( ...)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 panel was obtained from Clontech. Based on the LightCycler mRNA Quantification Kit concept, we have developed three new reagent kits for TP, DPD, and TS that are specially designed for usage with fresh-frozen tissues or PET in research applications. The kits are based on a two-step protocol for the quantification of target mRNA expression rates relative to a reference gene (glucose-6-phosphate dehydrogenase, G6PDH) using the LightCycler Instrument and the Relative Quantification Software.
In the first step (reverse transcription), gene-specific primers are utilized to obtain TP, DPD, TS, and G6PDH cDNA in separate reactions. Gene-specific priming is necessary as RNA from PET is degraded to fragments smaller than approximately 200 bp.
The cDNA is then used as a template for quantitative PCR using the LightCycler Hybridization Probe format. Additionally, calibrator RNA from an immortalized cell line is included in the kit and is used as an external standard to allow for relative quantification in each run.

Results and Applications

To validate the new kit concept we have performed three comparative studies:

Proof of principle specific priming

With the standard primer concentration (1 M), the amplification curves in cDNA synthesis tend to decline with decreasing template concentration (Figure 2). Reducing the concentration of the specific primers to 1/5 (0.2 M) leads to reproducible and stable curves in the reverse transcription- polymerase chain reaction (RT-PCR) dilution series, especially with low RNA concentrations (Figure 2). Template RNA in the range of 500 ng/l to as little as 32 pg/l was used. TP data are shown as being representative for DPD, TS, and G6PDH using calibrator RNA as a template.

The new primer concentration also extends the dynamic measuring range of the target and reference and improves sensitivity compared to random hexamer priming (Figure 3 a-c). Table 3d numerically shows the crossing point (Cp) ranges for all four genes TP, DPD, TS, and G6PDH.



The human RNA panel (healthy tissue)

We have also analyzed both priming concepts using a panel of 21 human RNA samples from healthy tissues. We observed a slight shift to smaller Cp values with specific priming for all four parameters TP, DPD, TS and G6PDH indicating the higher sensitivity of the new concept (Figure 4). These shifts depend on the parameter and the analyzed tissue type. Also, the comparison of the relative ratios reveals a tendency for slightly higher ratios with specific priming. This is caused by different Cp shifts between calibrator and sample RNA. Nevertheless, the expression rates are comparable and within a common range.



Comparison of fresh-frozen tissue vs. PET

For this study, we analyzed xenografts from human immortalized colon tumor cell lines HT29 and SW62 with the new kit concept. Total RNA was isolated from PET in 4 x 5-m slides and from matched fresh-frozen tissues in two 20-mg slices according to the High Pure RNA Paraffin Kit protocol. As depicted in Figure 5, the relative expression rates of TP, DPD, and TS are comparable in both tissue types.



The new kit concept with specific priming is superior to former hexamer protocols and shows comparable Cp values and ratios. The linear measuring ranges were extended and the sensitivity of analysis was improved for all three target genes, as well as for the reference gene G6PDH. The analysis of matched fresh-frozen tissues and PET yields nearly identical relative expression rates.

These preliminary and preclinical results suggest that the LightCycler Quantification KitsPLUS for TP, DPD and TS are well suited for the analysis of fresh-frozen tissue samples, as well as for retrospective investigations with PET samples in research applications.

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