Induction Of Thymidine Kinase Activity Research Articles

Inhibition of Mycoplasma pneumoniae growth by FDA-approved anticancer and antiviral nucleoside and nucleobase analogs.

BackgroundMycoplasma pneumoniae (Mpn) is a human pathogen that causes acute and chronic respiratory diseases and has been linked to many extrapulmonary diseases. Due to the lack of cell wall, Mpn is resistant to antibiotics targeting cell wall synthesis such as penicillin. During the last 10 years macrolide-resistant Mpn strains have been frequently reported in Asian countries and have been spreading to Europe and the United States. Therefore, new antibiotics are needed. In this study, 30 FDA-approved anticancer or antiviral drugs were screened for inhibitory effects on Mpn growth and selected analogs were further characterized by inhibition of target enzymes and metabolism of radiolabeled substrates.ResultsSixteen drugs showed varying inhibitory effects and seven showed strong inhibition of Mpn growth. The anticancer drug 6-thioguanine had a MIC (minimum inhibitory concentration required to cause 90% of growth inhibition) value of 0.20 μg ml-1, whereas trifluorothymidine, gemcitabine and dipyridamole had MIC values of approximately 2 μg ml-1. In wild type Mpn culture the presence of 6-thioguanine and dipyridamole strongly inhibited the uptake and metabolism of hypoxanthine and guanine while gemcitabine inhibited the uptake and metabolism of all nucleobases and thymidine. Trifluorothymidine and 5-fluorodeoxyuridine, however, stimulated the uptake and incorporation of radiolabeled thymidine and this stimulation was due to induction of thymidine kinase activity. Furthermore, Mpn hypoxanthine guanine phosphoribosyl transferase (HPRT) was cloned, expressed, and characterized. The 6-thioguanine, but not other purine analogs, strongly inhibited HPRT, which may in part explain the observed growth inhibition. Trifluorothymidine and 5-fluorodeoxyuridine were shown to be good substrates and inhibitors for thymidine kinase from human and Mycoplasma sources.ConclusionWe have shown that several anticancer and antiviral nucleoside and nucleobase analogs are potent inhibitors of Mpn growth and that the mechanism of inhibition are most likely due to inhibition of enzymes in the nucleotide biosynthesis pathway and nucleoside transporter. Our results suggest that enzymes in Mycoplasma nucleotide biosynthesis are potential targets for future design of antibiotics against Mycoplasma infection.

Induction of thymidine kinase activity and clonal growth of certain leukemic cell lines by a granulocyte-derived factor

Normal polymorphonuclear neutrophils (PMNs) constitutively secrete a mediator designated granulocyte-derived factor (GDF) that can enhance the uptake of 3H-thymidine (3- to 20-fold) by the molt-3, CTV-1, and K562 leukemic cell lines in a dose-dependent manner. GDF is heat labile (56 degrees C for 30 minutes) and acid labile (pH 2.0) and is sensitive to treatment with bacterial protease type IV. Our preliminary studies suggest that GDF is non-dialyzable (molecular weight cutoff, 12,000), binds to diethylaminoethyl (DEAE), and has an apparent molecular weight (mol wt) of about 40 Kd. Production of GDF is unaffected by treatment of PMN with activating agents (interferon gamma, OK432, phorbol ester, calcium ionophore, poly I:C) or metabolic inhibitors (actinomycin-D and cyclohexamide), suggesting that GDF is constitutively secreted. Despite the marked enhancement of 3H-thymidine uptake, cell number and the rate of DNA synthesis in GDF responsive cultures remain unchanged. In contrast, the clonogenic efficiency of the responsive cells is greatly increased in the presence of GDF. These phenomena occur in parallel to an amplification of the level of thymidine kinase activity in the sensitive cells. GDF is distinct from a panel of different lymphokines and monokines in antigenicity and biochemical and functional characteristics, and is possibly a novel cytokine that can alter the pattern of DNA synthesis and growth characteristics of certain hematopoietic cells. However, its biologic and physiologic significance remains to be determined.

