Stimulation of GATA-2 as a mechanism of hydrogen peroxide suppression in hypoxia-induced erythropoietin gene expression.

Hydrogen peroxide (H2O2) has previously been shown to inhibit the DNA binding activity of hypoxia inducible factor-1 (HIF-1), the accumulation of HIF-1alpha protein and erythropoietin (Epo) gene expression. Epo gene expression has been previously shown to be down-regulated through a GATA binding site at its promoter region. In this study, the effect of H2O2 on Epo gene expression under hypoxic conditions through a GATA transcription factor was investigated. Hypoxic induction was found to be inhibited upon the addition of H2O2, and this effect could be reversed through the addition of catalase. Hypoxic induction was found to be suppressed by co-transfection with a human GATA-2 cDNA expression plasmid. Transfection of Hep3B cells with a reporter gene bearing a mutation at the promoter GATA binding site was found to be only mildly affected by the addition of H2O2. Electrophoretic gel mobility shift assays (EMSAs), using the Epo promoter GATA site as a probe and the GATA-2 protein extracted from Hep3B cells, showed that addition of H2O2 enhanced the binding of GATA-2 while addition of catalase inhibited this binding. From these results, we conclude that H2O2 increases the binding activity of GATA-2 in a specific manner, thereby suppressing the activity of the Epo promoter and thus inhibiting Epo gene expression.

Sialyl-Tn- and neuron-specific enolase-positive minimally differentiated erythroleukemia.

Flow cytometric, immunochemical and molecular studies were performed on leukemic blasts from a patient with minimally differentiated erythroleukemia (AML-M6v). The blasts expressed CD36 and CD71 but not lymphoid antigens, myeloid antigens, CD41 or glycophorin A. Analysis of carbohydrate antigens showed that the blasts expressed the sialyl-Tn antigen. Immunochemistry revealed that the blasts had neuron-specific enolase (NSE). Serum sialyl-Tn and NSE levels were markedly increased. Finally, an erythroid lineage was confirmed in the presence of alpha-globin messages in the blasts. Sialyl-Tn and NSE expression in leukemic blasts may be useful to identify AML-M6v.

Essential thrombocythemia transformed to acute myelogenous leukemia with t(3;17)(p24; q12), del(5)(q13q34) after treatment with carboquone and hydroxyurea.

In 1991, a 52-year-old woman was diagnosed as having essential thrombocythemia (ET). She was doing well with continuous medication with carboquone (CQ) and subsequently hydroxyurea (HU). However, substantial leukocytosis with leukemic blast cells, anemia and thrombocytopenia developed in 1996. Analysis of peripheral blood showed 4.4 x 10(3)/microl white blood cells with 82% of leukemic blast cells. These blasts showed negative staining with myeloperoxidase by immunostaining, but the myeloperoxidase was positive by electron microscopic analysis. Cytogenetic analysis of bone marrow cells revealed a t(3;17)(p24; q12), del(5)(q13q34). On the basis of these findings, the leukemic blast cells were classified as acute myelogenous leukemia (AML:M0) in the FAB classification. The causative agent, CQ and HU in secondary leukemia from ET and chromosomal abnormality related to ET blastic crisis (BC) are discussed.

A de novo philadelphia chromosome-positive acute mixed-lineage leukemia with both major and minor BCR/ABL mRNA transcripts.

A patient with a Philadelphia chromosome (Ph)-positive acute mixed-lineage leukemia (AMLL) expressing both major and minor BCR/ABL mRNA transcripts is described. Phenotypic analysis of the leukemic blasts revealed positivity for both myeloid and B-cell lineages. Southern blot analysis showed a rearrangement of the immunoglobulin heavy chain (IgH) gene. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis showed the expression of both major and minor BCR/ABL mRNA transcripts. To our knowledge, this is the first report of AMLL expressing both major and minor BCR/ABL mRNA transcripts and rearrangement of the IgH gene.

N(G)-monomethyl-L-arginine inhibits erythropoietin gene expression by stimulating GATA-2.

N(G)-monomethyl-L-arginine (L-NMMA) has been reported to be elevated in uremic patients. Based on the hypothesis that the pathogenesis of the anemia of renal disease might be due to the perturbation of transcription factors of the erythropoietin (Epo) gene by L-NMMA, the present study was designed to investigate the effect of L-NMMA on Epo gene expression through the GATA transcription factor. L-NMMA caused decreased levels of NO, cyclic guanosine monophosphate (cGMP), and Epo protein in Hep3B cells. L-NAME (analogue of L-NMMA) also inhibited Epo production in anemic mice. Transfection of the Epo promoter-luciferase gene into Hep3B cells revealed that L-NMMA inhibited the Epo promoter activity. However, L-NMMA did not inhibit the Epo promoter activity when mutated Epo promoter (GATA to TATA) was transfected, and L-NMMA did not affect the enhancer activity. Electrophoretic mobility shift assays demonstrated the stimulation of GATA binding activity by L-NMMA. However, L-NMMA had no effect on the binding activity of hepatic nuclear factor-4, COUP-TF1, hypoxia-inducing factor-1, or NF-kappaB. Furthermore, cGMP inhibited the L-NMMA-induced GATA binding activity. L-NMMA also increased GATA-2 messenger RNA expression. These results demonstrate that L-NMMA suppresses Epo gene expression by up-regulation of the GATA transcription factor and support the hypothesis that L-NMMA is one of the candidate substances that underlie the pathogenesis of renal anemia. (Blood. 2000;96:1716-1722)

