PDZ-binding kinase inhibitor OTS514 suppresses the proliferation of oral squamous carcinoma cells.

OBJECTIVE: PDZ-binding kinase (PBK) has been reported as a poor prognostic factor and is a promising molecular target for anticancer therapeutics. Here, we aimed to investigate the effect of specific PBK inhibitor OTS514 on the survival of OSCC cells. METHODS: Four OSCC cell lines (HSC-2, HSC-3, SAS, and OSC-19) were used to examine the effect of OTS514 on cell survival and apoptosis. DNA microarray analysis was conducted to investigate the effect of OTS514 on gene expression in OSCC cells. Gene set enrichment analysis was performed to identify molecular signatures related to the antiproliferative effect of OTS514. RESULTS: OTS514 decreased the cell survival of OSCC cells dose-dependently, and administration of OTS514 readily suppressed the HSC-2-derived tumor growth in immunodeficient mice. Treatment with OTS514 significantly increased the number of apoptotic cells and caspase-3/7 activity. Importantly, OTS514 suppressed the expression of E2F target genes with a marked decrease in protein levels of E2F1, a transcriptional factor. Moreover, TP53 knockdown attenuated OTS514-induced apoptosis. CONCLUSION: OTS514 suppressed the proliferation of OSCC cells by downregulating the expression of E2F target genes and induced apoptosis by mediating the p53 signaling pathway. These results highlight the clinical application of PBK inhibitors in the development of molecular-targeted therapeutics against OSCC.

BCL6 inhibition ameliorates resistance to ruxolitinib in CRLF2-rearranged acute lymphoblastic leukemia.

Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) is an intractable disease and most cases harbor genetic alterations that activate JAK or ABL signaling. The commonest subtype of Ph-like ALL exhibits a CRLF2 gene rearrangement that brings about JAK1/2-STAT5 pathway activation. However, JAK1/2 inhibition alone is insufficient as a treatment, so combinatorial therapies targeting multiple signals are needed. To better understand the mechanisms underlying the insufficient efficacy of JAK inhibition, we explored gene expression changes upon treatment with a JAK1/2 inhibitor (ruxolitinib) and found that elevated BCL6 expression was one such mechanism. Upregulated BCL6 suppressed the expression of TP53 along with its downstream cell cycle inhibitor p21 (CDKN2A) and pro-apoptotic molecules, such as FAS, TNFRSF10B, BID, BAX, BAK, PUMA, and NOXA, conferring cells some degree of resistance to therapy. BCL6 inhibition (with FX1) alone was able to upregulate TP53 and restore the TP53 expression that ruxolitinib had diminished. In addition, ruxolitinib and FX1 concertedly downregulated MYC. As a result, FX1 treatment alone had growth-inhibitory and apoptosis- sensitizing effects, but the combination of ruxolitinib and FX1 more potently inhibited leukemia cell growth, enhanced apoptosis sensitivity, and prolonged the survival of xenografted mice. These findings provide one mechanism for the insufficiency of JAK inhibition for the treatment of CRLF2-rearranged ALL and indicate BCL6 inhibition as a potentially helpful adjunctive therapy combined with JAK inhibition.

Correction of a CD55 mutation to quantify the efficiency of targeted knock-in via flow cytometry.

