Normal Human Lymphocytes Research Articles

Life History of Normal Human Lymphocytes Revealed By Somatic Mutations

Introduction: Mature blood cells harbor a mixture of mutations inherited from ancestral hematopoietic stem cells (HSCs) and mutations accumulated after maturation. The landscape of these somatic mutations in normal blood is poorly mapped, with questions as simple as "how many mutations does a memory T cell accumulate throughout life?" remaining unanswered. This gap in our knowledge is particularly relevant for hematopoietic malignancy- while we know that lymphomas derive from lymphocytes of particular stages of differentiation, we do not know if the patterns we see are reflected in their normal counterparts. Results: In order to characterize normal somatic mutation in lymphocytes, we performed single-cell expansion and whole genome sequencing of over 600 T and B lymphocytes and 200 HSC and progenitor cells across 5 individuals (ages 0-85). All lymphocyte subsets show increased mutation burden with respect to HSCs across all classes of variants (Figure 1). Some of this increase can explained by lymphocyte-specific mutational processes, such as the activity of RAG, accounting for at least 20% of observed structural variants. We also find a striking variation in mutation burden within and between lymphocyte subsets. Microenvironment specific mutational processes dominate the observed differences. Examples of this include germinal center ("non-canonical AID") mutations in memory B cells and UV-like mutations in memory T cells (putatively skin resident cells). Naive B and T cells show a lack of variation in discrete mutational patterns relative to their memory counterparts, and have mutational profiles and mutation burdens more similar to that of HSCs. We also observe differences in the mutational patterns between B and T cells that are indicative of the increased divergence of T lymphocytes from the HSC pool. In general, the mutation burden we observe in normal lymphocytes approach those seen in lymphoma. Conclusions: Our work highlights the substantial genetic diversity in normal lymphocytes, with some cells accumulating thousands of mutations on top of those inherited from the HSC compartment. These mutations can be used to describe the life history of each individual lymphocyte including their exposure to specific microenvironments. Our findings shed light on the biology of these cells and will help differentiate between normal and disease processes. Figure 1 Disclosures No relevant conflicts of interest to declare.

Cytotoxic effect of green synthesized silver nanoparticles in MCF7 and MDA-MB-231 human breast cancer cells in vitro

With the incessant rise in the cancer burden worldwide it is a dire need to develop anticancer agents that will offer negligible or no side effects and at the same time will be economically feasible. In this study, we utilized the principle of green chemistry where tyrosine and chitosan were used as reducer and stabilizer respectively to synthesize biocompatible silver nanoparticles. They were characterized by ultraviolet–visible spectroscopy, transmission electron microscopy and dynamic light scattering technique and found to be spherical with average diameter of 13–22 nm. Their toxicity was evaluated in MCF7 and MDA-MB-231 human breast cancer cell lines. MTT assay revealed excellent cytotoxic effect with IC50 values as low as 6.4 and 6.56 ppb respectively after 48 h of treatment. Intriguingly, they showed minimum toxicity in normal human peripheral blood lymphocytes at these effective concentrations. Cytomorphological alteration, ROS generation (DCFDA analysis) and nuclear fragmentation (Hoechst staining) were pronounced in both cancer cell lines following treatment. These nanoparticles also promoted expression and nuclear translocation of Nrf2 as an antioxidant response which was revealed by Western blot and immunofluorescence studies respectively. ‘Apoptosis assay’ confirmed the presence of apoptosis and ‘Caspase-8 activity assay’ revealed absence of the extrinsic apoptosis pathway. Western blot data (upregulation of p21, Bax/Bcl2 ratio, Caspase-9, Caspase-3 and cleaved PARP1) established the occurrence of intrinsic apoptosis pathway following cell cycle arrest. To conclude, the green synthesized silver nanoparticles are cytotoxic to cancer cells and can be considered as effective and safe cytotoxic agents in breast cancer therapeutics.

Foeniculum Vulgare and Pelargonium Graveolens Essential Oil Mixture Triggers the Cell Cycle Arrest and Apoptosis in MCF-7 Cells.

