Concentrations Of Thymidine Research Articles

Modulation of RTX cytotoxicity by thymidine and dipyridamole in vitro: implications for chemotherapy.

The cytotoxicity of the thymidylate synthase (TS) inhibitor raltitrexed (RTX) is reversed by supraphysiologic thymidine concentrations. Dipyridamole (DP) blocks thymidine salvage (uptake) and potentiates several chemotherapeutic agents in vitro. The purpose of this study was to determine whether DP is capable of potentiating RTX cytotoxicity in the presence of physiologic thymidine concentrations. We examined the effect of DP on RTX cytotoxicity in the presence of various physiologic thymidine concentrations in eight colorectal adenocarcinoma cell lines by colony formation assay, and in p53-defective HL60 S and p53-competent HL60 SN3 promyelocytic leukemia cells by a tetrazolium reduction-based assay. In WiDr colon adenocarcinoma cells the cytotoxicity of 1.0 microM RTX (4 h) varied from 0% to >99% within the reported range of human serum thymidine concentrations from 500 to <50 nM, respectively. DP potentiated RTX cytotoxicity 2- to >93-fold within this range. Free DP concentrations of 10 to 20 nM, achievable with oral dosing, potentiated RTX at thymidine concentrations up to 100 nM. Potentiation at higher physiologic thymidine concentrations of >/=200 nM required DP concentrations of at least 50 nM, or about twice the steady-state DP concentration obtainable from oral and intravenous dosing, but well within that obtainable by intraperitoneal infusion. Maximal potentiation required 72 to 120 h of DP exposure. DP also potentiated RTX in the absence of exogenous thymidine in six of eight colon cell lines and HL60 S and SN3 cells suggesting a second, nonthymidine salvage-dependent mechanism of potentiation. Clinically achievable free DP concentrations potentiated RTX cytotoxicity within the range of physiologic serum thymidine concentrations. RTX/DP or DP analogue-based combination therapy should, therefore, be considered for clinical trial. Serum or tumor thymidine concentration determinations may aid in identification of patients likely to respond to TS and nucleoside salvage inhibitors versus alternate, non-TS-directed therapies.

A critical examination of substoichiometric isotope dilution analysis using thymidine and leucine

Isotope dilution analysis is used to determine the specific activity of radiolabelled precursors such as 3H-thymidine and 3H-leucine incorporated into DNA or protein in environmental samples. However, it is shown theoretically, experimentally, and using the results of published studies that the method can be misleading when one of its many requirements is not met. First, on the basis of our current understanding of thymidine metabolism, it is not only possible, but probable, that endogenous thymidine synthesis will not remain constant at different concentrations of external thymidine addition. Changes in the rate of thymidine biosynthesis will significantly bias the results of the standard analysis. Yet it is very difficult or impossible to know whether changes have occurred in any particular case. Second, published results show evidence of stimulation of total thymidine incorporation rate at higher thymidine concentrations. These changes in incorporation rate will also bias the conclusions drawn from the assay. Third, impurities common to heavily labelled precursors can produce misleading results. Finally, the current statistical methodology of isotope dilution experiments can be greatly improved with little or no extra effort. It is suggested that, if possible, isotopes should be used empirically to derive conversion factors based on incubations; theoretical calculations based on isotope dilution experiments are not always trustworthy.

A cell cycle model for the tachyzoite of Toxoplasma gondii using the Herpes simplex virus thymidine kinase

