MRP8 (ABCC11) is a recently identified cDNA that has been assigned to the multidrug resistance-associated protein (MRP) family of ATP-binding cassette transporters, but its functional characteristics have not been determined. Here we examine the functional properties of the protein using transfected LLC-PK1 cells. It is shown that ectopic expression of MRP8 reduces basal intracellular levels of cAMP and cGMP and enhances cellular extrusion of cyclic nucleotides in the presence or absence of stimulation with forskolin or SIN-1A. Analysis of the sensitivity of MRP8-overexpressing cells revealed that they are resistant to a range of clinically relevant nucleotide analogs, including the anticancer fluoropyrimidines 5-fluorouracil (ϳ3-fold), 5-fluoro-2-deoxyuridine (ϳ5-fold), and 5-fluoro-5-deoxyuridine (ϳ3-fold), the anti-human immunodeficiency virus agent 2,3-dideoxycytidine (ϳ6-fold) and the anti-hepatitis B agent 9-(2-phosphonylmethoxynyl)adenine (PMEA) (ϳ5-fold). By contrast, increased resistance was not observed for several natural product chemotherapeutic agents. In accord with the notion that MRP8 functions as a drug efflux pump for nucleotide analogs, MRP8-transfected cells exhibited reduced accumulation and increased efflux of radiolabeled PMEA. In addition, it is shown by the use of in vitro transport assays that MRP8 is able to confer resistance to fluoropyrimidines by mediating the MgATP-dependent transport of 5-fluoro-2-deoxyuridine monophosphate, the cytotoxic intracellular metabolite of this class of agents, but not of 5-fluorouracil or 5-fluoro-2-deoxyuridine. We conclude that MRP8 is an amphipathic anion transporter that is able to efflux cAMP and cGMP and to function as a resistance factor for commonly employed purine and pyrimidine nucleotide analogs.Cellular extrusion of cyclic nucleotides has been described in prokaryotic and eukaryotic cells (1-4). This process provides extracellular cAMP involved in intercellular signaling, as determined for Dictyostelium discoideum, in which cAMP effluxed by solitary amoebae under low nutrient conditions mediates cellular aggregation and differentiation, and has also been proposed as a potential mechanism that may contribute to the attenuation of intracellular signaling mediated by these second messengers (5). Investigations employing cultured cells and membrane vesicle preparations have established that cyclic nucleotide efflux is energy-dependent, and the susceptibility of this process to inhibition by antagonists of organic anion pumps indicates that it is mediated by amphipathic anion transporters (2, 3, 6 -16). Recently, insights into the identities of the cellular components that mediate cyclic nucleotide efflux have come from studies of the MRP 1 family of ABC transporters. MRP4 and MRP5, two members of this extended family of amphipathic anion transporters (17), have been determined to be competent in the transport of cyclic nucleotides (18 -20). By contrast, other characterized MRP family members are able to transport a variety of lipophilic anions, ...

Human multidrug resistance protein 7 (MRP7, ABCC10) is a recently described member of the C family of ATP binding cassette proteins (Cancer Lett 162:181-191, 2001). However, neither its biochemical activity nor physiological functions have been determined. Here we report the results of investigations of the in vitro transport properties of MRP7 using membrane vesicles prepared from human embryonic kidney 293 cells transfected with MRP7 expression vector. It is shown that expression of MRP7 is specifically associated with the MgATPdependent transport of 17␤-estradiol-(17-␤-D-glucuronide) (E 2 17␤G). E 2 17␤G transport was saturable, with K m and V max values of 57.8 Ϯ 15 M and 53.1 Ϯ 20 pmol/mg/min. By contrast, with E 2 17␤G, only modest enhancement of LTC 4 transport was observed and transport of several other established substrates of MRP family transporters was not detectable to any extent. In accord with the notion that MRP7 has a bipartite substrate binding pocket composed of sites for anionic and lipophilic moieties, transport of E 2 17␤G was susceptible to competitive inhibition by both amphiphiles, such as leukotriene C 4 (K i(app) , 1.5 M), glycolithocholate 3-sulfate (K i(app) , 34.2 M) and MK571 (K i(app) , 28.5 M), and lipophilic agents such as cyclosporine A (K i(app) , 14.4 M). Of the inhibitors tested, LTC 4 was the most potent, in agreement with the possibility that it is a substrate of the pump. The determination that MRP7 has the facility for mediating the transport of conjugates such as E 2 17␤G indicates that it is a lipophilic anion transporter involved in phase III (cellular extrusion) of detoxification.

