Rapid disappearance of deoxyribose-1-phosphate in platelet derived endothelial cell growth factor/thymidine phosphorylase overexpressing cells

doi:10.1016/S0006-291X(03)00022-6

Biochemical and Biophysical Research Communications

Volume 301, Issue 3, 14 February 2003, Pages 675-679

https://doi.org/10.1016/S0006-291X(03)00022-6 Get rights and content

Abstract

Platelet derived endothelial cell growth factor/thymidine phosphorylase (PD-ECGF/TP) catalyzes the phosphorolysis of thymidine (TdR) to thymine and deoxyribose-1-phosphate (dR-1-P) and has a pro-angiogenic effect for which dR-1-P may be responsible. Using a purine nucleoside phosphorylase based assay it was found that TdR incubation did not increase dR-1-P accumulation in colon cancer cell line Colo320 and its PD-ECGF/TP transfected variant Colo320TP1. The assay was linear up to 25,000 pmol dR-1-P with complete recovery of dR-1-P from cellular extracts. There was a huge discrepancy between thymine production and the measured dR-1-P level, 0.05% of the expected value for dR-1-P was found, indicating that there was a rapid disappearance of dR-1-P. However, in cellular extracts, TdR incubation increased dR-1-P, measurable by trapping, which was inhibited by a thymidine phosphorylase inhibitor. dR-1-P directly added to cellular extracts disappeared within 5–10 min. In conclusion, large amounts of dR-1-P are produced by Colo320TP1 cells, which rapidly disappear thus not resulting in a net accumulation of dR-1-P in these cells.

Section snippets

Materials and methods

Chemicals. Dulbecco’s modified Eagle’s medium (DMEM) and fetal calf serum (FCS) were obtained from Gibco-BRL (Life technology, Breda, The Netherlands). Deoxyribose-1-phosphate (dR-1-P), deoxyinosine (dino), and purine nucleoside phosphorylase (PNP) were purchased from Sigma chemicals (St. Louis, MO, USA). Ribose-1-phosphate (R-1-P), inosine (ino), hypoxanthine, and polyethylene imine (PEI) cellulose thin layer chromatography (TLC) sheets were purchased from Merck (Darmstadt, Germany). [8- $^{14} C$

Results

In order to study the effect of PD-ECGF/TP on cellular dR-1-P levels, cells overexpressing PD-ECGF/TP were incubated with thymidine after which dR-1-P and thymine levels were measured.

We checked linearity of the assay by addition of known amounts of dR-1-P to Colo320TP1 supernatants prepared in the dR-1-P buffer (pH 10) to construct a calibration line. This was performed with two concentrations of [8- $^{14} C$ ]hypoxanthine, 6.2 and 26 nmol (Fig. 1), to determine the optimal hypoxanthine

Discussion

Using a colon cancer cell line and its PD-ECGF/TP transfected variant, we observed that thymidine degradation was not associated with cellular dR-1-P accumulation. Apparently dR-1-P formed in this reaction was rapidly metabolized, either by degradation or conversion to other sugars, e.g., in the pentose phosphate pathway or glycolysis.

As expected, incubation with TdR did not result in a measurable production of thymine in the Colo320 cells, but in the transfected Colo320TP1 we demonstrated

Acknowledgements

This study was sponsored by the Spinoza award from NWO, received by Prof. Dr. H.M. Pinedo.