A case of bilateral heel ulcers associated with hydroxyurea therapy for chronic myelogenous leukemia.

Bilateral heel skin ulcers developed in a 50-year-old male in the chronic phase of chronic myelogenous leukemia who had been receiving hydroxyurea (HU) therapy for 3 years. Histological examination showed perivascular lymphocytic inflammation without vasculitis. After interruption of HU administration, the heel ulcers were completely resolved within 2 months. The clinical course strongly suggested that the heel ulcers were induced by long-term HU therapy.

All-trans-retinoic acid treatment for chemotherapy-resistant acute adult T-cell leukemia.

We report a case in which treatment with all-trans-retinoic acid (ATRA) improved the clinical features of a 47-year-old female patient with acute adult T-cell leukemia (ATL). The patient was first treated several times with combination chemotherapy. but the number of ATL cells increased and other clinical manifestations progressed. ATRA 60 mg was then administered daily. ATRA treatment dramatically improved the patient's clinical features. In vitro examination revealed that ATRA inhibited the growth of ATL cells from the patient. These findings suggest that ATRA may be a useful treatment for patients with chemotherapy-resistant acute ATL.

Factors affecting the ion-pair chromatography of water-soluble vitamins.

Factors controlling the separation of seven water-soluble vitamins on reversed-phase columns were systematically evaluated. Factors studied include both mobile phase constituents and column parameters. Data showed that a mobile phase containing hexanesulfonate (5 mM), methanol (15%), acetic acid (1%), and triethylamine (0.10-0.13%) yielded excellent separations with several C8 and C18 columns. Lowering the methanol concentration in the mobile phase enhanced the resolution of early eluting peaks, while the triethylamine level controlled the peak shape and retention of thiamine. The analytical precision, robustness, and sensitivity of the developed liquid chromatographic (LC) separation were evaluated. The stability of the LC separation was found to be satisfactory for over a 4-month period.

[Echocardiography of ischemic heart disease simulating dilated cardiomyopathy, with special reference to abnormal wall movement on the short-axis].

The purpose of this study was to determine whether two-dimensional echocardiography (2DE) can differentiate ischemic myocardial disease (IMD) from dilated cardiomyopathy (DCM). The subjects consisted of six cases of IMD which showed left ventricular dilatation (LVDd greater than or equal to 60 mm) and diffuse abnormality of wall motion, but did not show obvious localized myocardial infarction or left ventricular aneurysm on 2DE, and 16 cases of DCM. Two cases of IMD had previous myocardial infarction, and five cases of DCM had cardiomegaly following myocarditis. A short-axis image of the left ventricle was recorded at the chordal and the papillary muscle levels. Each image was divided into 4 segments, which were comprised of the septum, anterior wall, lateral (posterolateral) wall, and posterior (posteromedial) wall. Regional wall motion abnormality with reference to systolic thickening was analyzed qualitatively in each segment. The results were as follows: In ECG findings in IMD group, only one case showed abnormal Q waves and five cases showed left ventricular hypertrophy (LVH) similar to intraventricular conduction defect. On the other hand, in DCM group seven cases showed abnormal Q waves and five cases showed LVH. Two cases of IMD had two-vessel disease and four three-vessel disease, respectively. Left ventricular ejection fraction by cine-angiography ranged from 0.10 to 0.39 (mean 0.24) in IMD group and from 0.22 to 0.42 (mean 0.36) in DCM group. Mean LVDd showed no significant difference between these two groups. Five cases of DCM showed marked left ventricular dilatation (LVDd greater than or equal to 75 mm), but there were no such cases in IMD group. B-B' step was recognized in only one case of IMD, though it was present in eight cases in DCM. In regional wall motion, incidence of asynergy such as akinesis or dyskinesis was higher in IMD group than in DCM group. Left ventricular asynergy was more serious in the posteromedial wall than the posterolateral wall at the same image in five cases of IMD. However, in 12 cases of DCM, the degree of asynergy was equal at the both walls. In conclusion, it is recommended to examine echocardiographically the extent of severe asynergy in the posteromedial and posterolateral walls in order to differentiate IMD from DCM.