Takaaki Ono

Clinicopathological and prognostic characteristics of CD56‐negative multiple myeloma

Summary. We analysed CD56 expression in 70 patients with multiple myeloma (MM) to determine its clinicopathological and prognostic significance. Fifty‐five (79%) patients were CD56+. CD56– patients (n = 15) had higher β2 microglobulin levels and a higher incidence of extramedullary disease, Bence Jones protein, renal insufficiency and thrombocytopenia than CD56+ patients. Their myelomas more frequently had a plasmablastic morphology. Overall survival was significantly lower in CD56– than CD56+ patients (22 vs 63 months, P = 0·0002). We conclude that CD56– MM is a discrete entity associated with more aggressive disease. The higher incidence of plasmablastic cases suggested that CD56– MM may develop from a less mature plasma cell than CD56+ MM.

Light-Intensity-Dependent Expression of Lhc Gene Family Encoding Light-Harvesting Chlorophyll-a/b Proteins of Photosystem II in Chlamydomonas reinhardtii

Excessive light conditions repressed the levels of mRNAs accumulation of multiple Lhc genes encoding light-harvesting chlorophyll-a/b (LHC) proteins of photosystem (PS)II in the unicellular green alga, Chlamydomonas reinhardtii. The light intensity required for the repression tended to decrease with lowering temperature or CO2concentration. The responses of six LhcII genes encoding the major LHC (LHCII) proteins and two genes (Lhcb4 andLhcb5) encoding the minor LHC proteins of PSII (CP29 and CP26) were similar. The results indicate that the expression of theseLhc genes is coordinately repressed when the energy input through the antenna systems exceeds the requirement for CO2 assimilation. The Lhc mRNA level repressed under high-light conditions was partially recovered by adding the electron transport inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea, suggesting that redox signaling via photosynthetic electron carriers is involved in the gene regulation. However, the mRNA level was still considerably lower under high-light than under low-light conditions even in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Repression of theLhc genes by high light was prominent even in the mutants deficient in the reaction center(s) of PSII or both PSI and PSII. The results indicate that two alternative processes are involved in the repression of Lhc genes under high-light conditions, one of which is independent of the photosynthetic reaction centers and electron transport events.

The FOXM1 transcriptional factor promotes the proliferation of leukemia cells through modulation of cell cycle progression in acute myeloid leukemia

FOXM1 is an important cell cycle regulator and regulates cell proliferation. In addition, FOXM1 has been reported to contribute to oncogenesis in various cancers. However, it is not clearly understood how FOXM1 contributes to acute myeloid leukemia (AML) cell proliferation. In this study, we investigated the cellular and molecular function of FOXM1 in AML cells. The FOXM1 messenger RNA (mRNA) expressed in AML cell lines was predominantly the FOXM1B isoform, and its levels were significantly higher than in normal high aldehyde dehydrogenase activity (ALDH(hi)) cells. Reduction of FOXM1 expression in AML cells inhibited cell proliferation compared with control cells, through induction of G(2)/M cell cycle arrest, a decrease in the protein expression of Aurora kinase B, Survivin, Cyclin B1, S-phase kinase-associated protein 2 and Cdc25B and an increase in the protein expression of p21(Cip1) and p27(Kip1). FOXM1 messenger RNA (mRNA) was overexpressed in all 127 AML clinical specimens tested (n = 21, 56, 32 and 18 for M1, M2, M4 and M5 subtypes, respectively). Compared with normal ALDH(hi) cells, FOXM1 gene expression was 1.65- to 2.26-fold higher in AML cells. Moreover, the FOXM1 protein was more strongly expressed in AML-derived ALDH(hi) cells compared with normal ALDH(hi) cells. In addition, depletion of FOXM1 reduced colony formation of AML-derived ALDH(hi) cells due to inhibition of Cdc25B and Cyclin B1 expression. In summary, we found that FOXM1B mRNA is predominantly expressed in AML cells and that aberrant expression of FOXM1 induces AML cell proliferation through modulation of cell cycle progression. Thus, inhibition of FOXM1 expression represents an attractive target for AML therapy.

Spin-exchange interactions in the S2-state manganese tetramer in photosynthetic oxygen-evolving complex deduced from g=2 multiline EPR signal

Abstract Possible spin-exchange structures of the Mn(III,IV,IV,IV) cluster in an S 2 state of plant photosystem II were computer-searched, within the range compatible with X-ray absorption data, by diagonalizing each Heisenberg spin-exchange Hamiltonian and then by checking whether it can take the S =1/2 ground state capable of explaining the effective 55 Mn hyperfine constants determined from oriented multiline spectra and the first excited state with excitation energy around 20–50 cm −1 , or not. The possible spin-exchange structures were found to be distributed in those that contain only one strong-antiferromagnetic coupling and at most two intermediate coupling(s). The most probable structures are discussed in detail.