Fennel (Foeniculum vulgare) and rose geranium (Pelargonium graveolens) oils are known for their various biological effects including anticancer properties. This study aimed to evaluate the anticancer mechanism of fennel and geranium oils combined treatment on MCF-7 cells. The GC-MS method for essential oil characterization as well as the in vitro cytotoxicity, morphological changes, real-time PCR and immunocytochemical investigation for apoptosis-related markers, in addition, to flow cytometric cell cycle distribution analysis were done. The major constituents of both essential oils were anethole (55.33 %) and estragole (11.57 %) for fennel essential oil. However, cintronellol (34.40 %) and geraniol (8.67 %) were identified in geranium oil. The results revealed an IC50 of 220±5.7 and 60±2.1µg/ml for fennel and geranium oils, respectively. The mechanistic anticancer properties were investigated throughout the 70, 50, and 25µg/ml of oils mixture. The marked apoptotic morphology and the flow cytometric cell cycle distribution analysis in addition to the levels of apoptosisrelated makers such as p53, caspase-3, mir-21, mir-92a, Bcl-2, and ki-67 confirmed that fennel and geranium oils combination induced cell cycle arrest and apoptosis in MCF-7 cells. Moreover, the oils mixture did not exert any significant (P<0.01) toxicity on normal human peripheral blood lymphocytes in vitro. The findings showed that the mixture of oils exerted selective cytotoxicity towards MCF-7 cells through induction of cell cycle arrest and apoptosis which may be triggered by the synergistic effect between the active ingredients of fennel and geranium oils.

Triggering of apoptosis and cell cycle arrest by fennel and clove oils in Caco-2 cells: the role of combination

Background: Fennel (Foeniculum vulgare) and clove (Syzygium aromaticum) oils are known for their various biological effects, including anticancer properties.Objective: To investigate the anticancer effect of combined fennel and clove oil treatment on Caco-2 cells and normal human lymphocytes (NHL).Methods: GC-MS, in vitro cytotoxicity, morphological, apoptosis-related marker, and flow cytometric cell cycle distribution analyses were conducted.Results: Seventeen volatile compounds were identified in fennel oil, including trans-anethole (68.3%) and (+)-fenchone (8.1%). In clove oil, 22 compounds, including eugenol (71.4%) and caryophyllene (8.7%), were identified. IC50 of the fennel, clove, and oil mixture were 300 ± 5.0, 150 ± 4.0, and 73 ± 2.5 µg/mL, respectively with combination index (CI) < 1.0. Mechanistic anticancer properties were investigated using 30, 45, and 60 µg/mL oil mixture. Analysis of apoptotic morphology, flow cytometric cell cycle distribution, and apoptosis-related markers, such as Bcl-2 and Ki-67, confirmed cell cycle arrest and apoptosis induction in Caco-2 cells by the fennel and clove oil combination. Moreover, the oil mixture did not exert significant (p < 0.01) toxicity on NHL in vitro.Conclusion: The oil mixture exerted selective cytotoxicity towards Caco-2 cells through cell cycle arrest and apoptosis, which may occur through synergistic effects between fennel and clove oil active ingredients.

Cytotoxic and mutagenic effects of green silver nanoparticles in cancer and normal cells: a brief review

Applications of silver nanoparticles (AgNPs) have revolutionized the medicinal industry. Due to small size (1–100 nm) these nanoparticles are able to cross cell and nuclear membrane and induce cyto/genotoxicity. Green synthesized (plant based) AgNPs (GSAgNPs) have emerged as alternative antimicrobial agent to chemically synthesized nanoparticles assuming their methods of synthesis will be environment friendly and non toxic. In this review we report that the GSAgNPs were found to be cytotoxic and genotoxic to different cancer cell lines. They affected cell proliferation caused apoptosis and cell death of human cancer cells but did not cause such damages to normal human peripheral blood lymphocyte cells. This property can be utilized in cancer therapeutics to overcome the major drawbacks of chemotherapeutics with surface modifications of the nanoparticles by adding functionalized AgNPs.

