High expression of ABCG2 induced by EZH2 disruption has pivotal roles in MDS pathogenesis.

Both proto-oncogenic and tumor-suppressive functions have been reported for enhancer of zeste homolog 2 (EZH2). To investigate the effects of its inactivation, a mutant EZH2 lacking its catalytic domain was prepared (EZH2-dSET). In a mouse bone marrow transplant model, EZH2-dSET expression in bone marrow cells induced a myelodysplastic syndrome (MDS)-like disease in transplanted mice. Analysis of these mice identified Abcg2 as a direct target of EZH2. Intriguingly, Abcg2 expression alone induced the same disease in the transplanted mice, where stemness genes were enriched. Interestingly, ABCG2 expression is specifically high in MDS patients. The present results indicate that ABCG2 de-repression induced by EZH2 mutations have crucial roles in MDS pathogenesis.

Prospective and clinical validation of ALK immunohistochemistry: results from the phase I/II study of alectinib for ALK-positive lung cancer (AF-001JP study).

BACKGROUND: Anaplastic lymphoma kinase (ALK) fusions need to be accurately and efficiently detected for ALK inhibitor therapy. Fluorescence in situ hybridization (FISH) remains the reference test. Although increasing data are supporting that ALK immunohistochemistry (IHC) is highly concordant with FISH, IHC screening needed to be clinically and prospectively validated. PATIENTS AND METHODS: In the AF-001JP trial for alectinib, 436 patients were screened for ALK fusions through IHC (n = 384) confirmed with FISH (n = 181), multiplex RT-PCR (n = 68), or both (n = 16). IHC results were scored with iScore. RESULT: ALK fusion was positive in 137 patients and negative in 250 patients. Since the presence of cancer cells in the samples for RT-PCR was not confirmed, ALK fusion negativity could not be ascertained in 49 patients. IHC interpreted with iScore showed a 99.4% (173/174) concordance with FISH. All 41 patients who had iScore 3 and were enrolled in phase II showed at least 30% tumor reduction with 92.7% overall response rate. Two IHC-positive patients with an atypical FISH pattern responded to ALK inhibitor therapy. The reduction rate was not correlated with IHC staining intensity. CONCLUSIONS: Our study showed (i) that when sufficiently sensitive and appropriately interpreted, IHC can be a stand-alone diagnostic for ALK inhibitor therapies; (ii) that when atypical FISH patterns are accompanied by IHC positivity, the patients should be considered as candidates for ALK inhibitor therapies, and (iii) that the expression level of ALK fusion is not related to the level of response to ALK inhibitors and is thus not required for patient selection. REGISTRATION NUMBER: JapicCTI-101264 (This study is registered with the Japan Pharmaceutical Information Center).

Reduction of insulin signaling upregulates angiopoietin-like protein 4 through elevated free fatty acids in diabetic mice.

BACKGROUND: Angiopoietin-like protein 4 (Angptl4) is thought to cause an increase in serum triglyceride levels. In the present study, we elucidated Angptl4 expression in the mouse models of type 1 and type 2 diabetes mellitus, and investigated the possible mechanisms involved. METHODS: Type 1 diabetes was induced in C57BL/6 J mice by treating them with streptozotocin (STZ). Type 2 diabetes was induced by feeding the mice a high-fat diet (HFD) for 18 weeks. RESULTS: The levels of Angptl4 mRNA expression in liver, white adipose tissue (WAT), and brown adipose tissue (BAT) were found to increase in the STZ diabetic mice relative to control mice. This effect was attenuated by insulin administration. In the HFD diabetic mice, the Angptl4 mRNA expression levels were increased in liver, WAT, and BAT. Treatment with metformin for 4 weeks attenuated the increased levels of Angptl4 mRNA. Fatty acids (FAs) such as palmitate and linoleate induced Angptl4 mRNA expression in H4IIE hepatoma cells and 3T3-L1 adipocytes. Treatment with insulin but not metformin attenuated FA-induced Angptl4 mRNA expression in H4IIE. Both insulin and metformin did not influence the effect of FAs in 3T3-L1 cells. CONCLUSION: These observations demonstrated that Angptl4 mRNA expression was increased through the elevated free FAs in diabetic mice.

