Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.1.21 (thymidine kinase)
7,561 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The combination of cisplatin and AZT was synergistic in a subline (A2780DDP) of human ovarian carcinoma cells resistant to cisplatin, in contrast to the parental carcinoma cell line, A2780S. A2780DDP cells have elevated levels of enzymes necessary for DNA synthesis and repair. If A2780DDP cells respond to cisplatin with an increase in thymidine kinase activity, then AZT may chain terminate newly synthesized DNA. To test this hypothesis, a dual label [( 14C]-thymidine/[3H]-AZT) experiment was designed. A2780 cells were first incubated with [14C]-thymidine to label DNA and measure DNA degradation in response to cisplatin. These [14C]-thymidine labeled A2780 cells were then incubated for short intervals with [3H]-AZT to measure chain termination after cisplatin addition. A2780S cells responded to cisplatin with a modest increase in thymidine turnover and an increase in AZT incorporation. In contrast, A2780DDP cells initially responded to cisplatin treatment with a significant increase in thymidine turnover and a corresponding increase in [3H]-AZT incorporation. This was concomitantly associated with an increase in thymidine kinase mRNA within 2 hr after cisplatin treatment. Thus, the A2780DDP cells had the ability to rapidly turnover DNA, a property effectively exploited by the deoxythymidine analogue, AZT, utilizing the enhanced enzymes of the DNA synthesis and repair complex in A2780DDP cells.
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PMID:Potentiation of azidothymidine cytotoxicity in cisplatin-resistant human ovarian carcinoma cells. 169 26

Concanavalin A-activated lymphocytes were made polyamine deficient by treatment with alpha-difluoromethylornithine and ethylglyoxal bis(guanylhydrazone). Thymidine kinase activity in polyamine-deficient cells was 17% of the level in normal cells. Thymidine kinase mRNA increased with time after concanavalin A activation and reached a maximum at 36 h after concanavalin A addition. The amount of thymidine kinase mRNA in polyamine-deficient cells was approximately 75% of that in normal cells. The transcription of thymidine kinase gene in isolated nuclei of polyamine-deficient cells was also 75% of that from normal cells. The turnover rate of thymidine kinase mRNA in both normal and polyamine-deficient cells was nearly equal. In normal cells, 95% of thymidine kinase mRNA was polysome associated, while in polyamine-deficient cells, 60% of the mRNA was polysome associated. In addition, the size of polysomes associated with thymidine kinase mRNA in polyamine-deficient cells was smaller than that in normal cells. Synthesis of thymidine kinase was stimulated approximately seven-fold by 0.3 mM spermidine in a rabbit reticulocyte polyamine-free protein synthetic system. The half-life of thymidine kinase activity in both normal and polyamine-deficient cells was nearly equal. Thymidine kinase activity was not influenced significantly by 0.3 mM spermidine. These combined results suggested that the synthesis of thymidine kinase was mainly regulated by polyamines at the level of translation.
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PMID:Polyamine regulation of the synthesis of thymidine kinase in bovine lymphocytes. 210 6

The carboxyl-terminal one-third of human topoisomerase II polypeptide expressed in Escherichia coli was used as antigen to generate polyclonal antibodies in rabbits. With the use of antiserum, DNA topoisomerase II levels of phytohemagglutinin-stimulated human lymphocytes were measured by immunoblotting. Our results showed that the increase in intracellular topoisomerase II level paralleled the entry of cells into proliferation. We also found that the increase in the topoisomerase II level resulted from an increase in the amount of topoisomerase II mRNA. The time course study indicated that the appearance of topoisomerase II mRNA was first observed at 36 h after phytohemagglutinin stimulation. The maximal level of topoisomerase II mRNA was seen at 45 h after stimulation. The same RNA blot was rehybridized with a thymidine kinase probe. The maximal level of thymidine kinase mRNA was observed at 39 h after phytohemagglutinin stimulation. In a comparison of the time course of topoisomerase II gene expression with that of [3H]thymidine incorporation and thymidine kinase gene expression, it was found that the expression of the topoisomerase II gene was later than the onset of DNA replication. Thus, this study suggests that topoisomerase I, which is constantly expressed throughout the cell cycle, might participate in the initiation of DNA replication, while topoisomerase II is involved in solving the DNA topological problems accompanying DNA strand separation during DNA replication.
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PMID:Induction of topoisomerase II gene expression in human lymphocytes upon phytohemagglutinin stimulation. 216 62

