BrdU Uptake Research Articles

Abstract 495: Inactive status of cancer cells with suppression of AKT activity contributes to survival in chronic hypoxia

Abstract A hypoxic microenvironment in tumors has been recognized as a cause of malignancy or resistance to various cancer therapies. In contrast to the recent progress in understanding the acute response of cancer cells to hypoxia, the characteristics of tumor cells in chronic hypoxia remain elusive. We have identified a pancreatic cancer cell line, AsPC-1, which is exceptionally able to survive for weeks under hypoxic conditions (1%O2) without changing medium at all, while most of the tested cancer cell lines died after several days under these conditions. Under hypoxic conditions, the AsPC-1 cells entered an “inactive status” characterized by no proliferation or death and by metabolic suppression. They recovered to active status after being placed again in optimal culture conditions. Under chronic hypoxia, ATP turnover, an indicator of energy demand, was markedly decreased, as was AKT phosphorylation. Forced activation of AKT resulted in increased ATP turnover and massive cell death in vitro. Xenograft tumors generated with AKT over-expressing cells showed remarkable decrease of inactive zone, where BrdU uptake and S6 phosphorylation were eliminated without any sign of cell death. We recently developed a novel culture system for primary cancer cells. In contrast to most cancer cell lines, primary colorectal cancer cells entered inactive status with AKT suppression under hypoxia combined with growth factor-depleted conditions. The ability of cancer cells to survive in a deteriorated microenvironment by entering into an inactive status might be a common property among cancer cells. The cancer cells in inactive status of both primary cultured cancer cells and AsPC-1 cells were resistant to chemotherapy. Targeting the regulatory mechanism inducing this inactive status might provide a new strategy for overcoming resistance to various cancer therapies. Citation Format: Masahiro Inoue, Hiroaki Okuyama, Hiroko Endo, Masayuki Ohue. Inactive status of cancer cells with suppression of AKT activity contributes to survival in chronic hypoxia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 495. doi:10.1158/1538-7445.AM2014-495

Abstract MP03: Intracerebroventricular Administration Of Autologous Bone Marrow-derived Cells Attenuate Inflammation And Increase In AT1 Receptor In The Brain Of Angiotensin II Initiated Hypertensive Rats

Objectives: Bone marrow-derived cells (BMCs) include mesenchymal stem cells (MSC) play critical role in repairing damaged tissue and anti-inflammation. We reported that intracerebroventricular (icv) administration of autologous BMCs attenuate slow pressor Ang II initiated hypertension via attenuating Ang II mediated sympathetic activation in rats. However, mechanisms of this observation have not been elucidated. We hypothesized that BMCs in the brain would attenuate inflammation and modulate overactive renin angiotensin system (RAS) in the brain of Ang II initiated hypertensive rats. Therefore we examined characteristics of BMCs prepared with identical cultural condition to previous experiment and examined expression of inflammation related cytokines and RAS component in the brain of Ang II initiated hypertensive rats. Methods: Aspirated bone marrow cells underwent red blood cell lysis protocol were seeded in an individual dish in Dulbecco Modified Eagle Media with 10% fetal bovine serum. Floating cells were removed and plastic adherent cells were allowed to glow for 3 weeks at passage 3. The number of BMCs proliferated more than 10 6 were used for immunocytochemical, flowcytometric, BrdU uptake, and Western blot analysis. TGF-beta, IL-10 and AT1 receptor protein expression were examined in brains of Ang II initiated hypertensive rats. Results: Cultured BMCs expressed CD29, a marker of mesenchymal stem cell. Flowcytometric analysis revealed that 38% of BMCs were CD29+ CD34-. Endothelial cell (EC) proliferation assay exhibited 52% higher BrdU uptake with conditioned media from BMCs than the media from ECs. Expression of angiotensin converting enzyme (ACE) was 87% lower in BMCs than in ECs, whereas ACE2 expression was 142% higher in BMCs. Furthermore, Ang II infusion significantly increased expression of TGF-beta and AT1 receptor in the brain by 146% and 135% respectively and icv administration of BMCs attenuated the increases of both expressions (39%, 59% respectively). Ang II infusion with or without BMCs icv did not affect to IL-10 expression in the brain. Conclusion: BMCs in the brain may have protective role against Ang II induced sympathetic activation via attenuation of brain inflammation and increase in AT1 receptors.