Toxoplasma gondii (RH strain) tachyzoites were transfected with a plasmid containing a fusion of the chloramphenicol acetyl transferase and the Herpes simplex virus-2 thymidine kinase coding regions and transgenic parasites obtained by chloramphenicol selection. CTK11, a single high expressing clone was isolated based on immunofluorescence and contained approximately five integrated copies of the fusion sequence. Lysates prepared from this clone displayed thymidine kinase activity of 2.9 pmol min −1 μg −1 protein, whereas thymidine kinase activity was not detected in lysates from the parental RH strain. Growth of CTK11 tachyzoites was fully inhibited in 5 μM ganciclovir and thymidine and in 2.5 μM 5-bromo-2′-deoxyuridine. While the inhibitory effects of ganciclovir were lethal, low concentrations of thymidine (10 μM) were largely reversible. Asynchronously growing CTK11 tachyzoites were found to contain major G1 (1 N) and S phase (1 N+) distributions as determined by relative propidium iodide fluorescence and with reference to the haploid (1 N) DNA content of a T. gondii sporozoite population. CTK11 tachyzoites blocked 4 h in 10 μM thymidine exhibited mean fluorescence consistent with a 1 N complement of DNA indicating growth was arrested in G1. Following the removal of excess thymidine, parasites immediately entered S phase, thus confirming the late G1 block. Parasites with a 2 N complement of DNA (G2+M) first appear at 2 h post-release, while 1 N (G1) parasites re-appear at 3 h suggesting the length of S phase is ≤2 h and that of G2+M is ≤1 h. Within 7 h, parasites had transited G2+M and much of G1 and re-entered S of the subsequent cell cycle—a time consistent with the doubling of these parasites in culture. Thus, the CTK11 tachyzoite cell cycle is similar to those of higher eukaryotic cells and is characterized by major G1 and S phases and a relatively short G2+M.

Determining the optimal thymidine concentration for growing Thy- Escherichia coli strains.

Changes of thymidine concentration in the growth medium affect the chromosome replication time of Thy- strains without at the same time causing a detectable difference in the growth rate (R. H. Pritchard and A. Zaritsky, Nature 226:126-131, 1970). Consequently, the optimal thymidine concentration cannot be determined by ascertaining which concentration produces the highest growth rate. Here we present a method for determining the optimal thymidine concentration of any Thy- Escherichia coli strain. Using this method, we found that the E. coli "wild-type" strain MG1655 has a partial Thy- phenotype.

Cellular Localization and Expression of Template-Activating Factor I in Different Cell Types

Template-activating factors I (TAF-I) α and β have been identified as chromatin remodeling factors from human HeLa cells. TAF-Iβ corresponds to the protein encoded by thesetgene, which was found in an acute undifferentiated leukemia as a fusion version with thecangene via chromosomal translocation. To determine the localization of TAF-I, we raised both polyclonal and monoclonal antibodies against TAF-I. The proteins that react to the antibodies are present not only in human cells but also in mouse, frog, insect, and yeast cells. The mouse TAF-I homologue is ubiquitous in a variety of tissue cells, including liver, kidney, spleen, lung, heart, and brain. It is of interest that the amounts of TAF-Iα and β vary among hemopoietic cells and some specific cell types do not contain TAF-Iα. The level of the TAF-I proteins does not change significantly during the cell cycle progression in either HeLa cells synchronized with an excess concentration of thymidine or NIH 3T3 cells released from the serum-depleted state. TAF-I is predominantly located in nuclei, while TAF-I that is devoid of its acidic region, the region which is essential for the TAF-I activity, shows both nuclear and cytoplasmic localization. The localization of TAF-I in conjunction with the regulation of its activity is discussed.

