Percentage Of GFP-positive Cells Research Articles

Residues 140-142, 199-200, 222-223, and 262 in the Surface Glycoprotein of Subgroup A Avian Leukosis Virus Are the Key Sites Determining Tva Receptor Binding Affinity and Infectivity.

Subgroup A avian leukosis virus (ALV-A) invades cells through gp85-encoded surface glycoprotein (SU) via specifically recognizing the cellular receptor Tva. To identify the key residues of ALV-A SU that determine the Tva binding affinity and infectivity in DF-1 cells, a strategy of substituting corresponding residues of SU between ALV-A RSA and ALV-E ev-1 (using Tvb as the receptor) was adopted. A series of chimeric soluble gp85 proteins were expressed for co-immunoprecipitation (co-IP) analysis and blocking analysis of viral entry, and various recombinant viruses based on replication-competent avian retrovirus vectors containing Bryan polymerase (RCASBP) were constructed for transfection into DF-1 cells and measurement of the percentage of GFP-positive cells. The results revealed that the substitution of residues V138, W140, Y141, L142, S145, and L154 of host range region 1 (hr1), residues V199, G200, Q202, R222, and R223 of host range region 2 (hr2), and residue G262 of variable region 3 (vr3) reduced the viral infectivity and Tva binding affinity, which was similar to the effects of the −139S, −151N, −155PWVNPF, −201NFD, Δ214–215, and −266S mutations. Our study indicated that hr1 and hr2 contain the principal receptor interaction determinants, with new identified-vr3 also playing a key role in the receptor binding affinity of ALV-A.

Overexpression of Calreticulin Impairs Self-Renewal Capacity of Hematopoietic Stem Cells

Mutations in JAK2, c-MPL and CALR have all been identified as drivers of MPN pathogenesis. JAK2 and c-MPL are critical for hematopoietic stem cell self-renewal and differentiation, however CALR's physiologic role in hematopoiesis is unknown. To investigate CALR's role in normal and malignant hematopoiesis lethally irradiated C57B/6 mice were transplanted with bone marrow cells infected with CALRDEL, CALRWT or empty MSCV-IRES-hCD4 vector retrovirus. As expected, mice transplanted with CALRDEL developed thrombocytosis, mice transplanted with CALRWT and empty vector had normal peripheral blood counts. At 6 months post-transplant mice were sacrificed. The percentage of hCD4+ whole bone marrow cells (marks cells expressing gene of interest) was 76.6% for empty vector, 73.1% for CALRWT, and 8.6% for CALRDEL. hCD4+ hematopoietic stem/progenitor cells (LKS), common myeloid progenitors (CMP), granulocyte monocyte progenitors (GMP), and megakaryocyte erythroid progenitors (MEP) from empty vector, CALRWT and CALRDEL mice were sorted into methylcellulose supplemented with cytokines (mIL-3, hEPO, and mSCF). MEP from CALRWT mice formed fewer colonies compared to empty vector and CALRDEL (p>0.004). GMP, CMP, and LKS from empty vector, CALRWT and CALRDEL formed equivalent numbers of colonies. To assess self-renewal function colonies were amalgamated and serially replated into methylcellulose at a concentration of 10,000 cells/ml. Upon serial replating CALRWT LKS cells formed fewer colonies compared to CALRDEL and empty vector (p>0.001). To more specifically assess the effect of CALR on HSC self renewal potential whole bone marrow from mice transplanted with CALRDEL, CALRWT or empty MSCV-IRES-GFP (MIG) vector was serially transplanted into lethally irradiated C57B/6 secondary recipient mice and the percentage of GFP-positive cells in the peripheral blood was measured by flow cytometry over time. Mice transplanted with CALRWT bone marrow cells quickly lost GFP positive cells in the peripheral blood over time whereas MIG empty vector and CALRDEL mice retained GFP positive cells for at least 8 months post-transplant.Because mutant CALRDEL interacts with and activates the thrombopoietin receptor (c-MPL) to drive MPN and because the defect in hematopoietic stem cell self-renewal that we observed in mice overexpressing CALRWT is reminiscent of the phenotype of mice lacking c-MPL we hypothesize that CALRWT interacts with c-MPL and negatively affects its function. Using co- immunoprecipitation of Ba/F3 cells doubly transduced with GFP tagged c-MPL and hCD4 tagged empty vector, CALRWT or CALRDEL, we found that CALRWT and CALRDEL interacts with c- MPL.In summary, these data demonstrate that overexpression of CALRWT negatively affects the self-renewal capacity of hematopoietic stem cells, evidenced by impaired ability to reconstitute secondary hosts and reduced serial re-plating ability in methylcellulose. This may be due to CALRWT binding to c-MPL and negatively regulating its function. DisclosuresNo relevant conflicts of interest to declare.

RAD54 promotes alternative lengthening of telomeres by mediating branch migration.

Cancer cells can activate the alternative lengthening of telomeres (ALT) pathway to promote replicative immortality. The ALT pathway promotes telomere elongation through a homologous recombination pathway known as break‐induced replication (BIR), which is often engaged to repair single‐ended double‐stranded breaks (DSBs). Single‐ended DSBs are resected to promote strand invasion and facilitate the formation of a local displacement loop (D‐loop), which can trigger DNA synthesis, and ultimately promote telomere elongation. However, the exact proteins involved in the maturation, migration, and resolution of D‐loops at ALT telomeres are unclear. In vitro, the DNA translocase RAD54 both binds D‐loops and promotes branch migration suggesting that RAD54 may function to promote ALT activity. Here, we demonstrate that RAD54 is enriched at ALT telomeres and promotes telomeric DNA synthesis through its ATPase‐dependent branch migration activity. Loss of RAD54 leads to the formation of unresolved recombination intermediates at telomeres that form ultra‐fine anaphase bridges in mitosis. These data demonstrate an important role for RAD54 in promoting ALT‐mediated telomere synthesis.

