Significant Luciferase Activity Research Articles

The AP-1 Transcription Factor JunB Promotes Multiple Myeloma (MM) Cell Proliferation, Survival and Drug Resistance in the Bone Marrow Microenvironment

MEK/ERK and NF-kB signaling pathways have been reported to play a key role in multiple myeloma (MM) survival, proliferation and drug resistance. These pathways regulate the activity of numerous transcription factors. For example, the activator protein-1 (AP-1) transcription factor has been implicated in a multitude of physiologic processes, but also tumorigenesis. However, the function of AP-1 in MM is largely unknown. Our data show a vast variety of AP-1 (c-Jun, JunB, JunD, c-Maf and c-Fos) expression levels in MM cells. Importantly, co-culture of MM cells with bone marrow stromal cells (BMSCs), i.e. isotypic primary BMSCs as well as BMSC lines KM-105 and HS-27A, rapidly and strongly induces expression of JunB, but not other AP-1 members. Previous studies have shown that JunB exerts opposite functions depending on the cellular origin and the physiopathological context. For example, it serves as a gatekeeper in acute and chronic myeloid leukemia, but as a positive regulator in Hodgkin9s lymphomas and anaplastic large cell lymphomas. The relevance of JunB activity in MM growth, survival and drug resistance is elusive. First, our data demonstrate that induction of JunB is predominantly mediated by soluble factors secreted by BMSCs rather than direct MM-BMSC contact. Indeed, using cytokine arrays, we identified IL-6 among the most potent factors that trigger JunB expression. Mechanistically, JunB upregulation occurs at both transcriptional as well as translational level. Pharmacologic inhibition was used next in order to identify upstream signaling pathways, which mediate BMSC- induced JunB upregulation in MM cells. Our data show that activation of MEK/ERK or NF-kB is required for induction of JunB expression and AP-1 transcriptional activity. To delineate the specific functional role of JunB in MM pathogenesis, we transduced MM cells with pLKO.1-JunB shRNA or pLKO.1-scrambled shRNA (SCR). After puromycin- selection, effects of JunB knockdown on MM proliferation, survival and drug resistance were analyzed by 3H-thymidine incorporation, flow cytometry and western blot. Indeed, we observed significant inhibition of proliferation in MM/ JunB shRNA (decreased to ~ 25 – 40 %, p Furthermore, 4-hydroxytamoxifen (4-OHT) treatment of MM cell lines stably transduced with pMSCV-JunB-ER-IRES-GFP but not pMSCV-IRES-GFP induced significant AP-1 luciferase activity (~ 3.3 fold, p 1000 MM patient samples of different prognostic groups were compared to samples from healthy donors using the gene set enrichment analysis (GSEA). Our results further support a key role for JunB in MM pathogenesis. In summary, our data demonstrate for the first time an important role of JunB/AP-1 in MM tumorigenesis and strongly propose it as a novel therapeutic target in MM. Disclosures No relevant conflicts of interest to declare.

A TG-associated minor LPL haplotype supresses miR-29 binding on LPL 3'UTR

Aim: Identification and functional study of Lipoprotein Lipase (LPL) 3'UTR polymorphisms associated with triglyceridemia and predicted to modulate miRNA binding. Methods: We sequenced LPL 3'UTR in 271 type 2 diabetic patients with documented lipid phenotypes. Haplotype analysis was performed using PHASE software. These data were validated by meta-analysis across four French general population samples. In silico analysis identified putative miRNA binding-sites created by LPL polymorphisms. Both LPL 3'UTR wild-type (WT) and variant (VAR) sequences were cloned in luciferase reporter gene plasmid. Transfection experiments in HEK293T and co-transfection with candidate miRNA-precursor was performed to study the regulation of the LPL constructs by specific miRNA. Results: A specific haplotype combining 8 LPL polymorphisms including the rs328 and 3'UTR minor alleles was found significantly associated with lower triglycerides concentration both in type 2 diabetes and in general populations. This haplotype was predicted to disrupt several miR-29 binding-sites. In vitro, the LPL-WT construct co-transfected with miR-29 showed a significant luciferase activity decrease (-25±6%, p< 0.001), whereas no difference was observed in the presence of the LPL-VAR construct. Moreover in HEK293T cells expressing endogenously miR-29, LPL-WT haplotype expression was lower compared to LPL-VAR haplotype (-32±7%, p<0.0001). Finally, the introduction of either the rs328 minor or major alleles did not modify the expression of LPL-WT and LPL-VAR haplotype. Conclusion: We identify a 'gain of function' LPL haplotype significantly associated with lower TG concentration which may increase LPL expression and activity by disrupting miR-29 binding-sites. The miRNA regulation of this haplotype is independent of rs328.

