Reduction In Total Cell Number Research Articles

Abstract 1013: Mechanistic role of nelfinavir as drug-repurposing strategy against high-grade serous ovarian cancer

Abstract Platinum-based therapy following tumor-debulking surgery has been the backbone of treatment for high-grade serous ovarian cancer (HGSOC) since the 1970s; however, high recurrence of platinum-resistant disease necessitates the development of improved alternative therapies. Repurposing market-available drugs as cancer therapeutics carries the prospect of reducing the timeframe and cost of drug development, posing potential benefits to drug-resistant as well as financially underprivileged patients. Nelfinavir (NFV), an orally available anti-HIV drug, has shown promising effects against diverse cancers as demonstrated through a myriad of pre-clinical studies and clinical trials; however, its remedial benefits against HGSOC are still unclear. In this study, we explored the therapeutic efficacy of NFV on HGSOC cells generated from patients when platinum sensitive or resistant. Acute drug toxicity was assessed by total cell count, percent viability, and the level of hypo-diploid DNA content following 72 h of treatment with NFV. Living cells that tolerated 72 h of NFV treatment were subjected to further drug-free re-incubation for 14-21 days, to assess the residual anti-clonogenic potential of the drug. NFV triggered a dose-depended reduction of total cell number and viability, with a parallel increased hypo-diploid DNA content in both platinum-sensitive and resistant HGSOC cells. A dose-dependent reduction in the number of colonies - originating from cells that evaded acute toxicity - suggested long-term residual toxicity of NFV. Western blot analysis of underlying molecular mechanisms revealed phosphorylation of γH2AX, which suggested NFV-mediated induction of DNA damage. NFV also inhibited proliferation signals by reducing the phosphorylation of Akt and ERK. Moreover, NFV stimulated endoplasmic reticulum (ER) stress in a dose-dependent manner, which was evident from the enhanced expression of GRP78, IRE1α, ATF4, CHOP, and phosphorylated eIF2α. A time-dependent steady increase of ATF4 and CHOP suggested a progressive temporal apoptotic shift during NFV treatment. Finally, the cleavage of caspases-3 and -7 and their downstream effector PARP, in association with increased pro-apoptotic protein BAX, indicated caspase-associated cell-death during NFV-mediated cytotoxicity. In summary, we demonstrated that HGSOC cells of differential platinum sensitivities could be therapeutically targeted by NFV via multipronged mechanistic approaches, encouraging its prospective repurposing benefit against HGSOC. Citation Format: Mahbuba R. Subeha, Alicia A. Goyeneche, Carlos M. Telleria. Mechanistic role of nelfinavir as drug-repurposing strategy against high-grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1013.

The potential advantages of using a poly(HPMA) brush in urinary catheters: effects on biofilm cells and architecture

Infections related to bacterial colonization of medical devices are a growing concern given the socio-economical impacts in healthcare systems. Colonization of a device surface with bacteria usually triggers the development of a biofilm, which is more difficult to eradicate than free-floating or adhered bacteria and can act as a reservoir for subsequent infections. Biofilms often harbor Viable but nonculturable (VBNC) cells that are likely to be more resistant to antibiotic treatment and that can become active in more favorable conditions causing infection. Biofilm formation is dependent on different factors, chiefly the properties of the surface and of the surrounding medium, and the hydrodynamic conditions. In this work, the antifouling performance of a poly[N-(2-hydroxypropyl) methacrylamide] (poly(HPMA)) brush was evaluated in vitro in conditions that mimic a urinary catheter using Escherichia coli as a model organism. The results obtained with the brush were compared to those obtained with two control surfaces, polydimethylsiloxane (PDMS) (the most common material for catheters) and glass. A decrease in initial adhesion and surface coverage was observed on the brush. This antifouling behavior was maintained during biofilm maturation and even in a simulated post-bladder infection period when the reduction in total cell number reached 87 %. Biofilms were shown to adapt their architecture during that period and VBNC cells adsorbed weakly on the brushes and were completely washed away. Taken together, these results suggest that the use of the poly(HPMA) brush in urinary tract devices such as catheters and stents may reduce biofilm formation and possibly render the formed biofilms more susceptible to antibiotic treatment and with reduced infectivity potential.