Serum and glucocorticoid-regulated kinase 1 promotes vascular smooth muscle cell proliferation via regulation of β-catenin dynamics

In response to arterial intimal injury vascular smooth muscle cells (VSMCs) within the vessel wall proliferate upon exposure to growth factors, accumulate, and form a neointima that can occlude the vessel lumen. Serum and glucocorticoid inducible kinase 1 (SGK1) is a growth factor-responsive kinase; however its role in VSMC proliferation is not fully understood. Here, we examined growth factor-dependent regulation of SGK1 and defined a molecular role for SGK1 in stimulation of VSMC proliferation. We found that stimulation of VSMCs with the pro-proliferative growth factor, platelet-derived growth factor BB (PDGF) significantly increased SGK1 mRNA, protein, and kinase activity in aortic VSMCs in vitro. To test the hypothesis that activation of SGK1 activity promotes VSMC proliferation, we examined the effects of stable expression of constitutively active (S422D) and kinase-defective (S422A) mutants of SGK1 on VSMC growth. We found that activation of SGK1 increased, whereas interference of SGK1 signaling inhibited VSMC growth in vitro. Consistent with these findings, expression of the S422D mutant augmented both basal and PDGF-induced BrdU uptake in VSMCs. Conversely, PDGF-induced BrdU uptake was attenuated in VSMCs expressing S422A. Furthermore, we determined that activated SGK1 enhanced basal and PDGF-dependent G1→S cell cycle transition, whereas dominant-negative SGK1 abrogated G1→S cell cycle transition under similar conditions. Downstream signaling by active SGK1 induced basal and PDGF-induced phosphorylation of glycogen synthase kinase 3β, an effect which was attenuated when SGK1 activity was blocked by expression of the kinase-defective mutant, S422A. We also found that transfection of S422D enhanced β-catenin-nuclear localization and activation of the TOP/Flash and cyclin D1 transcriptional reporters. These effects were significantly blunted in VSMCs transfected with the S422A mutant. Our results provide compelling evidence of a role for SGK1 in stimulation of arterial VSMC growth via regulation of β-catenin dynamics and implicate SGK1 in the progression of intimal narrowing following arterial injury. Hence, the findings presented here point to inhibition of SGK1 activity as a novel therapeutic approach for the treatment of occlusive vascular diseases.

Activated CD8+ T lymphocytes inhibit neural stem/progenitor cell proliferation: role of interferon-gamma.

The ability of neural stem/progenitor cells (NSCs) to self-renew, migrate to damaged sites, and differentiate into neurons has renewed interest in using them in therapies for neurodegenerative disorders. Neurological diseases, including viral infections of the brain, are often accompanied by chronic inflammation, whose impact on NSC function remains unexplored. We have previously shown that chronic neuroinflammation, a hallmark of experimental herpes simplex encephalitis (HSE) in mice, is dominated by brain-infiltrating activated CD8 T-cells. In the present study, activated CD8 lymphocytes were found to suppress NSC proliferation profoundly. Luciferase positive (luc+) NSCs co-cultured with activated, MHC-matched, CD8+ lymphocytes (luc−) showed two- to five-fold lower luminescence than co-cultures with un-stimulated lymphocytes. On the other hand, similarly activated CD4+ lymphocytes did not suppress NSC growth. This differential lymphocyte effect on proliferation was confirmed by decreased BrdU uptake by NSC cultured with activated CD8 T-cells. Interestingly, neutralizing antibodies to interferon-gamma (IFN-γ) reversed the impact of CD8 lymphocytes on NSCs. Antibodies specific to the IFN-γ receptor-1 subunit complex abrogated the inhibitory effects of both CD8 lymphocytes and IFN-γ, indicating that the inhibitory effect of these cells was mediated by IFN-γ in a receptor-specific manner. In addition, activated CD8 lymphocytes decreased levels of nestin and Sox2 expression in NSCs while increasing GFAP expression, suggesting possible induction of an altered differentiation state. Furthermore, NSCs obtained from IFN-γ receptor-1 knock-out embryos were refractory to the inhibitory effects of activated CD8+ T lymphocytes on cell proliferation and Sox2 expression. Taken together, the studies presented here demonstrate a role for activated CD8 T-cells in regulating NSC function mediated through the production of IFN-γ. This cytokine may influence neuro-restorative processes and ultimately contribute to the long-term sequelae commonly seen following herpes encephalitis.

Isolation, expansion and transplantation of postnatal murine progenitor cells of the enteric nervous system.

Neural stem or progenitor cells have been proposed to restore gastrointestinal function in patients suffering from congenital or acquired defects of the enteric nervous system. Various, mainly embryonic cell sources have been identified for this purpose. However, immunological and ethical issues make a postnatal cell based therapy desirable. We therefore evaluated and quantified the potential of progenitor cells of the postnatal murine enteric nervous system to give rise to neurons and glial cells in vitro. Electrophysiological analysis and BrdU uptake studies provided direct evidence that generated neurons derive from expanded cells in vitro. Transplantation of isolated and expanded postnatal progenitor cells into the distal colon of adult mice demonstrated cell survival for 12 weeks (end of study). Implanted cells migrated within the gut wall and differentiated into neurons and glial cells, both of which were shown to derive from proliferated cells by BrdU uptake. This study indicates that progenitor cells isolated from the postnatal enteric nervous system might have the potential to serve as a source for a cell based therapy for neurogastrointestinal motility disorders. However, further studies are necessary to provide evidence that the generated cells are capable to positively influence the motility of the diseased gastrointestinal tract.

