Increase In Thymidine Kinase Activity Research Articles

Upregulation of thymidine kinase activity compensates for loss of thymidylate synthase activity in Mycoplasma pneumoniae

Thymidylate, an essential building block of DNA, is synthesized either from deoxyuridylate by thymidylate synthase (TS) or thymidine (dT) by thymidine kinase (TK). Thymidylate kinase (TMPK) phosphorylates dTMP to dTTP. Thymidine phosphorylase (TP) catalyses reversible phosphorolysis of dT. Using transposon mutagenesis M. pneumoniae TS gene (thyA/MPN320) was interrupted and requirement of these enzymes was studied. We found that TK activity and transcript levels and TP activity, but not TMPK or TS activity, are growth-phase-regulated, with induction at the exponential growth phase and a decline after the stationary phase. Inactivation of thyA results in upregulation of TK transcript and a 10-fold increase in TK activity, reduced TMPK level and it had no effect on TP activity. The level of [3H]-dT uptake and incorporation into DNA in the thyA mutant correlates with increases in TK activity, suggesting that dT uptake and metabolism is TK-dependent and that upregulation of TK activity in the thyA mutant compensates for the lack of ThyA activity. [3H]-dU uptake was low compared with dT, and incorporation of radioactivity into DNA in the thyA mutant indicates the presence of an alternative TS. Our results suggest that TK and TMPK are potential targets for the development of Mycoplasma-specific antibiotics.

Imaging with FLT-PET demonstrates that PF-477736, an inhibitor of CHK1 kinase, overcomes a cell cycle checkpoint induced by gemcitabine in PC-3 xenografts

3045 Background: The development of strategies to monitor the molecular and cellular response to novel agents that target the cell cycle is vital to provide proof of mechanism and biological activity of these compounds. The protein kinase CHK1 is activated following DNA damage in the S and G2-phases of the cell cycle and mediates cell cycle arrest. In vitro studies demonstrate that inhibition of CHK1 can overcome cell cycle arrest induced by DNA damage and enhance cytotoxic activity of DNA damaging agents. In vivo studies show that combining DNA damaging agents with a CHK1 inhibitor potentiates antitumor activity. We hypothesize that functional imaging with 18F-fluorine-L-thymidine (FLT), a PET-tracer where tumor uptake is maximal in the S and G2 phases of the cell cycle can be used to non-invasively monitor the induction and therapeutic inhibition of a cell cycle checkpoint in vivo. Methods: Nude mice harbouring PC-3 xenografts were treated with vehicle controls, gemcitabine, the CHK1-inhibitor PF-477736 or gemcitabine + PF-477736. FLT-PET scans were performed and tumors harvested for ex-vivo biomarkers to assess S-phase, M-phase and DNA-repair. Results: Gemcitabine induced a 8.3 ±0.8 fold increase in tumoral uptake of FLT at 21 hours that correlated with a 3.3 ±0.2-fold increase in thymidine kinase activity and S-phase arrest as demonstrated by BrdU incorporation and elevated expression of cyclin-A. Treatment with PF-477736 at 17 hours after gemcitabine abrogated the early FLT-flare at 21 hours by 82% (p<0.001). This was associated with both an increased fraction of cells in mitosis and G1-phase of the cell cycle as determined by phos-histone H3 and flow cytometry. Furthermore, the combination of gemcitabine and PF-477736 enhanced DNA damage as measured by phos-gamma-H2AX and significantly delayed tumor growth when compared to tumors treated with gemcitabine alone. Conclusion: These data clearly indicate that the CHK1-inhibitor PF-477736 can overcome the cell cycle checkpoint induced by gemcitabine and increase associated DNA damage in tumors in-vivo. The PET studies indicate that functional imaging with FLT-PET is a promising strategy to monitor responses to therapeutic agents that target cell cycle checkpoints. [Table: see text]

Increased Thymidine Kinase Activity in Human Thyroid Toxic Adenomas: Effects of Exposure to Epidermal Growth Factor In Vitro