Induction of Thymidine Kinase Activity and Clonal Growth of Certain Leukemic Cell Lines by a Granulocyte-Derived Factor

Normal polymorphonuclear neutrophils (PMNs) constitutively secrete a mediator designated granulocyte-derived factor (GDF) that can enhance the uptake of 3H-thymidine (3- to 20-fold) by the molt-3, CTV-1, and K562 leukemic cell lines in a dose-dependent manner. GDF is heat labile (56 degrees C for 30 minutes) and acid labile (pH 2.0) and is sensitive to treatment with bacterial protease type IV. Our preliminary studies suggest that GDF is non-dialyzable (molecular weight cutoff, 12,000), binds to diethylaminoethyl (DEAE), and has an apparent molecular weight (mol wt) of about 40 Kd. Production of GDF is unaffected by treatment of PMN with activating agents (interferon gamma, OK432, phorbol ester, calcium ionophore, poly I:C) or metabolic inhibitors (actinomycin-D and cyclohexamide), suggesting that GDF is constitutively secreted. Despite the marked enhancement of 3H-thymidine uptake, cell number and the rate of DNA synthesis in GDF responsive cultures remain unchanged. In contrast, the clonogenic efficiency of the responsive cells is greatly increased in the presence of GDF. These phenomena occur in parallel to an amplification of the level of thymidine kinase activity in the sensitive cells. GDF is distinct from a panel of different lymphokines and monokines in antigenicity and biochemical and functional characteristics, and is possibly a novel cytokine that can alter the pattern of DNA synthesis and growth characteristics of certain hematopoietic cells. However, its biologic and physiologic significance remains to be determined.

Human cytosolic thymidine kinase. Purification and physical characterization of the enzyme from HeLa cells.

The mammalian cytosolic thymidine kinase is one of a number of enzymes involved in DNA replication whose activities increase dramatically during S phase of the cell cycle. As a first step in defining the mechanisms that control the S phase induction of thymidine kinase activity, we have purified the human enzyme from HeLa cells and raised a specific immune serum against the purified protein. The enzyme was isolated from cells arrested in S phase by treatment with methotrexate and purified to near homogeneity by ion-exchange and affinity chromatography. Stabilization of the purified enzyme was achieved by the addition of digitonin. An electrophoretic Rm of 0.2 in nondenaturing gels characterizes the purified enzyme activity as cytosolic thymidine kinase. The enzyme has a Stoke's radius of 40 A determined by gel filtration and a sedimentation coefficient of 5.5 S determined by glycerol gradient sedimentation. Based on these hydrodynamic values, a native molecular weight of 96,000 was calculated for the purified enzyme. When electrophoresed in denaturing sodium dodecyl sulfate-polyacrylamide gels under reducing conditions, the most purified enzyme fraction was found to contain one predominant polypeptide of Mr = 24,000. Several lines of evidence indicate that this polypeptide is responsible for thymidine kinase enzymatic activity. 1) The Mr = 24,000 polypeptide co-migrates with thymidine kinase activity in electrophoretic and sedimentation analyses. 2) A subunit Mr = 25,504 is predicted by the nucleotide sequence of a recently isolated cDNA clone that encodes HeLa thymidine kinase. 3) Mouse LTK- cells transformed with this clone express a cytosolic thymidine kinase activity, as well as a novel Mr = 24,000 polypeptide detectable with immune serum raised against the purified human enzyme.

Chlordecone does not interfere with hepatic repair after carbon tetrachloride or partial hepatectomy

