Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.1.21 (thymidine kinase)
 7,561 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Our studies provide evidence for the presence of cyclin D1 in an early G1 cycle-specific DNA binding complex Yi1. Previously we identified several complexes including Yi and E2F that at different times during G0 to S transition bind to three distinct DNA sequences (MT1, MT2, MT3) located in the mouse thymidine kinase upstream promoter. These various complexes contain DNA binding proteins (Sp1, E2F, p110, p60), cyclins A and E, cyclin-dependent kinase 2 (cdk2), and retinoblastoma-related proteins (pRB, p107). Here we report that Yi1 is different from the E2F complexes. Yi1 contains cyclin D1/cdk2 kinase as shown by using specific antibodies to cyclins, cdks and the Yi1 DNA-binding protein in gel retardation, western blotting, and immunoprecipitation assays. Yi1 binding is specific to a consensus sequence different from that of E2F.
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PMID:Cyclin D1/cdk2 kinase is present in a G1 phase-specific protein complex Yi1 that binds to the mouse thymidine kinase gene promoter. 781 Dec 75

Production of thymidine kinase (TK) protein parallels the onset of DNA synthesis during the cell cycle. This process is regulated at transcriptional, posttranscriptional, and translational levels to cause a 40- to 50-fold increase in cytosolic enzymatic activity as cells progress from G1 to S phase. Transcriptional activation of the mouse TK gene through the cell cycle is dependent upon previously characterized cis elements of the proximal promoter, called MT1, MT2, and MT3, that bind at least two different complexes: TKE during the transition of cells from quiescence (G0) to G1, and Yi later at the G1/S boundary. To identify the transcription factors involved in this regulation, we screened a mouse fibroblast cDNA expression library with a labeled MT3 oligonucleotide probe and isolated a clone that encodes Egr-1, an immediate-early transcription factor, whose expression is regulated by serum or growth factors during the G0-to-G1 transition when cells reenter the cell cycle. Electrophoretic mobility shift assays demonstrate that Egr-1 is involved in the TKE complex that binds to the MT3 element and that expression of Egr-1 induces transcription of a mouse TK-chloramphenicol acetyltransferase reporter in transient transfections. These results suggest a role for Egr-1 in regulating expression of the TK gene at the G0-to-G1 transition.
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PMID:The immediate-early gene Egr-1 regulates the activity of the thymidine kinase promoter at the G0-to-G1 transition of the cell cycle. 803 3

By performing DNase I footprint analysis, we had identified three distinct protein binding sequences (MT1, MT2, and MT3) located on the mouse thymidine kinase (TK) upstream promoter (Dou, Q.-P., Fridovich-Keil, J. L., and Pardee, A.B. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 1157-1161). Here we report that MT2 includes an E2F-like binding site (GTTCGCGGGCAAA), as shown by the following evidence. (i) MT2 bound specifically to an affinity-purified fusion human E2F protein. (ii) Both MT2 and an authentic E2F site (TTTCGCGCGCTTT) bound specifically to similar or identical nuclear protein complexes. (iii) Formation of both these DNA-protein complexes were cell cycle-dependent: a G0/G1 phase-specific complex (E2F.G0/G1) was replaced by an S phase-specific complex(es) (E2F.S), whereas "free" E2F increased after the G1/S transition. (iv) Pulse inhibition of protein synthesis with cycloheximide interchanged these complexes with similar kinetics. (v) When MT2-shifted E2F.G0/G1, E2F.S, and free E2F were eluted and analyzed by Western blot assay using a specific antiserum to human E2F-1, two forms of murine E2F (62 and 66 kDa) were observed from all three complexes. The compositions of these MT2-bound complexes were also investigated. Studies using specific antibodies revealed that p107, a retinoblastoma-like protein, was present in both E2F-G0/G1 and E2F.S, whereas cyclin E.cyclin A.cdk2 were only present in E2F.S complex(es). These data suggest that removal of the p107-containing E2F.G0/G1 complex, a candidate repressor, from the MT2 site in late G1 may be essential for S phase-dependent transcription of the mouse TK gene.
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PMID:G1/S-regulated E2F-containing protein complexes bind to the mouse thymidine kinase gene promoter. 828 95