The activity of thymidine kinase (TK—EC 2.7.1.21)—an enzyme functioning as a part of the pyrimidine salvage pathway of DNA synthesis—is closely related to growth processes. The aim of the study was to measure TK activity in homogenates of human thyroid tissue of the following types: non‐toxic nodular goiter (NTNG)—macroscopically unchanged tissue, non‐toxic adenoma (NTA), and toxic adenoma (TA) (obtained from patients, who—before the surgery—had been treated with thyrostatic drugs for thyrotoxicosis). Thyroid tissue was obtained from female patients subjected to subtotal thyroidectomy at the Department of Endocrine Surgery, Medical University of Łódź. Thyroid homogenates were incubated in the presence of epidermal growth factor (EGF), used in five different concentrations (0.1 ng/ml, 1 ng/ml, 10 ng/ml, 100 ng/ml, 1000 ng/ml). TK activity was estimated by chromatographic measurements of the amount of the main reaction product—deoxythymidine monophosphate. Results: 1) We did not observe any significant difference between TK activity in the homogenates of the thyroid tissue collected from NTNG and NTA; TK activity was clearly higher in the homogenates of adenomatous tissue, collected from the patients with TA; 2) EGF increased TK activity in the homogenates of the macroscopically unchanged tissue, collected—during surgery—from the patients with NTNG, as well as in homogenates of thyroid tissue from NTA; 3) In case of hyperactive thyroid tissue, obtained from TA, EGF tended to increase TK activity, however, without any statistical differences. Our results confirm TK increased activity in hyperactive thyroid tissue. At the same time, the obtained data suggest a certain role of EGF in goiter formation in humans.

Evidence that dl-propranolol increases thymidine kinase activity, cell mitosis, and β-adrenoceptors during rat liver regeneration

Background. Adrenoceptor stimulation has been described as an important factor in liver regeneration. We examined the effects of the β-adrenoceptor antagonist, dl-propranolol, during early steps of regeneration after partial hepatectomy in rat. Methods. Thymidine kinase (TK) activity, cell mitosis, and β-adrenoceptors were measured after a single intraperitoneal (i.p.) injection of dl-propranolol (10 mg/kg) immediately following 70% partial hepatectomy. Enzyme activity was measured as [ 3H] thymidine (dThd) monophosphate; mitotic index was determined after tissue staining, and β-adrenoceptors were quantified by saturation binding of [ 3H] dihydroalprenolol. Liver regeneration was followed for 3 days. Results. dl-Propranolol increased TK activity as early as 12 h postsurgery, after which it leveled off at 24 h and declined at later times; mitotic index increased from 12 to 48 h after injection and β-adrenoceptors augmented at 24 h compared to partial hepatectomy alone. Conclusions. dl-Propranolol up-regulated β-adrenoceptors early after partial hepatectomy with an increase in TK activity and augmented hepatocyte mitosis. These data may suggest that dl-propranolol modulates, probably via β-adrenoceptor blockade, early steps of liver regeneration.

Positron emission tomography imaging of cardiac reporter gene expression in living rats.

Imaging reporter gene expression is useful for noninvasive monitoring of gene therapy. In this study, we imaged cardiac reporter gene expression in living rats using micro positron emission tomography (microPET). Rats (n=10) underwent intramyocardial injection with 1x10(9) pfu of adenovirus carrying cytomegalovirus promoter-driving herpes simplex virus type 1 mutant thymidine kinase (Ad-CMV-HSV1-sr39tk) as PET reporter gene. Control rats (n=4) received 1x10(9) pfu of adenovirus carrying cytomegalovirus promoter-driving firefly luciferase (Ad-CMV-Fluc). On days 2 to 4, microPET images were obtained after a tail vein injection of nitrogen-13 ammonia ([13N]-NH3) as myocardial perfusion tracer, followed by 9-(4-[18F]-fluoro-3 hydroxymethylbutyl) guanine ([18F]-FHBG) to assess HSV1-sr39tk expression. After imaging, hearts were removed for ex vivo [18F] gamma counting and thymidine kinase enzyme assay. Results show homogenous myocardial distribution of [13N]-NH3 on all microPET images. Rats injected with Ad-CMV-HSV1-sr39tk have significant [18F]-FHBG uptake in the anterolateral wall compared with background signal in controls. Gamma counting shows 20.0+/-4.4-fold increase of radioactivity, whereas enzyme assay shows 22.1+/-6.1-fold increase of thymidine kinase activity in Ad-CMV-HSV1-sr39tk injected rats (P<0.05). Successful imaging of cardiac HSV1-sr39tk expression was performed in living rats with microPET. The presence of [18F]-FHBG uptake is confirmed by gamma counting and the presence of HSV1-sr39TK protein by thymidine kinase enzyme assay. Cardiac reporter gene imaging by PET may eventually be applied toward human gene therapy studies.

