1.Chromatin and epigenetic regulation of the telomerase reverse transcriptase gene.
Jiyue ZHU ; Yuanjun ZHAO ; Shuwen WANG
Protein & Cell 2010;1(1):22-32
Telomerase expression and telomere maintenance are critical for long-term cell proliferation and survival, and they play important roles in development, aging, and cancer. Cumulating evidence has indicated that regulation of the rate-limiting subunit of human telomerase reverse transcriptase gene (hTERT) is a complex process in normal cells and many cancer cells. In addition to a number of transcriptional activators and repressors, the chromatin environment and epigenetic status of the endogenous hTERT locus are also pivotal for its regulation in normal human somatic cells and in tumorigenesis.
Animals
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Cell Transformation, Neoplastic
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Chromatin
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genetics
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metabolism
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DNA Methylation
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Epigenomics
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Gene Expression Regulation, Enzymologic
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Humans
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Mice
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Mice, Transgenic
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Promoter Regions, Genetic
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Telomerase
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genetics
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Telomere
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enzymology
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Transcription, Genetic
2.Studies on rat liver nuclear DNA damaged by chemical carcinogen (3'-Me DAB) and AP DNA endonuclease. II. Kinetic properties of AP DNA endonucleases in rat liver chromatin.
Yoon Soo KIM ; Jong Wook KIM ; Seo Eun LEE ; Sang Hwan OH
Journal of Korean Medical Science 1990;5(3):137-143
An experiment was designed to investigate the reaction mechanism of AP (apurinic or apyrimidinic) DNA endonucleases (APcI, APcII, APcIII) purified from rat liver chromatin. Sulfhydryl compounds (2-mercaptoethanol, dithiothreitol) brought about optimal activities of AP DNA endonucleases and N-ethylmaleimide or HgCl2 inhibited the enzyme activities, indicating the presence of sulfhydryl group at or near the active sites of the enzymes. Mg2+ was essential and 4mM of Mg2+ was sufficient for the optimal activities of AP DNA endonucleases. Km values of APcI, APcII and APcIII for the substrate (E. coli chromosomal AP DNA) were 0.53, 0.27 and 0.36 microM AP sites, respectively. AMP was the most potent inhibitor among adenine nucleotides tested and the inhibition was uncompetitive with respective to the substrate. The Ki values of APcI, APcII and APcIII were 0.35, 0.54 and 0.41mM, respectively. The degree of nick translation of AP DNAs nicked by APcI, APcII and APcIII with Klenow fragment in the presence and absence of T4 polynucleotide kinase or alkaline phosphatase were the same, suggesting that all 3 AP DNA endonucleases excise the phosphodiester bond of AP DNA strand to release 3-hydroxyl nucleotides and 5-phosphomonoester nucleotides.
Animals
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Binding Sites
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Chromatin/*enzymology
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DNA Damage/physiology
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DNA Repair/physiology
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DNA-(Apurinic or Apyrimidinic Site) Lyase
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Deoxyribonuclease IV (Phage T4-Induced)
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Endodeoxyribonucleases/antagonists & inhibitors/drug effects/*metabolism
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Kinetics
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Liver/drug effects/*enzymology
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Magnesium/pharmacology
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Rats
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Sulfhydryl Compounds/pharmacology
3.Studies on rat liver nuclear DNA damaged by chemical carcinogen (3'-Me DAB) and AP DNA endonuclease. I. Purification and some properties of AP DNA endonucleases in rat liver chromatin.
Yoon Soo KIM ; Jong Wook KIM ; Seo Eun LEE ; Sang Hwan OH
Journal of Korean Medical Science 1990;5(3):127-135
Three kinds of apurinic/apyrimidinic (AP) DNA endonuclease, APcI, APcII, APcIII, were purified from rat liver chromatin through 1M KCl extraction, DEAE-trisacryl ion exchange chromatography. Sephadex G-150 gel filtration and AP DNA cellulose affinity chromatography. Activities of the purified APcI, APcII and APcIII were 62.5, 83.3 and 52.0 EU/mg of protein, respectively. Molecular weights of APcI, APcII and APcIII, each consisting of a single polypeptide, were 30,000, 42,000 and 13,000, and isoelectric points of them were 7.2, 6.3 and 6.2, respectively. Three enzymes showed different substrate specificities; APcI acted only on AP DNA, and APcII acted on both AP DNA and UV DNA, while APcIII acted on 3'-methyl-4-monomethylaminoazobenzene (3'-Me MAB) DNA adduct as well as AP DNA and UV DNA. These results indicate that three kinds of AP DNA endonuclease present in rat liver chromatin have structural and functional diversities.
Animals
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Carcinogens
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Chromatin/*enzymology
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DNA Damage/*physiology
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DNA-(Apurinic or Apyrimidinic Site) Lyase
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Deoxyribonuclease IV (Phage T4-Induced)
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Electrophoresis, Polyacrylamide Gel
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Endodeoxyribonucleases/*isolation & purification/metabolism
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Isoelectric Focusing
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Liver/drug effects/*enzymology
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Male
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Rats
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Rats, Inbred Strains
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Substrate Specificity
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p-Dimethylaminoazobenzene
4.Effect of lidamycin on telomerase activity in human hepatoma BEL-7402 cells.