Detection of thymocytes apoptosis in mice induced by organochlorine pesticides methoxychlor.

The thymus has long been known to be vulnerable to atrophy when exposed to variety of stimuli, including hormones, immunosuppressive pharmaceuticals, and environmental chemicals. The organochlorine pesticide methoxychlor (MXC) is an immunosuppressive agent thought to affect thymic atrophy by inducing apoptosis of thymocyte T cells. We sought to develop an experimental protocol to detect in vivo thymocyte apoptosis induced by MXC in Balb/c mice. We treated the mice with 150-400 mg/kg MXC. We then measured thymus weight, cell counts, caspase activity (3/7, 8, and 9), annexin V labeling of phosphatidylserine (PS) and DNA fragmentation. In MXC-treated mice we observed decreases in thymus weight and cell counts and increases in caspase activity (3/7, 8, and 9), annexin V PS labeling and DNA fragmentation. These results suggest that MXC induces thymic atrophy caused by thymocyte apoptosis, and that our protocol may be useful for detecting in vivo thymocyte apoptosis induced by environmental chemicals in short-time.

Observation of an energetic radiation burst from mountain-top thunderclouds.

During thunderstorms on 20 September 2008, a simultaneous detection of gamma rays and electrons was made at a mountain observatory in Japan located 2770 m above sea level. Both emissions, lasting 90 sec, were associated with thunderclouds rather than lightning. The photon spectrum, extending to 10 MeV, can be interpreted as consisting of bremsstrahlung gamma rays arriving from a source which is 60-130 m in distance at 90% confidence level. The observed electrons are likely to be dominated by a primary population escaping from an acceleration region in the clouds.

Activation of Rac by cadherin through the c-Src-Rap1-phosphatidylinositol 3-kinase-Vav2 pathway.

Cadherin first forms homo-cis-dimers on the cell surface of the same cells, followed by formation of homo-trans-dimers (trans-interactions) in a Ca2+-dependent manner, eventually causing adherens junctions. In addition, trans-interacting cadherin induces activation of Rac small G protein, which stabilizes non-trans-interacting cadherin on the plasma membrane by inhibiting its endocytosis through the reorganization of the actin cytoskeleton. However, it has not fully been understood how cadherin induces the activation of Rac. We examined here the molecular mechanism of the activation of Rac by trans-interacting cadherin in fibroblasts and epithelial cells. Trans-interacting cadherin induced activation of c-Src locally at the cadherin-based cell-cell adhesion sites. c-Src then tyrosine-phosphorylated Vav2, one of the Rac-GDP/GTP exchange factors (GEFs), and induced activation of C3G, one of the Rap1-GEFs, through Crk adaptor protein, resulting in the activation of Rap1 locally at the cadherin-based cell-cell adhesion sites. The c-Src-catalysed tyrosine phosphorylation was not sufficient for the activation of Vav2 and the c-Src-induced activation of Rap1 was additionally necessary for it, although activated Rap1 alone was not sufficient for the activation of non-tyrosine-phosphorylated Vav2. This effect of Rap1 on Vav2 was mediated by phosphatidylinositol 3-kinase. We describe here the signaling pathway from trans-interacting cadherin to the activation of Rac.

MTG8 proto-oncoprotein interacts with the regulatory subunit of type II cyclic AMP-dependent protein kinase in lymphocytes.