The AP-1 Transcription Factor JunB Promotes Multiple Myeloma (MM) Cell Proliferation, Survival and Drug Resistance in the Bone Marrow Microenvironment

MEK/ERK and NF-kB signaling pathways have been reported to play a key role in multiple myeloma (MM) survival, proliferation and drug resistance. These pathways regulate the activity of numerous transcription factors. For example, the activator protein-1 (AP-1) transcription factor has been implicated in a multitude of physiologic processes, but also tumorigenesis. However, the function of AP-1 in MM is largely unknown.Our data show a vast variety of AP-1 (c-Jun, JunB, JunD, c-Maf and c-Fos) expression levels in MM cells. Importantly, co-culture of MM cells with bone marrow stromal cells (BMSCs), i.e. isotypic primary BMSCs as well as BMSC lines KM-105 and HS-27A, rapidly and strongly induces expression of JunB, but not other AP-1 members. Previous studies have shown that JunB exerts opposite functions depending on the cellular origin and the physiopathological context. For example, it serves as a gatekeeper in acute and chronic myeloid leukemia, but as a positive regulator in Hodgkin’s lymphomas and anaplastic large cell lymphomas. The relevance of JunB activity in MM growth, survival and drug resistance is elusive.First, our data demonstrate that induction of JunB is predominantly mediated by soluble factors secreted by BMSCs rather than direct MM-BMSC contact. Indeed, using cytokine arrays, we identified IL-6 among the most potent factors that trigger JunB expression. Mechanistically, JunB upregulation occurs at both transcriptional as well as translational level. Pharmacologic inhibition was used next in order to identify upstream signaling pathways, which mediate BMSC- induced JunB upregulation in MM cells. Our data show that activation of MEK/ERK or NF-kB is required for induction of JunB expression and AP-1 transcriptional activity.To delineate the specific functional role of JunB in MM pathogenesis, we transduced MM cells with pLKO.1-JunB shRNA or pLKO.1-scrambled shRNA (SCR). After puromycin- selection, effects of JunB knockdown on MM proliferation, survival and drug resistance were analyzed by 3H-thymidine incorporation, flow cytometry and western blot. Indeed, we observed significant inhibition of proliferation in MM/ JunB shRNA (decreased to ~ 25 – 40 %, p < 0.01) compared with MM/ SCR control cells, when co-cultured with BMSCs in particular. Moreover, our preliminary data show that knockdown of JunB overcomes resistance of MM cells against doxorubicin as well as melphalan.Furthermore, 4-hydroxytamoxifen (4-OHT) treatment of MM cell lines stably transduced with pMSCV-JunB-ER-IRES-GFP but not pMSCV-IRES-GFP induced significant AP-1 luciferase activity (~ 3.3 fold, p < 0.01) as well as MM cell proliferation. In ongoing experiments, the in vivo relevance of our in vitro data is evaluated in a xenograft mouse model inoculated with MM /JunB-ER-IRES-GFP and MM/ IRES-GFP cells. Finally, gene expression profiles on > 1000 MM patient samples of different prognostic groups were compared to samples from healthy donors using the gene set enrichment analysis (GSEA). Our results further support a key role for JunB in MM pathogenesis.In summary, our data demonstrate for the first time an important role of JunB/AP-1 in MM tumorigenesis and strongly propose it as a novel therapeutic target in MM. DisclosuresNo relevant conflicts of interest to declare.

Intestinal Helminth Colonization Regulates Lethal Graft Versus Host Disease and Preserves Graft Versus Tumor in Bone Marrow Transplanted Mice

