Heat-inactivated Fetal Bovine Serum Research Articles

The Skp1 Protein from Toxoplasma Is Modified by a Cytoplasmic Prolyl 4-Hydroxylase Associated with Oxygen Sensing in the Social Amoeba Dictyostelium

In diverse types of organisms, cellular hypoxic responses are mediated by prolyl 4-hydroxylases that use O(2) and α-ketoglutarate as substrates to hydroxylate conserved proline residues in target proteins. Whereas in metazoans these enzymes control the stability of the HIFα family of transcription factor subunits, the Dictyostelium enzyme (DdPhyA) contributes to O(2) regulation of development by a divergent mechanism involving hydroxylation and subsequent glycosylation of DdSkp1, an adaptor subunit in E3(SCF) ubiquitin ligases. Sequences related to DdPhyA, DdSkp1, and the glycosyltransferases that cap Skp1 hydroxyproline occur also in the genomes of Toxoplasma and other protists, suggesting that this O(2) sensing mechanism may be widespread. Here we show by disruption of the TgphyA locus that this enzyme is required for Skp1 glycosylation in Toxoplasma and that disrupted parasites grow slowly at physiological O(2) levels. Conservation of cellular function was tested by expression of TgPhyA in DdphyA-null cells. Simple gene replacement did not rescue Skp1 glycosylation, whereas overexpression not only corrected Skp1 modification but also restored the O(2) requirement to a level comparable to that of overexpressed DdPhyA. Bacterially expressed TgPhyA protein can prolyl hydroxylate both Toxoplasma and Dictyostelium Skp1s. Kinetic analyses showed that TgPhyA has similar properties to DdPhyA, including a superimposable dependence on the concentration of its co-substrate α-ketoglutarate. Remarkably, however, TgPhyA had a significantly higher apparent affinity for O(2). The findings suggest that Skp1 hydroxylation by PhyA is a conserved process among protists and that this biochemical pathway may indirectly sense O(2) by detecting the levels of O(2)-regulated metabolites such as α-ketoglutarate.

Nigericin-induced Impairment of Autophagic Flux in Neuronal Cells Is Inhibited by Overexpression of Bak

Bak is a prototypic pro-apoptotic Bcl-2 family protein expressed in a wide variety of tissues and cells. Recent studies have revealed that Bcl-2 family proteins regulate apoptosis as well as autophagy. To investigate whether and how Bak exerts a regulatory role on autophagy-related events, we treated independent cell lines, including MN9D neuronal cells, with nigericin, a K(+)/H(+) ionophore. Treatment of MN9D cells with nigericin led to an increase of LC3-II and p62 levels with concomitant activation of caspase. Ultrastructural examination revealed accumulation of autophagic vacuoles and swollen vacuoles in nigericin-treated cells. We further found that the LC3-II accumulated as a consequence of impaired autophagic flux and the disrupted degradation of LC3-II in nigericin-treated cells. In this cell death paradigm, both transient and stable overexpression of various forms of Bak exerted a protective role, whereas it did not inhibit the extent of nigericin-mediated activation of caspase-3. Subsequent biochemical and electron microscopic studies revealed that overexpressed Bak maintained autophagic flux and reduced the area occupied by swollen vacuoles in nigericin-treated cells. Similar results were obtained in nigericin-treated non-neuronal cells and another proton ionophore-induced cell death paradigm. Taken together, our study indicates that a protective role for Bak during ionophore-induced cell death may be closely associated with its regulatory effect on maintenance of autophagic flux and vacuole homeostasis.

