Interleukin-2 induces NF-kappaB activation through BCL10 and affects its subcellular localization in natural killer lymphoma cells.

Deregulation of nuclear factor (NF)-kappaB signalling is common in cancers and is essential for tumourigenesis. Constitutive NF-kappaB activation in extranodal natural killer (NK)-cell lymphoma, nasal type (ENKL) is known to be associated with aberrant nuclear translocation of BCL10. Here we investigated the mechanisms leading to NF-kappaB activation and BCL10 nuclear localization in ENKLs. Given that ENKLs are dependent on T-cell-derived interleukin-2 (IL2) for cytotoxicity and proliferation, we investigated whether IL2 modulates NF-kappaB activation and BCL10 subcellular localization in ENKLs. In the present study, IL2-activated NK lymphoma cells were found to induce NF-kappaB activation via the PI3K/Akt pathway, leading to an increase in the entry of G(2)/M phase and concomitant transcription of NF-kappaB-responsive genes. We also found that BCL10, a key mediator of NF-kappaB signalling, participates in the cytokine receptor-induced activation of NF-kappaB. Knockdown of BCL10 expression resulted in deficient NF-kappaB signalling, whereas Akt activation was unaffected. Our results suggest that BCL10 plays a role downstream of Akt in the IL2-triggered NF-kappaB signalling pathway. Moreover, the addition of IL2 to NK cells led to aberrant nuclear translocation of BCL10, which is a pathological feature of ENKLs. We further show that BCL10 can bind to BCL3, a transcriptional co-activator and nuclear protein. Up-regulation of BCL3 expression was observed in response to IL2. Similar to BCL10, the expression and nuclear translocation of BCL3 were induced by IL2 in an Akt-dependent manner. The nuclear translocation of BCL10 was also dependent on BCL3 because silencing BCL3 by RNA interference abrogated this translocation. We identified a critical role for BCL10 in the cytokine receptor-induced NF-kappaB signalling pathway, which is essential for NK cell activation. We also revealed the underlying mechanism that controls BCL10 nuclear translocation in NK cells. Our findings provide insight into a molecular network within the NF-kappaB signalling pathway that promotes the pathogenesis of NK cell lymphomas.

Frequent concomitant epigenetic silencing of the stress-responsive tumor suppressor gene CADM1, and its interacting partner DAL-1 in nasal NK/T-cell lymphoma.

Nasal NK/T-cell lymphoma (NL) is a rare but clinically important entity of lymphoma. Its preferential incidence in Orientals but not Caucasians suggests possible genetic predisposition. 11q deletion is common in NL, indicating certain tumor suppressor genes (TSGs) at this locus involved in its pathogenesis. We investigated the expression and methylation of an 11q23.2 TSG, CADM1 (or TSLC1), and its partner DAL-1 (or EPB41L3) in NL. Methylation and silencing of CADM1 were detected in 2 NL and 4 of 8 (50%) of non-Hodgkin lymphoma (NHL) cell lines, but not in normal NK cells and normal PBMC. Absence of CADM1 protein was also detected in NL cell lines. 5-aza-2'-deoxycytidine (Aza) demethylation or genetic knockout of both DNMT1 and 3B genes restored CADM1 and DAL-1 expression. CADM1 methylation was further detected in 36 of 45 (80%) of NL tumors. Concomitantly, DAL-1 was epigenetically inactivated in NL cell lines and virtually all the tumors with methylated CADM1. A significant correlation between the methylation of both genes was found (p < 0.0001). Homozygous deletion of CADM1 was detected in only 3 of 18 (17%) of tumors. The stress-response of CADM1 was abolished when its promoter becomes methylated. Our results demonstrate a frequent, predominant epigenetic silencing of CADM1 and DAL-1 in NL, which likely play a synergic role in NL pathogenesis.

Cell death by bortezomib-induced mitotic catastrophe in natural killer lymphoma cells.

