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Query: UMLS:C0027651 (tumor)
 685,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A one-step procedure which uses enzymes in a crude extract of herpes simplex virus (HSV) type 1-infected cells to synthesize 5-[125I]iodo-2'-deoxyuridine triphosphate [( 125I]dUTP) from [125I]dU is described. The design of a one-step procedure for the purification of the product is also presented. The recovery of [125I]dUTP from [125I]dU varied between 50 and 75%, the radiochemical purity of the product was greater than 90%, and both synthesis and purification were completed within 8 h. The sensitivity and specificity of [125I]dUTP as a substrate for both DNA-dependent DNA polymerase (DNAp) and RNA-dependent DNA polymerase (reverse transcriptase, RT) were evaluated and compared to those of [3H]dTTP for the following specimens: purified cloned Klenow fragment, crude extracts of HeLa-, BHK-, and HSV-2-infected BHK cells, purified avian myeloblastosis virus RT, and purified cloned human immunodeficiency virus (HIV) RT. The [125I]dUTP was accepted as a substrate equally as well [3H]dTTP by all of the specimens at all of the concentrations tested. When the same amount of radiolabel was used, [125I]dUTP gave a sensitivity 10- to 25-fold higher than that of [3H]dTTP. The gain in sensitivity was due to the higher specific activity and a higher counting efficiency of the 125I-label compound. The use of [125I]dUTP also offered technical advantages over alternative substrates available, such as product separation without acid precipitation and exclusion of the need for scintillation cocktails. The half-life of the nucleic also gives a reasonable shelf-life for use in routine assays. Activity of less than 0.3 pg of HIV RT could be detected when the new substrate was used, and this made it possible to quantitate HIV RT antibodies (abs) in diluted serum samples without purifying the immunoglobulin. Analysis of 31 HIV-infected individuals showed that all of them had anti-HIV RT ab and that the amount of serum needed for 50% inhibition of the HIV RT activity corresponded to an amount of immunoglobulin 100-fold smaller (i.e., 0.02-31.4 micrograms) than has been previously reported. With the substrate it was also possible to detect DNAp activity in serum from healthy individuals, although a long-duration assay was required. In a long-duration assay the DNAp activity found in sera from healthy individuals was linear with respect to time, whereas the DNAp activity found in many sera from tumor patients was not. [125I]dUTP is judged to be an excellent substrate for detecting and quantifying the activity of various DNA-synthesizing enzymes and their blocking abs.
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PMID:Improved assays for DNA-polymerizing enzymes by the use of enzymatically synthesized 5-[125I]iodo-2'-deoxyuridine triphosphate, illustrated by direct quantitation of anti-HIV reverse transcriptase antibody and by serum DNA polymerase analyses. 169 11

We now show that exposure of B16 melanoma cells to bromodeoxyuridine increases cell-substratum interactions concurrent with an increase in genome susceptibility to nucleases. Hypersensitive DNA was isolated after mild nicking of nuclei with DNase I followed by repair with DNA polymerase I in the presence of biotin-19-SS-dUTP and affinity chromatography on streptavidin-agarose. Dot blot studies showed that the hypersensitive DNA is enriched in c-myc sequences compared to total tumor genomic DNA, and hybridizes preferentially to the latter, compared to normal genomic DNA, particularly when prepared from BrdU-treated cells. Since hypersensitive DNA can hybridize with multiple Alu sequences in the genome, we postulate that one of the mechanisms for its differential reactivity may be by recognition of an unequal number of Alu repeats in normal and tumor genomic DNA.
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PMID:Tumor hypersensitive DNA is enriched in c-myc sequences and reacts differentially with normal and malignant genomic DNA. 219 5

