Spectrum of clinical and genetic features of patients with inherited platelet disorder with suspected predisposition to hematological malignancies: a nationwide survey in Japan.

BACKGROUND: Inherited thrombocytopenia (IT) contains several forms of familial thrombocytopenia and some of them have propensity to hematological malignancies. The etiological and genetic features of this heterogeneous syndrome have not yet been elucidated. PATIENTS AND METHODS: We conducted a nationwide survey to collect clinical information and samples from patients with familial thrombocytopenia and/or hematological malignancies in order to obtain a comprehensive understanding of IT. RESULTS: Among the 43 pedigrees with clinical samples, RUNX1 mutations were identified in 8 pedigrees (18.6%). While MYH9 and ANKRD26 mutations were identified in 2 and 1 pedigrees, respectively, no gene mutations were detected in the remaining 32 pedigrees from a panel of previously reported pathogenetic mutations. Clinical data were comparable between FPD/AML and non-FPD/AML probands. CONCLUSIONS: Our study clarified that it is unexpectedly difficult to diagnose FPD/AML based on clinical information alone, and thus, genetic testing is strongly recommended. Our survey also identified some pedigrees with a strong family history of myelodysplastic syndromes of unknown origin. Additionally, there were 14 pedigrees in which three or more members were affected by immune thrombocytopenia (ITP), and a computer-aided simulation suggested that such a distribution almost never happens by coincidence, which implicates a genetic predisposition to ITP.

Divergent cytotoxic effects of PKC412 in combination with conventional antileukemic agents in FLT3 mutation-positive versus -negative leukemia cell lines.

FMS-like tyrosine kinase-3 (FLT3) is a new therapeutic target for acute myelocytic leukemia (AML), because FLT3 mutations are the most common genetic alterations in AML and are directly related to leukemogenesis. We studied cytotoxic interactions of a FLT3 inhibitor, PKC412, with eight conventional antileukemic agents (cytarabine, doxorubicin, idarubicin, mitoxantrone, etoposide, 4-hydroperoxy-cyclophosphamide, methotrexate and vincristine) using three leukemia cell lines carrying FLT3 mutations (MOLM13, MOLM14 and MV4-11) and five leukemia cell lines without FLT3 mutations (KOPB-26, THP-1, BALL-1, KG-1 and U937). PKC412 showed synergistic effects with all agents studied except methotrexate for FLT3-mutated cell lines in isobologram analysis. In contrast, PKC412 was rather antagonistic to most drugs, except for 4-hydroperoxy-cyclophosphamide and vincristine, in leukemia cell lines without FLT3 mutations. Cell-cycle analysis revealed that PKC412 induced G1 arrest in leukemia cell lines carrying FLT3 mutations, whereas it arrested cells in G2/M phase in the absence of FLT3 mutations, which may underlie the divergent cytotoxic interactions. These results suggest that the simultaneous administration of PKC412 and other agents except methotrexate is clinically effective against FLT3 mutation-positive leukemias, whereas it would be of little benefit for FLT3 mutation-negative leukemias. Our findings may be of help for the design of PKC412-based combination chemotherapy.

Identification of myelodysplastic syndrome-specific genes by DNA microarray analysis with purified hematopoietic stem cell fraction.

Myelodysplastic syndrome (MDS) is a slowly progressing hematologic malignancy associated with a poor outcome. Despite the relatively high incidence of MDS in the elderly, differentiation of MDS from de novo acute myeloid leukemia (AML) still remains problematic. Identification of genes expressed in an MDS-specific manner would allow the molecular diagnosis of MDS. Toward this goal, AC133 surface marker-positive hematopoietic stem cell (HSC)-like fractions have been collected from a variety of leukemias in a large-scale and long-term genomics project, referred to as "Blast Bank," and transcriptome of these purified blasts from the patients with MDS were then compared with those from AML through the use of oligonucleotide microarrays. A number of genes were shown to be expressed in a disease-specific manner either to MDS or AML. Among the former found was the gene encoding the protein Delta-like (Dlk) that is distantly related to the Delta-Notch family of signaling proteins. Because overexpression of Dlk may play a role in the pathogenesis of MDS, the disease specificity of Dlk expression was tested by a quantitative "real-time" polymerase chain reaction analysis. Examination of the Blast Bank samples from 22 patients with MDS, 31 with AML, and 8 with chronic myeloid leukemia confirmed the highly selective expression of the Dlk gene in the individuals with MDS. Dlk could be the first candidate molecule to differentiate MDS from AML. The proposal is made that microarray analysis with the Blast Bank samples is an efficient approach to extract transcriptome data of clinical relevance for a wide range of hematologic disorders.

