Junjiro Tsuchiyama

Reduction of Hematopoietic Cell-Specific Tyrosine Phosphatase SHP-1 Gene Expression in Natural Killer Cell Lymphoma and Various Types of Lymphomas/Leukemias: Combination Analysis with cDNA Expression Array and Tissue Microarray

To investigate the lymphomagenesis of NK/T lymphoma, we comprehensively and systematically analyzed the expression pattern of the human NK/T cell line (NK-YS) genome by cDNA expression array and tissue microarray. We detected significant changes in the gene expression of NK-YS cell line: an increase in 18 and a decrease in 20 genes compared to normal NK cells or peripheral blood mononuclear cells. Among these genes, we found a strong decrease in hematopoietic cell specific protein-tyrosine-phosphatase SH-PTP1 (SHP1) mRNA by cDNA expression array and reverse transcriptase-polymerase chain reaction. Further analysis with standard immunohistochemistry and tissue microarray, which used 207 paraffin-embedded specimens of various kinds of malignant lymphomas, showed that 100% of NK/T lymphoma specimens and more than 95% of various types of malignant lymphoma were negative for SHP1 protein expression. On the other hand, SHP1 protein was strongly expressed in the mantle zone and interfollicular zone lymphocytes in reactive lymphoid hyperplasia specimens. In addition, various kinds of hematopoietic cell lines, particularly the highly aggressive lymphoma/leukemia lines, lacked SHP1 expression in vitro, suggesting that loss of SHP1 expression may be related to not only malignant transformation, but also tumor cell aggressiveness. SHP1 expression could not be induced in either of two NK/T cell lines by phorbol ester, suggesting that genetic impairment or modification with methylation of SHP1 DNA could be one of the critical events in the pathogenesis of NK/T lymphoma. This evidence strongly suggests that loss of SHP1 gene expression plays an important role in multistep tumorigenesis, possibly as an anti-oncogene in the wide range of lymphomas/leukemias as well as NK/T lymphomas.

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.

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.

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. [Mol Cancer Ther 2008;7(12):3807–15]

Antisense oligodeoxynucleotides to latent membrane protein 1 induce growth inhibition, apoptosis and Bcl-2 suppression in Epstein-Barr virus (EBV)-transformed B-lymphoblastoid cells, but not in EBV-positive natural killer cell lymphoma cells.

Epstein‐Barr virus (EBV)‐encoded latent membrane protein 1 (LMP‐1) is essential for immortalization of B cells by EBV, protects the infected cells from apoptotic cell death and induces Bcl‐2 expression. Suppression of LMP‐1 expression by antisense oligodeoxynucleotides (AS‐oligo) to LMP‐1 inhibits proliferation, promotes apoptosis and suppresses Bcl‐2 expression in EBV‐transformed B cells. However, the function of LMP‐1 expression in EBV‐positive natural killer (NK) cell lymphoma cells has not been reported previously. We examined the function of LMP‐1 in two EBV‐positive NK cell lymphoma cell lines (NK‐YS and YT) through suppressing LMP‐1 expression by AS‐oligo to LMP‐1. The AS‐oligo to LMP‐1 suppressed LMP‐1 mRNA and protein expression in two EBV‐positive NK cell lymphoma cell lines, as well as in an EBV‐transformed B‐cell line (CMG‐1). Proliferation was inhibited, apoptosis was induced and Bcl‐2 expression was suppressed in CMG‐1 cells, but none of these events were observed in NK‐YS or YT cells. These results suggest that proliferation, inhibition of apoptosis and Bcl‐2 expression in EBV‐positive NK cell lymphoma cells are not directly regulated by LMP‐1 as in EBV‐transformed B‐cell lines, but are probably mediated through other signal transducing systems.

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.

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.

Interleukin‐2 induces NF‐κB activation through BCL10 and affects its subcellular localization in natural killer lymphoma cells

Deregulation of nuclear factor (NF)‐κB signalling is common in cancers and is essential for tumourigenesis. Constitutive NF‐κB 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‐κB 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‐κB activation and BCL10 subcellular localization in ENKLs. In the present study, IL2‐activated NK lymphoma cells were found to induce NF‐κB activation via the PI3K/Akt pathway, leading to an increase in the entry of G2/M phase and concomitant transcription of NF‐κB‐responsive genes. We also found that BCL10, a key mediator of NF‐κB signalling, participates in the cytokine receptor‐induced activation of NF‐κB. Knockdown of BCL10 expression resulted in deficient NF‐κB signalling, whereas Akt activation was unaffected. Our results suggest that BCL10 plays a role downstream of Akt in the IL2‐triggered NF‐κB 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‐κB 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‐κB signalling pathway that promotes the pathogenesis of NK cell lymphomas. Copyright

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

Summary. 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.

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.