Induction of thymidine kinase in mouse skin exposed to 12-O-tetradecanoylphorbol-13-acetate

Topical application of 12-O-tetradecanoylphorbol-13-acetate (TPA) exhibited an increase in the epidermal thymidine kinase (TK) activity in a dose dependent manner. Maximum induction of TK was observed at a TPA concentration of 2.5 μg per animal. The induction of TK by TPA appeared to be a function of time with the maximum TK induction between 4 and 16 h after TPA application. Repeated applications of TPA every 24 h did not show any cumulative effect rather TK activity appeared to be normal after two applications. However, repeated applications of TPA at an interval of 48 h exhibited increased TK activity even after 16 applications. Cycloheximide and actinomycin D, the inhibitors of protein and RNA synthesis, inhibited the TPA induced activation of TK. Our results demonstrated that TPA induced the TK activity may be, by increasing de novo synthesis of enzyme protein and this induction might lead to increased de novo DNA synthesis after TPA application. DMBA was used as a reference compound. As far as the authors are aware, this is the first report on TK induction by topical application of TPA.

Impact of 9-(2-phosphonylmethoxyethyl)adenine on (deoxy)ribonucleotide metabolism and nucleic acid synthesis in tumor cells

Following exposure to 9-(2-phosphonylmethoxyethyl)adenine (an inhibitor of the cellular DNA polymerases α, δ and ϵ), human erythroleukemia K562, human T-lymphoid CEM and murine leukemia L1210 cells markedly accumulated in the S phase of the cell cycle. In contrast to DNA replication, RNA synthesis (transcription) and protein synthesis (mRNA translation) were not affected by 9-(2-phosphonylmethoxyethyl)adenine. The ribonucleoside triphosphate pools were slightly elevated, while the intracellular levels of all four deoxyribonucleoside triphosphates were 1.5–4-fold increased in 9-(2-phosphonylmethoxyethyl)adenine-treated K562, CEM and L1210 cells. The effect of 9-(2-phosphonylmethoxyethyl)adenine on de novo (thymidylate synthase-mediated) and salvage (thymidine kinase-mediated) dTTP synthesis was investigated using radiolabelled nucleoside precursors. The amount of thymidylate synthase-derived dTTP in the acid soluble pool was 2–4-fold higher in PMEA-treated than in untreated K562 cells, which is in accord with the 3–4-fold expansion of the global dTTP level in the presence of 9-(2-phosphonylmethoxyethyl)adenine. Strikingly, 2-derived dTTP accumulated to a much higher extent (i.e. 16–40-fold) in the soluble dTTP pool upon 9-(2-phosphonylmethoxyethyl)adenine treatment. In keeping with this finding, a markedly increased thymidine kinase activity could be demonstrated in extracts of 9-(2-phosphonylmethoxyethyl)adenine-treated K562 cell cultures. Also, in the presence of 200 μM 9-(2-phosphonylmethoxyethyl)adenine, 14-fold less thymidylate synthase-derived but only 3-fold less thymidine kinase-derived dTTP was incorporated into the DNA of the K562 cells. These data show that thymidine incorporation may be inappropriate as a cell proliferation marker in the presence of DNA synthesis inhibitors such as 9-(2-phosphonylmethoxyethyl)adenine. Our findings indicate that 9-(2-phosphonylmethoxyethyl)adenine causes a peculiar pattern of (deoxy)ribonucleotide metabolism deregulation in drug-treated tumor cells, as a result of the metabolic block imposed by the drug on the S phase of the cell cycle.

Differential effect of GM-CSF pretreatment on intracellular Ara-C metabolism in normal bone marrow mononuclear cells vs acute myeloid leukemia (AML) blasts.

