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. (Mol Cancer Res 2008;6(1):151 -64)

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.

The origin of drug-resistant cells in human cancers has been a fundamental problem of cancer pharmacology. Two major contrasting hypotheses (genetics versus epigenetics) have been proposed to elucidate the mechanisms of acquired drug resistance. In this study, we answer these fundamental questions through investigation of the genetic and epigenetic pathways that control the origin of ABCB1 (MDR1) gene activation with acquired multidrug resistance in drugsensitive human sarcoma (MES-SA cells). The genetic and epigenetic bases of this selected activation involve the initiation of transcription at a site 112 kb upstream of the ABCB1 proximal promoter (P1) in the drug-resistant cells. This activation was associated with a chromatin-remodeling process characterized by an increase in acetylated histone H3 within a 968-bp region 5V of the ABCB1 upstream promoter. These alterations provide both genetic and epigenetic susceptibility for ABCB1 expression in drug-resistant cells. Complete activation of the ABCB1 gene through the coding region was proposed by interactions of selected trans-alterations or epigenetic changes on the ABCB1 proximal promoter, which occurred during initial drug exposure. Thus, our data provide evidence for a major genomic alteration that changes the chromatin structure of the ABCB1 upstream promoter via acetylation of histone H3 initiating ABCB1 activation, further elucidating the genetic and epigenetic bases that determine chemotherapeutic response in drug-resistant derivatives of MES-SA cells. (Cancer Res 2005; 65(20): 9388-97)

Purpose: The mechanism of sensitivity and resistance to epidermal growth factor receptor (EGFR) inhibitors is incompletely understood, particularly in cancers other than non^small-cell lung cancer (NSCLC). To understand the variable response to this class of drugs, we used the NCI60 cancer cell lines. We aimed to determine if there are interactions between EGFR expression, mutations, polymorphisms, and gene amplification, and whether these factors are associated with variability in response to EGFR inhibitors. Experimental Design:The EGFRVIII and tyrosine kinase (TK) domain mutations were examined in the NCI60 cancer cell lines. Five polymorphisms, -216G/T, -191C/A, intron 1 (CA)n, R497K, and 2607A/G, were genotyped. EGFR amplification was also assessed with high-density single-nucleotide polymorphism chip and real-time PCR, respectively. The results were correlated with cytotoxicity data for erlotinib and other 11EGFR inhibitors, as well as other publicly available data for these lines. Results: All 12 inhibitors behaved similarly. No EGFRVIII but putative TK mutations in two cell lines were found. Both mutant cell lines were insensitive to all inhibitors. Meanwhile, response did not correlate with EGFR amplification but with EGFR gene expression, especially in the cell lines with relatively normal gene status. In addition, EGFR expression was associated with the -216G/T polymorphism but not with the intron 1 (CA)n polymorphism. A combination of -216G/Tand R497K polymorphisms was weakly associated with drug response. Conclusions: These observations suggest that in addition toTK mutations, germ-line variability may also contribute to the pharmacodynamics of EGFR inhibitors, particularly when EGFR is genetically normal.

Expression of the MDR-1/P-glycoprotein gene confers drug resistance both in vitro and in vivo. We previously reported that gene rearrangements resulting in a hybrid MDR-1 transcript represent a common mechanism for acquired activation of MDR-1/P-glycoprotein. We have identified hybrid MDR-1 transcripts in nine MDR-1-overexpressing cell lines and two patients with relapsed ALL. We characterize these rearrangements as follows. 1) Non-MDR-1 sequences in the hybrid MDR-1 transcripts are expressed in unselected cell lines, showing that these sequences are constitutively expressed. 2) The rearrangements occur randomly and involve partner genes (sequences) on chromosome 7 and on chromosomes other than 7. Breakpoints have been characterized in six cell lines. In one, the rearrangement occurred within intron 2 of MDR-1; in the other five, the rearrangement occurred 24 to >96 kb 5 of the normal start of transcription of MDR-1. In one cell line, homologous recombination involving an Alu repeat was observed. However, in the remaining five cell lines, nonhomologous recombination was observed. 3) The rearrangements arise during drug selection. The acquired rearrangements are not detected in parental cells. 4) Five of the six active promoters that captured MDR-1 controlled MDR-1 from a distance of 29 to more than 110 kb 5 to MDR-1. Transcription was initiated in an antegrade or retrograde direction. We conclude that drug selection with natural products targeting DNA or microtubules leads to DNA damage, nonhomologous recombination, and acquired drug resistance, wherein MDR-1 expression is driven by a random but constitutively active promoter.

