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Results for “Targeted expression”

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At least 649 records · Page 36Linked to original sources
PubMed2002

Prostate stem cell antigen as therapy target: tissue expression and in vivo efficacy of an immunoconjugate.

We conducted an expression analysis of prostate stem cell antigen (PSCA)in normal urogenital tissues, benign prostatic hyperplasia (n = 21), prostatic intraepithelial neoplasia (n = 33), and primary (n = 137) and metastatic (n = 42) prostate adenocarcinoma, using isotopic in situ hybridization on tissue microarrays. In normal prostate, we observe PSCA expression in the terminally differentiated, secretory epithelium; strong expression was also seen in normal urothelium. Forty-eight percent of primary and 64% of metastatic prostatic adenocarcinomas expressed PSCA RNA. Our studies did not confirm a positive correlation between level of PSCA RNA expression and high Gleason grade. We characterized monoclonal anti-PSCA antibodies that recognize PSCA expressed on the surface of live cells, are efficiently internalized after antigen recognition, and kill tumor cells in vitro in an antigen-specific fashion upon conjugation with maytansinoid. Unconjugated anti-PSCA antibodies demonstrated efficacy against PSCA-positive tumors by delaying progressive tumor growth in vivo. Maytansinoid-conjugated antibodies caused complete regression of established tumors in a large proportion of animals. Our results strongly suggest that maytansinoid-conjugated anti-PSCA monoclonal antibodies should be evaluated as a therapeutic modality for patients with advanced prostate cancer.

Adenocarcinoma↗
PubMed2002

Compartmentalization of central neurons in Drosophila: a new strategy of mosaic analysis reveals localization of presynaptic sites to specific segments of neurites.

Synaptogenesis in the CNS has received far less attention than the development of neuromuscular synapses, although only central synapses allow the study of neuronal postsynaptic mechanisms and display a greater variety of structural and functional features. This neglect is attributable mainly to the enormous complexity of the CNS, which makes the visualization of individual synapses on defined neuronal processes very difficult. We overcome this obstacle and demonstrate by confocal microscopy the specific arrangement of output synapses on individual neurites. These studies are performed via genetic mosaic strategies in the CNS of the fruitfly Drosophila melanogaster. First, we use targeted expression of synaptic proteins by the UAS/Gal4 system. Second, we apply a newly developed transplantation-based mosaic strategy that takes advantage of the intrinsic regulation and localization of synaptic proteins in single-cell clones. We propose the existence of three distinct neuritic compartments: (1) primary neurites that appear to form the main transport pathways and are mostly void of output synapses, (2) neuritic compartments that contain output synapses, and (3) neuritic compartments that are postsynaptic in nature. In addition we show that mutations of the kakapo gene have no obvious effect on the distribution of output synapses in the CNS, whereas neuromuscular synapses are severely reduced. This suggests that synaptogenic mechanisms in the CNS might differ from those at neuromuscular junctions.

Animals↗
PubMedDate not supplied

Targeting Survivin expression induces cell proliferation defect and subsequent cell death involving mitochondrial pathway in myeloid leukemic cells.

Survivin, a member of inhibitor of apoptosis family of proteins, plays important roles in both cell proliferation and cell death. We previously observed that Survivin is overexpressed in leukemic cell lines and blasts from patients with acute myelogenous leukemia (AML). To understand the roles of Survivin in AML and search for new approaches to the treatment of AML, we inhibited Survivin expression in HL-60 cells with a Survivin anti-sense oligonucleotide (sur-AS-ODN) (ISIS 23722). This blocked significant numbers of HL-60 cells in G2/M phase, and halted cell proliferation at 24 hrs and progressing over time. There was only a slight increase in the number of apoptotic cells at 24 hrs compared with cells treated with nonsense oligonucleotide (NS-ODN). At 48 hrs, however, there were significant increases in sub-G1 phase and annexin V+ cells, suggesting that cell division defects caused cell death. This was supported by the finding that a reduction in the Survivin protein by sur-AS-ODN in cells under serum-free medium did not induce G2/M block and cell death compared to cells treated with NS-ODN. The formation of polyploid cells was observed 48 hrs after sur-AS-ODN treatment, as was the activation of caspase 3, which suggested that apoptotic cell death had occurred. The mitochondrial release of cytochrome C and Smac and the nuclear translocation of the apoptosis-inducing factor were also detected. Our results suggest that Survivin is essential for cell cycle progression in leukemic cells. Reduced Survivin expression causes a cell-cycle defect that leads to cell death through a mitochondrial pathway. This finding has potential utility for therapy of patients with AML.

