3H Thymidine Research Articles

Sensitivity of Planktonic Aquatic Bacteria to Ciprofloxacin

Many anthropogenic compounds, such as antibiotics, are found at trace levels (-1) in aquatic and terrestrial systems. The effect of these compounds on the metabolism and function of microbes are difficult to assess because the assays used, such as the minimum inhibitory concentration (MIC) and the disk diffusion methods, lack the sensitivities to measure bacterial response to these very low levels of antibiotics on bacterial populations. Therefore, we theorized that the [3H] thymidine incorporation into DNA method might be sensitive in determining the effect of DNA inhibiting antibiotics on DNA production in planktonic bacteria in aquatic systems. Utilizing the 3H thymidine method, we measured the effects of ciprofloxacin on DNA production on planktonic bacteria in river and pond waters. Ciprofloxacin significantly (P -1 but significant inhibition (P -1in pond water. The very low concentration required to inhibit DNA production in river water bacteria indicates that bacteria are extremely sensitive to antibiotics at very low concentrations. A likely reason for the differences in inhibition between the two waters is due to ciprofloxacin becoming bound, and possibly becoming biologically inactive, in the pond water due to higher dissolved organic carbon content. This work demonstrates that bacteria in some aquatic systems can be significantly impacted by low concentrations of anthropogenic antibiotics finding their way into these systems and that our assumptions as to the concentrations at which antibiotics affect microbes are highly underestimated.

Comparison between the ameliorative potentials of canagliflozin and metformin on the testicular damage in diabetic rats

To evaluate the Renoprotective efficacy of melatonin against long term exposure induced changes of dimethylbenz(a)anthracene (DMBA), cell proliferation and DNA synthesis was determined in the epithelial cells of cortical and medullary renal tubules in female and male mice applying quantitative autoradiographic analysis and using 3H thymidine as a radioactive label. A total of 30 male and female adult albino mice were divided into 3 groups, each of 10 individuals: control (group C), DMBA exposed (group D) and DMBA/melatonin exposed (group D+M) mice. In female mice, long term exposure for 150 days to a single injection of DMBA (10mg/ 100g b.w.) stimulated the incorporation rate of 3H thymidine into the epithelium of cortical renal tubules by 6774% compared to control. The number of grains over labeled nuclei was reduced by 57.1%. Daily injected with melatonin (100 g/ 100g b.w.) during the last 60 days of exposure to a single dose of DMBA attenuated cell division rate of the epithelial cells by 80% compared to group D, but remained 1275% higher than that of group C. The mean grain count over labeled nuclei was increased by 59.5% compared to group D, but remained 31.5% lower than that of control. In the medullary portion of the renal tubules, DMBA induced changes were less pronounced than that in the cortical area. The cell division was stimulated by 833% compared to control and remained 8.1x lower than the percentage increase in the cortical part. The mean grain count over labeled nuclei was reduced by 40.4% compared to group C. Daily injected with melatonin (100 g/ 100g b.w.) during the last 60 days of exposure to a single dose of DMBA reduced the mitotic cell division by 83.9% compared to group D and thereby showing a similar effect as in the cortical part of the renal tubules. Compared to group C, the 3H labeling index remained 50% higher. In the cortical portion, the comparable value was 1275% higher than that of control. The mean grain count over labeled nuclei was increased by 26.2% compared to group D and remained 24.8% lower than in group C.

MiR-181b promotes chemoresistance in breast cancer by regulating Bim expression.

MicroRNAs are emerging as critical regulators of the initiation and progression of multiple types of human cancers, including breast cancer. In the present study, the expression of miR-181b in breast cancer patient serum and breast cancer cell lines was evaluated. It was demonstrated that the miR-181b level was significantly upregulated in patient serum and breast cancer cell lines compared with that in normal controls. The results of in vitro 3H thymidine incorporation and Transwell migration assay indicated that miR-181b overexpression markedly promoted the proliferation and metastasis of breast cancer cells. These data suggest that miR-181b is a tumor promoter in breast cancer. Furthermore, miR-181b expression was found to be upregulated in doxorubicin (DOX)-resistant T-47D cells (T-47D-R) compared with that in the parental T-47D cells, and upregulation of miR-181b expression decreased the anticancer effect of DOX in the T-47D cells. Mechanistic studies demonstrated that the Bim gene, an essential initiator of apoptosis, was inhibited by miR-181b overexpression. We observed that knockdown of miR-181b by its specific inhibitors significantly re-sensitized the T-47D-R cells to the cytotoxicity of DOX. Importantly, we demonstrated that miR-181b inhibitors increased the level of Bim in the T-47D-R cells, resulting in the loss of mitochondrial membrane potential (MMP) and the activation of caspases caused by DOX. In summary, the results of the present study suggest that miR-181b functions as an oncogene during breast cancer development, and the miR-181b/Bim pathway may be a novel target used to overcome the chemoresistance in breast cancer.

