Nobuo Nara

Expression of Notchl and Jaggedl Proteins in Acute Myeloid Leukemia Cells

Cell fate of hematopoietic progenitors is regulated by interaction between Notch proteins on progenitors and Notch ligands such as Jaggedl on stromal cells. Since acute myeloid leukemia (AML) originates from dysregulated hematopoietic progenitors, some abnormalities in the Notch-Jagged system may exist in AML cells. As the first step to clarify this, we examined the expression of Notchl and Jaggedl proteins in eight AML cell lines and 15 fresh AML samples by immunoblotting. In the Notchl protein, two bands, a 300 kDa band and a 120 kDa band, which appeared to be a full-length protein and a transmembrane fragment, respectively, were recognized in five AML cell lines and six fresh samples. In addition, three of the five cell lines showed a 110 kDa fragment, which appeared to be from an intracellular domain, namely an active form. One cell line showed aberrant sized fragments, which suggested a structural abnormality. Jaggedl protein was recognized in six cell lines and six fresh samples. In four cell lines and four fresh samples, both Notchl and Jaggedl proteins were observed. In these cells, Notchl and Jaggedl proteins may interact among themselves. We showed that Notchl and Jaggedl proteins are widely expressed in AML cells. We hypothesize that some abnormalities in the Notch-Jagged system which cause the excessive self-renewal and the block of differentiation, may be involved in the abnormal proliferation of AML cells.

Priming with G-CSF effectively enhances low-dose Ara-C-induced in vivo apoptosis in myeloid leukemia cells.

We investigated the role of apoptosis in chemotherapy for hematologic malignancies. Twelve consecutive patients with acute myelogenous leukemia (AML) or refractory anemia with excess of blasts in transformation (RAEB-t) who were not tolerable for standard-dose chemotherapy were treated with CAG regimen (low-dose cytosine arabinoside [Ara-C] plus aclarubicin with concurrent administration of granulocyte colony-stimulating factor [G-CSF]). Bone marrow mononuclear cells obtained before the commencement of the chemotherapy were cultured with various concentrations (0-10(-5) M) of Ara-C in the presence or absence of 10 ng/mL of G-CSF, and the resultant cell proliferation/cytotoxicity was assayed. In all but one patient, half killing concentration (LC50) of Ara-C was significantly reduced in the presence of G-CSF (by 400- and 1.45-fold, median: 21-fold). Furthermore, LC(50) values in responders assayed in the presence of 10 ng/mL of G-CSF were significantly lower than those in nonresponders (p = 0.02). In vitro killing tests using a G-CSF-dependent leukemic cell line suggested that addition of G-CSF potentiates Ara-C-induced cytotoxicity through the mechanism of apoptosis. We thus assayed apoptosis in peripheral blood leukemic cells during CAG chemotherapy by flow cytometry using 7-amino-actinomycin D. Peak percentages of apoptosis in responders were significantly higher than those in nonresponders (p = 0.02). These results collectively suggest that apoptosis plays an important role for eradicating leukemic cells by CAG chemo-therapy.

Effects of verapamil on the cellular accumulation of daunorubicin in blast cells and on the chemosensitivity of leukaemic blast progenitors in acute myelogenous leukaemia

Summary. Verapamil, a calcium channel blocker, was studied for its effects on the cellular daunorubicin (DNR) accumulation in blast cells and on the sensitivity of the blast progenitors to DNR in 30 acute myelogenous leukaemia (AML) patients. Using flow cytometry, verapamil was shown to increase the accumulation of DNR in blast cells. The effect was more prominent in the patients who showed poorer response to chemotherapy including DNR. The per cent increases of DNR content by verapamil were 6·4 ± 6·3% and 19·5 ± 23·1% in the 16 responders and the 14 nonresponders, respectively (P < 0·05). The data suggest the presence of enhanced efflux of DNR in nonresponders. Marked variation in the effects of verapamil among nonresponders suggests the heterogeneity of the mechanisms of drug resistance involved. Verapamil also enhanced the sensitivity of blast progenitors to DNR. The degree of increase of cellular DNR accumulation by verapamil correlated with the degree of increase in chemosensitivity of blast progenitors (nonresponders, P < 0·005; responders, P < 0·05). We conclude that enhanced efflux of DNR in blast progenitors may be related to remission induction failure in at least some of resistant AML patients.