Phenylbutyrate administration reduces changes in the cerebellar Purkinje cells population in PDC-deficient mice

In humans, pyruvate dehydrogenase complex (PDC) deficiency impairs brain energy metabolism by reducing the availability of the functional acetyl‑CoA pool. This "hypometabolic defect" results in congenital lactic acidosis and abnormalities of brain morphology and function, ranging from mild ataxia to profound psychomotor retardation. Our previous study showed reduction in total cell number and dendritic arbors in the cerebellar Purkinje cells in systemic PDC‑deficient mice. Phenylbutyrate has been shown to increase PDC activity in cultured fibroblasts from PDC‑deficient patients. Hence, we investigated the effects of postnatal (days 2‑35) phenylbutyrate administration on the cerebellar Purkinje cell population in PDC‑deficient female mice. Histological analyses of different regions of cerebellar cortex from the brain‑specific PDC‑deficient saline‑injected mice revealed statistically significant reduction in the Purkinje cell density and increased cell size of the individual Purkinje cell soma compared to control PDC‑normal, saline‑injected group. Administration of phenylbutyrate to control mice did not cause significant changes in the Purkinje cell density and cell size in the studied regions. In contrast, administration of phenylbutyrate variably lessened the ill effects of PDC deficiency on Purkinje cell populations in different areas of the cerebellum. Our results lend further support for the possible use of phenylbutyrate as a potential treatment for PDC deficiency.

Cyclophosphamide Pulse Regimen in the Treatment of Alveolitis in Systemic Sclerosis.

To evaluate, in a pilot study, the efficacy of a short term cyclophosphamide (CYC) pulse regimen on alveolitis in a cohort of patients with systemic sclerosis (SSc).Twenty-three patients with SSc (17 diffuse SSc and 6 limited SSc) were selected in 5 centers in Italy, based on the findings of an abnormal bronchoalveolar lavage (BAL) cell analysis in association with altered pulmonary function tests (PFT) or recent deterioration in flow volume curve (FVC). Patients were also evaluated by skin score (Rodnan), esophageal manometry and barium swallow radiography, and electrocardiography and 2-mode echocardiography. The pre-enrolment pulmonary evaluation and after 6 months of therapy included evaluation of the clinical status, PFT (FVC, FEV1, DLCO), BAL. standard chest radiograph, and chest high resolution computed tomography. All patients received i.v. CYC (1000 mg/m2 of body surface monthly for 6 mo) and oral prednisone (25 mg daily for the first month and subsequently 5 mg daily of maintenance dosage for the remaining 5 mo). A complete blood count and urinalysis were obtained at monthly intervals.After 6 months of therapy the values for FVC did not change significantly. Individually, 8 of 23 patients showed an improvement (> 15% increase) in FVC after 6 months, while FVC in 13 cases remained stable. Only 2 patients had an important decline in FVC after 6 months of therapy (17 and 24% decrease). Improvement in DLCO was noted in 15 of 23 patients after 6 months of therapy. Four patients were stable and 4 patients had a worsened DLCO at the end of the study. After therapy the mean value of BAL fluid recovery did not change. There was a reduction in total cell number although this value did not reach statistical significance. The levels of neutrophils, eosinophils, lymphocytes, and macrophages did not change significantly. Scans for patients with grades 1, 2, and 3 did not differ significantly after 6 months of therapy, and 14 patients were stable. Changes in appearance, in relation to changes in extent of disease, were seen in 8 patients and consisted of an extension of reticular pattern and transformation from grade 1 to 2 (6/8 patients). All patients showed a ground-glass appearance indicating an acute alveolitis. Improvement in ground-glass was noted in 10 of 23 patients after 6 mo therapy. At the end of the study, 8 patients were stable and 5 patients had a diffusion of the ground-glass to other segments. No side effects were experienced during the treatment except for mild nausea in 4 patients; no patients discontinued therapy during the study.CYC pulse regimen seems to stabilize alveolitis in the majority of cases. The association of CYC pulsed modality with prednisone may be useful in SSc patients to control disease evolution in the lung.

Dentofacial characteristics in a child with Meier–Gorlin syndrome: A rare case report

Meier–Gorlin syndrome (MGS) is a rare autosomal recessive disorder characterized by the triad of microtia, absent or small patellae and short stature. The other associated clinical features may include developmental delay, congenital pulmonary emphysema, gastro-esophageal reflux, urogenital anomalies, such as cryptorchidism and feeding problems. The facial characteristics during childhood are typical, comprising of a small mouth with full lips and micrognathia/retrognathia. The condition is rare affecting about one to nine individuals per million. Mutation in the genes of pre-replication complex involved in DNA-replication is detected in the majority of patients. This impedes the cellular proliferation resulting in a reduction of total cell number and thereby retardation of overall growth. This case report describe the typical dentofacial characteristics in a 5 years old child affected with Meier-Gorlin syndrome along with other associated anomalies and a multidisciplinary approach for their management.