The proteasome inhibitor bortezomib (PS-341/Velcade) is used for the treatment of relapsed and refractory multiple myeloma and mantle-cell lymphoma. We recently reported its therapeutic potential against natural killer (NK)-cell neoplasms. Here, we investigated the molecular mechanisms of bortezomib-induced cell death in NK lymphoma cells. NK lymphoma cell lines (SNK-6 and NK-YS) and primary cultures of NK lymphomas treated with bortezomib were examined for alterations in cell viability, apoptosis, cellular senescence, and cell cycle status. Bortezomib primarily induced mitochondrial apoptosis in NK-YS cells and in primary lymphoma cells at the same concentration as reported in myeloma cells. Unexpectedly, SNK-6 cells required a significantly higher median inhibitory concentration of bortezomib (23 nmol/L) than NK-YS and primary lymphoma cells (6-13 nmol/L). Apoptosis was limited in SNK-6 cells due to the extensively delayed turnover of Bcl-2 family members. These cells were killed by bortezomib, albeit at higher pharmacologic concentrations, via mitotic catastrophe-a mitotic cell death associated with M-phase arrest, cyclin B1 accumulation, and increased CDC2/CDK1 activity. Our results suggest that, in addition to cell death by apoptosis at lower bortezomib concentrations, NK lymphoma cells resistant to bortezomib-induced apoptosis can be killed via mitotic catastrophe, an alternative cell death mechanism, at higher pharmacologic concentrations of bortezomib. Hence, activating mitotic catastrophe by bortezomib may provide a novel therapeutic approach for treating apoptosis-resistant NK-cell malignancies and other cancers.

Plasmacytoid dendritic cell leukemia with potent antigen-presenting ability.

We report 2 patients with plasmacytoid dendritic cell leukemia (pDCL) expressing CD4, CD56, CD33, CD36, HLA-DR, CD123, CD86 and CD83 in the absence of lineage markers (myeloid, B, T or natural killer cells) except for CD33. Culturing leukemic blasts of both cases with IL-3 for 4 days increased the expression of surface molecules associated with antigen presentation, e.g. CD1a and CD40. Leukemic blasts of both cases possessed a considerable level of antigen-presenting ability to allogeneic lymphocytes in mixed leukocyte cultures. Culturing the blasts with IL-3 for 4 days markedly increased allogeneic antigen presenting ability. Combined with data showing evident graft-versus-leukemia effects without graft-versus-host disease in a cord blood stem cell transplanted pDCL case, leukemic cells in pDCL may act as potent antigen presenting cells in vivo, too.

Gene silencing of the tyrosine phosphatase SHP1 gene by aberrant methylation in leukemias/lymphomas.

High-frequent silencing of hematopoietic cell-specific protein-tyrosine phosphatase SHP1 gene by promoter methylation was detected in various kinds of leukemias and lymphomas, as well as in many hematopoietic cell lines, which is supported by our previous observation of strong decrease of SHP1 mRNA and protein. The promoter methylation of the SHP1 gene was clearly correlated with the clinical stage. Loss of heterozygosity with microsatellite markers near the SHP1 gene was shown in 79% of informative acute lymphoblastic leukemia cases. These results suggest that functional loss of SHP1 is associated with the pathogenesis of leukemias/lymphomas.

Acute T-lymphoblastic leukemia relapsed with the character of myeloid/natural killer cell precursor phenotype: a case report.

The leukemic lymphoblasts of a patient expressed CD7, CD13, CD33, CD34, HLA-DR and cytoplasmic CD3varepsilon. He was diagnosed with acute lymphoblastic leukemia (ALL), and successfully treated with a conventional chemotherapy for ALL. The disease relapsed three times, and the character of the cells gradually altered, i.e. CD56 expression increased and CD13, CD7 and cCD3 epsilon decreased. The phenotype of the relapsed ALL was, therefore, compatible with myeloid/natural killer cell precursor acute leukemia (M/NK-AL). Some of M/NK-AL may be closely related with T/myeloid-biphenotypic pro-T blasts, and both types of acute leukemia may develop a tendency to express myeloid antigens, and they may belong to the category of immature T lymphoid precursors.

Cutaneous lymphoblastic lymphoma of putative plasmacytoid dendritic cell-precursor origin: two cases.

