Expression of myeloperoxidase enhances the chemosensitivity of leukemia cells through the generation of reactive oxygen species and the nitration of protein.

Myeloperoxidase (MPO), a pivotal lineage marker for acute myeloid leukemia (AML), has been also shown to have a prognostic value: a high percentage of MPO-positive blasts correlates to favorable prognosis. To understand the relationship between the expression of MPO in leukemia cells and the response to chemotherapeutic agents, we established MPO-expressing K562 leukemia cell lines and then treated them with cytosine arabinocide (AraC). Cells expressing wild-type MPO, but not mutant MPO that could not mature, died earlier of apoptosis than control K562 cells. Reactive oxygen species (ROS) were generated more in leukemia cells expressing MPO, and the generation was abrogated by MPO inhibitors or antioxidants. Tyrosine nitration of cellular protein also increased more in MPO-expressing K562 cells than control cells after treatment with AraC. In clinical samples, CD34-positive AML cells from high-MPO cases showed a tendency to be sensitive to AraC in the colony-formation assay, and the generation of ROS and the nitration of protein were observed only when the percentage of MPO-expressing cells was high. These data suggest that MPO enhances the chemosensitivity of AML through the generation of ROS and the nitration of proteins.

Improvement of criteria for refractory cytopenia with multilineage dysplasia according to the WHO classification based on prognostic significance of morphological features in patients with refractory anemia according to the FAB classification.

In the criteria of refractory cytopenia with multilineage dysplasia (RCMD) according to the WHO (World Health Organization) classification, the frequency threshold concerning dysplasia of each lineage was defined as 10%. To predict overall survival (OS) and leukemia-free survival (LFS) for patients with refractory anemia (RA) according to the French-American-British (FAB) classification, we investigated prognostic factors based on the morphological features of 100 Japanese and 87 German FAB-RA patients, excluding 5q-syndrome. In the univariate analysis of all patients, pseudo-Pelger-Huet anomalies >or=10% (Pelger+), micromegakaryocytes >or=10% (mMgk+), dysgranulopoiesis (dys G) >or=10% and dysmegakaryopoiesis (dys Mgk) >or=40% were unfavorable prognostic factors for OS and LFS (OS; P<0.001, LFS; P<0.001). The prognostic effects of the morphological features were similar in both Japanese and German patients. However, dys Mgk >or=10% was not correlated with OS and LFS. In the multivariate analysis, mMgk+ and dys Mgk>or=40% were adverse prognostic factors for OS for all patients, and dys G >or=10% and dys Mgk>or=40% were adverse prognostic factors for LFS for all patients. On the basis of the present analysis, we propose the following modified morphological criteria for RCMD. Modified RCMD should be defined as FAB-RA, excluding 5q-syndrome with dys G >or=10%, dys Mgk>or=40% or mMgk+.

Gene analysis of Vibrio cholerae NAGV14 pilus and its distribution.

Adhesive pilus of Vibrio cholerae 034, strain NAGV14, was genetically analyzed. The deduced amino acid (aa) sequence of the major pilin structural gene (VcfA) was 67% homologous to the MshA pilin in the N-terminal region, but no homology was found in the C-terminal region which contained the antigenic epitopes. Upstream and downstream flanking regions examined were highly homologous to mshB and mshC of the MSHA (mannose-sensitive hemagglutinin) gene locus. A short leader sequence and a pair of cysteines near the C-terminus which are the characteristics of type 4a pilus family were found. The major pilin structural gene of NAGV14 was compared to that of a strain V10 producing non-adhesive pili. The deduced aa sequences showed 60% homology, and the distance between two cysteines in the C-terminal region was different. A total of 177 V. cholerae strains were investigated for the presence of a type 4 pilus gene locus by PCR, and 95% were positive. The major pilin gene of NAGV14 was detected in 4 of 93 V. cholerae non-O1, non-0139 strains tested, but none of the V. cholerae O1 and O139 (72 and 12 strains, respectively). Our result suggested that a type 4 pilus gene locus similar to the MSHA gene locus is widely distributed among V. cholerae strains. We proposed naming this type 4 pilus gene locus the VCF (for V. cholerae flexible pili) gene locus.

