R1 Cell Research Articles

IL-6 and IL-8 secretion by human glioma cells proliferating after Gamma-knife irradiation

One of the modern methods of treating patients with primary and recurrent brain tumors is radiosurgical irradiation using Gamma Knife, which allows therapeutic doses to be delivered to tumors not exceeding 2.5 cm in diameter in 1–2 sessions. Tumor cells on the periphery of this tissue volume that receive lower radiation doses can resume proliferation and serve as a source of recurrence. The increase of radiation dose may cause necroses formation and a worsening prognosis. The properties of glioblastoma cells that survive and resume proliferation long after stereotactic irradiation are still poorly known. The aim of the work was to evaluate the expression of IL-6 and IL-8 by glioblastoma A172, R1, T2, and T98G cell lines that resumed proliferation after sublethal Gamma Knife irradiation. Cells were irradiated once at doses ranging from 6 to 16 Gy, and then cultured for 40 days. Cell number was counted weekly; lethal and sublethal irradiation doses for each glioblastoma cell line were determined. In cultures descendant from proliferation of single most resistant cells, the level of IL-6 and IL-8 secretion after 96 hours cultivation (ng/1000 cells) was determined by ELISA. The cells of all four glioblastoma lines secreted IL-6 and IL-8 into culture medium. The highest production of cytokines, never before demonstrated for glioblastomas, was discovered in R1 cells. Glioblastoma T2 also had high interleukin production levels. In contrast to these lines, glioblastoma A172 (highly sensitive to the action of cytostatic drugs and radiation) secreted IL-6 at 30 times lower level than R1 cells. Glioblastoma T98G (highly resistant to the action of cytostatic drugs and radiation) also exhibited low interleukins production level. R1, T2, and T98G glioblastoma cells that resumed proliferation after irradiation had increased secretion of IL-6 and, to a lesser extent, IL-8. The dependence of cytokine production increase on irradiation dose for these cells was not linear. In contrast, A172 cells reduced IL-6 and IL-8 secretion under irradiation. The multidirectional changes in IL-6 and IL-8 production by cells of different glioblastoma lines were long-term and persisted for more than a month. The presented results cast doubt on the possibility to use IL-6 and IL-8 production by glioblastoma cells as potential biomarkers for early diagnosis, therapy monitoring as well as prognostic markers of the disease course.

Abstract 1687: Cyto R1 Platform enriches samples’ viability for single-cell sequencing and downstream assays

Abstract Single-cell level investigations such as DNA and RNA sequencing, protein analyses, and phenotypic studies require an input sample with a high rate of viability (>90%). If too many dead cells are present in the input, degraded proteins and ambient DNA or RNA can increase background noise that may lead to missing identification of crucial targets. Since the cost for single-cell sequencing experiments still remains high, it is critical to ensure the input sample is optimized for high viability to ensure cost-effective and reproducible data. Current bead assays or other sample viability enrichment techniques used in preparation for single-cell analyses typically result in a significant sample loss, sometimes sacrificing 80% of the starting sample, and these preparation steps require multiple passes (> 45 minutes time) and costly kits. These beads or kits can be biased and change the cell population, subject cells to unwanted stresses, and diminish sample integrity with the time needed for preparations. Here, we investigate sample cell viability enrichment on the Cyto R1 Platform, a label-free, cell enrichment, sorting, and recovery platform. At the core, the Cyto R1 uses Cyto Chips, microfluidic technologies with electrical fields, to phenotypically enrich and sort various cells based upon cells’ unique physical structures and subcellular features. Thus, the Cyto R1 ensures native cell recovery without any unwanted cell tagging to maximize sample integrity. In this work, a variety of single cell solutions derived from T cell lymphoma tumors, ascites fluid, and organ tissues were enriched for viable cells to obtain a final sample viability over 90%. Additionally, we aimed to achieve a minimum of 50,000 viable cells, a common target for single-cell sequencing, in as rapid a timeframe as possible. In one sample origin tested, murine T cell lymphomas, the average starting viability was 65 ± 6.5%, and the final viability after enrichment on the Cyto R1 Platform was 95.8 ± 1.8%. Similarly, murine ascites cell mixtures were enriched from average viabilities below 55% up to 91.6 ± 4.2%. For all samples tested, >50,000 viable cells were obtained in under 30 minutes. This work demonstrates that the Cyto R1 can effectively and efficiently enrich a variety of starting samples for high viability (>90%) as required for downstream assays and single-cell sequencing. Citation Format: Alexandra R. Hyler, Alicia M. Cole, Ridi Barua, Amiran K. Dzutsev, Eva M. Schmelz. Cyto R1 Platform enriches samples’ viability for single-cell sequencing and downstream assays [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1687.

