Genetic and chemical targeting of the ATPase complex TIP48 and 49 impairs acute myeloid leukemia.

The chromatin-associated AAA+ ATPases Tip48 and Tip49 are the core components of various complexes implicated in diverse nuclear events such as DNA repair and gene regulation. Although they are frequently overexpressed in many human cancers, their functional significance remains unclear. Here, we show that loss of Tip49 triggered p53-dependent apoptosis and inhibited leukemia development in vivo. To examine the impact of chemical inhibition of this complex on leukemia, we have developed the novel compound DS-4950, which interferes with the ATPase activity of the Tip48/49. Administration of DS-4950 was well-tolerated in healthy mice, and the drug effectively reduced tumor burden and improved survival. We also provide evidence that the dependency on Tip48/49 is widely conserved in non-hematologic malignancies with wild type p53. These results demonstrated that the Tip48/49 ATPases are functionally necessary and therapeutically targetable for the treatment of human cancers.

Ring1A and Ring1B inhibit expression of Glis2 to maintain murine MOZ-TIF2 AML stem cells.

Eradication of chemotherapy-resistant leukemia stem cells is expected to improve treatment outcomes in patients with acute myelogenous leukemia (AML). In a mouse model of AML expressing the MOZ-TIF2 fusion, we found that Ring1A and Ring1B, components of Polycomb repressive complex 1, play crucial roles in maintaining AML stem cells. Deletion of Ring1A and Ring1B (Ring1A/B) from MOZ-TIF2 AML cells diminished self-renewal capacity and induced the expression of numerous genes, including Glis2 Overexpression of Glis2 caused MOZ-TIF2 AML cells to differentiate into mature cells, whereas Glis2 knockdown in Ring1A/B-deficient MOZ-TIF2 cells inhibited differentiation. Thus, Ring1A/B regulate and maintain AML stem cells in part by repressing Glis2 expression, which promotes their differentiation. These findings provide new insights into the mechanism of AML stem cell homeostasis and reveal novel targets for cancer stem cell therapy.

The roles of Polycomb group proteins in hematopoietic stem cells and hematological malignancies.

Polycomb group (PcG) proteins are epigenetic regulatory factors that maintain the repression of target gene expression through histone modification. PcG proteins control the repression of genes that regulate differentiation and the cell cycle in the maintenance of hematopoietic stem cells (HSC). Moreover, abnormalities in expression level and mutations in PcG genes have been reported in various types of cancer, including hematological malignancies. In this review, we present an overview of the roles of PcG proteins in HSC and various types of hematological malignancies.

The increased expression of integrin α6 (ITGA6) enhances drug resistance in EVI1(high) leukemia.

Ecotropic viral integration site-1 (EVI1) is one of the candidate oncogenes for human acute myeloid leukemia (AML) with chromosomal alterations at 3q26. High EVI1 expression (EVI1(high)) is a risk factor for AML with poor outcome. Using DNA microarray analysis, we previously identified that integrin α6 (ITGA6) was upregulated over 10-fold in EVI1(high) leukemia cells. In this study, we determined whether the increased expression of ITGA6 is associated with drug-resistance and increased cell adhesion, resulting in poor prognosis. To this end, we first confirmed the expression pattern of a series of integrin genes using semi-quantitative PCR and fluorescence-activated cell sorter (FACS) analysis and determined the cell adhesion ability in EVI1(high) leukemia cells. We found that the adhesion ability of EVI1(high) leukemia cells to laminin increased with the increased expression of ITGA6 and integrin ß4 (ITGB4). The introduction of small-hairpin RNA against EVI1 (shEVI1) into EVI1(high) leukemia cells reduced the cell adhesion ability and downregulated the expression of ITGA6 and ITGB4. In addition, the overexpression of EVI1 in EVI1(low) leukemia cells enhanced their cell adhesion ability and increased the expression of ITGA6 and ITGB4. In a subsequent experiment, the introduction of shRNA against ITGA6 or ITGB4 into EVI1(high) AML cells downregulated their cell adhesion ability; however, the EVI1(high) AML cells transfected with shRNA against ITGA6 could not be maintained in culture. Moreover, treating EVI1(high) leukemia cells with neutralizing antibodies against ITGA6 or ITGB4 resulted in an enhanced responsiveness to anti-cancer drugs and a reduction of their cell adhesion ability. The expression of ITGA6 is significantly elevated in cells from relapsed and EVI1(high) AML cases; therefore, ITGA6 might represent an important therapeutic target for both refractory and EVI1(high) AML.

