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)

The present studies have examined the effects of 1-beta-D-arabinofuranosylcytosine (ara-C) on activation of the transcription factor kappa B (NF-kappa B). The results demonstrate that treatment of human KG-1 myeloid leukemia cells with ara-C is associated with induction of protein binding to the NF-kappa B consensus sequence. NF-kappa B binding was activated at 30 min and reached maximal levels of binding at 1-2 hr of ara-C treatment. The NF-kappa B consensus sequence was ligated to the heterologous thymidine kinase (TK) promoter and the human growth hormone (GH) reporter gene to determine whether ara-C-induced NF-kappa B activity includes an enhancer function. Ara-C treatment had little effect on transient expression of pTKGH in KG-1 cells but increased transcription of the p (NF-kappa B) TKGH vector by 8-fold. The results also demonstrate that ara-C transiently increases NF-kappa B mRNA levels. However, the finding that ara-C-induced binding of NF-kappa B to DNA occurs in the presence of cycloheximide indicates that this agent activates preexisting NF-kappa B protein. These results suggest that ara-C induces a cytoplasmic pathway that transduces signals to the nucleus by activation of NF-kappa B.
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PMID:Activation of the transcription factor kappa B in human KG-1 myeloid leukemia cells treated with 1-beta-D-arabinofuranosylcytosine. 173 23

Some features of the proliferative cells at the bottom of the ileal crypts in BDF1 mice have been studied in relation to the distribution of Paneth cells (PC) in an attempt to clarify the nature and function of these crypt base columnar cells (BCC) and to elucidate some aspects of the role of the microenvironment created by the PC. Longitudinal sections of crypts have shown that the ratio of PC to the BCC, which are scattered amongst the PC, is 2.7:1 in sections or approximately 29 PC and 9 BCC per whole crypt, i.e., a ratio of 3.2:1. The labelling index of BCC is about 35%, which is comparable to that of mid-crypt columnar cells. Although the BCC do become labeled, it is concluded that they cannot create vertical pairs or runs of several adjacent BCC since this would seriously disturb the distribution of Paneth cells. Only in dividing crypts are such runs (consisting of 3 to 5 cells) observed. The ability of BCC to synthesize DNA is not dependent on their position in the Paneth cell zone. In 95% of the crypts, the highest Paneth cell is below the 7th cell position from the bottom of the crypt, and the positions of the highest PC on either side of a given crypt are similar. The secreted granules or the cytoplasm of PC specifically bind pokeweed lectin, and this can be used for identification. Tracer doses of 3HTdR (37 kBq/gm body weight) result in the histological death of some BCC, and these damaged cells are evenly distributed throughout the Paneth cell zone. These tracer doses are somewhat selectively incorporated into BCC, i.e., the BCC have a higher grain count in autoradiographs, probably because they possess more thymidine kinase enzyme activity. This ability is very sensitive to the withdrawal of food, because 24 hr of fasting abolished the observed gradient in the intensity of labelling, which is very well correlated with the distribution of BCC. Regeneration of the crypts following cytotoxic exposure to Ara-C is initiated at the base of the crypt and hence may involve the BCC with possible help from the Paneth cells. The latter are insensitive to cytotoxic (S phase specific) agents and may help in the regeneration by preserving the architecture of the base of the crypt.
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PMID:Crypt base columnar cells in ileum of BDF1 male mice--their numbers and some features of their proliferation. 258 24

The effect of arabinosyl cytosine (ara-C) was studied on the uptake, phosphorylation and incorporation of 3H-thymidine in human tonsillar lymphocyte cultures is described along with its effect on the level of DNA polymerase and thymidine Kinase activities induced by phytohaemagglutinin (PHA). Freshly isolated tonsillar lymphocytes are stimulated cells with a remarkably high activity of DNA polymerase alpha and thymidine kinase. During in vitro culture, these stimulated cells are transformed to the resting state with low DNA polymerase and thymidine kinase activity. However, a new DNA synthesising cycle can be induced by PHA with maximum at 48 h. 10(-6) M ara-C inhibited the incorporation of 3H-thymidine by 90-95%. This inhibition may be reversed by rinsing the cells. The inhibition of the transport of 3H-thymidine seems to be only a consequence of the inhibitory effect of ara-C on the DNA polymerisation reaction, because at 10 degrees C, where DNA synthesis was arrested, ara-C does not influence the uptake and the phosphorylation of 3H-thymidine. Ara-C (10(-6) M) abolished also the PHA induced elevation of DNA polymerase alpha and thymidine kinase activities without influencing protein synthesis of the cell. This supports a coordinated regulation mechanism between DNA synthesis and the synthesis of enzymes involved in DNA replication.
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PMID:Effect of arabinosyl cytosine on the level of DNA polymerase and thymidine kinase activity in PHA-stimulated human tonsillar lymphocytes. 706 8

