Synthesis of DNA in the liver and testes of cadmium-tested partially hepatectomized rats.

Cadmium affects the induction of thymidine and thymidylate kinases in regenerating rat liver. EDTA administered simultaneously with cadmium reverses its inhibitory action on enzyme synthesis, and prevents the depression of thymidine incorporation into DNA observed in cadmium-treated animals. Zinc does not abolish the inhibitory action of cadmium on the synthesis of DNA in regenerating liver, and the incorporation of thymidine into DNA in the testes was inhibited more by intraperitoneal injection of cadmium plus zinc than by injection of cadmium alone. Inhibition of thymidine incorporation into DNA in the liver and testes was proportional to the amount of cadmium administered up to about 2 mg CdCl2/kg body weight, but surprisingly, higher doses of cadmium caused less inhibition.     

Association of thymidylate kinase activity with pyrimidine deoxyribonucleoside kinase induced by herpes simplex virus.

Thymidine kinase derived from LMTK+ does not exhibit thymidylate kinase activity. However, protein isolated by affinity column chromatography from thymidine kinase-deficient mouse cells (LMTK-) infected by herpes simplex virus type 1 shows thymidylate kinase activity in addition to thymidine kinase and deoxycytidine kinase activities. The virus-induced multifunctional enzyme has a molecular weight of 85,000, whereas the molecular weight of thymidylate kinase from uninfected LMTK- mouse cells is 71,000. The virus-induced enzyme has a Km for thymidine of 0.8 micromolar, and for thymidylate of 25 micromolar, and for thymidylate of 25 micromolar; the ratio of Vmax for thymidylate kinase to thymidine kinase is 1.7. When subjected to isoelectric focusing, thymidylate kinase activity is not separated from thymidine kinase activity, and even though four peaks of activity are observed they have a constant ratio of thymidylate kinase to thymidine kinase activity. The isoelectric points (pI) of these four peaks are 4.8, 5.8, 6.2, and 6.6, respectively. Thymidylate kinase, derived from uninfected cells when subjected to isoelectric focusing, separates into a major component with an isoelectric point at pH 8.2 and a minor component at pH 7.7. Although thymidine and thymidylate kinase activities derived from the virus-infected cells cannot be separated either by affinity column chromatography, glycerol density gradient centrifugation, or isoelectric focusing, there is a differential rate of inactivation when the enzyme is subjected to incubation at 37 degrees, with thymidylate kinase activity being more labile than thymidine kinase activity.     

Effect of the interferone inducer, polyriboinosinic.polyribocytidylic acid on rat liver regeneration following partial hepatectomy

The administration of the interferone inducer, polyriboinosinic.polyribocytidylic acid, inhibited the rise of activities of thymidylate synthase and thymidine kinase as well as DNA content in 24 h-regenerating rat liver in a dose dependent manner. The immunoblotting assay showed that the decrease of thymidylate synthase activity was due to inhibition of the induction of the enzyme. Co-administration of putrescine did not affect the inhibitory effect of polyriboinosinic.polyribocytidylic acid. Polyriboinosinic acid did not affect DNA synthesis in rat liver regeneration.     

Gamma-T4 hybrid bacteriophage carrying the thymidine kinase gene of bacteriophage T4.

Among 32 lambda-T4 recombinant phages selected for growth on a thymidylate synthetase-deficient (thyA) host, 2 were shown to carry the T4 thymidine kinase (tk) gene. The lambda-T4tk phages contain two T4 HindIII DNA fragments (2.0 and 1.5 kilobases) that hybridize to restriction fragments of T4 DNA, encompassing the tk locus at 60 kilobases on the T4 map. The T4tk insert compensates for the simultaneous host deficiencies of thymidine kinase and thymidylate synthetase in a thymidine kinase-deficient (tdk) host growing in the presence of fluorodeoxyuridine when provided with thymidine and uridine. The lambda-T4tk hybrid phages specified five polypeptides with Mrs of 22,000 (22K), 21K, 14K, 11K, and 9K.     

Stimulated DNA synthesis in livers and kidneys induced to proliferate associated with unchanged thymidine and thymidylate kinase activities.