Effect of recombinant canine interleukin‐6 and interleukin‐8 on tissue factor procoagulant activity in canine peripheral blood mononuclear cells and purified canine monocytes

Inflammation is a major cause of disseminated intravascular coagulation (DIC) in dogs, but underlying mechanisms for its initiation are unknown. We hypothesized that pro-inflammatory cytokines, interleukin (IL)-6 and IL-8, induce tissue factor (TF) expression on canine monocyte surfaces, which may contribute to DIC initiation. The objectives of this study were to determine if (1) IL-6 and IL-8 would induce TF activity on canine monocytes, (2) fetal bovine serum or autologous plasma was required for IL-6- or IL-8-induced TF responses in canine monocytes, and (3) these pro-inflammatory cytokines would enhance TF activity on canine monocytes in response to low concentrations of lipopolysaccharide (LPS). Canine monocytes were isolated from EDTA-anticoagulated blood as peripheral blood mononuclear cells (PBMC) by double-density gradient centrifugation and adhesion to plastic. Adherent cells were stimulated for 4 hours with recombinant canine (rc)-IL-6 or rc-IL-8 (10-5000 pg/mL) with or without 10% heat-inactivated (HI) fetal bovine serum, untreated autologous canine plasma (ACP), or HI-ACP. Lipopolysaccharide (100 ng/mL) served as a positive control. Cells were also costimulated with either cytokine (100 pg/mL) or low concentrations of LPS (0.1 and 1 ng/mL). Monocytes immunopurified from PBMC with anti-CD14 antibodies were also stimulated with both cytokines (100 and 5000 pg/mL). TF activity on cell surfaces was measured by a 2-stage amidolytic assay, based on activated factor X generation. Neither rc-IL-6 nor rc-IL-8 consistently stimulated TF procoagulant activity in canine PBMC or purified monocytes after 4 hours. Serum, plasma, or low concentrations of LPS did not enhance the TF response to these cytokines. IL-6 or IL-8 at evaluated concentrations may not play major roles in coagulation activation by induction of TF expression on monocytes in dogs with inflammation.

FTY720 Markedly Increases Alloengraftment but Does Not Eliminate Host Anti-Donor T Cells that Cause Graft Rejection on Its Withdrawal

The immunomodulator FTY720 (FTY) is beneficial in models of graft-versus-host disease, solid organ transplantation, and autoimmunity and has been approved for use in patients with multiple sclerosis. FTY modifies the homing and migration of many cell types. We report that FTY has profound positive and negative effects on allogeneic bone marrow (BM) engraftment in sublethally irradiated recipients. FTY increased donor hematopoietic progenitors in the BM, resulting in high donor engraftment in the B cell, myeloid cell, and natural killer cell, but not T cell, lineages. Donor T cell progenitors within the thymus of FTY-treated recipients were dramatically reduced, resulting in a lack of donor T cell reconstitution. In addition to preventing the ingress of donor (and host) T cell progenitors, FTY prevented the egress of fully functional host CD4+CD8- and CD4-CD8+ thymocytes that on cessation of FTY administration were able to exit from the thymus and contribute to a rapid and complete rejection of a well-established donor BM graft. When used in combination with anti-CD40L mAbs to block the CD40L:CD40 costimulatory pathway, FTY markedly enhanced anti-CD40L mAb-mediated alloengraftment promotion. In contrast to FTY alone, the combination of anti-CD40L mAb and FTY resulted in a surprisingly stable, multilineage, long-term donor chimerism. These data illustrate FTY's profound migration modulating effects and suggest a use in combinatorial therapy in achieving stable alloengraftment under nonmyeloablative conditions.

Epigenetic modifiers enhance the synergistic cytotoxicity of combined nucleoside analog-DNA alkylating agents in lymphoma cell lines