We achieved histological detection of the messenger RNAs coding for vasopressin, calcitonin, or calcitonin gene-related peptide by using biotinylated synthetic oligonucleotides, and defined the technical parameters enabling optimal detection of these mRNAs. Oligonucleotides labeled by fixation of one biotin at their 5' end or by addition of a biotin-11-dUTP tail at their 3' end can be used to detect mRNAs, although the latter are more sensitive. Streptavidin-alkaline phosphatase revealed with nitroblue tetrazolium-bromo-chloro-indolyl phosphate as substrate makes possible detection of the biotinylated oligonucleotides. Increasing formaldehyde concentration in the fixative decreases the signal intensity; 1% formaldehyde fixation provides the most intense signal. Several controls, including those with addition of unlabeled oligonucleotides to the hybridization buffer, confirm the specificity of mRNA detection. The sensitivity of the biotinylated probes is identical or lower as compared to the corresponding radiolabeled oligonucleotides. Histological and subcellular resolution is greatly enhanced with biotinylated probes. The rat vasopressin probes stain magnocellular neurons in the supraoptic and paraventricular nuclei and, under optimal conditions, parvocellular neurons in the suprachiasmatic nucleus. Vasopressin mRNA is present in the cytoplasm of the cell bodies and in the roots of certain processes. Calcitonin and calcitonin gene-related peptide mRNA are found co-localized in the cytoplasm of the same tumor cells in human medullary thyroid carcinoma.
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PMID:Histological detection of messenger RNAs with biotinylated synthetic oligonucleotide probes. 325 49

Human epidermal growth factor (EGF) receptor mRNA was detected in cryopreserved tissue sections adherent to whole glass slides using in situ reverse transcriptase polymerase chain reaction. EGF receptor cDNA was synthesized in situ by reverse transcription using an EGF receptor-specific oligonucleotide primer. In situ polymerase chain reaction amplification in the presence of digoxigenin-11-dUTP and subsequent binding with an antidigoxigenin antibody conjugated to alkaline phosphatase allowed direct visualization. Because DNase, RNase, or proteinase K are not required, tissue integrity is maintained. EGF receptor mRNA is expressed in the basal layer of normal human skin epithelium and is significantly overexpressed in squamous cell tumor specimens, which is consistent with conventional analysis of EGF receptor expression. The assay is semiquantitative, quicker, more sensitive, and void of the nonspecific binding associated with in situ hybridization. In situ reverse transcriptase polymerase chain reaction using whole glass slides is ideally suited for detecting moderate to infrequently expressed transcripts in biopsy specimens.
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PMID:Detection of epidermal growth factor receptor mRNA in tissue sections from biopsy specimens using in situ polymerase chain reaction. 808 54

The Fas antigen is a transmembrane receptor that can trigger apoptosis in a variety of tumor and hematopoietic cells. Ovarian follicular atresia and luteolysis are thought to occur by apoptosis. Using reverse transcriptase-polymerase chain reaction (RT-PCR) and flow cytometry, we demonstrated that human granulosa/luteal cells express the Fas antigen. An anti-human Fas antigen monoclonal antibody (Fas mAb; clone CH-11), which induces apoptosis in other cell types by binding to the Fas antigen, induced significant cell death (30%) in cultures pretreated with interferon gamma (IFN gamma). This agrees with studies on tumor cell lines showing that IFN gamma enhances cytotoxic effects of Fas mAb. Granulosa/luteal cells exhibited morphological characteristics typical of apoptosis, including membrane blebbing and condensed chromatin. DNA fragmentation into oligonucleosomal units of approximately 180 bp, typical of apoptosis, was detected at elevated levels in Fas mAb-treated cultures via 3' end-labeling and gel electrophoresis. Examination of cultured cells in situ for apoptotic DNA cleavage by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick end-labeling (TUNEL) indicated that more apoptotic death occurred in Fas mAb-treated cultures than in control cultures. Effects of hCG-induced luteinization of cultures on Fas mAb-induced cytotoxicity was examined: combined pretreatment with IFN gamma and hCG induced a synergistic increase in Fas mAb-induced cytotoxicity (40%) over that obtained with IFN gamma-pretreatment alone (15%). In summary, granulosa/luteal cells express the Fas antigen and are sensitive to Fas mAb-induced apoptosis. Human CG synergized with IFN gamma to increase Fas mAb-induced death.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Fas antigen-mediated apoptosis in human granulosa/luteal cells. 753 48