BACKGROUND: Targeted knock-in assisted by the CRISPR/Cas9 system is an advanced technology with promising applications in various research fields including medical and agricultural sciences. However, improvements in the efficiency, precision, and specificity of targeted knock-in are prerequisites to facilitate the practical application of this technology. To improve the efficiency of targeted knock-in, it is necessary to have a molecular system that allows sensitive monitoring of targeted knock-in events with simple procedures. METHODS AND RESULTS: We developed an assay, named CD55 correction assay, with which to monitor CD55 gene correction accomplished by targeted knock-in. To create the reporter clones used in this assay, we initially introduced a 7.7-kb heterozygous deletion covering CD55 exons 2-5, and then incorporated a truncating mutation within exon 4 of the remaining CD55 allele in human cell lines. The resultant reporter clones that lost the CD55 protein on the cell membrane were next transfected with Cas9 constructs along with a donor plasmid carrying wild-type CD55 exon 4. The cells were subsequently stained with fluorescence-labeled CD55 antibody and analyzed by flow cytometry to detect CD55-positive cells. These procedures allow high-throughput, quantitative detection of targeted gene correction events occurring in an endogenous human gene. CONCLUSIONS: The current study demonstrated the utility of the CD55 correction assay to sensitively quantify the efficiency of targeted knock-in. When used with the PIGA correction assay, the CD55 correction assay will help accurately determine the efficiency of targeted knock-in, precluding possible experimental biases caused by cell line-specific and locus-specific factors.

Cytotoxicity of Callerya speciosa Fractions against Myeloma and Lymphoma Cell Lines.

Callerya speciosa is widely distributed in tropical and subtropical countries and is traditionally used for preventing numerous disorders. In this study, a bioguided fractionation of ethyl acetate extract (SE) from C. speciosa root was carried out to target antioxidant and cytotoxic activities. Of the four fractions (SE1-SE4) obtained by column chromatography, SE4 had the strongest anti-radical ability in the DPPH and ABTS assays (IC50 = 0.05 and 0.17 mg/mL, respectively), with results close to butylated hydroxytoluene (BHT), a common antioxidant agent. The cytotoxic activities against the selected cells were analyzed in this study by MTT assay. Accordingly, SE2, SE3, and SE4 significantly inhibited the viability of multiple myeloma cell lines, comprising U266 (IC50 = 0.38, 0.09, and 0.11 mg/mL, respectively) and KMS11 (IC50 = 0.09, 0.17, and 0.15 mg/mL, respectively), mantle cell lymphoma Mino (IC50 = 0.08, 0.16, and 0.15 mg/mL, respectively), and the noncancerous cell line LCL (IC50 = 0.40, 0.32, and 0.21 mg/mL, respectively). At a concentration of 125 µg/mL, SE2, SE3, and SE4 induced the cell apoptosis of U266 (32.2%, 53.2%, and 55.6%, respectively), KMS11 (36.9%, 40.8%, and 47.9%, respectively), Mino (36.6%, 39.8%, and 22.0%, respectively), and LCL (12.4%, 17.5%, and 23.5%, respectively) via annexin V assay. The dominant compounds detected in fractions by high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS), were identified as isoflavones. This is the first report describing C. speciosa as a promising natural source of antileukemia and antimyeloma agents, which may be useful for the development of blood cancer treatments.

Transcriptome-wide analysis of intracranial artery in patients with moyamoya disease showing upregulation of immune response, and downregulation of oxidative phosphorylation and DNA repair.

OBJECTIVE: Moyamoya disease (MMD) is a rare cerebrovascular disease characterized by progressive occlusion of the internal carotid artery and the secondary formation of collateral vessels. Patients with MMD have ischemic attacks or intracranial bleeding, but the disease pathophysiology remains unknown. In this study, the authors aimed to identify a gene expression profile specific to the intracranial artery in MMD. METHODS: This was a single-center, prospectively sampled, retrospective cohort study. Microsamples of the middle cerebral artery (MCA) were collected from patients with MMD (n = 11) and from control patients (n = 9). Using microarray techniques, transcriptome-wide analysis was performed. RESULTS: Comparison of MCA gene expression between patients with MMD and control patients detected 62 and 26 genes whose expression was significantly (p < 0.001 and fold change > 2) up- or downregulated, respectively, in the MCA of MMD. Gene set enrichment analysis of genes expressed in the MCA of patients with MMD revealed positive correlations with genes involved in antigen processing and presentation, the dendritic cell pathway, cytokine pathway, and interleukin-12 pathway, and negative correlations with genes involved in oxidative phosphorylation and DNA repair. Microarray analysis was validated by quantitative polymerase chain reaction. CONCLUSIONS: Transcriptome-wide analysis showed upregulation of genes for immune responses and downregulation of genes for DNA repair and oxidative phosphorylation within the intracranial artery of patients with MMD. These findings may represent clues to the pathophysiology of MMD.