Th22 cells control colon tumorigenesis through STAT3 and Polycomb Repression complex 2 signaling

ABSTRACTTh22 cells traffic to and retain in the colon cancer microenvironment, and target core stem cell genes and promote colon cancer stemness via STAT3 and H3K79me2 signaling pathway and contribute to colon carcinogenesis. However, whether Th22 cells affect colon cancer cell proliferation and apoptosis remains unknown. We studied the interaction between Th22 cells and colon cancer cells in the colon cancer microenvironment. Colon cancer proliferation was examined by flow cytometry analysis and H3 thymidine incorporation. Cell cycle related genes were quantified by real-time PCR and Western blotting. We transfected colon cancer cells with lentiviral vector encoding specific gene shRNAs and used chromatin immunoprecipitation (ChIP) assay to determine the genetic signaling involved in interleukin (IL)-22-mediated colon cancer cell proliferation. We showed that Th22 cells released IL-22 and stimulated colon cancer proliferation. Mechanistically, IL-22 activated STAT3, and subsequently STAT3 bound to the promoter areas of the Polycomb Repression complex 2 (PRC2) components SUZ12 and EED, and stimulated the expression of PRC2. Consequently, the activated PRC2 catalyzed the promoters of the cell cycle check-point genes p16 and p21, and inhibited their expression through H3K27me3-mediated histone methylation, and ultimately caused colon cancer cell proliferation. Bioinformatics analysis revealed that the levels of IL-22 expression positively correlated with the levels of genes controlling cancer proliferation and cell cycling in colon cancer. In addition to controlling colon cancer stemness, Th22 cells support colon carcinogenesis via affecting colon cancer cell proliferation through a distinct histone modification.

Ubiquitin C-terminal hydrolase 1: A novel functional marker for liver myofibroblasts and a therapeutic target in chronic liver disease

Ubiquitination is a reversible protein modification involved in the major cellular processes that define cell phenotype and behaviour. Ubiquitin modifications are removed by a large family of proteases named deubiquitinases. The role of deubiquitinases in hepatic stellate cell (HSC) activation and their contribution to fibrogenesis are poorly defined. We have identified that the deubiquitinase ubiquitin C-terminal hydrolase 1 (UCHL1) is highly induced following HSC activation, determined its function in activated HSC and its potential as a therapeutic target for fibrosis. Deubiquitinase expression was determined in day 0 and day 10 HSC. Increased UCHL1 expression was confirmed in human HSC and in an alcoholic liver disease (ALD) patient liver. The importance of UCHL1 in hepatic fibrosis was investigated in CCl4 and bile duct ligation injured mice using a pharmacological inhibitor (LDN 57444). The effects of UCHL1 inhibition on HSC proliferation were confirmed by Western blot and 3H thymidine incorporation. Here we report that pharmacological inhibition of UCHL1 blocks progression of established fibrosis in CCl4 injured mice. UCHL1 siRNA knockdown, LDN 57444 treatment, or HSC isolated from UCHL1(-/-) mice show attenuated proliferation in response to the mitogen, platelet-derived growth factor. Additionally, we observed changes in the phosphorylation of the cell cycle regulator retinoblastoma protein (Rb) in the absence of UCHL1 highlighting a potential mechanism for the reduced proliferative response. UCHL1 expression is highly upregulated upon HSC activation and is involved in the regulation of HSC proliferation. This study highlights therapeutic opportunities for pharmacological targeting of UCHL1 in chronic liver disease.

Abstract 679: CF102 induces apoptosis of hepatocellular carcinoma

Abstract Background: The A3 Gi protein adenosine receptor (A3AR) is highly expressed in tumor compared to normal cells. A3AR agonists were shown to inhibit the growth of various tumor cell types via down-regulation of the Wnt and NF-κB signaling pathways. CF102 is a highly selective agonist of the A3AR, shown to have an anti-tumor effect in liver cancer in in vivo models. CF102 induced apoptosis via up-regulation of caspase-3 and the mitochondrial proteins Bax and Bad, demonstrating cell death in the tumor cells. In this study we explored the molecular mechanism involved with the effect of CF102 on human hepatocellular carcinoma cells. Methods: CF102 was added to a medium culture of Hep3b for 48 hours. CF102 effect was observed using Lactate dehydrogenase (LDH), MTT and H3 Thymidine proliferation assays. Cell mRNA and protein were extracted and were analyzed for different signaling pathways. Results: CF102 inhibits the proliferation of Hep3b cells and showed an apoptotic activity in MTT assay. A cytotoxicity effect of CF102 was observed when high levels of LDH were measured in the culture medium of CF102 treated cells compared to the control. Moreover, cyclin D1 expression levels were down-regulated and caspase-3 levels were up-regulated upon CF102 treatment. Both mRNA and protein expression levels of A3AR were down-regulated by CF102 with a subsequent decrease in all members of the JAK-STAT signaling pathway including pSTAT-1, p-JAK-2 and p-AKT along with up-regulation of SOCS-1 and SOCS-3. Conclusions: CF102 showed an anti-cancer activity via down-regulation of the A3AR and the JAK-STAT signaling pathway. CF102 induces cytotoxicity effect resulting in apoptosis of Hep3b cells, indicating that this drug may be considered as an anti-cancer drug for the treatment of human hepatocellular carcinoma. Note: This abstract was not presented at the meeting. Citation Format: Shira Cohen, Faina Barer, Salomon M. Stemmer, Pnina Fishman. CF102 induces apoptosis of hepatocellular carcinoma. [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 679. doi:10.1158/1538-7445.AM2015-679

Study on the immunosuppressive activities of myeloid derived suppressor cells in patients suffering from recurrent spontaneous abortion