Proliferation suppression by thyroid hormone requires thyroid hormone receptor in fetal cardiomyocytes (850.1)

Objectives: Ovine fetal cardiomyocyte (CM) maturation begins after 100 days gestation (dGA). At term (145dGA) ~60% of CMs are binucleated and cannot divide. Thyroid hormone (T3) rises near term and suppresses proliferation by insulin‐like growth factor‐1 (IGF‐1) in vitro. Despite T3 activation of extracellular regulatory kinase (ERK) and AKT we hypothesize that T3‐induced inhibition of proliferation is thyroid receptor (TR) mediated and reversed by the TR antagonist, NH3. Methods: Fetal CMs were isolated from 102±3 and 135±1dGA sheep (n=11/group). Cells were treated with T3 (0.37, 1.5nM), IGF‐1 (1ug/ml), NH3 (100nM) or a combination. Proliferation was quantified by BrdU uptake (10uM). Western blots measured ERK, AKT, TRa, and p21 expression. Results: IGF‐1 stimulated proliferation at both ages. T3 reduced this mitotic activity by ~50% in 100dGA and ~75% in 135dGA. At both ages T3 increased p21 (p<0.001), and NH3 blocked the T3+IGF‐1 reduction of BrdU uptake without altering ERK or AKT. In 100dGA CMs NH3 did not reduce T3‐induced p21 expression but upregulated TRa (p<0.05). In 135dGA CMs, NH3 alone reduced BrdU uptake as well as T3‐induced p21 (p<0.05). Conclusion: Fetal ovine CMs are sensitive to the effects of NH3 differently depending on maturation state. NH3 inhibition of proliferation suggests that TRs are needed for normal proliferation in the developing myocardium and that p21 may be the main effector. Grant Funding Source: Supported by NIH NHLBI and NICHD

Protective effects of calcium gluconate on osteoarthritis induced by anterior cruciate ligament transection and partial medial meniscectomy in Sprague–Dawley rats

BackgroundThis study aimed to determine whether calcium gluconate exerts protective effects on osteoarthritis (OA) induced by anterior cruciate ligament (ACL) transection and partial medial meniscectomy.MethodsCalcium gluconate was administered by mouth daily for 84 days to male ACL transected and partial medial meniscectomized Sprague–Dawley rats 1 week after operation.ResultsEighty-four days of treatment with 50 mg/kg calcium gluconate led to a lower degree of articular stiffness and cartilage damage compared to the OA control, possibly through inhibition of overexpressed cyclooxygenase (COX)-2 and related chondrocyte apoptosis. Similar favorable effects on stiffness and cartilage were detected in calcium gluconate-administered rats. Additionally, calcium gluconate increased 5-bromo-2′-deoxyuridine (BrdU) uptake based on observation of BrdU-immunoreactive cells on both the femur and tibia articular surface cartilages 84 days after intra-joint treatment with calcium gluconate.ConclusionsTaken together, our results demonstrate that calcium gluconate has a protective effect against OA through inhibition of COX-2 and related chondrocyte apoptosis.

In Vitro and In Vivo Activity of a Novel Locked Nucleic Acid (LNA)-Inhibitor-miR-221 against Multiple Myeloma Cells

Background & AimThe miR-221/222 cluster is upregulated in malignant plasma cells from multiple myeloma (MM) patients harboring the t(4;14) translocation. We previously reported that silencing of miR-221/222 by an antisense oligonucleotide induces anti-MM activity and upregulates canonical miR-221/222 targets. The in vivo anti-tumor activity occurred when miR-221/222 inhibitors were delivered directly into MM xenografts. The aim of the present study was to evaluate the anti-MM activity of a novel phosphorothioate modified backbone 13-mer locked nucleic acid (LNA)-Inhibitor-miR-221 (LNA-i-miR-221) specifically designed for systemic delivery.Methods In vitro anti-MM activity of LNA-i-miR-221 was evaluated by cell proliferation and BrdU uptake assays. In vivo studies were performed with non-obese diabetic/severe combined immunodeficient (NOD.SCID) mice bearing t(4;14) MM xenografts, which were intraperitoneally or intravenously treated with naked LNA-i-miR-221. RNA extracts from retrieved tumors were analyzed for miR-221 levels and modulation of canonical targets expression. H&E staining and immunohistochemistry were performed on retrieved tumors and mouse vital organs.Results In vitro, LNA-i-miR-221 exerted strong antagonistic activity against miR-221 and induced upregulation of the endogenous target p27Kip1. It had a marked anti-proliferative effect on t(4;14)-translocated MM cells but not on MM cells not carrying the translocation and not overexpressing miR-221. In vivo, systemic treatment with LNA-i-miR-221 triggered significant anti-tumor activity against t(4;14) MM xenografts; it also induced miR-221 downregulation, upregulated p27Kip1 and reduced Ki-67. No behavioral changes or organ-related toxicity were observed in mice as a consequence of treatments.ConclusionsLNA-i-miR-221 is a highly stable, effective agent against t(4;14) MM cells, and is suitable for systemic use. These data provide the rationale for the clinical development of LNA-i-miR-221 for the treatment of MM.