A new species of <i>Tephritis</i> Latreille (Diptera: Tephritidae) from Iran

Tephritis nozarii sp. n. from East Azerbaijan Province (NorthWest of Iran) is described and illustrated. It is similar to T. hendeliana Hering, T. hyosciami Linnaeus and T. multiguttata Becker in its pale brownish grey wing pattern, differing by the number of hyaline spots in marginal part of r1 cell and some other details of pattern. Cell r1 in T. nozarii sp. n. contains 3 subequally large spots (resembling some Campiglossa species), a character rarely encountered in Tephritis.

Abstract 2627: Inhibition of TGFβ in colorectal cancer cells is associated with a compensatory activation of AXL and p38 MAPK signaling pathways

Abstract According to the consensus molecular subtypes (CMS), almost 23% of human colorectal cancer (CRC) are classified in the CMS4 group, characterized by a mesenchymal signature and the activation of transforming growth factor β (TGF-β) signaling. AXL is a tyrosine kinase receptor involved in epithelial to mesenchymal transition, angiogenesis and immune modulation in CRC. We have previously demonstrated that AXL gene is amplified in approximately 5% of human CRC, and that AXL inhibition causes a significant blockade of CRC cell proliferation and migration. Here we have evaluated the role of TGF-β signaling and the potential interaction between TGF-β and AXL in human CRC cell lines. We assessed the expression and activation of TGF-β and AXL in a panel of six human CRC cell lines by western blot (WB) and real time PCR. We tested the sensitivity of HCT116 and LOVO cells to treatment with galunisertib ( LY21209761), a selective TGF-β R1 inhibitor, by MTT, cell invasion, wound healing and soft-agar colony forming assays. Further, intracellular CRC cell signaling was evaluated following galunisertib treatment by WB. TGF-β receptors 1 and 2 were expressed in all human CRC cell lines (HCT116, SW480, LOVO, LIM 1215, SW48 and CaCo2), whereas AXL expression was restricted to HCT116, SW480, LOVO cells. Treatment with galunisertib had little or no effect on cancer cell growth, whereas it partially reduced TGF-β induced cell migration, invasion and colony formation in HCT116 and LOVO cells (that co-expressed both TGF-β receptors and AXL). Interestingly, TGF-β inhibition resulted in a concomitant significant activation of AXL and p38 MAPK in both cell lines, that could represent a cancer cell adaptive mechanism of resistance to galunisertib treatment. These results suggest a potential functional cross talk between TGF-β, AXL and p38 MAPK signals in human CRC cells. In this respect, further experiments are ongoing to better understand this interaction with the aim of identifying potential novel anti-AXL and anti-TGF-β therapeutic strategies. Citation Format: Davide Ciardiello, Pietro Paolo Vitiello, Nunzia Matrone, Valentina Belli, Claudia Cardone, Luca Poliero, Carola Borrelli, Gianluca Arrichiello, Giulia Martini, Vincenza Ciaramella, Giusi Barra, Floriana Morgillo, Teresa Troiani, Davide Melisi, Fortunato Ciardiello, Erika Martinelli. Inhibition of TGFβ in colorectal cancer cells is associated with a compensatory activation of AXL and p38 MAPK signaling pathways [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 2627.

The Long Noncoding RNA Lncenc1 Maintains Naive States of Mouse ESCs by Promoting the Glycolysis Pathway.

SummaryThe naive embryonic stem cells (nESCs) display unique characteristics compared with the primed counterparts, but the underlying molecular mechanisms remain elusive. Here we investigate the functional roles of Lncenc1, a highly abundant long noncoding RNA in nESCs. Knockdown or knockout of Lncenc1 in mouse nESCs leads to a significantly decreased expression of core pluripotency genes and a significant reduction of colony formation capability. Furthermore, upon the depletion of Lncenc1, the expression of glycolysis-associated genes is significantly reduced, and the glycolytic activity is substantially impaired, as indicated by a more than 50% reduction in levels of glucose consumption, lactate production, and extracellular acidification rate. Mechanistically, Lncenc1 interacts with PTBP1 and HNRNPK, which regulate the transcription of glycolytic genes, thereby maintaining the self-renewal of nESCs. Our results demonstrate the functions of Lncenc1 in linking energy metabolism and naive state of ESCs, which may enhance our understanding of the molecular basis underlying naive pluripotency.