We have used site-specific mutagenesis and thymidine kinase (TK) promoter/reporter gene transfection experiments to investigate DNA sequences required for serum-responsive regulation of expression from the mouse thymidine kinase promoter. Mutations were targeted to each of three previously described protein binding domains (MT1, MT2, and MT3) upstream of the TK translation initiation site, as well as to sequences within the TK first exon in order to address each of the following three questions: (a) Do these sequences play any role in regulation? (b) Do all of these sites play the same role? and (c) If any controls are observed, do they act positively or negatively on gene expression? The results of these experiments indicated that, in the wild-type TK promoter, at least some of these sequences do play a role in regulation, that not all of these sites appear to play the same role, and that some of the targeted elements act positively on gene expression, whereas others appear to act negatively. In particular, mutagenesis of the Sp1 site within MT1 virtually eliminated promoter function, whereas mutations in either the MT2 site or the TK first exon rendered reporter gene expression nearly constitutive with respect to serum. Thus, both MT2 and sequences within the TK first exon appear to contain negatively acting elements. In contrast, mutation or deletion of the MT3 site produced a much less pronounced effect on reporter gene regulation. These results support recent observations from our laboratory (Q-P. Dou et al., manuscript in preparation) indicating that although the protein complexes that bind to these various sites are similar, they are not identical.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:DNA sequences required for serum-responsive regulation of expression from the mouse thymidine kinase promoter. 839 9

Adult T cell leukemia/lymphoma (ATL) is derived from CD4+ T cells and has a poor prognosis because of its resistance to chemotherapy. To evaluate the effectiveness of gene therapy for ATL, the effect of ganciclovir on ATL cell lines transfected with the thymidine kinase gene of herpes simplex virus type 1 (HSV-TK) was analyzed. To transfer the HSV-TK gene to ATL cells, a human immunodeficiency virus (HIV) vector that has specific infectivity to CD4+ cells was used. HSV-TK was inserted into the long terminal repeats of HIV-1 and driven by the SL3 promoter HXBSL3TK. HXBSL3TK was co-transfected with HXBCAT as a reporter into MT2 or HUT102 cells by DEAE-dextran. The cells were incubated with ganciclovir, and chloramphenicol acetyltransferase (CAT) activity was analyzed. The CAT activity of the MT2 cells and HUT102 cells transfected with HXBSL3TK decreased dose-dependently with ganciclovir. HXBSL3TK was also co-transfected into COS cells with an HIV-1 packaging vector that has gag, pol, and env driven by a cytomegalovirus promoter. The supernatant was transferred to MT2 cells or Raji cells and incubated with ganciclovir. Ninety percent of the MT2 cells transduced by HXBSL3TK and incubated with ganciclovir were killed, but Raji cells were not killed. In addition, HXBTK that expresses the HSV-TK gene and Tat gene driven by the LTR of HIV-1 was constructed. HXBTK had a higher expression of the HSV-TK gene and higher sensitivity to ganciclovir than did HXBSL3TK.
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PMID:Gene therapy for adult T cell leukemia using human immunodeficiency virus vector carrying the thymidine kinase gene of herpes simplex virus type 1. 895 10

We investigated the potential efficacy of treating adult T-cell leukemia (ATL) using a gene therapeutic approach involving the use of a herpes simplex virus-thymidine kinase (HSV-TK)-mediated suicide system. Human immunodeficiency virus (HIV)-based vectors containing the HSV-TK gene were constructed to achieve targeted gene transfer into CD4-positive ATL cells, after which the transduced cells were selectively killed by treatment with ganciclovir (GCV). To examine the utility of HIV vectors in vivo, ATL-NOD-SCID mice were prepared by intraperitoneal injection of 1 x 10(7) MT2 cells into NK-depleted nonobese diabetic/severely compromised immunodeficient (NOD-SCID) mice. Thereafter, 1 ml of concentrated HIV vector expressing HSV-TK (HXCTKN) or GFP (HXGFP) stock was injected into the intraperitoneal cavity, and GCV was administered twice a day for 5 days. Fluorescence-activated cell sorting (FACS) analysis showed that 7-11% of MT2 or HUT102 cells recovered from the peritoneal cavity were transduced with the HXGFP. After 3 weeks, plasma sIL2-R alpha levels were significantly lower in mice administered HXCTKN than in those administered HXGFP. Moreover, HXCTKN-injected mice survived significantly longer than HXGFP-injected mice. Taken together, these findings suggest that HIV vectors could be used for in vivo targeted gene transfer into ATL cells and could thus serve as the basis for the development of effective new therapies for the treatment of ATL.
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PMID:HIV vector-mediated targeted suicide gene therapy for adult T-cell leukemia. 1789 98