Crystal Protein of a Novel Bacillus thuringiensis Strain Inducing Cell Cycle Arrest and Apoptotic Cell Death in Human Leukemic Cells

Parasporal inclusions of a native non haemolytic Bacillus thuringiensis strain KAU 59 was screened for its cytotoxicity against human lymphocytic leukemic cell line jurkat and normal human lymphocytes. The cytotoxicity of proteinase activated and non activated solubilised parasporal inclusions against both cell lines was assessed by Cell Titer 96 Aqueous Non Radioactive Cell Proliferation Assay Kit using MTS. The 50 per cent effective concentration (EC50) values were deduced from log probit analysis at 48 h. Morphological changes associated with cytotoxicity were evaluated and molecular mechanisms of cell death were elucidated by TUNEL assay at 48 h post-inoculation. The fluorescence assisted cell sorting was done in the flow cytometer to assess the stage of cell cycle arrest. Relative quantification of caspase-3 expression in Jurkat cells treated with parasporal inclusion protein of KAU 59 was done by qRTPCR The results indicated that the protein was cytotoxic to jurkat cells at the same time non toxic to normal lymphocytes. Cytotoxicity was evident only after proteolytic activation. Apoptotic cell death was confirmed in the protein treated cells by TUNEL Assay and also up regulated caspase-3 gene expression (P < 0.001). S phase cell cycle arrest was confirmed by and fluorescence associated cell sorting.

Abstract 3509: An unexpected effect of low-dose arsenic on genomic integrity

Abstract Background: Arsenic trioxide is currently used to treat acute promyelocytic leukemia and is known as a cytotoxic agent. Arsenic is also known as a carcinogen. However, a cytoprotective effect of low-dose arsenic (LDA) has also been observed in some studies. Our previous studies have found that pretreatment with LDA before DNA damaging agents such as chemotherapy and radiation therapy protects normal cells without impairing the cancer killing effects. We have demonstrated that intact p53 function is required in this process, and therefore, LDA does not protect cancer cells as most of them have mutated or dysfunctional p53 pathway. We have also reported that LDA helps protect the telomere from enhanced erosion by DNA damaging agents in ConA-activated normal human lymphocytes. As enhanced telomere shortening is associated with genomic instability, we decided to investigate the effect of LDA in genomic integrity. Methods: Mouse embryonic stem cells (mESCs) with a mutator phenotype in DNA repair pathways (MSH2-/-, Rad51KA and Ku70null) and their wildtype counterparts were utilized for cell viability studies and HPRT minigene mutation assay. Cell viability under different doses of LDA (0-1000nM) and radiation (0-4Gy) was firstly examined by colony formation assay. DNA damage and DNA repair capacity were also measured by comet assay in MSH2-/- mESCs. TailDNA% was analyzed by OpenComet and used to describe the comet feature. Results: Arsenic had little effect on the cell viability at doses ≤200nM in all tested cell lines. In MSH2-/- mESCs, there was no significant difference between the tailDNA% of cells treated with 200nM LDA (11.41±0.52%) and the control (10.82±0.49%) immediately after radiation. However, LDA treatment significantly decreased the tailDNA% 30 minutes after radiation (4.68±0.35% vs. 7.29±0.51%, Student’s test P&amp;lt;0.0014). While HPRT mutation frequency was not detectable in radiation-treated Rad51KA, Rad51wt and MSH2wt mESCs, LDA decreased the fraction of 6-Thioguanine resistant cells after radiation in MSH2-/-, Ku70null and Ku70res mESCs, suggesting fewer radiation-induced mutants were formed after LDA treatment. Discussion: Our data suggest that brief use of LDA enhances DNA repair activities and provides protection from radiation-induced mutations in mESCs. The inability to correctly repair DNA damage from chemotherapy and radiation therapy has been attributed to the development of therapy related secondary malignancy. Helping maintain genomic integrity with LDA during chemotherapy and radiation therapy may have a role in reducing therapy related secondary malignancy. Further work is in progress to test the potential use of LDA as a genome protector in vivo. Citation Format: Hang Su, Meijun Long, Mi Young Son, Teresa C Marple, Paul Hasty, Chul Soo Ha. An unexpected effect of low-dose arsenic on genomic integrity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3509.

