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The multidrug resistance 1 gene (MDR1) encodes P-glycoprotein (Pgp), a member of the ATP-binding cassette (ABC) transporter family that confers tumor drug resistance by actively effluxing a number of antitumor agents. We had previously shown that MDR1 transcription is regulated by epigenetic events such as histone acetylation, and had identified the histone acetylase P/CAF and the transcription factor NF-Y as the factors mediating the enzymatic and DNA-anchoring functions, respectively, at the MDR1 promoter. It has also been shown that MDR1 activation is accompanied by increased methylation on lysine 4 of histone H3 (H3K4). In this study, we further investigated histone methylation in MDR1 regulation and function. We show that the mixed lineage leukemia 1 (MLL1) protein, a histone methyltransferase specific for H3K4, is required for MDR1 promoter methylation, as knockdown of MLL1 resulted in a decrease in MDR1 expression. The regulation of MDR1 by MLL1 has functional consequences in that downregulation of MLL1 led to increased retention of the Pgp-specific substrate DIOC(2)(3), as well as increased cellular sensitivity to several Pgp substrates. Regulation of MDR1 by MLL1 was dependent on the CCAAT box within the proximal MDR1 promoter, similar to what we had shown for MDR1 promoter acetylation, and also requires NF-Y. Finally, overexpression of the most prevalent MLL fusion protein, MLL-AF4, led to increased MDR1 expression. This is the first identification of a histone methyltransferase and its leukemogenic rearrangement that regulates expression of an ABC drug transporter, suggesting a new target for circumvention of tumor multidrug resistance., (©2010 AACR.)

Drug resistance caused by overexpression of P-glycoprotein (P-gp), the MDR1 (ABCB1) gene product, limits the therapeutic outcome. Expression of MDR1 can be induced by divergent stimuli, and involves a number of transcriptional factors. We found that the expression of CtBP1 (C-terminal-binding protein 1), a transcriptional co-regulator, was increased (approximately 4-fold) in human multidrug resistant (MDR) cancer cell lines, NCI/ADR-RES and A2780/DX, as compared to their sensitive counterparts. Silencing of CtBP1 expression by RNAi decreased the MDR1 mRNA and P-gp. Knockdown of CtBP1 also enhanced the sensitivity of MDR cells to chemotherapeutic drugs that are transported by P-gp and increased intracellular drug accumulation. In a reporter gene assay, co-transfection of MDR1 promoter constructs with a CtBP1 expression vector resulted in a approximately 2-4-fold induction of MDR1 promoter activity. CtBP1 appeared to contribute to the activation of MDR1 transcription through directly interacting with the MDR1 promoter, as evidenced by its physical binding to the promoter region of the MDR1 gene in chromatin immunoprecipitation and electromobility shift assays. Histone modifications at the MDR1 promoter, such as mono-methylation, di-methylation, and acetylation of histone H3, were not found to be affected by silencing of CtBP1 expression. Our results reveal a novel role for CtBP1 as an activator of MDR1 gene transcription, and suggest that CtBP1 might be one of the key transcription factors involved in the induction of MDR1 gene. Therefore, CtBP1 may represent a potentially new target for inhibiting drug resistance mediated by overexpression of the MDR1 gene.

Although the p53 family members share a similar structure and function, it has become clear that they differ with respect to their role in development and tumor progression. Because of the high degree of homology in their DNA binding domains (DBDs), it is not surprising that both p63 and p73 activate the majority of p53 target genes. However, recent studies have revealed some differences in a subset of the target genes affected, and the mechanism underlying this diversity has only recently come under investigation. Our laboratory has demonstrated previously that p53 represses transcription of the P-glycoprotein-encoding MDR1 gene via direct DNA binding through a novel p53 DNA-binding site (the HT site). By transient transfection analyses, we now show that p63 and p73 activate rather than repress MDR1 transcription, and they do so through an upstream promoter element (the alternative p63/p73 element (APE)) independent of the HT site. This activation is dependent on an intact DNA binding domain, because mutations within the p63DBD or p73DBD are sufficient to prevent APE-mediated activation. However, neither p63 nor p73 directly interact with the APE, suggesting an indirect mechanism of activation through this site. Most interestingly, when the p53DBD is replaced by the p63DBD, p53 is converted from a repressor working through the HT site to an activator working through the APE. Taken together, these data indicate that, despite considerable homology, the DBD of the p53 family members have unique properties and can differentially regulate gene targeting and transcriptional output by both DNA binding-dependent and -independent mechanisms.

Two decades ago, the overexpression of P-glycoprotein (Pgp) was first demonstrated to mediate the energy-dependent efflux of a variety of chemotherapeutic agents from tumor cells, resulting in the development of multidrug resistance (MDR). Not surprisingly, this discovery triggered an ongoing search for agents that would inhibit Pgp function, with the hope that by doing so the MDR phenotype could be reversed. As our understanding of Pgp function and pharmacokinetics has increased, this quest has become more urgent, as well as more complex.