The Effect of Fotemustine on Human Glioblastoma Cell Lines

Glioblastomas are a heterogeneous group of tumors of neuroectodermal origin. Tumor cell cultures obtained from patients with glioblastomas differ in morphology and phenotype, as well as genetic characteristics. In the current work we used glioblastoma cell lines A172 and T98G (known since the 1970s) and the new glioblastoma lines R1 and T2 (obtained in 2010 from surgical material of patients). The purpose of this study was to evaluate the effect of the alkylating antitumor drug fotemustine (FM) after single and repeated exposure on the survival and proliferation of these glioblastoma cells, as well as the effect of FM on gene expression of growth factors, extracellular matrix proteins, mesenchymal cell markers, and transcription factor Zeb1. The effect of FM on A172 and R1 glioblastoma cells differs from its effect on T98G and T2 cells. LD50 was in an amount of 60 μg/mL for A172 cells and 250 μg/mL for T98G cells. Single exposure of A172 and R1 cell lines to 100 μg/mL of FM resulted in the death of all cells in culture. A172 and R1 cell exposure to sublethal doses of FM caused decreased activity of all studied genes. After incubation of T98G and T2 glioblastoma cells with FM in sublethal doses we identified two cell populations: proliferating cells and nonproliferating growth-arrested cells. Cell exposure to FM in doses over 300 μg/mL led to the predominance of growth-arrested cells in populations. These cells remained viable for 1.5 months. The effect of FM on T98G and T2 cell lines was accompanied by increased activity of all the studied genes. In conclusion, a population of growth-arrested, drug-resistant cells has been revealed in T98G and T2 cell cultures after exposure to FM. These cells exhibit high expression of genes encoding factors associated with the epithelial–mesenchymal transition. We discuss the possible impact of nonproliferating cells actively producing growth factors on the tumor microenvironment.

Characterization of New Human Glioblastoma Cell Lines

Glioblastomas are tumors of neuroectodermal origin with a high degree of diversity. Tumor cells isolated from the surgical material of patients with glioblastomas are heterogeneous populations varying in morphology, phenotype, and genetic characteristics. This paper presents a description of two new glioblastoma cell lines, R1 and T2, isolated from tumor tissue of patients in 2010. We investigated their morphological and cytochemical cell characteristics and the expression of neuronal, mesenchymal, and endothelial markers, as well as the activity of genes encoding a number of growth factors, extracellular matrix proteins, and intracellular proteins typical for cells of mesenchymal origin. R1 and T2 cell lines are morphologically different. T2 cell line was characterized by the presence of multinuclear cells. In terms of the expression of β-tubulin III, MGMT, and p53 protein, R1 cell line was more heterogeneous than T2. R1 and T2 glioblastoma cell lines also differed in the presence and ratio of cell populations with mesenchymal, neuronal, and endothelial markers. Thus, neuronal markers CD133/2 and CD56 were detected only on R1 cells. Both lines were characterized by high activity of growth factor genes TGFβ1, VEGF, and FGF2(b), lower activity of EGF, and high expression of THBS1 and αSMA genes. However, the activity of most of the genes under study in R1 cells was higher than in T2 cells. The greatest difference lay in the expression of HGF, FAP, and TNC. Comparison of two new glioblastoma cell lines, R1 and T2, with the continuously cultivated lines А172 and T98G showed that R1 line had remarkable similarity with A172, while T2 glioblastoma resembled T98G cells. Apparently, the differences between R1 and T2 cell lines are determined by the properties of the initial tumors rather than the time of the cell cultivation.

Integrating Enhancer Mechanisms to Establish a Hierarchical Blood Development Program