Although the neoplasm of relatively mature type plasmacytoid dendritic cells (pDC) was recently reported, that of pDC-precursor has not yet been defined. We experienced two elderly male Japanese patients with reddish skin tumors. The histology of the tumors in both patients showed terminal deoxinucleotidyl transferase (TdT)-positive lymphoblastic lymphoma (LBL). The pathological cells did not express T, B or NK markers, and no rearranged bands were shown for immunoglobulin (Ig)-JH, T cell receptor (TCR)-C beta, J gamma, J delta1, and c-myc. In addition, no Epstein-Barr virus (EBV)-derived DNA was detected in either case. The cells were (CD45, CD43, CD74, CD10, and HLA-DR)-positive in both cases, and one of the cases showed (CD4, CD36, CD54, CD58 and CD86)-positive plasmacytoid lymphoblasts, which appeared to be compatible with intermediate cells between human bone marrow lymphoid precursors and mature lymphoid DC. The cutaneous LBL in the two cases may, therefore, have been of pDC-precursor origin.

CD56+, NKp46+ cell line (MZ93) expressing T-cell and myeloid antigens.

The MZ93 cell line, established from a patient with CML, expressed CD4, CD7, CD13, CD25, CD33, CD34, CD56 and NKp46. The additional karyotype abnormality of the Ph-positive leukemia cells in vivo, 6p+, was also observed in MZ93. The early passages of MZ93 expressed CD3 in the cytoplasm, but the late passages did not. The cells did not express mature NK-markers as expected. The messenger RNAs of CD2 and NKp46 were detected and those of CD3varepsilon and CD3zeta were absent in the cells. Therefore, the cell line has the immunophenotype likely to NK and/or T cell precursor.

Fc epsilon RI and CD22 mRNA are expressed in early B-lineage and myeloid leukemia cell lines.

CD22, one of the important markers for diagnosing B-lineage acute leukemia, was expressed in mature basophil granulocytes. We then investigated the expression of CD22 and other B cell- and basophil-related molecules in 25 human acute leukemia cell lines to find the phenotype of the virtual common progenitor of B and myeloid lineage. Surface and cytoplasmic expressions of antigens were analyzed using a flow cytometer and an essential antibody panel used for diagnosing acute leukemia as well as cytokine receptors and basophil-related enzymes. Messenger RNA expression of Fc epsilon R1 and CD22 was also analyzed. Peroxidase-positive and -negative myeloid leukemias showed eosinophil- and basophil-type expression of enzymes, respectively. Early myeloid and B-lineage cells expressed basically similar combinations of cytokine receptors and various combinations of mRNA listed above, while T-lineage cells did not. The virtual common progenitor of B and myeloid lineage cells may be defined as immature cells simultaneously expressing B and basophil phenotypes.

Physiological oxidants induce apoptosis and cell cycle arrest in a multidrug-resistant natural killer cell line, NK-YS.

Natural-killer (NK) cell-derived malignant tumors, such as angiocentric lymphoma, is often resistant to various chemotherapeutic agents and follows an aggressive clinical course. We report the effects of physiological oxidants (hydrogen peroxide, H2O2; sodium hypochlorite, NaOCl and monochloramine, NH2Cl) on the cell growth and cell death in a multidrug-resistant NK tumor cell line, NK-YS. Among the oxidants tested, NH2Cl was most cytotoxic, in which more than 90% of the cells died at 150 nmol/1 x 10(6) cells. H2O2 was less cytotoxic, whereas NaOCl showed no significant cell death at this dose. The cell death induced by NH2Cl was accompanied by DNA cleavage and caspase activation, which suggested apoptosis. In addition, lower dose of NH2Cl (70 nmol/1 x 10(6) cells) retarded cell growth and inhibited the cell cycle transition from G1 to S. This cell cycle arrest accompanied a decrease in the phosphorylation of retinoblastoma tumor suppressor protein at serine 795. These observations suggest that NH2Cl may induce apoptotic cell death and growth arrest in multidrug-resistant NK cell tumors.

Sensitive measurement of fragmented red cell population using flow cytometry, and its application for estimating thrombotic microangiopathy after stem cell transplantation.

