A molecular analysis of dihydropyrimidine dehydrogenase
Dihydropyrimidine dehydrogenase (DPD) is the rate-limiting enzyme in the reductive catabolism of the pyrimidine bases uracil and thymine. The clinical relevance of this enzyme is illustrated in individuals presenting with the inherited metabolic disorder thymine uraciluria. This syndrome is characterised by high plasma concentrations of thymine and uracil, and may result in clinical features including mental retardation and dysmorphia. DPD is also clinically relevant in the metabolism and subsequent inactivation of the chemotherapeutic agent 5- fluououracil (5FU). DPD activity has been shown to be highly variable in populations of healthy volunteers and cancer patients, but the mechanisms of regulation of DPD activity are as yet poorly understood. The extent of this variation may determine the efficacy or the severity of the side effects of this treatment. The aim of this research was to evaluate DPD in terms of mRNA expression, protein expression, and activity in a variety of normal and tumour tissues in an attempt to gain an insight into the regulation of DPD. Protein expression and catalytic activity were measured using the well-characterised techniques of Western blotting, and the HPLC separation of 5FU metabolites respectively. However, the method evaluating DPD mRNA expression needed to be developed and validated. After the appraisal of various mRNA detection and quantitation methodologies, competitive polymerase chain reaction (cPCR) was selected as the most suitable method for evaluating DPD transcription in these studies. The RNA samples are reverse transcribed into cDNA which then undergoes PCR amplification in the presence of known amounts of a synthetic template ('competitor') and competes for PCR primers with the target of interest. In each PCR reaction different quantities of target and competitor PCR product will be of both PCR products the concentration of the target template in the cDNA sample can be determined. Competitive PCR was demonstrated to be a highly sensitive and specific method for quantitating DPD mRNA expression, and could be used for tissues with both high and low levels of DPD (liver colon respectively). The technique was also found to be highly reproducible and reliable and was deemed to be suitable for use in further studies. To gain an understanding of the regulation of DPD in colorectal tumour, and the effect it may have upon the activity of 5FU in a specific location, the expression/activity profile of DPD was assessed in colorectal tumour, matched normal colorectal tissue, colorectal metastases to liver, and matched normnal liver. DPD activity, mRNA, and protein levels were all significantly higher in the normal liver than colon, and in the normal liver compared to liver metastases. In the colorectal tissues, mRNA levels were significantly lower in the colorectal tumour than normal colonic mucosa, however no significant difference could be determined between tissues for DPD protein and activity. A good relationship was determined between DPD activity and protein expression in colorectal tumour tissue (rs=0.61, p=0.01), whereas a weaker relationship was determined between DPD mRNA and activity for all colorectal tumour, metastases, and normal tissues (0.43, p 0.1). DPD activity has been detected in most tissues tested to date but appears to be tissue specific with higher levels observed in liver and peripheral blood mononuclear cells than other tissues. In these studies, DPD mRNA, protein, and activity were all found to be higher in the human liver tissue than normal colon.