Objective To investigate the roles of myeloid derived suppressor cells (MDSCs) in the maintenance of maternal immune tolerance and to analyze their associations with recurrent spontaneous abortion (RSA). Methods Peripheral blood samples were collected from sixty subjects including non-pregnant healthy subjects, normal pregnant women and women with recurrent spontaneous abortion aged 25 to 35 years (n=20). Flow cytometry analysis was performed to monitor the changes of MDSCs at the 12th, 24th and 36th weeks of gestational age in three normal pregnant women. The differences with the numbers of MDSCs during the same stage of pregnancy in peripheral blood samples were compared among the three groups by flow cytometry analysis. RT-PCR analysis was used for detection of Arg-1 in women with normal pregnancy and RSA. CD8+ T cells were separated from non-pregnant healthy subjects by using magnetic beads. The immunosuppressive effects of MDSCs, isolated from women with normal pregnancy and RSA, on CD8+ T cells were evaluated by using the 3H thymidine incorporation assay. Results The number of MDSCs increased during normal pregnancy and the differences among different weeks were significant (12th week vs 24th week: P=0.0442, 24th week vs 36th week: P=0.0233, 12th week vs 36th week: P=0.0017). The percentages of MDSCs in peripheral blood of the healthy non-pregnant women were the lowest [(0.937±0.127)%], followed by those in women with RSA [(2.151±0.185)%] and normal pregnant women [(3.777±0.427)%]. There was significant difference with the percentage of MDSCs between women with normal pregnancy and RSA (P=0.0250). Compared with women with normal pregnancy, those with RSA showed significantly decreased levels of Arg-1 (P=0.0474) and suppressed activities of MDSCs against CD8+ T cell proliferation (P=0.0230). Conclusion The MDSCs might play an important role in the maintenance of maternal immune tolerance. Decreased number and inhibited function of MDSCs in the peripheral blood of women with RSA might be responsible for the development of RSA. Key words: Myeloid derived suppressor cell; Maternal immune tolerance; Recurrent spontaneous abortion

IL-22 induces colon cancer proliferation through STAT3 and PRC2 signaling (TUM10P.1036)

Abstract Background: IL-22(interleukin-22) is a newly identified cytokine which has been recently highlighted owing to its biological significance in carcinogenesis. However, the role of IL-22 in colon cancer proliferation has remained unclear. Since both JAK-STAT signaling and Polycomb Repression complex (PRC) 2 play crucial roles in epithelial proliferation. We investigated the mechanism by which IL-22 induces colon cancer proliferation. Methods: Colon cancer cells proliferation was detected by FACS and H3 Thymidine Incorporation. Cell cycle related genes were tested by Real-Time PCR. Lentiviral vector encoding gene specific shRNAs (STAT3, EZH2, SUZ12) and scramble particles were used to transfect colon cancer cells. ChIP assays were performed to detect the binding of different proteins and DNA. Results: IL-22 significantly induces colon cancer cells proliferation through STAT3 signaling. This increased proliferation involved the inhibition of p16 and p18 through aberrant promoter histone methylation H3K27me3. PRC2 catalyzes promoter H3K27me3 of p16 and p18, which can be up-regulated by IL-22. Furthermore, STAT3 can bind to PRC2 members (SUZ12 and EED), which also can be up-regulated by IL-22 stimulation. Conclusion: IL-22 can induce human colon cancer cells proliferation by activation of STAT3, which was phosphorylated and directed bind to the PRC2 complex, especially SUZ12 and EED, and consequently catalyzed the H3K27m3 of cell cycle check-point genes p16 and p18.

Comparison of the duration of the cell cycle in successive generation of synchronously dividing antheridial filaments of Chara vulgaris L. as measured with 3H thymidine

Duration of the cell cycle in synchronously dividing cells of successive generation of antheridial filaments in <i>Chara vulgaris</i> L. was estimated on-the basis of labeling with <sup>3</sup>H thymidine. Duration of the cell cycle is proportional to the volume of cells and with their decreasing in the consequence1 of consecutive divisions, the cell cycle becomes shorter. In the 2-, 4-, 8-, and 16-cell generations the length of the S period remains constant, so the duration of the G2 period is gradually reduced.

Abstract 618: Implications of inhibition of steroid receptor co-activator-1 in human prostate cancer

Abstract Background: Prostate cancer (PCa) is still the most commonly diagnosed cancer and 2nd leading cause of death in the USA and the 3rd leading cause of death behind lung and colorectal cancers in Europe, respectively. Depending on the stage of the tumor different therapy options exist. Initially, those therapies show success and lead to tumor regression but eventually castration-resistant PCa (CRPC) develops. Androgen receptor (AR) is still active in CRPC through various mechanisms. One possibility of targeting the AR axis is inhibition of its co-regulators such as SRC-1, needed for efficient transcription of AR downstream genes. SRC-1, a member of the p160 coactivator family, is known to promote the transcriptional activity of steroid receptors like the AR as well as that of other non-steroidal transcription factors. Due to the fact that SRC-1 staining is increased in metastatic tissue of patients samples compared to clinically localized PCa our goal is to investigate the specific role of SRC-1 in PCa progression and metastatogenesis. Moreover, the question whether SRC-1 could be a target for a novel therapeutic approach will be addressed. Methods: Knock-down cell lines were generated using lentiviral-based RNAi technology. Western Blotting and qRT-PCR analysis were performed to verify knock-down efficiency. The proliferation of the different cell lines was addressed by 3H thymidine incorporation assays. Scratch (wound healing) and Boyden chamber assays were used to investigate the migratory and invasive behavior of the cell lines. Results: Immortalized benign and malignant prostate cell lines were screened for SRC-1 expression on mRNA and protein level, revealing that SRC-1 is expressed in all tested cell lines. Furthermore, knock down of SRC-1 in LNCaP and MDA PCa 2b (AR+) led to a markedly reduced AR transcriptional activity, which was verified by a reduced transcription of a downstream target gene prostate specific antigen. Whereas SRC-1 knockdown in MDA PCa 2b and PC3 cells did not show any effect on proliferation, depletion of SRC-1 negatively influenced cell proliferation of LNCaP cells. Moreover, we investigated the role of SRC-1 in the migratory and invasive behavior. While depletion of SRC-1 in PC3 and MDA PCa 2b cells significantly diminished migration, it had no effect on invasiveness. Conclusion: Together these findings demonstrate that SRC-1 is not only an important regulator of AR transcriptional activity but also affects other signaling pathways. Furthermore, SRC-1 seems to play a crucial role in migration but not in invasion and proliferation, depending on the cell lines used. Thus, SRC-1 may be a possible therapeutic target in metastatic prostate cancer, although its specific role has to be further investigated. Citation Format: Birgit Luef, Florian Handle, Frédéric R. Santer, Zoran Culig. Implications of inhibition of steroid receptor co-activator-1 in human prostate cancer. [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 618. doi:10.1158/1538-7445.AM2014-618