Biallelic loss of FAM46C triggers tumor growth with concomitant activation of Akt signaling in multiple myeloma cells.

Loss of heterozygosity or mutation of the family with sequence similarity 46, member C (FAM46C) gene on chromosome band 1p12 is associated with shorter overall survival of patients with multiple myeloma (MM). In this study, using human MM cell lines (KMS-11, OCI-My5, and ANBL-6), we generated FAM46C-/- cell clones and examined the effect of disruption of FAM46C on cell survival and cellular signaling. Cell proliferation assays showed increased clonogenicity of FAM46C-/- KMS-11 cells compared to WT cells. Xenograft experiments showed significantly shorter overall survival of mice harboring the FAM46C-/- cell-derived tumors than mice with the FAM46CWT cell-derived tumors. Notably, levels of phosphorylated Akt and its substrates increased both in vitro and in vivo in the FAM46C-/- cells compared to WT cells. In addition, caspase activities decreased in the FAM46C-/- cells. Results of gene set enrichment analysis showed that loss of FAM46C significantly activated serum-responsive genes while inactivating phosphatase and tensin homolog (PTEN)-related genes. Mechanistically, loss of FAM46C decreased the PTEN activity, number of apoptotic cells, and caspase activities. PF-04691502, a selective PI3K inhibitor, suppressed the augmented phosphorylation of Akt and its substrate FoxO3a. Treatment with afuresertib (a specific Akt inhibitor) in combination with bortezomib additively decreased FAM46C-/- MM cell survival. Collectively, this study is the first to report that loss of FAM46C triggers the concomitant activation of the PI3K-Akt signaling pathway, which might be a therapeutic target for MM with abnormalities in the FAM46C gene.

The plant alkaloid conophylline inhibits matrix formation of fibroblasts.

Conophylline is a Vinca alkaloid from leaves of the tropical plant Ervatamia microphylla and has been shown to mimic the effect of the growth and differentiation factor activin A on pancreatic progenitor cells. However, activin A stimulates fibrosis of pancreatic stellate cells, whereas conophylline inhibits it, suggesting that this compound may serve as an antifibrotic drug. Here we investigated the effects of conophylline on human foreskin fibroblasts, especially focusing on extracellular matrix (ECM) proteins. A gene microarray analysis revealed that conophylline remarkably suppressed expression of the gene for hyaluronan synthase 2 (HAS2) and of its antisense RNA, whereas the expression of collagen genes was unaffected. Of note, immunostaining experiments revealed that conophylline substantially inhibits incorporation of versican and collagens into the ECM in cells treated with transforming growth factor ß (TGFß), which promotes collagen synthesis, but not in cells not treated with TGFß. Moreover, a protein biosynthesis assay disclosed that conophylline decreases collagen biosynthesis, concomitant with a decrease in total protein biosynthesis, indicating that conophylline-mediated inhibition of fibrosis is not specific to collagen synthesis. Conophylline affected neither TGFß-induced nuclear translocation of SMAD family member 2/3 (SMAD2/3) nor phosphorylation of SMAD2. However, conophylline substantially inhibited phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), suggesting that conophylline inhibits HAS2 expression via TGFß-mediated activation of the ERK1/2 pathway. Taken together, our results indicate that conophylline may be a useful inhibitor of ECM formation in fibrosis.

Establishment and characterization of CRISPR/Cas9-mediated NF2-/- human mesothelial cell line: Molecular insight into fibroblast growth factor receptor 2 in malignant pleural mesothelioma.

