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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., (Copyright 2002 S. Karger AG, Basel)

The effects on incorporation into DNA of the deoxyribonucleotides dCTP and dTTP and the DNA synthesis rate after treatment with cisplatin (CDDP), 5-fluorouracil (5-FU) or a combination of CDDP and 5-FU were studied in ascites sarcoma (Bp8) growing in mice. Single administration of CDDP gave an early (1 h) transient increase in the DNA-synthesis followed by a decrease. 5-FU as single agent did increase the rate of DNA synthesis after 6 h with a maximum at 10 h. The combination of CDDP and 5-FU markedly increased the rate of DNA synthesis up to 6 h as compared to single drug treatment. Although the dCTP pool increased after combined treatment, while the dTTP pool was unchanged, no alterations in the proportions of dTTP and dCTP incorporated into DNA could be detected. Hence, misincorporation of pyrimidines is not the mechanism for the synergistic effect of the combination of CDDP and 5-FU.

In earlier studies we showed that a supra-additive cytotoxic effect was obtained with combined treatment with cisplatin and 5-fluorouracil (5-FU) of Bp8 ascites tumor growing in vivo in mice. We therefore studied the DNA interstrand cross-linking after single drug treatment with cisplatin, 5-FU and the drugs combined. The alkaline elution technique was used. 5-FU induced no measurable DNA cross-linking. Cisplatin caused a dose-dependent increase in DNA cross-links 6 h after treatment. When combining cisplatin and 5-FU, no significant change in the number of DNA cross-links was found for this time. The removal of DNA interstrand cross-links was studied after 24 and 36 h. There was no difference in the removal rate between single drug treatment with cisplatin as compared with the combined treatment with the two drugs. Also, the number of DNA single strand breaks (SSB) possibly created when the cisplatin induced cross-links or UV-induced DNA damage were repaired were measured with the alkaline elution technique. No increase in the amount of SSB was seen after combined treatment. Thus, we found no interaction of 5-FU and cisplatin in terms of DNA damage. Other mechanisms for the supra-additive effect found when combining cisplatin and 5-FU have to be considered.

We examined whether an increase in the salvage and/or the de novo synthesis of thymidine (TdR) can explain the elevated DNA synthesis rate found up to 15-20 h after combined treatment with cisplatin and 5-fluorouracil (5-FU), compared with single-drug regimen. The salvage and the de novo pathways of TdR in Bp8 mouse ascites tumor cells were reduced equally after the combined treatment and the single-drug treatments. The inhibition of the de novo pathway of TdR was confirmed by a reduced thymidylate synthase activity, as measured in cell extract. A marked imbalance of the deoxyribonucleotide triphosphates were found, in particular between the deoxypyrimidines. These imbalances were similar between the 5-FU single-drug treatment and combined treatment. We conclude that neither the extracellular TdR salvage nor the de novo synthesis of TdR explain the relatively elevated DNA synthesis rate after combined treatment. We suggest that the supra-additive effect of the combined treatment is due to an interaction between the elevated DNA synthesis, the imbalanced deoxyribonucleotides and the cisplatin-induced DNA cross-links, and possibly also due to a higher concentration of 5-FU incorporated into DNA.

The aim of this study was to elucidate the mechanism(s) behind the cellular toxicity of therapeutic concentrations of hydroxyurea (HU). Treatment of human T lymphoma cells (CCRF-CEM) with 60-100 microM of HU for 24 h decreased the growth rate by 90% due to accumulation of cells in early S phase. It induced a marked imbalance in both the DNA/protein cycle (as measured by two-parameter flow cytometry) and the deoxyribonucleotide (dNTP) pools. HU treatment did not enhance the frequency of DNA single-strand breaks (SSBs), as measured by the alkaline unwinding technique. Cell viability was unaffected. However, removal of HU led to 10-15% cell loss during the following 12 h period in parallel with increasing SSBs, and a rapid progression of cells through S and G2 stages. The unbalanced DNA to protein content per cell and the dNTP pools were normalized 6-12 and 24 h after removal of HU, respectively. These results show that marked changes in the DNA to protein ratio and dNTP pools alone are not directly lethal, but when combined with a high replicative DNA synthesis rate, as found after removal of HU, apparently lead to elevated cell death.