Possible Involvement of Opa-Interacting Protein 5 in Adipose Proliferation and Obesity

Obesity is an epidemic matter increasing risk for cardiovascular diseases and metabolic disorders such as type 2 diabetes. We recently examined the association between visceral fat adiposity and gene expression profile of peripheral blood cells in human subjects. In a series of studies, Opa (Neisseria gonorrhoeae opacity-associated)-interacting protein 5 (OIP5) was nominated as a molecule of unknown function in adipocytes and thus the present study was performed to investigate the role of OIP5 in obesity. Adenovirus overexpressing Oip5 (Ad-Oip5) was generated and infected to 3T3-L1 cells stably expressing Coxsackie-Adenovirus Receptor (CAR-3T3-L1) and to mouse subcutaneous fat. For a knockdown experiment, siRNA against Oip5 (Oip5-siRNA) was introduced into 3T3-L1 cells. Proliferation of adipose cells was measured by BrdU uptake, EdU-staining, and cell count. Significant increase of Oip5 mRNA level was observed in obese white adipose tissues and such increase was detected in both mature adipocytes fraction and stromal vascular cell fraction. Ad-Oip5-infected CAR-3T3-L1 preadipocytes and adipocytes proliferated rapidly, while a significant reduction of proliferation was observed in Oip5-siRNA-introduced 3T3-L1 preadipocytes. Fat weight and number of adipocytes were significantly increased in Ad-Oip5-administered fat tissues. Oip5 promotes proliferation of pre- and mature-adipocytes and contributes adipose hyperplasia. Increase of Oip5 may associate with development of obesity.

A novel method to follow meiotic progression in Arabidopsis using confocal microscopy and 5-ethynyl-2'-deoxyuridine labeling.

BackgroundMeiosis progression in the more recent past has been investigated using 5-bromo-2′-deoxyuridine (BrdU) uptake by S-phase meiocytes undergoing DNA replication. BrdU uptake is detected by reaction with BrdU antibody followed by epifluorescent microscopy examination of chromosome spreads and/or squashes. We here report using confocal microscopic examination of intact meiocytes in conjunction with the new thymidine analog 5-ethynyl-2′-deoxyuridine (EdU). The simplicity of the EdU detection coupled with confocal examination of anthers provides a more exact temporal description of meiotic prophase I progression in Arabidopsis and opens up the possibility of examining the coordination of microsporocyte development with the other tissues of the anther.ResultsUsing our time course protocol, we have determined the duration of wild type Arabidopsis leptotene to be 5 h, zygotene -6 h, pachytene -10 h and a diplotene duration of approximately 1 h. We estimate G2 duration to be approximately 7 h based on the timing of the initial appearance of EdU signal in early leptotene meiocytes. In addition we have found that DNA replication in meiocytes is not done synchronously with the associated tapetal layer of cells. The EdU labeling suggests that S-phase replication of meiocyte DNA precedes the duplication of tapetal cell DNA.ConclusionsThe increased number of meiotic staging criteria that can be assessed in our confocal analysis, as compared to chromosome spreading or squashing, makes the identification of even the early and late portions of the prophase I substages attainable. This enhanced staging coupled with the ability to easily generate large data sets at hourly time points makes it possible to more exactly determine substage duration and to detect modest temporal abnormalities involving meiocyte entrance into and/or exit from leptotene, zygotene and pachytene. Confocal analysis also makes it possible to study the relationships between different cell types within the flower bud as meiosis proceeds.Electronic supplementary materialThe online version of this article (doi:10.1186/1746-4811-10-33) contains supplementary material, which is available to authorized users.

Bone Marrow Stromal Cell-Derived Exosomes Facilitate Multiple Myeloma Cell Survival Through Inhibition Of The JNK Pathway

