Gerda Tyrsted

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 degrees 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 Km values of 14 microM and 0.5 microM, 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 G0/G1 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 Km values of 0.3-0.4 microM 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 Km values of TK1 from these cells were 45 microM for the ATP-free enzyme and 65 microM 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 Km values observed for the AMOL TK1 may be due to changes in post-translational regulatory mechanisms in acute monocytic cells.

The deoxyribonucleoside 5′-triphosphate (dATP and dTTP) pool in phytohemagglutinin-stimulated and non-stimulated human lymphocytes

Abstract The pool size of dATP and dTTP in human lymphocytes was studied in untreated and PHA-treated cells. Different methods of extracting the cellular content of dATP and dTTP have been investigated and extraction with 60% methanol was preferred. The pool size of dATP and dTTP in non-stimulated lymphocytes was about 0.2 and 0.05 pmoles/106 cells, respectively. After treatment with PHA for about 50 h the dATP and dTTP pools reached peak values representing increases in the pools of 20 and 170 fold, respectively. The variation in the pool sizes during transformation was paralleled by the variation of the rate of incorporation of labeled deoxy-thymidine into cellular DNA.

The pool size of deoxyguanosine 5'-triphosphate and deoxycytidine 5'-triphosphate in phytohemagglutinin-stimulated and non-stimulated human lymphocytes.

Abstract Requirements and optimal conditions have been studied for measurements of dGTP and dCTP in cellular extracts using the copolymer [d(1 − C)] as primer in a reaction catalysed by the large fragment of DNA polymerase from E. coli. The pool size of dGTP and dCTP in the human lymphocytes in the absence of PHA was found to be about 0.1 and 0.15 pmoles/106 cells, respectively. After treatment with PHA the pool size of both deoxynucleotides increased. The pool size of dCTP reached a maximum after 67 h simultaneously with the peak value of labelled deoxythymidine incorporation into DNA and the variation in these two parameters was very similar. The variation in the dGTP pool, however, was not so distinctly related to deoxythymidine incorporation as in the dCTP pool, since the increase in the dGTP pool was very small from 52–67 h. During transformation the dGTP pool was found to be the smallest pool. The relative cellular content of mono-, di- and triphosphate esters of deoxyadenosine, deoxyguanosine and deoxycytidine was studied.

Effect of hydroxyurea and 5-fluorodeoxy-uridine on deoxyribonucleoside triphosphate pools early in phytohemagglutinin-stimulated human lymphocytes

The induction of deoxyribonucleoside triphosphate pools was studied in phytohemagglutinin-stimulated human lymphocytes in the presence of metabolic inhibitors. The dATP pool was completely inhibited in cells treated with hydroxyurea, in contrast to the dTTP pool. However, 1-formyl-isoquinoline thiosemicarbazone inhibited the formation of these pools equally. During approximately 3 hr of treatment of stimulated cells with hydroxyurea, the dATP, dGTP and dCTP pools were depleted to the base levels observed in the cells before the pools were induced. The base level of the dTTP pool was achieved only in the presence of 5-fluorodeoxyuridine, but the inhibition was completely prevented by addition of thymidine. It is suggested that, when resting lymphocytes were stimulated to enter the growth cycle, the formation of deoxyribonucleoside triphosphates in the early transformation was due to the de novo pathway.

DNA polymerase activity in phytohemagglutinin-stimulated and non-stimulated human lymphocytes

Abstract Requirements and optimal conditions have been studied for the activity of DNA polymerase from phytohemagglutinin-stimulated and non-stimulated human lymphocytes. Differences were found in thermal stability and inhibitory effect of KC1 and p -chloromercuribenzoate. The relationship was determined between DNA polymerase activity, cellular pools of dATP, dTTP and incorporation of deoxythymidine into DNA during transformation. The increase in polymerase activity was paralleled by a similar increase in the pools of dATP and dTTP. The enzyme activity and the pool sizes of both nucleotides reached a maximum simultaneously with the peak of deoxythymidine incorporation into DNA. Studies in which protein synthesis was limited by cycloheximide showed that both the DNA polymerase activity and the rise in the pool sizes of both nucleotides were abolished. This implies that the de novo synthesis is required for the enzymes involved.

Metabolic studies on small molecular weight nuclear RNA components in human lymphocytes.

