Tomiteru Togano

Identification of the RelA domain responsible for action of a new NF-κB inhibitor DHMEQ

A new NF-kappaB inhibitor dehydroxymethylepoxyquinomicin (DHMEQ) has a potential to be applied to clinical medicine as an anti-cancer and anti-inflammatory agent. DHMEQ inhibits localization of NF-kappaB in the nucleus and the inhibitory effect by DHMEQ is more potent on p50/RelA than on p50 homodimer. However, a molecular target of DHMEQ is unknown. In this study, we identified residues CEGRSAGSI, which appear in RelA (amino acids 38-46), c-Rel (28-36), and RelB (144-152), but not in p50 and p52, as a target of DHMEQ. As a possible mechanism, we propose that DHMEQ accesses CEGRSAGSI domain recognizing RSAGSI structure and directly binds to cysteine. This target domain appears to be unique among mammalian proteins. The results obtained in this study may provide better understanding of the action of DHMEQ and a key for developing a new NF-kappaB inhibitor with more potent activity.

Epidemiology of visceral mycoses in autopsy cases in Japan: the data from 1989 to 2009 in the Annual of Pathological Autopsy Cases in Japan

To identify recent trends in the frequency of mycoses in autopsy cases, we analyzed, on a four-year basis, the 1989-2009 data in the Annual of Pathological Autopsy Cases in Japan. Of the 13,787 (9235 males) autopsies conducted in 2009, 4.5% (633/13,787) involved fungal infections and of the latter, 60.3% (368/633) were found to have severe clinical manifestations. Among the 610 (96.4%) cases involving a single etiologic angent, the predominant pathogens were Aspergillus (299 cases; 49%) and Candida (184 cases; 30.2%). However, it should be noted that the prevalence of severe aspergillosis and candidiasis has been decreasing. Although the frequency of cases involving zygomycetes seemed to be generally remaining stable from 1989-2009, we noted for the first time a peak in 2009 in such infections in patients less than one year old. Finally, deep-seated infections caused by unidentified fungi would appear to be decreasing over the time of the survey. Our finding, it is hoped, will encourage physicians to actively pursue viscerial fungal infections.

Splenic infarction after Epstein-Barr virus infection in a patient with hereditary spherocytosis.

We describe the first patient with hereditary spherocytosis (HS) known to have developed splenic infarction following infectious mononucleosis (IM). An 18-year-old Japanese man was referred to our hospital in November 2004 because of continuous fever and icterus. He had undergone cholecystectomy at the age of 14 years. On patient admission in November 2004, a physical examination showed marked hepatosplenomegaly, icterus, and jaundice. He had a white blood cell count of 14.9 x 109/L with 9.5% atypical lymphocytes, a red blood cell count of 2.93 x 1012/L, and a hemoglobin concentration of 7.8 g/dL. Microspherocytes were observed in the patient’s peripheral blood smear, and immunoglobulin M antibody to Epstein-Barr virus (EBV) viral capsid antigen was detected. The patient’s diagnosis was HS with IM. On day 4 of admission, the patient complained of severe abdominal pain. Abdominal computed tomography scanning revealed findings of splenic infarction.Two months after the occurrence of splenic infarction, a splenectomy was performed. A pathohistologic examination of the resected spleen revealed no evidence of thrombosis or arterial occlusion. We assume that the cause of splenic infarction was insufficient blood flow to oxygenate the entire spleen during the acute enlargement of the spleen.

Ets-1 activates overexpression of JunB and CD30 in Hodgkin's lymphoma and anaplastic large-cell lymphoma.

Overexpression of CD30 and JunB is a hallmark of tumor cells in Hodgkins lymphoma (HL) and anaplastic large-cell lymphoma (ALCL). We reported that CD30-extracellular signal-regulated kinase (ERK)1/2 mitogen-activated protein kinase (MAPK) signaling induces JunB, which maintains constitutive activation of the CD30 promoter. Herein, we localize a cis-acting enhancer in the JunB promoter that is regulated by Ets-1. We show that E26 transformation-specific-1 (Ets-1) (-146 to -137) enhances JunB promoter activation in a manner that is dependent on CD30 or the nucleophosmin-anaplastic lymphoma kinase (NPM-ALK)-ERK1/2 MAPK pathway. Ets-1 knockdown reduces the expression of both JunB and CD30, and CD30 knockdown significantly reduces JunB expression in HL and ALCL cell lines. NPM-ALK knockdown also reduces JunB expression in ALCL cell lines expressing NPM-ALK. Collectively, these results indicate that CD30 and NPM-ALK cooperate to activate the ERK1/2 MAPK-Ets-1 pathway. Ets-1, constitutively activated by ERK1/2-MAPK, plays a central role in the overexpression of JunB and CD30, which are both involved in the pathogenesis of HL and ALCL.

