X‐irradiation effects on thymidine kinase (TK): II. The significance of deoxythymidine triphosphate for inhibition of TK1 activity

He, Qing; Skog, Sven; Welander, Ingrid; Tribukait, Bernhard

doi:10.1046/j.1365-2184.2002.00227.x

Cited by 7 publications

(7 citation statements)

References 12 publications

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“…Both cell lines were obtained through American Type Culture Collection and maintained in Dulbecco's minimal essential medium (DMEM) supplemented with 10% fetal bovine serum, penicillin (100 U/ml) and streptomycin (100 μg/ml) at 37°C and with 5% CO 2 as described [33]. Lysates were prepared from cells growing exponentially ~24 h after re-plating (at 30–50% confluence) by a modification of established methods [34, 35]. In brief, trypsinized cells were washed twice with TBS-EDTA and lysed (4°C) at ~5×10 6 cells/ml in10 mM Tris-HCl, pH7.5, 0.5% P-40 ( v/v ) 1 mM DTT, 5 mM NaF, 5 mM MgCl 2 , with protease inhibitors, 1 μg/ml aprotinin and 1 mM phenylmethylsulfonylfluoride.…”

Section: Methodsmentioning

confidence: 99%

A Novel In Vitro Assay to Assess Phosphorylation of 3′-[18F]fluoro-3′-Deoxythymidine

et al. 2010

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Purpose 3′-[18F]fluoro-3’-deoxythymidine ([18F]FLT) is phosphorylated by thymidine kinase 1 (TK-1), a cell cycle regulated enzyme. Appropriate use of [18F]FLT tracer requires validation of the TK-1 activity. Here, we report development of a novel phosphoryl-transfer assay to assess phosphorylation of [18F]FLT both in tumor cell lysates and tumor cells. Procedures The intrinsic F-18 radioactivity was used to quantify both substrate and phosphorylated products using a rapid thin layer chromatography method. Phosphorylation kinetics of [18F]FLT in SW480 and DiFi tumor cell lysates and cellular uptake were measured. Results The apparent Michaelis–Menten kinetic parameters for [18F]FLT are Km = 4.8 ± 0.3 μM and Vmax=7.4 pmol min−1per 1×106 cells with ~2-fold higher TK-1 activity in DiFi versus SW480 lysates. Conclusions The apparent Km of [18F]FLT was comparable to the value reported with purified recombinant TK-1. The uptake of [18F]FLT by SW480 cells is inhibited by nitrobenzylthioinosine or dipyridamole indicating that uptake is mediated predominantly by the equilibrative nucleoside transporters in these tumor cells.

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Section: Methodsmentioning

confidence: 99%

A Novel In Vitro Assay to Assess Phosphorylation of 3′-[18F]fluoro-3′-Deoxythymidine

et al. 2010

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“…It is known that at least three different mechanisms are required for a non-equilibrium reaction: co-operativity, interconversion cycles and substrate cycles [21]. TK1 enzyme in at least EAT cells was not involved in co-operativity or interconversion cycles since the substrate (dTdR) kinetics of TK1 enzyme showed the Michaelis-Menten kinetics as represented by a straight line and a Hill constant of 1.0 [10,11]. This study indicates that TK1, as a regulatory kinase, together with TMPase, a nucleotidase, is involved in the substrate cycle, providing the proper balance of the required dTMP for DNA replication in the exponentially growing tumour cells.…”

Section: Discussionmentioning

confidence: 99%

“…Since the number of cells in the extract has been diluted to 250-fold, we tested that neither dTdR, dTMP, ATP nor ADP originating from the intact cells significantly affected the determination of the TK1 kinetics. We found that the substrate (TdR) kinetics of TK1 enzyme in EAT cells exhibited the Michaelis-Menten kinetics was represented by a straight line and a Hill constant of 1.0 [10,11], in agreement with the dTdR substrate kinetics of the purified TK1 in human stimulated lymphocyte as previously described by Munch-Petersen [12,13]. Thus, in this paper the substrate kinetics of enzyme is determined using the crude cell extract from the exponentially growing cells and the cells of stationary phase, respectively.…”

Section: Determination Of the Substrate Kinetics Of Enzymesmentioning

confidence: 99%

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Involvement of a Substrate Cycle Between Thymidine and Thymidylate in the Regulation of DNA Precursor Pool in Ehrlich Ascites Tumour