Poly(ADP-ribose) polymerase 1 (PARP1), a nuclear protein, utilizes NAD to synthesize poly(AD-Pribose) (pADPr), resulting in both automodification and the modification of acceptor proteins. Substantial amounts of PARP1 and pADPr (up to 50%) are localized to the nucleolus, a subnuclear organelle known as a region for ribosome biogenesis and maturation. At present, the functional significance of PARP1 protein inside the nucleolus remains unclear. Using PARP1 mutants, we investigated the function of PARP1, pADPr, and PARP1-interacting proteins in the maintenance of nucleolus structure and functions. Our analysis shows that disruption of PARP1 enzymatic activity caused nucleolar disintegration and aberrant localization of nucleolar-specific proteins. Additionally, PARP1 mutants have increased accumulation of rRNA intermediates and a decrease in ribosome levels. Together, our data suggests that PARP1 enzymatic activity is required for targeting nucleolar proteins to the proximity of precursor rRNA; hence, PARP1 controls precursor rRNA processing, post-transcriptional modification, and pre-ribosome assembly. Based on these findings, we propose a model that explains how PARP1 activity impacts nucleolar functions and, consequently, ribosomal biogenesis.

We previously determined that expression of human multidrug resistance protein (MRP) 8, a recently described member of the MRP family of ATP-binding cassette transporters, enhances cellular extrusion of cyclic nucleotides and confers resistance to nucleotide analogs (J Biol Chem 278: 29509 -29514, 2003). However, the in vitro transport characteristics of the pump have not been determined. In this study, the substrate selectivity and biochemical activity of MRP8 is investigated using membrane vesicles prepared from LLC-PK1 cells transfected with MRP8 expression vector. Expression of MRP8 is shown to stimulate the ATP-dependent uptake of a range of physiological and synthetic lipophilic anions, including the glutathione S-conjugates leukotriene C4 and dinitrophenyl S-glutathione, steroid sulfates such as dehydroepiandrosterone 3-sulfate (DHEAS) and estrone 3-sulfate, glucuronides such as estradiol 17-␤-Dglucuronide (E 2 17␤G), the monoanionic bile acids glycocholate and taurocholate, and methotrexate. In addition, MRP8 is competent in the in vitro transport of cAMP and cGMP, in accord with the results of our previously reported cellular studies. DHEAS, E 2 17␤G, and methotrexate were transported with K m and V max values of 13.0 Ϯ 0.8 M and 34.9 Ϯ 9.5 pmol/mg/min, 62.9 Ϯ 12 M and 62.0 Ϯ 5.2 pmol/mg/min, and 957 Ϯ 28 M and 317 Ϯ 17 pmol/mg/min, respectively. Based upon the stimulatory action of DHEAS on uptake of E 2 17␤G, the attenuation of this effect at high DHEAS concentrations and the lack of reciprocal promotion of DHEAS uptake by E 2 17␤G, a model involving nonreciprocal constructive interactions between some transport substrates is invoked. These results suggest that MRP8 participates in physiological processes involving bile acids, conjugated steroids, and cyclic nucleotides and indicate that the pump has complex interactions with its substrates.Investigations of members of the MRP family of ATPbinding cassette transporters have revealed a group of energy-dependent efflux pumps that are able to confer resistance to anticancer agents and transport a striking range of structurally diverse amphipathic anions Haimeur et al., 2004). Despite their conformity with respect to the transport of lipophilic anions, there are differences in the substrate ranges and functions of these pumps. MRP1 is a ubiquitous efflux pump for glutathione and glucuronate conjugates and plays a specific role in the extrusion of leukotriene C4 (LTC 4 ) from mast cells (Leier et al., 1994;Jedlitschky et al., 1996;Loe et al., 1996). The substrate range of MRP2 is similar to that of MRP1, but the former pump, by virtue of its expression in canalicular (apical) membranes of hepatocytes, is responsible for the extrusion of glutathione, bilirubin glucuronide, and a variety of pharmaceutical agents into the bile (Ito et al

Recently, the nuclear protein known as Poly (ADP-ribose) Polymerase1 (PARP1) was shown to play a key role in regulating transcription of a number of genes and controlling the nuclear sub-organelle nucleolus. PARP1 enzyme is known to catalyze the transfer of ADP-ribose to a variety of nuclear proteins. At present, however, while we do know that the main acceptor for pADPr in vivo is PARP1 protein itself, by PARP1 automodification, the significance of PARP1 automodification for in vivo processes is not clear. Therefore, we investigated the roles of PARP1 auto ADP-ribosylation in dynamic nuclear processes during development. Specifically, we discovered that PARP1 automodification is required for shuttling key proteins into Cajal body (CB) by protein non-covalent interaction with pADPr in vivo. We hypothesize that PARP1 protein shuttling follows a chain of events whereby, first, most unmodified PARP1 protein molecules bind to chromatin and accumulate in nucleoli, but then, second, upon automodification with poly(ADP-ribose), PARP1 interacts non-covalently with a number of nuclear proteins such that the resulting protein-pADPr complex dissociates from chromatin into CB.