Discovery of a novel oxadiazine derivative of glucocorticoids endowed with DNA binding activities and molecular docking studies

A series of new glucocorticoid oxadiazines (4–6) were synthesized by reacting glucocorticoids (1–3) with thiosemicarbazide and its derivatives. The structural assignment of products is confirmed on the basis of IR, 1H NMR, 13C NMR, MS and analytical data. The synthesized compounds (4–6) obeyed the Lipinski’s “Rule of Five” analysis based on a computational prediction of molecular and pharmacokinetic properties. The interaction studies of compounds (4–6) with DNA were carried out by employing single-cell gel electrophoresis (comet assay), UV-vis and fluorescence spectroscopy. Compounds (4–6) were found capable of cellular DNA degradation breakage in isolated normal human lymphocytes. Viscometric and steady-state measurements further correlated with the comet assay studies. Hence, it could be suggested that the glucocorticoid compounds bearing a core oxadiazine scaffold would be a potent biological agent. Molecular docking studies further characterize the interaction of the synthesized compounds with DNA.

Highly Multiplexed Fluorescence in Situ Hybridization for in Situ Genomics

The quantification of changes in gene copy number is critical to our understanding of tumor biology and for the clinical management of cancer patients. DNA fluorescence in situ hybridization is the gold standard method to detect copy number alterations, but it is limited by the number of genes one can quantify simultaneously. To increase the throughput of this informative technique, a fluorescent bar-code system for the unique labeling of dozens of genes and an automated image analysis algorithm that enabled their simultaneous hybridization for the quantification of gene copy numbers were devised. We demonstrate the reliability of this multiplex approach on normal human lymphocytes, metaphase spreads of transformed cell lines, and cultured circulating tumor cells. It also opens the door to the development of gene panels for more comprehensive analysis of copy number changes in tissue, including the study of heterogeneity and of high-throughput clinical assays that could provide rapid quantification of gene copy numbers in samples with limited cellularity, such as circulating tumor cells.

Manilkara zapota (L.) P. Royen Leaf Extract Derived Silver Nanoparticles Induce Apoptosis in Human Colorectal Carcinoma Cells Without Affecting Human Lymphocytes or Erythrocytes.

Plant-derived synthesis of silver nanoparticles (AgNPs) has found wide biomedical applications including cancer cure. This report deals with biosynthesis of silver nanoparticles (MZLAgNPs) employing leaf extracts of Manilkara zapota (L.) under optimized conditions. Characterization of MZLAgNPs using UV-Vis spectroscopy, FTIR, XRD, and FESEM analyses revealed that the particles were predominantly spherical averaging 24nm in size. Their cellular effects were assessed by MTT assay, fluorescence, and scanning electron microscopy of cells stained with propidium iodide, acridine orange/ethidium bromide, and annexin V-FITC to visualize signs of apoptosis. Evaluation of cell proliferation by clonogenic assay, wound healing ability by scratch assay and cell cycle distribution by flow-cytometry was also carried out. Apoptosis-related gene expressions were analyzed by RTq-PCR and western blot analysis. MZLAgNPs selectively inhibited growth of colorectal carcinoma HCT116, HeLa, and non-small lung carcinoma A549 cells, dose-dependently with IC50 concentrations of 8, 16, and 29μg/mL respectively, following 72-h treatment, without affecting growth of normal human lymphocytes and erythrocytes. Apoptosis induction was observed by fluorescence and scanning electron microscopy. Overproduction of reactive oxygen species (ROS), reduction of mitochondrial membrane potential, upregulation of apoptotic-related genes - PUMA, cas-3, cas-8, cas-9, and BAX, expression of caspase 3, and occurrence of PARP cleavage were observed in MZLAgNPs/cisplatin treated cells. Taken together, our results clearly demonstrate the therapeutic potential of biogenic MZLAgNPs as an effective agent for killing colorectal carcinoma cells by apoptosis induction.

