Takeshi Uchiumi

Genomic Structure of the Canalicular Multispecific Organic Anion–Transporter Gene (MRP2/cMOAT) and Mutations in the ATP-Binding–Cassette Region in Dubin-Johnson Syndrome

Summary Dubin-Johnson syndrome (DJS) is an autosomal recessive disease characterized by conjugated hyperbilirubinemia. Previous studies of the defects in the human canalicular multispecific organic anion transporter gene ( MRP2/cMOAT ) in patients with DJS have suggested that the gene defects are responsible for DJS. In this study, we determined the exon/intron structure of the human MRP2/cMOAT gene and further characterized mutations in patients with DJS. The human MRP2/cMOAT gene contains 32 exons, and it has a structure that is highly conserved with that of another ATP–binding–cassette gene, that for a multidrug resistance–associated protein. We then identified three mutations, including two novel ones. All mutations identified to date are in the cytoplasmic domain, which includes the two ATP-binding cassettes and the linker region, or adjacent putative transmembrane domain. Our results confirm that MRP2/cMOAT is the gene responsible for DJS. The finding that mutations are concentrated in the first ATP-binding–cassette domain strongly suggests that a disruption of this region is a critical route to loss of function.

Nuclear translocation of the Y‐box binding protein by ultraviolet irradiation

The Y‐box binding protein, YB‐1, is a member of a DNA binding protein family with a structurally and functionally conserved cold shock domain. Using Western blotting and immunohistochemical methods, larger amounts of YB‐1 were detected in the cytosol, particularly at the perinuclear region, than in the nucleus of human cancer cells. UV irradiation increased accumulation of YB‐1 in the nucleus at 20 min and thereafter. This translocation of YB‐1 into the nucleus by UV irradiation was blocked by the protein kinase inhibitor H‐7, but not HA‐1004. Both green fluorescent protein (GFP)‐YB‐1 and GFP‐YB‐1C with the C‐terminus (248–317) of YB‐1 were located mainly in the cytosol, but GFP‐YB‐1ΔC with a deletion at the C‐terminus of YB‐1 was located in the nucleus. YB‐1 is translocated into the nucleus by UV irradiation, possibly through a protein kinase C‐mediated signal transduction pathway, and the C‐terminal region of YB‐1 might be important for cytoplasmic retention of YB‐1.

Enhanced transport of anticancer agents and leukotriene C4 by the human canalicular multispecific organic anion transporter (cMOAT/MRP2)

We established stable human canalicular multispecific organic anion transporter (cMOAT/MRP2) cDNA transfectants, CHO/cMOAT from non‐polarized Chinese hamster ovary (CHO)‐K1 and LLC/cMOAT from polarized pig kidney epithelial LLC‐PK1. Human cMOAT was mainly localized in the plasma membrane of CHO/cMOAT and in the apical membrane of LLC/cMOAT. The ATP‐dependent uptake of leukotriene C4 (LTC4) into CHO/cMOAT membrane vesicles was enhanced compared with empty vector transfectants. K m values in CHO/cMOAT membrane vesicles were 0.24 μM for LTC4 and 175 μM for ATP. Drug sensitivity to vincristine and cisplatin in human cMOAT cDNA transfectants decreased, but not to etoposide. Cellular accumulation of vincristine and cisplatin in human cMOAT cDNA transfectants decreased, but not of etoposide. The uptake of LTC4 into CHO/cMOAT membrane vesicles was inhibited by exogenous administration of vincristine or cisplatin, but not that of etoposide. Moreover, this inhibition was more enhanced in the presence of glutathione. These consequences indicate that drug resistance to vincristine or cisplatin appears to be modulated by human cMOAT through transport of the agents, possibly in direct or indirect association with glutathione.

Mitophagy Plays an Essential Role in Reducing Mitochondrial Production of Reactive Oxygen Species and Mutation of Mitochondrial DNA by Maintaining Mitochondrial Quantity and Quality in Yeast