GATA-2 levels must be stringently regulated to ensure normal hematopoiesis, and human GATA-2 mutations cause hematologic disorders. GATA-2-regulated enhancers differentially control Gata2 expression in hematopoietic stem/progenitor cells and are essential for hematopoiesis and embryonic development. Mechanisms underlying how the enhancers control Gata2 expression and GATA-2 instigated genetic networks in a cell-specific manner are not completely understood. Targeted deletion of an intronic Gata2 enhancer 9.5 kb downstream of the transcription start site (+9.5) abrogates HSC genesis in the aorta-gonad-mesonephros (AGM) region (Gao et al., JEM, 2013). By contrast, the -77 kb enhancer (-77) activates transcription in myeloid progenitors, and its deletion impairs progenitor differentiation (Johnson et al., Science Advances, 2015). To dissect relationships between the enhancers, we developed a compound heterozygous (CH) mouse model bearing +9.5 and -77 enhancer mutations on different Gata2 alleles. While the CH embryos were alive at E13.5, nearly all died by E14.5 (p = 3.58 x 10-5). Flow cytometric analyses and embryo confocal imaging demonstrated that CH embryos have modestly reduced HSC numbers in the fetal liver (2.9-fold) and the AGM (41%, p = 7.8 x 10-5), which was comparable to +9.5+/- embryos. Thus, -77 does not genetically interact with +9.5 to control HSC emergence. Flow cytometric analysis revealed that Lin-Sca1-Kit+ myelo-erythroid progenitors were 6.6-fold lower in CH vs. WT embryos (p = 1.8 x 10-11), with the difference involving disproportionate losses of GMP (8.6-fold; p = 3.7 x 10-6) and MEP (379-fold; p = 3.2 x 10-9). By contrast, +9.5+/- fetal livers had 2-fold fewer myeloid progenitors, which involved similar reductions of CMP (2.1-fold; p = 1 x 10-6), GMP (2.6-fold; p = 0.0007) and MEP (1.9-fold; p = 0.002). Consistent with the myelo-erythroid progenitor reductions and MEP depletion, CH fetal livers lacked BFU-E (p < 0.001) and CFU-GEMM (p < 0.001) in a colony assay. These results illustrate a genetic interaction between +9.5 and -77 in progenitors, but not HSCs, and a new paradigm in which both enhancers must reside on a single allele to generate MEPs.As erythroid precursor cells express GATA-2, we tested whether the -77 deletion impairs erythroid maturation due to a reduction in myelo-erythroid progenitors or due to a cell-autonomous requirement of the enhancer in erythroid precursors. -77-/- E14.5 fetal livers were pale and smaller than WT counterparts, and -77-/- fetal liver cellularity was reduced 7.2-fold (5.3 x 10-4). When liver size was taken into account, there was little difference in the number of E14.5 R1 cells in -77-/- liver vs. WT littermates (p = 0.31). However, -77-/- R2-R5 cells declined sharply (R2, 8.2-fold, p = 0.004; R3, 14-fold, p < 10-5; R4, 9.7-fold, p = 0.002; R5, 14-fold, p = 0.087). The mutant R1 cells were defective in forming BFU-Es and CFU-Es. Analysis of transcriptomes of purified 77-/- and WT R1 cells from E14.5 fetal livers revealed 2805 and 2519 upregulated and downregulated (p < 0.05) genes, respectively, in -77-/- R1 cells. The -77 enhancer conferred GATA-2 expression, which strongly upregulated GATA-1 and therefore a large GATA-1 target gene cohort. A comparison of WT and -77-/- R1 cell transcriptomes with those of early (Tgbfr3low) and late (Tgbfr3high) BFU-Es (Gao et al., Blood, 2016) revealed a -77-/- R1 signature that correlated with the early BFU-E signature (r = 0.73, p < 10-4) and negatively correlated with the late BFU-E signature (r = -0.42, p = 4 x 10-4) differing from WT cells. In addition to GATA-1 target gene alterations, 253 of the -77-activated genes were not GATA-1-regulated in the G1E-ER-GATA-1 system. These genes included Ryk, which encodes a non-canonical Wnt receptor, and had not been studied in erythroid cells. Two Ryk shRNAs significantly decreased BFU-Es and CFU-GMs in lineage-depleted fetal liver cells. Ongoing studies are integrating Ryk function into signaling circuits that control erythroid maturation and analyzing other -77-regulated targets predicted to constitute new regulators of erythroid cell maturation/function. Thus, loss of the -77 enhancer creates multi-faceted defects in erythroid precursors, involving deficiencies of constituents of signaling and transcriptional circuitry required to enable and drive erythroid maturation. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Difference in suitable mechanical properties of three-dimensional, synthetic scaffolds for self-renewing mouse embryonic stem cells of different genetic backgrounds.

We evaluated whether the genetic background of embryonic stem cells (ESCs) affects the properties suitable for three-dimensional (3D) synthetic scaffolds for cell self-renewal. Inbred R1 and hybrid B6D2F1 mouse ESC lines were cultured for 7 days in hydrogel scaffolds with different properties derived from conjugating 7.5, 10, 12.5, or 15% (wt/vol) vinylsulfone-functionalized three-, four-, or eight-arm polyethylene glycol (PEG) with dicysteine-containing crosslinkers with an intervening matrix metalloproteinase-specific cleavage sites. Cell proliferation and expression of self-renewal-related genes and proteins by ESCs cultured in feeder-free or containing 2D culture plate or 3D hydrogel were monitored. As a preliminary experiment, the E14 ESC-customized synthetic 3D microenvironment did not maintain self-renewal of either the R1 or B6D2F1 ESCs. The best R1 cell proliferation (10.04 vs. 0.16-4.39; p < 0.0001) was observed in the four-arm 7.5% PEG-based hydrogels than those with other properties, whereas the F1 ESCs showed better proliferation when they were embedded in the three-arm 10% hydrogels. Self-renewal-related gene and protein expression by ESCs after feeder-free 3D culture was generally maintained compared with the feeder-containing 2D culture, but expression patterns and quantities differed. However, the feeder-free 3D culture yielded better expression than the feeder-free 2D culture. In conclusion, genetic background determined the suitability of hydrogel scaffolds for self-renewal of ESCs, which requires customization for the mechanical properties of each cell line. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 2261-2268, 2017.

Directed Differentiation of Dopamine-Secreting Cells from Nurr1/GPX1 Expressing Murine Embryonic Stem Cells Cultured on Matrigel-Coated PCL Scaffolds.

