Acknowledgments
The work was fi nancially supported by the DFG Excellence Cluster
“Infl ammation at Interfaces” and the “Doktor Robert Pfl eger Stiftung Bamberg.” D. Zapf is acknowledged for technical assistance.
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Mikhail A. Nikiforov (ed.), Oncogene-Induced Senescence: Methods and Protocols, Methods in Molecular Biology, vol. 1534, DOI 10.1007/978-1-4939-6670-7_16, © Springer Science+Business Media New York 2017
Chapter 16
Detection of Nucleotide Disbalance in Cells Undergoing Oncogene-Induced Senescence
Mikhail A. Nikiforov and Donna S. Shewach
Abstract
DNA damage response has been characterized as an important mediator of senescence phenotypes induced by activated oncogenes in normal human cells. Depletion of intracellular deoxyribonucleotide pools has been recently recognized as one of the major causes for DNA damage in these cells. Cells undergoing oncogene-induced senescence display decreased expression of several rate-limiting enzymes involved in the biosynthesis of deoxyribonucleotides, including thymidylate synthase (TS) and ribonucleotide reductase (RR). Individual depletion of these enzymes leads to premature senescence. Reciprocally, ectopic expres- sion of TS and RR or addition of deoxyribonucleosides resulted in suppression of senescence phenotypes in normal or tumor cells caused by overexpression of activated HRAS or depletion of C-MYC, respectively.
Therefore, in the current chapter, we will describe a methodology for the quantitative measurement of nucleotide pools in senescent cells.
Key words Thymidylate synthase (TS) , Ribonucleotide reductase (RR) , C-MYC , DNA damage
1 Introduction
Oncogene-induced senescence (OIS) in normal cells is character- ized by a set of intracellular processes ultimately resulting in sup- pression of proliferation of premalignant cells [ 1 – 3 ]. Due to this feature, OIS is considered a major impediment to oncogenic trans- formation [ 4 ]. Compelling data demonstrates that DNA damage response is the major factor contributing to OIS [ 5 – 7 ]. For a long time, oncogene-induced oxidative stress and hyper-replication of genomic DNA (multiple fi ring of the same origin of replication) were considered the two major causes for DNA damage in oncogene- expressing senescent cells. For instance, it has been demonstrated that generation of reactive oxygen species (ROS) via HRAS G12V -dependent induction of NADPH oxidase 4 (NOX4) is an important mediator of HRAS G12V -induced senescence [ 8 ]. On the other hand, ectopic expression of activated HRAS in human fi broblasts caused multiple fi ring of the same replication origin of
genomic DNA, which resulted in DNA replication fork-stalling, generation of single and double-stranded DNA breaks, and ulti- mately, activation of the DNA damage response [ 7 ].
However, since then experimental evidence has been accumu- lated suggesting that suppression of nucleotide biosynthesis repre- sents another major cause of DNA damage and OIS in normal and transformed cells. First, it has been demonstrated that upregulation of nucleotide metabolism is essential for proliferation promoting functions of oncoprotein C-MYC [ 9 , 10 ]. Genes encoding three major rate-limiting enzymes for nucleotide biosynthesis ( thymidylate synthase (TS) , inosine monophosphate dehydrogenase 2 ( IMPDH2 ), and phosphoribosyl pyrophosphate synthetase 2 (PRPS2) ) were identifi ed as bonafi de MYC targets [ 10 ]. Moreover, proliferation of cells affected by shRNA-mediated depletion of C-MYC was partially restored by coexpression of cDNAs for these genes [ 10 ]. In parallel, we demonstrated that upregulation of C-MYC is required for con- tinuous suppression of OIS in melanocytic cells suggesting a func- tional connection between nucleotide metabolism and OIS [ 11 , 12 ].
This connection was further elaborated on in a study demonstrating the activation of the Rb-E2F pathway by HPV-16 E6 and E7 or the overexpression of cyclin E resulting in depletion of intracellular nucleotide levels and DNA damage [ 13 ]. Furthermore, the overex- pression of C-MYC upregulated intracellular nucleotide pools and decreased replication- dependent DNA damage [ 13 ].
In follow-up studies, we and others established that depletion of deoxyribonucleotide pools plays a causative role in induction of DNA damage in cells undergoing OIS [ 14 – 16 ]. Overexpression of different combinations of cDNAs for enzymes required for deoxyri- bonucleotide biosynthesis ( TS , and ribonucleotide reductase sub- units M1 and M2 [RRM1 and RRM2]) resulted in elevation of dNTP pools originally depleted by ectopic expression of HRAS G12V in normal human fi broblasts [ 15 ]. Furthermore, fi broblasts overex- pressing the above enzymes demonstrated a substantial reduction in HRAS G12V -dependent senescence-associated phenotypes, including DNA damage [ 15 ]. Of note, proliferation arrest caused in human fi broblasts by serum deprivation ( quiescence ) also resulted in deple- tion of dNTP pools; however, no DNA damage was detected [ 14 ].
Accordingly, overexpression of the same enzymes was insuffi cient in promoting proliferation of serum-deprived fi broblasts, suggesting that, unlike quiescence , OIS occurs in cells where DNA replication is activated in the presence of depleted dNTP pools [ 15 ]. Indeed, generation of DNA strand breaks leading to activation of DNA damage response has been described as a major cytotoxicity pathway of several antineoplastic agents inhibiting nucleotide metabolism.
These agents include inhibitors of TS (5-Fluorouracil) and RR (hydroxyurea) [ 17 ]. Genetic inhibition of TS and RR also induces DNA damage and senescence-associated phenotypes in normal human fi broblasts [ 15 ]. Therefore, under conditions of low
nucleotide levels, an increase in the number of replication forks (due to overexpression of activated HRAS) will result in generation of higher levels of DNA damage than under normal conditions.
The molecular mechanisms governing induction or reactiva- tion of senescence programs in tumor cells still remain poorly understood. Downregulation of oncoprotein C-MYC has been identifi ed as one such mechanism [ 11 , 18 ]. Interestingly, depletion of C-MYC in tumor cells induces senescence phenotypes similar to those caused by depletion or pharmaceutical inhibition of TS or RR [ 11 , 12 ]. Moreover, senescence caused by downregulation of C-MYC in tumor cells can be abrogated by overexpression of TS and RR cDNAs; and similar effects can be achieved by supplement- ing cell media with deoxyribonucleosides [ 12 ]. Additionally, exog- enous deoxyribonucleosides are able to suppress senescence caused by activated HRAS in normal cells [ 15 , 16 ]. The latter observation may be important for prevention or early treatment of certain types of tumors such as melanomas . Approximately 30–50 % of melano- mas originate from benign melanocytic nevi, aggregates of senes- cent melanocytes most commonly expressing mutant BRAF V600E (~60 %) or NRAS Q61R (~20 %) [ 19 , 20 ]. Therefore, melanoma must develop mechanisms overcoming OIS and upregulation of nucleo- tide metabolism appears to be one of the most likely candidates.
In this chapter, we will describe a comprehensive methodology for measuring the intracellular amounts of ribo- and deoxyribo- nucleotides in control cells and cells undergoing HRAS G12V - induced senescence; however, the same methodology could be applied to any senescent cells.
2 Materials