The current study was undertaken to determine the relevance of leukemic blast cell proliferative activity, cellular parameters of Ara-C metabolism and the in vitro sensitivity to GM-CSF in association with the clinical response to TAD-9 induction therapy in 66 patients with de novo acute myeloid leukemia (AML). Proliferative activity was assessed by 3H-thymidine (3H-TdR) incorporation and thymidine kinase (TK) activity, parameters of Ara-C metabolism comprised the activities of deoxycytidine kinase (DCK) and DNA polymerase alpha (poly alpha) as well as Ara-CTP concentrations and 3H-Ara-C uptake into DNA. GM-CSF sensitivity was determined by in vitro incubation of blasts for 48 h with or without GM-CSF (100 U/ml) followed by an additional 4 h concurrent exposure to GM-CSF and 3H-TdR (0.5 microCi/ml). The following results were obtained as expressed by median values and ranges: 3H-TdR incorporation: 1.07 pmol/10(5) cells (0.0-10.1), TK: 7.3 pmol/min/mg protein (1.3-56.0), DCK: 9.3 pmol/min/mg protein (0.77-47.1), poly alpha: 1.7 pmol/min/mg protein (0.00-28.9), Ara-CTP: 53.3 ng/10(7) cells (13.3-211.0), 3H-Ara-C uptake: 0.06 pmol/10(5) cells (0.0-0.57). 3H-Ara-C uptake was correlated with 3H-TdR incorporation (r = 0.74) and with the (S-phase dependent) activities of TK (r = 0.73) and poly alpha (r = 0.71, but not with DCK activity or intracellular Ara-CTP content. Blast cells of 37 from 55 analyzed patients were found to be sensitive to GM-CSF stimulation as defined by an increase in 3H-TdR incorporation > or = 1.5-fold over control values after the 48 h GM-CSF exposure. In vitro data were related with clinical response to TAD-9 induction therapy in 43 patients with newly diagnosed AML, taking the blast cell reduction at day 10 or 16 to < 5% or > or = 5% residual blasts as early parameter for adequate or inadequate response, respectively. While neither 3H-Ara-C uptake, nor intracellular Ara-CTP concentration, TK nor DCK activity were predictive for response, a high 3H-TdR incorporation and a high poly alpha activity were associated with adequate blast cell reduction. Median values of 3H-TdR incorporation were 2.26 pmol/10(5) cells for patients with adequate blast cell clearance and 0.80 pmol/10(5) cells for patients with inadequate blast cell clearance (P = 0.11), the respective values for poly alpha were 3.22 pmol/min/mg protein for responders and 1.1 pmol/min/mg protein for non-responders (P = 0.0085).(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Blast cell proliferative activity and sensitivity to GM-CSF in vitro are associated with early response to TAD-9 induction therapy in acute myeloid leukemia. 747 75

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.
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PMID:Differential effect of GM-CSF pretreatment on intracellular Ara-C metabolism in normal bone marrow mononuclear cells vs acute myeloid leukemia (AML) blasts. 909 97