Enzymatic Synthesis of Unique Thymidine-Containing Polyphenols

Polynucleotides, in addition to their natural role as carriers of genetic information, offer a wide array of unique chemistries that can be exploited for use as therapeutic and diagnostic reagents,1 and as highly selective materials for biorecognition2 where specific binding to target molecules is desired. Unfortunately, native polynucleotides are susceptible to ubiquitous endoand exonucleases present in many biological systems. In addition, the natural phosphodiester linkage is particularly labile to harsh conditions often found in abiotic environments including extremes in pH and temperature. Conversely, replacing the phosphodiester linkage with unnatural bonds can result in a wide array of functionalized polymers. This strategy has been exploited to some extent for the chemical derivatization of thymine and related bases and nucleosides with vinyl groups to give functionalized vinyl polymers for use as selective absorbents.3,4 Polynucleotide analogues have also been synthesized using a variety of other polymer backbones including poly(trimethyleneimine),5 poly(vinyl alcohol)s,6 poly(ethylenimine),7 and poly(vinylamine).8 Nucleic acid analogues possessing high biostability and affinity based on polypeptide backbones have also been developed.9 In all cases, polymers with a broad range of sizes (103 to 105 Da) and controlled hydrophobicity/hydrophilicity, chain flexibility, and stability were produced for specific applications including aptamers that selectively bind organic ligands10 and polymeric drugs for chemotherapy.6,11 Nearly all of the aforementioned synthetic schemes require multiple chemical steps that provide little selectivity in the attachment of the nucleoside or nucleoside derivative to the polymeric backbone. Selectivity, if necessary, is achieved by complex site-specific chemistries involving blocking and deblocking steps. Such selectivity may be critical in the preparation of polymers with side chains occupying a specific orientation around the polymer backbone, thereby resulting in unique properties. In the present work, we have developed a unique, simple, and effective technique to prepare novel polynucelosides with unnatural polymeric backbones and with high inherent selectivity. Specifically, we have used a two-enzyme coupled reaction system to generate nucleoside-based polyphenols. The resulting polymers may provide highly selective and stable materials for therapeutic, diagnostic, and materials applications. The synthetic approach is shown in Scheme 1. Thymidine was used as a model nucleoside and a trifluoroethyl ester derivative of p-hydroxyphenylacetic acid (1)12 was used as the phenolic derivative. Regioselective acylation of thymidine at the 5′-hydroxyl was achieved in nearly anhydrous CH3CN using the lipase from Candida antarctica. This was followed by polymerization of the phenolic nucleoside derivative catalyzed by the peroxidase from soybean hulls (SBP). The lipase-catalyzed formation of the thymidine 5′p-hydroxyphenylacetate (2) was facile and over 95% of a 50 mM solution of 1 in CH3CN was consumed in 18 h to give almost entirely monoester 2.13 Purification of the monoester resulted in two distinct products in a ratio of ca. 5:1 with overall 70% isolated yield.13 1H and 13C NMR confirmed that the major product was the 5′ester (1.10 g, 59% isolated yield based on conversion of 1). The minor product was presumably the 3′-ester (0.21 g, 11% isolated yield); thus, the enzymatic transformation in CH3CN was highly regioselective. Similar specificity was observed for the Pseudomonas cepacia lipase-catalyzed acylation of thymidine with vinyl butyrate in tetrahydrofuran.14 The resulting thymidine monoesters were soluble in CH3CN and dimethyl sulfoxide (DMSO). The reaction was essentially catalyzed in a solid phase; the concentration of thymidine dissolved in CH3CN did not exceed 3 mM. Hence, C. antarctica lipase is effective in catalyzing the efficient regioselective acylation of a poorly organic solventsoluble nucleoside in CH3CN. SBP-catalyzed polymerization of 2 was carried out in the presence of H2O2 in a solution of aqueous buffer (50 mM Na phosphate, pH 7.0) containing 60% (v/v) CH3CN following a procedure developed previously for polyhydroquinone synthesis.15 Conversion of 2 reached 65% (as determined by reversed-phase HPLC) and the reaction was terminated by centrifuging the suspension and separating the reaction components into soluble and insoluble fractions. The soluble fraction was precipitated by adding to neat CH3CN. Extensive washing with CH3CN followed by water gave an isolated yield of 65 mg (28% of reacted 2) of poly(thymidine 5′-p-hydroxyphenylacetate) (3). 1H NMR in DMSO-d6 showed a clear presence of the thymidine (essentially identical with that in 2); however, the phenolic peaks were significantly broadened, indicating polyphenol synthesis (data not shown). Molecular weight determination16 provided an Mn ) 21 700 and an Mw/Mn ) 1.2. DSC17 of the polymer indicated the presence of a Tg at 60 °C and a broad melting point * To whom correspondence should be addressed. † Department of Chemical and Biochemical Engineering. ‡ Present address: Chemical Technology Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831-6226. § Division of Medicinal and Natural Products Chemistry. Scheme 1 941 Macromolecules 1998, 31, 941-943

Determinants of the therapeutic efficacy of thymidylate synthase inhibitors.

