Genji Yamaoka

Growth Hormone Prevents Fas-Induced Apoptosis in Lymphocytes through Modulation of Bcl-2 and Caspase-3

Objective: Growth hormone (GH) has been reported to have a potent effect on the immune system. However, the detailed mechanism of the effect of GH on the immune system has not yet been clarified. This study was designed to investigate the nature of this mechanism. Methods: In the present study, we investigated the effects of GH on the susceptibility of both human CEM/C7 lymphocytes and human IM-9 lymphocytes to Fas-induced apoptosis. Results: Both cell lines expressed GH receptor mRNA. GH rescued Fas-induced suppression of [3H]-thymidine incorporation into each cell line. GH prevented Fas-induced apoptosis in each cell line without changing Fas antigen expression. We next investigated the mechanisms of the prevention of Fas-induced apoptosis, by focusing on intracellular molecules related to the apoptotic signal. Bcl-2 expression was increased by GH treatment in both CEM/C7 and IM-9 lymphocytes. GH also downregulated caspase-3 expression and inhibited activation of caspase-3 in both cell lines. Conclusion: These findings suggest that GH regulates the human immune system through inhibition of Fas-induced apoptosis in activated T and B lymphocytes.

Specific skin manifestations in CD56 positive acute myeloid leukemia

We found 16 CD56+ cases (29.6%) among 54 acute myeloid leukemia (AML) patients; they showed significantly Frequent cutaneous involvement compared to CD56‐ cases (43.8% vs.15.8%, p<0.05). Four of the CD56+ AML cases with specific skin manifestations were reviewed histologieally. In all cases, cutaneous leukemic cells were seen in the dermis and subcutaneous tissue with accentuation around the adnexa/nerve, but sparing the epidermis. In addition, angiocentric/angiodestructive and prominent cohesive tumor cell growth were seen in two rases, respectively. These findings suggest that the expression of CD56 may often be associated with the cutaneous involvement in AML, and that the above histological findings should remind us of the possibility of specific skin manifestations in CD56+ AML.

Two cases of refractory thrombotic thrombocytopenic purpura associated with collagen vascular disease were significantly improved by rituximab treatment

Thrombotic thrombocytopenic purpura (TTP) is a rare disorder of small vessels. TTP is associated with deficiency of the von Willebrand factor-cleaving protease, ADAMTS13, and its inhibitor. Low ADAMTS13 activity is present in most of idiopathic TTP patients. The prognosis of TTP was improved by plasma exchange treatment, which replaces the ADAMTS13 and removes ADAMTS13 inhibitor. However, ADAMTS13 activity is normal in some TTP patients. These are found among the secondary TTP patients associated with collagen disease, hematopoietic stem cell transplantation, malignancy, or drugs. In addition, most of them do not respond to plasma exchange. On the other hand, several reports demonstrated that rituximab, which is an anti-CD20 monoclonal antibody, is effective for refractory TTP cases caused by ADAMTS13 deficiency. It is considered that the effect of rituximab is associated with disappearance of ADAMTS13 inhibitor. However, rituximab therapy was effective for the TTP patients with normal ADAMTS13 activity in our cases. We considered another mechanism of rituximab for TTP cases.

A novel mature B‐cell line (DOBIL‐6) producing both parathyroid hormone‐related protein and interleukin‐6 from a myeloma patient presenting with hypercalcaemia

A novel human EBV‐negative B‐cell line, designated DOBIL‐6, was established from a patient with non‐secretary myeloma. The DOBIL‐6 cell has cytoplasmic γ protein and expresses CD19, 20, 38, 45RO, VLA‐4 and PCA‐1 antigens, but lacks CD10, 45RA and VLA5 antigens. Chromosome analysis showed that DOBIL‐6 cells had many complex structural abnormalities, including t(11;14) (q13;q32), which were consistent with that of the fresh tumour cells. Interestingly, abundant interleukin‐6 (IL‐6) and parathyroid hormone‐related protein (PTHrP) accumulated in the culture supernatant of DOBIL‐6 cells. Hypercalcaemia and splenomegaly associated with plasma cell proliferations which resulted in the expansion of the light zones in the follicles were observed in DOBIL‐6 transplanted nude mice. RT‐PCR analysis detected mRNA for PTHrP, and IL‐6 as well as its receptor (GP80) in DOBIL‐6 cells. Treatment of the DOBIL‐6 cells with neutralizing anti‐IL‐6 antibody inhibited their growth in a dose‐dependent manner, whereas the addition of exogenous IL‐6 stimulated it in serum‐depleted conditions. These findings suggest that both IL‐6 and PTHrP are produced in DOBIL‐6 cells, and that IL‐6 promotes its growth by an autocrine mechanism. Since IL‐6 is known to stimulate not only the growth of B‐cell neoplasms but also osteoclastic bone resorption by cooperating with PTHrP, this simultaneous production of IL‐6 and PTHrP might be synergistically linked and play a role in the development of hypercalcaemia of the patient. The DOBIL‐6 cell is a useful tool to clarify the mechanism of hypercalcaemia associated with mature B‐cell neoplasms.