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In addition, the converted TdR was found in the cytoskeleton and to some extent in the cell membrane. Previously we demonstrated that dR-1-P formed from TdR rapidly disappeared from Colo320 TP1 cells [11]. dR-1-P can be isomerized to dR-5-P mediated by phosphopentomutase (E.C. 5.4.2.7) and is degraded to dR by a phosphatase [10].
Thymidine phosphorylase (TP) is often overexpressed in cancer and potentially plays a role in the stimulation of angiogenesis. The exact mechanism of angiogenesis induction is unclear, but is postulated to be related to thymidine-derived sugars. TP catalyzes the conversion of thymidine (TdR) to thymine and deoxyribose-1-phosphate (dR-1-P), which can be converted to dR-5-P, glyceraldehyde-3-phosphate (G3P) or deoxyribose (dR). However, it is unclear which sugar accumulates in this reaction. Therefore, in the TP overexpressing Colo320 TP1 and RT112/TP cells we determined by LC–MS/MS which sugars accumulated, their subcellular localization (using ³H-TdR) and whether dR was secreted from the cells. In both TP-overexpressing cell lines, dR-1-P and dR-5-P accumulated intracellularly at high levels and dR was secreted extensively by the cells. A specific inhibitor of TP completely blocked TdR conversion, and thus no sugars were formed. To examine whether these sugars may be used for the production of angiogenic factors or other products, we determined with ³H-TdR in which subcellular location these sugars accumulated. TdR-derived sugars accumulated in the cytoskeleton and to some extent in the cell membrane, while incorporation into the DNA was responsible for trapping in the nucleus. In conclusion, various metabolic routes were entered, of which the TdR-derived sugars accumulated in the cytoskeleton and membrane. Future studies should focus on which exact metabolic pathway is involved in the induction of angiogenesis.
Evaluation of 5′-deoxy-5′-[F-18]fluorothymidine as a tracer of intracellular thymidine phosphorylase activity
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Two human cell lines (A549 and U937) with cytosolic thymidine phosphorylase (TP) activity were used to evaluate the potential of 5′-deoxy-5′-[F-18]fluorothymidine ([F-18]DFT) as a tracer of intracellular TP expression. Cellular metabolism of DFT led to the production of 5-[F-18]fluoro-2,5-dideoxy-d-ribose-1α-phosphate ([F-18]FddR-1P), in analogy to the metabolism of thymidine, which produces 2-deoxy-d-ribose-1α-phosphate (dR-1P). A549 cells showed the highest production rate of FddR-1P. After A549 cells were exposed to [F-18]DFT for 40 min, the relative intracellular concentration of [F-18]FddR-1P was more than sevenfold higher in cells than its precursor in the incubating medium. For the same amount of time, a twofold concentration was seen in U937 cells. However, uptake ratios did not rank with the corresponding TP activities found in cell extracts [TP activity ratio (U937:A549)=1.6] that were independently determined with a labeled thymidine/thymine cleavage assay.
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Activity and substrate specificity of pyrimidine phosphorylases and their role in fluoropyrimidine sensitivity in colon cancer cell lines
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Thymidine phosphorylase (TP) and uridine phosphorylase (UP) are often upregulated in solid tumors and catalyze the phosphorolysis of natural (deoxy)nucleosides and a wide variety of fluorinated pyrimidine nucleosides. Because the relative contribution of each of the two enzymes to these reactions is still largely unknown, we investigated the substrate specificity of TP and UP in colon cancer cells for the (fluoro)pyrimidine nucleosides thymidine (TdR), uridine (Urd), 5′-deoxy-5-fluorouridine (5′DFUR), and 5FU. Specific inhibitors of TP (TPI) and UP (BAU) were used to determine the contribution of each enzyme in relation to their cytotoxic effect.
The high TP expressing Colo320TP1 cells were most sensitive to 5′DFUR and 5FU, with IC₅₀ values of 1.4 and 0.2 μM, respectively, while SW948 and SW1398 were insensitive to 5′DFUR (IC₅₀ > 150 μM for 5′DFUR). TPI and BAU only moderately affected sensitivity of Colo320, SW948, and SW1398, whereas TPI significantly increased IC₅₀ for 5′DFUR (50-fold) and 5FU (11-fold) in Colo320TP1 and BAU that in C26A (9-fold for 5′DFUR; p < 0.01). In the epithelial skin cell line HaCaT both inhibitors were able to decrease sensitivity to 5′DFUR and 5FU separately. HaCaT might be a model for 5′DFUR toxicity.
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Rapid disappearance of deoxyribose-1-phosphate in platelet derived endothelial cell growth factor/thymidine phosphorylase overexpressing cells

Abstract

Section snippets

Materials and methods

Results

Discussion

Acknowledgements

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

Biochem. Biophys. Res. Commun.

Biochem. Biophys. Res. Commun.

Biochem. Pharmacol.

Clin. Chim. Acta

Eur. J. Cancer Clin. Oncol.

J. Biochem. Biophys. Methods

Comp. Biochem. Physiol. B, Biochem. Mol. Biol.

Biochim. Biophys. Acta

Biochim. Biophys. Acta