Using a highly tumorigenic human breast cancer model (Ha-ras-transfected MCF7 cell line) we analyzed the efficacy of the differentiation-inducing agent sodium phenylacetate (NaPA), both in vitro and in vivo. NaPA-treated MCF7ras cells showed dose-dependent growth inhibition from 2.5 to 15 mM without apparent toxicity. Western blot analysis showed a Bcl-2 down-regulation after 48 h treatment with 5 mM NaPA, together with apparition of apoptotic nuclei by DAPI staining. Mice bearing MCF7ras xenografts (n = 40) were treated for 2 weeks through s.c.-delivering osmotic pumps, followed by 6 weeks of daily i.p. NaPA administration. After 3 weeks, the treated tumors showed growth arrest without regression for the whole observation time, e.g., 12 weeks. Immunohistochemical analysis showed Bcl-2 down-regulation and differentiation patterns: decrease of Ki-67 and increase of steroid receptors (estrogen and progesterone receptors) compared to controls. Cells cultured from treated tumors (II.b) displayed pseudotrabecular disposition as MCF7ras cells treated in vitro. They also showed a higher NaPA sensitivity, together with 70% Bcl-2 down-regulation as compared to the derived cells of untreated tumors (II.a). When reinjected into nude mice, II.b cells induced only one poorly vascularized, noninvasive tumor (8%) with lower proliferation index, 100% progesterone receptor positive cells, and 35% terminal deoxynucleotidyltransferase-mediated dUTP-X nick end labeling (+) nuclei, as compared to 100% induction of highly vascularized and invasive tumors with 3% terminal deoxynucleotidyltransferase-mediated dUTP-X nick end labeling (+) nuclei induced by II.a cells.
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PMID:Sodium phenylacetate induces growth inhibition and Bcl-2 down-regulation and apoptosis in MCF7ras cells in vitro and in nude mice. 758 64

Cancer gene therapy strategies for inducing apoptosis in solid tumors may allow contemporary medicine to reassess its management of these cancers. We demonstrated previously that overexpression of the wild-type p53 gene in squamous cell carcinoma of the head and neck cell lines via adenovirus-mediated gene transfer suppressed growth both in vitro and in vivo. Here, we characterize the mechanism of the growth suppression by the exogenous p53 gene as a consequence of programmed cell death (apoptosis). One of the cell lines used in this study, Tu-138, harbors a mutated p53 gene, whereas the other cell line, MDA 686LN, possesses a wild-type p53 gene. DNA fragmentation was detected by electrophoresis in both cell lines after infection with the wild-type p53 adenovirus, Ad5CMV-p53. With the use of the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling method, 4.4% of the remaining viable Tu-138 cell population was identified as apoptotic as early as 15 h after inoculation with Ad5CMV-p53. The percentage of apoptotic cells increased to 31% at 22 h. In contrast, only 10% of the viable MDA 686LN cells (wt-p53) had undergone apoptosis 30 h after Ad5CMV-p53 infection, although the percentage of apoptotic cells rapidly increased to 60% at 48 h after infection. For in vivo analysis of apoptosis, nude mice in which squamous cell carcinoma of the head and neck cell lines had been implanted s.c. had exogenous wt-p53 transiently introduced to the tumor cells via Ad5CMV-p53 2 days later. In situ end labeling clearly illustrated apoptosis in the tumor cells. These results suggest that wt-p53 plays an important role in the induction of apoptosis in human head and neck cancer cell lines and that selective induction of apoptosis in cancer cells can be further explored as a strategy for cancer gene therapy.
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PMID:Apoptosis induction mediated by wild-type p53 adenoviral gene transfer in squamous cell carcinoma of the head and neck. 760 33