Angiopoietin1 contributes to the maintenance of cell quiescence in EVI1(high) leukemia cells.

Ecotropic viral integration site-1 (EVI1) is an oncogenic transcription factor in human acute myeloid leukemia (AML) associated with poor prognosis. Because the drug-resistance of leukemia cells is partly dependent on cell quiescence in the bone marrow niche, EVI1 may be involved in cell cycle regulation in leukemia cells. As a candidate regulator of the cell cycle in leukemia cells with high EVI1 expression (EVI1(high)), we analyzed angiopoietin1 (Ang1), which is a down-regulated gene in EVI1-deficient mice and is involved in the quiescence of hematopoietic stem cells. The results of real-time PCR analyses showed that Ang1 is highly expressed in leukemia cell lines and primary AML cells with EVI1(high) expression. Introduction of shRNA against EVI1 into EVI1(high) leukemia cells down-regulated Ang1 expression. Moreover, knockdown of Ang1 in EVI1(high) leukemia cells promoted cell cycle progression and down-regulated the CDK inhibitor p18 (INK4c). Treatment with a decoy Tie2/Fc protein also down-regulated the expression of p18. These results suggest that Ang1/Tie2 signaling may suppress cell cycle progression via maintenance of G0/G1 phase through up-regulation of p18 expression. This mechanism may help to maintain EVI1(high) leukemia cells in the bone marrow niche and promote resistance to anti-cancer drugs.

Inhibition of carrier-mediated uptake of epirubicin reduces cytotoxicity in primary culture of rat hepatocytes.

Epirubicin, an antineoplastic drug, is considered to be taken up by tumor cells via a common carrier by facilitated diffusion and is then pumped out in an energy-dependent manner because epirubicin is a substrate for P-glycoprotein (P-gp). However, this study investigated the details of the influx mechanism of epirubicin and demonstrated that epirubicin uptake was mediated by active carrier systems in addition to facilitated diffusion in the primary culture of rat hepatocytes. The uptake of epirubicin gradually increased in a saturated manner when the concentrations were between 1 x 10(-7) M and 1 x 10(-6) M. In contrast, the uptake increased progressively in a linear manner when the concentration was high (greater than 1 x 10(-6) M). The uptake of epirubicin at a clinical concentration (7.5 x 10(-7) M) was significantly reduced at 4 degrees C and significantly inhibited when pretreated with metabolic inhibitors (carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), rotenone and sodium azide) by nearly 25%. Furthermore, an organic anion transporter inhibitor, namely, 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS); organic anion transport substrates, namely, para-aminohippurate (PAH), taurocholic acid and estradiol 17-beta-D-glucuronide; and organic cation transporter inhibitors, namely, verapamil and tetraethylammonium significantly reduced the uptake of epirubicin. Furthermore, pretreatment with verapamil and PAH significantly prevented epirubicin-induced reduction of proliferative activity in rat hepatocytes. These results indicated that the uptake of epirubicin was induced, at least in part, by the active transport protein in rat hepatocytes; the inhibition of the probable transport protein protected the intact normal cells from the injury induced by the cytotoxicity of epirubicin.

Hepatocyte growth factor increases uptake of estradiol 17beta-D-glucuronide and Oatp1 protein level in rat hepatocytes.

