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PubMed2005

Glucosylated polyethylenimine as a tumor-targeting gene carrier.

Glucosylated polyethylenimine (GPEI) was synthesized as a tumor-targeting gene carrier through facilitative glucose metabolism by tumor glucose transporter. Particle sizes of GPEI/DNA complex increased in proportion to glucose content of GPEI, whereas surface charge of the complex was not dependent on glucosylation, partially due to inefficient shielding of the short hydrophilic group introduced. GPEI with higher glucosylation (36 mol-%) had no cytotoxic effect on cells even at polymer concentrations higher than 200 microg/mL. Compared to unglucosylated PEI, glucosylation induced less than one-order decrease of transfection efficiency. Transfection of GPEI/DNA complex into tumor cells possibly occurred through specific interaction between glucose-related cell receptors and glucose moiety of GPEI. Gamma imaging technique revealed GPEI/DNA complex was distributed in liver, spleen, and tumors.

Carbohydrate Sequence↗
PubMed2004

Increased expression of a Myc target gene Mina53 in human colon cancer.

Mina53 is a novel Myc target gene that we previously demonstrated to be involved in cell proliferation. We studied, here, the expression of Mina53 in colon cancer to examine its possible role in carcinogenesis. We generated a specific monoclonal anti-human Mina53 antibody and found that colon tumor cell lines expressed Mina53 highly. We also found that expression of Mina53 was elevated in colon tumor tissues by immunoblotting analysis. Tissue sections of 23 surgical cases of adenocarcinoma and 1 case of adenoma were stained immunohistochemically, and the expression of Mina53 was found to be elevated in all of the adenocarcinomas compared to adjacent nonneoplastic tissues, which showed little staining. Deeply invading tumors as well as tumors that have invaded lymphatic vessels showed strong immunoreactivity against anti-Mina53 antibody. Mina53 was expressed in all pathological grades of cancer as well as in the adenoma. Staining patterns of Ki-67, a biomarker for cell proliferation, were similar to those of Mina53 in most cases, but the percentage of tumor cells stained by anti-Mina53 was higher. Although anti-Ki-67 antibody strongly stained some well-proliferating nonneoplastic cells including cells in the deeper part of the crypts and in lymphoid germinal centers, antibody to Mina53 rarely stained those cells. Suppression of mina53 expression severely suppressed proliferation of colon tumor cells in vitro. Together, our results indicate that the elevated expression of Mina53 is a characteristic feature in colon cancer, one that may have therapeutic applications.

Adenocarcinoma↗
PubMed2006

CK2 controls the recruitment of Wnt regulators to target genes in vivo.

Nuclear beta-catenin is a transcriptional coactivator of LEF-1/TCF DNA-binding proteins in the Wnt/Wg signaling pathway. Casein Kinase 2 (CK2), a positive regulator of Wnt signaling, is present in beta-catenin complexes and activated in Wnt-signaling cells. We show here that CK2 enhances beta-catenin:LEF-1 transactivation in vivo and in vitro and that beta-catenin and CK2 cycle on and off the DNA in an alternating manner with the TLE1 corepressor at Wnt target genes. Interestingly, CK2 phosphorylates hLEF-1 directly and stimulates binding and transactivation of beta-catenin:LEF-1 complexes on chromatin templates in vitro. In vitro, CK2 phosphorylation of hLEF-1 strongly enhances its affinity for beta-catenin and reduces its affinity for TLE1. MALDI-TOF mass spectrometry (MS) identified two CK2 phosphorylation sites (S42, S61) within the amino terminus of hLEF-1, and mutation of these sites reduced binding to beta-catenin in vitro and transactivation in vivo. Remarkably, treatment of cells with TBB, a pharmaceutical inhibitor of CK2, blocked the recruitment and cycling of beta-catenin and TLE1 at Wnt target genes in vivo. Taken together, these data indicate that CK2 is required for the assembly and cycling of Wnt-enhancer complexes in vivo.

Animals↗
PubMed2000

Mutational spectra for polycyclic aromatic hydrocarbons in the supF target gene.