Interplay between bone marrow stromal cells (BMSCs) and multiple myeloma (MM) cells plays a crucial role in MM pathogenesis by secreting growth factors, cytokines, and other functional components. Exosomes are 30-100nm diameter membranous vesicles constitutively released by several cell types including reticulocytes, cytotoxic T lymphocytes, B lymphocytes, epithelial and endothelial cells. Exosomes mediate local cell-cell communication by transferring mRNAs, miRNAs and proteins. Due to their ability to transfer functional components, exosomes play multiple roles by stimulating target cells, transferring membrane receptors, delivering proteins, and inducing epigenetic changes in recipient cells. Although the promotion of MM growth and survival induced by BMSCs has been studied, the role of BMSC-derived exosomes in this action remains unclear. Here, we investigated the effect and mechanisms of BMSC-derived exosomes on the proliferation and survival of MM cells using the murine 5T33MM model. This model mimics the human disease closely and of this model two lines exist: the 5T33MMvv model which is propagated in vivo and the 5T33MMvt line which is derived from 5T33MMvv cells but which can grow stroma-independently.Exosomes were isolated from conditioned medium using the ExoQuick-TC Exosome Precipitation Solution (System Biosciences) after culture of primary BMSCs obtained from naïve C57BL/KaLwRij mice or 5T33MM diseased mice. The size of exosomes derived from naïve BMSCs, 5T33 BMSCs and 5T33MMvt cells were confirmed using a NanoSight LM10. Several exosomal markers such as CD63, Flotillin-1, heat shock protein 90 (HSP90), and HSP70 were detected using Western blot. We co-cultured the BMSCs or MM cells with fluorescent dye-labeled exosomes to examine whether exosomes could be transferred into cells. The results showed that both naïve and 5T33 BMSC-derived exosomes could fuse with 5T33MMvt cells and that the uptake of 5T33MMvt cell-derived exosomes by BMSCs was also observed. As several cytokines were found to be present in BMSC- and MMvt cell-derived exosomes, this suggests that BMSCs and MM cells could exchange cytokines with each other through exosomes secretion and uptake. Furthermore, the cytokine composition of 5T33BMSC-derived exosomes compared to naïve BMSC-derived exosomes was different.We next performed luminescent cell viability assays, BrdU cell proliferation assays and 7-AAD/annexin-V stainings to examine the effects of BMSC-derived exosomes on MM cell viability, proliferation and survival, respectively. Both naïve and 5T33 BMSC-derived exosomes increased 5T33MMvt and MMvv cell viability in a dose- and time-dependently manner. BrdU uptake in 5T33MMvt and MMvv cells was also increased after treatment with BMSC-derived exosomes. Significantly reduced apoptosis of 5T33 MMvt and MMvv cells was observed when they were treated with BMSC-derived exosomes as judged by 7-AAD/annexin-V staining. 5T33MMvt and MMvv cells were treated with different amounts of BMSC-derived exosomes and apoptosis-related proteins Bcl-2, Bax, and caspase-3 were determined using western blot. Bcl-2 was increased slightly and activated (cleaved) caspase-3 was reduced after co-culture with exosomes, coinciding with the results of 7-AAD/annexin-V staining. To elucidate the mechanisms responsible for exosome-induced MM cell survival, we examined the activation of several proteins involved. Reduced phosphorylation of p53, p38MAPK and JNK were detected when 5T33MMvt were treated with naïve-BMSC-derived exosomes for 24h, whereas phosphorylated Erk1/2, Akt, and IGF1Rβ were not changed. Surprisingly, activation of p53 and p38MAPK were not changed after the treatment with 5T33 BMSC-derived exosomes. 5T33 BMSC-derived exosomes further decreased the activation of JNK, Bim expression and phosphorylated Bim compared to naïve BMSC-derived exosomes. As Bim is a pro-apoptosis protein, mainly regulated by the JNK pathway; promotion of MM cell survival likely results from the inhibition of the JNK pathway by BMSC-derived exosomes.In summary, our results demonstrate a positive role for BMSC-derived exosomes in induction of MM cell proliferation and survival. BMSC-derived exosomes could inhibit the JNK pathway, thereby reducing caspase-3 activation and protecting MM cells from apoptosis. Disclosures:No relevant conflicts of interest to declare.

The Jumanjii Histone Demethylase KDM2B controls EZH2 Expression In Myelodysplastic Syndromes (MDS) Via Mir Let-7b (let7b), a Pathway That Is Bypassed By The Histone Methylation Inhibitor DZNep