Introduction.Graft versus host disease (GVHD) is a frequent and severe complication of allogeneic bone marrow transplantation (BMT). Novel and less toxic treatment regimens are needed to suppress GVHD and preserve graft versus tumor (GVT). Self-limited intestinal helminth colonization stimulates host regulatory T cell (Treg) subsets. The intestine is a prime organ in GVHD generation, with Tregs playing a pivotal role in controlling GVHD. Treg-mediated suppression of GVHD preserves GVT. We hypothesized that self-limited intestinal colonization with helminths protects BMT mice from lethal acute GVHD and preserves the GVT.Methods. Three weeks after administering 3rd stage adult Heligmosomoides polygyrus larvae into 5-6 week old male WT Balb/C recipient mice (H2d), we initiated H2b→H2d MHC I/II mismatch model of acute GVHD by total body irradiation (TBI; 850 cGy) and administration of total splenic T cells and T cell depleted (TCD) bone marrow (BM) cells from male C57BL/6 WT (MHC: H2b) donors into uninfected control and helminth-infected male wild-type (WT) Balb/C (MHC: H2d) bone marrow recipients. In certain experiments, we used donor T cells from TGFβ RII dominant negative (DN) (TGFβ RII DN) mice (MHC: H2b) whose T cells are unresponsive to TGFβ-mediated immune regulation due to over-expression of a truncated TGFβ receptor II. Th1 (IFNγ, TNFα) cytokine generation was analyzed by ELISA. Splenic and mesenteric lymph node (MLN) cells were stained for H2b, H2d, T cell surface markers and FoxP3. Tissues were analyzed for GVHD-related inflammation. GVT was assessed in uninfected and helminth-infected BMT recipients by administration of luciferase expressing A20 leukemia/lymphoma cells (A20-luc; H2d) and IVIS imaging. Statistical and survival difference between groups was determined by Student’s t-test and Kaplan Meier curves, respectively.Results. Helminths increased ~3-fold CD4 T cell and FoxP3+ Treg TGFβ generation (p<0.01 btw uninfected and helminth-infected). Helminths suppressed donor T cell Th1 cytokine production (p<0.05 btw control and helminth-infected mice (N=3)) and regulated clinical as well as histopathological (colon and lung) GVHD score (p<0.05 between control and helminth-infected, for each parameter from multiple experiments). Helminths promoted the survival of recipient T cells after TBI (Spleen control: 1.4±0.9 x 104 vs. helminth-infected: 11.0±6.9 x 104 CD3+ T cells (N=8; p<0.05); MLN control: 0.09±0.09 x 104 vs. helminth-infected: 6.0±5.2 x 104 (N=8; p<0.001)). Surviving T cells in H. polygyrus-colonized mice were enriched for FoxP3+ Tregs (spleen: 1.16±0.6 x 104 and MLN: 0.65 ±0.56 x 104 FoxP3+ Tregs). Recipient T cells enriched for Tregs survived during GVHD, as analyzed 6 days after BMT (3.3±1.1% of splenic and 15.9±3.5% of MLN host T cells being FoxP3+ Tregs). The number of recipient T cells in uninfected BMT mice was low for further Treg analysis. Helminths increased the number of GVHD-regulating donor-derived H2b+ FoxP3+ Tregs (Spleen: 66±32 x 103 (control) vs. 206±73 x 103 (helminth-infected) Tregs per mouse (p<0.05; N=6); MLN: 1.6 ±1.1 x 103 (control) vs. 4.9±2.0 x 103(helminth-infected) Tregs per mouse (p<0.05; N=4). Helminth infection was associated with >15 weeks survival of 40% of recipients, while all uninfected BMT recipient mice died of GVHD (p<0.01 between uninfected (N=10) and helminth-infected (N=10) WT recipients). Helminths did not promote the survival of recipients, when T cells unresponsive to TGFβ were used as donor lymphocytes. Helminths preserved the GVT in BMT mice transferred A20-luc. All uninfected and helminth-infected TCD BM transferred recipients died within few weeks displaying significant luciferase activity, while no luciferase signal was evident at day 70 in surviving (5/9) helminth-infected mice administered TCD BM and splenic T cells along with A20-luc. Uninfected mice (9/9) transferred splenic T cells, TCD BM and A20-luc died of GVHD without showing luciferase activity.Conclusions. Our results show that (1) helminths stimulate TGFβ producing host T cells and FoxP3+ Tregs that survive BMT conditioning with TBI; (2) helminths regulate GVHD by inducing donor Tregs and in a TGFβ dependent manner; (3) helminths preserve GVT. With a safe side-effect profile, helminths or helminth products have the potential to be investigated as an alternative strategy to prevent or treat GVHD. DisclosuresNo relevant conflicts of interest to declare.

Differential response of the Senegalese sole (Solea senegalensis) Mx promoter to viral infections in two salmonid cell lines

Mx proteins are main effectors of the antiviral innate immune defence mediated by type I interferon (IFN I). The IFN I response is under a complex regulation; hence, one of the key issues in understanding virus-host interaction is the knowledge of the regulatory mechanisms governing this response. With this purpose, in this study Chinook salmon embryo cells (CHSE-214) and rainbow trout gonad cells (RTG-2) were transiently transfected with a vector containing the luciferase reporter gene under the control of the Senegalese sole Mx promoter. These transfected cells were infected with infectious pancreatic necrosis virus (IPNV), viral haemorrhagic septicaemia virus (VHSV) and epizootic haematopoietic necrosis virus (EHNV) at different doses in order to study the luciferase fold induction in response to viral infections. Transfected CHSE-214 cells infected with EHNV showed significant induction of the luciferase reporter gene, compared to control non-infected cells, at different times post infection (p.i.). The maximum expression was recorded at 24h p.i. in cells inoculated with 5×102TCID50/mL (2.17 folds compared to control cells). In these cells, the infection with IPNV and VHSV did not result in the luciferase expression at any time and doses tested. In transfected RTG-2 cells, VHSV stimulated luciferase expression, obtaining a maximum activity at 48h p.i. in cells infected with 5×102TCID50/mL (2.9 folds compared to control cells), whereas RTG-2 cells infected with IPNV and EHNV did not show significant luciferase activity at any time point. The different induction of the Senegalese sole Mx promoter in CHSE-214 and RTG-2 cells after infection with the same viruses indicates that cell-specific factors are significantly involved in the IFN-signalling response, and, probably, on the success of the strategies of these viruses to escape the IFN mechanisms. The use of these two different cellular systems might be an interesting approach to identify such cellular factors.

