arrows. Individual changes for clones 1 and 2 are labelled by arrows in darker green. For derivative chromosome 5, a whole chromosome paint (wcp) and a subtelomeric (st) probe for 5qter were applied. For the derivative chromosome 8, a centromeric probe (D8Z1) and a st probe for 14qter had been used. In (4C) to F, CEM/ADR5000 clones 1, 1a, 1b, 1b1 and 1c are depicted. The per clone acquired alterations are highlighted by coloured arrows as explained in the legend between (4A/B) and C/D. For clear visualisation of the inversion in chromosome 7, MCB 7 was applied as shown in (4C). In (4C), the only additional aberration present in clone 1a is depicted, i.e. a reciprocal translocation between chromosomes 6 and 14.

Figure 5. Summary of clonal evaluation of cell lines CCRF-CEM and CEM/ADR5000.

Figure 6. aCGH results of CCRF-CEM cells.

Figure 7. aCGH results of CEM/ADR5000 cells.

Figure 8

Comparison of chromosomal aberrations analysed in CCRF-CEM and CEM/ADR5000 cells in the year 2002 (A) with the results of the present study (B). Each dot represents an aberration, green: deletion, red: amplification. Some of the deregulated drug resistance linked genes are marked on the plots for the CEM/ADR5000 cells observed in the present study.