BACKGROUND: Thrombotic microangiopathy (TMA) is one of the lethal complications after hematopoietic stem cell transplantation (SCT). The levels of fragmented red cells (FRCs), thrombomodulin (TM), and factor VIII-related antigen in the blood are the most important markers for estimating TMA. However, the FRC level has been measured by using microscopy and the naked eye; therefore, an improvement in technology to objectively count FRC is necessary. METHODS: We established a novel technique to sensitively measure FRC as glycophorin A dull-positive small particles using a flow cytometer and estimated its reliability in patients treated with SCT. The blood level of FRC was compared with other clinical data in 257 blood samples in 16 clinical courses after SCT of 15 patients. RESULTS: Sorted glycophorin A dull-positive small particles morphologically showed FRC. Measured FRC percentage had a weak correlation with serum levels of lactate dehydrogenase (LDH) and total bilirubin but not with TM level, whereas TM showed a weak correlation with the levels of aspartate aminotransferase and LDH. In a patient with fulminant TMA, decrement of the FRC level led to improvement in liver parameters after treatment, presumably due to the rapid clearance of FRC, and increased simultaneously with the levels of LDH and bilirubin by the TMA recurrence. CONCLUSIONS: Levels of FRC percentage and TM were independent parameters of TMA. This novel technique may be used as a standard methodology in diagnosing TMA.

Successful treatment of KIT D816V-positive, imatinib-resistant systemic mastocytosis with interferon-alpha.

We describe a case of systemic mastocytosis associated with myelodysplastic syndrome. The bone marrow showed multifocal clusters of mast cells and myeloid dysplasia. Sequencing of the KIT DNA revealed a point mutation at codon 816 including a substitution of valine for aspartic acid (D816V). The patient's tumor did not respond to imatinib; however, interferon-alpha reduced the bone marrow mast cells and serum total tryptase. The patient remains alive at one year after the diagnosis without disease progression.

Long-term production of pre-existing alloantibodies to E and c after allogenic BMT in a patient with aplastic anemia resulting in delayed hemolytic anemia.

BACKGROUND: Delayed hemolysis mediated by long-term production of pre-existing recipient-derived antibodies directed against donor RBC antigens after allogenic BMT is an unusual complication of hematopoietic transplantation. CASE REPORT: A 26-year-old man with aplastic anemia had pre-existing alloantibodies to E and c. He received BMT from a donor, whose Rh phenotype was E+, c+. From about 1 month after the transplant, he showed mild hemolysis due to the antibodies. RESULTS: The patient was typed as group B, CCDee and had anti-E and c alloantibodies before BMT. The donor was typed as group O, ccDEE. Although MNCs from the donor marrow were infused into the patient, DAT became positive on Day 21, and the patient-origin antibodies remained detectable by both DAT and IAT even 20 months after BMT. However, immunomagnetically isolated peripheral circulating B cells were 100 percent donor origin. The patient received prednisolone from Day 221, and thereafter, the signs of hemolysis disappeared. CONCLUSION: It is likely that the long-term production of alloantibodies is due to the existence of long-lived recipient plasma cells, which survive the conditioning regimen. This case suggests that patients with pre- existing alloantibodies that do not belong to the ABO system should be carefully followed up after BMT.

Expression of cutaneous lymphocyte antigen is associated with a poor outcome of nasal-type natural killer-cell lymphoma.

Nasal and nasal-type natural killer (NK) lymphoma is a distinct clinicopathological entity mostly associated with Epstein-Barr virus. Cases that have widespread lesions are resistant to ordinary anti-cancer therapy and take a highly aggressive course. To date, there are no available data on the relationships between the localization, clinical outcome and expression of adhesion molecules in such cases. We examined the expression of cutaneous lymphocyte antigen (CLA) in 52 cases of NK-cell lymphoma. CLA was highly expressed in cutaneous cases. Also, the CLA+ group (n=29) had a much worse prognosis than the CLA- group (n=23), regardless of the primary site or clinical staging. Univariate analysis identified some significant prognostic factors, and multivariate analysis of these factors showed that the expression of CLA was an independent prognostic indicator. In conclusion, the present findings established that CLA is an independent and important prognostic factor in patients with NK-cell lymphomas.

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

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.