A functional role of mitogen-activated protein kinases, erk1 and erk2, in the differentiation of a human leukemia cell line, UT-7/GM: a possible key factor for cell fate determination toward erythroid and megakaryocytic lineages.

The mitogen-activated protein (MAP) kinase cascade is a key regulator of mammalian cell proliferation and differentiation. In this study, we examined the roles of 2 members of the MAP kinase family, extracellular signal-regulated kinase 1 (Erk1) and Erk2, in erythropoietin (EPO)-induced erythroid differentiation and thrombopoietin (TPO)-induced megakaryocytic differentiation. UT-7/GM was used as a model system because this cell line is an erythroid/megakaryocytic bipotent cell line that can be induced to differentiate into the erythroid and megakaryocytic lineages by EPO and TPO, respectively. The kinetics of activation of Erk1 and Erk2 were examined during erythroid and megakaryocytic differentiation of UT-7/GM cells. EPO induced a transient activation of these kinases, peaking after 1 minute of stimulation and then declining quickly almost to the basal level. In contrast, TPO-induced activation of the kinases peaked at 10 minutes and persisted for up to 60 minutes, similar to the activation by granulocyte-macrophage colony-stimulating factor. The percentage of EPO-induced hemoglobin-positive cells was elevated by the addition of PD98059, a specific inhibitor of MEK1 (MAP kinase/ERK kinase 1). In contrast, PD98059 clearly reduced the amount of glycoprotein IIb/IIIa antigens induced by TPO on UT-7/GM cells. Thus, inactivation of Erk1 and Erk2 kinases promoted EPO-induced erythroid differentiation and suppressed TPO-induced megakaryocytic differentiation of UT-7/GM cells. In conclusion, the activation of Erk1 and Erk2 kinases may be a critical event in the determination of cell fate and the differentiation processes of the erythroid and megakaryocytic lineages.

Forkhead family transcription factor FKHRL1 is expressed in human megakaryocytes. Regulation of cell cycling as a downstream molecule of thrombopoietin signaling.

FKHRL1, a member of the Forkhead transcription factor family, is one of the downstream molecules of phosphatidylinositol 3-kinase-Akt. This molecule is a mammalian homolog of DAF-16, which plays an important role in the longevity of Caenorhabditis elegans. In this study we found that Akt and FKHRL1 proteins were detectable in highly purified normal human megakaryocytes and that these molecules were actually phosphorylated by thrombopoietin (TPO). To clarify the functional role of FKHRL1 in TPO signaling, we established a tetracycline-inducible system in the human TPO-dependent leukemia cell line UT-7/TPO. Induced expression of active FKHRL1 led to cell cycle arrest at G0/G1 phase in this cell line. These results suggest that FKHRL1 plays an important role in the cell cycle of megakaryocytic cells as one of the downstream target molecules of phosphatidylinositol 3-kinase-Akt, presumably mediated through the activation or inactivation of cell cycle-associated gene(s).

Characterization of stage progression in chronic myeloid leukemia by DNA microarray with purified hematopoietic stem cells.

