Activation of Deoxycytidine Kinase During Inhibition of DNA Synthesis in Human Lymphocytes

We have studied the time course of disassembly of microtubules of resting and stimulated mouse lymphocytes caused by the drug colchicine, as well as the effect of this compound on DNA and RNA synthesis of human and mouse lymphocytes. Fine-structure studies with the electron microscope showed a great increase in number of microtubules resulting from stimulation of mouse lymphocytes by the mitogenic lectin Con A. The presence of a network of microtubules was demonstrated in resting lymphocytes by use of the technique of immunofluorescence; this technique was not effective for the study of the microtubules of stimulated lymphocytes in the blast stage. The disappearance of microtubular networks in some cells (approximately 25%) was caused by the protocol of colchicine treatment used in many laboratories (30 min at 10−6 M); a 6- to 8-h treatment was required to cause all cells to lose their microtubules. It is indicated in these findings that there is need for extreme caution in implicating microtubule disruption as the cause of certain colchicine effects, such as that on the Con A-induced inhibition of receptor–ligand migration. The addition of colchicine to stimulated cells at varying times of culture caused marked inhibition of DNA synthesis provided that sufficient time (approximately 20 h for maximum inhibition) elapsed between addition of the drug to the stimulated culture and assay of DNA synthesis. Our data on the time course of inhibition of DNA synthesis by α-methyl mannoside (αMM) and by colchicine do not exclude the possibility that the latter compound may act partly by affecting the commitment of stimulated lymphocytes to DNA synthesis but they show that it can inhibit well after commitment is complete. The later the time of assay of thymidine incorporation, the more disparate were the curves relating the effects of αMM and colchicine to DNA synthesis of human cells. In the case of mouse splenic lymphocytes, there was no resemblance between the time course of the αMM and of the colchicine effects. Synthesis of RNA after 12 h of culture of stimulated human lymphocytes was also sensitive to colchicine.

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Differences in flow cytometry and 3H-thymidine analyses of perturbed human lymphocytes.

Journal of Histochemistry & Cytochemistry ◽

10.1177/27.1.220326 ◽

1979 ◽

Vol 27 (1) ◽

pp. 486-490 ◽

Cited By ~ 13

Author(s):

A Pollack ◽

C B Bagwell ◽

J L Hudson ◽

G L Irvin

Keyword(s):

Flow Cytometry ◽

Dna Synthesis ◽

Human Peripheral Blood ◽

Tritiated Thymidine ◽

Human Lymphocytes ◽

Flow Cytometric Analysis ◽

Cyclic Adenosine Monophosphate ◽

Adenosine Monophosphate ◽

Peripheral Blood Lymphocytes ◽

Inhibition Of Dna Synthesis

A calf thymocyte crude aqueous extract was tested for DNA synthesis inhibitory activity using phytohemagglutinin-stimulated human peripheral blood lymphocytes. Inhibition of DNA synthesis was assayed using tritiated thymidine and flow cytometry. Although the calf thymocyte crude extract inhibited tritiated thymidine incorporation by over 50%, only very slight changes in the flow cytometric analysis were observed. When dibutyryl-cyclic adenosine monophosphate was used as an inhibitor, a correlation in terms of the inhibition of tritiated thymidine to the inhibition by flow cytometry was observed.

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Modulation of human deoxycytidine kinase activity as a response to cellular stress induced by NaF.

Acta Biochimica Polonica ◽

10.18388/abp.2001_5133 ◽

2001 ◽

Vol 48 (1) ◽

pp. 251-256 ◽

Cited By ~ 8

Author(s):

Z Csapó ◽

M Sasvári-Székely ◽

T Spasokoukotskaja ◽

M Staub

Keyword(s):

Dna Synthesis ◽

Cell Cultures ◽

Kinase Activity ◽

Protein Phosphatases ◽

Human Lymphocytes ◽

Deoxycytidine Kinase ◽

Post Translational Modification ◽

Key Enzymes ◽

Resting Lymphocytes ◽

Arabinosyl Cytosine

Deoxycytidine kinase (dCK) is one of the key enzymes of deoxynucleoside salvage supplying resting lymphocytes with DNA precursors for synthesis and repair. The level of dCK activity is especially important in chemotherapy with the use of deoxynucleoside analogues like arabinosyl cytosine (Citarabid, ara-C), or 2-chloro-deoxyadenosine (Cladribine, CdA). Previous results showed that Cladribine treatment of human lymphocytes increased several fold the activity of dCK without increasing the amount of dCK protein itself (Sasvári-Székely, et al., 1998, Biochem. Pharmacol. 56, 1175), and a possible post-translational modification was suggested. This theory was further investigated using NaF as an inhibitor of protein phosphatases. It was shown that NaF treatment of cells elevated dCK activity while inhibiting DNA synthesis. The possible mechanism of dCK activation/inactivation induced by exposure of cell cultures to different agents is discussed.

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