Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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Aphidicolin inhibits purified DNA polymerases-a and -d in vitro and inhibits mitosis in animal cells. The Chinese hamster V79 cell mutant, Aphr-4-2, was selected for its ability to form colonies in cultured medium supplemented with 1.0 microM aphidicolin. At this concentration, the parental wild-type V79 cells (clone 743x) have a survival rate of less than 10(-7). The mutant DNA polymerase-a is resistant to aphidicolin at concentrations that are inhibitory to the wild-type V79 DNA polymerase-a. The apparent Km for dCTP of the mutant DNA polymerase-a is consistently lower than that of the wild-type DNA polymerase-a. This mutant exhibits slow growth, mutator activity, hypersensitivity, and hypermutability to UV. We wanted to know the basis of UV hypersensitivity in this mutant. Using the antisera (UV2) raised against UV-induced thymidine dimers and a sensitive immunofluorescence assay to measure UV-induced thymidine dimers and with detection in ACAS 570 Workstation, we observed that 50% of the thymidine dimers disappeared within 5 h after irradiation and more than 80% of the dimers were removed within 24 h in both cell lines. These results indicate that the recognition, incision, and excision steps in nucleotide excision repair pathway are normal in the mutant. In order to know if there is a difference in DNA polymerase-a or -d activities in the parental V79(wt) and Aphr-4-2 cells, DNA polymerases were partially purified from the parental and the mutant cells using sequential centrifugation and column chromatographies on DEAE-cellulose (DE23 and DE52) to remove DNA polymerases-beta and -gamma. More than 90% of the enzymatic activities from both cells showed characteristics of DNA polymerase-a type on the basis of these criteria: sensitivity to butyl phenyl dGTP (1 microM) and to IgG raised against DNA polymerase-a (SJK 132-20). The results indicate that DNA replication involving a mutant DNA polymerase-a with altered affinity for dCTP may be responsible for the UV sensitivity and mutability of the mutant.
Somat Cell Mol Genet 1990 Jan
PMID:On the DNA polymerase-a mutant: immunofluorescence assay of UV-induced thymidine dimers in Aphr-4-2 cells. 210 26

Spontaneous mutants of mouse FM3A cells (AC1, AC2, and AC3), highly resistant to aphidicolin (3000-, 2500-, and 300-fold increase in resistance, respectively), were isolated by multistep selection. The DNA synthesizing activity in permeabilized cells of all three mutants was substantially resistant to aphidicolin, like that in intact cells. The DNA polymerase activity in nuclear extracts in AC1 and AC3, but not AC2, was resistant to aphidicolin. Partially purified DNA polymerase alpha from AC3, but not from AC1 or AC2, showed resistance to aphidicolin. The apparent Ki value for aphidicolin of AC3 polymerase alpha was three to four times that of the enzyme from the parent cells, but the apparent Km values of the enzyme for dCTP and dTTP were normal. All the mutants showed cross-resistance to both arabinofuranosyladenine and arabinofuranosylcytosine. The AC3 mutant had expanded deoxyribonucleoside triphosphate pools. On two-dimensional polyacrylamide gel electrophoresis, AC1 gave a new protein (mol wt 40 kDa). The aphidicolin-resistance trait was reversible in AC2, unlike in AC1 and AC3. These results show that in mammalian cells there are at least two mechanisms of aphidicolin-resistance that involve an altered DNA polymerase alpha that is resistant to aphidicolin and simultaneous expansion of the four DNA-precursor pools.
Somat Cell Mol Genet 1990 Sep
PMID:High level of aphidicolin resistance with multiple mutations in mouse FM3A cell mutants. 212 28

In situ hybridization (ISH) was used to study at the electron microscope level, the subcellular localization of oxytocin (OT) mRNA in the rat hypothalamic magnocellular neurons. Rat brains were fixed with paraformaldehyde and glutaraldehyde and vibratome slices were incubated with a 25-base synthetic oligonucleotide complementary to OT mRNA and labelled at the 3'-end with [3H]dCTP. Hybridized slices were embedded in Epon after post-fixation with osmium tetroxide and cut into ultrathin sections that were processed for ultrastructural radioautography. OT mRNA was observed in magnocellular neurons of supra-optic and paraventricular nuclei in the vibratome sections. On ultrathin sections, the cytological preservation appeared to be satisfactory. Except for a few silver grains over the nucleus, sometimes close to its membrane, most grains were localized over the cytoplasm of some magnocellular neurons, where they frequently overlapped the endoplasmic reticulum. To decrease exposure time, ISH was also performed with OT probes labelled with a long tritiated tail. In this case, clusters of silver grains occurred over the cell nuclei not only in magnocellular neurons but also in non-secretory neurons and even in glial cells. However, an excess of poly C added to the hybridization buffer strongly decreased this non-cytoplasmic labelling. In conclusion, the results obtained with the short-tailed oligonucleotides demonstrate that these synthetic oligonucleotides have possible applications for the ultrastructural localization of mRNAs and constitute a powerful tool for the dynamic study of cellular mRNA processing in several physiological and experimental conditions.
Brain Res Mol Brain Res 1990 Jun
PMID:Ultrastructural localization of oxytocin mRNA in the rat hypothalamus by in situ hybridization using a synthetic oligonucleotide. 216 99