The effect of chlordecone (CD) on hepatic repair, measured either as recovery of microsomal enzymatic functions or as the induction of cytosolic thymidine kinase (TK) activity, was evaluated in rats given carbon tetrachloride (CCl4). Carbon tetrachloride was administered to CD-potentiated and control animals using doses of this hepatotoxin which produce similar degrees of damage at 24 hours in both groups of animals (6 and 100 microliters CCl4 per 100 g body weight, respectively). Chlordecone had no significant effect on the time course of recovery of microsomal cytochrome P-450 content or glucose-6-phosphatase activity following CCl4 administration. Hepatic TK activity was measured 48 hours after CCl4 administration as a biochemical index of the hepatic regenerative response. Thymidine kinase activity was increased eightfold in CD-treated rats receiving 6 microliters CCl4 per 100 g body weight, whereas in controls a similar induction of TK activity was produced by 100 microliters CCl4 per 100 g body weight. Therefore, the TK response in CD-treated rats receiving CCl4 is appropriate for the amount of damage produced, suggesting that CD does not inhibit the hepatic regenerative response to CCl4-induced injury. The effect of CD on hepatic repair was also examined in rats receiving a two-thirds partial hepatectomy. Pretreatment of animals with CD had no significant effect on the increase in TK activity produced 24 hours after partial hepatectomy. These results offer no support for the idea that CD impairs hepatic repair after either partial hepatectomy or CCl4 administration.

Induction of thymidine kinase activity by viruses with group B DNA genomes: Bovine cytomegalovirus (bovine herpesvirus 4)

The bovine herpesvirus type 4 (BHV-4) group has a slow replication cycle, a narrow host range, and cytopathogenic effects characteristic of cytomegaloviruses (CMV), but a Group B genome structure similar to that of lymphotropic Herpesvirus saimiri (HVS). Reference BHV-4 strain DN599 and BHV-4 strains N124 and FHV-2 induced in the cytosol fraction of thymidine kinase-negative (TK −) rabbit skin (RAB-BU) cell mutants a novel TK activity. The BHV-4-induced thymidine kinase (TK) differed from the principal cytosol TK of mock-infected cells in PAGE mobility ( R m) under non-denaturing conditions and in the capacity to efficiently substitute CTP for ATP as a phosphate donor. The BHV-4 thymidine phosphorylating activity could also be distinguished from many common herpesvirus-induced TKs because it lacked iododeoxycytidine phosphorylating activity. lododeoxyuridine, trifluorothymidine and bromovinyldeoxyuridine inhibited [ 3H]thymidine (0.01 mM) phosphorylation by the BHV-4 enzyme in a dose-dependent manner, but arabinosylthymine and 2′-fluoro-5-methyl-arabinosyluracil (FMAU) were poor inhibitors of [ 3H]thymidine phosphorylation, and acyclovir and (dihydroxy-2-pro-poxymethyl)guanine (DHPG) did not inhibit at all at 60 and 40 times the concentrations of [ 3H]thymidine, respectively.

Regulation of uridine kinase activity in BALB/C-3T3 cells by serum components.

After the stimulation of quiescent density-inhibited BALB/c-3T3 cells with fresh bovine calf serum, uridine kinase activity measured in cellular extracts increased between hours 3 and 6 of incubation and remained elevated through 12 h after stimulation. The addition of either partially purified platelet-derived growth factor (PDGF) or platelet-poor plasma (PPP) also caused increased uridine kinase activity by 6 h, but the increased activity was not maintained and the activity returned to the prestimulated level by 12 h. However, when PDGF and PPP were added in combination an increased level of uridine kinase activity was maintained in a manner similar to that seen after the addition of serum. The components of PPP eluted in the void volume from Sephadex G-50 chromatography did not induce uridine kinase activity when present alone, although they did act synergistically with PDGF to allow the maintenance of elevated levels or uridine kinase activity over the period from 6 to 12 h after stimulation. Thymidine kinase activity was not induced by the addition of either PDGF or PPP alone, although either serum or the combination of PDGF and PPP did produce and induction of thymidine kinase activity in late G1.

Enzyme induction in a temperature-sensitive cell cycle mutant of Chinese hamster fibroblasts.

A temperature-sensitive (ts) cell cycle mutant of Chinese hamster fibroblasts with a block in G1 was investigated. Attention was on the expression of the activity of three enzymes: ornithine decarboxylase (ODC) S-adenosylmethionine decarboxylase (SAMDC), and thymidine kinase (TK). ODC and SAMDC activities are normally induced in the middle of, or late in, the G1 phase, while TK activity starts to appear at the G1/S boundary. In the ts mutant released from serum starvation at the nonpermissive temperature (40.8 degrees C), we find no effect on the expression of SAMDC activity, a significantly reduced level of ODC activity compared to the control at the permissive temperature (34 degrees C), and no induction of TK activity. Results presented here and in a previous publication (Landy-Otsuka and Scheffler, '78) suggest that the decrease in ODC activity is due to an effect of the nonpermissive temperature on a post-transcriptional step, possibly a very rapid inactivation of the enzyme. The absence of TK activity, on the other hand, appears to be due to a block in transcription at the nonpermissive temperature.