151 - Cinnamic Aldehyde as a Potential Therapeutic for Preventing Neointimal Hyperplasia in Diabetes

Background Diabetes Mellitus (DM) accelerates the rate of atherosclerosis development. Balloon angioplasty is a common surgical intervention for the treatment of atherosclerotic disease. However, angioplasty often fails due to restenosis secondary to neointimal hyperplasia. Chronic inflammation and redox dysfunction in DM leads to higher restenosis rates at earlier time points. Neointimal hyperplasia is the result of vascular smooth muscle cells (VSMC) and adventitial fibroblasts proliferating and migrating into the intima, in a process driven by reactive species. Activation of the Nrf2-KEAP1 pathway, a regulator of the antioxidant response, inhibits neointimal hyperplasia in animal models of vascular disease. Therefore we hypothesize that a Nrf2 activator, cinnamic aldehyde (CA), will inhibit VSMC migration and proliferation in vitro and in vivo in a rat model of DM thus preventing neointimal hyperplasia. Methods VSMC were isolated from the aorta of male Zucker Diabetic Fatty (ZDF) rats. Cell viability was measured by flow cytometry and the colorimetric MTT assay. Migration was analyzed using the scratch assay. Nrf2 pathway activation was assessed by western blot and confocal microscopy. The balloon carotid injury model was performed in male ZDF rats after onset of diabetes. CA (100 µM) in Pluronic F127 (100 µl) or vehicle alone was applied to the periadventitial surface of the carotid after balloon injury. Arteries were harvested 3 days after surgery for BrdU incorporation, and Nrf2 localization analysis. Neointimal hyperplasia was assessed by morphometric analysis of H&E-stained carotid cross-sections 2 weeks after injury. Results CA inhibited VSMC proliferation by MTT (EC50 = 230 ± 19 µM). By flow cytometry, CA (100 µM) caused a 30% reduction in total cell number (P Conclusion CA effectively inhibited neointimal hyperplasia in a rat model of DM. Localized Nrf2 activation may provide a new therapeutic target for inhibiting restenosis and improving surgery outcomes.

Abstract 3516: EZH2 overexpression in myeloma patients shortens survival and in-vitro data supports a potential new targeted treatment strategy

Abstract Enhancer of zeste homolog 2 (EZH2) induces methylation at histone 3 lysine 27 (H3K27), thought to have a repressive effect on target gene expression. Activating mutations in EZH2 have been shown to drive lymphomas and overexpression linked to poor prognosis in solid tumors. In normal plasma cell development EZH2 expression is downregulated on exit from the germinal centre but in myeloma (MM) expression has been shown to increase with disease progression. Recent work suggests high expression of the H3K36 methyltransferase MMSET sensitizes cells to EZH2 inhibition. We are seeking to further investigate the role of EZH2 in MM. Data were analyzed from newly diagnosed MM patients in several large clinical trials (Whole-exome sequencing, n = 463 UK Myeloma XI, Affymetrix gene expression array (GEP), n = 259 UK Myeloma IX and n = 1213 UAMS Total Therapy Protocols). Across the datasets, high expression of EZH2 mRNA shortened patient survival (e.g. UK IX median OS, 29.9 months vs 45.1 months, Log-rank p = 0.005), remaining significant on multivariate analyses and across GEP defined molecular subgroups. Of note, the proliferative subgroup had a higher mean expression of EZH2 (9.56 vs 8.48, t-test p = 5.953e-42) as did high risk patients defined by GEP 70 score (9.61 vs 8.46, t-test p = 1.993e-40) suggesting a link to a more aggressive disease phenotype. There were no mutations in EZH2. This contrasts with frequent mutations seen in malignancies at earlier stages of B cell development e.g. DLBCL. Of note, 3% of MM patients had a potentially inactivating mutation or deletion in the gene encoding the H3K27 demethylase, KDM6A and significantly shorter OS than those without (% alive 2 yr: Mutated 51% (CI 30-85) vs 80% (CI 77-84), Log-rank p = 0.0498). Such mutations may increase H3K27 methylation, potentially sensitizing patients to EZH2 inhibition. To study the biological implications of our results we tested the effect of inhibition of EZH2 (EZH2i), using a small molecule EPZ005687, in a panel of MM cell lines representing the epi/genetic diversity of MM. We saw a reduction in total cell number and% viability at 6 days of EZH2i (2-4uM) in 5/8 cell lines with evidence of dose dependent induction of apoptosis. All cell lines (except LP1) had similar levels of EZH2 protein at baseline and the responding cell lines had no unifying epi/genetic anomalies, suggesting response is not specific to either MMSET high or KDM6A mut/del cell lines. 6 days of EZH2i reduced H3K27me2/me3 on Western blotting in all cell lines regardless of viability response, demonstrating the epigenetic activity of the inhibitor. Further evaluation by Chip-seq and GEP is ongoing to identify potential differences, e.g. in focal methylation marks, that may account for differing viability responses. Our results highlight the prognostic significance of EZH2 in MM and demonstrate excellent in-vitro inhibitor activity suggesting EZH2 as a possible future target for treatment. Citation Format: Charlotte Pawlyn, Martin F. Kaiser, Caleb K. Stein, Christopher P. Wardell, Veronica Macleod, Rick Edmondson, Bart Barlogie, Brian Walker, Gareth J. Morgan, Faith E. Davies. EZH2 overexpression in myeloma patients shortens survival and in-vitro data supports a potential new targeted treatment strategy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3516. doi:10.1158/1538-7445.AM2015-3516