Capillary endothelial cells of the human blood–brain barrier (BBB) express high levels of P-glycoprotein (P-gp, encoded by ABCB1) and ABCG2 (encoded by ABCG2). However, little information is available regarding ATP-binding cassette transporters expressed at the zebrafish BBB, which has emerged as a potential model system. We report the characterization and tissue localization of two genes that are similar to ABCB1, zebrafish abcb4 and abcb5. When stably expressed in HEK293 cells, both Abcb4 and Abcb5 conferred resistance to P-gp substrates; however, Abcb5 poorly transported doxorubicin and mitoxantrone compared to zebrafish Abcb4. Additionally, Abcb5 did not transport the fluorescent P-gp probes BODIPY-ethylenediamine or LDS 751, while they were transported by Abcb4. High-throughput screening of 90 human P-gp substrates confirmed that Abcb4 has an overlapping substrate specificity profile with P-gp. In the brain vasculature, RNAscope probes for abcb4 colocalized with staining by the P-gp antibody C219, while abcb5 was not detected. The abcb4 probe also colocalized with claudin-5 in brain endothelial cells. Abcb4 and Abcb5 had different tissue localizations in multiple zebrafish tissues, potentially indicating different functions. The data suggest that zebrafish Abcb4 functionally phenocopies P-gp and that the zebrafish may serve as a model to study the role of P-gp at the BBB.

We sought to examine the effects of microtubule-targeting agents (MTA) on neural cells to better understand the problem of neurotoxicity, their principal side effect, and to possibly develop a model of clinical toxicity. Studies showed that microtubule-depolymerizing agents (MDA) not only disassembled microtubules in neural HCN2 cells but also led to rapid disappearance of tubulin, and that this was specific for MDAs. Tubulin levels decreased to 20% as early as 8 hours after adding vincristine, and to 1% to 30% (mean, 9.8 ± 7.6%; median of 7%) after 100 nmol/L vincristine for 24 hours. This disappearance was reversible. An increase in both glu-terminated and acetylated tubulin, markers of stable tubulin, preceded reaccumulation of soluble tubulin, suggesting a priority for stabilizing tubulin first as microtubules before replenishing the soluble pool. Similar results were shown with other MDAs. Furthermore, microtubule reassembly did not arise from a central focus but instead appeared to involve dispersed nucleation, as evidenced by the appearance of small, stable microtubule stubs throughout the cytoplasm. In contrast, experiments with four nonneural "normal" cell lines and four cancer cell lines resulted in microtubule destabilization but only modest tubulin degradation. Evidence for proteasome-mediated degradation was obtained by demonstrating that adding a proteasome inhibitor before vincristine prevented tubulin disappearance. In summary, MDAs lead to rapid disappearance of tubulin in neural but not in other normal or cancer cells. These results underscore the fine control that occurs in neural cells and may further our understanding of neurotoxicity following MDAs.

Purpose: Ixabepilone (Ixempra; BMS-247550) is an epothilone B analog and nontaxane microtubulestabilizing compound with clinical activity in a range of solid tumors. This phase II study was conducted to assess the efficacy and safety of ixabepilone in patients with metastatic renal cell carcinoma.Experimental Design: Patients with metastatic renal cell carcinoma who had measurable disease and had not received previous cytotoxic or targeted therapy were treated with 6 mg/m 2 ixabepilone i.v. daily for 5 days every 3 weeks. Levels of Glu-terminated and acetylated tubulin, markers of microtubule stabilization, were assessed by Western blot. VHL gene mutation status was determined by sequencing. Results: Eighty-seven patients received a total of 590 cycles, with a median of 5 cycles (range, 1-29). The overall response rate was 13% (Response Evaluation Criteria in Solid Tumor). One patient had a complete response, 10 patients had partial responses, and 59 patients had stable disease. The median duration of response was 5.5 months. The median overall survival of renal cell carcinoma Motzer grade 0 and 1 patients with clear cell histology was 19.25 months. Treatment-related adverse events were primarily alopecia, gastrointestinal toxicity, neuropathy, and fatigue. Biopsies were done at baseline and after five doses of ixabepilone. Microtubule target engagement was achieved in 84.6% to 92.3% of patients evaluated. No correlation was identified between the target engagement, VHL gene mutation status, and clinical response.Conclusion: Ixabepilone can cause tumor regression in some patients with metastatic renal cell carcinoma and could be considered in combination regimens with other therapies.