The present study was undertaken to assess the predictive value of pretherapeutic determinants of ara-C metabolism and proliferative activity of leukemic blasts for early response to antileukemic therapy in the setting of granulocyte-macrophage colony-stimulating factor (GM-CSF)-based priming before and during TAD-9 induction in 36 consecutive patients with de novo acute myeloid leukemia (AML). Ara-C metabolism was assessed by the activities of deoxycytidine kinase (DCK), deoxycytidine deaminase (DCD), DNA polymerase alpha (Poly alpha), and overall polymerase (overall Poly). The fraction of cells in S phase (%S phase) and thymidine kinase (TK) activity were determined as a measure of proliferative activity. Early response to therapy was defined by the percentage of leukemic blasts in the bone marrow 5 to 7 days after completion of TAD-9 with less than 5% signaling an adequate response and greater than 5% indicating an inadequate early reduction, respectively. While neither %S phase, DCK, nor overall Poly activity were predictive for early response, TK and Poly alpha activities were significantly higher for cases with adequate blast cell clearance. The respective median values were for TK 3.8 versus 1.85 pmol/min/mg protein (P = .012), and for Poly alpha 1.9 versus 0.69 pmol/min/mg protein (P = .014). An inverse relation was detected for DCD activity which was significantly lower in responding patients with a median of 0.33 nmol/min/mg protein (range, 0.0 to 29.5) as compared to a median of 5.1 nmol/min/mg protein (range, 0.11 to 8.45) in early nonresponders, (P = .009). Taking the respective median values as arbitrary cut-points for high or low enzyme activities, responders and nonresponders could be discriminated prospectively. Hence, 14 of 16 cases (88%) with DCD activities below the median of 1.56 nmol/min/mg protein responded as compared to only 3 of 14 (22%) patients with higher DCD activities (P = .0004). From the 15 patients with TK activity above the overall median of 3.2 pmol/min/mg protein, 11 cases (73%) achieved an adequate blast cell clearance while only 6 of 17 cases (35%) with lower values responded (P = .035). Similarly, 12 of 15 patients (80%) with high Poly alpha levels (>1.22 pmol/min/mg protein) responded to induction therapy as compared to only 5 of 14 patients (36%) with lower enzyme activities (P = .02). By logistic regression analysis of enzyme activities, DCD activity was found to be the most sensitive parameter to predict an adequate blast cell clearance (P = .032). Activities of DCD and TK were not only associated with initial response but were also found predictive for remission duration. Hence, from 11 patients with low TK levels 8 (73%) relapsed within 1 year, whereas only 2 of 11 (18%) patients with high TK activity experienced a recurrence of their disease (P = .015). Six of 9 (66%) patients with higher than median DCD levels relapsed within 1 year, whereas 10 of 14 patients (71%) with lower DCD levels had a longer remission duration (P = .085). Analysis of DCD gene expression at the mRNA level by a semi-quantitative reverse transcriptase-polymerase chain reaction method showed that a high transcription rate of the DCD gene was associated with high enzyme activities and vice versa. Hence, the observed intraindividual differences in DCD activity are a reflection of differences in gene activity and transcription rate rather than of variants in translation. Although further analyses are needed to elucidate the molecular mechanisms that determine the variation of enzyme activities in individual patients, the present study strongly suggests that pretherapeutic determination of TK and Poly alpha as well as of DCD allows to predict response to TAD-9 + GM-CSF induction therapy and may provide the means for the development of a risk adapted treatment strategy.
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PMID:Activity of thymidine kinase and of polymerase alpha as well as activity and gene expression of deoxycytidine deaminase in leukemic blasts are correlated with clinical response in the setting of granulocyte-macrophage colony-stimulating factor-based priming before and during TAD-9 induction therapy in acute myeloid leukemia. 929 31

The biological evaluation of mononucleotide prodrugs (pronucleotides) of various nucleoside reverse transcriptase inhibitors (NRTIs) such as zidovudine (AZT), zalcitabine (ddC) and lamivudine (3TC) was reported in human T-lymphoid MOLT-4/8 cells which were grown continuously for more than 1 year in a medium containing cytarabine (Ara-C). In this cell line, expression of deoxycytidine kinase (dCK) and thymidine kinase 1 (TK1) was decreased in comparison to parental cells (3.8 and 2.9-fold, respectively). The lower mRNA level of TK1 correlated significantly with lower enzyme activity, whereas no dCK activity was detectable. In Ara-C-resistant cells, anti-HIV-1 effects of ddC, 3TC and AZT were more than 100-fold lower compared with parental cells. In contrast, the corresponding mononucleoside phosphotriesters bearing S-acyl-2-thioethyl (SATE) groups as biolabile phosphate protection retained anti-HIV-1 activity due to their ability to bypass the first monophosphorylation step catalyzed by dCK or TK1. The results demonstrate that in vitro selection of T-lymphoid cells in the presence of Ara-C results in cross-resistance to deoxycytidine (ddC, 3TC) and thymidine (AZT) analogs and that these cellular resistance mechanisms can be bypassed by the use of bis(SATE) pronucleotides.
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PMID:S-acyl-2-thioethyl (SATE) pronucleotides are potent inhibitors of HIV-1 replication in T-lymphoid cells cross-resistant to deoxycytidine and thymidine analogs. 1175 Sep 40