Thymidylate synthase (TS) catalyses a critical step in the pathway of DNA synthesis.1,2 Using the co-substrate 5,10-methylene tetrahydrofolate (CH2-THF) as a methyl donor, thymidylate synthase converts dUMP by methylation to dTMP (Fig. 1). This process is the only de novo source of dTMP which is subsequently metabolized to dTTP, exclusively for incorporation into DNA during synthesis and repair. The other source for dTMP is thymidine kinase, which uses thymidine as its precursor.2 TS inhibition results in a thymineless state, which is lethal to most actively dividing cells. In addition, the circulating thymidine concentrations in humans may not be adequate to prevent the lethal thymineless state. TS is therefore an ideal target for anticancer therapy.1,2

Efficacy of trimethoprim in murine experimental infection with a thymidine kinase-deficient mutant of Escherichia coli.

The antimicrobial activity of trimethoprim is antagonized by thymidine in in vitro susceptibility tests. The purpose of this investigation was to determine whether this antagonism also occurred during experimental infection in mice, which have high serum thymidine concentrations. We derived a mutant strain of Escherichia coli, TT-48, incapable of utilizing exogenous thymidine from parent strain E. coli KC-14 and then investigated the in vitro and in vivo antimicrobial activities of trimethoprim, sulfamethoxazole, cefdinir, and ofloxacin against these strains. E. coli TT-48 lacked the activity of thymidine kinase, which catalyzes the conversion of thymidine to thymidylate, but its growth curve remained close to that of the parent strain. The MICs of all of the antimicrobial agents tested, except cefdinir, for the mutant strain were slightly inferior to those for the parent strain. The bactericidal effect of trimethoprim against the parent strain was antagonized by thymidine at concentrations of more than 1 microg/ml, while that against the mutant strain was not affected by thymidine even at the highest concentration (10 microg/ml). The therapeutic efficacy of trimethoprim in experimental murine infections was significantly higher when the mutant rather than the parent strain was used, whereas the therapeutic efficacy of cefdinir or ofloxacin, whose antimicrobial action is independent of folic acid synthesis, was the same with both strains. Unexpectedly, sulfamethoxazole also had similar efficacy against both strains. Thus, high thymidine concentrations antagonized the antimicrobial activity of trimethoprim in vitro and in vivo.

Modulation of the metabolism of beta-L-(-)-2',3'-dideoxy-3'-thiacytidine by thymidine, fludarabine, and nitrobenzylthioinosine.

beta-L-(-)-2',3'-Dideoxy-3'-thiacytidine (3TC) is a cytosine nucleoside analog that potently inhibits the replication of human and duck hepatitis B viruses and human immunodeficiency virus through the activity of its 5'-triphosphate ester metabolite. The present study examined the intracellular decay of 3TC 5'-phosphates and tested strategies for modulating the cellular content of those nucleotides in primary cultures of duck hepatocytes and in human hepatoma 2.2.15 cells and CCRF-CEM T lymphoblasts. Inhibition by deoxycytidine of the 5'-phosphorylation of 3TC in duck hepatocytes confirmed that, as in mammalian cells, deoxycytidine kinase catalyzed 3TC activation. The 5'-mono, 5'-di-, and 5'-triphosphates of 3TC underwent monoexponential elimination from duck hepatocytes and 2.2.15 cells (half-lives, 3.6 to 8.0 h). Thymidine and fludarabine, which are agents that enhance the activity of deoxycytidine kinase, were tested in strategies for increasing the cellular content of 3TC 5'-phosphates. Coordinate treatment of cells with 3TC and thymidine (50 microM) increased the content of 3TC 5'-monophosphate in duck hepatocytes and the content of 3TC 5'-di- and 5'-triphosphates in 2.2.15 cells, but enhancement of 3TC 5'-phosphate levels in CCRF-CEM cells required a higher thymidine concentration (100 microM). Fludarabine (5 microM) did not affect the contents of 3TC 5'-di- and 5'-triphosphates in duck hepatocytes, but modestly increased the contents of those nucleotides in 2.2.15 cells and CCRF-CEM cells. Nitrobenzylthioinosine (NBMPR), an inhibitor of the es facilitated diffusion nucleoside transporter, reduced the level of entry of 3TC into 2.2.15 cells and abolished inward fluxes of thymidine, adenosine, and deoxycytidine. In 2.2.15 cells and CCRF-CEM cells, NBMPR reduced the formation of 3TC 5'-di- and 5'-triphosphates and reversed the thymidine- and fludarabine-induced increases in the formation of those nucleotides. NBMPR protected against the cytotoxicity of 3TC in CCRF-CEM cells, whereas thymidine potentiated that toxicity, apparently by enhancing the formation of 3TC 5'-triphosphate. Taken together, these results indicate that deoxycytidine kinase and the es nucleoside transporter are targets for manipulation of the metabolism and activity of 3TC.