Malignant pleural mesothelioma (MPM), a highly refractory tumor, is currently incurable due to the lack of an early diagnosis method and medication, both of which are urgently needed to improve the survival and/or quality of life of patients. NF2 is a tumor suppressor gene and is frequently mutated in MPM. Using a CRISPR/Cas9 system, we generated an NF2-knockout human mesothelial cell line, MeT-5A (NF2-KO). In NF2-KO cell clones, cell growth, clonogenic activity, migration activity, and invasion activity significantly increased compared with those in NF2-WT cell clones. Complementary DNA microarray analysis clearly revealed the differences in global gene expression profile between NF2-WT and NF2-KO cell clones. Quantitative PCR analysis and western blot analysis showed that the upregulation of fibroblast growth factor receptor 2 (FGFR2) was concomitant with the increases in phosphorylation levels of JNK, c-Jun, and retinoblastoma (Rb) in NF2-KO cell clones. These increases were all abrogated by the exogenous expression of NF2 in the NF2-KO clone. In addition, the disruption of FGFR2 in the NF2-KO cell clone suppressed cell proliferation as well as the phosphorylation levels of JNK, c-Jun, and Rb. Notably, FGFR2 was found to be highly expressed in NF2-negative human mesothelioma tissues (11/12 cases, 91.7%) but less expressed in NF2-positive tissues. Collectively, these findings suggest that NF2 deficiency might play a role in the tumorigenesis of human mesothelium through mediating FGFR2 expression; FGFR2 would be a candidate molecule to develop therapeutic and diagnostic strategies for targeting MPM with NF2 loss.

Δ40p53α suppresses tumor cell proliferation and induces cellular senescence in hepatocellular carcinoma cells.

Splice variants of certain genes impact on genetic biodiversity in mammals. The tumor suppressor TP53 gene (encoding p53) plays an important role in the regulation of tumorigenesis in hepatocellular carcinoma (HCC). Δ40p53α is a naturally occurring p53 isoform that lacks the N-terminal transactivation domain, yet little is known about the role of Δ40p53α in the development of HCC. Here, we first report on the role of Δ40p53α in HCC cell lines. In the TP53+/Δ40 cell clones, clonogenic activity and cell survival dramatically decreased, whereas the percentage of senescence-associated ß-galactosidase (SA-ß-gal)-positive cells and p21 (also known as WAF1, CIP1 and CDKN1A) expression significantly increased. These observations were clearly attenuated in the TP53+/Δ40 cell clones after Δ40p53α knockdown. In addition, exogenous Δ40p53 expression significantly suppressed cell growth in HCC cells with wild-type TP53, and in those that were mutant or null for TP53 Notably, Δ40p53α-induced tumor suppressor activity was markedly attenuated in cells expressing the hot-spot mutant Δ40p53α-R175H, which lacks the transcription factor activity of p53. Moreover, Δ40p53α expression was associated with increased full-length p53 protein expression. These findings enhance the understanding of the molecular pathogenesis of HCC and show that Δ40p53α acts as an important tumor suppressor in HCC cells.

Versican A-subdomain is required for its adequate function in dermal development.

Versican, a large chondroitin sulfate (CS) proteoglycan, serves as a structural macromolecule of the extracellular matrix (ECM) and regulates cell behavior. We determined the function of versican in dermal development using VcanΔ3/Δ3 mutant mice expressing versican with deleted A-subdomain of the N-terminal G1 domain. The mutant versican showed a decreased hyaluronan (HA)-binding ability and failed to accumulate in the ECM. In the early developmental stage, VcanΔ3/Δ3 dermis showed a decrease in versican expression as compared with WT. As development proceeded, versican expression further decreased to a barely detectable level, and VcanΔ3/Δ3 mice died at the neonatal period (P0). At P0, VcanΔ3/Δ3 dermis exhibited an impaired ECM structure and decreased cell density. While the level of collagen deposition was similar in both genotypes, collagen biosynthesis significantly decreased in VcanΔ3/Δ3 fibroblasts as compared with that in wild type (WT). Transforming growth factor ß (TGFß) signaling mediated through the Smad2/3-dependent pathway was down-regulated in VcanΔ3/Δ3 fibroblasts and a reduced TGFß storage in the ECM was observed. Microarray analysis revealed a decrease in the expression levels of transcription factors, early growth response (Egr) 2 and 4, which act downstream of TGFß signaling. Thus, our results suggest that A-subdomain is necessary for adequate versican expression in dermis and that versican is involved in the formation of the ECM and regulation of TGFß signaling.

