Histone modifications at the ABCG2 promoter following treatment with histone deacetylase inhibitor mirror those in multidrug-resistant cells.

ABCG2 is a ubiquitous ATP-binding cassette transmembrane protein that is important in pharmacology and may play a role in stem cell biology and clinical drug resistance. To study the mechanism(s) regulating ABCG2 expression, we used ChIP to investigate the levels of acetylated histone H3, histone deacetylases (HDAC), histone acetyltransferases, and other transcription regulatory proteins associated with the ABCG2 promoter. Following selection for drug resistance and the subsequent overexpression of ABCG2, an increase in acetylated histone H3 but a decrease in class I HDACs associated with the ABCG2 promoter was observed. Permissive histone modifications, including an increase in histone H3 lysine 4 trimethylation (Me(3)-K4 H3) and histone H3 serine 10 phosphorylation (P-S10 H3), were observed accompanying development of the resistance phenotype. These changes mirrored those in some cell lines treated with a HDAC inhibitor, romidepsin. A repressive histone mark, trimethylated histone H3 lysine 9 (Me(3)-K9 H3), was found in untreated parental cells and cells that did not respond to HDAC inhibition with ABCG2 up-regulation. Interestingly, although all five studied cell lines showed global histone acetylation and MDR1 up-regulation upon HDAC inhibition, only those cells with removal of the repressive mark, and recruitment of RNA polymerase II and a chromatin remodeling factor Brg-1 from the ABCG2 promoter, showed increased ABCG2 expression. In the remaining cell lines, HDAC1 binding in association with the repressive Me3-K9 H3 mark apparently constrains the effect of HDAC inhibition on ABCG2 expression. These studies begin to address the differential effect of HDAC inhibitors widely observed in gene expression studies.

Localization of the ABCG2 mitoxantrone resistance-associated protein in normal tissues.

Reduced drug accumulation due to overexpression of individual members of the ATP binding cassette (ABC) superfamily of membrane transporters has been investigated as a cause of multidrug resistance and treatment failure in oncology. This study was designed to develop an immunohistochemical assay to determine the expression and localization of the 72kDa ABC half-transporter ABCG2 in normal tissues. Formalin-fixed, paraffin embedded archival tissue from 31 distinct normal tissues with an average of eight separate tissue samples of each were immunostained with rabbit-anti-ABCG2 antibody 405 using a modified avidin-biotin procedure. As a negative control, each sample was also stained with antibody pre-adsorbed with peptide to assess background staining. As a means of verification, selected tissues were also stained with the commercially available monoclonal antibody 5D3. ABCG2 positivity was consistently found in alveolar pneumocytes, sebaceous glands, transitional epithelium of bladder, interstitial cells of testes, prostate epithelium, endocervical cells of uterus, squamous epithelium of cervix, small and large intestinal mucosa/epithelial cells, islet and acinar cells of pancreas, zona reticularis layer of adrenal gland, kidney cortical tubules and hepatocytes. Placental syncytiotrophoblasts showed both cytoplasmic and surface staining. Our results support a hypothesis concluding that ABCG2 plays a role in the protection of organs from cytotoxins. However, many of the cell types expressing ABCG2 have a significant secretory function. These data suggest a dual function for ABCG2 in some tissues: the excretion of toxins and xenobiotics including anti-cancer agents and a potential, as-yet undefined role in the secretion of endogenous substrates.

Pheophorbide a is a specific probe for ABCG2 function and inhibition.