Candelariella vitellina extract triggers in vitro and in vivo cell death through induction of apoptosis: A novel anticancer agent

Candelariella vitellina is common green-yellow lichen found on barks, wood, and rocks in Japanese forests. To investigate the mechanism of its anticancer potential, C. vitellina (80% MeOH/H2O) extract was prepared. High-performance liquid chromatography–high-resolution electrospray ionization mass spectrometry analysis revealed seven new compounds and 11 natural compounds of terpenes and polyketides. In vitro cytotoxicity analysis of Caco-2 cells exhibited an IC50 of 125 ± 4.1 μg/mL. No significant cytotoxicity was observed in vitro in normal human peripheral lymphocytes. Both the IC25 and IC50 were determined to explore the potent anticancer potential in this study. C. vitellina exhibited a mitochondrial P53-independent apoptotic effect with negative P53 expression and an elevated BAX/BCL2 ratio as well as upregulated CASP3 mRNA expression. Similarly, in vivo analysis showed the same pattern of anticancer potential but was dependent on the P53 expression. Furthermore, C. vitellina induced antioxidative conditions in vitro and in vivo. The decreased invasion of tumor cells in vivo and increased apoptotic features in vitro and in vivo suggest the moderate to strong apoptotic anticancer potential of C. vitellina. However, further studies are needed to determine the extent and mechanism of action on different cell lines to support the anticancer properties of this lichen.

Development of a Potential Gallium-68-Labelled Radiotracer Based on DOTA-Curcumin for Colon-Rectal Carcinoma: From Synthesis to In Vivo Studies.

Colorectal cancer is the third most commonly occurring cancer in men and the second most commonly occurring cancer in women worldwide. We have recently reported that curcuminoid complexes labelled with gallium-68 have demonstrated preferential uptake in HT29 colorectal cancer and K562 lymphoma cell lines compared to normal human lymphocytes. In the present study, we report a new gallium-68-labelled curcumin derivative (68Ga-DOTA-C21) and its initial validation as marker for early detection of colorectal cancer. The precursor and non-radioactive complexes were synthesized and deeply characterized by analytical methods then the curcuminoid was radiolabelled with gallium-68. The in vitro stability, cell uptake, internalization and efflux properties of the probe were studied in HT29 cells, and the in vivo targeting ability and biodistribution were investigated in mice bearing HT29 subcutaneous tumour model. 68Ga-DOTA-C21 exhibits decent stability (57 ± 3% after 120 min of incubation) in physiological media and a curcumin-mediated cellular accumulation in colorectal cancer cell line (121 ± 4 KBq of radiotracer per mg of protein within 60 min of incubation). In HT29 tumour-bearing mice, the tumour uptake of 68Ga-DOTA-C21 is 3.57 ± 0.3% of the injected dose per gram of tissue after 90 min post injection with a tumour to muscle ratio of 2.2 ± 0.2. High amount of activity (12.73 ± 1.9% ID/g) is recorded in blood and significant uptake of the radiotracer occurs in the intestine (13.56 ± 3.3% ID/g), lungs (8.42 ± 0.8% ID/g), liver (5.81 ± 0.5% ID/g) and heart (4.70 ± 0.4% ID/g). Further studies are needed to understand the mechanism of accumulation and clearance; however, 68Ga-DOTA-C21 provides a productive base-structure to develop further radiotracers for imaging of colorectal cancer.

HPLC Analysis of Phenolic Acids, Antioxidant Activity and in vitro Effectiveness of Green and Roasted Caffea arabica Bean Extracts: A Comparative Study.

