![Metabolism of 5-FU, deoxyuridine, and dUTP in Brain Mitochondria: Implications for Current Treatment Options in GliomaKathleen McCann1,2, David Williams3, and Edward McKee1,2,31 Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, South Bend, IN 46617 USA2 Eck Institute for Global Health, University of Notre Dame, Notre Dame, IN 46556, USA3 Department of Biological Sciences, University of Notre Dame, Notre Dame, IN 46556 USA 5-FU (5-flourouracil) is currently in clinical trials for treatment of gliomas.1 In neoplastic cells, this drug is metabolized by two pathways. 5-FU is converted to 5-dUMP by thymidine phosphorylase and thymidine kinase. 5-FdUMP then competitively inhibits the conversion of dUMP to TMP by thymidylate synthase. This increases dUTP and depletes theTTP needed for DNA synthesis and repair, leading to apoptosis. 5-FdUMP is further phosphorylated to 5-FdUTP and is incorporated into DNA. In the second pathway, 5-FU is phosphorylated by actions of uridine phosphorylase and uridine kinase to 5-FUMP and then further phosphorylated to 5-FUTP and incorporated into RNA. However, the metabolism of 5-FU is as yet undefined in brain. 5-FU neurotoxicity may present as leukoencephalopathy, cerebellar signs, or acute demyelination. The use of thymidine infusions to reverse symptoms strongly suggested that the neurotoxicity is caused by dNTP pool imbalance. It is necessary to understand the underlying mechanisms to minimize neurotoxicity and judiciously use therapeutic thymidine. Deoxyuridine metabolism in the brain mitochondria is not fully understood. While a small amount is needed for RNA synthesis, incorporation of dUTP into mitochondrial DNA (mtDNA) can lead to mutagenesis and apoptosis, especially as base excision repair is known to be inefficient in brain mtDNA. These investigations aim to establish the activity of the enzymes necessary for regulation of dUTP pools in brain mitochondria: dUTPase, thymidine phosphorylase, and thymidine synthase. We also investigate the metabolism of 5-flourouricil (5-FU), the metabolite of which, 5-FdUMP, functions as a dUMP analogue. B A Thymidine Thymidine TMP TMP TTP TTP TDP TDP Figure 4. Phosphorylation of 3H-Thymidine (A) and phosphorylation of 3H Thymidine (B) quantified as pmol/mg protein/hour . The 5-FU did not appear to compete with thymidine for binding on enzyme thymidine kinase 2. There was no significant effect on the total amount of TTP created. B A dUTP dU dUMP dUMP dU dUDP dUDP dUTP Methods Figure 5. Phosphorylation of 3H-dU (A) and de-phosphorylation of 3H dUTP to dUMP (B) quantified as pmol/mg protein/hour . The enzyme dUTPase catabolizes much more dUTP to dUMP over the time course than the enzyme thymidine kinase 2 phosphorylates dU to dUMP. This is likely an important mechanism in maintaining low levels of dUTP in brain mitochondria. Mitochondria were isolated from freshly removed brains from adult Harlan Sprague Dawley rats. The protein content was measured by the method of Lowry and the intactness of the brain mitochondrial preparation was determined by measuring the respiratory control ratio (RCR). Mitochondria with RCR values over 5 were incubated at a final concentration of 4 mg protein /ml in media with labeled and unlabeled deoxynucleosides and deoxynucleoside analogs. Samples of the mitochondrial incubation were removed at specific time points and combined with an equal volume of 10% trichloroacetic acid to lyse mitochondria and precipitate the protein and nucleic acids. This mixture was placed on ice, centrifuged, and the supernatant extract neutralized by addition of AG-11A8 resin. Labeled deoxynucleosides and phosphorylated products in the filtered extracts were analyzed and quantitated by HPLC on an Alltech nucleoside-nucleotide reverse phase column coupled to an inline UV monitor and liquid scintillation counter as previously described.2Peaks were identified by comparison to standards. Discussion In terminally differentiated cells, mitochondria are now thought to play a key role in pathogenesis of neoplastic disease. Mitochondria in glioma have dysfunctional apoptotic pathways, altered energy metabolism, and abnormal counts of mtDNA. Many traditional chemotherapeutic agents act by inhibiting the synthesis of deoxypyrimidines, especially that of thymidine to TTP, and subsequently disrupt DNA