Pheophorbide a (PhA), a chlorophyll catabolite, was shown to be an ABCG2 substrate based on Abcg2(-/-) knockout mouse studies (J. W. Jonker et al., Proc. Natl. Acad. Sci. USA, 99: 15649-15654, 2002). We developed a functional assay for ABCG2 using PhA and the ABCG2 inhibitor fumitremorgin C. In selected cell lines expressing high levels of P-glycoprotein, multidrug resistance-associated protein 1, or ABCG2, PhA transport was observed only in cells expressing ABCG2. Fumitremorgin C-inhibitable PhA transport was found to correlate with cell surface ABCG2 expression as measured by the anti-ABCG2 antibody 5D3. We found that 100 micro M of the cyclin-dependent kinase inhibitor UCN-01 or 1 micro M of the P-glycoprotein inhibitor tariquidar inhibited ABCG2-mediated PhA transport. In 4-day cytotoxicity assays, ABCG2-mediated resistance to SN-38 and topotecan was abrogated in ABCG2-transfected HEK-293 cells treated with 1 micro M tariquidar, and ABCG2-transfected cells were 6-7-fold resistant to UCN-01. PhA is an ABCG2-specific substrate with potential value in measuring ABCG2 function and expression in clinical samples.

Single nucleotide polymorphisms modify the transporter activity of ABCG2.

Single nucleotide polymorphism (SNP) analyses of the ABCG2 gene have revealed three nonsynonymous SNPs resulting in the amino acid changes at V12M, Q141K and D620N. To determine whether the SNPs have an effect on drug transport, human embryonic kidney cells (HEK-293) were stably transfected with full length ABCG2 coding wild-type or SNP variants of ABCG2. In 4-day cytotoxicity assays with mitoxantrone, topotecan, SN-38 or diflomotecan, cells transfected with wild-type R482 ABCG2 showed IC50 values up to 1.2-fold to 5-fold higher than cells expressing comparable levels of Q141K ABCG2, suggesting that the Q141K SNP affects drug transport. FTC-inhibitable mitoxantrone efflux normalized to ABCG2 surface expression as assayed by the anti-ABCG2 antibody 5D3 was significantly lower in cells transfected with Q141K ABCG2 than in those transfected with wild-type R482 ABCG2 (P = 0.0048). Values for V12M and D620N ABCG2 were comparable to those for wild-type R482 ABCG2. The vanadate-sensitive ATPase activity of ABCG2 was assayed in Sf9 insect cells infected with wild-type or SNP variants of ABCG2. Basal ATPase activity in cells transfected with Q141K ABCG2 was 1.8-fold lower than in cells transfected with wild-type ABCG2, but was comparable among cells expressing wild-type, V12M or D620N ABCG2. Confocal studies of ABCG2 localization revealed higher intracellular staining in the Q141K transfectants than in cells transfected with wild-type or V12M ABCG2. Decreased transport of Hoechst 33342 was observed in Sf9 cells expressing V12M ABCG2; however, this was not true in HEK-293 cells expressing V12M ABCG2. These results suggest that the Q141K SNP affects the transport efficiency of ABCG2 and may result in altered pharmacokinetics or drug-resistance profiles in clinical oncology.

The role of breast cancer resistance protein in acute lymphoblastic leukemia.

PURPOSE: Overexpression of the transporter ABCG2, also known as breast cancer resistance protein and mitoxantrone resistance protein, can confer resistance to a variety of cytostatic drugs, such as mitoxantrone, topotecan, doxorubicin, and daunorubicin. This study analyzes the ABCG2 expression and activity in 46 human de novo acute lymphoblastic leukemia B- and T-lineage (ALL) samples. EXPERIMENTAL DESIGN: ABCG2 expression was measured flow cytometrically with the BXP-34 monoclonal antibody. ABCG2 functional activity was determined flow cytometrically by measuring mitoxantrone accumulation in combination with the ABCG2 inhibitor fumitremorgin C (FTC). To determine a possible effect of the transporters P-glycoprotein and multidrug resistance-associated protein (MRP1 and MRP2) on mitoxantrone accumulation, the accumulation was investigated in the presence of the P-glycoprotein inhibitor PSC 833 and MRP inhibitor MK-571. The ABCG2 gene was sequenced to investigate the amino acid at position 482. RESULTS: In B-lineage ALL (n = 23), the median BXP-34:IgG1 ratio was higher, namely 2.4 (range, 1.7-3.7), than in T-lineage ALL (n = 23; 1.9; range, 1.2-6.6; P = 0.003). The addition of FTC to mitoxantrone treatment caused a median increase in mitoxantrone accumulation of 21% (range, 0-140%) in B-lineage ALL. In T-lineage ALL, this FTC effect was less pronounced (5%; range, 0-256%; P = 0.013). The influence of FTC on mitoxantrone accumulation correlated with ABCG2 protein expression (r = 0.52; P < 0.001; n = 43). The increase in mitoxantrone accumulation, when FTC was added to cells treated with both PSC 833 and MK-571, correlated with the ABCG2 expression in B-lineage ALL but not in T-lineage ALL. Sequencing the ABCG2 gene revealed no ABCG2 mutation at position 482 in patients who accumulated more rhodamine after FTC. CONCLUSIONS: This study shows that ABCG2 is expressed higher and functionally more active in B-lineage than in T-lineage ALL.