Skog

2002

Cell Physiol Biochem

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In Ehrlich ascites tumour (EAT) cells the main route for dTTP required for DNA synthesis is closely related to thymidylate synthesis activity via the de novo pathway. However, more than 10-time of thymidylate (dTMP) is synthesised by cytosolic thymidine kinase (TK1) via the salvage pathway than needed for DNA synthesis in this cells. Therefore, this study focus to determine if a substrate cycle exists between thymidine (dTdR) and dTMP in the EAT cells. Results show that the ratio of K′eq/Q for the TK1 reaction is 264.2 and for the thymidylate 5′-phosphatase (dTMPase) reaction is 110.9 in the exponentially growing cells, respectively. Since the apparent ratios of K′eq/Q for both reactions are different from equality (ñ1) by two orders, it appears as a non-equilibrium reaction. This indicates that when TK1 and dTMPase are simultaneously active in the exponentially growing cells, a substrate cycle results. The regulation of the excess of non-essential products of dTdR/dTMP for DNA synthesis is involved in a substrate cycle for maintaining a balanced nucleotide pool, hence ensuring a balanced supply of the DNA precursor in the exponentially growing cells. As the tumours continue to grow, cells reached the stationary phase. The ratio of K′eq/Q for TK1 reaction is 7.7 and for the dTMPase reaction is 81.1, showing less than the equilibrium of two orders of magnitude. In this case, dTMPase could not act with TK1 together to form a pair of reaction, leading to an elevated concentration of intracellular dTMP and a dramatically excretion of dTdR into the ascites fluid.

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“…Interestingly, TK1 expression is related to regulation of radiation response in the acute lymphatic leukemia (ALL) HL60 cell line [12]. Recent papers reported the differences in the changes of TK1 and 2 phosphorylation after irradiation among spleen, thymus and ascites tumors [13][14]. Although TK1 is regulated at many levels, it is still unclear how TK1 regulates cell responses to radiation in radioresistant CML cells.…”

Section: Introductionmentioning

confidence: 99%

The modulation of radiation-induced cell death by genistein in K562 cells: Activation of thymidine kinase 1

et al. 2004

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Ionizing radiation is one of the most effective tools in cancer therapy. In a previous study, we reported that protein tyrosine kinase (PTK) inhibitors modulate the radiation responses in the human chronic myelogenous leukemia (CML) cell line K562. The receptor tyrosine kinase inhibitor, genistein, delayed radiation-induced cell death, while non-recepter tyrosine kinase inhibitor, herbimycin A (HMA) enhances radiation-induced apoptosis. In this study, we focused on the modulation of radiation-induced cell death by genistein and performed PCR-select suppression subtractive hybridization (SSH) to understand its molecular mechanism. We identified human thymidine kinase 1 (TK1), which is cell cycle regulatory gene and confirmed expression of TK1 mRNA by Northern blot analysis. Expression of TK1 mRNA and TK 1 enzymatic activity were parallel in their increase and decrease. TK1 is involved in G1-S phase transition of cell cycle progression. In cell cycle analysis, we showed that radiation induced G2 arrest in K562 cells but it was not able to sustain. However, the addition of genistein to irradiated cells sustained a prolonged G2 arrest up to 120 h. In addition, the expression of cell cycle-related proteins, cyclin A and cyclin B1, provided the evidences of G1/S progression and G2-arrest, and their relationship with TK1 in cells treated with radiation and genistein. These results suggest that the activation of TK1 may be critical to modulate the radiation-induced cell death and cell cycle progression in irradiated K562 cells.

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X‐irradiation effects on thymidine kinase (TK): II. The significance of deoxythymidine triphosphate for inhibition of TK1 activity

Cited by 7 publications

References 12 publications

A Novel In Vitro Assay to Assess Phosphorylation of 3′-[18F]fluoro-3′-Deoxythymidine

A Novel In Vitro Assay to Assess Phosphorylation of 3′-[18F]fluoro-3′-Deoxythymidine

Involvement of a Substrate Cycle Between Thymidine and Thymidylate in the Regulation of DNA Precursor Pool in Ehrlich Ascites Tumour

The modulation of radiation-induced cell death by genistein in K562 cells: Activation of thymidine kinase 1

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