Identification of novel residues involved in nuclear localization of a baculovirus polyhedrin protein.

A baculovirus polyhedrin protein has proven to possess a nuclear localization signal (NLS) sequence and a domain required for supramolecular assembly. Here we investigated five Bombyx mori nucleopolyhedrovirus (BmNPV) mutants that did not produce polyhedra. Two of five mutants were generated during routine baculoviral expression vector screening, and three were isolated by treatment with the mutagen 5-bromo-2'-deoxyuridine (BrdU). Marker rescue mapping and nucleotide sequence analysis showed that mutations in the polyhedrin gene caused the altered phenotype of these mutants. Biochemical fractionation indicated that cells infected with these mutants exhibited polyhedrin protein in both the nucleus and the cytoplasm. Electron microscopic observation revealed that polyhedrin produced by these mutants ocurred in both the nucleus and the cytoplasm, but did not form a crystalline lattice. Despite the incompleteness of polyhedrin nuclear localization, the NLSs of the five mutants were unchanged, although some of the mutations occurred within residues just outside of the domain reported to be required for polyhedron assembly (4). This result suggests that (a) the polyhedrin NLS directs polyhedrin to the nucleus, but the efficiency of this localization is regulated by regions other than the NLS (probably, polyhedrin conformation and its association with the nucleus are also involved), and (b) formation of a crystalline lattice may also be determined by several domains within polyhedrin.

Recovery of hematopoietic progenitor cells in patients with severe aplastic anemia who obtained good clinical response with a combination therapy of immunosuppressive agents and recombinant human granulocyte colony-stimulating factor.

Seventeen patients with newly diagnosed acquired severe aplastic anemia were treated with a combination of immunosuppressive drugs consisting of anti-lymphocyte or anti-thymocyte globulin, cyclosporin A (CyA), methylprednisolone, and recombinant human granulocyte colony-stimulating factor (G-CSF). Fourteen (82%) of the 17 patients achieved good response (GR), and 3 (18%) had no response. Among the 14 GR patients, 5 (36%) later evolved clonal diseases, 1 developed myelodysplastic syndrome, and 4 developed paroxysmal nocturnal hemoglobinuria. The numbers of granulocyte-macrophage colony-forming units (CFU-GM) and erythrocyte burst-forming units were markedly low or absent in all cases before therapy. After therapy, those numbers in 13 patients among 14 responders recovered to the level of the normal control at the time of GR. However, the CFU-GM number substantially declined after that but gradually recovered again to reach a normal level over longer clinical courses. The positive rate for HLA-DRB1*1501 was 60% (3/5) among 5 CyA-dependent patients, which tended to be higher than the 20% (1/5) among 5 CyA-independent patients. Thus, immunosuppressive therapy combined with G-CSF provides a high rate of good hematological response accompanied by the apparent recovery of the hematopoietic progenitor compartments.

Absolute number of circulating CD34+ cells is abnormally low in refractory anemias and extremely high in RAEB and RAEB-t; novel pathologic features of myelodysplastic syndromes identified by highly sensitive flow cytometry.

We scored absolute numbers of circulating CD34+ cells by a highly sensitive triple-color flow cytometric analysis using CD45 monoclonal antibody, CD34 monoclonal antibody and propidium iodide. Forty-one patients with MDS (RA: 27, RARS: 1, RAEB: 6, RAEB-t: 3,CMML: 4), 12 patients with aplastic anemia (AA) and 36 age-adjusted normal subjects were studied. RA had significantly decreased numbers of cells expressing CD34 (0.21 +/- 0.29 x 10(6)/l) compared with normal subjects (0.81 +/- 0.36 x 10(6)/l)(P < 0.001). This low number of CD34+ cells in RA resembles the case of AA (0.39 +/- 0.73 x 10(6)/l). In light-scatter analysis, the CD34+ cells of RA patients were distributed mainly in low forward scatter (FSC) (lymphocyte region). In contrast, the CD34+ cell counts were extremely high in patients with RAEB (46.54 +/- 71.37 x 10(6)/l) and RAEB-t (57.00 +/- 52.36 x 10(6)/l) (P < 0.001) and the CD34+ cells were observed in high FSC (blast region).CMML patients showed moderately increased numbers of CD34+ cells (3.69 +/- 4.64 x 10(6)/l). Thus, there was a distinct difference in cell size and number of circulating CD34+ cells between RA and RAEB/RAEB-t. In univariate and multivariate analysis, a high CD34+ cell count (> or = 1.0 x 10(6)/l) was a poor prognostic factor. This method allows one to distinguish RA from other MDS subtypes more reliably than by morphology alone and provides early signs of progression to acute leukemia.