The quantitation and kinetics of unscheduled (repair) DNA synthesis in ultraviolet-irradiated human skin by automated image analysis

Grain counting by eye is a tedious and time-consuming technique but one with great potential in cell kinetics and for the study of DNA excision repair activity (unscheduled DNA synthesis or UDS). We have been investigating the levels of UDS in human skin sections exposed in situ to ultraviolet radiation using a short-term incubation in tritiated thymidine and autoradiography and the decline in UDS levels with time (repair kinetics). We have adapted an automated image analysis system automatically to assess the number of grains over epidermal cell nuclei in autoradiographs of sections of epidermis. An excellent correlation was observed between visual counting and machine measurement of the area (in pixels) occupied by silver grains. The levels of UDS declined with time as lesions are progressively repaired. The half time (+/- standard deviation) for the reduction in UDS is 7.25 +/- 0.18 h. The grain counts can be significantly increased by increasing the autoradiographic exposure, by increasing the concentration of tritiated thymidine and by increasing the incubation time.

Nucleoside uptake in rat liver parenchymal cells

Rat liver parenchymal cells express Na(+)-dependent and Na(+)- independent nucleoside transport activity. The Na(+)-dependent component shows kinetic properties and substrate specificity similar to those reported for plasma membrane vesicles [Ruiz-Montasell, Casado, Felipe and Pastor-Anglada (1992) J. Membr. Biol. 128, 227-233]. This transport activity shows apparent K(m) values for uridine in the range 8-13 microM and a Vmax of 246 pmol of uridine per 3 min per 10(5) cells. Most nucleosides, including the analogue formycin B, cis-inhibit Na(+)-dependent uridine transport, although thymidine and cytidine are poor inhibitors. Inosine and adenosine inhibit Na(+)-dependent uridine uptake in a dose-dependent manner, reaching total inhibition. Guanosine also inhibits Na(+)-dependent uridine uptake, although there is some residual transport activity (35% of the control values) that is resistant to high concentrations of guanosine but may be inhibited by low concentrations of adenosine. The transport activity that is inhibited by high concentrations of thymidine is similar to the guanosine-resistant fraction. These observations are consistent with the presence of at least two Na(+)-dependent transport systems. Na(+)-dependent uridine uptake is sensitive to N-ethylmaleimide treatment, but Na(+)-independent transport is not. Nitrobenzylthioinosine (NBTI) stimulates Na(+)-dependent uridine uptake. The NBTI effect involves a change in Vmax, it is rapid, dose-dependent, does not need preincubation and can be abolished by depleting the Na+ transmembrane electrochemical gradient. Na(+)-independent uridine transport seems to be insensitive to NBTI. Under the same experimental conditions, NBTI effectively blocks most of the Na(+)-independent uridine uptake in hepatoma cells. Thus the stimulatory effect of NBTI on the concentrative nucleoside transporter of liver parenchymal cells cannot be explained by inhibition of nucleoside efflux.

Taenia multiceps (Cestoda): Ia antigen expression and prostaglandin secretion by parasite-modified, murine peritoneal macrophages.