Apoptosis↗
PubMed2004

[A transgenic mouse that targets the expression of Cre recombinase in hepatocyte].

OBJECTIVE: To construct a mouse that specifically expresses Cre recombinase in hepatocyte. METHODS: A hepatocyte specific transgenic construct containing mouse albumin promoter, the Cre recombinase gene and the poly (A) of human growth factor gene was generated. The linearized constructs were introduced into the fertilized eggs by microinjection to obtain the transgenic mice. The transcriptional specificity of Cre recombinase was detected by reverse transcription polymerase chain reaction (RT-PCR). The expression and function of Cre recombinase were detected by PCR and Southern Blot after crossing the Alb-Cre transgenic mice with the Smad4 conditional knockout mice. RESULTS: The linearized constructs were microinjected into 837 fertilized eggs, and then the 797 effective eggs of microinjected eggs were implanted into the oviducts of 27 pseudo pregnant mice. In the 53 offspring, there were 6 mice carrying the transgene identified by polymerase chain reaction (PCR) and Southern Blot. Cre recombinase transcripts were detected in the livers and testis of the Alb-Cre transgenic mice using RT-PCR. The Cre recombinase was expressed in the livers of the double heterozygous for Alb-Cre and Smad4 floxed allele, and the exon 8 floxed by loxP site was deleted. CONCLUSION: A hepatocyte-specific Cre transgenic mouse was generated successfully. The Cre recombinase expressed specifically in liver and could mediate the recombination between loxP sites in vivo.

Animals↗
PubMedDate not supplied

Ets2 transcription factor inhibits mineralization and affects target gene expression during osteoblast maturation.

Our goal is to understand how Ets family transcription factors affect the genetic programs that control bone development. Modest overexpression of Ets2 in transgenic mice leads to Down's syndrome-like bone abnormalities. We observed that in the MC3T3-E1 in vitro model of osteoblast development, mature osteoblasts have very high levels of Ets2 relative to the immature preosteoblasts. We hypothesized that overexpression of Ets2 could have noticeable effects on gene expression, and found that exogenous Ets2 expression results in a complete lack of mineralized matrix in stable Ets2 transfected cells. Our cDNA microarray-based expression profiling of preosteoblasts vs. differentiated osteoblasts revealed several genes previously unrecognized as having roles in osteoblast maturation and up-regulated only in the mature osteoblasts. The promoters of these genes and known osteoblast marker genes were examined for Ets transcription factor binding sites (EBSs). Interactions of these sites with Ets2 protein were tested by EMSA. In vitro expressed Ets2 protein was able to form a protein:DNA complex with both known (Bsp, Opn, ON) and novel (Btg2, CysC, Lum) bone-related genes. In addition, Cbfa1 was found to interact with Ets2, forming a complex on the Opn promoter.

Animals↗
PubMedDate not supplied

Ex vivo expansion of antitumor cytotoxic lymphocytes with tumor-associated antigen-loaded dendritic cells.

Cell therapy with lymphocytes is an attractive approach for cancer immunotherapy. Methods to generate ex vivo effector cells directed against whole autologous tumor antigens are under investigation. Our procedure involved stimulation of autologous lymphocytes with antigen-pulsed dendritic cells (DC). Experimental conditions were established with DC, matured with TNFa, LPS and CD40L, from healthy donors and the M74 melanoma cell line. DC were pulsed with either irradiated, apoptotic or necrotic tumor cells or fused with tumor cells. Increase of lymphocyte cytotoxicity and IFNy production were repeatedly observed with tumor cell-loaded DC. Stimulation of tumor-associated antigen-specific lymphocytes was clearly shown. MelanA-MART1 (dominant melanoma-associated antigen) tetramer staining revealed a high frequency of specific T cells. Lymphocytes were able to efficiently lyse MelanA-MART1-pulsed T2 target and MelanA-expressing target cells (M74) after CD56+ cells depletion. We confirmed with other tumor cell lines that this DC-mediated procedure induced activation of cytolytic lymphocytes.

Antigens, Neoplasm↗
PubMed2004

Glial-specific retrovirally mediated gas1 gene expression induces glioma cell apoptosis and inhibits tumor growth in vivo.