Ecteinascidin 743 (ET-743), a highly promising marine-based antitumor agent presently in phase II clinical trials, has been shown to interfere with the binding of minor-groove-interacting transcription factors, particularly NF-Y, with their cognate promoter elements in vitro. We have shown that NF-Y is a central mediator of activation of transcription of the human P glycoprotein gene (MDR1) by a variety of inducers and that NF-Y functions by recruiting the histone acetyltransferase PCAF to the MDR1 promoter. In the present study, we tested whether ET-743 could block activation of the MDR1 promoter by agents that mediate their effect through the NF-Y/PCAF complex. We report that physiologically relevant concentrations of ET-743 abrogate transcriptional activation of both the endogenous MDR1 gene and MDR1 reporter constructs by the histone deacetylase inhibitors as well as by UV light, with minimal effect on constitutive MDR1 transcription. Notably, this inhibition does not alter the promoter-associated histone hyperacetylation induced by histone deacetylase inhibitors, suggesting an in vivo molecular target downstream of NF-Y/PCAF binding. ET-743 is therefore the prototype for a distinct class of transcription-targeted chemotherapeutic agents and may be an efficacious adjuvant to the treatment of multidrug-resistant tumors.

Recent studies have shown that the histone-modifying enzymes histone acetyltransferase (HAT) and histone deacetylase (HDAC) are involved in transcriptional activation and repression, respectively. However, little is known about the endogenous genes that are regulated by these enzymes or how specificity is achieved. In the present report, we demonstrate that HAT and HDAC activities modulate transcription of the P-glycoprotein-encoding gene, MDR1. Incubation of human colon carcinoma SW620 cells in 100-ng/ml trichostatin A (TSA), a specific HDAC inhibitor, increased the steady-state level of MDR1 mRNA 20-fold. Furthermore, TSA treatment of cells transfected with a wild-type MDR1 promoter/luciferase construct resulted in a 10- to 15-fold induction of promoter activity. Deletion and point mutation analysis determined that an inverted CCAAT box was essential for this activation. Consistent with this observation, overexpression of p300/CREB binding protein-associated factor (P/CAF), a transcriptional coactivator with intrinsic HAT activity, activated the wild-type MDR1 promoter but not a promoter containing a mutation in the CCAAT box; deletion of the P/CAF HAT domain abolished activation. Gel shift and supershift analyses identified NF-Y as the CCAAT-box binding protein in these cells, and cotransfection of a dominant negative NF-Y expression vector decreased the activation of the MDR1 promoter by TSA. Moreover, NF-YA and P/CAF were shown to interact in vitro. This is the first report of a natural promoter that is modulated by HAT and HDAC activities in which the transcription factor mediating this regulation has been identified.

Cellular resistance to multiple chemotherapeutic agents is most often due to the overexpression of P-glycoprotein (Pgp). The mechanisms(s) underlying Pgp overexpression had not been determined, due, in part, to a failure to reproduce the overexpression in transient transfection assays. We now report that stable transfection of a Pgp (pgp1) promoter/luciferase construct in the drug-sensitive Chinese hamster cell line DC-3F and its drug-resistant sublines reproduced the overexpression phenotype, with up to 18-fold higher activity observed in the resistant cell lines compared with DC-3F. Moreover, mutation of a pgp1 promoter element, multiple start site element downstream (MED-1), decreased transcription in drug-resistant cells without affecting activity in drug-sensitive cells. This is the first report of a Pgp promoter element differentially regulated in drug-resistant cells. Moreover, these data suggest that the regulation of Pgp transcription is modulated by chromatin structure, and that stable transfection may be more suitable for identifying promoter elements important for overexpression in drug-resistant cells.

Although many TATA-less promoters transcribed by RNA polymerase II initiate transcription at multiple sites, the regulation of multiple start site utilization is not understood. Beginning with the prediction that multiple start site promoters may share regulatory features and using the P-glycoprotein promoter (which can utilize either a single or multiple transcription start site(s)) as a model, several promoters with analogous transcription windows were grouped and searched for the presence of a common DNA element. A downstream protein-binding sequence, MED-1 (Multiple start site Element Downstream), was found in the majority of promoters analyzed. Mutation of this element within the P-glycoprotein promoter reduced transcription by selectively decreasing utilization of downstream start sites. We propose that a new class of RNA polymerase II promoters, those that can utilize a distinctive window of multiple start sites, is defined by the presence of a downstream MED-1 element.

The overproduction of P-glycoprotein (Pgp) has been associated with the development and maintenance of the multidrug resistant (MDR) phenotype, although the regulatory events responsible have not yet been elucidated. We have analyzed the overexpression of the TATA-less hamster class-I Pgp-encoding gene (Pgp1) in several MDR Chinese hamster cell lines. The MDR lung cell line DC-3F/VCRd5L, as well as the MDR ovary cell line CHRC5, express a level of Pgp1 RNA commensurate with the increase in Pgp1 dosage; in contrast, the actinomycin D (ActD)-selected sublines of DC-3F overexpress Pgp1 mRNA without a concomitant increase in Pgp1 gene-copy number. Analysis of Pgp1 transcription start point (tsp) utilization revealed that drug-sensitive DC-3F cells, as well as DC-3F/VCRd5L and CHRC5 cells, utilize one major tsp; in contrast, the ActD-resistant sublines 'switch' to a more complex pattern, using four additional Pgp1 tsp 32, 42, 52, and 67 bp downstream from the major parental tsp (+1). This observation of a difference in the regulation of transcription of Pgp in MDR vs. drug-sensitive cells suggests that the 'switch' in tsp selection may be involved in the increased expression of Pgp1 mRNA. Interestingly, despite the existence of several hundred MDR cell lines, very few have been analyzed with respect to tsp selection; it is therefore possible that alternate tsp selection is a relatively common yet heretofore unobserved component of the MDR phenotype. Moreover, these cells provide an excellent system in which to evaluate the sequence elements and protein factors that govern the selection of tsp in TATA-less promoters.