Shigeyuki Yokokura

Phosphorylation of Numb regulates its interaction with the clathrin-associated adaptor AP-2

Numb is thought to participate in clathrin‐dependent endocytosis by directly interacting with the clathrin‐associated adaptor complex AP‐2, although the underlying mechanisms are unknown. Numb is also known to be phosphorylated at Ser264 in vitro and in vivo. Here, we found that Numb is phosphorylated in vitro by Ca2+/calmodulin‐dependent protein kinase I on Ser283. This phosphorylation was also observed in transfected COS‐7 cells, indicating its physiological relevance. Pull‐down experiments showed that the phosphorylation of Numb impaired its binding to the AP‐2 complex and simultaneously recruited 14–3–3 proteins in vitro. Based on experiments using Numb mutants, both the initial phosphorylation of Ser264 and the subsequent phosphorylation of Ser283 are sufficient to abolish the binding of Numb to AP‐2 and to promote the interaction with 14–3–3 protein. These findings suggest a novel mechanism for the regulation of Numb‐mediated endocytosis, namely through direct phosphorylation.

Potentiated activation of VLA-4 and VLA-5 accelerates proplatelet-like formation

Fibronectin (FN) plays important roles in the proliferation, differentiation, and maintenance of megakaryocytic-lineage cells through FN receptors. However, substantial role of FN receptors and their functional assignment in proplatelet-like formation (PPF) of megakaryocytes are not yet fully understood. Herein, we investigated the effects of FN receptors on PPF using the CHRF-288 human megakaryoblastic cell line, which expresses VLA-4 and VLA-5 as FN receptors. FN and phorbol 12-myristate 13-acetate (PMA) were essential for inducing PPF in CHRF-288 cells. Blocking experiments using anti-β1-integrin monoclonal antibodies indicated that the adhesive interaction with FN via VLA-4 and VLA-5 were required for PPF. PPF induced by FN plus PMA was accelerated when CHRF-288 cells were enforced adhering to FN by TNIIIA2, a peptide derived from tenascin-C, which we recently found to induce β1-integrin activation. Adhesion to FN enhanced PMA-stimulated activation of extracellular signal-regulated protein kinase 1 (ERK1)/2 and enforced adhesion to FN via VLA-4 and VLA-5 by TNIIIA2-accelerated activation of ERK1/2 with FN plus PMA. However, c-Jun amino-terminal kinase 1 (JNK1), p38, and phosphoinositide-3 kinase (PI3K)/Akt were not stimulated by FN plus PMA, even with TNIIIA2. Thus, the enhanced activation of ERK1/2 by FN, PMA plus TNIIIA2 was responsible for acceleration of PPF with FN plus PMA.

Confluence-dependent resistance to cisplatin in lung cancer cells is regulated by transforming growth factor-beta

ABSTRACT Purpose of the Study: Confluence-dependent resistance (CDR) is a phenomenon in which the efficacy of anti-cancer agents decreases when cell density increases. CDR in lung cancer has never been reported. The purpose of this study is to investigate if CDR can occur in NSCLC cells and to find a role for transforming growth factor (TGF)-β as a mechanism of CDR. Materials and Methods: Non–small cell lung cancer (NSCLC) cell lines A549 and H2228 were exposed to cisplatin in a variety of cell density conditions. RNA interference targeting TGF-β receptor I was performed to silence the TGF-β pathway. Results: CDR to cisplatin was induced in NSCLC cells, whereas CDR to crizotinib, an inhibitor of activin receptor-like kinase, was not observed. During confluent conditions, the TGF-β1 concentration in the culture medium was the highest. Exogenous TGF-β1 inhibited cell proliferation and reduced sensitivity to cisplatin. Inhibition of the TGF-β pathway increased in terms of sensitivity to cisplatin at confluency. Conclusions: CDR to cisplatin can occur in NSCLC cells, and the TGF-β pathway is associated with the regulation of CDR.

Calmodulin antagonists induce cell cycle arrest and apoptosis in vitro and inhibit tumor growth in vivo in human multiple myeloma