Cimetidine effects on the immunosuppression induced by burn injury.

Although many studies on the immune response following burn injuries have been reported, more attention has been given to the immunosuppression mechanism and mediators that shape the process of immune suppression. Specifically, information is not available concerning the immunomodulatory effects of the drugs which are involved in the immune response restoration. In this study, we investigated the effects of Cimetidine on the modulation of immune response in patients with burn injury of 20-60%. Two groups of patients were involved in this study; the patients in one group were treated with 15 mg/kg per day of Cimetidine while the patients in the other group were treated with placebo. Peripheral blood mononuclear cell (PBMC) expressing CD3, CD4, CD8, CD19 and CD3/HLA-DR was analyzed by flow cytometry. Cell proliferation assay using H3 thymidine was performed on PBMC samples. The proliferation assay showed a significant suppression of cell proliferation rate in post-burn patients (p = 0.001). We observed a significant reduction in the lymphocyte count (p = 0.001) and frequency of CD3 (p = 0.007) and CD4 (p = 0.001) T cells in post-burn patients. Also, the frequency of CD 19+ and HLA DR+ cells was increased compare to normal donors following burn injury. Treatment with Cimetidine increased the frequency of CD8+ T cells in the patient's peripheral blood. The PBMC proliferation rate was restored following the treatment with Cimetidine (p = 0.02). Our data indicates that Cimetidine may have beneficial effects on cell mediated immunity following burn injury.

Inhibition Of H3K27-Methylome As a Novel Therapeutic Strategy In Multiple Myeloma

Multiple myeloma (MM) remains incurable, despite recent therapeutic advances; newer insights into the pathogenic mechanisms that cause this disease and additional therapies are urgently needed. Recent studies of the epigenome and in particular the methylome, have shown that myeloma is characterized by widespread epigenetic changes. Epigenetic changes may precede genetic mutations and genomic instability; Ubiquitously Transcribed Tetratricopeptide Repeat Protein X (UTX), a histone H3K27 demethylase may represent such an example. Previous studies have shown inactivating somatic mutations in UTX (KDM6A) in 10% of MM cases. Aberrant methylation of core histone tails and deregulation of the corresponding enzymes such as UTX and JMJD3 have been implicated in leukemia as well as other types of cancers, but their role in MM remains unknown. We evaluated the activity of the selective Jumonji H3K27 demethylase (UTX/JMJD3) inhibitor, GSK-J4, in MM.In a panel of 15 human MM cell lines (HMCLs) including cell lines resistant to bortezomib and dexamethasone, GSK-J4 induced significant survival and proliferation inhibition, as measured by luminescence-based viability assay (CTG), MTT (inhibition>68% in 48 hours) and 3H thymidine uptake, with the exception of OPM2 that was resistant up to 5 uM concentration. GSK-J4 induced apoptosis as measured by flow cytometry upon staining with Annexin-V/Propidium Iodide. The compound did not induce cytotoxicity in PBMCs from healthy donors and normal human skin fibroblasts. The inhibitory effect of GSK-J4 was observed also in CD138+ primary plasma cells from newly diagnosed MM patients (n=5) compared to PBMCs from healthy donors (n=9) (p<0.001). The compound also resulted in significant inhibition of MM cell colony formation in soft agar after 2 weeks in compared to controls. Moreover, interaction between MM cells and bone marrow stromal cells from MM patients or HS-5 stromal cell line did not overcome the inhibitory effect of GSK-J4 in all the HMCLs (KMS-12BM, LP-1, MM1S, OPM2, RPMI-8226, H929 and INA6) tested. To further investigate the mechanism of GSK-J4-induced apoptosis, we evaluate the activation of caspases 3/7, 8 and 9, and observed significant activation of caspase 3/7 and 9, indicating the involvement of intrinsic apoptotic pathway into the GSK-J4 function, as also confirmed by western blot analysis of Bcl-xl, p53, and Bax.We evaluated the enzymatic activity of UTX/JMJD3 in HMCLs by using a fluometric-based assay. The sensitivity of the HMCLs to demethylase inhibitor was directly related with UTX/JMJD3 activity (p<0.05). As the methylation of H3K27 mark plays a major role in the maintenance of active and silent states of gene expression in important cellular processes, we next mapped H3K27me3 and H3K27me2 chromatin modifications by genome-wide chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq) analysis in RPMI8226 and KMS-12BM cells before and after GSK-J4 treatment and in UTX knockdown cells. Transcription factors of OSKM complex: Oct4, Sox2, and Nanog were found to be targets of aberrant demethylation after treatment with GSK-J4, indicating possible involvement of H3K27 demethylases in pathogenesis of MM through the regulation of “stemness” genes. Western blot analysis showed that the inhibitor reduced the expression of these genes.In conclusion, we demonstrate a significant beneficial impact of inhibition of H3K27 demethylation in MM. These results provide new insights into the mechanism of altering methylome as a potent epigenetic intervention in treatment of MM. Disclosures:Hideshima:Acetylon Pharmaceuticals: Consultancy. Anderson:celgene: Consultancy; onyx: Consultancy; gilead: Consultancy; sanofi aventis: Consultancy; oncopep: Equity Ownership; acetylon: Equity Ownership.

