Chemotherapeutic drug-induced ABCG2 promoter demethylation as a novel mechanism of acquired multidrug resistance

Abstract

ABCG2 is an efflux transporter conferring multidrug resistance (MDR) on cancer cells. However, the initial molecular events leading to its up-regulation in MDR tumor cells are poorly understood. Herein, we explored the impact of drug treatment on the methylation status of the ABCG2 promoter and consequent reactivation of ABCG2 gene expression in parental tumor cell lines and their MDR sublines. We demonstrate that ABCG2 promoter methylation is common in T-cell acute lymphoblastic leukemia (T-ALL) lines, also present in primary T-ALL lymphoblast specimens. Furthermore, drug selection with sulfasalazine and topotecan induced a complete demethylation of the ABCG2 promoter in the T-ALL and ovarian carcinoma model cell lines CCRF-CEM and IGROV1, respectively. This resulted in a dramatic induction of ABCG2 messenger RNA levels (235- and 743-fold, respectively) and consequent acquisition of an ABCG2-dependent MDR phenotype. Quantitative genomic polymerase chain reaction and ABCG2 promoter-luciferase reporter assay did not reveal ABCG2 gene amplification or differential transcriptional trans-activation, which could account for ABCG2 up-regulation in these MDR cells. Remarkably, mimicking cytotoxic bolus drug treatment through 12- to 24-hour pulse exposure of ABCG2-silenced leukemia cells, to clinically relevant concentrations of the chemotherapeutic agents daunorubicin and mitoxantrone, resulted in a marked transcriptional up-regulation of ABCG2. Our findings establish that antitumor drug-induced epigenetic reactivation of ABCG2 gene expression in cancer cells is an early molecular event leading to MDR. These findings have important implications for the emergence, clonal selection, and expansion of malignant cells with the MDR phenotype during chemotherapy.

Figure 1

ABCG2 mRNA levels in paired parental and MDR tumor cell lines assessed by QPCR. ABCG2 mRNA levels are expressed as fold of expression relative to the basal transcript levels of CCRF-CEM cells (black bars) or as fold of expression of the MDR sublines over their matched parental cells (gray bars).

Figure 2

Quantitative determination of promotermethylation status in parental and MDR cell lines assessed by COBRA and direct bisulfite sequencing. Methylation levels were assessed using a PCR-amplified 223-bp fragment of bisulfite-converted genomic DNA from within the CpG island of the abcg2 promoter of parental A549, MCF7, CCRF-CEM, and IGROV1 cells (A) or their corresponding A549/K1.5, MCF7/MR/FLV1000, CCRF-CEM/SSZ1.5/2.5, and IGROV1/T8/MX3 MDR cells (B). In addition, two consecutive fragments (termed A and B) from the abcg2 promoter's CpG island were subjected to direct bisulfite sequencing (C). Schematic representation of the sequenced genomic fragments including all CpG sites (line representation) is displayed (top). Eight representative clones from parental CCRF-CEM and IGROV1 cells and their MDR CCRF-CEM/SSZ1.5/2.5 and IGROV1/T8 sublines are presented in dot display (black dot represents a methylated CpG). Also, the promoter methylation status of the pcft and mdr1 genes in parental CCRF-CEM cells and their MDR CCRF-CEM/SSZ2.5 counterparts were assessed using a 414- and 223-bp PCR-amplified fragment of bisulfite-converted genomic DNA from within their CpG island, respectively (D). PCR fragments were subjected to 200-fold BstUI (abcg2 and pcft) or TaqI^α (mdr1) overdigestion and then compared to various dilutions of the uncut DNA.

Figure 3

The effect of epigenetic modifying agents on the status of ABCG2 promoter methylation and consequent transcription in parental cells and the correspondingMDR cells. QPCR analysis of ABCG2 transcript in CCRF-CEM(A), IGROV1 (B) cells, and their MDR sublines CCRF-CEM/SSZ2.5 (C) and IGROV1/T8 (D) that were treated for 72 hours with 2 µM of the demethylating agent 5-Aza-dC, in the presence or absence of 75 ng/ml of theHDACi-TSA. To some samples, 0.5 µg/ml of the transcription inhibitor ActDwas added in combination with the latter. abcg2 promoter methylation status was assessed using COBRA in parental CCRF-CEM and MDR CCRF-CEM/SSZ2.5 cells as well as CCRF-CEM cells treated with 5-Aza-dC+zebularine (E; see Materials and Methods for details). The corresponding ABCG2mRNA expression levels were assessed using QPCR (F); all QPCR results are expressed as fold of expression relative to the corresponding untreated or parental cells.

Figure 4

Effect of in vitro methylation on ABCG2 promoter activity. Methylated and nonmethylated -1285/+362 ABCG2 promoter luciferase reporter constructs were transiently transfected into parental IGROV1 and MDR IGROV1/T8 cells and the relative luciferase activity was determined. Results are expressed as the average (±SD) luciferase-to-Renilla ratio of three experiments.

Figure 5

Quantitative determination of abcg2 promoter methylation status in hematolymphoid cells. Genomic DNA was extracted from peripheral blood T cells of healthy volunteers (top lane 1–4), peripheral blood lymphoblasts fromadult T-ALL patients (middle lane 1–4), T-ALL cell lines CCRF-CEM, JURKAT, and MOLT4 (bottom lane 1–3), and the erythroleukemia cell line K562. A 223-bp fragment (fragment B) of bisulfite-converted genomic DNA from within the abcg2 promoter CpG island was PCR-amplified and subjected to COBRA (as described above).

Figure 6

The effect of short-term drug exposure on ABCG2 gene expression. CCRF-CEM cells were subjected to MX, Dox, and DNR in a dose- (A) and time-dependent (B) manner. Cells were harvested, and relative abcg2 mRNA levels were assessed using QPCR. Furthermore, the effect of functional inhibition of ABCG2 using the transport inhibitor FTC and transcriptional inhibition using Act D on drug-induced ABCG2 gene expression in CCRF-CEM cells was assessed (C). Drug-induced ABCG2 expression was evaluated in abcg2 methylated and nonmethylated cell lines Jurkat and K562, respectively (D). Drug-induced relative up-regulation of ABCG2 gene expression was expanded to other known silenced and nonsilenced genes in CCRF-CEM cells (E). Drug concentration and incubation times were set at an optimal induction capacity. QPCR expression levels are presented as fold expression relative to untreated cells.

Figure 7

Schematic representation of the putative dynamics of abcg2 promoter methylation in tumor progression and under anticancer drug treatment.