In cells with dysfunctional mitochondria (HeLa ρ0) a phenotype characterized by an imbalance in deoxyribonukleotid triphosphate (dNTP) pools and a decrease in the levels of pyrimidine dNTP is observed. In this context, chromosomal instability was also observed. The relationship between dysfunctional mitochondria and the observed phenotype is not yet clear. DHODHase, the fourth enzyme in de novo biosynthesis of pyrimidine, has been proposed as the link. DHODHase are located in the inner mitochondrial membrane and its activity depends on a functional electron transport chain. It is therefore proposed that dysfunctional mitochondria may affect DHODHase and results in the observed phenotype. By inhibiting DHODHase in vitro with the two modulators leflunomide (LFM) and its active metabolite A77 1726 it can be examined if an inhibition of DHODHase is involved in this phenotype. The purpose was to incubate a human cervical cancer cell line (HeLa ρ+) and an osteosarcoma cell line without thymidine kinase activity (TK- 143B) with LFM and A77 1726 and to measure dNTP pool levels and cell cycle distribution. Because of the time frame for this project an inhibition of DHODHase has only been studied in Hela ρ+ cells using LFM. The results show that 21 hours LFM treatment leads to a dNTP pool imbalance and a decrease in dTTP, dATP and dGTP pools. The dNTP purines were most sensitive. LFM did also have an inhibitory effect on cell cycle progression, since the percentage of Hela ρ+ cells in G0/G1 phase increased by LFM treatment. These differences in the cell cycle distribution do not seem to account for the observed changes in the dNTP pool levels in Hela ρ+ cells treated with LFM. Based on this study an inhibition of DHODHase does not seem to be the link between the decrease in dNTP pyrimidines observed in ρ0 cells. It is not possible to exclude if a more potent inhibition of DHODHase by A77 1726 would cause a decrease in the dNTP pyrimidines.
|Uddannelser||Molekylærbiologi, (Bachelor/kandidatuddannelse) Bachelor el. kandidat|
|Udgivelsesdato||25 jun. 2009|
- dNTP pool
- Dysfunctional mitochondria