Depletion of Deoxyribonucleotide Pools Is an Endogenous Source of DNA Damage in Cells Undergoing Oncogene-Induced Senescence

Abstract

In normal human cells, oncogene-induced senescence (OIS) depends on induction of DNA damage response. Oxidative stress and hyperreplication of genomic DNA have been proposed as major causes of DNA damage in OIS cells. Here, we report that down-regulation of deoxyribonucleoside pools is another endogenous source of DNA damage in normal human fibroblasts (NHFs) undergoing HRAS(G12V)-induced senescence. NHF-HRAS(G12V) cells underexpressed thymidylate synthase (TS) and ribonucleotide reductase (RR), two enzymes required for the entire de novo deoxyribonucleotide biosynthesis, and possessed low dNTP levels. Chromatin at the promoters of the genes encoding TS and RR was enriched with retinoblastoma tumor suppressor protein and histone H3 tri-methylated at lysine 9. Importantly, ectopic coexpression of TS and RR or addition of deoxyribonucleosides substantially suppressed DNA damage, senescence-associated phenotypes, and proliferation arrest in two types of NHF-expressing HRAS(G12V). Reciprocally, short hairpin RNA-mediated suppression of TS and RR caused DNA damage and senescence in NHFs, although less efficiently than HRAS(G12V). However, overexpression of TS and RR in quiescent NHFs did not overcome proliferation arrest, suggesting that unlike quiescence, OIS requires depletion of dNTP pools and activated DNA replication. Our data identify a previously unknown role of deoxyribonucleotides in regulation of OIS.

Pharmacological targeting of guanosine monophosphate synthase suppresses melanoma cell invasion and tumorigenicity

“…Lentiviral infection protocol was described previously. 50,51 All infected cells were briefly selected for resistance to puromycin and used in the described assays.…”

DNA replication stress-induced loss of reproductive capacity inS. cerevisiaeand its inhibition by caloric restriction

“…46 Similarly, activated Ras induces senescence in normal human fibroblasts by downregulating RNR and thymidylate synthase, which leads to a deficiency in dNTP pools and DNA damage. 47 It was also recently reported that the uncoordinated activation of growth signaling pathways that promote entry into S phase leads to depletion of dNTPs and genome instability in cultured human cells transformed by ectopic expression of cyclin E or oncogenic viral proteins. 48 Similar to mutations that activate oncogenes, elevated glucose activates PI3K/AKT/mTOR signaling and other oncogenic signaling pathways in cultured mammalian cells.…”

Oncogenes create a unique landscape of fragile sites

“…Additionally, oncogenic Ras expression 3 and overexpression of cyclin E induce replication stress, resulting in DNA DSBs and chromosomal rearrangements 4 . For both oncogenes insufficient nucleotide pools have been shown to be involved in the generation of replication stress 4,5 .…”

“…In early stages of cancer development, oncogene activation leads to replication stress [1][2][3][4] . Mechanisms by which oncogenes can induce replication stress include insufficient nucleotide pools to support extensive replication 4,5 , lack of other replication factors 6 and collision between replication and transcription 7 . In addition, a recent study showed that loss of FHIT expression encoded by the fragile histidine triad gene located in the fragile site locus FRA3B can initiate replication stress and continue genomic instability 8 .…”