Tritiated Thymidine Incorporation Assay Research Articles

Basigin is necessary for normal decidualization of human uterine stromal cells.

Does basigin (BSG) regulate human endometrial stromal cell (HESC) decidualization in vitro? BSG regulates HESCs proliferation and decidualization. Studies have shown that in the human endometrium, BSG expression is menstrual-cycle dependent and its expression was significantly lower in uterine endometrium during the luteal phase of women experiencing multiple implantation failures after IVF than in women with normal fertility. We utilized a telomerase-immortalized HESCs in an in vitro cell culture model system to investigate whether BSG regulates decidualization of stromal cells. Further, we used microarray analysis to identify changes in the gene expression profile of HESCs treated with BSG small interfering RNA (siRNA). All experiments were repeated at least three times. The effect of BSG knockdown (using siRNA) on HESC proliferation was determined by counting cell number and by tritiated thymidine incorporation assays. The effect of BSG on decidualization of HESCs was determined by RT-qPCR for the decidualization markers insulin-like growth factor-binding protein 1 (IGFBP1) and prolactin (PRL). Immunoblotting was used to determine the effect of BSG siRNA on the expression of MMP-2,3. Microarray analysis was used to identify BSG-regulated genes in HESCs at Day 6 of decidualization. Functional and pathway enrichment analyses were then carried out on the differentially expressed genes (DEGs). The STRING online database was used to analyze protein-protein interaction (PPI) between DEG-encoded proteins, and CytoScape software was used to visualize the interaction. MCODE and CytoHubba were used to construct functional modules and screen hub genes separately. Several BSG-regulated genes identified in the microarray analysis were confirmed by qPCR. Knockdown of BSG expression in cultured stromal cells by siRNA significantly (P < 0.05) inhibited HESC proliferation, disrupted cell decidualization and down-regulated MMP-2 and MMP-3 expression. Microarray analysis identified 721 genes that were down-regulated, and 484 genes up-regulated with P < 0.05 in BSG siRNA treated HESCs. GO term enrichment analysis showed that the DEGs were significantly enriched in cell communication, signaling transduction and regulation, response to stimulus, cell adhesion, anatomical structure morphogenesis, extracellular matrix organization, as well as other functional pathways. KEGG pathway analysis identified upregulated gene enriched in pathways such as the MAPK signaling pathway, colorectal cancer, melanoma and axon guidance. In contrast, downregulated genes were mainly enriched in pathways including ECM-receptor interaction, PI3K-Akt signaling pathway, pathways in cancer, antigen processing, type I diabetes mellitus and focal adhesion. The top 10 hub nodes were identified using 12 methods analyses. The hub genes that showed up in two methods were screened out. Among these genes, upregulated genes included EGFR, HSP90AA1, CCND1, PXN, PRKACB, MGAT4A, EVA1A, LGALS1, STC2, HSPA4; downregulated genes included WNT4/5, FOXO1, CDK1, PIK3R1, IGF1, JAK2, LAMB1, ITGAV, HGF, MXRA8, TMEM132A, UBE2C, QSOX1, ERBB2, GNB4, HSP90B1, LAMB2, LAMC1 and ITGA1. Hub genes and module genes involved in the top three modules of PPI analysis were analyzed through the string database. Analysis showed that hub and module genes were related mainly to the WNT signaling pathway, PI3K-AKT signaling pathway and pathways in cancer. The microarray data set generated in this study has been published online at databank.illinois.edu. Most of the findings were obtained using an in vitro cell culture system that may not necessarily reflect in vivo functions. Our results demonstrate that BSG plays a vital role in decidualization and that downregulation of BSG in the uterine endometrium may be associated with infertility in women. The identified hub genes and pathways increase our understanding of the genetic etiology and molecular mechanisms underlying the regulation of decidualization by BSG. This work was supported by the NIH U54 HD40093 (R.A.N.). The authors have no competing interests to declare.

Effect of surface modified reduced graphene oxide nanoparticles on cerebellar granule neurons

Graphene has been the cornerstone material in various disciplines owing to its captivating properties. But graphene also exerts some toxic effects apart from its extraordinary characteristics. Various surface functionalizations on graphene are developed in order to overcome the toxicity. In this background, pluronics functionalization of reduced graphene oxide was done as an effort to modify its colloidal stability. Effect of pluronics functionalized reduced graphene oxide (rGO-P) on cerebellar granule neurons (CGNs) was examined to infer on its neurotoxic potential. Male Wister rats were exposed to rGO-P and were analyzed for acute toxicity and toxicokinetic studies. Immunotoxicity was performed on rGO-P exposed female rats on the 7th day of gestation by tritiated thymidine incorporation assay. CGNs were also isolated from rat newborns born to female rats dams exposed with rGO-P and examined for any subsequent toxic reactions. The findings suggest that CGNs isolated from rat newborns pups born to particle exposed female rats dams showed normal cell yield and morphology. Findings from toxicokinetic studies using confocal Raman mapping revealed that rGO-P has got cleared off from the blood within 21 days. Overall results validate rGO-P as safe for in vivo systems. Hence rGO-P can be fine-tuned to serve the purpose of neurobiological applications promisingly for neuroregeneration.