Adaptation of Clostridium difficile toxin A for use as a protein translocation system

A cellular delivery system is a useful biotechnology tool, with many possible applications. Two derivatives of Clostridium difficile toxin A (TcdA) have been constructed (GFP-TcdA and Luc-TcdA), by fusing reporter genes to functional domains of TcdA, and evaluated for their ability to translocate their cargo into mammalian cells. The cysteine protease and receptor binding domains of TcdA have been examined and found to be functional when expressed in the chimeric construct. Whereas GFP failed to internalize in the context of the TcdA fusion, significant cellular luciferase activity was detected in vero cell lysates after treatment with Luc-TcdA. Treatment with bafilomycin A1, which inhibits endosomal acidification, traps the luciferase activity within endosomes. To further understand these results, clarified lysates were subjected to molecular weight sieving, demonstrating that active luciferase was released from Luc-TcdA after translocation and internal processing.

Asp-960/Glu-961 Controls the Movement of the C-terminal Tail of the Epidermal Growth Factor Receptor to Regulate Asymmetric Dimer Formation

The epidermal growth factor (EGF) receptor is a tyrosine kinase that dimerizes in response to ligand binding. Ligand-induced dimerization of the extracellular domain of the receptor promotes formation of an asymmetric dimer of the intracellular kinase domains, leading to stimulation of the tyrosine kinase activity of the receptor. We recently monitored ligand-promoted conformational changes within the EGF receptor in real time using luciferase fragment complementation imaging and showed that there was significant movement of the C-terminal tail of the EGF receptor following EGF binding (Yang, K. S., Ilagan, M. X. G., Piwnica-Worms, D., and Pike, L. J. (2009) J. Biol. Chem. 284, 7474-7482). To investigate the structural basis for this conformational change, we analyzed the effect of several mutations on the kinase activity and luciferase fragment complementation activity of the EGF receptor. Mutation of Asp-960 and Glu-961, two residues at the beginning of the C-terminal tail, to alanine resulted in a marked enhancement of EGF-stimulated kinase activity as well as enhanced downstream signaling. The side chain of Asp-960 interacts with that of Ser-787. Mutation of Ser-787 to Phe, which precludes this interaction, also leads to enhanced receptor kinase activity. Our data are consistent with the hypothesis that Asp-960/Glu-961 represents a hinge or fulcrum for the movement of the C-terminal tail of the EGF receptor. Mutation of these residues destabilizes this hinge, facilitating the formation of the activating asymmetric dimer and leading to enhanced receptor signaling.

Lipid-mediated delivery of RNA is more efficient than delivery of DNA in non-dividing cells

The design of appropriate gene delivery systems is essential for the successful application of gene therapy to clinical medicine. Cationic lipid-mediated delivery is a viable alternative to viral vector-mediated gene delivery in applications where transient gene expression is desirable. However, cationic lipid-mediated delivery of DNA to post-mitotic cells such as neurons is often reported to be of low efficiency, due to the presumed inability of the DNA to translocate to the nucleus. Lipid-mediated delivery of RNA is an attractive alternative to non-viral DNA delivery in some clinical applications, because transit across the nuclear membrane is not necessary. Here we report a comparative investigation of cationic lipid-mediated delivery of RNA versus DNA vectors encoding the reporter gene green fluorescent protein (GFP) in Chinese Hamster Ovary (CHO) and NIH3T3 cells following chemical inhibition of proliferation, and in primary mixed neuronal cell cultures. Using optimized formulations and transfection procedures, we assess gene expression by flow cytometry to specifically address some of the advantages and disadvantages of lipid-mediated RNA and DNA gene transfer. Despite inhibition of cell proliferation, over 45% of CHO cells express GFP after lipid-mediated transfection with RNA vectors. Transfection efficiency of DNA encoding GFP in proliferation-inhibited CHO cells was less than 5%. Detectable expression after RNA transfection occurs at least 3 h earlier than after DNA transfection, but DNA transfection eventually produces a mean level of per cell GFP expression (as assayed by flow cytometry) that is higher than after RNA transfection. Transfection of proliferation-inhibited NIH3T3 cells and primary mixed neuronal cultures produced similar results, with RNA encoded GFP expression in 2–4 times the number of cells as after DNA encoded GFP expression. These results demonstrate the increased efficiency of RNA transfection relative to DNA transfection in non-dividing cells. We used firefly luciferase encoded by RNA and DNA vectors to investigate the time course of gene expression after delivery of RNA or DNA to primary neuronal cortical cells. Delivery of mRNA resulted in rapid onset (within 1 h) of luciferase expression after transfection, a peak in expression 5–7 h after transfection, and a return to baseline within 12 h after transfection. After DNA delivery significant luciferase activity did not appear until 7 h after transfection, but peak luciferase expression was always at least one order of magnitude higher than after RNA delivery. The peak expression after luciferase-expressing DNA delivery occurred 36–48 h after transfection and remained at a significant level for at least one week before dropping to baseline. This observation is consistent with our in vivo delivery results, which are shown as well. RNA delivery may therefore be more suitable for short-term transient gene expression due to rapid onset, shorter duration of expression and greater efficiency, particularly in non-dividing cells. Higher mean levels of expression per cell obtained following DNA delivery and the longer duration of expression confirm a continuing role for DNA gene delivery in clinical applications that require longer term transient gene expression.