MRNA as a Tool for Gene Transfection in 3D Cell Culture for Future Regenerative Therapy.

A combination of three-dimensional (3D) cell culturing and non-viral gene transfection is promising in improving outcomes of cell transplantation therapy. Herein, gene transfection profiles in 3D cell culture were compared between plasmid DNA (pDNA) and messenger RNA (mRNA) introduction, using mesenchymal stem cell (MSC) 3D spheroids. Green fluorescence protein (GFP) mRNA induced GFP protein expression in 77% of the cells in the spheroids, whereas only 34% of the cells became GFP positive following pDNA introduction. In mechanistic analyses, most of the cells in MSC spheroids were non-dividing, and pDNA failed to induce GFP expression in most of the non-dividing cells. In contrast, both dividing and non-dividing cells became GFP-positive after mRNA introduction, which led to a high overall percentage of GFP-positive cells in the spheroids. Consequently, mRNA encoding an osteogenic factor, runt-related transcription factor 2 (Runx2), allowed in vitro osteogenic differentiation of MSCs in spheroids more efficiently compared to Runx2 pDNA. Conclusively, mRNA exhibits high potential in gene transfection in 3D cell culture, in which the cell division rate is lower than that in monolayer culture, and the combination of mRNA introduction and 3D cell culture is a promising approach to improve outcomes of cell transplantation in future regenerative therapy.

A novel screening system based on gene targeting to enrich the modified mammalian cells: without leaving selection marker and additional sequence

Gene targeting by homologous recombination (HR) has some disadvantages in screening modified cells that limits their use in targeting gene fragments in long exons. These disadvantages include retention of remaining selection marker after targeting, not removing cells with vector random integration, and leaving loxP sequences after removal of selection markers. Therefore, to overcome these disadvantages, we decided to design a eukaryotic two-step screening system to isolate the favorable, edited cells from undesirable cells in a gene targeting project. This system included two targeting plasmids containing one positive marker and two inducible negative markers. It was designed in such a way that, during the two-step HR and subsequent selection, only the well-edited cells survive and cells with vector random integration, and untargeted and episomal targeting plasmids are eliminated. The percentage of GFP-positive cells in two-step screening method (76.10 ± 3.50) was significantly higher than in the one-step screening method (0.90 ± 0.37) (p < 0.0001). GFP noise caused by the presence of the GFP-episomal expression plasmid had no significant effect on our results. We developed an efficient system to screen and enrich the HR-modified cells from undesired-HR and untargeted cells, without leaving the selection markers in mammalian cells. This method may be a promising method in ex vivo gene therapy approaches, especially when the target is a gene fragment within a large exon.

Abstract 5118: Development of new alternative in vivo models to study early metastasic dissemination in breast cancer

Abstract Breast cancer is the first cause of death from female cancer. After the treatments against the primary tumor, a few cells can prevail in a state of quiescence for a long period of time, becoming minimal residual disease. There is a clear need for alternative techniques to enable an early detection of the disease for a better understanding of it and the generation of therapies capable of acting with a smaller tumor burden. Although many models have been developed for the detection of macrometastasis, there is a limitation of in vivo models that allow the identification of disseminated cells, micrometastasis, or analyze another steps of the metastasic cascade, as embolization or cell arrest. The aim of this work is the generation of in vivo models to enable the detection of disseminated tumor cells (DTCs) We began our study by evaluating the presence of disseminated tumor cells in three in vivo models and quantification by FACS. To this end, we generated the F3II murine triple negative mammary carcinoma line expressing the fluorescent protein GFP (F3II GFP); the F3II line was used as control. In s.c heterotopic model, we observed the presence of positive cells in tumors and livers of mice inoculated with F3II GFP (Student's t test) 30 days post inoculation. In the s.c orthotopic model, we detected positive cells in tumors of mice inoculated with F3II GFP (Student's t test). In both models, we did not observe significant differences in the presence of lung DTCs. In experimental metastasis models, we detected DTCs 15 days post inoculation in lungs of mice injected i.v with F3II GFP (Mann Whitney test).We carried out the search for cells disseminated at 24 h in order to obtain an in vivo model that evaluates the establishment capacity of these cells, and we observed that the percentage of GFP positive cells was higher in those mice inoculated iv with F3II GFP (Student´s t test). In addition, the presence of DTCs was detected by fluorescence microscopy in blood smears of these mice, suggesting that at 24 h there are fluorescent tumor cells in both lung and blood. In conclusion, although the established models allowed us to detect DTCs, the experimental metastasis model was more efficient in the detection and quantification of disseminated tumor cells. These models allows the detection and quantification of DTCs in the lung and blood circulation within 24 hours and 15 days after inoculation and could be used to study the mechanisms of metastasic dissemination involved in these cells and to test the capacity of various anti-tumor drugs to the early stage of metastasis Citation Format: Johanna Elena Sidabra, Carla Sabrina Capobianco, Maria Florencia Gottardo, Daniel Fernando Alonso, Hernan Gabriel Farina. Development of new alternative in vivo models to study early metastasic dissemination in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5118.