Background and HypothesisDNA and histone methylation as well as microRNA (miR) expression are dysregulated in MDS. The histone demethylase KDM2B suppresses let7b, which post-transcriptionally controls EZH2, a histone-lysine N-methyl transferase that methylates lysine 27 on histone 3. We hypothesized that epigenetic dysregulation in MDS involves the KDM2B/ let7b/EZH2 axis (Figure). DZNep (cyclopentanyl analog of 3-deazaadenosine) is a novel compound, which results in loss of lysine27 methylation, re-expression of epigenetically silenced genes, and decreased cell proliferation.We propose that DZNep will bypass cellular dysregulation via the KDM2B/let7b/EZH2 axis and thereby enhance therapeutic efficacy when combined with azanucleosides. MethodsCD34+ MDS marrow cells were obtained by density separation and enriched by magnetic-activated cell sorting (MACS; Milteny Biotec, Auburn, CA); purity 95% to 98% by flow cytometry. For NanoString nCounter miR array total cell derived RNA or synthetic miR pools (IDT; 30 pmol per oligonucleotide) were used as input. Nano-string data were validated by real-time polymerase chain reaction (PCR). RNA was extracted using the RNeasy Mini kit (QIAGEN, Valencia, CA). Primers for let7b, U6, KDM2b and EZH2 were synthesized by Applied Biosystems. Total RNA was reverse transcribed with Taqman MiR Reverse Transcriptase kit (Applied Biosystems, Carlsbad, CA). Potential functional roles of KDM2B, let7b and EZH2 were determined in myeloid cell lines MDS-L, PL-21, KG1a,OCI-AML3 and ML1. Conditional knock-down of KDM2B was achieved with 2 short hairpins (open-biosystems). Expression of KDM2B was forced using pCMV-Myc-KDM2B (gift of Dr. Tzatsos) in cell lines with low/absent constitutive levels of KDM2B and high levels of let7b. miRZip plasmids (miRZip anti-sense) were used to knock-down let7b; pre-BAB-let7b (origene) served to overexpress let7b. Gene expression, protein levels and cell proliferation were analyzed by RQ-PCR, western blot and BrdU uptake. To determine the impact of modified let7b and KDM2B expression on treatment responses, MDS-L cells (high constitutive levels of EZH2) were treated with DZNep and methyltransferase inhibitor, 5-azacytidine, and subjected to the same analyses. ResultsLet7b levels were significantly higher in primary CD34+ MDS marrow cells (n=44) than in healthy controls (p= 0.0304), while KDM2B and EZH2 expression was reduced (n=21, p<0.0126, p<0.0001, respectively). Overexpression of let-7b in cells with low constitutive levels (PL21, ML1 and KG1a) reduced EZH2 and KDM2B protein levels, and decreased S-phase while increasing G0/G1 cells (p=0.0005); this was associated with a decrease of H3K27me3 and cyclin D1. Silencing of KDM2B in cells with high (MDS-L) and low (KG1a) constitutive levels of let-7b resulted in increased levels of let7b. This increase in let-7b was associated with reduced levels of EZH2 protein and H3K27me3. These findings were accompanied by reduced proliferation (fewer cells in S-phase, more cells in G0/G1). Addition of DZNep to the myeloid cell lines under study resulted in a decrease of let-7b expression, increased levels of EZH2, but reduced cell proliferation. When DZNep (1 uM/ml) was combined with 5-azacytidine (AZA, 5 uM/ml), levels of EZH2 increased in parallel with reduction of H3K27me3 and cell proliferation. KG1a cells overexpressing let-7b (to mimic conditions in primary MDS cells) showed further increase of EZH2 in response to DZNep and AZA. Summary and ConclusionThe KDM2B/ let7b/EZH2 axis is involved in epigenetic regulation in MDS. KDM2B, via let-7b/EZH2, promotes transcriptional repression in myeloid cell lines and primary MDS cells; KDM2B controls epigenetic modifications and cell proliferation. DZNep bypasses the “inhibitory” KDM2B/let7b/EZH2 axis by preventing H3K27 methylation and reducing cell proliferation, independent of EZH2 expression. Thus, the use of DZNep would add histone demethylation to the effect of azanucleosides on DNA methylation, thereby enhancing the therapeutic success in MDS. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Systems biology analysis of Drosophila in vivo screen data elucidates core networks for DNA damage repair in SCA1

DNA damage repair is implicated in neurodegenerative diseases; however, the relative contributions of various DNA repair systems to the pathology of these diseases have not been investigated systematically. In this study, we performed a systematic in vivo screen of all available Drosophila melanogaster homolog DNA repair genes, and we tested the effect of their overexpression on lifespan and developmental viability in Spinocerebellar Ataxia Type 1 (SCA1) Drosophila models expressing human mutant Ataxin-1 (Atxn1). We identified genes previously unknown to be involved in CAG-/polyQ-related pathogenesis that function in multiple DNA damage repair systems. Beyond the significance of each repair system, systems biology analyses unraveled the core networks connecting positive genes in the gene screen that could contribute to SCA1 pathology. In particular, RpA1, which had the largest effect on lifespan in the SCA1 fly model, was located at the hub position linked to such core repair systems, including homologous recombination (HR). We revealed that Atxn1 actually interacted with RpA1 and its essential partners BRCA1/2. Furthermore, mutant but not normal Atxn1 impaired the dynamics of RpA1 in the nucleus after DNA damage. Uptake of BrdU by Purkinje cells was observed in mutant Atxn1 knockin mice, suggesting their abnormal entry to the S-phase. In addition, chemical and genetic inhibitions of Chk1 elongated lifespan and recovered eye degeneration. Collectively, we elucidated core networks for DNA damage repair in SCA1 that might include the aberrant usage of HR.

Differential expression and regulatory roles of FABP5 and FABP7 in oligodendrocyte lineage cells

Fatty-acid-binding proteins (FABPs) are key intracellular molecules involved in the uptake, transportation and storage of fatty acids and in the mediation of signal transduction and gene transcription. However, little is known regarding their expression and function in the oligodendrocyte lineage. We evaluate the in vivo and in vitro expression of FABP5 and FABP7 in oligodendrocyte lineage cells in the cortex and corpus callosum of adult mice, mixed cortical culture and oligosphere culture by immunofluorescent counter-staining with major oligodendrocyte lineage markers. In all settings, FABP7 expression was detected in NG2(+)/PDGFRα(+) oligodendrocyte progenitor cells (OPCs) that did not express FABP5. FABP5 was detected in mature CC1(+)/MBP(+) oligodendrocytes that did not express FABP7. Analysis of cultured OPCs showed a significant decrease in the population of FABP7-knockout (KO) OPCs and their BrdU uptake compared with wild-type (WT) OPCs. Upon incubation of OPCs in oligodendrocyte differentiation medium, a significantly lower percentage of FABP7-KO OPCs differentiated into O4(+) oligodendrocytes. The percentage of mature MBP(+) oligodendrocytes relative to whole O4(+)/MBP(+) oligodendrocytes was significantly lower in FABP7-KO and FABP5-KO than in WT cell populations. The percentage of terminally mature oligodendrocytes with membrane sheet morphology was significantly lower in FABP5-KO compared with WT cell populations. Thus, FABP7 and FABP5 are differentially expressed in oligodendrocyte lineage cells and regulate their proliferation and/or differentiation. Our findings suggest the involvement of FABP7 and FABP5 in the pathophysiology of demyelinating disorders, neuropsychiatric disorder and glioma, conditions in which OPCs/oligodendrocytes play central roles.