A genotoxicity test system based on p53R2 gene expression in human cells: Assessment of its reactivity to various classes of genotoxic chemicals

The tumor suppressor, p53, plays an important role in DNA damage repair, by regulating the expression of target genes. One p53-target gene, p53R2, which encodes a subunit of ribonucleotide reductase, is activated by DNA damage. We have previously developed a genotoxicity test system, using human cell lines and a p53R2-dependent luciferase reporter gene assay. 80 chemicals have been examined with this system and 40 of 43 Ames-positive chemicals induced luciferase activity. Eight Ames-negative genotoxic chemicals also induced luciferase activity. Although this assay system could, potentially, be applied to the rapid screening of chemicals that are potentially genotoxic to humans, the ability of the assay to detect genotoxic effects was unclear. In this study, to evaluate the performance of this assay system, several different types of DNA damaging agents were screened. 27 chemicals, whose genotoxic mechanisms are well known, were screened. All genotoxic compounds, except for anti-metabolites and histone deacetylase HDAC inhibitors, showed significant luciferase activity with the following rank order of potency: topoisomerase II inhibitors, intercalaters > bleomycin > topoisomerase I inhibitors > alkylating agents = DNA cross-linking agents = polycyclic aromatic hydrocarbons > spindle poisons. This assay showed greater response to those genotoxic agents that induce DNA double strand break damage compared to those agents that cause other forms of DNA damage. DNA double strand breakage initiates genomic instability, a feature of carcinogenicity. These results indicate that this assay system could be a helpful tool for predicting chemical genotoxicity and carcinogenicity in humans.

Subtype-specific activation of estrogen receptors by a special extract of Rheum rhaponticum (ERr 731®), its aglycones and structurally related compounds in U2OS human osteosarcoma cells

The special extract ERr 731 from the roots of Rheum rhaponticum is the major constituent of Phytoestrol N which is used for the alleviation of menopausal symptoms. Recently, we demonstrated that ERr 731 and its aglycones trans-rhapontigenin and desoxyrhapontigenin as single test substances do not activate the estrogen receptors-alpha (ERalpha) in human endometrial adenoarcinoma cells. However, these substances together with the structurally related hydroxystilbenes cis-rhapontigenin, resveratrol and piceatannol activated the ERbeta-dependent reporter gene activity. To investigate if these substance are tissue selective ER activators, ERr 731 and the single test substances were examined in bone-derived U2OS cells stably expressing ERalpha or transiently expressing ERbeta. In the ERalpha expressing U2OS cells, a weak, but statistically significant ERalpha-coupled luciferase activity was detected with ERr 731 and desoxyrhapontigenin which was 10-times lower than with 10(8) M 17 beta-estradiol. In the ERbeta test system, all test substances significantly induced the luciferase activity in a magnitude comparable to 17beta-estradiol. All effects were abolished with the pure ER antagonist ICI 182 780, indicating an ER-specific effect. Intracellular actions were also examined with the glycosylated ERr 731 constituents rhaponticin and desoxyrhaponticin. Treatment of U2OS cells with defined mixtures of both glycosides resulted in a reporter gene activity comparable to that of ERr 731, thereby providing evidence for the existence of cellular uptake mechanisms for glycosylated hydroxystilbenes. This report confirms the strong ERbeta-dependent activity of ERr 731 and provides evidence for a tissue selective ER agonistic activity by ERr 731 and its aglycones, as demonstrated by the activation of ERalpha in bone cells but not in endometrial cells.

Human dialyzable leukocyte extracts (DLE) have ligands for TLR- 2 but not for TLR-4 (49.34)

Abstract Human dialyzable leukocyte extracts (DLE) have been used for the last 50 years to successfully treat different diseases. They are obtained by disruption of cells in buffy coats and subsequent dialysis (&amp;lt;12 KDa) of the extracts. They can transfer cellular immune response from an immune donor to a naïve recipient, and despite their immunomodulatory effect, until now little is known about their mechanism of action. One possibility is that they may contain ligands that activate TLRs. In this work using a reporter gene assay and HEK293 cells transfected with either TLR-2 or TLR-4 plasmids, we showed that different concentrations of DLE (100, 10 and 1 microg/mL) resulted in the activation of NF-kappaB (luciferase reporter system) in cells TLR-2 transfected cells with 10 or 1 microg/mL DLE. Interesting none of the DLE concentrations used induced a significant luciferase activity in the TLR-4 transfected cells. Our result can explain in part the immunomodulatory effects produced by DLE both in vivo and in vitro. Work will now focus in the identification of this ligand(s). This work was supported in part by SIP/IPN. 1Are fellows of COFAA, EDI and SNI.