Parkinson's disease (PD) is a progressive neurological disorder characterized by a large number of motor and non-motor features and is known as the second most common neurodegenerative disorder after Alzheimer's disease. The hallmark pathology of PD is the damage and death of dopamine-producing neurons in the substantia-nigra of midbrain. Intrastriatal transplants of fetal mesencephalon derived DAergic neurons have provided proof-of-principle for the cell replacement strategy and have demonstrated reinnervation of the denervated striatum. However, ethical, technical, and practical limitations of deploying fetal DAergic neurons as the source for cell therapy in PD have ceased the spread of this procedure into clinical practice. Embryonic stem (ES) cells have emerged as a therapeutic alternative that can proliferate extensively and generate dopamine-producing neurons. To this extent and to surmount the obstacles related to embryonic neural cells, many investigations have focused on using pluripotent stem cells for the derivation of DAergic neurons. In the present study, a mouse embryonic stem (mES) R1 cell line was generated which could stably co-express Nurr1 (an essential transcription factor in DAergic neuron development) and GPX-1 (a neuroprotective enzyme against oxidative stress). The Nurr1/GPX-1-expressing ES cells (Nurr1/GPX-1-ES) were differentiated into DAergic-like cells via a three-dimensional culture environment consisting of Poly-ε-Caprolactone (PCL) nanofibrous scaffolds embedded by Matrigel (Mtg) in the presence of specific signaling molecules. DAergic neuron-specific genes were highly expressed in ES-derived DAergic neurons cultured and differentiated on PCL/Mtg scaffolds. Reverse-phase HPLC confirmed that the Nurr1/GPX-1-ES-cells differentiated on PCL/Mtg electrospun scaffolds could efficiently and exclusively secrete dopamine in response to stimulus. In conclusion, our results demonstrated that PCL/Matrigel nanofibrous scaffolds could efficiently support and promote the generation of functional DAergic-like cells from Nurr1/GPX-1-ES cells. The results of this study may have an impact on future tissue engineering for cell therapy of PD.

Cistrome Control of Hematopoieitic Stem/Progenitor Cell Function

Cis-regulatory mechanisms control chromatin structure and cellular identity. At the GATA2 locus, two cis-elements are linked to human pathologies, including a primary immunodeficiency (MonoMAC syndrome) associated with multiple complex phenotypes, myelodysplastic syndrome, and acute myeloid leukemia (AML). Mutations that disrupt the function of an intronic GATA2 +9.5 element cause MonoMAC syndrome, while an inversion that relocates the distal GATA2 -77 element to the EVI1 locus induces AML. The +9.5 and -77 cis-elements are GATA-2-occupied and confer context-dependent enhancer activities in select hematopoietic cell types in vivo. In knockout mouse models, the Gata2 +9.5 cis-element is required for hematopoietic stem cell (HSC) genesis, whereas the Gata2 -77 cis-element governs a unique sector of the myeloid progenitor cell transcriptome without impacting HSC genesis. Three other GATA-2-occupied cis-elements (-1.8, -2.8 and -3.9) were not individually required for hematopoietic development, and had relatively mild effects on Gata2 expression; the -1.8 site was required to maintain Gata2 repression in late-stage erythroblasts, the -2.8 conferred maximal Gata2 expression, and the -3.9 had no effect on Gata2 expression. We predict that additional cis-elements exist in the genome with functions resembling the +9.5 and -77, and their analysis will provide important mechanistic and biological insights. We utilized the known properties of Gata2 cis-elements as learning tools to identify prospective constituents of a hematopoietic stem/progenitor cell (HSPC) regulatory cistrome genome-wide. Using sequence attributes shared with the critically-important +9.5 element, namely a CATCTG-8bp spacer-AGATAA, we generated a list of 797 candidate cis-elements ("+9.5-like" elements). This list was prioritized using chromatin occupancy by GATA-2 and Scl/TAL-1, among others, chromatin accessibility, evolutionary conservation, and histone modifications in a multitude of biologically-relevant cell types. Gene editing was used to delete three high-ranked elements (Samd14 +2.5, Bcl2l1 +12.2, and Dapp1 +23.5), revealing their importance for transcriptional activation, GATA-2 occupancy and chromatin accessibility, while deletion of two low-ranked elements (Mrps9 +17.6 and Mgmt +182) had no effect on gene transcription. One such cis-element (Samd14 +2.5) resided in Samd14, a gene with undescribed biological function. Samd14 has a conserved sterile α-motif and coiled-coil domain, and is highly expressed in hematopoietic progenitors and differentiated progeny. Mouse knockout of the Samd14 +2.5 element dramatically lowered expression of Samd14 in hematopoietic progenitors. We conducted loss-of-function analysis to elucidate Samd14 function in lineage-depleted (Lin-) E14.5 fetal liver cells infected with control or Samd14 shRNA-expressing retrovirus. In a CFU assay, Samd14 knockdown reduced BFU-E and CFU-GM colonies 3.4-fold. Early erythroid precursor R1 (CD71low, Ter119-) and R2 (CD71high, Ter119-) cell populations decreased ~2-fold, concomitant with increases in more mature R3 and R4/5 populations (Ter119+). In R1/R2 cells, Samd14 knockdown reduced surface c-Kit expression by 1.6-fold and prevented Stem Cell Factor/c-Kit activation of AKT. Cellular deficits resulting from Samd14 knockdown could be rescued by c-Kit. In -77-/- common myeloid progenitors, Samd14 was ~20-fold downregulated. Thus, the importance of Samd14 and the Samd14 +2.5 element on progenitor function and SCF/c-Kit signaling validates our strategy for identifying cis-elements relevant for hematopoiesis. Our findings demonstrate that +9.5-like elements control cell signaling (Samd14 +2.5) and apoptosis (Bcl2l1 +12.2), and we predict that additional cistrome constituents will control these and other important HSPC processes. I will discuss the mechanistic and biological properties of additional cis-elements analyzed from a cohort of 68 GATA-2-occupied elements and general principles arising from the HSPC cistrome analysis, which provide unique insights into the control of hematopoiesis and GATA-2-linked pathologies. DisclosuresNo relevant conflicts of interest to declare.