Excessive MET signaling causes acquired resistance and addiction to MET inhibitors in the MKN45 gastric cancer cell line

The clinical efficacy of MET tyrosine kinase inhibitors (MET-TKIs) is hindered by the emergence of acquired resistance, presenting an obstacle to drug discovery. To clarify the mechanisms underlying acquired resistance to MET-TKIs, we established resistance models by continuous exposure of the MET-amplified gastric cancer cell line MKN45 to MET-TKIs, PHA665752 (MKN45-PR) and GSK1363089 (MKN45-GR). Baseline expression and phosphorylation of MET were elevated in MKN45-PR and MKN45-GR compared to MKN45 cells, and higher concentrations of MET-TKIs were required to inhibit MET phosphorylation compared to parental cells. Alterations in MET previously associated with resistance to MET-TKIs were observed in resistant cells, including elevated MET copy number, observed in both resistant lines compared to MKN45 cells, and the Y1230H mutation, detected in MKN45-PR cells. Notably, the growth of resistant lines was lower in the absence of MET-TKIs, suggesting "addiction" to inhibitors. While MKN45-PR cells exhibited a higher S-phase fraction in the absence of PHA665752, bromodeoxyuridine (BrdU) uptake was identical. Baseline phosphorylation of ATR, Chk1 and p53 and p21(waf1/Cip1) expression was higher in MKN45-PR compared to MKN45 cells, and levels were reduced to those observed in untreated MKN45 cells following PHA665752 treatment. Furthermore, targeted knockdown of MET enhanced the growth of MKN45-PR cells. These findings suggest that alterations in MET leading to acquired MET-TKI resistance, may cause excessive MET signaling, subsequent replication stress and DNA damage response, and intra-S-phase arrest in the absence of MET-TKIs. Thus, partial MET inhibition is necessary for resistant cells to proliferate, a phenomenon we refer to as MET-TKI "addiction".

Mesenchymal stromal cell turnover in the normal adult lung revisited

We have employed a simple and robust noninvasive method of continuous in vivo long-term bromodeoxyuridine (BrdU) labeling to analyze lung mesenchymal stromal cell turnover in adult mice in the steady state. Mathematical modeling of BrdU uptake in flow cytometrically sorted CD45(neg)CD31(neg)Sca-1(pos) lung cells following long-term feeding of BrdU to mice in their drinking water reveals that lung mesenchymal stromal cells cycle continuously throughout life. Analysis of BrdU incorporation during long-term feeding and during chasing (delabeling) following replacement of BrdU-water with normal water shows that the CD45(neg)CD31(neg)Sca-1(pos) lung mesenchymal stromal cell compartment turns over at a rate of ∼2.26% per day with a time to half-cycled of 44 days, an estimated cell proliferation rate of 0.004/day, and a cell death rate of 0.018/day.

Attenuation of nucleoside and anti-cancer nucleoside analog drug uptake in prostate cancer cells by Cimicifuga racemosa extract BNO-1055

This study aimed to investigate the mechanisms underlying the anti-proliferative effects of the ethanolic Cimicifuga racemosa extract BNO-1055 on prostate cells and evaluate its therapeutic potential. BNO-1055 dose-dependently attenuated cellular uptake and incorporation of thymidine and BrdU and significantly inhibited cell growth after long-time exposure. Similar results were obtained using saponin-enriched sub-fractions of BNO-1055. These inhibitory effects of BNO-1055 could be mimicked using pharmacological inhibitors and isoform-specific siRNAs targeting the equilibrative nucleoside transporters ENT1 and ENT2. Moreover, BNO-1055 attenuated the uptake of clinically relevant nucleoside analogs, e.g. the anti-cancer drugs gemcitabine and fludarabine. Consistent with inhibition of the salvage nucleoside uptake pathway BNO-1055 potentiated the cytotoxicity of the de novo nucleotide synthesis inhibitor 5-FU without significantly altering its uptake. Collectively, these data show for the first time that the anti-proliferative effects of BNO-1055 result from hindered nucleoside uptake due to impaired ENT activity and demonstrate the potential therapeutic use of BNO-1055 for modulation of nucleoside transport.