Fate of toxic cyanobacterial genera from natural bloom events during ozonation

Intense accumulation of toxic cyanobacteria cells inside plants, unsuccessful removal of cells and consequent breakthrough of cells and toxins into treated water have been increasingly documented. Removal or destabilisation of cells in the pre-treatment stage using pre-ozonation could be an efficient practice as ozonation has been proven to be effective for the removal of cells and toxins. However, several unknowns including the ozone demand, the potential release of cell-bound toxins and organic matter and their impact on treatment train needs to be addressed. The general objective of this work was to study the impact of direct ozonation on different potentially toxic cyanobacteria genera from natural blooms. Water samples from five cyanobacterial bloom events in Lake Champlain (Canada) were ozonated using 2–5 mg/L O3 for a contact time of maximum 10 min. Cyanobacterial taxonomic enumeration, cyanotoxins, organic matter and post-chlorination disinfection by-product formation potential analyses were conducted on all samples. Anabaena, Aphanizomenon, Microcystis and Pseudanabaena were detected in bloom water samples. Total cell numbers varied between 197,000 and 1,282,000 cells/mL prior to ozonation. Direct ozonation lysed (reduction in total cell numbers) 41%–80% of cells and reduced released toxins to below detection limits. Microcystis was the genus the least affected by ozonation. However, DOC releases of 0.6–3.5 mg/L were observed leading to maximum 86.92 μg/L and 61.56 μg/L additional total THMs (four trihalomethanes) and HAA6 (six haloacetic acids) formation, respectively. The results of this study demonstrate that vigilant application of pre-ozonation under certain treatment conditions would help to avoid extreme toxic cells accumulation within water treatment plants.

Cerebral Developmental Abnormalities in a Mouse with Systemic Pyruvate Dehydrogenase Deficiency

Pyruvate dehydrogenase (PDH) complex (PDC) deficiency is an inborn error of pyruvate metabolism causing a variety of neurologic manifestations. Systematic analyses of development of affected brain structures and the cellular processes responsible for their impairment have not been performed due to the lack of an animal model for PDC deficiency. METHODS: In the present study we investigated a murine model of systemic PDC deficiency by interrupting the X-linked Pdha1 gene encoding the α subunit of PDH to study its role on brain development and behavioral studies. RESULTS: Male embryos died prenatally but heterozygous females were born. PDC activity was reduced in the brain and other tissues in female progeny compared to age-matched control females. Immunohistochemical analysis of several brain regions showed that approximately 40% of cells were PDH−. The oxidation of glucose to CO2 and incorporation of glucose-carbon into fatty acids were reduced in brain slices from 15 day-old PDC-deficient females. Histological analyses showed alterations in several structures in white and gray matters in 35 day-old PDC-deficient females. Reduction in total cell number and reduced dendritic arbors in Purkinje neurons were observed in PDC-deficient females. Furthermore, cell proliferation, migration and differentiation into neurons by newly generated cells were reduced in the affected females during pre- and postnatal periods. PDC-deficient mice had normal locomotor activity in a novel environment but displayed decreased startle responses to loud noises and there was evidence of abnormal pre-pulse inhibition of the startle reflex. CONCLUSIONS: The results show that a reduction in glucose metabolism resulting in deficit in energy production and fatty acid biosynthesis impairs cellular differentiation and brain development in PDC-deficient mice.

Changes in Airway Histone Deacetylase2 in Smokers and COPD with Inhaled Corticosteroids: A Randomized Controlled Trial

The expression of HDAC2 is reported as reduced in chronic obstructive pulmonary disease (COPD). We assessed HDAC2 expression within the airways of smokers and subjects with COPD and effects of inhaled corticosteroids (ICS), using immuno-histology to contrast with previous molecular methodology.Endobronchial biopsies (ebb) from current smokers with COPD (COPD-CS; n = 15), ex-smokers with COPD (COPD-ES; n = 17), smokers with normal lung function (NS; n = 16) and normal controls (NC; n = 9) were immunostained for HDAC2. A double-blinded, randomized, placebo-controlled 6 months intervention study assessed effects of ICS on HDAC2 in 34 COPD subjects.There was no difference in epithelial HDAC2 staining in all groups. There was a significant reduction in total cell numbers in the lamina propria (LP) in COPD-CS and NS (p<0.05). LP cellularity correlated inversely with smoking history in COPD-CS (R = −0.8, p<0.003). HDAC2 expression increased markedly in NS (p<0.001); in contrast COPD-CS was associated with suppressed signal (p<0.03), while normal in COPD-ES. ICS did not affect HDAC2 cell staining.Our findings suggest that airway HDAC2 expression is increased in the LP by smoking itself, but is reduced in COPD. Ex-smokers have normalised HDAC2 cell expression, but ICS had no effect. The paper emphasise the pit-falls of relying on molecular data alone to define airway changes.Clinical Trial Registration Information:Name of registryThe Australian New Zealand Clinical Trials Registry (ANZCTR)Registry number ACTRN12612001111864