CMC-544 (inotuzumab ozogamicin) shows less effect on multidrug resistant cells: analyses in cell lines and cells from patients with B-cell chronic lymphocytic leukaemia and lymphoma

The effect of CMC‐544, a calicheamicin‐conjugated anti‐CD22 monoclonal antibody, was analysed in relation to CD22 and P‐glycoprotein (P‐gp) in B‐cell chronic lymphocytic leukaemia (CLL) and non‐Hodgkin lymphoma (NHL) in vitro. The cell lines used were CD22‐positive parental Daudi and Raji, and their P‐gp positive sublines, Daudi/MDR and Raji/MDR. Cells obtained from 19 patients with B‐cell CLL or NHL were also used. The effect of CMC‐544 was analysed by viable cell count, morphology, annexin‐V staining, and cell cycle distribution. A dose‐dependent, selective cytotoxic effect of CMC‐544 was observed in cell lines that expressed CD22. CMC‐544 was not effective on Daudi/MDR and Raji/MDR cells compared with their parental cells. The MDR modifiers, PSC833 and MS209, restored the cytotoxic effect of CMC‐544 in P‐gp‐expressing sublines. In clinical samples, the cytotoxic effect of CMC‐544 was inversely related to the amount of P‐gp (P = 0·003), and to intracellular rhodamine‐123 accumulation (P < 0·001). On the other hand, the effect positively correlated with the amount of CD22 (P = 0·010). The effect of CMC‐544 depends on the levels of CD22 and P‐gp. Our findings will help to predict the clinical effectiveness of this drug on these B‐cell malignancies, suggesting a beneficial effect with combined use of CMC‐544 and MDR modifiers.

Ca(2+) function in photosynthetic oxygen evolution studied by alkali metal cations substitution.

Effects of adding monovalent alkali metal cations to Ca(2+)-depleted photosystem (PS)II membranes on the biochemical and spectroscopic properties of the oxygen-evolving complex were studied. The Ca(2+)-dependent oxygen evolution was competitively inhibited by K(+), Rb(+), and Cs(+), the ionic radii of which are larger than the radius of Ca(2+) but not inhibited significantly by Li(+) and Na(+), the ionic radii of which are smaller than that of Ca(2+). Ca(2+)-depleted membranes without metal cation supplementation showed normal S(2) multiline electron paramagnetic resonance (EPR) signal and an S(2)Q(A)(-) thermoluminescence (TL) band with a normal peak temperature after illumination under conditions for single turnover of PSII. Membranes supplemented with Li(+) or Na(+) showed properties similar to those of the Ca(2+)-depleted membranes, except for a small difference in the TL peak temperatures. The peak temperature of the TL band of membranes supplemented with K(+), Rb(+), or Cs(+) was elevated to approximately 38 degrees C which coincided with that of Y(D)(+)Q(A)(-) TL band, and no S(2) EPR signals were detected. The K(+)-induced high-temperature TL band and the S(2)Q(A)(-) TL band were interconvertible by the addition of K(+) or Ca(2+) in the dark. Both the Ca(2+)-depleted and the K(+)-substituted membranes showed the narrow EPR signal corresponding to the S(2)Y(Z)(+) state at g = 2 by illuminating the membranes under multiple turnover conditions. These results indicate that the ionic radii of the cations occupying Ca(2+)-binding site crucially affect the properties of the manganese cluster.

Changes in Structural and Functional Properties of Oxygen-evolving Complex Induced by Replacement of D1-Glutamate 189 with Glutamine in Photosystem II LIGATION OF GLUTAMATE 189 CARBOXYLATE TO THE MANGANESE CLUSTER

A carboxylate group of D1-Glu-189 in photosystem II has been proposed to serve as a direct ligand for the manganese cluster. Here we constructed a mutant that eliminates the carboxylate by replacing D1-Glu-189 with Gln in the cyanobacterium Synechocystis sp. PCC 6803, and we examined the resulting effects on the structural and functional properties of the oxygen-evolving complex (OEC) in photosystem II. The E189Q mutant grew photoautotrophically, and isolated photosystem II core particles evolved oxygen at ∼70% of the rate of control wild-type particles. The E189Q OEC showed typical S2 state electron spin resonance signals, and the spin center distance between the S2 state manganese cluster and the YD (D2-Tyr-160), detected by electron-electron double resonance spectroscopy, was not affected by this mutation. However, the redox potential of the E189Q OEC was considerably lower than that of the control OEC, as revealed by the elevated peak temperature of the S2 state thermoluminescence bands. The mutation resulted in specific changes to bands ascribed to the putative carboxylate ligands for the manganese cluster and to a few carbonyl bands in mid-frequency (1800 to 1100 cm-1) S2/S1 Fourier transform infrared difference spectrum. Notably, the low frequency (650 to 350 cm-1) S2/S1 Fourier transform infrared difference spectrum was also uniquely changed by this mutation in the frequencies for the manganese cluster core vibrations. These results suggested that the carboxylate group of D1-Glu-189 ligates the manganese ion, which is influenced by the redox change of the oxidizable manganese ion upon the S1 to S2 transition.

