SETDB1 deletion causes DNA demethylation and upregulation of multiple zinc-finger genes.

BACKGROUND: SETDB1 (SET domain bifurcated-1) is a histone H3-lysine 9 (H3K9)-specific methyltransferase that mediates heterochromatin formation and repression of target genes. Despite the assumed functional link between DNA methylation and SETDB1-mediated H3K9 trimethylations, several studies have shown that SETDB1 operates autonomously of DNA methylation in a region- and cell-specific manner. This study analyzes SETDB1-null HAP1 cells through a linked methylome and transcriptome analysis, intending to explore genes controlled by SETDB1-involved DNA methylation. METHODS AND RESULTS: We investigated SETDB1-mediated regulation of DNA methylation and gene transcription in human HAP1 cells using reduced-representation bisulfite sequencing (RRBS) and RNA sequencing. While two-thirds of differentially methylated CpGs (DMCs) in genic regions were hypomethylated in SETDB1-null cells, we detected a plethora of C2H2-type zinc-finger protein genes (C2H2-ZFP, 223 of 749) among the DMC-associated genes. Most C2H2-ZFPs with DMCs in their promoters were found hypomethylated in SETDB1-KO cells, while other non-ZFP genes with promoter DMCs were not. These C2H2-ZFPs with DMCs in their promoters were significantly upregulated in SETDB1-KO cells. Similarly, C2H2-ZFP genes were upregulated in SETDB1-null 293T cells, suggesting that SETDB1's function in ZFP gene repression is widespread. There are several C2H2-ZFP gene clusters on chromosome 19, which were selectively hypomethylated in SETDB1-KO cells. CONCLUSIONS: SETDB1 collectively and specifically represses a substantial fraction of the C2H2-ZFP gene family. Through the en-bloc silencing of a set of ZFP genes, SETDB1 may help establish a panel of ZFP proteins that are expressed cell-type specifically and thereby can serve as signature proteins for cellular identity.

Coordinated regulation of microRNA genes in C19MC by SETDB1.

The microRNA (miRNA) gene cluster on chromosome 19, C19MC, is the largest primate-specific miRNA gene cluster. The 46 homologous miRNA genes in C19MC are highly expressed in the placenta, but repressed in other tissues by DNA methylation. Here, we found that the SET domain bifurcated 1(SETDB1), a histone H3-lysine 9 (H3K9)-specific methyltransferase 1, transcriptionally controls C19MC miRNA genes in a coordinated manner in human HAP1 cells. SETDB1 knockout (KO) resulted in the overexpression of C19MC miRNA genes, which was accompanied by a reduction of H3K9 trimethylation (H3K9me3) in the cluster. We found that SETDB1 specifically binds to and modifies the upstream promoter locus of C19MC with H3K9me3, suggesting its role as a C19MC repressor. Overexpression of C19MC miRNA genes was not related to DNA methylation because cytosine methylation levels were not altered in the C19MC of SETDB1 KO cells, indicating that SETDB1 KO does not cause DNA demethylation in the C19MC promoter and body regions. In conclusion, our results suggest that SETDB1 binding and H3K9 methylation at the C19MC promoter and body regions are responsible for the coordinated regulation of miRNA genes in the cluster.

Dnmt1 binds and represses genomic retroelements via DNA methylation in mouse early embryos.

Genome-wide passive DNA demethylation in cleavage-stage mouse embryos is related to the cytoplasmic localization of the maintenance methyltransferase DNMT1. However, recent studies provided evidences of the nuclear localization of DNMT1 and its contribution to the maintenance of methylation levels of imprinted regions and other genomic loci in early embryos. Using the DNA adenine methylase identification method, we identified Dnmt1-binding regions in four- and eight-cell embryos. The unbiased distribution of Dnmt1 peaks in the genic regions (promoters and CpG islands) as well as the absence of a correlation between the Dnmt1 peaks and the expression levels of the peak-associated genes refutes the active participation of Dnmt1 in the transcriptional regulation of genes in the early developmental period. Instead, Dnmt1 was found to associate with genomic retroelements in a greatly biased fashion, particularly with the LINE1 (long interspersed nuclear elements) and ERVK (endogenous retrovirus type K) sequences. Transcriptomic analysis revealed that the transcripts of the Dnmt1-enriched retroelements were overrepresented in Dnmt1 knockdown embryos. Finally, methyl-CpG-binding domain sequencing proved that the Dnmt1-enriched retroelements, which were densely methylated in wild-type embryos, became demethylated in the Dnmt1-depleted embryos. Our results indicate that Dnmt1 is involved in the repression of retroelements through DNA methylation in early mouse development.