Hematopoietic stem cell transplantation is used for treatment of lymphoma. In an attempt to design an efficacious and safe prehematopoietic stem cell transplantation conditioning regimen, we investigated the cytotoxicity of the combination of busulfan (B), melphalan (M), and gemcitabine (G) in lymphoma cell lines in the absence or presence of drugs that induce epigenetic changes. Cells were exposed to drugs individually or in combination and analyzed by the MTT proliferation assay, flow cytometry, and Western blotting. We used ~IC(10) drug concentrations (57 μM B, 1 μM M and 0.02 μM G), which individually did not have major effects on cell proliferation. Their combination resulted in 50% inhibition of proliferation. Reduction to almost half concentration (20 μM B, 0.7 μM M and 0.01 μM G) did not have significant effects, but addition of the histone deacetylase inhibitor suberoylanilide hydroxamic acid (0.6 μM) to this combination resulted in a marked (~65%) growth inhibition. The cytotoxicity of these combinations correlates with the activation of the ataxia telangiectasia mutated-CHK2 pathway, phosphorylation of KRAB-associated protein-1, epigenetic changes such as methylation and acetylation of histone 3, and activation of apoptosis. The relevance of epigenetic changes is further shown by the induction of DNA methyltransferases in tumor cells with low constitutive levels of DNMT3A and DNMT3B. The addition of 5-aza-2'-deoxycytidine to (BMG+suberoylanilide hydroxamic acid) further enhances cell killing. Overall, BMG combinations are synergistically cytotoxic to lymphoma cells. Epigenetic changes induced by suberoylanilide hydroxamic acid and 5-aza-2'-deoxycytidine further enhance the cytotoxicity. This study provides a rationale for an ongoing clinical trial in our institution using (BMG+suberoylanilide hydroxamic acid) as pre-hematopoietic stem cell transplantation conditioning for lymphoma.

Cytoplasmic NANOG-Positive Stromal Cells Promote Human Cervical Cancer Progression

Tumor development has long been known to resemble abnormal embryogenesis. The embryonic stem cell gene NANOG, a divergent homeodomain transcription factor that is independent of leukemia inhibitory factor, has been reported to be expressed in germ cells and in several tumor types. However, the short-term expression and role of NANOG in cervical cancer remain unclear. In the present study, we demonstrate that NANOG exhibits cellular shuttling behavior and increasing stromal distribution during the progression of cervical cancer. Our molecular data using RT-PCR and restriction enzyme digestion show that NANOG is mainly transcribed from the NANOG gene in cervical cancer. In addition, IHC using confocal microscopy suggests that mesenchymal stem cells (MSCs) are one type of cytoplasmic NANOG-positive cells in cervical cancer stroma. Co-culture of cervical cancer-derived MSCs with SiHa cells showed increased proliferation characteristics in vitro and enhanced tumor growth in vivo. Our results show, for the first time to our knowledge, that MSCs are a source of cytoplasmic NANOG expression in the cervical cancer stroma and that they participate in the progression of cervical cancer both in vitro and in vivo. Our study provides evidence that NANOG is a cervical cancer progression marker and also serves as a starting point for a more extensive exploration of the cellular translocation of NANOG and the multifunctionality of the stromal microenvironment.

Establishment and characterization of a cell line from the mosquito Culex tritaeniorhynchus (Diptera: Culicidae)

We established a continuous cell line from the embryo of the mosquito Culex tritaeniorhynchus Giles (Diptera: Culicidae), a known major vector of the Japanese encephalitis virus (family Flaviviridae, genus Flavivirus) in Asia. The cell line, designated NIID-CTR, was serially subcultured in VP-12 medium supplemented with 10 % heat-inactivated fetal bovine serum (FBS). It continued to grow for more than 60 passages over a 750-d period. The NIID-CTR cell line mainly comprised two morphologically distinct types of cells with adhesive properties: spindle-shaped and round cells. Most of the NIID-CTR cells at the 45th passage were diploid (2n = 6). The growth kinetics of the NIID-CTR cells was significantly affected by the FBS concentration in the medium. The population doubling time of the NIID-CTR cells was 20 h in the presence of 10 % FBS and 76 h in its absence. The DNA sequence of the mitochondrial cytochrome oxidase I gene confirmed that the NIID-CTR cell line was derived from C. tritaeniorhynchus. The cells were highly susceptible to Japanese encephalitis and Dengue viruses, thus providing a valuable tool for the study of mosquito-borne flaviviruses.