Improved methods of AAV-mediated gene targeting for human cell lines using ribosome-skipping 2A peptide.

The adeno-associated virus (AAV)-based targeting vector has been one of the tools commonly used for genome modification in human cell lines. It allows for relatively efficient gene targeting associated with 1-4-log higher ratios of homologous-to-random integration of targeting vectors (H/R ratios) than plasmid-based targeting vectors, without actively introducing DNA double-strand breaks. In this study, we sought to improve the efficiency of AAV-mediated gene targeting by introducing a 2A-based promoter-trap system into targeting constructs. We generated three distinct AAV-based targeting vectors carrying 2A for promoter trapping, each targeting a GFP-based reporter module incorporated into the genome, PIGA exon 6 or PIGA intron 5. The absolute gene targeting efficiencies and H/R ratios attained using these vectors were assessed in multiple human cell lines and compared with those attained using targeting vectors carrying internal ribosome entry site (IRES) for promoter trapping. We found that the use of 2A for promoter trapping increased absolute gene targeting efficiencies by 3.4-28-fold and H/R ratios by 2-5-fold compared to values obtained with IRES. In CRISPR-Cas9-assisted gene targeting using plasmid-based targeting vectors, the use of 2A did not enhance the H/R ratios but did upregulate the absolute gene targeting efficiencies compared to the use of IRES.

Lipopolysaccharide augments the uptake of oxidized LDL by up-regulating lectin-like oxidized LDL receptor-1 in macrophages.

There is a growing body of evidence supporting an intimate association of immune activation with the pathogenesis of cardiovascular diseases, including atherosclerosis. Uptake of oxidized low-density lipoprotein (oxLDL) through scavenging receptors promotes the formation of mature lipid-laden macrophages, which subsequently leads to exacerbation of regional inflammation and atherosclerotic plaque formation. In this study, we first examined changes in the mRNA level of the lectin-like oxLDL receptor-1 (LOX-1) in the mouse macrophage cell line RAW264.7 and the human PMA-induced macrophage cell line THP-1 after LPS stimulation. LPS significantly up-regulated LOX-1 mRNA in RAW264.7 cells; LOX-1 cell-surface protein expression was also increased. Flow cytometry and fluorescence microscopy analyses showed that cellular uptake of fluorescence (Dil)-labeled oxLDL was significantly augmented with LPS stimulation. The augmented uptake of Dil-oxLDL was almost completely abrogated by treatment with an anti-LOX-1 antibody. Of note, knockdown of Erk1/2 resulted in a significant reduction of LPS-induced LOX-1 up-regulation. Treatment with U0126, a specific inhibitor of MEK, significantly suppressed LPS-induced expression of LOX-1 at both the mRNA and protein levels. Furthermore, LOX-1 promoter activity was significantly augmented by LPS stimulation; this augmentation was prevented by U0126 treatment. Similar results were also observed in human PMA-induced THP-1 macrophages. Taken together, our results indicate that LPS up-regulates LOX-1, at least in part through activation of the Erk1/2 signaling pathway, followed by augmented cellular oxLDL uptake, thus highlighting a critical role of TLR4-mediated aberrant LOX-1 signaling in the pathogenesis of atherosclerosis.

Array-comparative genomic hybridization profiling of immunohistochemical subgroups of diffuse large B-cell lymphoma shows distinct genomic alterations.