ß-Elemene Attenuates Renal Fibrosis in the Unilateral Ureteral Obstruction Model by Inhibition of STAT3 and Smad3 Signaling via Suppressing MyD88 Expression.

Renal fibrosis is a chronic pathological process that seriously endangers human health. However, the current therapeutic options for this disease are extremely limited. Previous studies have shown that signaling factors such as JAK2/STAT3, Smad3, and Myd88 play a regulatory role in renal fibrosis, and ß-elemene is a plant-derived sesquiterpenoid organic compound that has been shown to have anti-inflammatory, anti-cancer, and immunomodulatory effects. In the present study, the anti-fibrotic effect of ß-elemene was demonstrated by in vivo and in vitro experiments. It was shown that ß-elemene inhibited the synthesis of extracellular matrix-related proteins in unilateral ureteral obstruction mice, and TGF-ß stimulated rat interstitial fibroblast cells, including α-smooth muscle actin, vimentin, and connective tissue growth factor, etc. Further experiments showed that ß-elemene reduced the expression levels of the above-mentioned fibrosis-related proteins by blocking the phosphorylation of JAK2/STAT3, Smad3, and the expression or up-regulation of MyD88. Notably, knockdown of MyD88 attenuated the phosphorylation levels of STAT3 and Smad3 in TGF-ß stimulated NRK49F cell, which may be a novel molecular mechanism by which ß-elemene affects renal interstitial fibrosis. In conclusion, this study elucidated the anti-interstitial fibrosis effect of ß-elemene, which provides a new direction for future research and development of drugs related to chronic kidney disease.

A prognostic index for extranodal marginal-zone lymphoma based on the mucosa-associated lymphoid tissue International Prognostic Index and serum ß2-microglobulin levels.

The mucosa-associated lymphoid tissue (MALT) International Prognostic Index (IPI) was recently proposed as a prognostic index for patients with MALT lymphoma. We aimed to investigate the prognostic value of the serum ß2-microglobulin level in the context of MALT-IPI, and we proposed a new prognostic index. Survival outcomes were analysed with regard to ß2-microglobulin level, MALT-IPI, and the new prognostic index in MALT lymphoma patients (n = 571). The validity of the new prognostic index was assessed using an independent cohort (n = 216). Patients with high ß2-microglobulin levels had significantly worse progression-free survival (PFS) and overall survival (OS) outcomes. A high ß2-microglobulin level was independently associated with poor PFS and OS. ß2-microglobulin levels further stratified patients in the MALT-IPI intermediate-risk group in terms of PFS and OS. A new prognostic index based on the MALT-IPI and the ß2-microglobulin level, MALT-IPI-B, was proposed. The MALT-IPI-B was able to stratify patients into subgroups having distinct PFS and OS outcomes in both the training and validation cohorts. MALT-IPI-B enabled the identification of patients with poor survival outcomes who were classified into the intermediate-risk group by the MALT-IPI. In conclusion, this new ß2-microglobulin-based prognostic index may have the specific advantage of identifying high-risk patients who may require systemic treatment.

The Effect of Oxidative Stress and Memantine-Incorporated Reactive Oxygen Species-Sensitive Nanoparticles on the Expression of N-Methyl-d-aspartate Receptor Subunit 1 in Brain Cancer Cells for Alzheimer's Disease Application.