Elevated Cyclin G2 Expression Intersects with DNA Damage Checkpoint Signaling and Is Required for a Potent G2/M Checkpoint Arrest Response to Doxorubicin

To maintain genomic integrity DNA damage response (DDR), signaling pathways have evolved that restrict cellular replication and allow time for DNA repair. CCNG2 encodes an unconventional cyclin homolog, cyclin G2 (CycG2), linked to growth inhibition. Its expression is repressed by mitogens but up-regulated during cell cycle arrest responses to anti-proliferative signals. Here we investigate the potential link between elevated CycG2 expression and DDR signaling pathways. Expanding our previous finding that CycG2 overexpression induces a p53-dependent G(1)/S phase cell cycle arrest in HCT116 cells, we now demonstrate that this arrest response also requires the DDR checkpoint protein kinase Chk2. In accord with this finding we establish that ectopic CycG2 expression increases phosphorylation of Chk2 on threonine 68. We show that DNA double strand break-inducing chemotherapeutics stimulate CycG2 expression and correlate its up-regulation with checkpoint-induced cell cycle arrest and phospho-modification of proteins in the ataxia telangiectasia mutated (ATM) and ATM and Rad3-related (ATR) signaling pathways. Using pharmacological inhibitors and ATM-deficient cell lines, we delineate the DDR kinase pathway promoting CycG2 up-regulation in response to doxorubicin. Importantly, RNAi-mediated blunting of CycG2 attenuates doxorubicin-induced cell cycle checkpoint responses in multiple cell lines. Employing stable clones, we test the effect that CycG2 depletion has on DDR proteins and signals that enforce cell cycle checkpoint arrest. Our results suggest that CycG2 contributes to DNA damage-induced G(2)/M checkpoint by enforcing checkpoint inhibition of CycB1-Cdc2 complexes.

Striatal-enriched Protein-tyrosine Phosphatase (STEP) Regulates Pyk2 Kinase Activity

Proline-rich tyrosine kinase 2 (Pyk2) is a member of the focal adhesion kinase family and is highly expressed in brain and hematopoietic cells. Pyk2 plays diverse functions in cells, including the regulation of cell adhesion, migration, and cytoskeletal reorganization. In the brain, it is involved in the induction of long term potentiation through regulation of N-methyl-d-aspartate receptor trafficking. This occurs through the phosphorylation and activation of Src family tyrosine kinase members, such as Fyn, that phosphorylate GluN2B at Tyr(1472). Phosphorylation at this site leads to exocytosis of GluN1-GluN2B receptors to synaptic membranes. Pyk2 activity is modulated by phosphorylation at several critical tyrosine sites, including Tyr(402). In this study, we report that Pyk2 is a substrate of striatal-enriched protein-tyrosine phosphatase (STEP). STEP binds to and dephosphorylates Pyk2 at Tyr(402). STEP KO mice showed enhanced phosphorylation of Pyk2 at Tyr(402) and of the Pyk2 substrates paxillin and ASAP1. Functional studies indicated that STEP opposes Pyk2 activation after KCl depolarization of cortical slices and blocks Pyk2 translocation to postsynaptic densities, a key step required for Pyk2 activation and function. This is the first study to identify Pyk2 as a substrate for STEP.

Chick embryo tracheal organ: A new and effective in vitro culture model for Cryptosporidium baileyi

In the present study, chick embryo tracheal organ (TOCs) was used to cultivate oocysts or sporozoites of Cryptosporidium baileyi. Approximately 5×104 sporozoites and oocysts mixture for group I; 5×105, 1×106, 2×106 purified sporozoites for group II, group III and group IV, respectively, were inoculated into respective chick embryo tracheal rings maintained in RPMI 1640 supplemented with 5% heat-inactivated FBS, and cultivated in each well of the 24-well culture plate at 40°C and 5% CO2. The tracheal rings in four experimental groups (I–IV) were successfully infected with C. baileyi, and different stages of parasites were also observed under light and electron microscopy. Parasite infection and cytological alterations were noted as early as PI 72h. The Cryptosporidium were seen attached to the edge of the tracheal epithelium, with more number of parasites in group I than that in group II, group III and group IV. The moderate nuclear swelling and chromatin margination were also detected, and the normal vertical orientation and basilar location of the nuclei of the epithelial cells were almost lost. C. baileyi that has been passed by TOCs exhibited similar immunity and molecular features with parasites before intratracheal inoculation. These results suggest that chick embryo tracheal organ is a new and effective in vitro culture model for C. baileyi and other respiratory pathogens.

