Thymidine Kinase Activity In Extracts Research Articles

AZT: A Biochemical Response Modifier of Methotrexate and 5-Fluorouracil Cytotoxicity in Human Ovarian and Pancreatic Carcinoma Cells

In ovarian and pancreatic carcinoma cell lines, the activity of the salvage enzyme, thymidine kinase (EC 2.7.1.21), was 2- to 13-fold higher than that of the key enzyme of thymidylate de novo biosynthesis, thymidylate synthase (dTMP synthase, EC 2.1.1.45). AZT (3'-azido-3'-deoxythymidine, zidovudine) competitively inhibited thymidine kinase activity in extracts of human ovarian and pancreatic carcinoma cells, with Dixon plots yielding Ki = 1.1 microM in both cell lines. AZT (20 microM) yielded synergistic cytotoxicity with methotrexate (0.4 microM) in human pancreatic carcinoma cells in clonogenic assay and also with methotrexate (0.02 microM) in human ovarian carcinoma cells, as measured by cell counts. Thymidine (10 microM) and hypoxanthine (100 microM) reversed these inhibitions. AZT (20 or 40 microM) also provided synergistic cytotoxicity with 5-fluorouracil (0.5 and 1.0 microM) in human pancreatic carcinoma cells in clonogenic assay. These studies suggest a new role for AZT, which, as an inhibitor of thymidine salvage, should be useful as a biochemical response modifier to provide a synergistic clinical anticancer impact on de novo biosynthesis of thymidylates in conjunction with methotrexate or 5-fluorouracil.

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.

Genetic and biochemical characterization of the thymidine kinase gene from herpesvirus of turkeys

The thymidine kinase gene encoded by herpesvirus of turkeys has been identified and characterized. A viral mutant (ATR0) resistant to 1-beta-D-arabinofuranosylthymine was isolated. This mutant was also resistant to 1-(2-fluoro-2-deoxy-beta-D-arabinofuronosyl)-5-methyluracil and was unable to incorporate [125I]deoxycytidine into DNA. The mutant phenotype was rescued by a cloned region of the turkey herpesvirus genome whose DNA sequence was found to contain an open reading frame similar to that for known thymidine kinases from other viruses. When expressed in Escherichia coli, this open reading frame complemented a thymidine kinase-deficient strain and resulted in thymidine kinase activity in extracts assayed in vitro.

Mutant strains of Tetrahymena thermophila defective in thymidine kinase activity: biochemical and genetic characterization.

Three mutant strains, one conditional, of Tetrahymena thermophila were defective in thymidine phosphorylating activity in vivo and in thymidine kinase activity in vitro. Nucleoside phosphotransferase activity in mutant cell extracts approached wild-type levels, suggesting that thymidine kinase is responsible for most, if not all, thymidine phosphorylation in vivo. Thymidine kinase activity in extracts of the conditional mutant strain was deficient when the cells were grown or assayed or both at the permissive temperature, implying a structural enzyme defect. Analysis of the reaction products from in vitro assays with partially purified enzymes showed that phosphorylation by thymidine kinase and nucleoside phosphotransferase occurred at the 5' position. Genetic analyses showed that the mutant phenotype was recessive and that mutations in each of the three mutant strains did not complement, suggesting allelism.

Mutant strains of Tetrahymena thermophila defective in thymidine kinase activity: biochemical and genetic characterization.

Three mutant strains, one conditional, of Tetrahymena thermophila were defective in thymidine phosphorylating activity in vivo and in thymidine kinase activity in vitro. Nucleoside phosphotransferase activity in mutant cell extracts approached wild-type levels, suggesting that thymidine kinase is responsible for most, if not all, thymidine phosphorylation in vivo. Thymidine kinase activity in extracts of the conditional mutant strain was deficient when the cells were grown or assayed or both at the permissive temperature, implying a structural enzyme defect. Analysis of the reaction products from in vitro assays with partially purified enzymes showed that phosphorylation by thymidine kinase and nucleoside phosphotransferase occurred at the 5' position. Genetic analyses showed that the mutant phenotype was recessive and that mutations in each of the three mutant strains did not complement, suggesting allelism.

Regulation by Thymidine Monophosphate and Other Nucleotides of Thymidine Kinase Activity in Extracts from Primary Rabbit Kidney Cells Infected by HSV Types 1 and 2

In an attempt to differentiate between thymidine kinase (EC.2.4.1.21) induced by herpesvirus hominis type I (TK I) and type 2 (TK2), the different susceptibilities to the modifying effects of some thymidine analogues proved to be useful criteria: (I)2'-deoxythymidine-5'-triphosphate (dThd-5'-PPP) inhibits TK 2 at a concentration of 0-125 mM by 90%, whereas TK I is inhibited at 4-03 mM by 50%. (2) 2'-deoxythymidine-5'-monophosphate (dThd-5'-P) competitively inhibits TK 2 at all concentrations tested. On the other hand, the direction of its effect on TK I is concentration dependent: at 500 mum it stimulates and at 8 mM inhibits TK I activity. During enzyme kinetic studies, TK I displays substrate inhibition which is reversed by dThd-5'-P. This result explains the stimulating effect of dThd-5'-P at 500 muM. This phenomenon suggests the existence on the enzyme molecule of a second binding site for dThd which mediates substrate inhibition and which can be occupied also by dThd-5'-P. After polyacrylamide gel electrophoresis of TK I, the stimulation by dThd-5'-P disappears, suggesting the separation of the second binding site from the catalytic centre.

Isolation of mutants of bacteriophage T4 unable to induce thymidine kinase activity. II. Location of the structural gene for thymidine kinase.

Amber mutants of bacteriophage T4 have been isolated that induce thymidine kinase activity only after infection of a strain of Escherichia coli carrying a suppressor mutation. The activity induced when one of these mutants infected this suppressor strain is much more heat sensitive than the activity induced by wild-type T4. This indicates that this amber mutation lies within the structural gene for thymidine kinase. This gene is between fI and v on the standard T4 genetic map. A mutant of tt4 that is unable to induce thymidine kinase activity incorporates only about one-eighth as much thymidine into its DNA as phage that do induce thymidine kinase. This contrasts to the findings that the total thymidine kinase activity in extracts prepared from cells infected with phage able to induce thymidine kinase in only twice as great as the activity in cells infected with the mutant unable to induce the enzyme.

Persistence of thymidine kinase activity in mitochondria of a thymidine kinase-deficient derivative of mouse L cells.

The cell line LM(TK(-)) Cl 1D, a derivative of mouse L fibroblasts deficient in thymidine kinase (EC 2.7.1.21) that shows very little thymidine kinase activity in extracts of whole cells as compared to the parental line, and that does not incorporate thymidine or 5-bromodeoxyuridine into nuclear DNA, has maintained the capacity to incorporate these precursors into mitochondrial DNA at a substantial rate. The amount of [methyl-(3)H]thymidine incorporated into mitochondrial DNA of LM(TK(-)) Cl 1D cells after long-term labeling has been conservatively estimated in different experiments to be between 30 and 60% of that incorporated into mitochondrial DNA or nuclear DNA of strain A9, an L-cell derivative without any thymidine-kinase dificiency; by contrast, the incorporation of thymidine into nuclear DNA of Cl 1D cells is less than 1% of that in A9 cells. These results strongly suggest that the loss of thymidine kinase activity in the extramitochondrial compartment of LM(TK(-)) Cl 1D cells has not been accompanied by the loss of the mitochondria-associated enzyme activity, pointing to a different genetic or epigenetic control of the extramitochondrial and mitochondrial enzymes.