The aim of this study is to fabricate reactive oxygen species (ROS)-sensitive nanoparticles composed of succinyl ß-cyclodextrin (bCDsu), memantine and thioketal linkages for application in Alzheimer's disease, and to investigate the suppression of N-methyl-d-aspartate (NMDA) receptor 1 (NMDAR1) in cells. Thioketal diamine was attached to the carboxyl group of bCDsu to produce thioketal-decorated bCDsu conjugates (bCDsu-thioketal conjugates) and memantine was conjugated with thioketal dicarboxylic acid (memantine-thioketal carboxylic acid conjugates). Memantine-thioketal carboxylic acid conjugates were attached to bCDsu-thioketal conjugates to produce bCDsu-thioketal-memantine (bCDsuMema) conjugates. SH-SY5Y neuroblastoma cells and U87MG cells were used for NMDAR1 protein expression and cellular oxidative stress. Nanoparticles of bCDsuMema conjugates were prepared by means of a dialysis procedure. Nanoparticles of bCDsuMema conjugates had small particle sizes less than 100 nm and their morphology was found to be spherical in transmission electron microscopy observations (TEM). Nanoparticles of bCDsuMema conjugates responded to H2O2 and disintegrated or swelled in aqueous solution. Then, the nanoparticles rapidly released memantine according to the concentration of H2O2. In an in vivo animal imaging study, thioketal-decorated nanoparticles labelled with fluorescent dye such as chlorin e6 (Ce6) showed that the fluorescence intensity was stronger in the brain than in other organs, indicating that bCDsuMema nanoparticles can efficiently target the brain. When cells were exposed to H2O2, the viability of cells was time-dependently decreased. Memantine or bCDsuMema nanoparticles did not practically affect the viability of the cells. Furthermore, a western blot assay showed that the oxidative stress produced in cells using H2O2 increased the expression of NMDAR1 protein in both SH-SY5Y and U87MG cells. Memantine or bCDsuMema nanoparticles efficiently suppressed the NMDAR1 protein, which is deeply associated with Alzheimer's disease. Fluorescence microscopy also showed that H2O2 treatment induced green fluorescence intensity, which represents intracellular ROS levels. Furthermore, H2O2 treatment increased the red fluorescence intensity, which represents the NMDAR1 protein, i.e., oxidative stress increases the expression of NMDAR1 protein level in both SH-SY5Y and U87MG cells. When memantine or bCDsuMema nanoparticles were treated in cells, the oxidative stress-mediated expression of NMDAR1 protein in cells was significantly decreased, indicating that bCDsuMema nanoparticles have the capacity to suppress NMDAR1 expression in brain cells, which has relevance in terms of applications in Alzheimer's disease.

Src-mediated crosstalk between FXR and YAP protects against renal fibrosis.

Renal fibrosis is the common pathway of chronic kidney disease progression. The nuclear receptor farnesoid X receptor [FXR, NR1H4 (nuclear receptor subfamily 1 group member 4)], a multifunctional transcription factor, plays a pivotal role in protecting against fibrosis. However, the mechanisms underlying these antifibrotic actions of FXR in kidney disease are largely unknown. Here, we show that agonist GW4064-mediated FXR activation inhibits the activity of the nonreceptor tyrosine kinase Src (proto-oncogene tyrosine-protein kinase), which is critical for regulation of yes-associated protein (YAP) phosphorylation and nuclear localization in renal fibrosis. Activation of FXR suppressed renal fibrosis and Tyr416-Src phosphorylation in TGF-ß-treated human renal proximal tubule epithelial (HK2) cells. Moreover, GW4064 treatment in HK2 cells increased Ser127 phosphorylation, cytosolic accumulation of YAP, and interaction of the hippo core kinases (Ste20-like kinase 1, large tumor suppressor kinase 1, and salvador homolog 1). Inhibition of Src using PP2 (Src kinase inhibitor) prevented renal fibrosis and increased Ser127 phosphorylation and cytosolic accumulation of YAP. The expression of fibrosis markers, inflammatory genes, and YAP target genes was increased in the kidneys of FXR knockout mice compared with those of wild-type mice. In addition, GW4064 or WAY-362450 (turofexorate isopropyl) treatment protected against unilateral ureteral obstruction-induced renal fibrosis. Collectively, our data support the novel conclusion that Src-mediated crosstalk between FXR and YAP protects against renal fibrosis, making this pathway a possible therapeutic target for chronic kidney disease.-Kim, D.-H., Choi, H.-I., Park, J. S., Kim, C. S., Bae, E. H., Ma, S. K., Kim, S. W. Src-mediated crosstalk between FXR and YAP protects against renal fibrosis.