AML1-MTG8 chimeric oncogene is generated in acute myelogenous leukemia with t(8;21), and seems to be responsible for the pathogenesis of the disease. However, the role of MTG8 is ambiguous. Here we found that MTG8 interacted with the regulatory subunit of type II cyclic AMP-dependent protein kinase (PKA RIIalpha). The binding site of MTG8 was NHR3 domain, and that of RIIalpha was the N-terminus for interacting with PKA anchoring proteins (AKAPs). NHR3 contains a putative alpha-amphipathic helix which is characteristic in binding of AKAPs with RII. Indirect immunofluorescence microscopy showed that MTG8 and RIIalpha were overlapped at the centrosome-Golgi area in lymphocytes. These findings suggest that MTG8 may function as an AKAP at the centrosome-Golgi area in lymphocytes.

Intramolecular 1,3-dipolar cycloaddition strategy for enantioselective synthesis of FR-900482 analogues.

[reaction: see text] Enantioselective synthesis of FR-900482 analogues is described. The key reaction of the synthesis is intramolecular 1,3-dipolar cycloaddition of a highly functionalized nitrile oxide with complete stereo- and regioselectivities to construct the eight-membered benzazocine ring.

Dogger Bank Itch revisited: isolation of (2-hydroxyethyl) dimethylsulfoxonium chloride as a cytotoxic constituent from the marine sponge Theonella aff. mirabilis.

(2-Hydroxyethyl) dimethylsulfoxonium chloride (1), the well-known causative agent of Dogger Bank Itch, has been isolated as a cytotoxic constituent from the marine sponge Theonella aff. mirabilis. The structure of 1 was determined by spectral and X-ray crystallographic analyses.

Metastatic renal cancer arising from adenoid cystic carcinoma of the parotid gland: a case report.

A 40-year-old woman underwent excision of the right parotid gland tumor in 1988. The pathological examination showed adenoid cystic carcinoma. In 1993 she underwent excision of a recurrent tumor on the right face and was referred to our department because of an incidental finding of left renal tumors. She underwent nephrectomy and was diagnosed with left renal metastasis on pathological examination. In 1997 computerized tomography demonstrated multiple metastases in the right kidney, liver, lungs and brain. She died of cancer in 1998. Secondary carcinoma of the kidney is usually identified at autopsy and represents a late and poor manifestation of primary disease when diagnosed during life. The present case is unique in its primary site, pathology and clinical course.

Induction of MTG8-specific cytotoxic T-cell lines: MTG8 is probably a tumour antigen that is recognized by cytotoxic T cells in AML1-MTG8-fused gene-positive acute myelogenous leukaemia.

Several reports have demonstrated the persistent detection of AML1-MTG8 fusion products, representing minimal residual disease (MRD), in patients with t(8;21) acute myelogenous leukaemia (AML) who are in long-term remission. It is probable that immune-mediated mechanisms that are able to suppress the expansion of MRD may result in the continuance of remission. It was previously shown that some t(8;21) AML patients had high anti-MTG8 antibody titres. MTG8 expression in normal adult tissues is limited to the brain or heart in which human leucocyte antigen (HLA) class I cell-surface antigens are either not or are only faintly detectable. We hypothesized that the overexpression of the MTG8 gene in t(8;21) AML cells could act as a possible tumour antigen, which might be able to induce the immune-mediated suppression of the expansion of MRD. We were able to induce HLA-A0201-restricted cytotoxic T-lymphocyte (CTL) lines against an MTG8 peptide (MTG8b amino acids 182-191) using monocyte-derived dendritic cells from a healthy donor. T-cell receptor (TCR)Valpha17, TCRVbeta14 and 15, and TCRJbeta2.1 and 2.3 are predominantly used in these CTL lines. Our data, which suggest that the MTG8 protein could be one of the tumour antigens recognized by CTLs, may be helpful in further investigations of TCR analysis in t(8;21) AML patients with HLA-A0201 who are in long-term remission.

[Clinical evaluation of estramustine phosphate and vinblastine in patients with hormone-refractory prostate cancer].