Carbamazepine promotes liver regeneration and survival in mice

Carbamazepine (CBZ), a widely used anticonvulsant and mood stabilizer, activates multiple proliferative and pro-survival pathways. Here, we hypothesize that CBZ may promote hepatocellular proliferation and ameliorate liver regeneration. C57BL6/J mice were orally administered CBZ or vehicle and underwent a 70% partial hepatectomy (PHx), 85% PHx or treatment with carbon tetrachloride (CCl4). Liver regeneration was determined by liver to body weight ratio, hepatocyte proliferation markers, and activation of intracellular signalling pathways. Two to 5days after the 70% PHx, the liver to body weight ratio was significantly higher in the CBZ-treated mice than in the vehicle-treated mice. CBZ treatment upregulated the number of proliferative hepatocytes following PHx or CCl4 treatment, as assessed by intrahepatic Ki-67 staining, BrdU uptake, and PCNA protein expression. PHx surgery induced the expression of several cyclins and activated Akt/mTOR signalling pathways, all of which were enhanced by CBZ treatment. The administration of the mTOR inhibitor temsirolimus abrogated the hepato-proliferative effect of CBZ. CBZ treatment significantly improved the survival rate of the mice that underwent lethal 85% massive hepatectomy. CBZ demonstrated a novel hepato-proliferative effect through the activation of the mTOR signalling pathway in hepatectomised mice. CBZ has the potential to be a therapeutic option for facilitating efficient liver regeneration in patients subjected to liver surgery.

T-type Ca2 + channels regulate the exit of cardiac myocytes from the cell cycle after birth

T-type Ca2+ channels (TTCCs) are expressed in the fetal heart and then disappear from ventricular myocytes after birth. The hypothesis examined in this study was the α1G TTCCs' influence in myocyte maturation and their rapid withdrawal from the cell cycle after birth. MethodsCardiac myocytes were isolated from neonatal and adult wild type (WT), α1G−/− and α1G over expressing (α1GDT) mice. Bromodeoxyuridine (BrdU) uptake, myocyte nucleation, cell cycle analysis, and T-type Ca2+ currents were measured. ResultsAll myocytes were mono-nucleated at birth and 35% of WT myocytes expressed functional TTCCs. Very few neonatal myocytes had functional TTCCs in α1G−/− hearts. By the end of the first week after birth no WT or α1G−/− had functional TTCCs. During the first week after birth about 25% of WT myocytes were BrdU+ and became bi-nucleated. Significantly fewer α1G−/− myocytes became bi-nucleated and fewer of these myocytes were BrdU+. Neonatal α1G−/− myocytes were also smaller than WT. Adult WT and α1G−/− hearts were similar in size, but α1G−/− myocytes were smaller and a greater % were mono-nucleated. α1G over expressing hearts were smaller than WT but their myocytes were larger. ConclusionsThe studies performed show that loss of functional TTCCs is associated with bi-nucleation and myocyte withdrawal from the cell cycle. Loss of α1G TTCCs slowed the transition from mono- to bi-nucleation and resulted in an adult heart with a greater number of small cardiac myocytes. These results suggest that TTCCs are involved in the regulation of myocyte size and the exit of myocytes from the cell cycle during the first week after birth.

Effect of Small Molecules Modulating Androgen Receptor (SARMs) in Human Prostate Cancer Models

The management of hormone-refractory prostate cancer represents a major challenge in the therapy of this tumor, and identification of novel androgen receptor antagonists is needed to render treatment more effective. We analyzed the activity of two novel androgen receptor antagonists, (S)-11 and (R)-9, in in vitro and in vivo experimental models of hormone-sensitive or castration-resistant prostate cancer (CRPC). In vitro experiments were performed on LNCaP, LNCaP-AR, LNCaP-Rbic and VCaP human prostate cancer cells. Cytotoxic activity was assessed by SRB and BrdU uptake, AR transactivation by luciferase reporter assay and PSA levels by Real Time RT-PCR and ELISA assays. Cell cycle progression-related markers were evaluated by western blot. In vivo experiments were performed on SCID mice xenografted with cells with different sensitivity to hormonal treatment. In hormone-sensitive LNCaP and LNCaP-AR cells, the latter expressing high androgen receptor levels, (R)-9 and (S)-11 exhibited a higher cytotoxic effect compared to that of the reference compound ((R)-bicalutamide), also in the presence of the synthetic androgen R1881. Furthermore, the cytotoxic effect produced by (R)-9 was higher than that of (S)-11 in the two hormone-resistant LNCaP-AR and VCaP cells. A significant reduction in PSA levels was observed after exposure to both molecules. Moreover, (S)-11 and (R)-9 inhibited DNA synthesis by blocking the androgen-induced increase in cyclin D1 protein levels. In vivo studies on the toxicological profile of (R)-9 did not reveal the presence of adverse events. Furthermore, (R)-9 inhibited tumor growth in various in vivo models, especially LNCaP-Rbic xenografts, representative of recurrent disease. Our in vitro results highlight the antitumor activity of the two novel molecules (R)-9 and (S)-11, making them a potentially attractive option for the treatment of CRPC.