Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.1.21 (thymidine kinase)
7,561 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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.
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PMID:Effects of bone marrow stimulatory cytokines on human immunodeficiency virus replication and the antiviral activity of dideoxynucleosides in cultures of monocyte/macrophages. 137 54

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.
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PMID:Induction of thymidine kinase activity and clonal growth of certain leukemic cell lines by a granulocyte-derived factor. 235 May 81

We constructed human macrophage-monocyte hybridomas between a thymidine kinase-deficient human macrophage-like cell line, designated as TAM-2, and human peripheral blood monocytes in order for the study of cytokines from human monocytes. The hybrid and macrophage-monocyte nature of the growing cells was confirmed by the following facts: 1) All of the hybridomas established possess TK activity, whereas the TAM-2 cells are TK negative. 2) Most but not all of the hybridomas express the MaG-1 Ag which was shown to be a human macrophage-granulocyte specific Ag, but not T- and B-specific Ag. In the assay for cytokine, a few of the hybridomas produced a novel tumor-killing factor (TKF) after stimulation with PMA, polypeptone, and retinoic acid. Chemical nature of the TKF from the 3-63 hybrid clone was characterized and compared to those of well-known TNF and lymphotoxin. The TKF from a hybridoma was basic protein and had binding capacity to Con A-Sepharose, whereas TNF had an opposite nature. Moreover, TKF activity was not neutralized by both a murine monoclonal antibody against human TNF and rabbit antisera against human lymphotoxin. Thus, these results strongly indicate that the TKF is a novel TKF produced by human monocytic cells.
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PMID:Demonstration of a novel tumor-killing factor secreted from human macrophage-monocyte hybridomas. 318 86

Induction of granulocyte maturation in HL-60 leukemic cells by DMSO (1.2%) or RA (1 microM) is accompanied by a 50-60% decrease in cellular thymidine kinase activity. Similarly, the differentiation of HL-60 cells into monocyte-macrophage phenotype by the addition of PMA is paralleled by a 60-80% suppression of thymidine kinase specific activity. Measurement of thymidine kinase kinetic parameters shows that the Vmax decreases from 0.7 pmol/min in control cells to 0.43 pmol/min in PMA-treated cells and to 0.38 pmol/min in RA-treated cells. The Km of the enzyme is not affected by either inducing agent and remains at 2.1 microM. Studies with PMA analogs suggest that thymidine kinase modulation is coupled to HL-60 differentiation.
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PMID:Changes in thymidine kinase activity during differentiation of HL-60 leukemic cells. 357 10

Recombinant adeno-associated virus 2 (AAV) virions were constructed containing a gene for resistance to neomycin (neoR), under the control of either the herpesvirus thymidine kinase (TK) gene promoter (vTK-Neo), or the human parvovirus B19 p6 promoter (vB19-Neo), as well as those containing an upstream erythroid cell-specific enhancer (HS-2) from the locus control region of the human beta-globin gene cluster (vHS2-TK-Neo; vHS2-B19-Neo). These recombinant virions were used to infect either low density or highly enriched populations of CD34+ cells isolated from human umbilical cord blood. In clonogenic assays initiated with cells infected with the different recombinant AAV-Neo virions, equivalent high frequency transduction of the neoR gene into slow-cycling multipotential, erythroid, and granulocyte/macrophage (GM) progenitor cells, including those with high proliferative potential, was obtained without prestimulation with growth factors, indicating that these immature and mature hematopoietic progenitor cells were susceptible to infection by the recombinant AAV virions. Successful transduction did not require and was not enhanced by prestimulation of these cell populations with cytokines. The functional activity of the transduced neo gene was evident by the development of resistance to the drug G418, a neomycin analogue. Individual high and low proliferative colony-forming unit (CFU)-GM, burst-forming unit-erythroid, and CFU-granulocyte erythroid macrophage megakaryocyte colonies from mock-infected, or the recombinant virus-infected cultures were subjected to polymerase chain reaction analysis using a neo-specific synthetic oligonucleotide primer pair. A 276-bp DNA fragment that hybridized with a neo-specific DNA probe on Southern blots was only detected in those colonies cloned from the recombinant virus-infected cells, indicating stable integration of the transduced neo gene. These studies suggest that parvovirus-based vectors may prove to be a useful alternative to the more commonly used retroviral vectors for high efficiency gene transfer into slow or noncycling primitive hematopoietic progenitor cells, without the need for growth factor stimulation, which could potentially lead to differentiation of these cells before transplantation.
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PMID:Adeno-associated virus 2-mediated high efficiency gene transfer into immature and mature subsets of hematopoietic progenitor cells in human umbilical cord blood. 751 1