Dose optimization and indication of Linac radiosurgery for brain metastases.

PURPOSE: The authors have examined treatment effects of linear accelerator based radiosurgery for brain metastases. Optimal doses and indications were determined in an attempt to improve the quality of life for terminal cancer patients. METHODS AND MATERIALS: Ninety-two patients with 162 lesions were treated with Linac radiosurgery for brain metastases between April 1993 and September 1998. To determine prognostic factors, risk factors for recurrence, and appearance of new lesions, univariate and multivariate analyses were performed. To compare the local control between the high-dose (minimum dose > or =25 Gy: prescribed to the 50-80% isodose line) and low-dose (minimum dose <25 Gy) irradiated groups, matched-pairs analysis was performed. RESULTS: Median survival time was 11 months. In univariate analysis, extracranial tumor activity (p<0.001) and Karnofsky Performance Status (KPS) (p = 0.036) were two significant predictors of survival. In multivariate analysis, the status of an extracranial tumor was the single significant predictor of survival (p = 0.005). Minimum dose was the single most significant predictor of local recurrence in univariate, multivariate, and matched-pairs analyses (p<0.05). As to the appearance of new lesions, activity of extracranial tumors was a significant predictor (p<0.05). Side effects due to radiosurgery were experienced in 4 of 92 cases (4.3%). CONCLUSIONS: We concluded that brain metastases patients should be irradiated with > or =25 Gy, when extracranial lesions are stable and longer survival is expected. Combined surgery and conventional radiation may have little advantage over radiosurgery alone when metastatic brain tumors are small and extracranial tumors are well controlled. When extracranial tumors are progressive, the rate of appearance of new lesions in other nonirradiated locations becomes higher. In such cases, careful follow-up is required and a combination with whole brain irradiation should be considered.

Regulation of gamma-glutamylcysteine synthetase expression in response to oxidative stress.

Glutathione (GSH) is synthesized by the activity of two ATP-requiring GSH synthesizing enzymes. Gamma-glutamylcysteine synthetase (gamma-GCS) is the rate limiting enzyme for the GSH synthesis. Gamma-GCS is a heterodimer of heavy, catalytic subunit and light, regulatory subunit and responsive to many stresses, such as heat shock, oxidative stress or cytokines. To know the regulation of the expression of gamma-GCS gene, in the present study, we show evidences that gamma-GCS heavy subunit is upregulated by oxidative stress by ionizing radiation and TNF-alpha mediated by nuclear factor-kappaB (NF-kappaB), and impairment of the expression of gamma-GCS by TNF-alpha in diabetic condition. Furthermore we describe the importance of GSH in the regulation of NF-kappaB subunits.

Paroxysmal nocturnal haemoglobinuria clones in patients with myelodysplastic syndromes.