Taenia multiceps secretions modify accessory cell activity in macrophages. The present experiments were designed to elucidate the cellular mechanisms involved. While normal, murine peritoneal macrophages amplified mitogen-activated T-cell proliferation, macrophages modified by exposure to parasite secretions inhibited this proliferation. The modified behaviour was shown by glutaraldehyde-fixed as well as living macrophages, and modification was inducible by FPLC fraction 24 of coenurus fluid and was associated with an expanded population of 1a- macrophages. Secretory products of parasite-activated macrophages also inhibited T-cell proliferation, and secretion was prevented by indomethacin. The measurement of modified accessory activity was not influenced by the concentration of tritiated thymidine in lymphocyte proliferation assays. Consequently there is no evidence that the reported events are affected by macrophage-derived, cold thymidine secretion. It is concluded that T. multiceps si able to manipulate macrophage accessory function by mechanisms which involve altered histocompatibility antigen expression and the secretion of prostaglandin.

Complementary replication R- and G-band patterns induced by cell blocking at the R-band/G-band transition, a possible regulatory checkpoint within the S phase of the cell cycle.

The characteristic band patterns of replication banding (dynamic banding) were analyzed. High-resolution (550-1,250 bands per haploid genome) G- and R-band patterns were obtained after 5-bromo-2'-deoxyuridine (BrdU) incorporation during early or late S phase. Thymidine-BrdU permutation culture methods, which arrest DNA synthesis at the R-band/G-band transition, allow complementary BrdU substitution. The RBI (R bands by BrdU using immunological staining) and GBI (G bands by BrdU using immunological staining) band patterns were complementary for all chromosomes. There was no overlapping, and every part of each chromosome was positively stained by one of the two banding procedures. Comparative analysis of RBG (R bands by BrdU using Giemsa staining) and RBI band patterns, as well as GBG (G bands by BrdU using Giemsa staining) and GBI band patterns, showed good congruency, displaying a very good band-for-band match. The congruency and complementarity found for these band patterns show that high concentrations of both thymidine and BrdU blocked S-phase progression near the R-band to G-band replication transition within the S phase. They also prove that BrdU incorporation is complementary and, therefore, demonstrate the existence of the R/G transition: a possible regulatory checkpoint within the S phase of the cell cycle.

Optimal conditions for measurement of blastogenic responses of chickens to concanavalin A in whole blood assays

One-day-old chicks (Cobb 500) were raised in wire cages in clean isolated conditions and fed on a non-medicated diet. Heparinised peripheral blood samples were taken from the cutanea ulnaris (wing) vein. The effect of various parameters including concentration of concanavalin A (Con A), dilution of blood in medium, volume of blood dilution per culture, type of media, percentage of serum, incubation period, concentration of thymidine, pulse-labelling period, and age of chickens on the blastogenic response of chickens to Con A was evaluated. The results showed that despite a certain degree of individual variation among responses, the highest response was to 0.4 μg Con A per culture at a blood dilution of 1 50 in serum-free RPMI 1640 during incubation for 90 h at 40°C in a humidified atmosphere containing 5% CO 2. Cultures were pulsed with 0.2 μCi thymidine per culture for 18 h prior to harvesting. The stimulation response of chicken whole blood lymphocytes of chickens to Con A was prominent from 3 weeks of age onwards and the response measured in chickens 3 weeks of age was slightly than that of subsequent weeks. The possible applications of this technique in studying the pathogenesis of avian diseases and for quantitative assessment of circulating T cells of chickens are discussed.

Evaluation of cell death in EBV-transformed lymphocytes using agarose gel electrophoresis, light microscopy and electron microscopy. II. Induction of non-classic apoptosis ("para-apoptosis") by tritiated thymidine.