The effect of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) on the intracellular metabolism of cytosine arabinoside (Ara-C) was comparatively analyzed in normal bone marrow mononuclear cells (NBMMC) from eight healthy volunteers and in leukemic blasts from 50 patients with acute myeloid leukemia (AML). Pretreatment with GM-CSF (100 U/ml) for 48 h resulted in a significant enhancement of DNA synthesis in both cell types: 21 of 35 AML specimens were found to be responsive to GM-CSF as defined by an increase of 3H-TdR incorporation into the DNA > 1.5-fold while NBMMC from normal donors were responsive in all cases. In GM-CSF responsive AML blasts, overall DNA polymerase and DNA polymerase alpha activity increased from a median of 84.4 to 96.1 and from 3.45 to 5.2 pmol/min x mg as compared to a median of 96.7 to 189.9 and 1.2 to 2.2 pmol/min x mg in NBMMC (P < 0.05). Median Ara-C-mediated inhibition of DNA synthesis was significantly more effective in AML blasts as compared to NBMMC (76.5 vs 55.0% at 0.05 microM and 99.0 vs 96.0% at 5.0 microM Ara-C, P < 0.01) but was not influenced by GM-CSF pretreatment. Similarly, intracellular Ara-CTP levels were higher in AML blasts as compared to NBMMC (median of 46.9 vs 18.7 at 1 microM, 167.8 vs 48.0 at 10 microM and 337.5 vs 59.5 ng/10(7) cells at 100 microM extracellular Ara-C, P < 0.01) but showed no enhancement in the presence of GM-CSF. Median deoxycytidine (DCK) and thymidine kinase (TK) activity were only slightly increased in AML blasts after GM-CSF priming. In contrast, NBMMC revealed a significant increase in TK activity after GM-CSF pretreatment (from a median of 1.9 to 3.6 pmol/min x mg, P = 0.039). At low; intermediate and high extracellular Ara-C concentrations GM-CSF pretreatment resulted in a significant enhancement of the 3H-Ara-C incorporation into the DNA in both GM-CSF responsive AML blasts and NBMMC (median of 1.3 to 2.1- and 1.4 to 1.6-fold, P < 0.05). GM-CSF non-responsive AML blasts showed no change in 3H-Ara-C incorporation into the DNA in response to GM-CSF at low Ara-C concentrations but significant increases at intermediate and high extracellular Ara-C concentrations (median increases of 1.63-fold at 1.06 microM with P = 0.01 and 1.37-fold at 10 microM extracellular Ara-C with P = 0.0+005). NBMMC revealed significantly lower GM-CSF-induced increases of the 3H-Ara-C incorporation into the DNA as compared to the effect of GM-CSF priming on DNA synthesis (median increases of 1.4 to 1.7-fold vs 2.6-fold, P < 0.05). These data reveal a different effect of GM-CSF priming on the metabolism of Ara-C in normal vs leukemic cells which may cause a preferential increase in the antileukemic cytotoxicity of Ara-C in the presence of GM-CSF.

Promotion of proliferation of murine BALB/C3T3 fibroblasts mediated by nitric oxide at lower concentrations.

This study showed that nitric oxide (NO)-generating S-nitrosothiols, S-nitroso-N-acetylpenicillamine (SNAP) and S-nitrosoglutathione (GSNO), increased proliferation of BALB/c 3T3 (clone A31-1-1) fibroblasts in vitro. Treatment with SNAP at a relatively low concentration (0.005-0.02 mM) induced an increase in cell number compared to control. SNAP (0.005-0.02 mM) and GSNO (0.025-0.05 mM) both showed an increase of [3H]thymidine incorporation in exponentially growing cells in a dose-dependent manner. The maximal effect was observed at about 40 and 90% above control at 0.02 mM and 0.05 mM, respectively. The increase induced by 0.02 mM SNAP was abolished by the addition of 0.01 mM oxyhemoglobin, a scavenger of NO. [3H]Thymidine incorporation in stationary cells was also increased by SNAP. In addition, 0.02 mM SNAP produced a 1.8-3.2-fold increase of thymidine kinase activity in exponentially growing cells.