Biochemical signatures of in vitro radiation response in human lung, breast and prostate tumour cells observed with Raman spectroscopy

This work applies noninvasive single-cell Raman spectroscopy (RS) and principal component analysis (PCA) to analyze and correlate radiation-induced biochemical changes in a panel of human tumour cell lines that vary by tissue of origin, p53 status and intrinsic radiosensitivity. Six human tumour cell lines, derived from prostate (DU145, PC3 and LNCaP), breast (MDA-MB-231 and MCF7) and lung (H460), were irradiated in vitro with single fractions (15, 30 or 50 Gy) of 6 MV photons. Remaining live cells were harvested for RS analysis at 0, 24, 48 and 72 h post-irradiation, along with unirradiated controls. Single-cell Raman spectra were acquired from 20 cells per sample utilizing a 785 nm excitation laser. All spectra (200 per cell line) were individually post-processed using established methods and the total data set for each cell line was analyzed with PCA using standard algorithms. One radiation-induced PCA component was detected for each cell line by identification of statistically significant changes in the PCA score distributions for irradiated samples, as compared to unirradiated samples, in the first 24–72 h post-irradiation. These RS response signatures arise from radiation-induced changes in cellular concentrations of aromatic amino acids, conformational protein structures and certain nucleic acid and lipid functional groups. Correlation analysis between the radiation-induced PCA components separates the cell lines into three distinct RS response categories: R1 (H460 and MCF7), R2 (MDA-MB-231 and PC3) and R3 (DU145 and LNCaP). These RS categories partially segregate according to radiosensitivity, as the R1 and R2 cell lines are radioresistant (SF2 > 0.6) and the R3 cell lines are radiosensitive (SF2 < 0.5). The R1 and R2 cell lines further segregate according to p53 gene status, corroborated by cell cycle analysis post-irradiation. Potential radiation-induced biochemical response mechanisms underlying our RS observations are proposed, such as (1) the regulated synthesis and degradation of structured proteins and (2) the expression of anti-apoptosis factors or other survival signals. This study demonstrates the utility of RS for noninvasive radiobiological analysis of tumour cell radiation response, and indicates the potential for future RS studies designed to investigate, monitor or predict radiation response.

TH-C-220-12: Biochemical Radiation Response of Irradiated Human Tumour Cells Observed via Raman Spectroscopy

Purpose: To use non-invasive single-cell Raman spectroscopy (RS) and principal component analysis (PCA) to analyze radiation-induced biochemical changes in human tumour cell lines, and to correlate results with known radiosensitivity. Methods: Six human tumour cell lines, derived from prostate (DU145, PC3, and LNCaP), breast (MDA-MB-231 and MCF7) and lung (H460), were irradiated with single fractions (15, 30 or 50 Gy) of 6 MV photons. Remaining live cells were harvested for RS analysis at 0, 24, 48, and 72 hours post-irradiation, along with unirradiated controls. Single-cell Raman spectra were acquired from 20 cells per sample with a Raman microscope utilizing a 785 nm excitation laser. All spectra (200 per cell line) were post-processed, and the total data set for each cell line was analyzed with PCA using standard algorithms. Results: One unique radiation-induced PCA component was detected for each cell line by identification of statistically significant changes in the PCA score distributions for irradiated samples, as compared to unirradiated samples, in the first 24 to 72 hours post-irradiation. Correlation analysis between radiation-induced PCA components from the different cell lines shows that these RS radiation response signatures fall into 3 distinct RS categories: R1 (H460 and MCF7), R2 (MDA-MB-231 and PC3), and R3 (DU145 and LNCaP). These RS categories segregate according to radiosensitivity and p53 gene status. The R1 and R2 cell lines are radioresistant (SF2 > 0.6), whereas the R3 cell lines are radiosensitive (SF2 < 0.5). The R1 and R2 cell lines further segregate according to p53 status, as verified by cell cycle analysis post-irradiation. Conclusions: This study demonstrates that RS is a powerful tool for non-invasive radiobiological investigations, and is a promising technique for advancing the field of personalized radiation therapy.