According to the histone code hypothesis, histone variants and modified histones provide binding sites for proteins that change the chromatin state to either active or repressed. Here, we identify histone variants that regulate the targeting and enzymatic activity of poly(ADP-ribose) polymerase 1 (PARP1), a chromatin regulator in higher eukaryotes. We demonstrate that PARP1 is targeted to chromatin by association with the histone H2A variant (H2Av)-the Drosophila homolog of the mammalian histone H2A variants H2Az/H2Ax-and that subsequent phosphorylation of H2Av leads to PARP1 activation. This two-step mechanism of PARP1 activation controls transcription at specific loci in a signal-dependent manner. Our study establishes the mechanism through which histone variants and changes in the histone modification code control chromatin-directed PARP1 activity and the transcriptional activation of target genes.poly(ADP-ribosyl)ation | poly(ADP-ribose) glycohydrolase | nucleosome | Hsp70

Poly(ADP ribose) polymerase 1 (PARP1) is a nuclear protein that regulates chromatin remodeling and transcription as well as DNA repair and genome stability pathways. Recent studies have revealed a paradoxical dual role of PARP1 protein in transcription. Specifically, although PARP1 controls transcriptional activation of a subset of genes that are heat shock-or hormone-dependent, it also directly inactivates transcription, establishes heterochromatin domains, and silences retrotransposable elements. However, the domains required for these disparate functions are currently unknown. In this paper, we report the discovery of a previously undescribed mutation in the Drosophila Parp locus. We show that the mutants express a deletion mutant of PARP1 protein with an altered DNA binding domain that carries only the second Znfinger. We demonstrate that this alteration specifically excludes PARP1 protein from heterochromatin and makes PARP1 unable to maintain repression of retrotransposable elements. By characterizing the biological activity of this unique PARP1 mutant protein isoform, we have uncoupled the transactivation and transrepression functions of this protein.chromatin | poly(ADP ribose) polymerase | transcription P oly(ADP ribose) polymerase 1 (PARP1) protein has been known for decades as a nuclear protein that recognizes and binds nicks and ends of DNA and catalyses poly(ADP ribose) (pADPr) synthesis (1). The basic enzymatic reactions catalyzed by PARP1 involve transferring ADPr from nicotinamide-adenine dinucleotide to either a protein acceptor or an existing pADPr chain, the average length of which is 80 or more residues (2). PARP1 protein can modify numerous chromatin proteins in vivo and in vitro (3). A key role of PARP1 was shown in DNA repair and apoptosis (3), where PARP works as a trigger between the DNA repair (4) and apoptotic pathways (5). PARP1 enzymatic activity has also been shown to be required for normal assembly of higher order chromatin structures and for transcriptional activation (6). Moreover, it has been shown that PARP1 regulates the transcription of these genes by inducing chromatin loosening at targeted genetic loci (6, 7). Finally, PARP1 establishes silent chromatin domains and represses retrotransposable elements (8).The characterization of deletion mutants of PARP that distinguish among the varied functions of this protein is essential to establish a more complete understanding of PARP1 protein biology. At present, however, we have identified neither the mechanism of PARP protein targeting to specific chromatin domains nor the mechanism of local PARP activation. Closing these gaps in our current knowledge is complicated because the presence of 18 paralogous PARP proteins (9) in mammals most likely results in corresponding functional redundancies. The Drosophila genome (8, 10, 11) encodes only a single nuclear PARP (PARP1), making this animal an invaluable model system for the study of PARP functions.The PARP1 protein has three functionally defined domains conserved from human to Droso...

The poly (ADP-ribose) polymerase 1 (PARP1) enzyme is one of the promising molecular targets for the discovery of antitumor drugs. PARP1 is a common nuclear protein (1–2 million molecules per cell) serving as a “sensor” for DNA strand breaks. Increased PARP1 expression is sometimes observed in melanomas, breast cancer, lung cancer, and other neoplastic diseases. The PARP1 expression level is a prognostic indicator and is associated with a poor survival prognosis. There is evidence that high PARP1 expression and treatment-resistance of tumors are correlated. PARP1 inhibitors are promising antitumor agents, since they act as chemo- and radiosensitizers in the conventional therapy of malignant tumors. Furthermore, PARP1 inhibitors can be used as independent, effective drugs against tumors with broken DNA repair mechanisms. Currently, third-generation PARP1 inhibitors are being developed, many of which are undergoing Phase II clinical trials. In this review, we focus on the properties and features of the PARP1 inhibitors identified in preclinical and clinical trials. We also describe some problems associated with the application of PARP1 inhibitors. The possibility of developing new PARP1 inhibitors aimed at DNA binding and transcriptional activity rather than the catalytic domain of the protein is discussed.