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Gene-Expression Analysis of TP, DPD, and TS Using,,,the LightCycler mRNA Quantification KitsPLUS

In human cells, the enzymes thymidin phosphorylase (TP), dihydropyrimidine dehydrogenase (DPD), and thymidylate synthase (TS) play a pivotal role in the metabolism of pyrimidines. In cancer disease medical care, the cytotoxic compound 5-fluorouracil (5-FU) is the standard chemotherapy treatment for a wide range of solid tumors. Attempts to increase the efficacy and tolerability of fluoropyrimidine treatment has led to the development of Capecitabine (Xeloda), a fluoropyrimidine drug from Roche Pharmaceuticals. Capecitabine is not cytotoxic unless it is activated to 5-FU by TP a three-step mechanism. Therefore, it has several advantages over ordinary 5-FU by treatment [1, 2].



Capecitabine is preferentially activated and converted to 5-FU due to TP upregulation at the tumor site. 5-FU inhibits TS, an enzyme crucial for the de novo synthesis of thymidine nucleotides. The rate-limiting enzyme in the catabolism of 5-FU is DPD, which catalyzes the first step of pyrimidine degradation (Figure 1).

Efficacy and tolerability of Capecitabine or similar drugs may therefore largely depend on the expression rates of each of the three enzymes. These expression rates can be estimated by the detection of the relative mRNA expression levels of the three enzymes in tumor tissue or blood [3], using the new LightCycler TP, DPD, and TS mRNA Quantification KitsPLUS.

Materials and Methods

Fresh-frozen tissue and paraffin-embedded tissue (PET) slides (5 m) were taken from human xenografts in mouse models. Isolation of RNA was performed using the MagNA Lyser Instrument and the High Pure RNA Paraffin Kit. The human RNA
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... PP2A1, purified Formulation: 50mM Tris-HCl, ... Brij-35, 0.2mM PMSF, 1mM benzamidine, ... Properites: PP2A1 (Protein Phosphatase 2A1) ... ammonium sulfate precipitation, QSepharose,amino-hexyl agarose, ...
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