Chronic myeloid leukemia stem cells and molecular target therapies for overcoming resistance and disease persistence.

Chronic myeloid leukemia (CML) is effectively treated with tyrosine kinase inhibitors (TKI) targeted against BCR-ABL. We previously reported the investigation of residual CML diseases during TKI treatment using FACS-sorting and quantitative RT-PCR of BCR-ABL among each population; total mononuclear cells, hematopoietic stem cells, and myeloid progenitors. The observations also implied that the second-generation of ABL-tyrosine kinase inhibitors (2nd TKIs), dasatinib or nilotinib therapy can be more promising approach for efficient reduction of the CML stem cells. Moreover, we need to develop the evaluation method of the residual CML diseases to establish rational therapy-cessation strategies in CML.

GATA2 regulates differentiation of bone marrow-derived mesenchymal stem cells.

The bone marrow microenvironment comprises multiple cell niches derived from bone marrow mesenchymal stem cells. However, the molecular mechanism of bone marrow mesenchymal stem cell differentiation is poorly understood. The transcription factor GATA2 is indispensable for hematopoietic stem cell function as well as other hematopoietic lineages, suggesting that it may maintain bone marrow mesenchymal stem cells in an immature state and also contribute to their differentiation. To explore this possibility, we established bone marrow mesenchymal stem cells from GATA2 conditional knockout mice. Differentiation of GATA2-deficient bone marrow mesenchymal stem cells into adipocytes induced accelerated oil-drop formation. Further, GATA2 loss- and gain-of-function analyses based on human bone marrow mesenchymal stem cells confirmed that decreased and increased GATA2 expression accelerated and suppressed bone marrow mesenchymal stem cell differentiation to adipocytes, respectively. Microarray analysis of GATA2 knockdowned human bone marrow mesenchymal stem cells revealed that 90 and 189 genes were upregulated or downregulated by a factor of 2, respectively. Moreover, gene ontology analysis revealed significant enrichment of genes involved in cell cycle regulation, and the number of G1/G0 cells increased after GATA2 knockdown. Concomitantly, cell proliferation was decreased by GATA2 knockdown. When GATA2 knockdowned bone marrow mesenchymal stem cells as well as adipocytes were cocultured with CD34-positive cells, hematopoietic stem cell frequency and colony formation decreased. We confirmed the existence of pathological signals that decrease and increase hematopoietic cell and adipocyte numbers, respectively, characteristic of aplastic anemia, and that suppress GATA2 expression in hematopoietic stem cells and bone marrow mesenchymal stem cells.

3-Deazaneplanocin A (DZNep), an inhibitor of S-adenosylmethionine-dependent methyltransferase, promotes erythroid differentiation.

EZH2, a core component of polycomb repressive complex 2 (PRC2), plays a role in transcriptional repression through histone H3 Lys-27 trimethylation and is involved in various biological processes, including hematopoiesis. It is well known that 3-deazaneplanocin A (DZNep), an inhibitor of S-adenosylmethionine-dependent methyltransferase that targets the degradation of EZH2, preferentially induces apoptosis in various hematological malignancies, suggesting that EZH2 may be a new target for epigenetic treatment. Because PRC2 participates in epigenetic silencing of a subset of GATA-1 target genes during erythroid differentiation, inhibition of EZH2 may influence erythropoiesis. To explore this possibility, we evaluated the impact of DZNep on erythropoiesis. DZNep treatment significantly induced erythroid differentiation of K562 cells, as assessed by benzidine staining and quantitative RT-PCR analysis for representative erythroid-related genes, including globins. When we evaluated the effects of DZNep in human primary erythroblasts derived from cord blood CD34-positive cells, the treatment significantly induced erythroid-related genes, as observed in K562 cells, suggesting that DZNep induces erythroid differentiation. Unexpectedly, siRNA-mediated EZH2 knockdown had no significant effect on the expression of erythroid-related genes. Transcriptional profiling of DZNep-treated K562 cells revealed marked up-regulation of SLC4A1 and EPB42, previously reported as representative targets of the transcriptional corepressor ETO2. In addition, DZNep treatment reduced the protein level of ETO2. These data suggest that erythroid differentiation by DZNep may not be directly related to EZH2 inhibition but may be partly associated with reduced protein level of hematopoietic corepressor ETO2. These data provide a better understanding of the mechanism of action of DZNep, which may be exploited for therapeutic applications for hematological diseases, including anemia.

Elucidation of the role of LMO2 in human erythroid cells.