We recently reported that the targeted expression of growth arrest specific 1 (Gas1) induces apoptosis in glioma cells. Because the vast majority of gliomas present genetic alterations that reduce their ability to undergo apoptosis, a gene therapy strategy aimed at reinstating apoptotic processes in glioma cells is an interesting approach for the treatment of these tumors. We used a retroviral gene transfer system to transduce C6 glioma cells with a transgene in which the expression of a full-length human gas1 cDNA is under the transcriptional control of a human promoter of the glial fibrillary acidic protein (gfa2). In vitro experiments showed that the retroviral transfer of gas1 significantly reduces the number of viable cells, and induces apoptosis in C6 cells, through the activation of caspase-3. Furthermore, retroviral-mediated transfer of gas1 to gliomas implanted in nude mice induces a significant inhibition of tumor growth, accompanied by increased caspase-3 activation. In the present experiments, we have taken advantage of the property of retrovirus to transfer transgenes exclusively to proliferating cells, together with the use of a glial specific promoter, to selectively target the expression of gas1, a pro-apoptotic gene, to glioma cells.

Animals↗
PubMed2000

Site-specific gene targeting for gene expression in eukaryotes.

Major advances in the use of site-specific recombinases to facilitate sustained gene expression via chromosomal targeting have been made during the past year. New tools for genomic manipulations using this technology include the discovery of epitopes in recombinases that confer nuclear localization, crystal structures that show the precise topology of recombinase-DNA-substrate synaptic complexes, manipulations of the DNA recognition sequences that select for integration over excision of DNA, and manipulations that make changes in gene expression inducible by drug administration. In addition, endogenous eukaryotic and mammalian DNA sequences have been discovered that can support site-specific recombinase-mediated manipulations.

Animals↗
PubMed2002

Differential display analysis of breast carcinoma cells enriched by immunomagnetic target cell selection: gene expression profiles in bone marrow target cells.

The red bone marrow (BM) is an important indicator organ of hematogenous micrometastatic spread of carcinomas. Characterization of biological properties specific for BM micrometastatic cells, however, is technically challenging due to the limited number of target cells usually available for the purpose. This report provides referrals to qualitative gene expression profiling of BM micrometastatic cells enriched by immunomagnetic selection. First, an experimental strategy was used to study regulatory mechanisms involved when BM micrometastatic cells colonize distant organs. The MA-11 cells, originating from BM micrometastases in a breast cancer patient clinically devoid of overt metastatic disease, were injected into immunodeficient rats. Metastatic MA-11 cells were subsequently immunoselected from the resulting in vivo lesions. The selected cell populations were compared to the injected cells by differential display analysis, and several genes possibly involved in tumor cell invasion and proliferation were confirmed as differentially expressed among the various MA-11 cell populations. A direct approach to qualitative gene expression profiling of BM micrometastatic cells was also explored. Carcinoma cells were immunoselected from BM and axillary lymph nodes obtained from breast cancer patients, and the isolated cell populations were compared by differential display analysis. Two candidate genes, identified as factors involved in cellular growth control, appeared as differentially expressed by the target cells from BM. Our study provides detailed information on how to combine an immunomagnetic selection procedure and differential display analysis to reveal gene expression profiles that may characterize BM micrometastatic cells.

Animals↗
PubMed2005

Single protein production in living cells facilitated by an mRNA interferase.

We designed a single-protein production (SPP) system in living E. coli cells that exploits the unique properties of MazF, a bacterial toxin that is an ssRNA- and ACA-specific endoribonuclease. In effect, MazF functions as an "mRNA interferase," because it efficiently and selectively degrades all cellular mRNAs in vivo, resulting in a precipitous drop in total protein synthesis. Concomitant expression of MazF and a target gene engineered to encode an ACA-less mRNA results in sustained and high-level (up to 90%) target expression in the virtual absence of background cellular protein synthesis. Remarkably, target synthesis continues for at least 4 days, indicating that cells retain transcriptional and translational competence despite their growth arrest. SPP technology works well for E. coli (soluble and membrane), yeast, and human proteins. This expression system enables unparalleled signal to noise ratios that should dramatically simplify structural and functional studies of previously intractable but biologically important proteins.

Amino Acid Sequence↗
PubMed1997

Xath5 participates in a network of bHLH genes in the developing Xenopus retina.

We examined the function of basic-helix-loop-helix (bHLH) transcription factors during retinal neurogenesis. We identified Xath5, a Xenopus bHLH gene related to Drosophila atonal, which is expressed in the developing Xenopus retina. Targeted expression of Xath5 in retinal progenitor cells biased the differentiation of these cells toward a ganglion cell fate, suggesting that Xath5 can regulate the differentiation of retinal neurons. We examined the relationship between the three bHLH genes Xash3, NeuroD, and Xath5 during retinal neurogenesis and found that Xash3 is expressed in early retinoblasts, followed by coexpression of Xath5 and NeuroD in differentiating cells. We provide evidence that the expression of Xash3, NeuroD, and Xath5 is coupled and propose that these bHLH genes regulate successive stages of neuronal differentiation in the developing retina.