There is an extensive literature dating back to the late 1950's, on the damaging biological effects of radiolabeling DNA in vivo. Nonetheless, tritiated thymidine has often been used to label DNA in studies of programmed cell death (apoptosis). In the present study, we have investigated the effects of incorporation of tritiated thymidine into the DNA of an Epstein-Barr virus-transformed cell line (NC-37) in the absence of any other apoptosis-inducing agent. Cells were incubated in media containing 1-20 microCi/ml [methyl-3H]-thymidine ([3H]-TdR). At each concentration of tritiated thymidine used, cell proliferation ceased within 12 hours of incubation. The mode of cell death caused by tritiated thymidine incorporation was evaluated using DNA degradation patterns and cellular morphology. DNA degradation, in the absence of a "ladder" pattern, was shown by agarose gel electrophoresis. Electron microscopy was used as the "gold standard" to evaluate the specific morphologic type of cell death that accompanied the DNA degradation. Although some of the features of apoptosis were present, the cells lacked the early margination of the chromatin within an intact nucleus and surface blebbing leading to apoptotic body formation, two characteristic morphological features of apoptosis. We, therefore, coined the term "para-apoptosis" to be more precise about the morphologic type of cell death. The percent of para-apoptotic cells was quantitated by light microscopy using whole mount preparations (cytospins). The morphologic criteria of chromatin condensation, nuclear fragmentation, increase in cell density and cytoplasmic vacuolization were used for the evaluation of para-apoptosis by light microscopy of cytospin preparations. In the absence of tritiated thymidine, < 2% of the cells became apoptotic/para-apoptotic after 43 hours of incubation. However, at all concentrations of tritiated thymidine used in the incubation medium (1-20 microCi/ml), the number of para-apoptotic cells increased. In addition, we detected perturbations in the timing of the cell cycle of the surviving cells and an increase in the number of micronuclei after only one division cycle. The induction of para-apoptosis and micronuclei formation represent two distinct modes of cell death caused by tritiated thymidine incorporation. These studies emphasize the necessity for morphological examination in characterizing the induction of cell death in a new experimental system.

Human thymidine kinase 1. Regulation in normal and malignant cells

In mammalian cells, salvage pathway phosphorylation of thymidine is catalyzed by two thymidine kinases: the cell-cycle regulated cytoplasmic TK1 and the constitutively expressed mitochondrial TK2. Since TK1 is virtually absent in non-dividing cells, TK2 is probably the only thymidine kinase present in these cells. In cellular metabolism, TK1 and TK2 presumably serve to maintain sufficient dTTP for DNA replication and repair. TK1 purified from phytohemagglutinin-stimulated human lymphocytes is a dimer in the absence and a tetramer in the presence of ATP. In addition to the molecular weight transition, incubation with ATP at 4 °C or storage with ATP induces a reversible, enzyme concentration-dependent, kinetically slow transition from a low to a high affinity form of TK1, with K m values of 14 μ m and 0.5 μ m, respectively. This affinity difference implies that at cellular thymidine concentrations, the difference in catalytic activity between the two TK1 forms will be 3–5-fold. Calculations of cellular TK1 concentration suggested that the low affinity dimer form was dominant in G 0/G 1 cells and the high affinity tetramer form in S-phase cells. Hence, the transition may serve to fine-tune the cell-cycle regulation of thymidine kinase activity on the post-translational level. To study the ATP effect on the molecular level, an IPTG inducible T7 RNA polymerase-dependent expression system for the entire human TK1 polypeptide in E. coli was established. The recombinant TK1 has the same subunit mass and specific activity as the native enzyme. However, the recombinant TK1 solely displayed the kinetics of the high affinity form, with K m values of 0.3–0.4 μ m regardless of pre-exposure to ATP, indicating that the ATP effect may be dependent on post-translational modifications absent in E. coli. Surprisingly, we did not observe any effect of ATP on TK1 purified from bone-marrow cells from a patient with acute monocytic leukemia (AMOL). Furthermore, the K m values of TK1 from these cells were 45 μ m for the ATP-free enzyme and 65 μ m for the ATP-incubated enzyme. With TK1 purified from HL-60 cells, we obtained the same pattern and kinetic values as for TK1 from lymphocytes. In the light of the results with the recombinant TK1, we presume that the lack of ATP effect and very high K m values observed for the AMOL TK1 may be due to changes in post-translational regulatory mechanisms in acute monocytic cells.