LIM-only protein 2 (LMO2) is a non-DNA-binding component of a protein complex containing master regulators of hematopoiesis, including GATA-1, SCL/TAL1, and LDB1. However, the role of LMO2 in human erythroid differentiation is unclear. LMO2 knockdown in hemin-treated K562 cells reduced the benzidine-positive cell ratio, suggesting that LMO2 retards hemin-mediated K562 cell differentiation. Microarray analysis using K562 cells after siRNA-mediated LMO2 knockdown indicated that 177 and 78 genes were upregulated and downregulated (>1.5-fold), respectively. The downregulated gene ensemble contained prototypical erythroid genes (HBB, SLC4A1). Whereas LMO2 knockdown did not affect GATA-1 or SCL/TAL1 expression, it resulted in significantly reduced chromatin occupancy of GATA-1, SCL/TAL1, and LDB1 at the ß-globin locus control region and SLC4A1 locus in both K562 cells and human induced pluripotent stem cell-derived erythroid cells. Introduction of GATA-1 mutations, shown to impair direct interaction with LMO2, significantly diminished chromatin occupancy. On the other hand, knockdown of either SCL/TAL1 or LDB1 also resulted in significantly reduced chromatin occupancy of GATA-1 at endogenous loci, suggesting that impaired assembly of these components also affects GATA-1 chromatin occupancy. In an ex vivo model of erythroid differentiation from CD34(+) cells, LMO2 protein level peaked on day 5 and decreased at later stages of differentiation. The LMO2 expression pattern was similar to those of GATA-1 and SCL/TAL1. Furthermore, shRNA-mediated LMO2 knockdown in primary erythroblasts suggested that LMO2 regulates HBB, HBA, and SLC4A1 expression. LMO2 contributes to GATA-1 target gene expression by affecting assembly of the GATA-SCL/TAL1 complex components at endogenous loci.

Clinicopathological analysis of primary adrenal diffuse large B-cell lymphoma: effectiveness of rituximab-containing chemotherapy including central nervous system prophylaxis.

BACKGROUND: Primary adrenal lymphoma (PAL) is an extremely rare subtype of extranodal non-Hodgkin's lymphoma. Some researchers have reported some of the characteristics of PAL and its association with poor prognosis; however, the clinicopathological features of PAL remain to be elucidated. METHODS: From 2008 to 2011 we experienced seven cases of PAL in our institutions. We retrospectively analyzed the clinical and pathological features of these patients. RESULTS: The patients ranged in age from 50 to 85 years, with a median of 71 years. The overall male:female ratio was 6:1. All seven patients were diagnosed with diffuse large B-cell lymphoma (DLBCL) pathologically. Bilateral adrenal involvement was confirmed in five patients. The median largest tumor diameter at diagnosis was 58 mm. The Ki-67 index was generally high (>70%). All patients were treated with rituximab-containing chemotherapy, and central nervous system (CNS) prophylaxis was conducted for three patients. One patient with CNS involvement at the time of the diagnosis also received whole-brain radiation. The overall survival rate at two years was 57% (median follow-up; 24.8 months). It is noteworthy that the three patients who received a full course of the rituximab-containing regimen and CNS prophylaxis are currently alive without disease relapse, and that none of the seven patients died due to progression of lymphoma. CONCLUSIONS: Primary adrenal DLBCL can be a clinically aggressive disease entity. Rituximab-containing chemotherapy combined with CNS prophylaxis could be a reasonable option for the treatment of PAL; however, analyses of more PAL cases are needed for the establishment of this strategy.

Molecular determinants for small Maf protein control of platelet production.

MafG and p45 possess basic region-leucine zipper (bZip) domains and form a heterodimer called NF-E2, a key regulator of megakaryopoiesis. NF-E2 binds to the Maf recognition element (MARE) and activates transcription of many platelet genes. Since the bZip domain, which mediates DNA binding and heterodimerization, is the only functional domain established for MafG, it has been assumed that MafG is required only for p45 binding to MARE and to facilitate p45-mediated transcriptional activation. Analysis of the C-terminal region of MafG, which is distinct from the bZip domain, revealed that this region contains a nuclear matrix-targeting signal. We used a transgenic complementation rescue assay to delineate the function of the MafG C terminus in vivo. Transgenic mice expressing a mutant MafG protein lacking the C terminus (MafGΔC) were crossed into a MafG-null background. The compound mutant mice displayed severe thrombocytopenia and splenomegaly, which phenocopied p45-null mice. The MafG C terminus is essential for proplatelet formation and platelet gene activation but not for p45 binding to MARE. These results demonstrate that the MafG C terminus is required for NF-E2 function and suggest that efficient targeting of NF-E2 to a specific nuclear scaffold is important to achieve high-level activity.