An SV40-based shuttle vector system was used to identify the types of mutational changes and the sites of mutation within the supF DNA sequence generated by the four stereoisomers of benzo[c]phenanthrene 3,4-dihydrodiol 1,2-epoxide (B[c]PhDE), by racemic mixtures of bay or fjord region dihydrodiol epoxides (DE) of 5-methylchrysene, of 5, 6-dimethylchrysene, of benzo[g]chrysene and of 7-methylbenz[a]anthracene and by two direct acting polycyclic aromatic hydrocarbon carcinogens, 7-bromomethylbenz[a]anthracene (7-BrMeBA) and 7-bromomethyl-12-methylbenz[a]anthracene (7-BrMe-12-MeBA). The results of these studies demonstrated that the predominant type of mutation induced by these compounds is the base substitution. The chemical preference for reaction at deoxyadenosine (dAdo) or deoxyguanosine (dGuo) residues in DNA, which is in general correlated with the spatial structure (planar or non-planar) of the reactive polycyclic aromatic hydrocarbon, is reflected in the preference for mutation at A&z.ccirf;T or G&z.ccirf;C pairs. In addition, if the ability to react with DNA in vivo is taken into account, the relative mutagenic potencies of the B[c]PhDE stereoisomers are consistent with the higher tumorigenic activity associated with non-planar polycyclic aromatic hydrocarbons and their extensive reaction with dAdo residues in DNA. Comparison of the types of mutations generated by polycyclic aromatic hydrocarbons and other bulky carcinogens in this shuttle vector system suggests that all bulky lesions may be processed by a similar mechanism related to that involved in replication past apurinic sites. However, inspection of the distribution of mutations over the target gene induced by the different compounds demonstrated that individual polycyclic aromatic hydrocarbons induce unique patterns of mutational hotspots within the target gene. A polymerase arrest assay was used to determine the sequence specificity of the interaction of reactive polycyclic aromatic hydrocarbons with the shuttle vector DNA. The results of these assays revealed a divergence between mutational hotspots and polymerase arrest sites for all compounds investigated, i.e., sites of mutational hotspots do not correspond to sites where high levels of adduct formation occur, and suggested that some association between specific adducts and sequence context may be required to constitute a premutagenic lesion. A site-specific mutagenesis system employing a single-stranded vector (M13mp7L2) was used to investigate the mutational events a single benzo[a]pyrene or benzo[c]phenanthrene dihydrodiol epoxide-DNA adduct elicits within specific sequence contexts. These studies showed that sequence context can cause striking differences in mutagenic frequencies for given adducts. In addition, these sequence context effects do not originate only from nucleotides immediately adjacent to the adduct, but are also modulated by more distal nucleotides. The implications of these results for mechanisms of polycyclic aromatic hydrocarbon-induced mutagenesis and carcinogenesis are discussed.

Animals↗
PubMed2006

Gene targeting in vivo by adeno-associated virus vectors.

Therapeutic gene delivery typically involves the addition of a transgene expression cassette to mutant cells. This approach is complicated by transgene silencing, aberrant transcriptional regulation and insertional mutagenesis. An alternative strategy is to correct mutations through homologous recombination, allowing for normal regulation of gene expression from the endogenous locus. Adeno-associated virus (AAV) vectors containing single-stranded DNA efficiently transduce cells in vivo and have been shown to target homologous chromosomal sequences in cultured cells. To determine whether AAV-mediated gene targeting can occur in vivo, we developed a mouse model that contains a mutant, nuclear-localized lacZ gene inserted at the ubiquitously expressed ROSA26 locus. Foci of beta-galactosidase-positive hepatocytes were observed in these mice after injection with an AAV vector containing a lacZ gene fragment, and precise correction of the 4-bp deletion was demonstrated by gene sequencing. We also used AAV gene-targeting vectors to correct the naturally occurring GusB gene mutation responsible for murine mucopolysaccharidosis type VII.

Adenoviridae↗
PubMed2006

Expression of HIF-1alpha, HIF-2alpha (EPAS1), and their target genes in paraganglioma and pheochromocytoma with VHL and SDH mutations.