Coffee is a popular drink; it is one of the most commercialized food products and a rich source of biologically active compounds that are important for human health. This study aimed to prove the anticancer activity of Green Coffee (GC) and Roasted Coffee (RC) bean aqueous extracts (Coffea arabica) on breast cancer adenocarcinoma cell line (MCF-7) and the safety of both extracts on normal Human Peripheral Blood Lymphocytes culture (HPBL). Total phenolic content for GC and RC extracts was measured and result of both extracts were (0.308±0.016 & 0.233±0.013mg/g) respectively. The phenolic acids were screened by HPLC at the wavelength of 254& 278 and 300 nm and 5-caffeoylquinic acids (Chlorogenic acid), the predominant form of phenolic acids, was identified in GC and RC samples. Ferric Reducing Antioxidant Power (FRAP) as well as the free radical scavenging activity (DPPH) proved the antioxidant properties of both extracts. The DPPH IC50 mean values of GC and RC extracts were (2.4±0.08, 2.3±0.16 µg/ml) respectively. Cytotoxicity of both extracts on MCF-7 cells were evaluated by neutral red uptake assay which showed the IC50 mean values (377±5.7,500±8.1 µg/ml) for GC and RC extracts respectively. The safety of both extracts (0, 125, 250, 500 µg/ml) on HPBL was evaluated in vitro using trypan blue exclusion method and DNA single strand breaks (alkaline comet assay). Result revealed non-significant cytotoxic difference (P<0.001) between cultures especially at lower doses of GC and RC extracts except the highest dose of GC and RC extract which showed slightly significant damage (P<0.001). This study proved that GC and RC aqueous extracts were found to be selectively cytotoxic in vitro to cancerous cells (MCF-7 cell line) causing cell death with no cytotoxicity on normal human lymphocytes especially at lower doses.

In vitro cytobiological effects of phytochemicals from Artemisia turanica

Background: Phytochemicals from the plants have been used as traditional medicine and can cause many biological effects such as antimicrobial, anticancer, mutagenic and etc. Aims: The subject of this research was to determine the chemical constituents and evaluate the antimicrobial, anticancer, cytotoxicity, mutagenic and anti mutagenic activities of the volatile oil of Artemisia turanica Krasch. Material and Methods: Antimicrobial properties were measured by agar disc diffusion method. Cytotoxicity was determined through 3-(4,5-dimethylthiazolyl)-2,5-diphenyltetrazolium bromide method on human lymphocytes and cancer HeLa cells. Toxicity, mutagenesis, and anti-mutagenesis effects of the essential oil were measured by the Ames Salmonella mutagenicity method, in the existence or absence of S9 as a metabolic activation. Results: Oxygenated monoterpenes, especially 1,8-cineole (35.2 %), α-thujone (24.2 %) and cis-chrysanthenol (16.8 %) were the main components in this volatile oil. Bactericidal kinetics of the volatile oil of A. turanica indicated that Esherichia coli is the most vulnerable one (minimal inhibitory concentration = 2.5 mg/ml, D value = 6.43 min). Cytotoxicity of the A. turanica oil in the human tumor cell line (17.67 μg/ml) was more than that observed in normal human lymphocytes (3291.49 μg/ml). Volatile oil of A. turanica showed suitable antimutagenic potency on 2-nitrofluorene, in the strain of Salmonella typhimurium TA98, without the existence of metabolic activation S9. Conclusion: The results achieved by human cells and with S. typhimurium prove that the volatile oil of A. turanica may be exploited as a natural anticancer and antimutagenic agent with low adverse side effects.

Febuxostat mitigates genotoxicity induced by ionizing radiation in human normal lymphocytes

Febuxostat mitigates genotoxicity induced by ionizing radiation in human normal lymphocytes

In silico studies and evaluation of antiparasitic role of a novel pyruvate phosphate dikinase inhibitor in Leishmania donovani infected macrophages

The present work deals with the identification and characterization of a novel inhibitor Z220582104, specific to pyruvate phosphate dikinase, for leishmanicidal activities against free promastigotes and intracellular amastigotes. We have used structure-based drug designing approaches and performed homology modelling, virtual screening and molecular dynamics studies. Primary mouse macrophages and macrophage cell line J774A1 were infected with promastigotes of Leishmania donovani. Both promastigotes and infected macrophages were subjected to treatment with the varying concentrations of Z220582104 or miltefosine for assessment of leishmanicidal activity. The novel inhibitor Z220582104 demonstrated growth inhibitory potential and reduced the viability of the free promastigotes in a concentration- and time-dependent manner. Z220582104 was also effective against the intracellular form of the parasites and reduced the number of amastigotes in macrophages and also lowered the parasite index, compared with the untreated infected macrophages. Although less effective compared with the miltefosine, Z220582104 is well tolerated by the dividing cells and normal human lymphocytes and monocytes with no adverse effects on the growth kinetics or viability. Our in silico and in vitro studies suggested that Leishmania donovani pyruvate phosphate dikinase could be a potential new drug target.