Among acquired stem cell disorders, pathological links between myelodysplastic syndromes (MDS) and aplastic anaemia (AA), and paroxysmal nocturnal haemoglobinuria (PNH) and AA, have been often described, whereas the relationship between MDS and PNH is still unclear. We analysed blood cells of patients with MDS to determine the incidence of the PNH clone, and analysed the PIG-A gene to find mutations characteristic of the PNH clone in MDS. In four (10%) of 40 patients with MDS, flow cytometry showed affected erythrocytes and granulocytes negative for decay-accelerating factor (DAF) and CD59. The population of affected erythrocytes was smaller in MDS patients with PNH clone (MDS/PNH) than in patients with de novo PNH, and haemolysis was milder in the MDS/PNH patients. PIG-A mutations were found in granulocytes of all patients with MDS/PNH. In type and site, the PIG-A mutations were heterogeneous, similar to that observed in de novo PNH; i.e. no mutation specific to MDS/PNH was identified. Of note, three of four patients with MDS/PNH each had two PNH clones with different PIG-A mutations, suggesting that PIG-A is mutable in patients with MDS/PNH. In a MDS/PNH patient with trisomy 8, FISH detected a distinct karyotype in a portion of granulocytes with PNH phenotype, indicating that PNH and MDS partly shared affected cells. Thus, MDS predisposes to PNH by creating conditions favourable to the genesis of PNH clone. Considering the increasing prevalence and incidence of MDS, these disorders could be useful for investigating the mechanism by which PIG-A mutation is induced.

Unusual CT and MR appearance of an epidermoid tumor of the cerebellopontine angle.

We present a case of an unusual epidermoid tumor of the cerebellopontine angle that appeared hyperdense on CT scans and hyperintense on T1- and T2-weighted MR images. We believe that these imaging characteristics were caused by a high protein concentration within the contents of the cyst.

Nuclear factor kappa B dependent induction of gamma glutamylcysteine synthetase by ionizing radiation in T98G human glioblastoma cells.

Glioblastoma is one of the most malignant of all neoplasms, and often shows resistance to chemotherapy and radiation therapy. Ionizing radiation activates transcriptional factors, such as nuclear factor kappa-B (NF-kappa B). Previously we found that glutathione (GSH) synthesis is induced by cytokines mediated by NF-kappa B (Urata et al. J. Biol. Chem., 1996). Here, we present direct evidence that NF-kappa B activated by ionizing radiation induces the expression of gamma-glutamylcysteine synthetase (gamma-GCS), the rate limiting enzyme of GSH synthesis, using T98G human glioblastoma cells. T98G cells have approximately 14-times the level of intracellular GSH of NB9 cells, radiation-sensitive neuroblastoma cells. In T98G cells, 30-Gy of ionizing radiation was required for the activation of NF-kappa B on an electrophoretic mobility shift assay and the induction of gamma-GCS mRNA on Northern blots and a nuclear run-on assay. However, when T98G cells were treated with buthionine sulfoximine, 3-Gy of ionizing radiation stimulated the DNA-binding activity of NF-kappa B and the expression of gamma-GCS. We constructed chimeric genes containing various regions of gamma-GCS promoter gene and the coding region for Luciferase. T98G cells transiently transfected with a plasmid containing the gamma-GCS promoter-luciferase construct showed increased luciferase activity when treated with ionizing radiation. The luciferase activity stimulated by ionizing radiation was found in the gamma-GCS promoter containing the NF-kappa B binding site, whereas not in that containing its mutated site. These results suggest that GSH synthesis is upregulated by ionizing radiation mediated by NF-kappa B and a high concentration of GSH in T98G cells causes downregulation of the NF-kappa B-DNA binding activity in response to ionizing radiation. The irresponsiveness of the intracellular signal transduction cascade to irradiation may be a factor in the resistance of T98G cells to radiation therapy.

Protective role of glutathione synthesis on radiation-induced DNA damage in rabbit brain.

1. Radiotherapy has attracted increasing interest in recent years. It is known that ionizing radiation induces oxygen radical injury, whereas oxidative stress by the radiation can cause cellular responses to defense cellular injury. In this study, the metabolism of antioxidants in response to ionizing radiation to the brain was studied in the brain using experimental rabbits. 2. Ionizing radiation to the hemicerebrum caused an increase in the levels of glutathione (GSH) and the activity of a GSH synthesizing enzyme, gamma-glutamylcysteine synthetase (gamma-GCS), and Cu,Zn-superoxide dismutase (Cu,Zn-SOD). Ionizing radiation also induced DNA-damage estimated by the formation of 8-hydroxydeoxyguanosine. These changes were dependent on the radiation dose. 3. Previous intrathecal-administration of buthionine sulfoximine (100 microM), a specific inhibitor of gamma-GCS, increased DNA damage by radiation in the radiated hemicerebrum. That of S-methyl GSH, on the other hand, resulted in a significant reduction of DNA damage by radiation. 4. These results suggest that synthesis of GSH and Cu,Zn-SOD is responsive to ionizing radiation and this induction of antioxidants may play a role in reducing tissue damage in radiotherapy.

