Shuji Tohda

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.

Expression and function of the Delta-1/Notch-2/Hes-1 pathway during experimental acute kidney injury

The Notch signaling pathway consists of several receptors and their ligands Delta and Jagged and is important for embryogenesis, cellular differentiation and proliferation. Activation of Notch receptors causes their cleavage yielding cytoplastic domains that translocate into the nucleus to induce target proteins such as the basic-loop-helix proteins Hes and Hey. Here we sought to clarify the significance of the Notch signaling pathway in acute kidney injury using a rat ischemia-reperfusion injury model and cultured NRK-52E cells. Analysis of the whole kidney after injury showed increased expression of Delta-1 and Hes-1 mRNA and protein along with processed Notch-2. Confocal microscopy, using specific antibodies, showed that Delta-1, cleaved Notch-2 and Hes-1 colocalized in the same segments of the injured renal proximal tubules. Recombinant Delta-1 significantly stimulated NRK-52E cell proliferation. Our study suggests that the Delta-1/Notch-2/Hes-1 signaling pathway may regulate the regeneration and proliferation of renal tubules during acute kidney injury.

Human Vδ1 γδ T cells expanded from peripheral blood exhibit specific cytotoxicity against B-cell chronic lymphocytic leukemia-derived cells

BACKGROUND AIMS There is increasing interest in using γδ T cells (GDTC) for cancer immunotherapy. Most studies have been concerned with the Vδ2 subset in blood, for which several expansion protocols exist. We have developed a protocol to expand Vδ1 and Vδ2 preferentially from human blood. We have characterized these subsets and their specificities for leukemic targets. METHODS GDTC were isolated from the peripheral blood mononuclear cells (PBMC) of healthy donors via positive magnetic cell sorting; their proliferation in vitro was induced by exposure to the mitogen concanavalin A (Con A). CD107 and cytotoxicity (Cr(51)-release and flow cytometric) assays were performed. GDTC clones and target cells were immunophenotyped via flow cytometry. RESULTS Longer initial exposure to Con A typically resulted in higher Vδ1 prevalence. Vδ1 were activated by and cytotoxic to B-cell chronic lymphocytic leukemia (B-CLL)-derived MEC1 cells, whereas Vδ2 also responded to MEC1 but more so to the Philadelphia chromosome-positive [Ph+] leukemia cell line EM-enhanced green fluorescent protein (2eGFPluc). Vδ2 clone cytotoxicity against EM-2eGFPluc correlated with Vδ2 T-cell antigen receptor (TCR) and receptor found on Natural Killer cells and many T-cells (NKG2D), whereas Vδ1 clone cytotoxicity versus MEC1 correlated with Vδ1 TCR, CD56 and CD95 expression. Vδ1 also killed Epstein-Barr Virus (EBV)-negative B-CLL-derived TMD2 cells. Immunophenotyping revealed reduced HLA-ABC expression on EM-2eGFPluc, whereas MEC1 and TMD2 exhibited higher Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAILR1). CONCLUSIONS Our ability to expand peripheral Vδ1 cells and show their cytotoxicity to B-CLL-derived cell lines suggests that this novel approach to the cellular treatment of B-CLL may be feasible.

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.

Establishment of a double Philadelphia chromosome-positive acute lymphoblastic leukemia-derived cell line, TMD5: effects of cytokines and differentiation inducers on growth of the cells.

A double Philadelphia chromosome (Ph)-positive leukemia cell line with common-B cell phenotype, designated TMD5, was established from the blast cells of a patient with double Ph-positive acute lymphoblastic leukemia. TMD5 cells expressed 190 kDa BCR/ABL chimeric protein and 145 kDa ABL protein. The cells proliferated without added growth factors. Autocrine growth mechanism was not recognized. The addition of growth factors such as G-CSF, GM-CSF, IL-3, IL-6, or Stem Cell Factor did not affect the growth. Herbimycin A suppressed the growth of TMD5 cells at the low concentration that did not affect Ph-negative cells. It suppressed tyrosine phosphorylation of intracellular proteins in TMD5 cells. Dexamethasone and dibutyryl cyclic AMP also suppressed the growth. They, however, did not affect the phosphorylation significantly. Neither all-trans retinoic acid nor interferon-alpha affected the growth. TMD5 cells, characterized minutely here and rare in that they have double Ph chromosomes, will be a useful tool for the study of Ph-positive leukemia.

Negative feedback regulation of colitogenic CD4+ T cells by increased granulopoiesis

Background: Chronic inflammatory diseases are characterized by massive infiltration of innate and acquired immune cells in inflammatory sites. However, it remains unclear how these cells cooperate in the development of disease. Although bone marrow (BM) is a primary site for hematopoiesis of immune cells except T cells, BM recruits memory T cells from the periphery. We have recently demonstrated that colitogenic CD4+ memory T cells reside in BM of colitic CD4+CD45RBhigh T‐cell‐transferred SCID mice. Based on this background we here investigate whether granulocytes promote or suppress the expansion of colitogenic CD4+ T cells. Methods: First, we show that Gr‐1highCD11b+ granulocytes were significantly increased in colitic BM along with a significant increase of peripheral granulocytes. Consistently, the colony‐forming unit (CFU) assay revealed that granulocyte colony formation was dominantly induced by supernatants from anti‐CD3‐stimulated colitic BM CD4+ T cells. Results: Administration of granulocyte‐depleting anti‐Gr‐1 mAb to colitic mice did not ameliorate the colitis, but exacerbated the wasting disease with an increased expansion of systemic, but not lamina propria, CD4+ T cells with activated phenotype. Conclusions: These results suggest that the increased granulopoiesis by colitogenic BM CD4+ T cells represent a negative feedback mechanism to control systemic inflammation.

ITPA gene variation and ribavirin-induced anemia in patients with genotype 2 chronic hepatitis C treated with sofosbuvir plus ribavirin

Sofosbuvir (SOF) and ribavirin (RBV) combination therapy produces a sustained response in many patients with genotype 2 chronic hepatitis C. However, RBV‐induced anemia is a troublesome side‐effect that may limit this treatment. Genetic variation leading to inosine triphosphatase (ITPA) deficiency is known to protect against RBV‐induced hemolytic anemia. This study aimed to evaluate the relationships between the efficacy and safety of SOF/RBV treatment and ITPA gene variants.