Control of thymidine kinase during liver regeneration after partial hepatectomy

To elucidate the molecular basis for the periodic change of thymidine kinase (TK) activity, the expressions of TK protein and TK mRNA were examined during liver regeneration. TK protein level, quantified by immunoblotting assay using the polyclonal antiserum against rat TK polypeptide produced in Escherichia coli, increased 13-fold compared with the normal at 24 h after partial hepatectomy. This was closely correlated with a 11-fold increase in TK activity. Northern blot analysis showed that the partial hepatectomy caused 12 and 8-fold increase in 2.6 kb and 1.1 kb TK mRNA at 24 h after surgery, respectively. During next 12 h the levels of both TK mRNA species reduced to 5-fold of the normal base level. This reduction was coupled with a similar decrease in the activity as well as in the amount of TK protein. The TK mRNA levels were strictly proportional to the levels of TK activity and TK protein at 48 h and 72 h after partial hepatectomy. These results demonstrate that the change in TK activity is controlled at the mRNA level during liver regeneration. The injection of alpha-adrenoceptor antagonist, phenoxybenzamine, calcium channel blocker, nifedipine or calmodulin inhibitor, trifluoperazine was also found to inhibit TK activity by the repression of its mRNA level.

Stem Cell Factor Enhances Interleukin-3-Dependent Induction of 68-kD Calmodulin-Binding Protein and Thymidine Kinase Activity in NFS-60 Cells

Stem cell factor (SCF) is known to act synergistically with other hematopoietic factors in increasing the colony formation of hematopoietic progenitor cells. We have shown that interleukin-3 (IL-3)-dependent proliferation of NFS-60 cells is associated with the induction of a specific calmodulin-binding protein of about 68 kD (CaM-BP68). To evaluate the relationship between proliferative stimulation and the induction of CaM-BP68 by cytokines, we examined whether the increased proliferative potential of NFS-60 cells in response to SCF is reflected in an increased induction of the CaM-BP68. We observed that SCF alone has a limited effect on proliferative stimulation and on the induction of CaM-BP68 in factor-deprived NFS-60 cells. However, when combined with IL-3, granulocyte colony-stimulating factor (G-CSF), or IL-6, it caused a significant increase in cytokine-dependent proliferative stimulation, as well as in the induction of CaM-BP68. Furthermore, an increase in IL-3-dependent induction of CaM-BP68 in the presence of SCF coincided with a corresponding increase in thymidine kinase activity, whose expression is linked to G1/S transition of the cells. At low concentrations SCF caused a synergistic increase in IL-3-dependent induction of both CaM-BP68 and thymidine kinase activity. In contrast to the changes in CaM-BP68 and thymidine kinase activity, no significant changes in DNA polymerase alpha were observed in factor-deprived NFS-60 cells in response to IL-3 and/or SCF. These observations suggest an increased expression of CaM-BP68 and thymidine kinase are associated with the synergistic effect of SCF on factor-dependent proliferation of hematopoietic progenitor cells.

Analysis of thymidine kinase gene expression in preimplantation mouse embryos.

Thymidine kinase (TK) activity was examined during the development of preimplantation mouse embryos. TK activity was increased approximately 20-fold from day 2 embryos (2-cell) to day 5 embryos (late blastocyst). TK activity did not change along with the progression into S-phase of the first and the second cell cycles but increased sharply at S-phase of the third cell cycle. Analysis of TK mRNA with a reverse transcription-polymerase chain reaction (RT-PCR) method showed that the level of TK mRNA was low in ovulated eggs and 1-cell embryos and was hardly detectable in day 2 embryos (2-cell), but sharply increased in day 3 embryos (mixture of 5- to 8-cell and morula). The functional role of 5'-flanking sequence of TK gene was also investigated in preimplantation embryos after microinjection with the DNA construct of 5'-flanking sequence of TK (2.4 kb) linked to bacterial lacZ gene (TK2.5lacZ) into the pronucleus of 1-cell and subsequently by histochemical staining with X-gal. beta-Galactosidase activity was first detected in day 3 embryos (8-cell), and 30% of embryos were stained with X-gal in day 4 and day 5 embryos, respectively. These results show that an increase in TK activity occurred after 2-cell stage, and this increase was primarily due to the embryonic activation of TK gene expression. Also, it appears that the 5'-flanking sequence of TK may directly regulate the TK gene expression at the transcriptional level during preimplantation murine development.