Human immune-cell response towards diverse xenogeneic and allogeneic decellularized biomaterials

BackgroundImmunological knowledge on processed biological implants and mesh-prostheses is still mainly based on animal models, lacking information on the species-specific human immune response. We hypothesized that in contrast to human tissue even decellularized xenogenic specimens would lead to significant and tissue source dependent human immune reactions. MethodsSpecimens from processed allogenic and xenogenic pulmonary arteries, pericardium or dermis, were co-cultured with human peripheral blood mononuclear cells (PBMNC). Proliferative responses were measured in tritiated thymidine incorporation assays (n = 10). Stimulation indices (SI), calculated as counts-per-minute of co-cultured PBMNC divided by the cpm of basic cell proliferation, were compared. ResultsCompared to native porcine pulmonary artery tissue decellularization significantly reduced human PBMNC proliferation (mean SI: 48.7 vs. 18.0, p < 0.01), which was still higher compared to the human equivalent (SI: 0.7 vs. 1.7). Also the processed human dermal implant did not elicit immune response (SI: 1.5), whereas the decellularized and cross-linked porcine dermis lead to a significant human cell-proliferation (SI: 8.4, p < 0.01). Interestingly, both the processed human (SI: 15.2) and bovine pericardial patches (SI: 15.1) led to higher immune cell proliferation. ConclusionEven decellularized or cross-linked xenogenic cardiovascular and reconstructive biomaterials elicit increased human immune responses not seen in the majority of allogenic specimens tested.

Piperine blocks interleukin-2-driven cell cycle progression in CTLL-2 T lymphocytes by inhibiting multiple signal transduction pathways

Piperine, a pungent alkaloid found in the fruits of black pepper plants, has diverse physiological effects, including the ability to inhibit immune cell-mediated inflammation. Since the cytokine interleukin-2 (IL-2) is essential for the clonal expansion and differentiation of T lymphocytes, we investigated the effect of piperine on IL-2 signaling in IL-2-dependent mouse CTLL-2 T lymphocytes. Tritiated-thymidine incorporation assays and flow cytometric analysis of Oregon Green 488-stained cells showed that piperine inhibited IL-2-driven T lymphocyte proliferation; however, piperine did not cause T lymphocytes to die or decrease their expression of the high affinity IL-2 receptor, as determined by flow cytometry. Western blot analysis showed that piperine blocked the IL-2-induced phosphorylation of signal transducer and activator of transcription (STAT) 3 and STAT5 without affecting the upstream phosphorylation of Janus kinase (JAK) 1 and JAK3. In addition, piperine inhibited the IL-2-induced phosphorylation of extracellular signal-regulated kinase 1/2 and Akt, which are signaling molecules that regulate cell cycle progression. Piperine also suppressed the expression of cyclin-dependent kinase (Cdk) 1, Cdk4, Cdk6, cyclin B, cyclin D2, and Cdc25c protein phosphatase by IL-2-stimulated T lymphocytes, indicating G0/G1 and G2/M cell cycle arrest. Piperine-mediated inhibition of IL-2 signaling and cell cycle progression in CTLL-2 T lymphocytes suggests that piperine should be further investigated in animal models as a possible natural source treatment for T lymphocyte-mediated transplant rejection and autoimmune disease.

Evaluating Systemic Toxicity in Rabbits after Acute Ocular Exposure to Irritant Chemicals

Acute systemic toxicity via ocular exposure route is not a well understood aspect. Any material/drug/chemical that comes in contact with the eye can evade the first pass metabolism and enter the systemic circulation through the conjunctival blood vessels or via the nasolacrimal route. In this study, the effect of ocular irritant chemicals on the systemic toxicity was assessed in rabbit. Eyes of rabbits were exposed to known ocular irritant (cetyl pyridinium chloride, sodium salicylate, imidazole, acetaminophen, and nicotinamide) for 24 h and scored. After a period of 72 h, blood was collected from the animals for examining the hematological and biochemical parameters. The animals were then sacrificed and the eyes were collected for histopathology and cytokine analysis by ELISA. Splenocyte proliferation was assessed by tritiated thymidine incorporation assay. The liver and brain of the treated animals were retrieved for evaluating oxidative damage. The chemicals showed moderate to severe eye irritation. Inflammation was not evident in the histopathology but proinflammatory markers were significantly high. The splenocyte proliferation capacity was undeterred. And there was minimal oxidative stress in the brain and liver. In conclusion, acute exposure of ocular irritants was incapable of producing a prominent systemic side effect in the current scenario.

Mesenchymal stem cells exert anti-proliferative effect on lipopolysaccharide-stimulated BV2 microglia by reducing tumour necrosis factor-α levels.

BackgroundProgression of neurodegenerative diseases occurs when microglia, upon persistent activation, perpetuate a cycle of damage in the central nervous system. Use of mesenchymal stem cells (MSC) has been suggested as an approach to manage microglia activation based on their immunomodulatory functions. In the present study, we describe the mechanism through which bone marrow-derived MSC modulate the proliferative responses of lipopolysaccharide-stimulated BV2 microglia.MethodsBV2 microglia were cultured with MSC and stimulated with 1 μg/ml lipopolysaccharide. Using an inducible nitric oxide synthase inhibitor, tritiated thymidine (3H-TdR) incorporation assay was performed to determine the role of nitric oxide in the anti-proliferative effect of MSC. We also studied apoptosis and the cell cycle of both cell types using flow cytometry and explored their cytokine profile using protein and cytometric arrays. Moreover, the role of IL-6 and TNF-α in immunomodulation was deduced using specific blocking antibodies and recombinant proteins.ResultsMSC reduces microglia proliferation upon lipopolysaccharide stimulation by 21 to 28% and modulates the levels of nitric oxide, IL-6 and TNF-α. The role of nitric oxide in conferring the anti-proliferative effect of MSC was ruled out. Furthermore, we found that MSC exert their anti-proliferative effect by restoring the percentage of BV2 cells at S and G2/M phase to levels similar to unstimulated cells. MSC undergo a G0/G1 arrest while exerting this effect. We have also identified that MSC-mediated modulation of microglia is independent of IL-6, whilst reduction of TNF-α in co-culture is critical for inhibition of microglia proliferation.ConclusionsOur study demonstrates that MSC inhibit microglia proliferation independent of nitric oxide and IL-6, although reduction of TNF-α is critical for this effect. The inhibition of proliferation is through cell cycle modulation. These findings shed light on the mechanisms of microglial immunomodulation by MSC.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-014-0149-8) contains supplementary material, which is available to authorized users.