Targeted repression of overexpressed CD30 downregulates NF-kappaB and ERK1/2 pathway in Hodgkin lymphoma cell lines.

We previously reported that CD30 is induced during lymphocyte transformation and that overexpressed CD30 can transduce ligand-independent signals in Hodgkin lymphoma (HL) cells. However, its biological consequence is not fully addressed. In this study, we examined the effects of targeted repression of overexpressed CD30 on cell signaling and proliferation using small-interfering RNAs (siRNAs) in HL cell lines. Repression of CD30 inhibited cellular proliferation through reduced activation of IkappaB kinase (IKK) and extracellular signal-regulated kinase (ERK) 1/2 in both B- and T-HL cell lines. These HL cell lines bear one or more defects in negative regulators of nuclear factor (NF)-kappaB signaling, including A20, cylindromatosis tumor suppressor protein (CYLD), and IkappaBalpha, and when CD30 is repressed, they show reduced activation of the canonical NF-kappaB pathway. This suggests that CD30 governs NF-kappaB and ERK1/2 signaling pathways, and is involved in the proliferation of HL cells. Defective mutations in negative regulators of NF-kappaB signaling appear to promote CD30-initiated basal NF-kappaB activation. These results indicate that CD30 is involved in the tumorigenic process of HL, and that it may be useful as a therapeutic target for the treatment of HL.

Association of CD20 levels with clinicopathological parameters and its prognostic significance for patients with DLBCL

Diffuse large B-cell lymphomas (DLBCL) express CD20. CD20 expression is described as negative, weak, or normal as determined by flow cytometry (FCM) and is an important target for the treatment of DLBCL. However, the impact of CD20 levels at onset of the disease on patient prognosis has not been fully elucidated. We analyzed 174 DLBCL cases newly diagnosed between January 1998 and April 2010. The relationship of the association between CD20 levels and patients’ backgrounds and prognoses was analyzed using the Kaplan–Meier method and Cox proportional hazard regression. Of the 174 patients, three cases (1.7%) were defined as CD20 negative based on immunohistochemistry (IHC). Although the other 171 cases were positive by IHC, eight cases (4.7%) were defined as negative and 33 cases (19.3%) were defined as weak when analyzed by FCM. Of the 105 patients who received rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisolone therapy, those who were CD20 negative (FCM) showed significantly inferior overall (hazard ratios (HR): 6.79, 95% CI: 1.32–34.96, p = 0.04) and progression-free survival (HR: 7.3, 95% CI: 1.49–35.8, p = 0.04) compared to patients who were CD20 normal. Our findings indicate that the CD20 level (FCM) at onset is an independent predictor of the prognosis of patients with DLBCL.

Development of hepatosplenic γδ T-cell lymphoma with pancytopenia during early pregnancy: a case report and review of the literature

Abstract:  Lymphomas rarely develop during pregnancy, but hepatosplenic γδ T‐cell lymphoma (HSγδTCL) is extremely rare. We encountered a case of T‐cell intracellular antigen‐1 (TIA‐1) positive and granzyme B‐positive HSγδTCL that developed early during the course of pregnancy. The patient was a 31‐yr‐old female who was referred to our hospital because of pancytopenia and splenomegaly at the time of the14th week of her gestation. Her pancytopenia and hepatosplenomegaly worsened and she became fibril at the 27th week of gestation and Cesarean section was performed at the 29th week. Histopathological examination of the spleen, which was resected 28 d after delivery for a diagnostic purpose, revealed medium to large‐sized nodules composed of dense proliferation of lymphoid cells having round to oval‐shaped nuclei and abundant weakly eosinophilic cytoplasm. They were CD3ɛ+, mCD3+, CD4−, CD8−, CD56+, CD79a−, T‐cell receptor (TCR)‐γδ protein+, TIA‐1+, and granzyme B+ by either immunohistochemistry or flow cytometry. Clonal rearrangement of TCR‐γ genes without such rearrangement of TCR‐δ and TCR‐β genes was confirmed by Southern blot hybridization. Thus, the patient was diagnosed as having HSγδTCL, and combination chemotherapy was initiated. She is currently in partial remission. To our knowledge, this is the first case report of HSγδTCL that developed during pregnancy. Pathogenesis of pregnancy‐associated lymphoma is not known, but it is possible that maternal immunity during pregnancy or a hormonal imbalance, such as a change in the progesterone level, induces the development of lymphoma. Pregnancy‐associated lymphoma is resistant to standard chemotherapy and is associated with poor prognosis. Therefore, it is important to accumulate clinicopathologic data of such cases for the development of a treatment modality.