We have previously described the construction of a bicistronic retroviral vector using the picornavirus internal ribosome entry site (IRES), which allows two genes expression simultaneously from a single transcript. This vector transcribes RNA efficiently; however, in some cases the levels of protein production are low. In this report, we further modified the bicistronic vector by abolishing the functional viral gag initiation codon that is retained in the vector at 5' to the first initiation codon of transduced gene. Five different genes, human interleukin 2 (hIL-2), human interleukin 4 (hIL-4), human granulocyte macrophage stimulating factor (hGM-CSF), herpes simplex virus thymidine kinase (HSV-tk) gene, and hepatitis C virus (HCV) core gene (C190), were tested on this modified vector for gene transfer and expression. Our results demonstrated that the new bicistronic vector greatly increased the protein levels when compared with the original one. As the RNA levels and splicing patterns from these two vectors remained similar, the improvement was most likely resulted from the increased translational efficiency.
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PMID:Improved gene expression by a modified bicistronic retroviral vector. 757 63

We previously demonstrated stable integration of a transduced thymidine kinase (TK)-neo gene into immature and replatable stem and progenitor cells, as assessed by the presence of the gene in second-generation colonies. To evaluate whether this integration was still present in third- and fourth-generation colonies, nonadherent low-density T-lymphocyte-depleted (NALT-) cells from human umbilical cord blood were prestimulated with recombinant human (rhu) erythropoietin (Epo), steel factor (SLF), interleukin-3 (IL-3), granulocyte-macrophage (GM) colony-stimulating factor (CSF), and granulocyte (G)-CSF. Prestimulated NALT- cells were incubated with retroviral-containing supernatant obtained from TK-neo vector-producing cells, washed, and assayed for colony formation in the presence of Epo, SLF, IL-3, GM-CSF, and G-CSF -/+ G418. The results confirmed that the TK-neo gene could be efficiently introduced into hematopoietic progenitor cells without stromal cells as a source of virus. As previously reported, proviral integration was detected in primary G418R-colonies, and in second-generation replated colonies derived from G418R granulocyte erythroid macrophage megakaryocyte colony-forming units and high-proliferative potential colony-forming cells (HPP-CFCs). Moreover, we now document that proviral integration was apparent in cells from colonies derived from third- and fourth-generation replated HPP-CFC, suggesting a high degree of stable integration of the transduced gene.
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PMID:Stable integration of retrovirally transduced genes into human umbilical cord blood high-proliferative potential colony-forming cells (HPP-CFC) as assessed after multiple HPP-CFC colony replatings in vitro. 774 19

