- Authors: Basbous J. et al.
- Year: 2020
- Journal: Nucleic Acids Res 48 1886-1904
- Applications: in vitro / siRNA, siRNA and DNA cotransfection / INTERFERin, jetPEI, jetPRIME
- Cell types:
- Name: HCT 116
Description: Human colon carcinoma cells
Known as: HCT116
- Name: HEK-293T
Description: Human embryonic kidney Fibroblast
Known as: HEK293T, 293T
- Name: U-2 OS
Description: Human bone osteosarcoma
Known as: U2OS
- Name: HCT 116
SiRNAs were transfected with INTERFERin (Polyplus). Plasmids encoding cDNAs were transfected using jetPEI or jetPRIME reagent (Polyplus).
Imbalance in the level of the pyrimidine degradation products dihydrouracil and dihydrothymine is associated with cellular transformation and cancer progression. Dihydropyrimidines are degraded by dihydropyrimidinase (DHP), a zinc metalloenzyme that is upregulated in solid tumors but not in the corresponding normal tissues. How dihydropyrimidine metabolites affect cellular phenotypes remains elusive. Here we show that the accumulation of dihydropyrimidines induces the formation of DNA-protein crosslinks (DPCs) and causes DNA replication and transcriptional stress. We used Xenopus egg extracts to recapitulate DNA replication invitro. We found that dihydropyrimidines interfere directly with the replication of both plasmid and chromosomal DNA. Furthermore, we show that the plant flavonoid dihydromyricetin inhibits human DHP activity. Cellular exposure to dihydromyricetin triggered DPCs-dependent DNA replication stress in cancer cells. This study defines dihydropyrimidines as potentially cytotoxic metabolites that may offer an opportunity for therapeutic-targeting of DHP activity in solid tumors.