ELOVL4 protein preferentially elongates 20:5n3 to very long chain PUFAs over 20:4n6 and 22:6n3

We hypothesized that reduction/loss of very long chain PUFAs (VLC-PUFAs) due to mutations in the ELOngase of very long chain fatty acid-4 (ELOVL4) protein contributes to retinal degeneration in autosomal dominant Stargardt-like macular dystrophy (STGD3) and age-related macular degeneration; hence, increasing VLC-PUFA in the retina of these patients could provide some therapeutic benefits. Thus, we tested the efficiency of elongation of C20-C22 PUFA by the ELOVL4 protein to determine which substrates are the best precursors for biosynthesis of VLC-PUFA. The ELOVL4 protein was expressed in pheochromocytoma cells, while green fluorescent protein-expressing and nontransduced cells served as controls. The cells were treated with 20:5n3, 22:6n3, and 20:4n6, either individually or in equal combinations. Both transduced and control cells internalized and elongated the supplemented FAs to C22-C26 precursors. Only ELOVL4-expressing cells synthesized C28-C38 VLC-PUFA from these precursors. In general, 20:5n3 was more efficiently elongated to VLC-PUFA in the ELOVL4-expressing cells, regardless of whether it was in combination with 22:6n3 or with 20:4n6. In each FA treatment group, C34 and C36 VLC-PUFAs were the predominant VLC-PUFAs in the ELOVL4-expressing cells. In summary, 20:5n3, followed by 20:4n6, seems to be the best precursor for boosting the synthesis of VLC-PUFA by ELOVL4 protein.

Pivotal Role of ADP-ribosylation Factor 6 in Toll-like Receptor 9-mediated Immune Signaling

CpG oligodeoxynucleotide (CpG ODN) cellular uptake into endosomes, the rate-limiting step of Toll-like receptor 9 (TLR9) signaling, is critical in eliciting innate immune responses. ADP-ribosylation factor 6 (ARF6) is a member of the Ras superfamily, which is critical to a wide variety of cellular events including endocytosis. Here, we found that inhibition of ARF6 by dominant mutants and siRNA impaired CpG ODN-mediated responses, whereas cells expressing the constitutively active ARF6 mutant enhanced CpG ODN-induced cytokine production. Inhibition of ARF6 impaired TLR9 trafficking into endolysosomes, thereby inhibiting proceed functional cleavage of TLR9. Additional studies showed that CpG ODN uptake was increased in ARF6-activated cells but impaired in ARF6-defective cells. Furthermore, cells pretreated with CpG ODN but not GpC ODN had increased CpG ODN uptake due to CpG ODN-induced ARF6 activity. Further studies with ARF6-defective and ARF6-activated cells demonstrated that class III phosphatidylinositol 3-kinases (PI3K) was required for downstream ARF6 regulation of CpG ODN uptake. Together, our findings demonstrate that a novel class III PI3K-ARF6 axis pathway mediates TLR9 signaling by regulating the cellular uptake of CpG ODN.

Malin Regulates Wnt Signaling Pathway through Degradation of Dishevelled2

Using yeast-two hybrid screening followed by co-immunoprecipitation assay, we have found that the Lafora disease ubiquitin ligase malin interacts with dishevelled2, a key mediator of Wnt signaling pathway. Overexpression of malin enhances the degradation of dishevelled2 and inhibits Wnt signaling, which is evident from the down-regulation of β-catenin target genes and the decrease in β-catenin-mediated transcriptional activity. Partial knockdown of malin significantly increases the level of dishevelled2 and up-regulates Wnt signaling. Several malin mutants are found to be ineffective in degrading dishevelled2 and regulating the Wnt pathway. We have also found that malin enhances K48- and K63-linked ubiquitination of dishevelled2 that could lead to its degradation through both proteasome and autophagy. Altogether, our results indicate that malin regulates Wnt signaling pathway through the degradation of dishevelled2 and suggest possible deregulation of Wnt signaling in Lafora disease.

