Application of artificial neural network to investigate the effects of 5-fluorouracil on ribonucleotides and deoxyribonucleotides in HepG2 cells.

Jianru Guo,Zhihong Jiang,Qianqian Chen,Caiyun Wang,Wei Zhang,Christopher Wai Kei Lam,Fengguo Xu,Vincent Kam Wai Wong

doi:10.1038/srep16861

Abstract

Endogenous ribonucleotides and deoxyribonucleotides are essential metabolites that play important roles in a broad range of key cellular functions. Their intracellular levels could also reflect the action of nucleoside analogues. We investigated the effects of 5-fluorouracil (5-FU) on ribonucleotide and deoxyribonucleotide pool sizes in cells upon exposure to 5-FU for different durations. Unsupervised and supervised artificial neural networks were compared for comprehensive analysis of global responses to 5-FU. As expected, deoxyuridine monophosphate (dUMP) increased after 5-FU incubation due to the inhibition of thymine monophosphate (TMP) synthesis. Interestingly, the accumulation of dUMP could not lead to increased levels of deoxyuridine triphosphate (dUTP) and deoxyuridine diphosphate (dUDP). After the initial fall in intracellular deoxythymidine triphosphate (TTP) concentration, its level recovered and increased from 48 h exposure to 5-FU, although deoxythymidine diphosphate (TDP) and TMP continued to decrease compared with the control group. These findings suggest 5-FU treatment caused unexpected changes in intracellular purine polls, such as increases in deoxyadenosine triphosphate (dATP), adenosine-triphosphate (ATP), guanosine triphosphate (GTP) pools. Further elucidation of the mechanism of action of 5-FU in causing these changes should enhance development of strategies that will increase the anticancer activity of 5-FU while decreasing its resistance.

Highlights

Until recently, methods for assessment of RN and dRN pool sizes were not available
fluoro-2′ -deoxyuridine-5′ -monophosphate (FdUMP) binds to the nucleotide binding site of thymidylate synthase (TS) and blocks the binding of the normal substrate dUMP leading to inhibition of TMP synthesis[25,26]
Deoxyguanosine pool size decreased after incubation with 5-FU from 4 to 72 h, because the decrease in TTP represents the reduction of guanosine diphosphate (GDP), the deoxyguanosine pool level decreased according to the regulation mechanism of deoxyribouncleoside triphosphates (dNTP) synthesis (Fig. 13D—F)

Summary

Introduction

Methods for assessment of RN and dRN pool sizes were not available. We have developed such method to study the perturbation of RN and dRN in cancer cell lines incubated with hydroxyurea and aphidicolin[18]. A previous study has observed that 5-FU incubation of human colon carcinoma cells resulted in decrease of TTP and increase of dATP concentration without effect on deoxyguanosine triphosphate (dGTP) and deoxycytidine triphosphate (dCTP) concentration[27]. We investigated the effects of 5-FU incubation over different time-periods on RN and dRN pool sizes of a human hepatocarcinonma cancer (HepG2) cell line using HPLC/MS/MS methodology.

Results

Conclusion