Enhanced mitotic activity and stimulated DNA synthesis associated with unchanged activity of thymidine and thymidylate kinases were observed in mouse kidneys induced to proliferate by intracardiac injection of lead acetate, and in rat livers following repeated administration of 5-azacytidine. On the other hand, the enhanced thymidine kinase activity evoked by L-tryptophan given by intubation at later stages of liver regeneration was paralleled by the enhanced incorporation of thymidine into DNA only to a very small degree.     

Molecular cloning and expression of the human deoxythymidylate kinase gene in yeast.

(Deoxy)thymidylate (dTMP) kinase is an enzyme which phosphorylates dTMP to dTDP in the presence of ATP and magnesium. This enzyme is important in cellular DNA synthesis because the synthesis of dTTP, either via the de novo pathway or through the exogenous supply of thymidine, requires the activity of this enzyme. It has been suggested that the activities of the enzymes involved in DNA precursor biosynthesis, such as thymidine kinase, thymidylate synthase, thymidylate kinase, and dihydrofolate reductase, are subjected to cell cycle regulation. Here we describe the cloning of a human dTMP kinase cDNA by functional complementation of a yeast dTMP kinase temperature-sensitive mutant at the non-permissive temperature. The nucleotide sequence of the cloned human cDNA is predicted to encode a 24 KD protein that shows considerable homology with the yeast and vaccinia virus dTMP kinase enzymes. The human enzyme activity has been investigated by expressing it in yeast. In this work, we demonstrate that the cloned human cDNA, when expressed in yeast, produces dTMP kinase activity.     

Differentiation associated changes in thymidylate synthetase and thymidine kinase activities in intestinal cells.

Proliferative and mature intestinal cells of the jejunum and colon of rat, colon of man, and the surface cells of neoplastic colon lesions of man were assayed for thymidylate synthetase and thymidine kinase activities. Cells from the proliferative region of rat jejunal mucosa were found to have higher enzyme activities than cells from the non-proliferative region. Thymidylate synthetase activity was observed to decrease as cells migrated from base to upper crypt, whereas thymidine kinase activity increased during crypt migration and then declined as cells migrated onto villi. Thymidine kinase activity also remained elevated longer than thymidylate synthetase during cell migration in colonic mucosa of rat and man. High thymidine kinase: thymidylate synthetase ratios similar to those observed in flat mucosa before cells become fully mature were found in cells removed from expanding neoplastic lesions of man.     

Carbocyclic thymidine derivatives efficiently inhibit Plasmodium falciparum thymidylate kinase (PfTMK)

During the course of our research into new anti-malaria drugs, Plasmodium falciparum thymidylate kinase (PfTMK) has emerged as an important drug target because of its unique substrate specificity. Compared with human thymidylate kinase (HsTMK), PfTMK shows broader substrate specificity, which includes both purine and pyrimidine nucleotides. PfTMK accepts both 2'-deoxyguanosine monophosphate (dGMP) and thymidine monosphosphate (TMP) as substrates. We have evaluated the inhibitory activity of seven carbocyclic thymidine analogs and report the first structure-activity relationship for these inhibitors against PfTMK. The 2',3' dideoxycarbocyclic derivative of thymidine showed the most potent inhibition of the enzyme. The K(i)(dTMP) and K(i)(dGMP) values were 20 and 7 μM respectively. Thus, further modifications of carbocyclic thymidine analogs represent a good strategy for developing more powerful thymidylate kinase inhibitors.     

Human Thymidine Kinase Can Functionally Replace Herpes Simplex Virus Type 1 Thymidine Kinase for Viral Replication in Mouse Sensory Ganglia and Reactivation from Latency upon Explant

Herpes simplex virus type 1 thymidine kinase exhibits a strikingly broad substrate specificity. It is capable of phosphorylating deoxythymidine and deoxyuridine as does human thymidine kinase, deoxycytidine as does human deoxycytidine kinase, the cytosolic kinase whose amino acid sequence it most closely resembles, and thymidylate as does human thymidylate kinase. Following peripheral inoculation of mice, viral thymidine kinase is ordinarily required for viral replication in ganglia and for reactivation from latency following ganglionic explant. To determine which activity of the viral kinase is important for replication and reactivation in mouse ganglia, recombinant viruses lacking viral thymidine kinase but expressing individual human kinases were constructed. Each recombinant virus expressed the appropriate kinase activity with early kinetics following infection of cultured cells. The virus expressing human thymidine kinase exhibited thymidine phosphorylation activity equivalent to ~5% of that of wild-type virus in a quantitative plaque autoradiography assay. Nevertheless, it was competent for ganglionic replication and reactivation following corneal inoculation of mice. The virus expressing human thymidylate kinase was partially competent for these activities despite failing to express detectable thymidine kinase activity. The virus expressing human deoxycytidine kinase failed to replicate acutely in neurons or to reactivate from latency. Therefore, it appears that low levels of thymidine phosphorylation suffice to fulfill the role of the viral enzyme in ganglia and that this role can be partially fulfilled by thymidylate kinase activity alone.     