BackgroundHuman multiple myeloma (MM) is an incurable hematological malignancy for which novel therapeutic agents are needed. Calmodulin (CaM) antagonists have been reported to induce apoptosis and inhibit tumor cell invasion and metastasis in various tumor models. However, the antitumor effects of CaM antagonists on MM are poorly understood. In this study, we investigated the antitumor effects of naphthalenesulfonamide derivative selective CaM antagonists W-7 and W-13 on MM cell lines both in vitro and in vivo.MethodsThe proliferative ability was analyzed by the WST-8 assay. Cell cycle was evaluated by flow cytometry after staining of cells with PI. Apoptosis was quantified by flow cytometry after double-staining of cells by Annexin-V/PI. Molecular changes of cell cycle and apoptosis were determined by Western blot. Intracellular calcium levels and mitochondrial membrane potentials were determined using Fluo-4/AM dye and JC-10 dye, respectively. Moreover, we examined the in vivo anti-MM effects of CaM antagonists using a murine xenograft model of the human MM cell line.ResultsTreatment with W-7 and W-13 resulted in the dose-dependent inhibition of cell proliferation in various MM cell lines. W-7 and W-13 induced G1 phase cell cycle arrest by downregulating cyclins and upregulating p21cip1. In addition, W-7 and W-13 induced apoptosis via caspase activation; this occurred partly through the elevation of intracellular calcium levels and mitochondrial membrane potential depolarization and through inhibition of the STAT3 phosphorylation and subsequent downregulation of Mcl-1 protein. In tumor xenograft mouse models, tumor growth rates in CaM antagonist-treated groups were significantly reduced compared with those in the vehicle-treated groups.ConclusionsOur results demonstrate that CaM antagonists induce cell cycle arrest, induce apoptosis via caspase activation, and inhibit tumor growth in a murine MM model and raise the possibility that inhibition of CaM might be a useful therapeutic strategy for the treatment of MM.

Enteral Malakoplakia Prior to Helicobacter cinaedi Bacteremia

associated infectious pathogens are Klebsiella spp. and Escherichia coli ( 7 ), which colonize the mucosa. In a few cases, a rare opportunistic pathogen leads to the development of a systemic infection, which may occasionally become severe ( 7 ). A defective immune system, such as a cellular immune defect in macrophages, directly contributes to the pathological establishment of malakoplakia ( 8,9 ). In malakoplakia, a functional defect of monocytes or macrophages results in the formation of a well-characterized deposit in macrophages known as a Michaelis–Gutmann body ( 10 ). Moran et al. ( 11 ) reported that characteristic pathological fi ndings, such as Michaelis–Gutmann bodies, had been observed in cases of colonic cancer. Th is suggests that an underlying cancer is one of the diff erential diagnoses for defective immunity when malakoplakia is observed. We treated a 49-year-old man with relapsed and refractory multiple myeloma following autologous stem cell transplantation. Th e patient was admitted to our hospital because of febrile diarrhea. We performed colonoscopy and observed multiple focal white mucosal lesions ( Figure 1 ). Th ese lesions were diagnosed on pathological examination to be malakoplakia ( Figure 2 ). Th ree days aft er the colonoscopy, Helicobacter cinaedi was isolated in the peripheral blood culture, as identifi ed by mass spectrometry. H. cinaedi is a spiral, rod-shaped, fastidious bacteria in the Helicobacteraceae family and is one of the enterohepatic nonHelicobacter pylori Helicobacters ( 12 ). H. cinaedi resides in the gastrointestinal tract and results in bacteremia. In this case, we supposed it was translocated into the blood through the mucosal barrier. Another pathogenicity of H. cinaedi in humans has been recognized as a causative pathogen of cellulitis. Th us, H. cinaedi infections have various clinical manifestations, including bacteremia, cellulitis, gastroenteritis, and meningitis ( 12 ). Th is means that malakoplakia represents a systemic defect in the immune response, characteristically in cancer patients. A unique case report of malakoplakia in a patient with myeloma has been published ( 4 ). Th e authors of that case report speculated that malakoplakia was a predisposing condition, giving evidence of altered cellular immunity REFERENCES 1. Sbahi H , Di Palma JA . Faecal microbiota transplantation: applications and limitations in treating gastrointestinal disorders . BMJ Open Gastroenterol 2016 ; 3 : e000087 . 2. Lee CH , Belanger JE , Kazzam Z et al. Th e outcome and long-term follow-up of 94 patients with recurrent and refractory Clostridium diffi cile infection using single to multiple fecal microbiota transplantation via retention enema . Eur J Clin Microbiol Infect Dis 2014 ; 33 : 1425 – 8 . 3. Chang BW , Pimentel M , Chang C et al. Prevalence of excessive intestinal methane production and its variability with age and gender: a large-scale database analysis . Gastroenterology 2015 ; 148 : S-729 – 30 . 4. Rezaie A , Chang BW , Chua S et al. Accurate identifi cation of excessive methane gas producers by a single fasting measurement of exhaled methane: a large-scale database analysis . Am J Gastroenterol 2015 ; 110 : S759 . 5. Moayeddi P , Surette MG , Kim PT et al. Fecal microbiota transplantation induces remission in patients with active ulcerative colitis in a randomized controlled trial . Gastroenterology 2015 ; 149 : 102 – 9 .

