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)

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

Plasmid expression vectors combining human cytokine cDNAs and selectable marker genes on dicistronic transcription units were functionally characterized in vitro and in vivo. The internal ribosome entry sequence (IRES) of encephalomyocarditis virus mediated cap-independent translation of the downstream cistron. After cationic lipofection of cells with a dicistronic construct containing the Neor gene downstream of a human interleukin-2 (IL-2) cDNA, all G418-resistant clones secreted high amounts of IL-2. Reversal of the order of the cDNAs was associated with less efficient transgene expression and represented no advantage in comparison to separate expression cassettes. To combine direct in vitro selection of expression with in vivo elimination of cytokine-secreting cells, an improved chimeric cDNA of the Neor and herpes simplex virus (HSV) thymidine kinase (TK) genes was constructed and shown to confer sensitivity to ganciclovir concentrations that can be achieved in human patients. This chimeric marker was coupled on dicistronic constructs with a granulocyte colony-stimulating factor (G-CSF) cDNA as a molecule with easily detectable bioactivity in vivo. Subcutaneous implantation of pCMV.GCSF.ires TK/NEO-transfected CMS-5 cells into syngeneic BALB/c mice resulted in excessive leukocytosis and progressively growing tumors. Treatment with ganciclovir led to normalization of leukocyte counts in all animals, whereas complete regression of tumors was observed in only 3/5 mice. Hypermethylation of the transfected promoter was demonstrated in both ganciclovir-resistant tumors. Thus, transcription units combining selectable markers and genes of interest allow selection of high producer cells in vitro and efficient elimination of transgene-expressing cells in vivo. However, cells that hypermethylate transfected genes to terminate gene expression in vivo may escape conditional ablation.
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PMID:Systematic evaluation of chimeric marker genes on dicistronic transcription units for regulated expression of transgenes in vitro and in vivo. 889 74

The effects of macrophage (M) and granulocyte (G) colony-stimulating factors (CSFs) on the activity of thymidylate synthase and thymidine kinase, which are involved in de novo and salvage pathways for pyrimidine nucleotide synthesis, were investigated in the hematopoietic cells of rats treated with cyclophasphamide. Thymidine kinase activity, but not that of thymidylate synthase, was markedly enhanced in these cells by M- and G-CSF treatment (p < 0.05 and p < 0.01). G-CSF directly, and M-CSF indirectly stimulate myeloid cells and lead to S-phase predominantly via the salvage pathway for pyrimidine nucleotide synthesis. The present study indicates that these CSFs can be effective inducers of complete remission in acute leukemias when employed together with chemotherapy.
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PMID:Effects of macrophage- and granulocyte-colony stimulating factors on thymidylate synthase and thymidine kinase activity in rat hematopoietic cells. 917 25

It is known that colony stimulating factors (CSFs) stimulate the myeloid cells of bone marrow and splenic cells in rodents. The effects of macrophage (M)-CSF on the activities of thymidylate synthase and thymidine kinase, involved in de novo and salvage pathways for pyrimidine nucleotide synthesis, respectively, in haematopoietic cells of bone marrow and spleen were investigated in rats. A single M-CSF injection did not elevate the mRNA expression levels of the enzymes in bone marrow cells 6 h after treatment, but it enhanced the splenic thymidylate synthase mRNA expression. M-CSF stimulated the splenic thymidylate synthase activity without an increase of the peripheral granulocytes. The effect of M-CSF on granulocytes is considered to be weak compared with that of granulocyte (G)-CSF, because of the indirect secretion of endogenous G-CSF from the cells with M-CSF receptors stimulated by exogenous M-CSF. Since M-CSF was able temporarily to lead progenitor cells from long G1-phase into S-phase, M-CSF might accelerate the anticancer effects when used together with anticancer agents.
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PMID:Does macrophage-colony stimulating factor stimulate rat haematopoietic cells? 1076 79

In mammals, methylation of DNA within regulatory sites and histone deacetylase recruitment in transcriptional repressing domains are involved in the loss of the expression of retroviral DNA or repeat arrays transferred in cells for therapeutic purposes. Various investigation results suggest that methylation/deacetylation events are modulated by extracellular and cytoplasmic signal transduction pathways closely involved in regulating cell differentiation. To analyse gene silencing mechanisms and assess if potential pharmacological treatment affects gene silencing kinetics we transduced U937 myelomonocytic cells with a bicistronic retroviral construct carrying the herpes simplex virus thymidine kinase (HSV-TK) and beta-galactosidase (Lac-Z) genes. This vector can be employed in vivo and in vitro to render transduced cell populations susceptible to ganciclovir (GCV). We verified the effect of the histone deacetylase inhibitor Trichostatin A (TSA) alone or combined with 5'-azacytidine (5'aza-C) on transcription downmodulation. Our results indicate that in our in vitro model TSA is able to reactivate transgene expression, more efficiently and with quicker kinetics (12-24h) than 5'aza-C (36-48 h). The effect is dose dependent (between 1 and 50 nM), with no relevant toxicity. Treatment with both drugs is synergistic in gene reactivation in terms of extension and persistence, with low toxicity and no relevant differentiating effects. The cells in which transgene expression has been reactivated undergo progressive silencing, but once weekly drug treatment can maintain high transgene expression levels for more than 90 days with no evidence of selection. The results obtained by treating U937 transduced clones with TSA and/or 5'aza-C together with IL-3, G-CSF or GM-CSF cytokines suggest that transduced U937 differentiation levels do not affect basal expression, but render these cells more responsive to reactivation by TSA or TSA plus 5'aza-C, but not to 5'aza-C alone. In conclusion, the results suggest that in vitro inhibition of histone deacetylase by TSA can interfere with gene silencing mechanisms affecting 5' Moloney murine leukaemia virus long terminal repeat (MoMuLV-LTR) driven transgene expression thus providing the rationale for TSA and/or 5'aza-C administration in animal models for the translation on gene therapy applications.
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PMID:Effect of trichostatin a and 5'-azacytidine on transgene reactivation in U937 transduced cells. 1277 May 23