We evaluated the efficacy of the combination of estramustine phosphate and vinblastine in 13 patients with hormone-refractory prostate cancer. Of 12 patients with an elevated prostate specific antigen (PSA) level at the start of treatment, 5 (42%) had a greater than 50% decrease in PSA level. In a patient with cervical and mediastinal lymph node metastases, about a 57% decrease was observed in bidimensional measurement. Side effects were mild and manageable. The survival rate was not significantly different between patients who showed a greater than 50% decrease in PSA levels or regression of lymph node metastases versus the other patients.

[Small cell carcinoma of the urinary bladder: a case report].

A 64-year-old man presented with asymptomatic gross hematuria. Cystoscopy revealed a non-papillary broad-based tumor on the posterior wall of the bladder. He had no evidence of metastases. Urine cytology showed a cluster of molded tumor cells with naked hyperchromatic nuclei, which indicated small cell carcinoma. He received preoperative irradiation to the bladder and underwent partial cystectomy. Pathological examination of the specimen showed small cell carcinoma at the stage of pT2. Two years after the operation, he had no evidence of recurrence.

Ribonuclease H attack of leukaemic fused transcripts AML1-MTG8 (ETO) by DNA/RNA chimeric hammerhead ribozymes.

BACKGROUND: Catalytic anti-sense oligonucleotides might be useful tools for controlling specific gene expression. However, to obtain effective oligonucleotides of the desired function in vivo is still a difficult task. RESULTS: To evaluate the usefulness of synthesized DNA/RNA hammerhead ribozymes targeting AML1-MTG8 (ETO) leukaemic fusion transcripts in vivo, we analysed their effects on cell growth and the mechanism of action using isolated cell nuclei. These ribozymes inhibited the growth of leukaemic cell lines expressing the AML1 -MTG8 and degraded AML1-MTG8 mRNA in isolated nuclei of these cells. However, the reactions gave rise to additional cleavage products. Systematic cleavage analyses using an anti-sense oligonucleotide array revealed that the cleavage was induced by endogenous RNase H at specific sites, in accordance with their calculated melting temperature (Tm) values. With suppression of RNase H by sulfhydryl agents, the DNA/RNA ribozyme had a ribozyme catalytic activity. In addition, the ribozymes and anti-sense oligonucleotides suppressed the AML1-MTG8 protein in the leukaemic cells. CONCLUSIONS: The DNA/RNA ribozymes inhibited cell growth primarily via anti-sense effects, the main role of which was the activation of RNase H-digestion by their DNA arms. In addition, the isolated nuclei provided a useful assay system for modelling in vivo conditions for the quantitative evaluation of anti-sense/ribozyme activity.

Down-regulation of human telomeric protein TRF1 gene expression during myeloid differentiation in human hematopoietic cells.

The maintenance of telomere length is crucial for cell survival. Recently, it has been indicated that the human telomeric protein TRF1 is involved in the negative feedback mechanism that stabilizes telomere length. We studied TRF1 mRNA expression in hematopoietic cells to clarify the relation between TRF1 and telomerase by semiquantitative reverse transcriptase-polymerase chain reaction. In polymorphonuclear cells and monocytes isolated from peripheral blood, relatively low levels of TRF1 mRNA expression were seen, compared with those of lymphocytes and CD34+. We then assessed TRF1 mRNA expression in CD34+ cells cultured in vitro with growth factors. After 4 weeks of culture, all the cells showed myeloid differentiation, and telomerase activity was down-regulated. TRF1 mRNA was expressed in CD34+ cells but was down-regulated in cells cultured for 4 weeks. We conclude that TRF1 mRNA expression is down-regulated in accordance with telomerase down-regulation during the course of myeloid differentiation.

Proliferative involvement of ENX-1, a putative human polycomb group gene, in haematopoietic cells.