Chronic myeloid leukemia (CML) is characterized by the clonal expansion of hematopoietic stem cells (HSCs). Without effective treatment, individuals in the indolent, chronic phase (CP) of CML undergo blast crisis (BC), the prognosis for which is poor. It is therefore important to clarify the mechanism underlying stage progression in CML. DNA microarray is a versatile tool for such a purpose. However, simple comparison of bone marrow mononuclear cells from individuals at different disease stages is likely to result in the identification of pseudo-positive genes whose change in expression only reflects the different proportions of leukemic blasts in bone marrow. We have therefore compared with DNA microarray the expression profiles of 3456 genes in the purified HSC-like fractions that had been isolated from 13 CML patients and healthy volunteers. Interestingly, expression of the gene for PIASy, a potential inhibitor of STAT (signal transducer and activator of transcription) proteins, was down-regulated in association with stage progression in CML. Furthermore, forced expression of PIASy has induced apoptosis in a CML cell line. These data suggest that microarray analysis with background-matched samples is an efficient approach to identify molecular events underlying the stage progression in CML.

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.

A member of Forkhead family transcription factor, FKHRL1, is one of the downstream molecules of phosphatidylinositol 3-kinase-Akt activation pathway in erythropoietin signal transduction.

The phosphatidylinositol 3-kinase (PI3K) signaling pathway is important for the regulation of a number of cellular responses. Serine/threonine kinase Akt (protein kinase B; PKB) is downstream of PI3K and activated by growth factors. This study found that erythropoietin (EPO) induced tyrosine phosphorylation of Akt in a time- and dose-dependent manner in EPO-dependent human leukemia cell line UT-7/EPO. In vitro kinase assay using histone H2B and glucose synthase kinase as substrates demonstrated that Akt was actually activated by EPO. EPO-induced phosphorylation of Akt was completely blocked by a PI3K-specific inhibitor, LY294002, at 10 micromol/L, indicating that activation of Akt by EPO is dependent on PI3K activity. In addition, overexpression of the constitutively active form of Akt on UT-7/EPO cells partially blocked apoptosis induced by withdrawal of EPO from the culture medium. This finding suggested that the PI3K-Akt activation pathway plays some role in the antiapoptotic effect of EPO. EPO induced phosphorylation of a member of the trancription factor Forkhead family, FKHRL1, at threonine 32 and serine 253 in a dose- and time-dependent manner in UT-7/EPO cells. Moreover, results showed that Akt kinase activated by EPO directly phosphorylated FKHRL1 protein and that FKHRL1 phosphorylation was completely dependent on PI3K activity as is the case for Akt. In conjunction with the evidence that FKHRL1 is expressed in normal human erythroid progenitor cells and erythroblasts, the results suggest that FKHRL1 plays an important role in erythropoiesis as one of the downstream target molecules of PI3K-Akt.

GATA-1 and erythropoietin receptor genes are highly expressed in erythroleukemia.

We examined expression of the erythroid-associated genes GATA-1 and erythropoietin receptor (EPOR) in primary leukemia using the reverse transcriptase-polymerase chain reaction (RT-PCR). GATA-1 and EPOR mRNAs were detectable in all cases of erythroleukemia (French-American-British classification: M6) or early erythroblastic leukemia. In all other leukemia cases, including M2 through M5, stem cell leukemia, and adult T-cell leukemia, these gene transcripts were undetectable. GATA-2 was detectable in all the cases of primary leukemias examined in this study, except one case of M5. In one case, the phenotype switched from myeloid (M2) to erythroid (M6) and then back to myeloid. Northern blotting and RT-PCR revealed that GATA-1 and EPOR mRNAs were significantly upregulated at the M6 stage compared with the M2 stage. GATA-1 may be involved in the expression of an erythroid phenotype in acute leukemia. We generated HL-60 transfectants exogenously expressing GATA-1. The majority of HL-60 cells expressing GATA-1 lacked azurophilic granules, and electron microscopic analysis revealed that myeloperoxidase activity was negative. Platelet peroxidase activity, which was detectable in both megakaryoblasts and erythroid progenitors, was positive. However, EPOR and glycophorin A mRNAs were undetectable by RT-PCR. These findings suggest that besides GATA-1, a third factor may be required for the expression of mature erythroid phenotypes. In addition, our results indicate that GATA-1 is involved in inactivation of myeloperoxidase and activation of the platelet peroxidase.