UTX Affects Neural Stem Cell Proliferation and Differentiation through PTEN Signaling.

SummaryNeural stem cell (NSC) proliferation and differentiation in the developing brain is a complex process precisely regulated by intrinsic and extrinsic signals. Although epigenetic modification has been reportedly involved in the regulation of the cerebral cortex, whether UTX, an H3K27me3 demethylase, regulates the development of cerebral cortex during the embryonic period is unclear. In this study, we demonstrate that Utx deficiency by knockdown and conditional knockout increases NSC proliferation and decreases terminal mitosis and neuronal differentiation. Furthermore, we find that impairment of cortical development caused by lack of Utx is less significant in males than in females. In addition, UTX demethylates H3K27me3 at the Pten promoter and promotes Pten expression. P-AKT and P-mTOR levels are increased in the Utx conditional knockout cortices. Finally, Utx or Pten overexpression can rescue the impairment of brain development caused by Utx loss. These findings may provide important clues toward deciphering brain diseases.

Safe engineering of CAR T cells for adoptive cell therapy of cancer using long-term episomal genetransfer.

Chimeric antigen receptor (CAR) T‐cell therapy is a new successful treatment for refractory B‐cell leukemia. Successful therapeutic outcome depends on long‐term expression of CAR transgene in T cells, which is achieved by delivering transgene using integrating gamma retrovirus (RV) or lentivirus (LV). However, uncontrolled RV/LV integration in host cell genomes has the potential risk of causing insertional mutagenesis. Herein, we describe a novel episomal long‐term cell engineering method using non‐integrating lentiviral (NILV) vector containing a scaffold/matrix attachment region (S/MAR) element, for either expression of transgenes or silencing of target genes. The insertional events of this vector into the genome of host cells are below detection level. CD19 CAR T cells engineered with a NILV‐S/MAR vector have similar levels of CAR expression as T cells engineered with an integrating LV vector, even after numerous rounds of cell division. NILV‐S/MAR‐engineered CD19 CAR T cells exhibited similar cytotoxic capacity upon CD19+ target cell recognition as LV‐engineered T cells and are as effective in controlling tumor growth in vivo. We propose that NILV‐S/MAR vectors are superior to current options as they enable long‐term transgene expression without the risk of insertional mutagenesis and genotoxicity.

MP90-04 A ROLE FOR HISTONE DEMETHYALASE JMJD1A IN THE DNA DAMAGE RESPONSE OF PROSTATE CANCER CELLS

INTRODUCTION AND OBJECTIVES: The major therapies for prostate cancer (PCa) such as radio-, chemoor androgen deprivation therapy induce DNA damage. Thus the DNA damage response (DDR) and expression of DDR genes are key factors in determining outcome of PCa therapies. Histone demethylase JMJD1A regulates the gene expression by demethylating H3K9. Our previous work shows that JMJD1A is essential for the long-term proliferation and survival of PCa cells in vitro or in the xenograft prostate tumor model, partly via its effect on the activities of androgen receptor (AR) and c-Myc transcription factors. Here, we find a new role for JMJD1A in the expression of DDR genes and sensitivity of PCa cells to irradiation and androgen deprivation. METHODS: Real-time PCR was used to measure the mRNA levels of some DDR genes in the PCa cells (control or siRNA knockdown (KD) of JMJD1A, AR or c-Myc). Chromatin immunoprecipitation (ChIP) was used to evaluate the binding of JMJD1A to the AR or MYC binding sites on these DDR genes. JMJD1A-KD PCa cells were treated with or without ionizing radiation (IR), and analyzed by the r-H2AX staining (a widely used marker for the DNA double strand breaks (DSBs)), western blot analysis of DNA damage signaling proteins, flow cytometry analysis of cell cycle, and GFP reporter assays for the DSB repair via the homologous recombination (HR) or non-homologous end joining (NHEJ) pathways. The growth of these PCa cells was also measured under the IR and androgen deprivation conditions. RESULTS: JMJD1A was found to promote the expression of some DDR genes by co-activating AR and/or c-Myc. The DDR genes regulated by JMJD1A encode factors mediating the DSB repair by both HR and NHEJ pathways. JMJD1A KD led to increased r-H2AX foci in the non-irradiated cells, and the delayed clearance of r-H2AX foci in the irradiated cells. JMJD1A KD also increased the levels of phospho-DNAPK or phospho-CHEK2 in the western blot analysis and caused G2 cell cycle arrest. Consistently, JMJD1A KD reduced the percentage of GFPpositive cells that reflects the DSB repair by both HR and NHEJ pathways. JMJD1A-KD cells showed a reduced growth under IR and androgen deprivation conditions. CONCLUSIONS: JMJD1A promotes the DSB repair via regulation of DDR gene expression through AR and/or c-Myc. The role of JMJD1A in the DSB repair may contribute to the resistance of PCa cells to the radioand androgen deprivation therapies.