Homeobox genes have important roles in haematopoiesis and are regulated in an activated state by the trithorax group (trxG) of genes. In a repressed state, they are regulated by the Polycomb group (PcG) of genes. ENX-1, a putative human PcG gene product, interacts with the proto-oncogene product Vav. We report an investigation of the role of ENX-1 in human haematopoiesis. CD34+ cells mobilized to peripheral blood strongly expressed ENX-1. When stimulated to proliferate, both T and B lymphocytes rapidly up-regulated ENX-1. ENX-1 was expressed in all cell lines of the various lineages examined. When HL-60 cells were differentiated to mature granulocytes with all-trans retinoic acid, ENX-1 was down-regulated. Moreover, ENX-1 antisense oligodeoxynucleotide suppressed DNA synthesis in HL-60 cells. Our data indicate that ENX-1 is involved in the proliferation of both normal and malignant haematopoietic cells.

Direct carbonylation at a C-H bond in the benzene ring of 2-phenyloxazolines catalyzed by Ru(3)(CO)(12). Scope, limitations, and mechanistic aspects

The ruthenium-catalyzed carbonylation at a C-H bond in the benzene ring of a 2-phenyloxazoline is described. The reaction of 2-phenyloxazolines with CO and ethylene in toluene in the presence of a catalytic amount of Ru(3)(CO)(12) resulted in propionylation at an ortho C-H bond in the benzene ring. The presence of the oxazoline ring on the benzene ring is essential for the carbonylation to proceed. Other heterocycles, such as oxazine, oxazole, and thiazoline rings, also served as acceptable directing groups as did the oxazoline ring. A wide functional group compatibility was observed. The site selectivity of the carbonylation was examined using meta-substituted phenyloxazolines. It was found that the carbonylation took place exclusively at the less-hindered C-H bond, irrespective of the nature of substituents, indicating that the site selectivity was determined by steric factors. The reaction was also applicable, not only to a benzene ring, but also to naphthyl and thiophenyl rings. Olefins such as propene and trimethylvinylsilane in place of ethylene could also be used in the carbonylation reaction, while other olefins, such as 1-hexene, tert-butylethylene, vinylcyclohexane, isoprene, 1,5-hexadiene, cyclohexene, 1, 5-cyclooctadiene, styrene, methyl acrylate, vinyl acetate, allyltrimethylsilane, and triethoxyvinylsilane did not afford the coupling products. An equilibrium between 2-phenyloxazolines, carbon monoxide, and olefins exists on one hand and the corresponding ketones on the other hand, and product composition is governed by the equilibrium thermodynamics of the system. The results of deuterium labeling experiments suggest that the catalysis involves a reversible C-H bond cleavage and that the rate-determining step is not the cleavage of a C-H bond. The results of kinetic study of the effects of CO pressure show that the reaction rate accelerates with decreasing CO pressure.

[Lymphocytic infundibuloneurohypophysitis during the first remission in acute lymphoblastic leukemia].

A 15-year-old girl developed acute lymphoblastic leukemia (ALL). The patient was treated according to the 13th protocol of the Tokyo Children's Cancer Study Group, and thereafter remained free of disease. However, at the age of 20, she complained of polyuria, polydipsia and amenorrhea. Hematological or meningeal relapse was ruled out on the basis of clinical and laboratory findings. The plasma concentrations of GH, TSH, LH, FSH, ACTH and ADH were low or below the detectable limits. There was no increase in urine osmolarity after water deprivation. Arginine, LH-RH, TRH and CRH tolerance tests revealed no or low responses of GH, LH/FSH, TSH, and ACTH/cortisol, respectively. Magnetic resonance imaging demonstrated thickening of the pituitary stalk, which was homogeneously enhanced by gadolinium administration. A biopsy specimen showed fibrosis and infiltration of CD8-positive T lymphocytes in a portion of the pituitary stalk, whereas the adenohypophysis was normal. In addition, no leukemic cells were observed in the samples. Thus, a diagnosis of lymphocytic infundibuloneurohyophysitis (LIN) was established. All the symptoms were improved by treatment with hydrocortisone, L-thyroxine, desamino-8-d-arginine vasopressin, estrogen and gestagen. This is the first reported case of ALL complicated by LIN.