mRNA levels of related Abcb genes change opposite to each other upon histone deacetylase inhibition in drug-resistant rat hepatoma cells

Abstract

The multidrug-resistant phenotype of tumor cells is acquired via an increased capability of drug efflux by ABC transporters and causes serious problems in cancer treatment. With the aim to uncover whether changes induced by epigenetic mechanisms in the expression level of drug transporter genes correlates with changes in the drug resistance phenotypes of resistant cells, we studied the expression of drug transporters in rat hepatoma cell lines. We found that of the three major rat ABC transporter genes Abcb1a, Abcb1b and Abcc1 the activity of only Abcb1b increased significantly in colchicine-selected, drug-resistant cells. Increased transporter expression in drug-resistant cells results primarily from transcriptional activation. A change in histone modification at the regulatory regions of the chromosomally adjacent Abcb1a and Abcb1b genes differentially affects the levels of corresponding mRNAs. Transcriptional up- and down-regulation accompany an increase in acetylation levels of histone H3 lysine 9 at the promoter regions of Abcb1b and Abcb1a, respectively. Drug efflux activity, however, does not follow tightly the transcriptional activity of drug transporter genes in hepatoma cells. Our results point out the need for careful analysis of cause-and-effect relationships between changes in histone modification, drug transporter expression and drug resistance phenotypes.

Figure 1. Activity of drug transporters in drug-sensitive and drug-resistant cell lines.

MAF values reflecting ABCB1 (A), ABCC1 (B), ABCB1a (C) and ABCB1b (D) transporter activities were determined as described in the Materials and Methods and are shown as the percent of the total drug transporter activity of the indicated cell lines. (*p<0.05, Mann Whitney test).

Figure 2. Abcb1a and Abcb1b mRNA and protein levels in drug-sensitive and resistant rat hepatoma cells.

Relative mRNA levels of Abcb1a (A) and Abcb1b (B) in hepatoma cells were determined by quantitative real-time PCR. RNA samples were isolated from drug-sensitive parental (D12) and drug-resistant (col500 and col1000) cells, which were cultured for 3 days in the absence of colchicine. (*p<0.05, Mann Whitney test) The protein levels of ABCB1a (C) and ABCB1b (D) were determined by Western blot. Protein samples obtained from drug-sensitive and drug-resistant cells were separated on 10% SDS-polyacrylamide gels and transferred to immobilon membranes. Western blots were developed using ABCB1a and ABCB1b pan-specific primary antibodies. The loading control was developed with anti-tubulin Ab.

Figure 3. Abcb1a and Abcb1b gene copy numbers are unchanged, while the stability of Abcb1a- and Abcb1b-specific mRNAs differs slightly in drug-resistant cells.

Gene copy numbers of Abcb1a (A) and Abcb1b (B) in a drug-sensitive parental cell line (D12) and drug-resistant cells (col500, col1000) were determined by PCR using gene-specific primers (see Materials and Methods). Genomic DNA was isolated from cells grown for 3 days in the absence of colchicine. (*p<0.05, Mann Whitney test). Abcb1a and Abcb1b mRNA stability was compared by quantitative real-time PCR on cDNA templates prepared from RNA samples, which were obtained from cells treated with 20 µg/ml actinomicyn D for 6, 12, 24 and 36 h, as indicated. The specific mRNA levels were compared to the levels of an 18S rRNA internal control. The change of mRNA levels of Abcb1a and Abcb1b over a 36-h period compared to the amount detected at zero time are shown in panels C and D, respectively.

Figure 4. Abcb1 pre-mRNA levels and promoter activities in drug-sensitive and resistant cell lines.

The pre-mRNA levels of Abcb1a (A) and Abcb1b (B) were determined by quantitative PCR using specific primers hybridizing to intronic regions of the corresponding gene. RNA samples for cDNA preparation were obtained from cell cultured for 3 days in the absence of colchicine. The RNA levels were compared to the level of 18S rRNA internal control. (*p<0.05, Mann Whitney test). Luciferase activity determined in cell extracts prepared from D12, col500 and col1000 cells transiently transfected with the pGL3-Abcb1a-Luc or pGL3-Abcb1b-Luc reporter plasmids (C).

Figure 5. TSA treatment increases H3K9ac levels at the 5 ′ region of both rat Abcb1 genes.

(A) Detection of H3K9ac in drug-sensitive (D12) and resistant (col500, col1000) cells at the transcription initiation site (-inic) and 1500 nucleotides upstream of that (−1500) in the Abcb1a (a-) and Abcb1b (b-) regulatory regions by ChIP experiments. Primers and antibodies used are described in the Materials and Methods. (B) TSA treatment results in a comparable increase in acetylated histone H4 levels in both drug-sensitive and resistant cells. Protein samples obtained from TSA-treated (6 h, 50 ng/ml) and untreated, drug-sensitive and drug-resistant cells were separated on 15% SDS-polyacrylamide gels and transferred to immobilon membranes. Western blots were developed using acetylated histone H4 pan-specific primary antibody. The loading control was developed with anti-H3 Ab. (C–E) TSA treatment similarly affects H3K9ac levels in the Abcb1a and Abcb1b 5′ regions in drug-sensitive (D12; C) and drug-resistant (col500; D and col1000; E) cells. ChIP experiments were performed to detect H3K9ac levels as described in the Materials and Methods. (*p<0.05, Mann Whitney test).

Figure 6. Abcb1a and Abcb1b mRNA and pre-mRNA levels are differentially altered by TSA treatment.

Abcb1a (A and C) and Abcb1b (B and D) specific mRNA (A and B) and pre-mRNA (C and D) levels were compared in TSA-untreated and TSA-treated, drug-sensitive (D12) and drug-resistant (col500 and col1000) cells by quantitative PCR as described above. (*p<0.05, Mann Whitney test).

Figure 7. Summary of the drug efflux and drug-transporter expression changes observed in drug-selected rat hepatoma cells.

(A) The major contributor to the drug efflux increase in drug-resistant rat hepatoma cell lines is their increased ABCB1b activity. (B) The mRNA levels of the Abcb1a and Abcb1b transporters differ significantly in the parental D12 cells, and are up-regulated differently in drug-selected cell lines. (C) Changes in copy numbers of neither Abcb1a nor Abcb1b are responsible for their elevated expression. (D) Abcb1b mRNA stability is slightly higher in the drug-resistant col500 and col1000 cells compared to D12. (E) HDAC inhibition affects Abcb1 gene expression differently: upon TSA- treatment Abcb1a mRNA levels decrease, while Abcb1b mRNA levels increase both in drug-sensitive and drug-resistant cells.