Ochratoxin A Inhibits Mouse Embryonic Development by Activating a Mitochondrion-Dependent Apoptotic Signaling Pathway

Ochratoxin A (OTA), a mycotoxin found in many foods worldwide, causes nephrotoxicity, hepatotoxicity, and immunotoxicity, both in vitro and in vivo. In the present study, we explored the cytotoxic effects exerted by OTA on the blastocyst stage of mouse embryos, on subsequent embryonic attachment, on outgrowth in vitro, and following in vivo implantation via embryo transfer. Mouse blastocysts were incubated with or without OTA (1, 5, or 10 μM) for 24 h. Cell proliferation and growth were investigated using dual differential staining; apoptosis was measured using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay; and embryo implantation and post-implantation development were assessed by examination of in vitro growth and the outcome of in vivo embryo transfer, respectively. Blastocysts treated with 10 μM OTA displayed a significantly increased level of apoptosis and a reduction in total cell number. Interestingly, we observed no marked difference in implantation success rate between OTA-pretreated and control blastocysts either during in vitro embryonic development (following implantation in a fibronectin-coated culture dish) or after in vivo embryo transfer. However, in vitro treatment with 10 μM OTA was associated with increased resorption of post-implantation embryos by the mouse uterus, and decreased fetal weight upon embryo transfer. Our results collectively indicate that in vitro exposure to OTA triggers apoptosis and retards early post-implantation development after transfer of embryos to host mice. In addition, OTA induces apoptosis-mediated injury of mouse blastocysts, via reactive oxygen species (ROS) generation, and promotes mitochondrion-dependent apoptotic signaling processes that impair subsequent embryonic development.

Alternation of extracellular matrix remodeling and apoptosis by activation of the aryl hydrocarbon receptor pathway in human periodontal ligament cells

It is well known that the aryl hydrocarbon receptor (AhR) is involved in the toxicity of halogenated aromatic hydrocarbons (HAH) and polycyclic aromatic hydrocarbons (PAH). Recent experiments have shown the induction of impaired tooth and hard-tissue formation by AhR pathway activation, however, the effect on periodontal ligament (PDL) tissue remains unclear. Here, we investigated the effects of benzo(a)pyrene (BaP), a member of PAH, on the extracellular matrix (ECM) remodeling-related molecules, collagen type I (COL-I), matrix metalloproteinase-1 (MMP-1), alpha-smooth muscle actin (α-SMA) expression, and apoptosis in two different human periodontal ligament cells (HPDLCs). The transduction of AhR from the cytoplasm to the nucleus and the increase of AhR-responsive genes; that is, cytochrome P450 1A1 (CYP1A1), cytochrome P450 1B1 (CYP1B1), and aryl-hydrocarbon receptor repressor (AhRR), expression was induced by BaP exposure in both HPDLCs. BaP treatment significantly enhanced MMP-1 mRNA expression and MMP-1 protein production, while markedly suppressing COL-I and a-SMA mRNA expression in both HPDLCs. Furthermore, these BaP-treated HPDLCs fell into apoptotic cell death as evidenced by induction in annexin V and caspase-3/7 staining and reduction of total cell number and Bcl-2 mRNA expression. Thus, BaP exposure altered the expression of ECM-related molecules and induced apoptosis in HPDLCs through activation of the AhR pathway. Overactivity of the AhR pathway may induce an inappropriate turnover of PDL tissue via disordered ECM remodeling and apoptosis in PDL cells.

A functional role for the histone demethylase UTX in normal and malignant hematopoietic cells

Ubiquitously transcribed tetratricopeptide repeat, X chromosome (UTX), an H3K27Me2/3 demethylase, has been implicated in development, self-renewal, and differentiation of various organs and embryonic stem cells through chromatin modifications and transcriptional regulation of important developmentally related genes, such as Hox genes. However, the function of UTX in hematopoiesis is not well understood. To study the role of UTX in the mammalian hematopoietic system, we used lentiviral short hairpin RNA constructs to knockdown UTX in the murine hematopoietic progenitor cell line EML, in primary murine bone marrow cells and in leukemic cell lines. We report that Utx is highly expressed in the hematopoietic compartment and that it plays an important role in cell proliferation and homeostasis of hematopoietic cells in vitro. Knockdown of UTX in EML and primary murine bone marrow cells impairs their colony-forming ability. Moreover, knockdown of UTX affects expression of key genes that regulate hematopoietic differentiation such as Mll1, Runx1, and Scl in primary murine bone marrow cells. And we further demonstrate that UTX directly associates with the promoters of the Mll1, Runx1, and Scl genes and modulate their transcription by controlling H3K27me3 marks on respective promoter regions. In addition, UTX depletion severely impaired proliferation of several human leukemia cell lines. Together, these data demonstrate a functional role for UTX in normal and malignant hematopoiesis.