2',3'-Dideoxycytidine (ddCyd), a potent inhibitor of human immunodeficiency virus DNA replication, requires phosphorylation by cellular nucleoside kinases for antiviral activity. Deoxycytidine kinase (NTP:deoxycytidine 5'-phosphotransferase, EC 2.7.1.74) is responsible for the formation of dideoxycytidine monophosphate and this enzyme is controlled by feedback regulation by the natural endproduct, dCTP. We have examined whether a decrease in intracellular dCTP levels affects the growth inhibition caused by ddCyd, as well as the capacity to accumulate dideoxycytidine triphosphate (ddCTP), using human T lymphoblast (CEM) cells in culture. Subtoxic concentrations of thymidine were used to decrease the dCTP pool. The effects of 3'-azido-3'-deoxythymidine (AZT), alone or in combination with ddCyd, on cell growth, DNA precursor pools, and accumulation of ddCTP were also studied. The combination of ddCyd and thymidine led to growth inhibition of CEM cells that was twice what would be expected from addition, whereas the combination of AZT and ddCyd showed an additive effect. CEM cells accumulated ddCTP efficiently, so that with 10 microM ddCyd (corresponding to the EC50 value) and a 6-hr incubation the ddCTP pool was 3-fold higher than the dCTP pool. Simultaneous addition of thymidine (10 microM) increased the dTTP pool 2-fold and gave a 50% reduction in the dCTP level but only a 10% increase in ddCTP accumulation. The presence of AZT (300 microM, corresponding to the EC50 value) led, in contrast, to an elevation of dCTP and no significant change in the other DNA precursor pools. With this high concentration of AZT, the accumulation of ddCTP decreased 42%. It was also found that ddCyd is metabolized into two additional compounds, besides the dideoxycytidine mono-, di-, and triphosphate, i.e., the liponucleotides dideoxycytidine diphosphate-ethanolamine and dideoxycytidine diphosphate-choline, constituting 45 and 6% of the total phosphorylated ddCyd metabolites, respectively, whereas the mono-, di-, and triphosphate corresponded to 3, 21, and 25% of the phosphorylated dideoxynucleotides. These results indicate that the formation of dideoxycytidine monophosphate is not rate limiting in the synthesis of ddCTP in human lymphoblasts, which clearly differs from what was observed earlier in mouse cells (Mol Pharmacol 32:798-806 1988). Furthermore, growth inhibition by ddCyd seems to be related to the ratio between dCTP and ddCTP. There was no direct interference between ddCyd and AZT metabolism in clinically relevant concentrations, which may encourage the use of combination of these compounds for anti-human immunodeficiency virus treatment.
Mol Pharmacol 1990 Aug
PMID:2',3'-Dideoxycytidine toxicity in cultured human CEM T lymphoblasts: effects of combination with 3'-azido-3'-deoxythymidine and thymidine. 216 4

The new deoxycytidine analogue 2',2'-difluorodeoxycytidine (dFdC) is a specific inhibitor of DNA synthesis that has marked cytotoxicity and therapeutic activity. A 2-hr incubation with 0.1-10 microM dFdC decreased cellular viability 78-97%. This treatment reduced deoxynucleoside triphosphate pools, similar to the action of the ribonucleotide reductase inhibitor hydroxyurea. The most pronounced decrease occurred in the dCTP pool, quantitatively followed by the decrease of dATP, dGTP, and dTTP. In contrast, inhibition of DNA synthesis by arabinosylcytosine did not affect the dCTP level, whereas dATP, dGTP, and dTTP pools increased, but less than 2-fold. The incorporation of [5-3H]cytidine into the dCTP pool, a measure of ribonucleotide reductase activity in whole cells, was reduced to 3% of controls by 0.1 microM dFdC, but to only 40% by 0.1 microM ara-C. Each drug decreased incorporation of [5-3H]cytidine into DNA to a similar extent (greater than 94%), suggesting limitation by a reaction proximal to this step. The cellular concentration of dFdC 5'-diphosphate was 0.3 microM at 50% inhibition of the in situ activity of ribonucleotide reductase. Direct assays of partially purified ribonucleoside diphosphate reductase (EC 1.17.4.1) demonstrated 50% inhibition by 4 microM dFdC 5'-diphosphate; dFdC 5'-triphosphate was much less inhibitory. We conclude that dFdC 5'-diphosphate acts as an inhibitor of ribonucleoside diphosphate reductase.
Mol Pharmacol 1990 Oct
PMID:Inhibition of ribonucleotide reduction in CCRF-CEM cells by 2',2'-difluorodeoxycytidine. 223 93