Lifeguard Inhibits Fas Ligand-mediated Endoplasmic Reticulum-Calcium Release Mandatory for Apoptosis in Type II Apoptotic Cells

Death receptors are members of the tumor necrosis factor receptor superfamily involved in the extrinsic apoptotic pathway. Lifeguard (LFG) is a death receptor antagonist mainly expressed in the nervous system that specifically blocks Fas ligand (FasL)-induced apoptosis. To investigate its mechanism of action, we studied its subcellular localization and its interaction with members of the Bcl-2 family proteins. We performed an analysis of LFG subcellular localization in murine cortical neurons and found that LFG localizes mainly to the ER and Golgi. We confirmed these results with subcellular fractionation experiments. Moreover, we show by co-immunoprecipitation experiments that LFG interacts with Bcl-XL and Bcl-2, but not with Bax or Bak, and this interaction likely occurs in the endoplasmic reticulum. We further investigated the relationship between LFG and Bcl-XL in the inhibition of apoptosis and found that LFG protects only type II apoptotic cells from FasL-induced death in a Bcl-XL dependent manner. The observation that LFG itself is not located in mitochondria raises the question as to whether LFG in the ER participates in FasL-induced death. Indeed, we investigated the degree of calcium mobilization after FasL stimulation and found that LFG inhibits calcium release from the ER, a process that correlates with LFG blockage of cytochrome c release to the cytosol and caspase activation. On the basis of our observations, we propose that there is a required step in the induction of type II apoptotic cell death that involves calcium mobilization from the ER and that this step is modulated by LFG.

Use of magnetic nanoparticles and oscillating magnetic field for non-viral gene transfer into mouse cornea

BackgroundInherited, iatrogenic, and metabolic corneal disease could be potentially treated by supplying a functional gene or changing the expression levels of specific genes. Viral-based gene therapy is efficient, but restricted by evoking immune responses and inflammation. This study aimed to transfect mouse cornea with a non-viral based (oscillating magnetofection) method. MethodsCultured mouse corneas were treated with magnetic nanoparticles (MNP) tethered to CAG promoter and green fluorescent protein (GFP) reporter plasmids exposed to a 1 Hz, 2 Hz, and 4 Hz oscillating magnetic field for 30 min and 60 min and in three DNA:MNP ratios (1:2, 1:1, 2:3). Corneas were cultured for up to 3 days and their green fluorescent channel intensity and number of GFP-positive cells were recorded. Transfection efficiency was estimated as the percentage of GFP-positive cells per total cells in a microscopic field. FindingsControl experiments with absent magnetic exposure showed no GFP-positive cells. The optimum condition was recorded at 3:2 DNA:MNP ratio, 1 Hz magnetic oscillation, and 30 min duration of magnetic exposure (mean GFP-positive endothelial cell count 191·7 [SD 54·5], p=0·009; mean green fluorescent intensity 85·3 [SD 48·5]; and average transfection efficiency 23·3% [range 10·6–30·9]). InterpretationA novel non-viral method of transfecting cornea, magnetofection, is demonstrated and gives proof of principle for its translation into corneal gene therapy. FundingWelsh Clinical Academic Training Scheme.

Toll-like Receptor 1 Is a Candidate Therapeutic Target in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a fatal disease that contains rare immature cells with self-renewal and leukemia-initiating capacity, known as leukemia stem cells (LSCs). Because current therapies are inefficient in eradicating LSCs, new therapies are warranted that efficiently target this cell population. One strategy being explored towards new therapies is identification of novel therapeutic targets on the cell surface of AML stem cells.To identify a cell surface protein upregulated on LSC, we used flow cytometry to measure the expression level of 13 cell surface proteins on immature leukemic cells from 18 AML patients and bone marrow cells from 7 healthy controls. We identified Toll-like receptor 1 (TLR1) as significantly upregulated in the immature CD34+CD38-compartment compared to corresponding normal cells that were almost devoid of TLR1 expression. These findings are consistent with elevated TLR1 mRNA levels observed in MDS patients (Wei et al, Leukemia, 2013).To evaluate the role of Tlr1 on immature leukemic cells, we performed shRNA-mediated inhibition of Tlr1 in MLL-AF9-expressing murine c-Kit+ leukemic cells. By using lentiviral vectors expressing the Tlr1-shRNAs along with a puromycin resistance gene, we identified two unique shRNAs that successfully suppressed the Tlr1 transcript and protein expression in comparison to a shRNA control. We next co-expressed the two Tlr1-shRNAs along with GFP in leukemia cells and monitored the percentage of GFP positive cells over time. Expression of the Tlr1-shRNAs resulted in strong depletion of the leukemic cells both in vitro and in vivo relative to the control shRNA. These findings suggest that TLR1 is important for the growth and survival of leukemic cells.To further address the role of Tlr1 on leukemic cells, we stimulated the leukemic cells with Pam3CSK4, a specific Tlr1/2 agonist. Pam3CSK4 alone was added to in vitro cultures of leukemic cells for three days, leading to increased survival and a slight increased number of leukemic cells. However, flow cytometric analysis revealed a differentiation shift of cells stimulated with Pam3CSK4 indicated by a decreased expression of the immature cell surface marker c-Kit and an increased expression of the myeloid linage marker Mac-1. To evaluate how Pam3CSK4 affects LSCs, we added Pam3CSK4 to ex vivo-cultures of leukemic cells for 3 days and then transplanted the cells into sublethally irradiated mice. Blood samples after two weeks showed a decreased leukemic burden in mice receiving Pam3CSK4-stimulated cells compared to controls. These findings suggest that enforced TLR1/TLR2-signaling causes differentiation of LSCs.In summary, this study demonstrates that TLR1 is upregulated on AML-stem cell enriched patient cells and that TLR1 expression is finely balanced to maintain LSCs. More specifically, our data suggest that the leukemic cells require Tlr1-expression for survival, but enhanced Tlr1/Tlr2-activation force the LSCs into differentiation. Hence, our study suggests that approaches aiming either for inhibition or enforced activation of TLR1 in AML should be explored further towards a potential new AML therapy. Collectively, we here identify TLR1 as a novel and promising candidate therapeutic target in AML. DisclosuresNo relevant conflicts of interest to declare.