The side population, as a precursor of Hodgkin and Reed-Sternberg cells and a target for nuclear factor-κB inhibitors in Hodgkin's lymphoma

Although disturbed cytokinesis of mononuclear Hodgkin (H) cells is thought to generate Reed‐Sternberg (RS) cells, differentiation of Hodgkin’s lymphoma (HL) cells is not fully understood. Recent studies indicate that cells found in a side population (SP) share characteristics of cancer stem cells. In this study we identified an SP in the HL cell lines, KMH2 and L428. This SP almost entirely consists of distinct small mononuclear cells, whereas the non‐SP is a mixture of relatively large cells with H or RS cell‐like morphology. Culture of the small mononuclear cells in the SP from KMH2 generated a non‐SP. Single cell culture of the SP cells generated large cells with H or RS cell‐like morphology. We found that CD30 overexpression and constitutive nuclear factor‐κB (NF‐κB) activity, both of which are characteristics of HL cells, are shared between the SP and non‐SP cells for both KMH2 and L428. Inhibition of NF‐κB induced apoptosis in both fractions, whereas the SP cells were resistant to a conventional chemotherapeutic agent doxorubicin. The results show that HL cell lines contain an SP, that is enriched for distinct small mononuclear cells and generates larger cells with H and RS cell‐like morphology. The results also stress the significance of NF‐κB inhibition for eradication of HL cells. (Cancer Sci 2010; 101: 2490–2496)

Induction of oncogene addiction shift to NF-κB by camptothecin in solid tumor cells

The biological basis of the resistance of solid tumor cells to chemotherapy is not well understood. While addressing this problem, we found that gastric cancer cell line St-4/CPT, lung cancer cell line A549/CPT, and colon cancer cell line HT-29/CPT, all of which are resistant to camptothecin (CPT), showed strong and constitutive nuclear factor (NF)-kappaB activity driven by IkappaB kinase compared with their parental cell lines St-4, A549, and HT-29. A new NF-kappaB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ), reduced viability and induced apoptosis in St-4/CPT, A549/CPT, and HT-29/CPT cell lines, while their parental cell lines were resistant to DHMEQ. The results in this study present an example of the shift in signals that support the survival of solid tumor cells to NF-kappaB during the acquisition of resistance to CPT. The results also indicate that solid tumor cells that become resistant to chemotherapy may be more easily treated by NF-kappaB inhibitors.

A case of giardiasis expressing severe systemic symptoms and marked hypereosinophilia.

An 88-year-old Japanese woman was referred to our hospital due to a one-month history of face edema, aphagia, shortness of breath, and skin rush over almost her entire skin. She had no abdominal symptoms. Her peripheral blood count showed a white blood cell (WBC) count of 27.1x10(9)/L with 82.1% eosinophils. Serum non-specific Immunoglobulin E was within a normal range. Soluble interleukin-2 receptor was elevated to 4200U/mL. At first, her eosinophil count was so high that we suspected she had an eosinophilic leukemia or hypereosinophilic syndrome. After admission, cysts of Giardia duodenalis (G. duodenalis) were detected in the patients feces by microscopic analysis, then she was diagnosed with giardiasis, and 750mg per day of metronidazole was administered for seven days. Her WBC count decreased to 6.0x10(9)/L with 10% eosinophils, and her systemic symptoms improved. At that time her serum IL-5 was within a normal range. A few months later, the patient again complained of skin rush, and G. duodenalis was once again found in her feces. Her serum IL-5 was elevated to 751pg/mL. Metronidazole was administered for two weeks, and her eosinophil count decreased. G. duodenalis is a protozoan parasite, and it is one of the most common waterborne transmission gastrointestinal parasites in the world. G. duodenalis rarely causes hypereosinophilia. To our knowledge, this is the first case report of giardiasis with extreme hypereosinophilia and severe systemic symptoms.