Ritsuo Nishiuchi

Activated Proliferation of B-Cell Lymphomas/Leukemias with the SHP1 Gene Silencing by Aberrant CpG Methylation

Previously we showed reduced protein and mRNA expression of the SHP1 gene in lymphoma/leukemia cell lines and patient specimens by Northern blot, RT-PCR, Western blot, and immunohistochemical analyses. In this study, aberrant methylation in the SHP1 gene promoter was detected in many B-cell leukemia/lymphoma cell lines as well as in patient specimens, including diffuse large B-cell lymphoma (methylation frequency 93%), MALT lymphoma (82%), mantle cell lymphoma (75%), plasmacytoma (100%) and follicular lymphoma (96%) by methylation-specific PCR, bisulfite sequencing, and restriction enzyme-mediated PCR analyses. The methylation frequency was significantly higher in high-grade MALT lymphoma cases (100%) than in low-grade MALT lymphoma cases (70%), which correlated well with the frequency of no expression of SHP1 protein in high-grade (80%) and low-grade MALT lymphoma (54%). It suggests that the SHP1 gene silencing with aberrant CpG methylation relates to the lymphoma progression. SHP1 protein expression was recovered in B-cell lines after the treatment of the demethylating reagent: 5-aza-2′-deoxycytidine. Transfection of the intact SHP1 gene to the hematopoietic cultured cells, which show no expression of the SHP1 gene, induced growth inhibition, indicating that gene silencing of the SHP1 gene by aberrant methylation plays an important role to get the growth advantage of the malignant lymphoma/leukemia cells. The extraordinarily high frequency (75 to 100%) of CpG methylation of the SHP1 gene in B-cell lymphoma/leukemia patient specimens indicates that the SHP1 gene silencing is one of the critical events to the onset of malignant lymphomas/leukemias as well as important implications for the diagnostic or prognostic markers and the target of gene therapy. These data support the possibility that the SHP1 gene is one of the tumor suppressor genes.

Clonal analysis by polymerase chain reaction of B-cell lymphoma with late relapse: A report of five cases

Little is known about the clonal heterogeneity of non‐Hodgkins lymphoma between presentation and relapse, although several such reports have been published on acute lymphoblastic leukemia.

Expression of Fas ligand mRNA in germinal centres of the human tonsil

Fas ligand (FasL), a cell surface molecule belonging to the tumour necrosis family, induces apoptosis through its receptor, Fas antigen (Fas). Germinal centre B cells strongly express Fas, but the role of the Fas–FasL system in B‐cell selection in the germinal centre remains unclear. In the present study, FasL mRNA in the tonsils was examined by RNA in situ hybridization. FasL mRNA was detected in the lymphocytes of both germinal centres and interfollicular areas, but much more intensively in the former. The distribution of cells strongly expressing FasL mRNA in the germinal centres was quite similar to that of CD45RO‐positive T cells. Immunohistochemically, however, most of the germinal centre cells were positive for FasL. Flow cytometric analysis demonstrated that FasL‐positive cells of the tonsils included not only CD3‐positive/CD45RO‐positive T cells, but also CD19‐positive B cells. This finding therefore suggests either that germinal centre B cells can produce FasL, although the level of mRNA was equivocal, or that the soluble form of FasL may be released from FasL‐positive T cells in the germinal centres and then bind to Fas‐positive germinal centre B cells. Thus, the Fas–FasL system may participate in the positive selection of B cells.

The Fas antigen is detected on immature B cells and the representative cell lines show Fas‐mediated apoptosis

The expression and functions of Fas antigen, a major regulator of apoptosis, in T‐cell selection have been intensively investigated, but little is known about its expression in immature B cells which are also selected in the bone marrow, and plasma cells which are at the terminal stage of B‐cell differentiation and are designed to die. We examined bone marrow cells and found Fas antigen on these cells at low levels. Next, we analysed Fas expression and susceptibility to anti‐Fas antibody‐mediated apoptosis on B‐cell lines representing various stages of differentiation. We also examined the expression of Bcl‐2 and Bax on these lines, which were intimately related to apoptosis. Fas antigen was almost negative on pre‐pre‐B cell lines and was detected on pre‐B‐cell lines at low levels. All plasma cell lines expressed Fas at a low to moderate level. Some cell lines with peripheral B‐cell phenotype expressed Fas antigen. Except for the Burkitt cell lines and one plasma cell line, susceptibility to Fas‐mediated apoptosis depended on Fas expression. Bcl‐2 protein was detected on all but one cell line and Bax was detected on 15/23 lines, but neither were related to cellular differentiation or Fas expression.