RASSF2 Functions As a Tumor Suppressor in t(8;21) AML Via Promotion of Apoptosis

The t(8;21) chromosomal translocation is one of the most common chromosomal translocations associated with acute myeloid leukemia (AML), found in approximately 12% of de novo AML cases. The majority of these cases are classified as FAB-subtype M2 AML. The t(8;21) results in the stable fusion of the AML1 (RUNX1) and ETO (RUNX1T1) genes. The AML1-ETO fusion protein is composed of the N-terminal portion of AML1, which includes the DNA-binding Runt-homology domain, and nearly the full-length ETO protein. The primary accepted mechanism by which AML1-ETO promotes leukemia development is through the aberrant recruitment of transcriptional repression/activation complexes to normal AML1 target genes. Therefore, the identification of individual genes or biological pathways that are specifically disrupted in the presence of AML1-ETO will provide further molecular insight into the pathogenesis of t(8;21) AML and lead to the possibility for improved treatment for these patients.We identified RASSF2 as a gene that is specifically downregulated in (2-4 fold) in total bone marrow of t(8;21) patients compared to non-t(8;21) FAB-subtype M2 AML patients by analyzing publicly available gene expression datasets. Similarly, using a mouse model of t(8;21) AML we found Rassf2 mRNA levels to be nearly 30-fold lower in t(8;21) leukemia cells compared to wild-type Lin-Sca-cKit+ (LK) myeloid progenitors. Gene expression analysis by RT-qPCR in leukemia cell lines confirmed that RASSF2 mRNA levels are significantly downregulated (8-10-fold) in both Kasumi-1 and SKNO-1 t(8;21) cell lines as compared to a similar non-t(8;21) HL-60 cell line and to primary human CD34+ control cells. In addition, expression of AML1-ETO in HL-60 or CD34+ cells results in a decrease in RASSF2 mRNA expression, which further suggests that RASSF2 is a target for regulation by AML1-ETO. Assessment of published ChIP-seq data shows that AML1-ETO binds the RASSF2 gene locus at two distinct regions in both primary t(8;21) AML patient samples and in the Kasumi-1 and SKNO-1 cell lines. These regions are similarly bound by several important hematopoietic transcription factors in primary human CD34+ cells, including AML1, ERG, FLI1, and TCF7L2, implicating these two regions as important for the regulation of RASSF2 expression during blood cell differentiation. Overexpression of RASSF2 in human leukemia cell lines using an MSCV-IRES-GFP (MIG) construct revealed that RASSF2 has a strong negative effect on leukemia cell proliferation and viability. The overall percentage of GFP-positive cells in MIG-RASSF2 transduced cells markedly decreased compared to MIG-control transduced cells over a period of 14 days. This effect was primarily due to significantly increased apoptosis in the RASSF2 expressing cell populations. Similarly, we found that expression of RASSF2 significantly inhibits the long-term self-renewal capability of hematopoietic cells transduced with AML1-ETO in a serial replating/colony formation assay. AML1-ETO transduced hematopoietic cells were normally capable of serial replating for more than 6 weeks. However, AML1-ETO transduced cells co-expressing RASSF2 consistently had reduced colony number and lost their ability to replate after 3-4 weeks. This was due to a dramatically increased rate of apoptosis in RASSF2 expressing cells.RASSF2 is reported to be a tumor suppressor that is frequently downregulated at the transcriptional level by hypermethylation in primary tumor samples, but not healthy controls. Here we have identified RASSF2 as a target for repression, and demonstrated its tumor suppressive function in t(8;21) leukemia cells. Further insights into the molecular mechanisms of RASSF2 function in AML will continue to be explored. DisclosuresNo relevant conflicts of interest to declare.

Lentivirus vector-mediated Rho guanine nucleotide dissociation inhibitor 2 overexpression induces beta-2 adrenergic receptor desensitization in airway smooth muscle cells.

Beta-2 adrenergic receptor (β2AR) downregulation is critical to asthma rescue therapy; however, tolerance, also known as β2AR or bronchodilator desensitization, mechanisms potentially resulting in life-threatening rescue treatment failure remain poorly understood. Airway smooth muscle cells (ASMCs) from BALB/c mice were primarily cultured. The full-length Rho guanine nucleotide dissociation inhibitor 2 (RhoGDI2) gene from ASMCs was amplified by RT-PCR, and RhoGDI2 gene was subcloned into the digested PWPXL plasmid. The recombinant lentivirus PWPXL-RhoGDI2 expression plasmid was packaged into mature lentivirus by 293T cells and used to infect ASMCs. Fluorescent quantitation RT-PCR and Western Blot were used to detect the level of mRNA and protein expression of RhoGDI2, β2AR, guanine nucleotide exchange factor (GEF), GTPase-activating protein (GAP) and G protein-coupled receptor kinases (GRKs) in overexpression RhoGDI2-ASMCs group, negative GFP control ASMCs group and normal control ASMCs group. Membrane receptor numbers of β2AR was observed by radioligand receptor binding assay in overexpression RhoGDI2-ASMCs group, negative GFP control ASMCs group and normal control ASMCs group. RhoGDI2 vector successfully transfected ASMCs, with infection efficiency (the percentage of GFP-positive cells) >80%. RhoGDI2, GEF and G-protein-coupled receptor kinase 2 (GRK2) expressions significantly increased in the RhoGDI2 overexpression group compared to control and negative control groups (all P<0.05). Conversely, β2AR and GAP expressions were significantly lower in the RhoGDI2 overexpression group (both P<0.05), exhibiting an inverse correlation with RhoGDI2 expression. Control and negative control groups exhibiting β2AR density more than 2-fold higher than that observed in the RhoGDI2 overexpression group. RhoGDI2 reduces β2AR density, potentially by reducing β2AR and GAP expressions and increase GEF and GRK2 expressions. Thus, RhoGDI2 is central in cellular β2AR desensitization, though this full mechanism and intermediates merit further investigation.