Bendamustine and G-CSF support

We read the manuscript by Cerchione C et al. with particular interest [1]. The use of peg-filgrastim in patients treated with bendamustine in terms of the timing of filgrastim/pegfilgrastim is concerning. Prolonged neutropenia is sometimes anticipated during bendamustine chemotherapy. Cerchione C et al. reported the clinical benefits of peg-filgrastim as the primary prophylaxis for neutropenia [1]. This manuscript reported reduced neutropenic episodes and reduced febrile neutropenia (with or without documented infection) in patients treated with peg-filgrastim compared to those in the control group who were treated with filgrastim. This finding was hypothesized to reduce overall chemotherapy disruptions. We can acknowledge the importance of relative dose intensity (RDI) in the treatment of malignant lymphoma, which is supported by significant evidence. RDI is associated with a higher remission rate and better survival [2]. However, the relationship between RDI and survival in patients receiving bendamustine treatment and R-CHOP has not yet been fully revealed. In addition, grade 3–4 neutropenia occurred in only 44% of patients in a phase II study conducted by Czuczman MS et al. [3]. Grade 3–4 febrile neutropenia was observed in 6.1% of patients in another phase II study [4]. Thus, we might consider using peg-filgrastim in a Bsecondary-prophylaxis^ manner, according to the risk assessment of the NCCN Clinical Practice Guidelines (NCCN guidelines®) for myeloid growth factors. Here, we report the case of an 82-year-old male diagnosed with advanced mantle cell lymphoma and treated with rituximab–bendamustine (RB) therapy. He repeatedly received six courses of RB treatment until achieving complete remission. He did not initially receive G-CSF support during the chemotherapy. However, in his third course of RB therapy, prolonged neutropenia delayed the following course of chemotherapy (Fig. 1). He then received a single dose of each filgrastim and peg-filgrastim during the following chemotherapy course. Thereafter, his treatment course was not affected by chemotherapy disruption owing to the prolonged neutropenia after the fourth course of RB. Additionally, RDI was maintained throughout the following chemotherapy courses. Our case did not require G-CSF/peg-G-CSF support during the first two courses of RB. The first onset of grade 3 neutropenia was observed after the third course of chemotherapy. This suggests that primary prophylaxis with G-CSF/peg-G-CSF is not necessary for all the cases, even in those of elderly patients. Therefore, a secondary prophylactic use of peg-G-CSF seems more reasonable for the chemotherapy-induced neutropenia caused by bendmustine. However, the NCCN guidelines® for myeloid growth factors (Version 2.2017 revised on October 13, 2017) do not evaluate the use of G-CSF for bendamustine-containing regimens. Our case suggests that bendamustine causes prolonged neutropenia during the following course rather than during the initial course. The prevalence of severe neutropenia (grade 3– 4) complicated with febrile neutropenia was estimated to be < 10% according to several clinical studies [4] (up to 8.23% based on Japanese post-marketing surveillance data). This frequency of febrile neutropenia does not require routine prophylaxis of G-CSF/peg-G-CSF (NCCN guidelines). Further investigation to evaluate the efficacy of the primary or secondary prophylactic setting of G-CSF is warranted for the RB regimen. * Osamu Imataki oima@med.kagawa–u.ac.jp

Pure erythroid leukemia in a polymyositis patient treated with azathioprine

Acute erythroid leukemia, also known as acute myeloid leukemia-M6, may be associated with previous chemotherapy or immunosuppressive therapy. For 10 years, a 69-year-old Japanese female patient with pure erythroid leukemia (or acute myeloid leukemia-M6b) was treated for polymyositis with 50–100 mg/day azathioprine. She complained of dyspnea with low-grade fever and was diagnosed as having pure erythroid leukemia. Chromosomal analysis revealed a complex karyotype abnormality, with the deletion of 5q, -6, -7 and addition of 11q13. No morphological myelodysplastic changes were observed in her bone marrow cells. In this study, azathioprine accumulation was considered to be associated with the patient’s leukemogenesis.