CONTEXT: Activation of the hypoxia-inducible transcription factors HIF-1 and HIF-2 and a HIF-independent defect in developmental apoptosis have been implicated in the pathogenesis of pheochromocytoma (PCC) associated with VHL, SDHB, and SDHD mutations. OBJECTIVE: Our objective was to compare protein (HIF-1alpha, EPAS1, SDHB, JunB, CCND1, CD34, CLU) and gene (VEGF, BNIP3) expression patterns in VHL and SDHB/D associated tumors. RESULTS: Overexpression of HIF-2 was relatively more common in VHL than SDHB/D PCC (12 of 13 vs. 14 of 20, P = 0.02), whereas nuclear HIF-1 staining was relatively more frequent in SDHB/D PCC (19 of 20 vs. 13 of 16, P = 0.04). In addition, CCND1 and VEGF expression (HIF-2 target genes) was significantly higher in VHL than in SDHB/D PCC. These findings suggest that VHL inactivation leads to preferential HIF-2 activation and CCND1 expression as described previously in VHL-defective renal cell carcinoma cell lines but not in other cell types. These similarities between the downstream consequences of VHL inactivation and HIF dysregulation in renal cell carcinoma and PCC may explain how inactivation of the ubiquitously expressed VHL protein results in susceptibility to specific tumor types. Both VHL and SDHB/D PCC demonstrated reduced CLU and SDHB expression. SDHB PCC are associated with a high risk of malignancy, and expression of (proapototic) BNIP3 was significantly lower in SDHB than VHL PCC. CONCLUSION: Although inactivation of VHL and SDHB/D may disrupt similar HIF-dependent and HIF-independent signaling pathways, their effects on target gene expression are not identical, and this may explain the observed clinical differences in PCC and associated tumors seen with germline VHL and SDHB/D mutations.

Adrenal Gland Neoplasms↗
PubMed2004

Gene targeting in Drosophila.

DNA double-strand breaks provide a powerful means to modify the genome. This chapter describes how to generate and use these breaks to target specific sequences, or other modifications to the Drosophila genome. Both P element dependent gene conversion, in which the chromosomal DNA is broken, and the Rong and Golic gene-targeting technique, in which the targeting vector contains the DNA break are explained. The strengths and limitations of both methods are presented so that the user can choose the appropriate method for their particular situation. The efficiency of both methods depends upon the genomic location being modified, although few, if any, genomic locations are refractory to either method. It cannot be emphasized strongly enough that the investigator should be prepared to invest sufficient time into setting up and running these experiments properly.

Animals↗
PubMed2001

Mmip-2/Rnf-17 enhances c-Myc function and regulates some target genes in common with glucocorticoid hormones.

Members of the Mad family of basic-helix-loop-helix-leucine zipper proteins inhibit the transcriptional activity of the c-Myc oncoprotein. Mmip-2/Rnf-17 is a RING-finger protein that interacts with all four known Mad proteins, redistributes them to the cytoplasm, and thus enhances c-Myc function. We generated cell lines in which Mmip-2/Rnf-17 was rendered glucocorticoid (GC)-inducible. Stable expression of Mmip-/Rnf-17 resulted in the expected transport of the most abundant endogenous mad protein, Mxi1, to the cytoplasm. Compensatory increases in Mxi1 and Mad3 transcripts, similar to those previously described in Mad1 null hematopoietic cells, were also seen. Mmip-2/Rnf-17 also sensitized cells to several different pro-apoptotic stimuli and regulated a subset of c-Myc target genes. Unexpectedly, some of these genes were also found to be modulated solely by GCs. Thus, the inhibition of Mad proteins by Mmip-2/Rnf-17 modulates c-Myc function by enhancing its ability to regulate a subset of its potential target genes. Our results also identify a previously unrecognized overlap between genes regulated by c-Myc- and GCs and provide a potential molecular basis for their regulation of common cellular functions.

Animals↗
PubMed2000

Mechanisms involved in targeted gene replacement in mammalian cells.

