No abstract

ABCG2 is recognized as an important efflux transporter in clinical pharmacology and is potentially important in resistance to chemotherapeutic drugs. To identify epigenetic mechanisms regulating ABCG2 mRNA expression at its 3 untranslated region (3UTR), we performed 3 rapid amplification of cDNA ends with the S1 parental colon cancer cell line and its drug-resistant ABCG2-overexpressing counterpart. We found that the 3UTR is >1,500 bp longer in parental cells and, using the miRBase TARGETs database, identified a putative microRNA (miRNA) binding site, distinct from the recently reported hsa-miR520h site, in the portion of the 3UTR missing from ABCG2 mRNA in the resistant cells. We hypothesized that the binding of a putative miRNA at the 3UTR of ABCG2 suppresses the expression of ABCG2. In resistant S1MI80 cells, the miRNA cannot bind to ABCG2 mRNA because of the shorter 3UTR, and thus, mRNA degradation and/or repression on protein translation is relieved, contributing to overexpression of ABCG2. This hypothesis was rigorously tested by reporter gene assays, mutational analysis at the miRNA binding sites, and forced expression of miRNA inhibitors or mimics. The removal of this epigenetic regulation by miRNA could be involved in the overexpression of ABCG2 in drug-resistant cancer cells.ABCG2, a ubiquitous ATP-binding cassette (ABC) transporter, besides playing a significant role in absorption, distribution, and elimination of its substrate drugs, may also confer multidrug resistance in cancer cells (1). ABCG2 overexpression is frequently observed in human cancer cell lines selected with various anticancer drugs (12,34,38,44,52).The molecular mechanisms regulating ABCG2 expression are not well understood. Like most TATA-less gene promoters, the ABCG2 promoter contains numerous Sp1, AP1, and AP2 sites and a CCAAT box. ABCG2 also has a putative CpG island located upstream of the gene (50). To date, most studies examining the regulation of ABCG2 are focused at the transcriptional level. Two functional cis elements in the ABCG2 promoter, namely, the estrogen (15) and hypoxia (28) response elements, and a peroxisome proliferator-activated receptor response element upstream of the ABCG2 gene (48) have been reported. An aryl hydrocarbon receptor response element has been proposed, but the exact sequence has not been identified (13). In drug-resistant MCF-7 cells, alternative promoter use due to differential expression of splice variants at the 5Ј untranslated region (5ЈUTR) of ABCG2 mRNA has been observed (39). We recently reported that DNA methylation and histone modifications play important roles in the regulation of human ABCG2 in renal carcinoma cell lines (50) and in some drug-selected resistant cell lines (49), respectively.Less is known about the 3ЈUTR of ABCG2. In eukaryotic mRNAs, the 3ЈUTR plays an important role in regulating gene expression at the posttranscriptional level by modulating nucleocytoplasmic mRNA transport, polyadenylation status, subcellular targeting, translation efficiency, stability, and rate...

Depsipeptide (FK228) is a novel histone deacetylase inhibitor currently in clinical trials and the first to demonstrate clinical activity in patients. Responses have been observed in patients with T-cell lymphomas, despite prior treatment with multiple chemotherapeutic agents. To better understand the effects of histone deacetylase inhibitors on T-cell lymphoma, the human T-cell lymphoma cell line HUT78 was tested for sensitivity and molecular response to depsipeptide. Treatment with depsipeptide, as well as other histone deacetylase inhibitors, caused induction of histone acetylation, induction of p21 expression, and substantial apoptosis without significant cell cycle arrest. Treatment with the caspase inhibitor z-VADfmk significantly inhibited depsipeptideinduced apoptosis, enabling detection of cell cycle arrest. Treatment with depsipeptide increased expression of the interleukin-2 (IL-2) receptor, and combination with the IL-2 toxin conjugate denileukin diftitox resulted in more than additive toxicity. Cells selected for resistance to depsipeptide overexpressed the multidrug resistance pump, P-glycoprotein (Pgp).However, cells selected for resistance to depsipeptide in the presence of a Pgp inhibitor had a Pgp-independent mechanism of resistance. These studies confirm the activity of depsipeptide in a T-cell lymphoma model and suggest a general sensitivity of T-cell lymphoma to histone deacetylase inhibitors, an emerging new class of anticancer agents. IntroductionThe histone deacetylase inhibitors (HDIs) are a new class of antineoplastic agents currently being evaluated in clinical trials. HDIs induce growth arrest usually associated with cellular differentiation or apoptosis. Alterations in the enzymes controlling histone acetylation and deacetylation have been shown to be a direct mechanism of transformation in some malignancies. 1 The consequence of a decrease in histone acetylation is a decreased expression of cell cycle inhibitors and other genes involved in regulating a differentiated phenotype. 2 Depsipeptide (FK228) is an HDI that has in vitro and in vivo cytotoxic activity. 3 Several families of HDIs have been characterized. These include the short-chain fatty acids, such as sodium butyrate and valproic acid; the organic hydroxamic acids, such as trichostatin A (TSA) and suberanilohydroxamic acid (SAHA); the benzamides, such as CI-994 and MS-27-275; the cyclic tetrapeptides, such as trapoxin A; and the bicyclic depsipeptides, such as depsipeptide. Similar to other HDIs, depsipeptide has been shown to induce cell cycle arrest, cellular differentiation, and apoptosis. Depsipeptide induces a p53-independent/ p21-dependent G1 arrest and a p21-independent G2/M arrest. In addition, depsipeptide causes alterations in gene expression, including increased expression of p21 and cyclin E and decreased expression of cyclin D1 and c-myc. [4][5][6] T-cell lymphomas are composed of a spectrum of clinical phenotypes ranging from low-grade, cutaneous T-cell lymphomas (CTCLs) to highly aggressive peripheral T-cell...