JAK1/2 Inhibition Suppresses T Cell Proliferation and Inflammatory Cytokine Production In An Allogeneic Setting and Ameliorates Graft-Versus-Host Disease (GvHD) In a Murine GvHD Model

Graft-versus-host-disease (GvHD) constitutes a severe complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT). In GvHD, tissue damage is mediated by pro-inflammatory cytokines. Cytokine responses are mediated by activated Janus kinases (JAKs). We hypothesized that JAK1/2 inhibition might reduce T effector cell responses and inflammatory cytokine production in an allogeneic system, thereby ameliorating GvHD. We established an allogeneic (major mismatch) cell culture system using naive BALB/c CD4+ CD62Lhigh T cells that were co-cultured with C57BL/6 (B6) bone marrow derived dendritic cells (DC). JAK 1/2 signaling was specifically blocked using the small molecule inhibitor Ruxolitinib (INCB018424). By using a 3H Thymidine proliferation assay, we found that Ruxolitinib was able to inhibit the proliferation of allogeneic CD4+ effector T cells. In our intracellular cytokine staining experiments Ruxolitinib was able to impair the differentiation of naïve T cells into IFN-gamma and IL17A-producing T effector cells. Both cytokines – IFN-gamma and IL-17A – have been linked to aggravated courses of GvHD severity.In vivo administration of Ruxolitinib signficantly reduced lethal GvHD after allo-HSCT in a murine major mismatch model. BALB/c recipient mice were irradiated and received C57BL/6 (B6) T cell depleted bone marrow along with CD4+ and CD8+ T cells. Animals that were treated with an oral gavage of Ruxolitinib twice daily displayed significantly lower serum levels of IFN-gamma, TNF-alpha, IL-10 and IL-12p70, lower histological and clinical GvHD scores and improved overall survival compared to the control group.By using luciferase-positive (luc+) CD4+ and CD8+ T cells, we were able to visualize and quantify T cell migration after GvHD induction. The recipient mice received luc+ T cells along with conventional T cell depleted bone marrow. We were able to detect the luc+ T cell signal with a bioluminescence imaging system. At day 9 after allogeneic bone marrow transplantation, migration of donor T cells to GvHD target sites was significantly reduced in Ruxolitinib treated mice compared to control mice.In summary, our data demonstrate that suppression of inflammatory cytokine responses by JAK1/2 inhibitors modulates T effector cell responses in an allogeneic setting and improves survival in a murine major mismatch GvHD model. These observations suggest that JAK1/2 inhibition might be used for the treatment of GvHD following allo-HSCT. Disclosures:No relevant conflicts of interest to declare.

Concomitant, T-Independent TLR9-Mediated and BCR-Mediated Activation Provides Signals For Optimal Telomerase Induction In Chronic Lymphocytic Leukemia Cells Regardless Of IGHV Mutation Status