Aging↗
PubMed1992

Species specificity in the interaction of CD8 with the alpha 3 domain of MHC class I molecules.

The alpha 1 and alpha 2 domains of the class I MHC molecule constitute the putative binding site for processed peptides and the TCR, although the alpha 3 domain has been implicated as a binding site for the CD8 molecule. Species specificity in the binding of CD8 to the alpha 3 domain has been suggested as an explanation for the low xenogeneic T cell response to class I molecules, but results on this point have been conflicting and controversial. We have addressed this issue using CTL lines from HLA-A2.1 transgenic mice that specifically recognize and lyse A2.1-expressing cells infected with influenza A/PR/8 or pulsed with influenza matrix peptide M1(57-68). Species specificity was examined using transfectants that expressed hybrid molecules containing the alpha 1 and alpha 2 domains from HLA-A2.1 and the alpha 3 domain from a murine class I molecule. Lower levels of M1(57-68) peptide were required to sensitize L cell transfectants expressing a chimera that contained an H-2Dd alpha 3 domain than targets expressing the intact A2.1 molecule. However, at high doses of peptide, lysis of these two targets was similar. However, no reproducible difference in sensitization was observed using EL4 or Jurkat transfectants expressing A2.1 or A2.1 chimeric molecules that contained an H-2Kb alpha 3 domain. In all cases, however, lysis of peptide-pulsed A2.1 expressing targets was more sensitive to inhibition with anti-CD8 mAb than lysis of cells expressing these chimeric molecules. Thus, under suboptimal conditions such as low Ag density or in the presence of anti-CD8 mAb, these CTL preferentially recognize class I molecules with a murine alpha 3 domain. This suggests that there is some species specificity in the interaction of CD8 with the alpha 3 domain of the class I molecule. However, CTL recognition was inhibited by point mutations in the alpha 3 domain of HLA-A2.1 that have been shown to inhibit binding of human CD8 and recognition by human CTL, suggesting that murine CD8 interacts to some degree with human alpha 3 domains, and that similar alpha 3 domain residues may be important for murine and human CD8 binding. The relevance of these results to an understanding of low xenogeneic responses is discussed.

Animals↗
PubMed1994

Partial purification of murine tumor-associated peptide epitopes common to histologically distinct tumors, melanoma and sarcoma, that are presented by H-2Kb molecules and recognized by CD8+ tumor-infiltrating lymphocytes.

The B16/BL6 melanoma is a relatively nonimmunogenic tumor expressing low levels of MHC class I molecules. BL6 clones expressing transfected H-2Kb class I molecules were, by contrast, highly immunogenic in immunocompetent mice. Tumor-infiltrating lymphocytes (TILs) generated from the H-2Kb+ BL6 lesions (Thy 1.2+, CD8+, CD4-) efficiently lysed H-2Kb+ melanoma (CL8-1 and 2Kb38) and the H-2Kb+ nonrelated 3-methylcholanthrene (MCA)-105 sarcoma, but not the H-2Kb- parental melanoma BL6-8. This strongly suggests that CL8-1, 2Kb38, and MCA-105 express identical or cross-reactive T cell epitopes recognized by CL8-1 TILs in the context of the H-2Kb class I allele. To identify the T cell epitopes, peptides were acid-eluted from various cells, and fractionated by HPLC. Five HPLC fractions (F1mel-F5mel) of 70 tested contained peptides derived from H-2Kb+ CL8-1 melanoma (but not H-2Kb- melanomas) that were capable of conferring susceptibility to CL8-1 TIL lysis on H-2b-expressing target cells (EL4, C1R.Kb), but not on H-2d-expressing P815 target cells. CL8-1 TILs failed to recognize peptides derived from H-2Kb+ nonmelanoma targets such as EL4 or normal B6 splenocytes. Interestingly, CL8-1 TILs appeared to recognize peptide species contained in two HPLC fractions derived from the MCA-105 sarcoma (F1sar and F5sar). Conversely, TILs derived from MCA-105 lesions recognized MCA-105 and CL8-1 tumor cells, as well as F5mel and F5sar peptides presented by EL4 targets. These data support common murine tumor-associated peptide epitopes presented by H-2Kb and recognized by CD8+ CTLs derived from histologically distinct tumors, melanoma and sarcoma.