Autoantibody against platelet glycoprotein II b/ III a in a patient with non-Hodgkin's lymphoma

An autoantibody to platelet glycoprotein (GP) II b/III a was produced in a 38 year-old woman who had a previous history of the malignant lymphoma of the stomach. The aggregations of the patients platelets showed losses of the primary waves in response to ADP and epinephrine and marked hypoaggregation in response to collagen, while agglutination by ristocetin was normal. Crossed immuno-electrophoresis (CIE) of her platelets solubilized by 1% Triton X-100 revealed an abnormal biphasic precipitate line of GP II b/III a complex. Nine months later, she developed severe thrombocytopenia along with a relapse of the lymphoma in the cervical lymph nodes. The patients IgG, which was collected during her thrombocytopenic period and purified, inhibited ADP-, epinephrine- and collagen-induced aggregations of normal platelets. In CIE, the 125I-labelled IgG of the patient, inserted into the intermediate gel, was incorporated into the precipitation line of the GP II b/III a complex of normal platelets. Radiation treatment to the cervical lymph nodes dramatically normalized both the function and the count of the patients platelets. From these findings, it is suggested that an autoantibody to the GP II b and/or III a was produced by the lymphoma cells.

NOTCH1 mutations are rare in acute myeloid leukemia

Mutations in the NOTCH1 gene were investigated in 12 primary acute myeloid leukemia (AML) cell samples and eight AML cell lines. Mutations in the genomic DNA were screened using a nested PCR-SSCP analysis and confirmed by direct sequencing. A missense mutation, Pro2439Leu (7316C/T), was found in the PEST domain in one primary AML case. This mutation was different from those previously reported for T-cell acute lymphoblastic leukemia, in which more than half the cases had the mutations. This mutation was not detected in his sample in complete remission, which indicated that the mutation was not a single nucleotide polymorphism. The sample with the mutation expressed the intracellular Notch1 fragment by immunoblotting and HES1 mRNA by reverse transcription-polymerase chain reaction. This is the first paper to present an AML case with NOTCH1 mutation. The precise role of the mutation is to be determined.

Effects of Interleukin‐6 and Granulocyte Colony‐stimulating Factor on the Proliferation of Leukemic Blast Progenitors from Acute Myeloblastic Leukemia Patients

The effects of recombinant human interleukin‐6 (rh IL‐6), which has homology with rh granulocyte colony‐stimulating factor (rh G‐CSF) at the amino acid sequence level, and rh G‐CSF on normal human bone marrow cells, fresh leukemic blast progenitors from 16 acute myeloblastic leukemia (AML) patients, and G‐CSF‐dependent human AML cell line (OCI/AML 1a) were investigated. rh G‐CSF stimulated the proliferation of leukemic blast progenitors from 13 out of 16 AML patients tested. rh IL‐6 stimulated the proliferation of blasts from eight AML patients and enhanced the G‐CSF‐dependent proliferation of the fresh AML blasts from two out of eight patients tested. On the other hand, rh IL‐6 suppressed the blast colony formation from two AML patients and OCI/AML 1a cells and also reduced the G‐CSF‐dependent proliferation of the blast progenitors from one of the two patients and the cell line. rh IL‐6 had no effect on the colony formation of normal granulocyte‐macrophage colony‐forming units (CFU‐GM) with or without rh G‐CSF. Differentiation‐induction by rh IL‐6 was not observed in the fresh AML blasts but was observed in OCI/AML 1a. The effect of IL‐6 on the blast colony formation and G‐CSF‐dependent blast cell growth was complicated and heterogenous among the AML cases; IL‐6 stimulated blast colony formation in some cases and suppressed it in others. The heterogeneity of the response was supposed to be derived from the heterogeneity of the characteristics of AML cells. Although G‐CSF simply stimulated the blast colony formation, IL‐6 had a bimodulatory effect on the proliferation of leukemic blast progenitors from AML patients. IL‐6 might be involved in the regulation of the proliferation of AML cells in vivo as well as in vitro.