Herpes thymidine kinase mutants with altered catalytic efficiencies obtained by random sequence selection

We have obtained 190 active Herpes simplex virus type 1 thymidine kinase mutants by substituting a 33 nucleotide sequence with 20% degeneracy for a portion of the nucleotide sequence that encodes the putative thymidine binding site [K.M. Munir, D.C. French, D.K. Dube and L.A. Loeb (1992) J. Biol. Chem., 167, 6584-6589]. In order to classify these mutants with respect to thymidine kinase activity we determined the ability of Escherichia coli harboring these mutants to form colonies in the presence of varying concentrations of thymidine. Escherichia coli harboring one of the mutant enzymes was able to form colonies at a concentration of thymidine lower than did the wild type. It was able to phosphorylate thymidine more rapidly than the wild type both in vivo and in vitro. The increased thymidine kinase activity was manifested by (i) a 42% enhanced uptake of [methyl-3H]thymidine into E. coli, (ii) a 2.4 times higher rate of [methyl-3H]thymidine incorporation into acid-insoluble material and (iii) a 5-fold increase in the kcat of the purified enzyme compared to the wild type. Herpes thymidine kinase purified from other mutants that formed colonies at higher thymidine concentrations than that of the wild type exhibited a decrease in kcat. The kcat of one of these mutant thymidine kinases was 10(-4) of that of the wild type enzyme. This study demonstrates that a spectrum of mutant enzymes with different catalytic properties can be obtained by selection from a plasmid with random sequence substitutions and this can be done in the absence of rational protein design.

Effects of bone marrow stimulatory cytokines on human immunodeficiency virus replication and the antiviral activity of dideoxynucleosides in cultures of monocyte/macrophages

Cells of the monocyte lineage are important targets for the replication of human immunodeficiency virus (HIV). Our group and others have previously shown that granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulates HIV replication in monocyte/macrophages, but that it also enhances the anti-HIV activity of 2′,3′-dideoxy-3′- azidothymidine (AZT). In the present study, we have explored the effects of other bone marrow stimulatory cytokines on the replication of HIV and on the anti-HIV activity of certain dideoxynucleosides in human peripheral blood monocyte/macrophages (M/M). Like GM-CSF, macrophage CSF (M-CSF) enhanced HIV replication in M/M. In contrast, granulocyte CSF (G-CSF) and erythropoietin (Epo) had no such effects. The anti-HIV activity of zidovudine (AZT) was increased in M/M exposed to GM-CSF. In contrast, the anti-HIV activity of AZT was unchanged in M/M exposed to M-CSF, and the activities of 2′,3′-dideoxycytidine (ddC) and 2′,3′-dideoxyinosine (ddl) were unchanged or slightly diminished in M/M stimulated with GM-CSF or M-CSF. These differential activities of AZT and ddC were paralleled by differential effects of the cytokines on the anabolism of these drugs to their active 5′-triphosphate moieties. GM-CSF increased the levels of AZT-5′-triphosphate (at least in part through an increase in thymidine kinase activity) and overall induced an increase in the ratio of AZT-5′-triphosphate/thymidine-5′- triphosphate. In contrast, M-CSF-induced increases in AZT-5′- triphosphate were roughly matched by increases in thymidine-5′- triphosphate. Also, GM-CSF- or M-CSF-induced increases in the levels of ddC-5′-triphosphate were associated with parallel increases in the levels of deoxycytidine-5′-triphosphate (the physiologic nucleoside that competes at the level of reverse transcriptase), so that there was relatively little net change in the ddC-5′-triphosphate/deoxycytidine- 5′-triphosphate ratio. Thus, bone marrow stimulatory cytokines may have a variety of effects on HIV replication and on the activity and metabolism of dideoxynucleosides in M/M.

Effects of Bone Marrow Stimulatory Cytokines on Human Immunodeficiency Virus Replication and the Antiviral Activity of Dideoxynucleosides in Cultures of Monocyte/Macrophages