Enzymatic measurement of phosphatidylserine in cultured cells

Phosphatidylserine (PS) is a quantitatively minor membrane phospholipid involved in diverse cellular functions. In this study, we developed a new fluorometric method for measuring PS using combinations of specific enzymes and Amplex Red. The calibration curve for PS measurement was linear and hyperbolic at low (0-50 µM) and high (50-1000 µM) concentrations, respectively, and the detection limit was 5 µM (50 pmol in the reaction mixture). This assay quantified PS regardless of the chain length and the number of double bonds. We applied this new method to the determination of PS content in HEK293 cells, which was validated by a recovery study and comparison with TLC-phosphorus assay. We showed that the PS content was high in sparse cells. The overexpression of PS synthase 1 elevated not only the cellular PS content but also the phosphatidylcholine (PC) and phosphatidylethanolamine (PE) contents, suggesting the conversion of PS into PE and the enhancement of PC production. This new assay for PS measurement is simple, specific, sensitive, and high throughput, and it will be useful to clarify the metabolism and biological functions of PS.

Thermal Stability of siRNA Modulates Aptamer- conjugated siRNA Inhibition

Oligonucleotide aptamer-mediated in vivo cell targeting of small interfering RNAs (siRNAs) is emerging as a useful approach to enhance the efficacy and reduce the adverse effects resulting from siRNA-mediated genetic interference. A current main impediment in aptamer-mediated siRNA targeting is that the activity of the siRNA is often compromised when conjugated to an aptamer, often requiring labor intensive and time consuming design and testing of multiple configurations to identify a conjugate in which the siRNA activity has not been significantly reduced. Here, we show that the thermal stability of the siRNA is an important parameter of siRNA activity in its conjugated form, and that siRNAs with lower melting temperature (Tm) are not or are minimally affected when conjugated to the 3′ end of 2′F-pyrimidine-modified aptamers. In addition, the configuration of the aptamer-siRNA conjugate retains activity comparable with the free siRNA duplex when the passenger strand is co-transcribed with the aptamer and 3′ overhangs on the passenger strand are removed. The approach described in this paper significantly reduces the time and effort necessary to screening siRNA sequences that retain biological activity upon aptamer conjugation, facilitating the process of identifying candidate aptamer-siRNA conjugates suitable for in vivo testing.

Pro-survival Effects of 17β-Estradiol on Osteocytes Are Mediated by Nitric Oxide/cGMP via Differential Actions of cGMP-dependent Protein Kinases I and II

Estrogens promote bone health in part by increasing osteocyte survival, an effect that requires activation of the protein kinases Akt and ERK1/2, but the molecular mechanisms involved are only partly understood. Because estrogens increase nitric oxide (NO) synthesis and NO can have anti-apoptotic effects, we examined the role of NO/cGMP signaling in estrogen regulation of osteocyte survival. Etoposide-induced death of MLO-Y4 osteocyte-like cells, assessed by trypan blue staining, caspase-3 cleavage, and TUNEL assays, was completely prevented when cells were pre-treated with 17β-estradiol. This protective effect was mimicked when cells were pre-treated with a membrane-permeable cGMP analog and blocked by pharmacological inhibitors of NO synthase, soluble guanylate cyclase, or cGMP-dependent protein kinases (PKGs), supporting a requirement for NO/cGMP/PKG signaling downstream of 17β-estradiol. siRNA-mediated knockdown and viral reconstitution of individual PKG isoforms demonstrated that the anti-apoptotic effects of estradiol and cGMP were mediated by PKG Iα and PKG II. Akt and ERK1/2 activation by 17β-estradiol required PKG II, and cGMP mimicked the effects of estradiol on Akt and ERK, including induction of ERK nuclear translocation. cGMP induced BAD phosphorylation on several sites, and experiments with phosphorylation-deficient BAD mutants demonstrated that the anti-apoptotic effects of cGMP and 17β-estradiol required BAD phosphorylation on Ser(136) and Ser(155); these sites were targeted by Akt and PKG I, respectively, and regulate BAD interaction with Bcl-2. In conclusion, 17β-estradiol protects osteocytes against apoptosis by activating the NO/cGMP/PKG cascade; PKG II is required for estradiol-induced activation of ERK and Akt, and PKG Iα contributes to pro-survival signaling by directly phosphorylating BAD.