Chronic lymphocytic leukemia (CLL) is characterized by the clonal amplification of CD5-expressing B cells that appear to develop and evolve based on signals from the microenvironment. In vitro and in vivo evidence suggests that the B-cell antigen receptor (BCR) and Toll-like receptors (TLRs) may be keys to this stimulation. Because clonal turnover can lead to the release of naked nuclear material into the cellular microenvironment, these remnants of dying/dead cells may contribute to disease progression by repeated low level T-independent activation of CLL cells through the combination of the BCR and TLRs. To test this hypothesis, we assessed TLR9-driven or BCR + TLR9-driven CLL B-cell activation, focusing on its impact on telomerase activation in CLL cells, which is known to be important in the disease and which we have shown to be selectively activated by BCR stimulation in Ig V-unmutated (U-CLL) clones but not in Ig V-mutated (M-CLL) clones.B cells, isolated by negative selection from peripheral blood of IgM+ CLL patients and cryopreserved until use, were cultured for 16 hr without/ with TLR9 agonist, ODN 2006, alone and were assayed for apoptosis using Annexin V and flow cytometry. To study the relative contribution of simultaneous TLR9 activation and BCR activation, B cells were exposed to ODN2006 alone or HB57dex (monoclonal anti IgM Ab conjugated onto dextran) alone or a combination of the two reagents. Extracts from cells cultured for a period of 3 days were assayed for functional telomerase activity using TRAP. Parallel cultures of B cells exposed to the same stimuli were harvested at day 3 and assayed for cell activation and proliferation, which was assessed by 3H thymidine incorporation. CLL cells cultured with ODN2006 exhibited significant apoptosis within 16 hours in 6/12 cases. However at day 3, the same stimulus elicited significant increases in percentages of CD69-expressing cells and densities of HLA-DR in all CLL cases studied. As compared to BCR activation, which upregulates telomerase activity in U-CLL only, TLR9-mediated activation of CLL induced telomerase activation in all CLL cases. Furthermore, ODN2006 elicited significantly higher induction of telomerase activity in M-CLL cases compared to U-CLL cases (p=0.01). In addition, in M-CLL cases, simultaneous activation via TLR9 and BCR significantly upregulated the telomerase activity (p=0.05) that was induced by TLR9 activation alone. IRAK-1/4 inhibitor down modulated both TLR9 mediated and TLR9 +BCR mediated telomerase activity to a greater extent in M-CLL cases than in U-CLL cases. TLR9 activation of CLL cells induced a 3.75 + 0.8 fold (range 1.1 to 19.6; n=32) increase in cell proliferation. When segregated by Ig V mutation, U-CLL cells (n=16) responded significantly better (6.0 + 1.6 fold) compared to M-CLL cells (2.1 + 0.3 fold, n=16; p=0.03). However, co-stimulation of cells via their BCR significantly increased TLR-mediated responses only in M-CLL cases (from 2.3 + 0.4 fold to 5.4 + 1.7 fold; p=0.05). IRAK-1/4 inhibitor did not exert a significant effect on TLR9 mediated cell proliferation in either the U-CLL or M-CLL cases. Co-culture of CLL cells with human stromal cells, HS5, further upregulated the concerted TLR9 + BCR induced proliferative responses in 70% of the cases studied.Together, these results indicate that simultaneous stimulation of CLL cells via both their TLR9 and BCR molecules positively impacts on telomerase activity in all patients studied. Since telomerase is crucial in maintaining longevity of repeatedly stimulated cells, this could represent a mechanism for worse clinical outcome in CLL. These studies stress the need for devising therapeutic agents or combinations thereof to effectively target multiple pathways downstream of these signaling receptors and to ultimately eradicate newly evolving CLL cells. Disclosures:No relevant conflicts of interest to declare.

Protective Mechanism of Melatonin on Kainic Acid Induced Immune Modulatory Effect on Lymphocytes Derived from Mouse Spleen

Toxicity occurs when cells develop inflammation due to exposure to several toxic substances. The study was aimed to assess the damage (toxicity) induced by kainic acid on splenic lymphocyte and enriched T lymphocyte under in vitro conditions and the protective role of exogenous melatonin against this damage (toxicity). The study included assessment of inflammatory mechanism by the expression of immune modulatory cytokine mediators using real time PCR. Oxidative stress (reactive oxygen species) and nitrosative stress (reactive nitrogen species) were also studied to determine the free radical production. Interestingly, kainic acid caused severe splenic lymphocytes and enriched T lymphocyte damage which was evident from deleterious alterations in various parameters. Kainic acid treatment (1 mM) resulted in increased mRNA expression of cytokines like tumour necrosis factor-beta, interleukin 6, interleukin 1, interferon gamma, mitogen-activated protein kinase gene-14, inducible nitric oxide synthase and decreased interleukin 10 mRNA expression. Tritiated (3H) Thymidine Incorporation study signifies that kainic acid treatment (1 mM) increased the proliferation of splenic and enriched T lymphocytes. These changes were normalized by exogenous administration of melatonin (0.25-1.0 mM) in combination with kainic acid. Flow Cytometry Analysis using Annexin V apoptosis assay kit revealed an increase in apoptotic and necrosis (double positive cells) in splenic lymphocytes treated with kainic acid alone. However, melatonin treated in combination with kainic acid, showed attenuation to apoptosis and necrosis on splenic lymphocytes. This study depicts that kainic acid induced inflammatory toxicity could be attenuated by exogenous melatonin treatment as evident by the decreased levels of the inflammatory cytokines, immune reactions and free radical production.

CK2 Basal Activity Regulates IL-7-Mediated JAK/STAT and PI3K/Akt Pathway Activation and Pro-Survival Effects in T-Cell Acute Lymphoblastic Leukemia Cells