Different Susceptibility to Neurodegeneration of Dorsal and Ventral Hippocampal Dentate Gyrus: A Study with Transgenic Mice Overexpressing GSK3β

Dorsal hippocampal regions are involved in memory and learning processes, while ventral areas are related to emotional and anxiety processes. Hippocampal dependent memory and behaviour alterations do not always come out in neurodegenerative diseases at the same time. In this study we have tested the hypothesis that dorsal and ventral dentate gyrus (DG) regions respond in a different manner to increased glycogen synthase kinase-3β (GSK3β) levels in GSK3β transgenic mice, a genetic model of neurodegeneration. Reactive astrocytosis indicate tissue stress in dorsal DG, while ventral area does not show that marker. These changes occurred with a significant reduction of total cell number and with a significantly higher level of cell death in dorsal area than in ventral one as measured by fractin-positive cells. Biochemistry analysis showed higher levels of phosphorylated GSK3β in those residues that inactivate the enzyme in hippocampal ventral areas compared with dorsal area suggesting that the observed susceptibility is in part due to different GSK3 regulation. Previous studies carried out with this animal model had demonstrated impairment in Morris Water Maze and Object recognition tests point out to dorsal hippocampal atrophy. Here, we show that two tests used to evaluate emotional status, the light–dark box and the novelty suppressed feeding test, suggest that GSK3β mice do not show any anxiety-related disorder. Thus, our results demonstrate that in vivo overexpression of GSK3β results in dorsal but not ventral hippocampal DG neurodegeneration and suggest that both areas do not behave in a similar manner in neurodegenerative processes.

Direct Inhibition of Osteoclast Formation and Activity by the Vitamin E Isomer gamma-Tocotrienol

Vitamin E homologues, specifically tocotrienols, have been shown to have favorable effects on bone. They possess properties that are indicative of anti-resorptive activity, suggesting the potential for vitamin E in preventing bone loss. To investigate the anti-resorptive activity of the various vitamin E homologues, we cultured human osteoclasts from blood-derived CD14+ cells on collagen, dentin, and calcium phosphate substrates, with some samples supplemented with vitamin E homologues in their cell culture medium. These were compared to the clinically used bisphosphonate, pamidronate. Compounds were either added at the start of culture to study effects on osteoclast formation, or at the start of osteoclastic resorption to determine their effects on activity. The alpha- and gamma-tocotrienol isomers inhibited osteoclast formation without consequent reduction in total cell number. Only gamma-tocotrienol inhibited osteoclast activity without toxicity. Gamma-tocotrienol was the most potent inhibitor of both osteoclast formation and activity and requires further investigation into its anti-resorptive effects on bone.

The miR-143/145 Cluster Is a Novel Transcriptional Target of Jagged-1/Notch Signaling in Vascular Smooth Muscle Cells

Activation of Notch signaling by Jagged-1 (Jag-1) in vascular smooth muscle cells (VSMC) promotes a differentiated phenotype characterized by increased expression of contractile proteins. Recent studies show that microRNAs (miR)-143/145 regulates VSMC phenotype. The serum response factor (SRF)/myocardin complex binds to CArG sequences to activate miR-143/145 transcription, but no other regulators are known in VSMC. Using miR arrays, we found miR-143/145 induced following expression of a constitutively active Notch1 intracellular domain (N1ICD). We hypothesized that miR-143/145 is required for Jag-1/Notch-induced VSMC differentiation. Activation of Notch receptors by Jag-1 caused CBF1-dependent up-regulation of miR-143/145, increased differentiation, and decreased proliferation. Conversely, inhibiting basal Notch signaling decreased steady state levels of miR-143/145. Using SRF knockdown, we found that Jag-1/Notch induction of miR-143/145 is SRF independent, although full acquisition of contractile markers requires SRF. Using miR-143/145 promoter reporter constructs we show Jag-1/Notch increases promoter activity, and this is dependent on intact CBF1 consensus sites within the promoter. Chromatin immunoprecipitation (ChIP) assays revealed that N1ICD-containing complexes bind to CBF1 sites in the miR-143/145 promoter. We also identified N1ICD complex binding to CBF1 sites within the endogenous human miR-143/145 promoter. Using miR-143/145-interfering oligonucleotides, we demonstrate that Jag-1/Notch signaling requires induction of both miR-143 and miR-145 to promote the VSMC contractile phenotype. Thus, miR-143/145 is a novel transcriptional target of Jag-1/Notch signaling in VSMC. We propose miR-143/145 as activated independently by Jag-1/Notch and SRF in parallel pathways. Multiple pathways converging on miR-143/145 provides potential for fine-tuning or amplification of VSMC differentiation signals.