Development of a novel β-cell specific promoter system for the identification of insulin-producing cells in in vitro cell cultures

Recently, it has been reported that islet transplantation into patients with Type 1 diabetes may achieve insulin independence for a year or longer [Shapiro et al., Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen, N Engl J Med . 343 (2000) 230–238]. However, the amount of donor islet tissue is limited, therefore, multiple approaches are being explored to generate insulin-producing cells in vitro. Some promising results have been obtained using mouse and human stem cells and progenitor cells [Soria et al., From stem cells to beta cells: new strategies in cell therapy of diabetes mellitus, Diabetologia. 4 (2001) 407–415; Lechner et al., Stem/progenitor cells derived from adult tissues: potential for the treatment of diabetes mellitus, Am J Physiol Endocrinol Metab. 284 (2003) 259–266; Bonner-Weir et al., In vitro cultivation of human islets from expanded ductal tissue, Proc Natl Acad Sci U S A , 97 (2000) 7999–8004; Assady et al., Insulin production by human embryonic stem cells, 50 (2001) Diabetes 1691–1697]. However, the efficiency of obtaining populations with high numbers of differentiated cells has been poor. In order to improve the efficiency of producing and selecting insulin-producing cells from undifferentiated cells, we have designed a novel β-cell specific and glucose responsive promoter system designated pGL3.hINS-363 3×. This artificial promoter system exhibits significant luciferase activity not only in insulin-producing MIN6 m9 cells but also in isolated human islets. The pGL3.hINS-363 3× construct shows no activity in non-insulin-producing cells in low glucose conditions (2 mM glucose) but demonstrates significant activity and β-cell specificity in high glucose conditions (16 mM glucose). Furthermore, pGL3.hINS-363 3× shows significant promoter activity in differentiated AR42J cells that can produce insulin after activin A and betacellulin treatment. Here, we describe a novel β-cell specific and glucose responsive artificial promoter system designed for analyzing and sorting β-like insulin-producing cells that have differentiated from stem cells or other progenitor cells.

Structure and functional regulation of the CD38 promoter

CD38 has multiple roles in biology, including T lymphocyte signaling, neutrophil migration, neurotransmission, cell proliferation, apoptosis, and bone remodeling. To study the transcriptional control of the CD38 gene, we cloned a putative 1.8 kb promoter fragment from a rabbit genomic DNA library. Primer extension analysis indicated two transcription start sites consistent with the absence of a TATA box. Sequence analysis revealed several AP-1, AP-4, myo-D, GATA, and SP-1 sequences. MC3T3.E1 (osteoblast) or RAW-C3 (osteoclast precursor macrophage) cells were then transfected with the CD38 promoter or its deletion fragments ligated to the luciferase reporter gene. Except for the shortest 41 bp fragment, all fragments showed significant luciferase activity. There was a marked stimulation of basal activity in the 93 bp fragment that contained a GC box and SP-1 site. Furthermore, there were significant differences in the activity of the fragments in MC3T3.E1 and RAW-C3 cells. Intracellular Ca 2+ elevations by ionomycin (10 μM) in MC3T3.E1 cells inhibited promoter activity, except in the short 41 bp. In contrast, cAMP elevation by exposure to forskolin (100 μM) inhibited activation of all fragments, except the 0.6 and 1.2 kb fragments. Finally, TNF-α stimulated promoter activity in RAW-C3 cells transfected with the 93 bp and 1.0 kb fragments, consistent with the stimulation of CD38 mRNA by TNF-α. Physiologically, therefore, modulation of the expression of the NAD +-sensing enzyme, CD38, by Ca 2+, cAMP, and cytokines, such as TNF-α may contribute to coupling the intense metabolic activity of osteoclasts and osteoblasts to their respective bone-resorbing and bone-forming functions.

Delivery of Condensed DNA by Liposomal Non-viral Gene Delivery System into Nucleus of Dendritic Cells

In this study, we developed novel double-membranous non-viral gene delivery system modified with SV-40 T antigen-derived nuclear localization signal (NLS-DMEND) for delivery of luciferase plasmid DNA to nucleus of non-dividing mouse bone marrow-derived dendritic cells (BMDC). Intracellular trafficking and gene expression of NLS-DMEND in the BMDC were evaluated. Condensed DNA was observed in the nucleus by confocal laser scanning microscopy, and the NLS-DMEND induced significant luciferase activity in the BMDC. It was suggested that the condensed DNA particle transferred into nucleus via energy dependent manner, since the nuclear transfer was inhibited by metabolic inhibitors. In conclusion, condensed plasmid DNA was delivered into the nucleus of non-dividing BMDC by NLS-DMEND.