Evolutionary origin of the Otx2 enhancer for its expression in visceral endoderm.

In the mouse, the Otx2 gene has been shown to play essential roles in the visceral endoderm during anterior-posterior axis formation and head induction. While these are primary processes in vertebrate embryogenesis, the visceral endoderm is a tissue unique to mammals. Two enhancers (VE and CM) have been previously found to direct Otx2 expression during early embryogenesis. This study demonstrates that in anterior visceral endoderm the CM enhancer does not have an activity by itself, but enhances the activity of the VE enhancer. These two enhancers also cooperate for the activities in anterior mesendoderm and cephalic mesenchyme. Comparative studies suggest that VE enhancer function was most likely established before the divergence of sarcopterygians into Actinistia, Dipnoi and tetrapods, while the nucleotide sequence corresponding to the VE enhancer was already present in the last common ancestor of bony fishes. The CM enhancer sequence and function would have been also established in ancestral sarcopterygians. The VE/CM enhancers and their gene cascades in the ancestral sarcopterygian head organizer would then have been co-opted by amphibian deep endoderm cells and mammalian visceral endoderm cells for the head development.

NF-E2 domination over Nrf2 promotes ROS accumulation and megakaryocytic maturation.

In megakaryocytes, the maturation process and oxidative stress response appear to be closely related. It has been suggested that increased oxygen tension and reactive oxygen species (ROS) promote megakaryopoiesis and that the expression of stress-responsive genes responsible for ROS elimination declines during megakaryocytic maturation. NF-E2 p45 is an essential regulator of megakaryopoiesis, whereas Nrf2 is a key activator of stress-responsive genes. Because p45 and Nrf2 have similar DNA-binding specificities, we hypothesized that p45 competes with Nrf2 to repress stress-responsive genes and achieves favorable intracellular conditions to allow ROS to be efficiently used as signaling molecules. We conducted comprehensive gene expression profiling with wild-type and p45-null megakaryocytes and examined the functional relationship between p45 and Nrf2. We found that 2 characteristic gene clusters are defined within p45 target genes: platelet genes and cytoprotective genes. The former are unique targets activated by p45, whereas the latter are common targets of p45 and Nrf2. Further analysis suggested that, as a less efficacious activator, p45 maintains moderate expression of cytoprotective genes through competing with Nrf2 and promotes ROS accumulation. Increased ROS enhanced platelet gene expression. These results suggest that p45 dominates over Nrf2 to enhance megakaryocytic maturation by promoting ROS accumulation.

Budd-Chiari syndrome associated with hypereosinophilic syndrome; a case report.

A 27-year-old man was admitted due to abdominal fullness. He had ascites and subcutaneous nodules on his head, with liver dysfunction and eosinophilia. Abdominal imaging revealed obstruction of the hepatic veins and stenosis of the inferior vena cava. Histological diagnosis of a subcutaneous nodule revealed obstructive thrombophlebitis with eosinophils. Tyrosine kinase created by fusion of the FIP1L1 and PDGFRA genes, which is characteristic of hypereosinophilic syndrome (HES), was detected. He was diagnosed with Budd-Chiari syndrome associated with HES. Liver function tests improved after interventional therapy followed by steroid therapy. It is important to diagnose the cause of Budd-Chiari syndrome.

Metabolic syndrome, diabetes and subclinical atherosclerosis as assessed by carotid intima-media thickness.