Toll-Like Receptor Signaling Inhibits Eosinophilopoiesis.

AbstractAbstract 1558Eosinophils and their progenitors are robustly elevated in the bone marrow in response to diverse stimuli including helminth infection and allergic triggers. In contrast, bacterial infection results in diminution in the number of circulating eosinophils. Markedly reduced peripheral blood eosinophils of sudden onset but prolonged duration is generally a consequence of acute bacterial infection. Further, eosinopenia has been shown to be a sensitive marker of sepsis and correlates with severity of disease in critically ill patients. While the initial eosinopenic response is believed to be secondary to rapid migration of circulating eosinophils to the site of infection, the mechanism for prolonged eosinophil depletion with bacterial infection remains undefined. As Toll-like receptors (TLRs) have been shown to be expressed by early hematopoietic progenitors and by mature eosinophils, we initially investigated TLR expression during eosinophil differentiation. In a culture system we developed to differentiate mature eosinophils from low density bone marrow progenitors, IL-5 stimulation of progenitors resulted in induced expression of seven TLRs, including TLR1 (4-fold), TLR4 (5-fold), TLR6 (2-fold), TLR7 (6-fold), TLR8 (4-fold), TLR9 (3-fold), and TLR13 (8-fold), when compared to expression in the progenitors prior to IL-5-induced differentiation. Notably, a majority of the TLRs (TLR1, TLR4, TLR7, TLR8, TLR9, and TLR13) were induced specifically early in eosinophil differentiation after 4 days of IL-5 stimulation and prior to expression of surface markers, including Siglec-F and CCR3, associated with mature eosinophils. To study the effects of TLR signal transduction on eosinophil development, we exposed bone marrow progenitors to TLR agonists for 18 hours after 4 days of IL-5 stimulation and then subsequently induced further eosinophil differentiation with IL-5 stimulation for another 8 days. We measured effects of TLR signaling on cell number, eosinophil differentiation, cytokine production and protease activity. LPS stimulation resulted in a reduction in total cell numbers by more than 65% with immature eosinophils a predominant cell type. In addition, LPS stimulation of progenitors resulted in significantly increased cytokine and chemokine production, including IL-6 (80-fold) and CCL22 (2-fold), and protease activity (2-fold) after 4–6 days of subsequent IL-5 stimulation compared to IL-5 stimulation alone. Similarly, stimulation of eosinophil progenitors with the TLR1/TLR2 agonist PAM3CSK4 or TLR6/TLR2 agonist PAM2CSK4 for 18 hours resulted in markedly decreased total cell numbers in a dose-dependent manner after 3 days of subsequent IL-5 stimulation. Together, these data suggest that TLR signaling in progenitors results in inhibition of IL-5-stimulated eosinophil development with aberrant cytokine, chemokine and protease production. As eosinophil granules have been shown to have anti-bacterial properties and eosinophilia improves survival rate in an experimental bacterial peritonitis model, delineation of the mechanism of infection-induced eosinopenia may lead to novel adjuvant therapeutics for bacteremic critically ill patients. Disclosures:No relevant conflicts of interest to declare.

Abstract 1405: Radiosensitization of breast cancer cells by Vitamin D3 and Vitamin D analogs

Abstract Introduction: Previous studies in our laboratory have demonstrated that the active form of vitamin D, 1,25-di hydroxy vitamin D3 (1,25 D3), or its analogs such as EB 1089, can confer enhanced sensitivity to ionizing radiation (IR). Sensitization by EB 1089 appeared to be a consequence of the promotion of autophagic cell death. The current work was designed to extend these findings to ZR-75-1 breast tumor cells, also a p53 wild type ER positive cell line, utilizing 1,25- D3 combined with IR. Experimental Procedure : In vitro studies assessed the effects of 100nM 1,25 D3 alone, fractionated IR (5 × 2 Gy) delivered over a period of 3 days, and the combination treatment of 100nM 1,25 D3 72hrs prior to IR. Reults: Radiation alone produced an approximately 30% reduction in total cell number. Pretreatment with 1,25D3 enhanced radiation sensitivity such that an approximately 75% reduction in total cell number was observed. There was no evidence of apoptosis in cells treated with IR alone or pretreated with 1,25 D3 (lack of nuclear fragmentation or chromatin condensation or cleavage of caspase-3). Cell death was succeeded by a prolonged interval of growth arrest with either IR alone or IR preceded by 1,25 D3. Positive β-galactosidase staining, cell flattening and enlargement indicated that residual surviving cells (with IR alone or IR preceded by 1,25 D3) were in a state of senescence. Acridine orange staining and p62 degradation were indicative of autophagy with IR alone; however, autophagy was reduced when IR was preceded by pretreatment with 1,25 D3; there was minimal evidence of autophagy with 1,25 D3 alone. Clearly, autophagy does not appear to be responsible for radiation sensitization in this experimental model. Unexpectedly, the formation of binucleated cells with micronuclei, morphology indicative of mitotic catastrophe, was evident primarily in cells pretreated with 1,25 D3 prior to IR but not in cells treated with IR alone. Conclusion: Pretreatment with vitamin D3 has the capacity to radiosensitize ZR-75-1 breast cancer cells by increasing the rate and extent of cell killing. Residual surviving cells after both radiation treatment alone and vitamin D pre-treatment appear to be in a state of prolonged growth arrest/senescence. Furthermore, in this system autophagy may prove to be cytoprotective instead of cytotoxic and could be involved in regulating senescence. While the initial mode of cell death has yet to be determined, it appears that mitotic catastrophe may play a role in vitamin D3's radiosensitization of ZR-75-1 breast cancer cells. Implications. Taken together with our previous work, these studies support the concept that 1,25 D3 and/or its analogs enhance radiation sensitivity in breast tumor cells through the promotion of multiple cell death pathways. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1405.