A novel function of Stat1 and Stat3 proteins in erythropoietin-induced erythroid differentiation of a human leukemia cell line.

We recently determined that erythropoietin (EPO) activates 3 members of the signal transducer and activator of transcription (STAT) family, Stat1alpha, Stat3, and Stat5, in the human EPO-dependent cell lines, UT-7 and UT-7/EPO (Kirito et al, J Biol Chem 272:16507, 1997). In addition, we have shown that Stat1alpha, but not Stat3, is involved in EPO-induced cellular proliferation. In this study, we examined the roles of Stat1alpha and Stat3 in EPO-induced erythroid differentiation. UT-7/GM was used as a model system, because this cell line can differentiate into erythroid-lineage cells with EPO treatment (Komatsu et al, Blood 89:4021, 1997). We found that EPO did not activate Stat1alpha or Stat3 in UT-7/GM cells. Transfection experiments showed that both Stat1alpha and Stat3 inhibited the induction by EPO of gamma-globin and erythroid-specific 5-aminolevulinate synthetase transcripts, resulting in a reduction of the percentage of hemoglobin-positive cells. Dominant negative forms of Stat1alpha or Stat3 promoted the EPO-induced erythroid differentiation of UT-7/GM cells, even in the presence of granulocyte-macrophage colony-stimulating factor, although this cytokine never induced erythroid differentiation of the parent UT-7/GM cells with or without EPO. A cell cycle analysis showed that the constitutive activation of Stat1alpha, but not Stat3, shortened the period of G0/G1 prolongation caused by EPO stimulation. Taken together, our data suggest that Stat1alpha and Stat3 act as negative regulators in EPO-induced erythroid differentiation. Specifically, Stat1alpha may activate a cell cycle-associated gene(s), leading to the entry of cells into the cell cycle.

Thrombopoietin supports in vitro erythroid differentiation via its specific receptor c-Mpl in a human leukemia cell line.

Thrombopoietin (TPO) acts on megakaryopoiesis and erythropoiesis in vitro and in vivo. We isolated a novel subline, UT-7/GMT, from the human leukemia cell line UT-7/GM (N. Komatsu, et al., Blood, 89: 4021-4033, 1997). A small population of UT-7/GM cells positively stained for hemoglobin (Hb) after a 7-day exposure to TPO. More than 50% of TPO-treated UT-7/GMT cells positively stained for Hb. Using UT-7/GMT cells, we examined how TPO promotes hemoglobinization. TPO induced tyrosine phosphorylation of the TPO receptor but not the erythropoietin (EPO) receptor. There was no competition between TPO and EPO for binding to EPO receptor. These findings suggest that TPO has a direct effect on hemoglobinization via a specific receptor on UT-7/GMT cells. Isoelectric focusing demonstrated that TPO induced fetal and adult Hb synthesis, whereas EPO induced embryonic, fetal, and adult Hb synthesis. Thus, our data suggest that TPO has a distinct action on erythropoiesis.

Regulation of both erythroid and megakaryocytic differentiation of a human leukemia cell line, UT-7.