A novel cell line derived from de novo acute myeloblastic leukaemia with trilineage myelodysplasia which proliferates in response to a Notch ligand, Delta-1 protein

Summary. A novel human leukaemia cell line, designated TMD7, was established from blast cells of a patient with de novo acute myeloblastic leukaemia with trilineage myelodysplasia (AML/TLD). As seen in the original blast cells, TMD7 cells expressed CD7, CD13, CD33 and CD34 and showed an abnormal karyotype containing −5, −7, −8, der(16)t(10;16)(q22;q13). The cells proliferated without added growth factors. Growth was stimulated with the addition of granulocyte colony‐stimulating factor (G‐CSF), granulocyte–macrophage CSF (GM‐CSF) and interleukin 3. Differentiation was not observed with the addition of various cytokines. As a cell line derived from AML/TLD has not been reported, TMD7 will be a useful tool as a model of AML/TLD cells. Recently, it was reported that the Notch system has crucial roles to regulate the self‐renewal and differentiation of haematopoietic stem cells. We found that TMD7 cells expressed Notch‐1 and Notch‐2 mRNA. The exposure to recombinant Delta‐1 protein, which was one of the Notch ligands, significantly stimulated the growth of TMD7 cells. This is the first human cell line which was shown to proliferate in response to Delta‐1, without artificially expressed Notch protein. Therefore, TMD7 will also be a useful tool to study the mechanism of the Notch–Notch ligand interaction.

Effects of recombinant interleukin 4 on the growth and differentiation of blast progenitors stimulated with G-CSF, GM-CSF and IL-3 from acute myeloblastic leukaemia patients

Summary The effects of human recombinant interleukin 4 (rIL‐4) on the growth of leukaemic blast progenitors were investigated. Cells obtained from 20 acute myeloblastic leukaemia (AML) patients were evaluated using the blast colony assay in methylcellulose and suspension cultures. While rIL‐4 by itself did not show any colony stimulatory activity in the blast colony assay, it suppressed the blast colony formation in methylcellulose stimulated with G‐CSF, GM‐CSF or IL‐3 in 14 patients. In another six patients, rIL‐4 enhanced blast colony growth in four patients or did not show any significant effect with any CSF in two patients. In suspension cultures of 12 cases studied, the effects of rIL‐4 on the clonogenic cell recoveries were essentially similar to the results of the blast colony assay in each case. In three patients, rIL‐4 augmented the differentiation of the leukaemic cells to monocyte lineage. Further, the clinical outcome was significantly different between the patients whose blast progenitors were stimulated by rIL‐4 and the patients whose blast progenitors were suppressed by rIL‐4 (P<0.05): three out of four patients in the former group failed in achieving complete remission (CR), while 12 out of 14 patients in the latter group achieved CR. The results show that the effects of IL‐4 on leukaemic blast progenitors were diverse and the responsiveness to IL‐4 may be correlated with the prognoses of the patients.

Reduction of Granulocyte-Macrophage Progenitor Cells (CFU-C) and Fibroblastoid Colony-Forming Units (CFU-F) by Leukemic Cells in Human and Murine Leukemia

The number of bone marrow granulocyte-macrophage progenitor cells (CFU-C) and fibroblastoid colony-forming units (CFU-F) was significantly smaller in acute leukemia patients. To analyze the mechanism by which CFU-C and CFU-F were reduced, the study on murine myeloid leukemia was carried out. In murine leukemia, bone marrow CFU-C and CFU-F decreased in number in correlation with the proliferation of leukemic cells. Murine leukemic cells suppressed the growth of CFU-C and CFU-F in the mixed culture of leukemic cells and normal murine marrow cells. Furthermore, leukemic cell-conditioned medium inhibited the growth of CFU-F. The results show that murine leukemic cells themselves and/or humoral factors produced by them give inhibitory effects on the growth of not only CFU-C but also CFU-F.

Effect of chest wall vibration on dyspnea during exercise in chronic obstructive pulmonary disease

To elucidate the effect of in-phase chest wall vibration (IPV) during exercise, 17 COPD male patients performed two constant-load exercise tests on a cycle ergometer with and without IPV. The Borg dyspnea score significantly decreased from IPV (-) to IPV (+) (from 13.6+/-2.9 to 12.5+/-2.9, P<0.01). IPV elicited a significant increase in V(O(2)) (P<0.005) and significant decreases in both VE/V(O(2)) (P<0.05) and respiratory frequency (P<0.05), but it did not elicit any changes in VE. The change in Borg score between IPV (+) and IPV (-) showed a significant positive correlation with % predicted V(O(2),max) (r=0.71) and FEV(1)/FVC (r=0.69). Patients in the responsive group (n=11) showed significantly lower FEV(1) (P<0.05) and higher DeltaN(2)/L (P<0.01) than patients in the non-responsive group (n=6). We conclude that IPV reduces dyspnea and improves respiratory efficiency during aerobic exercise in severe COPD.