Induction and characterization of cutaneous lymphocyte antigen on natural killer cells.

Summary. Cutaneous lymphocyte antigen (CLA) has been reported to be expressed mainly by memory/effector T lymphocytes infiltrating inflammatory skin lesions and cutaneous T‐cell lymphoma. It has been suggested that CLA is a specific homing receptor, facilitating the T‐cell migration into skin lesions, and also an indicator of the skin‐homing T‐cell subset. In the present study, we investigated the expression of CLA in natural killer (NK) cells defined phenotypically as surface CD3– and CD56+ cells in peripheral blood. CLA was definitely expressed on CD3–CD56+ cells at a level comparable to CD3+ cells in peripheral blood of normal Japanese volunteers. After in vitro stimulation of peripheral blood mononuclear cells with interleukin 2 (IL‐2) and IL‐12, there was a significant increase in the number and percentage of CLA+ NK cells but not CLA+ T cells (P < 0·01). To analyse the characteristics of CLA expressed by NK cells, we investigated a CLA+ NK‐leukaemia cell line, NK‐YS, established from a patient with NK leukaemia/lymphoma with skin infiltration. In the in vitro study, the CLA‐expressing NK‐leukaemic cell line bound to E‐selectin‐transfected cells and was inhibited by HECA 452 antibody or neuraminidase treatment of leukaemic cells. These findings suggest that CLA expressed by NK cells is a homing receptor for the E‐selectin molecule and may explain skin infiltration by NK cells and NK lymphoma cells analogous to T cells. An NK‐cell subset expressing CLA must play an important role in host defence against microorganisms and neoplasms in skin lesions.

CD95 LIGAND IS EXPRESSED IN REED-STERNBERG CELLS OF HODGKIN'S DISEASE

Reed–Sternberg (RS) cells and their mononuclear variants, Hodgkin’s (H) cells, are considered to be the neoplastic cells of Hodgkin’s disease (HD). The cellular origin of H‐RS cells remains the subject of considerable controversy, although most recent papers have claimed that H‐RS cells are of B cell origin. Recently, however, it has been reported that some H‐RS cells express granzyme B, as observed in cytotoxic T cells and/or natural killer cells, which also express CD95 ligand (FasL/APO‐1L). In the present study, the expression of CD95L and granzyme B in H‐RS cells of HD was investigated. CD95L was detected in H‐RS cells in five of nine HD cases (one case of lymphocyte‐rich classical HD, two of these cases of nodular sclerosis type, and two of four cases of mixed cellularity type). All three examined HD cell lines expressed CD95L in the cytoplasm, although cell surface expression was seen only in L428 cells. Three HD cases expressed both CD95L and granzyme B. It was concluded that CD95L is frequently expressed in H‐RS cells, which is one of their notable characteristics; albeit it seems to be irrespective of cell lineage.

Homotypic Cell Aggregation via Conformational Change of CD44 Molecule Induced by Anti-CD44 Monoclonal Antibodies