In this study DNA-binding and gene transfer experiments were performed to examine a potential glucocorticoid regulatory element (GRE) in the human growth hormone gene. As assayed by nitrocellulose filter binding, only two regions of the human growth hormone gene, the 5'-flanking sequences and a fragment containing part of the first intron, were retained preferentially by purified glucocorticoid-receptor complexes. The relative binding by the transcribed sequences was three times greater than the relative binding by the 5'-flanking sequences, but less than the relative binding by a fragment containing the human metallothionein-IIA gene GRE. The intron, but not the 5'-flanking sequences, generated a "footprint" when the receptor complex was used to protect the segments against exonuclease III digestion; the protected sequence spanned nucleotides +86 to +115 in the first intron and contained a structure homologous in 14 of 16 nucleotides to a 16-nucleotide consensus GRE. The hexanucleotide 5'-TGTCCT-3', thought to be important for GRE activity, not only was found in this sequence and in the 5'-flanking region, but also was present twice in the 3' end of the gene that did not show specific receptor binding. The latter results suggest that the hexanucleotide alone is not sufficient to generate specific receptor binding tight enough to be assayed in this way. To test the biological activity of the intron binding site, a fragment containing these sequences was fused 5' to the human metallothionein-IIA gene promoter depleted of its GRE and linked to the structural sequences of the herpes simplex virus thymidine kinase (TK) gene. When this hybrid gene was transfected into Rat 2 TK- cells, its expression was induced threefold by the glucocorticoid dexamethasone, as assessed by transfection efficiency and RNA blotting analyses. Expression of the same gene without the human growth hormone gene segment was not affected by the steroid, whereas the wild-type human metallothionein-IIA gene promoter containing its GRE responded to the hormone by a sixfold increase in thymidine kinase mRNA. These results indicate that the human growth hormone gene contains a structure within its first intron that can function as a GRE.
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PMID:Glucocorticoid receptor binding and activation of a heterologous promoter by dexamethasone by the first intron of the human growth hormone gene. 301 91

Present experimental data show that the synthesis of ribosomal protein S1 and PI protein was stimulated greatly by polyamines at the early stage after addition of putrescine in polyamine-requiring mutants of E. coli. No macromolecular synthesis was stimulated at this stage. Polyamine stimulation of the synthesis of these proteins probably plays an important role for cell growth. In polyamine-deficient bovine lymphocytes, protein synthesis became perturbed before RNA and DNA synthesis. Among enzymes concerned with DNA replication, thymidine kinase activity was most strongly influenced by polyamines. The activity in polyamine-deficient cells was only 7% of the level in normal cells. Judging from the amount of thymidine kinase mRNA and its distribution in polysomes, it was concluded that polyamines mainly regulate the synthesis of thymidine kinase at the level of initiation of protein synthesis. A polyamine-free protein synthetic system, established from components of rabbit reticulocytes, consisted of globin mRNA, salt-washed ribosomes, partially purified initiation factors, and pH 5 enzymes. Spermidine added to this system not only lowered the optimal magnesium concentration required for globin synthesis, but it also stimulated the globin synthesis 8- to 10-fold. The optimal spermidine concentration was 0.4 to 0.6 mM, a concentration similar to that in intact rabbit reticulocytes. The ratio of alpha to beta globin chains synthesized in the presence of spermidine and Mg2+ was approximately 1.0, while the ratio in the presence of only Mg2+ was approximately 1.5. The results strongly suggest that polyamines play an important role in rabbit reticulocyte protein synthesis.
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PMID:Regulation of protein synthesis by polyamines. 307 28