Possible association between adult T-cell leukemia/lymphoma and acute myeloid leukemia.

BACKGROUND: To the authors' knowledge, an association between adult T-cell leukemia/lymphoma (ATL) and acute myeloid leukemia (AML) has been reported only in four patients. The authors identified five additional patients with both neoplasms. METHODS: A review of the clinical records of patients with AML, ATL, or lymphoid neoplasms other than ATL diagnosed between 1986 and 1995 was performed. Cytokine levels were assayed in selected patients. The authors searched for reports from other institutions using MEDLINE and the proceedings of two Japanese hematology societies. RESULTS: ATL was diagnosed in 134 patients, whereas 180 had AML. Five patients with both neoplasms were identified (3.7% of ATL patients and 2.8% of AML patients). In seven of the nine patients (including four patients in the literature) with ATL and AML, the ATL was diagnosed prior to the AML, whereas in the remaining two patients both neoplasms were diagnosed simultaneously. Six of the nine cases were therapy-related (t)-AML, which developed after chemotherapy for ATL. Monoclonal integration of proviral human T-lymphotropic virus type 1 was detected in ATL cells but not in AML cells in the six patients examined. The plasma levels of macrophage colony-stimulating factor (M-CSF), granulocyte-colony stimulating factor, and granulocyte-macrophage-colony stimulating factor (GM-CSF) were elevated in 3, 1, and 1, respectively, of the 4 patients examined at AML onset who had active ATL. In one case, the levels of several cytokines, including GM-CSF and M-CSF, in the supernatant fluid of short term cultured ATL cells were elevated. Three patients with de novo ATL and AML received remission induction therapy, and two achieved a complete remission (CR) of both diseases. Among the four patients who received chemotherapy for t-AML, two achieved CR. CONCLUSIONS: ATL patients also can develop AML, irrespective of treatment with chemotherapy for ATL. This association does not indicate exclusive chemoresistance of both neoplasms. Cytokines produced by ATL cells may support the growth of AML cells.

Molecular analysis of a filamentous phage (fsl) of Vibrio cholerae O139.

A filamentous bacteriophage from Vibrio cholerae O139 strain A1-4450 was isolated (fsl). The phage fsl had a ssDNA genome and dsDNA as a replicative form (RF) in lysogenic host cell. The DNA sequence of fsl RF was determined. It consisted of 6340 bp and had a G + C content of 43.5%. Fifteen possible ORFs were found in fsl. One of them, ORF384, was estimated to encode 384 amino acid residues (44.6 kDa) and had homologous regions with the zot gene of V. cholerae and gene I of the coliphage group. ORF104, located upstream of ORF384, was homologous to gene 93 protein of Pf3 (filamentous phage of Pseudomonas sp.) corresponding to gene VI of coliphage. Other than ORF384 and ORF104, the ORF81, ORF44, ORF29, and ORF193 were speculated to correspond to gene V, gene VII, gene IX, and gene III, respectively, in the order as reported on f1 phage.

Cyclin-D1-gene amplification is a more potent prognostic factor than its protein over-expression in human head-and-neck squamous-cell carcinoma.

To evaluate the prognostic significance of cyclin D1 protein/gene expressions in human head-and-neck squamous-cell carcinoma (HNSCC), we examined amplification of the cyclin-D1 gene (CCND1) by the differential PCR method and over-expression of cyclin-D1 protein by immunohistochemistry in 45 paraffin-embedded sections from HNSCC. Amplification of CCND1 was found in 10 (22%) cases and over-expression of cyclin D1 was found in 24 (53%) cases. CCND1 amplification was also found in 3 (25%) of 12 cases of dysplastic lesions adjacent to HNSCC. The overall 5-year survival of patients with CCND1 amplification or with protein over-production was significantly lower than that of patients without (p < 0.0001 and p < 0.05, respectively). However, with multivariate analysis, only amplification of CCND1 retained an independent prognostic value (p = 0.0018). These suggest that CCND1 amplification occurs at early stages of HNSCC tumorigenesis and is a more useful prognostic factor than over-expression of cyclin D1 in HNSCC.