AIM: Carotid intima-media thickness (IMT) is a useful surrogate marker of cardiovascular disease and is associated with cardiac events. We investigated cross-sectionally the association between carotid intima-media thickness (IMT), confounding risk factors, and metabolic syndrome (MetS) using the modified Japanese criteria. METHODS: Carotid IMT was evaluated on B-mode ultrasonography in 918 patients (394 men aged 66 +/- 15 years and 524 women aged 72 +/- 13 years). RESULTS: Among our 918 patients, 74 (8.1%) had no metabolic abnormalities, 478 (52.1%) had a metabolic abnormality with neither type 2 diabetes or MetS, and 127 had MetS without diabetes. Of the patients with type 2 diabetes, 132 (14.4%) did not have MetS and 107 (11.7%) had both type 2 diabetes and MetS. The carotid IMT values in the four groups with any metabolic abnormalities were significantly greater than the IMT of the group with neither condition (p=0.001), respectively. In syndrome model, type 2 diabetes was significantly associated with carotid atherosclerosis (p= 0.006), but MetS was borderline significant. In the component model of MetS, there was a significant association with hypertension (p<0.001) and dyslipidemia (p=0.006). Multiple logistic regression analysis for carotid atherosclerosis compared to neither condition demonstrated that subjects with both MetS and diabetes (OR, 5.58; 95% CI, 2.64-11.8), those with type 2 diabetes without MetS (OR, 3.00; 95% CI, 1.45-6.22), and those with MetS without type 2 diabetes (OR, 2.58; 75% CI, 1.24-5.39) showed a higher odds ratio after adjustment for covariates. CONCLUSION: Even after taking into account each individual component of MetS, the clustering of visceral obesity with at least 2 of the 3 components, and diabetes are independently associated with increased carotid IMT. This suggests that the components of MetS and type 2 diabetes interact to affect vascular thickness synergistically.

Metabolic syndrome may be a risk factor for early carotid atherosclerosis in women but not in men.

AIM: Metabolic syndrome (MetS) is a cluster of metabolic abnormalities and a predictor of both type 2 diabetes mellitus and adverse cardiovascular events. Whether there are gender differences in the association between early atherosclerosis and MetS has not yet been thoroughly elucidated. METHODS: The subjects consisted of 388 men aged 64+/-16 years and 480 women aged 70+/-13 years. Early atherosclerosis was assessed by carotid intima-media thickness (IMT) on B-mode ultrasonography. RESULTS: Carotid IMT values were significantly greater in both male (p=0.007) and female (p=0.002) subjects with MetS. After adjusting for established risk factors, the difference persisted on a significant level in women (p=0.003), but was weak in men (p=0.013). Multiple regression analysis using IMT as an objective variable, with adjustment for various risk factors as explanatory variables, showed that MetS (p=0.016) was a significant independent contributing factor along with known risk factors only in women. Among the components of MetS, hypertension (p=0.036) and dyslipidemia (p=0.008) had a strong impact on carotid IMT in men, whereas hypertension (p=0.003) ranked first in women. CONCLUSION: The effect of MetS in early carotid atherosclerosis is more pronounced in women than in men, and the impact of MetS components on carotid IMT differs between men and women.

Preperitoneal fat thickness by ultrasonography and obesity-related disorders.

PURPOSE: To determine optimal cutoff values for preperitoneal fat thickness measured by ultrasonography as indicators for obesity-related disorders. METHODS: We studied 276 men aged 60 ± 13 years and 307 women aged 64 ± 11 years. Participants were consecutively enrolled from inpatients aged ≤75 years. Demographic data were collected and maximal preperitoneal fat thickness (PFTmax) and carotid intima-media thickness were evaluated on B-mode ultrasonography. Receiver operating characteristic (ROC) curve analysis was performed to determine optimal cutoff values for PFTmax. RESULTS: Multiple regression analysis using one or more obesity-related disorders as an objective variable showed that the tertile on the basis of PFTmax was a significant independent contributing factor in both men and women. Receiver operating characteristic curve analysis identified the cutoff points of 6.1 mm for PFTmax in men (sensitivity, 66.7%; specificity, 62.5%) and 8.7 mm for PFTmax in women (sensitivity, 56.6%; specificity, 63.6%) as discriminator values corresponding to the presence of one or more obesity-related disorders. Using the new criteria to diagnose visceral obesity, we found that adjusted carotid intima-media thickness was significantly higher in men and women with visceral obesity and two or more obesity-related disorders than in those without them. CONCLUSIONS: These findings suggested that PFTmax measured on ultrasonography was useful in screening for indicators of cardiovascular risk factors.

Evolutionary constraint on Otx2 neuroectoderm enhancers-deep conservation from skate to mouse and unique divergence in teleost.