UT-7 is a human megakaryoblastic leukemia cell line with absolute dependence on interleukin-3, granulocyte-macrophage colony-stimulating factor (GM-CSF), or erythropoietin (EPO) for growth and survival. Among its sublines, UT-7/GM, which remains undifferentiated in the presence of GM-CSF, has a bipotency showing differentiation into erythroid or megakaryocytic cell lineages in the presence of EPO or thrombopoietin (TPO), respectively. To investigate the mechanism underlying determination of cell differentiation, we investigated the role of signal transducers and activators of transcription (STAT) in EPO-induced erythroid differentiation. UT-7 cells grow in response to GM-CSF and EPO but do not differentiate into mature cells. UT-7/GM cells grow in response to GM-CSF and differentiate into erythroid cells by EPO. In UT-7 cells, both GM-CSF and EPO induced the activation of Stat1 alpha, Stat3 and Stat5. In UT-7/GM cells, EPO activated Stat5 alone, although the activation of Stat1 alpha, Stat3, and Stat5 was induced by GM-CSF or TPO. In addition, GM-CSF inhibited EPO-induced erythroid differentiation and concomitantly activated Stat1 alpha and Stat3 in UT-7/GM cells even in the presence of EPO. Further we transfected Stat1 alpha, Stat3 cDNA or both into UT-7/GM cells. Hemoglobin-positive cells were decreased in the stable transfectants stimulated with EPO. These results indicate that Stat1 alpha and Stat3 have an inhibitory effect on the EPO-induced erythroid differentiation, and more complicated combination of transcription factors may play an important role in the decision of cell differentiation.

[Retrospective analysis of elderly patients > or = 60 years of age with acute leukemia].

A retrospective analysis was performed on 76 consecutive elderly patients with acute leukemia aged 60 years or more (48 men, 28 women). Forty patients were 60-69 years old, 28 were 70-79 years old and 8 were > or = 80 years old. There were 55 patients with acute myelogenous leukemia (AML), 13 acute lymphoblastic leukemia (ALL) and 8 AML from myelodysplastic syndrome (MDS/AML). Patients were treated with the JALSG protocol, CAG regimen, or low-dose Ara-C regimen for AML, and DVP/M-CHOP protocol for ALL. The complete remission (CR) rates were 52.7% (29 of 55) in AML, 61.5% (8 of 13) in ALL, and 0% in MDS/AML. The median CR durations were 226, 85, 0 days, and the median survivals were 204, 177, 99 days, respectively. CR rates were 65.3% for the JALSG protocol, 62.5% for the CAG regimen and 25.0% for low-dose Ara-C regimen. According to age, CR was obtained 62.5% in patients aged 60-69 years and 33.3% in patients over 70 years old. Our results indicated that patients aged 60-69 years should be treated with intensive chemotherapy.

In vitro development of erythroid and megakaryocytic cells from a UT-7 subline, UT-7/GM.

UT-7 is a human megakaryoblastic leukemia cell line with absolute dependence on interleukin-3, granulocyte-macrophage colony-stimulating factor (GM-CSF), or erythropoietin (EPO) for growth and survival. We isolated a novel subline, UT-7/GM after long-term culture of UT-7 with GM-CSF. The hemoglobin concentration and gamma-globin and EPO-receptor mRNA levels were significantly higher in EPO-treated UT-7/GM cells than in untreated cells. In contrast, the platelet factor 4 and glycoprotein IIb mRNA levels were much higher in thrombopoietin (TPO)-treated UT-7/GM cells than in untreated cells. Some TPO-treated cells had morphologically mature megakaryocytic characteristics such as a developed demarcation membrane in the cytoplasm and multilobular nuclei. These findings indicate that UT-7/GM is a bipotential cell line that can be induced to differentiate into erythroid and megakaryocytic lineages by EPO and TPO, respectively. Moreover, a minority of UT-7/GM cells acquired a high hemoglobin concentration by treatment with TPO, suggesting that TPO in part induced the erythroid differentiation of the UT-7/GM cells. Interestingly, GM-CSF inhibited the EPO- or TPO-induced erythroid differentiation and the TPO-induced megakaryocytic differentiation of UT-7/GM cells. These results support the hypothesis that cytokines influence the programming of gene expression required for lineage commitment or differentiation.

A distinct function of STAT proteins in erythropoietin signal transduction.