284 CHARACTERIZATION OF GOAT MESENCHYMAL STEM CELLS DERIVED FROM BONE MARROW AND ADIPOSE TISSUE

Gene modification of cells in vitro followed by somatic cell nuclear transfer (SCNT) currently offers the best route for creating genetically modified livestock species. However, low cloning efficiencies in differentiated somatic cells have been attributed to the possibility of improper nuclear reprogramming. Adult stem cells may have greater developmental potential and better nuclear reprogramming potential following cloning. There is considerable interest in using goats as models for genetically engineering dairy animals and for using stem cells as therapeutics for bone and cartilage repair. Mesenchymal stem cells (MSC) are adult stem cells that that have been isolated and characterised from various species, but are poorly characterised in goats. Three MSC lines were isolated from bone marrow (9004 BM-MSC, 9003 BM-MSC) and adipose tissue (9003 A-MSC) of neonatal goats. In this study, these MSC lines were characterised to verify MSC-specific characteristics and assess their amenability to genetic modification in vitro. Passage 5 cells were evaluated for capacity to differentiate into osteogenic, adipogenic, and chondrogenic lineages, as well as for colony-forming efficiency after 10 days of culture from low-density plating. Expression of MSC-specific positive cell surface markers CD90, CD73, and CD105, as well as pluripotency markers Nanog, Oct-4, and Sox-2, was examined by RT-PCR. Oct-4 protein localization was examined by immunofluorescence. The MSC were also assessed for their potential for gene modification by nucleofection with circular and linearized plasmids expressing green fluorescent protein (GFP) and neomycin resistance. Differences between cell lines were statistically analysed using ANOVA. The 9003 BM-MSC cells were also utilised for SCNT. All 3 MSC lines showed a normal karyotype. The MSC lines were capable of undergoing osteogenic, adipogenic, and chondrogenic differentiation, with observed differences in capacities between the BM-MSC and A-MSC lines, as shown by staining with Alizarin Red S, Oil Red O, and Alcian Blue. Expression of CD90, CD73, CD105, Nanog, Oct-4, and Sox-2 was detected, and Oct-4 was localised in the cytoplasm. There were significant differences in clonability between the cell lines, with 9004 BM-MSC showing the highest colony-forming efficiency (61% ± 5.4; P &lt; 0.05). There were no significant differences in the percentage of GFP-positive cells from transfections done with the circular plasmid, but 9003 BM-MSC yielded a significantly lower number of integrant colonies per 500 000 cells transfected with the linear plasmid and G418 selection (12.75 ± 3.24; P &lt; 0.05). Somatic cell nuclear transfer was able to reprogram 9003 BM-MSC and produce pregnancies. One hundred forty-four embryos were reconstructed, 101 embryos were transferred into 8 recipients, and the resulting pregnancy rate was 73%. Our findings provide characterisation information on goat MSC, and show that significant differences can exist between MSC isolated from different tissues and from within the same tissue.