Background: The physiological importance of mitophagy in yeast has been largely unexplored. Results: Mitochondrial DNA deletion frequently occurs in mitophagy-deficient cells during nitrogen starvation because of overproduction of the reactive oxygen species from unregulated mitochondria. Conclusion: Mitophagy prevents excess reactive oxygen species production and mitochondrial DNA mutation. Significance: Our findings provide insight into mitophagy-related disorders such as Parkinson disease. In mammalian cells, the autophagy-dependent degradation of mitochondria (mitophagy) is thought to maintain mitochondrial quality by eliminating damaged mitochondria. However, the physiological importance of mitophagy has not been clarified in yeast. Here, we investigated the physiological role of mitophagy in yeast using mitophagy-deficient atg32- or atg11-knock-out cells. When wild-type yeast cells in respiratory growth encounter nitrogen starvation, mitophagy is initiated, excess mitochondria are degraded, and reactive oxygen species (ROS) production from mitochondria is suppressed; as a result, the mitochondria escape oxidative damage. On the other hand, in nitrogen-starved mitophagy-deficient yeast, excess mitochondria are not degraded and the undegraded mitochondria spontaneously age and produce surplus ROS. The surplus ROS damage the mitochondria themselves and the damaged mitochondria produce more ROS in a vicious circle, ultimately leading to mitochondrial DNA deletion and the so-called “petite-mutant” phenotype. Cells strictly regulate mitochondrial quantity and quality because mitochondria produce both necessary energy and harmful ROS. Mitophagy contributes to this process by eliminating the mitochondria to a basal level to fulfill cellular energy requirements and preventing excess ROS production.

Increased Expression of an ATP-binding cassette Superfamily Transporter,Multidrug Resistance Protein 2,in Human Colorectal Carcinomas

The expression of ATP-binding cassette superfamily transporter genes, such as P-glycoprotein/multidrug resistance (MDR) 1 and MDR protein (MRP) 1, is often up-regulated in various tumor types and is involved in responses to some anticancer chemotherapeutic agents. Five human MRP subfamily members (MRP2-6) with structural similarities to MRP1 have been identified. The relationships between MRP2-6 mRNA levels and drug resistance are not well understood. Data on 45 patients with colorectal cancer were analyzed. Of the ATP-binding cassette superfamily genes, we asked whether mRNA levels of MDR1, MRP1, MRP2, and MRP3 correlated with drug resistance to anticancer agents. For this analysis, we used quantitative reverse transcription-PCR, and the sensitivity to anticancer agents in surgically resected colon carcinomas was determined using the in vitro succinate dehydrogenase inhibition test. MDR1, MRP1, and MRP3 were highly expressed in normal colorectal mucosa, and the relative mRNA levels of MDR1, MRP1, and MRP3 in cancerous tissues compared with noncancerous tissues were decreased or unchanged. By contrast, MRP2 mRNA expression was low in normal colorectal mucosa and specifically increased in cancer regions compared with noncancerous regions. Of the anticancer agents prescribed for patients with colorectal cancers, including doxorubicin, mitomycin C, cisplatin, 5-fluorouracil, etoposide, and a camptothecin derivative, mRNA expression of MRP2 was significantly associated with resistance to cisplatin. MRP2 may be important for resistance to cisplatin treatment in colorectal cancer.

Direct interaction of p53 with the Y-box binding protein, YB-1: a mechanism for regulation of human gene expression

The Y-box binding protein, YB-1, belongs to a family of multifunctional proteins which regulate gene expression on both transcriptional and translational levels. The tumor suppressor gene p53 displays growth suppressive properties by regulating gene expression through transcriptional regulation. We now demonstrate that YB-1 directly interacts with p53 using an in vitro pull-down assay. Using immunochemical co-precipitation methods, we also found that the two proteins are bound in vivo. Deletion analysis showed that three independent domains of YB-1, one at the N-terminal and two at the C-terminal, interact with p53. Conversely, a 14 amino acid sequence at the C-terminal of p53 was required for its interaction with YB-1. Gel mobility shift assays showed that the interaction of YB-1 with p53 stimulated the sequence-specific DNA binding of p53 to its consensus sequence. By contrast, this interaction inhibited the binding of YB-1. Using a p53-responsive p21 promoter linked to a reporter gene, it can be shown that antisense expression of YB-1 inhibits the induction of this promoter by p53 in transient transfection assays. These findings delineate a straightforward mechanism for gene expression through p53-YB-1 interaction.

Human placental transport of vinblastine, vincristine, digoxin and progesterone: contribution of P-glycoprotein.