AbstractAbstract 1242Interleukin 7 (IL-7) and its receptor (IL-7R) are essential for normal T-cell development and homeostasis. However, IL-7/IL-7R-mediated signaling may also partake in leukemia development, as we recently demonstrated by the identification of IL-7Rα gain-of-function mutations in around 9% of T-cell acute lymphoblastic leukemia (T-ALL) patients (Zenatti et al, Nat Genet 2011). Moreover, we have shown that IL-7 produced in the leukemia milieu by stromal cells can accelerate leukemia progression in vivo (Silva et al, Cancer Res 2011) and have a relevant contribution to the viability and proliferation of T-ALL cells via activation of PI3K/Akt signaling pathway (Barata et al, J Exp Med 2004). On the other hand, the pleiotropic serine/threonine Casein Kinase 2 (CK2) is overexpressed and hyperactivated in leukemia, including in T-ALL. CK2 phosphorylates and thereby inactivates PTEN, contributing to constitutive activation of PI3K/Akt pathway in the vast majority of primary T-ALL cases (Silva et al, J Clin Invest 2008). Here, we show for the first time, that CK2 activity crosstalks with IL-7-mediated signaling in T-ALL. To determine the involvement of CK2 in IL-7 mediated signaling, we treated HPB-ALL (IL-7-responsive) and TAIL7 (IL-7-dependent) cells, with two CK2-specific pharmacological inhibitors: 4,5,6,7-tetrabromobenzotriazole (TBB) and CX-4945, the latter being in phase I clinical trials for several cancers. The efficacy of the inhibitors was first confirmed by assessing the phosphorylation of Akt at S129, a CK2-specific phosphorylation site, or by CK2 in vitro kinase activity assays, after lysis of TAIL7 cells treated with or without each drug. Stimulation of T-ALL cell lines with IL-7 had a minor positive effect on CK2 activity, which was not statistically significant. However, treatment with TBB or CX-4945 revealed that CK2 inhibition completely abrogated IL-7-mediated activation of PI3K/Akt pathway, as demonstrated by Akt S473 phosphorylation. Remarkably, CK2 inhibition also prevented STAT5 activation, as measured by STAT5 Y694/Y699 phosphorylation. These results suggest that although CK2 activity is not regulated by IL-7, it is fundamental for the activation of two major IL-7-dependent survival pathways. In agreement, we found that both TBB and CX-4945 completely prevented IL-7 viability effects not only in T-ALL cell lines but also in primary leukemia cells collected from patients at diagnosis, as determined by flow cytometry analysis of FSCxSSC and Annexin V-PE/7AAD staining. Accordingly, IL-7-induced Bcl-2 upregulation and maintenance of mitochondrial transmembrane potential were both reversed by treatment with the CK2 antagonists. Furthermore, CK2 inhibition completely abrogated T-ALL cell proliferation, as determined by evaluation of DNA synthesis upon 3H thymidine incorporation. In summary, our study contributes to the elucidation of the mechanisms involved in IL-7-induced viability and proliferation in T-ALL, identifying CK2 as a master regulator not only of cell-intrinsic but also of growth factor-dependent activation of pro-survival pathways in this malignancy. Disclosures:No relevant conflicts of interest to declare.

Luteolin exerts anti-tumor activity through the suppression of epidermal growth factor receptor-mediated pathway in MDA-MB-231 ER-negative breast cancer cells

This study investigated the inhibitory effect of luteolin on MDA-MB-231 estrogen receptor (ER) negative breast tumor growth both in vitro and in vivo. Study results showed that luteolin suppresses 3H thymidine incorporation indicating cell growth inhibition, and this was accompanied by cell cycle arrest at the G2/M and S stages and apoptotic activity. Further analyses showed that luteolin exhibited cell cycle arrest and apoptotic activity by decreasing AKT, PLK1, cyclin B1, cyclin A, CDC2, CDK2, and Bcl-xL expression and increasing p21 and Bax expression. Underlying mechanisms of action exerted by luteolin included the down-regulation. EGFR mRNA expression followed by the inhibition of EGF-induced MAPK activation, including the phosphorylation of ERK, p38 and AKT. Luteolin-supplementation at 0.01% or 0.05% significantly reduced tumor burden in nude mice inoculated with MDA-MB-231 cells. In conclusion, luteolin effectively suppresses MDA-MB-231 ER-negative breast cancer cell growth, and its anticancer activity may be partly derived from inhibitory effects on EGFR-mediated cell survival.

P181 IL-36 signaling amplifies Th1 responses by enhancing proliferation and Th1 polarization of naïve CD4+ T cells

Introduction The interleukin (IL)-1 superfamily of cytokines comprises a set of pivotal mediators of inflammation, playing an important role in innate and adaptive immune responses. Since the cloning of IL-1 α and IL-1 β , a number of additional members of this family have been discovered, including IL-18, IL-33, IL-36a, IL-36b, and IL-36g. While IL-1, IL-18 and IL-33 are well recognized for their immunomodulatory effects, the action of IL-36 cytokines in immune responses has remained elusive. We have previously shown that IL-36 cytokines were able to stimulate the production of cytokines by dendritic cells (DC) and that IL-36b acts as an adjuvant to stimulate T helper (Th)1 responses in vivo. In the present study, we investigated more precisely the function of IL-36 in Th1 immune responses. Methods Th0 (CD62L hi CD44 lo CD4 + T) cells were isolated from spleen of WT or IL-36R KO mice by sorting on a FacsVantage and were activated with plate-bound anti-mouse CD3/CD28 antibodies before cytokines stimulation. The expression of receptors and IL-36R ligands were assessed by RT-qPCR. Th0 cell proliferation was determined by 3H thymidine incorporation and FACS (CFSE) analysis. The role of IL-36 in Th1 differentiation was determined using co-culture systems and cytokine combination assays. Cytokine protein levels were monitored by ELISA, Milliplex assay or FACS. The involvement of IL-2 was studied by using anti-IL-2 and anti-IL-2Ra (CD25) neutralizing antibodies. Mycobacterial infection was induced in WT and IL-36R deficient mice by intravenous injection of 10 7 living M. bovis BCG Connaught. Results IL-36R is predominantly expressed on nai¨ve CD4 + T (Th0) cells, while other IL-1R family members are predominantly expressed in other Th subsets. IL-36 cytokines act directly on Th0 cells by enhancing both cell expansion and IL-2 secretion. The specificity of the stimulatory effects of IL-36 cytokines was confirmed by using IL-36R −/− Th0 cells.IL-36R signaling is required both on BMDC and Th0 cells via IL-12 and IL-2 production respectively, to induce IFN- γ production. Like IL-18, IL-36 β acts as a co-polarization factor with IL-12 to promote Th1 response inducing IL-12R β 2, T-bet mRNA expression and IFN- γ production. Using IL-2 neutralizing conditions, we observed that IL-2 is not required for the effects of IL-36 onnai¨ve T cell survival and proliferation, whereas the effect of IL-36 on Th1 differentiation is IL-2 dependent. Moreover, we showed that IL-36 β produced endogenously by Th0 cells acts as an autocrine inducer of IFN- γ production. Finally, the effects of IL-36 on Th1 cell polarization in vitro were confirmed in vivo by using a systemic model of mycobacterial infection. After 4 weeks of BCG infection, there was a significant impairment of IFN- γ , IL-6, TNF- α and nitrite oxide production by ex vivo restimulated splenocytes of IL-36R deficient mice compared to WT mice. Conclusion Our findings point towards a critical function of IL-36 in the initiation and maintenance of Th1 responses in vitro, via IL-12 and IL-2 signaling and in adaptive immunity in a model of mycobacterial infection in vivo.