Pharmacokinetic and pharmacodynamic study of lenograstim for hematopoietic stem cell mobilization: a prospective randomized study for optimal apheresis.

BACKGROUND: This study evaluated the correlation between the pharmacokinetics and pharmacodynamics of granulocyte colony-stimulating factor (lenograstim) and the impact of initiation time of apheresis on stem cell mobilization in patients with multiple myeloma. STUDY DESIGN AND METHODS: Twenty-four patients with multiple myeloma were randomized into one of the two groups (early vs. late). Lenograstim at 10 µg/kg/day once daily was injected for at least 4 consecutive days. Apheresis was initiated 2 hours after the fourth dose of lenograstim in the early collection group and 16 hours after the fourth dose of lenograstim in the late collection group. Blood sampling for pharmacokinetics was performed within 30 minutes before, and 1, 2, 6, and 24 hours after the fourth dose of lenograstim. RESULTS: Overall, the two groups (early vs. late, n = 10 vs. 14) exhibited similar baseline characteristics including age, sex, subtype of myeloma, stage distribution, and myeloma-associated symptoms. No correlation was found between plasma lenograstim concentration and peripheral blood (PB) CD34+ cell counts or hematopoietic progenitor cells. In the late collection group, the median number of apheresis procedures for minimal collection was significantly lower (early vs. late: 2 vs. 1; p = 0.04) and there was a higher number of total collected PB CD34+ cells in a single session of apheresis (1.4 vs. 3.1; p = 0.06). There were no differences in median overall PB stem cell collection efficiency. CONCLUSION: Late collection positively impacted the number of apheresis procedures for minimal collection, with numerically improved PB stem cell collection efficiency at first apheresis in patients with multiple myeloma.

Thiotepa, busulfan, and cyclophosphamide or busulfan, cyclophosphamide, and etoposide high-dose chemotherapy followed by autologous stem cell transplantation for consolidation of primary central nervous system lymphoma.

Primary central nervous system lymphoma (PCNSL) is a rare extranodal non-Hodgkin lymphoma for which standard treatment has yet to be established. High-dose chemotherapy followed by autologous stem cell transplantation (ASCT) is a suitable consolidation strategy for patients who respond to induction chemotherapy. The purpose of this study was to compare the outcome and toxicity profile of the combination of busulfan, cyclophosphamide, and etoposide (BuCyE) with that of the combination of thiotepa, busulfan, and cyclophosphamide (TBC) as conditioning regimens of upfront ASCT for consolidation therapy in PCNSL. The PCNSL registry data set, prospectively collected from March 1993 to May 2017 at Asan Medical Center, was reviewed retrospectively. Patients with objective response to induction chemotherapy who received BuCyE or TBC as conditioning regimen for ASCT were included in the analysis. Primary endpoints were overall survival (OS) and progression-free survival (PFS). Among 241 patients with a diagnosis of PCNSL, 53 received ASCT as upfront consolidation therapy with TBC (28 patients) or BuCyE (25 patients) as conditioning regimen. No median OS or PFS was reached in the TBC group, while the BuCyE group reached a median OS of 4.9 years (p = 0.02) and median PFS of 1.1 years (p = 0.007). The incidence of oral mucositis, nausea, and vomiting was higher with TBC than BuCyE. The median admission duration and days to engraftment were similar between the two groups. Despite the greater incidence of adverse events, TBC showed better outcomes than BuCyE in terms of survival.

RON Receptor Tyrosine Kinase Regulates Epithelial Mesenchymal Transition and the Expression of Pro-Fibrotic Markers via Src/Smad Signaling in HK-2 and NRK49F Cells.

