Hiroshi Tsutani

The role of protein-tyrosine phosphorylation and gelatinase production in the migration and proliferation of smooth muscle cells.

BACKGROUND It has been reported that matrix metalloproteinase (MMP) was expressed in coronary arterial atherosclerotic lesions. However, not much is known about the relationship between the production of MMP and the progression of atherosclerosis. PURPOSE AND METHOD To demonstrate the association between the protein-tyrosine phosphorylation (PTP) and the activation of extracellular MMP in the proliferation and migration of vascular smooth muscle cells (VSMCs), the effect of platelet-derived growth factor (PDGF) and vanadate (an inhibitor of protein-tyrosine phosphatase and an activator of certain protein-tyrosine kinases) on mitogenesis ([3H]thymidine incorporation after 24 hours), migration, PTP (Western blot analysis using anti-phosphotyrosine antibodies), and production of MMP (gelatin zymography) was examined in cultured VSMCs. RESULTS Both vanadate (1-5 micromol/l) and PDGF (1-10 ng/ml) caused a dose-dependent increase in thymidine incorporation and migration and produced 72-kDa type IV gelatinase (MMP-2) in VSMCs. The combination of vanadate and PDGF resulted in a dose-dependent synergistic effect on thymidine incorporation and MMP-2 production. Western blot analysis revealed that PDGF caused an increase in PTP, extracellular signal-regulated kinases (ERK1, ERK2) and PDGF receptor in VSMCs. Vanadate given together with PDGF induced a marked increase in the intensity of tyrosine phosphorylation in these proteins. Tyrosine kinase inhibitors (genistein and herbimycin A) and a synthetic inhibitor of MMP (1,10-phenanthroline) and an anti-MMP-2 neutralizing antibody inhibited the mitogenic effect induced by vanadate and/or PDGF. CONCLUSIONS The data suggest that the proliferation and migration of cultured VSMCs was closely related to the stimulation of MMP-2 production that was induced through activation of PTK.

Establishment and characterization of a novel myeloid cell line from the bone marrow of a patient with the myelodysplastic syndrome

SUMMARY. A novel long‐term cultured myeloid cell line was established from the bone marrow of a patient with myelodysplastic syndrome (MDS). This cell line, designated MDS92. proliferated in the presence of interleukin‐3 or granulocyte‐macrophage colony‐stimulating factor and transiently in the presence of Steel factor, with a tendency for gradual maturation, and formed myeloid colonies in the semi‐solid culture condition.

Reduction of leukemia cell growth in a patient with acute promyelocytic leukemia treated by retinol palmitate

A 67-year-old woman with acute promyelocytic leukemia (APL) showed a marked decrease in leukemic promyelocytes with concomitant maturation of leukemic cells during treatment with retinol palmitate. A culture study in vitro revealed that retinol, which is the main metabolite of retinol palmitate detected in plasma, induced morphological and functional maturation of leukemic promyelocytes. These findings may indicate that retinol palmitate induces cell differentiation and slows proliferation of leukemic cells in vivo, and that the reduction in cell growth is the key phenomenon in the clearing of leukemic cells, rather than the maturation phenomenon itself.

Myeloid differentiation antigen and cytokine receptor expression on acute myelocytic leukaemia cells with t(16;21)(p11;q22): frequent expression of CD56 and interleukin‐2 receptor α chain

We report the cellular characteristics of cells from three patients with de novo acute myelocytic leukaemia (AML) with t(16;21)(p11;q22), two M4 and one M5a according to the FAB classification, and two permanent cell lines with t(16;21)(p11;q22), TSU1621MT and YNH‐1. The FUS/ERG fusion mRNA was demonstrated in all cases by reverse transcriptase‐polymerase chain reaction (RT‐PCR). The immunophenotypes of the AML cells, and YNH‐1 and TSU1621MT cell lines with t(16;21) were characterized as CD34+CD33+CD13+CD11b+CD18+CD56+ HLA‐DR−/+. Cells from all samples strongly expressed c‐kit, granulocyte colony‐stimulating factor receptor (G‐CSFR), c‐fms (macrophage colony‐stimulating factor receptor), interleukin‐3 receptor α chain (IL‐3Rα), and granulocyte macrophage colony‐stimulating factor receptor α chain (GM‐CSFRα), and these data corresponded well to the growth responsiveness to the cytokines. IL‐2Rα expression was also found in all t(16;21) samples, but IL‐2 did not act on the proliferation of the leukaemic cells in in vitro cultures. G‐CSF distinctly promoted the proliferation of leukaemic cells of t(16;21) AML, but did not enhance the expression of MPO and neutrophil differentiation of these cells. Our findings indicate that AML cells with t(16;21) preserve stem cell properties such as CD34 and c‐kit expression, and suggest that they have the potential to differentiate into a monocytic lineage. The relationship between the unique cellular characteristics (especially CD56 and IL‐2Rα expression) and FUS/ERG protein remains undetermined.

Pharmacological studies of retinol palmitate and its clinical effect in patients with acute non-lymphocytic leukemia

The pharmacokinetics of retinol palmitate were studied, and a therapeutic trial was performed in patients with ANLL. In the pharmacokinetics study, retinol was the only metabolite that was detected in plasma, and the peak concentration was 332 micrograms/dl (about 1.2 x 10(-5) M) 2.1 h after administration of retinol palmitate. Five patients with ANLL (4 with ANLL-M3 and one with ANLL-M2) were treated with retinol palmitate. In all patients with ANLL-M3, bone marrow suspension culture studies revealed that retinol induced both morphological and functional differentiation of immature leukemic cells. During the course of the treatment with retinol palmitate, morphological differentiation of bone marrow immature leukemic cells was observed in all patients with ANLL-M3 within 3-4 days after initiation of the therapy. In three of the four patients who underwent conventional chemotherapy, the sequential treatment with retinol palmitate resulted in a complete remission: controlling residual bone marrow leukemic cells. None of the patients showed any signs of aggravation of DIC in the coagulation parameters. These findings suggest the possibility that retinol palmitate functions as salvage therapy by inducing maturation and slowing proliferation, there by clearing out the residual leukemic cells following conventional chemotherapy.