Aqueous extract of unfermented honeybush (Cyclopia maculata) attenuates STZ-induced diabetes and β-cell cytotoxicity.

New strategies, which include β-cell protection, are required in the treatment of T2D, as current drugs demonstrate little or no capacity to directly protect the vulnerable β-cell against diabetes-induced cytotoxicity. In this study we investigated the ameliorative effect of pre-treatment with an aqueous extract of unfermented Cyclopia maculata (honeybush) on STZ-induced diabetes and pancreatic β-cell cytotoxicity in Wistar rats after demonstrating a protective effect in vitro in RIN-5F cells. The amelioration of STZ-induced diabetes was seen in the reduction of the area under the curve, determined by the oral glucose tolerance test, as well as fasting glucose levels in extract-treated rats. Pre-treatment with extract also improved serum triglyceride levels and the glucose-to-insulin ratio. Pre-treatment with the extract or the drug, metformin, increased the β-cell area in islets, with a concomitant increase in β-cell proliferation at the higher extract dose (300 mg/kg/d), but not the lower dose (30 mg/kg/d). Subsequently, the in vitro tritiated thymidine incorporation assay showed that the extract was not mitogenic in RIN-5F cells. STZ-induced elevation of plasma nitrite levels was reduced in extract-treated rats, but no changes were observed in their serum catalase, serum glutathione, liver lipid peroxidation and liver nitrotyrosine levels. Pre-treating the rats with extract ameliorated the diabetic effect of STZ in Wistar rats, with evidence of pancreatic β-cells protection, attributed to the presence of high levels of antioxidants such as the xanthones, mangiferin and isomangiferin.

The anti-osteoporotic effect of velvet antler polypeptides from Cervus elaphus Linnaeus in ovariectomized rats

Ethnopharmacological relevanceThe deer velvet antler is well known for its traditional medicinal value, and is widely used in the clinic. It is recorded in the Compendium of Materia Medica that the deer velvet antler replenishes vital essence and strengthens the bone. Aim of the studyThe goal of this study was to investigate the anti-osteoporotic effect of total velvet antler polypeptides from Cervus elaphus Linnaeus (TVAPL) on ovariectomized rats (OVX), and their possible mechanism of the action. Materials and methodsWistar rats were divided into five groups: sham-operated group, OVX group, and OVX rats treated with 20, 40, or 60mk/kg TVAPL for 12 weeks. Calcium and phosphorus levels, bone weight coefficient (BWC), bone mineral density (BMD), and bone mineral content (BMC) were evaluated. The MTT assay was used to measure the activities of interleukin-1 (IL-1) and interleukin-6 (IL-6). In addition, cartilage cells and osteoblast-like cells were exposed to TVAPL, natural velvet antler polypeptides (nVAP), and synthetic velvet antler polypeptides (sVAP), to determine their effects on cell proliferation using the tritiated thymidine incorporation assay. Finally, the enzyme-linked immunosorbent assay was used to determine the effects of nVAP and sVAP on cytokines related to bone metabolism. ResultsThe administration of TVAPL for 12 weeks significantly reversed osteoporosis in OVX rats, thereby improving the BWC, BMD, BMC, and bone microarchitecture. IL-1 and IL-6 were significantly activated in the OVX group, and their activation was inhibited by TVAPL. In addition, nVAP and sVAP promoted the proliferation of cartilage and osteoblast-like cells (p<0.01 or p<0.001), and inhibited the secretion of IL-1α from THP-1 monocytic cells in vitro. ConclusionThese results suggest that TVAPL are effective in preventing bone loss in OVX rats. The effect of TVAPL on osteoporosis is due to inhibition of IL-1 and IL-6 by nVAP, and promotion of mitosis. sVAP has similar bioactivity as nVAP. Thus, both TVAPL and sVAP may be potential therapeutic agents for the treatment of postmenopausal osteoporosis.

Propyl-2-(8-(3,4-Difluorobenzyl)-2′,5′-Dioxo-8-Azaspiro[Bicyclo[3.2.1] Octane-3,4′-Imidazolidine]-1′-yl) Acetate Induces Apoptosis in Human Leukemia Cells through Mitochondrial Pathway following Cell Cycle Arrest

BackgroundDue to the functional defects in apoptosis signaling molecules or deficient activation of apoptosis pathways, leukemia has become an aggressive disease with poor prognosis. Although the majority of leukemia patients initially respond to chemotherapy, relapse is still the leading cause of death. Hence targeting apoptosis pathway would be a promising strategy for the improved treatment of leukemia. Hydantoin derivatives possess a wide range of important biological and pharmacological properties including anticancer properties. Here we investigated the antileukemic activity and mechanism of action of one of the potent azaspiro hydantoin derivative, (ASHD).Materials and MethodsTo investigate the antileukemic efficacy of ASHD, we have used MTT assay, cell cycle analysis by FACS, tritiated thymidine incorporation assay, Annexin V staining, JC1 staining and western blot analysis.ResultsResults showed that ASHD was approximately 3-fold more potent than the parent compounds in inducing cytotoxicity. Tritiated thymidine assay in conjunction with cell cycle analysis suggests that ASHD inhibited the growth of leukemic cells. The limited effect of ASHD on cell viability of normal cells indicated that it may be specifically directed to cancer cells. Translocation of phosphatidyl serine, activation of caspase 3, caspase 9, PARP, alteration in the ratio of BCL2/BAD protein expression as well as the loss of mitochondrial membrane potential suggests activation of the intrinsic pathway of apoptosis.ConclusionThese results could facilitate the future development of novel hydantoin derivatives as chemotherapeutic agents for leukemia.

