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

In normal human cells, oncogene-induced senescence (OIS) depends on induction of DNA damage response (DDR). Oxidative stress and hyper-replication 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 (NHF) undergoing HRAS[superscript G12V]-induced senescence. NHF-HRAS[superscript G12V] cells under-expressed 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 RB tumor suppressor protein and histone H3 tri-methylated at lysine 9. Importantly, ectopic co-expression 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[superscript G12V]. Reciprocally, shRNA-mediated suppression of TS and RR caused DNA damage and senescence in NHF although less efficiently than HRAS[superscript G12V]. However, overexpression of TS and RR in quiescent NHF 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 oncogene-induced senescence.This is the author's peer-reviewed final manuscript, as accepted by the publisher. The published article is copyrighted by Elsevier and can be found at: http://www.journals.elsevier.com/the-american-journal-of-pathology/

Ribonucleotide reductase and thymidylate synthase or exogenous deoxyribonucleosides reduce DNA damage and senescence caused by C‐MYC depletion

The down‐regulation of dominant oncogenes, including C‐MYC, in tumor cells often leads to the induction of senescence via mechanisms that are not completely identified. In the current study, we demonstrate that MYC‐depleted melanoma cells undergo extensive DNA damage that is caused by the underexpression of thymidylate synthase (TS) and ribonucleotide reductase (RR) and subsequent depletion of deoxyribonucleoside triphosphate pools. Simultaneous genetic inhibition of TS and RR in melanoma cells induced DNA damage and senescence phenotypes very similar to the ones caused by MYC‐depletion. Reciprocally, overexpression of TS and RR in melanoma cells or addition of deoxyribonucleosides to culture media substantially inhibited DNA damage and senescence‐associated phenotypes caused by C‐MYC depletion. Our data demonstrate the essential role of TS and RR in C‐MYC‐dependent suppression of senescence in melanoma cells.Keywords: ribonucleotide reductase, oncogene‐induced senescence, dNTP, myc, melanoma, thymidylate synthas

A Purine Nucleotide Biosynthesis Enzyme Guanosine Monophosphate Reductase Is a Suppressor of Melanoma Invasion

Melanoma is one of the most aggressive types of human cancers, and the mechanisms underlying melanoma invasive phenotype are not completely understood. Here, we report that expression of guanosine monophosphate reductase (GMPR), an enzyme involved in de novo biosynthesis of purine nucleotides, was downregulated in the invasive stages of human melanoma. Loss- and gain-of-function experiments revealed that GMPR downregulates the amounts of several GTP-bound (active) Rho-GTPases and suppresses the ability of melanoma cells to form invadopodia, degrade extracellular matrix, invade in vitro, and grow as tumor xenografts in vivo. Mechanistically, we demonstrated that GMPR partially depletes intracellular GTP pools. Pharmacological inhibition of de novo GTP biosynthesis suppressed whereas addition of exogenous guanosine increased invasion of melanoma cells as well as cells from other cancer types. Our data identify GMPR as a melanoma invasion suppressor and establish a link between guanosine metabolism and Rho-GTPase-dependent melanoma cell invasion