Diffuse large B-cell lymphoma (DLBCL) displays striking heterogeneity at the clinical, genetic and molecular levels. Subtypes include germinal center B-cell-like (GCB) DLBCL and activated B-cell-like (ABC) DLBCL, according to microarray analysis, and germinal center type or non-germinal center type by immunohistochemistry. Although some reports have described genomic aberrations based upon microarray classification system, genomic aberrations based upon immunohistochemical classifications have rarely been reported. The present study aimed to ascertain the relationship between genomic aberrations and subtypes identified by immunohistochemistry, and to study the pathogenetic character of Chinese DLBCL. We conducted immunohistochemistry using antibodies against CD10, BCL6 and MUM1 in 59 samples of DLBCL from Chinese patients, and then performed microarray-based comparative genomic hybridization for each case. Characteristic genomic differences were found between GCB and non-GCB DLBCL from the array data. The GCB type was characterized by more gains at 7q (7q22.1, P < 0.05) and losses at 16q (P ≤ 0.05), while the non-GCB type was characterized by gains at 11q24.3 and 3q13.2 (P < 0.05). We found completely different mutations in BCL6+ and BCL6- non-GCB type DLBCL, whereby the BCL6- group had a higher number of gains at 1q and a loss at 14q32.13 (P ≤ 0.005), while the BCL6+ group showed a higher number of gains at 14q23.1 (P = 0.15) and losses at 6q (P = 0.07). The BCL6- group had a higher frequency of genomic imbalances compared to the BCL6+ group. In conclusion, the BCL6+ and BCL6- non-GCB type of DLBCL appear to have different mechanisms of pathogenesis.

Clonal heterogeneity of lymphoid malignancies correlates with poor prognosis.

Clonal heterogeneity in lymphoid malignancies has been recently reported in adult T-cell lymphoma/leukemia, peripheral T-cell lymphoma, not otherwise specified, and mantle cell lymphoma. Our analysis was extended to other types of lymphoma including marginal zone lymphoma, follicular lymphoma and diffuse large B-cell lymphoma. To determine the presence of clonal heterogeneity, 332 cases were examined using array comparative genomic hybridization analysis. Results showed that incidence of clonal heterogeneity varied from 25% to 69% among different types of lymphoma. Survival analysis revealed that mantle cell lymphoma and diffuse large B-cell lymphoma with clonal heterogeneity showed significantly poorer prognosis, and that clonal heterogeneity was confirmed as an independent predictor of poor prognosis for both types of lymphoma. Interestingly, 8q24.1 (MYC) gain, 9p21.3 (CDKN2A/2B) loss and 17p13 (TP53, ATP1B2, SAT2, SHBG) loss were recurrent genomic lesions among various types of lymphoma with clonal heterogeneity, suggesting at least in part that alterations of these genes may play a role in clonal heterogeneity.

Mutation of a single allele of the cancer susceptibility gene BRCA1 leads to genomic instability in human breast epithelial cells.

Biallelic inactivation of cancer susceptibility gene BRCA1 leads to breast and ovarian carcinogenesis. Paradoxically, BRCA1 deficiency in mice results in early embryonic lethality, and similarly, lack of BRCA1 in human cells is thought to result in cellular lethality in view of BRCA1's essential function. To survive homozygous BRCA1 inactivation during tumorigenesis, precancerous cells must accumulate additional genetic alterations, such as p53 mutations, but this requirement for an extra genetic "hit" contradicts the two-hit theory for the accelerated carcinogenesis associated with familial cancer syndromes. Here, we show that heterozygous BRCA1 inactivation results in genomic instability in nontumorigenic human breast epithelial cells in vitro and in vivo. Using somatic cell gene targeting, we demonstrated that a heterozygous BRCA1 185delAG mutation confers impaired homology-mediated DNA repair and hypersensitivity to genotoxic stress. Heterozygous mutant BRCA1 cell clones also showed a higher degree of gene copy number loss and loss of heterozygosity in SNP array analyses. In BRCA1 heterozygous clones and nontumorigenic breast epithelial tissues from BRCA mutation carriers, FISH revealed elevated genomic instability when compared with their respective controls. Thus, BRCA1 haploinsufficiency may accelerate hereditary breast carcinogenesis by facilitating additional genetic alterations.