AbstractCells of the monocyte lineage are important targets for the replication of human immunodeficiency virus (HIV). Our group and others have previously shown that granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulates HIV replication in monocyte/macrophages, but that it also enhances the anti-HIV activity of 2′,3′-dideoxy-3′- azidothymidine (AZT). In the present study, we have explored the effects of other bone marrow stimulatory cytokines on the replication of HIV and on the anti-HIV activity of certain dideoxynucleosides in human peripheral blood monocyte/macrophages (M/M). Like GM-CSF, macrophage CSF (M-CSF) enhanced HIV replication in M/M. In contrast, granulocyte CSF (G-CSF) and erythropoietin (Epo) had no such effects. The anti-HIV activity of zidovudine (AZT) was increased in M/M exposed to GM-CSF. In contrast, the anti-HIV activity of AZT was unchanged in M/M exposed to M-CSF, and the activities of 2′,3′-dideoxycytidine (ddC) and 2′,3′-dideoxyinosine (ddl) were unchanged or slightly diminished in M/M stimulated with GM-CSF or M-CSF. These differential activities of AZT and ddC were paralleled by differential effects of the cytokines on the anabolism of these drugs to their active 5′-triphosphate moieties. GM-CSF increased the levels of AZT-5′-triphosphate (at least in part through an increase in thymidine kinase activity) and overall induced an increase in the ratio of AZT-5′-triphosphate/thymidine-5′- triphosphate. In contrast, M-CSF-induced increases in AZT-5′- triphosphate were roughly matched by increases in thymidine-5′- triphosphate. Also, GM-CSF- or M-CSF-induced increases in the levels of ddC-5′-triphosphate were associated with parallel increases in the levels of deoxycytidine-5′-triphosphate (the physiologic nucleoside that competes at the level of reverse transcriptase), so that there was relatively little net change in the ddC-5′-triphosphate/deoxycytidine- 5′-triphosphate ratio. Thus, bone marrow stimulatory cytokines may have a variety of effects on HIV replication and on the activity and metabolism of dideoxynucleosides in M/M.

Liver regeneration after partial hepatectomy in rats with defective bilirubin conjugation or biliary excretion

The role of conjugated bilirubin in liver regeneration after partial hepatectomy (PH) was studied in Gunn rats, in transport-mutant (TR-) rats, and in rats with extrahepatic biliary obstruction. Ornithine decarboxylase (ODC) and thymidine kinase (TK) activities in liver homogenates and immunohistochemistry of in vivo bromodeoxyuridine (BrdU) incorporation in hepatic DNA were followed as regeneration parameters at 24 and 48 hr after PH. The results relative to TK activity and BrdU incorporation were consistent with significantly delayed hepatic DNA synthesis in Gunn rats in comparison to control Wistar and TR- rats. This delay in DNA synthesis was not reflected in the hepatic ODC activity. After one week of complete common bile duct obstruction (CBO), an increased TK activity and BrdU incorporation was seen. PH following CBO resulted in a further increase in ODC activity and BrdU incorporation. TK activity did not change, however. These data relative to the regulation of hepatic DNA synthesis after PH in Gunn rats and in rats with extrahepatic biliary obstruction suggest a possible stimulatory role for conjugated bilirubin in hepatic regeneration; however, the normal hepatic DNA synthesis in TR- rats studied before PH and the subnormal DNA synthesis seen 24 hr after PH in TR- rats and in rats with CBO indicate that conjugated bilirubin does not stimulate hepatic DNA synthesis.

Effects of granulocyte-macrophage colony-stimulating factor on 3′-azido-3′-deoxythymidine uptake, phosphorylation and nucleotide retention in human U-937 cells

Previous studies have demonstrated that granulocyte-macrophage colony-stimulating factor (GM-CSF) both increases and decreases levels of 3′-azido-3′-deoxythymidine (AZT) nucleotides in certain human myeloid cells. The present studies have examined the effects of GM-CSF on AZT metabolism in U-937 cells. The results demonstrate that GM-CSF stimulated AZT nucleotide formation in these cells. This stimulation was detectable during concurrent exposure to GM-CSF and AZT or as a result of pretreatment with GM-CSF. The GM-CSF-induced enhancement in AZT nucleotide formation was associated with a 4-fold increase in AZT uptake. The finding that uptake of AZT into U-937 cells was only partially sensitive to 6-[(4-nitrobenzyl)thio]-9-β- d-ribofuranosylpurine (NBMPR) suggested a process primarily involving nonfacilitated diffusion. The results also demonstrate that treatment of U-937 cells with GM-CSF was associated with nearly a 2-fold increase in thymidine kinase activity. Moreover, the findings indicate that retention of AZT-MP and AZP-TP was prolonged significantly (P < 0.05 and P < 0.01 respectively) in association with GM-CSF treatment. Taken together, these results suggest that GM-CSF enhances the formation of AZT nucleotides by increasing AZT uptake and phosphorylation, as well as increasing retention of phosphorylated derivatives.