Recombinant human adeno-associated virus 2 (AAV) virions were constructed containing a gene for resistance to neomycin (neoR), under the control of either the herpesvirus thymidine kinase (TK) promoter (vTK-Neo), the murine colony-stimulating factor-1 (CSF-1) promoter (vCSF1-Neo) or the CSF-1 promoter plus an upstream human erythroid cell-specific enhancer, HS-2 (vHS2-CSF1-Neo). Recombinant virions were used to infect low-density murine primary bone marrow cells. In hematopoietic progenitor cell assays initiated with cells infected with these recombinant virions, myeloid as well as erythroid cell colonies resistant to the drug G418, a neomycin analogue, were readily obtained, indicating that the murine hematopoietic progenitor cells were susceptible to infection by the recombinant AAV virions and that the transduced neo gene was functionally active in these cells. Whereas only approximately 10% of the colony-forming unit-granulocyte/macrophage (CFU-GM) colonies cloned from mock-infected cells survived the G418-selection at a final active concentration of 250 micrograms/mL of the drug, the extent of the CFU-GM colony formation initiated with the recombinant AAV-Neo virions was as follows: 15% with vTK-Neo, 22% with vCSF1-Neo and 49% with vHS2-CSF1-Neo. In addition, only 14% of the burst-forming unit-erythroid (BFU-E) colonies from mock-infected cells were resistant to G418, whereas 82% of the BFU-E colonies initiated with cells infected with vHS2-CSF1-Neo virions survived the drug selection, suggesting that a human erythroid cell-specific enhancer was able to potentiate expression of the transduced neoR gene from a murine promoter. Individual CFU-GM and BFU-E colonies from mock-infected or recombinant AAV-Neo virus-infected cultures were subjected to polymerase chain reaction (PCR) analysis using a neo-specific synthetic oligonucleotide primer-pair. A 276 bp DNA fragment that hybridized with a neo-specific DNA probe on Southern blots was detected only in colonies cloned from the recombinant virus-infected cells, indicating stable integration of the transduced neo gene. These studies suggest the feasibility of using the AAV-based vector system in an animal model as a prelude to evaluating its safety and efficacy in human gene therapy.
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PMID:Adeno-associated virus 2-mediated gene transfer in murine hematopoietic progenitor cells. 839 71

The effectiveness of combination therapy using a suicide gene and cytokine genes for the treatment of metastatic colon carcinoma in the mouse liver was investigated. Pre-established hepatic tumors treated with a recombinant adenoviral vector containing the herpes simplex virus thymidine kinase gene(tk) exhibited substantial regression, although all treated animals suffered from subsequent relapses. Although cotreatment with a mouse interleukin 2 (mIL-2)-containing adenoviral vector induced an effective antitumor immune response, the immunity waned with time, and the treated animals eventually succumbed to hepatic tumor relapse or distant metastases. In this study, mouse granulocyte macrophage colony-stimulating factor (mGM-CSF) gene was tested for its ability to further enhance and prolong the antitumoral cellular immunity. A fraction of the animals treated with tk + mIL-2 + mGM-CSF developed long-term antitumor immunity and survived for more than 4 months without recurrence. This long-term antitumor immunity could be enhanced further by subsequent "vaccination" with mIL-2-expressing parental tumor cells. The results indicate that local expression of GM-CSF in the hepatic tumors and prolonged mIL-2 expression are necessary to generate persistent antitumor immunity that is essential for the prevention of tumor recurrence and long-term animal survival.
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PMID:Combination suicide and cytokine gene therapy for hepatic metastases of colon carcinoma: sustained antitumor immunity prolongs animal survival. 870 21

In this work we have explored the use of adenoviral vectors for the purging of cancer cells from hematopoietic stem cell (HSC) autografts. We showed that a recombinant adenovirus expressing the herpes simplex-1 thymidine kinase gene (AD-tk) plus ganciclovir (GCV) killed HELA cells more effectively than did GCV alone. HELA cells were then mixed with human HSCs and exposed to AD-tk/GCV. AD-tk/GCV reduced the number of HELA colonies to 4% of control values, with no detectable reduction in the hematopoietic progenitor, colony forming unit-granulocyte/monocyte (CFU-GM). Similar studies of the JB6 non-Hodgkins lymphoma cell line showed a reduction to 5% of controls; studies of MCF-7, a breast carcinoma cell line, showed a reduction to 30% of controls, with no CFU-GM toxicity. Thus, AD-tk mediated selective killing of contaminating tumor cells. We also evaluated a recombinant adenovirus encoding the tumor suppressor gene p53 (AD-p53). AD-p53 was able to selectively kill all three cell lines (reducing tumor colonies approximately 100-fold) without any toxicity to CFU-GM. Although both AD-tk/GCV and AD-p53 were effective in these experiments, AD-p53 seemed to be more potent. Adenoviral vectors show promise for selectively targeting cancer cells that contaminate HSC autografts.
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PMID:Selective elimination (purging) of contaminating malignant cells from hematopoietic stem cell autografts using recombinant adenovirus. 885 51


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