Establishment and Characterization of a Novel Human Cell Line of Triple Hit Lymphoma, AMU-ML1, Carrying t(2;18)(p11.2;q21) and t(3;8;14)(q27;q24;q32) That Involve BCL2, BCL6 and MYC

Abstract 5210Mature B-cell lymphomas with both BCL2 and MYC translocations to IG loci are rarely identified and most of them are classified as B-cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma (IL) in the new World Health Organization classification.In this study, we established a novel human lymphoma cell line, AMU-ML1, from pericardial effusion (PE) of a patient with IL before the initiation of chemotherapy and analyzed its characters. A 60-year-old male was admitted to our hospital because of PE and diagnosed as having IL from the atypical lymphocytes in PE. He was treated with rituximab plus hyper-CVAD and other regimens but died of lymphoma approximately 10 months after diagnosis without reaching complete remission.The cells from patient's PE at diagnosis were cultured in RPMI 1640 supplemented with 20% heat-inactivated fetal bovine serum (FBS). After 5 months of culture, cell proliferation became continuous with 10% FBS and the cell line was designated as AMU-ML1 (Aichi Medical University, malignant lymphoma, no. 1) after confirmation that the cells began growing again after the conventional freeze-thaw procedure.AMU-ML1 cells were positive for CD10, CD19, CD20, CD79a, HLA-DR and cytoplasmic lambda chain and negative for CD3, CD4, CD5, CD8, CD13, CD23, CD33 and CD56 by flow cytometry analysis and showed a complex karyotypes including t(2;18)(p11.2;q21) and t(3;8;14)(q27;q24;q32) by G-banding analysis. This profile is consistent with the profile of the patient's cells. Spectral karyotyping and fluorescent in situ hybridization analysis of AMU-ML1 cells revealed that t(2;18)(p11.2;q21) was IGL/BCL2 and t(3;8;14)(q27;q24;q32) was BCL6/MYC, MYC/IGH and IGH/BCL6.Subcutaneous transplantation of AMU-ML1 cells into NOD/scid mice treated with anti-asialo GM1 antibody resulted in formation of primary tumors.Thus, the AMU-ML1 cell line is useful for studying the biological consequences of IL with triple hit of BCL2, BCL6 and MYC, and possibly invasion to PE of lymphoma. Disclosures:No relevant conflicts of interest to declare.

Abstract B45: The role of combined inhibition of DNA methylation and histone deacetylation in CTCL treatment.