Overexpression of NF-kappa B reportedly plays anti-apoptotic roles in the growth of AML cells. Control of AML cell growth was attempted using a replication-defective herpes simplex virus-1 vector, T0I kappa B alpha, overexpressing mutant I kappa B alpha to inhibit NF-kappa B in vitro. T0I kappa B alpha displays defective ICP4/ICP22/ICP27, isogenic thymidine kinase, and mutant I kappa B alpha. T0Z.1 expressing lacZ instead of I kappa B was used for controls. Infection of T0I kappa B alpha at 15 multiplicity of infection (MOI) with cells of AML lines, HL60, K562, and NB4 displaying >90% infection efficiency and tumor killing in vitro. Use of 10 microM of Ara-C alone was clinically equivalent to high-dose Ara-C, displaying 11% tumor killing. Neither ganciclovir (GCV) nor Ara-C enhanced T0I kappa B- alpha mediated tumor killing. Attenuation of NF-kappa B by T0I kappa B alpha was confirmed by EMSA. T0I kappa B alpha induced caspase-3 activity, with subsequent apoptosis confirmed by colorimetric and TUNEL assays. Fresh AML cells from 8 patients were infected with T0I kappa B alpha at 3 MOI, with or without GCV or 10 microM of Ara-C in vitro. Infection efficiency was 10%. T0I kappa B alpha displayed 8-15% tumor killing, superior to Ara-C in 6 of the 8 patients. Administration of Ara-C enhanced tumor killing in 5 of these 6 cases. Our results suggest that T0I kappa B alpha-mediated gene therapy induces apoptosis of AML cells in vitro.
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PMID:I kappa B-mediated apoptotic gene therapy against acute myelogenous leukemia using replication-defective HSV-1 vector expressing TK and mutant I kappa B alpha. 1617 66

Refractoriness of acute myeloid leukemia (AML) cells to chemotherapeutics represents a major clinical barrier. Suicide gene therapy for cancer has been attractive but with limited clinical efficacy. In this study, we investigated the potential application of herpes simplex virus thymidine kinase/ganciclovir (HSV-TK/GCV) based system to inhibit chemoresistant AML cells. We first generated Ara-C resistant K562 cells and doxorubicin-resistant THP-1 cells. We found that the HSV-TK/GCV anticancer system suppressed drug resistant leukemic cells in culture. Chemoresistant AML cell lines displayed similar sensitivity to HSV-TK/GCV. Moreover, HSV-TK/GCV killing of leukemic cells was augmented to a mild but significant extent by all-trans retinoic acid (ATRA) with concomitant upregulation of Connexin 43, a major component of gap junctions. Interestingly, HSV-TK/GCV killing was enhanced by expression of vesicular stomatitis virus G glycoprotein (VSV-G), a fusogenic membrane protein, which also increased leukemic cell fusion. Co-culture resistant cells expressing HSV-TK and cells stably transduced with VSV-G showed that expression of VSV-G could promote the bystander killing effect of HSV-TK/GCV. Furthermore, combination of HSV-TK/GCV with VSV-G plus ATRA produced more pronounced antileukemia effect. These results suggest that the HSV-TK/GCV system in combination with fusogenic membrane proteins and/or ATRA could provide a strategy to mitigate the chemoresistance of AML.
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PMID:Vesicular Stomatitis Virus G Glycoprotein and ATRA Enhanced Bystander Killing of Chemoresistant Leukemic Cells by Herpes Simplex Virus Thymidine Kinase/Ganciclovir. 2475 16