Bcl-2 protooncogene, originally discovered at the chromosomal breakpoint of the t(14;18) in follicular lymphoma, is known to regulate the process of programmed cell death or apoptosis. The inhibition of apoptosis is thought to be one of the mechanisms involved in the development of tumors. To investigate the possible association of bcl-2 protooncogene with the tumorigenesis of neuroblastomas, the authors examined bcl-2 expression by immunohistochemistry in 49 neuroblastomas and 7 ganglioneuromas. The distribution of apoptotic cells was also examined by the TUNEL method (terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling). Bcl-2 oncoprotein was detected in the cytoplasm in 40 of 49 neuroblastomas (81.6%). There was no correlation between bcl-2 oncoprotein expression and the clinical features of neuroblastoma. The incidence of bcl-2-positive tumors in ganglioneuroma was significantly lower than that in neuroblastoma (28.6%) (P < .01). TUNEL stained the nuclei of tumor cells in 11 of 34 (32.4%) neuroblastomas. TUNEL-positive cells tended to be located around calcifications in neuroblastomas in patients less than 1 year old. Examination of serial sections showed that apoptotic cells were distributed in the area where bcl-2 oncoprotein was not expressed. What we have observed indicates that apoptosis of neuroblastoma cells may be regulated by bcl-2 expression. Our observations suggest that the survival of neuroblastoma cells might be promoted by bcl-2 expression and that bcl-2 might be associated with the tumorigenesis of neuroblastomas.
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PMID:Bcl-2 oncoprotein expression and apoptosis in neuroblastoma. 766 11

A method combining flow sorting and molecular cytogenetic techniques for the identification of unknown marker chromosomes is described. In this study, the bladder tumor cell line J82 was used, which was known to carry a marker chromosome of the size of chromosome 7 in every cell. From the cytogenetic analysis of Q-banded metaphase cells, it was shown to be composed of approximately 40% presumably the greater part of chromosome 20 and for the rest microscopically unidentifiable material. This marker chromosome was found using flow cytometric analysis to form an independent peak and hence was suitable for isolation using dual-parameter sorting after staining with Hoechst 33258 and chromomycin A3. Subsequently, the marker was isolated by dual-parameter sorting. DNA amplification of 300 isolated chromosomes by polymerase chain reaction (PCR) using the Alu-primer Bk33 and the LINES-primer LH5 was carried out. After purification of the amplified product, a yield of 5 microns of DNA was obtained. The DNA was labelled using Bio-11-dUTP and applied to human lymphocyte metaphase cells in a suppressive in situ hybridization procedure. Fluorescence was visible over chromosome 20 and over the distal one-half of 6p. Together the fluorescent regions accounted for only approximately 60% of the marker length, indicating a possible duplication of chromosome 20 material. This was confirmed by applying bicolor in situ hybridization using chromosome 6- and 20-specific DNA libraries to metaphase cells of the J82 cells.
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PMID:Identification of a tumor marker chromosome by flow sorting, DNA amplification in vitro, and in situ hybridization of the amplified product. 768 Feb 16

The concentrations of bases, nucleosides, and nucleosides mono-, di- and tri-phosphate are compared for about 600 published values. The data are predominantly from mammalian cells and fluids. For the most important ribonucleotides, average concentrations +/- SD (microM) are: ATP, 3,152 +/- 1,698; GTP, 468 +/- 224; UTP, 567 +/- 460 and CTP, 278 +/- 242. For deoxynucleosides-triphosphate (dNTP), the concentrations in dividing cells are: dATP, 24 +/- 22; dGTP, 5.2 +/- 4.5; dCTP, 29 +/- 19 and dTTP 37 +/- 30. By comparison, dUTP is usually about 0.2 microM. For the 4 dNTPs, tumor cells have concentrations of 6-11 fold over normal cells, and for the 4 NTPs, tumor cells also have concentrations 1.2-5 fold over the normal cells. By comparison, the concentrations of NTPs are significantly lower in various types of blood cells. The average concentration of bases and nucleosides in plasma and other extracellular fluids is generally in the range of 0.4-6 microM; these values are usually lower than corresponding intracellular concentrations. For phosphate compounds, average cellular concentrations are: Pi, 4400; ribose-1-P, 55; ribose-5-P, 70 and P-ribose-PP, 9.0. The metal ion magnesium, important for coordinating phosphates in nucleotides, has values (mM) of: free Mg2+, 1.1; complexed-Mg, 8.0. Consideration of experiments on the intracellular compartmentation of nucleotides shows support for this process between the cytoplasm and mitochondria, but not between the cytoplasm and the nucleus.
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PMID:Physiological concentrations of purines and pyrimidines. 787 93


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