Abstract 5602: Cyclin D2- mediated cardiomyocyte cell cycle activity reverses doxorubicin-induced cardiotoxicity.

Abstract Introduction: Doxorubicin (DOX) is an effective anticancer agent which also induces acute and chronic cardiotoxicities. We have generated a juvenile mouse model to study DOX-induced cardiotoxicity in children. Cardiomyocyte apoptosis with an ensuing loss of myocardial function is observed in this model. Here, we hypothesize that activation of cardiomyocyte cell cycle protects against DOX-induced cardiotoxicity. Methods: Two week old MHC-cylin D2 (D2) mice and their non-transgenic (NON-TXG) littermates were given weekly DOX injections for 5 weeks (25 mg/kg total dose). Animals were studied 1 week (acute stage) or 13 weeks (late stage) after the last DOX injection. Cardiac function was measured with echocardiography and cardiomyocyte (CM) cell cycle was quantified using a tritiated thymidine incorporation assay. Results: Our results showed that CM cell cycle was increased in D2 mice as compared to NON-TXG mice (thymidine positive CM nuclei, 0.03±0.01% vs. 0.002±0.002 in acute stage, p&amp;lt;0.01; and 0.03±0.01% vs. 0.001±0.001 in late stage, p&amp;lt;0.01). In the absence of cell cycle activity, cardiac function remained depressed in late stage vs. acute stage (Fractional Shortening, FS, 41.4±3.1% vs. 44.0±3.2%, p&amp;gt;0.05), whereas in the presence of CM cell cycle activity, cardiac function improved in late stage vs. acute stage (FS = 60.1±1.3% vs. 47.1±3.3%, p&amp;lt;0.01). CM apoptosis (0.02±0.01/mm2 vs. 0.05±0.01/mm2, p&amp;lt;0.01) and fibrosis (6.2±0.9% vs. 12.0±1.7%, p&amp;lt;0.01) was reduced in D2 mice as compared to NON-TXG mice in late stage. Conclusion: For the first time, our data suggest activation of CM cell cycle in adult mice reverses DOX-induced cardiotoxicity. Citation Format: Wuqiang Zhu, Mark H. Soonpaa, Wenjun Zhang, Weinian Shou, Mark Payne, Loren J. Field. Cyclin D2- mediated cardiomyocyte cell cycle activity reverses doxorubicin-induced cardiotoxicity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5602. doi:10.1158/1538-7445.AM2013-5602

Cyclin D2‐mediated cardiomyocyte cell cycle activity reverses doxorubicin‐induced cardiotoxicity

Doxorubicin (DOX) is an effective anticancer agent which also induces acute and chronic cardiotoxicities. We hypothesize that activation of cardiomyocyte (CM) cell cycle protects against DOX‐induced cardiotoxicity. Two week old MHC‐cyclin D2 (D2) mice and their non‐transgenic (NON‐TXG) littermates were given weekly DOX injections for 5 weeks (25 mg/kg total dose). Animals were studied 1 week (acute stage) or 13 weeks (late stage) after the last DOX injection. Cardiac function was measured with echocardiography and CM cell cycle was quantified using a tritiated thymidine incorporation assay. Our results showed that CM cell cycle was increased in D2 mice as compared to NON‐TXG mice (thymidine positive CM nuclei, 0.03±0.01% vs. 0.002±0.002 in acute stage, p&lt;0.01; and 0.03±0.01% vs. 0.001±0.001 in late stage, p&lt;0.01). In the absence of cell cycle activity, cardiac function remained depressed in late stage vs. acute stage (Fractional Shortening, FS, 41.4±3.1% vs. 44.0±3.2%, p&gt;;0.05), whereas in the presence of CM cell cycle activity, cardiac function improved in late stage vs. acute stage (FS = 60.1±1.3% vs. 47.1±3.3%, p&lt;0.01). CM apoptosis (0.02±0.01/mm2 vs. 0.05±0.01/mm2, p&lt;0.01) and fibrosis (6.2±0.9% vs. 12.0±1.7%, p&lt;0.01) was reduced in D2 mice as compared to NON‐TXG mice in late stage. For the first time, our data suggest activation of CM cell cycle in adult mice reverses DOX‐induced cardiotoxicity.

In Vitro and in Vivo Anticancer Activity of Copper Bis(thiosemicarbazone) Complexes

Neutral and cationic copper bis(thiosemicarbazone) complexes bearing methyl, phenyl, and hydrogen, on the diketo-backbone of the ligand have been synthesized. All of them were characterized by spectroscopic methods and in three cases by X-ray crystallography. In vitro cytotoxicity studies revealed that they are cytotoxic unlike the corresponding zinc complexes. Copper complexes Cu(GTSC) and Cu(GTSCHCl) derived from glyoxal-bis(4-methyl-4-phenyl-3-thiosemicarbazone) (GTSCH(2)) are the most cytotoxic complexes against various human cancer cell lines, with a potency similar to that of the anticancer drug adriamycin and up to 1000 fold higher than that of the corresponding zinc complex. Tritiated thymidine incorporation assay revealed that Cu(GTSC) and Cu(GTSCHCl) inhibit DNA synthesis substantially. Cell cycle analyses showed that Cu(GTSC) and Cu(GTSCHCl) induce apoptosis in HCT116 cells. The Cu(GTSCHCl) complex caused distinct DNA cleavage and Topo IIα inhibition unlike that for Cu(GTSC). In vivo administration of Cu(GTSC) significantly inhibits tumor growth in HCT116 xenografts in nude mice.