Abstract Introduction: Change in the epigenetic pathways can alter level of gene expressions and trigger neoplastic transformation.1DNA methylation and histone modifications are two main epigenetic mechanisms that regulate gene expression. DNA demethylation results in reexpression of tumor suppressor genes silenced via hypermethylation of their promoter regions and HDACi, approved for cutaneous T cell lymphoma (CTCL) treatment, are shown to synergize with demethylating agents to relieve transcriptional repression2,3. We combined Romidepsin and Azacitidine treatment on CTCL primary cell cultures to investigate cell viability and apoptosis. Methods: Human CTCL cell lines were grown in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), glutamine (2 mM) and streptomycin (100 μg/ml) at 37°C, 5% CO2 and 95% humidity. The CTCL cell cultures were treated with Romidepsin (0.25nM-10uM) and/or Azacitidine (500nM-10uM). After 24 to 72 hrs of combination drug treatment level of apoptosis and necrosis was measured by flow cytometry with Annexin V-FITC detection kit. With the above combination treatment the gene expression levels of cell cycle regulatory genes, HDACs (HDAC1, HDAC2, HDAC3, HDAC6,) and DNA methyltransferases (DNMT1, DNMT3a, DNMT3b) were examined by RT-PCR. The induction of Histone acetylation of H3 and H4 and cell cycle regulatory genes were analyzed by western blotting. Results: The combination treatment showed a noticeable change of cell viability due to an increase in apoptosis and necrosis in a time and dose dependent manner. Furthermore, the RT-PCR results were notable for change in the expression of several of the genes mentioned above, suggesting that the combination of DNA demethylation and histone acetylation has a more selective effect on the induction of apoptosis in CTCL cells. We observed a more pronounced increase cell cycle arrest by induction of cell cycle regulators such as p21 and in acetylation of H3 in the combination treatment than each drug alone suggesting that the this combination treatment, counteracts loss of cell cycle control in CTCL more efficiently. This not only provides a rationale for future clinical trials with combined Romidepsin and Azacitidine treatment but also has initiated us to further explore the possible effects of this combination in HDAC inhibitor resistant CTCL. Conclusion: In the light of our current investigation's outcome, we plan to explore the impact of the cumulative chromatin changes by this treatment combination and try to understand their specific mechanism of cell death induction in cutaneous T cell lymphomas. Further confirmation will be followed by experiments on other CTCL cell cultures and lymphocytes of mycosis fongiodes/Sezary syndrome patients.

Bortezomib-resistant nuclear factor κB expression in stem-like cells in mantle cell lymphoma

Mantle cell lymphoma (MCL) is a subtype of B-cell Non-Hodgkin's Lymphoma (NHL) and accounts for approximately 6% of all lymphomas. Unlike small lymphocytic lymphoma and chronic lymphocytic lymphoma, which are relatively sensitive to chemotherapy, MCL is highly refractory to most chemotherapy, and has the worst survival rate among NHL patients. Stem-like cells in MCL, which we have termed mantle cell lymphoma-initiating cells (MCL-ICs), enriched in the population that are lack of prototypic B-cell marker CD19. These cells were able to self-renew upon serial transplantation and are highly tumorigenic. Importantly, these stem-like cells confer chemotherapeutic resistance to MCL. In this report, we show that stem-like MCL-ICs are resistant to bortezomib, as well as chemotherapeutic regimens containing bortezomib, despite constitutive nuclear factor-κB (NF-κB) expression. Interestingly, bortezomib treatment induced MCL-IC differentiation in plasma-like cells with upregulated expression of CD38 and CD138. This process was accompanied by expression of plasma cell differentiation transcriptional factors, BLIMP-1 and IRF4. This article is the first to show that stem-like MCL cells utilize constitutive NF-κB expression for survival. Given that the NF-κB expression in MCL-ICs is resistant to bortezomib, it will be important to find alternative therapeutic strategies to inhibit NF-κB expression.

Identification of Human Fumarylacetoacetate Hydrolase Domain-containing Protein 1 (FAHD1) as a Novel Mitochondrial Acylpyruvase

The human fumarylacetoacetate hydrolase (FAH) domain-containing protein 1 (FAHD1) is part of the FAH protein superfamily, but its enzymatic function is unknown. In the quest for a putative enzymatic function of FAHD1, we found that FAHD1 exhibits acylpyruvase activity, demonstrated by the hydrolysis of acetylpyruvate and fumarylpyruvate in vitro, whereas several structurally related compounds were not hydrolyzed as efficiently. Conserved amino acids Asp-102 and Arg-106 of FAHD1 were found important for its catalytic activity, and Mg(2+) was required for maximal enzyme activity. FAHD1 was found expressed in all tested murine tissues, with highest expression in liver and kidney. FAHD1 was also found in several human cell lines, where it localized to mitochondria. In summary, the current work identified mammalian FAHD1 as a novel mitochondrial enzyme with acylpyruvate hydrolase activity.