TFL Deletion Induces Incredible CXCL13 Secretion and Cachexia in Vavp-Bcl2Transgenic Mice

Diffuse large B-cell lymphomas (DLBCL) are heterogeneous diseases caused by several genetic aberrations. The novel post-transcriptional regulator gene called transformed follicular lymphoma (TFL) was first identified from t(2;6)(p12;q23), which appeared during the transformation of FL to DLBCL (Minagawa et al. Br J Haematol 2007). Normal human lymphocytes generally express TFL, but it is defective in some leukemia/lymphoma cell lines. TFL overexpression in such cell lines inhibited cell growth, suggesting that TFL functions as a tumor suppressor (Minagawa et al. Mol Cancer Res 2009). TFL locates in mRNA processing body in the cytoplasm and has the unique CCCH-type zinc finger motif functioning as RNase. TFL regulates several cytokines, including IL-2, IL-6, IL-10, TNF-α, and IL-17a, via mRNA degradation. In an experimental autoimmune encephalitis model, TFL null mice (TFL-/-) demonstrated persistent paralysis, resulting from more infiltration of Th17 cells into CNS with markedly increased IL-17a mRNA levels. Therefore, a TFL-driven feedback mechanism for excessive inflammation is indispensable to suppress T-cell-mediated autoimmune diseases (Minagawa et al. J Immunol 2014).TFL deletion examined by FISH using a 110kbp DNA probe containing an entireTFL locus was found in 12.8% of mature B-cell lymphomas (n=86, FL=30, DLBCL=40). However, the pathological significance of TFL deletion has not yet been clarified. To investigate how TFL loss affects lymphoma biology, we developed VavP-bcl2 transgenic (Bcl2-Tg)/TFL-/-mice. Although the survival of TFL-/- was comparable to the wild-type, Bcl2-Tg/TFL-/- died about 19 weeks earlier than Bcl2-Tg (Fig. 1). Both strains developed lymphadenopathy and splenomegaly similarly. No different microscopic finding was noted in lymph nodes, spleen, or bone marrow (BM). No additional malignancy was found in Bcl2-Tg/TFL-/- on autopsy. However, significant body weight loss appeared by 30 weeks in Bcl2-Tg/TFL-/- but not in Bcl2-Tg (Fig. 3).To identify what causes earlier death in Bcl2-Tg/TFL-/-, we carefully examined the phenotypic change of BM lymphocytes. We found a unique B220-IgM+ population in Bcl2-Tg BM, which was not found in wild-type. We speculated that TFL deficiency in this population might drive the deterioration in Bcl2-Tg/TFL-/-. To identify which mRNA was dysregulated by TFL deficiency, we comprehensively analyzed mRNA expression profiles in B220-IgM+ cells in both strains using cDNA microarray chip. Among several genes upregulated at least threefold in Bcl2-Tg/TFL-/- than Bcl2-Tg, we paid attention to CXCL13, the mRNA expression of which in Bcl2-Tg/TFL-/- was 4.19-fold higher than that in Bcl2-Tg (p=0.03). In fact, CXCL13 concentration in BM extracellular fluid as well as plasma in Bcl2-Tg/TFL-/- showed incredible increase in a logarithmic scale (Fig. 2). As a noteworthy event, body weight loss in Bcl2-Tg/TFL-/- followed the increase of CXCL13 in plasma by 30 weeks (Fig. 3). To confirm that TFL post-transcriptionally regulates CXCL13 mRNA through the degradation of its 3′UTR, we performed a reporter assay with a plasmid vector containing 3′UTR of CXCL13 mRNA. Co-transfection with a TFL expression vector showed decreased luciferase activity compared to the control. This suggests that TFL directly regulates CXCL13 mRNA via its 3′UTR degradation. This regulation occurs more prominently in B-cell lineage rather than myeloid or T-cell lineage, whereas IL-2 mRNA regulation occurs promiscuously.CXCL13 secretion was significantly increased in the culture supernatant of BM cells but not spleen cells derived from Bcl2-Tg/TFL-/-. We further sorted several cell populations, including B220-IgM+ in BM, and cultured them for 96 h. CXCL13 secretion from B220-IgM+ population was increased significantly compared to other populations. Thus, we concluded that B220-IgM+ cells in BM are the main producer of CXCL13 in Bcl2-Tg/TFL-/-. Loss of TFL-driven attenuation for excessive inflammation in lymphoma-bearing mice could contribute to the short survival. It is of interest whether high plasma CXCL13 directly affects cachexia and early death in Bcl2-Tg/TFL-/-.TFL deletion in human lymphoma might contribute not only to malignant transformation but also to a major B symptom, i.e., weight loss. Our findings may open a new window for the predictive factor on the prognosis of B-cell lymphoma and/or new therapeutic intervention by targeting CXCL13. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Revealing a human p53 universe.