The "ends-out" or omega (Omega)-form gene replacement vector is used routinely to perform targeted genome modification in a variety of species and has the potential to be an effective vehicle for gene therapy. However, in mammalian cells, the frequency of this reaction is low and the mechanism unknown. Understanding molecular features associated with gene replacement is important and may lead to an increase in the efficiency of the process. In this study, we investigated gene replacement in mammalian cells using a powerful assay system that permits efficient recovery of the product(s) of individual recombination events at the haploid, chromosomal mu-delta locus in a murine hybridoma cell line. The results showed that (i) heteroduplex DNA (hDNA) is formed during mammalian gene replacement; (ii) mismatches in hDNA are usually efficiently repaired before DNA replication and cell division; (iii) the gene replacement reaction occurs with fidelity; (iv) the presence of multiple markers in one homologous flanking arm in the replacement vector did not affect the efficiency of gene replacement; and (v) in comparison to a genomic fragment bearing contiguous homology to the chromosomal target, gene targeting was only slightly inhibited by internal heterology (pSV2neo sequences) in the replacement vector.

Animals↗
PubMed2001

Amphiregulin is a vitamin D3 target gene in squamous cell and breast carcinoma.

1alpha,25-Dihydroxyvitamin D(3) [1,25(OH)2D3] inhibits growth of cells derived from a variety of tumors in vitro and in vivo. Proliferation in vitro of human SCC25 cells, derived from a primary squamous cell carcinoma (SCC) of the tongue, was blocked by 1,25(OH)2D3 and its analog EB1089. A similar effect was observed with 13-cis retinoic acid (RA), which has been used in chemoprevention of SCC. We identified amphiregulin, a member of the epidermal growth factor family, as a 1,25(OH)2D3 target gene in SCC25 cells. Induction of amphiregulin mRNA by 1,25(OH)2D3 was rapid and sustained over 48 h, and was unaffected by cycloheximide. 1,25(OH)2D3 also induced amphiregulin mRNA in estrogen receptor-positive and -negative human breast cancer cell lines, but not in LNCaP human prostate cancer cells. RAR- or RXR-specific retinoids did not affect amphiregulin mRNA levels in SCC25 cells; however, 13-cis RA partially blocked the response to 1,25(OH)2D3. Amphiregulin partially inhibited growth of SCC25 cells in culture. Our data show that amphiregulin is a 1,25(OH)2D3 target gene, and suggest that its induction may contribute to the growth inhibitory effects of 1,25(OH)2D3.

Amphiregulin↗
PubMed2001

Specificity of signaling by STAT1 depends on SH2 and C-terminal domains that regulate Ser727 phosphorylation, differentially affecting specific target gene expression.

Complete activation of signal transducer and activator of transcription 1 (STAT1) requires phosphorylation at both Y701 and a conserved PMS(727)P sequence. S727 phosphorylation of STAT1 in interferon-gamma (IFN-gamma)-treated mouse fibroblasts occurred without a need for p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinases 1 and 2 or c-Jun kinases, and required both an intact SH2 domain and phosphorylation of Y701. In contrast, UV irradiation-induced STAT1 phosphorylation on S727 required p38MAPK, but no SH2 domain- phosphotyrosine interactions. Mutation of S727 differentially affected IFN-gamma target genes, at the level of both basal and induced expression. Particularly strong effects were noted for the GBP1 and TAP1 genes. The PMS(727)P motif of STAT3 was phosphorylated by stimuli and signaling pathways different from those for STAT1 S727. Transfer of the STAT3 C-terminus to STAT1 changed the stimulus and pathway specificity of STAT1 S727 phosphorylation to that of STAT3. Our data suggest that STAT C-termini contribute to the specificity of cellular responses by linking individual STATs to different serine kinase pathways and through an intrinsically different requirement for serine phosphorylation at different target gene promoters.

3T3 Cells↗
PubMed1996

Analysis of glucocorticoid and mineralocorticoid signalling by gene targeting.

To get a better understanding of the role of glucocorticoid and mineralocorticoid signalling during development and in whole animal physiology, we have disrupted the mouse glucocorticoid and mineralocorticoid receptor gene by gene targeting. Most of the mice with a disrupted glucocorticoid receptor gene die within the first hours after birth due to severe lung atelectasis. Perinatal induction of gluconeogenic enzymes in the liver is impaired. Feed back control of the glucocorticoid synthesis via the hypothalamic-pituitary-adrenal axis is perturbed leading to increased plasma levels of corticosterone and adrenocorticotrophic hormone. Increased activity of the hypothalamic-pituitary-adrenal axis results in extensive hypertrophy and hyperplasia of the cortical zones of the adrenal and induction of genes involved in steroid biosynthesis. The adrenal medulla is disorganized and severely reduced in size; cells capable of adrenaline synthesis are missing. Mineralocorticoid receptor deficient mice die around day 10 after birth. Weight loss precedes death of homozygous mutant mice and is correlated with an increase in the haematocrit. As a consequence of this mutation plasma levels of renin, angiotensin II, and aldosterone are highly elevated.

