Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: DrugBank:BIOD00001 (DNase I)
 8,324 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The nuclear matrix is concealed by a much larger mass of chromatin, which can be removed selectively by digesting nuclei with DNase I followed by elution of chromatin with 0.25 M ammonium sulfate. This mild procedure removes chromatin almost completely and preserves nuclear matrix morphology. The complete nuclear matrix consists of a nuclear lamina with an interior matrix composed of thick, polymorphic fibers and large masses that resemble remnant nucleoli. Further extraction of the nuclear matrices of HeLa or MCF-7 cells with 2 M sodium chloride uncovered a network of core filaments. A few dark masses remained enmeshed in the filament network and may be remnants of the nuclear matrix thick fibers and nucleoli. The highly branched core filaments had diameters of 9 and 13 nm measured relative to the intermediate filaments. They may serve as the core structure around which the matrix is constructed. The core filaments retained 70% of nuclear RNA. This RNA consisted both of ribosomal RNA precursors and of very high molecular weight hnRNA with a modal size of 20 kb. Treatment with RNase A removed the core filaments. When 2 M sodium chloride was used directly to remove chromatin after DNase I digestion without a preceding 0.25 M ammonium sulfate extraction, the core filaments were not revealed. Instead, the nuclear interior was filled with amorphous masses that may cover the filaments. This reflected a requirement for a stepwise increase in ionic strength because gradual addition of sodium chloride to a final concentration of 2 M without an 0.25 M ammonium sulfate extraction uncovered core filaments.
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PMID:Core filaments of the nuclear matrix. 230

The physical-chemical properties of the nuclear estrogen receptor released by DNase I were characterized. Nuclei were isolated from MCF-7 cells previously exposed to 10-nM-[3H]estradiol. The parameters determined were: sedimentation coefficients (S) on a sucrose gradient, Stokes radii (Rs) by gel filtration on a Sephadex G-200 column and the binding ability to a DNA-cellulose column. The molecular weights (Mr) and frictional ratios (f/fo) were calculated from the S and Rs values. The properties of the receptor released by DNase I obtained from Worthington were compared to the properties of the receptor released by DNase I obtained from Sigma. Digestion with DNase I (Worthington) excised a receptor form which could be solubilized from nuclei by EDTA. This form sedimented at 5.2S with a Rs = 7.08 nm and a calculated Mr = 152.000. About 40% of this receptor form bound to a DNA-cellulose column. 0.4 M KCl dissociated this receptor form into a smaller form sedimenting at 4.2S with Rs = 4.64 nm and a calculated Mr = 80.000. The properties of the receptor solubilized by micrococcal nuclease followed by DNase I (Worthington) digestion were identical to the properties of the DNase I (Worthington) released receptor. Digestion with DNase I (Sigma) released a 3.2S receptor form, which diffused through the nuclear membrane and a 4-5S form which could be extracted from nuclei by EDTA. The 3.2S receptor had a Rs = 2.41 nm, a calculated Mr = 32.000 and less than 5% of it bound to a DNA-cellulose column. Digestion with micrococcal nuclease followed by DNase I (Sigma) solubilized a receptor form with identical properties to the 3.2S receptor. These results suggest that DNase I (Worthington) released a receptor form still associated with some molecules, probably chromatin proteins, which complexed it to DNA, while DNase I (Sigma) released the estradiol binding fragment of the receptor (meroreceptor) as a result of a proteolytic activity present in this preparation.
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PMID:Physical-chemical properties of the estrogen receptor released by deoxyribonuclease I. 372 48

The physical-chemical properties of the nuclear estrogen receptor from MCF-7 cells were determined. The receptor was solubilized by micrococcal nuclease. Nuclei were isolated from cells previously exposed to 10 nM [3H]estradiol. The amount of receptor released was parallel to the extent of chromatin solubilized, which suggested that the receptor is homogeneously distributed on the chromatin. Following mild nuclease digestion the excised receptor sedimented as an abundant 6-7 S form and as a less abundant approximately 12 S species. The 6-7 S form represented the receptor excised in association with linker DNA, while the approximately 12 S may represent receptor bound to linker DNA which remained associated with the nucleosome. Increasing the extensiveness of digestion resulted in one receptor form sedimenting at 5.6 S. Additional digestion with DNase I did not affect the sedimentation coefficient of the receptor. Sedimentation of the micrococcal nuclease hydrolysate in a 0.4 M KCl sucrose gradient resulted in a 4.2 S receptor form. The same receptor form was extracted from undigested nuclei with 0.4 M KCl. Using Sephadex G-200 column chromatography we have determined the Stokes radii (Rs), molecular weight (Mr) and frictional ratio (f/fo) for the 5.6 S and 4.2 S receptor forms. For the 5.6 S form: Rs = 7.04 nm, Mr = 163,000 and (f/fo) = 1.80. For the 4.2 S receptor, Rs = 4.45 nm, Mr = 77,000 and (f/fo) = 1.46. The ability of the nuclease solubilized 5.6 S receptor to bind DNA was tested using DNA-cellulose column and highly polymerized DNA. About 40% of the applied receptor bound to the column and could be eluted by high salt concentrated buffer. The 5.6 S receptor form was sedimented on sucrose gradient by the highly polymerized DNA. These results suggested that the receptor is bound in chromatin as a dimer or as a monomer in association with other protein(s) which complexed it with DNA.
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PMID:Physical-chemical properties of the estrogen receptor solubilized by micrococcal nuclease. 403 15