Induction of endogenous virus and of thymidine kinase by bromodeoxyuridine in cell cultures transformed by Friend virus.

Thymidine kinase positive (TK(+)) N type cell lines that had been transformed by spleen focus-forming virus were established by transformation with NB tropic Friend virus complex. Thymidine kinase deficient (TK(-)) cell clones were isolated. Some of these cell clones release 1000- to 100,000-fold reduced amounts of Friend virus complex as compared to the TK(+) parental cell clone. TK(-) clones were grown in medium without BrdUrd. Some of these TK(-) clones can be induced to release endogenous helper virus and transforming spleen focus-forming virus on reexposure to 10(-6)-10(-4) M BrdUrd. The induced Friend virus complex is of N host range as expected with induced endogenous virus in N-type cells. Before the induction of the endogenous virus spleen focus-forming virus complex, an induction of thymidine kinase (ATP:thymidine 5'-phosphotransferase, EC 2.7.1.75) activity is observed. The latter is possibly a prerequisite for the induction of endogenous virus in TK(-) cells. Induction of thymidine kinase activity and of endogenous virus is transient and always correlated. The role of BrdUrd and another thymidine analogue, azidothymidine, in interfering with C-type virus release in virus positive cells is discussed. Azidothymidine is unable to induce endogenous virus. Induction of endogenous virus by BrdUrd and inhibition of virus release in virus positive cells is apparently not caused by the same mechanism.

Effects of 5-fluorouracil, actinomycin D and mitomycin C on the induction of thymidine kinase by vaccinia-infected L-cells

The effects of actinomycin D, 5-fluorouracil, 5-fluorodeoxyuridine and mitomycin C on the induction of thymidine kinase in vaccinia-infected mouse fibroblasts were investigated. When cells were treated with high levels of actinomycin D 1 hr prior to the addition of vaccinia, thymidine kinase induction was prevented. However, when cells were pretreated for 8 hr with 1 μ g/ml. actinomycin D 24 hr before the inoculation of virus, thymidine kinase induction was reduced to only about half the control level, although the incorporation of [ 3 H]uridine into RNA was inhibited by about 95%. 5-Fluorouracil prevented the incorporation of [ 3 H]uridine into the DNA-thymine of both non-infected and vaccinia-infected cells. Either in the absence or in the presence of 5-fluorouracil or 5-fluorodeoxyuridine, there was no detectable incorporation of [ 3 H]thymidine into the DNA of non-infected L-M (TK−) cells. Following infection of L-M (TK−) cells with vaccinia, however, 5-fluorouracil and 5-fluorodeoxyuridine enhanced the incorporation of [ 3 H]thymidine into DNA. 5-Fluorouracil (100 to 300 μ g/ml.) caused only moderate reductions in the induction of thymidine kinase activity in vaccinia-infected cells. Antecedent treatment of non-infected L-cells for 1 hr with 50 μ g/ml. mitomycin C or for 17 hr with 15 μ g/ml. mitomycin C, respectively, reduced the incorporation for 1 hr of [ 3 H]thymidine into DNA by 95% and 99%. However, high levels of mitomycin C did not prevent thymidine kinase induction in vaccinia-infected cells. When L-M (TK−) cells were pretreated for 16 hr with 15 μ ,g/ml. mitomycin C, infected with vaccinia virus, and further treated with 48·5 μ g/ml. mitomycin C at 1 hr after inoculation, thymidine kinase induction was only reduced by about 46%. The results support the concept that vaccinia DNA-dependent RNA synthesis is needed for thymidine kinase induction, but that the suppression of DNA synthesis or host-cell DNA-dependent RNA synthesis does not prevent enzyme induction.