Animals↗
PubMed2006

Pregnane X receptor activation ameliorates DSS-induced inflammatory bowel disease via inhibition of NF-kappaB target gene expression.

Pregnane X receptor (PXR) expression was shown to be protective in inflammatory bowel disease (IBD). However, the mechanism by which PXR provides protection remains unclear. Wild-type and Pxr-null mice were treated with the PXR agonist pregnenolone-16alpha-carbonitrile or vehicle and administered 2.5% dextran sulfate sodium (DSS) in drinking water to induce IBD. Typical clinical symptoms were evaluated on a daily basis. In vivo intestinal permeability assays and proinflammatory cytokine analysis were performed. PXR agonist-treated mice were protected from DSS-induced colitis compared with vehicle-treated mice, as defined by body weight loss, diarrhea, rectal bleeding, colon length, and histology. Pregnenolone-16alpha-carbonitrile did not decrease the severity of IBD in Pxr-null mice. PXR agonist treatment did not increase epithelial barrier function but did decrease mRNA expression of several NF-kappaB target genes in a PXR-dependent manner. The present study clearly demonstrates a protective role for PXR agonist in DSS-induced IBD. The data suggest that PXR-mediated repression of NF-kappaB target genes in the colon is a critical mechanism by which PXR activation decreases the susceptibility of mice to DSS-induced IBD.

ATP-Binding Cassette Transporters↗
PubMedDate not supplied

Novel shielded transferrin-polyethylene glycol-polyethylenimine/DNA complexes for systemic tumor-targeted gene transfer.

Tumor-targeting DNA complexes which can readily be generated by the mixing of stable components and freeze-thawed would be very advantageous for their subsequent application as medical products. Complexes were generated by the mixing of plasmid DNA, linear polyethylenimine (PEI22, 22 kDa) as the main DNA condensing agent, PEG-PEI (poly(ethylene glycol)-conjugated PEI) for surface shielding, and Tf-PEG-PEI (transferrin-PEG-PEI) to provide a ligand for receptor-mediated cell uptake. Within the shielding conjugates, PEG chains of varying size (5, 20, or 40 kDa) were conjugated with either linear PEI22 (22 kDa) or branched PEI25 (25 kDa). The three polymer components were mixed together at various ratios with DNA; particle size, surface charge, in vitro transfection activity, and systemic gene delivery to tumors was investigated. In general, increasing the proportion of shielding conjugate in the complex reduced surface charge, particle size, and in vitro transfection efficiency in transferrin receptor-rich K562 cells. The particle size or surface charge of the complexes containing the PEG-PEI conjugate did not significantly change after freeze-thawing, while complexes without the shielding conjugate aggregated. Complexes containing PEG-PEI conjugate efficiently transfected K562 cells after freeze-thawing. Furthermore the systemic application of freeze-thawed complexes exhibited in vivo tumor targeted expression. For complexes containing the luciferase reporter gene the highest expression was found in tumor tissue of mice. An optimum formulation for in vivo application, PEI22/Tf-PEG-PEI/PEI22-PEG5, containing plasmid DNA encoding for the tumor necrosis factor (TNF-alpha), inhibited tumor growth in three different murine tumor models. These new DNA complexes offer simplicity and convenience, with tumor targeting activity in vivo after freeze-thawing.

Animals↗
PubMedDate not supplied

Ets-2 interacts with co-repressor BS69 to repress target gene expression.