p53 transcriptional networks are well-characterized in many organisms. However, a global understanding of requirements for in vivo p53 interactions with DNA and relationships with transcription across human biological systems in response to various p53 activating situations remains limited. Using a common analysis pipeline, we analyzed 41 data sets from genome-wide ChIP-seq studies of which 16 have associated gene expression data, including our recent primary data with normal human lymphocytes. The resulting extensive analysis, accessible at p53 BAER hub via the UCSC browser, provides a robust platform to characterize p53 binding throughout the human genome including direct influence on gene expression and underlying mechanisms. We establish the impact of spacers and mismatches from consensus on p53 binding in vivo and propose that once bound, neither significantly influences the likelihood of expression. Our rigorous approach revealed a large p53 genome-wide cistrome composed of >900 genes directly targeted by p53. Importantly, we identify a core cistrome signature composed of genes appearing in over half the data sets, and we identify signatures that are treatment- or cell-specific, demonstrating new functions for p53 in cell biology. Our analysis reveals a broad homeostatic role for human p53 that is relevant to both basic and translational studies.

Overexpression of p53 and Bax mediating apoptosis in cancer cell lines induced by a bioactive compound from Bacillus endophyticus JUPR15

Abstract Anticancer agents from microbial sources are gaining importance based on their safety and also due to the broad range of novel metabolites produced by them. In the present study, anti-cancer potential of a pink coloured bioactive fraction B5, extracted from Bacillus endophyticus JUPR15 was assessed on the cancer cells HeLa, HepG2, MCF-7 and toxicity was tested on normal human lymphocytes. Treatment with B5 resulted in a significant reduction in the survival of the cancer cells with the IC50 value ranging from 6.5 to 13.2 μg/ml. At the molecular level, the apoptotic cell death mediated by B5 was correlated with the upregulation of p53 and Bax genes along with the downregulation of Bcl-2 as per the results of flow cytometry and qRT-PCR. In response to the apoptosis-inducing stimulus of B5, activation of caspases along with cell cycle arrest at G1 phase were evidenced in the cancer cells. Non-toxic nature of B5 to normal human peripheral lymphocytes was also confirmed throughout the study. Characterization of the compound through GC–MS method indicated the presence of 9-Octadecenoic acid as one of the major components along with a thiophene derivative and a dinitrobenzamide derivative.

Evaluation of Cytotoxicity and Genotoxicity of Micromeria pulegium (Rochel) Benth Extract in Human Lymphocytes and Gr-M Melanoma Cells in vitro

Micromeria pulegium (Rochel) Benth. is an endemic species of Lamiacea family that includes frequently used plants in culinary and folk medicine. As cytotoxic potential of some species of Micromeria genus has been confirmed, this study aimed to test unknown antiproliferative and genotoxic potential of M. pulegium, endemic bh species, aqueous leaf extract in normal (human lymphocytes) and cancer (human melanoma GR-M) cells in order to protect small populations of native M. pulegium populations or promote its controlled micropropagation or cultivation. Cytokinesis-block micronucleus cytome assay was applied for human lymphocyte cultures, while trypan blue exclusion assay was used for evaluation of cytotoxicity in human GR-M melanoma cells. Results demonstrate no genotoxic effects up to concentration of 0.2 mg/ml in human lymphocyte in vitro but significant reduction of cell viability in human GR-M melanoma cell line cultures treated with 0.3 mg/ml of Micromeria extract.