Cross-Talk Between Tumor B Cells, Follicular Dendritic Cells, and Follicular Helper T Cells: Implications for Follicular Lymphoma Pathogenesis and Therapy,

Abstract 3696 Background:The stromal microenvironment is thought to play a critical role in supporting the survival and growth of follicular lymphoma (FL) tumor cells. However, the key stromal factors involved are not well characterized. We hypothesized that FL tumor cells hijack the survival and growth mechanisms that are usually used by normal B cells in the lymph node microenvironment. For example, follicular dendritic cells (FDC) residing in germinal centers of lymph nodes play a vital role in supporting normal B cell survival and proliferation through adhesion molecules and by secreting chemokines and cytokines. In normal lymph nodes, lymphotoxin-alpha (LTA, TNFSF1) secreted by B cells activates FDC through LTbR and induces secretion of BAFF (TNFSF13B) and CXCL13. CXCL13 recruits follicular helper T cells (Tfh) into the B cell zone and CD40L (expressed by Tfh) together with BAFF prevents apoptosis and promotes proliferation of B cells. Here, we determined whether a similar cross-talk exists between FL tumor B cells, FDC, and Tfh. Methods:FL tumor samples were processed into single-cell suspension and cryopreserved in aliquots for later use. Primary tumor B cells were isolated by magnetic cell separation from single-cell suspensions of FL. The FDC-like cell line HK derived from human tonsil was used in place of primary FDC. Levels of BAFF, CXCL13, and LTA in culture supernatants were determined by ELISA. Apoptosis of tumor cells was determined by Annexin V assay and proliferation by tritiated thymidine incorporation assay. Activation of NFkB was determined by the TransAM NFkB p52 ELISA kit. Results:We developed a co-culture model to mimic the FL tumor microenvironment to study the interactions between tumor cells, FDC and Tfh. We observed that FL tumor cells spontaneously produced low levels of LTA but its production was significantly enhanced when tumor cells were cultured with either recombinant BAFF or CD40L. Dose response curves from FL tumor cells treated with both BAFF and CD40L recombinant proteins showed synergistic increase in LTA secretion. Recombinant BAFF and CD40L also synergistically enhanced the survival and proliferation of FL tumor cells. HK cells spontaneously produced low levels of BAFF and CXCL13 but their production was significantly enhanced by recombinant LTA in a dose-dependent manner.Consistent with the above studies, co-culturing FL tumor cells with HK cells significantly enhanced LTA secretion, possibly due to the effects of BAFF secreted by HK cells. In addition, in this co-culture, we also observed enhanced production of BAFF and CXCL13 possibly due to the effects of LTA secreted by the tumor cells. The production of BAFF and CXCL13 in this co-culture could be inhibited by an LTA neutralizing antibody in a dose-dependent manner suggesting the role of LTA in this mechanism. Co-culturing FL tumor cells with HK cells also significantly enhanced the survival and proliferation of FL tumor cells greater than two-fold compared with FL tumor cells alone. This effect also could be inhibited by LTA neutralizing antibody but not isotype control antibody. The survival benefit for tumor cells was further enhanced upon co-culture with autologous Tfh cells FACSorted from the same tumor sample. We also studied the downstream NFkB signaling mediated by BAFF on tumor B cells and by LTA on HK cells in the co-culture. We found that both tumor B cells and HK cells had significantly higher NFkB activity upon co-culture compared to cells alone. Conclusions:Our results suggest a model where LTA secreted by tumor B cells forms the afferent axis and induces production of BAFF and CXCL13 by FDC. CXCL13 preferentially recruits CXCR5-expressing Tfh into the tumor microenvironment and CD40L expressed by Tfh and BAFF secreted by FDC form the efferent axis and promote further production of LTA by tumor cells. In addition, CD40L and BAFF synergistically enhance the survival and proliferation of FL tumor cells via activation of NFkB. Our results suggest that because of their redundant functions, blocking BAFF-BAFF-R pathway alone or CD40-CD40L pathway alone with monoclonal antibodies as is being done in ongoing clinical trials may not be optimal. In contrast, blocking both pathways simultaneously is likely to significantly enhance the clinical responses. Our results also suggest that neutralizing LTA or blocking its receptor may be another novel therapeutic strategy for management of FL. Disclosures:No relevant conflicts of interest to declare.

Abstract A52: Overexpression of Sp2 increases susceptibility to wound- and carcinogen-induced papillomas in vivo and inhibits cell cycle progression and induces apoptosis of epidermal stem cells in vitro