584. Gene Delivery to Primary Neuronal Cells

Top of pageAbstract Neuronal protection under numerous clinical circumstances (i.e. trauma brain injury, stroke, ischemia and circulation arrested surgery, etc.) remains a tough challenge due to the paucity of effective clinical options. Transient gene delivery to CNS neuronal cells may offer an alternative approach to induce neuronal protection. We have previously shown widespread distribution, uptake, and expression in rat brain after non-viral, cationic lipid-mediated delivery of reporter gene vectors to the lateral ventricle 1,2. In order to further develop techniques for, and understand the mechanisms of, non-viral gene delivery, we conducted cationic lipid mediated gene delivery of fire fly luciferase mRNA and DNA to primary rat cortical cells to study the post-transfection transgene expression profiles. The delivery of mRNA resulted in a rapid onset of luciferase expression 1 hour after transfection, peaked at 5- 6 hours post transfection, and diminished to base line 12 hours after transfection. Compared to the delivery of mRNA, DNA delivery resulted in a much later onset of gene expression. Although the transgene expression after DNA delivery was generally one order of magnitude higher than after mRNA delivery, the earliest significant luciferase activity after DNA transfection did not appear until 7 hours after transfection. Peak expression following DNA delivery was 36-48 hours after transfection, and transgene expression persisted at a significant level for at least one week before it dropped to base line. Based on this observation, we attempted the delivery of mRNA and DNA vectors that encode for the heat shock protein gene HSP70. The inducible HSP70 is a chaperone protein and its expression has protective effects under stress conditions. HSP70 was delivered using non-viral cationic lipid-mediated techniques that we have developed to rat primary cortical cells, and analyzed by flow cytometry and immunohistochemistry. Consistent with the luciferase activity results, we detected evenly stained HSP70 expression 5 hours after the delivery of HSP70 mRNA, while expression after the delivery of HSP70 DNA appeared to peak at 36-48 hours.

Mechanisms of Leukemogenesis by MLL-CALM: Characterization of a CALM-Derived Transcriptional Regulatory Domain.

Background:MLL translocations are common in infant leukemias, and >50 distinct translocation partners have been described. We recently identified the CALM gene as a novel MLL partner in an infant with aggressive AML. Interestingly, CALM was first discovered as a translocation partner for AF10, which had previously been identified as an MLL fusion partner in aggressive leukemias and lymphomas. The native CALM protein exhibits predominantly cytoplasmic localization, and participates in clathrin-dependent endocytosis and intracellular vesicle transport. We have previously shown that expression of MLL-CALM immortalizes murine hematopoietic progenitors, and that fusion of the carboxy terminus of CALM to MLL alters MLL transcriptional activity. We hypothesize that CALM possesses a specific transcriptional activation domain (TAD) which modulates MLL transcriptional activity of HOX genes, thereby contributing to leukemogenesis.Objectives: 1) To determine whether native CALM localizes to the nucleus, 2) To delineate specific CALM domains which constitute the CALM TAD, and 3) To determine whether MLL-CALM activates transcription through the murine HOXA7 promoter.Methods: Human fibroblast cells were treated with Leptomycin B (an antifungal antibiotic which specifically inhibits nuclear export) and stained with an anti-CALM antibody. We prepared a set of expression vectors in which various portions of CALM are fused to a GAL4 DNA-binding domain. These vectors were co-transfected with a GAL4-luciferase reporter plasmid into COS7 cells, and luciferase activity was measured 48 hours after transient transfection. Luciferase assays were also performed using MSCV-MLL-CALM or MSCV-CALM plasmids co-transfected with a HOXA7 promoter-luciferase reporter construct.Results: After inhibition of nuclear export, native CALM localized to both the nucleus and cytoplasm. Significant luciferase activity was only observed with constructs containing distal CALM carboxy amino acids (aa 436–660). Mutation of an NR (Nuclear Receptor) Box motif (aa 510–514) did not affect CALM-dependent transcription. We found that two endocytosis-related NPF domains play opposite roles: deletion of NPF#1 (aa 437–439) dramatically reduced, while mutation of NPF#2 (aa 639–641) increased transcriptional activity. Expression constructs lacking GAL4 DNA binding domains had no effect on transcription, and GAL4 binding sites were required for luciferase activity in this system. Finally, MLL-CALM activated transcription of the murine HOXA7 promoter in comparison with native CALM or empty vector.Conclusions: We have confirmed that native CALM is able to localize to the nucleus, and we have begun to identify specific critical residues in the CALM TAD. The presence of a CALM TAD in MLL-CALM suggests that altered transcriptional regulation of MLL-dependent HOX genes may play an important role in MLL-CALM dependent transformation. Our observations raise the possibility that other MLL partners with native cytoplasmic localization may possess unrecognized transcriptional activity, and provide new insight into both MLL-CALM and CALM-AF10 mediated leukemogenesis.