Rui-Juan GAO ; Yue-Xin LIANG ; Dian-Dong LI ; Hong-Yin ZHANG ; Yong-Su ZHEN
Biomedical and Environmental Sciences 2007;20(3):189-197
OBJECTIVETo investigate the effect of lidamycin (LDM) on telomerase activity in human hepatoma BEL-7402 cells under the condition of LDM inducing mitotic cell death and senescence.
METHODSChromatin condensation was detected by co-staining with Hoechst 33342 and PI. Cell multinucleation was observed by Giemsa staining and genomic DNA was separated by agarose gel electrophoresis. Fluorescent intensity of Rho123 was determined for mitochondrial membrane potential. MTT assay and SA-beta-gal staining were employed to analyze the senescence-like phenotype. The expression of proteins was analyzed by Western blot. Telomerase activity was assayed by telomerase PCR-ELISA.
RESULTSMitotic cell death occurred in LDM-treated cells characterized by unique and atypical chromatin condensation, multinucleation and increased mitochondrial membrane potential. However, no apoptotic bodies or DNA ladders were found. In addition, apoptosis-related proteins remained nearly unaltered. Senescence-like phenotype was identified by increased and elongated size of cells, growth retardation, enhanced SA-beta-gal activity and the changes of senescence-related protein expression. Telomerase activity markedly decreased (P<0.01) in LDM-treated hepatoma BEL-7402 cells.
CONCLUSIONMitotic cell death and senescence could be triggered simultaneously or sequentially after exposure of hepatoma BEL-7402 cells to LDM. The decrease in telomerase activity may play a key role in the defective mitosis and aging morphology. Further investigation of detailed mechanism is needed.
Aminoglycosides ; pharmacology ; Antibiotics, Antineoplastic ; pharmacology ; Apoptosis ; drug effects ; Azure Stains ; Benzimidazoles ; Carcinoma, Hepatocellular ; enzymology ; pathology ; Cell Nucleus ; drug effects ; metabolism ; Cellular Senescence ; drug effects ; Chromatin ; metabolism ; DNA, Neoplasm ; analysis ; Dose-Response Relationship, Drug ; Enediynes ; pharmacology ; Genome, Human ; genetics ; Humans ; Liver Neoplasms ; enzymology ; pathology ; Membrane Potential, Mitochondrial ; drug effects ; Mitosis ; drug effects ; Phenotype ; Propidium ; Telomerase ; metabolism ; Time Factors ; beta-Galactosidase ; metabolism
5.Dual androgen-response elements mediate androgen regulation of MMP-2 expression in prostate cancer cells.
Ben-Yi LI ; Xin-Bo LIAO ; Atsuya FUJITO ; J Brantley THRASHER ; Fang-Yun SHEN ; Ping-Yi XU
Asian Journal of Andrology 2007;9(1):41-50
AIMTo characterize the matrix metalloproteinases (MMP)-2 promoter and to identify androgen response elements (AREs) involved in androgen-induced MMP-2 expression.
METHODSMMP-2 mRNA levels was determined by reverse transcription-polymerase chain reaction (RT-PCR). MMP-2 promoter-driven luciferase assays were used to determine the fragments responsible for androgen-induced activity. Chromatin-immunoprecipitation assay and electrophoretic mobility shift assays (EMSA) were used to verify the identified AREs in the MMP-2 promoter.
RESULTSAndrogen significantly induced MMP-2 expression at the mRNA level, which was blocked by the androgen antagonist bicalutamide. Deletion of a region encompassing base pairs -1591 to -1259 (relative to the start codon) of the MMP-2 promoter led to a significant loss of androgen-induced reporter activity. Additional deletion of the 5'-region up to -562 bp further reduced the androgen-induced MMP-2 promoter activity. Sequence analysis of these two regions revealed two putative ARE motifs. Introducing mutations in the putative ARE motifs by site-directed mutagenesis approach resulted in a dramatic loss of androgen-induced MMP-2 promoter activity, indicating that the putative ARE motifs are required for androgen-stimulated MMP-2 expression. Most importantly, the androgen receptor (AR) interacted with both motif-containing promoter regions in vivo in a chromatin immunoprecipitation assay after androgen treatment. Furthermore, the AR specifically bound to the wild-type but not mutated ARE motifs-containing probes in an in vitro EMSA assay.
CONCLUSIONTwo ARE motifs were identified to be responsible for androgen-induced MMP-2 expression in prostate cancer cells.
Androgens ; pharmacology ; Cell Line, Tumor ; Chromatin ; genetics ; DNA Primers ; Gene Expression Regulation, Enzymologic ; Gene Expression Regulation, Neoplastic ; Genes, Reporter ; Humans ; Luciferases ; genetics ; Male ; Matrix Metalloproteinase 2 ; genetics ; metabolism ; Mutagenesis, Site-Directed ; Promoter Regions, Genetic ; Prostatic Neoplasms ; enzymology ; RNA, Messenger ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Sequence Deletion