PPARγ agonists regulate the expression of stemness and differentiation genes in brain tumour stem cells

Background:Brain tumour stem cells (BTSCs) are a small population of cancer cells that exhibit self-renewal, multi-drug resistance, and recurrence properties. We have shown earlier that peroxisome proliferator-activated receptor gamma (PPARγ) agonists inhibit the expansion of BTSCs in T98G and U87MG glioma. In this study, we analysed the influence of PPARγ agonists on the expression of stemness and differentiation genes in BTSCs.Methods:The BTSCs were isolated from T98G and DB29 glioma cells, and cultured in neurobasal medium with epidermal growth factor+basic fibroblast growth factor. Proliferation was measured by WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2 H-5-tetrazolio]-1,3-benzene disulphonate) and 3H thymidine uptake assays, and gene expression was analysed by quantitative reverse--transcription PCR and Taqman array. The expression of CD133, SRY box 2, and nanog homeobox (Nanog) was also evaluated by western blotting, immunostaining, and flow cytometry.Results:We found that PPARγ agonists, ciglitazone and 15-deoxy-Δ12,14-ProstaglandinJ2, inhibited cell viability and proliferation of T98G- and DB29-BTSCs. The PPARγ agonists reduced the expansion of CD133+ BTSCs and altered the expression of stemness and differentiation genes. They also inhibited Sox2 while enhancing Nanog expression in BTSCs.Conclusion:These findings highlight that PPARγ agonists inhibit BTSC proliferation in association with altered expression of Sox2, Nanog, and other stemness genes. Therefore, targeting stemness genes in BTSCs could be a novel strategy in the treatment of glioblastoma.

Abstract 5266: Anti-cancer drug development using bioactive compounds from Tillandsia recurvata in mouse models of human cancer

Abstract Background: The Jamaican Tillandsia recurvata species is being fully investigated scientifically for medicinal properties and has demonstrated powerful anti-cancer properties in vitro and in vivo. Aim The scope of this study was to investigate the anti-tumor and anti-HIV effects of Ball Moss extracts, isolates and synthesized molecules both in vivo and in vitro in five histogenic cell lines: Melanoma, Prostate, Breast, Kaposi sacrcoma and B16-Lymphoma cancer cell lines. Methodology/Experimentation: The bioactive compounds from Ball Moss were first extracted, isolated and identified. The fresh plant material was collected from power lines in Jamaica after which it was thorough washed, dried in a steam oven at 70°C, then milled and extracted with methanol (MeOH). Isolation of the bioactive compounds was done in two phases. Firstly, isolates from the crude extract was isolated by column chromatography and sub-fractions processed by bio-guided fractionation. During phase two, advanced separation technologies, including HPLC, supercritical fluid chromatography and capillary electrophoresis, were used followed by LC/MS and finally NMR. The bioassays of the isolated fractions were tested against the five different histogenic cancer cell lines in vitro using trypan blue exclusion and 3H Thymidine incorporation assays. The in vivo studies included 10 nude mice per group and using the crude form of the extract at 10mg per mice per day for 7 days. The extracts were tested in vivo against the same tumor cell lines and compared to controls administered normal saline treatment. Results: Utilizing the bioassay-guided fractionation process, the bioactive moiety was isolated at 98% purity. This purified compound tested in vitro demonstrated to be highly effective at a rate of in excess of 95% cell kill in the 5 different histogenic cell lines. The in vivo studies were equally impressive utilizing the crude extract. All 5 different tumors responded to the treatment by reducing the tumor size from 0.4mm X 0.4mm to almost non-existent on gross examinations of tumor cell lines (see images below). While the histological assessment using immuno-histochemical staining revealed that 90 to 95% of the tumors had undergone cell death cell which was due to apoptosis. There were no observations in the animals of any indications of any toxicity in the in vivo studies. The representative sample of gross tumors from a xenograft of the growth of Kaposi Sarcoma in nude mice and the histological results showing extensive necrosis following the use of Ball Moss extract and the immune-histochemistry, demonstrated cell death by apoptosis. It is to be noted that the treatments of all 5 cancer cell demonstrated the same level of bioactivity. Discussion/Conclusion: The findings from this study strongly indicate that these newly extracted compounds have significant anti-cancer / anti-HIV properties. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5266. doi:1538-7445.AM2012-5266