Thymidylate synthetase during synchronous nuclear division cycle and differentiation of Physarum polycephalum

Thymidylate synthetase and thymidine kinase activities in wild type strain M₃b and in thymidine kinase-deficient mutant TU63 of Physarum polycephalum are studied. Whenever nuclear division occurs in macroplasmodia of wild type, thymidine kinase and thymidylate synthetase activities sharply increase, although the increase of thymidylate synthetase activity is less pronounced than thymidine kinase activity. This is also true for other investigated nuclear divisions during the life cycle of P. polycephalum. It is shown for the first time that thymidylate synthetase is a periodically fluctuating enzyme during the naturally synchronous nuclear division cycle of P. polycephalum with a peak of specific activity in the S phase. In macroplasmodia, as well as after germination of microsclerotia of M₃b, thymidine kinase is the dominant enzyme, whereas at the time of the precleavage mitosis in sporulating macroplasmodia thymidylate synthetase is the predominant enzyme. This study describes and compares both dTMP-synthesizing enzymes during proliferation and differentiation of the same organism.     

Deoxycytidine transport and pyrimidine deoxynucleotide metabolism in phytohaemagglutinin-stimulated pig lymphocytes.

Increased entry of deoxy[3H]cytidine begins at about 12h after addition of phytohaemagglutinin to peripheral pig lymphocyte cultures, and is accompanied by a parallel stimulation of deoxycytidine kinase up to the beginning of DNA synthesis at 24h. The increased deoxycytidine uptake is characterized by an increase in Vmax. without alteration of the apparent Km (0.7 +/- 0.11 muM). Although the entries of both nucleosides are promoted at the same time, the stimulation of deoxycytidine uptake is less than that of thymidine, and the two nucleosides are transported by separate systems. In addition to deoxycytidien kinase, the synthesis of deoxycytidylate deaminase and thymidylate synthetase are stimulated after addition of phytohaemagglutinin, but to a lesser extent than that of thymidine kinase. The importance of the latter enzyme in forming dTMP, and of thymidylate kinase in providing dTTP, is discussed.     

Thymidine kinase, thymidylate synthase, and endothelial cell growth under hyperoxia

To determine the respective role of thymidine kinase and thymidylate synthase activities in the hyperoxia-induced decrease in DNA synthesis and their relationship with cell replication, we measured these two enzyme activities in primary cultures of porcine aortic endothelial cells under different O2 concentrations for various durations. In confluent cells, exposure to 95% O2 for 5 days reduced thymidine kinase activity to 15% of control values; thymidylate synthase activity was unaffected. In preconfluent cells exposed to 95% O2 for 2 days, similar results were obtained, together with evidence for arrest in cell proliferation. Thymidylate synthase activity could therefore not be related to decreased cell proliferation under hyperoxia. [3H]thymidine incorporation into DNA, thymidine kinase activity, and cell proliferation were all similarly affected under exposure to graded O2 concentration for 2 days. Thymidine kinase appears to be a key enzyme in the modulation of DNA synthesis from thymidine and in its replication in endothelial cells.     

The sex difference in the regulation of liver regeneration after partial hepatectomy in the rat

The increases in the activity of hepatic thymidylate synthetase and thymidine kinase, which catalyzes the formation of thymidylate via the de novo and salvage pathways, respectively, were significantly suppressed 24 h after 70% partial hepatectomy in female rats administered either alpha- or beta-adrenoreceptor antagonists. The injection of beta-antagonist to male or ovariectomized female rats had no effect on the activities of these enzymes. Only alpha-adrenoceptor antagonist depressed these enzymatic activities of 24-h-regenerating liver in male and ovariectomized female rats. The decrease of the activities of thymidylate synthetase and thymidine kinase was accompanied by a concomitant reduction of DNA content in 24-h-regenerating liver. It is concluded that catecholamine regulates the female rat liver regeneration through both alpha- and beta-adrenergic pathways by the inductions of thymidylate synthase and thymidine kinase, while in adult male and ovariectomized female rats, only the alpha-mediated pathway is involved.     