The Janus kinase (JAK)-signal transducers and activators of transcription (STAT) pathway is an important signaling pathway of interferons and cytokines. We examined the activation of STAT proteins induced by interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), or erythropoietin (EPO) using the human leukemia cell line, UT-7, which requires these cytokines for growth. IL-3, GM-CSF, and EPO induced DNA-binding activity to the oligonucleotides corresponding to the sis-inducible elements (SIE) of c-fos, in addition to the beta-casein promoter (beta-CAP), SIE- and beta-CAP-binding proteins were identical to Stat1alpha and Stat3 complex and to Stat5 protein, respectively. This indicates that IL-3, GM-CSF, and EPO commonly activated Stat1alpha, Stat3, and Stat5 proteins in UT-7. However, EPO hardly activated Stat1alpha and Stat3 in UT-7/GM, which is a subline of UT-7 that grows slightly in response to EPO. Transfection studies revealed that UT-7/GM cells constitutively expressing Stat1alpha, but not Stat3, can grow as well in response to EPO as GM-CSF, suggesting that Stat1alpha is involved in the EPO-induced proliferation of UT-7. Thus, although Stat1alpha, Stat3, and Stat5 proteins are activated by GM-CSF, IL-3, and EPO, our data suggest that each STAT protein has a distinctive role in the actions of cytokines.

Cell-cycle-dependent regulation of erythropoietin receptor gene.

To understand the regulatory mechanism of erythropoietin (EPO) receptor (EPOR) gene expression, the effect of EPO on the steady-state level of EPOR mRNA was examined using the human EPO-dependent cell line UT-7 as a model system. We found that the treatment of UT-7 cells with EPO resulted in a transient decrease of the EPOR mRNA level. This transient downregulation was also induced by stimulation with granulocyte-macrophage colony-stimulating factor (GM-CSF), another stimulator of UT-7 cell growth. These results raised the possibility that EPOR gene expression is in part related to cell growth. Moreover, it was found that EPO-induced downregulation of EPOR mRNA level was preceded by a transient downregulation of GATA-1 mRNA. To examine the relationship between the expression of EPOR, GATA-1, and GATA-2 mRNA levels and the cell cycle, logarithmically growing UT-7 cells were centrifugically fractionated according to the cell-cycle phase. Both EPOR and GATA-1 mRNA levels, but not the GATA-2 mRNA level, concomitantly decreased at the G0/G1 phase and increased at the S and G2/M phases. An electrophoretic mobility shift assay (EMSA) showed that in EPO-stimulated UT-7 cells, the dynamic changes in EPOR gene expression paralleled the GATA-1 DNA-binding activity to the oligonucleotide probe containing a GATA-binding site located at the promoter region of the EPOR gene. These findings suggest that the regulation of EPOR mRNA level is mainly associated with GATA-1 gene expression in UT-7 cells undergoing proliferation, and that these serial events are under the control of, or related to, the cell cycle.

Interleukin-3-associated ganglioside GD1a is induced independently of normal interleukin-3 receptor in murine myelogenous leukaemia NFS60 cells transfected with the interleukin-3 gene.

The mechanism of interleukin-3 (IL-3) independent cell growth and of IL-3-associated ganglioside expression was analysed using the IL-3 dependent murine myelogenous leukaemia cell line NFS60-I7 and IL-3 gene-transfected sublines. The transfected cell lines showed autonomous cell growth, tumorigenicity, and IL-3 associated ganglioside GD1a expression in spite of their IL-3 production. While the parental NFS60-I7 cells did not express significant amounts of GD1a, exogenous recombinant IL-3 (rIL-3) was demonstrated to induce IL-3-associated ganglioside GD1a expression in NFS60-I7 cells. Furthermore, the growth potential of the transfected cells was not blocked by anti-IL-3 antibody and expression of GD1a was not affected by anti-IL-3 antibody. These findings suggest that IL-3 expressed intracellularly by gene transfection might act independently of the normal IL-3 receptor on autonomous cell growth and on IL-3-associated GD1a expression in murine myelogenous leukaemia NFS60 cells.

Interleukin-3-associated expression of gangliosides in mouse myelogenous leukemia NFS60 cells introduced with interleukin-3 gene: expression of ganglioside GD1a and key involvement of CMP-NeuAc:lactosylceramide alpha 2-->3-sialyltransferase in GD1a expression.

