X-irradiation effects on thymidine kinase (TK): I. TK1 and 2 in normal and malignant cells.

The effect of radiation on TK is more complicated than would be expected from earlier results on bone marrow cells (Feinendegen et al. 1984, Int. J. Radiat. Biol. 45, 205). TK activity increased at 0.01 Gy and then decreased up to 1 Gy in mouse spleen. In contrast to the results for the spleen, an increase in activity at 0.1 Gy was seen in mouse thymus. The activity of dephosphorylated TK1 (TK1a) in both spleen and thymus was reduced to 50% after irradiation at 0.5-1 Gy. The degree of phosphorylation (TK1b/TK1a ratio) changed in spleen, but not in thymus. The activity of TK2 in mouse liver increased at 3 h after 5 Gy by about 60%. In mouse ascites tumour, a dose-independent (1-5 Gy) oscillating TK1 activity was found up to 24 h, especially for TK1a and TK1b. The amount of TK1 was unchanged up to 12 h, but decreased at 24 h. This suggests that the differences in the changes in the degree of phosphorylation of TK1 after irradiation among spleen, thymus and ascites tumour further underline the complexity of the response of TK1 activity to irradiation. The dramatic change in the activities of TK1a and TK1b may illustrate that both of them are more radiosensitive than TK-h, a variant with mixed TK1 and TK2 properties.

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

The purpose of this study was to investigate the mechanism behind the high sensitivity of thymidine kinase 1 (TK1) to X-irradiation. The deoxythymidine triphosphate (dTTP) pool was studied in mouse ascites tumour cells 1-24 h after X-irradiation with 5 Gy. Irradiation changed the Michaelis-Menten kinetics of TK1 from linear to biphasic, showing a negative co-operativity. These changes were closely related to changes in the dTTP pool. Addition of dTTP to the cell extract of non-irradiated cells, or thymidine (dTdR) to the culture medium, resulted in changes very similar to the kinetics found in the irradiated cells. Addition of 5 cent-amino-5 cent-deoxythymidine (5 cent-AdTdR), a thymidine analogue that eliminated the inhibitory effect of dTTP on TK1 activity, completely abolished the irradiation-induced inhibition of TK1 activity. We suggest that the reduced TK1 activity is mainly due to an elevated intracellular concentration of dTTP.

Improved measurement of tyrosine-phosphorylated DNA-PKcs by isoelectric focusing as an indirect determination of DNA-PK activity in vivo.

UNLABELLED: Isolation of various phosphorylated forms of DNA-PKcs by SDS gel electrophoresis is difficult because of the same molecular weight. By isoelectric focusing (IEF), we confirmed a de-phosphorylated (pI 9.0) and a phosphorylated (pI 6.9) form of DNA-PKcs. When cells were damaged by X-irradiation (5 Gy), an additional tyrosine-phosphorylated (p-Tyr) DNA-PKcs form (pI 6.3) appeared in both nuclear and cytoplasmic fractions, after 30 minsutes. CONCLUSION: IEF in combination with Western blotting of p-Tyr-DNA-PKcs results in an appropriate separation of the p-Tyr form from other phosphorylated forms of DNA-PKcs, which may permit an indirect measurement of changes in DNA-PK activity in irradiated cells.

Cisplatin-induced inhibition of p34cdc2 is abolished by 5-fluorouracil.

Clinical (Dimery and Hong, J Nat Cancer Inst 1993; 85: 95- 111) and experimental studies (Scanlon et al., Proc Natl Acad Sci USA 1986; 83: 8923-5; Lewin et al., In Vivo 1990; 4: 277-82) have indicated an increased cytotoxic effect, when cisplatin (CDDP) is combined with 5-fluorouracil (5-FU). Addition of 5-FU abolishes the G2-arrest induced by CDDP (Lewin et al., In Vivo 1990; 4: 277-82; Nylén et al., Acta Oncol 1996; 35: 229 35). The mechanism for the synergy is unclear. Activation of p34cdc2 is necessary for progression from G2 to mitosis (Lewin et al., Anti-Cancer Drugs 1995; 6: 465-70). The aim was to study p34cdc2, cdc25C and weel after treatment of mammalian tumour cells in vivo with CDDP as single agent or in combination with 5-FU. CDDP prevented activation of p34cdc2 by keeping cdc25C inactive and weel active. Addition of 5-FU to CDDP decreased the expression of weel and promoted cdc25C-activation. p34cdc2 was dephosphorylated by cdc25C and activated. Alterations in activity of cdc25C and weel after drug combination were due to changes in the protein amount, rather than to changes in the phosphorylation degree.

Enzyme kinetics of thymidine kinase isoenzymes of Ehrlich ascites tumour.

Three isoenzymes of thymidine kinase (TK) identified on Ehrlich ascites tumour corresponding to pl values of 5.3, and 6.9 and 8.3 were studied with respect to their kinetic properties. The isoenzymes were separated by thin-layer isoelectric focusing and measured in the presence of thymidine (TdR) as substrate, adenosinetriphosphate (ATP) as phosphor donor and deoxythymidinetriphosphate (dTTP) as inhibitor. Exponentially growing cells and growth inhibited cells were used with high proportions of the isoenzymes at pl values of 6.9 and 8.3, and 5.3 respectively. The concentrations of the former were further enhanced by the use of S-phase cells isolated by elutriator centrifugation. The isoenzymes were identified as TK1-onc, earlier found in human leukemia cells. In order to confirm the existence of TK1, particular at pl value 5.3, the ability of the isoenzymes to use different phosphor donors (ATP and uridinetriphosphate (UTP)) and substrates (TdR and deoxycytidine (dCdR) was also studied. These results showed that the isoenzymes at pl 6.9 and 8.3 correspond to TK1, while the isoenzyme at pl 5.3 is heterogeneous. Further, high resolution isoelectric focusing confirmed the existence of two isoenzymes at pl values of 5.1 and 5.3, representing mitochondrial (TK2) and cytoplasmic (TK1) isoenzymes.