331 OPTIMIZATION OF BRANCHED 25kDa POLYETHYLENIMINE FOR EFFICIENT GENE DELIVERY IN BOVINE FETAL FIBROBLASTS

Cost-effective, highly efficient, and noncytotoxic transfection of bovine fetal fibroblasts (BFF) has proven difficult to achieve by regular chemical and physical methods. The aims of this study were to evaluate transient transfection efficiency and toxicity of commercially available branched 25 kDa polyethylenimine (25 kDa PEI, Sigma-Aldrich, St. Louis, MO, USA) and to optimize the transfection conditions leading to high percentages of PEI-transfected fibroblasts with minimum cytotoxic effects. Bovine fetal fibroblast (BFF) cells were seeded a day before transfection in 24-well plates at a density of 3 × 104 cells per well in DMEM with antibiotics and 10% SFB. When 70 to 90% confluence was reached, cells were washed with PBS and incubated in DMEM without antibiotics or SFB. For the transfection-mix preparation, increasing amounts of plasmidic DNA (pZsGreen1; 2 to 6 µg) were added to 50 µL of DMEM without antibiotics or SFB, incubated for 5 min at room temperature, and complexed with 0.5 to 4 µg of PEI (from 1 mg mL–1 solution) in 50 µL of PBS for 10 min. This transfection mix was added to the cell cultures and, 2 h later, 500 µL of DMEM with antibiotics and 10% SFB was added to each well. Detection of green fluorescent protein (GFP) expression by flow cytometry (reported as percentage of green fluorescent cells) was performed 48 h after transfection. Results were analysed by ANOVA and Tukey test and expressed as mean ± SEM (P &lt; 0.05). We found no significant difference between the percentage of GFP-positive cells transfected with 1 or 2 µg of 25 kDa PEI at 2 µg of DNA/well (15.2 ± 1.3 v. 16.9 ± 0.9%, respectively; P &gt; 0.05), whereas cells transfected with 1 or 2 µg of low-molecular-weight PEI (2 kDa) showed extremely low transfection efficiencies. Increasing the DNA load up to 6 µg significantly enhanced cell transfection (3.5- and 6-fold comparing 2 µg v. 4 µg and 6 µg of DNA, respectively; P &lt; 0.05) at 1 and 2 µg of 25 kDa PEI/well. In order to evaluate the cytotoxic effect of PEI, cell viability was determined using the MTT assay in 96-well plates (cells/well), with each condition scaled down to replicate the effect of 2 kDa or 25 kDa PEI in a 24-well plate. The MTT results (expressed as % of the control) indicated that PEI became cytotoxic at concentrations equivalent to 2 and 4 µg/well (54.7 ± 3.4 and 18.5 ± 5.7, respectively), whereas 1 µg/well produced a slight detrimental effect on cell viability (90.0 ± 2.6). No evidence of cytotoxicity was observed when the BFF were incubated with 0.5 µg/well of 25 kDa PEI and 1 or 2 µg/well of 2 kDa PEI. To study if a combination of low- and high-molecular-weight PEI could improve transfection efficiency and reduce toxicity, we tested a mixture (1 : 1) of 2 kDa and 25 kDa PEI. Even though the 1 : 1 mixture was less cytotoxic, the efficiency of gene delivery was not improved. We conclude that, under our experimental conditions, the highest percentage of GFP-expressing cells with good viability was obtained when 1 µg of 25 kDa PEI was added per well. Therefore, branched 25 kDa PEI transfection represents an efficient, simple, and cost-effective alternative for gene delivery in bovine fibroblast cells in culture.

Abstract 3348: Pax8-PPARδ fusion protein activates β-catenin/TCF pathways

Abstract Pax8-PPARδ Fusion Protein (PPFP) occurs in ∼35% of follicular thyroid carcinomas. Expression of PPFP in the non-transformed rat thyroid cell line PCCL3 conferred on the cells the ability to invade through matrigel and to form colonies in anchorage independent conditions. PPFP also increased the percentage of cells that have activated β-catenin/TCF. We transfected control and PPFP expressing PCCL3 thyroid cell lines with two different β-catenin/TCF-GFP reporter systems. We then sorted a population of GFP positive cells and expanded them to obtain 100% transfected cell lines. These GFP positive cells gave rise to a new heterogenous population with a stable equilibrium between GFP positive, β-catenin/TCF active cells and GFP negative, β-catenin/TCF inactive cells. PPFP expressing cell lines contained more than twice the percentage of GFP positive cells compared to control cell lines. GFP positive PPFP expressing thyroid cells were enriched for cells exhibiting the transformed phenotypes previously observed in the unsorted, untransfected cell lines. Sorted β-catenin/TCF active single cells gave rise to heterogenous clonal populations containing both β-catenin/TCF active and negative cells. Clonal populations derived from β-catenin/TCF inactive cells remained purely negative. Clonal populations derived from GFP positive cells were twice as enriched for anchorage independent colony forming cells than GFP negative clones. More strikingly, GFP positive clones were 5-10 times more invasive than negative clones, which exhibited the same invasive potential as control cells. Treatment with PPARδ full or partial agonists further increased the β-catenin/TCF population in PPFP expressing cells. Again, the effects were confined to populations containing β-catenin/TCF active cells. Co-treatment with PPARδ antagonists reversed the effects of PPARδ agonists. These data suggest that β-catenin/TCF active cells have properties of cancer stem cells and that PPFP acts on this population to expand the stem cell like fraction. 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 3348. doi:1538-7445.AM2012-3348

Abstract 1344: ZAP-70 enhances migration of malignant B lymphocytes toward lymphoid organs in a Burkitt lymphoma xenograft model

Abstract Chronic Lymphocytic Leukemia (CLL) is a lymphoproliferative disease characterized by the accumulation and proliferation of mature B-lymphocytes in the blood, bone marrow and secondary lymphoid organs. CLL patients with adverse outcome prognosis can be identified by the presence of a high ZAP-70 expression. ZAP-70, a protein tyrosine kinase that plays a crucial role in cellular activation in T and NK cells, has been related to aggressive features of the CLL cells, such as higher migrative capacity in vitro. In order to analyze the consequences of ZAP-70 ectopic expression in an in vivo model, we stably transfected a Burkitt cell line (Raji) with a vector expressing a ZAP-70-GFP fusion protein. Raji transfectants showed constitutively active ZAP-70 protein. Subsequently, thirty-four 8-week old SCID mice were inoculated intravenously with 5 x106 cells from each cell line (control Raji-GFP, 12 mice; Raji-ZAP-70-GFP, 9 mice). Mice were euthanized when hind legs paralysis, dyspnea, or tumor growth was observed. Organs were obtained to quantify the percentage of GFP-positive cells present in each organ by flow cytometry. Median survival of mice injected with the ZAP-70 cell line did not differ from that observed in the control mice (16 days, p=0.658). Percentage of GFP positive cells was analyzed by flow cytometry in different tissue compartments (Table 1). We observed a significantly higher percentage of infiltrating GFP-positive cells in the bone marrow from mice injected with ZAP-70 expressing cell line (58.8%±6.08 vs 4.2%±1.4, p&amp;lt;0.001), whereas in the rest of organs the infiltration was comparable (Table 1). Raji-ZAP-70-GFP has a differential expression of CCR7 and CD49d among others (Calpe et al, Blood 2011). In conclusion, malignant B-lymphocytes with ectopic expression of activated ZAP-70 protein showed enhanced ability to migrate toward and infiltrate the bone marrow, where soluble factors are produced to mediate homing, survival, and proliferation of tumor cells. 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 1344. doi:1538-7445.AM2012-1344