Potentiation of Azidothymidine Cytotoxicity in Cisplatin-resistant Human Ovarian Carcinoma Cells

The combination of cisplatin and AZT was synergistic in a subline (A2780DDP) of human ovarian carcinoma cells resistant to cisplatin, in contrast to the parental carcinoma cell line, A2780S. A2780DDP cells have elevated levels of enzymes necessary for DNA synthesis and repair. If A2780DDP cells respond to cisplatin with an increase in thymidine kinase activity, then AZT may chain terminate newly synthesized DNA. To test this hypothesis, a dual label [( 14C]-thymidine/[3H]-AZT) experiment was designed. A2780 cells were first incubated with [14C]-thymidine to label DNA and measure DNA degradation in response to cisplatin. These [14C]-thymidine labeled A2780 cells were then incubated for short intervals with [3H]-AZT to measure chain termination after cisplatin addition. A2780S cells responded to cisplatin with a modest increase in thymidine turnover and an increase in AZT incorporation. In contrast, A2780DDP cells initially responded to cisplatin treatment with a significant increase in thymidine turnover and a corresponding increase in [3H]-AZT incorporation. This was concomitantly associated with an increase in thymidine kinase mRNA within 2 hr after cisplatin treatment. Thus, the A2780DDP cells had the ability to rapidly turnover DNA, a property effectively exploited by the deoxythymidine analogue, AZT, utilizing the enhanced enzymes of the DNA synthesis and repair complex in A2780DDP cells.

The replication of viral and cellular DNA in human herpesvirus 6-infected cells

Human herpesvirus 6 (HHV-6) is a newly identified lymphotropic herpesvirus. We have analyzed viral and host DNA replication in peripheral blood lymphocytes infected in the absence of drugs or infected in the presence of phosphonoacetic acid (PAA) or acyclovir (ACV). The results revealed the following: (i) Infection with HHV-6 resulted in the shutoff of host DNA replication. (ii) PAA at concentrations of 100 and 300 μg/ml significantly reduced virus replication. The drug inhibited viral DNA replication, whereas host cell DNA replication was not affected. This strongly suggests that HHV-6 encodes a PAA sensitive viral DNA polymerase. (iii) ACV at 20 μ M did not interfere with virus production and virus spread. ACV at 100 μ M only partly interfered with virus replication, whereas at 400 μ M the block was more complete. Viral DNA replication was not affected by ACV at 20 μ M. However, approximately 60 and 85% inhibition in viral DNA replication was observed in the presence of 100 and 400 μ M of ACV. (iv) Assays for viral thymidine kinase (TK) revealed no significant increase in TK activity, whereas increased TK activity was noted following infection of the same peripheral blood lymphocytes with herpes simplex virus. Thus, either HHV-6 does not encode a tk enzyme which can phosphorylate ACV or the inefficient block may reflect lower sensitivity of the HHV-6 DNA polymerase to the drug.

Control of ribonucleotide reductase in heat- and cold-sensitive mammalian cell-cycle mutants

Two heat-sensitive (reversibly arrested in G1 phase at 39.5δC, multiplying at 33°C) and two cold-sensitive (reversibly arrested in G1 phase at 33°C, multiplying at 39.5°C) cell-cycle mutants of the P-815-X2 murine mastocytoma line were tested for ribonucleotide reductase activity, using cells made permeable to necleotides. After trasfer of the heat-sensitive mutant cells to 39.5° C, ribonucleotide reductase activity, similar to thymidine kinase (Schneider, E., Müller, B. and Schindler, R. (1985) Biochim. Biophys. Acta 741, 77–85), but unlike DNA polymerase α (Schneider, E., Müller, B. and Schindler, R. (1985) Biochim. Biophys. Acta 825, 375–383), decreased rapidly and in parallel with numbers of cells in S phase, whereas in the cold-sensitive mutant cells brought to 33° C, ribonucleotide reductase activity decreased approx. 8 h later than numbers of DNA-synthesizing cells. When arrested heat- or cold-sensitive mutant cells were returned to the permissive temperature, ribonucleotide reductase activities, similar to DNA polymerase α and to thymidine kinase in heat-sensitive mutants, increased essentially in parallel with reentry of cells into S phase, whereas the increase in thymidine kinase activity in the cold-sensitive mutants was previously shown to occur approx. one cell-cycle time later. This indicates that ribonucleotide reductase and thymidine kinase are coordinately expressed in the heat-sensitive, but independently regulated in the cold-sensitive mutants.