Leucine zipper protein 1 (LUZP1) regulates the constriction velocity of the contractile ring during cytokinesis.

There has been a great deal of research on cell division and its mechanisms; however, its processes still have many unknowns. To find novel proteins that regulate cell division, we performed the screening using siRNAs and/or the expression plasmid of the target genes and identified leucine zipper protein 1 (LUZP1). Recent studies have shown that LUZP1 interacts with various proteins and stabilizes the actin cytoskeleton; however, the function of LUZP1 in mitosis is not known. In this study, we found that LUZP1 colocalized with the chromosomal passenger complex (CPC) at the centromere in metaphase and at the central spindle in anaphase and that these LUZP1 localizations were regulated by CPC activity and kinesin family member 20A (KIF20A). Mass spectrometry analysis identified that LUZP1 interacted with death-associated protein kinase 3 (DAPK3), one regulator of the cleavage furrow ingression in cytokinesis. In addition, we found that LUZP1 also interacted with myosin light chain 9 (MYL9), a substrate of DAPK3, and comprehensively inhibited MYL9 phosphorylation by DAPK3. In line with a known role for MYL9 in the actin-myosin contraction, LUZP1 suppression accelerated the constriction velocity at the division plane in our time-lapse analysis. Our study indicates that LUZP1 is a novel regulator for cytokinesis that regulates the constriction velocity of the contractile ring.

ARK5 enhances cell survival associated with mitochondrial morphological dynamics from fusion to fission in human multiple myeloma cells.

5' adenosine monophosphate-activated protein kinase-related kinase 5 (ARK5) is involved in mitochondrial ATP production and associated with poor prognosis of multiple myeloma (MM). However, the molecular mechanisms of ARK5 in MM remain largely unknown. This study examined the pathogenic role of ARK5 in mitochondria by using genetically modified isogenic cell clones with or without ARK5 in human myeloma cell lines, KMS-11 and Sachi, which overexpress ARK5. The biallelic knockout of ARK5 (ARK5-KO) inhibited cell proliferation, colony formation, and migration with increased apoptosis. Mitochondrial fusion was enhanced in ARK5-KO cells, unlike in ARK5 wild-type (ARK5-WT) cells, which exhibited increased mitochondrial fission. Furthermore, ARK5-KO cells demonstrated a lower phosphorylated dynamin-related protein 1 at serine 616, higher protein expression of mitofusin-1 (MFN1) and MFN2, optic atrophy 1 with a lower level of ATP, and higher levels of lactate and reactive oxygen species than ARK5-WT cells. Our findings suggest that ARK5-enhanced myeloma cells can survive associated mitochondrial fission and activity. This study first revealed the relationship between ARK5 and mitochondrial morphological dynamics. Thus, our outcomes show novel aspects of mitochondrial biology of ARK5, which can afford a more advanced treatment approach for unfavorable MM expressing ARK5.

Arsenic trioxide prevents nitric oxide production in lipopolysaccharide -stimulated RAW 264.7 by inhibiting a TRIF-dependent pathway.