Abstract Some metabolic properties of small molecular weight nuclear RNA (snRNA) components have been studied in human lymphocytes cultured with PHA. Pulse-labelling experiments with 3H-uridine in 3 h-intervals around the onset of DNA synthesis showed no qualitative or quantitative differences in the snRNA labelling pattern. Long labelling experiment with 3H-methionine demonstrated the following relative degrees of methylation: tRNA (1.0), 5S RNA (0), D (0.3), 5.5S RNA (0.2), C (0.6), A (0.2), L (0) and rRNA (0.2). Chase-experiments with 3H-methionine showed that the snRNA components D, C and A are metabolically stable with half-lives of not less than 30 h. Actinomycin D (0.05 μg/ml) reduced markedly the synthesis of rRNA and 5 S RNA whereas the synthesis of D, C, A and L was unaffected or only slightly affected. Actinomycin D at a concentration of 0.25 μg/ml inhibited the synthesis of D, C and A. Cycloheximide (0.19 μg/ml) reduced the synthesis of D, C and rRNA to about 50% of control whereas 5S RNA synthesis was only slightly inhibited and tRNA synthesis was unaffected.

Cytidine 5′-diphosphate reductase activity in phytohemagglutinin stimulated human lymphocytes

The optimal conditions and the effect of deoxyribonucleoside triphosphates were determined for CDP reductase activity in PHA-stimulated lymphocytes. The enzymatic reaction showed an absolute requirement for ATP. In the absence of ATP, only dATP showed a minor stimulation of the reduction of CDP to dCDP. During transformation the CDP reductase activity reached a maximum at the same time as the four deoxyribonucleoside triphosphate pools, corresponding to mid S-phase at about 50 h after PHA addition. The DNA polymerase activity reached a maximum at 57 h.

Thymidine kinase in human leukemia expression of the lymphoblastic isoenzyme in three patients with acute myelocytic leukemia

The dominating thymidine kinase activity in mononuclear white blood cells from three patients with untreated acute myelocytic leukemia (AML) was compared with TK 1 from phytohemagglutinin-stimulated and TK 2 from unstimulated, normal lymphocytes. The enzyme activity in the AML cells and the stimulated lymphocytes was found to be in the same range. Regarding the combined thymidine and dTTP kinetics, the enzymes from the three AML patients resembled TK 1, but the ATP kinetics were different and the molecular weights were lower, as previously found for thymidine kinases from other leukemic cells. Therefore, the designation TK-1-onc is suggested for the thymidine kinases from the AML cells.

Thymidine kinase isoenzymes in human acute and chronic lymphatic leukemia

The dominating thymidine kinase isoenzyme was examined in mononuclear leucocytes from two patients, one with acute and one with chronic lymphatic leukemia. The two isoenzymes exhibited Michaelis-Menten substrate kinetics with ATP and cooperative inhibition kinetics with dTTP. The substrate kinetics with thymidine were different. According to the enzymatic properties the isoenzymes from the acute and chronic lymphatic cells were designated TK 3 and TK 4, respectively. Comparison with the isoenzymes in normal lymphocytes (TK 1, TK 2) and in acute monocytic leukemic cells (TK 3, TK 4) indicated the existence of three thymidine kinase isoenzymes in human leukemic cells which differed from the two isoenzymes in normal human lymphocytes.

Low-molecular weight nuclear RNA components in human lymphocytes cultured without or with phytohaemagglutinin☆

Abstract Purified human lymphocytes were cultured without or with phytohaemagglutinin (PHA) in the presence of radioactive RNA precursors. RNA was extracted with phenol at 0°, 40° or 62°C and separated on polyacrylamide gels. RNA extracted with phenol either in presence or absence of the RNAse inhibitor diethylpyrocarbonate showed no sign of degradation when separated on 2.6 or 3% polyacrylamide gels. Ten percent gel profiles of whole cell or nuclear RNA showed a a number of small mol. wt RNA components (K, L, M, N, A, B, C, D, F) apart from tRNA, 5 S RNA and 5.5 S RNA. Profiles of cytoplasmic RNA showed only components K and L apart from tRNA, 5 S RNA and 5.5 S RNA. L, C, D and F have an electrophoretic mobility similar to the corresponding components in various ascites cells, while M, N and B may be unique for human cells. The low-molecular wt nuclear RNA components (snRNA) are found in non-stimulated as well as in PHA-stimulated cells and the relative amounts of the snRNA components are not changed during PHA-induced transformation. It is therefore concluded that the relative amounts of the different snRNA components are not related to the dynamic state of the cell.