Promoter elements accounting for HER2 (c-erbB-2/neu) overexpression were searched for in several human breast cancer cell lines (MDA-453, BT-474, ZR-75-1, MCF-7) known to express constitutively a 30-fold range in HER2 transcripts per gene copy. HER2 overexpressing cells showed a single prominent DNase I hypersensitive site near a conserved and hitherto unrecognized ets response element (GAGGAA), located 38 bases down-stream from the CAAT box and directly 5' of the TATA box in the human HER2 promoter. Transient transfection of HER2 promoter constructs (0.125, 0.5, and 2.0 kilobase pairs (kb)) demonstrated that the most proximal promoter region (0.125 kb) was capable of conferring up to 30-fold enhanced activity in HER2-overexpressing cell lines relative to low HER2-expressing control lines. Site-directed mutagenesis of the ets response element (GAGGAA-->GAGAGA) caused a > or = 60% reduction in promoter activity affecting at least 0.5 kb of upstream HER2 regulatory sequence. Gel-shift assays with nuclear extracts and oligonucleotide sequences spanning the 0.125-kb promoter region detected an ETS-immunoreactive complex, present most abundantly in cells overexpressing HER2, whose high-affinity binding depended on the GAGGAA response element. Methylation interference confirmed the ETS-specific pattern of protein binding by this complex to guanine bases in the ets response element. UV cross-linking and immunoprecipitation implicate a approximately 60-kDa ETS protein, and candidate ETS genes expressed in these breast cancer cells include GABP alpha, elk-1, elf-1, and PEA3.
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PMID:Binding of an ETS-related protein within the DNase I hypersensitive site of the HER2/neu promoter in human breast cancer cells. 791 92

The human oestrogen receptor (hER) gene has recently been shown to be transcribed from two different promoters. In order to study the hER gene promoter region we initially isolated and sequenced 3.3 kb of the 5' region of the hER gene. DNase I hypersensitivity experiments with nuclei from the ER-expressing cell line MCF-7 mapped three different hypersensitive sites, located in the vicinity of the two hER promoters previously identified. However, in another ER-positive cell line, ZR-75-1, DNase I hypersensitivity was observed only in the region of the downstream promoter, suggesting differences between the two cell lines in promoter usage. Polymerase chain reaction-mediated detection of mRNAs transcribed from the two different promoters demonstrated transcripts from the downstream promoter in both the cell lines studied. However, mRNAs transcribed from the upstream promoter could only be detected in MCF-7 cells. Thus, the pattern of DNase I hypersensitivity correlates with the transcriptional activity of the two promoters in the hER gene. Based on these data, we suggest that differential promoter usage might be a novel level of regulation of the hER gene.
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PMID:Localization of DNase I hypersensitive sites in the human oestrogen receptor gene correlates with the transcriptional activity of two differentially used promoters. 837 11

DNase I hypersensitivity regions correlate with genetic regulatory loci and binding sites for sequence-specific DNA-binding proteins. We present data supporting the presence of novel DNase 1 hypersensitive sites (which we have designated sites VI-IX) in both the body of the human c-myc gene downstream from exon 2 and the 3'-flanking region of the c-myc gene in HL-60 cells. All of these novel DH sites are markedly decreased when HL-60 cells are treated with either dimethyl sulfoxide (DMSO) or retinoic acid. Moreover, a similar pattern of DNase I hypersensitive sites in this region of c-myc was present in MCF-7 human breast cancer cells growing in culture. Our results suggest a potential role for these sites in transcriptional regulation of the human c-myc gene.
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PMID:Novel DNase I hypersensitive sites in the 3'-flanking region of the human c-myc gene. 875 35

The induction of cell death along with cell-cycle arrest is one of the foremost mechanisms regulating cell growth. In the human breast carcinoma cell line MCF-7 we investigated two chemotherapeutic agents, the antiestrogen tamoxifen and the DNA-damaging drug cisplatin, for the relative contribution of these mechanisms to growth inhibition in culture. Growth kinetics and flow cytometry confirmed that tamoxifen at 1 microM acts mainly by arresting cells in the G0/G1 phase of the cell cycle. Compared to untreated controls, only a few more cells were detached from the monolayer and dead after a 5-day incubation. On the other hand, cisplatin at 1 microM did not induce the well-defined G2/M-arrest reported for other cell types, but resulted in a marked increase in the rate of cell death. A morphological feature observed, especially with cisplatin-treated MCF-7 cells, was the formation of numerous micronuclei (in up to 30% of the cells) and an increase in the number of binucleate cells (up to 20%). In both tamoxifen- and cisplatin- treated cultures, cell death appeared to occur by apoptosis, as indicated morphologically by cellular and nuclear shrinkage accompanied by DNA-condensation and ultimately the formation of DNA containing apoptotic bodies. However, no internucleosomal DNA degradation or endogenous endonuclease activity could be detected in the cells of the monolayer or in the mainly dead and detached cells of the culture supernatant. DNA fragmentation was only observed when isolated MCF-7 nuclei were incubated with exogenous endonucleases. However, as determined by reverse transcriptase/polymerase chain reaction amplification, MCF-7 cells do express the mRNA for DNase I, an endonuclease known to be involved in apoptosis. Thus, apoptosis is part of the growth-inhibitory process and occurs without apparent internucleosomal DNA fragmentation in MCF-7 cell cultures.
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PMID:Cell-cycle arrest, micronucleus formation, and cell death in growth inhibition of MCF-7 breast cancer cells by tamoxifen and cisplatin. 887 58