Differentiation and Reduction of Intracellular GTP Levels in Hl-60 and U937 Cells Upon Treatment with IMP Dehydrogenase Inhibitors

Inosine-5′-monophosphate dehydrogenase (IMP dehydrogenase, EC 1.1.1.205) is a rate-limiting enzyme of de novo guanylate biosynthesis and catalyzes the formation of XMP from IMP (1). IMP dehydrogenase have two isoforms. Type I transcript is the major form found in normal leukocytes (2), whereas type II is predominant in malignant cells (3), thus type II IMP dehydrogenase is a target for antineoplastic agents. In addition, a variety of human leukemic cells, including K562 erythroid leukemia cells (4), and HL-60 myeloid leukemia cells (5) as well as human breast cancer cells (6) which are treated with IMP dehydrogenase inhibitors, decreases in type II IMP dehydrogenase and the level of guanine nucleotide, especially GTP, are shown to be associated with cell maturation (7,8).

Reduced intensity allogeneic stem cell transplantation for systemic primary amyloidosis refractory to high-dose melphalan.

Complete elimination of the plasma cell dyscrasia is a rational therapeutic goal, as intercepting supply of precursor protein is a necessary condition for a major regression of amyloid deposits. High‐dose melphalan with autologous stem cell transplantation has shown the ability to induce complete hematological response (HR) along with recovery of organ dysfunction. However, the rate of HR with this treatment rarely exceeds 40%. We describe here the first known case of successful reduced intensity allogeneic stem cell transplantation (RIST) for a patient with primary amyloidosis complicated with nephrotic syndrome but without cardiac disease, who had obtained only partial HR by high‐dose melphalan with autologous stem cell transplantation. RIST may be feasible and be capable of achieving complete HR along with recovery from nephrotic syndrome with acceptable toxicity.

Induction of Cell Differentiation by IMPDH Antisense Oligomer in HL-60 and K562 Human Leukemia Cell Lines

Inosine-5’-monophosphate dehydrogenase (IMPDH, EC 1.1.1.205) is the enzyme that catalyzes the formation of xanthosine-5’-monophosphate from inosine-5’-monophosphate (IMP). In the purine de novo synthetic pathway, IMPDH is positioned at the branch point in the synthesis of adenine and guanine nucleotides and is thus the rate-limiting enzyme in the de novo synthesis of guanine nucleotides. Alterations in the activity of IMPDH and the levels of guanine nucleotides have been implicated in the regulation of cell growth and differentiation[1]. The addition of IMPDH inhibitors, such as mycophenolic acid and tiazofurin, to cultures induces terminal differentiation in a variety of human leukemia cells, including K562 erythroid leukemia cells, and HL-60 myeloid leukemia cells as well as human breast cancer cells and melanoma cells[2–4]. To verify the role of this enzyme in controlling differentiation, we determined the ability of specific IMPDH antisense oligomers to induce maturation in the K562 and HL-60 human leukemia cell lines. From these experiments, we concluded that a specific reduction in IMPDH results in inhibition of cell replication and induction of terminal differentiation in the two human leukemia cell lines.

Monitoring of intracellular 1-beta-D-arabinofuranosylcytosine 5'-triphosphate in 1-beta-D-arabinofuranosylcytosine therapy at low and conventional doses.

1‐β‐D‐Arabinofuranosylcytosine (ara‐C) is used empirically at a low, conventional, or high dose. Ara‐C therapy may be optimal if it is directed by the clinical pharmacokinetics of the intracellular active metabolite of ara‐C, 1‐β‐D‐arabinofuranosylcytosine 5′‐triphosphate (ara‐CTP). However, ara‐CTP has seldom been monitored during low‐ and conventional‐dose ara‐C therapies because detection methods were insufficiently sensitive. Here, with the use of our newly established method (Cancer Res., 56, 1800‐1804 (1996), ara‐CTP was monitored in leukemic cells from acute myelog‐enous leukemia patients receiving low‐ or conventional‐dose ara‐C [subcutaneous ara‐C administration (10 mg/m2) (3 patients), continuous ara‐C infusion (20 or 70 mg/m2/24 h) (7 patients), 2‐h ara‐C infusion (70 mg/m2) (4 patients), and 2‐h infusion of N4‐behenoyl‐l‐β‐D‐arabinofuranosylcy‐tosine, a deaminase‐resistant ara‐C derivative (70 mg/m2) (6 patients)]. Ara‐CTP could be determined at levels under 1μM. There was a close correlation between the elimination half‐life values of the plasma ara‐C and the intracellular ara‐CTP. The presence of ara‐C in the plasma was important to maintain ara‐CTP. The continuous ara‐C and the 2‐h N4‐behenoyl‐l‐β‐D‐arabinofura‐nosylcytosine infusions maintained ara‐CTP and the plasma ara‐C longer than the subcutaneous ara‐C or the 2‐h ara‐C infusion. They also afforded relatively higher ara‐CTP concentrations, and consequently produced ara‐CTP more efficiently than the 2‐h ara‐C infusion. Different administration methods produced different quantities of ara‐CTP even at the same dose.

Successful treatment of Aspergillus spondylodiscitis with high-dose itraconazole in a patient with acute myelogenous leukemia.

Successful treatment of Aspergillus spondylodiscitis with high-dose itraconazole in a patient with acute myelogenous leukemia