Abstract Transcription factors regulate gene expression and de-regulation of their activities can transform normal cells into tumor cells. The Sp-family of transcription factors consists of nine distinct genes (Sp1–9) that are evolutionarily-conserved and share a common DNA-binding domain. Sp2 is one of several poorly-characterized members of this transcription factor family. Inactivation of Sp2 function in zebrafish and mice indicates that Sp2 is required for the completion of gastrulation and embryogenesis, respectively, and thus Sp2 is an essential gene. A causal relationship between Sp2 and tumorigenesis is also suggested by the observation that Sp2 expression is correlated directly with the progression of human prostate cancers and murine cutaneous squamous cell carcinomas. To determine if this correlation is functionally meaningful we created transgenic mice that over-express mouse Sp2 in basal cells of stratified epithelia. We reported recently that these animals (Sp2-C) exhibit increased susceptibility to wound- and carcinogen (DMBA/TPA)-induced tumorigenesis, and we concluded that Sp2 over-expression is oncogenic. To determine whether wound-induced papillomas depend on infiltrating T-cells for growth we treated transgenic animals with Tacrolimus, a well-characterized T-cell inhibitor, before and after surgical wounding. We find that Tacrolimus treatment in Sp2-C, but not wild-type animals, increased wound-induced papillomagenesis dramatically indicating that a T-cell mediated immune response limits the outgrowth of these lesions. To determine if Sp2 DNA-binding activity is required for the increased susceptibility to wound- and carcinogen-induced tumorigenesis, we generated a transgenic mouse line (Sp2-Not1) that over-expresses a mutated Sp2 cDNA lacking a functional DNA binding domain. Interestingly, the incidence of papillomagenesis in Sp2-Not1 mice is half that of wild-type littermates when treated with DMBA/TPA. Moreover, Sp2-Not1 mice do not develop wound-induced neoplasms. Instead Sp2-Not1 mice exhibit a decreased capacity to heal surgical wounds. We interpret these results to indicate that the transgene carried by Sp2-Not1 mice negatively regulates keratinocyte proliferation and/or viability in vivo. To determine the molecular mechanisms underlying the phenotypes presented by our transgenic strains we have prepared primary keratinocyte cultures and analyzed rates of proliferation, expression of differentiation-specific markers, cell-cycle progression, and the incidence of apoptosis. CyQUANT® cell proliferation and tritiated-thymidine incorporation assays showed that keratinocytes obtained from Sp2-C and Sp2-Not1 animals proliferate poorly in vitro compared to wild-type keratinocytes. Flow cytometric analyses indicated that Sp2-C and Sp2-Not1 keratinocytes progress through the cell cycle inefficiently and accumulate in all cell-cycle compartments. Flow cytometry following annexin V/PI staining, immunohistochemistry for cleaved caspases-3, and genomic DNA laddering assays indicate increased numbers of apoptotic cells in Sp2-C and Sp2-Not1 cultures relative to cultures prepared from wild-type littermates. In summary, (1) Sp2 over-expression in epidermal progenitor cells is oncogenic and requires Sp2 DNA-binding activity. (2) Wound-induced papillomagenesis in Sp2 transgenic mice is inhibited by infiltrating T-cells. (3) Sp2 over-expression inhibits progression through all cell-cycle compartments in vitro and induces programmed cell death. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr A52.

Mechanisms of Genistein Protection on Pancreas Cell Damage in High Glucose Condition

An adequate β cell number is important to prevent the onset and development of type 2 diabetes. The aim of this study was to determine if phytoestrogen gesintein has protective effects against high glucose-induced cell apoptosis in human pancreas cells, and to try to determine the possible mechanism for this protection. Human pancreatic β cells were subjected to normal (5 mM) or high glucose (25 mM) with and without the presence of 100 nM genistein, and also in the presence and absence of the pure anti-estrogen ICI-182780 (100 nM). Bcl-2 siRNA transfection was performed to investigate if the effect of genistein was also Bcl-2 dependent. Cell proliferation and apoptosis were determined by Tritiated Thymidine Incorporation Assay and Cell Apoptosis Detection ELISA. Estrogen receptor and Bcl-2 mRNA expression was measured by Real-time Quantitative PCR. High glucose concentration caused cell proliferation inhibition and apoptosis in cultured human pancreatic β cells, and these effects were significantly reversed by genistein (P<0.01). Estrogen receptor beta was expressed in the cultured cells, and genistein protection was blocked by ICI-182780 administration as well as Bcl-2 siRNA transfection. Phytoestrogen gave protection against high glucose-induced pancreatic cell damage through estrogen receptor beta and Bcl-2 dependent pathways.

A New BCR-ABL1 Mutation (L248R) Is Highly Resistant to Imatinib, Bosutinib, Nilotinib and Dasatinib, but Can Be Inhibited by AP-24534 and DCC-2036.