Anemia and hypogammaglobulinemia caused by Ménétrier’s disease

was performed. Histopathological examination did not reveal any malignancy. H. pylori was detected in the stomach. Cytomegalovirus (CMV) infection was not detected in the gastric mucosa. His immunoglobulin levels decreased to 543 mg/dL (normal range 870–1700 mg/dL). In addition, he exhibited mild hypoproteinemia, his total protein level was 6.4 g/dL, and serum albumin level was 4.1 g/dL. Anti-CMV antibody was negative. Therefore, Ménétrier’s disease was diagnosed. Protein-losing gastroenteropathy was confirmed by 99mTc-human serum albumin scintigraphy (Fig. 2), and stool was examined for α-1-antitrypsin. The daily volume of fecal α1-antitrypsin was 36.4 mg/day, and the clearance of fecal α1-antitrypsin was 29.1 ml/day (normal range 0–22 ml/day) [1]. Ménétrier’s disease is categorized as a protein-losing gastroenteropathy, and its pathogenesis is hypothesized to be associated with epidermal growth factor receptor. Eradication therapy was administered for H. pylori infection, and anemia did not recur. Therefore, Ménétrier’s disease should be recognized as a cause of anemia in adolescents and young adults. A 13-year-old boy with anemia, identified during a health check-up at his junior high school, visited his primary physician clinic. His first laboratory results indicated a hemoglobin (Hb) level of 9.8 g/dL. After 2 months, a subsequent consultation with his primary physician showed that his Hb had decreased to 7.1 g/dL. Next, he presented at our hospital. His blood results revealed microcytic hypochromic anemia. Reticulocyte count was elevated to 29.23 × 104/μL (normal range 3 × 104–9 × 104/μL). Serum iron level was 526 μg/ dL (80–140 μg/mL), serum ferritin level was 137.8 ng/ mL (39–765 ng/mL), vitamin B12 level was 503 pg/mL (233–914 pg/mL), folate level was 5.4 ng/mL (3.6–129 ng/ mL), serum zinc level was 84 μg/dL (64–111 μg/dL), and serum copper level was 97 μg/dL (78–130 μg/dL). In addition, his erythropoietin level had increased to 43.9 mIU/mL (4.2–23.7). Although the patient did not report any episode of enteral bleeding, we suspected this based on his laboratory results. Gastroendoscopy revealed a fibrous mucoid coagulation in the stomach with enlarged gastric folds (Fig. 1). Atrophic mucosa suggested a Helicobacter pylori infection, and a biopsy of the hyperplastic mucosal lesion

Paradoxical thrombosis in idiopathic thrombocytopenic purpura.

a platelet count of 17.0 × 10/μL and rhabdomyolysis, showing creatinine kinase elevation to 6798 U/L (normal range 40–200 U/L). After recovery of the platelet count, he underwent catheter intervention that restored the blood flow (day 83) (Fig. 1c). Thromboembolism in the setting of thrombocytopenia is a hematological conundrum. The treatment of thromboembolism in thrombocytopenic patients is problematic due to its paradoxical pathophysiology. In ITP patients, both venous thromboembolism and arterial thrombus are more prevalent in patients with more comorbidities [1]. This risk is similar both before and after treatment. These apparently contradictory conditions can be understood based on the classical rule for thrombus formation, as hypothesized by Virchow’s triad: (1) hypercoagulopathy occurs with low platelet numbers and excessive platelet turnover; (2) endothelial cell injury occurs when platelet numbers are decreased; and (3) disturbed blood flow is caused by a precedent condition, for instance arteriosclerosis, as was seen in the present case. Our patient did not have glucose intolerance and hyperlipidemia, but had a smoking history (40 cigarettes/day × 48 years) as a risk factor for thromboembolism. The determinant risk posed by co-existing vascular conditions can be evaluated before the treatment, for A 77-year-old male patient was referred to our hospital with frailty, accompanied with muscle weakness and gait disturbance, and bilateral lower leg resting pain. Blood tests showed severe thrombocytopenia without remarkable coagulopathy, and cranial CT revealed multiple focal hemorrhages (Fig. 1a), which suggested a potential cause for the gait disturbance. At the onset of intracranial hemorrhage, the patient’s platelet count was 0.3 × 10/μL. Diagnostic testing for thrombocytopenia was performed by examining bone marrow aspirate. We diagnosed idiopathic thrombocytopenic purpura (ITP) based on the unrepressed proliferation of megakaryocytes in the hyperplastic bone marrow (day 0). His presentation was not consistent with any other possible differential diagnosis. We did not observe any immunological abnormalities in the blood tests, including anti-cardiolipin antibody and lupus anticoagulant (antiCL–β2GP1 complex). Soon after the diagnosis of ITP, the patient was treated with prednisolone at 1.0 mg/kg/day. His platelet count immediately recovered from 0.2 × 10 to 13.8 × 10/μL 10 days after corticosteroid treatment. Seventeen days after prednisolone treatment for ITP, the pain in the patient’s right leg was exacerbated, and ischemia and cyanosis were observed (day 17). CT angiography revealed arterial sclerosis obliterans in the right leg (Fig. 1b), with

Methionine PET Might Be Reliable for the Detection of Low M-Protein–Producing Myelomas

C-methionine (MET) positron emission tomography (PET) is more sensitive than F-fluorodeoxyglucose (FDG)-PET for detecting myeloma lesion. Many clinical studies support this evidence mainly for multiple myeloma cases of IgG or IgA subtypes. However, this is not confirmed for low monoclonal protein-producing myelomas, such as IgD, IgE, and nonsecretory type. We encountered a 71-year-old man with IgD λ-type myeloma. In this patient, FDG-PET did not reveal any abnormal uptake lesions, whereas MET-PET revealed diffuse bone marrow uptake that relieved after initial chemotherapy. We speculate that the determinants for high serological activity of protein metabolism are transporter system activity or proliferation index.