Telomerase Reverse Transcriptase Related with Telomerase Activity Regulates Tumorigenic Potential of Mouse Embryonic Stem Cells

Embryonic stem cell (ESC) research gave rise to the possibility that stem cell therapy could be used in the treatment of incurable diseases such as neurodegenerative disorders. However, problems related to the tumorigenicity of undifferentiated ESCs must be resolved before such cells can be used in the application of cell replacement therapies. In the present study, we attempted to determine biomarkers that predicted tumor formation of undifferentiated ESCs in vivo. We differentiated mouse ESCs (R1 cell line) into neural lineage using a 5-step method, and evaluated the expression of oncogenes (p53, Bax, c-myc, Bcl2, K-ras), telomerase-related genes (TERT, TRF), and telomerase activity and telomere length during differentiation of ESCs. The expression of oncogenes did not show a significant change during differentiation steps, but the expression of telomerase reverse transcriptase (TERT) and telomerase activity correlated with mouse ESCs differentiation. To investigate the possibility of mouse TERT (mTERT) as a biomarker of tumorigenicity of undifferentiated ESCs, we established mTERT knockdown ESCs using the shRNA lentivirus vector and evaluated its tumorigenicity in vivo using nude mice. Tumor volumes significantly decreased, and appearances of tumor formation in mice were delayed in the TERT-knockdown ESC treated group compared with the undifferentiated ESC treated group. Altogether, these results suggested that mTERT might be potentially beneficial as a biomarker, rather than oncogenes of somatic cells, for the assessment of ESCs tumorigenicity.

SONICC: A Novel Nonlinear Optical Detection Technique for 2D Celluar Crystallography

2D crystallization is one of the most powerful methods to study the structure and function of membrane proteins in their native lipid bilayer environment, yet obtaining high resolution diffraction protein crystals is the most difficult and time consuming step. Traditional techniques for protein crystal detection mostly depend on optical microscopy and the birefringence property of crystals. This situation becomes even more difficult for 2D crystallization because a negative stain check on electron microscope is required which makes it nearly impossible for high-throughput screening. Recently, UV spectroscopy has also been applied to distinguish protein crystals from salt ones. However, all these methods has detection limit which depends on the crystal size and crystallization condition. The Second Order Nonlinear Imaging of Chiral Crystals (SONICC) therefore is developed to overcome this difficulty. SONICC could selectively detect large or small non-centrosymmetric 2D and 3D protein crystals (<1um) with high signal noise ratio. Using this method, we successfully detected purple membrane (ie 2-D crystal of bacteriohrodopsin). We could even detect the crystal patch in a single living H. Salinarium R1 cell. By removing retinals from purple membrane, the signal dramatically decreased (∼5 fold) due to the distortion of the crystalline order and the absence of pigment. Considering the difficulty of growing 2D membrane crystals, SONICC not only solved the current detection limitation, but also provided a new opportunity for direct detection membrane crystals in situ formed naturally or artificially by overexpression.

Microcalorimetric Studies of the Biological Effects of Ho(III) on Halobacterium halobium R1

The biological effect of Ho3+ on Halobacterium halobium R1 growth was analyzed using the microcalorimetric method. Using the LKB-2277 Bioactivity Monitor with the ampoule method at 37 degrees C, the thermogenic curves of the growth of H. halobium R1 were obtained. Then, the maximum power (P (m)) and the growth rate constants (k) were determined, and the values of P (m) and k were linked to the concentration of Ho3+. In all, the addition of Ho3+ cause a decrease in the maximum heat production and growth rate constants. To confirm the results, the shapes of H. halobium R1 cell addition with Ho3+ using a transmission electron microscope (TEM) were observed. According to the thermogenic curves and TEM photos of H. halobium R1 under different conditions, it is clear that the metabolic mechanism of H. halobium R1 growth has been changed with the addition of Ho3+.

Expression of conserved signalling pathway genes during spontaneous vascular differentiation of R1 embryonic stem cells and in Py-4-1 endothelial cells

Embryonic stem (ES) cells are an invaluable model for identifying subtle phenotypes as well as severe outcomes of perturbing gene function that may otherwise result in lethality. However,though ES cells of different origins are regarded as equally pluripotent,their in vitro differentiation potential varies, suggesting that their response to developmental signals is different. The R1 cell line is widely used for gene manipulation due to its good growth characteristics and highly efficient germline transmission. Hence, we analysed the expression of Notch, Wnt and Sonic Hedgehog (Shh) pathway genes during differentiation of R1 cells into early vascular lineages. Notch-, Wnt- and Shh-mediated signalling is important during embryonic development. Regulation of gene expression through these signalling molecules is a frequently used theme, resulting in context-dependent outcomes during development. Perturbing these pathways can result in severe and possibly lethal developmental phenotypes often due to primary cardiovascular defects. We report that during early spontaneous differentiation of R1 cells, Notch-1 and the Wnt target Brachyury are active whereas the Shh receptor is not detected. This expression pattern is similar to that seen in a mouse endothelial cell line. This temporal study of expression of genes representative of all three pathways in ES cell differentiation will aid in further analysis of cell signalling during vascular development.