The metabolism and cytostatic effects of 3'-azido-3'-deoxythymidine (AZT), one of the most effective agents being used in the treatment of acquired immunodeficiency syndrome, were investigated in the CCRF-CEM line of human T lymphoid cells. The concentration of drug required to inhibit cell growth by 50% (CD50) was significantly lower when the cells were exposed to AZT for 24 hr (CD50 = 50 microM), as compared with 48 or 96 hr (CD50 = 225 and greater than 300 microM, respectively). AZT at 25 microM blocked the progression of cells in S phase for about 12 hr, but this effect was reversed by 24 hr, despite the continued presence of drug in the medium. At this drug concentration, the level of dTTP decreased to about 75% of the control level by 4 hr but rebounded to 30% above normal by 8 hr of drug exposure. dGTP and dATP pool sizes were unchanged, whereas the dCTP pool increased 5-fold. The time course of these biochemical changes indicated that the onset of S phase arrest was not directly related to the decrease in deoxynucleoside triphosphate pools. CCRF-CEM cells incubated with 25 microM AZT accumulated about 0.9 mM 5'-monophosphate (AZTMP) after 4 hr whereas levels of the 5'-di- and 5'-triphosphates (AZTDP and AZTTP) plateaued at about 2 and 5 microM, respectively. After this period, there was a rapid decrease in AZTMP accumulation, to one third its initial level by 24 hr, whereas AZTDP and AZTTP pools decreased to only about 70%. The loss in AZT nucleotide formation with time of drug exposure was associated with a concomitant accumulation of AZTMP in the medium. Cellular excretion of AZTMP was not associated with any detectable cell lysis or leakage of other cellular metabolites. The ability of CCRF-CEM cells to excrete AZTMP may be an important factor limiting the biochemical and biological effects of the drug.
Mol Pharmacol 1990 May
PMID:Relationship of deoxynucleotide changes to inhibition of DNA synthesis induced by the antiretroviral agent 3'-azido-3'-deoxythymidine and release of its monophosphate by human lymphoid cells (CCRF-CEM). 233 44

1-beta-D-arabinofuranosyl-5-aza-cytosine (ara-5-aza-Cyd) is an analog of 1-beta-D-arabinofuranosylcytosine (ara-C), which resembles ara-C in anabolic metabolism, incorporation into DNA, and inhibition of DNA replication. Human T-lymphoblastic cells (Molt-4) incorporate three- to fivefold more ara-5-aza-Cyd than ara-C into DNA during 5-8 hr exposure. Although ara-5-aza-Cyd and its triphosphate metabolite are unstable in aqueous solution, the aza-analog was much more stable in solution when incorporated into native DNA isolated from Molt-4 cells. By using gapped duplex DNA as a substrate for purified human DNA polymerases alpha and beta, inhibition of [3H]-dCTP incorporation by ara-5-aza-CTP and ara-CTP was competitive, with Ki values for alpha of 11 and 1.5 microM, respectively. Ki values for polymerase beta were 39 and 7.6 microM, respectively. A DNA elongation assay was adapted from DNA sequencing technology, using singly primed bacteriophage M13mp19 or M13mp9 (+)-DNA. Elongation of 5'-[32P]-labeled primer by polymerase alpha is slowed considerably by incorporation of one ara-CMP and to a lesser extent after incorporation of one ara-5-aza-CMP. Neither analog significantly affected elongation by polymerase beta after a single incorporation. However, neither polymerase alone could appreciably extend the growing chain if two consecutive ara-5-aza-CMP or ara-CMP analogs were incorporated. Thus, if similar mechanisms are operant in intact cells, the greater incorporation of ara-5-aza-Cyd than ara-C into DNA may be due to a more facile elongation of the nascent DNA strand by polymerase alpha after incorporation of a single analog. The effect in vitro of incorporation of either analog on DNA chain elongation is widely variable, depending on the identity of the polymerase involved and the sequence of the DNA template being copied.
Mol Pharmacol 1987 Sep
PMID:Sequence-specific effects of ara-5-aza-CTP and ara-CTP on DNA synthesis by purified human DNA polymerases in vitro: visualization of chain elongation on a defined template. 244 69