As an approach to defining the molecular basis for the periodic expression of thymidine kinase activity during the cell cycle, we have examined properties of the cytosolic enzyme in cycling HeLa cells synchronized by centrifugal elutriation and mitotic selection. By immunoblot analyses with a specific antiserum raised against the purified HeLa enzyme, we have demonstrated that changes in the levels of thymidine kinase activity reflect similar changes in the levels of thymidine kinase polypeptide. In contrast, the steady state levels of thymidine kinase mRNA show relatively small changes during the cell cycle. Using pulse labeling methods, we have shown that the synthetic rate of thymidine kinase protein is about 10-fold greater in S phase than in G1 phase, indicating that the efficiency of translation of thymidine kinase mRNA increases as cells begin DNA replication. In addition, the stability of thymidine kinase protein dramatically decreases upon cell division, resulting in the rapid clearance of the enzyme from newly divided G1 cells. Thus, two different post-transcriptional mechanisms largely account for the periodic behavior of the enzyme activity during the cell cycle.
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PMID:Regulation of human thymidine kinase during the cell cycle. 337 30

Aging of IMR-90 human diploid fibroblasts in vitro is accompanied by significant changes of polyamine metabolism, most notably, a 5-fold decrease of serum-induced activity of ornithine decarboxylase, the key enzyme in the biosynthesis of polyamines (Chen, K. Y., Chang, Z. F., and Liu, A. Y.-C. (1986) J. Cell. Physiol. 129, 142-146). In this paper, we employed Northern blot hybridization and affinity radiolabeling techniques to investigate the molecular basis of this age-associated change of ornithine decarboxylase activity. Since the induction of ornithine decarboxylase by serum is a mid-G1 event, we also examined expressions of other cell cycle-dependent genes that are induced before and after the mid-G1 phase to determine if their expressions may also be age-dependent. Our results demonstrated a 3-fold decrease of the amount of active ornithine decarboxylase molecules that can be labeled by alpha-difluoromethyl[3H]ornithine in senescent IMR-90 cells (population doubling level (PDL) = 52) as compared to young cells (PDL = 22). However, the levels and kinetics of induction of ornithine decarboxylase mRNA in both young and senescent IMR-90 cells were found to be identical throughout a 24-h time period after serum stimulation. The time course and the magnitude of the expression of c-myc, an early G1 gene, were quite similar in young and senescent IMR-90 cells and appeared to be PDL-independent. In contrast, the expression of thymidine kinase, a late G1/S gene, was significantly reduced in senescent IMR-90 cells. Levels of thymidine kinase mRNA and thymidine kinase activity in senescent IMR-90 cells were 6- and 8-fold less than those in young cells, respectively. Based on these data, we proposed that impairment of cell cycling in senescent IMR-90 cells may occur at the late G1/S phase and that decreases of ornithine decarboxylase activity and putrescine accumulation during cell senescence may contribute to this impairment.
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PMID:Regulation of ornithine decarboxylase and other cell cycle-dependent genes during senescence of IMR-90 human diploid fibroblasts. 340 38