Oxidation of the Mn Cluster Induces Structural Changes of NO3− Functionally Bound to the Cl− Site in the Oxygen-Evolving Complex of Photosystem II

Cl(-) is an indispensable cofactor for photosynthetic O(2) evolution and is functionally replaced by NO(3)(-). Structural changes of an isotopically labeled NO(3)(-) ion, induced by the oxidation of the Mn cluster (S(1)-to-S(2)), were detected by FTIR spectroscopy. NO(3)(-)-substituted photosystem II core particles showed (14)N(16)O(3)(-)/(15)N(16)O(3)(-) and (14)N(16)O(3)(-)/(14)N(18)O(3)(-) isotopic bands in the S(2)/S(1) spectra with markedly high signal/noise ratio. These bands appeared only in the region from 1415 to 1284 cm(-1), indicating that the bands do not arise from a metal-bound NO(3)(-) but from an ionic NO(3)(-). The intensity of the bands exhibited a quantitatively proportional relationship with the O(2) activity. These results demonstrate that the NO(3)(-) functionally bound to the Cl(-) site couples to the Mn cluster structurally, but is not associated with the cluster as a direct ligand. Comparison of the bands for two isotopes ((15)N and (18)O) and their simulations enable us to assign each band to the S(1) and S(2) states. The results indicate that the NO(3)(-) ion bound to the Cl(-) site is highly asymmetric in S(1) but rather symmetric in S(2). Since NO(3)(-) functionally replaces Cl(-), most of the conclusions drawn from this study will be also applicable to Cl(-).

CMC-544 (inotuzumab ozogamicin), an anti-CD22 immuno-conjugate of calicheamicin, alters the levels of target molecules of malignant B-cells.

We studied the effect of CMC-544, the calicheamicin-conjugated anti-CD22 monoclonal antibody, used alone and in combination with rituximab, analyzing the quantitative alteration of target molecules, that is, CD20, CD22, CD55 and CD59, in Daudi and Raji cells as well as in cells obtained from patients with B-cell malignancies (BCM). Antibody inducing direct antiproliferative and apoptotic effect, complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) were tested separately. In Daudi and Raji cells, the CDC effect of rituximab significantly increased within 12 h following incubation with CMC-544. The levels of CD22 and CD55 were significantly reduced (P<0.001 in both cells) after incubation with CMC-544, but CD20 level remained constant or increased for 12 h. Similar results were obtained in cells from 12 patients with BCM. The antiproliferative and apoptotic effect of CMC-544 were greater than that of rituximab. The ADCC of rituximab was not enhanced by CMC-544. Thus, the combination of CMC-544 and rituximab increased the in vitro cytotoxic effect in BCM cells, and sequential administration for 12 h proceeded by CMC-544 was more effective. The reduction of CD55 and the preservation of CD20 after incubation with CMC-544 support the rationale for the combined use of CMC-544 and rituximab.

Depletion of Pleckstrin Homology Domain Leucine-rich Repeat Protein Phosphatases 1 and 2 by Bcr-Abl Promotes Chronic Myelogenous Leukemia Cell Proliferation through Continuous Phosphorylation of Akt Isoforms

The constitutive activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway commonly occurs in cancers and is a crucial event in tumorigenesis. Chronic myelogenous leukemia (CML) is characterized by a reciprocal chromosomal translocation (9;22) that generates the Bcr-Abl fusion gene. The PI3K/Akt pathway is activated by Bcr-Abl chimera protein and mediates the leukemogenesis in CML. However, the mechanism by which Bcr-Abl activates the PI3K/Akt pathway is not completely understood. In the present study, we found that pleckstrin homology domain leucine-rich repeat protein phosphatases 1 and 2 (PHLPP1 and PHLPP2) were depleted in CML cells. We investigated the interaction between PHLPPs and Bcr-Abl in CML cell lines and Bcr-Abl+ progenitor cells from CML patients. The Abl kinase inhibitors and depletion of Bcr-Abl induced the expression of PHLPP1 and PHLPP2, which dephosphorylated Ser-473 on Akt1, -2, and -3, resulting in inhibited proliferation of CML cells. The reduction of PHLPP1 and PHLPP2 expression by short interfering RNA in CML cells weakened the Abl kinase inhibitor-mediated inhibition of proliferation. In colony-forming unit-granulocyte, erythroid, macrophage, megakaryocyte; colony-forming unit-granulocyte, macrophage; and burst-forming unit-erythroid, treatment with the Abl kinase inhibitors and depletion of Bcr-Abl induced PHLPP1 and PHLPP2 expression and inhibited colony formation of Bcr-Abl+ progenitor cells, whereas depletion of PHLPP1 and PHLPP2 weakened the inhibition of colony formation activity by the Abl kinase inhibitors in Bcr-Abl+ progenitor cells. Thus, Bcr-Abl represses the expression of PHLPP1 and PHLPP2 and continuously activates Akt1, -2, and -3 via phosphorylation on Ser-473, resulting in the proliferation of CML cells.