Thymidine salvage in Pseudomonas stutzeri and Pseudomonas aeruginosa provided by heterologous expression of Escherichia coli thymidine kinase gene.

Unlike enteric bacteria, Pseudomonas spp. generally lack thymidine phosphorylase and thymidine kinase activities, thus preventing their utilization of exogenous thymine or thymidine and precluding specific radioactive labeling of their DNA in vivo. To overcome this limitation, a DNA fragment encoding thymidine kinase (EC 2.7.1.21) from Escherichia coli was cloned into pKT230, a small, broad-host-range plasmid derived from plasmid RSF1010. From transformed E. coli colonies, the recombinant plasmid bearing the thymidine kinase gene was conjugally transferred to Pseudomonas stutzeri, Pseudomonas aeruginosa, Pseudomonas mendocina, Pseudomonas alcaligenes, and Pseudomonas pseudoalcaligenes. Thymidine kinase activity was expressed in all of these species, and all gained the ability to incorporate exogenous [2-14C]thymidine into their DNA. Thymidine incorporation into P. stutzeri was enhanced 12-fold more in mutants lacking thymidylate synthetase activity. These mutants produced higher levels of thymidine kinase and were thymidine auxotrophs; thymineless death resulted from removal of thymidine from a growing culture.     

The activities of thymidine metabolising enzymes during the cell cycle of a human lymphocyte cell line LAZ-007 synchronised by centrifugal elutriation

The activities throughout the cell cycle of thymidine kinase (EC 2.7.1.21), dihydrothymine dehydrogenase (EC 1.3.1.2), thymidine phosphorylase (EC 2.4.2.4) and dTMP phosphatase (EC 3.1.3.35) were measured in the Epstein-Barr virally transformed human B lymphocyte line LAZ-007. Cells were synchronised at different stages of the cell cycle using the technique of centrifugal elutriation. The degree of synchrony in each cycle-stage cell population was determined by flow microfluorimetric analysis of DNA content and by measurement of thymidine incorporation into DNA. The activity of the anabolic enzyme thymidine kinase was low in the G₁ phase cells, but increased many-fold during the S and G₂ phases, reaching a maximum after the peak of DNA synthesis, then decreasing in late G₂ + M phase. By contrast, the specific activities of the enzymes involved in thymidine and thymidylate catabolism, dihydrothymine dehydrogenase, thymidine phosphorylase and dTMP phosphatase remained essentially constant throughout the cell cycle, indicating that the fate of thymidine at different stages of the cell cycle is governed primarily by regulation of the level of the anabolic enzyme thymidine kinase and not by regulation of the levels of thymidine catabolising enzymes.     

Metabolism of 4'-azidothymidine. A compound with potent and selective activity against the human immunodeficiency virus.