Receptor tyrosine kinases (RTKs) play important roles in the pathogenic processes of kidney fibrosis. However, the pathophysiological roles of recepteur d'origine nantais (RON), one of the receptor tyrosine kinases, have not yet been defined. We investigated whether the activation or sequence-specific small interfering RNA (siRNA) suppression of RON could regulate epithelial mesenchymal transition (EMT) and the expression of pro-fibrotic markers, and its underlying molecular mechanisms. Stable cell lines and transient transfection for RON and the transfected cells of siRNA for RON were developed to investigate the molecular mechanisms in human kidney proximal tubular epithelial (HK-2) and interstitial fibroblasts (NRK49F) cells. RON overexpression induced EMT and increased expression of fibrosis-related proteins such as N-cadherin, vimentin, transforming growth factor-ß (TGFß), αSMA, and fibronectin in HK-2 and NRK49F cells. RON overexpression increased various RTKs and the phosphorylation of Src (Y416) and Smad, while inhibition of RON by siRNA attenuated the expression of EMT- and fibrosis-related proteins and decreased RTKs such as insulin-like growth factor receptor (IGFR), fibroblast growth factor receptor 1 (FGFR1), vascular endothelial growth factor receptor (VEGFR), and platelet-derived growth factor receptor (PDGFR), as well as the phosphorylation of Src and Smad pathways. siRNA silencing of Src also attenuated the expression of IGFR, FGFR1, VEGFR, and PDGFR. Inhibition of RON can exert an anti-fibrotic effect by the inhibition of EMT and other RTKs through control of Src and Smad pathways in HK-2 and NRK49F cells.

PGC-1α Suppresses the Activation of TGF-ß/Smad Signaling via Targeting TGFßRI Downregulation by let-7b/c Upregulation.

TGF-ß/Smad signaling is a major pathway in progressive fibrotic processes, and further studies on the molecular mechanisms of TGF-ß/Smad signaling are still needed for their therapeutic targeting. Recently, peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) was shown to improve renal fibrosis, making it an attractive target for chronic kidney diseases (CKDs). Here, we show the mechanism by which PGC-1α regulates the TGF-ß/Smad signaling pathway using HK-2 cell lines stably overexpressing empty vector (mock cells) or human PGC1α (PGC1α cells). Stable PGC-1α overexpression negatively regulated the expression of TGF-ß-induced epithelial-mesenchymal transition (EMT) markers (fibronectin, E-cadherin, vimentin, and α-SMA) and EMT-related transcription factors (Snail and Slug) compared to mock cells, inhibiting fibrotic progression. Interestingly, among molecules upstream of Smad2/3 activation, the gene expression of only TGFßRI, but not TGFßRII, was downregulated in PGC-1α cells. In addition, the downregulation of TGFßRI by PGC-1α was associated with the upregulation of let-7b/c, miRNA for which the 3' untranslated region (UTR) of TGFßRI contains a binding site. In conclusion, PGC-1α suppresses TGF-ß/Smad signaling activation via targeting TGFßRI downregulation by let-7b/c upregulation.

Efficacy and Survival Outcome Associated with the Use of Novel Agents and Autologous Stem Cell Transplantation in Cases of Immunoglobulin D Multiple Myeloma in Korea.

BACKGROUND: Immunoglobulin D multiple myeloma (IgD MM) is characterized by a poor prognosis. Data are lacking on the survival benefits associated with the use of novel agents followed by autologous stem cell transplantation (ASCT) in IgD MM patients. We evaluated the clinical outcomes of induction treatment with novel agents followed by ASCT. METHODS: This was a single-center, retrospective study of 22 IgD MM patients who underwent ASCT between 1995 and 2016. Of these, 10 (45.4%) received novel agents and 12 (54.6%) received nonnovel agents. Clinical features and survival outcomes were examined. RESULTS: Median overall survival (OS) was 37.7 months in the 22 patients. Those in the novel-agents group received bortezomib or thalidomide-based regimens, whereas 91.7% of the nonnovel-agents group received a vincristine-based regimen. The median progression-free survival and OS in the novel-agent/nonnovel-agent groups were 8.3/7.4 and 38.6/12.5 months, respectively. The median OS of patients receiving maintenance therapy was not reached. CONCLUSION: This study showed improved survival outcomes compared to our previous study (37.7 vs. 12 months), suggesting that the use of a novel agent as induction and maintenance therapy may be beneficial in patients with IgD MM who undergo ASCT.

Methotrexate elimination and toxicity: MTHFR 677C>T polymorphism in patients with primary CNS lymphoma treated with high-dose methotrexate.