The growth of MCF-7 cells was arrested by 24 h of isoleucine deprivation. Following replenishment of the medium, the incorporation of uridine and thymidine into trichloroacetic acid-precipitable material began to increase slowly and gradually rose to the level of cycling cells. The addition of 5 X 10(-9) M estradiol to growth-arrested cells dramatically shortened the time of onset of macromolecular synthesis and increased the overall amount of precursor incorporation 2- to 4-fold over the level obtained by arrested control cells. The increase in uridine incorporation preceded the increase in thymidine incorporation by 6 h. Inhibition of protein synthesis with cycloheximide blocked the recovery of macromolecular synthesis in both control and estrogen-treated cells. Actinomycin D was ineffective in blocking the estrogen-stimulated recovery of macromolecular synthesis at concentrations known to inhibit pre-rRNA synthesis (10(-8) M). At higher concentrations, uridine and thymidine incorporation were inhibited in a dose-dependent manner. Inhibition of RNA polymerase II activity with alpha-amanitin similarly blocked both the recovery of the cells from isoleucine starvation and the potentiation of this by estradiol. Dihydrofolate reductase and thymidine kinase activities are both stimulated by estradiol in MCF-7 cells. In cycling cells, estrogen stimulates a 2-fold increase in their messenger RNAs (mRNAs) within 24 h. The level of dihydrofolate reductase mRNA is unaffected by isoleucine starvation, and estrogen caused no change in dihydrofolate reductase mRNA levels over a 24-h period following reversal of growth arrest. Similar results were observed for the 600-nucleotide pS2 mRNA that has been identified as an estrogen-induced RNA in MCF-7 cells. In contrast, thymidine kinase mRNA was found to be increased by estrogen at 24 h, but not at 12 h, following reversal of growth arrest. This increase correlates with increases in thymidine, but not uridine incorporation. These data indicate that the estrogen-stimulated increase in thymidine incorporation following release from growth arrest is dependent on new RNA synthesis. However, the hormone did not increase the levels of three estrogen-regulated mRNAs coordinately with the increases observed in uridine incorporation.
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PMID:Relationship between the expression of estrogen-regulated genes and estrogen-stimulated proliferation of MCF-7 mammary tumor cells. 398 99

An 8.5-kilobase segment of cloned human DNA including the complete G gamma-globin gene was introduced into LMTK- cells by the calcium phosphate precipitation method in the presence or absence of carrier DNA. Transfectants containing one or more copies of intact G gamma-globin genes were obtained either by ligation of the human DNA segment to a plasmid containing the herpes simplex virus thymidine kinase gene or by nonligated cotransfer. The integrity of the integrated gamma-globin gene was established by Southern blotting experiments. Expression of the herpes simplex virus thymidine kinase and human gamma-globin genes was evaluated by Northern blotting and solution hybridization. Of 23 transfectants analyzed, 21 produced a 9S gamma-globin RNA migrating like authentic gamma-globin mRNA on denaturing agarose gels. The gamma-globin RNA is polyadenylated and present in the cytoplasm of the transfected cells; it accumulates to a level 10 times that of thymidine kinase mRNA, or about 5 to 50 molecules per transfected cell. By using plasmids in which the gamma-gene is inserted in either transcriptional orientation with respect to the thymidine kinase gene, it was possible to show that transcription occurred from the gamma-gene promoter.
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PMID:Introduction and expression of a fetal human globin gene in mouse fibroblasts. 618 Mar 5

We have determined the complete nucleotide sequence of the thymidine kinase (ATP:thymidine 5' phosphotransferase, EC 2.7.1.21) gene of herpes simplex virus type 1 strain CL101 from a plasmid clone of viral DNA derived by Enquist et al. [Enquist, L. W., Vande Woude, G. F., Wagner, M., Smiley, J. R. & Summers, W. C. (1979) Gene 7, 335-342]. A cDNA copy of the 5' end of thymidine kinase mRNA was also analyzed to locate the transcribed sequences. The transcribed portion of the gene is approximately 1300 nucleotides in length and appears to contain no intervening sequences. There is an untranslated region of 107 nucleotides at the 5' end of the mRNA followed by an open reading frame of 1128 nucleotides. The gene is thus capable of coding for a protein of 376 amino acids. Sequences similar to those thought to be involved in control of transcription and translation of a variety of eukaryotic and viral genes such as a "Hogness box" and A-A-T-A-A-A polyadenylylation signals are also present in the herpesvirus thymidine kinase gene.
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PMID:Nucleotide sequence of the thymidine kinase gene of herpes simplex virus type 1. 626 99


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