THROMBOPOIETIN MODULATES PLATELET ACTIVATION IN VITRO THROUGH PROTEIN-TYROSINE PHOSPHORYLATION

To determine the roles of thrombopoietin (TPO) in platelet function in vitro, we examined the effects of TPO on platelet aggregation. Although several proteins in platelets were tyrosine‐phosphorylated by TPO treatment, TPO alone was unable to induce platelet aggregation. However, the secondary wave of platelet aggregation induced by adenosine diphosphate (ADP) was enhanced by TPO in a dose‐dependent manner. TPO in conjunction with ADP augmented tyrosine phosphorylation of platelet proteins, including tyrosine‐phosphorylated proteins induced by TPO alone. Genistein inhibited protein‐tyrosine phosphorylation in platelets induced by TPO with ADP and suppressed TPOenhanced platelet aggregation. Moreover, tyrosine phosphorylation of MAP‐kinases induced by TPO alone and TPO with ADP was consistent with TPO‐enhanced platelet aggregation. These findings in the present study suggest that signal transduction involved in TPO‐enhanced platelet aggregation is mediated in part by tyrosine‐phosphorylated proteins, including MAP‐kinases, in platelets through TPO‐stimulated c‐Mpl, TPO receptor.

Alterations of p53 and Rb genes in a novel human GM-CSF-dependent myeloid cell line (OHN-GM) established from therapy-related leukaemia

A novel GM‐CSF‐dependent myeloid cell line, OHN‐GM, was established from a patient who developed acute myelogenous leukaemia (AML) as a consequence of myelodysplastic syndrome (MDS). As the patient had previously received cytotoxic chemotherapy for Hodgkins disease, the MDS and AML were probably related to such therapy. Sequential karyotypic analysis established a del(5q) as the initial cytogenetic abnormality. Additional alterations, including t(10;13)(q24;q14), had developed subsequently during disease progression. Southern blot analysis of OHN‐GM cells suggested deletion of one allele of the IRF‐1 gene, although no aberrant transcripts were detected. Fluorescence in situ hybridization analysis revealed the deletion of the Rb gene due to the t(10;13)(q24;q14) translocation, and Western blot analysis demonstrated the absence of Rb protein in OHN‐GM cells. Finally, the OHN‐GM cells exhibited two missense point mutations in highly conserved regions of the p53 gene. These observations suggest that a multistep process, involving alterations of Rb and p53 genes, may have contributed to the patients disease development and progression. To our knowledge, OHN‐GM is the first cell line derived from a therapy‐related AML. These cells may aid the investigation of leukaemogenesis as well as the biology of secondary leukaemia.

Secondary myeloid/natural killer cell precursor acute leukemia following essential thrombocythemia

The de novo leukemic transformation of essential thrombocythemia is a rare event, and usually associated with previous treatments. We describe a patient who received treatments with nitrosourea for long-standing essential thrombocythemia and subsequently developed extramedullary tumors, tentatively diagnosed as lymphoblastic lymphoma. Combination chemotherapy was initially successful, but relapsed with marked bone marrow involvement. Surface marker analysis revealed that the tumor cells had CD5, CD7, CD33, CD34, and CD56 antigens but lacked other T-cell, and B-cell markers. Immunogenotypical studies revealed germline configurations for both T-cell receptors and immunoglobulin genes. These clinical and phenotypical features are consistent with a myeloid/natural killer cell precursor leukemia, a recently proposed distinct clinical entity. To our knowledge, this is the first report of secondary leukemia of myeloid/ natural killer cell precursor origin, and suggest that myeloid/natural killer cell precursor might be a potent target of therapy-related leukemia.