Animals↗
PubMed1988

The organization of the rat GRP78 gene and A23187-induced expression of fusion gene products targeted intracellularly.

Expression of the glucose-regulated protein, GRP78, is markedly increased when cells are placed in a variety of stressful environments (i.e., low glucose medium, calcium ionophore treatment). In this report, the genomic organization of the rat GRP78 gene is described. This gene comprises eight exons and encodes a protein which is highly hydrophilic with the notable exception of several short hydrophobic domains. The first hydrophobic region, 18 amino acids at the N-terminus of the protein, putatively acts as a signal sequence to target GRP78 into the endoplasmic reticulum (ER). By ligating portions of the GRP78 gene and its promoter to the bacterial gene encoding chloramphenicol acetyltransferase (CAT), we created heterologous CAT genes inducible by calcium ionophore A23187. Through immunofluorescence analysis, the intracellular localizations of endogenous GRP78 and fusion CAT proteins under normal growth and A23187-induced conditions are identified. By fusing the GRP78 signal sequence to CAT, we influence intracellular targeting of the CAT protein into the ER.

Acetyltransferases↗
PubMed2006

Targeted in vivo expression of IFN-gamma-inducible protein 10 induces specific antitumor activity.

Although it is known that the chemoattractant effect of IFN-gamma inducible protein 10 (IP-10), a CXC chemokine (CXCL10), plays an important role in T cell-mediated antitumor immunity in vivo, whether IP-10 is involved in modulating the proliferation, survival and functional activation of tumor-specific T cells remains poorly investigated. Using an experimental mouse tumor model, we demonstrated that the in vivo growth of 4T1 tumor cells harboring IP-10 gene (4T1-IP-10) was inhibited. Mice inoculated with 4T1-IP-10 tumor cells expressing functional IP-10 survived over 90 days, whereas mice injected with control parental 4T1 cells and mice of control 4T1 cells transduced with control plasmid all succumbed to the tumor by day 38 after tumor inoculation. Mechanical analysis showed that targeted expression of IP-10 in 4T1 tumor cells markedly enhanced the infiltration of tumor-specific T cells into the 4T1-IP-10 tumor. These tumor infiltrating T lymphocytes (TILs) recruited by IP-10 were potent cytolytic killers against 4T1 tumor cells and were able to proliferate and produce high levels of IFN-gamma in response to 4T1 cells. In vivo administration of IP-10-recruited TILs induced vigorous proliferation of these TILs in situ in the 4T1-IP-10 tumor but not in the 4T1-pcDNA3 and parental 4T1 tumors. Furthermore, culture of TILs together with recombinant IP-10 significantly enhanced the proliferation and expansion of IP-10-recruited TILs in response to 4T1 tumor antigens. These results suggest that IP-10 is not only able to chemoattract tumor-specific T cells into the local tissue, but also enhance the proliferation, survival, and functional activation of these TILs, leading to the tumor regression. Thus, targeted expression of IP-10 in vivo will allow for the development of a novel approach for immunotherapy of tumor.

Adoptive Transfer↗
PubMed2004

Direct comparison of the insulating properties of two genetic elements in an adenoviral vector containing two different expression cassettes.

Targeted gene expression can be achieved through the use of cell-selective promoters. However, when the expression cassette is delivered by an adenovirus, "promoter interference," resulting in the loss of specificity, has been reported. To overcome this problem, insulator elements (the bovine growth hormone transcription stop signal or HS4 chromatin insulators of the chicken beta-globin locus) have been used. The present study examines the efficacy of these insulators elements, when two independent expression cassettes (one in which a strong, ubiquitous promoter drives the expression of the green fluorescent protein and another in which the "cancer-selective" ERBB2 promoter drives the expression of the herpes simplex virus thymidine kinase [HSVtk] gene) are incorporated into the same recombinant adenovirus. As expected, the presence of either insulator does not alter the expression of HSVtk in ERBB2-positive cells, measured through sensitization of the cells to ganciclovir. When ERBB2-negative cells were infected at a multiplicity of infection (MOI) of 10, the uninsulated virus sensitized ERBB2-negative cells to the same extent as it did for ERBB2-positive cells; partial sensitization was observed when transcriptional terminators were used, indicating a partial insulating effect; and complete insulation (no sensitization to ganciclovir) was observed when HS4 chromatin insulators were used. However, this complete insulation was lost when the MOI of virus was increased to 100. Our study demonstrates the possibility of insulating a conditionally expressed transgene in the vicinity of another expression cassette, but this insulating effect is lost when the multiplicity of infection is increased.

Adenoviridae↗