Arsenic trioxide (ATO) is one of the most potent drugs in cancer chemotherapy, and is highly effective in treating both newly diagnosed and relapse patients with acute promyelocytic leukemia (APL). Despite a number of reports regarding the molecular mechanisms by which ATO promotes anti-tumor or pro-apoptotic activity in hematological and other solid malignancies, the effects of ATO on immune responses remain poorly understood. To further understand and clarify the effects of ATO on immune responses, we sought to examine whether ATO affects the production of nitric oxide (NO) in a lipopolysaccharide (LPS)-stimulated mouse macrophage cell line, RAW 264.7. Arsenic trioxide was found to prevent NO production in a dose-dependent manner. Arsenic trioxide significantly inhibited the increase in inducible nitric oxide synthase (iNOS) at both the mRNA and protein levels. Furthermore, our analyses revealed that the inhibitory effect of ATO on iNOS expression was ascribed to the prevention of IRF3 phosphorylation, interferon (IFN)-ß expression, and STAT1 phosphorylation, but not the prevention of the MyD88-dependent pathway. Taken together, our results indicate that ATO prevents NO production by inhibiting the TIR-domain-containing adaptor protein inducing IFN-ß (TRIF)-dependent pathway, thus highlighting an anti-inflammatory property of ATO in innate immunity.

Genomic profiling combined with gene expression profiling in primary central nervous system lymphoma.

Of the genetic changes in primary central nervous system lymphoma (PCNSL), little is known. To detect copy number alterations and differentially expressed genes in PCNSL, we analyzed a total of 12 PCNSL samples with high-resolution array-based comparative genomic hybridization and performed expression profiling in 7 of the 12 samples. The most frequent deletion found in 8 patients (66.7%) occurred in 9p21.3 containing CDKN2A. We compiled the top 96 genes (family-wise error rate, P < .05) showing the greatest differential expression between PCNSL and normal lymph node tissues. From these, we selected 8 candidate genes (NPFFR2, C4orf7, OSMR, EMCN, TPO, FNDC1, COL12A1, and MSC) in which expression changes were associated with copy number aberrations. All 8 genes showed both down-regulation in expression microarray and deletion in array-based comparative genomic hybridization analyses. These genes participate in cell signaling or cell adhesion. In addition, low mRNA expression of C4orf7 was significantly associated with poor survival (P = .0425). Using gene set enrichment analysis, we identified several signal transduction pathways, such as Janus kinase-signal transducers and activators of transcription pathway and adhesion-related pathways, which may be involved in pathogenesis of PCNSL. In conclusion, this study identified novel tumor suppressor genes that may serve as therapeutic targets of PCNSL.

Arsenic augments the uptake of oxidized LDL by upregulating the expression of lectin-like oxidized LDL receptor in mouse aortic endothelial cells.

Although chronic arsenic exposure is a well-known risk factor for cardiovascular diseases, including atherosclerosis, the molecular mechanism underlying arsenic-induced atherosclerosis remains obscure. Therefore, this study aimed to elucidate this molecular mechanism. We examined changes in the mRNA level of the lectin-like oxidized LDL (oxLDL) receptor (LOX-1) in a mouse aortic endothelial cell line, END-D, after sodium arsenite (SA) treatment. SA treatment significantly upregulated LOX-1 mRNA expression; this finding was also verified at the protein expression level. Flow cytometry and fluorescence microscopy analyses showed that the cellular uptake of fluorescence (Dil)-labeled oxLDL was significantly augmented with SA treatment. In addition, an anti-LOX-1 antibody completely abrogated the augmented uptake of Dil-oxLDL. We observed that SA increased the levels of the phosphorylated forms of nuclear factor of kappa light polypeptide gene enhancer in B cells (NF-κB)/p65. SA-induced upregulation of LOX-1 protein expression was clearly prevented by treatment with an antioxidant, N-acetylcysteine (NAC), or an NF-κB inhibitor, caffeic acid phenethylester (CAPE). Furthermore, SA-augmented uptake of Dil-oxLDL was also prevented by treatment with NAC or CAPE. Taken together, our results indicate that arsenic upregulates LOX-1 expression through the reactive oxygen species-mediated NF-κB signaling pathway, followed by augmented cellular oxLDL uptake, thus highlighting a critical role of the aberrant LOX-1 signaling pathway in the pathogenesis of arsenic-induced atherosclerosis.