Otx2 is a paired type homeobox gene that plays essential roles in each step and site of head development in vertebrates. In the mouse, Otx2 expression in the anterior neuroectoderm is regulated primarily by two distinct enhancers: anterior neuroectoderm (AN) and forebrain/midbrain (FM) enhancers at 92 kb and 75 kb 5'of the Otx2 locus, respectively. The AN enhancer has activity in the entire anterior neuroectoderm at headfold and early somite stages, whereas the FM enhancer is subsequently active in the future caudal forebrain and midbrain ectoderm. In tetrapods, both AN and FM enhancers are conserved, whereas the AN region is missing in teleosts, despite overt Otx2 expression in the anterior neuroectoderm. Here, we show that zebrafish and fugu FM regions drive expression not only in the forebrain and midbrain but also in the anterior neuroectoderm at headfold stage. The analysis of coelacanth and skate genomic Otx2 orthologues suggests that the utilization of the two enhancers, AN and FM, is an ancestral condition. In contrast, the AN enhancer has been specifically lost in the teleost lineage with a compensatory establishment of AN activity within the FM enhancer. Furthermore, the AN activity in the fish FM enhancer was established by recruiting upstream factors different from those that direct the tetrapod AN enhancer, yet zebrafish FM enhancer is active in both mouse and zebrafish anterior neuroectoderm at the headfold stage.

Impact of C-reactive protein on the likelihood of carotid atherosclerosis in Japanese adults.

OBJECTIVE: Several cohort studies have shown a link between serum C-reactive protein (CRP) and subsequent cardiovascular disease; however, the role of CRP as an independent risk factor remains controversial. We therefore investigated the association between CRP and sclerotic lesions of common carotid atherosclerosis. PATIENTS AND METHODS: We evaluated sclerotic lesions of common carotid arterial intima-media thickness (IMT) by ultrasonography in 139 men aged 67 +/- 15 years and 201 women aged 75 +/- 10 years. To investigate the relation between CRP and various confounding factors, subjects were divided into four groups based on the quartile of CPR. RESULTS: Carotid IMT values were significantly higher in groups with higher CRP (p = 0.022). To identify the possible CRP level and risk factor interactions for IMT, multiple regression analysis for IMT was performed based on risk factors in subjects with a specific CRP level. It was shown that age, smoking status, systolic blood pressure (SBP) and LDL cholesterol were significantly associated with IMT in subjects in the lower CRP groups (CRP-1 approximately CRP-3), and age, SBP and presence of diabetes mellitus in the highest CRP group (CRP-4). To further investigate whether the interaction between CRP and conventional risk factors could influence IMT, a general linear model demonstrated that interaction between CRP and the presence of diabetes mellitus (F = 4.754 p = 0.030) was significantly associated with IMT, in addition to sex, age, SBP, antihypertensive drug use, LDL cholesterol and HDL cholesterol. CONCLUSIONS: This finding indicates that the association between CRP and IMT significantly differed between subjects with and without diabetes mellitus.

A new serine/threonine protein kinase, Omphk1, essential to ventral body wall formation.

Here, we report a new serine/threonine protein kinase of the SNF1 subfamily Omphk1. Two Omphk homologues exist in each vertebrate species, and one homologue exists in Drosophila and Caenorhabditis elegans; the kinase domain is highly conserved among these homologues, and several domains are conserved among vertebrate Omphk. Omphk1 expression dynamically changes in the developing central nervous system, is found ubiquitously in epidermis, and is present uniquely in several other tissues. Its expression is also found in each tissue associated with the ventral body wall closure: the primary body wall composed of primitive ectoderm and each component of the secondary body wall. Concomitantly, its null mutant exhibits omphalocele with a failure in closure of the secondary body wall. There are no apparent gross morphological defects in brain, however, despite the unique Omphk1 expression in this tissue.

Argon plasma coagulation for the treatment of early gastric cancer.

BACKGROUND/AIMS: Endoscopic mucosal resection (EMR) is widely used to treat early gastric cancer and is considered safe and effective. However, its indication range is limited. Other endoscopic treatment options are needed for patients with surgical risks. The aim of the study was to evaluate the safety and effectiveness of argon plasma coagulation (APC) for the treatment of early gastric cancer. METHODOLOGY: APC was done in 23 patients (mean age, 77.5 years) with early gastric cancer. The depth of tumor invasion, estimated primarily by endoscopic ultrasonography, was mucosal in 19 patients and submucosal in 4. EMR was not indicated in 4 cases, and 14 cases were not successfully treated by EMR alone. All patients were followed up for more than 12 months (median, 42.0+/-20.8 months). RESULTS: Fifteen patients had no recurrence and survived. Four patients had no recurrence, but died of causes other than gastric cancer. Cancer recurred in four patients. Recurrence was managed by repeated treatment with APC, with no technical problems. No serious complications occurred. CONCLUSIONS: APC is useful for follow-up treatment of early gastric cancer after EMR. APC may also be effective for radical treatment of early gastric cancer, especially in elderly patients and patients in whom surgical intervention is contraindicated.