We previously demonstrated an enhancer-like positive regulatory element within a 259-bp sequence (-2352 to -2094 bp) of the human CYP1A2 gene in HepG2 cells. Three protein binding sites were identified by DNase I footprinting analyses within the 259-bp sequence: protected region A PRA; -2283 to -2243 bp), PRB (-2218 to -2187 bp), and PRC (-2124 to -2098 bp) (I. Chung and E. Bresnick, Mol. Pharmacol. 47, 677-685, 1995). In the present study, the functional significance of those protected regions was examined. Transfection experiments with deletion and substitution mutants defined the PRB and PRC as containing positive and negative regulatory elements, respectively. Human breast carcinoma MCF-7 cells were cotransfected with a hepatocyte nuclear factor-1 (HNF-1) expression vector and CYP1A2 promoter- or thymidine kinase promoter-luciferase reporter gene constructs. HNF-1, which contributes to the liver specificity of genes, enhanced reporter gene activity in a PRC sequence-dependent manner. These results suggested that PRC could exist bound to a repressor which was displaceable by other transcription factors such as HNF-1. Results obtained by transfection of HepG2 hepatoma cells with various PRB substitution mutant-luciferase gene fusion constructs indicated that the entire sequence of PRB was necessary for promoter activity. Consequently, the regulation of CYP1A2 expression is very complex, requiring a number of both positive and negative regulatory factors.
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PMID:Identification of positive and negative regulatory elements of the human cytochrome P4501A2 (CYP1A2) gene. 902 75

The positioning of nucleosomes on a promoter is a significant determinant in its responsiveness to inducing signals. We have mapped the chromatin structure of the human, estrogen-responsive pS2 promoter at nucleotide level resolution within the context of its normal genomic location in human mammary epithelial cells. In vivo digestion by nucleases followed by ligation-mediated polymerase chain reaction analysis revealed two rotationally phased and translationally positioned nucleosomes within the promoter between nucleotide positions -450 and +7. The estrogen response elements at -400 and TATAA box at -35 are each located at the edge of a nucleosome. The two precisely positioned nucleosomes exist in both transformed and nontransformed human mammary epithelial cells, regardless of estrogen receptor status or transcriptional activity of the gene. However, two structural alterations correlate with the transcriptional potential of the promoter. In MCF-7 cells, in which the pS2 promoter is inducible, the chromatin exhibits an increased sensitivity to DNase I in a region of DNA adjacent to the TATAA box and an additional micrococcal nuclease-hypersensitive site in the linker DNA between the two positioned nucleosomes. We were also able to demonstrate that nucleotides -1100 to +10 of the pS2 promoter are sufficient to determine the positioning of these two nucleosomes. Our results establish the structural features of the chromatin covering the pS2 promoter as well as transcriptionally associated alterations, suggesting how the nucleosomal template influences transcriptional regulation by estrogen receptor.
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PMID:Nucleosome positioning and transcription-associated chromatin alterations on the human estrogen-responsive pS2 promoter. 938 65

Connexin (Cx) 26, a major gap junction protein expressed in mammary epithelial cells, has been considered to be a tumor suppressor gene candidate. This study investigated the molecular mechanism of transcriptional up-regulation of Cx26 by phorbol ester (TPA) in human immortalized MCF-10 mammary epithelial cells and MDA-MB-231 mammary cancer cells. Such up-regulation was mediated through the protein kinase C pathway and could be blocked by the PKC inhibitor, calphostin C. Based on the results of the nuclear run-on assay, there was a TPA-induced increase in the rate of transcriptional initiation. We identified a TPA-induced DNase I hypersensitivity (DH) region approximately 1 kb 5' upstream of the ATG translation starting site. Sequence analysis revealed that this DH region was located in intron 1 and contained two TRE-like TGAT/ATCA elements, two 5'TTCA3' motifs and a 5'AGGAAG3' PEA3 motif. Both TRE-like elements were capable of binding AP1. TPA inducibility of this DH region was seen by the CAT reporter assay and appeared to be direction-dependent suggesting a functional cooperation between PEA3/TTCA and TRE.
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PMID:Gap junction Cx26 gene modulation by phorbol esters in benign and malignant human mammary cells. 952 50


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