The effect of increasing temperature up to 43 degrees C on thymidine (TdR) salvage, i.e. uptake of TdR, thymidine kinase (TK) activity, intracellular amount of thymidinetriphosphate (dTTP) and specific activity of 3H-dTTP and incorporation of dTTP into DNA, was studied in ascites tumour cells in vitro. While the uptake of [3H]TdR was almost unchanged up to 40.5 degrees C and 2.4 h followed by a decrease at later times and higher temperatures, TK activity declined already after 30 min in a temperature-dependent way, leading to a decrease in cellular amount of dTTP reaching almost undetectable values at 43 degrees C and 6.0 h. The specific activity of [3H]dTTP in the cell was elevated at 39 degrees C and 43 degrees C, but somewhat reduced at 40.5 degrees C. Incorporation into DNA of [3H]TdR decreased in a temperature-dependent way with increasing incubation time. After correction for specific activity of [3H]dTTP, however, the incorporation of [3H]TdR into DNA at 39 degrees C and 40.5 degrees C was unchanged up to 2.4 h and for 40.5 degrees C up to 6.0 h, while incorporation into DNA at 43 degrees C was still reduced. The overall protein synthesis was also reduced in a temperature-dependent way. We concluded that phosphorylation of TdR to dTMP was more sensitive to moderately elevated temperatures than uptake of TdR and DNA synthesis per se, caused by a reduced TK activity. The decline in TK activity was probably due to a decrease in TK polypeptide synthesis.

Thymidine kinase (TK) and its isoenzymes were studied in relation to age of Ehrlich ascites tumour cells growing in vivo. Various steps of the pathway of thymidine through deoxynucleotide metabolism were studied: [3H]-thymidine cellular uptake and incorporation into DNA; the cellular nucleotide pools; and the concentration of thymidine in ascites. In addition, the proportion of cells in the various parts of the cell cycle and the bromodeoxyuridine labelling index were determined. Four isoenzymes at pI 4.1, 5.3, 6.9 and 8.3 were identified using isoelectric focusing. The TK activity declined with age of the tumour by about 90%, mostly due to a decrease of the isoenzyme at pI 8.3. However, this decline was neither related to the changes in DNA synthesis rate of the cells with tumour age, nor to the proportion of cells in S-phase or the bromodeoxyuridine (BrdU) labelling index. In contrast, the contribution of DNA synthesis via the thymidine salvage pathway relative to the total DNA synthesis increased from less than 1% at exponential growth to about 15% at plateau phase of growth. Blocking of DNA synthesis by aphidicolin did not change the TK activity. We therefore conclude that changes in TK activity and changes in cell growth are epiphenomena rather than causally related to each other. All nucleotide pools decreased with tumour age. The inhibition of TK by an increase in the deoxythymidine triphosphate pool could therefore be excluded. With a decrease of the TK activity during tumour growth, increasing amounts of TdR were excreted by the cells and accumulated in the ascites fluid. To explain our results on TK activity we propose a substrate cycle in which thymidine monophosphate supplied by de novo synthesis is dephosphorylated and is then either phosphorylated by TK to thymidine monophosphate or excreted by the cell.

Melphalan inhibits the incorporation of 3H-thymidine and 3H-uridine significantly more in phytohaemagglutinin (PHA)-stimulated human lymphocytes than in a human melanoma cell line (RPMI 8322). Melphalan - induced total DNA cross-linking was 1.7 times higher and DNA interstrand cross-linking was 1.8 times higher in the PHA-stimulated lymphocytes than in the melanoma cells. A higher level of DNA cross-linking was required in melanoma cells than in PHA-stimulated lymphocytes to obtain similar levels of inhibition of incorporation of 3H-thymidine and 3H-uridine. The outflow of cells from G1 to S phase was significantly more inhibited by melphalan in the lymphocytes than in the melanoma cells. Thus the melanoma cells can replicate and transcribe DNA in the presence of levels of DNA damage, which in PHA-stimulated lymphocytes strongly inhibit DNA and RNA synthesis.

Cellular uptake of [3H]thymidine [( 3H]TdR) and incorporation into DNA of Ehrlich ascites tumour cells were studied in relation to the cell cycle by measuring the activity in the acid-soluble and insoluble parts of the cell material. Cells were synchronized at various stages of the cell cycle using centrifugal elutriation. The degree of synchrony of the various cell fractions was measured by flow-cytofluorometric DNA analysis. From the cellular uptake, the TdR triphosphate (dTTP) concentration of a mean cell in an unseparated cell population was calculated to be 20 X 10(-18) mol/cell. The pool activity of G1 cells was unmeasurable but rose to maximum values at the border of the G1-S phase. It decreased again during G2. The [3H]TdR incorporation into DNA was low during early S phase, reached a maximum value at two-thirds of the S phase and decreased again during late S phase. These changes in DNA synthesis were not due to changes in the dTTP pool being a limiting factor. During maximum DNA synthesis, 10% X min-1 of the dTTP pool was utilized, at which time the pool size also decreased by about 30%. Changes in pool size during the cell cycle have to be taken into account when the results of incorporation of radioactive TdR into DNA are discussed.