The genetic association of the methylenetetrahydrofolate reductase gene (MTHFR) 677C>T polymorphism with methotrexate (MTX)-associated toxicity has been evaluated and conflicting results have been reported. The substantial heterogeneity of the studied population was suggested to be a possible explanation because ethnicity, MTX dose, coadministered chemotherapeutic agents, and folinate rescue dosage regimen could alter the MTX toxicity profile. The patient population was homogenized by limiting the cancer type to primary central nervous system lymphoma and chemotherapy protocol to a high-dose MTX monotherapy regimen. A total of 111 patients with 402 chemotherapy courses were analyzed. MTHFR 677C>T polymorphism was identified as an independent predictive marker for MTX-associated hematologic toxicity (odds ratio, 2.60; 95% confidence interval, 1.32-5.09; P = .0055). Clinically significant nephrotoxicity occurred in patients without delayed elimination, suggesting roles for factors other than serum MTX levels. MTX-induced hepatotoxicity and oral mucositis occurred independently of plasma MTX levels.

Interim 18F-FGD PET/CT may not predict the outcome in primary central nervous system lymphoma patients treated with sequential treatment with methotrexate and cytarabine.

18F-fluoro-2-dexoy-D-glucose-positron emission tomography (PET)/computed tomography (CT) is a useful imaging technique for monitoring the treatment response in lymphoma cases. We investigated the value of interim brain PET/CT (I-PET/CT) for monitoring the response to intensive methotrexate-based chemotherapy in primary central nervous system lymphoma (PCNSL) patients with diffuse large B cell lymphoma (DLBCL). Of the 76 PCNSL patients treated with intensive methotrexate and cytarabine chemotherapy between September 2006 and December 2012, 66 patients with DLBCL were included in this study. The patient cohort of 66 individuals comprised 43 men and 23 women with a median age of 59 years (range, 17-75 years). During chemotherapy, 36 patients (54.5%) showed a negative metabolism on I-PET/CT, and 47 (71.2%) were negative on final (F) PET/CT. The baseline characteristics were similar between I-PET/CT-negative (n = 36) and I-PET/CT-positive patients (n = 30) except ECOG performance status. After a median follow-up of 27.5 months, there was no difference in the progression-free survival (PFS; P = 0.701) or overall survival (OS; P = 0.620) between the I-PET/CT-negative and I-PET/CT-positive groups. However, PFS in the F-PET/CT-negative group was significantly longer than that in the F-PET/CT-positive group (P < 0.001) without a significant difference in OS (P = 0.892). I-PET/CT may not predict the survival outcome of PCNSL patients with DLBCL treated with intensive methotrexate and cytarabine chemotherapy. Prospective trials are required to fully evaluate the role of I-PET/CT.

Pretreatment whole blood Epstein-Barr virus-DNA is a significant prognostic marker in patients with Hodgkin lymphoma.

Epstein-Barr virus (EBV) in the peripheral blood has become a significant predictor of clinical outcomes in EBV-associated Hodgkin lymphoma (HL). However, due to its relative rarity, prevalence and prognostic role of circulating EBV-DNA has not been well established in Asian patients. Seventy patients with newly diagnosed HL were prospectively registered between October 2007 and January 2013, and underwent pretreatment whole blood (WB) EBV-DNA quantitation using real-time polymerase chain reaction (RT-PCR). WB EBV-DNA in baseline and serial RT-PCR within 1 year were investigated. Clinicopathologic parameters of the patients according to pretreatment WB EBV-DNA were also explored. Twelve patients (17.1 %) demonstrated WB EBV-DNA(+), which was significantly associated to older age, advanced stages, frequent involvements of extranodal sites, low serum albumin and hemoglobin levels, and high international prognostic scores ≥2. Three-year event-free survival (EFS) and overall survival (OS) were significantly inferior in patients with pretreatment WB EBV-DNA(+) (53.5 vs 67.0 and 65.6 vs 90.2 %) (p < 0.032 and <0.01). Negatively conversed EBV-DNA within 1 year after chemotherapy also significantly affected favorable EFS (p < 0.01). Taken together, pretreatment WB EBV-DNA(+) may be a significant predictor of inferior EFS and OS over EBV-encoded RNA in situ hybridization (EBER-ISH)(+) in Korean patients with HL. Serial EBV-DNA monitoring following chemotherapy also seems helpful to predict survival outcomes.