Murine interleukin-3 (IL-3)-associated expression of gangliosides has been investigated using a gene transfection technique. A murine IL-3 cDNA was introduced into the parental NFS60-17 cells that was exclusively dependent on IL-3. We analyzed the glycosphingolipids from the parental cells and the transfected cells by fast atom bombardment mass spectrometry analyses and/or immunostaining techniques using specific antibodies. Two major gangliosides, IV3NeuAc-GgOse4Cer (GM1b) and IV3-NeuAc,III6NeuAc-GgOse4Cer (GD1 alpha), were expressed, in the parental cells. By contrast, in the IL-3 gene-transfected cells, a ganglioside IV3NeuAc,II3NeuAc-GgOse4Cer (GD1a) was strikingly expressed, in addition to GM1b and GD1 alpha that were already present in the parental cells. In spite of various IL-3-secreting capabilities, all transfectants investigated have exhibited the same ganglioside patterns and expressed GD1a. Furthermore, the appearance of GD1a was a consequence of the up-regulation of a single glycosyltransferase, CMP-NeuAc:lactosylceramide alpha 2-->3-sialytransferase (GM3 synthase). Activities of the other downstream glycosyltransferases that were involved in GD1a synthesis were not significantly different between the parental and the transfected cells. According to these data, the progression of tumor stage by the acquisition of autonomous cell growth ability after IL-3 gene transfection resulted in dramatic changes in cell surface gangliosides and their biosynthetic pathways. GD1a could be considered as an IL-3-associated ganglioside and was expressed in a tight connection with a single glycosyltransferase (GM3 synthase) up-regulation and with IL-3 expression in murine myelogenous leukemia cells.

[HTLV-I negative adult T cell leukemia; a case report of acute type].

We described a case of adult T cell leukemia (ATL) not associated with human T-cell leukemia virus type I (HTLV-I), a clinical entity that was first reported by Shimoyama et al. A 79-year-old male was admitted with anorexia and fever in October, 1989. Physical examination revealed marked hepatosplenomegaly and superficial lymphadenopathies. Hematological examination revealed marked leukocytosis (136,300/microliters) with abnormal lymphoid cells showing highly lobulated nuclei. Hypercalcemia (11.2 mg/dl) and elevation of lactic dehydrogenase were also recognized. Surface marker analysis showed that the abnormal lymphoid cells in the peripheral blood were positive for CD2 and CD4 but negative for CD8. Southern blot analysis of the DNA from peripheral blood leukemic cells revealed monoclonal rearrangement of T-cell receptor beta-chain gene. The clinical and hematological findings of the patient were compatible with those of acute type ATL, however, serum anti-HTLV-I antibody was negative and HTLV-I proviral DNA was not detected in the leukemic cells by Southern blot analysis. Furthermore, the polymerase chain reaction showed no integration of the HTLV-I proviral DNA in the leukemic cells.

[Sick sinus syndrome caused by amyloidosis associated with multiple myeloma].

A 65-year-old man, who had been treated for multiple myeloma (MM) since 1986, was admitted because of loss of consciousness in September 1989. An electrocardiogram taken just before admission showed a sinus arrest, junctional escaped rhythm, and marked bradycardia. The diagnosis of sick sinus syndrome (SSS) was made. Soon a temporary pacemaker was inserted, and the dyspnea ameliorated. However on the second day in the hospital, he had a high fever and Staphylococcus aureus was detected in the cultured blood. A diagnosis of septicemia was made, and the pacemaker was removed. He was then treated with beta-stimulants, but died in November 1989. Necropsy revealed cardiomegaly and microscopic examination showed amyloid deposits in the sinoatrial node, and the walls of the ventricles and coronary arteries. Although amyloidosis is often a complication of MM and the heart is frequently affected, SSS caused by amyloidosis associated with MM is quite unusual. In such patients, the use of a pacemaker is controversial, because amyloid deposits are occasionally accelerated by insertion of a pacemaker and for patients with hematological disorders, septicemia associated with pacemaker insertion may prove fatal.