Single-nucleotide polymorphism (SNP) analysis in the ABC half-transporter ABCG2 (MXR/BCRP/ABCP1).

Variations in the amino acid sequence of ABC transporters have been shown to impact substrate specificity. We identified two acquired mutations in ABCG2, the ABC half-transporter overexpressed in mitoxantrone-resistant cell lines. These mutations confer differences in substrate specificity and suggest that naturally occurring variants could also affect substrate specificity. To search for the existence of single nucleotide polymorphisms (SNPs) in ABCG2, we sequenced 90 ethnically diverse DNAs from the Single Nucleotide Polymorphism Discovery Resource representing the spectrum of human genotypes. We identified 3 noncoding SNPs in the untranslated regions, 3 nonsynonymous and 2 synonymous SNPs in the coding region and 7 SNPs in the intron sequences adjacent to the sixteen ABCG2 exons. Nonsynonymous SNPs at nucleotide 238 (V12M; exon 2) and nucleotide 625 (Q141K; exon 5) showed a greater frequency of heterozygosity (22.2% and 10%) than the SNP at 2062 (D620N; exon 16). Heterozygous changes at nucleotide 238 are in linkage disequilibrium with an SNP observed 36 bases downstream from the end of exon 2. No polymorphism at amino acid 482 was identified to correspond to the R to G or R to T mutations previously found in two drug resistant cell lines. Among 23 drug resistant sublines for which sequence at position 482 was determined, no additional mutations were found. Heterozygosity at amino acid 12 allowed us to identify overexpression of a single allele in a subset of drug resistant cell lines, a feature that could be exploited clinically in evaluating the significance of ABCG2 expression in malignancy. We conclude that ABCG2 is well conserved and that described amino acid polymorphisms seem unlikely to alter transporter stability or function.

Technetium-99m sestamibi single photon emission computed tomography findings correlated with P-glycoprotein expression, encoded by the multidrug resistance gene-1 messenger ribonucleic acid, in intracranial meningiomas.

The present study evaluated whether technetium-99m sestamibi (99mTc-MIBI) single photon emission computed tomography (SPECT) characteristics of intracranial meningioma are correlated with the histological malignancy, proliferative potential, and P-glycoprotein (Pgp) expression, encoded by the multidrug resistance gene-1 (MDR-1) messenger ribonucleic acid (mRNA). Twenty-one patients with intracranial meningiomas, including 17 benign and four nonbenign meningiomas, underwent 99mTc-MIBI SPECT imaging at 15 minutes (early) and 3 hours (delayed) after injection. The tumor-to-normal pituitary gland ratio was calculated on both early (ER) and delayed (DR) images. Retention index (RI) was calculated using the following formula: (DR - ER)/ER x 100%. Meningioma specimens were examined by immunohistochemistry using anti-Pgp and MIB-1 monoclonal antibody. MDR-1 mRNA expression was also investigated using reverse transcription-polymerase chain reaction assay. 99mTc-MIBI was highly accumulated and retained in the tumors. 99mTc-MIBI SPECT findings were not related to MIB-1 labeling index. 99mTc-MIBI SPECT RI of the Pgp-positive group (-9.12 +/- 22.27%) was significantly lower than that of the Pgp-negative group (28.79 +/- 22.80%) (p = 0.0016). No significant difference was seen in ER and DR between the positive and negative groups. These results show that 99mTc-MIBI may not be useful for determining proliferative potential and histological malignancy, but could predict anticancer drug resistance related to the expression of MDR-1 mRNA and its gene product Pgp in patients with intracranial meningiomas.