AGO2 and SETDB1 cooperate in promoter-targeted transcriptional silencing of the androgen receptor gene.

In mammals, RNA interference is primarily a post-transcriptional mechanism. Evidence has accumulated for additional role in transcriptional gene silencing (TGS) but the question for a good paradigm for small interfering antigene RNA (agRNA)-induced chromatin modification remains unanswered. Here, we show that SETDB1, a histone H3-lysine 9 (H3K9)-specific methyltransferase, cooperates with Argonaute-2 (AGO2) and plays an essential role in agRNA-induced TGS. The androgen receptor (AR) gene was transcriptionally silenced by agRNA targeted to its promoter, and we show that this repression was mitigated by knockdown of SETDB1 or AGO2. Chromatin immunoprecipitation demonstrated that agRNA-driven AGO2 was first targeted to the AR promoter, followed by SETDB1. SIN3A and HDAC1/2, the components of the SIN3-HDAC complex, immunoprecipitated with SETDB1, and localized at the agRNA-targeted promoter. Agreeing with the presence of SETDB1, trimethyl-H3K9 was enriched in the AR promoter. Both EZH2 and trimethyl-H3K27 were also present in the targeted locus; accordingly, EZH2 immunoprecipitated with SETDB1. DNA methylation level was not significantly changed, suggesting the absence of de novo methylating activity in agRNA-induced AR promoter. Our results demonstrate that SETDB1, together with AGO2, plays an essential role in TGS through recruiting chromatin remodeler and/or other modifiers, consequently creating a repressive chromatin milieu at the targeted promoter.

MicroRNA-375 Functions as a Tumor-Suppressor Gene in Gastric Cancer by Targeting Recepteur d'Origine Nantais.

Emerging evidence supports a fundamental role for microRNAs (miRNA) in regulating cancer metastasis. Recently, microRNA-375 (miR-375) was reported to be downregulated in many types of cancers, including gastric cancer. Increase in the expression of Recepteur d'Origine Nantais (RON), a receptor tyrosine kinase, has been reported in tumors. However, the function of miR-375 and RON expression in gastric cancer metastasis has not been sufficiently studied. In silico analysis identified miR-375 binding sites in the 3'-untranslated regions (3'-UTR) of the RON-encoding gene. Expression of miR-375 resulted in reduced activity of a luciferase reporter containing the 3'-UTR fragments of RON-encoding mRNA, confirming that miR-375 directly targets the 3'-UTR of RON mRNA. Moreover, we found that overexpression of miR-375 inhibited mRNA and protein expression of RON, which was accompanied by the suppression of cell proliferation, migration, and invasion in gastric cancer AGS and MKN-28 cells. Ectopic miR-375 expression also induced G1 cell cycle arrest through a decrease in the expression of cyclin D1, cyclin D3, and in the phosphorylation of retinoblastoma (Rb). Knockdown of RON by RNAi, similar to miR-375 overexpression, suppressed tumorigenic properties and induced G1 arrest through a decrease in the expression of cyclin D1, cyclin D3, and in the phosphorylation of Rb. Thus, our study provides evidence that miR-375 acts as a suppressor of metastasis in gastric cancer by targeting RON, and might represent a new potential therapeutic target for gastric cancer.

Regulated nuclear entry of over-expressed Setdb1.

Setdb1 is a histone H3-lysine 9 (H3K9)-specific methyltransferase that interacts with various transcriptional regulators to induce local heterochromatin formation and participates as an indispensable component in building promyelocytic leukemia nuclear body (PML-NB), which is involved in various biological processes. We studied the effects of Setdb1 over-expression. We unexpectedly observed that exogenously expressed GFP-Setdb1 was retained in the cytoplasm, whereas endogenous Setdb1 showed a punctate nuclear signal. Leptomycin B (LMB) treatment, which blocks protein export from the nucleus, showed that entry of GFP-Setdb1 to the nucleus was regulated and that GFP-Setdb1 in the nucleus could localize at PML-NB as endogenous Setdb1. An analysis of Setdb1 deletion constructs showed that the N-terminal region was related to the nuclear export of Setdb1; supporting this, we detected two nuclear export signal motifs in this region. This N-terminal region had a SUMO interaction motif (SIM) whose mutation greatly reduced the ability of Setdb1 to associate with PML-NB and thus resulted in the disaggregation of PML-NB structure. We therefore presume that the cytoplasmic retention of over-expressed Setdb1 occurs as part of a regulatory mechanism to set a tight limit on the nuclear activity of Setdb1, whose excess activity might result in random and haphazard chromatin modifications that cause globally aberrant gene expression.