BACKGROUND: The ETS-family of proteins consists of over 30 members that regulate the growth, differentiation and survival of both normal and tumor cells. How specificity is achieved within this family remains largely unresolved. One mechanism for attaining specificity is through the action of signaling pathways on specific family members. For example, Ets-2 is an activator modulated by ras-dependent phosphorylation of a single residue in the conserved pointed domain of this factor. We hypothesized that phosphorylation of the pointed domain regulates the proteins that can interact with ets-2 in the cell nucleus, resulting in regulation of target genes. MATERIALS AND METHODS: We used a combination of biochemical assays, yeast two-hybrid screens and transfection assays to identify and characterize proteins interacting with the pointed domain. RESULTS: BS69, a known co-repressor, was identified in a yeast two hybrid screen as an ets-2 interacting partner. BS69 can interact with ets-2 in vivo and phosphorylation of the ets-2 pointed domain decreased the interaction with BS69 in vitro. In transfection assays, co-expression of ets-2 and BS69 resulted in repression of defined ets-2 target genes. CONCLUSION: These results support a role for ets-2 as a repressor and indicate that BS69 is required as co-repressor. Phosphorylation of ets-2 may switch its activity from repressor to activator by interfering with formation of the BS69 complex.

3T3 Cells↗
PubMed1998

Analysis of WT1 target gene expression in stably transfected cell lines.

The Wilms' tumour suppressor gene WT1 encodes a zinc finger protein that is mutated in a subset of Wilms' tumours. Mutation screening and animal studies revealed essential roles during development and later function of the kidneys and the entire genitourinary system. Sequence similarity suggested a possible role for WT1 as a transcription factor. Indeed, sequence specific DNA binding and transcriptional activation or repression potential could be demonstrated in transient transfection assays with various reporter constructs. To identify endogenous WT1 target genes we established HEK293 cell lines expressing the different WT1 isoforms in a tetracycline dependent manner. Differential display PCR (ddPCR) was performed on RNA from stable WT1 transfected HEK293 cell lines and two other WT1 transfected lines (G401 and Saos-2). In an extended survey of several thousand ddPCR bands only few differences in intensity were seen and none of these could unambiguously be verified as being WT1 regulated by subsequent Northern blot analysis. In addition, almost none of the WT1 target genes identified to date in transient co-transfection assays could be confirmed by either ddPCR or Northern hybridization in the three stable transfected cell lines. Among the nine genes expressed, the only exceptions were CSF1 and to a lesser extent IGF1R being induced in Saos-2/G401 and HEK293 cells, respectively. At least two of the cell lines tested had previously shown clear biological effects though -- either WT1 dependent apoptosis (Saos-2) or greatly reduced tumorigenicity (G401). This suggests that WT1 may regulate only a very small set of genes that escape the detection methods used or it may not act as a transcription factor that influences steady state levels of mRNA.

Blotting, Northern↗
PubMed1994

Gene targeting of the immune system: the surprises continue.

To date, an impressive number of mutant mice strains have been generated by targeted mutagenesis of the immune system. During the past year, such knockout mice have been particularly valuable in revealing the biological functions of certain cytokines and their receptors, and also in identifying cell surface molecules critical for T-cell activation. Advances in targeting technologies also figure prominently in the accomplishments of the past year, with cell type specific gene targeting representing a major refinement of current methodologies.

Animals↗
PubMed2004

Activation of REST/NRSF target genes in neural stem cells is sufficient to cause neuronal differentiation.

REST/NRSF is a transcriptional repressor that acts at the terminal stage of the neuronal differentiation pathway and blocks the transcription of several differentiation genes. REST/NRSF is generally downregulated during induction of neuronal differentiation. The recombinant transcription factor REST-VP16 binds to the same DNA binding site as does REST/NRSF but functions as an activator instead of a repressor and can directly activate the transcription of REST/NRSF target genes. However, it is not known whether REST-VP16 expression is sufficient to cause formation of functional neurons from neural stem cells (NSCs). Here we show that regulated expression of REST-VP16 in a physiologically relevant NSC line growing under cycling conditions converted the cells rapidly to the mature neuronal phenotype. Furthermore, when grown in the presence of retinoic acid, REST-VP16-expressing NSCs activated their target, as well as other differentiation genes that are not their direct target, converting them to the mature neuronal phenotype and enabling them to survive in the presence of mitotic inhibitors, which is a characteristic of mature neurons. In addition, these neuronal cells were physiologically active. These results showed that direct activation of REST/NRSF target genes in NSCs with a single transgene, REST-VP16, is sufficient to cause neuronal differentiation, and the findings suggested that direct activation of genes involved in the terminal stage of differentiation may cause neuronal differentiation of NSCs.

Animals↗