Narrow photon beam dosimetry for linear accelerator radiosurgery.

The dosimetric characteristics of linear accelerator radiosurgery for 10-MV X-ray were measured. Measurement of the relative output factor and tissue maximum ratio with a microchamber produced results equivalent to those of measurement with X-ray film. The 80% isodose level width measured with the microchamber was significantly smaller than that measured with the X-ray film. For the measurement of relative output factor and tissue maximum ratio, a microchamber seems to be the more appropriate choice. X-Ray film was found to be suitable for beam profile measurement.

Re-examination of the local control by nerve growth factor of the outgrowth of neurites in PC12D cells.

We have examined the local control by nerve growth factor (NGF) of the outgrowth of neurites from clonal cells, PC12D, a subline whose phenotype resembles that of the parent PC12 cell line in the NGF-primed state. We show here that (i) the outgrowth of neurites, and their survival can be induced by NGF in enucleated PC12D cells (ii) individual neurites of a single 'giant cell', produced by cell fusion of PC12D cells, can respond independently to the NGF in the local environment, (iii) dissected neurites from giant cells survive for longer in medium that contains NGF than in medium that does not, (iv) in PC12D cells, the rapid formation of ruffles in response to NGF, which appears to be based on increased cell-substratum adhesion, leads to the subsequent formation of neurites, and (v) upon addition of NGF, the movement of short processes displaces polylysine-coated beads in the vicinity of neurites. These observations suggest that the NGF-dependent maintenance or extension of neurites might be controlled within the neurites themselves and might not require the direct involvement of the cell body, even in PC12 cells. It seems possible that any NGF-induced changes that promote an increase in cell-substratum adhesion might be responsible for the initiation and elongation of neurites. It also seems possible that the growth of neurites towards a source of NGF might be based on repeated rounds of extension and retraction of filopodia and neurites in a manner that depends on the concentration of NGF.

[Traumatic atlanto-occipital dislocation: a case report with survival].

We reported a case of a patient who survived anterior atlanto-occipital dislocation. A seven-year-old girl was admitted to our hospital due to disturbed consciousness after being involved in a traffic accident. With gradual improvement of consciousness, she showed bilateral abducens nerve palsy, quadriparesis and cervical instability. Lateral plain film showed anterior atlanto-occipital dislocation. MRI showed severe compression of the cervical cord and disruption of supporting ligaments of the cervicomedullary junction. After 4 months, cervical instability continued and posterior fixation was carried out with bone grafts. Her postoperative course was uneventful.

The activation and nuclear translocation of extracellular signal-regulated kinases (ERK-1 and -2) appear not to be required for elongation of neurites in PC12D cells.

The outgrowth of neurites was induced in PC12D cells, a subline of PC12 cells, that were treated not only with NGF but also with dbcAMP, staurosporine or bFGF. Simultaneous activation and rapid nuclear translocation of MAP kinases (ERK-1 and ERK-2) were observed in cells treated with NGF or bFGF. But staurosporine and dbcAMP induced no or only slight activation of the kinases. The nuclear translocation of the MAP kinases was not induced by the latter agents. These observations suggest a close relationship between the activation and the nuclear translocation of MAP kinases and, moreover, that stimulation and relocalization of MAP kinases might not be required for the outgrowth of neurites from PC12D cells. Staurosporine and dbcAMP may stimulate a down-stream step of the NGF pathway, or a parallel pathway(s) to the MAP kinase cascade in promoting neurite formation from PC12D cells. These agents mimic the effects of NGF in promoting neurite outgrowth in cultured sympathetic neurons, but not in conventional PC12 cells. Because of the similarity between PC12D cells and primed cells, it seems possible that activation and nuclear translocation of MAP kinases might be required for the transcription-dependent differentiation step but might not be necessary for the elongation of neurites at least in response to staurosporine or to dbcAMP.