Preparation and Characterization of Polyclonal and Monoclonal Antibodies Specific for Covalently Linked DNA/RNA Cross Sections

Covalently linked cross sections refer to structures that mimic hydrogen-bonded purine-pyrimidine, purine-purine, and pyrimidine-pyrimidine duplexes. Cross sections dA [symbol:see text] U and A [symbol: see text] dT, which have been synthesized chemically, have molecular dimensions similar to purine-pyrimidine base pairs in a double helix. We propose that antibodies to such covalent cross sections might facilitate the study of the pathogenesis of specific diseases or of biochemical processes in which base pair involvement is suspected and/or demonstrated. We have made polyclonal antibodies against "A:U" and "A:T" cross sections by immunizing rabbits with dA [symbol: see text] U and A [symbol: see text] dT, each conjugated to keyhole limpet hemocyanin (KLH). The antibodies were found to be highly specific for the cross sections and to cross react minimally to single nucleosides. Hybridomas secreting monoclonal antibodies to "A:T" were then generated from spleen cells of mice immunized with A [symbol: see text] dT conjugated to KLH. The MAbs produced were also found to be highly specific for "A:T" among various nucleosides. In fact, the binding of most of the monoclonal antibodies to "A:T" was only partially inhibited by high concentrations of adenosine or thymidine. All monoclonal antibodies to "A:T" cross react, but with lower affinity, to "A:U." Selected MAbs showed greater inhibition of binding to "A:T"-BSA by A + T than by A or T alone.

Thymidine incorporation in Lake Cisó: Problems in estimating bacterial secondary production across oxic-anoxic interfaces

Heterotrophic bacterial activity was measured by means of the 3H-thymidine (3H-TdR) incorporation technique in Lake Ciso, a small holomictic lake with anoxic hypolimnion. We tested several methodological questions across the vertical profile: TdR concentration at which maximal incorporation is reached, linearity of incorporation and isotope dilution, during holomixis and stratification periods. The TdR concentration at which maximal incorporation is reached changed seasonally and vertically. During holomixis, maximal incorporation was not always reached at concentrations up to 40 nM. Uptake was always linear in short incubation times and decreased from epi- to hypolimnion. The isotope dilution technique indicated a degree of participation in DNA synthesis higher than 50%, although a linear relationship between the inverse of 3H-TdR incorporation and increasing ‘cold’ thymidine concentration was not always observed. Autoradiographic experiments showed a low percentage of bacteria taking up 3H-TdR in both aerobic and anaerobic samples. The percentage of total labeled bacteria seemed to be generally higher in the metalimnion (11% maximal value) than in the hypolimnion. Labeled Amoebobacter and Chromatium cells were detected in field samples. Amoebobacter cells photoassimilated TdR in culture. Therefore, our results show that 3H-TdR incorporation is not an appropriate technique to estimate bacterial secondary production in anaerobic systems and in oxic-anoxic interfaces.

Destruction of WiDr multicellular tumor spheroids?with the novel thymidylate synthase inhibitor 1843U89?at physiological thymidine concentrations

Destruction of WiDr multicellular tumor spheroids?with the novel thymidylate synthase inhibitor 1843U89?at physiological thymidine concentrations

Effect of 3 H Decays on DNA Partition during Cell Division

Normal human embryonic lung fibroblasts were grown in the presence of different concentrations of tritium-labeled thymidine ([3H]dThd) for various periods of time, and the flow of cells from metaphase to anaphase was followed. Mitotic indices were evaluated as a function of the percentage of cells in metaphase and anaphase. They revealed that the presence of [3H]dThd induces a disturbance of the metaphase-anaphase transition. The DNA content of cells in metaphase and anaphase was measured with cytophotometry after the cells were grown in the presence of the radioactive precursor. It was found that the latter interfered with the flow of cells from metaphase to anaphase, impairing the symmetric distribution of DNA between daughter cells.