synthesis. Neurotoxicity is a factor limiting the use of many agents. This may be because normal brain mitochondria are vulnerable to dNTP pool imbalances, disrupting mtDNA synthesis and repair; the dNTP pool in brain mitochondria is only about 1% of that found in an actively replicating cell. In the small mitochondrial dNTP pool, the availability of TTP is not sufficient to protect against incorporation of dUTP into mtDNA, especially since mtDNA polymerases do not readily correct U:A mismatches. The dNTP ratios must be strictly controlled. Here, we demonstrate the presence of a highly active and specific dUTPase It plays a critical role in the metabolism of dU, which seems to be a process of futile cycling: The dU is phosphorylated to dUMP and any further phosphorylation to dUTP occurs at a slower rate than the dephosphorylation back to dUMP, as seen in Figure 5. This is also seen in Figure 1 where there is actually no detectable dUTP. There was no evidence of thymidine phosphorylase activity in brain mitochondria (Figures 1, 2, & 3). Thymidine phosphorylase catabolizes thymidine to thymine, as well as deoxyuridine to uracil. It also converts 5-FU to 5-FdURd, the precursor for 5-FdUMP. Lack of this enzyme may make brain mitochondria more vulnerable to dNTP pool imbalances. There was no TMP detected in these studies (Figures 1 & 3). The lack of TMP indicates an absence of the enzyme thymidylate synthetase. In replicating cells, thymidylate synthetase maintains dUTP:TTP ratios by catalyzing the reductive methylation of dUMP to TMP and dihydrofolate in the de novo synthesis pathway. This is This lab has previously demonstrated that brain mitochondria rely on the salvage pathway for maintenance of dNTP pools. The current trials using 5-FU specifically target thymidylate synthetase, known to be upregulated in high grade glioma. We demonstrate, however,as seen in Figure 2 that 5-FU is phosphoryated to 5-FUTP, and can cause mutagenesis by incorporation into RNA. This suggests are more likely mechanism for neurotoxicity than thymidine depletion. This is further supported by the data shown in Figure 4, in which 5-FU had no effect on mitochondrial TTP synthesis. Results Figure 1. HPLC results of samples taken from incubations at 180 minutes. Isolated rat brain mitochondria were able to transport dU across the inner membrane into the matrix. dU is phosphorylated to dUMP by thymidine kinase 2. Of note, there is no evidence of conversion of dUMP to TMP. There was no breakdown of dU to uracil. DPM Column Retention Time in Minutes Figure 2. HPLC results of samples taken from incubations at 180 minutes. Isolated rat brain mitochondria were able to transport 5-FU across the inner membrane into the matrix. 5-FU is likely phosphorylated to 5-FUMP by the activity of uridine phosphorylase and uridine kinase. 5-FUMP is then phosphorylated to 5-FUDP by nucleotide monophosphate kinase and then to 5-FUTP by nucleoside diphosphokinase. There is no evidence of conversion 5-FdUrd or 5-FdUMP. DPM Conclusions Column Retention Time in Minutes ,[object Object]](https://image.slidesharecdn.com/5fuposter-13059186846443-phpapp02-110520143146-phpapp02/85/Metabolism-of-5-FU-deoxyuridine-and-dUTP-in-Brain-Mitochondria-1-320.jpg)
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Brain Mitochondria Metabolism of 5-FU, dU, and dUTP in Glioma Treatment
- 2. dU metabolism in brain mitochondria is likely a process of futile cycling, and dUTP levels in brain mitochondria are regulated by a highly active dUTPase enzyme to limit mutagenic incorporation into mtNDA.
- 3. There is no evidence of thymidine phosphorylase or thymidylate synthetase activity in brain mitochondria. Figure 3. HPLC results of samples taken from incubations at 180 minutes. Isolated rat brain mitochondria were able to transport dUTP across the inner membrane into the matrix. dUTP was rapidly and nearly completely dephosphorylated to dUMP. dUMP was then dephosphorylated to dU by the enzyme 5’-nucleotidase. As above, there was no conversion to TMP nor was there breakdown of dU to uracil. DPM References: 1 ClinicalTrials.gov Identifier: NCT00717197 2 McKee, E.E., et al., Phosphorylation of thymidine and AZT in heart mitochondria: elucidation of a novel mechanism of AZT cardiotoxicity. CardiovascToxicol, 2004. 4(2): p. 155-67. Column Retention Time in Minutes