AbstractAbstract 3398Chronic Myeloid Leukemia (CML) treatment was radically modified by the discovery of imatinib (IM), a selective inhibitor of the fusion kinase Bcr-Abl. Second generation ATP-competitive tyrosine kinase inhibitors (TKIs) bosutinib (BOS), nilotinib (NIL) and dasatinib (DAS) further improved CML therapy. However, resistance to TKIs occurs in a variable proportion of patients; it can arise from different mechanisms but in the majority of cases it is due to Bcr-Abl point mutations that alter directly or indirectly the drug-protein binding. Over 70 mutations have been described in patients, with T315I showing resistance to IM, NIL, BOS and DAS. Here we report the discovery of a new P-loop mutant (L248R) that is highly resistant to all currently available inhibitors. L248R was identified in a lymphoid Blast Crisis CML patient. The patient initially presented with an IM-resistant F359I mutation. A cytogenetic and molecular response was obtained with BOS, but after 1 year haematological relapse developed; at this point a F359I/L248R clone was identified. L248R was previously reported only in an in vitro during a mutagenesis screen involving IM (Bradeen et al. 2006) and in a study with the T315I inhibitor SGX393 (O'Hare et al. 2008). L248R was never isolated from a clinical sample. Activity profile of BOS, IM, DAS and NIL against L248R, F359I and F359I/L248R was performed (Table). Stable transfectant Ba/F3 cells were generated and the TKIs anti-proliferative activity was determined. The relative IC50 increase over wild type Bcr-Abl (Relative Resistance, RR) was calculated. We classified RR values in four categories: sensitive (RR≤ 2), moderately resistant (RR between 2.1 and 4), resistant (RR between 4.1 and 10) or highly resistant (RR>10). In all cases a RR >10 was obtained, for L248R. Recently new compounds were developed to overcome resistance generated by the T315I mutant. Among them AP-24534 (AP), a panBcr-Abl inhibitor (O'Hare et al. 2009) and the switch pocket inhibitor DCC-2036 (DCC) were reported as potently active against the T315I mutation. Both compounds showed activity against L248R (RR 6.2 and 0.4), although the activity against the double mutant F359I/L248R was reduced especially for AP (RR 17.7 and 1.0). The activity profile of AP and DCC against L248R and a panel of 26 mutated forms of Bcr/Abl covering the most common mutations is also presented (Table). Activity of BOS, IM, DAS and NIL is also reported for comparison. According to our data, only mutation E255V is classified as highly resistant to AP, in addition to F359I/L248R: 6/26 mutations are considered “resistant” to AP (L248R, G250E, G252H, E255K, F359V, H396R) and 3/26 to DCC (D276G, E279K, F317V). The huge difference in the IC50 values for AP between Bcr/Abl wild type (2.1 nM) and parental Ba/F3 cells (>1000 nM) could render some of the mutants with high RR values to AP still sensitive to this inhibitor. It is also important to note that, according to our data, every mutation analysed shows sensitivity to at least one of the tested TKIs. In conclusion we describe a novel mutation that is highly resistant to the commonly used Bcr-Abl TKIs but which is inhibited by AP and DCC. Modelling data on the L248R mutant in complex with different TKIs will also be presented [Display omitted] IC 50 values are based on tritiated thymidine incorporation assay. Results are an average of at least 3 independent experiments Disclosures:Wise:Deciphera Pharmaceuticals: Employment. Flynn:Deciphera Pharmaceuticals: Employment. Gambacorti-Passerini:Pfizer pharmaceuticals: Research Funding.

Rhizoctonia bataticola lectin (RBL) induces mitogenesis and cytokine production in human PBMC via p38 MAPK and STAT-5 signaling pathways

Background Rhizoctonia bataticola lectin (RBL), purified from phytopathogenic fungus Rhizoctonia bataticola is highly mitogenic towards human peripheral blood mononuclear cells (PBMC). The lectin has sugar specificity towards N-glycans and binds to glycoproteins containing complex N-glycans (Nagre et al., Glycoconj J. 2010). In this study, we investigated the role of Mitogen Activated Protein Kinase (MAPK) and Signal Transducers and Activators of Transcription (STAT)-5 signaling in RBL-induced proliferation and production of Th1/Th2 cytokines. Methods Human PBMC were stimulated with RBL and proliferation was determined by tritiated thymidine incorporation assay, cytokine profiles by ELISA and activation of MAPK and STAT-5 by western blotting. RBL binding was monitored by immunofluorescence staining. Expression of IL-2Rα (CD25) was measured by flow cytometry. Results The binding and mitogenic activities of RBL were inhibited by glycoproteins– mucin, asialofetuin and fetuin. RBL stimulated expression of IL-2Rα and production of Th1/Th2 cytokines– IL-2, IFN-γ, IL-4 and IL-10. RBL-induced phosphorylation of ERK1/2 and p38 MAPK was detected at 1 h and 3 h respectively. Significant phosphorylation of STAT-5 (tyr 694) was observed at 12 h. Pharmacological inhibitors of p38 MAPK (SB203580) and JAK/STAT (AG490) but not ERK (PD98059) abrogated proliferation. RBL-induced expression of IL-2Rα and secretion of cytokines were drastically inhibited by SB203580 and AG490. Conclusions RBL-induced proliferation and production of Th1/Th2 cytokines are mediated via p38 MAPK and STAT-5 signaling. General significance RBL, a lectin with complex sugar specificity, is strongly mitogenic to human PBMC and stimulates the production of Th1 and Th2 cytokines. The results identified the signaling mechanism underlying the immunostimulatory activity of RBL.

Inhibitory effects of Agaricus blazei extracts on human myeloid leukemia cells

Ethnopharmacological relevance Agaricus blazei has been used as an adjuvant in cancer chemotherapy and is found to inhibit the growth of various types of tumor cells. Aim of the study Our study has adopted a systematic and bioassay-guided approach to optimize the extraction of Agaricus blazei for anti-leukemic bioactive components. The tumor-selective growth inhibitory activity of the extracts on leukemic cell lines was evaluated in vitro and in vivo using tumor-bearing nude mice. Materials and methods Agaricus blazei extracts were prepared using different methods. MTT and tritiated thymidine incorporation assays were used to evaluate the in vitro anti-leukemic effects. The most potent extract was further investigated using NB-4 cells-bearing nude mice and mechanistic studies using DNA fragmentation assay and cell death detection ELISA. Results The JAB80E70 extract showed the most potent tumor-selective growth inhibitory activity against human leukemia NB-4 and K-562 cells. This is the first report of anti-leukemic activity of JAB80E70 in athymic nude mice bearing NB-4 cells. Using DNA fragmentation assays and cell death detection ELISA, JAB80E70 was found to induce apoptosis in NB-4 cells. However, the polysaccharide enriched fractions failed to show significant cytotoxicity on NB-4 cells in vitro. Conclusions The JAB80E70 extract exhibited potent anti-leukemic effect in vitro and in vivo. The effect can be attributed, at least in part, to the induction of apoptosis. Besides, polysaccharides in Agaricus blazei may not possess direct anti-leukemic activity in vitro.

