Effect of inhibitors of protein phosphatase 1 and 2A on erythroid colony formation: an investigation of the specificities of inhibitors.

To investigate the possible involvement of protein phosphatase (PP)1 and PP2A in the process of erythropoiesis, we assessed the effect of PP1 and PP2A inhibitors on erythroid colony formation using an in vitro colony formation assay. Okadaic acid (OKA), calyculin A (Cal-A) and tautomycin suppressed colony formation but 1-nor-okadaone did not. These results suggest that PP1 and PP2A both play an important role in erythropoiesis. Furthermore, higher concentrations of tautomycin were needed to suppress colony formation compared to concentrations of OKA and Cal-A. The target enzyme of inhibitors in erythropoiesis may be PP2A.

Improvement of extrathymic T cell type of large granular lymphocyte (LGL) leukemia by cyclosporin A: the serum level of Fas ligand is a marker of LGL leukemia activity.

We report a case of gammadelta T-cell-type large granular lymphocyte (LGL) leukemia (CD3 +,CD8 +, CD57 +,TCR gammadelta+), which was accompanied by pure red cell aplasia, neutropenia and thrombocytosis. Southern blotting analysis of the T-cell receptor beta gene showed the germline configuration, but clonal TCR J gamma rearrangements were identified. These granular lymphocytes demonstrated non-major histocompatibility complex-restricted cytotoxicitity. The serum-soluble FasL (sFasL) concentration of this patient was very high, whereas the serum levels of tumor necrosis factor alpha (TNF-alpha), interferon gamma (IFN-gamma), interleukin-1 beta (IL-1beta), interleukin-2 (IL-2) and thrombopoietin were normal. After treatment with cyclosporin A, anemia and thrombocytosis were improved, and LGL and the elevated sFasL concentration decreased. These observations suggested that FasL may have played a role in the establishment of the clinical symptoms of this patient and could be useful as an indicator of disease activity.

Aminopeptidase inhibitor ubenimex inhibits the growth of leukaemic cell lines and myeloma cells through its cytotoxicity.

We investigated the effect of ubenimex, which has been demonstrated to have immunomodulating activities, on the proliferation of human leukaemic cell lines (HL 60 and K 562, myelogenic lines, Jurkat, a T-cell line and RPMI 8226, a multiple myeloma cell line) and on human bone marrow mononuclear cells from haematopoietic malignancy patients with acute myeloblastic leukaemia, chronic lymphocytic leukaemia or multiple myeloma. The growth of the myeloid cell lines and of a multiple myeloma cell line, but not the T-cell line, was significantly inhibited by cocultivation with ubenimex in a dose-dependent manner. The results of the experiment with haematopoietic malignancy cells showed inhibitory effects on tumour cells from patients with acute myeloblastic leukaemia and multiple myeloma, but not on those from patients with chronic lymphocytic leukaemia. These findings suggest the usefulness of ubenimex for the treatment of multiple myeloma.

Rhabdomyosarcoma in a patient with mosaic Klinefelter syndrome and transformation of immature teratoma.

A 27-year-old man was found to have a mediastinal tumour and the histological diagnosis was immature teratoma. Remission was achieved by chemotherapy and total resection. However, he developed anaemia and leukoerythroblastosis after 2 years of remission, and was referred to our hospital. Rhabdomyosarcoma cells were detected in the bone marrow and pleural effusion. Moreover, karyotype analysis of peripheral blood and bone marrow cells revealed mosaic-type Klinefelter syndrome. We diagnosed the case as transformation of teratoma into rhabdomyosarcoma in Klinefelter syndrome. Although intensive chemotherapy was performed, the patient died with meningeal infiltration.

[Myelodysplastic syndrome accompanied by i(17) (q10) anomaly following pure red cell aplasia and transient myeloproliferative stage].

A 71-year-old man was given a diagnosis of pure red cell aplasia (PRCA) in May 1995. However, immunosuppressive agents, including prednisolone, azathioprine, and cyclosporin A, were not effective, and he required frequent red cell transfusions. In September 1995, leukocytosis and thrombocytosis developed (peaking at 10,100/microliter white cells and 98.1 x 10(4)/microliter platelets, respectively, in November 1996). Conversely, the patient's peripheral blood count began to decrease in July 1996, and pancytopenia progressed thereafter i(17) (q10) chromosomal abnormality of bone marrow cells was detected in November 1996. The patient was readmitted due to the progression of thrombocytopenia (1.2 x 10(4)/microliter). His bone marrow has 16.6% blasts, and a diagnosis of myelodysplastic syndrome (MDS) was made. The patient died in November 1997. His hematological state demonstrated significant changes in a relatively short period and severe hypoerythropoiesis and eosinophilia of the bone marrow persisted throughout the clinical course. These findings suggested that a common deranged stem cell was the origin of 3 different states; PRCA, chromic myeloproliferative disorder, and MDS. The i(17) (q10) anomaly may have caused the acute proliferation of blasts and pancytopenia.