4'-Azidothymidine (ADRT) is a novel nucleoside analog, that selectively inhibits human immunodeficiency virus replication in human lymphocytes. Unlike the dideoxyribonucleoside analogs and 3'-azido-2',3'-dideoxythymidine (AZT), ADRT retains the 3'-hydroxy group. The pathways of ADRT metabolism were elucidated by determining: (i) the kinetics of the interactions of ADRT and its metabolites with enzymes of thymidine metabolic pathways, (ii) the pool sizes of phosphorylated metabolites, and (iii) the nature of ADRT incorporation into human DNA. ADRT is not a substrate for thymidine phosphorylase, but is metabolized by kinases. Thymidine kinase phosphorylates ADRT to ADRT monophosphate (ADRT-MP). For this enzyme, ADRT has a Ki value of 5.2 microM, in comparison to a Km value of 0.7 microM for thymidine. The Km value of ADRT toward thymidine kinase is 8.3 microM and the rate of ADRT phosphorylation is 1.4% that of thymidine phosphorylation. ADRT-MP has a low affinity toward thymidylate kinase (a Ki value of 28.9 microM versus a Km value of 0.56 microM for thymidylate), and toward thymidylate synthase (a Ki value of 180 microM versus a Km value of 8 microM for deoxyuridylate). The results suggest that ADRT can be activated effectively by cellular kinases without significant interference of normal thymidine metabolism. In cultured human lymphocytes (A3.01, H9, and U937 cells), ADRT was phosphorylated efficiently to ADRT 5'-triphosphate (ADRT-TP), which is the major metabolite of ADRT. The intracellular concentrations of ADRT-TP ranged from 1 to 3.3 microM after 24 h of incubation with 2 microM of ADRT and the half-life of ADRT-TP varied from 3 to 6 h. Although ADRT-TP is a poor competitive inhibitor against dTTP toward DNA polymerases alpha and beta with Ki values of 62.5 and 150 microM, respectively. ADRT-MP was found to be internally incorporated into cellular DNA. The extent of ADRT-MP substitution for dTMP in DNA was 1 in 6979 for A3.01 cells incubated with 2.9 microM ADRT for 24 h. Internal incorporation of ADRT-MP contrasts with the mechanism of other 2',3'-dideoxynucleoside analogs (i.e. AZT, ddC, ddI, d4T...), which are DNA chain terminators. This finding indicates that a 3'-deoxy structure in a nucleoside analog is not a prerequisite for anti-human immunodeficiency virus activity.     

Acquisition of thymidylate by the obligate intracytoplasmic bacterium Rickettsia prowazekii.

The pathway for the acquisition of thymidylate in the obligate bacterial parasite Rickettsia prowazekii was determined. R. prowazekii growing in host cells with or without thymidine kinase failed to incorporate into its DNA the [3H]thymidine added to the culture. In the thymidine kinase-negative host cells, the label available to the rickettsiae in the host cell cytoplasm would have been thymidine, and in the thymidine kinase-positive host cells, it would have been both thymidine and TMP. Further support for the inability to utilize thymidine was the lack of thymidine kinase activity in extracts of R. prowazekii. However, [3H]uridine incorporation into the DNA of R. prowazekii was demonstrable (973 +/- 57 dpm/3 x 10(8) rickettsiae). This labeling of rickettsial DNA suggests the transport of uracil, uridine, uridine phosphates (UXP), or 2'-deoxyuridine phosphates, the conversion of the labeled precursor to thymidylate, and subsequent incorporation into DNA. This is supported by the demonstration of thymidylate synthase activity in extracts of R. prowazekii. The enzyme was determined to have a specific activity of 310 +/- 40 pmol/min/mg of protein and was inhibited greater than or equal to 70% by 5-fluoro-dUMP. The inability of R. prowazekii to utilize uracil was suggested by undetectable uracil phosphoribosyltransferase activity and by its inability to grow (less than 10% of control) in a uridine-starved mutant cell line (Urd-A) supplemented with 50 microM to 1 mM uracil. In contrast, the rickettsiae were able to grow in Urd-A cells that were uridine starved and supplemented with 20 microM uridine (117% of control). However, no measurable uridine kinase activity could be measured in extracts of R. prowazekii. Normal rickettsial growth (92% of control) was observed when the host cell was blocked with thymidine so that the host cell's dUXP pool was depressed to a level inadequate for growth and DNA synthesis in the host cell. Taken together, these data strongly suggest that rickettsiae transport UXP from the host cell's cytoplasm and that they synthesize TTP from UXP.     

Biochemical Aspects of Chick Embryo Retina Development: The Effects of Glucocorticoids

In chick embryo retina during development, DNA synthesis and the activities of DNA polymerase, thymidine kinase, thymidylate synthetase, and ornithine decarboxylase (ODC) declined in parallel from day 7 to 12. The administration in ovo of hydrocortisone reduced significantly, particularly at 8-10 days of incubation, both DNA synthesis and the four enzyme activities tested. The effect was dose dependent, reaching the maximum with 50-100 nmol of hydrocortisone, 8-16 h after treatment. The highest inhibition was found for ODC activity (70%), followed by thymidine kinase activity (62%) and DNA synthesis (45%), whereas activities of DNA polymerase and thymidylate synthetase were reduced only by 30%. The inhibitory effect was exerted by all the glucocorticoids tested, with dexamethasone and hydrocortisone being the most efficacious. The results support the view that glucocorticoids reduce the proliferative events in chick embryo retina, particularly at 8-10 days of embryonic life.