Hepatocyte growth factor (HGF) ameliorates liver injuries in hepatectomized cholestatic rats. On the other hand, the protein level of organic anion-transporting polypeptide (Oatp1), which is responsible for the uptake of bile salts into hepatocytes, decreases in cholestatic humans and rats. However, the relationship between the ameliorative effects of HGF and the decrease in Oatp1 levels in cholestasis remains to be understood. Therefore, in order to investigate this relationship, we evaluated the effects of HGF on the function and protein level of Oatp1. HGF treatment significantly increased the uptake of radiolabeled estradiol 17beta-d-glucuronide ([(3)H]E(2)17betaG), a predominant Oatp1 substrate, in primary cultured rat hepatocytes. Additionally, there was an increase in the Oatp1 protein levels. The increased [(3)H]E(2)17betaG uptake was significantly inhibited by simultaneous incubation with the HGF receptor antibody and treatment with non-radiolabeled E(2)17betaG. However, inhibition by taurocholic acid, a Na(+)-taurocholate co-transporting polypeptide (Ntcp) substrate, was weaker than that caused by non-radiolabeled E(2)17betaG. Further, the increase was not altered by replacing Na(+) in the medium with Li(+). In the inhibition study, the increased [(3)H]E(2)17betaG uptake was inhibited by Oatp1 substrates, including bromosulfophthalein, ochratoxin A, and ouabain, but not by digoxin, which is an Oatp2-specific substrate. Furthermore, HGF did not alter the Oatp1 mRNA expression. In contrast, HGF treatment suppressed the ubiquitination of Oatp1 protein. In conclusion, this is the first report suggesting that HGF regulates Oatp1 protein level and that the ameliorative effects of HGF in cholestasis was induced, at least in part, by correcting the down-regulation of the Oatp1 protein level.

D-3-hydroxybutyrate dehydrogenase from Pseudomonas fragi: molecular cloning of the enzyme gene and crystal structure of the enzyme.

The gene coding for d-3-hydroxybutyrate dehydrogenase (HBDH) was cloned from Pseudomonas fragi. The nucleotide sequence contained a 780 bp open reading frame encoding a 260 amino acid residue protein. The recombinant enzyme was efficiently expressed in Escherichia coli cells harboring pHBDH11 and was purified to homogeneity as judged by SDS-PAGE. The enzyme showed a strict stereospecificity to the D-enantiomer (3R-configuration) of 3-hydroxybutyrate as a substrate. Crystals of the ligand-free HBDH and of the enzyme-NAD+ complex were obtained using the hanging-drop, vapor-diffusion method. The crystal structure of the HBDH was solved by the multiwavelength anomalous diffraction method using the SeMet-substituted enzyme and was refined to 2.0 A resolution. The overall structure of P.fragi HBDH, including the catalytic tetrad of Asn114, Ser142, Tyr155, and Lys159, shows obvious relationships with other members of the short-chain dehydrogenase/reductase (SDR) family. A cacodylate anion was observed in both the ligand-free enzyme and the enzyme-NAD+ complex, and was located near the catalytic tetrad. It was shown that the cacodylate inhibited the NAD+-dependent D-3-hydroxybutyrate dehydrogenation competitively, with a Ki value of 5.6 mM. From the interactions between cacodylate and the enzyme, it is predicted that substrate specificity is achieved through the recognition of the 3-methyl and carboxyl groups of the substrate.

Crystallization and preliminary X-ray characterization of D-3-hydroxybutyrate dehydrogenase from Pseudomonas fragi.

A recombinant form of D-3-hydroxybutyrate dehydrogenase (EC 1.1.1.30) from Pseudomonas fragi has been crystallized by the hanging-drop method using PEG 3000 as a precipitating agent. The crystals belong to the orthorhombic group P2(1)2(1)2, with unit-cell parameters a = 64.3, b = 99.0, c = 110.2 A. The crystals are most likely to contain two tetrameric subunits in the asymmetric unit, with a VM value of 3.29 A3 Da(-1). Diffraction data were collected to a 2.0 A resolution using synchrotron radiation at the BL6A station of the Photon Factory.