[Autoimmune neutropenia accompanied by Tolosa-Hunt syndrome].

A 47-year-old woman presented with severe neutropenia accompanied by diplopia and orbital pain. Her bone marrow was normal except for the absence of segmented neutrophils. Because the administration of granulocyte colony-stimulating factor (G-CSF) at a dose of 1 microgram/kg/day was not sufficiently effective and neutropenia developed, the patient was admitted to our hospital. Physical examination revealed painful ophthalmoplegia and hypoalgesia in the first region of trigeminal nerve, suggestive of Tolosa-Hunt syndrome. Severe neutropenia was observed in both peripheral blood and bone marrow, together with mild anemia and thrombocytopenia. The life span of red cells and platelets was shortened. High PAIgG levels, a positive Coombs test, and a positive test for anti-NA1 antibody suggested that blood cells were being destroyed by an autoimmune mechanism. Corticosteroid hormone therapy preceded by the administration of G-CSF at 5 micrograms/kg/day was effective for both neutropenia and in improving the patient's neurological findings.

Fulminant, CMV-associated, haemophagocytic syndrome following unrelated bone marrow transplantation.

We report a case of haemophagocytic syndrome (HPS) occurring after allogeneic bone marrow transplantation (BMT) for acute promyelocytic leukaemia (APL) in a patient in fourth complete remission (CR). Anti-cytomegalovirus (CMV) antibody (Ab) was negative in this patient before BMT. BMT was performed from an HLA-identical unrelated donor who was positive for CMV Ab. After bone marrow engraftment and haematological recovery, severe acute graft-versus-host disease (GVHD) developed. This patient was treated with methylprednisolone in addition to cyclosporin A (CsA). Acute GVHD showed partial improvement, but CMV antigenaemia was observed. Despite administration of gancyclovir and immunoglobulin, CMV antigenaemia showed no improvement and HPS developed. As no other infections or malignancies were observed, we suspect that CMV infection was the trigger for development of HPS.

Changes in granulocyte-macrophage colony formation in relation to chemotherapy and clinical progress in acute myeloblastic leukaemia patients.

To investigate the correlation between granulocyte-macrophage colony formation and the prognosis of acute myelocytic leukaemia, an in vitro colony formation assay using a practical method was performed at diagnosis in 50 patients newly diagnosed with acute myelocytic leukaemia. Granulocyte-macrophage colony counts were significantly lower in acute myelocytic leukaemia patients than in the control group (n = 5). The diminished colony formation was restored to within the normal range in 15 patients after complete remission was achieved. In eight patients, serial evaluation of granulocyte-macrophage colony formation was performed, and suppression of colony formation was observed at relapse. The comparison of granulocyte-macrophage colony counts at diagnosis between a group of patients with complete remission (n = 26) and a group of treatment failure cases (n = 24) revealed a significant difference suggesting that high counts of granulocyte-macrophage colony formation are predictive of a favourable prognosis for acute myelocytic leukaemia.

Changes in fluorescence energy transfer between sulfhydryl fluorescent residues during ouabain sensitive Na+,K+-ATP hydrolysis.

Na+,K+-ATPase from pig kidney was sequentially modified with two different sulfhydryl fluorescent reagents, N-[p-(2-benzimidazolyl)phenyl]maleimide (BIPM) and N-[7-dimethylamino 4-coumarinyl]maleimide (DACM). The preparation thus obtained contained 3 and 2 moles of each residue in the alpha-chain. When the BIPM residues were excited at 313 nm, ouabain sensitive decrease and increase in the fluorescence intensity at not only 365 nm (BIPM fluorescence) but also 455 nm (DACM fluorescence) were observed, which were dependent on the amounts of reaction intermediates accumulated. When DACM residues were excited directly at 390 nm, only the decrease in the fluorescence intensity was observed irrespective of the intermediates accumulated. The data suggest that at least two DACM residues which differently change their microenvironments during ouabain sensitive Na+,K+-ATPase reaction are present. One is located close enough and the other is located too far to accept the energy from BIPM residue(s) in the three dimensional structure of Na+,K+-ATPase. Addition of sodium dodecyl sulfate (SDS) remarkably inhibited the energy transfer from BIPM to DACM residues. Limited proteolysis suggested that BIPM residues are located mainly in the peptides which are assumed to contain ATP binding sites and that DACM residues are located near the phosphorylation sites.