Microcalorimetric Studies of the Biological Effect of Holmium (III) on Halobacterium halobium R1 Growth

The biological effect of Ho3+ on Halobacterium halobium R1 growth was analyzed by a microcalorimetric technique. By means of LKB-2277 Bioactivity Monitor, ampoule method at 37 degrees C, we obtained the thermogenic curves of H. halobium R1 growth. To analyze the results, the maximum power (Pm) and the growth rate constants (k) were determined, which show that values of Pm and k are linked to the concentration of Ho3+. In all, the addition of Ho3+ causes a decrease of the maximum heat production and growth rate constants. For comparison, we observed the shapes of H. halobium R1 cell by means of transmission electron microscope (TEM). According to the thermogenic curves and TEM photos of H. halobium R1 under different conditions, it is clear that metabolic mechanism of H. halobium R1 growth has been changed with the addition of Ho3+.

172 PRIMORDIAL GERM CELL DIFFERENTIATION FROM ES CELLS IN VITRO IN MOUSE

The primordial germ cells (PGC) in the genital ridge of the embryo are the progenitors of sperm and eggs. The goal of the present study was to derive PGC cells from embryonic stem (ES) cells and compare their gene expression with that of primary PGC cultures. R1 (Nagy A et al. 1990 Development 110, 815–821) and Oct4-GiP (Ying QL et al. 2002 Nature 416, 545–548) ES cell lines were differentiated into PGCs. For in vitro differentiation, the modified method of Geijsen N et al. (2004 Nature 427, 148–154) was used. In brief, ES cell suspension was put into hanging drops (400 cells per drop) for two days, where they formed embryoid bodies (EBs). The medium consisted of Iscove's Modified Dulbecco's Media (Gibco) supplemented with 10% FBS (Hyclone, Logan, UT, USA), 30 μg mL−1 iron saturated transferrin (Gibco), 1 mM sodium pyruvate (Gibco), 0.1 mM 2-mercaptoethanol (Sigma, Hungary), non-essential amino acids (Sigma), 4.5 mM monothioglycerol (Sigma), 50 μg mL−1 ascorbic acid (Sigma), 2 mM glutamine (Gibco), and antibiotics. The EB clumps were differentiated in suspension culture for 2 or 5 days, and then dissociated with collagenase treatment. Cells positive for SSEA-1 were isolated from dissociated EBs by immunomagnetic bead sorting and plated into gelatinized plates in the presence of 2 μM retinoic acid (Sigma). After 7 days of culture, individual PGC colonies were isolated and subcloned. The subcloned PGCs were cultured in PGC medium consisting of Dulbecco's Modified Eagle Media (Gibco) supplemented with 15% FBS, 2 mM glutamine, 1 mM sodium pyruvate, 1000 U recombinant mouse leukaemia inhibitor factor (ESGRO®, Chemicon International, Inc., Temecula, CA, USA), 20 ng mL−1 basic fibroblast growth factor (Sigma), 60 ng mL−1 soluble mouse stem cell factor (Sigma), and antibiotics. The gene expression profile was monitored using semi-quantitative RT-PCR. The gene expression of Oct4, Nanog, Stella, Piwil2, Rnf17, and Tex14 were analyzed during the differentiation. Primary PGC cultures were also isolated from (C57BL/6 × DBA)F1 embryos of age 8.5 and 11.5 days post-coitum, and differentiated in vitro. The previously described PGC medium was used to proliferate the isolated cells. The gene expression profile of PGCs and ES-derived PGC lines were compared. There were no great differences between the gene expression profiles of PGCs and ES cell-derived PGC cells. SSEA-1 and alkaline phosphatase staining of cells did not show differences between the two cell populations. We have shown here the two PGC populations do not differ from each other in gene expression of the selected genes. Further investigation is needed to differentiate the PGCs into gametes and to analyze the gene expression of other genes involved in gamete differentiation. The authors would like to acknowledge Gyorgyi Kungl for the technical help. This research was supported by OTKA T046171 grant.

Role of caveolin-1 in etoposide resistance development in A549 lung cancer cells

Caveolin 1 expression is down-regulated in various cancer cell lines. Interestingly, in several drug-resistant cancer cells, a strong induction of caveolin 1 expression has been reported suggesting a role for caveolin 1 in the acquisition and/or the maintenance of multidrug resistance phenotype. In addition, it was reported that p-glycoprotein localized to caveolin-rich membrane domains in these cells. In this study, we progressively exposed A549 lung adenocarcinoma cells to increasing doses of etoposide. Both R1 and R2 cell lines had greatly increased levels of p-glycoprotein expression while mrp expression levels were moderately increased but only R2 cells had raised caveolin levels compared to control A549 cells. Both caveolin-1 and p-glycoprotein co-localize in Triton-insoluble membrane domains in all our cell lines but only caveolins-1 was solubilized by the addition of octylglucoside at 4C suggesting that these two proteins are located in different membrane domains. Using an anti-caveolin-1 antibody, we did not succeed to immunoprecipitate p-glycoprotein. Interestingly, total cellular cholesterol (the major lipid component of caveolae and triton-insoluble domains) was greatly increased in both R1 and R2 cell lines compared to naïve A549 cells.