Determination of the Activity Profile of Bosutinib, Dasatinib and Nilotinib against 18 Imatinib Resistant Bcr/Abl Mutants

The treatment of Chronic Myeloid Leukemia (CML) has been radically modified by the discovery of imatinib (IM), a selective inhibitor of the fusion protein Bcr-Abl, the cause of the disease. A variable portion of CML patients experience resistance to IM therapy. Resistance can arise from different mechanisms but in the vast majority of cases is due to point mutations into the protein sequence that alter directly or indirectly the drug-protein binding. Mutation sites can be schematically clustered in four region: the P loop, the IM binding site, the catalytic domain and the activation loop (A loop). At present more than 70 mutations conferring different levels of resistance have been found in CML patients. Recently, several new inhibitors have been developed in order to obtain an increased potency and a broad range of activity against IM resistant mutants. Nilotinib (NIL) is an IM derivative about 30-fold more potent than IM. Dasatinib (DAS) is a dual-specific Src/Abl inhibitor, structurally unrelated to IM and characterized by an activity 100 to 300-fold higher than IM. Bosutinib (BOS) is a dual Src/Abl inhibitor that shows an activity 10 to 30-fold higher than IM. It is known that resistance to second generation TKIs can also arise and the analysis of mutation profiles reveals substantial differences among different TKIs. Presently the choice of a TKI to treat a patient resistant to IM is mostly based on an empirical basis, e.g. the fact that a certain patient has not been previously exposed to that particular TKI. The possibility to directly compare the different activities of TKIs against a given mutation is of remarkable importance in clinical practice. Such a tool could be used similarly to an antibiogram for bacterial diseases, guiding the choice of the most appropriate inhibitor for each patient.In our study, we investigated the activity of BOS, DAS, IM and NIL against a panel of 18 mutated forms of BCR/ABL chosen to cover the most common mutations found in patients. Stable Ba/F3 transfectant cell lines were generated and the TKIs antiproliferative activity was determined by tritiated thymidine incorporation assay. The relative IC50 increase over wild type BCR/ABL (Relative Resistance RR) was calculated. We classified the RR values in three categories: sensitive (RR≤2), resistant (between 2.01 and 10) or highly resistant (>10) as presented in the table.IC50-fold increase (WT=1)ImatinibBosutinibDasatinibNilotinibParental10.7838.31>5038.45WT1111P-LOOPL248V3.542.975.112.80G250E6.864.314.454.56Q252H1.390.813.052.64Y253F3.580.961.583.23E255K6.029.475.616.69E255V16.995.533.4410.31D276G2.180.601.442.00C-HelixE279K3.550.951.642.05V299L1.5426.108.651.34Active siteT315117.5045.4275.0339.41F317L2.602.424.462.22SH2-contactM351T1.760.700.880.44Active siteF359V2.860.931.495.16A-LOOPL384M1.280.472.212.33H396P2.430.431.072.41H396R3.910.811.633.10G398R0.351.160.690.49C terminal lobeF486S8.102.313.041.85Sensitive≤2Resistant2.01–10Highly resistant>10(Updated table available online at http://www.dimep.medicina.unimib.it/en/staff_174.php?docente_id=32)Our study points out at the differences in the activity spectrum of the 4 TKIs against the 18 Bcr/Abl mutations considered. The activity pattern presented in this work will help to reach a rational and tailored therapy offering to physicians a tool to use the new TKIs in the most efficient way for their patients.

Anticancer activities of sesquiterpene lactones from Cyathocline purpurea in vitro

Cyathocline purpurea has been traditionally used to treat various diseases including cancers for many years. However, these applications of C. purpurea have not been supported by pharmacological investigation. The objective of this study is to investigate the anticancer activities of three main constituents such as santamarine, 9beta-acetoxycostunolide and 9beta-acetoxyparthenolide isolated from C. purpurea in vitro. Cell viability was determined by trypan blue exclusion and methylene blue assays. Colony formation was assessed by microtitration cloning assay. DNA synthesis was determined by tritiated thymidine incorporation assay. Cell cycle analysis was carried out by flow cytometry. Apoptosis was observed by DAPI staining assay and Caspase 3/7 activities was measured using Caspase-Glo 3/7 assay kit. Santamarine, 9beta-acetoxycostunolide and 9beta-acetoxyparthenolide inhibited the growth of L1210 murine leukaemia, CCRF-CEM human leukaemia, KB human nasopharyngeal carcinoma, LS174T human colon adenocarcinoma and MCF 7 human breast adenocarcinoma cells in vitro, with IC(50) in the range of 0.16-1.3 microg/mL. In L1210 model, santamarine and 9beta-acetoxycostunolide inhibited L1210 cell growth, colony formation and [(3)H]-thymidine incorporation in time- and concentration-dependent manners. Flow cytometry studies showed that santamarine and 9beta-acetoxycostunolide blocked L1210 cells in the G(2)/M phase of the cell cycle. DAPI staining and caspase activity assays showed santamarine and 9beta-acetoxycostunolide induced apoptosis and activated caspase 3 in L1210 cells. These results indicated that santamarine, 9beta-acetoxycostunolide and 9beta-acetoxyparthenolide exhibit significant anticancer activities in vitro. The inhibitory effects of santamarine and 9beta-acetoxycostunolide on L1210 cells are cytotoxic rather than just cytostatic. They block mitosis and reduce uptake of thymidine. The mechanism of the cytotoxicity of santamarine and 9beta-acetoxycostunolide to L1210 cells could be related to alkylation of the sulfhydryl enzymes involved in nucleic acids and protein synthesis, as previously found for other sesquiterpenes with the alpha-methylene-gamma-lactone moiety present in santamarine, 9beta-acetoxycostunolide and 9beta-acetoxyparthenolide. It may also be related to suppression of microtubular proteins. Santamarine and 9beta-acetoxycostunolide induced apoptosis of L1210 cells via activation of caspase 3.