To investigate the molecular basis of the regulatory mechanisms responsible for the orderly replication of the mammalian genome, we have developed an experimental system by which the replication order of various genes can be defined with relative ease and precision. Exponentially growing CHO-K1 cells were separated into populations representing various stages of the cell cycle by centrifugal elutriation and analyzed for cell cycle status flow cytometry. The replication of specific genes in each elutriated fraction was measured by labeling with 5-mercuri-dCTP and [3H]dTPP under conditions of optimal DNA synthesis after cell permeabilization with lysolecithin. Newly synthesized mercurated DNA from each elutriated fraction was purified by affinity chromatography on thiol-agarose and replicated with the large fragment of Escherichia coli DNA polymerase I by using [alpha-32P]dATP and random primers. The 32P-labeled DNA representative of various stages of the cell cycle was then hybridized with dot blots of plasmid DNA containing specific cloned genes. From these results, it was possible to deduce the nuclear DNA content at the time each specific gene replicated during S phase (C value). The C values of 29 genes, which included single-copy genes, multifamily genes, oncogenes, and repetitive sequences, were determined and found to be distributed over the entire S phase. Of the 28 genes studied, 19 had been examined by others using in vivo labeling techniques, with results which agreed with the replication pattern observed in this study. The replication times of nine other genes are described here for the first time. Our method of analysis is sensitive enough to determine the replication time of single-copy genes. The replication times of various genes and their levels of expression in exponentially growing CHO cells were compared. Although there was a general correlation between transcriptional activity and replication in the first half of S phase, examination of specific genes revealed a number of exceptions. Approximately 25% of total poly(A) RNA was transcribed from the late-replicating DNA.
Mol Cell Biol 1989 Jul
PMID:Temporal order of gene replication in Chinese hamster ovary cells. 247 59

Aphidicolin is a specific inhibitor of DNA polymerase-alpha and -delta from eukaryotic cells. Because of the specificity of this inhibitor, it is potentially a useful probe for the detailed studies of the function of these polymerases. DNA polymerase-alpha mutants isolated on the basis of resistance to aphidicolin have been described. We have isolated four variants that exhibit hypersensitivities to aphidicolin (Aphhs) from Chinese hamster V79/743X fibroblasts. These variants are designated aphhs-1, aphhs-2, aphhs-3 and aphhs-4. We reported here results of studies involving immunochemical characterization. The Aphhs phenotype in all mutants was stable for at least 30 days in the absence of selection pressure. The dCTP pools in the 743X and Aphhs cell lines were not significantly different. The level of total DNA polymerase activity in the crude extract from aphhs-2 cells was 30% of that observed in the parental 743X clone. We developed a method to quantitate DNA polymerase-alpha antigen at single cells in situ using monoclonal antibody SJK 132-20 and fluorescence pseudocolor image. We found that the antigen of DNA polymerase-alpha in aphhs-2 was 30-50% of that in the parental 743X cells. The underproduction of the antigen of DNA polymerase-alpha provides a basis for the observed Aphhs phenotype. Possible mechanisms for the underproduction of DNA polymerase-alpha in aphhs-2 clone are presented.
Somat Cell Mol Genet 1989 Jul
PMID:Aphidicolin hypersensitive mutant of Chinese hamster V79 fibroblasts that underproduces DNA polymerase-alpha antigen. 250 94

The modification of tyrosine residues of the human placenta DNA-polymerase alpha by N-acetylimidazole was investigated. The poly(dT)-template and the r(pA)10-primer a each added separately or simultaneously do not influence the rate of enzyme inactivation. In the presence of poly(dT)-r(pA)10 no effect of dCTP and dTTP (noncomplementary to template) and of dAMP and dADP (complementary to template) on the rate and the level of the enzyme inactivation was found. However dATP revealed practically complete protection. Orthophosphate, pyrophosphate each taken separately do not influence the rate of enzyme inactivation with this reagent. The presence of dADP with either ortho- or pyrophosphate, or dAMP with the one of these ligands leads to half protective action in comparison with dATP. Imidazolides of phosphonoacetic acid and 5'-adenylyl++ 1(phosphonoacetic acid) do not inactivate DNA-polymerase alpha from human placenta and the Klenov fragment of DNA-polymerase I from E. coli. All data obtained allow to suggest that the tyrosine residue in the dNTP binding site of DNA-polymerase reveals stacking with the nucleotide only if dNTP is complementary to the template.
Mol Biol (Mosk)
PMID:[Modification of human DNA polymerase alpha by N-acetylimidazole]. 254 93


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