Inhibition of Notch pathway enhances photoreceptor commitment from cultured retinal stem cells

Abstract Purpose Consequently to the principle that photoreceptors have to be at a very precise development stage to be successfully transplanted (MacLaren 2006), we are trying to mimic this development stage in vitro using retinal stem cells. The latter were isolated from the newborn mouse retina, derived from the radial glia population, and were previously isolated and characterized in our laboratory. We developed a protocol to commit these cells to the photoreceptor fate, but even if the percentage of cells expressing photoreceptor markers is high (30%), the differentiation process is incomplete so far (Merhi‐Soussi 2006). Methods To ameliorate photoreceptor differentiation, we hypothesized that the Notch pathway may interfere with this process by either promoting glia commitment, or maintaining an undifferentiated state. We are thus using a gamma‐secretase inhibitor (DAPT), which inhibits Notch activation, during the in vitro differentiation process. Results The constant presence of DAPT i) leads to a 233% increase in peripherin/RDS‐positive (photoreceptor marker) cells, compared to controls (no DAPT, n=3, P&lt;0.02) along with a 68% decrease in GFAP‐ positive cells (n=3, P&lt;0.04), ii) modifies the ratio between uni‐/bi‐ (23%) and multi‐ (77%) polar peripherin/RDS‐positive cells to 45% and 55%, respectively, and iii) reduces by 50% the total cell number during the whole differentiation process. Conclusion We are now exploring whether this reduction in total cell number is due to inhibition of cell proliferation or to cell death and whether photoreceptor differentiation is promoted instead of glial induction. Such protocol may help to better mimic photoreceptor development, but this needs to be confirmed by genomic and proteomic profile analyses.

152. INVOLVEMENT OF TRP53 IN SIRT1 FUNCTION DURING EMBRYO DEVELOPMENT

Sirtuin 1 (SIRT1) is an NAD+-dependant deacetylase with significant functions in cell survival and metabolism, including glucose homeostasis and mitochondrial physiology. TRP53 is a universal effector of cellular stress responses and is an important target of SIRT1. Transcriptional activity of TRP53 in the pre-implantation embryo is associated with retarded development, however examination of SIRT1 function and how it relates to TRP53 activity remains to be elucidated. We therefore assessed whether SIRT1 is involved in pre-implantation embryo development and determined whether TRP53 interacts with SIRT1 function. Zygotes were collected from superovulated female mice and cultured to the blastocyst stage in optimised conditions (F1 mouse strain, G1/G2 series sequential media, 5%CO2, 5%O2, 90%N2, group culture) or compromised conditions (C57Bl/6 strain, mHTF static media, 5%CO2 in air, individual culture). Embryo development and blastocyst cell number was assessed following exposure to a SIRT1 inhibitor (0, 1, 10, 100 or 1000µM sirtinol). In subsequent experiments, embryos were cultured in a 2x2 factorial design (±1µM sirtinol and ±30µM pifithrin-α (TRP53 inhibitor)) and embryo development and cell number determined. Sirtinol caused a dose-dependent reduction in total cell number in blastocysts during culture in both optimised and compromised conditions (p&lt;0.05), while the rate of development of zygotes was reduced for embryos in compromised but not optimised conditions (p&lt;0.05). When SIRT1 was inhibited, in the presence or absence of TRP53 inhibition, blastocyst development and cell number for embryos in optimal conditions was unchanged. However, blastocyst development (83% vs 55%, p&lt;0.05), and cell number (39 vs 54 cells, p&lt;0.05) was reduced when SIRT1 was inhibited in compromised embryos, and in the absence of TRP53, development was resolved to control levels. These results show that SIRT1 is important for embryo development, particularly under compromised conditions, and that TRP53 is a likely target for SIRT1 deacetylase activity in the mammalian embryo.