Remobilization of hematopoietic stem cells with high-dose methotrexate and cytarabine in patients with non-Hodgkin's lymphoma and multiple myeloma after failure to mobilize with chemotherapy and cytokines.

BACKGROUND: High-dose chemotherapy supported by autologous stem cell transplantation is an effective treatment for patients with relapsed or refractory non-Hodgkin's lymphomas (NHLs) and fit patients with multiple myeloma (MM). However, failure rates of hematopoietic stem cell mobilization are estimated to be between 5 and 30%, respectively. Thus, we investigated the efficacy of the combination chemotherapy of high-dose methotrexate (MTX) and cytarabine with granulocyte-colony-stimulating factor (G-CSF) as a remobilization method in those who failed a prior mobilization and collection with chemotherapy and G-CSF. STUDY DESIGN AND METHODS: Mobilization failure was defined as a collection of fewer than 5 × 10(6) CD34+ cells after three to five apheresis procedures. MTX (3500 mg/m(2) in a 120-min infusion) on Day 1 and cytarabine (3000 mg/m(2) infusion for 120 min) on Day 4 and Day 5 were followed by G-CSF (10 µg/kg daily). RESULTS: A total of eight patients (six NHL and two MM; median age, 55 years) who had failed in prior mobilization with conventional chemotherapy and G-CSF underwent the second mobilization as described in the method. Successful collection of CD34+ cells (> 5 × 10(6) /kg) was achieved in six patients (75%) with three to five apheresis procedures. The total yield of CD34+ cells/kg body weight was 6.28 × 10(6) /kg (median; range, 1.53 × 10(6) -10.09 × 10(6) /kg). CONCLUSIONS: This preliminary result warrants further investigation of high-dose MTX and cytarabine plus G-CSF as a means to remobilize stem cells in those with prior failure to mobilize stem cells with chemotherapy and G-CSF.

Role of APE1/Ref-1 in hydrogen peroxide-induced apoptosis in human renal HK-2 cells.

BACKGROUND: Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) is a multipotent protein that plays essential roles in cellular responses to oxidative stress. METHODS: To examine the role of APE1/Ref-1 in ischemia-reperfusion (I/R) injuries and hydrogen peroxide (H2O2)-induced renal tubular apoptosis, we studied male C57BL6 mice and human proximal tubular epithelial (HK-2) cells treated with H2O2 at different concentrations. The colocalization of APE1/Ref-1 in the proximal tubule, distal tubule, thick ascending limb, and collecting duct was observed with confocal microscopy. The overexpression of APE1/Ref-1 with knockdown cell lines using an APE1/Ref-1-specific DNA or small interfering RNA (siRNA) was used for the apoptosis assay. The promotor activity of nuclear factor kappa B (NF-κB) was assessed and electrophoretic mobility shift assay was conducted. RESULTS: APE1/Ref-1 was predominantly localized to the renal tubule nucleus. In renal I/R injuries, the levels of APE1/Ref-1 protein were increased compared with those in kidneys subjected to sham operations. The overexpression of APE1/Ref-1 in HK-2 cells enhanced the Bax/Bcl-2 ratio as a marker of apoptosis. Conversely, the suppression of APE1/Ref-1 expression by siRNA in 1-mM H2O2-treated HK-2 cells decreased the Bax/Bcl-2 ratio, the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2, p38, c-Jun N-terminal kinase (JNK) 1/2, and NF-κB. In HK-2 cells, the promoter activity of NF-κB increased following H2O2 exposure, and this effect was further enhanced by APE1/Ref-1 transfection. CONCLUSION: The inhibition of APE1/Ref-1 with siRNA attenuated H2O2-induced apoptosis through the modulation of mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38 and the nuclear activation of NF-κB and proapoptotic factors.