Geminin Is Targeted for Repression by the Retinoblastoma Tumor Suppressor Pathway through Intragenic E2F Sites

The geminin protein is a critical regulator of DNA replication. It functions to control replication fidelity by blocking the assembly of prereplication complexes in the S and G(2) phases of the cell cycle. Geminin protein levels, which are low in G(0)/G(1) and increase at the G(1)/S transition, are controlled through coordinate transcriptional and proteolytic regulation. Here we show that geminin is regulated transcriptionally by the retinoblastoma tumor suppressor (RB)/E2F pathway. Initially, we observed that the activation of RB led to the repression of geminin transcription. Conversely, Rb-null mouse embryonic fibroblasts have enhanced the expression of geminin relative to wild type mouse embryonic fibroblasts. Similarly, an acute loss of Rb in mouse adult fibroblasts deregulated geminin RNA and protein levels. To delineate the responsible regulatory motifs, luciferase reporter constructs containing fragments of the geminin promoter were generated. An analysis of the critical regulatory cis-acting elements in the geminin promoter indicated that intragenic E2F sites down-stream of the first exon were responsible for RB-mediated repression of geminin. The direct analysis of the endogenous geminin promoter revealed that these intragenic E2F sites are occupied by E2F proteins, and the mutation of these sites eliminates responsiveness to RB. Together, these data link the expression of geminin to the RB/E2F pathway and represent the first promoter analysis of this important regulator of DNA replication.

A Renilla luciferase-Aequorea GFP (ruc-gfp) fusion gene construct permits real-time detection of promoter activation by exogenously administered mifepristone in vivo.

In this study, we used a steroid-induced promoter activation system as a molecular switch to study the exogenous activation of transgene expression. This promoter activation system consists of three components: (1) a steroidal inducer drug, mifepristone (RU486), which binds to (2) a chimeric transcription factor complex, consisting of the mutant human progesterone receptor fused to the yeast GAL4 DNA-binding domain and the activation domain of the herpes simplex virus protein VP16, and (3) a synthetic promoter, consisting of a series of GAL4 recognition sequences upstream of the adenovirus major late E1B TATA box, linked to a gene construct (ruc-gfp) encoding a Renilla luciferase- Aequorea green fluorescent protein (GFP) fusion protein. Transcription of the promoter-marker gene cassette is activated by the drug (mifepristone)-bound chimeric transcription factor complex. Monitoring of induced gene expression was carried out using a low-light video camera and a UV microscope to detect luciferase and GFP, respectively. Using this activation system, we observed a 10- to 25-fold activation, depending on the inducer dose, of both luciferase and GFP expression in transiently transfected cells in comparison to cells that were not exposed to mifepristone. We further demonstrated activation of gene expression from the promoter activation system in live animals. The plasmids PAP CMV-GL914VPc'SV, carrying the chimeric transcription factor cassette, and plasmid p17x4-TATA-ruc-gfp, carrying the ruc-gfp reporter gene construct, were co-injected into limb muscles of nude mice. Following DNA injection, mifepristone (50 micro g/kg) was delivered by intraperitoneal injection. Thirty-six hours after DNA and mifepristone injection, significant Renilla luciferase activity was detectable in the limb muscles. The promoter activation system was also demonstrated in limb muscles and livers of nude mice that had received transplants of ex vivo-modified cells, which were transiently transformed with both the chimeric activator plasmid and the ruc-gfp reporter plasmid prior to implantation. Significant Renilla activity and GFP fluorescence were detected externally in limb muscles and in the livers of anesthetized animals that had received an intraperitoneal injection of inducer. This external monitoring method for observing inducible gene expression in live animals will facilitate experimental studies of fundamental questions of biological and therapeutic relevance. It will be especially valuable for the analysis of gene function at specific stages of animal development. The method should also be of general use in gene therapy, since it permits simultaneous monitoring of the expression levels of light-emitting proteins and therapeutic proteins originating from the activation of identical promoters.

Molecular cloning of a murine type III sodium-dependent phosphate cotransporter (Pit-2) gene promoter

We report the novel cloning and preliminary characterization of a murine type III sodium-dependent phosphate cotransporter (Pit-2) gene promoter. Five promoter/luciferase reporter gene constructs, −1816/+61, −1620/+61, −1223/+61, −600/+61 and −225/+61, showed significant luciferase activity (6–14-fold over background) when transfected into human colon carcinoma (Caco-2) and opossum kidney (OKP) cells.

Molecular cloning of murine sodium–phosphate cotransporter type IIb (Na/P i-IIb) gene promoter and characterization of gene structure

We report the cloning of the murine Na/P i-IIb cotransporter gene, which spans more than 18 kilobases and consists of 12 introns and 13 exons. Three promoter/reporter gene constructs, −159/+73, −429/+73 and −954/+73, showed significant luciferase activity (22–82-fold over background) when transfected into in rat intestinal epithelial (RIE-1) cells.