Mutational studies of G553 in TM5 of ABCG2: a residue potentially involved in dimerization.

ABCG2 is an ATP-binding cassette half-transporter conferring resistance to chemotherapeutic agents such as mitoxantrone, irinotecan, and flavopiridol. With its one transmembrane and one ATP-binding domain, ABCG2 is thought to homodimerize for function. One conserved region potentially involved in dimerization is a three-amino acid sequence in transmembrane segment 5 (residues 552-554). Mutations in the corresponding residues in the Drosophila white protein (an orthologue of ABCG2) are thought to disrupt heterodimerization. We substituted glycine 553 with leucine (G553L) followed by stable transfection in HEK 293 cells. The mutant was not detectable on the cell surface, and markedly reduced protein expression levels were observed by immunoblotting. A deficiency in N-linked glycosylation was suggested by a reduction in molecular mass compared to that of the 72 kDa wild-type ABCG2. Similar results were observed with the G553E mutant. Confocal microscopy demonstrated mostly ER localization of the G553L mutant in HEK 293 cells, even when coexpressed with the wild-type protein. Despite its altered localization, the G553L and G553E mutants were cross-linked using amine-reactive cross-linkers with multiple arm lengths, suggesting that the monomers are in the proximity of each other but are unable to complete normal trafficking. Interestingly, when expressed in Sf9 insect cells, G553L moves to the cell membrane but is unable to hydrolyze ATP or transport the Hoechst dye. Still, when coexpressed, the mutant interferes with the Hoechst transport activity of the wild-type protein. These data show that glycine 553 is important for protein trafficking and are consistent with, but do not yet prove, its involvement in ABCG2 homodimerization.

Mutational analysis of ABCG2: role of the GXXXG motif.

ABCG2 (BCRP/MXR/ABCP) is a half-transporter associated with multidrug resistance that presumably homodimerizes for function. It has a conserved GXXXG motif in its first transmembrane segment, a motif that has been linked with dimerization in other proteins, e.g., glycophorin A. We substituted either or both glycines of this GXXXG motif with leucines to evaluate the impact on drug transport, ATP hydrolysis, cross-linking, and susceptibility to degradation. All mutants also carried the R482G gain-of-function mutation, and all migrated to the cell surface. The mutations resulted in lost transport for rhodamine 123 and impaired mitoxantrone, pheophorbide a, and BODIPY-prazosin transport, particularly in the double leucine mutant (G406L/G410L). Basal ATPase activity of the G406L/G410L mutant was comparable to the empty vector transfected cells with no substrate induction. Despite impaired function, the mutants retained susceptibility to cross-linking using either disuccinimidyl suberate (DSS) or the reducible dithiobis(succinimidyl propionate) (DSP) and demonstrated a high molecular weight complex under nonreducing conditions. Mutations to alanine at the same positions yielded fully functional transporters. Finally, we exposed cells to mitoxantrone to promote folding and processing of the mutant proteins, which in the leucine mutants resulted in increased amounts detected on immunoblot and by immunofluorescence. These studies support a hypothesis that the GXXXG motif promotes proper packing of the transmembrane segments in the functional ABCG2 homodimer, although it does not solely arbitrate dimerization.