To elucidate the role of P-glycoprotein in human placenta, we examined its expression in placenta, and the transcellular transport and uptake of P-glycoprotein substrates in cultured human placental choriocarcinoma epithelial cells (BeWo cells). The uptake of [(3)H]vinblastine and [(3)H]vincristine into BeWo cells was increased in the presence of a metabolic inhibitor, sodium azide. The basolateral-to-apical transcellular transport of [(3)H]vinblastine, [(3)H]vincristine and [(3)H]digoxin was greater than the apical-to-basolateral transcellular transport. In the presence of cyclosporin A, the basolateral-to-apical transcellular transport of [(3)H]vinblastine, [(3)H]vincristine and [(3)H]digoxin was significantly increased, and the apical-to-basolateral transcellular transport was decreased. The uptake of [(3)H]vinblastine, [(3)H]vincristine and [(3)H]digoxin into BeWo cells was significantly enhanced in the presence of several inhibitors, such as verapamil or mouse monoclonal antibody anti-P-glycoprotein MX-MDR (MRK16) as well as cyclosporin A. Although progesterone significantly enhanced the uptake of [(3)H]vinblastine, [(3)H]vincristine and [(3)H]digoxin into BeWo cells, the uptake of [(3)H]progesterone was not affected by these inhibitors. Immunoblot analysis revealed that P-glycoprotein with a molecular weight of 172 kDa was expressed in BeWo cells and isolated trophoblast cells. Furthermore, P-glycoprotein was detected in human placental brush-border membrane vesicles, but not in human placental basolateral membrane vesicles. In conclusion, these data suggest that P-glycoprotein is expressed on the brush-border membrane (maternal side) of human placental trophoblast cells. P-Glycoprotein is considered to regulate the transfer of several substances including vinblastine, vincristine and digoxin from mother to fetus, and to protect the fetus from toxic substances.

Effects of grapefruit juice and orange juice components on P‐glycoprotein‐ and MRP2‐mediated drug efflux

We investigated the effects of grapefruit juice (GFJ) and orange juice (OJ) on drug transport by MDR1 P‐glycoprotein (P‐gp) and multidrug resistance protein 2 (MRP2), which are efflux transporters expressed in human small intestine. We examined the transcellular transport and uptake of [3H]vinblastine (VBL) and [14C]saquinavir in a human colon carcinoma cell line (Caco‐2) and in porcine kidney epithelial cell lines transfected with human MDR1 cDNA and human MRP2 cDNA, LLC‐GA5‐COL150, and LLC‐MRP2, respectively. In Caco‐2 cells, the basal‐to‐apical transports of [3H]VBL and [14C]saquinavir were greater than those in the opposite direction. The ratio of basal‐to‐apical transport to apical‐to‐basal transport of [3H]VBL and [14C]saquinavir by Caco‐2 cells was reduced in the presence of MK571 (MRPs inhibitor), verapamil (P‐gp inhibitor), cyclosporin A (inhibitor of both), 50% ethyl acetate extracts of GFJ and OJ, or their components (6′,7′‐dihydroxybergamottin, bergamottin, tangeretin, hepatomethoxyflavone, and nobiletin). Studies of transport and uptake of [3H]VBL and [14C]saquinavir with MDR1 and MRP2 transfectants showed that VBL and saquinavir are transported by both P‐gp and MRP2. GFJ and OJ components inhibited the transport by MRP2 as well as P‐gp. However, their inhibitory potencies for P‐gp or MRP2 were substrate‐dependent. The present study has revealed that GFJ and OJ interact with not only P‐gp but also MRP2, both of which are expressed at apical membranes and limit the apical‐to‐basal transport of VBL and saquinavir in Caco‐2 cells.

Phosphorylation of Serine 114 on Atg32 mediates mitophagy

Mitophagy, which selectively degrades mitochondria via autophagy, has a significant role in mitochondrial quality control. When autophagy selects mitochondria as a cargo, Atg32 is bound by Atg11. It is shown that the phosphorylation of Atg32, especially phosphorylation of Ser-114 on Atg32, mediates the Atg11–Atg32 interaction and mitophagy.

Basal Membrane Localization of MRP1 in Human Placental Trophoblast

The placental trophoblast is considered to act as a barrier between mother and fetus, mediating the exchange of various materials across the placenta. ATP-binding cassette (ABC) transporters such as P-glycoprotein (P-gp) and multidrug-resistance protein (MRP) are expressed in the placenta and function as efflux transport systems for xenobiotics. In the present study, we aimed to determine the localization of MRP1 in the human placenta in comparison with that of P-gp. Western blotting analysis with human placental membrane vesicles indicated that P-gp and MRP1 are localized on the brush-border membranes and basal membranes, respectively. Immunohistochemical analysis with human normal full-term placenta showed that anti-P-gp monoclonal antibody F4 stained the brush-border side of the trophoblast cells, whereas anti-MRP1 monoclonal antibody MRPr1 stained the basal side. These results confirm that P-gp and MRP1 are located on the brush-border membranes and basal membranes, respectively, of human full-term placental trophoblast. MRP1 was also detected on the abluminal side of blood vessels in the villi. Accordingly, MRP1 may play a role distinct from that of P-gp, which is considered to restrict the influx of xenobiotics into the fetus.