The homotypic cell aggregation of leukocytes is an unique adhesive event which is caused by cellular activation. Anti-CD44 monoclonal antibody (mAb) induces homotypic cell aggregation of hematopoietic cell lines expressing CD44, but the mechanism of homotypic cell aggregation is poorly understood. We used four mAbs against CD44: TL-1 which was newly developed and seemed to react with a non-hyaluronate binding site, OS/37 and BU52 which recognized a hyaluronate binding site, and Hermes-3 which recognized a non-hyaluronate binding site. TL-1 treatment induced strong homotypic cell aggregation in several types of cell lines including a B cell line from a patient with leukocyte adhesion deficiency syndromes (LAD) and normal peripheral blood lymphocytes (PBL). OS/37 and BU52 also induced weak homotypic cell aggregation. None of these anti-CD44 mAbs-induced homotypic cell aggregations was blocked by antibodies against LFA-1, ICAM-1, VLA-4, or L-selectin. Interestingly, the TL-1-induced homotypic cell aggregation was blocked by Hermes-3 or OS/37, but not by BU52. BU52-induced homotypic cell aggregation was blocked by Hermes-3 or OS/37, but not by TL-1. OS/37-induced homotypic cell aggregation was blocked by Hermes-3, TL-1 or BU52. The blocking experiments with anti-metabolic agents revealed that the induced homotypic cell aggregation was energy-dependent and associated with intracytoplasmic actin filaments. This homotypic cell aggregation did not require de novo protein synthesis, because it was not affected by pretreatment with either cycloheximide or actinomycin D. FACS analysis revealed that TL-1 binding did not affect the intensity of expression of the CD44 molecule on the cell surface.

Analysis of linear growth in survivors of childhood acute lymphoblastic leukemia.

Abstract. Therapy for childhood acute lymphoblastic leukemia (ALL) is entering a new era in terms of quality-of-life. In the current study, 21 patients with childhood-onset ALL were assessed for linear growth, bone mineral density (BMD), and endocrinological status, focusing especially on longitudinal analysis of the growth of each patient. Linear growth was uniformly attenuated during therapy in all patients. In contrast, after the cessation of therapy, the growth of each patient varied widely from attenuated to dramatic catch-up growth. In pubertal survivors who had received chemotherapy and cranial irradiation during prepuberty, the degree of growth after the cessation of therapy was negatively correlated with changes in height Z scores during therapy (r = −0.76, P = 0.004). One of the factors involved in catch-up growth, urinary N-telopeptide/creatinine (U-NTx/Cr), was significantly higher in patients whose Z scores decreased after cessation of therapy (P = 0.01), despite normal pubertal development and normal endocrinological assessments. The present study revealed individual differences in linear growth after the cessation of therapy and suggests the importance of catch-up growth during puberty.

Persistence of TEL‐AML1 transcript in acute lymphoblastic leukemia in long‐term remission

Background : It has recently been shown that t (12;21) (p13;q 22) is the most common molecular genetic abnormality in childhood acute lymphoblastic leukemia (ALL). We have analyzed this translocation in an attempt to evaluate its incidence and to monitor minimal residual disease (MRD) with t (12; 21) rearrangement by detection of TEL‐AML1 transcript in patients with childhood ALL.

ABL kinase mutation and relapse in 4 pediatric Philadelphia chromosome-positive acute lymphoblastic leukemia cases

The tyrosine kinase inhibitor (TKI) imatinib mesylate (IM) revolutionized the treatment of Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph-ALL), which had showed poor prognosis before the dawn of IM treatment. However, if Ph-ALL patients showed IM resistance due to ABL kinase mutation, second-generation TKI, dasatinib or nilotinib, was recommended. We treated 4 pediatric Ph-ALL patients with both IM and bone marrow transplantation (BMT); however, 3 relapsed. We retrospectively examined the existence of ABL kinase mutation using PCR and direct sequencing methods, but there was no such mutation in all 4 diagnostic samples. Interestingly, two relapsed samples from patients who were not treated with IM before relapse did not show ABL kinase mutation and IM was still effective even after relapse. On the other hand, one patient who showed resistance to 3 TKI acquired dual ABL kinase mutations, F359C at the IM-resistant phase and F317I at the dasatinib-resistant phase, simultaneously. In summary, Ph-ALL patients relapsed with or without ABL kinase mutation. Furthermore, ABL kinase mutation was only found after IM treatment, so an IM-resistant clone might have been selected during the IM treatment and intensive chemotherapy. The appropriate combination of TKI and BMT must be discussed to cure Ph-ALL patients.