Development of Direct Intra-Bone Marrow Gene Transfer of Lentiviral Vectors Containing a B-Domain Variant of Human Factor VIII for Hemophilia A Treatment

Abstract 4710Lentiviral vectors (LVs) directly delivered to the bone marrow by intra-bone marrow (iBM) injection can efficiently transduce hematopoietic stem cells (HSCs) for treating genetic blood disorders. Our previous study showed that FVIII activity in the plasma of hemophilia A (HemA) mice achieved nearly normal levels one week after being treated with iBM injection of LVs containing a B-domain variant human factor VIII (hFVIII/N6) gene driven by a human elongation factor-1 promoter (E-LV). However, plasma FVIII activity fell very quickly with concomitant generation of anti-FVIII antibodies. In this study, to prevent anti-FVIII immune responses in the treated HemA mice, we treated three groups of mice with intraperitoneal injection of 200 μL of immunomodulatory protocols including IL-2/IL-2 mAb complexes (IL-2 Complex, 1 μg IL-2 mixed with 5 μg anti-IL-2 mAb per mouse on day −5, −4, and −3), anti-mouse CD3ε mAb (20 μg per mouse on day −2, −1, 0, 1 and 2), and rabbit anti-mouse thymocyte IgG (ATG, 0.6 mg per mouse on day −1 and 0), respectively. The mice were treated with 5 μL E-LV (7 × 107 TU/mL) on day 0. The peak plasma FVIII levels in 0/4 E-LV only treated mice, 3/5 IL-2 Complex pretreated mice, 2/3 anti-CD3ε pretreated mice and 4/5 ATS pretreated mice showed up one month after treatment. 2/4 E-LV only treated mice, 2/5 IL-2 Complex pretreated mice, 1/3 anti-CD3ε pretreated mice and 3/5 ATS pretreated mice generated anti-FVIII antibodies two months after treatment. These data indicated that all three immunomodulatory protocols protected E-LV transduced cells from eradication mediated by cytotoxic T cells in the early stage after treatment; however these protocols did not prevent the production of anti-FVIII antibodies at later time points. Next, we employed 10 HemA/Foxp3-Tg mice in which all T cells over-expressed Foxp3 as an immunosuppressive mouse model and treated them with low (5 μL) or high (25 μL) dosage of E-LVs, respectively. None of the 10 treated HemA/Foxp3-Tg produced anti-FVIII antibodies however plasma FVIII levels in all 10 mice fell to low levels one month after treatment. Thus, increase of regulatory T cells could not totally inhibit cytotoxic T cells to lyse transduced cells, whereas they prevented anti-FVIII antibody production. In order to examine whether LV-transduced cells were destroyed in the early stage post-injection, we treated HemA mice with 3 μL of LVs (4.6 × 1010 TU/mL) containing GFP gene driven by a modified myeloid proliferative sarcoma virus (MND) promoter and tracked GFP expression in bone marrow cells overtime. The percentage of GFP positive cells reached highest on day 15 post-injection, and then fell quickly afterwards, while GFP positive cells (0.04% of total bone marrow cells) were still detectable on day 94. Therefore, in order to achieve persistent transgene expression of hFVIII in the HemA mice treated with intra-bone marrow injection of E-LVs, cytotoxic-T-cell-mediated elimination of the transduced cells needs to be inhibited in the early stage and formation of anti-FVIII antibody can be eliminated by increasing FVIII-specific Treg cell populations. Disclosures:No relevant conflicts of interest to declare.

Inhibitory effect of aqueous dandelion extract on HIV-1 replication and reverse transcriptase activity

BackgroundAcquired immunodeficiency syndrome (AIDS), which is caused by the human immunodeficiency virus (HIV), is an immunosuppressive disease that results in life-threatening opportunistic infections. The general problems in current therapy include the constant emergence of drug-resistant HIV strains, adverse side effects and the unavailability of treatments in developing countries. Natural products from herbs with the abilities to inhibit HIV-1 life cycle at different stages, have served as excellent sources of new anti-HIV-1 drugs. In this study, we aimed to investigate the anti-HIV-1 activity of aqueous dandelion extract.MethodsThe pseudotyped HIV-1 virus has been utilized to explore the anti-HIV-1 activity of dandelion, the level of HIV-1 replication was assessed by the percentage of GFP-positive cells. The inhibitory effect of the dandelion extract on reverse transcriptase activity was assessed by the reverse transcriptase assay kit.ResultsCompared to control values obtained from cells infected without treatment, the level of HIV-1 replication and reverse transcriptase activity were decreased in a dose-dependent manner. The data suggest that dandelion extract has a potent inhibitory activity against HIV-1 replication and reverse transcriptase activity. The identification of HIV-1 antiviral compounds from Taraxacum officinale should be pursued.ConclusionsThe dandelion extract showed strong activity against HIV-1 RT and inhibited both the HIV-1 vector and the hybrid-MoMuLV/MoMuSV retrovirus replication. These findings provide additional support for the potential therapeutic efficacy of Taraxacum officinale. Extracts from this plant may be regarded as another starting point for the development of an antiretroviral therapy with fewer side effects.