Lineage switch from precursor B cell acute lymphoblastic leukemia to acute monocytic leukemia at relapse

A lineage switch in leukemia, in which the leukemic cell lineage at onset converts to another lineage at a later time, is an uncommon type of hybrid (mixed) leukemia regarded as a variation of bilineage leukemia. We present a case of a 60-year-old female diagnosed with precursor B cell acute lymphoblastic leukemia (ALL), whose markers in flow cytometry shifted from their original status of CD19+, 22+, 79a+, 13+, HLA-DR+, and TdT+. Although her bone marrow achieved remission after induction therapy, there was a small residual population of leukemic cells in the liver. Residual disease was proved by biopsy and pathologically shown to have an immature phenotype of CD5+, CD10−, CD20−, CD79a− and myeloperoxidase negativity. Two weeks after liver biopsy, blast cells progressively appeared in the peripheral blood; these cells had a monocytoid morphology and phenotype (CD13, 14) but were accompanied by myeloid (CD33) and lymphoid (CD2, 4, 20) cells. Markers CD7, 10 and 19 were negative by flow cytometry. This phenotypical conversion from B-ALL to hybrid leukemia featuring monocytoid characteristics is known as a lineage switch. This case suggests that leukemic subclones tend to carry out de-differentiation, occasionally in extramedullary sites, which serve as a hotbed for the selection of resistant clones.

Establishment and characterization of a Kaposi's sarcoma- associated herpesvirus- and Epstein-Barr virus-negative malignant lymphoma cell line (OHK) with primary effusion lymphoma immunophenotype

Summary. A novel cell line, designated OHK, was established from ascites of a 59‐year‐old Japanese woman with diffuse large B‐cell lymphoma showing a peculiar serosal tropism, as seen in primary effusion lymphomas (PEL). OHK exhibited a large pleomorphic morphology with irregular nuclei and distinct nucleoli, and included immunoblastic and Reed–Sternberg‐like giant cells. On ultrastructural examination, rich intermediate filaments, and well‐developed Golgi apparati and rough endoplasmic reticulum, were seen. Immunophenotypically, OHK lacked T and B cell‐associated antigens, and had CD10, CD30, CD33 and CD138 antigens. Although OHK cells did not express immunoglobulin (Ig) protein, Southern blot analysis demonstrated clonal rearrangements of Ig heavy and light chain genes. These observations suggest that OHK cells are derived from preterminally differentiated B cells, and that they have features of PEL. Kaposis sarcoma‐associated herpesvirus and Epstein–Barr virus were not detected. OHK displayed hyperploid karyotypes with multiple structural abnormalities, and produced some cytokines such as macrophage‐colony‐stimulating factor (M‐CSF), granulocyte‐CSF, interleukin 6 and transforming growth factor β1. In particular, vascular endothelial growth factor (VEGF), whose stimulation of vascular permeability is thought to be critical to the pathogenesis of PEL, was also produced in large quantities. These results indicate that OHK may be a useful tool for the investigation of PEL.

Gab1 transduces PI3K-mediated erythropoietin signals to the Erk pathway and regulates erythropoietin-dependent proliferation and survival of erythroid cells

In this study, we examined the biological functions of Gab1 in erythropoietin receptor (EPOR)-mediated signaling in vivo. Knockdown of Gab1 by the introduction of the Gab1 siRNA expression vector into F-36P human erythroleukemia (F-36P-Gab1-siRNA) cells resulted in a reduction of cell proliferation and survival in response to EPO. EPO-induced activation of Erk1/2 but not of Akt was significantly suppressed in F-36P-Gab1-siRNA cells compared with mock-transfected F-36P cells. The co-immunoprecipitation experiments revealed an EPO-enhanced association of Gab1 with the Grb2-SOS1 complex and SHP-2 in F-36P cells. A selective inhibitor of phosphatidylinositol 3-kinase (PI3K) LY294002 and short interfering RNA (siRNA) duplexes targeting the p85 regulatory subunit of PI3K (p85-siRNA) independently suppressed tyrosine phosphorylation of Gab1; its association with Grb2, SHP-2 and p85; and the activation of Erk in EPO-treated F-36P cells. LY294002 inhibited EPO-induced tyrosine phosphorylation of Gab1 and its association with Grb2 in human primary EPO-sensitive erythroid cells. The co-immunoprecipitation experiments using the Jak inhibitor AG490 or siRNA duplexes targeting Jak2 and in vitro binding experiments demonstrated that Jak2 regulated Gab1-mediated Erk activation through tyrosine phosphorylation of Gab1. Taken together, these results suggest that Gab1 couples PI3K-mediated EPO signals with the Ras/Erk pathway and that Gab1 plays an important role in EPOR-mediated signal transduction involved in the proliferation and survival of erythroid cells.